Bacterical/permability-increasing protein(BPI) deletion analogs

a technology of permability and protein, applied in the field of bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-bpi-b

Inactive Publication Date: 2005-06-16
XOMA CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0017] The present invention provides novel biologically active BPI deletion analogs and preparations thereof characterized by enhanced stability and homogeneity, including for example, resistance to dimerization and cysteine adduct formation and reduced amino-terminal and carboxy-terminal heterogeneity of the recombinant product, as well as by enhanced in vivo biological activity, properties which render it highly suitable for therapeutic and diagnostic uses. Novel BPI deletion analogs are the expression product of DNA encoding amino acid residues 10 through 193 of mature human BPI (SEQ ID NO: 2), in which the cysteine at position 132 has been replaced with a different amino acid, preferably a non-polar amino acid such as serine or alanine. In a preferred embodiment, designated “rBPI(10-193)C132A” or “rBPI(10-193)ala132,” the cysteine at position 132 is replaced with an alanine.

Problems solved by technology

In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death.

Method used

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  • Bacterical/permability-increasing protein(BPI) deletion analogs
  • Bacterical/permability-increasing protein(BPI) deletion analogs

Examples

Experimental program
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Effect test

example 1

Construction of Expression Vector pING1742

[0034] The rBPI(10-193)C132A expression vector, pING1742, was constructed as follows. The expression vector pING4155 was first constructed by ligating a BamHI-BsaI fragment containing the neo gene from pING3174 with a BsaI-XhoI fragment containing the CMV promoter and rBPI21 gene from pING4144 and an XhoI-BamHI fragment containing the mouse (kappa) light chain 3′ untranslated region from pING4537 (pING3174, pING4144 and pING4537 are described in U.S. Pat. No. 5,420,019, incorporated by reference). The resulting pING4155 vector contains the gene encoding rBPI21 fused to the human IgG enhancer, the human CMV promoter and the mouse (kappa) light chain 3′ untranslated region. It also contains the neo gene encoding neomycin phosphotransferase, for selection of transfectants resistant to the antibiotic Geneticin® (G418).

[0035] The vector pING1732 was produced by deleting the 0.7 kbp HindIII-HindIII fragment of pING4155 containing the human Ig en...

example 2

Transformation of CHO Cells with pING1742

[0037] CHO-K1 cells (American Type Culture Collection (ATCC) Accession No. CCL61) were adapted to growth in serum-free Ex-Cell 301 medium as follows. CHO-K1 cells grown in Ham's F12 medium were trypsinized, centrifuged and resuspended in Ex-Cell 301 medium. Cells were grown in a 125-ml flask at 100 rpm and passaged every two to three days in either a 125-ml or 250-ml flask.

[0038] These Ex-Cell 301-adapted CHO cells were transfected by electroporation with pING1742. Prior to transfection, pING1742 was digested with NotI, which linearizes the plasmid. Following a 48-hour recovery, cells were plated at approximately 104 cells / well into 96-well plates containing Ex-Cell 301 medium supplemented with 0.6 mg / mL G418 (Life Technologies, Gaithersburg, Md.). At approximately 2 weeks, supernatants from approximately 250 wells containing single colonies were screened by ELISA for the presence of BPI-reactive protein using an anti-BPI monoclonal antibod...

example 3

Production and Purification of rBPI(10-193)C132A

[0041] Large quantities of rBPI(10-193)C132A were produced for characterization by growing Clone 139 cells in 2-liter research fermenters (Biolafitte, St. Germain en Laye, France) and then in a 500 liter ABEC fermenter (ABEC, Allentown, Pa.). Protein product obtained from the 2-liter fermenters was used for the in vitro studies described below, while product obtained from the 500 liter fermenter was used for animal toxicology and efficacy studies.

[0042] A. Growth in Two-Liter Fermenters

[0043] Clone 139 cells were passaged in spinner flasks of increasing volumes containing Ex-Cell medium supplemented with 1% FBS until sufficient volume and cell density was achieved to inoculate the 2 liter bioreactors at approximately 2×105 cells / mL. Cells were grown in three 2-liter fermenters in Ex-Cell medium supplemented with 1% FBS, at 37° C., pH 7.2, 150 rpm with dissolved oxygen maintained at 5-10%. Large sterile SP-Sepharose beads (Pharmacia ...

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Abstract

Novel BPI deletion analogs are provided that consist of amino acid residues 10 through 193 of mature human BPI wherein the cysteine residue at BPI amino acid position 132 is replaced by another amino acid. Fusion proteins comprising these analogs are also provided, as are polynucleotides encoding these products, materials and methods for their recombinant production, compositions and medicaments of these products, and therapeutic uses for these products.

Description

[0001] This application is a continuation-in-part of U.S. Ser. No. 09 / 099,725 filed Jun. 19, 1998.BACKGROUND OF THE INVENTION [0002] The present invention provides preparations of novel biologically active deletion analogs of bactericidal / permeability-increasing protein (BPI) characterized by improved stability and homogeneity as well as by enhanced in vivo activity, and pharmaceutical compositions containing the same. [0003] BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. BPI is known to bind to lipopolysaccharide, a major component of the outer membrane of gram-negative bacteria that stimulates a potent inflammatory response which can lead to septic shock. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254: 11000 (1979)] or E. coli affinity chromatogr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/09A01N37/18A61K38/00A61K38/16A61P31/04C07H21/04C07K14/00C07K14/47C12N5/00C12N5/10C12N9/48C12N15/31C12N15/74C12P21/02C12P21/06C12R1/91
CPCC07K14/4742A61K38/17A61P31/04
Inventor HORWITZ, ARNOLDCARROLL, STEPHENBURKE, DAVID
Owner XOMA CORP
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