EP0720652A1 - Sequence de proteines ou facteurs de reconnaissance de recepteurs et leur procede d'utilisation - Google Patents

Sequence de proteines ou facteurs de reconnaissance de recepteurs et leur procede d'utilisation

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Publication number
EP0720652A1
EP0720652A1 EP94931767A EP94931767A EP0720652A1 EP 0720652 A1 EP0720652 A1 EP 0720652A1 EP 94931767 A EP94931767 A EP 94931767A EP 94931767 A EP94931767 A EP 94931767A EP 0720652 A1 EP0720652 A1 EP 0720652A1
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European Patent Office
Prior art keywords
leu
glu
gin
ser
lys
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EP94931767A
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German (de)
English (en)
Inventor
James E. Darnell, Jr.
Christian W. Schindler
Ke Shuai
Zilong Wen
Zhong Zhong
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Rockefeller University
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Rockefeller University
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Priority claimed from US08/212,185 external-priority patent/US6605442B1/en
Application filed by Rockefeller University filed Critical Rockefeller University
Publication of EP0720652A1 publication Critical patent/EP0720652A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Kessler et al. "IFN ⁇ Regulates Nuclear Translocation and DNA-Binding Affinity of ISGF3, A Multimeric Transcriptional Activator” GENES AND DEVELOPMENT, 4: 1753 (1990); (4) C. Schindler et al., “Interferon-Dependent Tyrosine Phosphorylation of a Latent Cytoplasmic Transcription Factor” Science, 257:809-812 (1992); (5) Ke Shuai et al. , "Interferon- ⁇ triggers transcription through cytoplasmic tyrosine phosphorylation of a 91 kD DNA binding protein” Science, 258: 1808 (1992); and (6) International Patent Publication No. WO 93/19179, "IFN RECEPTORS RECOGNITION FACTORS, PROTEIN SEQUENCES AND METHODS OF USE THEREOF, " published 30 September 1993.
  • the present invention relates generally to intracellular receptor recognition proteins or factors (i.e. groups of proteins), and to methods and compositions including such factors or the antibodies reactive toward them, or analogs thereof in assays and for diagnosing, preventing and/or treating cellular debilitation, derangement or dysfunction. More particularly, the present invention relates to particular molecules that exhibit both receptor recognition and message delivery via DNA binding in an interferon-dependent manner, and specifically that directly participate both in the interaction with the liganded receptor at the cell surface and in the activity of transcription in the nucleus as a DNA binding protein. The invention likewise relates to the antibodies and other entities that are specific to this factor and that would thereby selectively modulate its activity.
  • interferons activate sets of other genes entirely. Even IFN ⁇ and IFN 7 , whose presence results in the slowing of cell growth and in an increased resistance to viruses (Tamm et al., 1987) do not activate exactly the same set of genes (Larner et al. , 1984; Friedman et al., 1984; Celis et al. , 1987, 1985; Larner et al. , 1986).
  • Second messengers cAMP, diacyl glycerol, phosphoinositides, and Ca 2+ are the most prominently discussed
  • cAMP diacyl glycerol, phosphoinositides, and Ca 2+
  • a cell can be called upon to respond simultaneously to two or more different ligands with an individually specific transcriptional response each involving a different set of target genes.
  • IL-2 interleukin-2
  • IFN ⁇ Uze et al. , 1990
  • IFN 7 IFN 7
  • NGF Johnson et al. , 1986
  • growth hormone Leung et al. , 1987
  • Stat84 was found to be a truncated form of Stat91. There is 42% amino acid sequence similarity between Statl 13 and Stat91/84 in an overlapping 715 amino acid sequence, including four leucine and one valine heptad repeats in the middle helix region, and several tyrosine residues were conserved near the ends of both proteins.
  • the receptor recognition proteins thus possess multiple properties, among them: 1) recognizing and being activated during such recognition by receptors; 2) being translocated to the nucleus by an inhibitable process (e.g. , NaF inhibits translocation); and 3) combining with transcription activating proteins or acting themselves as transcription activation proteins, and that all of these properties are possessed by the proteins described herein.
  • the proteins are activated by binding of interferons to receptors on cells, in particular interferon- ⁇ (all three Stat proteins) and interferon- ⁇ (Stat91).
  • receptor recognition factors also termed herein signal transducers and activators of transcription - STAT
  • signal transducers and activators of transcription - STAT have been further characterized that appear to interact directly with receptors that have been occupied by their ligand on cellular surfaces, and which in turn either become active transcription factors, or activate or directly associate with transcription factors that enter die cells' nucleus and specifically binds on predetermined sites and thereby activates the genes.
  • the receptor recognition proteins thus possess multiple properties, among them: 1) recognizing and being activated during such recognition by receptors; 2) being translocated to the nucleus by an inhibitable process (eg. NaF inhibits translocation); and 3) combining with transcription activating proteins or acting themselves as transcription activation proteins, and that all of these properties are possessed by the proteins described herein.
  • a further property of the receptor recognition factors is dimerization to form homodimers or heterodimers upon activation by phosphorylation of tyrosine.
  • infra, Stat91 and Stat84 form homodimers and a Stat91- Stat84 heterodimer. Accordingly, the present invention is directed to such dimers, which can form spontaneously by phosphorylation of the STAT protein, or which can be prepared synthetically by chemically cross-linking two like or unlike STAT proteins.
  • the present invention further relates to receptor recognition factors that are functionally active fragments of the 91 kD receptor recognition factor, particularly such fragments that contain an amino acid residue corresponding to the tyrosine 701 residue, and preferably that contain a corresponding phosphotyrosine residue.
  • the functionally active fragments further comprises the SH2 domain, particularly the SH2 domain that has a residue corresponding to an arginine-602 residue. It is envisioned that such functionally active receptor recognition factors comprise at least about 8 amino acid residues.
  • the invention contemplates inhibitory fragments of the 91 kD protein.
  • the SH2 domain of the 91 kD protein can competitively inhibit phosphorylation of the whole protein or fragment thereof containing tyrosine 701.
  • an inhibitory fragment can compete with the 91 kD protein for binding to a tyrosine kinase.
  • Such an inhibitory fragment may contain a residue corresponding to tyrosine 701.
  • the receptor recognition factor is proteinaceous in composition and is believed to be present in the cytoplasm. The recognition factor is not demonstrably affected by concentrations of second messengers, however does exhibit direct interaction with tyrosine kinase domains, although it exhibits no apparent interaction with G- proteins.
  • the 91 kD human interferon (IFN)- ⁇ factor (hence, formerly also termed "GAF"), represented by SEQ ID NO:4 directly interacts with DNA after acquiring phosphate on tyrosine located at position 701 of the amino acid sequence.
  • the recognition factor is now known to comprise several proteinaceous substituents, in the instance of IFN ⁇ and IFN ⁇ .
  • Three proteins derived from the factor ISGF-3 have been successfully sequenced and their sequences are set forth in SEQ ID NOS: l , 2; SEQ ID NOS:3, 4; and SEQ ID NOS:5, 6, herein (see International Patent Publication No. WO 93/19179).
