Lenalidomide (LEN), a second-generation immunomodulatory small-molecule drug used to patients with treat multiple myeloma (MM) [7], mantle cell lymphoma [8], and 5q syndromes [9], improves the function of CAR T cells in hematological and solid tumors [10, 11]. Works et al. have shown that LEN treatment enhances the production of effector cytokines interferon (IFN)-γ, interleukin (IL)-2, and tumor necrosis factor (TNF)-α in BCMA CAR T cells, which correlates with improved anti-myeloma responses both in vitro and in vivo [10]. To elucidate the mechanism of action of LEN, they conducted RNA-sequencing and CITE-sequencing analyses. These assays revealed that LEN upregulates pathways associated with T helper 1 cell responses, cytokine production, T-cell activation, and cytoskeletal remodeling [10]. Wang et al. also demonstrated that LEN enhances the functionality of other anti-myeloma CAR T cells, such as anti-CS1 CAR T cells, both in vitro and in vivo [12]. RNA-sequencing analysis of CD8+ anti-CS1 CAR T cells, both untreated and treated with LEN, showed that LEN-treated CAR T cells expressed genes linked to stem memory or less differentiated phenotypes (e.g., IL7, ACTIN1, KIT, WNT5B, IL9, MYB), enhanced effector functions (e.g., IRF4, IFI44), and improved homing capacity (e.g., integrins ITGA4 and ITGB3) [12].

In clinical settings, LEN maintenance after CD19/CD20/CD22 CAR-T therapy has been shown to be safe and to improve overall survival in patients with refractory/relapsed (r/r) diffuse large B-cell lymphoma (DLBCL) [13]. Thieblemont et al. demonstrated that LEN treatment at the time of relapse post-CAR-T therapy can enhance the anti-tumor response [14]. In a case report, a patient received LEN on the day prior to a BCMA CAR-T infusion [15]. This patient achieved a very good partial response 14 days after CAR-T treatment, which was maintained for over 8 months [15]. Additionally, LEN-treated CAR T cells exhibit higher anti-tumor responses in solid tumors, such as glioblastoma (GBM) and breast cancer. Kuramitsu et al. demonstrated that anti-epidermal growth factor receptor (EGFR) variant III CAR T cells treated with LEN showed increased intracellular IFN-γ production and enhanced cytotoxic activity against the GBM cell line U87, both in vitro and in intracranial GBM xenograft models [11]. They also found that LEN treatment improved EGFR variant III CAR-T-cell proliferation, immune synapse formation, and migration into tumor cells [11]. Similar results have been observed with LEN-treated CAR T cells targeting other antigens, such as CD133 and HER2, in GBM and breast cancer cell lines [16].

The findings summarized here highlight the significant potential of LEN to boost CAR-T therapies, which have transformed cancer treatment by specifically targeting tumor cells. Lenalidomide enhances CAR T-cell function by modulating multiple biological pathways, including T-cell activation, effector cytokine production, and immune cell differentiation. These effects lead to better tumor eradication in both hematologic and solid tumors, addressing one of the main limitations of CAR-T therapy, particularly in solid tumors where the TME often impairs immune responses. Moreover, the use of LEN alongside CAR-T therapy in clinical settings provides evidence of improved overall survival and tumor control, with minimal added toxicity. This is particularly notable in hematologic malignancies such as MM and lymphomas. The potential for LEN to work in solid tumors such as GBM and breast cancer opens new possibilities for expanding the use of CAR-T therapies in these harder-to-treat cancers.

Other immunomodulatory drugs, such as all-trans retinoic acid (ATRA), approved for treating acne and acute promyelocytic leukemia, enhance the anti-tumor efficacy of CAR T cells in both hematological and solid tumors. It has been demonstrated that ATRA increases BCMA [17] and CD38 [18] expression in MM, thereby enhancing the anti-tumor efficacy of anti-BCMA and anti-CD38 CAR T cells in pre-clinical models. In addition, ATRA modulates the TME [19]. In sarcoma, myeloid-derived suppressor cells create an immunosuppressive environment that leads to the failure of immunotherapy in this entity. Treating sarcoma-bearing xenograft mice with ATRA leads to eradication of these myeloid-derived suppressor cells, increasing the efficacy of GD2-targeting CAR T cells [19]. The effects of ATRA on myeloid-derived suppressor cells have also been addressed in clinical trials, where combining ATRA with the immune checkpoint inhibitor pembrolizumab was well tolerated and induced a high overall response rate of 71% in metastatic melanoma [20]. This combined approach may help to overcome some of the resistance mechanisms that limit the effectiveness of CAR T cells in certain cancers, especially those with a highly immunosuppressive TME, such as sarcoma. Overall, the synergistic use of ATRA represents a promising strategy to enhance the durability and effectiveness of CAR-T therapies across a broader spectrum of cancers.

