Schmitt et al. developed a method of creating higher-affinity TCRs by in vitro maturation of hematopoietic progenitor cells that were transduced with only the TCRα chain of a known antigen-specific TCR on antigen-expressing feeder cells. Following T cell differentiation and natural Tcrb gene rearrangement, TCRβ chains were isolated from cells pre-enriched for target antigen-specificity and paired with the parental TCRα to create enhanced-affinity TCRs.
Many promising targets for T-cell-based cancer immunotherapies are self-antigens. During thymic selection, T cells bearing T cell receptors (TCRs) with high affinity for self-antigen are eliminated. The affinity of the remaining low-avidity TCRs can be improved to increase their antitumor efficacy, but conventional saturation mutagenesis approaches are labor intensive, and the resulting TCRs may be cross-reactive. Here we describe the in vitro maturation and selection of mouse and human T cells on antigen-expressing feeder cells to develop higher-affinity TCRs. The approach takes advantage of natural Tcrb gene rearrangement to generate diversity in the length and composition of CDR3beta. In vitro differentiation of progenitors transduced with a known Tcra gene in the presence of antigen drives differentiation of cells with a distinct agonist-selected phenotype. We purified these cells to generate TCRbeta chain libraries pre-enriched for target antigen specificity. Several TCRbeta chains paired with a transgenic TCRalpha chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.