  • the present invention is therefore particularly directed to additional members of the STAT family, including a murine gene encoding the 91 kD protein (SEQ ID NO:4) has been identified and sequenced.
  • the nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO: 8) of the murine homolog of SEQ ID NO:4 are shown in FIGURE lA-lC.
  • murine genes encoding homologs of the recognition factor have been successfully sequenced and cloned into plasmids.
  • a gene in plasmid 13sfl has the nucleotide sequence (SEQ ID NO:9) and deduced amino acid sequence (SEQ ID NO: 10) as shown in FIGURE 2A-D.
  • a gene in plasmid 19sf6 has the nucleotide sequence (SEQ ID NO: 11) and deduced amino acid sequence (SEQ ID NO: 12) shown in FIGURE 3A-E.
  • the protein sequence of SEQ ID NO: 2 and the sequence of the proteins of SEQ ID NO:4 and SEQ ID NO:6 derive, respectively, from two different but related genes.
  • the protein sequence of FIGURE 1 (SEQ ID NO: 8) derives from a murine gene that is analogous to the gene encoding the protein of SEQ ID NO:4.
  • the protein sequences of FIGURES 2 (SEQ ID NO: 10) and 3 (SEQ ID NO: 12) derive from two genes that are different from, but related to, the protein of FIGURE 1 (FIG ID NO:8).
  • the capacity of such family members to function in the manner of the receptor recognition factors disclosed, herein may be assessed by determining those ligand that cause the phosphorylation of the particular family members.
  • the present invention extends to a receptor recognition factor implicated in the transcriptional stimulation of genes in target cells in response to the binding of a specific polypeptide ligand to its cellular receptor on said target cell, said receptor recognition factor having the following characteristics: a) apparent direct interaction with the ligand-bound receptor complex and activation of one or more transcription factors capable of binding with a specific gene; b) an activity demonstrably unaffected by the presence or concentration of second messengers; c) direct interaction with tyrosine kinase domains; and d) a perceived absence of interaction with G-proteins.
  • the receptor recognition (STAT) protein forms a dimer upon activation by phosphorylation.
  • the receptor recognition factor represented by SEQ ID NO:4 possesses the added capability of acting as a translation protein and, in particular, as a DNA binding protein in response to interferon-7 stimulation.
  • This discovery presages an expanded role for the proteins in question, and other proteins and like factors that have heretofore been characterized as receptor recognition factors. It is therefore apparent that a single factor may indeed provide the nexus between the liganded receptor at the cell surface and direct participation in DNA transcriptional activity in the nucleus.
  • This pleiotypic factor has the following characteristics: a) It interacts with an interferon- ⁇ -bound receptor kinase complex; b) It is a tyrosine kinase substrate; and c) When phosphorylated, it serves as a DNA binding protein.
  • the factor represented by SEQ ID NO:4 is interferon-dependent in its activity and is responsive to interferon stimulation, particularly that of interferon- ⁇ . It has further been discovered that activation of the factor represented by SEQ ID NO:4 requires phosphorylation of tyrosine-701 of the protein. In particular, phosphorylation of tyrosine-701 is required for nuclear transport, DNA binding, and transcription activation. Furthermore, tyrosine phosphorylation requires the presence of a functionally active SH2 domain in the protein. Preferably, such SH2 domain contains an amino acid residue corresponding to an arginine at position 602 of the protein.
  • the present invention extends to a receptor recognition factor interactive with a liganded interferon receptor, which receptor recognition factor possesses the following characteristics: a) it is present in cytoplasm; b) it undergoes tyrosine phosphorylation upon treatment of cells with IFN ⁇ or IFN 7 ; c) it activates transcription of an interferon stimulated gene; d) it stimulates either an ISRE-dependent or a gamma activated site (GAS)-dependent transcription in vivo; e) it interacts with IFN cellular receptors, and f) it undergoes nuclear translocation upon stimulation of the IFN cellular receptors with IFN.
  • a receptor recognition factor interactive with a liganded interferon receptor which receptor recognition factor possesses the following characteristics: a) it is present in cytoplasm; b) it undergoes tyrosine phosphorylation upon treatment of cells with IFN ⁇ or IFN 7 ; c) it activates transcription of an interferon stimulated gene; d
  • the factor of the invention represented by SEQ ID NO:4 appears to act in similar fashion to an earlier determined site-specific DNA binding protein that is interferon-7 dependent and that has been earlier called the 7 activating factor (GAF). Specifically, interferon-7-dependent activation of this factor occurs without new protein synthesis and appears within minutes of interferon-7 treatment, achieves maximum extent between 15 and 30 minutes thereafter, and then disappears after 2-3 hours. These further characteristics of identification and action assist in the evaluation of the present factor for applications having both diagnostic and therapeutic significance.
  • the present invention relates to all members of the herein disclosed family of receptor recognition factors, specifically the proteins whose sequences are represented by one or more of SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a receptor recognition factor, or a fragment thereof, that possesses a molecular weight of about 113 kD and an amino acid sequence set forth in FIGURE 1 (SEQ ID NO:8).
  • the receptor recognition factor has an amino acid sequence set forth in FIGURE 2 (SEQ ID NO: 10); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 2 (SEQ ID NO:9).
  • the receptor recognition factor has an amino acid sequence set forth in FIGURE 3 (SEQ ID NO: 12); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 3 (SEQ ID NO: 1 1).
  • the human and murine DNA sequences of the receptor recognition factors of the present invention or portions thereof may be prepared as probes to screen for complementary sequences and genomic clones in the same or alternate species.
  • the present invention extends to probes so prepared that may be provided for screening cDNA and genomic libraries for the receptor recognition factors.
  • the probes may be prepared with a variety of known vectors, such as the phage ⁇ vector.
  • the present invention also includes the preparation of plasmids including such vectors, and the use of the DNA sequences to construct vectors expressing antisense RNA or ribozymes which would attack the mRNAs of any or all of the DNA sequences set forth in FIGURES 1 , 2, and 3 (SEQ ID NOS:7, 9, and 11 , respectively).
  • SEQ ID NOS:7, 9, and 11 are included herein.
  • the present invention also includes receptor recognition factor proteins having the activities noted herein, and that display the amino acid sequences set forth and described above and selected from SEQ ID NO:8, SEQ ID NO: 10 and SEQ ID NO:
  • the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host.
  • the invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present receptor recognition factor(s), and more particularly, the complete DNA sequence determined from the sequences set forth above and in SEQ ID NO:7, SEQ ID NO:9 and SEQ ID NO- 11.
  • a recombinant expression system is provided to produce biologically active animal or human receptor recognition factor.
  • the present invention naturally contemplates several means for preparation of the recognition factor, including as illustrated herein known recombinant techniques, and the invention is accordingly intended to cover such synthetic preparations within its scope.
  • the isolation of the cDNA amino acid sequences disclosed herein facilitates the reproduction of the recognition factor by such recombinant techniques, and accordingly, the invention extends to expression vectors prepared from the disclosed DNA sequences for expression in host systems by recombinant DNA techniques, and to the resulting transformed hosts.
  • the invention includes an assay system for screening of potential drugs effective to modulate transcriptional activity of target mammalian cells by interrupting or potentiating the recognition factor or factors.
