Background Chimeric antigen receptor (CAR)-T cell therapy is a revolutionary new pillar in cancer treatment. In research CAR-T-cells are often detected through tags added to the CAR construct. However, these methods lack sensitivity and often do not address CAR receptors recognition of its target. Detection methods using the targeted ligand (ie: CD19 + fluorescent antibody) exist but they are indirect and laborious. In this study we developed a direct CD19 CAR-T cells detection method using CD19 multimers reagents taking advantage of its high avidity to detect both high and low affinity CAR T cells.

Methods Six different CD19 multimer reagents (#1 to #6) were generated. The six reagents were composed of PE fluorescent multimer and CD19 proteins of different structural format and in different stochiometric ratios. CD19 multimer reagents + controls were incubated together with CD19 antibody coated beads, washed and acquired on FACS to assess CD19 correct refolding and functionality. Functional reagents were selected for further analysis.

Cells expressing CD19 specific CAR construct were directly stained with CD19 multimer or a control , washed and acquired on FACS to evaluate CD19 multimer reagent CAR-T detection ability.

Results Out of six CD19 multimer reagents tested on the bead assay, all six were able to recognize anti-CD19 coated beads, validating CD19 proteins correct refolding and ability to be recognized by anti-CD19 antibody. The six reagents, however, gave different signal-to-noise ratios and did not work equally well highlighting the importance of (i) CD19 protein construct (ii) CD19 protein purity and (iii) multimer:CD19 protein ratio.

The best CD19 multimer reagents successfully detected CAR-T cells expressing CD19-specific CAR constructs of different affinities and expression level. All six reagents worked to some degree but not equally well.

Conclusions CD19 multimer allows direct and specific detection of CD19 CAR-T cells of with different overall avidity. This new reagent allows simple, quick and sensitive detection of CAR-T-cells and opens new possibilities for directly detecting and monitoring CAR-T-cells without being dependent on tags. It also offers a method to demonstrate cell surface expression of a functional CD19 binding CAR of CAR T cells, which a tag doesn´t reveal. This technology has been proven to work on transduced cells do have the potential to measure persistency of CAR-T Cells in patient blood samples.