Development of an in Situ CAR-T Cell Protocol Through Optical and PSMA-Targeted PET Imaging

Chimeric antigen receptor (CAR)-T cell therapy has transformed the treatment landscape for hematologic malignancies. Despite its clinical success, the conventional CAR-T manufacturing process remains complex and costly, involving ex vivo manipulation and expansion of patient-derived T cells, and patients may face severe side effects and safety issues.

To overcome these limitations, in vivo CAR-T cell engineering has emerged as a next-generation approach. This strategy utilizes targeted delivery of CAR-encoding mRNA using lipid nanoparticles (LNPs) to program T cells directly within the body, bypassing the need for lymphodepletion and allowing for repeated dosing. However, the absence of ex vivo characterization necessitates robust noninvasive tools to monitor T cell generation, distribution, and tumor engagement in real time.

Positron emission tomography (PET) offers high sensitivity for tracking T cells labeled with molecular tags. Among various imaging targets, prostate-specific membrane antigen (PSMA) is especially attractive due to its clinical availability and well-established PET imaging probes, including FDA-approved agents like 18F-DCFPyL (PYLARIFY®, 2021) and 68Ga-PSMA-11 (LOCAMETZ®, 2022). While primarily used for prostate cancer, the technique of PSMA-targeted PET imaging can also be applied for CAR-T cell surveillance, especially for the assessment of in situ engineered CAR-T cells.

In our recent study, we established a protocol for in vivo CD19 CAR-T cell therapy and collaborated with Dr. Ronald Levy, a renowned oncologist and expert in lymphoma immunotherapy, to treat B cell lymphoma. Anti-CD5 antibody-conjugated LNPs (aCD5 LNPs) were developed for co-delivering CD19 CAR mRNA (mCAR) and PSMA mRNA (mPSMA) to T cells in vivo, enabling both therapeutic function and imaging capability. Working with Dr. Martin G. Pomper, a pioneer in developing imaging and therapeutic agents targeting PSMA, we employed a truncated PSMA variant (tPSMA-N9del), which lacks intracellular signaling domains to avoid interference with T cell function. Following administration of aCD5 LNPs in mice, CAR and PSMA were co-expressed on T cells. PSMA-targeted PET imaging of 68Ga-PSMA-617 was applied to noninvasively detect PSMA-expressing T cells. In PET/CT imaging of healthy BALB/c mice, tracer accumulation increased in spleens with high T cell density. In a lymphoma mouse model, mice treated with aCD5 LNPs encoding both mCAR19 and mPSMA exhibited significantly higher 68Ga-PSMA-617 uptake in tumors, confirming infiltration of in situ engineered CAR-T cells tagged with PMSA. Further, aCD5 LNP-mPSMA/mCAR19 treatments significantly suppressed lymphoma growth and extended survival.

In summary, co-delivery of CAR and PSMA reporter mRNAs using targeted LNPs enables PET imaging of in vivo-engineered T cells and therapeutic efficacy against tumors. This imaging-guided approach provides a powerful tool for assessing in vivo CAR-T cell therapy, with promising implications for clinical translation.

Written by: Nisi Zhang,¹ Jai Woong Seo,¹ Elise Robinson,¹ Angelie Rivera-Rodriguez,¹ James Wang,¹ Yutong Guo,¹ Debra K. Czerwinski,² Ha Rin Kim,² Spencer K. Tumbale,¹ Marina N. Raie,¹ Basit L. Jan,¹ Gokce Engudar,¹ Adrienne Sallets,² Il Minn,³ Martin G. Pomper,³ Ronald Levy,² Katherine W. Ferrara¹

Molecular Imaging Program at Stanford, Department of Radiology, School of Medicine, Stanford University, Stanford, CA.
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA.
Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX.

Read the Abstract

Login