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WO2000063253A1 - Compositions de proteine hybride agp-1 et procedes connexes - Google Patents

Compositions de proteine hybride agp-1 et procedes connexes Download PDF

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Publication number
WO2000063253A1
WO2000063253A1 PCT/US2000/008004 US0008004W WO0063253A1 WO 2000063253 A1 WO2000063253 A1 WO 2000063253A1 US 0008004 W US0008004 W US 0008004W WO 0063253 A1 WO0063253 A1 WO 0063253A1
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WO
WIPO (PCT)
Prior art keywords
protein
gly
ala
derivative
agp
Prior art date
Application number
PCT/US2000/008004
Other languages
English (en)
Inventor
Hailing Hsu
Shi-Yuan Meng
Original Assignee
Amgen Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amgen Inc. filed Critical Amgen Inc.
Priority to AU39230/00A priority Critical patent/AU3923000A/en
Publication of WO2000063253A1 publication Critical patent/WO2000063253A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention has a number of aspects relating to the modification of proteins via fusion of an Fc region to an AGP-1 protein (or variant, fragments or derivative thereof), as well as, specific modifications, preparations and methods of use thereof.
  • the present invention provides for a protein having a formula selected from the group consisting of: R. - R 2 and R 1 - L - R 2 , wherein R. is a Fc protein, or a variant or fragment thereof, R 2 is an AGP-1 protein, or variant or fragment thereof, and L is a linker.
  • the invention provides for genetic or chemical linkages of the Rl and R2 moieties as described herein.
  • Figure 3 shows the nucleotide and amino acid sequence of Fc-huAGP-1 (95- 281) . Amino acids corresponding to OPG signal peptide are underlined. Amino acids corresponding to human AGP-1 (95-281) are bracketed.
  • Figure 5 shows the nucleotide and amino acid sequence of Fc-muAGP-1 (99- 291) . Amino acids corresponding to OPG signal peptide are underlined. Amino acids corresponding to murine AGP-1 (99-291) are bracketed.
  • the invention provides for FcAGP-1 fusion polypeptides and compositions thereof. Fusions of an Fc region to an AGP-1 polypeptide are advantageously made at the amino terminus of AGP-1, that is, the carboxy terminus of an Fc region is fused to the amino terminus of AGP-1.
  • These fusion proteins (and nucleic acids encoding same) are designated herein as FcAGP-1. However, it is also contemplated that, in certain instances, it may be desirable to fuse the carboxy terminus of AGP-1 to the amino terminus of an Fc region.
  • the fusion proteins (and nucleic acids encoding same) are designated herein as AGP-lFc.
  • Fc variants may also be made which show reduced binding to Fc receptors which trigger effector functions such as antibody dependent cellular cytotoxicity (ADCC) and activation of complement.
  • ADCC antibody dependent cellular cytotoxicity
  • Such variants may include leucine at position 20 deleted or substituted with a glutamine residue, glutamate at position 103 deleted or substituted with an alanine residue, and lysines at positions 105 and 107 deleted or substituted with alanine residues (following the numbering as set forth in Figure 1) .
  • One or more of such substitutions are contemplated.
  • Fc variants include one or more tyrosine residues replaced with, for example, phenyalanine residues.
  • other variant amino acid insertions, deletions and/or substitutions are also contemplated and are within the scope of the present invention. Examples include Fc variants disclosed in W096/32478 and WO97/34630 hereby incorporated by reference.
  • alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
  • the Fc protein may be also linked to the AGP- 1 proteins of the Fc-AGP-1 protein by "linker" moieties whether chemical or amino acids of varying lengths.
  • a nucleic acid molecule encoding an Fc-AGP-1 fusion protein is inserted into an appropriate expression vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur) .
  • a nucleic acid molecule encoding an FcAGP-1 protein may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend in part on whether an Fc-AGP-1 protein is to be post-translationally modified (e.g, glycosylated and/or phosphorylated) . If so, yeast, insect, or mammalian host cells are preferable.
  • a heterologous signal sequence is the OPG signal sequence as described in W097/23614.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • the native AGP-1 signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders.
  • the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.
  • the recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, or other known techniques. After the vector has been constructed and a nucleic acid molecule encoding an AGP-1 polypeptide has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vi tro culture of primary tissue, as well as primary explants, are also suitable. Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • Other suitable mammalian cell lines include but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. Each of these cell lines is known by and available to those skilled in the art.
  • Similarly useful as host cells suitable for the present invention are bacterial cells . For example, the various strains of E.
  • Such procedures include, without limitation, ion exchange chromatography, molecular sieve chromatography, HPLC, native gel electrophoresis in combination with gel elution, and preparative isoelectric focusing ("Isoprime” machine/technique, Hoefer Scientific) . In some cases, two or more of these techniques may be combined to achieve increased purity.
  • the pellet material can then be treated at pH extremes or with chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • An AGP-1 polypeptide in its now soluble form can then be analyzed using gel electrophoresis, immunoprecipitation or the like. If it is desired to isolate an AGP-1 polypeptide, isolation may be accomplished using standard methods such as those set forth below and in Marston et al . (Meth. Enz . , 182, 264-275 (1990)).
  • polyaminoacids may be selected from the group consisting of serum album (such as human serum albumin), or other polyaminoacids, e.g. lysines. As indicated below, the location of attachment of the polyaminoacid may be at the N-terminus of the Fc-AGP-1 protein moiety, or C-terminus, or other places in between, and also may be connected by a chemical "linker" moiety to the Fc-AGP-1 protein.
  • serum album such as human serum albumin
  • polyaminoacids e.g. lysines
  • the optimum ratio in terms of efficiency of reaction in that there is no excess unreacted protein or polymer
  • the desired degree of derivatization e.g., mono, di-, tri-, etc.
  • the molecular weight of the polymer selected whether the polymer is branched or unbranched, and the reaction conditions.
  • N-terminal pegylation ensures a homogenous product as characterization of the product is simplified relative to di-, tri- or other multi-pegylated products.
  • the use of the above reductive alkylation process for preparation of an N-terminal product is preferred for ease in commercial manufacturing.
  • Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e. a. . Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form. Implantable sustained release formulations are also contemplated, as are transdermal formulations.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • pulmonary delivery of the present protein (or derivatives thereof) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Adjei et al . Pharmaceutical Research 1 565-569 (1990); Adjei et al . , International Journal of Pharmaceutics ,6_3: 135-144 (1990) (leuprolide acetate); Braquet et al . , Journal of Cardiovascular Pharmacology 13 (suppl. 5): s.143-146 (1989) (endothelin-1) ;Hubbard et al . , Annals of Internal Medicine 3: 206-212 (1989) ( ⁇ l-antitrypsin) ; Smith et al . , J. Clin. Invest. 84: 1145-1146
  • the protein (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns) , most preferably 0.5 to 5 ⁇ m, for most effective delivery to the distal lung.
  • the major capsid protein promoter sequence (GenBank Ace No. M22978) was PCR amplified from the Bac-N-Blue ⁇ r linear AcMNPV DNA purchased from Invitrogen (Carlsbad, CA) with the following primers:
  • Fc-AGP-1 fusion proteins were purified using Pharmacia Protein A Sepharose. The resin was equilibrated with TBS containing 20mM Tris pH7.0 and 150mM NaCl before applying the media. Complete protease inhibitor cocktail (Boehringer-Mannheim) was added to the media according to the manufacturer's instructions. The media was loaded, the column washed with TBS, and protein was eluted using Gentle Elution buffer (Pierce, Rockford, IL) . Protein containing fractions were pooled and submitted for in vitro analysis .
  • EXAMPLE 4 Biological Activity of AGP-1 and AGP-1 Fusion Proteins
  • Jurkat cells (ATCC No. ) were maintained in RPMI medium 1640 containing 10% fetal calf serum, 100 mg/ml penicillin G, and 100 mg/ml streptomycin (GIBCO) .
  • GEBCO streptomycin
  • 250 ml of Jurkat cells (5 x 105 cells/ml) were seeded to each well of a 96 well plate.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
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Abstract

