Immunotherapy with chimeric antigen receptor (CAR)–engineered T cells can be effective against advanced malignancies. CAR T cells are “living drugs” that require technologies to enable physicians (and patients) to maintain control over the infused cell product. Here, we demonstrate that the tyrosine kinase inhibitor dasatinib interferes with the lymphocyte-specific protein tyrosine kinase (LCK) and thereby inhibits phosphorylation of CD3ζ and ζ-chain of T cell receptor–associated protein kinase 70 kDa (ZAP70), ablating signaling in CAR constructs containing either CD28_CD3ζ or 4-1BB_CD3ζ activation modules. As a consequence, dasatinib induces a function-off state in CD8+ and CD4+ CAR T cells that is of immediate onset and can be sustained for several days without affecting T cell viability. We show that treatment with dasatinib halts cytolytic activity, cytokine production, and proliferation of CAR T cells in vitro and in vivo. The dose of dasatinib can be titrated to achieve partial or complete inhibition of CAR T cell function. Upon discontinuation of dasatinib, the inhibitory effect is rapidly and completely reversed, and CAR T cells resume their antitumor function. The favorable pharmacodynamic attributes of dasatinib can be exploited to steer the activity of CAR T cells in “function-on-off-on” sequences in real time. In a mouse model of cytokine release syndrome (CRS), we demonstrated that a short treatment course of dasatinib, administered early after CAR T cell infusion, protects a proportion of mice from otherwise fatal CRS. Our data introduce dasatinib as a broadly applicable pharmacologic on/off switch for CAR T cells.

Objective: Integrins are heterodimeric receptors that convey cell-to-cell and cell-to-matrix interactions. Integrin αvβ3 is expressed in several tumour entities including melanoma, glioblastoma, breast, pancreatic and prostate cancer, where it promotes tumour cell survival and metastasis. Here, we generated αvβ3-specific chimeric antigen receptor (CAR) T-cells and analysed their antitumour function in pre-clinical models in vitro and in vivo. Methods: αvβ3-CARs comprising a super-humanised hLM609 targeting domain with either high or low affinity (hLM609v7, Kd = 3 nM vs. hLM609v11, Kd = 160 nM) and equipped with either a long or a short IgG4-Fc extracellular spacer (229 vs. 12 amino acids) were expressed in CD8+ and CD4+ T-cells through lentiviral transduction. Results: αvβ3-CAR T-cells eliminated αvβ3-positive tumour cells rapidly and specifically, produced IFN-γ and IL-2 (CD4+ > CD8+) and exhibited productive proliferation. In vitro, we observed the strongest reactivity with the higher-affinity hLM609v7 αvβ3-CAR in the short spacer configuration, consistent with the tumour membrane-distal localization of the hLM609 epitope. In a murine xenograft model of metastatic A-375 melanoma, the strongest antitumour effect was mediated by the lower-affinity hLM609v11 αvβ3-CAR. Notably, a single administration of hLM609v11 αvβ3-CAR T-cells was able to induce complete elimination of melanoma lesions, leading to long-term tumour-free survival. Conclusions: These data establish αvβ3 integrin as a novel target for CAR T-cell immunotherapy, and affirm our previous notion that binding domain affinity and spacer length can be calibrated to augment CAR reactivity. Clinical implications: αvβ3-CAR T-cells have therapeutic potential in several prevalent solid tumours, including melanoma and triple-negative breast cancer.

Acute myeloid leukemia (AML) is attractive for the development of CAR T-cell immunotherapy because AML blasts are susceptible to T-cell-mediated elimination. Here, we introduce sialic-acid-binding immunoglobulin-like lectin (Siglec)-6 as a novel target for CAR T-cells in AML. We designed a Siglec-6-specific CAR with a targeting-domain derived from a human monoclonal antibody JML‑1. We found that Siglec-6 is prevalently expressed on AML cell lines and primary AML blasts, including the subpopulation of AML stem cells. Treatment with Siglec-6-CAR T-cells confers specific anti-leukemia reactivity that correlates with Siglec-6-expression in pre-clinical models, including induction of complete remission in a xenograft AML model in immunodeficient mice (NSG/U937). In addition, we confirmed Siglec-6-expression on transformed B-cells in chronic lymphocytic leukemia (CLL) and show specific anti-CLL-reactivity of Siglec-6-CAR T-cells in vitro. Of particular interest, we found that Siglec-6 is not detectable on normal hematopoietic stem and progenitor cells (HSC/P) and that treatment with Siglec-6-CAR T-cells does not affect their viability and lineage differentiation in colony-formation assays. These data suggest that Siglec-6-CAR T-cell therapy may be used to effectively treat AML without a need for subsequent allogeneic hematopoietic stem cell transplantation. In mature normal hematopoietic cells, we detected Siglec-6 in a proportion of memory (and naïve) B-cells and basophilic granulocytes, suggesting the potential for limited on-target/off-tumor reactivity. The lacking expression of Siglec-6 on normal HSC/P is a key differentiator from other Siglec-family members (e.g. Siglec-3=CD33) and other CAR target antigens, e.g. CD123, that are under investigation in AML and warrants the clinical investigation of Siglec-6-CAR T-cell therapy.