  • the test drug could be administered to a cellular sample with the ligand that activates the receptor recognition factor, or an extract containing the activated recognition factor, to determine its effect upon the binding activity of the recognition factor to any chemical sample (including DNA), or to the test drug, by comparison with a control.
  • the assay system could more importantly be adapted to identify drugs or other entities that are capable of binding to the receptor recognition and/or transcription factors or proteins, either in the cytoplasm or in the nucleus, thereby inhibiting or potentiating transcriptional activity.
  • Such assay would be useful in the development of drugs that would be specific against particular cellular activity, or that would potentiate such activity, in time or in level of activity.
  • drugs might be used to modulate cellular response to shock, or to treat other pathologies, as for example, in making IFN more potent against cancer.
  • the invention contemplates antagonists of the activity of a receptor recognition factor (STAT).
  • STAT receptor recognition factor
  • an agent or molecule that inhibits dimerization can be used to block transcription activation effected by an activated, phosphorylated STAT protein.
  • the antagonist can be a peptide having the sequence of a portion of an SH2 domain of a STAT protein, or the phosphotyrosine domain of a STAT protein, or both. If the peptide contains both regions, preferably the regions are located in tandem, more preferably with the SH2 domain portion N-terminal to the phosphotyrosine portion. In a specific example, infra, such peptides are shown to be capable of disrupting dimerization of STAT proteins.
  • the diagnostic utility of the present invention extends to the use of the present receptor recognition factors in assays to screen for tyrosine kinase inhibitors. Because the activity of the receptor recognition-transcriptional activation proteins described herein must maintain tyrosine phosphorylation, they can and presumably are dephosphorylated by specific tyrosine phosphatases. Blocking of the specific phosphatase is therefore an avenue of pharmacological intervention that would potentiate the activity of the receptor recognition proteins.
  • the present invention likewise extends to the development of antibodies against the receptor recognition factor(s), including naturally raised and recombinantly prepared antibodies.
  • the antibodies could be used to screen expression libraries to obtain the gene or genes that encode the receptor recognition factor(s).
  • Such antibodies could include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi- specific (chimeric) antibodies, and antibodies including other functionalities suiting them for additional diagnostic use conjunctive with their capability of modulating transcriptional activity.
  • antibodies against specifically phosphorylated factors can be selected and are included within the scope of the present invention for their particular ability in following activated protein.
  • activity of the recognition factors or of the specific polypeptides believed to be causally connected thereto may therefore be followed directly by the assay techniques discussed later on, through the use of an appropriately labeled quantity of the recognition factor or antibodies or analogs thereof.
  • the receptor recognition factors are capable of use in connection with various diagnostic techniques, including immunoassays, such as a radioimmunoassay, using for example, an antibody to the receptor recognition factor that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • immunoassays such as a radioimmunoassay, using for example, an antibody to the receptor recognition factor that has been labeled by either radioactive addition, reduction with sodium borohydride, or radioiodination.
  • the present invention relates to certain therapeutic methods which would be based upon the activity of the recognition factor(s), its (or their) subunits, or active fragments thereof, or upon agents or other drugs determined to possess the same activity.
  • a first therapeutic method is associated with the prevention of the manifestations of conditions causally related to or following from the binding activity of the recognition factor or its subunits, and comprises administering an agent capable of modulating the production and/or activity of the recognition factor or subunits thereof, either individually or in mixture with each other in an amount effective to prevent the development of those conditions in the host.
  • drugs or other binding partners to the receptor recognition/transcription factors or proteins may be administered to inhibit or potentiate transcriptional activity, as in the potentiation of interferon in cancer therapy.
  • the blockade of the action of specific tyrosine phosphatases in the dephosphorylation of activated (phosphorylated) recognition/transcription factors or proteins presents a method for potentiating the activity of the receptor recognition factor or protein that would concomitantly potentiate therapies based on receptor recognition factor /protein activation.
  • the therapeutic method generally referred to herein could include the method for the treatment of various pathologies or other cellular dysfunctions and derangements by the administration of pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of the recognition factor or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • pharmaceutical compositions that may comprise effective inhibitors or enhancers of activation of the recognition factor or its subunits, or other equally effective drugs developed for instance by a drug screening assay prepared and used in accordance with a further aspect of the present invention.
  • drugs or other binding partners to the receptor recognition/transcription factor or proteins as represented by SEQ ID NO:8, 10, or 12, may be administered to inhibit or potentiate transcriptional activity, as in the potentiation of interferon in cancer therapy.
  • the blockade of the action of specific tyrosine phosphatases in the dephosphorylation of activated (phosphorylated) recognition/transcription factor or protein presents a method for potentiating the activity of the receptor recognition factor or protein that would concomitantly potentiate therapies based on receptor recognition factor/protein activation.
  • the inhibition or blockade of the activation or binding of the recognition/transcription factor would affect MHC Class II expression and consequently, would promote immunosuppression. Materials exhibiting this activity, as illustrated later on herein by staurosporine,. may be useful in instances such as the treatment of autoimmune diseases and graft rejection, where a degree of immunosuppression is desirable.
  • the proteins of ISGF-3 whose sequences are presented in SEQ ID NOS:8, 10, or 12 herein, their antibodies, agonists, antagonists, or active fragments thereof, could be prepared in pharmaceutical formulations for administration in instances wherein interferon therapy is appropriate, such as to treat chronic viral hepatitis, hairy cell leukemia, and for use of interferon in adjuvant therapy.
  • interferon therapy such as to treat chronic viral hepatitis, hairy cell leukemia, and for use of interferon in adjuvant therapy.
  • the specificity of the receptor proteins hereof would make it possible to better manage the aftereffects of current interferon therapy, and would thereby make it possible to apply interferon as a general antiviral agent.
  • compositions for use in therapeutic methods which comprise or are based upon the recognition factor, its subunits, their binding partner(s), or upon agents or drugs that control the production, or that mimic or antagonize the activities of the recognition factors.
  • FIGURE 1 depicts (A) the deduced amino acid sequence (SEQ ID NO: 8) of and (B-C) the DNA sequence (SEQ ID NO: 7) encoding the murine 91 kD intracellular receptor recognition factor.
  • FIGURE 2 depicts (A) the deduced amino acid sequence (SEQ ID NO: 10) of and (B-D) the DNA sequence (SEQ ID NO: 9) encoding the 13sfl intracellular receptor recognition factor.
  • FIGURE 3 depicts (A) the deduced amino acid sequence (SEQ ID NO: 12) of and (B-E) the DNA sequence (SEQ ID NO: 1 1) encoding the 19sf6 intracellular receptor recognition factor.
  • FIGURE 4 presents identification of the phosphotyrosine residue in the 91 kd protein.
  • A Tryptic phosphopeptide map of 32 P-91 kD protein from IFN-7-treated FS2 cells. Phosphoamino acid analysis indicated that only peptide X contains phosphotyrosine (31).
  • B Edman degradation of peptide X (32). The position of the PTH-P-Tyr marker detected by ultraviolet light is indicated.
