Optimized T-cell receptor-mimic chimeric antigen receptor T cells directed toward the intracellular Wilms Tumor 1 antigen

S Rafiq, T J Purdon, A F Daniyan, M Koneru, T Dao, C Liu, D A Scheinberg, R J Brentjens, S Rafiq, T J Purdon, A F Daniyan, M Koneru, T Dao, C Liu, D A Scheinberg, R J Brentjens

Abstract

CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy.

Conflict of interest statement

CONFLICT OF INTEREST

RJB is a co-founder, stockholder and consultant for Juno Therapeutics Inc. DAS and TD are inventors of TCRm technology owned by MSKCC, licensed to Novartis.

Figures

Figure 1
Figure 1
Generation and validation of WT1-28z CAR T cells. (a) Schematic representation of the WT1-28z constructs. WT1/HLA-A*02:01-specific scFv derived from the heavy (VH) and light (VL) chain variable regions of the ESK1 antibody; CD28: human CD28 transmembrane and cytoplasmic signaling domains; z-chain: human TCR zeta chain cytoplasmic signaling domain; black box: κ leader sequence; gray box: (Gly4Ser)3 linker. (b) Flow cytometry histograms depicting the expression of WT1-28z in retrovirally transduced into primary human T cells, as detected by binding to a fluorescently-labeled WT1/HLA-A*02:01 Tetramer (representative figure of n > 10). (c) WT1-28z CAR T cells are more toxic than control irrelevant antigen-specific control CAR T cells 4H11-28z or 19-28z CAR T cells against AML-14, BV173 and OVCAR3 in standard 51Cr release assays (representative figures, n =3 for each cell line). (d) WT1-28z CAR T cells when co-cultured with AML-14, BV173, or OVCAR3 cell lines for 24 h have significantly enhanced release of IFN-γ (*P = <0.0001, 0.03, or 0.0013 respectively) and IL-2 (*P ≤ 0.0001, 0.013, or <0.0001 respectively), as compared with control 4H11-28z or 19-28z CAR T cells (n =3 for each cell line). (e) Standard 51Cr release assays with antigen-specific WT1-28z CAR T cells showed enhanced cytotoxicity against primary AML samples as compared with a control irrelevant antigen-specific 4H11-28z CAR T cells (n =5) (*P =0.029). (f) WT1-28z CAR T cells have significantly enhanced release of IFN-γ and IL-2 (*P = 0.04) when co-cultured with AML patient samples for 24 h, as compared with 4H11-28z CAR T cells (n =3).
Figure 2
Figure 2
Generation and validation of WT1-28z/IL-12 CAR T cells. (a) Schematic representation of the WT1-28z/IL-12 CAR constructs. WT1/HLA-A*02:01-specific scFv derived from heavy (VH) and light (VL) chain variable regions of the ESK1 antibody; CD28: human CD28 transmembrane and cytoplasmic signaling domains; z-chain: human TCR zeta chain cytoplasmic signaling domain; flexi human IL-12 (hIL-12 f); LTR: 50 and 30 long terminal repeat; black box: κ leader sequence; gray box: (Gly4Ser)3 linker. (b) IL-12 expression by viral producer cell lines stably transduced with CAR constructs show that only 293Galv9 cells transduced with WT1-28z/IL-12 express IL-12 (n =3 independent experiments) (P =0.01). (c) The IL-12 produced by the WT1-28z/IL-12 CAR in 293Galv9 is functional, as determined by increases in levels of IFN-γ produced by peripheral blood mononuclear cell cultured in supernatant (n =3 independent experiments) (P =0.03). (d) Flow cytometry histograms depicting the expression of WT1-28z/IL-12 CARs in retrovirally transduced primary human T cells, as detected by binding to a fluorescently-labeled WT1/HLA-A*02:01 tetramer (representative figure of n > 10). (e) WT1-28z/IL-12 CAR T cells have significantly enhanced release of IFN-γ (*P =0.008, 0.01, respectively) and IL-2 (*P = 0.002, 0.002, respectively), when co-cultured with Set2 cells for 24 hours, as compared to 19-28z or 19-28z/IL-12 CAR T cells (n =4). (f) WT1-28z/IL-12 CAR T cells proliferate after co-culture with Set2 cells (n =4).
Figure 3
Figure 3
