TCRαβ/CD3 disruption enables CD3-specific antileukemic T cell immunotherapy

Abstract

T cells engineered to express chimeric antigen receptors (CARs) against B cell antigens are being investigated as cellular immunotherapies. Similar approaches designed to target T cell malignancies have been hampered by the critical issue of T-on-T cytotoxicity, whereby fratricide or self-destruction of healthy T cells prohibits cell product manufacture. To date, there have been no reports of T cells engineered to target the definitive T cell marker, CD3 (3CAR). Recent improvements in gene editing now provide access to efficient disruption of such molecules on T cells, and this has provided a route to generation of 3CAR, CD3-specific CAR T cells. T cells were transduced with a lentiviral vector incorporating an anti-CD3ε CAR derived from OKT3, either before or after TALEN-mediated disruption of the endogenous TCRαβ/CD3 complex. Only transduction after disrupting assembly of TCRαβ/CD3 yielded viable 3CAR T cells, and these cultures were found to undergo self-enrichment for 3CAR+TCR-CD3- T cells without any further processing. Specific cytotoxicity against CD3ε was demonstrated against primary T cells and against childhood T cell acute lymphoblastic leukemia (T-ALL). 3CAR T cells mediated potent antileukemic effects in a human/murine chimeric model, supporting the application of cellular immunotherapy strategies against T cell malignancies. 3CAR provides a bridging strategy to achieve T cell eradication and leukemic remission ahead of conditioned allogeneic stem cell transplantation.

Keywords: Cell Biology; Gene therapy; Immunology; T cells.

Figure 1. Successful generation of 3CAR requires disruption of CD3ε expression prior to lentiviral 3CAR transduction.

(A) Vector configuration showing codon optimized single-chain variable fragment (scFv) derived from OKT3 with CD8 stalk and 41BB/CD3ζ activation domains, all under the control of an internal human phosphoglycerate kinase (hPGK) promoter in a third-generation self-inactivating (SIN) lentivirus with HIV-1–derived reverse response element (RRE), central polypurine tract (cPPT), and mutated woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Human cytomegalovirus (CMV); Repeat region (R); Unique 5’ region (U5); Psi (Ψ); and modified unique 3’ region (ΔU3). (B) Percentage of CAR⁺ cells with and without TRAC-KO prior to lentivirus (LV). Data are presented as mean ± SEM. n = 3 donors. (C) Schema of event precedence required for successful 3CAR T cell production. CD3/CD28 activation with TransAct for 48 hours was followed by electroporation of mRNA encoding TRAC-specific TALENs ahead of LV transduction by 72 hours. (D) Summary of CD3⁺ cells following TRAC-KO (n = 3 donors), mean ± SEM. ***P < 0.0005, by unpaired, 2-tailed Student’s t test. (E) Representative flow cytometry plots of day 7 cells. In cultures with 3CAR expression, there were no surviving CD3⁺ cells compared with 20% residual expression in the absence of 3CAR transduction (n = 3 donors). (F) Representative flow cytometry plots showing 3CAR T cells retained CD4 and CD8, but not TCRαβ, expression; n = 3 donors. (G) For comparison, control transductions with LV CAR19 mediated 65% transduction but required further processing by column-mediated TCRαβ depletion to yield TRAC-KO CAR19⁺ T cells; n = 3 donors. (H) Summary of CAR⁺ cells following TRAC-KO (n = 3 donors); mean ± SEM. Two-tailed Student’s t test.

Figure 2. 3CAR T cell expansion and self-enrichment.

(A) Primary T cells were activated, and following TALEN TRAC mRNA exposure, 2 × 10⁶ T cells were transduced and expanded in a G-Rex flask over 17 days; n = 2 donors. (B) Representative expression of CD3/TCR in TRAC-KO untransduced (top panel) and 3CAR T cells (lower panel) on day 7 and 13, confirming the absence of CD3/TCRαβ cells after 3CAR lentiviral expression; n = 3 donors. (C) Tracking indels by decomposition PCR (TIDE-PCR) confirmed 62.9% of alleles harbored molecular signatures of nonhomologous end joining across the TRAC locus (n = 2). (D) Representative flow cytometry plots at end of manufacture for CD20 and CD56 expression and exhaustion markers PD-1, LAG-3, and TIM-3 (gated on CAR⁺ and CAR^– populations); n = 3 donors. (E) The percentage of memory T cell markers on expanded day 17 cells; n = 3 donors, mean ± SEM.

Figure 3. 3CAR T cells mediate potent killing of CD3⁺ cells in vitro.

