Figure 4.

Enhanced CD8HTM CAR affinity compared to CD28HTM CAR. (a-d) purified recombinant GUCY2C extracellular domain was incubated with CD8HTM and CD28HTM CAR-T cells produced from n = 3 donors at varying concentrations (0-300 nM). Bound GUCY2C was detected with a fluorescent secondary antibody and cells were analyzed by flow cytometry. (a) Representative flow cytometry plots comparing CD8HTM and CD28HTM CARs. (b) affinity and EC50 determination for CD8HTM and CD28HTM CARs among CD8⁺ and CD4⁺ CAR-T cells. (c) maximum binding (bmax) determination for CD8HTM and CD28HTM CARs among CD8⁺ and CD4⁺ CAR-T cells. (d) comparison of CAR affinities between CD8⁺ and CD4⁺ T cells for each CAR design. Non-linear regression analysis was performed in b-d with p-value testing for matching curves.