Fig. 1: An experimental framework de novo identifies on-target binding of the functional MAGE-A4-specific TCB recognizing the peptide GVYDGREHTV presented by HLA-A*02:01.

A Schematic of the MAGE-A4 TCB and its mode of action. B Average ratio of endogenous GVYDGREHTV peptide area under the curve (AUC) by comparison to spiked-in isotopically labeled GVYDGREHTV peptide AUC (n = 4), measured in elution fractions from HLA-A2 immunoprecipitation in A375 tumor cells lines and MAGE-A4 KO (A375-KO) and equivalent lines in a xenograft model (XA375 and XA375-KO). Horizontal bars depict the mean of quadruplicate analyses. The estimated copy number of endogenous GVYDGREHTV per cell (shown on top of each bar graph) is calculated by comparison to a spiked-in isotopically labeled GVYDGREHTV peptide, assuming 100% recovery for both standard and endogenous peptides at the end of the workflow. C A375 wild-type cells and A375-KO cells were incubated with human PBMCs at an E:T ratio of 10:1. Depicted are dose-response curves of tumor cell lysis after 24 h of incubation with different concentrations of MAGE-A4 TCB as determined by quantification of lactate dehydrogenase release in the supernatant (plotted are the mean of triplicate repeat analyses and error bars depict the SD). D Schematic workflow using TCR-like antibodies to enrich interacting HLA-peptide complexes. Tissues were lysed, and solubilized HLA-peptide complexes were immunoprecipitated using the MAGE-A4 tool antibody as a bait. Peptides were enriched in acidic conditions through a 5 kDa molecular weight cut-off (MWKO) filter and analyzed by LC-MS². E Peptide intensity as measured in three biological replicate analyses demonstrates significant enrichment (p = 0.0015, unpaired t-test, two-tailed) of the target peptide GVYDGREHTV in XA375, and not in MAGE-KO equivalent tissues (XA375-KO) nor with a control TCB (CTRL) (F) LC-MS spectrum leading to identification of the target peptide GVYDGREHTV in XA375 after enrichment using the MAGE-A4 antibody. The spectral identifier, the measured mass of the precursor peptide ion (m/z), charge state (z), and retention time (RT), at which the peptide ion was selected for fragmentation, are stated within the spectral panel. C-terminal fragment ions are indicated as y, N-terminal fragments are designated b, pre: unfragmented precursor peptide, +: singly charged ion, ++: doubly charged ion.