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WO1996001276A1 - Expression of a functional human type i interferon receptor - Google Patents

Expression of a functional human type i interferon receptor 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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ifn
cells
receptor
human
yac
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PCT/US1995/008456
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French (fr)
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Sidney Pestka
Thomas M. Mariano
Jaemog Soh
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University Of Medicine & Dentistry Of New Jersey
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    • 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

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  • 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

This invention relates to a yeast artificial chromosome, YAC F136C5, containing a segment of human Chromosome 21 which when introduced into Chinese hamster ovary (CHO) cells confers upon these cells a greatly enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Hu-IFN-omega, Hu-IFN-αA/D (Bgl), and Hu-IFN-β. This invention also relates to a functional human type I interferon receptor expressed from the yeast artificial chromosome, YAC F136C5.

Description

EXPRESSION OF A FUNCTIONAL HUMAN TYPE I INTERFERON RECEPTOR
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of application serial no. 08/110,119, filed 20 August 1993.
Statement Of Government Interest
This invention was made with government support under United
States Public Health Services Grant ROl CA46465 from the National Cancer Institute. The Government has certain rights in this invention.
1. Field Of The Invention
This invention relates to a yeast artificial chromosome, YAC F136C5, containing a segment of human Chromosome 21 which when introduced into Chinese hamster ovary (CHO) cells confers upon these cells a greatly- enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Hu-IFN-ømegα, Hu-IFN-αA/D (Bgl), and Hu-IFN-β. This invention also relates to a functional human type I interferon receptor expressed from the yeast artificial chromosome, YAC F136C5. 2. Description of the Background
The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference and, for convenience, are referenced in the following text and respectively grouped in the appended bibliography.
Expression of a Functional Human Type I Interferon Receptor
Type I human interferons (the multiple Hu-IFN-α species, one Hu- IFN-β, and one Uu-IFN-omega) are a family of cytokines that induce a variety of physiological responses. These effects include antiviral and antiproliferative activities, stimulation of cytotoxic activity in lymphocytes, natural killer cells and macrophages, modulation of cellular differentiation, and stimulation of class I MHC antigens and other surface markers (Lengyel, 1982; Pestka et al. 1987). Like most cytokines and growth factors, the actions of Type I IFNs are mediated by interaction with specific cell-surface receptors (Friedman, 1967; Aguet et al. 1980). Competition binding studies demonstrated that Type I IFNs share the same receptor complex, whereas Type II IFN (ΪFN-gamma) binds to a distinct receptor (Branca and Baglioni, 1981; Pestka et al , 1987; Flores et al , 1991). Cloning of a human IFN-α/β receptor (IFN-αRl) cDNA was reported on the basis of rendering mouse cells sensitive to Hu-IFN-αB2 (Uzέ et al , 1990). However, when mouse cells were transfected with the Hu-IFN-αRl cDNA, they did not exhibit binding and antiviral protection with Type I IFN subtypes other than Hu-IFN-αB2; and Chinese hamster ovary (CHO-K1) cells transfected with the cloned Hu-IFN-αRl cDNA displayed no induction of 2' -5' A synthetase in response to Hu-IFN-αA and Hu- IFN-αB2 (Revel et al , 1991). Similarly, human cells transfected with the homologous cloned Mu-IFN-αRl receptor cDNA showed antiviral protection only with Mu-IFN-αll. However, the expression of this Mu-IFN-αRl cDNA in murine cells (L1210 R101; cells resistant to type I IFNs) lacking mRNA for this Mu-IFN- αRl receptor component showed antiviral protection in response to all Type I Mu- IFNs tested (Uze et al. , 1992).
I OC
In affinity cross-linking experiments, [l iJI]IFN: receptor complexes with Mr 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 Mr of 140,000-150,000 or sometimes as a doublet at 110,000 and 130,000 (Colamonici et al , 1992). These observations suggest that other subunits, components or accessory proteins are involved in ligand binding and subsequent signal transduction of all Type I IFNs (reviewed by Colamonici and Pfeffer, 1991; Mariano et al. , 1992), as described for the ΪFN-gamma receptor (Jung et al. , 1987; Cook et al , 1992, 1994; Soh et al , 1993, 1994a). Bazan (1990a, 1990b) proposed that the IFN receptors as well as other cytokine receptors of the same superfamily are composed of two folding domains that comprise the ligand binding site which, at least in some cases, resides in the crevice between the folds. The primary cytokine-receptor interaction was suggested to involve one face of the ligand while another face of the bound cytokine can interact with accessory binding components.
Somatic cell genetic studies with human x rodent hybrid cells containing various combinations of human chromosomes have provided evidence that the presence of human Chromosome 21 confers sensitivity of the rodent cells to various human Type I IFNs (Tan et al , 1973; Slate et al , 1978; Epstein et al , 1982; Raziuddin et al , 1984). It was also demonstrated that antibodies to human Chromosome 21 -encoded cell-surface components were able to block the action or binding of Hu-IFN-α to cells (Revel et al , 1976; Shulman et al , 1984). Furthermore, Langer et al (1990) demonstrated that 3xlS irradiation-reduced hamster x human somatic hybrid cells containing about 3 mb of Chromosome 21q around 21q22.1 (Jung, 1991; Soh et al , 1994a) were able to bind [32P]Hu-IFN-αA and generate a complex of about 150 kDa when cross-linked to the cell surface. In addition, the cloned Hu-IFN-αRl receptor gene was mapped to the 3xlS region (21q22.1) (Lutfalla et al , 1990). The paradoxical observation that CHO 3xlS cells could bind Hu-IFN-αA whereas the expression of the cloned receptor cDNA in mouse cells did not confer binding to Hu-IFN-αA and expression in CHO cells did not induce 2,5'-A synthetase activity in response to Hu-IFN-αA suggested that the 3xlS region of human Chromosome 21 contains other subunits of the receptor complex (Langer et al. , 1990; Revel et al. , 1991). This assumption was supported by the identification of two separate components following immunoprecipitation of [12 I]Hu-IFN-αA:receptor complexes from 3xlS cell extracts with anti-IFN-α receptor antibody (Colamonici and Domanski, 1993; Colamonici et al , 1990, 1992). These two subunits seem to differ from the cloned Hu-IFN-αRl. The monoclonal antibodies (MAb) against one subunit (110 kDa) and the recombinant Hu-IFN-αRl receptor block the biological activity of Type I interferons while MAb against the second subunit does not (Colamonici and Domanski, 1993; Benoit et al. 1993), suggesting that the Type I IFN receptor consists of at least three different subunits. In addition, antibodies to the Hu-IFN-αRl block the activity of various Type I interferons on human cells (Uze et al. , 1991 ; Benoit et al. , 1993). All these observations indicate that the Hu-IFN-αRl molecule is only one component of the Type I receptor.
A 1.5kb cDNA, coding for another IFN-α/β receptor component, has been isolated and expressed. Its 331 amino acid sequence includes a leader and a transmembrane region and the ectodomain corresponds to p40, a soluble form found in urine (Novick et al. , 1994).
Yeast artificial chromosome (YAC) cloning techniques allow the cloning of DNA fragments up to 2000 kb (Burke et al. , 1987; Chumakov et al. , 1992a). The large insert size can facilitate not only physical mapping of chromosomes (Chumakov et al , 1992b), but also permits expression of very large genes not possible by conventional cloning procedures and regions of chromosomes containing multiple genes (Soh et al , 1993, 1994b; Cook et al , 1994). It has been demonstrated that 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).
Knockout and Reconstitution of a Functional Human Type I Interferon Receptor Complex
The full complement of the components of the human Type I interferon receptor have been sought for several years. In 1986, a series of genomic clones were isolated after transfection of mouse NIH3T3 cells with total human DNA that responded to Type I interferons (human interferon alpha, Hu-IFN-α; human interferon beta, Hu-IFN-β); yet a specific cosmid or cDNA clone was not isolated at that time (Jung and Pestka, 1986; Jung, 1991). Using a similar procedure, Uzέ et al. (1990) reported the cloning of a cDNA they considered to encode the human interferon alpha receptor. However, this cDNA clone (designated Hu-IFN-αRl here) exhibited activity in response only to Hu-IFN-αB2 (=Hu-IFN-α8).
Results with antibodies to Hu-IFN-αRl and other Type I interferon receptor components have suggested that Hu-IFN-αRl is one of two or more components that comprise the functional Hu-IFN-α receptor complex. This conclusion was supported by the identification of two separate components following chemical cross-linking and/or immunoprecipitation of [125I]Hu-IFN- αA: receptor complexes with an anti-IFN-α receptor antibody (Raziuddin and Gupta, 1985; Colamonici and Pfeffer, 1991; Colamonici and Domanski, 1993; Colamonici et al , 1990, 1992). These two components seem to differ from the cloned Hu-IFN-αRl. The monoclonal antibodies (MAb) against one subunit (110 kDa) and the recombinant Hu-IFN-αRl block the biological activity of Type I interferons while a MAb against the second subunit (210 kDa) does not (Uzέ et al. , 1991; Colamonici and Domanski, 1993; Benoit et al. 1993), suggesting that the Type I IFN receptor consists of at least three different subunits. Furthermore, functional differences among the Hu-IFN-α species have been described in a variety of assays and have suggested the existence of multiple components in the specific ligand-receptor interactions (Evinger et al , 1981; Rehberg et al , 1982; Ortaldo et al , 1983; 1984; Pestka, 1983, 1984; Pestka et al , 1987; Hu et al , 1993). All these observations suggested that the Hu-IFN-αR l molecule is one component of the Type I receptor complex.