B-cell maturation antigen (BCMA) is the lead antigen for CAR T-cell therapy in multiple myeloma (MM). A challenge is inter- and intra-patient heterogeneity in BCMA expression on MM cells and BCMA downmodulation under therapeutic pressure. Accordingly, there is a desire to augment and sustain BCMA expression on MM cells in patients that receive BCMA-CAR T-cell therapy. We used all-trans retinoic acid (ATRA) to augment BCMA expression on MM cells and to increase the efficacy of BCMA-CAR T-cells in pre-clinical models. We show that ATRA treatment leads to an increase in BCMA transcripts by quantitative PCR and an increase in BCMA protein expression by flow cytometry in MM cell lines and primary MM cells. Analyses with super-resolution microscopy confirmed increased BCMA protein expression and revealed an even distribution of non-clustered BCMA molecules on the MM cell membrane after ATRA treatment. The enhanced BCMA expression on MM cells after ATRA treatment led to enhanced cytolysis, cytokine secretion and proliferation of BCMA-CAR T-cells in vitro, and increased efficacy of BCMA-CAR T-cell therapy in a murine xenograft model of MM in vivo (NSG/MM1.S). Combination treatment of MM cells with ATRA and the γ-secretase inhibitor crenigacestat further enhanced BCMA expression and the efficacy of BCMA-CAR T-cell therapy in vitro and in vivo. Taken together, the data show that ATRA treatment leads to enhanced BCMA expression on MM cells and consecutively, enhanced reactivity of BCMA-CAR T-cells. The data support the clinical evaluation of ATRA in combination with BCMA-CAR T-cell therapy and potentially, other BCMA-directed immunotherapies.

Background: Immunotherapy with CAR-T-cells (CAR-T) is a powerful novel treatment for hematologic malignancies, but also bound with significant acute and chronic side effects, including potentially life-threatening cytokine release syndrome (CRS) and on-target recognition of normal cells expressing the targeted antigen. This toxicity limits clinical utility and is at least in part caused by the inability to effectively control CAR-T function following infusion. Here, we present a novel strategy of pharmacologic “ON/OFF” switch to precisely control CAR-T function in real-time, which we demonstrate to modulate T-cell activity in vitro and in vivo. Methods: We considered that an effective way for controlling CAR-T function was to interfere with signal transduction though the CAR. We assembled a library of clinically approved drug compounds and screened for their ability to reversible block CAR-T function without affecting CAR-T viability. We performed functional testing with CD8+ and CD4+ CAR-T (n=3 donors) in the presence of titrated doses of the lead compound, and employed CD19- and ROR1-specific CARs comprising 4-1BB or CD28 costimulatory moieties. Results: We identified a lead compound, TCI-1, that stood out through its ability to confer a dose-dependent (partial at lower, complete at higher doses) blockade of all CAR-T effector functions, i.e., cytolytic activity, cytokine secretion and proliferation. We confirmed TCI-1 was effective in both CD8+ and CD4+ T-cells, and in each of the 3 CAR constructs. The onset of CAR-T blockade was immediate after exposure to TCI-1 and was caused by interference with early phosphorylation events in the CAR signaling cascade as demonstrated by Western blot, and interference with the induction of transcription factors, as demonstrated with an NFAT-inducible reporter gene. Intriguingly, blockade of CAR-T function was effective for several days if exposure to TCI-1 was sustained and instantaneously and fully reversible after removal of the compound. Short- and long-term exposure to TCI-1 did not induce a reduction of CAR-T viability, and did not hinder the subsequent ability of CAR-T to exert their functions. We considered that in patients with CRS, CAR-T are in an activated state, and performed comprehensive testing to show that TCI-1 was able to arrest CAR-T that are in the process of executing their effector functions. In addition, we employed a xenograft model in immunodeficient mice (NSG/Raji) to determine whether TCI-1 was capable of controlling the function of CD19 CAR-T-cells in vivo. Indeed, we demonstrate that administration of TCI-1 conferred a functional arrest of CAR-T, and that CAR-T resumed their antitumor function once administration of TCI-1 was discontinued. Conclusions: Our data show that TCI-1 is capable to exert real-time, on/off control over CAR-T function, suggesting the potential to prevent or mitigate side effects of CAR-T therapy in a clinical setting. The reversible complete inhibition of CAR-T function through TCI-1 without compromise to CAR-T viability surpasses the qualities of steroids that are toxic to T-cells and provide only incomplete functional control, and complements suicide-gene strategies that effectively control chronic side effects but also abrogate the antitumor effect of CAR-T. Citation Format: Katrin Mestermann, Rydzek Julian, Frenz Silke, Einsele Hermann, Michael Hudecek. A novel pharmacologic “ON/OFF” switch to modulate CAR-T-cell function in vitro and in vivo [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A037.