CA2114168A1 - Chimeric human interferon-gamma-receptor/immunoglobulin polypeptides - Google Patents

Abstract

Abstract The present invention relates to chimeric inferon-.gamma. receptor/
immunoglobulin polypeptides, to DNA sequences coding for these chimeric polypeptides and methods for making these.

Description

.AN 4100174 C~IIMERllC HUMAN INTERFERON~
RECEPTOR/IMMUNOGLOBULIN POLYPEPTIDES ~;

s .: , Interferon~ ) is a protein produced by NK cells and activated helper T lymphocytes which has antiviral and antiproliferative activity and which plays a multipotent role in the 0 control of the immune system and of the inflammatory response. It regulates aneibodies formation and T lymphocytes or NK cells differentiation. It enhances the expression of Major Histocompatibility Complex (MHC) class I and II structures on several cell types, -amplifying their antigen presentation (accessory cells) capacity. Acting 5 on phagocytes, fibroblasts, epithelial and endothelial cells, IFN~ plays a central role in the activation of non-specific defense mechanisms. I~ is the main activator of microbicidal and tumoricidal properties of ~ ~-macrophages, enhances phagocytosis, induces ADCC ~Antibody Dependent Cellular Cytotoxicity) and modulates the release of 20 polypeptides encoded by MHC class III genes (i.e. complement components and TNF), proteinase inhibitors (Cl inhibitor), IL19 GM~
CSF, fibronectin, arachidonic acid metabolites, 1125-hydroxy-vitamin - -D3, lysosomal enzymes (IP-30, hydrolases, neutral proteinases, i:e:
uPA), and chemotactic factors (IP-10). Finally, I~N~ promotes cell-to-2s cell interac~ion, migration of phagocytes through connective tissue, chemotaxis and adhesion to ex~acellular matrix glycoproteins (Landolfo and Garotta, J. Immunol. Res. 3, 81-94 [l991~

Several animal experiments suggest that IFN~ plays a crucial role `~ ~ `
30 in either th~ induction or the progression of insulin-dependent diabetes, systemic lupus erythematosus, thyroiditis, multiple sclerosis, ~;
fulminant hepatitis, allograft rejection, thrombosis and hemorrhage that follows the generalized Shwartzman type reaction. Additionally, Ar/15.12.93 ; ;~

IFN~ plays a role in the progression of Kawasaki disease (mucocutaneous lymph node syndrome) and in the hypercalcemia - observed in some of sarcoidosis patients (IFN~ induces the elevation of l,a-sterol-hydroxylase that converts vitamin D3 into its most active s form 1,25-dihydroxy-vitamin D3) (Garotta et al., Pha~nacol. Res. 21, 5-17 [1989]; Landolfo and Garotta, supra; Gilles et al., Hepatology 16, 655-663 11992~). In the pathogenesis of AIDS, tissue macrophages play a role in the establishment of a sta~le of chronic infection because they represent an important reservoir for human immunodeficiency 0 virus (HIV). In vitro experiments show that IFN~ enhances the expression of mature HIV by infected macrophages and can exacerbate the pr~gression of the disease (Ganser et al., Onkologie 9, 163-166 [1986], Biswas et al., J. Exp. Med., 176, 739-750 [19921).
Finally, IFN~ plays an important role in inflammatory neurological 5 diseases or in neurological cornplications of AIDS, poliovirus infections, Lyme disease and septicemia. IFN~ activated macrophages convert L-tryptophan into the neurotoxin quinolinic acid (Heyes et al., Biochem. J.
2837 633-635 Cl99~]).

The existence of a specific receptor for IFNy (IFN~R) on various cells was demonstrated by cross-linking experiments, by binding of radiolabeled IFN~ and by specific competition with unlabeled IFN~.
Cross-linked complexes between human IFN~ (hI~N~y) and human IFN~
receptor (hIFN~R) with a molecular weight (Mr) ranging from 70,000 to 165,000 have been described (Rubinstein et al., Immunol. Rev. 97, 29-50 [1987]). Despite this apparent heterogeneity, binding and -structural studies on different cell types revealed only one IFN~
binding protein with 90 kDa of apparent Mr (p90) and a single class of binding site with a dissociation constant of about 10-1l to 10-10 M
(Sarkar and Gupta, Proc. Natl. Acad. Sci. USA 81, 5160-5164 [1984];
Aguet and Merlin, J. Exp. Med. 165, 988-999 [1987]; Calderon et al., - ~ `
Proc. Natl. Acad. Sci. USA 85, 4837-4841 [1988]; Fountoulakis et al., J.
Immunol., 143, 3266-3276 [1989]; van Loon A.P.G.M. et al., J.
Leukocyte Biol., 49, 462-473 [1991]). An additional 50 Kd (pS0) 35 componenl of hIFN~R that can be detected is a proteolytic degradation .' .- " :' - '~

3 - i~ 3 8 product of the p90 protein (Aguet and Merlin, supra, Sheehan et al., J.
Immunol. 140, 4231-4237 [1988]; Fountoulakis et al., supra; van Loon A.P.G.M. et al., supra). Since ~his p50 component of hIFN~R includes the extracellular domain, the transmembrane region and a portion of the s intracellular domain, it is not a natural occurring soluble form of hIFNrR (Fountoulakis et al., supra; van Loon A.P.G.M. et al., supra). In general, the IFN~R is an ubiquitous membrane anchored protein (Yalente et al., Eur. J. Immunol. 22, 2403-2412 [19921) that seems to be expressed to a lesser extent on normal cells 1UP to 103 sites per 10 cell) than on tumor cells. Thus, some human carcinoma and B-cell lines were reported to express in the order of 104 binding sites per cell (Uecer et al., Cancer Res. 46, 5339-5343 [1986]; Ague~ and Merlin, supra).
Molecular cloning and expression of the hIFN~R has been described (Aguet et al., Cell 55, 273-280 [1988]). However, because the natural IFN~R as well as the recombinant IFN~R are membrane anchored proteins, they have the disadvantage that they are insoluble in physiological solution. Consequently, search or design of an IFN~
20 antagonist, and administration of purified IPN~R to mammals to inhibit IFN~ binding to its specific receptor thereby preventing, suppressing and/or modulating the course of autoimmune disorders, chronic -inflammations, delayed hypersensitivities and allotransplant rejections was difficult to accomplish. -The soluble forms of human [shIFN~R] and mouse IFN~yR
[smIFN~R] were engineered by culturing transformants carrying an expression vector containing a DNA sequence coding for a soluble form of the respecd~e IFN~R and isolating such a soluble IFN~R. The 30 said shIFN~R includes the whole extracellular domain of the natural IFN~R from the N-terminal portion ~o the transmembrane region (at least amino acids 26-246 of the natural IFN~R sequence), lacks the cytoplasmic and transmembrane domains of the natural I~
receptor and is capable of specifically binding I~N~ (Fountoulakis et 35 al., J. Biol. Chem. 265, 13268-13275 [1990], Gentz et al., Eur. J.

Biochem. 210, 545-5~4 [1992] and Furopean Patent Application, Publication No. 393 502). This soluble form of the IFN~R has a molecular mass of about 30-32 kDa, binds IFN~ with an affinity of about 10-10 M, is active and stable in vivo, is not immunogenic and 5 can be applied as a drug that speci~lcally neutralizes the endogenous IFN~. Accordingly, it can be used for the therapy of insulin-dependent diabetes, systemic lupus erythematosus, multiple sclerosis, fulminant hepatitis, thyroiditis, allowgraft rejection, septic peritonotis and Shwartzman-type reaction and inflammatory 0 neurological diseases produced by the neurotoxin quinolinic acid released by IFN~ activated macrophages. Additionally, the soluble form OI the IFN~R can be applied to AIDS patients to delay the chronicization of the infection, to prolong the asymptomatic phase of the disease and to prevent neurological complications. The soluble ~ -5 form of I~ receptor can be applied as IFN~ antagonist to prevent neurological complications of poliovirus infections, Lyme disease and septicemia. However, the soluble form of the I~N~ shows a ;~
persistency of only 1-3 hours in the blood and of 6 hours in the Iymphoid organs (O~men et al., J. Chemotherapy 3, Suppl. 3, 99-102 20 [1991]; Ozmen et al., J. Immunol. Methods 147, 261-270 [1992];
Gentz et al, supra). ;
. :
By combining the soluble fonn of the mouse IFN~R with parts of the constant domain of mouse immunoglobulins resulting in ch;meric 2S mouse IFN~R-immunoglobulin polypeptides (Kurschner et al., J. Biol. ` ~- Chem. 267, 9354-9360 [1992]) increased mouse IFN~R persistency in the blood and the Iymphold organs could be achieved (Kurschner et al..
J. Immunol. 149, 4096-4100 [1992]).

