MXPA99006595A - Novel ifn receptor 1 binding proteins, dna encoding them, and methods of modulating cellular response to interferons - Google Patents

Abstract

Novel proteins IR1B1 and IR1B4 have been isolated which bind to the type I IFN receptor IFNAR1 and function in the cellular response to IFNs. DNA encoding such proteins in either the sense or anti-sense orientation can be administered to either enhance or inhibit the cellular response to IFNs. Antibodies to the proteins can be used for isolation of the new protein or for immunodetection thereof.

Description

NOVEDOUS PROTEINS OF UNION OF IFN RECEIVER 1, DNA THAT CODIFIES THEM, AND METHODS TO MODULATE THE CELLULAR RESPONSE TO INTERFERONS FIELD OF THE INVENTION The present invention relates generally to the molecular mechanisms of action of interferon, and more specifically, to novel interferon receptor 1 binding proteins, recombinant DNA molecules encoding them, and methods for modulating the cellular response to interferon.

BACKGROUND OF THE INVENTION Type I interferons (subtypes of IFN-a and -ß) produce pleiotropic effects on cells, such as inhibition of viral replication (antiviral effect), inhibition of cell proliferation (antitumor effects), and modulation of activities immune cells (immunoregulatory effects). These multiple effects of interferons (IFNs) are correlated with morphological and biochemical modifications of cells (Revel, 1984, for review). Interferons exert their activities through species-specific receptors. Two chains of transmembrane receptors have been identified for type I IFNs: IFNAR1 (Uze et al., 1990) and IFNAR2-2 (or IFNAR2-C, Domanski et al., 1995). The transduction of the signal generated by IFN-a and -ß,? involves tyrosine kinases from the Janus kinase family (Jak), and transcription factors from the Stat family (Darnell et al., 1994). The proteins of the Jak-Stat pathways are activated by binding to the intracytoplasmic (IC) domains of the IFNAR1 and IFNAR2 receptor chains. Among the proteins that bind to the IFNAR1-IC domain are tyk2 and Stat2 (Abramovich et al., 1994). Stat2 would then recruit Statl to form the ISGF3 transcription complex induced by IFN, which activates genes induced by IFN (Leung et al., 1995). Transcription complexes containing Stat3 are also induced by IFN-β (Harroch et al., 1994), and binding of Stat3 to IFNAR1-IC dependent on IFN was observed (Yang et al., 1996). Tyrosine phosphatase PTP1C and D is reversibly associated with IFNAR1 after the addition of IFN (David et al., 1995a). In addition, two serine / threonine protein kinases, the 48 kDa ERK2 MAP kinase (David and others 1995b), and AMPc-activated protein kinase A (David et al., 1996) bind to the next 50 IFNAR1-IC residues to the isolated membrane. Therefore, the IC domains of the IFN receptor type I function as the coupling sites for multiple proteins that function to generate and regulate the biological effects of the IFNs on the cells. The analysis of two hybrids in yeast is a powerful method to identify new proteins that bind to defined polypeptide sequences (Fields and Song, 1989). Briefly stated, the analysis of two hybrids is carried out by transfecting yeast cells with (a) a plasmid DNA in which the defined polypeptide (bait) is fused with the DNA binding domain of the transcription factor Ga14, and (b) a cDNA library is fused to the activation domain of Ga14 in a pACT plasmid. Yeast cells transfected with a cDNA encoding a protein that binds to the polypeptide bait will then reconstitute the activity of Ga14. The presence of said protein that binds to the polypeptide bait is revealed by the expression of the activity of an enzyme, such as β-galactosidase, from a GAL1-lacZ construct that is preferably introduced into the genome of the yeast. From yeast clones that are positive in this test, it is possible to isolate the pACT plasmid, to determine the nucleotide sequence of its insertion and to identify the protein it encodes. This method has allowed the identification of novel proteins that interact with the IC domain of cytokine receptors (Boldin et al., 1995). It is not intended that the citation of any document hereof be an acknowledgment that said document is pertinent to the prior art, or that it be considered as a material for the patentability of any claim of the present application. Any statement regarding the content, or the date of any document, is based on the information available to applicants on the filing date, and does not constitute an acknowledgment of the accuracy of that statement.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to two novel human proteins, designated herein as IR1B1 and IR1B4, which have been identified as binding proteins of IFN receptor 1 (IFNAR1), and to the DNA encoding these two proteins. Each of the IR1B1 and IR1B4 proteins interacts with the intracytoplasmic domain (IC) of IFNAR1, and mediates cellular responses to interferon. The present invention is directed to a recombinant DNA molecule that contains a nucleotide sequence that codes for any of the IR1B1 or IR1B4 proteins, or fragments thereof, as well as the proteins encoded thereby. In recombinant DNA molecules, the nucleotide sequence encoding the IR1B1 or IR1 B4 protein, or fragments thereof, is operably linked to a promoter in a sense orientation or an antisense orientation. By administering the recombinant DNA molecule containing a promoter operably linked to the nucleotide sequence coding for a novel binding protein of IFNAR1 in the sense orientation directly in tumors, the response to therapy with exogenous interferon in the cancer treatment. In addition, the present invention also relates to a method for prolonging the survival of tissue grafts by introducing the recombinant molecule containing a promoter operably linked to the nucleotide sequence encoding a novel IFNAR1 binding protein, or fragments thereof. , in the antisense orientation in the graft tissue before grafting it into the patient. Thus, the present invention also relates to pharmaceutical compositions containing said DNA, RNA or protein, and therapeutic methods for using same. The present invention also relates to antibodies specific for the novel proteins of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the interaction of IR1 B1 with the IFNAR1-IC domain, measured by the genetic interaction analysis of two hybrids in yeast. In the lower portion enclosed in a box of the figure, the insertion of cDNA in pACT is indicated, when it is combined with several "baits". Figure 2 shows the interaction of IR1B4 with the IFNAR1-IC domain, measured by genetic interaction analysis of two hybrids in yeast. In the lower portion enclosed in a box of the figure, the insertion of cDNA in pACT is indicated, when it is combined with several "baits". Figure 3 shows the nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of IR1B1. Figure 4 shows the homology and alignment of the amino acid sequence of IR1B1 (SEQ ID NO: 2) with the amino acid sequences of two calcium binding proteins, calcineurin B (abbreviated as CALB; SEQ ID NO: 3) and calternin (abbreviated as CATR; SEQ ID NO: 4). The identical amino acids in IR1B1 and CALB or between CALB and CATR are shown by the symbol "|" between them. The identity between IR1B1 and CATR, but not with CALB, is shown by the symbol ":" between them. The regions shown in bold are the loop-like EF helical-loop-helix domains. Figure 5 shows Northern blots of the IR1 B1 messenger RNA and 18S rRNA (lower band) in human myeloma U266S cells hybridized with IR1B1 cDNA, and the rapid and transient induction of IR1B1 after treating the cells with IFN-a8 or IFN -β for 2 hours or 18 hours. The first band is a control without IFN treatment after 2 hours. Figures 6A and 6B are SDS-PAGE bands showing the in vitro interaction of IR1B4 with the isolated IFNAR1-IC domain (FIG 6A), and with membrane extracts of human U266S and U266R cells (Fig. 6B). In Figure 6A, translation products marked with [35 S] methionine with or without flag-IR1B4 transcripts in vitro were immunoprecipitated (10 μl) with M2 anti-flag links (bands 1 and 4), or were reacted (50 μl) with glutathione links coupled to GST fused to the IFNAR1-IC domain of 100 amino acids in length (lanes 2 and 5), or coupled only to GST (lanes 3 and 6). After incubation overnight at 4 ° C (final volume of 100 μl), the links were washed, and the proteins eluted with SDS were boiled under reducing conditions before SDS-PAGE. In Figure 6B, U266S cells (lane 1) or U266R cells (lane 2) were removed with Brij pH regulator and antiproteases (Abramovich et al., 1994), and 0.35 ml (107 cells) were incubated with 75. translation marked with [35S] methionine transcribed flag-IR1 B4 overnight at 4 ° C. Anti-IFNAR1 mAb R3 immobilized on G protein links (25 μl) was added for 2.5 hours, washed in Brij pH buffer, and eluted with SDS, boiled, and the reduced proteins analyzed by SDS-PAGE. A control with M2 anti-flag links was run as shown previously (band 3). The dehydrated gels were visualized in a Phosphor-lmager apparatus. In the first three bands of Figure 6a, IR1B4 messenger RNA was not added to the in vitro translation reaction. In the second three bands of Figure 6a, the messenger RNA encoding the IR1 B4 protein fused to the flag protein was translated into an in vitro system. Figure 7 shows the nucleotide sequence (SEQ ID NO: 7) and the deduced amino acid sequence (SEQ ID NO: 8) of IR1 B4. Figure 8 shows the amino acid alignment of IR1 B4 (SEQ ID NO: 8) and PRMT1 (SEQ ID NO: 9), and their differences. Figure 9 shows the amino acid alignment of IR1 B4 and HCP-1 (SEQ ID NO: 10), and their differences. Figure 10 shows a methyltransferase test. Extracts of U266S cells were reacted with links coated with protein A and anti-IFNAR1 antibody (band 1) or with protein A alone (band 2). The activity of methyltransferase was measured by labeling the histones with 14C (methyl) -S-adenosyl methionine and analyzing the radioactivity in the histone band by electrophoresis on SDS-PAGE. Figure 11 shows a test of protein-arginine methyltransferase activity in U266S cells. In band 1, the protein-arginine methyltransferase activity of human U266S cells was measured by methylation of peptide R1, having the sequence of SEQ ID NO: 11. In band 2, an antisense oligonucleotide of SEQ ID NO: 12, complementary to nucleotide sequence 12 to 33 around the initiation codon of the IR1B4 cDNA, was added. In band 3, the corresponding sense oligonucleotide was added. It is noted that the antisense oligonucleotide substantially inhibits the activity of protein-arginine methyltransferase, while the sense sense oligonucleotide has little effect. Figure 12 is a graph showing growth inhibition of human U266S cells in response to treatment with IFN-β in the presence or absence of the antisense oligonucleotide used in Figure 11 (AS-1), the corresponding sense oligonucleotide (S-3) ), and another antisense oligonucleotide directed to the middle part of the IR1B4 cDNA (AS-2). The cell density was quantified by a color test with Alamar blue (see Example 7), and the reduction in cell density was calculated in percentage of untreated control wells, and plotted as growth inhibition.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery of two novel human proteins that interact with the intracytoplasmic domain (IC) of the IFNAR1 chain of interferon-type 1 receptor (IFN-α, β or co), and which are designated herein as IFN receptor binding protein 1 (IR1 B1) and IFN receptor binding protein 4 (IR1B4). The interaction of these two novel proteins with IFNAR1 was demonstrated in a genetic test of two hybrids in yeast where the transfection of strain SFY526 of yeast reporter (Bartel et al., 1993) with cDNA of IR1B1 or IR1B4 fused to the activation domain of Gal4, resulted in β-galactosidase activity only when the domain of IFNAR1-IC (fused to the Gal4 DNA binding domain) was used as a bait. The sequence of the IR1B1 cDNA codes for a 191 amino acid polypeptide. Searches of computer sequence databases revealed that IR1B1 is a novel protein showing remarkable homology, eg, calcium binding sites (EF loops), with calcium binding proteins, calcineurin ß and caltactrin Calcineurin ß (Guerini et al., 1989) is a 19 kDa subunit of a serine / threonine phosphatase that plays a key role in the activation of the translocation of transcription factor NFAT into the nucleus of T lymphocytes, and which is inhibited by drugs immunosuppressants such as cyclosporin. Caltactrine (Lee and Huang, 1993), a 21 kDa protein, is a protein associated with the cytoskeleton and present in centrosomes, and intervenes in the movement of chromosomes during mitosis, and more generally in the centers of organization of the microtubules Thus, the novel IR1 B1 protein is a new member of the calcineurin and caltactrine family of calcium-regulated proteins. It was surprisingly found that the gene for IR1B1 is rapidly activated in human cells by treatment with IFN. Thus, this is the first example of a calcium binding protein that is induced by IFN. Since calcium ions regulate cell morphology, adhesion and division, the modulation of IR1 B1 activity in cells can affect the response of normal and malignant cells to IFN. The function of IR1B1 to mediate the action of IFN in cells is supported by the interaction of IR1B1 with the IC domain of an IFN receptor chain. Although it was found that IR1 B4, like IR1 B1, is a novel protein determined by searching computer sequence databases, it was also found that IR1B4 has sequence homology with enzymes that use S-adenosyl methionine to methylate arginine residues in proteins, which are designated as arginine protein methyltransferases (PRMT1; Kagan and Clarke, 1994; Lin et al., 1996). It was found that IR1B4 binds directly to the IC domain of IFNAR1 in vitro, and the constitutive association of PRMT activity with the IFNAR chain of the IFN-a receptor, β isolated from human cells was demonstrated by histone methylation. When antisense oligodeoxynucleotides were added from the IR1B4 cDNA to cultures of human cells, depletion of PRMT activity was observed in the cell culture. The human myeloma cells that were treated in this manner showed a very reduced response to IFN when this was measured by growth inhibition. Therefore, IR1B4 / PRMT intervenes in the pathway by which the IFN receptor causes growth inhibition in tumor cells, and also intervenes in other functions of the IFN receptor. Known substrates of PRMT include numerous RNA and DNA binding proteins, and in particular heterologous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are involved in the transport of messenger RNA from the nucleus to the cytoplasm, the alternative splicing of the pre-mRNA, and posttranscription controls (Liu and Dreyfuss)., nineteen ninety five). Accordingly, novel human IR1B4 / PRMT proteins and cDNAs, which were discovered by their association with the IFN receptor, can be used to modify the response of human or animal cells to IFN. A recombinant DNA molecule according to the present invention contains a nucleotide sequence encoding the IR1B1 or IR1B4 protein, or a fragment thereof, and can be used to increase the cellular response to IFN by increasing the expression of the IR1B1 cDNA. or IR1B4 or to decrease the cellular response to IFN by decreasing the expression of IR1 B1 or IR1 B4 proteins with antisense RNA molecules. Increased in vivo expression of the IR1B1 or IR1B4 cDNA would be useful in cancer therapy, where the increased cellular response to IFN would result in a decrease in the growth of malignant cells and an improved response to therapy with exogenous IFN. To obtain increased in vivo expression of IR1 B1 or IR1 B4 at the target site for an increased cellular response to IFN, expression vectors containing IR1 B1 or IR1B4 cDNA operably linked in a sense orientation can be directly injected into the target site. to a strong constitutive promoter, as occurs in brain tumors or metastatic tumor nodules (for example, melanomas or breast cancer). Conversely, decreased in vivo expression of IR1B1 or IR1B4 proteins would be useful for prolonging the survival of tissue grafts, since the rejection of these grafts in the host is mediated by histocompatibility antigens (MHC class I), whose synthesis depends on the stimulus of IFN. When the IR1B1 or IR1B4 cDNA, or fragments thereof, carried in a suitable vector and operably linked in the antisense orientation to a promoter, are introduced into the cells of the tissue to be grafted, the expression of the antisense RNA leads to the degradation of the Messenger RNA of IR1B1 or IR1B4 (or RNA sense for IR1 B1 / IR1 B4), and a consequent decrease in cellular levels of protein IR1B1 or IR1 B4. The antisense RNA is transcribed from a promoter to the 5 'end operably linked to a coding sequence oriented in the antisense direction, ie, opposite to the normal direction or sense of the DNA and its sense RNA transcribed (messenger RNA). The expression of antisense RNA complementary to sense RNA is a powerful way to regulate the biological function of RNA molecules. By forming a stable duplex between the sense RNA and the antisense RNA, the normal or sense RNA transcript becomes inactive or non-translatable. Recombinant DNA molecules, as embodiments of the present invention, contain the IR1 B1 or IR1B4 cDNA, or fragments thereof, operably linked to a promoter in a sense or antisense orientation. The term "promoter" is used to encompass a double-stranded DNA or RNA sequence, which is capable of binding to RNA polymerase and promoting the transcription of an "operably linked" nucleic acid sequence. Thus, a DNA sequence would be operably linked to a promoter sequence if the promoter is capable of transcribing the DNA sequence, regardless of the orientation of the DNA sequence. The types of promoters that are used to control transcription can be any of those that are functional in the target / host cells. Examples of functional promoters in mammalian cells include the SV40 early promoter, the adenovirus major late promoter, the herpes simplex thymidine kinase (HSV) promoter, the Rous sarcoma virus (RSV) LTR promoter, the immediate early promoter of human cytomegalovirus (CMV), the LTR promoter of the mouse mammary tumor virus (MMTV), the interferon-β promoter, the heat shock protein 70 promoter (hsp70), as well as many others well known in the art. A promoter operably linked to the cDNA of IR1B1 or IR1B4 in sense orientation for the expression of the IR1B1 or IR1B4 protein, is preferably a strong constitutive promoter. This allows a high level of IR1 B1 or IR1B4, regardless of the presence of endogenous cellular mechanisms to regulate the expression of IR1B1 or IR1 B4. Similarly, the promoter, which is operably linked to the cDNA of IR1B1 or IR1 B4 in the antisense orientation, is preferably a strong promoter, such as the promoter present in the Epstein-Barr virus (EBV) that regulates the region that allows high levels of antisense RNA expression (Deiss and Kimchi, 1991). The antisense sequence is preferably only expressible in the host / target cells, which are preferably human cells, and the expressed antisense RNA must be stable (i.e., not undergo rapid degradation). The antisense RNA should specifically hybridize only with the sense messenger RNA expressed in the target / host cells, and form a stable double-stranded RNA molecule that is essentially non-translatable. In other words, the antisense RNA expressed in host / target cells prevents expressed sense messenger RNA from being translated into the IR1B1 or IR1B4 proteins. The antisense sequence carried by the vector may carry the complete cDNA sequence of 1R1B1 or IR1B4, or only a portion thereof, as long as the antisense portion is capable of hybridizing to the sense messenger RNA and preventing its translation into the IR1B1 proteins or IR1B4. Accordingly, an "antisense" sequence as used throughout the specification and claims is defined as the entire antisense sequence or a portion thereof that is capable of being expressed in transformed / transfected cells, and which is also capable of specifically hybridizing with the "sense" messenger RNA of IR1 B1 or IR1 B4 to form a non-translatable double-stranded RNA molecule. It is not necessary that the antisense sequence hybridizes to the full length of the messenger RNA of IR1 B1 or IR1 B4. Rather, it can hybridize to selected regions, such as the 5 'untranslated non-coding sequence, the coding sequence, or the 3' untranslated sequence of the "sense" messenger RNA. Preferably, the antisense sequence hybridizes to the 5 'coding sequence and / or the 5' non-coding region, such as occurs at cap and initiation codon sites, since it has been observed with many examples of antisense oligonucleotides to be identified the initiation codon is more effective, while identifying the internal sequences within the coding region is not as effective (Wickstrom, 1991). The efficacy of an antisense sequence to prevent translation of the sense messenger RNA from IR1B4 can be easily tested in a test for protein-arginine methyltransferase activity in U266S cells, as described in example 7. In view of the size of the genome of mammals, the antisense sequence of IR1B1 or IR1B4 is preferably at least 17, more preferably at least 30 pairs in length. However, the shorter sequences may still be useful, ie, fortuitously they do not hybridize with other mammalian sequences, or said "cross-hybridization" does not interfere with the metabolism of the cell in a way and to a degree that prevents the achievement of the objectives of this invention.

