Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell

Abstract

The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets in vitro is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.

Immune inactivation of anti-simian immunodeficiency virus chimeric antigen receptor T cells in rhesus macaques

“…The killing potency of anti-SIV CAR T cells was evaluated using an In-cuCyte cytotoxic assay that records in real time the disappearance of CD4 + target cells infected with a SIVmac239 virus carrying an enhanced green fluorescent protein (EGFP) gene (SIVGFP). 18 Although CAR T cells with a V H -V L orientation combined with a medium spacer were slightly more potent, all combinations of spacers and scFv domains generated CAR T cells with similar killing potency toward SIV-infected targets (Figure 1B). No significant killing of control EGFR T cells was observed.…”

“…The production of recombinant lentiviruses was performed as previously described. 18 Briefly, Lenti-XTM 293T cells (Takara Bio) in DMEM media containing 10% fetal bovine serum (FBS) and 100 U/mL Pen/Strep were transfected using the standard calcium phosphate method with 15 mg of the CAR transfer vector together with 6 mg of the pCAG-SIVgprre plasmid carrying the gag/pol and rev responsive element (RRE), 4 mg of the rev/tat expression plasmid pCAG4-RTR-SIV, and 3 mg of the pMD2.CocalG containing the glycoprotein G of the cocal virus. 47 The next day, fresh media was added after washing the HEK293T cells with phosphatebuffered saline (PBS).…”

“…The preparation and transduction of T lymphocytes from frozen PBMCs from RMs (Macaca mulatta) and their analysis by flow cytometry were performed as previously described. 18 Briefly, CD4 + and CD8 + T cells were enriched from PBMCs by negative selection using immunomagnetic beads (Easy Sep nonhuman primate [NHP], STEMCELL Technologies). T cells were cultured in X-vivo 15 media (Lonza) including 10% FBS, 100 U/mL Pen/Strep, 1Â glutamax, 50 mM b-mercaptoethanol, 50 IU/mL human recombinant IL-2, 5 ng/mL of human interleukin-7 (IL-7), and 5 ng/mL IL-15 (PeproTech) at 37 C in a humidified 5% CO 2 atmosphere.…”

Novel engineered chimeric engulfment receptors trigger T-cell effector functions against SIV infected CD4+ T cells

“…Transduction of RM CD4+ and CD8+ T cells with CER21 or EGFR lentivirus led to high levels of EGFR expression ( Figure 2B ). We developed a real-time fluorescence assay to evaluate CER T-cell potency against freshly SIVGFP infected target cells expressing surface-exposed PS 23 . A significant decrease in the number of GFP+ infected T cells were detected over time in the presence of CD4+ CER T cells but not CD8+ CER T cells or EGFR T cells, indicating the potency of CD4 CER T cells in killing SIV-infected cells ( Figure 2C ).…”

CAR T Cell Locomotion in Solid Tumor Microenvironment

“…In particular, the complex TME of solid tumors highlights the need for three-dimensional (3D) in vitro models to better study cell–cell and cell–extracellular matrix (ECM) interactions. We discuss how CAR T cells can be engineered to eradicate antigen-specific cancer cells that are heterogeneously expressed, evolving, and shielded by layers of biochemical and physical barriers using novel 3D in vitro models and platforms [ 11 , 12 , 13 ].…”