Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging

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

doi:10.1073/pnas.2504950122

Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging

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

Isotope Applications Research

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

In situ T cell transfection methods overcome the complexity and high costs associated with conventional chimeric antigen receptor (CAR)-T therapy. However, the in situ CAR-T cell approach operates within the patient’s complex immune environment and bypasses preinfusion ex vivo cellular quality controls, necessitating advanced imaging techniques to track immune cell migration and function. Positron emission tomography (PET) can detect biochemical processes in patients and, when combined with a radiotracer specific for the engineered cells, can monitor CAR-T cell trafficking. Herein, we develop an approach for in situ T cell generation, tracking, and functional assessment using anti-CD5-conjugated lipid nanoparticles for codelivering CD19 CAR mRNA (mCAR19) and a prostate-specific membrane antigen mRNA (mPSMA) tag. With interleukin-7 (IL-7) preconditioning and repeated administration, this approach achieves tumor-free survival in 75% of B cell lymphoma-bearing mice (similar efficacy to ex vivo approaches), and through PET imaging of ⁶⁸Ga-PSMA-617, the generation and tumor infiltration of in situ-engineered PSMA-tagged CD19 CAR-T cells is validated.

Original language	English
Article number	e2504950122
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	122
Issue number	24
DOIs	https://doi.org/10.1073/pnas.2504950122
State	Published - Jun 17 2025

Funding

We acknowledge the support of NIH R01CA253316, NIH T32CA118681, P41EB024495, and R01CA134675, and Leukemia and Lymphoma Society 8022-20. We also thank Dr. James Kochendorfer at the National Cancer Institute for providing the sequences of the CD19 CAR mRNA constructs. ACKNOWLEDGMENTS. We acknowledge the support of NIH R01CA253316, NIH T32CA118681,P41EB024495,and R01CA134675,and Leukemia and Lymphoma Society 8022-20. We also thank Dr. James Kochendorfer at the National Cancer Institute for providing the sequences of the CD19 CAR mRNA constructs.

Keywords

CAR-T therapy
immunotherapy
lipid nanoparticles
mRNA therapeutics
PET

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10.1073/pnas.2504950122

Cite this

Zhang, N., Seo, J. W., Robinson, E., Rivera-Rodriguez, A., Wang, J., Guo, Y., Czerwinski, D. K., Kim, H. R., Tumbale, S. K., Raie, M. N., Jan, B. L., Engudar, G., Sallets, A., Minn, I., Pomper, M. G., Levy, R., & Ferrara, K. W. (2025). Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging. Proceedings of the National Academy of Sciences of the United States of America, 122(24), Article e2504950122. https://doi.org/10.1073/pnas.2504950122

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title = "Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging",

abstract = "In situ T cell transfection methods overcome the complexity and high costs associated with conventional chimeric antigen receptor (CAR)-T therapy. However, the in situ CAR-T cell approach operates within the patient{\textquoteright}s complex immune environment and bypasses preinfusion ex vivo cellular quality controls, necessitating advanced imaging techniques to track immune cell migration and function. Positron emission tomography (PET) can detect biochemical processes in patients and, when combined with a radiotracer specific for the engineered cells, can monitor CAR-T cell trafficking. Herein, we develop an approach for in situ T cell generation, tracking, and functional assessment using anti-CD5-conjugated lipid nanoparticles for codelivering CD19 CAR mRNA (mCAR19) and a prostate-specific membrane antigen mRNA (mPSMA) tag. With interleukin-7 (IL-7) preconditioning and repeated administration, this approach achieves tumor-free survival in 75\% of B cell lymphoma-bearing mice (similar efficacy to ex vivo approaches), and through PET imaging of 68Ga-PSMA-617, the generation and tumor infiltration of in situ-engineered PSMA-tagged CD19 CAR-T cells is validated.",

keywords = "CAR-T therapy, immunotherapy, lipid nanoparticles, mRNA therapeutics, PET",

author = "Nisi Zhang and Seo, \{Jai Woong\} and Elise Robinson and Angelie Rivera-Rodriguez and James Wang and Yutong Guo and Czerwinski, \{Debra K.\} and Kim, \{Ha Rin\} and Tumbale, \{Spencer K.\} and Raie, \{Marina N.\} and Jan, \{Basit L.\} and Gokce Engudar and Adrienne Sallets and Il Minn and Pomper, \{Martin G.\} and Ronald Levy and Ferrara, \{Katherine W.\}",

