Intra-Domain Cysteines (IDC), a New Strategy for the Development of Original Antibody Fragment-Drug Conjugates (FDCs)

Antibody-drug conjugates (ADC) are the pinnacle of vectorized chemotherapy against cancer, with 14 approved ADCs worldwide and hundreds more under clinical trials. However, most ADCs are derived from a full immunoglobulin-G (IgG) and are associated with suboptimal solid tumor penetration and Fc-mediated toxicities. Antibody fragment-drug conjugates (FDC) could thus be an alternative. Nevertheless, innovative solutions are needed to implant conjugation sites on the single chain fragment variable (scFv) format, which is the backbone from which many other antibody formats are built. In addition, the bioconjugation site has an utmost importance to optimize bioconjugates.

Considering these observations, here we assess the impact of different locations of a site-specific bioconjugation motif on variable domains in eight engineered anti-HER2 scFv (H0C2-S1 to H0C2-S8) produced in CHO cells. For this purpose, we explored different positions for the incorporation of two cysteines in these scFv using two strategies (linear vs conformational). We evaluated on each scFv the impact on proteins characteristics and the controlled bioconjugation process of a single monomethyl auristatin F (MMAF) molecule.

Our results showed no proteolysis issue and satisfying production yields in CHO cells for new scFv H0C2.S1 to H0C2.S8. Four scFv (H0C2.S3 to H0C.S6) displayed excellent bioconjugation ability, on which we evaluated the integrity, the affinity to HER2, the stability, and the cytotoxicity in vitro, on both native and conjugated forms. In comparison to previously described 4D5.2-MMAF, two new fragment-drug conjugates (FDCs), H0C2.S4-MMAF and H0C2.S6-MMAF, exhibited enhanced ability to kill in vitro HER2-positive SK-BR-3 cells at picomolar concentrations while sparing HER2-low MDA-MB-231 cells.

Until recently most innovation in the ADC field involved the chemical part of the ADC. This work represents an important optimization step in the design of more complex and effective conjugates towards future in vivo translation to evaluate their full potential. Protein engineering for control bioconjugation and high loading payload are on the rise, in particular for new vector format based antibody.