PT  - JOURNAL ARTICLE
AU  - Fang, Karen Kai-Lin
AU  - Lee, Jongbok
AU  - Khatri, Ismat
AU  - Na, Yoosu
AU  - Zhang, Li
TI  - Targeting T-cell malignancies using allogeneic double-negative CD4-CAR-T cells
AID  - 10.1136/jitc-2023-007277
DP  - 2023 Sep 01
TA  - Journal for ImmunoTherapy of Cancer
PG  - e007277
VI  - 11
IP  - 9
4099  - http://jitc.bmj.com/content/11/9/e007277.short
4100  - http://jitc.bmj.com/content/11/9/e007277.full
SO  - J Immunother Cancer2023 Sep 01; 11
AB  - Background Patients with relapsed/refractory T-cell malignancies have limited treatment options. The use of chimeric antigen receptor (CAR)-T cell therapy for T-cell malignancies is challenging due to possible blast contamination of autologous T-cell products and fratricide of CAR-T cells targeting T-lineage antigens. Recently, allogeneic double-negative T cells (DNTs) have been shown to be safe as an off-the-shelf adoptive cell therapy and to be amendable for CAR transduction. Here, we explore the antitumor activity of allogeneic DNTs against T-cell malignancies and the potential of using anti-CD4-CAR (CAR4)-DNTs as adoptive cell therapy for T-cell malignancies.Methods Healthy donor-derived allogeneic DNTs were ex vivo expanded with or without CAR4 transduction. The antitumor activity of DNTs and CAR4-DNTs against T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphoma (PTCL) were examined using flow cytometry-based cytotoxicity assays and xenograft models. Mechanisms of action were investigated using transwell assays and blocking assays.Results Allogeneic DNTs induced endogenous antitumor cytotoxicity against T-ALL and PTCL in vitro, but high doses of DNTs were required to attain therapeutic effects in vivo. The potency of DNTs against T-cell malignancies was significantly enhanced by transducing DNTs with a third-generation CAR4. CAR4-DNTs were manufactured without fratricide and showed superior cytotoxicity against CD4+ T-ALL and PTCL in vitro and in vivo relative to empty-vector transduced-DNTs. CAR4-DNTs eliminated T-ALL and PTCL cell lines and primary T-ALL blasts in vitro. CAR4-DNTs effectively infiltrated tumors, delayed tumor progression, and prolonged the survival of T-ALL and PTCL xenografts. Further, pretreatment of CAR4-DNTs with PI3Kδ inhibitor idelalisib promoted memory phenotype of CAR4-DNTs and enhanced their persistence and antileukemic efficacy in vivo. Mechanistically, LFA-1, NKG2D, and perforin/granzyme B degranulation pathways were involved in the DNT-mediated and CAR4-DNT-mediated killing of T-ALL and PTCL.Conclusions These results demonstrate that CAR4-DNTs can effectively target T-ALL and PTCL and support allogeneic CAR4-DNTs as adoptive cell therapy for T-cell malignancies.Data are available upon reasonable request. All data used and analyzed in the current study are available from the corresponding author upon request.