Glycoprotein hormone long-acting superagonists

Inventors

Szkudlinski, Mariusz • Weintraub, Bruce D.

Assignees

Trophogen Inc

Abstract

This invention provides long-acting, superactive analogs of glycoprotein hormones demonstrating enhanced bioactivity both in vitro and in vivo as compared to wild type counterparts. The analogs are particularly useful for treating subjects showing low receptor expression or poor receptor responsiveness, and for the treatment of any condition associated with glycoprotein hormone activity.

Core Innovation

The invention provides engineered bovine, human, ovine, porcine, and equine follicle-stimulating hormone alpha-subunit variants as modified alpha subunits with conservative basic residue substitutions and inserted peptides at specific positions in the alpha subunit. The modified alpha subunits pair with wild-type beta subunits to form modified FSH, CG, LH, and TSH constructs, and the engineering is directed to improve potency and biological half-life versus wild type.

The disclosed alpha subunit modifications include conservative basic amino acid substitutions at positions exemplified by Q13, E14, P16, Q20 and K15, K17, K20, K24, as well as arginine, histidine, and lysine substitutions. Example inserted peptides include VNVTINVT (SEQ ID NO:20) and NVTINV (SEQ ID NO:1), and related peptide inserts, with potential glycosylation sites and neoglycosylation sites introduced by inserted peptides.

The document also describes modified glycoprotein hormones, including modified FSH and CG analogs, with evaluation of activity, potency, maximal efficacy, receptor binding, cAMP stimulation, and other functional assays. It further describes increasing serum half-life by adding N-/O-glycosylation motifs, pegylation, conjugates, fusion proteins, and increasing negatively charged residues, together with sequence-specific binding and half-life enhancement for the insert, including removal of amino terminal valine.

In particular examples, designed human and bovine alpha analog variants are described, and characterization is reported using receptor assays, immunoassay options, isoelectric focusing, western blot, and in vivo superovulation studies in cattle using an rFSH analog with outcomes for follicles, ovulation, embryos, and transferable embryos. The document links superactive FSH and potent luteinizing hormone-like activities from CG analogs to improved oocyte outcomes using in vitro and in vivo endpoints, including oocyte formation, fertilization, and blastocyst formation.

Claims Coverage

The provided claims include one independent claim directed to a modified bovine alpha subunit polypeptide comprising SEQ ID NO:7, with dependent claims defining a modified glycoprotein hormone paired with the wild-type bovine FSH beta subunit and bovine methods for stimulating the FSH receptor and stimulating ovulation, including specified administration regimens. The claim set covers six inventive features.

The claims cover the modified bovine alpha subunit polypeptide comprising SEQ ID NO:7, the corresponding modified glycoprotein hormone formed with the wild-type bovine FSH beta subunit, and bovine methods that stimulate the FSH receptor and induce ovulation using that modified glycoprotein hormone, with two specified dosing regimens.

Stated Advantages

Documented Applications