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These chimeric mouse IFN~R-immunoglobulin polypeptides have, however, the disadvantage that they cannot be used for administration to humans to inhibit IFNy binding to its specific receptor. To solve this problem l~NA sequences coding for chimeric s human IFN~R-human immunoglobulin polypeptides have been constructed.

More precisely, the present invention provides DNA sequences comprising a combination of two partial DNA sequences, wherein one 10 of said partial sequences is coding for a fragment of the hIFN~R ~
binding hIFN~, whereby a fragment of the hIFN~ with the whole or a ~ s part of the sequence as shown in Figure 1 is preferred, and the other partial sequence is coding for part or all of the cons~ant domains of human immunoglobulin heavy or light chains, whereby heavy chains, 5 especially all domains except the first domain of the constant domain of human immunoglobulins, such as IgG, IgA, IgM or IgE and specifically IgG, e.g. IgGI and IgG3, are preferred. A p~eferred const3nt domain of human immunoglobulin light chains is, e.g. C~

The present invention is also concerned with the recombinant ~
chimeric polypeptides coded by such DNA sequences, such as hIFN~R- ~ -HGl, hIFN~R-HG3 or hIFN~R-HC,c. The chimenc polypeptides may contain amino acid substitutions that do not significantly change the --activity of the proteins. Amino acid substitutions in proteins and 25 peptides which do not generally alter the activity of such molecules are known in the art and are described, e.g. by H. Neurath and R.L. Hill in "The Proteins" (Academic Press, New York, 1979, see especially Pigure 6, page 14). The most commonly occurring exchanges are~
Ala/Ser, ValtIle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, 30 Ala/Val~ Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, AspJAsn, Leu/Ile, Leu/Val, Ala/~}lu, Asp/Gly and vice versa. - ~;

The recombinant chimeric polypeptides of the present invention can additionally contain sequences of several amino acids whicb are 3s coded for by "linker" sequences. These sequences arise as a result :;' ~.~':' - 6 - 2 1 ~

from the expression vectors used for expression of the recombinant chimeric polypeptides.

The recombinant chimeric polypepticles of the present invention s can also contain specific sequences that perferably bind to an affinity carrier material. Examples of such sequences are sequences containing at least two adjacent histidine residues (see in this respect European Patent Application Publication No. 282 042). Such sequences bind selectively to nitrilotriacetic acid nickel chelate resins (Hochuli and 10 Dobeli, Biol. Chem. Hoppe-Seyler 368, 748 (1987); European Patent No.
253 303). Recombinant chimeric polypeptides which contain such a ~-specific sequence can, therefore, be separated selectively from the remaining polypeptides. The specific sequence can be linked either to the C-terminus or the N-terminus of the amino acid sequence of the, ~ ;
l s chimeric polypeptide.

Such chimeric polypeptides could have increased half-life in ~ivo. Increased half-life in vivo has been shown, e.g., for chimeric polypeptides consisting of the first two domains or parts thereof of the 20 human CD4-molecule and different domains of the constant regions of the heavy chain or ~he light chain of a mammalian immunoglobulin ~see Traunecker et al., Nature 331, 84-86 [1988~ and European Patent ~--Application, Publication No. 394 827). Additionally, as already -mentioned hereinbefore, chimeric mouse IFN7R-immunoglobulin ~
25 polypeptides show increased half-life in the blood and in the lymphoid ~ i -..
organs. ~ ~ ~ .. .~ ., -,-.~.
Because the complete DNA sequence of the gene coding for the natural IFNyR is known (Ague~ et al., supra) a DNA sequence coding for 30 a fragment of the hIFN~R can be chemically synthesized using standard methods known in the art, preferably solid state methods, such as the methods of Merrifield (J. Am. Chem. Soc. 85, 2149-2154 -[1963]). Alternatively, fragments of the hIFN~R can be produced from DNA encoding the hIFNyR using methods of DNA recombinant 35 technology (Sambrook et al. in "Molecular Cloning-A Laboratory ~ `~

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Manual", 2nd. ed., Cold Spring Harbor Laboratory [1989]).

Preferably, fragments are prepared including the signal sequence and the extracellular portion of the hIFN~R by polymerase chain s reaction (PCR) using plasmids encoding the hIFN~ as described in detail in Examples 5, 6 and 7.
.. . .
Plasmids suitable for amplification of DNA sequences coding for the IFN~R by PCR are described, for exa]mple, in European Patent 10 Appliation, Publication No. 393 502. An especially suitable plasmid is plasmid phIFN~R (Example 2).

The DNA sequences coding for fragments of the hIFN~R can then be integrated into suitable expression vectors containing DNA
15 sequences coding for part or all of the constant domains of human immunoglobulin (Ig) heavy or light chains using known methods (Sambrook et al., supra). The Ig constant domains can be provided by expression vectors which have been used to express CD4 as hybrid molecules with Ig constant domains. Such expression vectors include 20 but are not limited to pSV2-derived vectors (see for example German, C. in "DNA Cloning", Vol. II., edt. by Glover, D.M., IRL Press, Oxford, 1985), like pCD4-H~l, pCD4-H~l, pCD4-H~3 (described in detail in -~
European Patent Application, Publication No. 394 827). ~' 2s The specification of European Patent Application, Publication No. ~ ~ `
394 827 and the Traunecker et al. reference cited supra contain also data with respect to the further use of these vectors for the expressio of chimeric proteins and for the construction of vectors for the expression of chimeric proteins with other immunoglobulin fragments.
30 For the purpose of the present invention the CD4 coding part in these vectors is replaced by a DNA sequence coding for a fragment of the hIFN~R by methods known in the art and described, e.g., in Sambrook et al. (supra), resulting, for example, in plasmids phuIFN~R-HGl (Example 5), phuIFN~R-HG3 (Example 6) and phulFN~R-HC,c (Example 35 7).

., Suitable expression vectors include, for example, vectors such as pBC12MI [ATCC 67109], pSV2dhfr lATCC 37146], pSVL [Pharmacia, Uppsala, Sweden], pRSVcat [ATCC 37152] and pMSG [Pharmacia, s Uppsala]. Preferred vectors for the expression of the chimeric hIFN7R- -~
immunoglobulin polypeptides are pN316 and pN345 type vectors (see ~ ~-Figures 2 and 3) resulting in expression vectors pN316-huIFN~R-HGl, pN316-huIFN7R-HG3 and pN346-huIFNyR-HClc (see Examples 5, 6 and 7), which have been deposited transformed in E. coli K803 under the lo conditions of the Budapest Treaty for patent purposes at the Deutsche Sammlung Yon Mikroorganismen und Zellkulturen GmbH (DSM) in Braunschweig, Federal Republic of Germany on January 26, 1993, ~ --under accession numbers DSM 7421, 7419, 7420 respectively. These vectors are preferably introduced into suitable mammalian host cells, 15 for example, by transfection.