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year = "2025",

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doi = "10.1073/pnas.2504950122",

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Zhang, N, Seo, JW, Robinson, E, Rivera-Rodriguez, A, Wang, J, Guo, Y, Czerwinski, DK, Kim, HR, Tumbale, SK, Raie, MN, Jan, BL, Engudar, G, Sallets, A, Minn, I, Pomper, MG, Levy, R & Ferrara, KW 2025, 'Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging', Proceedings of the National Academy of Sciences of the United States of America, vol. 122, no. 24, e2504950122. https://doi.org/10.1073/pnas.2504950122

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T1 - Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging

AU - Zhang, Nisi

AU - Seo, Jai Woong

AU - Robinson, Elise

AU - Rivera-Rodriguez, Angelie

AU - Wang, James

AU - Guo, Yutong

AU - Czerwinski, Debra K.

AU - Kim, Ha Rin

AU - Tumbale, Spencer K.

AU - Raie, Marina N.

AU - Jan, Basit L.

AU - Engudar, Gokce

AU - Sallets, Adrienne

AU - Minn, Il

AU - Pomper, Martin G.

AU - Levy, Ronald

AU - Ferrara, Katherine W.

PY - 2025/6/17

Y1 - 2025/6/17

N2 - In situ T cell transfection methods overcome the complexity and high costs associated with conventional chimeric antigen receptor (CAR)-T therapy. However, the in situ CAR-T cell approach operates within the patient’s complex immune environment and bypasses preinfusion ex vivo cellular quality controls, necessitating advanced imaging techniques to track immune cell migration and function. Positron emission tomography (PET) can detect biochemical processes in patients and, when combined with a radiotracer specific for the engineered cells, can monitor CAR-T cell trafficking. Herein, we develop an approach for in situ T cell generation, tracking, and functional assessment using anti-CD5-conjugated lipid nanoparticles for codelivering CD19 CAR mRNA (mCAR19) and a prostate-specific membrane antigen mRNA (mPSMA) tag. With interleukin-7 (IL-7) preconditioning and repeated administration, this approach achieves tumor-free survival in 75% of B cell lymphoma-bearing mice (similar efficacy to ex vivo approaches), and through PET imaging of 68Ga-PSMA-617, the generation and tumor infiltration of in situ-engineered PSMA-tagged CD19 CAR-T cells is validated.

AB - In situ T cell transfection methods overcome the complexity and high costs associated with conventional chimeric antigen receptor (CAR)-T therapy. However, the in situ CAR-T cell approach operates within the patient’s complex immune environment and bypasses preinfusion ex vivo cellular quality controls, necessitating advanced imaging techniques to track immune cell migration and function. Positron emission tomography (PET) can detect biochemical processes in patients and, when combined with a radiotracer specific for the engineered cells, can monitor CAR-T cell trafficking. Herein, we develop an approach for in situ T cell generation, tracking, and functional assessment using anti-CD5-conjugated lipid nanoparticles for codelivering CD19 CAR mRNA (mCAR19) and a prostate-specific membrane antigen mRNA (mPSMA) tag. With interleukin-7 (IL-7) preconditioning and repeated administration, this approach achieves tumor-free survival in 75% of B cell lymphoma-bearing mice (similar efficacy to ex vivo approaches), and through PET imaging of 68Ga-PSMA-617, the generation and tumor infiltration of in situ-engineered PSMA-tagged CD19 CAR-T cells is validated.

KW - CAR-T therapy

KW - immunotherapy

KW - lipid nanoparticles

KW - mRNA therapeutics

KW - PET

UR - https://www.scopus.com/pages/publications/105008394165

U2 - 10.1073/pnas.2504950122

DO - 10.1073/pnas.2504950122

M3 - Article

C2 - 40493195

AN - SCOPUS:105008394165

SN - 0027-8424

VL - 122

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

IS - 24

M1 - e2504950122

ER -

Development of an in situ CAR-T cell protocol through optical and PSMA-targeted PET imaging

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