Engineering and characterization of a long half-life relaxin receptor RXFP1 agonist

Abstract

Relaxin-2 is a peptide hormone with important roles in human cardiovascular and reproductive biology. Its ability to activate cellular responses such as vasodilation, angiogenesis, and anti-inflammatory and anti-fibrotic effects have led to significant interest in using relaxin-2 as a therapeutic for heart failure and several fibrotic conditions. However, recombinant relaxin-2 has a very short serum half-life, limiting its clinical applications. Here we present protein engineering efforts targeting the relaxin-2 hormone in order to increase its serum half-life, while maintaining its ability to activate the G protein-coupled receptor RXFP1. To achieve this, we optimized a fusion between relaxin-2 and an antibody Fc fragment, generating a version of the hormone with a circulating half-life of up to five days in mice while retaining potent agonist activity at the RXFP1 receptor both in vitro and in vivo.

The relaxin receptor RXFP1 signals through a mechanism of autoinhibition

“…The binding of the catalytically inactive mini-G s protein blocks RXFP1 signaling through endogenous G s proteins and likely stabilizes the receptor, leading to higher purification yields. The fusion protein of RXFP1 and mini-G s was purified in complex with human G protein β 1 and γ 2 subunits, the camelid antibody VHH fragment nanobody 35 (Nb35) 16 , and an engineered version of the agonist relaxin-2 (SE001) 17 to form an agonist–GPCR–G protein complex (hereafter referred to as RXFP1–G s ).…”

“…The model also predicted that one of the relaxin binding cassette residues, Arg17 B-chain , interacts with Glu206, a residue on the LRRs identified by CLMS that was not previously known to be involved in the binding site. To verify this interaction, we mutated Glu206 to Ala and tested the effect of the mutation on binding of an Fc-tagged relaxin-2 protein (SE301) 17 . The Glu206 to Ala mutant expressed at equivalent levels to wild type receptor, but the single mutant reduced relaxin-2 binding to 42% of wild type levels ( Fig.…”

“…These single-polypeptide relaxin-2 constructs utilize linkers to connect relaxin-2’s B-chain and A-chain. Sequences for these proteins can be found in Table S8 and their design and characterization are described in detail elsewhere 17 .…”

“…Single-chain relaxin proteins were purified as previously described 17 . Briefly, His-tagged single-chain relaxin-2 (SE001) was expressed as a secreted protein from inducible Expi293F tetR cells.…”

“…A flow cytometry assay was used to measure the binding of an Fc-tagged relaxin-2 protein, SE301 17 , to Expi293F cells transfected with human RXFP1 or empty pcDNA-Zeo-tetO vector. Expi293F tetR cells were grown in Expi293 media and transfected using FectoPRO (Polyplus), according to the manufacturer’s protocols.…”