T-cell specific transcriptional control eliminates CAR-mediated off-target transduction by CD3-retargeted lentiviral vectors, enabling safe in vivo generation of potent alphaBCMA-CAR T cells

CAR T-cell therapy is a transformative treatment for hematologic malignancies. A key milestone to expand access to this technology is to enable CAR-T generation directly within the patient. For in vivo therapy, the CAR delivery vehicle must resist serum neutralization, target T cells and exhibit minimal or no off-target effects. We recently developed the CD3-targeted lentiviral vector LV-169 which delivers a BCMA CAR transgene to lymph node-resident primary T cells in vivo. LV169 incorporates a VSV-G protein that has been detargeted (blinded) from its natural LDL receptor and re-targeted to T cells via a displayed CD3-specific scFv. However, CAR proteins incorporated into the LV-169 envelope during packaging can induce off-target transduction of malignant plasma cells. Here, we reversed the orientation of the CAR transgene and placed it downstream of the human distal Lck promoter. In contrast to the EF1alpha promoter, the Lck promoter was transcriptionally silenced in 293 suspension cells but was insufficiently active in Jurkat cells and primary T cells. We therefore inserted a TATA box and a CMV enhancer into the Lck promoter (Lck1.3). The reverse-orientation Lck1.3-driven CAR construct (R-Lck1.3) did not express the BCMA-CAR protein in transfected 293 LV producer cells but was highly active in LV-transduced Jurkat cells and primary human T cells. Western blotting confirmed the high abundance of CAR protein in EF1a-driven vector particles and its absence in R-Lck1.3 particles. Therapeutic efficacy of LV169-R-Lck1.3 vectors encoding the BCMA-CAR was evaluated in human PBMC-engrafted NSG-DKO mice with advanced, disseminated human multiple myeloma. Mice were engrafted with OPM-2-FLuc cells and humanized with PBMCs one week prior to vector infusion. Tumor burden was monitored weekly via luciferase imaging. A single intravenous dose of LV169 (1×10 9 LVP) resulted in complete tumor clearance within 14 days. Circulating anti-BCMA CAR-T cells were detectable 14 days after LV injection but became undetectable once the tumor was eliminated. No severe toxicity was observed, based on stable body weight and cytokine levels in the blood. No tumor growth was observed in treated mice that were re-challenged with OPM-2 on days 22 and 30. In summary, our newly developed LV169-R-Lck1.3 platform encoding the BCMA-CAR demonstrates potent therapeutic activity, and its further clinical translation is underway.