  • C Schematic diagram showing the site of the phosphotyrosine residue in the 91 kD protein. HR, heptapeptide repeat; SH2, Src homology domain 2; and SH3, Src homology domain 3.
  • the synthetic peptide (10 ⁇ g) (obtained from Genetics) was incubated with 1 U of p45 v abl (Oncogene Science), in 50 mM Hepes (pH 7.4), 0.1 mM EDTA, 0.015 % Brij 35, 0.1 mM ATP, 10 mM MgCl 2 and 2 ⁇ Cl of [ 7 - 32 P]ATP for 30 min. at 30°C.
  • the 32 P- labeled peptide was subjected to electrophoresis at pH 3.5 on a thin layer chromatography plate and purified. Tryptic digestion of 32 P-labeled peptide was done as described (32).
  • Human FS2 cells were labeled with [ 32 P]orthophosphate (Du Pont) for 3 hours in phosphate-free medium and subsequently treated with IFN-7 for 10 min.
  • Cell lysates were immunoprecipitated with antiserum to the COOH-terminal 35 amino acids of 91 kD (anti-91T) and separated by SDA-polyacrylamide gel electrophoresis (PAGE) (7% gel).
  • the 32 P-labeled 91 kD band was excised and subjected to tryptic mapping (31). Edman degradation was done as described (32, 33) with minor modifications.
  • Peptide X 600 counts per minute was taken through five cycles of Edman degradation. Samples from each cycle and an equivalent amount of untreated peptide X were analyzed by electrophoresis at pH 3.5.
  • the PTH-P-Tyr marker was synthesized as described (31).
  • FIGURE 5 presents an analysis of phosphorylation of the 91 and 84 kD proteins in established cell lines.
  • A Protein immunoblot analysis with antiserum to the 91 kD protein (anti-91) of whole-cell extracts from parental 2fTGH cells (lanes 1 and 4); mutant U3 cells lacking the 91 and 84 kD proteins (lanes 2 and 3); U3 cells expressing the 91 kD protein (C91 , lane 6), the 84 kD protein (C84, lane 7), or the Tyr 7w mutant MNC-ty (Cty, lane 5).
  • B Tryptic peptide map of the 84 kD protein.
  • C84 cells were labeled with [ 32 P]orthophosphate for 3 hours and then treated with IFN-7 for 10 min.; immunoprecipitation with anti-91 and tryptic peptide mapping of the 32 P-labeled 84-kDa protein was done as described
  • FIG. 4 Proteins in whole-cell lysates from 2fTGH (lanes 3, 4, 7 and 8) and Cty (lanes 1 , 2, 5 and 6) cells were immunoprecipitated with anti-91T (31) and separated by SDA-PAGE (7% gel). The blot was then probed with a mAb to phosphotyrosine 4G10 (UB1, lanes 1 through 4). The blot was stripped and reprobed with anti-91T (lanes 5 through 8). U3A cells (5 x 10 5 ) (30) were transfected with 4 ⁇ g of expression vector and 16 ⁇ g of pBSK (Strategene) plasmid by the calcium phosphate procedure (35).
  • FIGURE 6 presents data relating to DNA binding and nuclear localization of the 91 and 84 kD proteins.
  • A DNA binding and translocation to the nucleus of the 91 and 84 kD proteins.
  • a 21 nucleotide oligomer containing the GAS sequence from the Ly-6E gene (34) was labeled and used as a probe for shift assays as described (31).
  • B Nuclear localization tested by immunofluorescence.
  • FIGURE 7 presents an analysis of transcriptional activation.
  • An oligonucleotide corresponding to the herpes simplex virus thymidine kinase (TK) promoter from - 35 to + 10 was fused to the Hindlll site of pZLUC, a luciferase reporter construct (TK-LUC).
  • TK-LUC a luciferase reporter construct
  • One copy of the 91 kD binding site [a 21 nucleotide oligomer from the Ly-6E gene (34)] was inserted into the BamHl cloning site of TK-LUC (GAS- LUC).
  • U3 cells were transfected by the calcium phosphate method as described (FIGURE 5) with 4 ⁇ g of each construct.
  • the cells were also transfected with 4 ⁇ g of pMNC alone (35) (MNC) or pMNC encoding the 91 kD protein (MNC-91 ) or the 84 kD protein (MNC-84) or the Tyr 701 mutant of the 91 kD protein (MNC- ty).
  • Lane 1 MNC-91 + GAS-LUC; lane 2, MNC-84 + GAS-LUC; lane 3, MNC + GAS-LUC; lane 4, MNC-ty + GAS-LUC; lane 4 MNC-91 + TK-LUC; and lane 6, GAS-LUC.
  • FIGURE 8 demonstrates that R 6U2 in the 91 kD protein SH2 domain is required for tyrosine phosphorylation.
  • Antibody used was anti-91 , which recognizes both the 91 and 84 kD proteins (15, 31).
  • FIGURE 9 Determination of molecular weights of Stat91 and phospho Stat91 by native gel analysis.
  • B Native gel analysis.
  • Phosphorylated Stat91 (the AO.8 fraction from A) and u ⁇ phosphorylated Stat91 (the Flow fraction from A) were analyzed on 4.5 % , 5.5 % , 6.5 % and 7.5 % native polyacrylamide gels followed by immunoblotting with anti-91T. The top of gels (TOP) and the migration position of bromophenol blue (BPB) are indicated. C) Ferguson plots. The relative mobilities (Rm) of the Stat91 and phospho Stat91 were obtained from Figure IB (see Experimental Procedures).
  • FIGURE 10 Determination of molecular weights by glycerol gradients.
  • FIGURE 11 Stat91 in cell extracts binds DNA as a dimer.
  • FIGURE 12 Formation of heterodimer by denaturation and renaturation.
  • Cytoplasmic (Left Panel) or nuclear extracts (Right Panel) from IFN-7-treated cell lines expressing either Stat84 (C84) or Stat91 (C91) were analyzed by gel mobility shift assays. +: with addition; -: without addition; D/R: samples were subjected to guanidinium hydrochloride denaturation and renaturation treatment.
  • FIGURE 13 Diagrammatic representation of dissociation and reassociation analysis.
  • FIGURE 14 Dissociation-reassociation analysis with peptides.
  • 91-Y unphosphorylated peptide from Stat91 (LDGPKGTGYIKTELI) (SEQ ID NO: 15); 91Y-p, phosphotyrosyl peptide from Stat91 (GY*IKTE) (SEQ ID NO: 16); 113Y- p, phosphotyrosyl peptide with high binding affinity to Src SH2 domain (EPQY*EEIPIYL, Songyang et al. , 1993, Cell 72:767-778) (SEQ ID NO: 18).
  • FIGURE 15 Dissociation-reassociation analysis with GST fusion proteins.
  • Final concentrations of fusion proteins added are 0.5 ⁇ M (lanes 2, 5, 8, 11 , 14), 2.5 ⁇ M (lanes 3, 6, 9, 12, 15) and 5 ⁇ M (lanes 4, 7, 10, 13, 17, 18). +: with addition; -: without addition; FP: fusion proteins.
  • FIGURE 16 Comparison of Stat91 SH2 structure with known SH2 structures.
