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WO1996001276A1 - Expression d'un recepteur fonctionnel de l'interferon humain de type i - Google Patents

Expression d'un recepteur fonctionnel de l'interferon humain de type i Download PDF

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Publication number
WO1996001276A1
WO1996001276A1 PCT/US1995/008456 US9508456W WO9601276A1 WO 1996001276 A1 WO1996001276 A1 WO 1996001276A1 US 9508456 W US9508456 W US 9508456W WO 9601276 A1 WO9601276 A1 WO 9601276A1
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Prior art keywords
ifn
cells
receptor
human
yac
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PCT/US1995/008456
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English (en)
Inventor
Sidney Pestka
Thomas M. Mariano
Jaemog Soh
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Rutgers State University of New Jersey
Rutgers Health
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University of Medicine and Dentistry of New Jersey
Rutgers State University of New Jersey
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • mice when mouse cells were transfected with the Hu-IFN- ⁇ Rl cDNA, they did not exhibit binding and antiviral protection with Type I IFN subtypes other than Hu-IFN- ⁇ B2; and Chinese hamster ovary (CHO-K1) cells transfected with the cloned Hu-IFN- ⁇ Rl cDNA displayed no induction of 2' -5' A synthetase in response to Hu-IFN- ⁇ A and Hu- IFN- ⁇ B2 (Revel et al , 1991). Similarly, human cells transfected with the homologous cloned Mu-IFN- ⁇ Rl receptor cDNA showed antiviral protection only with Mu-IFN- ⁇ ll.
  • [ l iJ I]IFN receptor complexes with M r of 80,000 (Hannigan et al , 1986), 210,000 (Colamonici et al , 1992), 260,000 (Vanden Broeke et al , 1988), or 300,000 (Raziuddin and Gupta, 1985) were observed in addition to the major complex which migrates as a broad band with a M r of 140,000-150,000 or sometimes as a doublet at 110,000 and 130,000 (Colamonici et al , 1992).
  • YAC clones can be used for expression and phenotypic mapping of genes after fusion of appropriate yeast spheroplasts with mammalian cells (Soh et al , 1993; Cook et al , 1994) in the absence of any specific DNA mapping or sequence information. In this way, a YAC clone was isolated which contains the gene for an accessory factor required for the function of the human interferon gamma receptor (Soh et al , 1993).
  • 2,5-A n (2'-5')-oligoadenylate: (2'-5')-oligo(adenylic) acid; oligoadenylic acid with 2', 5'-phosphodiester linkages: also abbreviated as (2'-5')-oligio(A), pppA(2'p5'A) n , pppA(2'-5')A expect, or (2'-5')p3A3; 2-5A is used as well as 2,5-p3A n .
  • BP Base pairs; usually used as lower case bp.
  • BSA Bovine serum albumin
  • DNA Deoxyribonucleic acid
  • cDNA Complementary DNA
  • dsDNA Double-stranded DNA
  • FBS Fetal bovine serum
  • PBL Peripheral blood leukocytes
  • PBMC Peripheral blood mononuclear cells
  • PBS Phosphate-buffered saline
  • PFC Plaque-forming cell
  • RNA Ribonucleic acid dsRNA: double-stranded RNA
  • VSV Vesicular stomatitis virus
  • Figure 2 illustrates the antiviral activity of interferons.
  • the data in Panel A represent the reciprocal of the IFN- ⁇ A and - ⁇ B2 titer (units/mL) for 50% protection of cells (ED50) against EMCV.
  • the data in Panel B represent the reciprocal of the IFN- ⁇ A and - ⁇ B2 titer (units/ mL) for 50% protection of cells (ED50) against VSV.
  • the data in Panel C represent the reciprocal of the IFN- ⁇ titer (units/mL) for 50% protection of cells (ED50) against EMCV.
  • the data in Panel D represent the reciprocal of the IFN- ⁇ titer (units/mL) for 50% protection of cells (ED50) against VSV.
  • transfected and YAC-fused cell lines were maintained in F-12 nutrient mixture (Sigma) containing 10% fetal calf serum (Sigma) and 450 ⁇ g/ml of antibiotic G418 (GIBCO).
  • Human x hamster hybrid cells, 153B7-8, containing human Chromosome 21q were grown in F-12D nutrient mixture (GIBCO) supplemented with 10% dialyzed fetal calf serum (Jung et al , 1990).
  • Binding data were analyzed by the method of Scatchard (19).
  • F143C3 (lane 3) were digested with EcoRI and the blot was probed with the cloned Hu-IFN- ⁇ Rl receptor cDNA.
  • the YAC 524 F143C3 does not contain any of the EcoRI fragments corresponding to the receptor gene while YACs IFNAR B49F1 and 524 F136C5 have part or all of the EcoRI fragments corresponding to the gene (Lutfalla et al , 1992), respectively (see text).
  • Molecular weight markers are shown on the right of the figure.
  • Figure IB illustrates a pulsed-field gel electrophoresis of ne ⁇ r -targeted YACs and Southern hybridization.
  • the agarose plugs from neo ⁇ Lys + transformants derived from YAC 524 F136C5 were analyzed by PFGE and the blot was probed with the labelled neo ⁇ gene fragment.
  • the YACs 524 F136C5.neo.6 (lane 2) and neo.10 (lane 4) clones contain smaller YACs than the original one whereas F136C5.neo.3 (lane 1) and neo.9 (lane 3) are of a size similar to the original YAC.
  • FIG. 2 illustrates the antiviral activity of interferons.
  • the data in Panel A represent the reciprocal of the IFN- ⁇ A and ⁇ B2 titer (units/mL) for 50% protection of cells (ED50) against EMCV.
  • the 16-9 cell line is a human x hamster hybrid containing the long arm of human Chromosome 6 and a transfected HLA-B7 gene (Soh et al , 1993).
