Thesis
TGF-[beta] dominant negative receptor equipped Nectin-4 CAR-iT and iNK cells for enhanced killing in bladder cancer tumor microenvironment
Master of Science (M.S.), Drexel University
May 2025
DOI:
https://doi.org/10.17918/00011085
Abstract
Chimeric Antigen Receptor (CAR)-T cell therapy is a therapeutic approach whereby T-cells are engineered with a synthetic receptor for increased tumor targeting specificity and tumor cytotoxicity. [1]. CAR-T cell therapies have demonstrated efficacy in treating hematological cancers including lymphomas, however, similar clinical success has not yet been demonstrated in solid tumor malignancies. One challenge in solid tumors is the harsh tumor microenvironment (TME) that maintains immune suppressive conditions throughout the area directly surrounding tumors [2]. Literature suggests that transforming growth factor beta (TGF-[beta]) is a critical factor in maintaining immunosuppressive conditions within the TME and that disruption of the TGF-[beta] signaling cascade can improve T-cell proliferation and tumor killing [3]. The objective of this work is to design dominant negative TGF-[beta] receptor type II (TGF-[beta]RII DNR) protein variants, engineer the receptors into IPSC-derived Natural Killer (iNK) and T-cells (iT) containing a solid tumor targeting CAR, demonstrate inhibition of the TGF-[beta] signaling cascade thus armoring the iT-cells against the inhibitory effects of TGF-[beta], and enhancing the ability to destroy solid tumors within the TGF-[beta]-rich tumor microenvironment. The ability of the TME to dysregulate immune cells can cripple CAR engineered immune effector cells limiting their ability to irradicate the disease. The novel technology of disrupting the TGF-[beta] receptor signaling cascade through a DNR is intended to improve efficacy of cell therapies in the TME. Specifically, this project focuses on bladder cancer as a solid tumor model, by demonstrating improved function of Nectin-4 targeted CAR engineered iT and iNK Cells in the presence of soluble TGF-[beta] when cells are equipped with TGF-[beta]RII DNR. IPSC-derived iT and iNK cells were engineered to express the TGF-[beta]RII DNR through lentiviral transductions for the purpose of screening construct designs and through homology directed repair (HDR) to align with the cell engineering methods used to generate therapeutic cell lines at Century Therapeutics. Multiple TGF-[beta]RII DNR designs were screened, and one design was selected and characterized in cytotoxicity assays in both iT and iNK cell platforms. The data demonstrate that the DNR engineering provides effective protection to the cells through TGF-[beta] signaling inhibition and supports enhanced tumor cell killing which will lead to more efficacious therapies against solid tumors with TGF-[beta]-rich TME in the clinic. The TGF-[beta]RII DNR was engineered and expressed on IPSC cells that were differentiated into Nectin-4 targeting CAR iT and iNK cells without observable impact on the differentiation profile. Future work may include in vivo testing of the DNR equipped cells, optimizing the spheroid assay for improved quantification of the behavior of CAR iT and iNK cells, and adapting the DNR design to prevent signaling of other inhibitory factors at the tumor site.
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Details
- Title
- TGF-[beta] dominant negative receptor equipped Nectin-4 CAR-iT and iNK cells for enhanced killing in bladder cancer tumor microenvironment
- Creators
- Olivia Anne Carton
- Contributors
- Kara L. Spiller (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 64 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- School of Biomedical Engineering, Science, and Health Systems (1997-2026); Drexel University
- Other Identifier
- 991022059033404721