About the Project
Background: Cell therapies using patients’ own T cells engineered to express chimeric antibody receptors (CAR-T) have had unprecedented success for haematological malignancies. This has initiated the search for cancer-specific CARs, as well as conventional HLA-restricted T cell receptors (TCRs), suitable for the generation of CAR-T or TCR-T cells in order to treat patients with solid, vascularised cancers. CAR-T cell therapy for leukaemias involves injecting engineered T cells into the bloodstream, where they have easy access to their blood-borne targets. However, cell therapies for solid cancers using CAR-T or TCR-T cells will depend on injected T cells being able to find and infiltrate cancerous tissues in sufficient numbers to kill the cancer. L-selectin is an important adhesion molecule that controls the recruitment of T cells from the bloodstream into lymph nodes and other vascularised organs. L-selectin is a candidate T cell homing molecule for solid cancers because ligands for L-selectin are induced on tumour blood vessels and L-selectin ligand expression correlates with improved patient outcome. Lentiviral or retroviral vectors are currently used to express CARs in leukaemia patients’ own T cells. T cell proliferation is required for efficient viral transduction, as well as the subsequent expansion of CAR-T cells to generate sufficient numbers of cells to treat patients. The techniques used to transduce and expand T cells for patient therapy are known to downregulate L-selectin expression. This occurs both by ectodomain proteolytic shedding of cell surface protein and transcriptional silencing of the gene for L-selectin. We hypothesised that the loss of L-selectin that accompanies T cell expansion will reduce the efficacy of T cell therapies for solid, vascularised cancers because injected T cells are unable to infiltrate and kill cancerous tissues. We have tested this hypothesis by generating ‘L-selectin enhanced’ tumour-specific T cells expressing a shedding-resistant mutant of L-selectin driven by a heterologous promoter that resists shedding and gene silencing such that expression is maintained on activated T cells. In mouse models of adoptive cell therapies for solid cancers, L-selectin-enhanced T cells are better at reducing cancer growth than wildtype T cells that downregulate L-selectin expression. We predict that L-selectin-enhanced cancer-specific T cells will be more effective in adoptive T cell therapies for solid cancers because they are able to infiltrate and kill cancerous tissues. The aim of the project is to determine the impact of enhanced L-selectin expression on cancer-specific CAR-T and TCR-T cells targeting for the same cancerous tissues.
Aims
1. To develop L-selectin-enhanced cancer-specific CAR-T and TCR-T cells for testing in preclinical models of adoptive T cell therapy of solid cancers
2. To determine whether enhanced L-selectin regulates the activation, differentiation and cytotoxicity of cancer-specific CAR-T and TCR-T cells
3. To determine the impact of enhanced L-selectin expression on cancer-specific CAR-T and TCR-T cell homing in cancer-bearing mice
Methods
HLA-A2-restricted TCRs and CARs against cancer cells will be expressed alongside wiltype and protease-resistant forms of human L-selectin in human T cells using lentiviral transduction. The impact of co-expressed wildtype or protease-resistant L-selectins on T cell activation and cytotoxicity will be determined using multiparameter flow cytometry. Humanised mouse models will be used to determine the impact of L-selectins on the recruitment of TCR-T or CAR-T cells into solid vascularised cancers using PET imaging to track injected T cells and determine the impact of wildtype and mutant L-selectins on the outcome of adoptive T cell therapy.
How results will be used
This proposal will determine whether enhanced expression of L-selectin could be used to improve the outcome of adoptive T cell immunotherapies. This approach complements and, therefore, can be combined with other therapies that aim to increase the number of cancer-specific T cells inside cancerous tissues, such as vaccination, checkpoint blockade inhibition and tumour blood vessel normalisation.
References
1. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol 2012, 12(4): 269-281.
2. Gattinoni L, Klebanoff CA, Palmer DC, Wrzesinski C, Kerstann K, Yu Z, et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells. J Clin Invest 2005, 115(6): 1616-1626.
3. Sampson JH, Choi BD, Sanchez-Perez L, Suryadevara CM, Snyder DJ, Flores CT, et al. EGFRvIII mCAR-modified T-cell therapy cures mice with established intracerebral glioma and generates host immunity against tumor-antigen loss. Clin Cancer Res 2014, 20(4): 972-984.
4. Mohammed RN, Watson HA, Vigar M, Ohme J, Thomson A, Humphreys IR, et al. L-selectin Is Essential for Delivery of Activated CD8(+) T Cells to Virus-Infected Organs for Protective Immunity. Cell Reports 2016, 14(4): 760-771.
5. Galkina E, Tanousis K, Preece G, Tolaini M, Kioussis D, Florey O, et al. L-selectin shedding does not regulate constitutive T cell trafficking but controls the migration pathways of antigen-activated T lymphocytes. J Exp Med 2003, 198(9): 1323-1335.
6. Richards H, Longhi MP, Wright K, Gallimore A, Ager A. CD62L (L-Selectin) Down-Regulation Does Not Affect Memory T Cell Distribution but Failure to Shed Compromises Anti-Viral Immunity. J Immunol 2008, 180(1): 198-206.
7. Yang S, Liu F, Wang QJ, Rosenberg SA, Morgan RA. The shedding of CD62L (L-selectin) regulates the acquisition of lytic activity in human tumor reactive T lymphocytes. PLoS One 2011, 6(7): e22560.
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