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

Abstract

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 CDR3β. 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 TCRβ chain libraries pre-enriched for target antigen specificity. Several TCRβ chains paired with a transgenic TCRα chain to produce a TCR with higher affinity than the parental TCR for target antigen, without evidence of cross-reactivity.

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Figure 1: Agonist signaling drives CD4/CD8 DN TCRαβ+ γδ-like T cell development in vitro.
Figure 2: Ectopic expression of an antigen-specific TCRα chain before β-selection.
Figure 3: Analysis of enhanced-affinity TCRs recovered from the agonist-selected TCRβ library screen.
Figure 4: High-affinity WT1-specific T cells develop in 3D-PYYα retrogenic mice.
Figure 5: Agonist-selection of in vitro-derived human T cells with enhanced affinity for antigen.
Figure 6: Analysis of human enhanced-affinity TCRs.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) (P01 CA18029-40 and R01 CA033084-32, P.D.G.; NIH CA178844, D.M.K.) and the Guillot Family Fund. T.M.S. is supported by the Jose Carreras International Leukemia Foundation 2013 E.D. Thomas Post-Doctoral Fellowship. We would also like to thank C. Delaney for providing cord blood samples, L. Badenhorst for technical assistance, S. Tan for help with in vivo experiments, and N. Duerkopp for administrative assistance.

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T.M.S. conceptualized, designed and performed the research, interpreted results and wrote the manuscript; D.H.A. contributed to experimental design and developed the 3D-PYY TCR; K.I.-T. performed and optimized experiments; S.O. contributed to experimental design; D.M.K. designed experiments and interpreted results and P.D.G. designed the experiments, assisted in writing the manuscript and supervised the study.

Corresponding author

Correspondence to Philip D Greenberg.

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P.D.G. is a co-founder of Juno therapeutics, which is developing T-cell-based immunotherapies including TCR gene therapy. P.D.G. and T.M.S. are co-inventors on a patent application covering the technology described here.

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Schmitt, T., Aggen, D., Ishida-Tsubota, K. et al. Generation of higher affinity T cell receptors by antigen-driven differentiation of progenitor T cells in vitro. Nat Biotechnol 35, 1188–1195 (2017). https://doi.org/10.1038/nbt.4004

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