Novel allogeneic BCMA CAR-expressing iNKT cells with PD1 deletion demonstrate superior tumor control in a multiple myeloma preclinical model

Abstract

Abstract Background: Two BCMA-directed CAR-T cell products, Idecabtagene vicleucel (ide-cel) and Ciltacabtagene autoleucel (cilta-cel), have received FDA approval for use in patients with relapsed/refractory multiple myeloma (R/R MM) and demonstrated outstanding benefit. However, the use of autologous CAR T cells being manufactured in a centralized production site poses several challenges, including prolonged manufacturing timelines, higher costs, delayed treatment initiation, and reliance on the patient's own T cell quality and functionality. Here we use invariant natural killer T (iNKT) cells as the basis for our allogeneic “off-the-shelf” cell therapy platform. iNKT cells share characteristics of T cells and Natural Killer (NK) cells, expressing both an invariant TCR (Va24Ja18) and canonical NK receptors. They can be activated by recognition of lipid antigens in the context of CD1d through the TCR and recognition of stress ligands via NKRs. Moreover, iNKT cells do not cause Graft versus Host Disease, making them an ideal platform for allogeneic CAR cell therapy. Here we describe a novel allogeneic PD1-deleted iNKT product targeting BCMA designed to promote the efficacy of adoptive T cell therapy. Methods: We sought to identify the improvement of CAR designs that would enhance iNKT cells. The VHH CAR construct targeting BCMA and its modified variant incorporating additional PD1 intracellular retention (a non-gene editing technique to remove membrane proteins) were developed to assess and compare their in vivo therapeutic efficacy of eliminating malignant myeloma cells. In the study, 3×10⁶ of H929-luc cells per mouse were inoculated subcutaneously into NOG-hIL15Tg mice, allowed to engraft for 5 days. Subsequently, the mice were treated with one or three injections of 4.5×10⁶ CAR+ iNKT cells per mouse on days 5, 10, and 15 respectively. This experimental setup enabled comparison of anti-tumor activity between the two CAR designs and assessment of dosing frequency impact. Results: The CAR-iNKT with PD1 deletion demonstrated a much better long-term cytotoxicity against H929 cells than the unmodified CAR-iNKT in vitro in a 50-hour real-time killing assay by Incucyte at an E:T ratio of 3:1. To further evaluate the efficacies of both BCMA CAR-iNKT cells in vivo, we deployed a xenograft myeloma model using H929 luciferase-expressing cells. Both CAR-iNKT cells along with their controls were i.v transferred to myeloma-bearing mice at one or three injections and disease burden was monitored by bioluminescence. As a result, the CAR-iNKT with PD1 deletion in three doses showed much better tumor control in the animal model study until D35. Conclusions:We are advancing a novel allogeneic CAR-iNKT cell therapy by integrating our proprietary PD-1 intracellular retention technology to boost the efficacy of adoptive cell treatment. Leveraging an iNKT platform naturally devoid of alloreactivity, we enable rapid engineering and expansion of an “off-the-shelf” CAR product. Additionally, the inherent capabilities of iNKT cells, such as bone marrow homing and BCMA targeting, reinforced by CD1d and NK receptor-ligand interactions, offer unique advantages. We believe this approach could deliver significant benefits to patients with R/R MM, surpassing the efficacy of existing therapies.