Mammalian host cells that could be used include, e.g., human Hela, - ~
H9 and Jurkat cells, mouse NIH3T3 and C127 cells, CVl African green ; .
monkey kidney cells, quail QC1-3 cells, Chinese hamster ovary (CHO) 20 cells, mouse L cells and ~he COS cell lines. The CHO cell line (ATCC CCL
61) is preferred.

The DNA sequences coding for the chimeric hIFN7R-immuno-globulin polypeptides can also be integrated into suitable vectors for 25 expression in yeast or insect cells. For the production in insect cells, e.g., ~he baculovirus-insect cell vector system can be used (for review see Luclow and Summers, BiolTechnology 6, 47-55 ~19881). The chimeric polypeptides produced in insect cells infected with recombinant baculovirus can undergo post-translational processing 30 including N-glycosylation (Smith et al., Proc. Na~. Acad. Sci. USA 82, 8404-8408) and O-glycosylation (Thomsen et al., 12. International Herpesvirus Workshop, University of Philadelphia, Pennsylvania). ;

The manner in which the expression of the chimeric hIFN7R- ~ ~
35 immunoglobulin polypeptides of the present invention is ca~ried out ~ ~;
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~, _ 9 ~ i depends on the chosen expression vector/host cell system. -Usually, mammalian host cells which contain a desired expression vector are grown under conditions which are optimal for s the growth of the mammalian host cells. A typical expression vector contains the promoter element, which mediates the transcription of `
mRNA, the protein coding sequence, and the signals required for efficient termination and polyadenylation of the transcript. Additional elements may include enhancers and intervening sequences bounded ~ -10 by spliced donor and acceptor sites.

Most of the vectors used for the transient expression of a given coding sequence carry the SV40 origin of replication, which allows them to replicate to high copy numbers in cells ~e.g. COS cells) that I s constitutively express the T antigen required to initiate viral DNA
synthesis. Transient expression is not limited to COS cells. Any mammalian cell line that can be transfected can be utilized for this purpose. Elements that control a high efficient transcnption include the early or the late promoters from SV40 and the the long terminal 20 repeats (LTRs) from retroviruses, e.g. RSV, HIV, HTLVI. However, also cellular signals can be used ~e.g. human-,B-actin-promoter).

Alternatively, stable cell lines carrying a gene of interest integrated into the chromosome can be selected upon co-transfection 25 with a selectable marker such as gp~, dhfr, neomycin or hygromycin.

Now, the trans~ected gene can be amplified to express large quantities of a foreign protein. The dihydrofolate reductase (DHPR~ is a useful marker to develop lines of cells carrying more than 1000 copies 30 of ,the gene of interest. The mammalian cells are grown in increasing amounts of methotrexate. Subsequently, when the methotrexate is ~ -withdrawn, cell lines contain the amplified gene integrated into the chromosome. In the expression vectors used in the preferred embodiments of the present invention, the expression is controlled by 35 the Rous sarcoma virus LTR promoter.

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1 o The host cells transfected with a suitable expression vector as well as the expression vectors used for their transfection and expressing the recombinant chimeric polypeptides are also an object of s the present invention. ;

The chimeric polypeptides of the present invention can be punfied from the cell mass or the culture supernatants according ~o methods of protein chemistry which are known in ~he art such as, for 0 example, precipitation, e.g., with ammonium sulfate, dialysis, ultrafiltration, gelfiltration, ion-exchange chromatography, SDS-PAGE, isoelectric focusing, af~inity chromatography like immunoaffinity chromatography, HPLC on normal or reverse systems or the like.
Preferably, the chimeric hIFN~R-immunoglobulin polypepffdes s expressed in mammalian host cells are obtained after affinity chromatography. `

The chimeric polypeptides of the present invention as well as their physiologically compatible salts, can be used for the treatment 20 of illnesses in which IFN~ is involved in their course, e.g., for the treatment of autoimmune diseases, e.g. type I diabetes or lupus erythematosus or rheumatoid arthritis, chronic inflammations, e.g.
Shwartzman Reaction, delayed hypersensitivity, allotransplant rejections, multiple sclerosis, fulminant hepatitis and inflammatory 2S neurological diseases produced by the neurotoxin quinolinic acid `
released by IFN~ activated macrophages. Additionally, the chimeric polypeptides can be applied to AIDS patients to delay the ~-;
chronicization of the infection, to prolong the asymptomatic phase of the disease and to prevent neurological complications. The chimeric 30 polypeptides can be also used as IFNlr antagonist to prevent neurological complications of poliovirus infections, Lyme disease and ~ ~septicemia and/or the production of corresponding pharmaceutical ~ !,' ` . ' preparations. They nnay be administered in pharmaceutically acceptable oral, injectable or topical compositions and modes. Dosage `
35 and dose rate may parallel that currently being used in clinical applications of the known IFNs. The pharmaceutical compositions of the present invention contain the chimeric polypeptides or their physiologically compatible salts ~hereof in association with a compatible pharmaceutically acceptable carrier material. Any -s conventional carrier material can be utilized. The carrier material can be an organic or inorganic one suitable for enteral, percutaneous or parenteral administration. Suitable carriers include water, gelatine, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, petroleum jelly and the like. Furthermore, the - ~
10 pharmaceutical preparations may contain other pharmaceutically ~ `
active agents. Additional additives such as flavouring agents, preser- ~ ~
vatives, stabilizers, emulsifying agents, buffers and the like may be ~ -added in accordance with accepted practices of pharmaceutical compounding.
I S
The pharmaceutical preparations can be made up in any conventional form including: a) a solid form for oral administration --such as tablets, capsules, pills, powders, granules and the like; b) a ~ ~ -liquid form for oral adminis~ration such as solutions, syrups, 20 suspensions, elixirs and the like; c) preparations for parenteral administration such as sterile solutions, suspensions or emulsions; and ~
d) preparations for topical administrations such as solu~ions, ;
suspensions, ointments, creams, gels, micronized powders, aerosols and the like. The pharmaceutical preparations may be sterilized and/or 25 may contain adjuvants such as preservatives, stabilizers, wet~ing agents, emulsifiers, salts for varying the osmotic pressure and/or buffers . ~ -Parenteral dosage forms may be infusions or lnjectable solutions 30 which can be injected intravenously or intramuscularly. These preparations can also contain other medicinally active substances.
Additional additives such as preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.

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Such pharmaceutical preparations and the use of ~he compounds of the present invention for therapeutical purposes are also an object of the presen~ invention.

s Having now generally described this invention, the same will become better understood by reference to the specific examples, which are included hçrein for purpose of illustration only and are not intended to be limited unless otherwise specified, in connection with -;~
the following figure:
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Fi~ure 1 displays the nucleic acid sequence of shIFN~R cDNA -(SEQ. ID No.2) and the amino acid sequence deduced therefrom (SEQ.
ID No. l ). Sequences are represented by standard abbreviations for nucleotides and amino acids. ~ -Figure 2 is a schematic drawing of the expression vector pN316. ~ -The abbreviations and symbols used are: RSV = rous sarcoma virus ~ ~
LTR sequence (promo~er); rppi 3' intron + poly A = rat preproinsulin 3' ~ ;
intron and polyadenylation site; PSV 40 = SV40 viral promoter; mDH~
20 = mouse DHFR; SV40 poly A = SV40 virus polyadenylation site -sequence; ampicillin = ~-lactamase gene that confers resistance to the antibiotic. Restriction enzyme sites which are inactivated by the sub- ;
cloning procedures described in the Examples are indicated in brackets.
2s E~ure ~ is a schematic drawing of the expression vector pN346.
The abbreviations and symbols used are: RSV = rous sarcoma virus LTR sequence (promoter); rppi 3' intron + poly A = rat preproinsulin 3' intron and polyadenylation site; PSV40 = SV40 viral promoter; mDHPR
30 = mouse DHFR; SV40 poly A = SV40 virus polyadenylation site sequence; ampicillin =,B-lactamase gene that confers resistance to the antibiotic; CMV = part of Cy~omegalovirus enhancer sequence.
Restriction enzyme sites which are inac~ivated by the subcloning procedures described in the Examples are indicated in braskets.
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Fi~ure 4 is a schematic drawing of the expression vector pN316- ~
huIFN~R-HGl. For abbreviations and symbols see legend to Fig. 2. ~-Fi~ure 5 is a schematic drawing of the expression vector pN316-s huIFN~R-HG3. For abbreviations and symbols see legend ~o Fig. 2.

