Combinatorial Effects of BH3 Mimetics and CAR T Cells Targeting T-Cell Lymphomas

T-cell lymphomas (TCL) represent a significant unmet need for improved therapeutic strategies. Recent advances in disease modeling and molecular characterization have greatly increased the potential for advancement. We previously showed that a subset of TCL cell lines, patient-derived xenografts (PDXs), and primary samples express high and uniform levels of the tetraspanin CD37 (PMID: 30089630). Selective expression on malignant T cells distinguishes CD37 from other CAR T cell targets under development against TCL and led TCL inclusion in our phase 1 clinical trial of autologous CD37-directed CAR T cells (CAR-37; NCT04136275).

CAR-37 T cells specifically lysed CD37+ TCL cell lines in vitro. In an aggressive T cell prolymphocytic leukemia PDX model, we observed >100-fold reduction in disease burden in vivo with CAR-37 T cells, but all mice eventually relapsed. We transplanted the relapsed tumor cells and re-treated with CAR-37 T cells. All recipients experienced a second response, with relapse in only a subset. Thus, a third dose of CAR-37 T cells was administered, but only a subset of mice underwent a third response. At the time of relapse/progression, CD37 expression was detected on tumor cells from each mouse. In addition, a small percentage of CAR-37 T cells were present in the tumor. Based on these results, we sought to evaluate combinatorial strategies that potentiate the tumoricidal activity of CAR-37 T cells and increase the rate of complete disease eradication.

CAR T cells kill primarily through driving the intrinsic pathway of apoptosis. We previously showed that BH3 mimetics of BCL2, BCL-xL, and MCL1 can selectively induce apoptosis in TCLs based on predicted BH3 protein dependencies (PMID: 30498064). Thus, to define novel therapeutic combinations with CAR-37 T cells, we performed BH3 profiling on CD37-expressing TCL cell lines. Corresponding to the predicted dependencies via BH3 profiling, FEPD and SMZ1 were most sensitive to the MCL1 inhibitor AZD5991, whereas HUT78 was most sensitive to the BCL-2/BCL-xL inhibitor navitoclax. We observed concordant sensitivity patterns to the BH3 mimetics by viability/apoptosis assay and decreased active mitochondrial membrane potential. Importantly, CD37 antigen density was unaffected by exposure to BH3 mimetics.

To evaluate the feasibility of combining BH3 mimetics and CAR T cells, we determined the impact of BH3 mimetics on CAR-37 T cell phenotype. First, we did not observe a reduction in CAR-37 T cell viability when cultured in vitro in the presence of TCL target cells and either venetoclax 0.5μM, navitoclax 0.5μM, or AZD5991 0.1μM for 48 hours. The same culture conditions also did not affect the differentiation or subset distribution of CAR-37 T cells. Nearly 60% of the CAR-37 T cells had an effector memory phenotype, whereas approximately 20% had a central memory phenotype and the remaining were either effector memory T cells re-expressing CD45RA or naïve. In addition, cytokine production (GMCSF and IFN-γ) and expression of T-cell exhaustion markers (PD-1, TIM3, and LAG3) was not affected by co-culture with BH3 mimetics.

Since BH3 mimetics did not alter CAR-37 T cell activity, we evaluated the cytotoxicity effect of their combination against target TCL cell lines. Using a luminescence based killing assay and xCELLigence RTCA (ACEA), we found that all 3 BH3 mimetics in combination with CAR-37 T cells resulted in greater specific lysis of FEPD tumor cells. The most significant increase occurred in combination with AZD5991, which corresponds to the predicted MCL1 dependence of FEPD cells. Next, we evaluated the combination of CAR-37 T cells and AZD5991 in NSG mice engrafted with FEPD. CAR-37 T cells were administered upon a total flux (photons/s) ≥10⁶ and the combination cohort subsequently received AZD5991 (100 mg/kg) on days 1 and 2, 8 and 9, and 15 and 16 post-CAR T cells. We observed significantly reduced tumor burden and prolonged survival in the combination cohort (median 42 days) compared to CAR-37 T cell (median 18 days) or AZD5991 (median 31 days) alone.

Together, our findings demonstrate that BH3 mimetics with predicted activity against individual TCLs can enhance the cytotoxic activity of CAR-37 T cells. These results could be used to nominate and/or deprioritize future combination strategies both for TCL and for other patients being treated with effector T cells for a range of hematologic malignancies.

AZD5991 was kindly provided by AstraZeneca.

Weinstock:Abcuro: Research Funding; Merck: Current Employment; Daiichi Sankyo: Research Funding; Ajax: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Travera: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bantam: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Secura: Research Funding. Maus:Century Therapeutics: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; 2SeventyBio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties: held by University of Pennsylvania; Promab: Patents & Royalties: held by Massachusetts General Hospital; Servier: Research Funding; WindMIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity: Consultancy; TCR2: Consultancy, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy; Oncternal: Consultancy, Current holder of stock options in a privately-held company; Novartis: Consultancy, Research Funding; Neximmune: Consultancy, Current equity holder in publicly-traded company; Micromedicine/BendBio: Consultancy; Kite Pharma: Consultancy, Research Funding; GSK: Consultancy; Intellia: Consultancy; In8bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding; Cellectis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy; Bayer: Consultancy; Atara: Consultancy; AstraZeneca: Consultancy; Astellas: Consultancy; Arcellx: Consultancy; Adaptimmune: Consultancy; Agenus: Consultancy; Allogene: Consultancy.