The preferred hybridization target and the preferred antisense sequence length are easily determined by systematic experimentation. Standard methods such as those described in Ausubel et al., Eds. Current Protocols in Molecular Biology, Greene Publishing Assoc., New York, NY, 1987-1996, and Sambroo et al., Molecular Cloninq: A Laboratorv Manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), they can be used to systematically remove an increasingly large portion of the antisense sequence of the vector. In addition to the full-length antisense sequence, a series of stepped deletions may be generated, preferably at the 5 'end of the antisense sequence. This creates a series of truncated antisense sequences that continue to be still complementary to preferably the 5 'end of the sense messenger RNA and, as a result, still forms an RNA molecule that is double stranded at the 5' end of sense messenger RNA (complements the 3 'end of an antisense RNA), and it remains non-translatable. In addition, they can be chemically synthesized and easily introduce antisense oligonucleotides, such as oligonucleotide AS-1 (SEQ ID NO: 12), into a vector operable with a promoter to be used to decrease in vivo cell expression of IR1B1 O proteins. IR1B4. The vectors of the present invention can be any suitable eukaryotic or prokaryotic vector normally used to transfect mammalian cells, such as episomal, replicable or chromosomally integrable vectors well known in the art. A particularly preferred vector for the expression of the antisense RNA of IR1 B1 or IR1B4 is the episomal plasmid containing the regulatory region of the Epstein-Barr virus (Deiss and Kimchi, 1991) to function as the promoter that is operably linked to the cDNA of IR1 B1 or IR1 B4 arranged in an antisense orientation with respect to this regulatory region. The use of antisense vectors and oligonucleotide phosphorothioates is described in Annals of the New York of Sciences: Gene Therapy for Neoplastic Diseases, eds. B. E. Huber and J. S. Lazo, Vol. 716, 1994 (see for example, Milligan et al., Pp. 228-241). In accordance with the present invention, the survival of grafted tissues or organs in a patient in need of said graft can be prolonged by decreasing the cellular response to IFN. The rejection of the graft tissue is mediated by histocompatibility antigens, the synthesis of these MHC class I antigens being dependent on the IFN stimulation. Thus, a decrease in the cellular response to the stimulation by IFN will prolong the survival of the graft tissue. The method for prolonging the survival of the tissue graft according to the present invention involves introducing into the cells of a tissue or organ to be grafted into a patient, a recombinant DNA molecule containing a cDNA sequence of IR1 B1 or IR1 B4. , or fragment thereof, operably linked to a promoter in the antisense orientation, whereby the antisense RNA of IR1B1 or IR1B4 is expressed in said transfected / transformed cells. The recombinant DNA molecule can be introduced into the cells of a tissue or organ in a manner well known in the art which will be suitable for this purpose.

