Development of a novel CAR-T cell therapy using a multimodal approach to target T cell lymphoma

Abstract INTRODUCTION Relapsed/refractory T cell lymphoma (TCL) patients (both peripheral and cutaneous TCL) are aggressive malignancies with limited treatment options and dismal prognoses (<30% survival at 5 years). Chimeric antigen receptor (CAR)- T cell therapy has transformed the treatment landscape for refractory relapsed B cell malignancies. However, there are no approved CAR-T cell therapies for TCL, and progress is hindered by several formidable challenges: 1) overlap of T cell antigens between healthy and malignant cells leading to on-target off-tumor toxicities, 2) few healthy T cells in patients complicating autologous CAR-T cell manufacturing, and 3) antigen escape. Therefore, there is an urgent clinical need to develop tumor-specific CAR-T cell therapy for TCL that overcome these barriers. METHODS To develop a novel CAR-T cell therapy for TCL that addresses current challenges, we adopted a modular approach. This included selecting a tumor-specific target, employing a new allogeneic CAR-T cell technology, and conducting a screen to identify drugs that can be combined with CAR-T cells to prevent antigen escape. The KIR3DL2 receptor is highly expressed on TCL tumor cells and rarely expressed on healthy T and NK cells. Therefore, we tested several CAR-T constructs that target KIR3DL2 and tested their function against TCL cell lines and isogenic KIR-negative cell lines. We evaluated the efficacy of the best CAR-T cell candidates in vivo using localised and systemic mouse TCL models. To circumvent the issues of obtaining healthy T cells from TCL patients, we generated allogeneic CAR-T cells using a novel double knock-in/knockout strategy via CRISPR-Homology Direct Repair (HDR) technology, where we knocked in the CAR gene at the TRAC locus and the HLA-E gene in the B2M locus. Lastly, we conducted an epigenetic drug screen to increase KIR3DL2 expression on tumor T cells to further augment our CAR-T cell efficacy against antigen-low tumor cells. RESULTS Two out of the seven CAR-T cell constructs tested demonstrated superior effector function, as evidenced by cytotoxicity, proliferation, and cytokine secretion against TCL lines in comparison to isogenic KIR-negative lines. Importantly, KIR3DL2-CAR-T cells demonstrated superior tumor control in both in vivo localised and systemic CTCL and PTCL models compared to tumor-bearing mice that received control T cells. Remarkably, more than 70% of mice with aggressive CTCL leukemia showed complete tumor resolution after treatment with KIR-CAR-T cells. Additionally, the CRISPR-HDR approach successfully generated allogeneic CAR-T cells with a double-knock-in efficiency of greater than 70% and demonstrated significant anti-tumor function comparable to autologous CAR-T cells. Through our drug screen, we also identified drugs that can specifically upregulate KIR3DL2 expression on the tumor cells, which will be further tested in combination with CAR-T cells. CONCLUSION Through a multifaceted strategy that integrates careful target selection, CRISPR-HDR technology, and synergistic drug screening, we have developed an off-the-shelf CAR-T cell therapy for TCL. Our KIR3DL2 CAR-T cells demonstrated superior efficacy and tumor specificity in preclinical models compared to other candidate antigens. Furthermore, our epigenetic drug screening expands the applicability of our CAR-T cell therapy across diverse TCL subtypes. These findings support the potential of this platform to deliver a novel and much-needed treatment for TCL patients, potentially improving their clinical outcomes.