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318 Building A Better CAR: Improving CAR-T Trafficking in Cancer Therapy

Published online by Cambridge University Press:  19 April 2022

Yeonsun Hong
Affiliation:
University of Rochester School of Medicine and Dentistry
Hye-Ran Kim
Affiliation:
University of Rochester School of Medicine and Dentistry
Brandon L. Walling
Affiliation:
University of Rochester School of Medicine and Dentistry
John Lozada
Affiliation:
University of Rochester School of Medicine and Dentistry
Andrea M. Amitrano
Affiliation:
University of Rochester School of Medicine and Dentistry
Cooper J. Sailer
Affiliation:
University of Rochester School of Medicine and Dentistry
Kihong Lim
Affiliation:
University of Rochester School of Medicine and Dentistry
Raj Kumar Mongre
Affiliation:
University of Rochester School of Medicine and Dentistry
Kyun-Do Kim
Affiliation:
University of Rochester School of Medicine and Dentistry
Tara Capece
Affiliation:
University of Rochester School of Medicine and Dentistry
Elena B. Lomakina
Affiliation:
University of Rochester School of Medicine and Dentistry
Nicholas S. Reilly
Affiliation:
University of Rochester School of Medicine and Dentistry
Richard E. Waugh
Affiliation:
University of Rochester School of Medicine and Dentistry
Patrick M. Reagan
Affiliation:
University of Rochester School of Medicine and Dentistry
Minsoo Kim
Affiliation:
University of Rochester School of Medicine and Dentistry
Patrick W. Oakes
Affiliation:
Loyola University
Chang-Duk Jun
Affiliation:
GIST
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Abstract

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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.

Type
Valued Approaches
Creative Commons
Creative Common License - CCCreative Common License - BYCreative Common License - NCCreative Common License - ND
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.
Copyright
© The Author(s), 2022. The Association for Clinical and Translational Science