A Novel Cell-based Luciferase Reporter Platform for the Development and Characterization of T-Cell Redirecting Therapies and Vaccine Development

Abstract

T-cell immunotherapies are promising strategies to generate T-cell responses towards tumor-derived or pathogen-derived antigens. Adoptive transfer of T cells genetically modified to express antigen receptor transgenes has shown promise for the treatment of cancer. However, the development of T-cell redirecting therapies relies on the use of primary immune cells and is hampered by the lack of easy-to-use model systems and sensitive readouts to facilitate candidate screening and development. Particularly, testing T-cell receptor (TCR)-specific responses in primary T cells and immortalized T cells is confounded by the presence of endogenous TCR expression which results in mixed alpha/beta TCR pairings and compresses assay readouts. Herein, we describe the development of a novel cell-based TCR knockout (TCR-KO) reporter assay platform for the development and characterization of T-cell redirecting therapies. CRISPR/Cas9 was used to knockout the endogenous TCR chains in Jurkat cells stably expressing a human interleukin-2 promoter-driven luciferase reporter gene to measure TCR signaling. Reintroduction of a transgenic TCR into the TCR-KO reporter cells results in robust antigen-specific reporter activation compared with parental reporter cells. The further development of CD4/CD8 double-positive and double-negative versions enabled low-avidity and high-avidity TCR screening with or without major histocompatibility complex bias. Furthermore, stable TCR-expressing reporter cells generated from TCR-KO reporter cells exhibit sufficient sensitivity to probe in vitro T-cell immunogenicity of protein and nucleic acid-based vaccines. Therefore, our data demonstrated that TCR-KO reporter cells can be a useful tool for the discovery, characterization, and deployment of T-cell immunotherapy.

Temperature Dependence of Membrane Permeability Parameters for Five Cell Types Using Nonideal Thermodynamic Assumptions to Mathematically Model Cryopreservation Protocols

“…Transplantation of engineered corneal tissue created through endothelial cell expansion ,, has also been explored as an alternative treatment for corneal endothelial disorders. − Jurkat cells are an immortalized line of human T lymphocytes, first derived from the peripheral blood of a 14-year-old boy suffering from T-cell leukemia . Because Jurkat cells can simulate the function of T lymphocytes, they are frequently used in in vitro studies of T-cell signal transduction, cytokines, and receptor expression, and can provide reference in cell therapy, cancer treatment, blood research, and studies of differentiation, apoptosis, and cell survival. − The human cerebral microvascular endothelial cell (hCMEC)/D3 line is widely used in in vitro models of the human blood–brain barrier for drug delivery studies. , The blood–brain barrier regulates the passage of ions, molecules, and cells between the blood vessels, lined mainly with continuous endothelial cells, and the brain . Optimization of the hCMEC cryopreservation process , is important for blood–brain barrier research. , …”