Inactivation of TGF-β signaling in CAR-T cells

Accumulating evidence suggests that the transforming growth factor (TGF)-β pathway might be an ideal target for optimizing chimeric antigen receptor (CAR)-T-cell therapy for solid tumors. In Cellular & Molecular Immunology, Liang et al. (2024) reported that CAR-T cells expressing SMAD7 displayed favorable antitumor efficacy and a reduced risk of systemic toxicity.

CAR-T cells have demonstrated promising efficacy in treating hematologic malignancies. However, this approach still faces multiple challenges in eliminating solid tumors. One challenge regarding CAR-T cell treatment for solid tumors is the immunosuppressive tumor microenvironment (TME), which is characterized by high levels of multiple inhibitory factors involving the second and third signals of T-cell activation. To overcome the immunosuppressive TME, one viable strategy is the removal or blockade of coinhibitory pathways, such as PD-1, on CAR-T cells to enhance CAR-T-cell responses and restore tumor-infiltrating lymphocytes (TIL) function [1]. Another approach entails enhancing the third signal of T-cell activation. This is achieved by the transgenic expression of cytokines or engineered cytokine receptors on CAR-T cells. This strategy not only enhances CAR-T-cell expansion and persistence but also bolsters their functionality within the immunosuppressive TME [2]. Another approach focuses on modulating the response of CAR-T cells to inhibitory TME cytokines, among which TGF-β is the most prominent. TGF-β is recognized as a pivotal immunosuppressive factor due to its impact on both T-cell and macrophage functions [3].

In the 1990s, studies demonstrated that TGF-β signaling could be blocked by using the dominant-negative TGFβ receptor type II (dnTGFβRII), which is a truncated protein lacking the intracellular domain necessary for downstream signaling [4]. Subsequent investigations revealed that dnTGFβRII expression driven by a T-cell-specific promoter specifically ablated TGFβ signaling in T cells. Consequently, the abrogation of TGFβ signaling in T cells results in their differentiation into effector T cells, eliciting inflammatory cell infiltration in multiple organs and stimulating immunopathological responses [5]. Utilizing the dnTGFβRII transgenic model, Leonid et al. demonstrated that T-cell-specific blockade of TGF-β signaling facilitated an immune response capable of eradicating tumors in mice [6]. These pivotal findings underscore the potential therapeutic implications of targeted modulation of TGF-β signaling pathways in T cells for enhancing antitumor immune responses (Fig. 1).

Fig. 1.

Strategies for targeting the TGF signaling pathway in CAR-T cells. A TGF-β signaling can be blocked by introducing dnTGFβRII or knocking out TGFβRII. B Endogenous suppressive signals from the TME can be converted into positive signals. C Coexpressing SMAD7 can inhibit TGF-β signaling and reduce the production of inflammatory cytokines

In light of the findings of these studies on TGF-β signaling in T cells, researchers have explored diverse strategies to attenuate or change the TGF signaling pathway in CAR-T cells. In addition, a recent study reported that CRISPR/Cas9-mediated knockout of TGFβRII could suppress the growth of solid tumors [7]  (Fig. 1A). Another investigation involved the incorporation of a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) into CD19 CAR-T cells. This engineered receptor was designed to convert TGF-β signaling into immune-activating IL-7 signaling. In animal models, mice treated with CD19 CAR-tTRII-I7R-T cells exhibited significantly prolonged overall survival compared to control mice [8]. Gracell Biotechnologies have developed a similar type of CAR-T cells named Suppressive Molecule Activated and Rejuvenated T cells (SMART CAR-T cells), which not only block endogenous TGF-β signals but also convert negative signals into positive signals (Fig. 1B). Additionally, the introduction of dnTGFβRII to CAR-T cells increased proliferation, augmented cytokine secretion, and induced tumor eradication in aggressive human prostate cancer mouse models. These notable outcomes in preclinical models prompted Narayan and colleagues to progress to clinical testing. They engineered and evaluated PSMA CAR-T cells with dnTGFβRII in patients with metastatic castration-resistant prostate cancer (mCRPC) (NCT03089203) [9]. However, in the clinical context, infusion of dnTGFβRII-CAR-T-PSMA cells in the therapeutic window was associated with cytokine release syndrome (CRS) , so the outcomes of dnTGFβRII-CAR-T-cell therapy were suboptimal [10]. These findings emphasize the inherent and nuanced challenges and complexities in translating promising preclinical results to clinical efficacy in the context of CAR-T-cell therapy for prostate cancer.

CRS represents a severe complication of acute systemic inflammation involving massive T-cell activation and cytokine release. In contrast to the dnTGFβRII-CAR-T-cell design, Liang et al. demonstrated that CAR-T cells expressing SMAD7 exhibited favorable antitumor efficacy and a reduced risk of systemic toxicity [11]. SMAD7 is a multifaceted protein that acts as a general inhibitor of both canonical and noncanonical TGF-β signaling pathways. The primary mechanism involves interference with the TGF-β receptor complex, preventing the downstream activation of SMAD2/3. Consistent with previous findings, SMAD7 binding to the adapters TAB2/TAB3 blocks the recruitment of TAK1 to the TNF receptor signalosome and inhibits IκB phosphorylation [12]. The study established that overexpression of SMAD7-CAR-T cells impairs cytokine production through NF-κB pathway inhibition (Fig. 1C).

Therefore, SMAD7-CAR-T cells are a potentially safer treatment option. Moreover, a recent study reported that the overexpression of SMAD7-CAR-T cells results in increased antitumor activity but also increased cytokine secretion and upregulated total IκB, demonstrating that the complex crosstalk between the TGF-β and NF-κB pathways remains to be determined [13]. Despite these promising findings, additional studies are needed to determine the safety of SMAD7-CAR-T cells for clinical application, especially for potential toxicity. Another pertinent question is how the overexpression of SMAD7 reduces the levels of cytokines or killing molecules while still exhibiting a notable antitumor function? How can this paradox be explained? One plausible explanation could be that enhanced CAR-T cells differentiate into a memory phenotype, but this mechanism requires further investigation. Tu et al. reported that strong TCR stimulation downregulated TGF-βRI expression and consequently abolished TGF-β signaling in T cells. The TGF-βRI level has been reported to be a crucial determinant of T-cell quiescence and activation [14]. It remains unclear whether the activation of CAR-T cells also negates TGF-β signaling and whether the expression of SMAD7 is dispensable. Therefore, additional studies are needed to elucidate whether SMAD7 interacts with signaling events that regulate T-cell activation, exhaustion, and differentiation.

Competing interests

The authors declare no competing interests.