High-throughput cloning reveals diverse properties of T-cell receptors targeting minor histocompatibility antigens

Hematopoietically restricted minor histocompatibility antigens (miHAs) presented on HLA-class I molecules are ideal targets for adoptive T-cell immunotherapy in the context of an allogeneic stem cell transplant (alloSCT). This is because CD8 cells that recognize these miHAs can mediate potent antileukemia effects with a low risk for graft-versus-host disease (GVHD). A barrier to translating this concept broadly to the clinic has been the difficulty and expense of cloning potentially high-value T-cell receptors (TCRs). Here, we describe the isolation of anti-miHA TCRs using a novel high-throughput platform (TCXpress) that enables the rapid cloning and characterization of TCRs from single cells without nucleic acid sequencing or gene synthesis. We cloned 7 unique TCRs recognizing the miHA HA-1H from 50 mL of blood from an HLA-A∗02:01 HA-1R/R woman who was immunized against HA-1H during pregnancy. After in vitro expansion, 13 additional unique anti-HA-1H TCRs were cloned from 740 HA-1H-dextramer+ cells, with the screen completed in 35 days. We also cloned 12 unique anti-HA-2V TCRs from 771 HA-2V-tetramer+ cells from a patient with myelodysplastic syndrome (MDS) that had relapsed after alloSCT and who received a donor leukocyte infusion. Ten anti-HA-2V TCRs were isolated from an unmanipulated sample collected at GVHD-onset, and 8 were isolated when GVHD and MDS were in remission. Anti-HA-1H and -HA-2V TCRs had a wide range of avidities measured by activation of cell lines expressing them. Taken together, our results validate a new method for TCR isolation and characterization with potentially broad applications, and provide insights into the nature of anti-miHA responses.