Figure 1.

(a) Schematic design of the pMHCI-IgG (top) and the TCB fusion proteins (bottom). Both formats consist of a full monoclonal antibody in an IgG format (light green: antibody light chain, dark green: antibody heavy chain variable domain and CH1, gray/black: constant domain of the antibody heavy chain in the knob and hole format or with charge mutations to drive heterodimerization). A single pMHCI complex consisting of the antigenic peptide (magenta), MHC class I heavy chain lacking the transmembrane domain (purple) and beta-2-microglobulin (blue) or a single anti-CD3 binding CrossMab Fab (crossed heavy and light chain in yellow and red) are fused to one of the antibody heavy chains. (b) Frequency of IFN-γ expressing CD8 T cells measured in flow cytometry after exposure to compound-treated target cells. Incubation of MC38-FAP tumor cells loaded with MCMV-derived M38 peptide (blue), T cell bispecific TCB-αFAP (green), pMHCI-IgG M38-αFAP (red) or pMHCI-IgG IE3-αFAP (pink) and unloaded tumor cells (cyan) with splenocytes from M38 vaccinated mice. Splenocytes were isolated nine days after start of vaccination. All graphs show mean of replicates (n = 2) with error bars indicating standard deviation. (c) Specific tumor cell lysis mediated by compounds after incubation with splenocytes from M38 vaccinated mice. Number of viable cells is measured with xCELLigence as electrical impedance mediated by adherent cells on the culture dish bottom. Color code: IE3-αFAP (purple), M38 peptide (blue), TCB-αFAP (green), M38-αFAP (red). Kinetics of cell lysis at a compound concentration of 25 nM is shown (left). Tumor cell elimination after 40 hours is shown for all concentrations tested (right). All graphs show mean of replicates (n = 3) with error bars indicating standard deviation. Two-sided t-test for significance evaluation was applied. P-values from 0.01 to 0.05 were considered as significant (*), p-values from 0.001 to < 0.01 were considered as very significant (**) and p-values < 0.001 were considered as extremely significant (***). (d) Relative expression of endogenous MHC class I compared to pMHCI-IgG delivered recombinant MHC class I. FAP expressing B16 and MC38 tumor cells were loaded with Ova257-264 peptide (light green) or pMHCI-IgG Ova257-264-αFAP (dark green) and SIINFEKL peptide-MHC class I complexes were detected with the monoclonal antibody anti-Ova257-264 – mouse H-2Kb antibody (25-D1.16) in flow cytometry