(A) TCR⁺ and TCR^– Jurkat target cells were stained for TCR expression prior to coculture experiments. (B) ⁵¹Cr-labeled CD3⁺TCR⁺ (white symbols) or CD3^–TCR^– (black symbols) Jurkat leukemia cells were cocultured with either 3CAR T cells (solid lines) or untransduced controls (dotted lines); (n = 3) from 3 donors. (C) Upper panel, 3CAR T cells were cocultured with either GFP⁺CD3⁺TCR⁺ or GPP⁺CD3^–TCR^– Jurkat leukemia cells and, in the lower panel, cocultured with either CFSE-loaded primary CD3⁺ or CD3^– cells at the effector target ratio of 1:1 for 24 hours. Representative flow cytometry plots of gated on GFP⁺ or CFSE⁺ cells at the end of coculture demonstrate target-specific killing. (D) Summary of 3CAR cytotoxicity as in C where data are presented as mean ± SEM; n = 3 per group. ***P < 0.0005, by unpaired, 2-tailed Student’s t test.

Figure 4. 3CAR T cell functionality against healthy donor PBMC.

(A) ⁵¹Cr-labeled peripheral blood mononuclear cell (PBMC) in autologous (dotted lines) or allogenic (solid lines) coculture. PBMC were cultured with either 3CAR T cells (white symbols) or untransduced controls (black symbols) (n = 3). (B) 3CAR, CAR19, or untransduced T cells were cocultured with CSFE-loaded healthy donor PBMC at the effector target ratio of 1:1 for 24 hours. Top panel shows representative frequency of gated CSFE⁺ target cells at the end of coculture. Flow cytometry plots below show representative frequency of surface antigen markers CD3, CD19, and CD7 on gated CSFE⁺ tumor cells, displaying the specificity in mixed cell populations; n = 3 donors. (C) Cytokine production by 3CAR T cells when cocultured with healthy donor PBMC target cells were comparable with untransduced cells; n = 3 donors. (D) Mixed lymphocyte reactions of 3CAR or untransduced T cells cultured with irradiated (*) allogeneic PBMC or CD3^– cells. ³H-thymidine proliferation responses are consistent with specific targeting of CD3 rather than alloreactive proliferation); mean ± SEM, 4 experimental replicates (n = 2 donors). ^#P < 0.05, by unpaired, 2-tailed Student’s t test. Corrected counters per minute, CCPM.

Figure 5. 3CAR T cells specifically target CD3 in childhood T-ALL patient cells.

(A) Representative flow cytometry plots gated on CSFE⁺ T-ALL tumor cells. Lower panel shows representative frequency of surface antigen CD3 and CD19 of gated CSFE⁺ tumor cells. (B) Percentage of CD3⁺ (n = 6) or CD19⁺ (n = 4) cells as in A from T-ALL donors. *P < 0.05, by unpaired, 2-tailed Student’s t test. (C) 3CAR (lower panels) or untransduced T cells (upper panels) were cocultured with CSFE-loaded T-ALL patient donors at the effector target ratio of 1:1 for 24 hours. Flow cytometry plots show the frequency of CD3 and CD34 cells gated on CSFE⁺ T-ALL tumor cells. Four of 6 patient samples were positive for CD34 expression.

Figure 6. Antileukemic responses by 3CAR effector T cells against CD3⁺TCR⁺ GFP/luciferase human leukemia in immunodeficient mice.

(A) Serial bioluminescence imaging (BLI) of NSG mice (representative images from each cohort) following i.p. administration of D-luciferin substrate showing elimination of CD3⁺ leukemia by 3CAR T cells but not by untransduced T cells. (B) Kinetics of systemic leukemia progression in mice following administration PBS (n = 2), untransduced T cells (n = 4), or 3CAR (n = 5) effectors. Error bars represent median with interquartile range. Linear regression analysis showed significance between 3CAR vs. untransduced (****P < 0.0001) and 3CAR vs. PBS (****P < 0.0001) groups. (C) Average radiance values at termination (day 18) indicated significant difference in disease burden between untransduced and 3CAR effector–injected groups (*P = 0.0159 by Mann-Whitney U test). Error bars represent median with interquartile range. (D) Upper panel, representative flow cytometry plots of T cells (gated on hCD45⁺ cells) in BM. Middle panel, plots of leukemic T cell (CD3⁺TCR⁺GFP⁺ Jurkat targets) after effector challenge (gated on hCD45⁺CD2⁺ cells). Lower panel, plots of CAR T cells population (gated on hCD45⁺CD2⁺GFP^– expression). (E) Proportion of GFP⁺ Jurkat leukemia or GFP^– effector T cells out of CD45⁺CD2⁺ population in BM of untransduced or 3CAR-injected mice. Red marking highlights an animal that exhibited selective outgrowth of GFP⁺CD3^– Jurkat cells after 3CAR therapy.