Several protein kinases have been identified as critical drivers of tumorigenicity. Over the past decade, the pharmaceutical industry has developed numerous protein kinase inhibitors, many of which are now in clinical use to treat cancer [21]. Beyond their direct effects on cancer cells, these inhibitors significantly impact the immune function of T cells and dendritic cells, either enhancing or suppressing their immune responses [21]. This section explores how small-molecule inhibitors targeting specific protein kinases or signaling pathways can enhance the anti-tumor efficacy of CAR T cells in both hematological and solid cancers.

Approximately 30% of patients with acute myeloid leukemia (AML) harbor a mutation within the tyrosine kinase receptor FLT3, which is associated with a poor prognosis [22,23,24]. Over the past decade, pharmaceutical companies have developed various multi-kinase inhibitors targeting the FLT3 signaling pathway. These FLT3 inhibitors are classified into type I and type II based on their interaction with the FLT3 receptor.

Type I inhibitors, such as sunitinib (first generation) [25], midostaurin (first generation) [26], lestaurtinib (first generation) [27], crenolanib (second generation) [28], and gilteritinib (second generation) [29], bind to the ATP-binding site when the receptor is in its active conformation. In contrast, type II inhibitors, including sorafenib (first generation) [30], ponatinib (first generation) [31], and quizartinib (second generation) [32], bind to a hydrophobic region adjacent to the ATP-binding domain, which is accessible only when the receptor is inactive, thereby preventing receptor activation.

We and others have shown that while FLT3 inhibitors block the intracellular FLT3 signaling, they simultaneously enhance its surface expression in FLT3 mutant AML cells [33, 34]. This increase in surface expression presents an opportunity to enhance the engagement of anti-FLT3 CARs. Indeed, we demonstrated that this approach significantly improved the anti-tumor efficacy of FLT3 CAR T cells in vitro and in MOLM-13 xenograft models [35]. In another study, Li et al. showed that the FLT3 inhibitor gilteritinib upregulates immune activation NKG2DL ligands, including MICA/B, ULBP1, and ULBP2 through the NF-kB2/Rel B signaling pathway [36]. To leverage this effect, they developed a dual FLT3 scFv/NKG2D CAR T cell. The combination of gilteritinib and bispecific FLT3/NKG2D CAR T cells demonstrated synergistic anti-tumor efficacy in vitro and in MOLM-13 xenograft models [36].

Multikinase inhibitors are also used in the treatment of solid tumors. For example, sunitinib is employed in treating renal cell carcinoma [37]. Li et al. demonstrated that combining sunitinib with anti-CAIX CAR T cells induced synergistic anti-tumor efficacy in renal cell carcinoma xenograft models [38]. The mechanism behind this effect involves sunitinib upregulating CAIX expression on tumor cells, improving T-cell infiltration, and reducing the frequency of immunosuppressive myeloid-derived suppressor cells within the TME [38].

Overall, multi-target tyrosine kinase inhibitors can increase the surface expression of some CAR targets and immune activation ligands, leading to an enhanced anti-tumor response of CAR T cells in hematologic and solid tumor xenograft models in vivo. These findings warrant further investigation through clinical trials to evaluate their efficacy and safety in patients with cancer.

We and others have demonstrated that the tyrosine kinase inhibitor dasatinib, clinically approved for the treatment of chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, impacts the function of unmodified and modified T cells [39,40,41]. Dasatinib specifically binds to the ATP-binding site of Lck and other Src family kinases, thereby inhibiting Lck signaling and preventing subsequent T-cell activation after an antigen encounter [40, 41]. As a result, all activation-dependent functions such as target cell lysis, cytokine production, and cell proliferation are inhibited in a dose-dependent manner. When administered in vivo at adequate doses, dasatinib suppresses the secretion of IFN-γ and other proinflammatory cytokines and thereby disrupts the activation of immune cells, including macrophages. Thus, the development of CRS can be prevented [40], which has been supported by case reports of patients receiving dasatinib for the treatment of CRS and other CAR-related toxicities [42, 43]. Additionally, the defined administration of dasatinib might also prevent the exhaustion of CAR T cells during manufacturing [44], for example, in tonic signaling CARs or when targeting antigens that are also expressed on T cells (suppression of fratricide), or after an infusion.

