Modal CAR T cells: A synthetic cytokine circuit to overcome TGFβ-mediated immunosuppression in AML

Abstract Chimeric antigen receptor (CAR) T cells have shown clinical success in B and plasma cell derived hematologic malignancies, but durable responses remain limited due to T cell exhaustion, poor trafficking, and suppressive tumor microenvironments (TME). Furthermore, efforts to harness CAR T cells in acute myeloid leukemia (AML) have been largely unsuccessful. Transforming growth factor beta (TGFβ) is a dominant immunosuppressive cytokine in multiple tumors including AML, which impairs T cell function, proliferation, and persistence. Rather than blocking TGFβ receptor, we hypothesized that elevated TGFβ in the AML (and possibly other tumors) could be harnessed as an input to drive local, therapeutic cytokine expression. We designed a synthetic TGFβ-sensing circuit to conditionally express IL-12 in response to tumor derived TGFβ. Two SMAD-responsive sensors (sensor 1 and 2), each paired with either SV40 or MLP promoters, were cloned into lentiviral vectors and tested in Jurkat cells. Sensor 1 consistently induced the highest eGFP expression following TGFβ1 stimulation. We then evaluated this sensor to drive expression of CD70 CAR and secreted IL-12. All configurations showed TGFβ-inducible CAR and IL-12 expression but also demonstrated baseline (“leaky”) expression. To mitigate IL-12 leakage, we reconfigured the construct with the TGFβ sensor and IL-12 cassette on the reverse strand, while CD70CAR remained on the forward strand. This “bidirectional” design markedly reduced baseline IL-12 by ~87% (43 vs. 362 pg/mL, p=0.0019) without impairing CAR expression or T cell function. These CAR T cells displayed improved cytotoxicity against CD70+ AML lines (OCI-AML2, OCI-AML3, MOLM13), though TGFβ still dampened killing, suggesting that inducible IL-12 alone was insufficient to fully overcome suppression. To further enhance efficacy, we introduced secretory IL-18 downstream of the CD70CAR on the forward strand, generating what we call MODAL (MOdular Design with Asymmetric Logic) CAR T cells. In this platform, IL-18 is constitutively secreted, while IL-12 remains tightly regulated via TGFβ-sensing on the reverse strand (MODAL12/18 configuration). Primary T cells transduced with CD70-MODAL12/18 constructs showed robust CAR expression, high IL-18 secretion (~2000 pg/mL), and inducible IL-12 expression (13 vs. 78 pg/mL ± TGFβ1, p=0.0057). CD70-MODAL12/18 CAR-T cells exhibited enhanced proliferation and cytotoxicity in vitro across CD70+ AML lines (OCI-AML2, OCI-AML3 and MOLM13), fully overcoming TGFβ-mediated suppression. In NSG mice injected intravenously (tail vein) with MOLM13-luciferase tumors, CD70-MODAL12/18 CAR T cells led to significantly reduced tumor burden (ongoing survival analysis) and improved expansion compared to CD70CAR, CD70CAR-IL-12, or CD70CAR-IL-18 arms. Flow cytometry analysis revealed higher frequencies of central memory CD4+ and CD8+ T cells and reduced PD-1 expression upon bleeding the mice on day 14. No treatment-related toxicities were observed, while the cytokine release syndrome (CRS) relevant markers are currently under evaluation. In summary, we present a novel synthetic platform, MODAL CAR T cells,that integrates constitutive IL-18 and TGFβ-inducible IL-12 expression in a bidirectional gene circuit. This asymmetric design enables context-specific cytokine delivery to counteract immune suppression and enhance CAR T cell function. The MODAL12/18 strategy is modular and generalizable, offering a blueprint for next-generation T cell therapies targeting immunosuppressive TMEs in both hematologic and solid tumors.