iPSC-Derived Chimeric Antigen Receptor T Cells That Originally Lack CD5 Expression Exhibits Robust Cytotoxicity Against T-Cell Malignancies

While CD19-directed chimeric antigen receptor T (CAR-T) therapy has demonstrated promising results for R/R B-cell lymphomas, the prognosis for patients with T-cell malignancies remains poor.

CD5 is expressed on many T-cell malignancies, suggesting a good target for refractory and relapsed (R/R) T cell lymphoma / leukemia. However, CAR-T therapies targeting CD5 T-cell malignancies are more challenging because of fratricide, caused by shared target antigen between conventional T-cells and T-cell malignancies, which induces exhaustion and poor proliferation.

We have developed a platform to generate antigen-specific cytotoxic T lymphocytes (CTLs) from iPSCs. These CTLs (rejTs) showed younger memory phenotype and stronger cytotoxicity than the original CTLs, functionally rejuvenated (Cell Stem Cell 2013, Stem Cell Reports 2015). Additionally, we have demonstrated that LMP2-specific rejTs (LMP2-rejTs) robustly suppress EBV-associated lymphoma in vivo with remarkable persistence for more than seven months (Haematologica 2020). Next, we generated dual-antigen receptor T cells from iPSCs by introduction of CD19-directed CAR into LMP2-rejTs which have already demonstrated in vivo persistence. These cells recognize dual antigens via CAR and native T cell receptor, resulting in cooperative antitumor effects in vivo (Molecular Therapy 2021).

We confirmed that these iPSC-derived LMP2-rejTs lacked CD5 expression by flow cytometry. Based on this, we hypothesized that CD5-CAR-introduced rejTs (CD5-CARrejTs) could exhibit robust cytotoxicity without fratricide, making them a promising therapeutic approach.

In this study, we developed CD5-CARrejTs by incorporating CD5-CAR into LMP2-rejTs. We reprogrammed a healthy donor-derived LMP2-CTL clone into iPSCs, followed by transduction with a lentiviral CD5-CAR vector. CAR transgene expression and LMP2 antigen-specificity in CD5-CARrejTs were evaluated by flow cytometry, resulting in nearly 100% for both. As a control, we used CD5KO CARTs (KO-CD5-CARTs) generated using CRISPR/Cas9 gene editing technology, which has been reported to overcome fratricide.

To compare the cytotoxicity of CD5-CARrejTs with CD5-CARTs against T cell leukemia cells, we performed ⁵¹Cr release assays. The cytotoxicity of CD5-CARrejTs against Jurkat cells was higher (86.2%) than that of wild type (WT)-CD5-CARTs (25.0%) and KO-CD5-CARTs (41.0%), at an effector to target ratio of 20:1. Similar results were obtained using other T-cell leukemia / lymphoma cell lines (CCRF-CEM, SU-DHL1, and MT-2).

To investigate the differences in phenotypes between CD5-CARTs and CD5-CARrejTs, we performed flow cytometry. CD5-CARrejTs showed higher expression of perforin and granzyme B than CD5-CARTs. Additionally, cytometric bead array revealed that CD5-CARrejTs secreted significantly higher levels of IFN-γ and TNF-α (both, p<0.0001) compared to CD5-CARTs. Moreover, single-cell RNA-sequencing analysis indicated that CD5-CARrejTs exhibited higher expression of genes associated with cytotoxicity (IFNG, GZMB, GNLY) and TCR downstream signals (LYN, SRC) than CD5-CARTs.

Finally, to investigate antitumor effect of CD5-CARrejTs in vivo, Jurkat cells labeled with firefly luciferase were intravenously injected into NOG mice. Three days after tumor injection, the mice were divided into untreated and 3 treated groups (CD5-CARrejTs, WT-CD5-CARTs, and KO-CD5-CARTs). As a result, CD5-CARrejTs significantly prolonged the survival of mice compared to WT-CD5-CARTs (p=0.0008), and KO-CD5-CARTs (p = 0.0227).

In conclusion, iPSC-derived CD5-CARrejTs, which originally lacked CD5 expression exhibit robust cytotoxicity against T-cell malignancies without fratricide. The most significant advantage of this therapy is the ability to supply unlimited amounts of therapeutic cells, which can be used as “off-the-shelf” CAR-T therapy. These results suggest that CD5-CARrejTs represent a promising novel approach to overcome R/R T-cell malignancies.