These observations have led to the development of clinical trials to assess the feasibility to modulate CAR T-cell function by dasatinib. The clinical trial NCT04603872 uses CAR T cells targeting either BCMA or CD19 for the treatment of patients with r/r B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin’s lymphoma, or MM. In this trial, multiple dasatinib treatment regimens are evaluated, such as during manufacturing, after an infusion for the management of side effects, or to accelerate T-cell contraction. In addition, a phase Ib clinical trial has the aim to assess the feasibility of oral dasatinib pulses (3 consecutive days per week) during the first month following an infusion of brexucabtagene autoleucel (Tecartus®) in adults with r/r B-ALL (NCT05993949). A third study evaluates the supplementation of a culture medium during manufacturing of B7-H3 CAR T cells in solid tumors (NCT06500819). So far, recruitment is ongoing, and no results have been reported yet.

Interestingly, Marcos-Jiménez et al. [45] reported that dasatinib induces spleen contractions and lymphocyte egress from the spleen. This finding could give another spin to the potential combination of dasatinib and CAR-T therapy, as dasatinib treatment before leukapheresis might increase the amount of collectable T cells during leukapheresis. Additionally, administering dasatinib after a CAR-T infusion may increase the number of T cells in circulation, which can (a) enhance the likelihood of T cells encountering tumor cells or (b) improve the chances of T cells migrating to tumor-containing areas rather than homing to the spleen. Thus, the spleen resizing and lymphocyte mobilization induced by dasatinib open new possibilities for clinical application. Of note, dasatinib has also been shown to increase the expression of ROR1 on the surface of acute lymphoblastic leukemia tumor cells [46], which is a target of CAR-T under clinical evaluation for the treatment of solid and hematologic malignancies (e.g., NCT02706392, NCT05588440)

In summary, evaluating pre-clinical data, dasatinib seems to be an all-rounder in regard to T-cell control. By applying different dose and treatment schedules, inhibition can be either used to boost CAR T-cell function by preventing exhaustion or to mitigate CAR-T-related side effects. Being clinically approved, dasatinib has a known safety and toxicity profile, with side effects mostly being relevant during long-term treatment. However, relevant questions with regard to the transferability of data from in vitro and in vivo settings to a human application need to be addressed in clinical trials. In particular, the optimal treatment schedule and the dosage required for efficient control need to be evaluated in order to preserve subsequent CAR T-cell function and efficiency. There is widespread concern that a complete shutdown of T-cell activity, such as during the treatment of severe CRS, could lead to uncontrolled growth of aggressive tumors. As a consequence, a more specific shutdown of single CAR T-cell functions might be more adequate to address side effects than a general shut down.

The Bruton’s tyrosine kinase inhibitor ibrutinib has demonstrated potent anti-cancer activity across various types of leukemia, most notably in chronic lymphocytic leukemia (CLL) [47] and mantle cell lymphoma [48]. In CLL, T-cell fitness is impaired because of intrinsic defects, leading to a differentiated phenotype, which reduces CAR-T expansion. This dampens the efficacy of CAR-T therapy in patients with CLL in comparison to DLBCL or ALL. Beyond its direct anti-tumor efficacy, ibrutinib significantly inhibits the IL-2-inducible T-cell kinase, which is involved in T-cell differentiation, thereby enhancing T helper 1-based immune responses [49]. Recent studies have shown that treating peripheral blood mononuclear cells derived from patients with CLL in vitro before/during CAR-T manufacturing improved T-cell expansion and viability, supported a less differentiated naïve-like phenotype, reduced the expression of exhaustion markers, and rescued cytokine release after antigen recognition [50].