After introducing the recombinant DNA molecule into the cells of the tissue or organ, the tissue or organ can be grafted onto the patient in need of said graft. A pharmaceutical composition containing a recombinant DNA molecule, which is an expression vector and possesses IR1B1 or IR1B4 cDNA operably linked to a promoter in a sense orientation, can be directly injected into tumors, e.g., brain tumors and tumor nodules metastatic, to make cells within these tumors exhibit a better response to therapy with exogenous IFN as cancer treatment. The improved cellular response to exogenous IFN therapy would lead to the inhibition of malignant cell growth. Gene transfer in vivo or ex vivo is well reported, that is, in Annals of the New York Academy of Sciences: Gene Therapy for Neoplastic Diseases, Vol. 716, 1994; see, for example, "Direct Gene Transfer for the Understanding and Treatment of Human Disease" by GE Plautz on pages 144-153, and "Mechanisms of Action of the p53 Tumor Suppressor and Prospects for Cancer Gene Therapy by Reconstitution of p53 Function" by Roemer and others, on pages 265-282. Methods for inserting recombinant DNA molecules into cells of a tissue or organ to be grafted, or of a tumor, include adenovirus, retrovirus, adeno-associated virus (AAV) vectors, as well as direct injection of DNA or injection of oligonucleotides -liposomes. Clinical tests in which retroviral vectors are injected into brain tumors, or where adenoviruses are used to infect the cells of the upper respiratory tract of a patient with cystic fibrosis are well known. It is intended that pharmaceutical compositions comprising the recombinant DNA molecule encoding the IR1 B1 or IR1 B4 cDNA, or a fragment thereof, in a sense or antisense orientation with respect to an operably linked promoter, include all compositions in where the recombinant DNA molecule is contained in an effective amount to achieve its intended purpose. In addition, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers or excipients which stabilize the recombinant DNA molecule, or facilitate its administration. Another embodiment of the present invention is directed to molecules that include the antigen-binding portion of an antibody specific for IR1 B1 or IR1B4 binding proteins to IFNAR1, or fragments thereof, for use in diagnostics, such as immunodetection methods for to test the level of IR1 B1 or IR1 B4 proteins in tumor tissue obtained from biopsies, or to be used in the purification of the protein by affinity chromatography. The term "antibody" means that it includes polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies (anti-ld), single chain antibodies, and humanized antibodies produced in recombinant form, as well as active fractions of they are provided by any known technique such as, but not limited to, enzymatic digestion, peptide synthesis or recombinant techniques. Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antigen-specific antibodies, the population of which contains substantially similar epitope-binding sites. The mAbs can be obtained by methods well known to those skilled in the art. See for example Kohler and Milstein, Nature 256: 495-497 (1975); patent of E.U.A. No. 4,376,110; Ausubel et al., Eds., Cited above, Hariow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y. (1992, 1993), the contents of which are hereby incorporated by reference in their entirety. Said antibodies can be of any class of immunoglobulin including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma that produces a mAb of the present invention can be cultured in vitro, in situ or in vivo. The production of high titers of mAbs in vivo or in situ makes this the currently preferred production method. Chimeric antibodies are molecules whose different portions are derived from different animal species, such as those having the variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are used primarily to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher hybridoma yields, but greater immunogenicity in humans, so that human chimeric mAbs are used / murine Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Nati, Acad. Sci. USA 81: 3273-3277 (1984); Morrison et al., Proc. Nati Acad. Sci. USA 81: 6851-6855 (1984); Boulianne et al., Nature 312: 643-646 (1984); Cabiliy others, European patent application 125023 (published November 14, 1984); Neuberger et al., Nature 314: 268-270 (1985); Taniguchi et al., European patent application 171496 (published February 19, 1985); Morrison et al., European patent application 173494 (published March 5, 1986); Neuberger et al., PCT application WO 8601533 (published March 13, 1986); Kudo et al., European patent application 184187 (published June 11, 1986); Morrison et al., European patent application 173494 (published March 5, 1986); Sahagan et al., J. Immunol. 137: 1066-1074 (1986); Robinson et al., International patent application WO 9702671 (published May 7, 1987); Liu and others, Proc. Nati Acad. Sci. USA 84: 3439-3443 (1987); Sun and others, Proc. Nati Acad. Sci. USA 84: 214-218 (1987); Better et al., Science 240: 1041-1043 (1988); and Harlow and Lane, Antibodies: A Laboratory Manual, cited above. An anti-idiotypic antibody (anti-ld) is an antibody that recognizes unique determinants generally associated with the antigen binding site of an antibody. An Id antibody can be prepared by immunizing an animal of the same species and the same genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-ld is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody, producing an antibody to said idiotypic determinants (the anti-ld antibody). See, for example, the patent of E.U.A. No. 4,699,880. The anti-ld antibody can also be used as a "immunogen" to induce an immune response in yet another animal, producing the so-called anti-anti-ld antibody. The anti-anti-ld can be epitotically identical mAb that induced the anti-ld. In this way, by using antibodies to the diotypic determinants of a mAb, it is possible to identify other clones that express antibodies of identical specificity. It should be understood that the antibodies of the present invention can be intact antibodies, such as monoclonal antibodies, but that it is the binding site of the epitope of the antibody that provides the desired function. In this way, apart from the intact antibody, proteolytic fragments thereof can be used, such as the Fab or F (ab ') 2 fragments. The Fab and F (ab ') 2 fragments lack the Fc fragment of the intact antibody, are more rapidly removed from the circulation and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucí, Med. : 316-325 (1983)). Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments) - In addition, the DNA encoding the variable region of the antibody can be inserted in other antibodies to produce chimeric antibodies (see, for example, US Pat. No. 4,816,567) or in T cell receptors to produce T cells with the same broad specificity (see Eshhar, Z. et al., Br. J. Cancer Suppl., 10: 27-9, 1990; Gross, G. et al., Proc. Nati, Acad. Sci. USA, 86: 10024-8, 1989). Individual chain antibodies can also be produced and used. The single chain antibodies can be single chain mixed polypeptides having antigen binding capabilities and comprising a pair of amino acid sequences homologous or analogous to the variable regions of a heavy and light immunoglobulin chain (VH-V | linked or individual chain Fv). Both VH and VL can copy natural sequences of monoclana.es antibodies or one or both of the chains can comprise a CDR-FR construct of the type described in the US patent. 5,092,513. Separate polypeptides analogous to the variable regions of the light and heavy chains are held together by a polypeptide linker. Methods of producing such individual antibodies, particularly when the DNA encoding the polypeptide structures of the V and VL chains are known, can be achieved according to the methods described, for example, in the US patents. 4,946,778, 5,091, 513 and 5,096,815. Thus, with the term "a molecule that includes the antigen-binding portion of an antibody" is intended to include not only immunoglobulin molecules intact from any isotope and generated by any animal cell line or microorganism, but also the fraction t / 24 reactive thereof, including, but not limited to, the Fab fragment, the Fab 'fragment, the F (ab') 2 fragment, the variable portion of the light and / or heavy chains thereof and the antibodies chimeric or individual chain that incorporate said reactive fraction, as well as any other type of molecule - or a cell in which said antibody reactive fraction, such as a chimeric T cell receptor or a T cell having said receptor, or molecules developed to provide therapeutic portions by means of a portion of the molecule that has been physically inserted. contains said reactive fraction. Having now completely described the invention, it is will more readily understand by reference to the following examples, which are provided by way of illustration and are not intended to limit the present invention.

EXAMPLE 1 15 Binding of two human proteins, IR1 B1 and IR1B4. to the IFN receiver A cDNA fragment encoding the entire IFNAR1-IC domain amplified by PCR using a sense BamHl primer (5'ctgaggatccAAAGTCTTCTTGAGATGCATC (SEQ ID NO: 5)) and an EcoRI antisense primer (5'tgacgaattcctaTCATACAAAGTC (SEQ ID NO: 6) )), was cloned into a BlueScript vector (BS-SK +, Stratagene). The BamHl-Sall fragment of this BS-IFNAR1-IC was introduced into a pGBTio vector (CloneTech) and fused in phase after the Gal4 DNA binding domain (pGBTl0-IFNAR1-IC) for the investigation of two hybrids. The two hybrid research method (Fields and Song, 1989) was carried out with the modified procedure of Durfee et al. (1993) using the pACT plasmid cDNA library of human B lymphocytes of Epstein-Barr virus (EBV) - transformed to cotransform the yeast Y153 reporter strain with pGBT-? 0-IFNAR1-IC. Y153 strain of yeast has two reporter genes under the control of activation sequences towards the 5 'end (UAS) of GAL1 that are transcribed only if the activity of the transcription factor of GaL4 is reconstituted. This requires that the fusion protein encoded by the pACT plasmid that was introduced is this particular yeast clone having affinity for the 1FNAR1-IC domain of the pGBT-io plasmid. One of the reporter genes is GAL1 His3, which allows growth in a medium lacking histidine; the other reporter gene is GAL-LacZ, which provides β-galactosidase activity. further, the pACT plasmids have the Leu2 gene and the pGBTio plasmid has the TRP1 gene that allows growth in a medium lacking leucine and tryptophan, respectively. The colonies were selected in synthetic medium SC minus Trp, Leu, His in the presence of 3-aminotriazole at 25 mM (which also selects for histidine prototrophy). The growth colonies were then tested for β-galactosidase activity using the X-gal filter test (Breeden and Naysmithm 1985). Nine positive yeast clones were obtained and their pACT plasmids were recovered by transfection into HB101 of E. coli and selection for leu + transformants. For each yeast DNA two HB101 clones of E. coli were isolated. The partial DNA sequence determination of the pACT plasmids of these E. coli clones showed that they fell within two groups of cDNA sequences that were designated IR1B1 and 1R1 B4. The pACT plasmids of groups IR1 B1 and IR1 B4 were subjected to specificity tests by cotransformation of yeast SFY526 reporter strain (Bartel et al., 1993) with pAS plasmids carrying lamin, cdk2 and p53 or other control inserts (CloneTech). The colonies that grew in -trp, -leu were tested for β-galactosidase expression. Of the specifically positive pACT plasmids, inserts were extracted with XhoI, cloned into BS-KS (Stratagene) and subjected to sequence determination from the T7 and T3 promoters using the DyeDeoxy terminator cycle sequence determination equipment in a DNA sequence determinant 373A (Applied Biosystems). Figure 1 shows the results for clone IR1B1 of pACT cotransfected in yeast SFY526 with different plasmid baits of pAS or pGBTio. The yeast cells grew in the selective medium SC of -trp, -leu in lines 1 to 9 of the filter. Staining by X-gal reagent (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was positive only on lines 2 and 4. As indicated in figure 1, line 4 is a yeast of control with an active lacZ gene. Line 2 is the combination of the IR1B1 and IFNAR1-IC fusion proteins. IR1B1 alone (line 9), or any other combination other than IR1 B1 and IFNAR1-IC, exhibited no β-galactosidase activity. Therefore, IR1B1 is specifically capable of combining with the 1C domain of the IFN receptor chain of IFNAR1. Similarly, Figure 2 shows the results for the clone IR1 B4 from pACT cotransfected in yeast SFY526 with different plasmid baits from pAS or pGBT10. Yeast cells cultured in SC, -trp, leu medium in lines 1 to 8 of the filter and staining by X-gal reagent were positive only in lines 3 and 7. As indicated in the portion of the lower table of the Figure 2, line 7 is a control yeast with an active lacZ gene. Line 3 is the combination of fusion proteins 1R1 B4 and IFNAR1-IC. Like the results obtained with IR1B1, IR1B4 alone (line 1), any other combination other than IR1 B4 and IFNAR1-IC, did not exhibit β-galactosidase activity.

