Engineering a tumor-selective prodrug T-cell engager bispecific antibody for safer immunotherapy

Figures & data

Figure 1. Crystal structure of E10 antibody with CD3ε peptide (PDB 8VY4) informs design of pro-TCE. (a) Top-down view of the isolated CDR loops (green) from anti-CD3 antibody E10 in contact with the CD3ε peptide antigen (pink). Key binding interaction features are highlighted from left to right respectively: N-terminal pyroglutamate, CDRH3 contacts with CD3ε, and CDRL3 contacts with CD3ε. (b) Fluorescence polarization binding data with WT and alanine mutant CD3ε peptides with anti-CD3 hE10 fab. The table below shows two K_D values for WT. “WT*” represents our best estimate for K_D and associated error from all replicates, while “WT*” and all other K_D values in the table are the fitted values and errors to the fit from the specific experiment shown in the figure. (c) PyRosetta Model of designed pro-TCE anti-CD3 antibody fragment and sequence of CD3ε peptide (pink) prepended with MMP-2 cleavage sites (blue). (d) Fluorescence polarization binding data with pro-TCE antibody fragment before and after MMP-2 proteolysis. K_D with reported standard deviation of the mean is shown for two replicate measurements. *Q undergoes cyclization to pyroglutamic acid (Pyr) under physiological conditions.

Figure 2. Recombinant expression, assembly and biophysical characterization of pro-TCE. (a) Interface mutations across the antibody fab and Fc framework regions facilitate proper recombinant assembly of IgG bispecific antibodies. (b) SDS-PAGE gel electrophoresis showing recombinant MMP-2 before and after activation with APMA, and the pro-TCE before and after proteolysis with the active MMP-2. Protein samples treated with βMe. (c) Fluorescence polarization binding data with pro-TCE IgG before and after MMP-2 proteolysis. The reported K_D values are averages with errors reflecting the standard deviation of the mean for three replicates. (d) Biolayer interferometry data of the immobilized pro-TCE and parental anti-HER2 pertuzumab binding to HER2 extracellular domain before proteolysis. (e) Biolayer interferometry data of the immobilized pro-TCE and parental anti-CD3 hE10 mAb binding to CD3γε extracellular domain before and after proteolysis. In (d-e) the reported K_D values are averages along with the standard deviation of the mean for two replicates.

Figure 3. Preclinical functional validation of pro-TCE with recombinant MMP-2 protease treatment. All experiments were performed at an effector cell to target cell ratio of 1:1. Each point represents the mean value of triplicates. (a) T cell activation induced by pro-TCE after proteolysis using a Jurkat NFAT-Luciferase T cell model. (b) HER2⁺ target cell killing by hPBMCs induced by pro-TCE after proteolysis after 48 hours. (c–d) IL-2 and IFN-γ secretion by hPBMCs induced by pro-TCE after 48 hours. The reported errors for the EC₅₀ values are the 95% confidence intervals from fitting each individual experiment.

Figure 4. NCI-N87 tumor cell secreted proteases trigger pro-TCE activity. SDS-PAGE gel electrophoresis of pro-TCE before and after incubation with NCI-N87 cellular supernatant for 1 h (left). Jurkat NFAT-Luciferase T cell activation assay with NCI-N87 target cells (right). The reported EC₅₀ value is an average of three replicates with errors reflecting the 95% confidence intervals of the average measurements.

Figure 5. Expanding the pro-TCE conditionally active T cell engager platform to clone SP34. (a) Anti-CD3 mAbs hE10 and SP34 both bind the N-terminus of CD3ε. (b) SDS-PAGE gel electrophoresis of SP34_Pertuzumab pro-TCE before and after proteolysis. (c) Designed pro-TCE using anti-CD3 mAb with SP34 CD3-binding arm activates Jurkat NFAT-Luciferase T cells when proteolyzed but not when masked. The reported EC₅₀ value is an average of three replicates with errors reflecting the 95% confidence intervals of the average measurements.

Data availability statement

Coordinates and structure factors for E10 fab complexed to the CD3ε-N7 peptide at pH 6.5 have been deposited in the Protein Data Bank under accession code 8VY4. All other data are available in the manuscript or supplementary materials.