Figure 2.

Enhancing TCR Affinity toward MART-1 Leads to Increased Cross-Reactivity and Ultimately Decreased T Cell Potency

(A) Percent transduced CD8⁺ T cells expressing intracellular IFN-γ after co-culturing TCR-transduced PBMCs with T2 cells pulsed with the MART-1 peptides and each of the MART-1 homologs in Table 1. As affinity toward MART-1 nonamer or decamer is enhanced, as indicated in the insets, TCR cross-reactivity increases. Data are averages of six sets of experiments (two independent repeats from three donors), normalized to the values for the MART-1 decamer. Error bars indicate SEM. (B) Wild-type DMF5 binds MART-1 homologs weaker than it does the MART-1 nonamer or decamer, as measured by surface plasmon resonance. nq, binding detectable, but too weak to quantify. (C) Strengthening affinity toward MART-1 nonamer or decamer strengthens affinity toward the homologs, as shown by the increase in favorable binding free energy. Shaded bars represent minimum ΔG° estimates where binding was detectable but too weak to quantify. Error bars represent fitting errors from global fitting of multiple datasets, as described in the Materials and Methods, propagated from errors in K_D. (D) For those complexes that could be generated recombinantly, functional responses from (A) correlate well with binding free energies from (C).