Antibody-drug conjugates (ADCs) have been proven clinically to be more effective anti-cancer agents than native antibodies. However, the classical conjugation chemistries to prepare ADCs by targeting primary amines or hinge disulfides have a number of shortcomings including heterogeneous product profiles and linkage instability. We have developed a novel site-specific conjugation method by targeting the native glycosylation site on antibodies as an approach to address these limitations. The native glycans on Asn-297 of antibodies were enzymatically remodeled in vitro using galactosyl and sialyltransferases to introduce terminal sialic acids. Periodate oxidation of these sialic acids yielded aldehyde groups which were subsequently used to conjugate aminooxy functionalized cytotoxic agents via oxime ligation. The process has been successfully demonstrated with three antibodies including trastuzumab and two cytotoxic agents. Hydrophobic interaction chromatography and LC-MS analyses revealed the incorporation of ~1.6 cytotoxic agents per antibody molecule, approximating the number of sialic acid residues. These glyco-conjugated ADCs exhibited target-dependent antiproliferative activity toward antigen-positive tumor cells and significantly greater antitumor efficacy than naked antibody in a Her2-positive tumor xenograft model. These findings suggest that enzymatic remodeling combined with oxime ligation of the native glycans of antibodies offers an attractive approach to generate ADCs with well-defined product profiles. The site-specific conjugation approach presented here provides a viable alternative to other methods, which involve a need to either re-engineer the antibody sequence or develop a highly controlled chemical process to ensure reproducible drug loading.

Purpose: Gasdermin B (GSDMB) overexpression/amplification occurs in about 60% of HER2 breast cancers, where it promotes cell migration, resistance to anti-HER2 therapies, and poor clinical outcome. Thus, we tackle GSDMB cytoplasmic overexpression as a new therapeutic target in HER2 breast cancers. Experimental Design: We have developed a new targeted nanomedicine based on hyaluronic acid-biocompatible nanocapsules, which allow the intracellular delivery of a specific anti-GSDMB antibody into HER2 breast cancer cells both in vitro and in vivo. Results: Using different models of HER2 breast cancer cells, we show that anti-GSDMB antibody loaded to nanocapsules has significant and specific effects on GSDMBoverexpressing cancer cells' behavior in ways such as (i) lowering the in vitro cell migration induced by GSDMB; (ii) enhancing the sensitivity to trastuzumab; (iii) reducing tumor growth by increasing apoptotic rate in orthotopic breast cancer xenografts; and (iv) diminishing lung metastasis in MDA-MB-231-HER2 cells in vivo. Moreover, at a mechanistic level, we have shown that AbGB increases GSDMB binding to sulfatides and consequently decreases migratory cell behavior and may upregulate the potential intrinsic procell death activity of GSDMB. Conclusions: Our findings portray the first evidence of the effectiveness and specificity of an antibody-based nanomedicine that targets an intracellular oncoprotein. We have proved that intracellular-delivered anti-GSDMB reduces diverse protumor GSDMB functions (migration, metastasis, and resistance to therapy) in an efficient and specific way, thus providing a new targeted therapeutic strategy in aggressive HER2 cancers with poor prognosis.

Metal-complexing polymer matrices capable of specific recognition and binding to metalcoordinating substrates have been prepared by template-directed polymerization. The synthesis of these materials involves preorganization of a copper-containing vinyl monomer, copper(I1) [N-(Gvinylbenzy1)-iminoldiacetic acid (l), with bifunctional bis(imidazo1e) templates (2-5) of varying geometry and subsequent polymerization with a large excess of ethylene glycol dimethacrylate as the cross-linking agent. Complexation of metal-chelating monomers with the template during polymerization directs the positioning of metal ions in the polymer matrices, while a high degree of cross-linking stabilizes the functional group arrangement. In equilibrium binding experiments with single substrates and selected substrate pairs, the polymers preferentially bind their own templates, with separation factors (a) of 1.17-1.35 and binding constants that range from 1800 to 3800 M-l. The capacities and affinities of the polymers for different substrates and ESR spectral analyses of the polymers loaded with substrate suggest a defined arrangement of metal ion sites in the templated materials that is absent in nontemplated polymers. The substrate selectivity likely involves some cooperative two-site coordination of the bis(imidazo1es) as well as steric interactions with the binding cavities ("cavity fitting"). This template polymerization strategy is discussed from the viewpoint of designing highly specific abiotic receptors for recognition of delicate and complex biomolecules.

High-purity milk exosomes were engineered with surface PEGylation and siRNA encapsulation for enhanced oral drug delivery.