Vγ9Vδ2 T cells (Vδ2 T cells hereafter) are an attractive platform for allogeneic chimeric antigen receptor (CAR)-modified cell therapy, as their invariant TCR minimizes their risk of graft-versus-host disease. However, the susceptibility of Vδ2 T cells to activation-induced cell death (AICD) and dependency on exogenous cytokine supplementation for prolonged persistence limit their in vivo efficacy. Addressing these limitations in would increase therapeutic potential of engineered Vδ2 T cells.

Fas signaling is critical for AICD induction in Vδ2 T cells. To attenuate AICD, we generated a truncated Fas receptor lacking a signaling endodomain (tFas), to prevent downstream activation of apoptosis. Incubation with soluble FasL for 24h induced apoptosis in unmodified Vδ2 T cells and CD19.CAR-expressing Vδ2 T cells, while tFas-armed CD19.CAR-Vδ2 T cells resisted FasL-induced cell death.

In extended (9-day) coculture experiments with CD19(+) NALM6 target cells at a 1:2 E:T ratio without exogenous cytokines, tFas-armed CD19.CAR-Vδ2 T cells produced enhanced anti-tumor control at early time points, compared to unarmed CD19.CAR-Vδ2 T cells which exhibited only transient activity, resulting in tumor regrowth. However, the lack of cytokine support limited long-term expansion of tFas/CD19.CAR-Vδ2 T cells resulting in tumor regrowth after 6 days of coculture.

IL-18 signaling via MyD88 stimulates Vδ2 T cells, promoting their growth. Hence, we hypothesized combining IL-18/MyD88 signaling with AICD suppression would synergize to improve anti-tumor function of Vδ2 T cells. We generated a chimeric receptor consisting of the extracellular domain of Fas linked with intracellular MyD88 (Fas88) aiming to both prevent AICD and transmit MyD88 signaling upon engagement with FasL. Fas88-armed CD19.CAR-Vδ2 T cells resisted FasL-induced apoptosis similar to those armed with a tFas construct. Furthermore, in cocultures with NALM6 cells, Fas88/CD19.CAR-Vδ2 T cells resisted AICD and, in contrast to tFas/CD19.CAR-Vδ2 T cells, expanded over 9 days in the absence of exogenous cytokines and mediated a significantly superior tumor control.

Next, we investigated the activity of armed CD19.CAR-Vδ2 T cells in a xenograft mouse model, where NALM6 cells (0.5 x 10 6 cells/mouse) were intravenously infused into NSG mice followed 5 days later by a single injection of Vδ2 T cells (4 x 10 6 cells/mouse). Both tumor and T cell populations were tracked via dual bioluminescent imaging. Unmodified CD19.CAR-Vδ2 T cells extended median survival from 18 days (control group) to 30 days but demonstrated limited persistence. tFas-armed CD19.CAR-Vδ2 T cells produced comparable tumor control, with a median survival of 31.5 days, suggesting that AICD suppression alone is insufficient for the long-term activity of CD19.CAR-Vδ2 T cells in vivo. In contrast, Fas88/CD19.CAR-Vδ2 T cells persisted and expanded in tumor-bearing mice improving the median survival to 47 days (p = 0.0478), indicating the ability of Fas88 to sustain functional persistence of CD19.CAR-Vδ2 T cells. This data shows that combining AICD suppression with MyD88 signaling improves the therapeutic potency of CAR-engineered Vδ2 T cells, demonstrating a means for exploiting Vδ2 T cells for allogeneic adoptive immunotherapy. Current work is focused on evaluating this approach for clinical translation.

Heslop:Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Research Support; Marker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; PACT Pharma: Membership on an entity's Board of Directors or advisory committees; Mesoblast: Membership on an entity's Board of Directors or advisory committees; Ankyra Therapeutics: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in private company; Fresh Wind Biotechnologies: Current equity holder in private company; CoRegen Inc.: Current equity holder in private company; Kuur Therapeutics: Other: Research Support. Brenner:Allovir: Current equity holder in private company, Current holder of stock options in a privately-held company; Marker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Tessa Therapeutics: Consultancy, Current equity holder in private company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Allogene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Kuur: Consultancy, Membership on an entity's Board of Directors or advisory committees; Turnstone Biologics: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Posedia: Membership on an entity's Board of Directors or advisory committees; Tscan: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Adaptimmune Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adintus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onkimure: Membership on an entity's Board of Directors or advisory committees; Triumvira Immunologics: Membership on an entity's Board of Directors or advisory committees; Allogene: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Bellicum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen, LLC: Consultancy, Membership on an entity's Board of Directors or advisory committees; Brooklyn Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Coya: Membership on an entity's Board of Directors or advisory committees; Astrazeneca Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Takeda: Patents & Royalties. Watanabe:Allogene Therapeutics, Inc: Patents & Royalties.

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2023
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