Throughout the specification and claims, the following abbreviations will stand for the words and phrases set out below.
A, Ala; C, Cys; D, Asp; E, Glu; F, Phe, G, Gly; H, His; I, He; K, Lys; L,
Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
2,5-An: (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„, or (2'-5')p3A3; 2-5A is used as well as 2,5-p3An.
A26O unit: The quantity of material that yields an absorbance of 1.0 when measured at 260nm in a cuvette with a path length of 1.0 cm. Analogously, Agoo represents measurements at 600 nm, A550 at 550 nm, etc. This quantity of material is also sometimes designated as OD26O' OD550, OD600, etc. Act D: Actinomycin D
BES: N,N-bis[2-Hydroxyl]-2-aminoethanesulfonic acid; 2-[bis(2- hydroxethyl)amino]-ethanesulfonic acid
BP: Base pairs; usually used as lower case bp.
Bq: Becquerel; 1 Bq = 1 dps or 60dpm; 1 Ci = 3.7 x 1010Bq; TBq = 1012Bq
BSA: Bovine serum albumin
Con A: Concanavalin A
CPD: Citrate phosphate dextrose solution
CPE: Cytopathic effect DEAE Cellulose: Diethylaminoethylcellulose
DHFR: Dihydrofolate reductase
DNA: Deoxyribonucleic acid cDNA: Complementary DNA dsDNA: Double-stranded DNA
DRB : 5 , 6-Dichloro- 1 -beta-D-ήbof uranosy 1 benzimidazole dsRNA: Double-stranded RNA
DTT: Dithiothreitol
EDTA: Ethylenediaminetetraacetic acid EGTA: Ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid
EID50: Egg infectious dose; concentration at which half the eggs are infected.
EMCV: Encephalomyocarditis virus
EMEM: Eagle's minimal essential medium ESS: Earle's salt solution
FBS: Fetal bovine serum
FCS: Fetal calf serum
GPT: Guanosine phosphoribosyltransferase (recommended name is guanosine phosphorylase; systematic name is guanosine: orthophosphate ribosyltransferase
HBSS: Hanks' balanced salt solution
HEPES: N-2-Hydroxethylpiperazine-N'-2-ethanesulfonic acid
IU: Units of interferon with respect to the appropriate international reference standard. KBP: Kilobase pairs; usually used as lower case kbp.
MDMP: 2-(4-Methyl-2, 6-dinitroanilino)-N-methyl-propionamide
MEM: Minimal essential medium (EMEM, unless otherwise noted).
Mg(OAc)2: Magnesium acetate
MOI: Multiplicity of infection NAD: Nicotinamide adenine dinucleotide
NaOAc: Sodium acetate
Natural interferon: This term refers to interferon produced by animal cells after induction in contrast to interferon produced by recombinant DNA technology. In some cases the two (i.e. , the interferon produced by animal cells and the same species by recombinant DNA technology) may be chemically identical.
NDV: Newcastle disease virus
PBL: Peripheral blood leukocytes
PBMC: Peripheral blood mononuclear cells
PBS: Phosphate-buffered saline PFC: Plaque-forming cell
PFU: Plaque-forming unit
PHA: Phytohemagglutinin
Poly (I) . poly(C): Polyinosinic acid . polycytidylic acid, double stranded synthetic homopolymers [alternatively poly(rl) . poly(rC)]
RNA: Ribonucleic acid dsRNA: double-stranded RNA
SDS: Sodium dodecyl sulfate
SDS-Page: SDS-Polyacrylamide gel electrophoresis SEA: Staphylococcal enterotoxin A
SEB: Staphylococcal enterotoxin B
SSC: Standard saline citrate solution, 0.15 M NaCl/0.015 M sodium citrate, adjusted to pH 7.0 with NaOH.
TCA: Trichloroacetic acid TCID50: Tissue culture infectious dose; concentration at which half the cultures are infected.
TEMED: N,N,N' ,N'-Tetramethylethylenediamine
TES: N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid
Tricine: N-Tris(hydroxymethyl)methylglycine TPA: The phorbol ester 12-0-tetradecanoylphorbol-13-acetate.
Tris: Tris(hydroxymethyl)aminomethane
VSV: Vesicular stomatitis virus
Other Abbreviations: YAC, yeast artificial chromosome; PFGE, pulsed-field gel electrophoresis; Hu-ϊFN-gamma, human interferon gamma; HLA, human leucocyte antigen; CHO, Chinese hamster ovary; GART, phosphoribosylglycinamide formyltransferase; EMCV, encephalomyocarditis virus;
VSV, vesicular stomatitis virus.
BRIEF DESCRIPTION OF THE FIGURES
Figure IA illustrates a Southern Hybridization of YAC DNA probed with labeled Hu-IFN-αRl/cDNA. Figure IB illustrates a pulsed-field gel electrophoresis of neør-targeted YACs and Southern hybridization.
Figure 2 illustrates the antiviral activity of interferons. The data in Panel A represent the reciprocal of the IFN-αA and -αB2 titer (units/mL) for 50% protection of cells (ED50) against EMCV. The data in Panel B represent the reciprocal of the IFN-αA and -αB2 titer (units/ mL) for 50% protection of cells (ED50) against VSV. The data in Panel C represent the reciprocal of the IFN-β titer (units/mL) for 50% protection of cells (ED50) against EMCV. The data in Panel D represent the reciprocal of the IFN-β titer (units/mL) for 50% protection of cells (ED50) against VSV. The data in Panel E represent the reciprocal of the IFN- omega titer (units/mL) for 50% protection of cells (ED50) against EMCV. The data in Panel F represent the reciprocal of the ϊFN-omega titer (units/mL) for 50% protection of cells (ED50) against VSV.
Figure 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- αRl (pVADN123).
Figure 4 illustrates the binding of[32P] Hu-IFN-αA and [32P]Hu-IFN- αB2 to Cells. Figure 4A illustrates the binding of [32P]Hu-IFN-αA-Pl to various cells. Figure 4B illustrates the binding of [ 2P]Hu-IFN-αB2 to the cells described in Panel A.
Figure 5 illustrates the competition of unlabeled Hu-IFN-αA, Hu-IFN- αB2, Hu-IFN-β and Ru-ΪFN-omega with [ 2P]Hu-IFN-αA.
Figure 6 illustrates the covalent cross-linking of [ P]Hu-IFN-αB2 and [32P]Hu-IFN-αA to the Receptors on Cells.
Figure 7 is a map of the Hu-IFN-αRl gene showing the location of the insertion of pJSl.
Figure 8 illustrates the induction of Class I MHC cell surface antigen expression by interferons.
Figure 9 illustrates the antiviral activity of Hu-IFN-αA.
Figure 10 illustrates the antiviral activity of Hu-IFN-αB2. SUMMARY OF THE INVENTION
This invention relates to a yeast artificial chromosome, YAC F136C5, containing a segment of human Chromosome 21 which when introduced into Chinese hamster ovary (CHO) cells confers upon these cells a greatly- enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Hu-IFN-ømegα, Hu-IFN-αA/D (Bgl), and Hu-IFN-β. This invention also relates to a functional human type I interferon receptor expressed from the yeast artificial chromosome, YAC F136C5.
DETAILED DESCRIPTION OF THE INVENTION
Expression of a Functional Human Type I Interferon Receptor
The previously-cloned human interferon α/β (Hu-IFN-α/β; Type I interferon) receptor cDNA appears to be only one component of a receptor complex since expression of the cDNA in mouse cells confers sensitivity only to Hu-IFN- αB2, but a monoclonal antibody against this cloned receptor subunit inhibits biological activities of Hu-IFN-αA, Hu-IFN-αB2, Hu-WN-omega, and Hu-IFN-β. Here we report that a yeast artificial chromosome (YAC) containing a segment of human Chromosome 21 introduced into Chinese hamster ovary (CHO) cells confers upon these cells greatly-enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Uu-IFN-omega , Hu-IFN-αA/D (Bgl), and Hu-IFN-β. These responses were measured by induction of class I MHC antigens and by protection against encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV). Furthermore, these cells exhibit specific high-affinity binding of Hu- IFN-αA and Hu-IFN-αB2, Hu-IFN-β and Hu-ΪFN-omega. The results indicate that all the genes necessary to reconstitute a biologically-active Type I human IFN receptor complex are located within the human DNA insert of this YAC clone.