  • the Stat91 sequence is disclosed herein (SEQ ID NO:4).
  • the structures used for the other SH2s are Src (Waksman et al., 1992, Nature 358:646-653) (SEQ ID NO:4).
  • receptor recognition factor means “receptor recognition factor”, “receptor recognition-tyrosine kinase factor”, “receptor recognition factor /tyrosine kinase substrate”, “receptor recognition/transcription factor”, “recognition factor” , “recognition factor protein(s)”, “signal transducers and activators of transcription”, “STAT”, and any variants not specifically listed, may be used herein interchangeably, and as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in FIGURE 1 (SEQ ID NO:8), FIGURE 2 (SEQ ID NO: 10), and in FIGURE 3 (SEQ ID NO: 12), and the profile of activities set forth herein and in the Claims.
  • proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms "receptor recognition factor”, “recognition factor”, “recognition factor protein(s)”, “signal transducers and activators of transcription”, and “STAT” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations.
  • amino acid residues described herein are preferred to be in the "L” isomeric form.
  • residues in the "D” isomeric form can be substituted for any L- amino acid residue, as long as the desired functional property of immunoglobulin- binding is retained by the polypeptide.
  • NH2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide.
  • amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues.
  • the above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.
  • a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double- stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g. , restriction fragments), viruses, plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e. , the strand having a sequence homologous to the mRNA).
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • RNA polymerase a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA” boxes and “CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence, e.g. , and enhancer or suppressor element.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g. , ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • standard hybridization conditions refers to salt and temperature conditions substantially equivalent to 5 x SSC and 65 °C for both hybridization and wash.
  • a "signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
  • oligonucleotide as used herein in referring to the probe of the present invention, is defined as a molecule comprised of two or more ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
  • primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e. , in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
  • the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent.
  • the exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the primers herein are selected to be “substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product.
  • a cell has been "transformed” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • Two DNA sequences are "substantially homologous" when at least about 75 % (preferably at least about 80% , and most preferably at least about 90 or 95 %) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g. , Maniatis et al., supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • an “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope.
  • the term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Patent Nos. 4,816,397 and 4,816,567.
  • An “antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen.
  • the phrase "antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.
  • Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope. including those portions known in the art as Fab, Fab', F(ab') 2 and F(v), which portions are preferred for use in the therapeutic methods described herein.
  • the phrase "monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts.
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • terapéuticaally effective amount is used herein to mean an amount sufficient to prevent, and preferably reduce by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, a clinically significant change in the S phase activity of a target cellular mass, or other feature of pathology such as for example, elevated blood pressure, fever or white cell count as may attend its presence and activity.
  • the present invention concerns the identification of novel receptor recognition factors, and the isolation and sequencing of a particular receptor recognition factor proteins, that are believed to be present in cytoplasm and that serves as a signal transducer between a particular cellular receptor having bound thereto an equally specific polypeptide ligand, and the comparably specific transcription factor that enters the nucleus of the cell and interacts with a specific DNA binding site for the activation of the gene to promote the predetermined response to the particular polypeptide stimulus.
  • the present disclosure confirms that specific and individual receptor recognition factors exist that correspond to known stimuli such as tumor necrosis factor, nerve growth factor, platelet-derived growth factor and the like. Specific evidence of this is set forth herein with respect to the interferons ⁇ and y (IFN ⁇ and IFN7).
  • a further property of the receptor recognition factors is dimerization to form homodimers or heterodimers upon activation by phosphorylation of tyrosine.
  • infra, Stat91 and Stat84 form homodimers and a Stat91- Stat84 heterodimer. Accordingly, the present invention is directed to such dimers, which can form spontaneously by phosphorylation of the STAT protein, or which can be prepared synthetically by chemically cross-linking two like or unlike STAT proteins.
  • the present receptor recognition factor is likewise noteworthy in that it appears not to be demonstrably affected by fluctuations in second messenger activity and concentration.
  • the receptor recognition factor proteins appear to act as a substrate for tyrosine kinase domains, however do not appear to interact with G-proteins, and therefore do not appear to be second messengers.
  • a particular receptor recognition factor identified herein by SEQ ID NO:4, Stat91 or Statl ⁇ has been determined to be present in cytoplasm and serves as a signal transducer and a specific transcription factor in response to IFN-7 stimulation that enters the nucleus of the cell and interacts directly with a specific DNA binding site for the activation of the gene to promote the predetermined response to the particular polypeptide stimulus.
  • This particular factor also acts as a translation protein and, in particular, as a DNA binding protein in response to interferon-7 stimulation.
  • This factor is likewise noteworthy in that it has the following characteristics: a) It interacts with an interferon-7-bound receptor kinase complex; b) It is a tyrosine kinase substrate; and c) When phosphorylated, it serves as a DNA binding protein.
  • the factor of SEQ ID NO:4 directly interacts with DNA after acquiring phosphate on tyrosine located at position 701 of the amino acid sequence. Also, interferon-7-dependent activation of this factor occurs without new protein synthesis and appears within minutes of interferon-7 treatment, achieves maximum extent between 15 and 30 minutes thereafter, and then disappears after 2-3 hours.
  • Stat 91 is more particularly characterized by at least one of the following additional characteristics: d) Phosphorylation of tyrosine-701 is required for nuclear transport; e) Phosphorylation of tyrosine-701 is required for DNA binding; f) Phosphorylation of tyrosine-701 is required for transcription activation; g) A functional SH2 domain is required for tyrosine-701 phosphorylation.
  • a further property of the present factor is its ability to dimerize when phosphorylated.
  • a further property of the receptor recognition factors (also termed herein signal transducers and activators of transcription ⁇ STAT) is dimerization to form homodimers or heterodimers upon activation by phosphorylation of tyrosine.
  • infra, Stat91 and Stat84 form homodimers and a Stat91-Stat84 heterodimer.
  • the present invention is directed to such dimers. which can form spontaneously by phosphorylation of the STAT protein, or which can be prepared synthetically by chemically cross-linking two like or unlike STAT proteins.
  • the present invention further relates to receptor recognition factors that are functionally active fragments, e.g.. as exemplified herein with fragments of the 91 kD receptor recognition factor, particularly such fragments that contain an amino acid residue corresponding to the tyrosine 701 residue, and preferably that contain a corresponding phosphotyrosine residue.
  • the functionally active fragments further comprises the SH2 domain, particularly the SH2 domain that has a residue corresponding to an arginine-602 residue of the 91- kD receptor recognition factor. It is envisioned that such functionally active receptor recognition factors comprise at least about 8 amino acid residues.
  • the invention contemplates inhibitory fragments of such receptor recognition proteins, e.g.
  • the SH2 domain of the 91 kD protein can competitively inhibit phosphorylation of the whole protein or fragment thereof containing tyrosine 701.
  • an inhibitory fragment can compete with the 91 kD protein for binding to a tyrosine kinase. Such an inhibitory fragment may contain a residue corresponding to tyrosine 701.
  • the invention contemplates antagonists of the activity of a receptor recognition factor (STAT).
