Advances in CD137-Enriched Adoptive T Cell Therapy for Acute Myeloid Leukemia Via Ex Vivo Immune Cell Priming with DC/AML Fusion Vaccine

Background: We have pioneered a vaccine by fusion of patient-derived tumor with autologous dendritic cells (DCs) that presents an array of tumor antigens generating a polyclonal immune response. In a phase II clinical trial, DC/AML vaccination led to expansion of leukemia-specific T cells with durable disease remission. Ex vivo generation of vaccine-educated T cells (VETc) is a potent approach for adoptive cell therapy that selectively captures tumor heterogeneity. In murine AML and MM models, enrichment of VETc for activated antigen-specific effector cells via agonistic CD137 antibody-based selection further enhances for cytotoxicity, T cell activation and memory phenotype (Rallis et al., Blood (2023) 142, Supplement 1). Here, we report updates for CD137+ VETc in murine models, findings for CD137+ VETc in primary human AML cells, and validation of TGF-β signaling inhibition in VETc via SMAD2 knockout to counter immunosuppression.

Methods: DC/AML vaccines were generated by fusing C57BL/6J mice DCs and syngeneic C1498 mCherry/luciferase+ AML cells or primary human DCs and patient derived AML cells. Splenic murine T cells or human T cells isolated from peripheral blood were co-cultured with autologous irradiated DC/AML fusions in presence of IL-2/7/15. Selection with biotinylated agonistic CD137 antibody was performed on VETc followed by expansion and activation via CD3/CD28. T cells were phenotyped for activation (CD25/CD69), immune checkpoints (PD1/LAG3/TIM3) and memory (CD44+CD62L- or CCR7+CD45RA-). Cytotoxicity was evaluated by luminescence. Mice inoculated with C1498 and injected with T cells after 7 days were imaged by BLI and monitored for survival. Human tumor reactive T (TRT) cells were enriched for CD137 and expanded in a closed system using the CliniMACS Prodigy instrument (Miltenyi). CRISPR-Cas9 ribonucleoproteins (RNPs) were generated by incubating three SMAD2 sgRNAs (Synthego) with Cas9 protein (Macrolabs, UC Berkeley) in nuclease-free duplex buffer (IDT). The effect of TGF-β on cell proliferation was assessed on human VETc electroporated in the presence [SMAD2 knockout (KO)] or absence [SMAD2 wildtype (WT)] of RNPs and cultured in the presence or absence of TGF-β (50 ng/mL). Cells were stained with Cell Trace Violet dye (Thermo Fisher) and allowed to proliferate in culture for 5 days.

Results: In the C1498 model, VETc selected based on CD137 expression demonstrate enhanced immune activation (CD25+CD69+) and memory (CD44+CD62L-) phenotype compared to unstimulated naïve T-cell controls (TN) (9.3-fold, p=0.0009; 4.4-fold, p=0.0013, respectively). The CD137+ VETc showed 11.2-fold enhanced cytotoxicity compared to TN at 10:1 E:T (p<0.0001), while VETc showed 44-fold increase (p=0.0020). Anti-tumor activity and specificity was maintained following CD3/CD28-mediated expansion. In vivo, a survival benefit was observed for mice treated with CD137+ VETc (100 days mOS not reached, p=0.0082) and VETc (mOS=39 days, p=0.0082) compared to mice treated with TN (mOS=28 days). In the human model, CD137+ VETc demonstrate enhanced immune activation compared to TN (15.8-fold, p=0.0060). A 16-fold target cell expansion was observed across 10 days with a cell viability of 85-100% in the CliniMACS Prodigy. SMAD2 KO resulted in indel efficiency of 100% and predicted KO score of 99% calculated by synthego inference of CRISPR edits analysis. Western blot validation confirmed significant reduction in SMAD2 protein levels in KO cells, with near complete loss of protein expression. SMAD2 KO T cells exhibited increased proliferation in culture media containing TGF-β compared to WT.

Conclusion: VETc enriched for CD137 via agonistic antibody selection represents a unique approach for adoptive immunotherapy in AML with enhanced cytotoxicity, activation, and memory cell phenotype. We report a first institutional experience using a closed system to manufacture TRT cells as an adoptive cell therapy for patients with AML. Knockout of SMAD2 was validated in human T cells as a strategy to mitigate cytostasis by immunosuppressive TGF-β signaling. Ongoing investigations are evaluating the effects of lymphodepletion and immune boosting with DC/AML vaccination on T cell persistence and expansion in vivo using a C57BL/6-CD45.1/2 immunocompetent murine model for adoptive T cell tracking. Future directions include clinical trial investigation of the proposed cellular therapy in AML patients.