Knockout and Reconstitution of a Functional Human Type I Interferon Receptor Complex
By functional screening of yeast artificial chromosomes (YACs) (Soh et al , 1993, 1994a,b; Cook et al , 1993, 1994) similar to that which led to the cloning of the accessory chain of the Hu-\FN- gam ma receptor (accessory factor- 1, AF-1; a β-like receptor chain), we were able to isolate a functional YAC clone that, when expressed in hamster cells, exhibited the full complement of Type I interferon receptor activity in response to all Type I interferons tested (Soh et al. , 1994c). Because this YAC clone (αYAC or αRy9A-2 in figures) contained the gene for Hu- IFN-αRl (Uzέ et al , 1990; Lutfalla et al , 1992; Mariano et al , 1992), we were able to determine if the Hu-IFN-αRl, indeed, is required for Type I interferon receptor activity. To accomplish this, we used homologous recombination into the αYAC with a vector containing human repetitive DNA Alu sequences to delete regions between Alu sequences (Soh et al , 1993, 1994b) and provide a knockout of all or part of the Hu-IFN-αRl gene. A clone was obtained (delta αYAC, noted as αRylOA) in which exon II of the Hu-IFN-αRl gene was deleted (Figure 7).
We then determined the MHC Class I antigen induction and antiviral activity in hamster cells containing this delta αYAC in response to human Type I interferons. This deletion effectively eliminates the MHC Class I antigen induction and antiviral activity previously reported for this fully functional parental YAC clone (Soh et al , 1994c). We have successfully reconstituted this activity by expression of the cDNA encoding the Hu-IFN-αRl component (Uzέ et al , 1990) in cells containing the YAC with this deletion. The Hu-IFN-αRl subunit thus plays a critical role in the functional human Type I IFN receptor complex, whose components are encoded on this YAC. In addition, as binding of ligands is retained in the cells containing the YAC with the deletion, it is clear a second subunit encoded on the YAC is responsible for ligand binding activity. This system will now allow the identification of additional subunits involved in the response to the Type I IFNs and the functional significance of each.
The present invention is further illustrated by the following examples which are not intended to limit the effective scope of the claims. All parts and percentages in the examples and throughout the specification and claims are by weight of the final composition unless otherwise specified.
EXPERIMENTAL PROCEDURES
Expression of a Functional Human Type I Interferon Receptor
Cells and Media
Three YAC clones, F136C5 and F143C3 (from Drs. Chumakov and Cohen, Centre d'Etude du Polymorphisme Humain, Paris, France; screened by probe 524-5P) and B49F1 (Washington University Library; screened by probe IFNAR), were obtained through the Chromosome 21 Joint YAC Screening Effort (directed by Dr. David Patterson, Eleanor Roosevelt Institute, Denver, CO. USA). All YACs were derived from Saccharomyces cerevisiae AB1380 (MATa, ura3, trpl, ade2-l, canl-100, lys2-l and his5) and grown in AHC media. YACs F136C5.neo.3 and F136C5.neo.9 are derivatives of YAC F136C5 that contained an integrated neomycin phosphotransferase gene (neo) as described (Soh et al , 1993, 1994b). Chinese hamster ovary Kl (CHO-K1) cells were obtained from the American Type Culture Collection. 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). CHO-Kl/αRc4 and 16-9/αRc5 cell lines were obtained by transfecting pVADN123 (from Gilles Uze), a plasmid containing the Hu-IFN-αRl cDNA, into CHO-K1 and 16-9 cells, respectively. CHO-Kl/αRy9-4 and 16- 9/αRy9-2 cell lines were obtained by fusing the spheroplasts from YAC F136C5.neo.9 with CHO-K1 and 16-9 cells, respectively. These transfected and YAC-fused cell lines were maintained in F-12 nutrient mixture (Sigma) containing 10% fetal calf serum (Sigma) and 450 μg/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).
Construction of YACs Containing the Antibiotic G418 Resistance Marker
Transformation of yeast cells was carried out as described (Rose et al. 1990) with the following modifications. Fifty milliliters of each YAC clone were grown to an absorbance of 1.0 unit/ml at 600 nm in AHC liquid media, harvested, and suspended in 5 mL of 1 M sorbitol. Fifty microliters of 10 mg/ml Zymolyase 20T (ICN) were added to the cell suspension, which was then incubated at 37°C for 15-30 minutes to obtain greater than 95% conversion to spheroplasts. The spheroplasts were washed three times with 1 M sorbitol, collected by centrifugation (500 x g, 5 minutes) and resuspended in STC medium (1 M sorbitol, 10 mM Tris/Cl pH 7.5, 10 mM CaCl2) at a density of 5 x 108 spheroplasts/mL. Five micrograms of linearized plasmid pJSl (Soh et al , 1993, 1994b) and 10 μg of salmon testes DNA as a carrier were put into 100 μL of the spheroplast suspension in a polypropylene tube, and the mixture incubated for 5 minutes at room temperature. After addition of 4 mL of polyethylene glycol (PEG) solution (20% w/v PEG 8000, 10 mM CaCl2, 10 mM Tris/Cl, pH 7.5), the mixture was incubated for 10 minutes at room temperature, then centrifuged (500 x g, 5 minutes) to pellet the spheroplasts. The spheroplasts were resuspended in 300 μL of SOS medium (1 M sorbitol, 5 mM CaCl2 in YPD medium consisting of 10 g of yeast extract, 20 g of peptone, and 20 g of glucose per liter), and incubated for 20 minutes at 30°C with shaking. Finally, the spheroplasts were mixed with 6 mL of top agar and the mixture was overlaid on a synthetic dextrose minimal medium plate supplemented with adenine sulfate (10 μg/ml) and histidine (20 μg/ml) to select Lys+ transformants.
Electrophoresis and Hybridization Procedures
Agarose plugs were prepared as described (Smith et al , 1987). Yeast cells were grown to stationary phase at 30°C in 50 mL of AHC media, washed twice with 25 mL of 50 mM EDTA (pH 8.0) by centrifugation at 3500 x g for 5 minutes, followed by resuspension to a density of 1 x 1010 cells/ml in 50 mM EDTA. An equal volume of 1 % (w/v) Insert agarose (FMC BioProducts) in 50 mM EDTA (pH 8.0) and 20 μL of 10 g/mL Zymolyase 20T per milliliter of agarose solution were added to the cell suspension and the mixture was put into the plug mould. Spheroplasts were made by pushing plugs out of the mould into 1 mL of 0.5 M EDTA (pH 8.0) containing 7.5% β-mercaptoethanol per plug and incubating overnight at 37°C with gentle shaking. The solution was removed and the plugs were rinsed twice with 50 mM EDTA (pH 9.25). The spheroplasts were lysed by transferring to Solution ESP [0.5 M of EDTA (pH 9.25), 1 % Sarkosyl (IBI), and 1 mg/mL proteinase K (Sigma)] and incubating for 24 hours at 50°C. The agarose plugs were analyzed by pulsed-field gel electrophoresis (PFGE) (CHEF, OWL Scientific Plastics, Ine). The gels measured 13 cm x 13 cm, consisted of 120 mL of 1 % agarose in TBE (0.045 M Tris/borate, pH 8.0, 2.5 mM EDTA), and were run at 170 V for 24 hours at 15°C with a pulse time of 70 seconds. The DNAs were blotted onto Nytran (Schleicher & Schuell) by standard Southern blot procedures. DNA probes were labeled with [α-32P]dCTP (NEN, 3000 Ci/mmol) with random hexadeoxynucleotides as primers (Feinberg and Vogelstein, 1983).
Fusion and Transfection
The spheroplasts from YACs F136C5.neo.3 and F136C5.neo.9 were fused to CHO-K1 and 16-9 cells by procedures already described (Pavan et al , 1990; Soh et al. , 1994b). Thirty-five milliliters of each clone were grown in AHC media to stationary phase. After being washed twice with 20 mL of 1 M sorbitol, cells were resuspended in 5 mL of a solution which contained 1 M sorbitol, 100 mM sodium citrate (pH 5.8), 10 mM EDTA, and 30 mM β-mercaptoethanol. Eighty microliters of a Zymolyase 20T stock solution (10 mg/mL) were added, and the mixture was incubated at 37°C for 20 minutes until 95% of the cells were spheroplasts. The spheroplasts were pelleted and washed twice in 5 mL of a solution of 1 M sorbitol in 10 mM Tris/Cl (pH 7.5), resuspended in 5 mL of the same solution, and counted. While the spheroplasts were being washed, the CHO- Kl or 16-9 cells were harvested by trypsinization, resuspended in serum-free F-12 medium, and counted. Aliquots containing 4 x 10' spheroplasts were placed in 15- mL tubes and centrifuged at 500 x g for 5 minutes. The supernatant was removed from the spheroplast pellet and 5 ml of the cell suspension containing 2 x 10° mammalian cells was added carefully. The cells were then centrifuged at 500 x g for 5 minutes. The supernatant was removed and 50 μL of serum-free F12 medium was added to resuspend the pellets. Five hundred microliters of 45% PEG 1500 solution (Boehringer Mannheim Biochemicals) containing 5% DMSO, 10 μM β- mercaptoethanol, and 5 mM CaCl2 were added to the mixed cell suspension. The cells were mixed by tapping the tube briefly, incubated for 2 minutes at room temperature, and diluted with 5 mL of serum-free F12 medium. This cell suspension was left at room temperature for 20 minutes, then centrifuged at 600 x g for 6 minutes. The resulting pellet was resuspended in 50 mL of complete F12 medium (F12 plus 10% heat-inactivated fetal bovine serum) and plated at 10" mammalian cells per 150 mm plate. Thirty-six hours later, plates were washed with phosphate-buffered saline (PBS) to remove dead cells and yeast, then refed with complete F12 medium containing 450 μg/mL of antibiotic G418. Cells were fed as necessary, and resistant colonies typically appeared after 10 to 14 days. The G418-resistant colonies were pooled and expanded for further analysis. After initial assay of pools of colonies, five subclones from each pool were obtained by the limiting dilution technique. CHO-K1 and 16-9 cells were also transfected with Hu- IFN-αRl cDNA clone pVADN123, as described (Hibino et al , 1992). Five individual G418-resistant colonies from each transfection were tested for various functions and binding activity.
