Preparation of well-defined antibody-drug conjugates through glycan remodeling and strain-promoted azide-alkyne cycloadditions

Abstract

Antibody-drug conjugates hold considerable promise as anticancer agents, however, producing them remains a challenge and there is a need for mild, broadly applicable, site-specific conjugation methods that yield homogenous products. It was envisaged that enzymatic remodeling of the oligosaccharides of an antibody would enable the introduction of reactive groups that can be exploited for the site-specific attachment of cytotoxic drugs. This is based on the observation that glycosyltransferases often tolerate chemical modifications in their sugar nucleotide substrates, thus allowing the installation of reactive functionalities. An azide was incorporated because this functional group is virtually absent in biological systems and can be reacted by strain-promoted alkyne-azide cycloaddition. This method, which does not require genetic engineering, was used to produce an anti-CD22 antibody modified with doxorubicin to selectively target and kill lymphoma cells.

Keywords: antibodies; click chemistry; glycan remodeling; glycosyltransferases; sialic acid.

Figure 1

Mass spectrometry-based determination of glycan structures of IgG control antibody. (A) N-Glycans isolated from the immunoglobulin G. (B) Galactosylation of the IgG antibody resulted in primarily a di-galactosylated glycan. (C) Galactosylation followed by sialylation of the IgG antibody using ST6Gal I resulted primarily in a bis-sialylated glycan.

Figure 2

Confirmation of IgG labeling of CMP-Neu5Ac9N₃ by ST6Gal I before and after remodeling with galactosyltransferase. (A) Western blotting using an anti-biotin antibody conjugated to HRP. (B) Total protein staining by Coomassie blue.

Figure 3

Cytotoxicity of anti-CD22 antibody conjugated to Dox. Daudi B cells were incubated with various concentrations of Dox, Dox-DIBO, IgG-Dox-DIBO, and CD22-Dox-DIBO for 48 h at 37 °C. Cell viability was assessed using the MTT assay. Data were fitted using Prism nonlinear regression software. EC₅₀ values for Dox, Dox-DIBO, and CD22-Dox-DIBO were found to be 0.1, 1.5, and 1.4 μM, respectively.

Scheme 1

Glycan remodeled of IgG antibodies to produce a homogenous glycoform that have azido moieties for strain promoted cycloadditions with compound 2-4. Reagents and conditions: (i) UDP-Gal, galactosyltransferase, MOPS buffer, pH 7.2; (ii) Compound 1, sialyltransferase, cacodylate buffer pH 7.6; (iii) Compound 2, 3, or 4 in cacodylate buffer pH 7.6.