Complement dependent cytotoxicity activity of therapeutic antibody fragments is acquired by immunogenic glycan coupling

Oligosaccharides are implicated in the development of the immune response notably in complement activation. Anti-tumoural immunotherapy by monoclonal antibodies (mAbs) offers some advantages to chemotherapy including cell targeting but some of them are inefficient to generate cytotoxicity dependent complement (CDC) known to be important in the antibody’s efficacy. The aim of this study is to give a CDC activity of mAb by linkage of a complement activating oligosaccharide to this antibody via a hetero-bifunctional linker allowing control of the conjugation reaction. We worked on non Hodgkin Burkitt’s lymphoma as cancer source, Fab fragments of rituximab devoid of complement activity as mAb and the trisaccharide Gal alpha(1→3)Gal beta(1→4)GlcNAc as immunogenic glycan. The bioconjugate Fab-Gal was characterized by biochemical methods and we demonstrated that the α-Gal epitope was recognized by seric immunoglobulins. After checking the recognition capacity of the Fab-Gal conjugate for the CD20 epitope, in vitro assays were performed to evaluate the activation of the complement cascade by the Fab-Gal conjugate. The effect of this bioconjugate was confirmed by the evaluation of the proliferation response of Burkitt’s cell line. The relative facility realization of this strategy represents new approaches to increase activities of mAbs.

Norfloxacin-poly(L-lysine citramide imide) conjugates and structure-dependence of the drug release.

Norfloxacin (Nflx), an antibiotic which is active against some intracellular bacteria, was coupled to a polymeric carrier, namely poly(L-lysine citramide) via a lysine or an ethylcarbamate spacer to obtain a macromolecular prodrug. The carrier, which derived from the two metabolites citric acid and L-lysine, is known to be biocompatible and slowly degradable under slight acidic conditions. Conjugates were characterised by UV, 1H and 13C NMR and SEC. The presence of Norfloxacin and the lysine type spacer caused chain aggregation, due to a probable physical cure. The release of Norfloxacin from these prodrugs and from a prodrug where Norfloxacin is bound to the carrier backbone without spacer arm was investigated comparatively in vitro. Conjugation via a carbamate-type linkage appeared as a method to achieve the release of Norfloxacin from a PLCA-type conjugate at neutral.

Improved brain uptake and pharmacological activity of dalargin using a peptide-vector-mediated strategy.

The blood-brain barrier restricts the passage of substances into the brain. Neuropeptides, such as enkephalins, cannot be delivered into the brain when given systemically because of this barrier. Therefore, there is a need to develop efficient transport systems to deliver these drugs to the brain. Recently, we have demonstrated that conjugation of doxorubicin or penicillin to peptide vectors significantly enhances their brain uptake. In this study, we have conjugated the enkephalin analog dalargin with two different peptide vectors, SynB1 and SynB3, to improve its brain delivery and its pharmacological effect. We show by in situ brain perfusion that vectorization markedly enhances the brain uptake of dalargin. We also show using the hot-plate model that this enhancement in brain uptake results in a significant improvement in the observed antinociceptive effect of dalargin. These results support the usefulness of peptide-mediated strategies for improving the availability and efficacy of central nervous system drugs.