Cytofluorographic Analysis of Cells for Expression of Class I MHC Surface
Antigens
Cells were seeded in 24- well plates at a density of about 25,000 cells/well (1 mL/well) and were treated with the indicated concentrations and types of IFN for about 72 hours by which time the cells were nearly confluent. Cells were trypsinized, transferred to 1.5 mL tubes, and washed with complete F12 medium. HLA-B7 antigens on 16-9 cells were detected by incubating the cells with 15 μL of culture supernatant from the hybridoma line producing mouse monoclonal anti-HLA antibody W6/32 (Jung et al , 1988; Hibino et al , 1992) for 30 minutes at 4°C. Cells were washed with complete medium and resuspended in 15 μL of fluorescein isothiocyanate-conjugated (FITC -conjugated) goat anti-mouse IgG (Cappel) diluted to 80 μg/mL and incubated for 30 minutes at 4°C, after which they were washed with complete medium and resuspended in 200 μL of cold complete medium for the immediate analysis of live cells. If cells had to be fixed for future analysis, they were washed twice with PBS, resuspended in 15 μL of 3% (w/v) paraformaldehyde in PBS, and incubated from 1 to 16 hours at 4°C. The fixed cells were washed with PBS and finally resuspended in 200 μL PBS. Samples were analyzed on a Coulter Epics Profile Cytofluorograph. For each analysis, 10,000 events were accumulated and analyzed on CytoLogic software.
Interferons and Antiviral Assay
Hu-IFN-αA, Hu-IFN-αA/D, and Hu-IFN-β were prepared as previously reported (Staehelin et al , 1981 ; Rehberg et al , 1982; Moschera et al , 1986) and Ηxx-ΪFN-omega was a gift from Dr. G. Bodo (Ernst-Boehringer Institute fur Arzneimittelforschung, Austria). Hu-IFN-αA-Pl and Hu-IFN-αB2-P were prepared as described (Li et al , 1989; Wang et al , 1994). Hu-IFN-β activity was measured by a cytopathic-effect inhibition assay on human WISH cells with vesicular stomatitis virus (VSV); the activity of all other Type I human interferons was measured on bovine MDBK cells with VSV (Familletti et al , 1981). Parental and transfected CHO-K1 and 16-9 cells were also assayed for resistance to encephalomyocarditis virus (EMCV) or VSV infection by a cytopathic-effect inhibition assay (Familletti et al , 1981).
Genetically-engineered phosphorylatable Hu-IFN-αA (Hu-IFN- A-Pl) and Hu-IFN-αB2 (Hu-IFN-αB2-P) were phosphorylated with the catalytic subunit of bovine heart cAMP-dependent protein kinase and [α- P]ATP as described (Li et al. , 1989; Wang et al. , 1994). The specific activity of the labeled IFN was 3-5 x 10" cpm/pmole at the time used.
IFN Binding
For binding studies, cells were treated with trypsin and collected from
75 cm^ tissue culture flasks. Binding of IFNs to cells was performed in a volume of 0.1 ml containing 0.5- 1 x 10" cells as noted in the appropriate legends to the figures and tables. The [J ,P]IFN bound to cells was separated from the unbound [ 2P]IFN by sedimentation through a cushion of 10% sucrose in PBS (Langer et al , 1986; Langer and Pestka, 1986). Nonspecific binding of [32P]Hu-IFN-αA-Pl and [ 2P]Hu-IFN-αB2-P was determined by the addition of a 200-fold excess of unlabeled recombinant Hu-IFN-αA. The nonspecific binding was subtracted from the total radioactivity in each case to yield the cpm bound specifically.
For competition studies, cells were collected from tissue culture flasks and resuspended at 2 x 10 cells/ml for Daudi cells, and 8 x 10" cells/ml for CHO-
Kl/αRy9-4 and 16-9/αRy9-2 cells. To 100 μL of cells was added 10 μL of non- radioactive Hu-IFN-αs to produce final concentrations in the range of 2 x \QT 1l9Δ M to 4 x 10"8 M, and 1 μL [32P]Hu-IFN-αA-Pl (final concentration, 1 x 10"10 M;
0.8 x l(r cpm). After incubation at room temperature for 1 hour with intermittent shaking, bound [ 2P]Hu-IFN-αA-Pl was measured as described above.
Covalent Cross-linking of [32P]Hu-IFN-αA-Pl and [32P]Hu-IFN-αB2-P To Cell-Surface Receptors
Cells were harvested, pelleted and resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum at a density of 1 x 10 cells/ml. Cells were incubated with 5 x 10^ cpm/ml of [32P]Hu-IFN-αA-Pl or [ 2P]Hu-IFN-αB2-P at room temperature for 1 hour in a volume of 0.2 ml with or without a 200-fold excess of unlabeled IFN-αA. The cells were washed and resuspended in 0.1 ml of cold PBS (pH adjusted to 8.0 with 1 M potassium borate) and the chemical cross-linker disuccinimidyl suberate (Pierce) in dimethyl sulfoxide was added to a final concentration of 500 μM (Rashidbaigi et al , 1985; Langer et al , 1986). After incubation on ice for 30 minutes, 50 mM Tris/HCl (pH 8.0) was added to quench the reaction for 5 minutes. The cells were washed with ice-cold PBS and pelleted. [J- P]IFN: receptor complexes were extracted with 0.1 ml of 1 % (v/v) Triton X-100 in PBS containing protease inhibitors (Ronnett et al , 1984). The detergent extracts were then analyzed on 7.5% polyacrylamide gels in the presence of SDS (Laemmli, 1970). Gels were dried under vacuum and autoradiographed . Table 1
Interferon Concentrations for 50% Protection (ED50) of Cells from EMCV Infection
Cell Line
Hu-IFN CHO-K1 CHO-Kl/αRc4 CHO-Kl/αRy9-4 16-9 16-9/αRc5 16-9/αRy9-2
A/D 88 88 44 625 442 28 αA 442 313 2 2500 1768 28 ά&2 >10,000 >10,000 111 >10,000 7072 55 omega 177 177 44 >2000 >2000 707 β 11 11 8 55 55 <5
The values Table 1 represent the IFN titer (units/mL) for 50% protection of cells (ED50). Hu-IFN-α A/O(Bgl) is a chimeric Hu-IFN-α that is active on CHO and most mammalian species tested (Rehberg et a , 1982) and is used as a positive control. The two sets of cell lines (i.e. , derived from CHO-K1 and from 16-9 cells) were not assayed at the same time so the precise endpoints between the two sets are not directly comparable. However, each set of cells was assayed at one time.
Table 2
Interferon Concentrations for 50% Protection (ED50) of Cells from VSV
Infection
Cell Line
Hu-IFN CHO-K1 CHO-Kl/αRc4 CHO-Kl/αRy9-4 16-9 16-9/αRc5 16-9/αRy9-2
αA/D 157 111 39 884 442 55 αA 884 625 156 5000 3536 78 αB2 > 10,000 > 10,000 78 > 10,000 > 10,000 78 omega 1414 1414 177 >2000 >2000 500 β 10 10 <5 111 55 4
The values in the table represent the IFN titer (units/mL) for 50% protection of cells (ED50). The two sets of cell lines were assayed at the same time. See legend to Table 1 for additional details.
