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Fusion protein comprising leptin and methods for producing and using the same

A technology of fusion protein and leptin protein, applied in the field of fusion protein, can solve the problems of low production efficiency, complicated purification process, short serum half-life, etc.

Pending Publication Date: 2019-09-20
ASKGENE PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the resulting leptin "derivatives" may be immunogenic in subjects
[0014] Due to the large dose required for treatment, low production efficiency, short serum half-life, and extremely complicated purification process, there is an urgent need for a method that can increase the production yield of leptin

Method used

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  • Fusion protein comprising leptin and methods for producing and using the same
  • Fusion protein comprising leptin and methods for producing and using the same
  • Fusion protein comprising leptin and methods for producing and using the same

Examples

Experimental program
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Effect test

Embodiment 1

[0087] Example 1: Expression and purification of leptin fusion proteins by CHO cells. The DNA of the chimeric molecule, containing the Fc-leptin fusion protein (SEQ ID NO: 42, designated ASKB-O42), was synthesized and cloned into an expression vector. DNA sequencing confirmed that it contained the DNA gene of the complete expression construct. The expression vector was amplified by transforming DH10B E. coli and growing overnight. The DNA of the expression vector was prepared and passed through an endogenous plasmid-free kit (from )purification.

[0088] The expression vector was introduced into GS- / - Chinese hamster ovary cells (CHO) by electroporation using glutamine-free selective medium ( CD CHO Fusion Growth Medium) to screen transfected CHO cells to obtain a cell line stably expressing ASKB-O42. Thirty-two or more stable mini-mix clones were established in this manner, and major mini-mix clones were selected based on their expression levels in batch and fed-batch c...

Embodiment 2

[0090] Example 2: E. coli expression of Fc-leptin fusion protein. Fc-leptin fusion proteins A, B and C were expressed by E. coli BL21DE3 strain. Schematic diagrams of the Fc-leptin fusion proteins A, B and C are presented in Figure 4 , 5, and 6. The gene sequences contained in the plasmids are as described in SEQ ID NOs: 50, and 52, and were synthesized by DNA 2.0. The plasmid sequence containing the Fc-leptin fusion protein B gene is shown in SEQ ID NO: 51, which is derived from the mutation of SEQ ID NO: 50 (Fc-leptin fusion protein A). E. coli was transformed, plated and positive clones were selected. Overexpression was performed in shake flasks with LB medium and expression was induced with 1 mM IPTG. Cells were harvested approximately 5 hours to overnight after induction. The expression levels of Fc-leptin fusion protein at different time points after IPTG induction are shown in Figure 7 . The results showed that the expression level was stable after about 5 hour...

Embodiment 3

[0091] Example 3: Harvesting of inclusion bodies. About 15 grams (wet weight) of the cell pellet was resuspended in about 60 ml of distilled water. The mixture was sonicated on ice using a Fisher Scientific Model FB50 sonicator at an amplitude of about 85 for 20-30 s, three times with 1 min between each sonication. The resulting cell lysate was centrifuged in a Sorvall RC 3BP centrifuge at 300 RPM for 20 minutes. The pellet was resuspended in 60 mL of distilled water and washed 2 times by centrifugation. The fusion protein-containing inclusion body pellet obtained by the third centrifugation was directly frozen at -80°C until subsequent use.

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Abstract

The present invention provides fusion proteins comprising leptin and a second protein. The presence of the second protein provides increased biological activity and / or increased half-life in vivo. The present invention also provides human, canine and feline leptin molecules fused to peptides, antibodies or antibody fragments which enhances the abilities of the leptin molecules to transport through the blood-brain-barrier (BBB). The present invention also provides fusion proteins further comprising a peptide agonist that is capable of binding to and stimulate one, two or all three of the following receptors: GLP-1 receptor, Glucagon receptor, and GIP receptor. Also disclosed is a method of production such fusion proteins through recombinant technologies. The invention further discloses a pharmaceutical composition comprising one of the fusion proteins as an active intergradient as well as a method for using such a pharmaceutical composition to treat diseases in dogs, cats and humans.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims priority to US Provisional Application No. 62 / 360,271 filed on July 8, 2016, and the entire contents of which are incorporated herein by reference. technical field [0003] The present invention relates to a fusion (ie, chimeric) protein comprising an antibody or fragment thereof and a leptin protein. The present invention also relates to a pharmaceutical combination comprising the fusion protein and methods of making and using the same. In particular, the present invention relates to the preparation and use of an Fc region and a leptin ("leptin") fusion protein comprising an immunoglobulin. Background technique [0004] Metabolic disorders in pets are associated with important physiological disorders such as diabetes, hypertension, heart disease and certain types of cancer as well as other metabolic disorders. For example, in the United States alone, it is estimated that approximately 52.7% or...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/22A61K38/26A61K38/28A61K47/68C07K14/575C07K14/605
CPCA61K38/00C07K14/5759C07K14/76C07K2319/30C07K2319/31C07K7/08
Inventor 越峰·吕卢建丰杨岚李路K·肖恩贝克刘磊
Owner ASKGENE PHARM INC
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