Site-Specific Conjugation of Auristatins onto Engineered scFv Using Second Generation Maleimide to Target HER2-positive Breast Cancer in Vitro

Antibody-drug conjugates (ADC) are spearheading vectorized chemotherapy against cancer, with 4 FDA-approved ADCs and 79 in clinical trials. However, most ADCs are produced using a stochastic bioconjugation method, target hematological cancers, and are derived from a full immunoglobulin-G (IgG). These factors limit their efficacy, especially against solid tumors which remain difficult to treat. Here we report the site-specific conjugation of a single auristatin derivative onto an engineered anti-HER2 single chain fragment variable (scFv) of the trastuzumab antibody, generating new scFv-drug conjugates (SDC). Two cysteines were judiciously incorporated at the beginning of the scFv hexahistidine tag, in order to allow controlled bioconjugation of a heterobifunctional linker including a second generation maleimide (SGM), either cleavable (for monomethyl auristatin E) or noncleavable (for monomethyl auristatin F). Our data indicated that both SDCs conserved their affinity to HER2 in comparison to the native scFv, and were efficiently able to kill in vitro HER2-positive SK-BR-3 cells at subnanomolar concentrations (EC50 of 0.68 nM and 0.32 nM). No effect was observed on HER2-negative MCF-7 cells. Ours results showed efficient targeting of site-specific SDCs against HER2-positive breast cancer cells. This work represents a first important step in the design of more effective small conjugates, paving the way for future in vivo translation to evaluate their full potential.