Table 3
Induction of HLA B7 Antigen by Human Type I IFNs
IFN Cell Line
Units/mL Type 16-9 16-9/αRc5 16-9/αRy9-2
0 None 1.0 1.0 1.0
1 αA 1.0 0.9 2.0
3 αA 0.8 0.7 1.4
10 αA 0.8 1.0 2.2
30 αA 0.9 0.8 2.0
100 αA 1.0 0.9 2.6
1 αB2 0.9 0.8 1.4
3 αB2 0.8 0.8 2.1
10 αB2 0.8 0.8 1.7
30 αB2 0.6 1.0 2.5
100 αB2 0.9 0.6 2.8
1 omega 1.2 1.0 1.7
3 omega 1.0 1.2 3.0
10 omega 0.9 1.1 3.0
30 omega 0.7 1.3 3.6
100 omega 0.8 1 -4 4.3
1 β 0.9 1.1 1.8
3 β 1.0 0.8 1.9
10 β 1.0 1.0 2.0
30 β 1.0 1.2 2.4
100 β 1.6 1.5 2.0
The values in the table represent the average fluorescence displayed by the treated cells divided by the average fluorescence displayed by untreated cells, which were defined as having a relative average fluorescence of 1.0. The assays were all performed at the same time except for the results with Hu-IFN-ømegα which were performed in a separate experiment. Table 4
Summary of Comparison of [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2 Binding to CHO-Kl/αRy9-4 and Daudi Cells
Hu-IFN-αA Hu-IFN-αB2
Cell Line Kd (M) Binding Sites/Cell Kj (M) Binding Sites/Cell
CHO-Kl/αRy9-4 l.l x lO"9 2.3 x lO3 3.6 x 10"10 1.7 x 103
Daudi 2.1 x lO"10 2.3 x lO4 1.1 x lO"10 6.2 x lO3
Binding data were analyzed by the method of Scatchard (19).
Figure IA illustrates a Southern hybridization of YAC DNA. Yeast chromosomal DNAs from IFNAR B49F1 (lane 1), 524 F136C5 (lane 2), and 524
F143C3 (lane 3) were digested with EcoRI and the blot was probed with the cloned Hu-IFN-αRl 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 neør-targeted YACs and Southern hybridization. The agarose plugs from neoτ Lys+ transformants derived from YAC 524 F136C5 were analyzed by PFGE and the blot was probed with the labelled neoτ 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.
Figure 2 illustrates the antiviral activity of interferons. The data in Panel A represent the reciprocal of the IFN-αA and αB2 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). "αRl" denotes 16-9 cells stably transfected with the plasmid containing the IFN-αRl cDNA; "αYAC" denotes 16-9 cells containing YAC F136C5/neo/9.
The data for both Hu-IFN-αA and Hu-IFN-αB2 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-αA and IFN-αB2 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-αA and Hu-IFN-αB2 are shown.
The data in Panel C represent the reciprocal of the IFN-β titer (units/ mL) for 50% protection of cells (ED50) against EMCV. The experiments were performed as described in the legend to Figure 2, Panel A except that Hu- IFN-β was used instead of the alpha interferons. The value for the (ED50) for the cells containing the αYAC is > 0.20 shown in the figure as maximal protection was obtained at 5 units/ml of Hu-IFN-β, the lowest Hu-IFN-β concentration tested: therefore, the endpoint (ED50) was <5 units/ml and thus the (ED50) is >0.20. Accordingly, the value for the cells containing the αYAC as shown is a minimal value.
The data in Panel D represent the reciprocal of the IFN-β 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-β was used instead of the alpha interferons.
The data in Panel E represent the reciprocal of the ΪFN-omega titer (units/mL) for 50% protection of cells (ED50) against EMCV. The experiments were performed as described in the legend to Figure 2, Panel A except Hu-IFN- omega was used instead of the alpha interferons. The value for the (ED50) for the 16-9 cells and 16-9/αRc5 cells is < 0.0005 shown in the figure as the maximal Ru-ΪFN-omega concentration tested was 2000 units/ml of Jlu-IFN-omega. Therefore, the endpoint (ED50) was > 2000 units/ml and thus the (ED50)'* is < 0.0005. Accordingly, the values for these two cell lines are maximum values.
The data in Panel F represent the reciprocal of the ΪFN-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. In addition, 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- αRl cDNA (αRl) and CHO-K1 cells containing the αYAC. The second three values of the histogram (right) represent parental 16-9 cells, 16-9 cells transfected with the Hu-IFN-αRl cDNA (αRl) and 16-9 cells containing the αYAC. The value for the (ED50)"1 for the 16-9 cells and 16-9/αRc5 cells is <0.0005 shown in the figure as the maximal ϊiu-ΪFN-omega concentration tested was 2000 units/ml of Uu-TFN-omega. Therefore, the endpoint (ED50) was > 2000 units/ml and thus the (ED50) is < 0.0005. Accordingly, the values for these two cell lines are maximum values. The values for the CHO-K1, αRl and αYAC in CHO-K1 cells (first three values shown in the figure) are correct as the endpoints of the assays fell within the range tested.
Figure 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- αRl (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-αRl cDNA (16-
9/αRc5 cells) and panels G, H, and I subclone 2 of 16-9 cells fused to the
F136C5.neo.9 YAC (16-9/αRy9-2 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-αA, panels B, E, and H treatment with Hu-IFN-αB2, and panels C,
F, and I treatment with Η.u-ΪFN-omega. Relative fluorescence values are shown on a log scale as described (Hibino et al , 1992).
Figure 4 illustrates the binding of [ 2P]Hu-IFN-αA and [32P]Hu-IFN- αB2 to cells. In a volume of 100 μL, 16-9, 16-9/αRc5, 16-9/αRy9-2 and 16-
9/YAC-JS2 cells at a density of 1 x 107 cells/ml were incubated with 300,000 cpm of [32P]Hu-IFN-αA (A) or [32P]Hu-IFN-αB2 (B) at room temperature for 1 hour with or without addition of a 200-fold excess unlabeled Hu-IFN-αA. The nonspecific binding was subtracted as indicated under "Experimental Procedures." The data shown represent the specific binding of [32P]Hu-IFN-αA and [32P]Hu- IFN-αB2 to 1 x 106 cells.
Figure 4A illustrates the binding of [32P]Hu-IFN-αA-Pl to various cells. The 16-9 cells are the parental cells for all the other transformants. Other cells are as follows: YAC JS2, 16-9 cells fused to the irrelevant control YAC JS2 (Soh et al , 1993); "αRl," 16-9 cells transfected with the Hu-IFN-αRl cDNA clone (Uzέ et al , 1990); αYAC, CHO 16-9 cells fused to the YAC encompassing the Type I interferon receptor complex. At the concentration of [J P]Hu-IFN- A tested, the specific cpm bound were as follows: Daudi cells, 26048 cpm; 16- 9/αRy9-2, 7126 cpm; 16-9/αRc5, -413 cpm; 16-9 cells, 50 cpm; 16-9/YAC-JS2 cells, 239 cpm. The data for Daudi cells are not shown in the histogram.
Figure 4B illustrates the binding of [32P]Hu-IFN-αB2 to the cells described in Panel A. At the concentration of [J^P]Hu-IFN-αB tested, the specific cpm bound were: 16-9/αRy9-2, 7850 cpm; 16-9/αRc5, 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 5 illustrates the competition of unlabeled Hu-IFN-αA, Hu-IFN- αB2, Hu-IFN-β and ϊiu-lFN-omega with [32P]Hu-IFN-αA. Various concentrations of the unlabeled Type I IFNs were added to cells and [32P]Hu-IFN- αA as described under "Experimental Procedures." After an incubation at room temperature for 1 hour, the bound [ P] Hu-IFN-αA was measured. Binding of [32P]Hu-IFN-αA in the absence of competitor (average of 4 determinations) is indicated on the ordinate axis (open box). The nonradioactive ligands are: Hu-IFN- αA (closed circle), Hu-IFN-αB2 (open circle), Hu-IFN-β (closed triangle) and Hu- WN-omega (open triangle).
Figure 6 illustrates the covalent cross-linking of [ 2P]Hu-IFN-αB2 and
[32P]Hu-IFN-αA to the receptors on cells. Cells were harvested and were incubated with [32P]Hu-IFN-αB2 (A) or [32P]Hu-IFN-αA (B) for 1 hour with (+) or without (-) addition of a 200-fold excess of unlabeled Hu-IFNαA and cross- linked as indicated under "Experimental Procedures." The extracted ligand: receptor complexes were mixed with sample buffer containing 10% β- 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.
Knockout and Reconstitution of a Functional Human Type I Interferon
Receptor Complex
As shown in Figure 8, cells containing the delta αYAC do not respond to Hu-IFN-αA or Hu-IFN-αB2 in MHC Class I antigen induction, whereas cells containing the fully functional αYAC do. Parental hamster cells do not respond to these human interferons at the concentrations tested. As shown in Figures 9 and 10, hamster cells containing the αYAC exhibit significantly enhanced protection against EMCV, whereas hamster cells containing the delta αYAC exhibit no more protection against EMCV than the parental hamster cells in response to both of these interferons. We thus conclude that the activity of the Hu-IFN-αRl is required to construct a fully functional receptor.
In order to reconstitute these biological activities, the Hu-IFN-αRl cDNA clone (see legend to Figure 8) was then introduced into the cells containing the delta αYAC. The transfected cells were then able to respond to both Hu-IFN- αA and Hu-IFN-αB2 in both MHC Class I antigen induction (Figure 8) and antiviral assays (Figures 9 and 10).
