Chimeric fibroblast growth factor 21 proteins and methods of use

A fibroblast, chimeric protein technology, applied in the direction of fibroblast growth factor, growth factor/inducing factor, peptide/protein component, etc., can solve the problem of thiazoline dione losing popularity and so on

Active Publication Date: 2015-06-24
NEW YORK UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, thiazolidinediones lost their popularity

Method used

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  • Chimeric fibroblast growth factor 21 proteins and methods of use
  • Chimeric fibroblast growth factor 21 proteins and methods of use
  • Chimeric fibroblast growth factor 21 proteins and methods of use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-F

[0254] Purification of Example 1-FGF, FGFR and Klotho Proteins

[0255] In vitro refolding of human FGF19 (SEQ ID NO:337) (R23 to K216), human FGF21 (SEQ ID NO:233) (H29 to S209; Figure 5A ) and human FGF23 (SEQ ID NO:351 ) from bacterial inclusion bodies (Y25 to I251; FIG. 5A ) N-terminal hexahistidine-tagged mature form by published protocols (Ibrahimi et al., Hum. Mol. Genet. 13:2313-2324 (2004); Plotnikov et al., Cell 101 : 413-424 (2000), which is hereby incorporated by reference in its entirety) to purify it. The amino acid sequence (SEQ ID NO:351) of human FGF23 (GenBank Accession No. AAG09917, which is hereby incorporated by reference in its entirety) is as follows:

[0256]

[0257] HS-binding site mutants of FGF19 (K149A) and FGF23 (R140A / R143A) were purified from bacterial inclusion bodies by a similar protocol to the wild-type proteins. To minimize proteolysis of FGF23 wild-type and mutant proteins, glutamine was used to replace the proteolytic cleavage site ...

Embodiment 2

[0259] Example 2 - Analysis of the Interaction of FGF-Heparin and FGF-FGFR-α / βKlotho Using Surface Plasmon Resonance Spectroscopy

[0260] Surface plasmon resonance (SPR) experiments were performed on a Biacore 2000 instrument (Biacore AB), at 25°C in HBS-EP buffer (10mM HEPES-NaOH, pH 7.4, 150mM NaCl, 3mM EDTA, 0.005% (v / v) The interaction was studied in polysorbate 20). To study endocrine FGF-heparin interactions, heparin chips were prepared by immobilizing biotinylated heparin (Sigma-Aldrich) on the flow channels of research-grade streptavidin chips (Biacore AB). The coupling density is ~5 fmol mm -2 the runner. To measure the binding of the chimeric FGF2 protein to heparin, biotinylated heparin was coupled to a streptavidin chip at a density as low as about 1 / 4 as judged based on the binding response obtained for FGF1. To study the FGF-FGFR-α / βKlotho interaction, FGF chips were prepared by covalently coupling FGF proteins via their free amino groups to the flow channel ...

Embodiment 3

[0266] Example 3 - Analysis of Phosphorylation of FRS2α and 44 / 42 MAP Kinases in Hepatoma and Epithelial Cell Lines

[0267] To examine FGF19 K149A and FGF23 R140A / R143A Whether mutants can activate FGFR in an α / βK1 otho-dependent manner, induction of tyrosine phosphorylation of FGFR substrate 2α (FRS2α) and downstream activation of the MAP kinase cascade were used as readouts for FGFR activation. Subconfluent cells of the H4II E rat hepatoma cell line (which endogenously express βKlotho (Kurosu et al., J. Biol. Chem. 282:26687-26695 (2007), which is hereby incorporated by reference in its entirety) ) serum starvation for 16h, followed by FGF19 K149A Mutant or wild-type FGF19 (0.2ng ml -1 to 2.0 μg ml -1 ) stimulation for 10 min. Similarly, with FGF23 R140A / R143A Mutant or wild-type FGF23 (0.1 to 100ng ml -1 ) treatment of subconfluent cells of the HEK293 cell line ectopically expressing the transmembrane isoform of murine αKlotho (Kurosu et al., J. Biol. Chem. 281:6120...

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Abstract

The present invention relates to a chimeric protein that includes an N-terminus coupled to a C-terminus, where the N-terminus includes a portion of a paracrine fibroblast growth factor (''FGF'') and the C-terminus includes a C-terminal portion of an FGF21 molecule. The portion of the paracrine FGF is modified to decrease binding affinity for heparin and / or heparan sulfate compared to the portion without the modification. The present invention also relates to pharmaceutical compositions including chimeric proteins according to the present invention, methods for treating a subject suffering from diabetes, obesity, or metabolic syndrome, and methods of screening for compounds with enhanced binding affinity for the betaKappaIotaomicronthomicron-FGF receptor complex involving the use of chimeric proteins of the present invention.

Description

[0001] This application claims U.S. Provisional Patent Application No. 61 / 656,778 filed June 7, 2012, U.S. Provisional Patent Application No. 61 / 664,081 filed June 25, 2012, and U.S. Patent Application No. 61 / 664,081 filed June 25, 2013 Priority benefit of Serial No. 13 / 837,880, each of which is incorporated herein by reference in its entirety. [0002] This invention was made with government support under Grant Nos. DE13686, DK077276, AG019712, DK091392, and DK067158 awarded by the National Institutes of Health. The Government has certain rights in this invention. field of invention [0003] The present invention relates to chimeric fibroblast growth factor ("FGF") proteins and uses thereof. Background of the invention [0004] Type 2 diabetes is a chronic progressive disorder caused by end-organ resistance to the action of insulin combined with insufficient insulin secretion by the pancreas. Metabolic abnormalities associated with insulin resistance and secretion defects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/50A61K38/18C12P21/04A61K38/00A61K38/24C07K5/00
CPCA61K38/1825A61K45/06A61P3/00A61P3/10C07K14/50C07K2319/00G01N2333/50G01N2500/02G01N2800/042A61K2300/00A61K31/00C07K14/501C07K14/503
Inventor M·莫哈马蒂R·戈茨R·M·埃文斯M·道恩斯J·M·徐
Owner NEW YORK UNIV
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