  • STAT receptor recognition factor
  • an agent or molecule that inhibits dimerization (homodimerization or heterodimerization) can be used to block transcription activation effected by an activated, phosphorylated STAT protein.
  • the antagonist can be a peptide having the sequence of a portion of an SH2 domain of a STAT protein, or the phosphotyrosine domain of a STAT protein, or both. If the peptide contains both regions, preferably the regions are located in tandem, more preferably with the SH2 domain portion N-terminal to the phosphotyrosine portion. In a specific example, infra, such peptides are shown to be capable of disrupting dimerization of STAT proteins.
  • each member of the family of receptor recognition factors is designated by the apparent molecular weight (e.g. , Statl 13, Stat91 , Stat84. etc.), or by the order in which it has been identified (e.g. , Statl ⁇ [Stat91], StatljS [Stat84], Stat2 [Statl 13], Stat3 [a murine protein also termed 19sf6],, and Stat4 [a murine STAT protein also termed 13sfl]).
  • the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes a receptor recognition factor, or a fragment thereof, that has a molecular weight of about 91 kD and the amino acid sequence set forth in FIGURE 1 (SEQ ID NO:8); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 91 kD receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 1 (SEQ ID NO: 8).
  • the receptor recognition factor has an amino acid sequence set forth in FIGURE 2 (SEQ ID NO: 10); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 2 (SEQ ID NO:9).
  • the receptor recognition factor has an amino acid sequence set forth in FIGURE 3 (SEQ ID NO: 12); preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding such receptor recognition factor has a nucleotide sequence or is complementary to a DNA sequence shown in FIGURE 3 (SEQ ID NO: 11).
  • the present invention contemplates pharmaceutical intervention in the cascade of reactions in which the receptor recognition factor is implicated, to modulate the activity initiated by the stimulus bound to the cellular receptor.
  • an appropriate inhibitor of the receptor recognition factor could be introduced to block the interaction of the receptor recognition factor with those factors causally connected with gene activation.
  • instances where insufficient gene activation is taking place could be remedied by the introduction of additional quantities of the receptor recognition factor or its chemical or pharmaceutical cognates, analogs, fragments and the like.
  • the recognition factors or their binding partners or other ligands or agents exhibiting either mimicry or antagonism to the recognition factors or control over their production may be prepared in pharmaceutical compositions, with a suitable carrier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated specific transcriptional stimulation for the treatment thereof.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. Average quantities of the recognition factors or their subunits may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the recognition factors and/or their subunits may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as viral infection or the like.
  • the recognition factor or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by such well known techniques as immunization of rabbit using Complete and Incomplete Freund's Adjuvant and the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells, respectively.
  • small molecules that mimic or antagonize the activity(ies) of the receptor recognition factors of the invention may be discovered or synthesized, and may be used in diagnostic and/or therapeutic protocols.
  • the diagnostic method of the present invention comprises examining a cellular sample or medium by means of an assay including an effective amount of an antagonist to a receptor recognition factor/protein, such as an anti-recognition factor antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • a receptor recognition factor/protein such as an anti-recognition factor antibody, preferably an affinity-purified polyclonal antibody, and more preferably a mAb.
  • the anti- recognition factor antibody molecules used herein be in the form of Fab, Fab', F(ab') 2 or F(v) portions or whole antibody molecules.
  • patients capable of benefiting from this method include those suffering from cancer, a pre-cancerous lesion, a viral infection or other like pathological derangement.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and one or more of a receptor recognition factor, polypeptide analog thereof or fragment thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific binding of the present recognition factor within a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or active fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount.
  • the quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of inhibition or neutralization of recognition factor binding capacity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably one to several, milligrams of active ingredient per kilogram body weight of individual per day and depend on the route of administration. Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed by repeated doses at one or more hour intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations of ten nanomolar to ten micromolar in the blood are contemplated.
  • the therapeutic compositions may further include an effective amount of the factor/factor synthesis promoter antagonist or analog thereof, and one or more of the following active ingredients: an antibiotic, a steroid.
  • active ingredients an antibiotic, a steroid.
  • Exemplary formulations are well known in the art, e.g., as disclosed in International Patent Publication WO 93/19179.
  • DNA sequences disclosed herein may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and Synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g. , E. coli plasmids col El, pCRl , pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g. , M13 and
  • Filamentous single stranded phage DNA such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAS, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • any of a wide variety of expression control sequences - sequences that control the expression of a DNA sequence operatively linked to it ⁇ may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early or late promoters of SV40,
  • CMV CMV, vaccinia, polyoma or adenovirus
  • lac system the trp system, the TAC system, the 7RC system, the LTR system, the major operator and promoter regions of phage ⁇ , the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g. , Pho5), the promoters of the yeast ⁇ -mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, BSC1 , BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
  • eukaryotic and prokaryotic hosts such as strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1 , COS 7, BSC
  • a genes encoding a receptor recognition factor of the invention may be incorporated in a transgenic expression vector, e.g. , one of the well known retroviral vectors, for in vivo or ex vivo transfection of cells for gene therapy.
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products.
  • receptor recognition factor analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention.
  • Analogs, such as fragments may be produced, for example, by pepsin digestion of receptor recognition factor material.
  • Other analogs, such as muteins can be produced by standard site-directed mutagenesis of receptor recognition factor coding sequences.
  • Analogs exhibiting "receptor recognition factor activity" such as small molecules, whether functioning as promoters or inhibitors, may be identified by known in vivo and/or in vitro assays.
  • a DNA sequence encoding receptor recognition factor can be prepared synthetically rather than cloned.
  • the DNA sequence can be designed with the appropriate codons for the receptor recognition factor amino acid sequence. In general, one will select preferred codons for the intended host if the sequence will be used for expression.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature, 292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J. Biol. Chem. , 259:6311 (1984).
  • muteins allow convenient construction of genes which will express receptor recognition factor analogs or "muteins".
  • DNA encoding muteins can be made by site-directed mutagenesis of native receptor recognition factor genes or cDNAs, and muteins can be made directly using conventional polypeptide synthesis.
  • the present invention extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the receptor recognition proteins at the translational level.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense and ribozyme technology are well known in the art, and have been described in many publications, e.g. , International Patent Publication WO 93/19179.
  • the present invention also relates to a variety of diagnostic applications, including methods for detecting the presence of stimuli such as the earlier referenced polypeptide ligands, by reference to their ability to elicit the activities which are mediated by the present receptor recognition factor.
  • the receptor recognition factor can be used to produce antibodies to itself by a variety of known techniques, and such antibodies could then be isolated and utilized as in tests for the presence of particular transcriptional activity in suspect target cells.
  • Many assay procedures, or formats, are well known in the art.
  • the "competitive" procedure is described in U.S. Patent Nos. 3,654,090 and 3,850,752.
  • the "sandwich” procedure is described in U.S. Patent Nos. RE 31,006 and 4,016,043. Still other procedures are known such as the "double antibody", or "DASP" procedure.
  • the receptor recognition factor forms complexes with one or more antibody(ies) or binding partners and one member of the complex is labeled with a detectable label.