These results confirm that Hu-IFN-αRl 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-αRl cDNA reconstitutes these biological activities.
As we reported (Soh et al , 1994c), the αYAC 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-αRl protein is one component of this receptor complex. With the use of mice with a deletion of the Mu-IFN-αRl gene, Mϋller et al. (1994) have also concluded that the IFN-αRl protein is required for Type I interferon receptor function. Direct, although weak, participation of the Hu-IFN-αRl polypeptide in IFN-α ligand binding was suggested by the observation that the human and bovine IFN-αRl polypeptide expressed in Xenopus laevis oocytes can bind and cross-link radiolabeled Hu-IFN-αA and Hu-IFN-αB2 to a small extent (J.-K. Lim and J.A. Langer, personal communication). Nevertheless, when we examined the binding of Hu-IFN-αA and Hu-IFN-αB2 to pools of hamster cells containing the delta αYAC, there was substantial binding of these ligands (J.-K. Lim, J.A. Langer, personal communication); yet there was little binding to hamster cells expressing only Hu-IFN-αRl (Soh et al , 1994c). We can thus also conclude that a second subunit of the receptor complex other than Hu- IFN-αRl is responsible for ligand binding activity. Additional analysis of this αYAC clone will permit us to identify the remaining components of the functional Type I interferon receptor. Figure 7 is a map of the Hu-IFN-αRl gene showing the location of the insertion of pJSl. The EcøRI sites of the 32.9 kb Hu-IFN-αR gene are shown above the intron-exon structural map (exons are boxed, introns are heavy lines; Lutfalla, et al , 1992; Mariano, et al , 1992). The exons are numbered I - XI. The size of the EcoTZI fragments determined from the DNA sequence that contain exon sequences are 8331 bp (exons II and III), 10699 bp (exons IV-VIII), 4898 bp (exons IX-XI) and a five prime end fragment greater than 4026 base pairs (exon I) which appears at approximately 12 kb on a southern blot of genomic DNA. The insertion vector pJSl (9.6 kb; Soh, et al , 1994a,b,c) is shown as a linear fragment as would be the structure following a Clal digestion resulting in the Alu sequences at the ends of the fragment. The insertion of the vector at Alu sequences located at 1890 - 2135 bp (1) and at 14507 - 14829 bp (2) of the gene results in the introduction of EcøRI sites at new positions increasing the size of the 12 kb fragment by 3 kb and reducing the 8331 bp fragment to 3.5 kb (exon III). The shaded area of the gene is the location of the deletion. Enzymes: E, EcøRI; C,
Clal. Ε* indicates the location of several clustered EcøRI sites in the gene.
Figure 8 illustrates the induction of class I MHC cell surface antigen expression by interferons. All cell lines were maintained in F12 medium containing 10% FBS. Parental 16-9 cells are CHO-K1 cells containing the gene encoding the human Class I HLA B7 antigen and human chromosome 6 (Soh et al , 1993); 16- 9/αRy9A-2 (αYAC) cells (16-9/9-2) are 16-9 cells transfected with the αYAC containing the IFNAR locus as described (Soh et al , 1994c); 16-9/αRylOA (16- 9/10A) are 16-9 cells transfected with the delta αYAC containing a deletion of exon II of the Hu-IFN-αRl gene. Cell lines carrying YACs were maintained in medium containing 0.25 mg/mL antibiotic G418. The Hu-IFN-αRl cDNA was excised from plasmid pYH12 (Y. Hibino and S. Pestka, unpublished data) and cloned into the vector p8942 (gift of G. Mark and B. Daugherty, Merck Sharp & Dohme Research Laboratories, NJ.) for eukaryotic expression under the control of the adenovirus 2 major late promoter. This plasmid was then transfected into 16- 9/αRyl0A cells. Clones were selected in medium containing 0.4 mg/mL hygromycin and designated 16-9/αRylOA + Hu-αRl (delta αYAC reconstituted with the Hu-IFN-αR 1 expression vector, the far right panels). Isolates were expanded and maintained in medium containing 0.25 mg/mL each of antibiotic G418 and hygromycin. To test for induction of Class I MHC antigen expression, cells were treated with Hu-IFN-αA or Hu-IFN-αB2 as indicated in the panels of the figure at 100 units/mL in the absence of antibiotic for 3 days as described (Hibino et al , 1992; Soh et al , 1994a). Cells were harvested by trypsinization and treated with mouse anti-HLA monoclonal antibody (W6/32), washed and treated with FITC-conjugated goat anti-mouse IgG. Cells were washed of excess antibody and analyzed by cytofluorography. The shaded regions represent MHC Class I antigen expression of IFN treated cells; unshaded regions represent that of untreated cells.
Figure 9 illustrates the antiviral activity of Hu-IFN-αA. The data in the figure represent the reciprocal of the IFN 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 (Cook et al , 1994; Soh et al , 1993, 1994a); αYAC represents 16-9 cells containing the αYAC (Soh et al , 1994c); KO, 16-9 cells containing the delta αYAC (αRylOA) with exon II deleted; KO + αRl, represents the KO cells reconstituted by transfection with the Hu-IFN-αRl cDNA clone. The data for Hu- IFN-αA are shown.
Figure 10 illustrates the antiviral activity of Hu-IFN-αB2. The experiments were performed as described in the legend to Figure 9 except that Hu- IFN-αB2 was substituted for Hu-IFN-αA. The data for 16-9 and KO cells are maximal values as the endpoint titer was > 10,000 units/ml.
RESULTS
YAC F136C5 contains the gene for the cloned Hu-IFN-α/β receptor (Hu-IFN- αRl)
Two YAC clones (F136C5 and F143C3) were identified by screening with a sequence tagged site (STS) made from the 524-5P (D21S58) probe located in the vicinity of 21q22.1. YAC B49F1 (IFNAR YAC) was identified by a PCR primer pair generated from the exon VH-intron VI junction of the Hu-IFN-αRl gene (Mariano et al , 1992). Physical mapping data showed that the IFNAR, GART, and D21S58 loci were located in the same 400 kb Mlul fragment, and furthermore that the D2IS58 and IFNAR loci mapped within a 170 kb MlullNotl fragment (Tassone et al. , 1990; Cheng et al , 1993). Therefore, it is likely that the YACs screened with the 524-5P (D21S58) probe contain the entire gene for the cloned Hu-IFN-αRl receptor if the YACs are not chimeric. To test this point, yeast chromosomal DNAs from the F136C5, F143C3 and B49F1 YACs were digested with EcoRI restriction endonuclease and the blot was probed with cDNA for the Hu-IFN-αRl receptor. Exon I and part of the next long intron, and a fragment of about 5 kb corresponding to the gene segment encoding the intracellular domain of the protein were missing from YAC B49F1 (J. Soh, unpublished data). As shown in Figure IA, the F136C5 YAC contained four EcoRI fragments hybridizing to the cDNA, which indicates that the YAC contains the entire 30 kb Hu-IFN-αRl receptor gene, based on the sequence and mapping of the gene (Lutfalla et al , 1992; Mariano et al , 1992). Therefore two additional bands, 15 kb and 4.8 kb, in YAC F136C5 must correspond to exon I and part of the intron I sequence, and to the intracellular region of the cloned Hu-IFN-αRl gene, respectively.
Integration of the Nrør gene into YACs
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 neoτ gene fragment. Four of 12 transformants had the neoτ gene targeted into the YAC while one clone had the neor gene integrated in yeast chromosome II (data not shown). As shown in Figure IB, YACs F136C5.neo.6 and F136C5.neo.10 have a reduced size compared to the original YAC (430 Kb), indicating that a fairly large genomic fragment located between two Λ/α-targeted sites was deleted (Soh et al , 1994b). Therefore, YACs F136C5.neo.3 and F136C5.neo.9, which had no apparent deletions, were selected for fusion with CHO-K1 or 16-9 cells.
After selection of antibiotic G418-resistant colonies following fusion between yeast spheroplasts from YACs F136C5.neo.3 or F136C5.neo.9 on the one hand and CHO-K1 or 16-9 cells on the other, the resistant colonies were pooled and expanded for further analysis. The pools of colonies from YAC-fused CHO-K1 and 16-9 cells were referred to as CHO-K l/αRy3 or CHO-Kl/αRy9 and 16-9/αRy3 or 16-9/αRy9, respectively, where 3 and 9 represents different neor gene-targeted
YACs.