L'invention concerne des compositions de protéine hybride Fc-AGP-1, des procédés de préparation de ces compositions et leurs applications. L'invention concerne plus particulièrement une protéine hybride génétique ou chimique comprenant la région de l'immunoglobuline Fc, de son dérivé ou analogue fusionnée avec la partie N-terminale de la protéine AGP-1, de son déviré ou analogue.
PCT/US2000/008004 1999-04-16 2000-03-24 Compositions de proteine hybride agp-1 et procedes connexes WO2000063253A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU39230/00A AU3923000A (en) 1999-04-16 2000-03-24 Agp-1 fusion protein compositions and methods

Applications Claiming Priority (2)

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US29324599A 1999-04-16 1999-04-16
US09/293,245 1999-04-16

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001096395A3 (fr) * 2000-06-13 2002-05-16 Childrens Medical Center Molecules oncolytiques biosynthetiques et leur utilisation
US6521228B1 (en) 1995-06-29 2003-02-18 Immunex Corporation Antibodies directed against trail
WO2003089450A3 (fr) * 2002-04-22 2004-06-03 Absorber Ab Compositions et procedes d'inhibition de l'adherence microbienne
WO2003105908A3 (fr) * 2002-03-15 2004-10-28 Dep Veterans Affairs Rehab R&D Methodes et compositions faisant appel a des asialodeterminants cellulaires et a des glycoconjugues pour fournir des cellules a des tissus et a des organes
EP1870464A3 (fr) * 1999-06-02 2008-03-12 Genentech, Inc. Procédés et compositions d'inhibition de la croissance de cellules néoplasiques
WO2008075833A1 (fr) 2006-12-21 2008-06-26 Mogam Biotechnology Research Institute Protéine chimère comprenant une région fc d'immunoglobuline et un fragment kringle d'apolipoprotéine (a) humaine
US7736657B2 (en) * 2002-02-10 2010-06-15 Apoxis S.A. Fusion constructs containing active sections on TNF ligands
US8188244B2 (en) 2004-02-11 2012-05-29 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Carcinoembryonic antigen fusions and uses thereof
US9388230B2 (en) 2010-09-28 2016-07-12 Kahr Medical(2005) Ltd Compositions and methods for treatment of hematological malignancies
US10913790B2 (en) 2003-08-26 2021-02-09 The Regents Of The University Of Colorado, A Body Corporate Compositions of, and methods for, alpha-1 anti trypsin Fc fusion molecules

