Two engineered site-specific antibody-drug conjugates, HLmD4 and HLvM4, have potent therapeutic activity in two DLL4-positive tumour xenograft models.

The humanized Delta-like 4 (DLL4) monoclonal antibody H3L2 with a quite high affinity for hrDLL4 inhibits the DLL4-mediated human umbilical vein endothelial cell (HUVEC) phenotype, inducing dysfunctional angiogenesis and tumour cell apoptosis, which effectively arrests breast cancer cell growth in vivo. To develop a more effective therapy, an engineered cysteine residue at alanine 121 (Kabat numbering) on each H3L2 heavy chain or at valine 207 (Kabat numbering) on each H3L2 light chain was established by site-directed mutagenesis. Three engineered antibodies, THL4, TH2 and TL2, were identified, and the specific-site antibody-drug conjugates (ADCs) THL4-mpeoDM1 (named HLmD4), TH2-mpeoDM1 (named HmD2), TL2-mpeoDM1 (named LmD2) and THL4-vcMMAE (named HLvM4), were produced, which exhibit much more potent antitumour activity than the naked antibody. The engineered ADCs can be directed against DLL4 and effectively internalized, followed by the release of small molecule cytotoxic agents, e.g., DM1 or MMAE, into the cytosol, which inhibit the synthesis of microtubules and induce G2/M phase growth arrest and cell death through the induction of apoptosis. ADC-conjugated DM1 was highly potent against DLL4-expressing cells in vitro. We systematically compared the in vitro potency and the in vivo preclinical efficacy and safety profiles of the heterogeneous conventional ADC, H3L2-mpeoDM1 (named JmD4) with that of the homogeneous engineered conjugate HLmD4. The engineered anti-DLL4 ADCs, particularly HLmD4, showed more potent antitumour activity than Docetaxel and superior safety compared with JmD4 in two xenograft tumour models. Our findings indicate that engineered ADCs have promising potential as effective preclinical therapies for cancers.