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312 IL7 increases targeted lipid nanoparticle-mediated mRNA protein expression in T cells in vitro and in situ by enhancing T cell translation
  1. Caitlin M Tilsed1,
  2. Barzan A Sadiq2,
  3. Tyler E Papp1,
  4. Phurin Areesawangkit1,
  5. Estela Noguera-Ortega1,
  6. Kenji Kimura1,
  7. Nicholas Cerda1,
  8. Haig Aghajanian3,
  9. Adrian Bot2,
  10. Barbara Mui4,
  11. Ying Tam4,
  12. Drew Weissman1,
  13. Steven M Albelda1 and
  14. Hamideh Parhiz1
  1. 1University of Pennsylvania, Philadelphia, PA, USA
  2. 2Capstan Therapeutics, San Diego, CA, USA
  3. 3Capstan Therapeutics, Philadelphia, PA, USA
  4. 4Acuitas Therapeutics, Vancouver, BC, Canada
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Chimeric Antigen Receptor T cells (CARTs) are a powerful anti-cancer therapy, demonstrating success in hematologic malignancies. The development of targeted lipid nanoparticle-mRNA (tLNP-mRNA) therapeutics has allowed for the generation of CARTs in situ and may resolve several challenges of conventional ex vivo viral-engineered CAR T cell products including scalability, access and mulitplexing. The in situ T cell transfection rate achieved by tLNP-mRNA varies from 4–20% of T cells expressing the protein of interest. As tLNP-mRNA platform efficacy may critically depend on the number, metabolic state, and localization of engineered T cells, we investigated whether cytokines could enhance protein expression.

Methods We used tLNPs that target CD5, a marker expressed highly on mouse and human T cells. These tLNPs carried the mRNA for the reporter protein mCherry or encoded a fibroblast activated protein (FAP) targeted CAR. Mouse and human T cells were cultured with IL2, IL7, IL15 or activated using αCD3/CD28. CD5-mCherry-tLNPs or CD5-FAPCAR-tLNPs were added and protein expression was detected using flow cytometry. For in vivo studies, C57BL/6 mice were pretreated with IL7, injected with tLNPs and sacrificed 24 hours later. To analyze the T cell transcriptome, CD8+ T cells were isolated from mouse spleens and cultured with IL2, IL7 or IL15 for 48 hours before being sequenced.

Results We found that CD5-mCherry-tLNPs induced protein expression on 10% of resting T cells in vivo and ~15% of T cells in vivo. Culturing mouse and human T cells with IL7 significantly improved CD5-mCherry-tLNPs protein expression in vitro. This also occurred in the in vivo setting as pre-treating mice with IL7 elevated both the proportion and total number of mCherry expressing T cells. FAPCAR expression was also increased by combining CD5-FAPCAR-tLNPs with recombinant IL7. Transcriptomic analysis showed IL7 selectively increased pathways associated with protein translation. The significance of these transcriptomic changes was demonstrated by showing that after electroporation with mRNA, T cells cultured in IL7 produced more protein compared to IL2 or IL15.

Conclusions T cells can be engineered in situ using CD5-targeted tLNPs and IL7 increases the protein expression induced by tLNPs. Our data suggests that the upregulation of translation-associated pathways in T cells by IL7 could be exploited to improve the expression of proteins in situ after tLNP administration. This provides a novel paradigm through which a T cell activating cytokine, instead of lymphodepletion, can potentiate in situ CAR T cell therapy.

Ethics Approval This study was approved by The University of Pennsylvania’s Ethics Board; approval number 806099.

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