  • the fact that a complex has formed and, if desired, the amount thereof, can be determined by known methods applicable to the detection of labels.
  • the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
  • a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine and auramine.
  • the receptor recognition factor or its binding partner(s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3 H, 14 C, 3 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 9 Y, 125 I, 13I I, and 186 Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • a particular assay system developed and utilized in accordance with the present invention is known as a receptor assay.
  • the material to be assayed is appropriately labeled and then certain cellular test colonies are inoculated with a quantity of both the labeled and unlabeled material after which binding studies are conducted to determine the extent to which the labeled material binds to the cell receptors. In this way, differences in affinity between materials can be ascertained.
  • a purified quantity of the receptor recognition factor may be . radiolabeled and combined, for example, with antibodies or other inhibitors thereto, after which binding studies would be carried out. Solutions would then be prepared that contain various quantities of labeled and unlabeled uncombined receptor recognition factor, and cell samples would then be inoculated and thereafter incubated. The resulting cell monolayers are then washed, solubilized and then counted in a gamma counter for a length of time sufficient to yield a standard error of ⁇ 5 % . These data are then subjected to Scatchard analysis after which observations and conclusions regarding material activity can be drawn. While the foregoing is exemplary, it illustrates the manner in which a receptor assay may be performed and utilized, in the instance where the cellular binding ability of the assayed material may serve as a distinguishing characteristic.
  • an assay useful and contemplated in accordance with the present invention is known as a "cis/trans” assay. Briefly, this assay employs two genetic constructs, one of which is typically a plasmid that continually expresses a particular receptor of interest when transfected into an appropriate cell line, and the second of which is a plasmid that expresses a reporter such as luciferase, under the control of a receptor/1 igand complex.
  • one of the plasmids would be a construct that results in expression of the receptor in the chosen cell line, while the second plasmid would possess a promoter linked to the luciferase gene in which the response element to the particular receptor is inserted.
  • the compound under test is an agonist for the receptor
  • the ligand will complex with the receptor, and the resulting complex will bind the response element and initiate transcription of the luciferase gene.
  • the resulting chemiluminescence is then measured photometrically, and dose response curves are obtained and compared to those of known ligands.
  • the foregoing protocol is described in detail in U.S. Patent No. 4,981 ,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is referred.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or absence of predetermined transcriptional activity or predetermined transcriptional activity capability in suspected target cells.
  • one class of such kits will contain at least the labeled receptor recognition factor or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive", “sandwich”, “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • test kit may be prepared for the demonstration of the presence or capability of cells for predetermined transcriptional activity, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. "competitive”, “sandwich”, “double antibody” , etc.), and comprises:
  • a labeled component which has been obtained by coupling the receptor recognition factor to a detectable label;
  • an assay system for screening potential drugs effective to modulate the activity of the receptor recognition factor may be prepared.
  • the receptor recognition factor may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the transcriptional activity of the cells, due either to the addition of the prospective drug alone, or- due to the effect of added quantities of the known receptor recognition factor.
  • a fragment of the gene encoding the human 91 kD protein was used to screen a murine thymus and spleen cDNA library for homologous proteins.
  • the screening assay yielded a highly homologous gene encoding a murine polypeptide that is greater than 95 % homologous to the human 91 kD protein.
  • the nucleic acid and deduced amino acid sequence of the murine 91 kD protein are shown in Figure 1A-1C, and SEQ ID NO:7 (nucleotide sequence) and SEQ ID NO: 8 (amino acid sequence).
  • EXAMPLE 2 ADDITIONAL MEMBERS OF THE STAT PROTEIN FAMILY
  • murine genes encoding two additional members of the 113-91 family of receptor recognition factor proteins were isolated from a murine splenic/thymic cDNA library according to the method of Sambrook et al. (1989, Molecular Cloning, A Laboratory Manual, 2nd. ed., Cold Spring Harbor Press: Cold Spring Harbor, New York) constructed in the ZAP vector. Hybridization was carried out at 42 °C and washed at 42 °C before the first exposure (Church and Gilbert, 1984, Proc. Natl. Acad. Sci. USA 81 : 1991-95).
  • This probe was chosen to screen for other STAT family members because, while Statl and Stat2 SH2 domains are quite similar over the entire 100 to 120 amino acid region, only the amino terminal half of the STAT SH2 domains strongly resemble the SH2 regions found in other proteins.
  • the two genes have been cloned into plasmids 13sfl and 19sf6.
  • the nucleotide sequence, and deduced amino acid sequence, for the 13sfl and 19sf6 genes are shown in Figures 2 and 3, respectively.
  • These proteins are alternatively termed Stat4 and Stat3, respectively.
  • Stat91 Statl
  • Statl 13 Stat2
  • the conserved amino acid stretches likely point to conserved domains that enable these proteins to carry out transcription activation functions.
  • Stat3 like Statl (Stat91), is widely expressed, while Stat4 expression is limited to the testes, thymus, and spleen.
  • Stat3 has been found to be activated as a DNA binding protein through phosphorylation on tyrosine in cells treated with EGF or IL-6, but not after IFN- 7 , treatment.
  • Both the 13sfl and 19sf6 genes share a significant homology with the genes encoding the human and murine 91 kD protein. There is corresponding homology between the deduced amino acid sequences of the 13sfl and 19sf6 proteins and the amino acid sequences of the human and murine 91 kD proteins, although not the greater than 95 % amino acid homology that is found between the murine and human 91 kD proteins. Thus, though clearly of the same family as the 91 kD protein, the 13sfl and 19sf6 genes encode distinct proteins.
  • the chromosomal locations of the murine STAT proteins (1-4) have been determined: Statl and Stat4 are located in the centromeric region of mouse chromosome 1 (corresponding to human 2q 32-34q); the two other genes are on other chromosomes.
  • Northern analysis demonstrates that there is variation in the tissue distribution of expression of the mRNAs encoded by these genes.
  • the variation and tissue distribution indicates that the specific genes encode proteins that are responsive to different factors, as would be expected in accordance with the present invention.
  • the actual ligand, the binding of which induces phosphorylation of the newly discovered factors, will be readily determinable based on the tissue distribution evidence described above.
  • the antisera were obtained by subcloning amino acids 688 to 727 of Stat3 and 678 to 743 of Stat4 to pGEXl ⁇ t (Pharmacia) by PCR with oligonucleotides based on the boundary sequence plus restriction sites (BamHI at the 5' end and EcoRI at the 3' end), allowing for in-frame fusion with GST.
  • One milligram of each antigen was used for the immunization and three booster injections were given 4 weeks apart.
  • Anti- Stat3 and anti-Stat4 sera were used 1 : 1000 in Western blots using standard protocols. To avoid cross reactivity of the antisera, antibodies were raised against the C-terminal of Stat3 and Stat4, the less homologous region of the protein.
  • Protein expression was checked in several cell lines as well. A protein of 89 kD reactive with Stat4 antiserum was expressed in 70Z cells, a preB cell line, but not in many other cell lines. Stat3 was highly expressed, predominantly as a 97 kD protein, in 70Z, HT2 (a mouse helper T cell clone), and U937 (a macrophage-derived cell).