WT1-28z and WT1-28z/IL-12 CAR T cells are specific to WT1/HLA-A*02:01. (a and b) WT1-28z or WT1-28z/IL-12 CAR T cells are significantly more toxic than control irrelevant antigen-specific CAR T cells 4H11-28z and 4H11-28z/IL-12 CAR T cells against Set2 and KARPAS-620 in standard 51Cr release assays (representative figures, n =3 for each cell line). WT1-28z significantly more lysed Set2 cells than 4H11-28z CAR T cells at all the effector to target ratios tested (P ≤ 0.005). WT1-28z/IL-12 significantly more lysed Set2 cells than 4H11-28z/IL-12 CAR T cells at 40:1, 20:1 and 10:1 effector to target ratios tested (P<0.0006). WT1-28z significantly more lysed KARPAS-620 than 4H11-28z CAR T cells at all the effector to target ratios tested (P<0.007). WT1-28z/IL-12 significantly more lysed KARPAS-620 than 4H11-28z/IL-12 CAR T cells at 40:1, 20:1, and 10:1 effector to target ratios tested (P<0.0003). (c) WT1-28z and WT1-28z/IL-12 CAR T cells show no toxicity toward the HLA-A*02:01-positive, WT1-negative cell lines SKLY-16 compared with control 4H11-28z or 4H11-28z/IL-12 CAR T cells in standard 51Cr release assays (n =3). (d) WT1-28z and WT1-28z/IL-12 CAR T cells show no toxicity toward the WT1-positive, HLA-A*02:01-negative cell line HL-60 as compared with control 4H11-28z or 4H11-28z/IL-12 CAR T cells in standard 51Cr release assays (n =3).
Figure 4
Figure 4
WT1-28z/IL-12 CAR T cells eradicates established tumors in a human xenograft mouse models. (a) SCID/Beige mice were injected i.v. with 5E6 Set2 cells and treated with i.v. injection of 2E7 CAR-positive primary human T cells 7 days post-tumor injection. WT1-28z CAR T cells significantly improved survival of mice compared to untreated mice (*P =0.006) or mice treated with control 4H11-28z CAR T cells (*P =0.01). WT1-28z/IL-12 CAR T cells had significant survival over mice treated with 4H11-28z/IL-12 CAR T cells (*P =0.004). (n =3 separate experiments). (b) SCID/Beige mice were i.v. injected with 5E6 Set2 cells and treated with i.v. injection of 2E7 CAR-positive primary human T cells 14 days post-tumor injection. WT1-28z/IL-12 CAR T cells significantly enhanced survival of mice in a more established disease model compared with WT1-28z CAR T cells (*P = 0.006). (c) SCID/Beige mice were i.p. injected with 1E7 OVCAR3 cells and treated 41 days post-tumor injection with i.p. injection of 5E6 CAR-positive primary human T cells. WT1-28z CAR T cells mediated significantly enhanced survival compared with treatment with control 19-28z CAR T cells (*P =0.009) or untreated mice (*P =0.0034). A single dose of WT1-28z/IL-12 CAR T cells mediated significantly enhanced survival compared with treatment with control 19-28z/IL-12 CAR T cells (*P ≤ 0.0001). Mice treated with WT1-28z/IL-12 CAR T cells exhibited enhanced survival as compared with WT1-28z CAR T cells, but this difference was not statistically significant (P =0.23) (n =2 independent experiments). (d) Bioluminescent imaging of mice on day 58 after OVCAR3 tumor injection show eradication of tumor in mice treated with WT1-28z or WT1-28z/IL-12 CAR T cells.
Figure 5
Figure 5
Differential gene expression of WT1-28z/IL-12 CAR T cells compared with WT1-28z CAR T cells when stimulated with OVCAR3 tumor cells for 48 hs. (a) Heat map of statistically significant (P ≤ 0.05) differential gene expression of cytokines between four normal donor T cells expressing WT1-28z/IL-12 CAR or WT1-28z CAR. (b) Heat map of statistically significant (P ≤ 0.05) differential expression of genes involved in chemokine or chemotaxis functions between four normal donor T cells expressing WT1-28z/IL-12 CAR or WT1-28z CAR. (c) Heat map of statistically significant (P ≤ 0.05) genes showing a distinct transcriptional profile between four normal donor T cells expressing WT1-28z/IL-12 CAR or WT1-28z CAR.

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