Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Antibodies which activate an erythropoietin receptor

a technology of erythropoietin and antibodies, which is applied in the field of antibodies which recognize erythropoietin receptors, can solve the problems of not being able to isolate a stable multimeric form of purified epo soluble receptors, not being able to complete cell activation by itself, and not being able to dimerize epor

Inactive Publication Date: 2008-07-31
AMGEN INC
View PDF13 Cites 25 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Decreased production of EPO, which commonly occurs in adults as a result of renal failure, leads to anemia.
However, isolation of a stable multimeric form of purified EPO soluble receptor has not been reported.
In addition, dimerization of EPOR may be required, but not by itself be sufficient for complete activation of cells.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Antibodies which activate an erythropoietin receptor
  • Antibodies which activate an erythropoietin receptor
  • Antibodies which activate an erythropoietin receptor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Production of Soluble Human Erythropoietin Receptor

A. Isolation of Clones for Expression of Soluble Human Erythropoietin Receptor.

[0040]Using a clone containing the human erythropoietin receptor as described by Jones et al. supra, the PCR technique was used to obtain a clone for expression of soluble human erythropoietin receptor (sHuEPOR). Primers for PCR amplification of human erthropoietin receptor were:

5′ primer:(SEQ. ID NO: 1)5′ -CTC CAA GCT TGC CGT CAC CAT GGA CCA CCT CGGGGC GTC CCT-3′;and3′ primer:(SEQ. ID NO: 2)5′-CAG GTC TAG ATT ACT AGG GAT CCA GGT CGC TAGGC-3′.

[0041]PCR reactions were carried out using 2.5 ng of a plasmid containing human EPOR, 5 pmol of each of the above oligonucleotide primers, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM Mg Cl2, 200 μM each dNTP and 1 unit of Taq polymerase. Amplification was for 5 cycles of 30 sec. at 94° C., 1 min. at 50° C., 1 min at 72° C., followed by 20 cycles of 30 sec. at 94° C., 1 min. at 55° C., 1 min at 72° C. DNA was purified ...

example 2

Purification of Soluble Human Erythropoietin Receptor

[0047]Four different preparations of soluble recombinant human EPOR were made. In the first preparation, Epoxy-activated SEPHAROSE 6B (Pharmacia, Piscataway, N.J.) is coupled with recombinant human erythropoietin (rHuEPO) essentially as per manufacturer's instructions. 218 mg of rHuEPO in 4.5 mL of 32 mM ZnCl2 is added to 7.2 g of Epoxy-activated SEPHAROSE 6 B previously hydrated and washed with H2O. This slurry is titrated to pH 10.8 then mixed overnight at room temperature. Any remaining reactive groups are then blocked by addition of ethanolamine to a final concentration of 1 M and mixed for 4 hours at room temperature. The subsequent steps are performed at 8°±2° C. The coupled resin (Epoxy-EPO) is packed into a column and washed with alternating cycles of 0.5 M NaCl / 0.1 M HOAc pH 4 and 0.5 M NaCl / 0.1 M Borate pH 8. The column is equilibrated with 140 mM NaCl / 10 mM Tris pH 7.6 (TBS). It is loaded with 1560 mL of roller bottle p...

example 3

Preparation and Screening of Hybridoma Cell Lines

A. Enzyme-Linked Immunosorbent Assay (EIA)

[0051]EIAs were initially performed to determine serum antibody (Ab) titres of individual animals, and later for screening of potential hybridomas. Flat bottom, high-binding, 96-well microtitration EIA / RIA plates (Costar Corporation, Cambridge, Mass.) were coated with purified sHuEPOR at 5 ug per ml carbonate-bicarbonate buffer, pH 9.2 (0.015 M Na2CO3, 0.035 M NaHCO3). Fifty μl of the Ab were added to each well. Plates were then covered with acetate film (ICN Biomedicals, Inc., Costa Mesa, Calif.) and were incubated at room temperature (RT) on a rocking platform for 2 hours or over-night at 4° C. sHuEPOR lot #1 was used after the first and second boost, lot #2 was used after the third boost. sHuEPOR lots #3 and 4 were used for screening of hybridomas. Plates were blocked for 30 minutes at RT with 250 μl per well 5% BSA solution prepared by mixing 1 part BSA diluent / blocking solution concentrat...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

Antibodies and fragments thereof which activate an erythropoietin receptor and stimulate erythropoiesis are described. Also described are hybridoma cell lines which produce the antibodies and methods and compositions for the treatment of anemia.

Description

[0001]This application is a divisional of U.S. patent application Ser. No. 11 / 406,835, filed Apr. 18, 2006, which is a divisional of U.S. patent application Ser. No. 10 / 364,276, filed Feb. 10, 2003, now U.S. Pat. No. 7,081,523, which is a continuation of U.S. patent application Ser. No. 09 / 640,090, filed Aug. 17, 2000, now abandoned, which is a divisional of U.S. patent application Ser. No. 09 / 092,671, filed Jun. 5, 1998, now abandoned, which is a divisional of U.S. patent application Ser. No. 08 / 280,864, filed Jul. 26, 1994, now U.S. Pat. No. 5,885,574; all of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]This invention relates to antibodies which recognize an erythropoietin receptor. More particularly, the invention relates to antibodies which activate an erythropoietin receptor and stimulate erythropoiesis.BACKGROUND OF THE INVENTION[0003]Erythropoietin (EPO) is a glycoprotein hormone involved in the growth and maturation of erythroid pr...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): C07K16/28C12N15/02A61K38/00A61K39/00A61K39/395A61P7/06C07K14/71C12N5/00C12N5/10C12N5/20C12P21/08C12R1/91G01N33/00G01N33/53G01N33/566G01N33/577
CPCA61K38/00A61K2039/505C07K2317/73C07K16/2863C07K2316/95C07K14/71C07K2317/74A61P7/06
Inventor ELLIOTT, STEVEN G.
Owner AMGEN INC
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com