Therefore, IR1B4 is also specifically capable of combining with the IC domain of the IFNAR1 IFN receptor chain.

EXAMPLE 2 The sequence of the IR1B1 protein shows EF calcium binding sites The cDNA insert of the plasmids pACT-IR1B1 was excised with the restriction enzyme Xhol, cloned into a BS-KS Bluescript vector and subjected to the sequence determination from the T7 and T3 promoters using the sequence determination equipment of DyeDeoxy terminator cycle in a DNA sequence determinant 373A (Applied Biosystems). The longest plasmid had a sequence of 830 nucleotides (Fig. 3) following the activation domain of GaL4 and the linker sequence of plasmid pACT and an open reading frame of 191 amino acids was found there (Fig. 3). An online search of the protein databases was carried out using the Blastp algorithm (Alignment et al., 1990), as well as the alignment with bioaccelerator (Henikoff and Henikoff, 1992). The highest scores were obtained for calternin (CATRJHUMAN, accession Swiss Protein SW New P41208) with 62.1% similarity and 32.4% identity of amino acids 52 to 173, and for calcineurin B (CALB NAEGR, accession Swiss Protein P42322; CALBJHUMAN, accession P06705) with 59.8% similarity and 32.5% identity of amino acids 50 to171. Figure 4 shows the alignment of IR1B1 with human calcineurin B (CALB) and calcinin (CATR). The Ef-asa helix-loop-helix calcium-binding domains are shown in bold and underlined characters. IR1 B1 has two EF-asa sites but the first two EF-asa domains are not conserved. IR1B1 shows homology for both calcineurin B (represented by the vertical lines in figure 4) and for calternin (represented by the two points in figure 4). However, IR1 B1 is clearly a novel and different human protein that had not previously been identified. / 29 EXAMPLE 3 IR1B1 is a gene product induced by IFN Human myeloma U266S cells (approximately 3 x 106 * 5 cells in 5 ml suspension cultures) were treated with recombinant IFN-a8 (2 x 108 IU / mg of bacteria) or with recombinant IFN-β (3 x 108 IU / mg of CHO cells) at 750 IU / ml for 2 hours or for 18 hours. After treatment with IFN, the cells were harvested and extracted with Tri-reagent (Molecular Research Center, Cincinnati, Ohio), which is a product containing 0 guanidinium thiocyanate and phenol. The extracted RNA was precipitated with ethanol, denatured with formaldehyde, analyzed by electrophoresis in formaldehyde-agarose gels (10 μg RNA / slot), and blotted on GeneScreen Plus (Dupont, New England Nuclear, Billerica, MA). The Northern blot was reacted with 106 cpm of IR1B1 cDNA labeled with the Rediprime kit (Amersham, UK) using 32P-dCTP and random initiation. Figure 5 shows that the cDNA of IR1B1 hydrated to a 1.1 kb RNA. The amount of IR1 B1 mRNA was markedly increased 2 hours after treatment with IFN-ß of U266S cells. However, 18 hours after treatment with IFN, the IR1 B1 mRNA had disappeared from the 0 cells, indicating that the induction is both rapid and transient. Many IFN-induced mRNAs continue to accumulate in the cells for more than 24 hours after treatment with IFN (Revel and Chebath, 1986).

It was verified that the same amount of RNA was present in each band. As shown in the lower part of Figure 5, the hybridization of the same U266S RNA (rich in IFN receptor) to an 18S ribosomal cDNA probe reveals the same amount of 18S rRNA in each band (only part of the blot where 18S rRNA runs are indicated). In another experiment using 1,200 U / ml of IFN for induction, ANRm of IR1 B1 was also observed with IFN-a8 at 2 hours, but not at 30 minutes (not shown). It was found that the IR1 B1 mRNA had the same size of 1.1 kb in different human cells (U266, Daudi and THP-1 cells). It is noteworthy that this size is close to the ANRm of calcitin but not to the ANRm of calcinerium B (2.5 kb). The small size of the mRNA is consistent with IR1B1 which is a small protein of approximately 20 kDa.

EXAMPLE 4 In vitro binding of the IR1B4 protein to IFNAR1 The binding of IR1B4 to the IC domain of IFNAR1 was tested by synthesizing the IR1B4 protein with a protein marker (flag sequence) using an in vitro translation in reticulocyte lysates and reacting this protein with a recombinant IFNAR1-IC fusion protein in E. coli The DNA of pACT-iR1B4 of Example 1, cut with XhoI and filled by Klenow enzyme, was cloned into the PECE-flag expression vector (Ellis et al., 1986) cut with EcoRI and filled. The Notl-BamHI fragment containing the flag-iRIB4 fusion in frame was recloned in BS-SK cut with Notl-BamHI and towards the 3 'end of the T3 promoters. The sequence of the flag fusion was verified by determining the sequence of the T3 promoter. In vitro transcription (Promega team) was done with T3 polymerase and 1 μg of Bs-flag-IR1 B4 DNA linearized with BamHl. The * 5 in vitro translation was carried out in rabbit reticulocyte lysates (Promega kit) with [35 S] methionine (Amersham) and 5 μg of RNA transcripts for 1 hour at 30 ° C. The products were treated with RNase before use. The GST-IFNAR1-IC fusion protein was prepared by cloning the BamHI-EcoRI, the insert of BS-IFNAR1-IC (see above) at the same sites of pGEX2 (Pharmacia Biotech). GST and GST-IFNAR1-IC were expressed in E. coli and recovered bound to glutathione-agarose spheres (Sigma). The M2 anti-flag agarose spheres were from Kodak Scientific Imaging Systems. The monoclonal antibodies IFNaR3 to component a of the IFN receptor (IFNAR1) were a nice gift from Dr. O. Colamonici , 15 (Colamonici et al., 1990) and used at a 1: 100 dilution. Rabbit antibodies were prepared to the C-terminal peptide of IFNAR1-IC (Ab 631) and used for the immunoprecipitation of IFNAR1 from Brij extracts (0.75 ml) of 2 x 10 7 U266S cells and U266R of human myelone with previously detailed antiproteases ( Ambrovich et al., 1994) except that the G spheres of Protein (Pharmacia) was used with mAb, IFNar3, SDS-PAGE and analyzes on a Fujix BAS1000 Phosphor-lmager were as before (Harroch et al., 1994). It was first verified that a protein product of approximately 32 kDa is obtained when the products of the translation were immunoprecipitated by anti-flag antibodies (Figs 6a and 6B). In Figures 6A and 6B, whenever the use of antiflag antibodies is noted (by + sign), it means that the radioactive translation product of the mRNA of the 1R1B4-flag fusion (transcribed in vitro from the corresponding DNA construct) reacted with the M2 anti-flag antibody bound to agarose spheres (product of Kodak Scientific Imaging Systems). The translated protein containing IR1 B4 fused to the flag amino acid sequence was bound to these anti-flag antibody spheres and after spheres centrifuged, the protein was eluted with SDS pH regulator and applied on SDS-PAGE. These reactions serve as a control to show that the expected fusion protein is present. The glutathione-sepharose spheres (Sigma), to which glutathione S-transferase (GST) was fused to IFNAR1-TC, were added to the reticulocyte lysate translation reaction. The spheres were centrifuged and washed, and the proteins bound to GST spheres were released by sodium dodecylsulfate (SDS at 1%) and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE). It was observed that the 32 kDa protein marked by 35S-methionine was bound to GST-IFNAR1-IC but not to GST alone (Fig. 6A). This demonstrates that IR1B4 binds directly to the isolated IFNAR1-IC peptide region. To verify that IR1B4 interacts with the IFNAR1 protein present in membranes of human cells, detergent extracts of human myeloma U266 cells were mixed with the 35S-methionine labeled translation products of IR1 B4 mRNA from the reticulocyte lysates.

The IFNAR1 protein was immunoprecipitated by a monoclonal antibody IFNaR3 specific to the ectodomain of IFNAR1 (from Colamonici et al., 1990). Analysis by SDS-PAGE showed the presence of the IR1B4-flag of 32 kDa (Fig. 6B) when the detergent extracts originated from U266S (rich in IFN receptor), but not when they originated from U266R cells-a line of mutant, IFN-α, ß-resistant mutant cells of U266 deficient in IFN receptors (Abramovich et al., 1994). Similarly, it was observed that the 32 kDa band when U266S extracts reacted with Ab 631 against the C-terminal peptide of IFNAR1 and IFNAR1 was precipitated by anti-flag when Cos-7 cells were transferred by Flag-IR1 B4 and human IFNAR1 cDNAs. These results demonstrated that IRIB4 binds to intact IFNAR1 of human cells in a specific manner.

EXAMPLE 5 IR1B4 cDNA sequences and proteins The nucleotide sequence of the IR1B4 cDNA has an open reading frame that codes for a 361 amino acid long protein (Fig. 7). This human cDNA recognized a poly-A + mRNA constitutively expressed as 1.5 kb in several human cells including U266 myeloma cells. An on-line search of the protein databases was carried out using the BlastP algorithm (Altschul et al., 1990) as well as the alignment with Bioaccelerator (Henikoff and Henikoff, 1992), and it was found that IR1 B4 is a unique member of the protein-arginine methyltransferase family. The rat PRMT1 cDNA described by Lin et al (1996, Genbank sequence I.D. 1390024; Accession U60882) is only 81.4% homologous when analyzed by the ALIGN computer program. At the amino acid level (Fig. 8), the human IRIB4 / PRMT differs clearly at its amino terminus from PRMT1, the first 19 amino acids being completely different. The sequence determination of the N-terminus of IRIB4 alone could not have provided any indication that IRIB4 is homologous to PRMT1. It was also found that another human protein that had been described, HCP-1 (Nikawa et al., 1996; Genbank accession D66904) had homology to IRIB4. However, HCP-1 has a different amino acid sequence from residues 147-175 (Fig. 9). HCP-1 was originally identified on the basis of its ability to complement the ire15 mutation in yeast, and its enzymatic function was not previously identified (Nikawa et al., 1996). Therefore, IR1B4 is a novel human protein.

EXAMPLE 6 The IR1B4 protein bound to IFNAR1-IC has methyltransferase activity The methyltransferase activity could be co-immunoprecipitated from human cell extracts with the IFNAR1 receptor. The Brij-detergent extracts of U266S cells were reacted overnight at 4 ° C with or without anti-IFNAR1 Ab 631 antibody (Abramovich et al., 1994). Protein A spheres (40 μl of a 50% fast flow of IPA-400, Repligen) were added for 1 hour. The beads were washed and incubated in 0.1 ml of Tris-HCL at 25 mM, pH 7.5, EDTA at 1 mM, EGTA at 1 mM, 50 μM (0.25 μCi) of 14C- (methyl) -S- adenosyl-methionine ( Amersham) and 100 μg of histones (NA type of calf thymus, Sigma) for 30 minutes at 30 ° C. In vitro methylation of the histones was carried out under the conditions described by Lin et al. (1996). The radioactivity of the histone band was analyzed after SDS-PAGE (15% acrylamide) and exposure was made in the Phosphor-imager. A 14C-methyl labeling of the histones was observed with the beads that were coated with anti-IFNAR1, but not with those in the control reaction (Fig. 10). Therefore, the methyltransferase activity of the protein is constitutively associated with the IFN receptor chain of these human cells. A similar enzymatic activity was recovered when IFNAR1 was immunoprecipitated five minutes after the addition of IFN-β to the U266S cells.

