Background & Aim The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Methods, Results & Conclusion Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis. To prevent off-target effects caused by the mispairing of endogenous and recombinant TCR chains, the endogenous TCR knockout was made using gRNA/Cas9 ribonucleoprotein complexes. Transgenic alpha and beta-chain constant domains were modified to be resistant to Cas9 cleavage and to form more stable TCR. Antigen-specificity was measured by binding to MHC-tetramers loaded with the MiHA peptide and by TCR-mediated activation of the Jurkat 76 triple parameter reporter cell line upon antigen stimulation. Most of the generated cell lines were specific to the desired antigen, and none were activated in response to irrelevant peptides presented in the same HLA. After analysis of cross-reactivity to allogeneic HLA on the panel of Epstein-Barr virus-transformed lymphoblastoid cell lines, the best receptors would be selected for further therapeutic application. Adoptive transfer of MiHA-specific genetically modified T cells holds great promise for the treatment of leukemia relapse after allo-HSCT. The work was supported by the Russian Foundation for Basic Research grant 19-29-04156. The relapse of acute leukemia occurs in one third of the patients after HLA-matched allogeneic stem cell transplantation (HSCT), resulting in high mortality. Disparities in minor histocompatibility antigens (MiHA, MHC-presented peptides derived from polymorphic self-proteins) can result in the development of allogeneic immune response. Several MiHA (HA-1, HA-2, and ACC-1Y) are encoded by the genes predominantly expressed in hematopoietic tissues. They represent promising targets for post-HSCT relapse therapy. The immune response towards them selectively targets patient hematopoietic cells sparing hematopoietic cells of donor origin and patients healthy tissues. Naïve CD8+ T cells from MiHA-negative donors were expanded in vitro using autologous dendritic cells, pulsed with MiHA peptides. Antigen-specific cells were sorted, and the sequences of T-cell receptor (TCR) alpha and beta chains were determined by NGS. 8, 10, and 1 TCRs specific for HA-1, HA-2, and ACC-1Y antigens, respectively, were cloned in the lentiviral vectors containing coreceptor CD8. T cell lines were generated for further analysis.