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...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Cambridge University Press
2022-04-01
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| Series: | Journal of Clinical and Translational Science |
| Online Access: | https://www.cambridge.org/core/product/identifier/S2059866122001777/type/journal_article |
| _version_ | 1811155260492742656 |
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| author | Yeonsun Hong Hye-Ran Kim Brandon L. Walling John Lozada Andrea M. Amitrano Cooper J. Sailer Kihong Lim Raj Kumar Mongre Kyun-Do Kim Tara Capece Elena B. Lomakina Nicholas S. Reilly Richard E. Waugh Patrick M. Reagan Minsoo Kim Patrick W. Oakes Chang-Duk Jun |
| author_facet | Yeonsun Hong Hye-Ran Kim Brandon L. Walling John Lozada Andrea M. Amitrano Cooper J. Sailer Kihong Lim Raj Kumar Mongre Kyun-Do Kim Tara Capece Elena B. Lomakina Nicholas S. Reilly Richard E. Waugh Patrick M. Reagan Minsoo Kim Patrick W. Oakes Chang-Duk Jun |
| author_sort | Yeonsun Hong |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-10T04:31:13Z |
| format | Article |
| id | doaj.art-f605ef69391e4c20b5889316e4a19c02 |
| institution | Directory Open Access Journal |
| issn | 2059-8661 |
| language | English |
| last_indexed | 2024-04-10T04:31:13Z |
| publishDate | 2022-04-01 |
| publisher | Cambridge University Press |
| record_format | Article |
| series | Journal of Clinical and Translational Science |
| spelling | doaj.art-f605ef69391e4c20b5889316e4a19c022023-03-10T07:53:49ZengCambridge University PressJournal of Clinical and Translational Science2059-86612022-04-016575710.1017/cts.2022.177318 Building A Better CAR: Improving CAR-T Trafficking in Cancer TherapyYeonsun Hong0Hye-Ran Kim1Brandon L. Walling2John Lozada3Andrea M. Amitrano4Cooper J. Sailer5Kihong Lim6Raj Kumar Mongre7Kyun-Do Kim8Tara Capece9Elena B. Lomakina10Nicholas S. Reilly11Richard E. Waugh12Patrick M. Reagan13Minsoo Kim14Patrick W. Oakes15Chang-Duk Jun16University of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryUniversity of Rochester School of Medicine and DentistryLoyola UniversityGISTOBJECTIVES/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.https://www.cambridge.org/core/product/identifier/S2059866122001777/type/journal_article |
| spellingShingle | Yeonsun Hong Hye-Ran Kim Brandon L. Walling John Lozada Andrea M. Amitrano Cooper J. Sailer Kihong Lim Raj Kumar Mongre Kyun-Do Kim Tara Capece Elena B. Lomakina Nicholas S. Reilly Richard E. Waugh Patrick M. Reagan Minsoo Kim Patrick W. Oakes Chang-Duk Jun 318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy Journal of Clinical and Translational Science |
| 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 |
| url | https://www.cambridge.org/core/product/identifier/S2059866122001777/type/journal_article |
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