EXAMPLE 7 Implication of IR1B4 / PRMT1 in the IFN action An anti-sense oligodeoxynucleotide phosphorothioate (Steina et al., 1989) complementary to nucleotide sequence 12-33 around the cDNA initiation codon of IR1B4 (AS-1, anti-sense sequence 5'-GGCTACAAAATTCTCCATGATG-3) was chemically synthesized. '; SEQ ID NO: 12). The oligonucleotides were added to U266S cells cultured in 96-well microplates (8000 cells / well / 0.2 ml RPMI, 10% FCS) at a final concentration of 10 μm on day 0, and re-added at 5 μm per day 2. IFN-β was added at 64 or 125 IU / ml on day 0. After 3 days of culture, 20 μl of Alamar blue, a colorimetric cell density indicator based on oxide-reduction (BioSource, Camarillo, CA) was added. ) to each cavity and incubation continued for 6-7 hours. The color was measured in a microplate ELISA reader (530 nm test filter, 630 nm reference filter) with multiple reading of the duplicated cavities. The correlation of the growth curves was verified by the number of living cells and by OD. To measure methyltranferase, the cells of the pooled cavities were used by freeze-thawing in 25 μl / cavity of Tris-HCl at 25 mM, pH 7.4, EDTA at 1 mM, EGTA at 1 mM, 40 μl / ml of leupeptin and aprotinin , 20 μg / ml of pepstain and 1 μM of phenylethylsulfonyl fluoride (PMSF). Reactions were in 50 μl with 25 μl of cell extracts, 100 μM of peptide R1 (Najbauer et al., 1993, obtained from Genosys, Cambridge, UK) and 3 μCi of [3 H] (methyl) S-adenosylmethionine (Amersham 73 Ci / mmol) for 30 minutes at 30 ° C. After electrophoresis in SDS-polyacrylamide gel (16%), fixation in 50% methanol, 10% acetic acid and treatment by Amplify® (Amersham), autoradiography was carried out for 8 days. This AS-1 antisense DNA is able to strongly reduce the protein-arginine methyltransferase activity in U266S cells as measured by the incorporation of tritiated methyl groups to the substrate of the R1 peptide (Fig. 11), and was used to investigate the role of this enzyme You can play in the IFN action. The growth inhibitory activity of IFN was chosen because it can be quantified very directly on the cells and because an interaction of rat PRMT1 with growth-related gel products has been observed (Lin et al., 1996). The addition of antisense-1 oligonucleotide AS-1, which is complementary to the sequence around the cDNA initiation codon of IR1B4 / PRMT, reduced the growth inhibitory effect of IFN-β on human myeloma U266S cells (Figure 12) . This means that, in the presence of antisense AS-1, cells treated with IFN exhibited a higher growth (excluding any toxic effect of phosphorothioates). Growth in the absence of IFN was not significantly affected. The S-3 sense oligonucleotide corresponding to the same cDNA region had only a small effect (S-3, Fig. 12) compared to the antisense-1. S-3 sense also had only a slight inhibitory effect on the activity level of the enzyme (Figure 11). Another antisense phosphorothioate oligonucleotide AS-2 (SEQ ID NO: 13), directed to the intermediate part of the cDNA and complementary to the nucleotides 572-592 of SEQ ID NO: 7, had almost no effect (Figure 12). The up to 5-fold reduction in the growth inhibitory effect of IFN-β on myeloma cells, which became partially deficient in PRMT activity by the antisense-1 oligonucleotide, demonstrates that the association of the IR1B4 / PRMT enzyme with the domain IC of the IFNAR1 receptor is functionally significant for the action of IFN in cells. These experiments also demonstrate that the IR1B4 protein methylates the peptide substrates of the PRMT class of enzymes, such as the R1 peptide Gly-Gly-Phe-Gly-Gly-Arg-Gly-Gly-Phe-Gly (SEQ ID NO: 11 Najbauer et al., 1993), which was used in the experiment illustrated in Figure 11. The methylation of the proteins in arginine residues close to glycine residues (e.g., as in the previous peptide) can be a type of protein modification that, like phosphorylation, can be used to translate signals into the cell. The hnRNP group of proteins is a target for PRMT enzymes, and since these proteins affect the processing, separation, transport and stability of mRNA (Liu and Dreyfuss, 1995), their methylation may play a role in post-control -transcriptional gene expression. The IR1B4 / PRMT protein, discovered here as binding to an IFN receptor chain, could mediate changes in gene expression in response to IFN. Other protein substrates can be methylated through the IFN receptor, including other components of the IFN receptor complex and transcription factors. Lin et al. (1996) have observed that the binding of rat PRMT1 to proteins induced with active growth factor PRMT1 and modifies its substrate specificity, possibly through the removal of certain inhibitory proteins associated with PRMT1 in the cytoplasm of cells. A similar activation of IR1B4 bound to the IFNAR1 chain of the IFN receptor can be expected.

Conclusions A new IR1B1 protein is described that interacts with the intracytoplasmic domain of the IFNAR1 chain of the interferon-type I receptor. This protein is induced very rapidly and transiently after treatment with IFN from human cells. IR1B1 is characterized by the presence of loop-helix-loop EF-helical sites that are characteristic of calcium-binding proteins. Calcium ion fluxes have been implicated in the mechanism of action of IFNs, and in particular for initial cell responses and changes in cell morphology and cytoskeletal organization (Tamm et al., 1987). Enzymes activated by calcium ion can produce second messengers, such as diacyl-glycerol, in response to IFNs. In addition, calmodulin-like proteins regulate a number of protein kinases, and it has been observed that these pathways function in cells treated with IFN (Tamm et al., 1987). It is possible that the IR1B1 protein binding to the IFN receptor is involved in said Ca ++ -dependent effects of the IFNs in cells. The investigation of two hybrids for proteins that interact with the IFNAR1-IC domain also identified another IR1 B4 protein, which turned out to be a member of the protein-arginine enzyme family! transferase (PRMT1; Lin et al., 1996). This enzyme is known to methylate a number of RNA and DNA binding proteins, in particular heterologous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are involved in transporting mRNA from the nucleus to the cytoplasm, the alternative separation of pre-mRNA, and post-transcriptional controls (Liu and Dreyfuss, 1995). The IR1 B1 and IR1B4 / PRMT1 proteins that accumulate on the IFNAR1-1C domain reveal novel signaling mechanisms of IFNs that exist apart from the Jak-Stat trajectories known and described by Darnell et al. (1994).

Having now fully described this invention, it will be appreciated by those skilled in the art that it can be carried out within a wide range of equivalent parameters, concentrations and conditions, without departing from the spirit and scope of the invention and without undue experimentation. Although this invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications. This application is designed to cover any variations, uses or adaptations of the invention, following, in general, the principles of the invention and including departures from the present description that are within the known or common practice in the art to which the invention belongs. invention and that could be applied to the essential features set forth above as follows in the scope of the appended claims. All references cited herein, including articles or summaries of reports, published or corresponding to US patent applications. or foreign, US patents. or foreign issued, or any other references, are fully incorporated by reference herein, including all data, tables, figures and text presented in the references cited. In addition, the complete contents of the references cited within the references cited herein are also incorporated completely by way of reference.

