High-Affinity TCRs Generated by Phage Display Provide CD4+ T Cells with the Ability to Recognize and Kill Tumor Cell Lines

Abstract

We examined the activity of human T cells engineered to express variants of a single TCR (1G4) specific for the cancer/testis Ag NY-ESO-1, generated by bacteriophage display with a wide range of affinities (from 4 μM to 26 pM). CD8+ T cells expressing intermediate- and high-affinity 1G4 TCR variants bound NY-ESO-1/HLA-A2 tetramers with high avidity and Ag specificity, but increased affinity was associated with a loss of target cell specificity of the TCR gene-modified cells. T cells expressing the highest affinity TCR (KD value of 26 pM) completely lost Ag specificity. The TCRs with affinities in the midrange, KD 5 and 85 nM, showed specificity only when CD8 was absent or blocked, while the variant TCRs with affinities in the intermediate range—with KD values of 450 nM and 4 μM—demonstrated Ag-specific recognition. Although the biological activity of these two relatively low-affinity TCRs was comparable to wild-type reactivity in CD8+ T cells, introduction of these TCR dramatically increased the reactivity of CD4+ T cells to tumor cell lines.

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

“…A 10-to 30-fold increase in affinity is seen, ranging from the WT 1G4 TCR (9.3 M), across ␤52:I (1.1 M) and ␣95:LY variants (730 nM) to the ␤51:AI 1G4 TCR (280 nM). These responses by substituted 1G4 TCRs concur with those seen previously with the more complex 1G4 TCR wt/ c59 and c12/c2 mutants, having respective Ag complex affinities of 4 M and 450 nM (21). Taken together, an elevated response by TCR-transfected CD4 ϩ T cells is seen over a TCR affinity range of ϳ5 M to ϳ500 nM, a 2-to 20-fold higher affinity than the 1G4 WT TCR.…”

“…These results, taken together with those presented in an earlier study of modified 1G4 TCR variants (21), demonstrate that the TCR variants that are capable of mediating enhanced tumor recognition by TCR gene-modified CD4 ϩ T cells possess higher affinities for the NY-ESO-1/HLA-A*02 complex than the WT TCR but suggest that TCRs with affinities equal to or greater than the WT ␣/␤51:AI TCR (280 nM) can lead to impaired function in CD8 ϩ T cells.…”

“…When this TCR and other mutant1G4 TCRs were transfected into CD8 ϩ T cells, Ag cross-reactivity with NY-ESO-1-negative target cell lines was observed with the highest affinity TCR and with those with Ag affinities ranging from 20 to 50 pM to 84 nM. However, when CD8 ϩ T cells were transfected with mutant 1G4 TCRs with affinities 450 nM and weaker were Ag specific (21). In agreement with these findings, the ␤51:AI 1G4 TCR variant (280 nM affinity) was also associated with a loss of Ag specificity in CD8 ϩ T cells, as these cells were stimulated by the NY-ESO-1-negative 2661R tumor cell line.…”

“…In addition, binding studies indicate that soluble monomeric complexes of this high-affinity 1G4 TCR can be used to probe cells that endogenously express the NY-ESO-1/HLA-A*02 Ag complex (20). Recent studies analyzing the function of T cells that express 1G4 variants have indicated that high-affinity (5-84 nM) variants that lead to the Ag-specific activation of TCR gene-modified CD4 ϩ T cells result in nonspecific activation of TCR gene-modified CD8 ϩ T cells, whereas TCR variants with affinities nearer to that of the WT 1G4 TCR show enhanced reactivity in CD4 ϩ T cells without any apparent loss of specificity in CD8 ϩ T cells (21).…”

Phenotypic models of T cell activation

“…This would mean that at a low dose, one antigen will outperform another at activating a T cell, whereas at a high dose their performance would be reversed (see figure 4 in REF 13). Although not explicitly stated, previous work suggests that dose-response curves may intersect such that the relative activity of antigens does not simply depend on their binding properties but also on the dose at which they are presented 2,6,38 . In its present formulation, the kinetic proofreading with negative feedback model is unable to reproduce an optimal dissociation time but does predict an optimal pMHC dose for T cell activation (FIG.…”