  • ⁇ Rl denotes 16-9 cells stably transfected with the plasmid containing the IFN- ⁇ Rl cDNA;
  • ⁇ YAC denotes 16-9 cells containing YAC F136C5/neo/9.
  • the data for both Hu-IFN- ⁇ A and Hu-IFN- ⁇ B2 are shown here with the 16-9 cells. Similar data were obtained with parental CHO-K1 hamster cells.
  • the data in Panel B represent the reciprocal of the IFN- ⁇ A and IFN- ⁇ B2 titer (units/mL) for 50% protection of cells (ED50) against VSV. The experiments were performed as described in the legend to Figure 2, Panel A except that VSV was used instead of EMCV.
  • the data for both Hu-IFN- ⁇ A and Hu-IFN- ⁇ B2 are shown.
  • Panel F represent the reciprocal of the ⁇ FN-omega titer (units/mL) for 50% protection of cells (ED50) against VSV.
  • the experiments were performed as described in the legend to Figure 2, Panel B except that Hu-IFN- omega was used instead of the alpha interferons.
  • data with both CHO- Kl and 16-9 cells are shown for illustration.
  • the first three values of the histogram (left) represent parental CHO-K1 cells, CHO-K1 cells transfected with the Hu-IFN- ⁇ Rl cDNA ( ⁇ Rl) and CHO-K1 cells containing the ⁇ YAC.
  • FIG. 3 illustrates the induction of HLA-B7 surface antigen of 16-9 cells transfected with F136C5.neo.9 YAC and the cDNA for the cloned Hu-IFN- ⁇ Rl (pVADN123).
  • HLA-B7 antigen was detected by treatment of cells with mouse anti-HLA monoclonal antibody (W6/32) followed by treatment with FITC- conjugated goat anti-mouse IgG. The cells were then analyzed by cytofluorography.
  • Panels A, B, and C represent the parental 16-9 cells, panels D, E, and F subclone 5 of 16-9 cells transfected with the Hu-IFN- ⁇ Rl cDNA (16-
  • F136C5.neo.9 YAC (16-9/ ⁇ Ry9-2 cells).
  • the light lines (unfilled areas) represent cells not treated with IFN, and the dark lines (filled areas) represent cells treated with 100 units/mL of the indicated Hu-IFNs.
  • Panels A, D, and G show treatment with Hu-IFN- ⁇ A, panels B, E, and H treatment with Hu-IFN- ⁇ B2, and panels C,
  • Figure 4B illustrates the binding of [ 32 P]Hu-IFN- ⁇ B2 to the cells described in Panel A.
  • the specific cpm bound were: 16-9/ ⁇ Ry9-2, 7850 cpm; 16-9/ ⁇ Rc5, 1191 cpm; 16-9 cells, 282 cpm; 16-9/YAC-JS2 cells, 239 cpm. Binding to Daudi cells was not measured in this experiment.
  • Figure 6 illustrates the covalent cross-linking of [ 2 P]Hu-IFN- ⁇ B2 and
  • [ 32 P]Hu-IFN- ⁇ A to the receptors on cells were harvested and were incubated with [ 32 P]Hu-IFN- ⁇ B2 (A) or [ 32 P]Hu-IFN- ⁇ A (B) for 1 hour with (+) or without (-) addition of a 200-fold excess of unlabeled Hu-IFN ⁇ A and cross- linked as indicated under "Experimental Procedures.”
  • the extracted ligand: receptor complexes were mixed with sample buffer containing 10% ⁇ - mercaptoethanol and heated at 55 °C for 22 minutes. Samples were run on 7.5% SDS-polyacrylamide gels. Dried gels were autoradiographed for 10 days. The two specific cross-linked bands formed on Daudi cells are indicated with arrows.
  • Hu-IFN- ⁇ Rl is an important component of the human Type I receptor complex. Deletion of this gene from a YAC containing the fully functional receptor results in loss of biological responses to the IFNs tested. Transfection of Hu-IFN- ⁇ Rl cDNA reconstitutes these biological activities.
  • the ⁇ YAC contains all the components for a functional Type I interferon receptor complex.
  • the results in this disclosure allow us to conclude that the Hu-IFN- ⁇ Rl protein is one component of this receptor complex.
  • M ⁇ ller et al. (1994) have also concluded that the IFN- ⁇ Rl protein is required for Type I interferon receptor function.
  • YAC F136C5 contains the gene for the cloned Hu-IFN- ⁇ / ⁇ receptor (Hu-IFN- ⁇ Rl)
  • a YAC integration plasmid pJSl for the S. cerevisiae strain AB1380 commonly used for YAC library construction was described (Soh et al , 1993, 1994b). After transformation of YAC F136C5 with pJSl linearized with Clal restriction endonuclease, twelve Lys transformants were selected for further analysis. In order to confirm that the plasmid is targeted into the YAC, agarose plugs from these 12 clones were run on PFGE gel and the blot was probed with a neo ⁇ gene fragment.
  • Subclones of CHO-K1 and 16-9 cells fused to YAC F136C5.neo.9 or transfected with the Hu-IFN- ⁇ Rl cDNA were tested for class I MHC induction. Due to the relatively high endogenous background of hamster class I MHC antigens on the CHO-K1 cells, treatment of transformed CHO-K1 cells showed little or no hamster class I MHC antigen induction as detected with MAb K204 which reacts with mouse MHC class I antigens, and which also reacts with hamster MHC class I antigens on CHO-K1 cells (data not shown).
  • [ 2 P]Hu-IFN- ⁇ B2 with the cells by affinity cross-linking of the ligands to cells and by analyzing the complexes by SDS-PAGE.
  • [ 32 P]Hu-IFN- ⁇ B2 two strong bands at -130 kDa and -150 kDa are observed with the CHO-Kl/ ⁇ Ry9-4 cells, which co-migrate with the major complexes formed on human Daudi cells ( Figure 6A). These bands are not seen when excess non-radioactive Hu-IFN- ⁇ A is included in the binding reaction (lanes labeled " + "), thus demonstrating their specificity.
  • Somewhat lighter 130 and 150 kDa bands are also formed with 153B7- 8 cells containing human chromosome 21q.