The reference to known method steps, steps of conventional methods, known methods or conventional methods is in no way an admission that any aspect, description or embodiment of the present invention is described, taught or suggested in the relevant art. The above description of the specific modalities will so completely reveal the general nature of the invention that others may, applying knowledge within the technique (including the contents of the references cited herein), modify and / or easily adapt for various applications said specific modalities, without undue experimentation, without departing from the general concept of the present invention. Therefore, these adaptations and modifications are designed to be within the meaning and scale of equivalents of the described modalities, based on the teaching and guidance presented here. It should be understood that the phraseology or terminology of the present has only the reasons for description and not for limitation, such that the terminology or phraseology of the present description should be interpreted by the person skilled in the art in light of the teachings and guides presented. in the present, in combination with the knowledge of a person skilled in the art.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: REVEL, Michael CHEBATH, Judith ABRAMOVICH, Carolina (I) TITLE OF THE INVENTION: NOVEDOUS PROTEINS OF UNION OF THE IFN RECEIVER 1, DNA THAT CODIFIES THEM, AND METHODS TO MODULATE THE CELLULAR RESPONSE TO INTERFERONES (iii) SEQUENCE NUMBER: 13 (iv) CORRESPONDENCE ADDRESS: (A) NAME: BROWDY AND NEIMARK (B) STREET: 419 Seventh Street, N.W., Suite 300 (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: E.U.A. (F) POSTAL CODE: 20004 (V) COMPUTER LEADABLE FORM: (A): TYPE OF MEDIUM: Flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0, Version # 1.30 (vi) CURRENT INFORMATION ON THE APPLICATION: (A) APPLICATION NUMBER: US (B) SUBMISSION DATE: (vii) PREVIOUS INFORMATION OF THE APPLICATION: (A) APPLICATION NUMBER: US 60 / 035,636 (B) SUBMISSION DATE: January 15, 1997 (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: BROWDY, Roger L. (B) REGISTRATION NUMBER: 25,618 (C) REFERENCE CASE NUMBER: REVEL = 14 PCT (¡X) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202-628-5197 (B) TELEFAX: 202-737-3528 (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 830 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCALIZATION: 43..615 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 CGTCTCGAGG CGAGTTGGCG GAGCTGTGCG CGCGGCGGGG CG ATG GGG GGC TCG, 54. -. "r, -? -; Met7'Gly-Gly: Ser" '' '* "1. GGC AGT CGC CTG TCC AAG GAG CTG CTG GCC GAC TAC CAG GAC TTG ACG 102 Gly Ser Arg Leu Ser Lys Glu Leu Leu Wing Glu Tyr Gln Asp Leu Thr 5 10 15 20 TTC CTG ACG AAG CAG GAG ATC CTC CTA GCC CAC AGG CGG TTT TGT GAG 150 Phe Leu Thr Lys Gln Glu He Leu Leu Wing His Arg Arg Phe Cys Glu 25 30 35 CTG CTT CCC CAG GAG CAG CGG AGC GTG GAG TCG TCA CTT CGG GCA CA 198 Leu Leu Pro Gln Glu Gln Arg Ser Val Glu Ser Ser Leu Arg Ala Gln 40 45 50 GTG CCC TTC GAG CAG ATT CTC AGC CTT CCA GAG CTC AAG GCC AAC CCC 246 Val Pro Phe Glu Gln lie Leu Ser Leu Pro Glu Leu Lys Wing Asn Pro 55 60 65 TTC AAG GAG CGA ATC TGC AGG GTC TTC TCC TCC CCA CAC GCC AAA GAC 294 Phe Lys Glu Arg He Cys Arg Val Phe Ser Thr Ser Pro Ala Lys Asp 70 75 ~ 80 - "'AGC CTT AGC TTT GAG GAC TTC CTG GAT CTC CTC AGT GTG TTC AGT GAC 342 Ser Leu Ser Phe Glu Asp Phe Leu. Asp Leu Leu Ser Val Phe - Ser Asp 85 90 * 95 100 ACA GCC ACG CCA GAC ATC AAG TCC CAT TAT GCC TTC CGC ATC TTT GAC 390 Thr Wing Thr Pro Asp He Lys Ser His Tyr Wing Phe Arg He Phe Asp 105 ' 110 _ .. ". L-5 TTT GAT GAT GAC GGA ACC TTG AAC AGA GAA GAC CTG AGC CGG CTG GTG 438 Phe Asp Asp Asp Gly Thr Leu Asn Arg Glu Asp Leu Ser Arg Leu Val 120 125 130 AAC TGC CTC ACG GGA GAG GGC GAG GAC ACA CGG CTT AGT GCG TCT GAG 486 Asn Cys Leu Thr Gly Glu Glu Glu Asp Thr Arg Leu Ser Wing Glu 135 140 145 ATG AAG CAG CTC ATC GAC TAC ATC CTG GAA GAG TCT GAC ATT GAC AGG 534 Het Lys Gln Leu He Asp Tyr He Leu Glu Glu Be Asp He Asp Arg 150 155 160 GAT GGA ACC ATC AAC CTC TCT GAG TTC CAG CAC GTC ATC TCC CGT TCT 582 Asp Gly Thr He Asn Leu Ser Glu Phe Gln His Val He Ser Arg Ser 165 170 175 180 CCA GAC TTT GCC AGC TCC TTT AAG ATT GTC CTG TGACAGCAGC CCCAGCGTGT 635 Pro Asp Phe Wing Ser Ser Phe Lys He Val Leu 185 190 GTCCTGGCAC CCTGTCCAAG AACCTTTCTA CTGCTGAGCT GTGGCCAAGG TCAAGCCTGT 695 GTTGCCAGTG CGGGCCAAGC TGGCCCAGCC TGGAGCTGGC GCTGTGCAGC CTCACCCCGG 755 GCAGGGGCGG CCCTCGTTGT CAGGGCCTCT CCTCACTGCT GTTGTCATTG CTCCGTTTGT 815 GGCCCCTTCGT GGCCA. 830 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 191 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 Met Gly Gly Ser Gly Ser Arg Leu Ser Lys Glu Leu Leu Ala Glu Tyr 1 5 10 15 Gln Aep Leu Thr Phe Leu Thr Lys Gln Glu He Leu Leu Wing Hie Arg 20 25 30 Arg Phe Cys Glu Leu Pro Gln Glu Gln Arg Ser Val Glu Ser Ser 35 40 45 Leu Arg Ala Gln Val Pro Phe Glu Gln He Leu Ser Leu Pro Glu Leu 50 55 60 Lys Wing Asn Pro Phe Lys Glu Arg He Cye Arg Val Phe Ser Thr Ser 65 70 75 80 Pro Ala Lys Asp Ser Leu Ser Phe Glu Asp Phe Leu Asp Leu Leu Ser 85 »• 90 -95 Val Phe Ser Asp Thr Ala Thr Pro Asp He. Lys Ser His Tyr Wing Phe 100 .105 110 Arg He Phe Asp Phe Asp Asp Asp Gly Thr Leu Asn Arg Glu Aep Leu 115 120 125 Ser Arg Leu Val Asn Cys Leu Thr Gly Glu Gly Glu Asp Thr Arg Leu 130 135 140 Ser Ala Ser Glu Met Lys Gln Leu He Asp Tyr He Leu Glu Glu Ser 145 150 155 160 Asp He Asp Arg Asp Gly Thr He Asn Leu Ser Glu Phe Gln His Val 165 170 • 175 He Ser Arg Ser Pro A.sp Phe Ala Ser Ser Phe Lys He Val Leu 180 - 185 '190 (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 170 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 3 Met Gly Asn Glu Wing Ser Tyr Pro Leu Glu Met Cys Ser His Phe Asp 1 5 10 15 Wing Asp Glu He Lys Arg Leu Gly Lys Arg Phe Lys Lys Leu Asp Leu 20 25 30 Asp Asn Ser Gly Ser Leu Ser Val Glu Glu Phe Met Ser Leu Pro Glu 35 40 45 Leu Gln Gln Asn Pro Leu Val Gln Arg Val He Asp He Phe Asp Thr 50 55 -;: - ': -, ... 60 • > ~, "? - '. •• .- - Asp Gly Asn Gly Glu Val Asp Phe Lys.'. Glu-.PheMle .Glu-Gly Val Ser 65 70 •• - • 75 .v -: - r : - '80 Gln Phe Ser Val Lys Gly Aep Lys Glu .Gln Lys Leu -Arg Phe Ala Phe 85 90'-:; : '_' "';'. ': •"' '95 Arg He Tyr Asp Met Asp Lys Asp Gly Tyr He Ser Asn Gly Glu Leu 100 105 110 Phe Gln Val Leu Lys Met Met Val Gly Asn Asn Leu Lys Asp Thr Gln 115 120 125 Leu Gln Gln He Val Asp Lys Thr He He Asn Ala Asp Lys Asp Gly 130 135 140 Asp Gly Arg He Ser Phe Glu Glu Phe Cys Wing Val Val Gly Gly Leu 145 150 155 160 Asp He His Lye Lys Met Val Val Asp Val 165 170 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 172 amino acids (B) TYPE: amino acid * 5 (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (I) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Met Wing Being Asn Phe Lys Lys Wing Asn Met Wing Being Ser Gln Arg 1 5 10 15 Lys Arg Met Ser Pro Lys Pro Glu Leu Thr Glu Glu Gln Lys Gln Glu 20.. 25 30 He Arg Glu Wing Phe Asp Leu Phe Asp Wing Aep Gly Thr Gly Thr He 35 40 45 -? C Asp Val Lys Glu Leu Lys Val Wing Met Arg Wing Leu Gly Phe Glu Pro * 50 55 60 Lys Lys Glu Glu He Lys Lys Met He Ser Glu He Asp Lys Glu Gly 65 70 75 80 Thr Gly Lys Met Asn Phe Gly Asp Phe Leu Thr Val Met Thr Gln Lys 85 90 95 Met Ser Glu Lys Asp Thr Lys Glu Glu He Leu Lys Wing Phe Lye Leu 100 105 110 Phe Asp Asp Asp Glu Thr Gly Lys He Ser Phe Lye Asn Leu Lys Arg 115 120 125 20 Val Wing Lys Glu Leu Gly Glu Aen Leu Thr Asp Glu Glu Leu Gln Glu 130 135 140 Met He Asp Glu Wing Aep Arg Asp Gly Asp Gly Glu Val Ser Glu Gln 145 150 155"'160 Glu Phe Leu Arg He Met Lys Lys Thr Ser Leu Tyr 165 170 (2) INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid »5 (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: 31 CTGAGGATCC AAR.GTCTTCT TGAGATGCAT C (2) INFORMATION FOR SEQ ID NO. 6:, (i) CHARACTERISTICS OF THE SEQUENCE: '15 (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TGACGAATTC CTATCATACA AAGTC 25 (2) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1308 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) ) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 16..1098 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: GCCGCGAACT GCATC ATG GAG AAT TTT GTA GCC ACC TTG GCT AAT GGG ATG 51 Met Glu Asn Phe Val Wing Thr Leu Wing Asn Gly Met 195 200 AGC CTC CAG CCG CCT CTT GAA GAA GTG TCC TGT GGC CAG GCG GAA AGC 99 Ser Leu Gln Pro Pro Leu Glu Glu Val Ser Cys Gly Gln Wing Glu Ser 205 210 215 AGT GAG AAG CCC AAC GCT GAG GAC ATG ACA TCC AAA GAT TAC TAC TTT 147 Ser Glu Lys Pro Asn Wing Glu Asp Met Thr Ser Lys Asp Tyr Tyr Phe 220 225 230 235 GAC TCC TAC GCA CAC TTT GGC ATC CAC GAG GAG ATG CTG AAG GAC GAG 195 Asp Ser Tyr Wing His Phe Gly He His Glu Glu Met Leu Lys Asp Glu 240 245 250 GTG CGC ACC CTC ACT TAC CGC AAC TCC ATG TTT CAT AAC CGG CAC CTC 243 Val Arg Thr Leu Thr Tyr Arg Asn Ser Met Phe His Asn Arg Hie Leu 255 .260 265 TTC AAG GAC AAG GTG GTG CTG GAC GTC GGC TCG GGC ACC GGC ATC CTC 291 Phe Lys Asp Lys Val Val Leu Asp Val Gly Ser Gly Thr Gly He Leu 270 275 '280' TGC ATG -TTT GCT GCC AAG GCC GGG 'GCC .CGC- AAG -GTC ATC GGG'ATC GAG 339 Cys Met "Phe Ala Ala Lys Ala Gly Ala Arg Lys Val lie.Gly He Glu *, .., 2 &; S -J * f --._ i ..? I fy :: ¡.: - and-y? 7.290,:. .. { . - ^ * '* - • 295 - - - * TGT TCC-AGT-ATCTTCT (-. GAT? TAT GCG': GTG AAG ATC "- 'GTC-'AAA" GCC "AC' AÁG" "" ' 387 Cys Ser 'Ser He Ser Asp Tyr Wing Val Lys He Val Lys Wing.Asn, Lys, - v..y.,:. <, ~ r 300 305' 310 • '••' "'' ''! -315 'TTA GAC CAC GTG GTG ACC ATC ATC AAG GGG AAG GTG GAG GAG GTG GAG 435 Leu Asp His Val Val Thr He He Lys Gly Lys Val Glu Vallu Glu c 5 320 325 330 CTC CGA GTG GAG AAG GTG GAC ATC ATC ATC AGC GAG TGG ATG GGC TAC 483 Leu Pro Val Glu Lys Val Asp He He He Ser Glu Trp Met Gly Tyr 335 340 345 TGC CTC TTC TAC GAG TCC ATG CTC AAC ACC GTG CTC TAT GCC CGG GAC 531 Cys Leu Phe Tyr Glu Ser Met Leu Asn Thr Val Leu Tyr Wing Arg Asp 350 355 360 AAG TGG CTG GCG CCC GAT GGC CTC ATC TTC CCA GAC CGG GCC ACG CTG 579 Lys Trp Leu Wing Pro Asp Gly Leu He Phe Pro Asp Arg Wing Thr Leu 365 370 375 10 TAT GTG ACG GCC ATC GAG GAC CGC CAG TAC AAA GAC TAC AAG ATC CAC 627 Tyr Val Thr Wing He Glu Asp Arg Gln Tyr Lys Asp Tyr Lys He His 380 385 390 •• - • '- 395 TGG TGG GAG AAC GTG TAT GGC TTC GAC ATG TCT- TGC ATC AAA GAT GTG 675 Trp Trp Glu Asn Val Tyr Gly Phe, Asp Met Ser Cys He Lys Asp Val 400 • '405 • * -' 410 GCC ATT AAG GAG CCC CTA GTG GAT GTC GTG GAC CCC AAA CAG CTG GTC 723 Wing He Lys Glu Pro Leu Val Asp Val Val Asp Pro Lys Gln Leu Val 415.,., 420 425 * ACC AAC GCC TGC CTC ATA AAG GAG GTG GAC ATC TAT • ACC GTC AAG GTG 771 Thr Asn Wing Cys Leu He Lys Glu Val Asp He Tyr Thr Val Lvs Val '15 430 435 440 GAA GAC CTG ACC TTC CC TCC CCG TTC TGC CTG CA GTG AAG CGG AAT 819 Glu Asp Leu Thr Phe Thr Ser Pro Phe Cys Leu Gln Val Lys Arg Asn 445 450 455 GAC TAC GTG CAC GCC CTG GTG GCC TAC TTC AAC ATC GAG TTC ACA CGC 867 Asp Tyr Val His Wing Leu Val Wing Tyr Phe Asn He Glu Phß Thr Arg 460 465 470 475 TGC CAC AAG AGG ACC GGC TTC TCC ACC AGC CCC GAG TCC CCG TAC ACG 915 Cys Hie Lys Arg Thr Gly Phe Ser Thr Ser Pro Glu Ser Pro Tyr Thr 480 485 490 20 CAC TGG AAG CAG ACG GTG TTC TAC ATG GAG GAC TAC CTG ACC GTG AAG • 963 His Trp Lys Gln Thr Val Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys 495 500 505 ACG GGC GAG GAG ATC TTC GGC ACC ATC GGC ATG CGG CCC AAC GCC AAG 1011 Thr Gly Glu Glu He Phe Gly Thr He Gly Met Arg Pro Asn Ala Lys 510 515 520 AAC AAC CGG GAC CTG GAC TTC ACC ATC GAC CTG GAC TTC AAG GGC CAG 1059 Asn Asn Arg ASO Leu Aso Phe Thr He Aep Leu Asp Phe Lys Gly Gln 525 530 535 CTG TGC GAG CTG TCC TGC TCC ACC G AC TAC CGG ATG CGC TGAGGCCCGG 1108 Leu Cye Glu Leu Ser Cys Ser Thr Asp Tyr A.rg Met Arg 540 545 • 550 CTCTCCCGCC CTGCACGAGC .CCAGGGGCTG AGCGTTCCTA GGCGGTTTCG 'GGGCTCCCCC "' '1168 TTCCTCTCCC TCCCTCCCGC AGAÁGGGGGT TTTAGGGGCC TGGGCTGGGG GGATGGGGAG "'1228 GGCACATTGG._GACTGTGTTT -TTCATAAATT. ATGTTTTTAT.ATGGTTGCAT TTAATGCCÁA"' - 1288 -..- •, - • •• .. cv *. '.. ... "••• _ * '* i "ry • -" • - • -' '•'. '' cl '- - •' - "•" "- * '" "" "r -'- t. TAAATCCTCA GCTGGGGAAA '•;.; .v '"" V. 1308 (2) INFORMATION FOR SEQ ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 361 amino acids 10 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 8: Met Glu Asn Phe Val Wing Thr Leu Wing Asn Gly Met Being Leu Gln Pro * 1 5 10 '15"15"? Le Glu Glu Val Ser Cys Gly Gln Wing Glu Ser Ser Glu Lys Pro i 20 25 30 Asn Wing Glu Asp Met Thr Ser Lys Asp Tyr Tyr Phe Asp "Ser Tyr Ala 35 40 45 His Phe Gly He His Glu Glu Met. Leu Lys Asp Glu Val Arg Thr Leu 50 55"60, Thr Tyr Arg Asn Ser Met Phe His Aen Arg His Leu Phe Lys Asp Lys 65 70 75 80 Val Val Leu Asp Val Gly Ser Gly Thr Gly He Leu Cys Met Phe Wing 85 90 95 20 Ala Lys Ala Gly Ala Arg Lys Val He Gly He Glu Cys Ser Ser He 100 105 110 Ser Asp Tyr Ala Val Lys He Val Lys Ala Asn Lys Leu Asp His Val 115 120 125 Val Thr He He Lys Gly Lys Val Glu Val Glu Glu Leu Pro Val Glu 130 135 140 Lys Val Aep He He Ser Glu Trp Met Gly Tyr Cys Leu Phe Tyr _ 145 150 155 160 O Glu Ser Met Leu Asn Thr Val Leu Tyr Wing Arg Asp Lys Trp Leu Wing 165 170 175 Pro Asp Gly Leu He Phe Pro Asp Arg Wing Thr Leu Tyr Val Thr Wing 180 185 190 He Glu Asp Arg Gln Tyr Lys Aep Tyr Lys He His Trp Trp Glu Asn 195 200 205 Val Tyr Gly Phe Asp Met Ser Cys He Lys Asp Val Wing He Lys Glu 210 215 220 Pro Leu Val Asp Val Val Asp Pro Lys Gln Leu Val Thr Asn Ala Cys 10 225 230 235 240 Leu He Lye Glu Val Asp He Tyr Thr Val Lys Val Glu Asp Leu Thr .245 -250"255 Phe Thr Ser Pro Phe Cys Leu Gln Val Lys Arg Asn Asp Tyr Val His .260 .- -. 265 •• •" 270 Ala Leu Val Ala "Tyr Phe Asn He GÍu Phe Thr Arg Cys His Lys Arg 275. * - ..., ** .- *., --- 280t .- ••, - --.- * •, .-- ::. - -_85 - "" < • * - * • ' Thr Gly Phe Ser Thr Ser Pro Glu Ser Pro Tyr Thr His Trp Lys Gln 290 295 300 '15 Thr Val Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys Thr Gly Glu Glu 305 310 315 320 He Phe Gly Thr He Gly Met Arg Pro Asn Wing Lys Asn Asn Arg Asp 325 330 335 Leu Asp Phe Thr He Asp Leu Asp Phe Lye Gly Gln Leu Cys Glu Leu 340 345 350 Ser Cys Ser Thr Asp Tyr Arg Met Arg 355 360 (2) INFORMATION FOR SEQ ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 353 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Met Wing Wing Wing Glu Wing Wing Asn Cys He Met Glu Val Ser Cys Gly 3- 5 10 15 Gln Wing Glu Ser. Ser Glu Lys Pro Asn Wing Glu Asp Met Thr Ser Lys 20 25 30 Asp Tyr Tyr Phe Asp Ser Tyr Wing Hie Phe Gly He His Glu Glu Met 35 40 45 Leu Lys Asp Glu Val Arg Thr Leu Thr Tyr Arg Asn Ser Met Phe Hie 50 - 55 G0 Asn Arg His Leu Phe Lys Asp Lys Val Val Leu Asp Val Gly Ser Gly 65 70 75 80 Thr Gly He Leu Cys Met Phe Ala Ala Lys Ala Gly Ala Arg Lys Val 85 90 95 He Gly He Glu Cys Ser Ser He As Asp Tyr Ala Val Lys He Val 100 105 110 Lys Ala Asn Lye Leu Asp His Val Val Thr He He Lys Gly Lys Val 115 120 125 Glu Glu Val Glu Leu Pro Val Glu Lys Val Asp He He He Ser Glu 130 135 140 Trp Met Gly Tyr Cys Leu Phe Tyr Glu Ser Met Leu Asn Thr Val Leu 3-45 150 155 160 His Ala Arg Asp Lys Trp Leu Wing Pro Asp Gly Leu lie "Phe Pro Asp 165 170 _ .. 175 Arg Wing Thr Leu Tyr Val Thr Wing He Glu Asp Arg Gln Tyr Lys Asp 180 185 ... 190 ... .. Tyr LYe --e Hie Trp -Trp Glu Asn Val Tyr "Gly Phe Asp Met Ser Cys? 5 195 200 205 He Lys Asp Val Wing He Lys Glu Pro Leu Val Asp Val Val Asp Pro 210 215 220 Lys Gln Leu Val Thr Asn Wing Cys Leu He Lys Glu Val Asp He Tyr 225 230 235 240 Thr Val Lys Val Glu Asp Leu Thr Phe Thr Ser Pro Phe Cys Leu Gln 245 250 255 Val Lys Arg Asn Asp Tyr Val His Wing Leu Val Wing Ala Tyr Phe Asn He 260 265 270 10 Glu Phe Thr Arg Cys His Lys Arg Thr Gly Phe Ser Thr Ser Pro Glu 275 • 280 285 Ser Pro Tyr Thr His Trp Lys Gln Thr Val Phe Tyr Met Glu Asp Tyr 290 295 300 Leu Thr Val Lye Thr Gly Glu Glu He Phe Gly Thr He Gly Met Arg 305 310 315 320 Pro Asn Ala- Lys Asn Aen Arg Aep Leu Asp Phe Thr * He Aep Leu Asp 325 • 330 335 Phe Lys Gly Gln Leu Cys Glu Leu Ser Cys Ser Thr Asp Tyr Arg Met 340 345 350 '15 * -. ' > Arg (2) INFORMATION FOR SEQ ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 360 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Met Glu Asn Phe Val Wing Thr Leu Wing Asn Gly Met Ser Leu Gln Pro 1 5 10 15 Pro Leu Glu Glu Val Ser Cys Gly Gln Ala Glu Ser Ser Glu Lys Pro 20 25 30 Asn Ala Glu Asp Met Thr Ser Lys ASO Tyr Tyr Phe Asp Ser Tyr Ala 35 40"45 Kis Phe Gly He His Glu Glu Met Leu Lys Asp Glu Val Arg Thr Leu 50 55 60 Thr Tyr Arg Asn Ser Met Phe Hie Asn Arg His Leu Phe Lys Asp Lys 65 70 75 80 Val Val Leu Aep Val Gly Ser Gly Thr Gly He Leu Cys Met Phe Wing • 85 90 95 Ala Lys Ala Gly Ala Arg Lys Val He Gly He Val Cys Ser Ser He? Oo 105 -. > • "•, v, .... • - •" -. •; -. , \; no - r.i -. .

