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A Universal CAR Construct
A Universal CAR Construct
Our CAR construct differs from the traditional CAR in that we have a universal R-phycoerythrin (PE) receptor. Antibodies specific to the tumor antigen are conjugated to PE and introduced to the tumor prior to introducing the CAR T cells. The PE-conjugated antibody binds to the tumor, and the CAR binds to the PE, which leads to CAR T cell activation.
CAR Construct Design
CAR Construct Design
- PEbody is the specialized PE receptor.
- The transmembrane domain is a spacer that links the extracellular and intracellular regions of the CAR.
- The co-stimulatory domain enhances binding specificity.
- The signaling domain triggers downstream signaling events after CAR Activation
- EGFP is a marker used for our experiments
CAR T Cell - Experimental Validation
CAR T Cell - Experimental Validation
Step One: Generate PE CAR T Cells
Step One: Generate PE CAR T Cells
Viral Transfection
Viral Transfection
All of our experiments begin with production of our PE CAR T Cells. HEK 293T producer cells are transfected with the PE CAR plasmid, and the cells assemble and release the CAR viral vector.
The virus is then concentrated and used to transfect T cells. Via reverse transcription, the CAR construct is incorporated into the cell’s genome. Transcription and translation result in the production of the CAR protein, which is then expressed on the cell surface. A variety of methods such as FACS, MACS, and puromycin selection may be used to enrich the CAR T cells.
Expression Validation
Expression Validation
Before continuing with our experiements, we must verify that our T cells are truly expressing the CAR protein on the surface.
To do this, we first stain CAR T cells with fluorescent PE-conjugated antibody. We use a Flow Cytometer to measure fluorescent signal per cell. This allows us to measure the number of cells expressing the CAR as well as the number of CAR expressed per cell.
Step Two: Assess the Efficacy of the CAR T Cell
Step Two: Assess the Efficacy of the CAR T Cell
CAR T Cell Activation
CAR T Cell Activation
The first metric for assessing our design is CAR activation. We need to ensure that binding to the PE-conjugated antibody will trigger CAR T cell activation and the resulting immune response. To do this, we use flow cytometry to measure CD69, which is one of the earliest cell surface antigens expressed by T cells following activation.
CAR T Cell Killing Ability
CAR T Cell Killing Ability
The second metric for assessing our design is CAR T cell killing ability. We co-culture our PE CAR T cells with the appropriate PE-conjugated antibody and cancer cells. Then, we use a dual-luciferase reporter assay to measure the percentage of dead cancer cells.
The cancer cells we use are engineered to have the luciferase in their genome. When the cells are lysed, luciferase will bind to the added substrate in the medium and fluoresce. This fluorescence is measured with flow cytometry and used to quantify cancer cell death.
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