Enhanced anti-tumor activity by zinc finger repressor-driven epigenetic silencing of immune checkpoints and TGFBR2 in CAR-T cells and TILs

Abstract

Chimeric antigen receptor T (CAR-T) therapies have shown remarkable success in treating hematological malignancies. However, effectiveness against solid tumors remains limited due to the immunosuppressive tumor microenvironment (TME), such as transforming growth factor β (TGF-β) signaling and upregulated immune checkpoints (ICs). Furthermore, identifying universal, tumor-specific targets for CAR-T cells in solid tumors is challenging, but using reinvigorated, immunosuppressive-resistant tumor-infiltrating lymphocytes (TILs) could be a promising alternative approach. Unlike nucleases, which may induce genotoxic DNA double-strand breaks, multiplexed zinc finger repressors (ZFRs) offer a safer alternative for knocking out TME-related immunosuppressive factors. We epigenetically repressed PD-1 expression both in CAR-T cells and TILs from colorectal liver metastases. PD-1 repression did not affect T cell viability, proliferation, or functionality. In a murine B cell lymphoma model, PD-1-repressed CD19-CAR-T cells exhibited enhanced anti-tumor activity and improved survival. Notably, PD-1 repression alone did not increase cytotoxicity against a PD-L1-positive colorectal cell line in vitro. To further increase anti-tumor potency in this context, ZFR-expressing lentiviral vectors (LVs) targeting PD-1 and other ICs (LAG-3, TIM-3, and TIGIT) or TGFBR2 were developed, improving significantly the cytotoxic activity in TILs. This strategy highlights the potential to enhance tumor-reactive T cells and improve anti-cancer immunotherapies by epigenetically repressing immunosuppressive factors in the TME using multiplexed ZFRs.

Keywords: CAR-T; KRAB; TILs; TME; epigenetic; immune checkpoints; repressor; zinc finger protein.

Graphical abstract

Figure 1

T cell phenotype and functionality after ZFR-mediated repression of PD-1 (A and B) Human T cells were transduced with LV constructs expressing the CD19-CAR alone or in combination with the PD-1 ZFR. CAR expression (A) and PD-1 repression efficiency (B) were monitored in T cells using flow cytometry (D3 post-LV) two-way ANOVA Friedman test (∗∗∗p < 0.001). (C) Kinetic of PD-1 repression efficiency in CAR-T cells. Cells were cultured for 14 days with an anti-CD3/CD28 reactivation step at D7. (D) Repression efficiency of PD-1 following TCR activation (anti-CD3/CD28 beads) or CD19-CAR activation after co-culture with a CD19-expressing cell line (Nalm6-L-PD-L1) two-way ANOVA Tukey test (∗∗∗p < 0.001). (E) Cytotoxic capacity of T cells, CAR-T, and PD-1 ZFR-CAR-T cells after overnight co-culture with NALM-6-L-PD-L1 cells at the indicated effector: target (E:T) ratios. Results were obtained from three independent donors. Error bars indicate mean ± SD. (F) Activation (left) and degranulation (right) of T cells, CAR-T, and PD-1 ZFR-CAR-T cells were assessed by measuring CD69 and CD107a expression respectively, following 24 h co-culture with NALM-6-L-PD-L1 cells at 1:3 E:T ratio. Two-way ANOVA Sidak test (∗∗∗p < 0.001). (G) Expression of IFN-γ (left), IL-2 (middle), and TNF-α (right) in CAR-T and PD-1 ZFR-CAR-T cells was assessed by flow cytometry following a 24 h co-culture with NALM-6-L-PD-L1 cells at 1:3 E:T ratio. Results were obtained from three independent donors. Data are represented as mean ± SEM.

Figure 2

Efficient and durable PD-1 repression in TILs from CRLM patients (A) Schematic representation of the isolation, transduction, and expansion of TILs isolated from CRLMs patients. After tissue dissection, enriched CD3⁺ TILs were activated, transduced, and expanded for three weeks. Transduction efficacy was determined seven days and functionality 21 days after transduction. (B) PD-1 expression on dNGFR-enriched TILs (n = 13) at day 7 and day 14 post-LV. Two-way ANOVA Sidak test (∗∗∗p < 0.001). (C) Killing of SNU-C5-GFP-Luc cells by control or PD-1 ZFR transduced TILs in absence or presence of anti-CD3E-EpCAM antibody. Specific cancer cell line lysis was defined as reduction in viable GFP⁺ cancer cells after 48 h co-culture with TILs (n = 8). (D and E) After three weeks of culture, the phenotype (D, n = 9 donors) and the functionality (E, n = 8 donors) of expanded NGFR⁺ CD4⁺ and CD8⁺ TILs expressing or not expressing PD-1 ZFR were assessed by flow cytometry. Functionality was determined by monitoring TNF-α, IFN-γ, CD40L, and CD107a expressions before and after stimulation with anti-CD3/CD28 dynabeads for 24 h. Abbreviations are as follows: CM, central memory T cell; EM, effector memory T cell; Temra, EM CD45RA + T cell; MPEC, memory precursor effector cells; SLEC, short-lived effector cells; Trm, tissue-resident memory T cells. Data are represented as mean ± SEM.

Figure 3

PD-1 downregulation enhances the anti-tumor activity of CAR-T cells in vivo (A) Schematic of the in vivo experimental protocol (BioRender). During six months following CAR-T or PD-1 ZFR-CAR-T cells injection, Nalm6-L-PD-L1 tumor cell proliferation was evaluated by monitoring the in vivo bioluminescence imaging (BLI) signal (photons/sec). (B) Representative mice images showing BLI from tumor cells at different time points in the different groups (n = 3–5/group). (C–F) The total BLI signal of the tumor (C), the body weight loss (D), the clinical score progression (E), and the survival rate (F) were monitored in the different mice groups over time. (G) PD-1 expression levels (MFI) in CAR-T versus PD-1 ZFR-CAR-T cells in organs (BL, blood; SP, spleen, BM, bone marrow) of individual mice (left) or pulled altogether (right, n = 4–8) or by organs in blood (H, n = 2–4), spleen (I, n = 1–2), and bone marrow (J, n = 1–2). Data are represented as mean ± SEM and two-tailed Mann-Whitney test (∗∗p < 0.01).

Figure 4

Multiplexed ICs and TGBR2 repression improve anti-cancer TIL cytotoxicity (A) Transduction efficiency of single and multiplexed ZFR-expressing LVs detected by flow cytometry (dNGFR⁺ cells) 7 days post-LV. (B) Expansion of NGFR-enriched TILs after transduction with single PD-1 ZFR or multiplexed ZFRs. (C and D) Expression of PD-1, TIM-3, LAG-3, TIGIT, or TGFBR2 on dNGFR⁺ TILs after transduction with single PD-1 ZFR or respective multiplexed ZFRs (n = 14 donors). Two-way ANOVA Friedman test (∗∗∗p < 0.001). (E) Anti-CD3E-EpCAM-mediated killing of SNU-C5-GFP-Luc cells by TILs (n = 6–8 donors) transduced with single PD-1 ZFR or multiplexed ZFRs. Specific cancer killing was defined by reduction of viable GFP⁺ cancer cells compared with cell count obtained in unstimulated co-cultures in the control condition. Wilcoxon test (∗p < 0.05). Data are represented as mean ± SEM.