Fi~ure 6 is schematic drawing of the expression vector pN346-huIFN~R-HC,c. For abbrevia~ions and symbols see legend to Fig. 3.

to Figure 7 displays standard curves for shIFN~R and hIFN~R-HG3.

Example 1 ~ . .-,.
Sequencin g All cDNAs or fragments thereof obtained by suitable restriction enzyme digests were subcloned in pUC18/19 type vectors (Pharmacia, Uppsala, Sweden~. Sequencing was performed using a protocol based ~-on the Sanger procedure and involving Taq polymerase and double 20 stranded DNA. -Example 2 (~n~ction of expression vectors pN346 and pN316 1. Preparation of the fragment containing part of the CMV enhancer.
A fragment containing 232 basepairs of the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al., Cell 41, 30 521-530 [1985]) was obtained using the Polymerase Chain Reaction (PCR) (Saiki et al., Science 230, 1350-1354 [1985]).

PCR is based on the enzymatic amplification of a DNA fragment: Two oligonucleotide primers that are oriented with their 3' ends towards 35 each other are hybridized to opposite strands of the target sequence.

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Repeated cycles of heat denaturation of the template, annealing of the primers to their complementary se~quences and extension of the annealed primers with a DNA polymerase result in the amplification of -the segment defined by the 5' ends of the PCR primers. The addition of s restriction enzyme sites to the 5' end of each primer facilitates cloning ~ -of the ~mal PCR product (Scharf et al., Science 233, 1076-1078 [1986]). -The following oligonucleotides have been used to amplify the DNA -- ~
fragment containing part of the CMV enhancer: :

1) 5'- GGGCTCGAG AC~ ATGGGACITICCIACITGG 3' (SEQ. ID No. 8) (forward primer), and 1 s 2) 5'- CCCGTCGAC CCIACCGCCCA l-l l GCGTCAATG 3' (SEQ. ID No. 9) (reYerse primer). ~ ~-': :': '', ':.-The ampli~led fragment was isolated from an agarose gel using a commercial available kit ("Genecleann, BIO 101 Inc., La Jolla, Ca.). The 20 fragment was then digested with the endonucleases Xhol and Sall (restriction sites in the above primers underlined) and then purified again as described above (Fragment 1).
2. Preparation of the vector fragment ;~
Ihe expression vector pK21 which contains the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, March 1985, 438-447) and a polylinker -region of the following sequence:

5'-AAGCTTGGCCAGGATCCAGCTG ACTGACTGATCGCGAGATC3 "SEQ.ID N~. 10) ~ ~
3'-TTCGAACCGGTCCTAGGTCGAC TGACTGACTAGCGCTCTAG5' (SEQ.ID No. 11) ~ .
that allows the integration of genes of interest (PvuII site underlined) `
3S was used. Downstreann the cloning sites for the genes this vector contains the 3' intron, the polyadenylation site and termination signal of ~ ~ ~

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the rat preproinsulin gene.

Plasmid pK21 was digested with XhoI and then treated with calf intestinal alkaline phosphatase to remove the phosphates from the 5' s ends of the DNA fragments. The dephoshorylated vector was then isolated from an agarose gel as described above (Fragment Vl).

3. Preparation of plasmid pN340 10 Fragment 1 was ligated with fragment Vl using T4 ligase. ; ;

E.coli HB101 cells were then transformed and transformants which contained the enhancer fragment in the proper orientation (same orientation as in the CMV enhancer) were identified by restriction 15 enzyme mapping and sequencing. The resulting plasmids were named pN340.

4. Construction of the expression vector pN346.

2û Preparation of fragment F2 Plasmid pN340 was digested with Xhol and Xbal. The XhoI and XbaI
fragment contains the promoter (CMV-enhancer fragment plus RSV-LTR), the polylinker region shown above and the 3' intron plus the 25 polyadenylation signal of the rat preproinsulin gene. The fragment was isolated from an agarose gel as described above using C~enclean.
The s~icky ends of this fragment were then fflled in using Klenow enzyme (Fragment 2).

30 Preparation of the vector ~ragment V2.

Plasmid pN308 which is a derivative of the expression vector pSV2-DHFR (supra). missing the region between the single EcoRI and BamHI
restriction sites was digested with PvuII and then treated with calf 35 intestinal alkaline phosphatase. The dephosphorylated vector was then isolated as described (Fragment V2).
Ligation of fragmene F2 and vector V2. ~ -s The blunt ended vector fragment Y2 and the blunt ended fragment F2 were ligated with T4 ligase. - -~

E.coli HB101 cells were then transformed and ~ransformants which i contained the Fragment 2 in the desired orientation were identified by ;~
I o restriction enzyme analysis and sequencing. The resulting plasmids were named pN346 (Figure 3). `

5. Construction of the plasmid pN3 16 s The construction of plasmid pN3 16 was the same as described under paragraph 4 above with the exception that instead of plasmid pN340, plasmid pK21 was used. As a result, plasmid pN316 lacks Fragment 1 (described in paragraph 1) and has only the RSV LTR promoter element (Figure 2).
Construction of plasmid "phlFN~yR"

Plasmid constructions were carried out as described in the -~
following paragraphs. In case that no speci~1c references or details of 2s preparation are given standard methodology according to Sambrook et al. in "Molecular Cloning - A Laboratory Manual" (2nd ed.), Cold Spring Harbor Laboratory [1989], was used.
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The insert from phage ~gtll-hIFN~R-16 (Aguet et al., Cell 55, :~ -30 273-280 [1988]) was excised using EcoRl restriction enzyme under conditions where a partial digestion of the DNA was obtained. The complete insert of about 2,2 kb was ligated into plasmid pUC18 and one of the resulting constructs, plasmid phIFN~R, was chosen for further analysis.
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- 17 - 21~ 8 Example 3 ~ -Transformation of E.coli K803 s Transformation of E.coli K803 cells with plasmid phIFNyR
~Example 2) was achieved by the heat shock procedure as described in Sambrook et al., supra.
Example 4 lo Isolation of Plasmid DNA

Plasmid DNA from transformed E.coli K803 cells (Example 3) was prepared using a standard procedure /Birnboim and Doly, Nucl. Acids Res. 7, 1513 (1979); Sambrook et al., 1989, supra). The insert coding s for soluble hIFN~R was cut out of plasmid phIFNyR and sequenced as described in Example 1. The complete nucleic acid sequence of shIFN~R
and the amino acid sequence deduced therefrom are shown in Fig. 1.

Example 5 Constr~ctiQn of a chimenc human IFN~R-I~Gl (HG 1~ molecule In the first step, a polymerase chain reaction (PCR) was performed using plasmid phIFN~R as a template and the following 2s primers:

1) 5'-AA~CGAGCICGTAGCAGCATGGCICICCI~ 1 lC 3' (SEQ. ID
NO.3) matching 24 nucleotides of the hIFN~R cDNA sequence (8 residues of the 5' untranslated (5'-UT3 and 16 nucleotides of the 30 hIFN~ coding region), containing a SacI restric~ion enzyme site, and 2) 5'-l~AAGCTI ACI~ACC ACCl~ATACI GCTAl~GA-3' (SEQ. }D.
NO.4) matching the last 20 nucleotides of the coding region of shIFN~R -and containing in addition 15 residues among which three nucleotides 35 code for the first amino acid residue of the hinge region in the HG~l ~

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molecule, and the others a splicing site followed by a HindIII
recognition site.