Antiviral Protection of Cells by Interferons
The CHO-Kl/αRy9 cells (CHO-K1 cells fused with YAC
F136C5.neo.9) showed increased sensitivity to Hu-IFΝ-αA and Hu-IFΝ-αB2 as measured by antiviral protection against EMCV when compared with parental CHO-K1 cells or CHO-K1 cells transfected with the Hu-IFN-αRl receptor cDNA construct pVADN123 (CHO-Kl/αRc4). Also 16-9/αRy9 cells showed a similar increased sensitivity to Hu-IFN-αA and Hu-IFN-αB2. After fusing the spheroplasts from YAC F136C5.neo.3 to CHO-K1 and to 16-9 cells (CHO-Kl/αRy3 and 16- 9/αRy3), pools of G418-resistant cells (data not shown) showed the same phenotype as CHO-Kl/αRy9 and 16-9/αRy9. The subclones (CHO-Kl/αRy9-4 and 16- 9/αRy9-2 derived from CHO-Kl/αRy9 and 16-9/αRy9, respectively) were assayed for antiviral protection against EMCV (Table 1) and VSV (Table 2). CHO- Kl/αRc4 and 16-9/αRc5 were individual cell lines from CHO-K1 and 16-9 cells, respectively, transfected with Hu-IFN-αRl receptor cDNA (pVADN123). Five subclones each from CHO-K1 and 16-9 cells transfected with the Hu-IFN-αRl cDNA were tested and all of them showed the same sensitivity as the parental cells.
The CHO-K1 and 16-9 cells transfected with the Hu-IFN-αRl cDNA showed a slight increase in protection from EMCV in response to Hu-IFN-αB2, but no increased sensitivity in response to Hu-IFN-αA, -omega, -β or -A/D (Figure 2; Table 1). However, cells containing the YAC F136C5.neo.9 exhibited 89-fold to greater than 200-fold increases in sensitivity to Hu-IFN-αA and Hu-IFN-αB2. There was even an increase in sensitivity to Hu-IFN-αA/D that interacts well with the hamster Type I receptor. Furthermore, in both of these YAC-containing cells there was significant increase in sensitivity to Hu-IFN-ø/negα; 16-9/αRy9-2 cells showed a significant increase in sensitivity to Hu-IFN-β (> 11 -fold). Decreased sensitivity of 16-9 cells compared to the parental CHO-K1 cells to Ku-ϊFN-omega was observed (> 2,000 vs 177 units/ml).
When the ED50 of the IFNs on cells challenged with VSV was measured, cells with the YAC F136C5.neo.9 showed substantial increase in sensitivity to all these interferons, ranging from about 3-fold to greater than 100- fold (Table 2). There was little or no increased sensitivity of the comparable cells containing the cloned Hu-IFN-αRl cDNA.
All these antiviral data with 16-9 cells are graphically summarized in
Figure 2. In all cases, sensitivity of 16-9 cells containing the αYAC was greater than parental 16-9 cells or 16-9 cells containing the Hu-IFN-αRl cDNA. The sensitivity of cells to the interferons was expressed as the reciprocal of the ED50 (Figure 2).
Induction of Class I MHC Surface Antigens
Subclones of CHO-K1 and 16-9 cells fused to YAC F136C5.neo.9 or transfected with the Hu-IFN-αRl 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). However, subclones isolated from transformed 16-9 cells, which express the human HLA B7 antigen, showed very good HLA B7 induction (Figure 3 and Table 3). Figure 3 shows treatment of cells with 100 units/mL of Hu-IFN-αA, -αB2, and -omega, which gave the most significant increases in the HLA-B7 antigen expression. Table 3 summarizes the HLA-B7 induction as a function of IFN concentration for all of the Type I IFNs tested. At 100 units/mL, Hu-IFN-αA, Hu-IFN-αB2, and Hu-IFN-ømegfl had little or no effect on the parental 16-9 cells (Figure 3, panels A, B, and C, respectively; Table 3) or 16-9 cells transfected with the Hu-IFN-αRl cDNA (Figure 3, panels D, E, and F, respectively; Table 3). However, 16-9 cells fused to YAC F136C5.neo.9 (16-9/αRy9-2 cells) showed enhanced levels of HLA-B7 antigens in response to Hu-IFN-αA, Hu-IFN-αB2, and Uu-ΪFN-omega (Figure 3, panels G, H, and I, respectively); and to Hu-IFN-β (Table 3). Treatment of 16-9/αRy9-2 cells with as little as 1-30 units/mL of Hu-IFN-αA, Hu-IFN-αB2, Hu-IFN-omegfl, and Hu-IFN-β gave a significant HLA-B7 induction whereas 16-9 cells or 16-9 cells transfected with the Hu-IFN-αR cDNA (16-9/αRc5 cells) did not show any significant induction at these levels (Table 3).
CHO-K1 and 16-9 Cells Containing YAC F136C5 Bind Diverse Type I
Interferons
Antiviral protection and class I MHC antigen induction exhibited by Type I interferons in 16-9 cells containing YAC F136C5 suggested that 16-9/αRy9- 2 cells could bind the Type I interferons. To test this, 16-9, 16-9/αRc5, 16- 9/αRy9-2, 16-9/YAC-JS2 and human Daudi cells were incubated with [32P]Hu- IFN-αA and [J,ώP]Hu-IFN-αB2 at room temperature and binding activity was assayed. [The 16-9/YAC-JS2 cells are 16-9 cells fused with the irrelevant GART YAC D142H8.neo.18 (Soh et al , 1993).] As shown in Figure 4, 16-9/αRy9-2 cells bound [32P]Hu-IFN-αA-Pl and [32P]Hu-IFN-αB2-P quite well. There was a low level of specific binding of [32P]Hu-IFN-αB2-P to cells containing the Hu- IFN-αRl cDNA, but no binding of [ P] Hu-IFN- A-Pl was seen. However, no significant specific binding of [32P]Hu-IFN-αA-Pl and [32P]Hu-IFN-αB2-P2 to either 16-9 or 16-9/YAC-JS2 control cells was observed. Daudi cells were used as a positive control for the binding assay. The level of IFN binding to the 16- 9/αRy9-2 cells was about 1/4 to 1/3 of that of Daudi cells, consistent with the lower level of receptors on 16-9/αRy9-2 cells (Figure 4A, legend).
Scatchard analyses of the Hu-IFN-αA and Hu-IFN-αB2 binding data are summarized in Table 4. Human Daudi cells had 4-10 fold more binding sites per cell than did CHO-Kl/αRy9-4 cells. The binding was of high affinity, with dissociation constants (K j) for the binding of the IFNs to CHO-Kl/αRy9-4 cells 3- 5 fold higher than that to Daudi cells. The specific binding of Hu-IFN-αA and Hu- IFN-αB2 to CHO-K1 and 16-9 cells containing YAC F136C5 (Figure 4; Table 4) indicates that the human DNA insert in this YAC contains the genes encoding the subunits comprising a functional Type I receptor.
The specificity of the binding of Type I IFNs by the receptor expressed on cells containing YAC F136C5 was further examined by testing the ability of Hu- IFN-αA, Hu-IFN-αB2, Hu-lFN-omega and Hu-IFN-β to compete with [32P]Hu- IFN-αA and [32P]Hu-IFN-αB2. As seen (Figure 5), all the Type I IFNs compete with [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2 for binding to CHO-K1 or 16-9 cells containing the YAC F136C5. This further supports the thesis that YAC F136C5 contains all the genes encoding components of the functional Type I interferon receptor.
Cross-Unking of [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2 to the Receptor
Complex
We further characterized the interaction of [32P]Hu-IFN-αA and
[ 2P]Hu-IFN-αB2 with the cells by affinity cross-linking of the ligands to cells and by analyzing the complexes by SDS-PAGE. With [32P]Hu-IFN-αB2, two strong bands at -130 kDa and -150 kDa are observed with the CHO-Kl/αRy9-4 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-αA 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. This observation is consistent with the lower levels of binding of [ 2P]Hu-IFN-αB2 to 153B7-8 cells relative to CHO- Kl/αRy9-4 cells. Crosslinked bands at 130 and 150 kDa, which co-migrate with the Daudi bands, are not detected when parental CHO-K1 cells, or CHO-Kl/αRc4 cells (containing the Hu-IFN-αR 1 cDNA) are used in the various CHO-K1 parental and transfected cells. However, faint bands at > 150 kD and -125 kDa (as well as several faster migrating bands) are seen with [JZ'P]Hu-IFN-αB2; these represent low-level crosslinking with endogenous CHO-Kl components whose nature is not known.
The cross-linking of [ 2P]Hu-IFN-αA to Daudi cells also shows two characteristic strong diffuse bands migrating similarly (-120-125 kDa and -140-145 kDa) to those seen with [32P]Hu-IFN-αB2 (Figure 6B). The slight increase in the migration of the complexes formed with Hu-IFN-αA, relative to those formed with Hu-IFN-αB2, may be attributable to the anomalously slow migration of Hu-IFN- αB2 (Wang et al , 1994). The 125 kDa and 145 kDa bands are also formed with CHO-Kl/αRy9-4 cells, but the bands are barely detectable with 153B7-8 cells. These specific bands are not detectable on CHO-Kl or CHO-Kl /αRc4 cells, which are indistinguishable from each other and on which several bands in the region of 84-116 kDa are seen.
Thus, the major ligand/receptor bands characteristic of human Daudi cells are also formed on CHO-Kl cells carrying YAC F136C5, but not on cells carrying the Hu-IFN-αRl cDNA. This is consistent with the strong binding and functional activity of various Type I IFN-αs on these cells. There are variations between Daudi cells and CHO-Kl /αRy9-4 cells in the relative intensity of the two major bands and in the width of the upper band; a full understanding of these more subtle features will probably require specific antibodies and/or DNA probes for each receptor subunit.
