Antitumor Activity of Antibody-Drug Conjugates Targeting Cancer-Expressed EGFR in Preclinical Models

Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.