The PCR was performed using Taq Polymerase, under conditions 5 as described by the manufacturer (Perkin-Elmer, Cetus~ USA).

After phenol extraction and ethanol precipitation, the PCR product was resuspended in an appropriate buffer, and was digested by the SacI and HindIlI restriction enzymes by methods described in the art.
"~' " ~ '' '' The PCR fragment was then ligated into SacI-HindIII digested and -~
gel punfied pCD4-H~1 vector generating plasmid phuIFN~R-HGl.

Plasmid phuIFN~R-HG1 was cleaved with SacI and KpnI
I s enzymes. Then it was resuspended in an appropriate buffer and blunt-ended by Klenow Polymerase using standard procedures. The resulting ~ ~-fragment was purified and ligated into PvuII opened expression vector pN3 16. HindIII restriction enzyme digests of the resulting plasmids were performed and a construct harbouring approximately ~o 2.55 and 0.95 kb fragments was selected. This construct was designated pN3 1 6-huIFN~R-HGl . A schematic drawing of this construct is shown in Fig. 4. -~ -:. ' .~ ' .
Example 6 Construction of a chimeric human IFNyR-IgG3 (HG3! molecule ':
The same protocol as described in Example 5 was used with 30 the following exceptions:
:. :
The PCR 3' linker used was:

5'-AAl~CGAGG~ ACCITITATACTGCTA~ GA (SEQ ID NO.5) SacI
. . ..... .
:, ' ...:-:,..:, . :

- 19 21~

which creates one extra ~estriction site as indicated. The first 18 nucleotides downstream the SacI site match with the last 6 amino acids of soluble hIFN~R (positions 245 to 240 of the hIFN~R sequence, starting with Met as No. 1, as indicated in Aguet et al., supra~. The last two nucleotides match with the3rd and 2nd nucleotide of the codon for the amino acid in the position No. 239 of the hIFN~R.

Furthermore, a SacI digest was used instead of a SacI/HindIII
0 double digest of the PCR product and pCD4-H~3 was used as a source of the immunoglobulin gene part. The pCD4-H~3 was digested with SacI
and then the PCR fragment was ligated into the SaoI digested pCD4-H~3 vector. A construct containing an approximately 7.2 Kb KpnI /
SacII fragment was selected and designated phuIFN~R-HG3.
In addition, for ligation with expression vector pN3 16 the hII~N~R -IgG3 chimeric gene construet was digested with KpnI (which cleaves the DNA several nucleotides upstream of the SacI site which was originally introduced by the 5'-PCR primer; see Example 5 and SphI
20 prior to endpolishing with T4 DNA Polymerase.

HindIII / SacII restriction enzyme double digests of the resulting plasmids were performed and a construct yielding a 2.3 kb fragment ~;
among other bands was selected. This contruct was designated pN3 16-25 huIFN~R-HG3. A schematic drawing of this construct is shown in Fig. 5.

Example 7 Construction of a chimeric human ~FNyR-HClc molecule The same protocol as described in Example S was used with the following exceptions~

A human genomic ~-phage library prepared as described by 3S Sarnbrook et al., supra was used as a source of the genomic : ~, - ~ - 21~ 8 ~ ~
. .. ..
immunoglobulin c light chain constan~ region. A ~-clone containing the ~ `
human immunoglobulin K light chain gene was digested with HindIII
and BamHI enzymes and a 5.4 kb fragment containing a genomic C~c gene segment was purified and subsequently ligated into the s Bluescrip~ KS- plasmid (Stratagene, La Jolla~ Ca.) generating plasmid pBS-HC~
: .: ' , The plasmid phuIFN~R-HGl as desc,ribed in Example 5 was used ~ -as a source for the hIFN~ fragment. This plasmid was opened at the ~ -10 SacI site and blunt ends were created using T4 DNA polymerase. XhoI
recognition sites were added to the blunt ends by ligation of 2 synthetic palindromic oligonucleotides having the sequences: ~
~.
5'-CCGCICGAGCGG (Seq. ID No.6) 3'-GGCGAGCICGCC (Seq. ID No.7) These oligonucleotides were obtained from Promega, Madison.
The plasmids obtained after ligation of the XhoI recognition site containing the hIFN~R fragment was then digested with XhoI and -~0 HindIII.

The resulLing fragment was gel-purified and subsequently ligated into XhoI/HindIII-opened plasmid pBS-HClc described above generating plasmid phuIFN~R-HCx. -2s Plasmid phuIFN~R-HCK was digested with XhoI and BamHI
thereby excising the hybrid gene construct of about 6,2 kb in length.
This fragment was blunt ended and purified as described. ~-The resulting hIFN~R-HC1c chimeric gene construct was then ~;
ligated with PvuII-opened pN346 vector DNA yielding plasmid pN346-huIFNyR-HCk. A schematic drawing of this construct is shown in Fig. 6.
. i 3s Selection of the plasmid constructs with preferred orientation of ~;

'''.',~' ~"

- 21 - 2~

the inseIt was done as in Example 5, taking - into account differences in the fragmen~ length: the preferred construct being characterized by 0.85 and 6.8 kb, but not by 2.25 and 5.4 kb fragments (the remaining fragment being constant).
s Example B
Transfection of CHO-dhfr-cells and expression of chimeric hIPN~R-Ig polypeptides CHO-dhfr-cells were cultured in alpha-minimal essential medium (alpha - MEM, GIBCO/BRL, Paisley, Scotland) containing ~% Fetal Bovine Serum (F13S). The cells were plated into dishes (35 mm in diameter) to give a monolayer of approximately 90% confluence on the l5 day of transfection. Prior to transfection the culture medium was aspirated and the monolayers washed with phosphate buffered saline (PBS). pH7,2, containing 0.14 M NaCl and 15 mM phosphate. One ml of alpha-~M without PBS was added and the plates maintained at 37C
for three hours. The transfection mixture consisted of 10 ~I Lipofectin `~
20 (GIBCO, Gaithersburg, MD, U.S.A.~ g pSV2-Neo plasmid DNA
(Southern and Berg, J. Mol. Appl. Genet. 1, 327-341 ~1982]) and 5 llg of either huIFN~R-HGl, huIFN~R-HG3 or huIFN~R-HC,cDNA. The mixtures were added to the cells and after 5 hours I ml of alpha-MEM
containing 10% FBS was added to each dish. Then the cells were 25 cultured for 24 hours. For selection, the cells were trypsinized and resuspended in selection medium consisting of alpha minus MEM
(alpha-MEM missing nucleosides) and 1 mg/ml of G41g (GIBCOBRL, Paisley, Scotland) and plated into culture dishes (10û mm diameter). `~
The dishes were incubated for 12 days. Well developed colonies were 30 picked and transferred into 12-multiwell dishes. The supernatants of these clones were analyzed for the presence of the hIFN~R-Ig polypeptides by a specific ELISA (for details see Example 10, Figure 7 and Table I). The best producing clones were then amplified by successive passage in increasing concentrations of methotrexate (MTX, 35 Sigma, St. Louis, MO), beginning at 0.01 ~lM up to 80 IlM MTX. The : ,. i .
, . .... . .
., :

