Generation of TCRs of higher affinity by antigen-driven differentiation of progenitor T cells in vitro

Many promising targets for T cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing TCRs with high affinity for self-antigen are eliminated. The affinity of the remaining low avidity TCRs can be improved to increase their anti-tumor efficacy, but conventional saturation mutagenesis approaches are labor intense and the resulting TCRs may be cross-reactive. Here we report an in vitro T cell maturation and selection system on antigen-expressing feeder cells for developing high affinity antigen-specific TCRs, which takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3β. In vitro differentiation of progenitors transduced with a known Tcra in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. These cells are then purified to generate TCRβ chain libraries pre-enriched for target antigen-specificity. Several TCRβ chains were identified that paired with a transgenic TCRα chain to produce a TCR with higher affinity for target antigen compared to the parental TCR.