Non-hematotoxic Tim-3/CD70 dual-targeting CAR T cells promote potent anti-leukemia activity against acute myeloid leukemia

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy for acute myeloid leukemia (AML) faces major challenges due to the lack of target antigens that are uniformly and specifically expressed on AML cells. AML-associated antigens exhibit heterogeneous expression, increasing the risk of antigen-negative leukemia escape when CAR T cells target a single antigen. Furthermore, expression of target antigens on normal myeloid cells can cause on-target/off-tumor hematotoxicity. Tim-3 and CD70 have emerged as AML-associated antigens with frequent expression on AML cells and minimal expression on healthy counterparts. This study explores Tim-3/CD70 dual-targeting CAR T cells in syngeneic mouse models and their translational potential. METHODS To assess Tim-3 and CD70 expression, we immunophenotyped bone marrow samples of patients with newly diagnosed (n=17) or relapsed/refractory AML (n=17) and healthy controls (n=5). We developed a spectral flow cytometry panel quantifying absolute Tim-3 and CD70 surface molecules. To evaluate Tim-3/CD70 dual-targeting CAR T cells, we constructed second-generation CARs targeting mouse Tim-3 using a single chain variable fragment and targeting mouse CD70 using its natural receptor CD27. Both CARs used CD28 co-stimulation and included CD3 zeta with a single active membrane-proximal immunoreceptor tyrosine-based activation motif (1XX ITAM) to reduce potential T cell exhaustion. Murine T cells were retrovirally transduced and enriched for dual-transduced CAR T cells using Zip-sort technology (James et al., Nat Biomed Eng, 2024). To support translational development, we generated and screened ten novel nanobody-based CARs targeting human Tim-3 to identify a lead candidate. We also generated and validated a CD27-based CAR targeting human CD70. RESULTS Immunophenotyping of primary patient samples confirmed higher Tim-3 and CD70 expression on AML bulk cells and leukemic stem cells compared to hematopoietic stem/progenitor cells and T cells from healthy controls, supporting their potential as CAR targets. Using a 1:1 mix of Tim-3+ and CD70+ tumor cells to reflect antigen heterogeneity, Tim-3 or CD70 single-CAR T cells failed to eliminate tumor cells expressing the alternate antigen, while dual-CAR T cells overcame antigen-negative escape and eradicated leukemia cells in vitro and in vivo in two syngeneic leukemia models (BM185 and C1498). Single cell RNA sequencing of bone marrow and gene set enrichment analysis revealed significant enrichment of E2F and MYC target genes in T cells of dual-CAR T cell-treated mice, indicating an enhanced proliferative transcriptional profile. Dual-CAR T cells persisted for over three months in the bone marrow and spleen after tumor clearance. Additionally, dual-CAR T cells demonstrated improved control of tumor growth compared to single-CAR T cells in mice inoculated with leukemia cells co-expressing attenuated levels of both Tim-3 and CD70. These cells were engineered to reflect the antigen-low expression pattern characteristic of a significant number of patients, highlighting the therapeutic advantage of a dual-targeting strategy even at suboptimal antigen expression levels. To assess potential on-target/off-tumor toxicity, mice received a four-fold higher dose of Tim-3/CD70-CAR T cells to accentuate T cell-driven effects. Transient pancytopenia observed during the first two weeks was fully attributable to the effects of irradiation as a conditioning regimen, confirmed by irradiation-only controls. No clinical signs of CAR T cell-related toxicity including weight loss were detected. Blood counts recovered after one month, and necropsy of long-term survivors on day 90 showed no evidence of sustained hematotoxicity or organ damage, supporting a favorable safety profile. Subsequently, we transduced human T cells with a nanobody-based Tim-3-CAR or a CD27-based CD70-CAR. These CAR T cells exhibited potent cytotoxicity against C1498 cells overexpressing human Tim-3 or CD70 in vitro, respectively, and ongoing studies are evaluating the combination of these CARs. CONCLUSIONS In summary, Tim-3/CD70 dual-CAR T cells represent a promising and previously unexplored therapeutic strategy for AML. They demonstrate potent anti-leukemia activity in vitro and in vivo without hematotoxicity or organ damage and with effective clearance of antigen-low disease. Further studies are evaluating Tim-3 and CD70-CARs targeting human antigens to support clinical translation of these findings.