Herein we describe the synthesis and evaluation of four novel HER2-targeting, cathepsin B-sensitive antibody-drug conjugates bearing a monomethylauristatin E (MMAE) cytotoxic payload, constructed via the conjugation of cleavable linkers to trastuzumab using a site-specific bioconjugation methodology. These linkers vary by both cleavable trigger motif and hydrophilicity, containing one of two cathepsin B sensitive dipeptides (Val-Cit and Val-Ala), and engendered with either hydrophilic or hydrophobic character via application of a PEG spacer. Through evaluation of physical properties, in vitro cytotoxicity, and receptor affinity of the resulting antibody-drug conjugates (ADCs), we have demonstrated that while both dipeptide triggers are effective, the increased hydrophobicity of the Val-Ala pair limits its utility within this type of linker. In addition, while PEGylation augments linker hydrophilicity, this change does not translate to more favourable ADC hydrophilicity or potency. While all described structures demonstrated excellent and similar in vitro cytotoxicity, the ADC with the ValCitPABMMAE linker shows the most promising combination of in vitro potency, structural homogeneity, and hydrophilicity, warranting further evaluation into its therapeutic potential.

Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.

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.

Inhibition of protein–DNA interactions represents an attractive strategy to modulate essential cellular functions. We reported the synthesis of unique oligoamide-based foldamers that adopt single helical conformations and mimic the negatively charged phosphate moieties of B-DNA. These mimics alter the activity of DNA interacting enzymes used as targets for cancer treatment, such as DNA topoisomerase I, and they are cytotoxic only in the presence of a transfection agent. The aim of our study was to improve internalization and selective delivery of these highly charged molecules to cancer cells. For this purpose, we synthesized an antibody-drug conjugate (ADC) using a DNA mimic as a payload to specifically target cancer cells overexpressing HER2. We report the bioconjugation of a 16-mer DNA mimic with trastuzumab and its functional validation in breast and ovarian cancer cells expressing various levels of HER2. Binding of the ADC to HER2 increased with the expression of the receptor. The ADC was internalized into cells and was more efficient than trastuzumab at inhibiting their growth in vitro. These results provide proof of concept that it is possible to site-specifically graft high molecular weight payloads such as DNA mimics onto monoclonal antibodies to improve their selective internalization and delivery in cancer cells.