Being Asp Tyr Ala Val Lys He Val Lys Al -Asn 'Lys-.Leu' sp His Val 115 120, - ,,. ". '. . =: '. i -.-.;: *. 125.-....-. ? - • Val Thr He He Lys Gly Lys Val Glu Glu Val Glu Leu Pro Val Glu 130 135 140 Lys Val Wing Ser Ser Wing Ser Gly Trp Wing Thr Wing Ser Ser Thr 145 150 155 160 Ser Pro Cys Ser Thr Pro Cys Ser Met Pro Gly Thr Ser Val Wing Pro 165 170 175 Asp Gly Leu He Phe Pro Asp Arg Wing Thr Leu Tyr Val Thr Wing He 180 185 190 * 5 Glu Asp Arg Gln Tyr Lys Aep Tyr Lys He Hie Trp Trp Glu Asn Val 195 200 205 Tyr Gly Phe Asp Met Ser Cys He Lys Asp Val Wing He Lys Glu Pro 210 215 220 Leu Val Asp Val Val Asp Pro Lys Gln Leu Val Thr Asn Ala Cys Leu 225 230 235 240 He Lys Glu Val Asp He Tyr. Thr Val Lys -Val Glu Asp Leu Thr Phe '245 250 255 Thr Ser Pro Phe Cys Leu.Gln Val Lys Arg Asn Asp Tyr Val Hie Wing 260 265 270 10 •' Leu Val Ala Tyr Phe Asn He Glu Phe Thr Arg Cys His Lye Arg Thr 275 280 285 Gly Phe Ser Thr Ser Pro Glu Ser Pro Tyr Thr His Trp Lys Gln Thr 290 295 300 Val Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys Thr 'Gly Glu Glu He 305 310 315 320 Phe Gly Thr He Gly Met Arg Pro Asn Wing Lys Asn Asn Arg Aep Leu 325 330 335 * Asp Phe Thr He Aep Leu Asp Phe Lys Gly Gln Leu Cys Glu Leu Ser 15 340 345 350 «Cys Ser Thr Asp Tyr Arg Met Arg 355 360 (2) INFORMATION FOR SEQ ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: 20 (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (i) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: Gly Gly Phe Gly Gly Arg Gly Gly Phe Gly 1 • 5"'10 (2) INFORMATION FOR SEQ ID NO. 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid 10 (C) CHAIN TYPE: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ? - 15 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: * GGCTACAAAA TTCTCCATGA TG 22 (2) INFORMATION FOR SEQ ID NO. 13: (i) CHARACTERISTICS OF THE SEQUENCE: 20 (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: TGGCCGTCAC ATACAGCGTG G 21 REFERENCES Abramovich, C, Shulman, L.M., Ratovitski, E., Harroch, S., Tovey, M, Eid, P. and Revel, M. (1994). Differential tyrosine phosphorylation of the INFAR chain of the type I Interferon receptor and of an associated surface protein response to IFN- and IFN-β. EMBO J .. 13: 5871-5877. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990). Basic local alignment research tool. J. Mol. Biol .. 215: 403-410. Barter, P.L., Chien, C.T., Stemglanz, R. and Fields, S. (1993). Elimination of false positives thatarise using the two-hybrid system. BioTechniques 14: 920-924. Bolding, M.P. Varfolomeev, E.E. Pancer, Z. Mett, I.L. Camonis J.H. and Wallach D. (1995). A novel protein that interactions with the death domain of Fas / AP01 contains a sequence motif related to the death domain. J. Biol Chem. 270: 7795-7798. Breeden, L. and Naysmith, K. (1995). Regulation of the yeast HO gene. Cold Spring Harbor Svmp. Quant. Biol .. 50: 643-650. Colamonici, O.R., D'Allessandro, F., Diaz, M.O., Gregory, S.a., Necker, L.M. and Nordan, R. (1990). Characterization of three monoclonal antibodies that recognize the receptor-a2 receptor. Proc. Nati Acad. Sci. USA 87, 7230-7234.

