Carbohydrate-Mediated Polyethylene Glycol Conjugation of TSH Improves Its Pharmacological Properties

Abstract

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.

Engineering Multi-Walled Carbon Nanotube Therapeutic Bionanofluids to Selectively Target Papillary Thyroid Cancer Cells

“…Both Thyrotropin and Thyrogen bound with high affinity to the TSHR, as reflected in the percentage of cell death. Our data implies that TSHR antibody may bind with a lower affinity to the TSHR, as less cell killing is observed [ 51 , 52 ]. The natural TSH, as well as the recombinant one have the same amino acid sequence, attaching to the TSHR within a binding pocket, made from a combination of the α and β subunits [ 53 , 54 ].…”

Site-Specific PEGylation of Therapeutic Proteins

“…Recently, Park et al used a combination of sialic acid and galactose-mediated chemistry to glycosylate thyrogen (rh-TSH) and then used either a chemical or enzymatic method to add an aldehyde group to the protein, which was then used to conjugate an aminooxy–PEG group [ 114 ]. This method, like the previous glycosylation methods, has the dual advantages that it is both site-specific and that the site of PEGylation occurs at a site of normal modification; hence it is less likely to interfere the protein’s activity.…”

Carbohydrate PEGylation, an approach to improve pharmacological potency

“…As galactose is usually substituted with sialic acid, the latter procedure was applied before or after sialidase treatment [38]. …”

“…(A) Enzymatic modification of the glycan; (B) Chemical modification of the glycan, adapted from [38]. …”

“…The effect of PEG size and mono- vs multi-PEGylation was compared both in vitro and in vivo. The best performing of the products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 5-fold lower affinity which was however compensated by a 23-fold increase of circulation half-life [38]. …”