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318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy
OBJECTIVES/GOALS: #NAME? METHODS/STUDY POPULATION: Cell culture & protein identification: human T cells were purified from healthy blood, then activated & cultured for 5d. CAR-T cells were collected from infusion bags of cancer patients undergoing CAR-T. Silver staining of naive & activa...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Cambridge University Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209174/ http://dx.doi.org/10.1017/cts.2022.177 |
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author | Hong, Yeonsun Kim, Hye-Ran Walling, Brandon L. Lozada, John Amitrano, Andrea M. Sailer, Cooper J. Lim, Kihong Mongre, Raj Kumar Kim, Kyun-Do Capece, Tara Lomakina, Elena B. Reilly, Nicholas S. Waugh, Richard E. Reagan, Patrick M. Kim, Minsoo Oakes, Patrick W. Jun, Chang-Duk |
author_facet | Hong, Yeonsun Kim, Hye-Ran Walling, Brandon L. Lozada, John Amitrano, Andrea M. Sailer, Cooper J. Lim, Kihong Mongre, Raj Kumar Kim, Kyun-Do Capece, Tara Lomakina, Elena B. Reilly, Nicholas S. Waugh, Richard E. Reagan, Patrick M. Kim, Minsoo Oakes, Patrick W. Jun, Chang-Duk |
author_sort | Hong, Yeonsun |
collection | PubMed |
description | OBJECTIVES/GOALS: #NAME? METHODS/STUDY POPULATION: Cell culture & protein identification: human T cells were purified from healthy blood, then activated & cultured for 5d. CAR-T cells were collected from infusion bags of cancer patients undergoing CAR-T. Silver staining of naive & activated healthy T-cell lysates was compared; B-II spectrin was upregulated and confirmed by Western blot. Migration assays: naive & activated T-cells were imaged during migration on ICAM-1 and ICAM-1 + CXCL12 coated plates. T-cells were transfected with BII-spectrin cDNA & the chemokine dependence of migration was compared with controls. In-vivo studies: in a melanoma mouse model, BII-spectrin transfected or control T-cells were injected; tumors were followed with serial imaging. Human patient records were examined to correlate endogenous BII-spectrin levels and CAR-T response. RESULTS/ANTICIPATED RESULTS: Activated T-cells downregulate the cytoskeletal protein B-II spectrin compared to naive cells, leading to chemokine-independent migration in in vitro assays and off-target trafficking when CAR-T cells are given in vivo. Restoration of B-II spectrin levels via transfection restores chemokine-dependence of activated T-cells. In a mouse melanoma model, control mice injected with standard activated T-cells showed fewer cells in the tumor site and more cells in the off-target organs (spleen, lungs) when compared to mice injected with B-II spectrin transfected cells. Furthermore, among 3 human patients undergoing CAR-T therapy, those with higher endogenous B-II spectrin levels experienced fewer side-effects, measured by the neurotoxicity and cytokine release syndrome grades. DISCUSSION/SIGNIFICANCE: A major hurdle to widespread CAR-T therapy for cancer is significant, often fatal side-effects. Our work shows that the protein B-II spectrin is downregulated during CAR-T production, and that restoring B-II spectrin levels decreases side-effects while increasing tumor clearance--hopefully translating to better CAR-T regimens for the future. |
format | Online Article Text |
id | pubmed-9209174 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Cambridge University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-92091742022-07-01 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy Hong, Yeonsun Kim, Hye-Ran Walling, Brandon L. Lozada, John Amitrano, Andrea M. Sailer, Cooper J. Lim, Kihong Mongre, Raj Kumar Kim, Kyun-Do Capece, Tara Lomakina, Elena B. Reilly, Nicholas S. Waugh, Richard E. Reagan, Patrick M. Kim, Minsoo Oakes, Patrick W. Jun, Chang-Duk J Clin Transl Sci Valued Approaches OBJECTIVES/GOALS: #NAME? METHODS/STUDY POPULATION: Cell culture & protein identification: human T cells were purified from healthy blood, then activated & cultured for 5d. CAR-T cells were collected from infusion bags of cancer patients undergoing CAR-T. Silver staining of naive & activated healthy T-cell lysates was compared; B-II spectrin was upregulated and confirmed by Western blot. Migration assays: naive & activated T-cells were imaged during migration on ICAM-1 and ICAM-1 + CXCL12 coated plates. T-cells were transfected with BII-spectrin cDNA & the chemokine dependence of migration was compared with controls. In-vivo studies: in a melanoma mouse model, BII-spectrin transfected or control T-cells were injected; tumors were followed with serial imaging. Human patient records were examined to correlate endogenous BII-spectrin levels and CAR-T response. RESULTS/ANTICIPATED RESULTS: Activated T-cells downregulate the cytoskeletal protein B-II spectrin compared to naive cells, leading to chemokine-independent migration in in vitro assays and off-target trafficking when CAR-T cells are given in vivo. Restoration of B-II spectrin levels via transfection restores chemokine-dependence of activated T-cells. In a mouse melanoma model, control mice injected with standard activated T-cells showed fewer cells in the tumor site and more cells in the off-target organs (spleen, lungs) when compared to mice injected with B-II spectrin transfected cells. Furthermore, among 3 human patients undergoing CAR-T therapy, those with higher endogenous B-II spectrin levels experienced fewer side-effects, measured by the neurotoxicity and cytokine release syndrome grades. DISCUSSION/SIGNIFICANCE: A major hurdle to widespread CAR-T therapy for cancer is significant, often fatal side-effects. Our work shows that the protein B-II spectrin is downregulated during CAR-T production, and that restoring B-II spectrin levels decreases side-effects while increasing tumor clearance--hopefully translating to better CAR-T regimens for the future. Cambridge University Press 2022-04-19 /pmc/articles/PMC9209174/ http://dx.doi.org/10.1017/cts.2022.177 Text en © The Association for Clinical and Translational Science 2022 https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article, distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is unaltered and is properly cited. The written permission of Cambridge University Press must be obtained for commercial re-use or in order to create a derivative work. |
spellingShingle | Valued Approaches Hong, Yeonsun Kim, Hye-Ran Walling, Brandon L. Lozada, John Amitrano, Andrea M. Sailer, Cooper J. Lim, Kihong Mongre, Raj Kumar Kim, Kyun-Do Capece, Tara Lomakina, Elena B. Reilly, Nicholas S. Waugh, Richard E. Reagan, Patrick M. Kim, Minsoo Oakes, Patrick W. Jun, Chang-Duk 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title | 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title_full | 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title_fullStr | 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title_full_unstemmed | 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title_short | 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy |
title_sort | 318 building a better car: improving car-t trafficking in cancer therapy |
topic | Valued Approaches |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9209174/ http://dx.doi.org/10.1017/cts.2022.177 |
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