The enhanced biological response of CHO-Kl and 16-9 cells containing YAC F136C5 (CHO-Kl /αRy9-4 and 16-9/αRy9-2) to diverse Type I IFNs as well as the specific high-affinity binding of [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2 indicates that the human DNA insert in this YAC contains the genes encoding the subunits comprising a functional Type I receptor. Since the Hu-IFN-αRl gene is included in this DNA insert, one or more additional genes complementing the cloned Hu-IFN-αRl receptor must be encoded on YAC F136C5.
DISCUSSION
Human Chromosome 21 is the smallest and one of the best characterized human chromosomes. Genetic diseases such as Down's syndrome and Alzheimer's disease are associated with this chromosome (Cox and Shimizu, 1990). Although overlapping clones spanning the entire human Chromosome 21 will facilitate the understanding of the structure of this chromosome (Chumakov et al , 1992b), the methods to identify the function of genes and isolated cDNAs encoded in YACs need to be improved (Duyk et al , 1990; Lovett et al , 1991; Elvin et al. , 1990). This study and previous reports (Soh et al. , 1993; Cook et al. , 1994) demonstrate that expression of YAC clones in eukaryotic cells can be used to identify the function of genes in YACs as long as genetic linkage maps and/or cytogenetic studies provide information to permit selection of appropriate YAC clones. If the efficiency of YAC fusion to cells could be increased dramatically, then it might be possible to analyze YAC libraries as cosmid and phage libraries by selecting or screening for specific expressed markers.
Langer et al. (1990) sublocalized the genes for the Hu-IFN-α/β (Type
I) receptor complex to about 3 mb in the vicinity of the 21q22.1 band (3xlS region) with irradiation-reduced somatic hybrid 3xlS cells. Later it was shown that 3xlS cells encode at least two additional subunits which form affinity cross-linked complexes with [ 119'6J5I]Hu-IFN-αA that are distinct from the cloned Hu-IFN-αRl receptor subunit (Colamonici and Domanski, 1993). The ability of antibodies against the cloned Hu-IFN-αR 1 receptor subunit to block the binding of various Type I IFNs suggested that the Hu-IFN-αR 1 polypeptide is one component necessary to form a functional Type I receptor (Uzέ et al , 1991; Benoit et al , 1993). Although the cloned Hu-IFN-αR 1 receptor subunit can bind only Hu-IFN- αB2 when expressed in murine or hamster cells (Uzέ et al , 1990; Figure 4), expression in Xenopus oocytes could generate binding to both Hu-IFN-αB2 and Hu-IFN-αA, albeit the latter at a very low level (Lim et al , 1994).
Several IFN-related genes have been mapped to the 3xlS region or to YACs containing DNA inserts in this region. The gene encoding the Hu-IFN- gamma receptor accessory factor- 1 (AF-1) required for class I MHC induction was mapped to the 3xlS region (Langer et al , 1990), and further localized within the 540 kb GART D142H8 YAC (Soh et al , 1993). A gene encoding a class 2 cytokine receptor protein (CRF2-4) of unknown function was described and found to be about 35 kb from the gene for the cloned Hu-IFN-αRl receptor cDNA (Lutfalla et al , 1993). These observations suggest that the 3xlS region is rich in genes encoding proteins involved in cytokine function, including the IFN-αRl receptor, one or more additional components of the Type I IFN receptor, the IFN- gamma receptor accessory factor-1 and, perhaps, others.
On the basis of these observations, YACs in the 3xlS region were examined by functional assays. We tested two YACs initially screened by a primer pair derived from the 524-5P probe (21S58 locus), which is within 170 kb of the cloned Hu-IFN-αRl (IFNAR) receptor gene (Tassone et al , 1990). YAC F136C5 (430 kb) containing the gene for the cloned Hu-IFN-αRl receptor cDNA was selected for expression in CHO-Kl and 16-9 cells based on the assumption that all of the genes involved in forming a functional Type I IFN receptor might be encompassed on this YAC.
The YAC F136C5 in CHO-Kl and 16-9 cells provides genes which are necessary and sufficient to encode a functional Type I interferon receptor complex as measured by three distinct biological assays, the specific binding of both [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2, and competition of diverse Type I interferons for ligand binding. As monoclonal antibodies against the cloned Hu- IFN-αRl receptor subunit can inhibit the biological activity of the Type I IFNs, it is likely that this subunit is a part of the receptor complex on human cells (Benoit et al , 1993; Uzέ et al , 1991). Since this YAC contains the entire gene for the cloned Hu-IFN-αRl receptor subunit (-30 kb), it is likely that there are other genes responsible for the formation of the receptor complex present in this YAC. The results presented in this study suggest that a high-affinity receptor might be composed of the cloned Hu-IFN-αR 1 receptor subunit (and/or some alternatively spliced subunit of Hu-IFN-αR 1; Geary et al , 1992) and one or more additional subunits encoded by genes within this YAC. To identify all the components of the Type I IFN receptor, it is necessary to characterize all the relevant genes contributing to functional reconstitution of the Type I receptor. Inactivation of the genes for the cloned Hu-IFN-αRl receptor subunit and other genes on the YAC by homologous recombination (Soh et al , 1994b) may help to define the total number of components involved in the Type I IFN receptor.
Cross-linking studies of CHO-Kl /αRy9-4 cells with [32P]Hu-IFN-αB2 have suggested that the high affinity receptor is comprised of subunits which have similar molecular weights (Mr of -150 kd and - 130 kd) to those on Daudi cells. However, the pattern of cross-linking with [ YJ )P] Hu-IFN-αA was different from that of [ 2P]Hu-IFN-αB2, suggesting that these IFNs interact with the receptor complex differently. This may be consistent with functional differences between these interferons (Tables 1-3; Figures 2 and 3). Differences in function among the Hu-IFN-α species have been described in a variety of assays (Evinger et al , 1981; Rehberg et al , 1982; Ortaldo et al. , 1983; 1984; Pestka, 1983, 1984; Pestka et al , 1987; Hu et al , 1993). These results suggest that these functional differences may be reflected in the specific ligand-receptor interactions. It should be kept in mind that differences in cross-linking patterns may also reflect chemical constraints to one extent or another. The 153B7-8 (CHO-Kl /HuCh21q) somatic hybrid cells showed much lower binding and cross-linking to [32P]Hu-IFN-αA and [32P]Hu-IFN-αB2 than did the CHO-Kl cells containing YAC F136C5. This hybrid cell exhibited only a 16-fold increase in sensitivity to Hu-IFN-αB2 in antiviral protection against VSV and no increase in sensitivity to Hu-IFN-αA (data not shown). In addition, 3xlS cells containing 3 mb of the human Chromosome 21q showed a pattern of antiviral protection similar to that of the 153B7-8 cells. Mutations in at least one of the genes involved in formation of the receptor complex in the 153B7-8 cells and in the 3xlS cells might be responsible for the low biological activity. Alternatively, expression of all the Type I receptor components may not be balanced in these hamster x human somatic cell hybrids.
The reconstitution of the panoply of Type I IFN receptor activities not previously obtained with discrete genomic or cDNA clones provides the foundation to isolate the individual components involved. The use of functional YAC screening to identify this complex receptor is illustrative of the power of this procedure that previously permitted us to identify the accessory factor- 1 required for ΪFN-gamma receptor function (Soh et al , 1993, 1994b; Cook et al , 1994). This functional YAC screening technology provides a new dimension for the use of large DNA inserts in identification, characterization and localization of genes.
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The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims.

Claims

We claim:
1. A yeast artificial chromosome, YAC F136C5, containing a segment of human Chromosome 21 which when introduced into cells confers upon the cells a greatly-enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Hu-IFN-ømega, Hu-IFN-αA/D (Bgl), and Hu- IFN-β.
2. The yeast artificial chromosome according to claim 1, wherein the cells are Chinese hamster ovary (CHO) cells.
3. A yeast artificial chromosome, YAC F136C5.neo.9, containing a segment of human Chromosome 21 which when introduced into (CHO) cells confers upon these cells a greatly-enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Hu-IFN-ømegα, Hu-IFN- αA/D (Bgl), and Hu-IFN-β.
4. The yeast artificial chromosome according to claim 3, wherein the cells are Chinese hamster ovary (CHO) cells.
5. A functional human type I interferon receptor expressed from a yeast artificial chromosome, YAC F136C5, wherein the chromosome contains a segment of human Chromosome 21 which when introduced into cells confers upon the cells a greatly-enhanced response to Hu-IFN-αA and Hu-IFN-αB2 as well as increased response to Uu-lFN-omega, Hu-IFN-αA/D (Bgl), and Hu- IFN-β.
6. The functional human type I interferon receptor according to claim 5, wherein the cells are Chinese hamster ovary (CHO) cells.
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