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Apelin fusion proteins and uses thereof

A palintide-receptor binding technology, applied in the field of fusion proteins, can solve the problems of deficiency and reduced cardiovascular stress ability

Inactive Publication Date: 2015-11-04
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Apelene in mice (Apelene - / - ) and APJ (APJ - / - ) gene knockout studies have shown that deficiency of the endogenous APALIN-APJ pathway results in a reduced ability to respond to cardiovascular stress such as exercise (Charo et al., 2009, Am J Physiol. Heart Circ. Physiol., 297: H1904 -1913)

Method used

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  • Apelin fusion proteins and uses thereof
  • Apelin fusion proteins and uses thereof
  • Apelin fusion proteins and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0214] Example 1 - Cloning of expression constructs

[0215] A synthetic gene fragment was used to generate N-terminal and C-terminal hFc fusions with Apelin-13. The DNA encoding the resulting fusions hFc-apalin 13 (SEQ ID NO: 1 ) and apelin 13-hFc (SEQ ID NO: 3) were inserted into an expression vector downstream of the CMV promoter using standard molecular cloning techniques. CHO stable cell lines were generated and used to produce fusion proteins which were then purified by affinity methods. N-terminal hFc-apalin 13 and C-terminal apelin 13-hFc fusion proteins migrated on SDS-PAGE gels consistent with their predicted masses. (see Figure 3A . ) Western blot analysis performed with anti-apalin antibody (Abcam, #ab59469) was used to confirm the presence of apelin on hFc-apalin 13 and apelin 13-hFc. (see Figure 3B . )

Embodiment 2

[0216] Example 2 - Potency and therapeutic effect of Apelin Fc fusion protein in cAMP-reporter gene assay

[0217] Modulation of intracellular cAMP levels by unmodified apelin peptide (Bachem, #H-4568.0001) and the apelin 13 fusion protein of the invention was assessed using a bioassay developed to detect activation of hAPLNR. The HEK293 cell line was transfected together with a luciferase reporter gene [cAMP response element (CRE, 4X)-luciferase] to stably express full-length human hAPLNR (amino acids 1-380 of accession number NP_005152.1). The resulting cell line HEK293 / CRE-luc / hAPLNR was maintained in DMEM containing 10% FBS, NEAA, penicillin / streptomycin, and 100 μg / mL hygromycin B. For the bioassay, HEK293 / CRE-luc / hAPLNR cells were seeded on 96-well assay plates at 20,000 cells / well in 80 μL OPTIMEM supplemented with 0.1% FBS and penicillin / streptomycin / L-glutamine and at 37°C in 5% CO 2 Incubate for 16 hours. The next morning, to measure the inhibition of forskolin-in...

Embodiment 3

[0219] Example 3 - Potency and therapeutic effect of Fc fusion proteins in the β-arrestin assay

[0220] DiscoverX The platform is based on the recruitment of β-arrestin to GPCRs in response to treatment with relevant ligands. In this assay format, β-arrestin is fused to an N-terminal deletion mutant of β-galactosidase (β-gal) and is stably expressed in cells, while the GPCR is fused to a smaller (42 amino acid) , a weakly complementary β-gal fragment. Ligand stimulation of the GPCR in this assay results in the recruitment of β-arrestin to the GPCR, prompting the complementarity of the two β-gal fragments and resulting in the formation of a functional enzyme that converts the substrate into a detectable signal ( DiscoverX Corporation, Fremont, CA, USA).

[0221] For the assay, CHO-K1 / hAPLNR DiscoverX cells were plated at 10,000 cells / well in assay medium (DiscoverX Corporation; #93-0250E2) and incubated at 37°C in 5% CO 2 Incubate for 48 hours. Cells were then treated wi...

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Abstract

The invention provides a fusion protein or polypeptide comprising an apelin peptide fused to a multimerizing component. The invention also provides a fusion protein or polypeptide comprising an apelin peptide fused to an Fc domain, a fragment of an Fc domain, or a variant of an Fc domain. Apelin Fc-fusion polypeptides are capable of binding to the apelin receptor (APLNR). Apelin Fc-fusion polypeptides are capable of activating the APLNR and have improved pharmacokinetic properties compared to apelin peptides that are not fused to an Fc or an Fc fragment. Apelin Fc-fusion polypeptides are useful in diseases and conditions related to cardiovascular function, diabetes, cancer, obesity and other apelin-related conditions.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 786,172, filed March 14, 2013, under 35 USC §119(e), and claims the benefit of U.S. Provisional Patent Application, filed November 20, 2013, under 35 USC §119(e) 61 / 906,567, each of which is expressly incorporated herein by reference in its entirety. [0003] sequence listing [0004] This application incorporates by reference the Sequence Listing filed in computer readable form as file 8050WO_ST25.txt (43,420 bytes) created on March 13, 2014. field of invention [0005] The present invention relates to fusion proteins engineered with a multimeric component, such as a human immunoglobulin Fc domain, fused to the N- or C-terminus of an apelin peptide. The recombinant protein and its composition of the present invention are suitable for treating cardiovascular disease, ischemia-reperfusion, diabetes and other Apelene-related therapies. Ba...

Claims

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Application Information

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IPC IPC(8): C07K14/47A61K38/10A61K38/17C07K7/08
CPCC07K14/435A61K38/00C07K7/08C07K14/47C07K2319/30A61P3/04A61P3/12A61P31/18A61P35/00A61P35/04A61P9/00A61P9/04A61P9/10A61P9/12A61P3/10
Inventor P·史蒂维斯J·科洛马达A·墨菲
Owner REGENERON PHARM INC
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