Conception d'espaceurs pour relever les défis de bioconjugaison

One of the major problems of oral or intravenous administration of drugs is the fact that the active pharmaceutical ingredient will distribute itself in the whole body, without any specificity towards the diseased site. In this project, the working hypothesis is that conjugating these active pharmaceutical ingredients with targeted ligands will direct them towards therapeutic targets, to improve therapeutic efficacy or intestinal permeability. For this purpose, a series of spacers with diverse properties (length, hydrophily, cleavability) were developed to conjugate the active pharmaceutical ingredient to a targeting moiety such as a peptide or a protein.  Firstly, poorly permeable active pharmaceutical ingredients were conjugated to ligands recognizing receptor GM1 such as peptides or the β subunit of cholera toxin (CTB), in the purpose to improve intestinal permeability. Indeed, CTB is internalized in enterocytes by binding to receptor GM1. Following optimization of spacers, the affinity of the peptide conjugates with receptor GM1 was preserved as shown by isothermal titration calorimetry (ITC) and microscale thermophoresis (MST). Internalisation of conjugates was evaluated by flow cytometry in Caco-2 and T84 intestinal cells. Peptide conjugates were internalized in Caco-2 and T84 cells, but the internalization was limited and did not increase in presence of GM1 supplementation.  On the other hand, an albumin-CTB conjugate (CTB-BSA) was internalized more significantly and the mechanism depended on GM1. Finally, the permeability was evaluated on a monolayer model of Caco-2 and T84 cells. The peptide conjugates demonstrated modest permeability across the monolayer, whereas CTB-BSA had superior permeability against controls.  This study shows that conjugating CTB to a macromolecule improves its permeability across a monolayer of intestinal cells.   Secondly, an immunostimulating drug was conjugated to an antibody targeting the HER2 receptor of breast cancer to form an antibody drug conjugate (ADC). The objective of this approach was to improve the anticancer efficacy of the antibody targeting HER2 currently used in the treatment of breast cancer (Trastuzumab), by combining the immunostimulating properties of the drug. The anti-HER2 murine antibody was conjugated to the drug using a cleavable pH-sensitive spacer. The degree of conjugation and colloidal stability of the resulting ADCs were evaluated by hydrophobic interaction chromatography (HIC) and size exclusion chromatography (SEC) respectively. It was possible to graft 4 or 8 molecules of drug to the antibody while keeping an adequate colloidal stability. The ADC retained its affinity towards cells presenting HER2 receptor such as TUBO cells and H2N100 cells. The drug grafted to the ADC also conserved its therapeutic activity in vitro on these cells, as demonstrated by the evaluation of demethylation and gene expression of CXCL9 and CXCL10. These conjugates were promising in vitro et the assessment of the anticancer activity in vivo on a mouse model of breast is currently being studied in Stagg’s laboratory.

Keyphrases: GM1, CTB, Spacer, Conjugation, Bioconjugates, Permeability, Oral bioavailability, Antibody drug conjugate