Using Electroporation to Determine Function of a Chimeric Antigen Receptor in T Cell and Macrophage Cell Lines

ABSTRACT

Chimeric antigen receptors (CARs) have been used extensively in adoptive immunotherapy to modify T lymphocytes (CTLs), conferring the effector abilities of these cells towards a tumor-associated antigen (TAA). However, the ability of a CAR to redirect the effector function in a range of immune cells has not been well characterized, mainly due to the limitations in current genetic modification techniques. To overcome these limitations, we used the rapid technique of electroporation to transiently modify a macrophage, CD4⁺ T and CD8⁺ T cell line to express a CAR. This CAR consisted of an anti-ErbB2 extracellular single chain variable fragment region linked to the intracellular signaling domain comprising CD28-CD3ζ. The ability of three different promoters (CMV, Vav and LTR) to drive CAR expression was compared within each of the cell lines. CAR expression was highest under the CMV promoter in all cell lines, with expression ranging from 20-80%. We subsequently performed functional analysis of these CMV-CAR expressing cells, and observed antigen- specific release of IFN-γ from the CD8⁺ and CD4⁺ T cell lines. In addition, antigen-specific release of both IL-2 and IL-17α was also detected from the CD4⁺ T cell line, EL4. Overall this investigation demonstrated the feasibility of electroporation to compare promoter activity, induce rapid expression and subsequent function of a CAR in a number of hematopoietic cell lines.

Keywords:

Electroporation, genetic engineering, chimeric receptor, T cell, hematopoietic cells, vector screen, IL-17α.