22 ~ 8 ; :j yield of the secreted hIF~R-Ig polypeptides reached the level of 10~
20 ~g/ml. ~ `-Exarnple 9 . ~ .
Purification of the hIFNyR-I~ polypeptides ~ ~
-; .-The hIFN~R-HG1 and huIFNyR-HG3 hybrid proteins were purified 0 from supernatants of CHO cells using a protein G column (5 ml Protein ~
G Sepharose 4 Fast Flow, Pharmacia LKB~ Uppsala). Hybrid protein - - ~ ~-hIFNyR-HCk was purified using an anti-human IFN~R receptor column which had been produced by coupling 60 mg of monoclonal antibody -yR46 to 3g CNBr activated Sepharose 4B (Pharmacia LKB~ Uppsala) 15 according to the manufacturer's instructions and packed into a column.
Alterna~ively, hIFN~R-HCk was affinity purified on an anti-human 1C Ig light chain affinity column produced as described above. Volumes of liters supernatant were loaded at a flow rate of 50 ml per hour. The proteins were eluded with 0.1 M Glycin-HCI, pH2.8. Fractions of l.S ml 20 were collected, analyzed by the ELISA for hIFN~R-Ig hybrid molecules ~ `;and by SDS-PAGE under reduced conditions. ~ - `
,: ~ .. ~ .. ...
Example 1 0 2s De¢eçtion of hlFNyR-I~_polypeptides bv ELISA
. .' .~ : ~ .-Supernatants of transfected CHO cells were assayed for thepresence of the hIFNyR-Ig polypeptides by using the monoclonal anti~
bodies yR46 and ~R89 in an ELISA that detects hIFNyR. These mono-30 clonal antibodies had been produced against the native human IFN~receptor as described by Garotta et al., in J. Biol. Chem. 265, 6908-6915 [199Oj. Both monoclonal antibodies bound only the non-reduced form of the human IFN~ receptor, recognized structural epitopes of the ~ ~ "
extracellular region of human IFNy receptor and in- hibited IFNy ~ `
35 binding to cell-bound human IFNy receptor. The antibody ~R46 is an ., ~

~, - 23 ~ 8 IgM that detects an epitope between amino acids 26-133 while the antibody yR89 is an IgGl that reacts with another epitope located between amino acids 70-210. Monoclonal antibodies ~R46 were affinity purified on an Anti-Mouse-IgM-Agarose column (Sigma, s St.Louis, MO) according to the manufacturer's instructions. Monoclonal antibodies ~R89 wese affinity purified on protein G column (Pharmacia LKB, Uppsala) according to the manufacturer's instructions.

A stock solution of antibody ~R46 was diluted with O.lM Na-10 phosphate buffer, pH 6.5 to give 10 llg/ml of pure antibody solution.
100 111 of this solution containing 1 ~g of pure antibody were distributed in Maxisorp microtiter plates (Nunc. Naperville, Il) to coat the flat-bottomed plastic wells. The coating reaction was performed ovemight at 15-25C. The residual binding capacity of plastic was then blocked by adding to each well 250 111 of blocking buffer (TRIS/HCl 0.2 M, pH 7.5) containing 10 mg/ml of Bovine serum albumine (BSA) and -~
by incubating the microtiter plates at room temperature for 24 hours (h) at 15-25C. The supernatants containing the hIFN^yR-Ig poly-peptides hIFN~R-HG3, hIFN~R-HGl or hI~R-HC,c expressed by trans~
fected CHO cells, were diluted 1:5, 1:50, 1:500, 1:5000 with test bu~fer ;
(TRIS/Acetate 0.05 M, pH 6.2) containing 5% Foetal Bovine serum (FSA). The standard solutions were diluted with test buffer to have 30 ng/ml, 10 ng/ml, 3 ng/ml, 1 ng/ml, 0.3 ng/ml and 0.1 ng/ml of soluble -hIFN~R, hI~R-HG3, hIFNyR-HG1 or hIFN~R-HC,c. Before the test, the 25 blocking buffer was poured off from the coated plates and a volume of 200 111 from each sample or standard dilution was distributed in 3 -wells of coated plates. Finally, 50 111 (100 ng/ml) ~R89 antibody con-jugated with peroxidase (Gallati et al., J. Clin. Chem. Biochem. 20, 907-914, [1982~) were added to each well. After 16-24 h at 15-25C, the plates were washed 6-8 times with deionized water and 200 ,ul of a -~
tetramethylbenzidine/H202 solution (O.OlM 3,3',5,5'-tetramethyl-benzidine, 0.08 M H22 in 10% aceton, 90% ethanol) dilu~ed 1:20 with substrate buffer (K-citrate, 0.03 M, pH 4.1) were added into each well.
After 10 minutes at 15-25C, the enzymatic reaction was stopped by 3S adding 100 111 of 5% sulphuric acid to each well and the colour was ~~" 24- '~

then red at 540 nm wave length using a photometer (Titertek Multiskan MC, from Flow Laboratories, Woodcock Hill, U.K.). Ihe concentration of hIFN yR-Ig polypeptides expressed by transfected CHO
cells was determined on the basis of the proper s~andard curve with S shIFN~R (for results see Table I below ana Fig.7).

Table I

DETER~ATION OF hI~yR-Ig POLYPEPIIDE CONCEN~TION BY A
SPECIFIC ELISA : .:

Receptor SampleReciprocal mOD llg/ml ::
protein of Dilution 450 nm : .
hIFN~R-HG3(*) 21 200 981 0.40 221,000 1 09 1 2.24 23S,000 578 5.60 :. .
hIFN~R-HGl(~) 11 200 862 3.48 :
121,000 288 4.~4 135,000 822 8.27 ~ -hIFN3~R-HC,c(*) 31 200 1 27 1 5.28 321,000 ~62 1.304 335,000 1 035 10.61 - ~
25shIFN~R(~) 1 200 437 0.11 -.
21,000 598 0.78 325,000 871 2.85 :~ .
~) Data calculated on hIFN~R-HG3 standard curve 30 ~) Data calculated on shIFN~R standard curve ,:

- 25 - ;~

Example l 1 Analysi~ of ~hIENyR~Ig polypeptides by~_SDS-PAGE and Imm~n~blo~ ~ -S
5DS-PAGE was performed on 7.5% gels. The samples were loaded in sample buffer with or without reducing agents for reducing or non- -reducing SDS-PAGE, respectively. Bands were stained wi~h Coomassie blue R-250 (Sigma, St.Louis, MO) or blotted to nitrocellulose and 10 visualized by monoclonal antibodies 3~R46 or ~R89 (Garotta et al., supra) and iodinated sheep Ig anti-mouse Ig (Amersham, Little Chalfont, U.K.) or by affinity purified an~ human IFN~ receptor rabbit antibodies (Valente et al., Eur. J. Immunol. 22, 2403-2412 [1992]) and iodinated donkey Ig anti-rabbit Ig (Amersham, Little Chalfont, U.K.).
5 The molecular weights of each of the hIFN^yR-Ig polypeptides are summarized in Table II and III below.

Example 12 20 Inhibition of IFNy binding to Raji cells bv hlFNyR-T~ polvpeptides The binding capacity of the hIFN~R-Ig polypeptides expressed in CHO cells was determined by inhibition of IFN~ binding to Raji cell ;
surface receptor. Solutions containing the hIFN yR-Ig polypeptides ~ ~ ~
25 (hI~N~R-HG3, hIFN~R-HGl and hIFN~R-HCk) were serially diluted wi~h ;
HBSS (Hank's basal salt solution; GIBCO/BRL, Paisley, U.K. and supplemented with 1% BSA and 15 mM HEPES) and 50 ~1 of each solution were distributed in triplicate into the wells of polystyrene U-bo~tomed microtiter plates (Costar, Cambridge, MA). 50 Ill iodinated 30 IFNy 2ng (200 U) of a preparation with 2x104 dpm/ng was added to each well after dilution in HBSS. Then 100 ~ll of HBSS medium containing 106 Raji cells (ATCC CCL 86) were added. After incubation for ~û minutes at 0C, the plates were centrifuged for S minutes at 100 x g, the supernatant with unbound radioactive IFN~ was discarded 3S and the cells were washed twice with washing buffer (Hank's basal ;

21~ .6g salt solution additioned with 0.1% BSA, 15 mM HEPES and 0.01% Triton X 100). Then the cells were resuspended in 200 ~,~l washing buffer~
transferred to flexible U-bottomed PVC microtiter plates ~Dynatech, Chantilly, VA) and once more centrifuged. The flexible plates were cu~
s and the radioactivity in each well was determined. IFN~ was iodinated using the chloramine T procedure (Greenwood et al., Biochem. J. 89, 1 1~-123 [1963]).
.':; ~.
Scatchard's analysis (Scatchard Ann. N.Y. Acad. Sci. 51, 660-672 lO [1949]) was performed with incleasing doses ~0.4-9 ng) of iodinated IFN~, incubated with 106 Raji cells. The amount of non-specific binding was determined in the presence of an excess of cold IFN~(10 ',Ig per ml). The capacity to inhibit the binding of IFN~to Raji cells of the ~ -differen~ receptor proteins is summarized in Table II below. -1s . ~: Table II

Inhibition Of IFN~ Binding To Raji Cells Receptor proteinMW Binding IC50 IC50 expressed kDa sites ng/ml nM
_ _ hIFNyR-HG3 184 2 24 0.13 -~
hIFN~R-HGl 170 2 29 0.17 ~ `
hIFN~R-HCx 82 1 42 0.51 -shIFN~R 31 1 100 3.23 , 30 MW is the molecular mass evaluated by non-reducing SDS-PAGE.

