Human Relaxin 2 - Fc Fusion Proteins for Interrogating Relaxin Signaling

Introduction:

Relaxin-2 is a hormone comprised of two peptide chains of 24 and 29 amino acids linked by disulfide bridges. It is a pleiotropic hormone with anti-fibrotic, vasodilatory, ECM remodeling and pro-angiogenic properties, making it therapeutically relevant in many indications. A recombinant form of relaxin-2, SERELAXIN, was studied for treatment acute heart failure in clinical trials, but had a limited effect due to its short in vivo half-life and unfavorable pharmacodynamics that requires continuous administration of the drug. We propose to study Relaxin-2 Fc fusion proteins as a mean to increase the stability and half-life of the hormone, and evaluate its potential in tissue fibrosis related models.

Methods:

The pre-pro form of relaxin-2 was grafted onto an Fc domain of an antibody. The protein was produced in a mammalian cell expression system and purified by an affinity column. Western blot analysis and mass spectrometry were used to characterize the fusion protein. The ability of the fusion protein to signal via the relaxin-2 receptor was determined by a luciferase based cell assay, measuring transcription factor activation downstream to the cAMP signaling pathway.

Results:

The fusion protein was composed of three isomers. Mass spectrometry showed the existence of the precursor form of Relaxin – Pro-relaxin in all three isomers. The cell-based assay confirmed the fusion protein to be an agonist with similar properties to recombinant relaxin.

Conclusions:

A new recombinant form of relaxin-2 was easily produced and demonstrated similar signaling to the original protein, with suggested higher stability and longer half-life.