Site-Selective Bioconjugation to Albumin using Next Generation Maleimides

The serum half-life of a drug can be increased by conjugation to various entities.[1] In general, strategies operate by increasing the size of the overall construct to minimise renal clearance or through enabling recycling via the neonatal Fc receptor (FcRn).[1] Thus, human serum albumin is an excellent candidate for serum half-life extension as it offers both of these features (half-lifetime albumin cca 19 days).[2-4] As albumin has a single free thiol (Cys-34), covalent conjugation via reaction at this position has proved to be a very popular strategy for site-selective attachment of drugs.[2-4] Classical maleimide conjugation has been widely used to facilitate this attachment (Scheme 1). However, it has recently come to light that the thioether bond on the resultant succinimide is not robust in vivo, leading to undesirable release of the maleimide-drug which can react with other nucleophiles in the blood.[5] To supersede conventional maleimide-bioconjugation we have described the use of Next Generation Maleimide conjugation (Scheme 1). These reagents can be employed in the highly efficient, site-selective bioconjugation of cysteine residues or disulfide bonds.[6- 10] The resultant products can be quantitatively hydrolysed to afford serum stable maleamic acids, thus precluding the problems associated with classical maleimides. We have recently reported the first examples of the use bromomaleimides (parent members of the NGM class) for the construction of serum stable albumin conjugates.[11]
In this studentship project we aim to extend the NGM conjugation methodology to enable the development of a platform for the construction of a serum stable albumin conjugates. The project will include the following components; 1). The design and synthesis of NGM reagents, and associated pyridazinediones (PDs), to optimise the conjugation to albumin (Scheme 2). Aims include; efficient conjugation to Cys-34; selective conjugation to a Cys mutant in preference to Cys-34 (which is situated in a shallow, anionic crevice); dual conjugation to albumin. 2.) Optimise linker design to facilitate one-step conjugation and hydrolysis at pH 7.4. Install cleavable motifs to enable controlled release of the drugs in vivo.
3.) Apply optimised conjugation reagents to the construction of albumin-drug conjugates and demonstrate serum stability and in vivo life-time extension. 4.) Develop trifunctional linkers to access unprecedented albumin triconjugates e.g. albumin-antibody fragment-drug conjugates as a new class of targeted therapeutics.