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.

Author Info: (1) Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. (2) Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. (3) Fred Hu

Author Info: (1) Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. (2) Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. (3) Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. (4) Department of Hematology, Oncology, and Tumor Immunology, Charite Campus Benjamin Franklin, Berlin, Germany. (5) Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA. (6) Fred Hutchinson Cancer Research Center, Seattle, Washington, USA. Departments of Immunology and Medicine, University of Washington, Seattle, Washington, USA.