IC50 is the concentration that produces 50% inhibition of the binding of 3 ng of iodinated IFN~ to 106 Raji cells.

:

- 2~

Example 1 3 Inhibi~on of the an~iviral activitv of IFNy by hlFN~R-I~ polvpeptides ~ `~
s The binding capacity of hIFN~R-Ig polypeptides expressed in CH0 cells was determined by the inhibition of antiviral activity exerted by IFNy. Solutions containing hIFN~R-Ig polypeptides (hIFN~R-HG3, hIFN~R-HGl or hIFN~R-HC~) were serially diluted with Minimal 0 Essential Medium (MEM) from GIBC0/BRL supplemented with S% FBS.
50 111 of each dilution were distributed in triplicate to wells of tissue culture grade flat-bottomed microtiter plates (Costar). Then 25 solution containing 0.25 pg of IFN~ (1 U/ml of a preparation of IFN~
with a specific activity of 108 U/mg) were added to each well. After 1 s h at room temperature the IFN~ activity therein was determined by ~ `
the cytopathic effect (CPE) reading method according to Armstrong (Methods in Enzymology, S. Pestka ed., Vol. 78, page 38, Academic Press New York [1981]). Accordingly, 50 111 of a WISH cell (ATCC CCL
25) suspension (4x105 cells/ml) in 5% FBS-containing MEM were 20 placed in each well of the 96-well microplates and incubation was conducted in a carbon dioxide gas incubator (5% C02) at 37C. After 24 h the culture supernatants were discarded and 100 1ll of MEM medium -with 2% ~:BS and 1000 plaque forming units of Encephalomyocarditis (EMC) Virus (ATCC VR-129B) were added to each well. The plates were 25 incubated for 90 minutes at 37C, the medium discarded and -~-substituted with 100 ~11 2% FBS-containing MEM. About 24 h later, when the cells in wells not containing IFN~ show lOO~o CPE, the medium was discarded and the remaining adherent cells were washed with PBS. Then 100 111 of a crystal violet solution (1% crystal violet, 30 10% ethanol in water) were a~ded to each well. After 5 minutes, the unbound dye was removed by washlng with water before the stained monolayers were dried. Finally, the dye associated with the cells was extracted in 100 111 methylcellosolve and quantitated by reading the absorbance at 550 nm in a Titertek Multiskan MC (Flow Laboratories, 3~ Woodcock Hill, U.K.). Since the amount of dye is proportional to the : ..: . ' .. ... : .
~. ,.,.~

~` - 28 -number of cells present per well, the obtained absorbance readings :
are a measure for the protection of IFN~ against the viral cytopatic ;
effect. The capacity of the hIFN~R-Ig polypeptides hIFN~R-HG3, hIFN~R-HGl or hIFN~R-HC,~ to inhibit the antiviral activity exerted by ::
s IFNy is summarized in Table III below. ~ `-:
Ta~le ITI ~ :-Inhibition Of IFN~ AntiYiral Activity !: ' ~ ' _. ' '~ :. .
Receptor protein MW Binding IC50 IC50 ~ -expressed kDa sites llg/ml nM

~ ~
hIFNyR-HG318 4 2 2.0 1 1 hIFNyR-HGl 170 2 4.4 26 hIFNyR-HC,c82 1 nd nd shIFN~R 31 16.7 216 MW is the molecular mass evaluated by non-reducing SDS-PAGE

IC50 is the concentration that produces 50% inhibition of antiviral activity exerted by 10 U/ml (5 ng/ml) of IFNy. ~ .
2s nd = not done ' '~' ;~:''~' - ...
. . , ~.

29 - 211~ ~8 SEQUENCE LISTING

(1) GENERAL INFORMATION: ~ :
, S
~i~ APPLICANT:
~A) NAME: F. HOFFMANN-LA ROCHE AG ~.
(B) STREET: Grenzacher~tras~e 124 :
(C) CITY: Basle :: ; p (D) STATE: BS . ~
(E) COUNTRY: Switzerland :-(F) POSTAL CODE (ZIP): CH-4002 (G) TELEPHONE: 061 - 688 42 56 - m~
(H) TELEFAX: 061 - 68B 13 95 (I) TELEX: 962292t965542 hlr ch (ii) TITLE OF INVENTION: Chimeric Human Interferon-Gamma Receptor/Immunoglobulin Polypeptide~
(iii) NUMBER OF SEQUENCES: ll ~ -(iv) COMPUTER READABLE FORM: .~
(A) MEDIUM TYPE: Floppy disk : ~ :
(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Relea~e ~1.0, Ver~ion #1.25 (EPO) (vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: EP 93810170.6 (B) FILING DATE: 05-MAR-1993 . . ~ .
(2) INFORMATION FOR SEQ ID NO: 1: .~ ~
(i) SEQUENCE CHARACTERISTICS: . ~i -(A) LENGTH: 245 amino acid~
(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide :.
~iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO .. :
::
(v) FRAGMENT TYPE: N-terminal .-(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Met Ala Leu Leu Phe Leu Leu Pro Leu Val Met Gln Gly Val Ser Arg ~ :

Ala Glu Met Gly Thr Ala A~p Leu Gly Pro Ser Ser Val Pro Thr Pro . .
20 25 30 : `~
Thr A~n Val Thr Ile Glu Ser Tyr Aan Met A~n Pro Ile Val Tyr Trp ' '`~ '~,;
:.., "

' :, ,' ,' '' :

- ~ - 30 - 21 ~

Glu Tyr Gln Ile Met Pro Gln Val Pro Val Phe Thr Val Glu Val Lyq 50 55 60 :.
Asn Tyr Gly Val Ly~ A~n Ser Glu Trp Ile A~p Ala Cy~ Ile Asn Leu 65 70 75 80 .
Ser Hi~ Hi~ Tyr Cy3 Aqn Ile Ser A~p Hi~ Val Gly A~p Pro Ser A~n ' '' ' ' Ser Leu Trp Val Arg Val hy~ Glu Arg Val Gly Gln Ly~ Glu Ser Ala Tyr Ala Lys Ser Glu Glu Phe Ala Val Cy~ Arg Asp Gly Lys Ile Gly Pro Pro Lyq Leu A.~p Ile Arg Ly~ Glu Glu Ly~ Gln Ile Met Ile A~p 20 Ile Phe Hi~ Pro Ser Val Phe Val A~n Gly A~p Glu Gln Glu Val A~p ~
145 150 155 160 ~ ~ :
Tyr A3p Pro Glu Thr Thr Cy9 Tyr Ile Arg Val Tyr Asn Val Tyr Val 165 1~0 175 -:
Arg Met A~n Gly Ser Glu Ile Gln Tyr Ly~ Ile Leu Thr Gln Ly~ Glu .i.
180 185 190 ~::
A-~p Aqp Cy3 A~p Glu Ile Gln Cy~ Gln Leu Ala Ile Pro Val Ser Ser . .
195 200 205 ~ ~
Leu Asn Ser Gln Tyr Cy~ Val Ser Ala Glu Gly Val Leu Hi~ Val Trp ~ -.- 210 215 220 35 Gly Val Thr Thr Glu Ly~ Ser Lyq Glu Val Cy~ Ile Thr Ile Phe Asn 225 230 235 240 ::~

.
:
(2) INFORMATION FOR SEQ ID NO: 2: :
~, :
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 735 ba~e pair~
~B) TYPE: nucleic acid ~C) STRANDEDNESS: qingle (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(viii) POSITION IN GENOME:
(C) UNITS: bp - 31 - ;

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: .
- 5 ATGGCTCTCC TCTTTCTCCT ACCCCTTGTC ATGQGGGTG TGAGcAGGGc TGAGATGGGC60 ACCGCGGATC TGGGGCCGTC CTCAGTGCCT ACACQACTA ATGTTACAAT TGAATCCTAT 120 :.

