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Modified erythropoietin (epo)polypeptides that exhibit increased protease resistance and pharmaceutical compositions thereof

A technology of erythropoietin and composition, which is applied in the direction of erythropoietin, drug combination, peptide/protein composition, etc., can solve the problems of highly variable, difficult, and expensive preparation of glycosylated therapeutic protein

Inactive Publication Date: 2011-09-14
韩诺生物制约株式会社
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Additionally, many therapeutic proteins are glycosylated, and making glycosylated therapeutic proteins is expensive, highly variable, and difficult to perform

Method used

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  • Modified erythropoietin (epo)polypeptides that exhibit increased protease resistance and pharmaceutical compositions thereof
  • Modified erythropoietin (epo)polypeptides that exhibit increased protease resistance and pharmaceutical compositions thereof
  • Modified erythropoietin (epo)polypeptides that exhibit increased protease resistance and pharmaceutical compositions thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0720] Cloning and production of EPO mutants

[0721] 1. Cloning of cDNA encoding EPO and inserting into mammalian expression vector

[0722] Use the following primers from GeneStorm using standard techniques known in the art Human clone IDRG001720 (from the ResGen ORF positive collection-catalog number #H-K1000, Invitrogen; the sequence is carried in the mammalian expression plasmid pcDNA3.1 / GS, Invitrogen, SEQ ID NO: 230) by polymerase chain reaction (PCR) ) Amplify the nucleotide sequence including the coding sequence of human erythropoiesis factor (SEQ ID NO: 229):

[0723] EPO BamHI forward primer:

[0724] 5'-GGGAATTCCATATGGGGGTGCACGAATGTCCTGCCTGG-3' (SEQ ID NO: 231) and

[0725] EPO NdeI reverse primer:

[0726] 5'-CGGGATCCTCATCTGTCCCCTGTCCTGCAGGCCTCCC-3' (SEQ ID NO:232).

[0727] The amplified sequence human erythropoietin cDNA sequence was cloned into pTOPO-TA vector (Invitrogen) to generate plasmid pTOPO-TA-hEPO. The sequence of the EPO cDNA was detected by automated DNA sequ...

Embodiment 2

[0742] Production and yield determination of natural and modified human EPO polypeptides (proteins) in mammalian cells

[0743] Chinese hamster ovary (CHO) cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (FCS). On the day before transfection, take 5x10 per well 5 The density of the cells, in DMEM containing 10% FCS (without antibiotics), spread the CHO cells in a 6-well plate at 37° C. in a humidified atmosphere containing 7% CO2 for the next few days after transfection Achieve 50-90% confluence every day.

[0744] The Perfectin reagent (Ozyme) was used to transfect CHO cells with 2 μg of EPO mutant DNA according to the manufacturer's instructions. After transfection, plate the cells in Opti-MEM Reduced serum medium (Invitrogen) at 37°C containing 7% CO 2 The ingredients are incubated for 4 hours in a humid atmosphere. After incubation, the transfection medium was replaced with fresh 1 ml DMEM medium containing 1% FCS. After 96 hours, the ...

Embodiment 3

[0747] Measure the specific activity of human EPO by TF-1 proliferation test

[0748] Proliferation test was performed on each aliquot sampled at different time points of the protease degradation kinetics in the human erythroleukemia cell line TF-1 bioassay to determine the residual proliferation activity (EC 50 ).

[0749] In RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, 2mM L-glutamine, and 2ng / ml human recombinant GM-CSF, at 37°C and containing 7% CO 2 / 95% air content in a humid atmosphere, under T175 (175cm 2 ) Maintain the TF-1 cell line in a polystyrene tissue culture flask and divide twice a week. Twenty-four hours before use in the proliferation test, the cells were washed twice in ice-cold PBS, and resuspended in GM-CSF-free RPMI medium supplemented with 2mM glutamine and 10% FCS for 16 hours .

[0750] Divide TF1 cells at 4x10 per well 4 The cells were plated in a 96-well plate in 70 μl of GM-CSF-free RPMI medium supplemented with 2mM glutamine and 10% FCS. Ea...

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Abstract

Modified erythropoietin (EPO) polypeptides and other modified therapeutic polypeptides are provided. The EPO polypeptides and other therapeutic polypeptides are modified to exhibit physical properties and activities that differ from the unmodified EPO polypeptides and other unmodified therapeutic polypeptides, respectively. Nucleic acid molecules encoding these polypeptides also are provided. Also provided are methods of treatment and diagnosis using the polypeptides.

Description

[0001] Related application [0002] Claim the priority of the U.S. Provisional Application 61 / 130,376 entitled "Modified Erythropoietin (EPO) Polypeptides that Exhibit Increased Protease Resistance and Pharmaceutical Compositions Thereof" filed on May 29, 2008. The applicant of this provisional application is Thierry Guyon, Giles Borrelly , Xavier Gallet, Lila Drittanti and Manuel Vega. When permitted, the subject of the above-mentioned application shall be incorporated by reference in its entirety. [0003] This application relates to the US application 11 / 998,387 entitled "MODIFIED ERYTHROPOIETIN POLYPEPTIDES AND USES THEREOF" filed on November 28, 2007. The applicants are Thierry Guyon, Giles Borrelly, Xavier Gallet, Lila Drittanti and Manuel Vega. This application also involves questions. The international application PCT / GB2007 / 004520 for "MODIFIED ERYTHROPOIETIN POLYPEPTIDES AND USES THEREOF", the applicants are Thierry Guyon, Giles Borrelly, Xavier Gallet, Lila Drittanti and...

Claims

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Application Information

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IPC IPC(8): C07K14/505A61K38/18C12N15/12
CPCC07K14/505A61K38/00A61P7/06
Inventor T·居永G·伯雷利X·加莱L·德里坦提M·维嘉
Owner 韩诺生物制约株式会社
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