T-cell receptors (TCRs)

D19 chimeric antigen receptor (CAR) T-cell therapy frequently induces complete remission in acute B-lymphoblastic leukemia (B-ALL), dramatically improving the outcome for this patient group. However, approximately 40–50% of patients relapse1,2, most frequently due to loss of CD19 (refs. 3–5). CAR T-cell therapy directed at other B-cell-specific cell-surface molecules has shown a higher tendency to relapse6, possibly because of lower expression levels than CD19 on leukemia-propagating cells. Cell-based immunotherapies targeting alternative molecules expressed early during B-cell differentiation would thus be desirable. In spite of a multitude of T-cell-specific markers, no CAR therapy is approved for T-ALL that efficiently targets malignant cells across all T-ALL subtypes while at the same time sparing mature, normal T cells. This would be crucial to avoid depletion of norm T cells, which is prohibitively toxic or even incompatible with life. Moreover, no tumor-specific target has been identified in T-ALL and no immunotherapy has yet proven effective in clinical trials. In the event of failed chemotherapy (15–20%), T-ALL has a dismal prognosis with overall survival <25% (refs. 7,8). We hypothesized that the enzyme terminal deoxynucleotidyl transferase (TdT) might be an ideal target of immunotherapy in B-ALL and T-ALL, for several reasons. First, as the function of TdT is to add N-nucleotides to the V-D-J junctions during recombination of the TCR and B-cell receptor9, its expression is confined to the B and T lineages. Second, TdT is overexpressed in 80–94% of ALL and lymphoblastic lymphoma of B- and T-cell origin10–12, making it a widely used diagnostic marker13. Third, expression of TdT is restricted to a narrow window during

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Results We determined the sequence of TdT-derived peptides that were naturally processed and presented on HLA-A2 from HLA-A2pos Epstein–Barr virus-transformed lymphoblastoid cells (EBV-LCL) transduced with full-length TdT, by mass spectrometry (MS). Two peptides identified as HLA-A2 binders (ALYDKTKRI (peptide-1) and ALYDKTKRIFL (peptide−3)) were further characterized (Fig. 1b, Supplementary Fig. 1a,b and Supplementary Table 1). Naïve T cells from HLA-A2neg donors were cocultured with monocyte-derived dendritic cells (MoDCs) generated from HLA-A2pos donors that were electroporated with TdT-encoding messenger RNA (Fig. 1c)16. T cells staining positively with peptide–major histocompatibility complex (pMHC)-multimers (Fig. 1d) were sorted as single cells to generate T-cell clones. Clones reactive to peptide-1 and peptide-3 stained positively with the relevant multimers (Fig. 1e) and were activated by peptide-pulsed HLA-A2pos EBV-LCL, and by a B-ALL cell line endogenously expressing TdT (Fig. 1f). One TCR sequence was identified from clones reactive to each peptide, named T1 (reactive to peptide 1) and T3 (reactive to peptide-3). Both TCRs were efficiently expressed in third-party peripheral blood (PB) CD8+ T cells, as demonstrated by staining with either