1 0 ~" ' "
GTAGAGGTAA AGAACTATGG TGTTAAGAAT TCAGAATGGA TTGATGCCTG CATCA~TCTT 240 GTATGCCGAG ATGGAAAAAT TGGACCACCT AAACTGGATA T Q GAAAGGA GGAGAAGCAA 420 ~ .
AT QTGATTG A Q TATTTCA CCCTTCAGTT TTTGTAAATG GAGACGAGCA GGAAGTCG~T 480 '~
TATGATCCCG AAACTACCTG TTACATTAGG GTGTACAATG TGTATGTGAG A~TGAACGGA 540 AGTGAGATCC AGTATAAAAT ACTCACGCAG AAGGAAGATG ATTGTGACGA GATTCAGTGC 600 ~ :... ~.

TTACATGTGT GGGGTGTTAC AACTGAaAAG TCA~AAGAAG TTTGTATTAC CATTTTCAAT 720 ~ .
AG QGTATAA AAGGT 735 ~:. :

(2) INFORMATION FOR SEQ ID NO: 3:
~i) SEQUENCE CHARACTERISTICS~
~A) LENGTH: 35 base pair~ ~
~B) TYPE: nucleic acid ~ .
~C) STRANDEDNESS: ~ingle .: :.
~D) TOPOLOGY: linear ~.~ ... .
~ii) MOLECULE TYPE: DNA ~genomic) ~iii) ANTI-SENSE: YES :~
~viii) POSITION IN GENOME:
tC) UNITS: bp ..
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
AATTCGAGCT CGTAGCAGQ TGGCTCTCCT CTTTC 35 .. :
~2) INFORMATION FOR SEQ ID NO: 4:
~i) SEQUENCE CHARACTERISTICS: .:. .
(A) LENGTH: 35 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single . ;.~ :
(D) TOPOLOGY: linear . .
:,.. .
': , :' ...

-~ - 32 - ,~

~ii) MOLECULE TYPE: DNA (genomic~
(iii~ HYPOTHETICAL: NO
S : :
(iii) ANTI-SENSE: YES .~
. ~ .
(viii) POSITION IN GENOME:
(C) UNITS: bp (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 4: .-(2) INFORMATION FOR SEQ ID NO: 5:
ti) SEQUENCE CHARACTERISTICS: : -(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: 3ingle . . -(D) TOPOLOGY: linear :~ :

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ;~
(iii) ANTI-SENSE: YES .

(viii~ POSITION IN GENOME:
(C~ UNITS: bp - .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: ;

(2) INFORMATION FOR SEQ ID NO: 6:
4~
~i~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 12 ba~e pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) . ~(iii) HYPOTHETICAL: NO
~iii) ANTI-SENSE: YES

(viii) POSITION IN GENOME:
~C) UNITS: bp (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

^``` 33 2 ~ i A ~

(2) INFORMATION FOR SEQ ID NO: 7:
S ~ . .
~i) SEQUENCE CHARACTERISTICS: :~
~A~ LENGTH: 12 ba3e pairs : .:
tB) TYPE: nucleic acid ~C) STR~NDEDNESS: single .. ;:::
(D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) (iii) HYPOTHETICAL: NO . - .
' ~iii) ANTI-SENSE: YES .
(viii) POSITION IN GENOME:
(C) UNITS: bp '~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: . :`~

(2) INFoRMaTIoN FOR SEQ ID NO: 8:
~i) SEQUENCE CHARACTERISTICS: .
(A) LENGTH: 33 base pairs : .
~B) TYPE: nucleic acid ~C) STR~DEDNESS: ~ingle ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic~
(iii) HYPOTHETICAL: NO
(iii) ANTI~SENSE: NO
(v$ii) POSITION IN GENOME:
~C) UNITS: bp ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: :~
GGGCTCGAGA-CCTTATGGGA CTTTCCTACT TGG 33 ..
: :~
~2) INFORMATION FOR SEQ ID NO: 9:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 32 base pairs ~B) TYPE: nucleic acid : : :
~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) 5 5 ' : '- ,: :
(iii) HYPOTHETICAL: NO

', .."'~' .

: .,.~: :.' ~ ; . ~ '; . ~ !

- 34 ~ 3 ..
(iii) ANTI-SENSE: NO
(viii) POSITION IN GENOME:
(C) UNITS: bp (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 ba~e pair~
(B~ TYPE: nucleic acid (C) STRANDEDNESS: aingle (D) TOPOLOGY: linear -(ii) MOLECULE TYPE: DNA (genomic) .
~iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO

(viii) POSITION IN GENOME:
(C~ UNITS: bp (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 10: ~ -(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 baqe pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
4S `
~iii) ANTI-SENSE: NO
(viii) POSITION IN GENOME:
(C) ,UNITS: bp (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

~ :

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Claims (13)

1. A DNA sequence comprising a combination of two partial DNA
sequences, one partial sequence coding for a fragment of the human interferon-.gamma. receptor which fragment binds human interferon-.gamma. and the other partial sequence coding for part or all of the constant domains of human immunoglobulin heavy or light chains such as IgG, IgA, IgM, IgE or Ck.

2. A DNA sequence according to claim 1, wherein one partial sequence codes for a fragment of the human interferon-.gamma. receptor which binds human interferon-.gamma. and the other partial sequence codes for part or all of the constant domain of IgG, preferably IgG1 and IgG3, or for part or all of the constant domain of Ck.

3. A vector comprising a DNA sequence as claimed in claim 1 or 2.

4. A vector as claimed in claim 3 capable of directing expression in a compatible prokaryotic, insect or mammalian host cell.

5. A prokaryotic, mammalian or insect host cell transformed with a vector as claimed in claim 3 or 4.

6. A recombinant protein coded for by a DNA sequence as claimed in claim 1 or 2.

7. A recombinant protein as claimed in claim 6 selected from the group consisting of hIFN.gamma.R-HG1, hIFN.gamma.R-HG3 or hIFN.gamma.R-HCx.

8. A process for the production of a protein as claimed in claim 6 or 7 which process comprises cultivating a transformed host as claimed in claim 5 in a suitable medium and isolating said protein.

9. A pharmaceutical composition which contains one or more compounds according to claim 6 or 7, if desired in combination with additional pharmaceutically active agents and pharmaceutically acceptable carrier materials.

10. The use of a compound according to claim 6 or 7 for the preparation of pharmaceutical compositions.

11. The use of a compound according to claim 6 or 7 for the preparation of a medicament for the treatment of illnesses especially autoimmune diseases, chronic inflammations, delayed hypersensitivity, allotransplant rejections, multiple sclerosis and fulminant hepatitis, inflammatory neurological diseases, and neurological complications of AIDS, poliovirus infections, lyme disease and septicemia.

12. A recombinant protein according to claim 6 or 7 whenever prepared by a process as claimed in claim 8 or by an obvious equivalent thereof.

13. The invention as hereinbefore described, especially with reference to the examples.