Chemoenzymatic Site-Selective Attachment to Antibodies: Using Glycosylation Sites to Post-Translationally Position Cargo and to Program Function

Targeted delivery of molecules through conjugation of probes and/or cytotoxics to antibodies represents a promising approach to therapy and diagnosis for many diseases, including but not limited to cancer. More than 30 IgG-type antibodies have been approved for therapy yet the use of conjugates has lagged behind. Current methods for generating Ab conjugates typically rely on non-specific chemistries, which create heterogeneous mixtures of modified products with variable profiles. These can also alter Ab function detrimentally, due to direct modification of functional amino acids. Site-selective strategies based on new linking chemistries to study function are needed in order to generate homogenous conjugates.

The Davis Group have recently discovered and demonstrated an enzymatic transformation using the newly-discovered synthetic activity of the enzyme EndoS that is highly efficient, site-selective and does not target the amino acid side chains as with other antibody conjugate but instead allows attachment to the N-linked glycosylation site that is found in immunoglobulins. This biocatalytic activity now allows processing of previously refractory antibodies including eukaryotic proteins and antibodies from human sources. This novel chemistry will convert the glycan side chain to a site for attachment of payloads, such as labels, imaging motifs, functional peptides or toxins and allow the modulation of Ab function via glycans. Advantageously, this chemistry will also increase the purity of the mixtures of carbohydrates found at this site creating purer therapeutic product. Finally, it will create a linkage that is highly stable in blood due to the high chemical stability of glycosidic linkages but will be readily degraded in the lysosome of cells by endogenous glycosidases, which are present for normal degradation and carbohydrate recycling.

Aims and objectives:
- Generate carbohydrate reagents that will allow the site-selective alteration of glycans in Abs
- Test EndoS-catalyzed attachment of these reagents to representative Abs
- Use these reagents in 2 Ab alteration strategies:
a) direct attachment of payload to Ab glycosylation site (direct enzymatic)
b) creation of an unnatural reactive functional group in the carbohydrate that can be used as a 'tag' for subsequent attachment (two-step chemoenzymatic conjugation)
- Site-selectively attach payloads (e.g. model toxins or peptide antigens) using both strategies to representative antibodies (isolated & recombinant sources)
- Study & explore release modes through degradation of carbohydrate linkages by lysosomal glycosidases
- Selection of an Ab for a validated biomarker target & application of methods to that therapeutically-relevant Ab
- The study of the use of this construct in cellulo and evaluation of efficacy in model e.g., xenograft, in vivo systems