Darnell, J.E., Kerr, I.M. and Stark, G.R. (1994). Jak-Stat pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins. Science, 264: 1415-1421. David, M., Chen, H.E., Goelz, S., Lamer, A.C. and Neel, B.G. (1995a). 5 Differential regulation of the alpha / beta Interferon-stimulated Jak / Stat pathway by the SH2 domain-containing tyrosine phosphatase SHPTP1. Mol. Cell. Biol .. 15: 7050-7058. David, M., Petricoin, E. lll, Benjamin, C, Pine, R., Weber, M.J. and Lamer, A.C. (1995b). Requirement for MAP kinase (ERK2) activity ¡n Interferon at 10 and Interferon ß-stimulated gene expression through Stat proteins. Science. 269: 1721-1723. David, M. Petricoin, E. lll and Lamer, A.C. (1996) Activation of Protein kinase A nhibits Interferon induction of the Jak / Stat pathway in U266 cells. J Biol. Chem .. 271: 4585-4588. ? * 15 Deiss, L.P. and Kimchi, A. (199). A genetic tool used to identify thiroredoxin as a mediator of a growth inhibitory signal. Science 252, 117-20. Domanski, P., Witte, M., Kellum, M., Rubinstein, M., Hackett, R., Pitha, P. and Colamonici, O.R. (nineteen ninety five). Cloning and expression of a long form of the beta subunit of the Interferon alpha beta receptor that is required for signaling. 20 Biol. Chem. 270: 21606-21611. Durfee, T., Becherer, K, Chen, P.-L., Yeh, S-H, Yang, Y., Kilbum, A.E., Lee, W.-H. and Elledge, S. (1993). The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes & Devpt. 7: 555-569.

Ellis, L., Clauser, E., Morgan, D.O., Edery, M., Roth, R.A. and Rutter, W.J. (1986). Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell, 45, 721-731. Fields, S. and Song, O. (1989). A novel genetic system to detect protein-protein interactions. Nature 340: 245-246. Guerini, D. and others (1989). DNA 8: 675-682. Harroch, S., Revel, M. and Chebath, J. (1994). lnterleukin-6 signaling via four transcription factors binding palindromic enhancers of different genes. Biol. Chem .. 269: 26191-26195. Henikoff, S. and Henikoff, J.G. (1992). Proc. Nati Acad. Sci. USA .. 89: 10915-10919. Kagan, R.M. and Clarke, S. (1994). Widespread occurrence of three sequence motifs in diverse S-adenosyl methionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys., 310, 417-427. Lee, V.D. and Huang, B. (1993). Proc. Nati Acad. Sci. USA 90: 11039-11043. Leung, S., Qureshi, S.A., Kerr, I.M., Darnell, J.E. and Stark, G.R. (nineteen ninety five). Role of Stat2 in the alpha Interferon signaling pathway. Mol. Cell. Biol .. 15: 1312-1317. Lin, W.-J., Gary, J.D., Yang, M.C., Clarke, S. and Herschman, H.R. (nineteen ninety six). The mammalian immediate-early T1S21 protein and the leukemia-associated BTG1 protein interact with a Protein-arginine Methyltransferase. J. Biol Chem .. 271: 15034-15044. Liu, Q. and Dreyfuss, G. (1995). In vivo and in vitro arginine methylation of RNA-binding proteins. Mol. Cell. Biol., 15: 2800-2808. Najbauer, J., Johnson, B.A., Young, A.L. and Asward, D.W. (1993).

Peptides with sequences similar to glycine arginine rich motifs n proteins interacting with RNA are recognized by methyltransferases modifying arginine n numerous proteins. J. Biol. Chem., 268, 10501-10509. Nikawa, J.-l., Nakano, H. and Ohi, N. (1996). Structural and functional conservation of human yeast HCPI genes which can suppress the growth defect of the Saccharomvces cerevisiae 5 mutant. Gene, 171, 107-111. Revel, M. (1984). The Interferon system ¡n man: nature of the Interferon molecules and mode of action. In Becker I. (ed.), Antiviral Drugs and Interferon. The molecular basis of their activity. Martinus Nijhoff Publ., Boston, p. 357-433. Revel, M. and Chebath, J. (1986). Interferon-activated genes. Trends Biochem. Sci .. 11: 166-170. Stein, C.A., Subasinghi, C, Shinozuka, K. and Cohen, J.S. (1989). Physicochemical properties of phosphorothionate oligodeoxynucleotides. Nucleic Acids Res., 16, 3209-3221. Tamm, I., Lin, S.L., Pfeffer, L.M. and Sehgal, P.B. (1987). Interferons a and ß as cellular regulatory molecules. In Gresser, I. (ed.), Interferon 9, Acad. Press, London, pgs. 14-74.

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«E * Wickstrom, E. (1991). In: Prospects for Antisense Nucleic Acid 5 Therapy of Cancer and AIDS. p. 7-24, Wiley-Liss, New York. Yang, C.H., Shi, W, Basu, L, Murti, A., Constantinescu, S.N., Blatt, L., Croze, E., Mullersman, J.E. and Pfeffer, L.M. (nineteen ninety six). Direct association of Stat3 with the TFNAR1- chain of the human type I Interferon receptor. J. Biol. Chem. 271: 8057-8061.

Claims (1)

NOVELTY OF THE INVENTION CLAIMS * 5 1.- A recombinant DNA molecule, characterized in that it comprises a nucleotide sequence that encodes an IFNAR1 receptor binding protein capable of being induced by IFN- or IFN-β and having the amino acid sequence of SEQ ID NO. : 2 or SEQ ID NO: 8. 2.- The recombinant DNA molecule in accordance with the
1. Claim 1, further characterized in that it comprises a promoter operably linked to said sequence encoding an IFNAR1 receptor binding protein in a sense orientation, such that said promoter is capable of expressing said protein when the recombinant DNA molecule is F- in an appropriate expression host. 3. The recombinant DNA molecule according to claim 2, further characterized in that said promoter is a strong constitutive promoter in human cells. 4. The recombinant DNA molecule according to claim 1, further characterized in that said nucleotide sequence is SEQ ID NO: 1 or SEQ ID NO: 7. 5. A recombinant RNA or DNA molecule comprising a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 7 in sense or antisense orientation, or a fragment thereof, capable of hybridizing with mRNA encoding an IFNAR1 receptor binding protein inducible by interferon and thus preventing its expression, or an RNA molecule or a sequence corresponding to that of said DNA molecule. 6. The RNA molecule or recombinant DNA according to claim 5, further characterized in that it comprises an RNA molecule. 7. The recombinant RNA or DNA molecule according to claim 5, further characterized in that said sequence is in the sense orientation. 8. The RNA molecule or recombinant DNA according to claim 5, further characterized in that said sequence is in the antisense orientation. 9. The RNA or recombinant DNA molecule according to claim 8, further characterized in that it comprises a recombinant DNA molecule further comprising a promoter operably linked to said sequence in an antisense orientation, such that said promoter is capable of expressing an antisense RNA complementary to a full sense RNA or part thereof encoding an IFNAR1 receptor binding protein induced by interferon. 10. The recombinant DNA molecule according to claim 9, further characterized in that said nucleotide sequence is u? 5 'end segment of SEQ ID NO: 1 or SEQ ID NO: 7. 11. - The recombinant DNA molecule according to any of claims 2, 9 and 10, further characterized in that said promoter is an interferon-inducible promoter. * 12.- The recombinant DNA molecule in accordance with * * Any of claims 1 to 5 and 7 to 10, further characterized in that it is an expression vector. 13. A host cell capable of expressing an antisense RNA complementary to a sense RNA that codes for an interferon-inducible IFNAR1 receptor binding protein, which cell includes an expression vector according to claim 12. 14.- The Use of an expression vector comprising the recombinant DNA molecule of any of claims 8 to 10 in the manufacture of a medicament for prolonging the survival of tissue grafts, characterized in that the cells of a tissue or organ are grafted into a * 15 patient. 15. The use of an expression vector comprising the recombinant DNA molecule according to claims 3 or 4, and a pharmaceutically acceptable excipient for the manufacture of a pharmaceutical composition that is administered directly into a tumor of a patient for 20 improve the response to exogenous interferon in the treatment of cancer. 16. An IFNAR1 binding protein having the sequence of SEQ ID NO: 2 or SEQ ID NO: 8.17. - A molecule comprising the antigen binding portion of an antibody specific for an IFNAR1 binding protein according to claim 16. 18. The molecule according to claim 17, further characterized in that it consists of a monoclonal antibody. 19. A method for providing an IFNAR1 receptor binding protein, characterized in that it comprises placing a DNA according to claim 1, in an appropriate expression host in a form by which the expression of said protein can be obtained after cultivate said host, and cultivate said host to obtain the expression of said protein.