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Abstract

L'invention concerne un chromosome de levure artificielle, YAC F136C5, contenant un segment du chromosome humain 21 qui, lorsqu'il est introduit dans des cellules ovariennes de hamster chinois, confère à ces dernières une réponse sensiblement améliorée à Hu-IFN-αA et Hu-IFN-αB2 ainsi qu'une réponse accrue à Hu-IFN-omega, Hu-IFN-αA/D (Bgl) et Hu-IFN-β. L'invention porte également sur un récepteur fonctionnel de l'interféron de type I humain exprimé à partir du chromosome de levure artificielle YAC F136C5.
PCT/US1995/008456 1994-07-06 1995-07-06 Expression d'un recepteur fonctionnel de l'interferon humain de type i Ceased WO1996001276A1 (fr)

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US08/271,337 1994-07-06

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF INTERFERON RESEARCH, Vol. 11 Supplement, issued 1991, REVEL et al., "Components of the Human Type I IFN Receptor System", page S61, Abstract No. 27. *
NATURE, Vol. 359, issued 01 October 1992, CHUMAKOV et al., "Continuum of Overlapping Clones Spanning the Entire Human Chromosome 21q", pages 380-387. *
PHARMACOLOGY AND THERAPEUTICS, Vol. 52, issued 1991, COLAMONICI et al., "Structure of the Human Interferon alpha Receptor", pages 227-233. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 89, issued May 1992, UZE et al., "Behavior of a Cloned Murine Interferon alpha/beta Receptor Expressed in Homospecific or Heterospecific Background", pages 4774-4778. *
PROC. NATL. ACAD. SCI. U.S.A., Vol. 90, issued September 1993, SOH et al., "Identification of a Yeast Artificial Chromosome Clone Encoding an Accessory Factor for the Human Interferon gamma Receptor: Evidence for Multiple Accessory Factors", pages 8737-8741. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2527456A1 (fr) 2004-10-22 2012-11-28 Revivicor Inc. Porcs transgéniques déficients en chaîne légère d'immunoglobuline endogène
WO2010051288A1 (fr) 2008-10-27 2010-05-06 Revivicor, Inc. Ongulés immunodéprimés

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