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1997001633A1 (fr) * 1995-06-29 1997-01-16 Immunex Corporation Cytokine inductrice d'apoptose
WO1997046686A2 (fr) * 1996-06-07 1997-12-11 Amgen Inc. Polypeptide apparente au facteur de necrose tumorale

Patent Citations (2)

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WO1997001633A1 (fr) * 1995-06-29 1997-01-16 Immunex Corporation Cytokine inductrice d'apoptose
WO1997046686A2 (fr) * 1996-06-07 1997-12-11 Amgen Inc. Polypeptide apparente au facteur de necrose tumorale

Non-Patent Citations (3)

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Title
DANILENKO D M (REPRINT) ET AL: "AGP - 1, a novel member of the tumor necrosis factor family, induces hepatic necrosis and inflammation in transgenic mice.", FASEB JOURNAL, (28 FEB 1997) VOL. 11, NO. 3, PP. 2951-2951, XP002141678 *
KIM J -K ET AL: "IDENTIFYING AMINO ACID RESIDUES THAT INFLUENCE PLASMA CLEARANCE OF MURINE IGG1 FRAGMENTS BY SITE-DIRECTED MUTAGENESIS", EUROPEAN JOURNAL OF IMMUNOLOGY,DE,WEINHEIM, vol. 24, no. 3, 1 January 1994 (1994-01-01), pages 542 - 548, XP000590871, ISSN: 0014-2980 *
SHERIDAN J P ET AL: "Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors", SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 277, 8 August 1997 (1997-08-08), pages 818 - 821, XP002065023, ISSN: 0036-8075 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6521228B1 (en) 1995-06-29 2003-02-18 Immunex Corporation Antibodies directed against trail
EP1870464A3 (fr) * 1999-06-02 2008-03-12 Genentech, Inc. Procédés et compositions d'inhibition de la croissance de cellules néoplasiques
WO2001096395A3 (fr) * 2000-06-13 2002-05-16 Childrens Medical Center Molecules oncolytiques biosynthetiques et leur utilisation
US8501177B2 (en) 2002-02-10 2013-08-06 Topotarget Switzerland Sa Treatment of ectodermal dysplasia with EDA1 fusion proteins
US8895003B2 (en) 2002-02-10 2014-11-25 Topotarget Switzerland Sa Pharmaceutical composition comprising EDA-1 Fusion Protein
US7736657B2 (en) * 2002-02-10 2010-06-15 Apoxis S.A. Fusion constructs containing active sections on TNF ligands
US7563459B2 (en) 2002-03-15 2009-07-21 The United States of America as represented by the Department of Vetrans Affairs Methods and compositions for regenerating tissue
WO2003105908A3 (fr) * 2002-03-15 2004-10-28 Dep Veterans Affairs Rehab R&D Methodes et compositions faisant appel a des asialodeterminants cellulaires et a des glycoconjugues pour fournir des cellules a des tissus et a des organes
WO2003089450A3 (fr) * 2002-04-22 2004-06-03 Absorber Ab Compositions et procedes d'inhibition de l'adherence microbienne
US10913790B2 (en) 2003-08-26 2021-02-09 The Regents Of The University Of Colorado, A Body Corporate Compositions of, and methods for, alpha-1 anti trypsin Fc fusion molecules
US8188244B2 (en) 2004-02-11 2012-05-29 Istituto Di Ricerche Di Biologia Molecolare P. Angeletti Spa Carcinoembryonic antigen fusions and uses thereof
EP2097456A1 (fr) * 2006-12-21 2009-09-09 Mogam Biotechnology Research Institute Proteine chimere comprenant une region fc d'immunoglobuline et un fragment kringle d'apolipoproteine (a) humaine
EP2097456A4 (fr) * 2006-12-21 2010-07-28 Mogam Biotech Res Inst Protéine chimère comprenant une région fc d'immunoglobuline et un fragment kringle d'apolipoprotéine (a) humaine
JP2010513471A (ja) * 2006-12-21 2010-04-30 モガム バイオテクノロジー リサーチ インスティチュート 免疫グロブリンＦｃ及びヒトアポリポタンパク質クリングル断片の融合タンパク質
WO2008075833A1 (fr) 2006-12-21 2008-06-26 Mogam Biotechnology Research Institute Protéine chimère comprenant une région fc d'immunoglobuline et un fragment kringle d'apolipoprotéine (a) humaine
US9388230B2 (en) 2010-09-28 2016-07-12 Kahr Medical(2005) Ltd Compositions and methods for treatment of hematological malignancies
US10000549B2 (en) 2010-09-28 2018-06-19 Kahr Medical Ltd. Compositions and methods for treatment of hematological malignancies

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