These findings were confirmed by a clinical observation reported by Fraietta et al. who described that more than five cycles of ibrutinib treatment prior to leukapheresis improved ex vivo and in patient T-cell proliferation in patients with CLL, a condition where T-cell proliferation is typically impaired [51]. Notably, ibrutinib has been crucial for enhancing the manufacturing and expansion of CD19 CAR T cells used in treating B-cell malignancies [51]. Additionally, continuous ibrutinib treatment has been shown to improve the efficacy of CD19 CAR T cells in drug-resistant OSU-CLL and Nalm-6 (acute lymphoblastic leukemia) xenograft models. Prolonged ibrutinib treatment led to greater expansion of CD19 CAR T cells in peripheral blood and reduced levels of the immunosuppressive marker programmed cell death protein 1, resulting in significantly prolonged survival in mice [51]. The synergistic effects of combining ibrutinib with CD19 CAR T cells have prompted clinical trials: a phase I clinical trial assessed the feasibility and safety of administering ibrutinib alongside CD19 CAR T cells in patients with CLL (NCT01865617) [27]. This trial involved 19 patients with CLL, with a median of five prior therapies and 17 patients (89%) with high-risk cytogenetics, including the 17p deletion or complex karyotype. The results indicated that concurrent treatment with ibrutinib and CD19 CAR T cells was feasible for 18 of the 19 patients, although six patients required a reduced dose of ibrutinib [52]. Furthermore, ibrutinib treatment enhanced CD19 CAR T-cell expansion, reduced cytokine concentrations associated with CRS after a CAR-T infusion, and resulted in high rates of minimal residual disease negativity as assessed by IGH sequencing [52]. In addition, a phase II trial was conducted to evaluate the combinatory effects of ibrutinib and CD19 CAR-T therapy (CTL019) in 20 patients with r/r mantle cell lymphoma [53] (NCT04234061). In this study, ibrutinib was administered before leukapheresis, and treatment was maintained until at least 6 months after a CAR-T infusion. At a 13-month median follow-up, all patients were alive with a progression-free survival of 75% with manageable toxicities. Thus, the combination of ibrutinib and CAR-T therapy seems to be a feasible combination that will be further evaluated.

In summary, Bruton’s tyrosine kinase inhibition by ibrutinib resets the T-cell phenotype in patients with intrinsic malignancy-related T-cell defects. Even though clinical data are promising, the underlying mechanism is not completely understood, as Bruton’s tyrosine kinase expression varies in between T-cell subsets, with increased expression in activated and regulatory T cells (Tregs) [54]. Therefore, more research is required to fully understand the effects of ibrutinib on CAR T-cell efficacy.

The PI3K/AKT/mammalian target of rapamycin (mTOR) signaling pathway is frequently hyper-activated in cancer, playing a critical role in tumor cell growth and survival [55]. This pathway also significantly influences the T-cell phenotype [56, 57]. Therefore, targeting this pathway with small molecules presents a promising strategy to enhance CAR T-cell fitness and improve their anti-tumor efficacy.

Recent studies have shown that, in general, ex vivo expansion of CAR T cells leads to a reduction in naïve and stem memory populations, while increasing effector subsets compared with vector-transduced control cells, ultimately decreasing CAR T-cell persistence in vivo [58]. This phenomenon has been linked to tonic signaling through the immune receptor tyrosine-based activation motifs of the CAR-CD3ζ [59], which activate the downstream PI3K pathway in CAR T cells [60]. Interestingly, the use of the PI3K inhibitor LY294002 ex vivo resulted in a less differentiated T-cell phenotype in CD33 CAR T cells [60]. Additionally, LY294002-treated CD33 CAR T cells exhibited increased persistence, enhanced anti-tumor efficacy, and prolonged survival in mice bearing MOLM-13 AML cells [60].

In patients with CLL, where T cells are often exhausted and highly differentiated, the PI3Kδ/γ dual inhibitor duvelisib was effective in reversing T-cell differentiation and exhaustion. The addition of duvelisib during manufacturing promoted CD19 CAR T cells with a stem cell memory T-cell phenotype and reduced the exhaustion marker TIM-3, thereby improving CD8+ CAR-T-cell generation [61]. Duvelisib also increased CD19 CAR-T expansion and anti-CLL activity in an OSU CLL xenograft model [61]. Additionally, duvelisib enhanced mitochondrial fusion in CD19 CAR T cells, a feature previously associated with improved clinical responses [61].

In lymphoma, similar to CLL, CD19 CAR T cells derived from heavily pre-treated patient T cells exhibited lower ex vivo expansion rates and poorer clinical responses [62]. The combination of the PI3Kδ inhibitor idelalisib with the vasoactive intestinal peptide (VIPhyb) signaling antagonist reduced T-cell differentiation [62]. Adding VIPhyb and idelalisib to T-cell cultures significantly enhanced the expansion, transduction, and cytotoxicity of human anti-CD5 CAR T cells against CD5+ lymphoma [62]. T cells from patients with DLBCL, expanded with idelalisib and VIPhyb, showed improved persistence in vivo in immune-deficient mice [62]. A novel approach using VIP antagonist-secreting CAR T cells targeting Muc16 in pancreatic ductal adenocarcinoma has shown promising results. These armored CAR T cells, which counteract VIP-mediated inhibition, have demonstrated an improved phenotype and function [