  • the present disclosure is illustrated by the results of work on protein factors that govern transcriptional control of IFN ⁇ -stimulated genes, as well as more recent data on the regulation of transcription of genes stimulated by IFN7.
  • the present disclosure is further illustrated by the identification of related genes encoding protein factors responsive to as yet unknown factors. It is expected that the murine 91 kD protein is responsive to IFN-7.
  • the above represents evidence that the 91 kD protein is the tyrosine kinase target when IFN7 is the ligand.
  • the 91 kD protein is the tyrosine kinase target when IFN7 is the ligand.
  • two different ligands acting through two different receptors both use these family members.
  • this family of proteins With only a . modest number of family members and combinatorial use in response to different ligands, this family of proteins becomes an even more likely possibility to represent a general link between ligand-occupied receptors and transcriptional control of specific genes in the nucleus.
  • the 91 kD protein is an IFN-7 dependent tyrosine kinase substrate as indeed it had earlier proved to be in response to IFN- ⁇ (15). 5) The 91 kD protein but not the 113 kD protein moved to the nucleus in response to IFN-7 treatment. None of these experiments prove but do strongly suggest that the same 91 kD protein acts differently in different DNA binding complexes that are triggered by either IFN- ⁇ or IFN-7.
  • the trk protein which has an intracellular tyrosine kinase domain, associates with the NGF receptor when that receptor is occupied (23).
  • the lck protein a member of the src family of tyrosine kinases, is co-precipitated with the T cell receptor (24). It is possible to predict that signal transduction to the nucleus through these two receptors could involve latent cytoplasmic substrates that form part of activated transcription factors. In any event, it seems possible that there are kinases like trk or lck associated with the IFN-7 receptor or with IFN- ⁇ receptor.
  • the 91 kD protein is specifically translocated the nucleus in the wake of IFN-7 stimulation.
  • EXAMPLE 3 TYROSINE 701 IS PHOSPHORYLATED IN THE 91 kD PROTEIN
  • IFN-7 stimulates phosphorylation of the 91 kD protein.
  • Thermolysin digestion of 32 P-labeled 91 kD protein from IFN-7-treated cells yielded a single peptide labeled on tyrosine.
  • the 91 kD protein contains 19 tyrosines (12).
  • a tryptic digest of 32 P-labeled 91 kD protein from IFN-7-treated cells (FIGURE 4 A) was examined.
  • IFN-7 induced phosphorylation of a single tryptic peptide (X) on tyrosine.
  • Peptide X was recovered and stepwise Edman degradation done.
  • the labeled phosphotyrosine was released in the fourth degradative cycle (FIGURE 4B).
  • Computer alignment of all the potential tryptic peptides showed a single peptide (amino acids 698 to 703) in which tyrosine was the fourth amino acid, revealing this peptide as the major candidate for IFN-7- stimulated tyrosine kinase action (FIGURE 4C).
  • the original sequence of the 91 kD protein omitted an 11 amino acid segment from residues 261 to 271.
  • the putative phosphorylated peptide contained a single tyrosine at residue 701 , confirming the expectation of phosphorylation at tyrosine 690 under the incorrect numbering system.
  • a synthetic peptide corresponding to amino acids 693 to 707 was prepared. This peptide was exposed to purified p43 v abl protein kinase [Oncogene Science (27)] and [7- 32 P]adenosine triphosphate (ATP). Although labeling was inefficient, only tyrosine was phosphorylated. The labeled synthetic phosphopeptide was cleaved with trypsin, and the resulting peptide migrated identically with peptide X during 2D peptide mapping. Thus, we conclude that Tyr 701 is the single residue in the 91 kD protein that is tyrosine phosphorylated in response to IFN-7.
  • TAT codon for tyrosine was changed to TTT, which encodes phenylalanine.
  • the wild-type and mutant DNAs were inserted into an expression vector.
  • the gene encoding the 91 kD protein produces two mRNAs with different 3' ends (12).
  • the two mRNAs are translated to produce the 91 kD protein and the 84 kD protein, respectively.
  • An expression vector containing complementary DNA (cDNA) encoding the 84 kD protein was also constructed.
  • constructs were introduced by permanent transfection into U3A cells, which do not respond to IFN- ⁇ or IFN-7 (28, 29) because they do not express the 84 kD protein or the 91 kD protein.
  • Full-length 91 kD protein restores the ability of these cells to respond to IFN- ⁇ and IFN-7, as tested by IFN-induced accumulation of mRNA from endogenous genes.
  • the 84 kD protein restores the accumulation of IFN- ⁇ -responsive mRNA but not IFN-7-responsive mRNA (30).
  • C91 expressing the 91 kD protein
  • Cty expressing the 91 kD protein in which Tyr 7 " 1 was changed to Phe
  • C84 expressing the 84 kD protein
  • a monoclonal antibody (mAb) to phosphotyrosine was used to detect IFN-7-dependent tyrosine phosphorylation in protein immunoblots.
  • the mutant 91 kD protein was not phosphorylated on tyrosine in response to IFN-7, whereas the 91 kD protein from either the wild- type parental cell (2fTGH) or the C91 cell was phosphorylated on tyrosine when treated with IFN-7 (FIGURE 5C).
  • This experiment confirmed that residue 701 is the sole site on the 91 kD that is phosphorylated on tyrosine in response to IFN-7.
  • the function of the 91 kD protein and the 84 kD proteins and the Tyr 701 ⁇ Phe 701 mutant was tested in various steps in the signal transduction pathway that results in IFN-7-dependent gene activation. Removal of phosphate from the 91 kD protein phosphoprotein by calf intestinal phosphatase or inhibition of in vivo phosphorylation with staurosporine abolishes the 91 kD protein DNA binding activity.
  • the IFN-7-dependent DNA protein complex, GAF was detected in the wild-type parental cells (2fTGH) and in C91 cells (FIGURE 6A).
  • the C84 cells also responded to IFN-7, yielding a DNA-protein complex that migrated somewhat faster, as would be expected for a smaller protein (FIGURE 6A). In contrast, cells expressing the Tyr 701 mutant (Cty) failed to produce an IFN-7-dependent DNA binding protein.
  • IFN-7-induced translocation to the nucleus was also tested. Immunofluorescence in C91 or C84 cells detected throughout the cell before IFN-7 treatment increased in the nucleus after IFN-7 treatment (FIGURE 6). In contrast, the Tyr 701 mutant protein did not move to the nucleus in response to IFN-7, suggesting that phosphorylation on Tyr 7 " 1 is required for the nuclear translocation of the 91 kD protein (FIGURE 6). U3 cells were transiently transfected with the 91 and 84 kD proteins, and the Tyr 7 " 1 mutant protein, and the transcriptional response to IFN-7 was measured in these cells.
  • a target gene was constructed containing luciferase as the reporter and bearing one copy of the binding site for the 91 kD phosphoprotein upstream of an RNA start site otherwise lacking promoter elements.
  • Cells transfected with the target gene and the wild-type 91 kD protein expression vector showed a 5- to 10- fold stimulation of luciferase expression when treated with IFN-7 (FIGURE 7).
  • the IFN-7-dependent transcriptional activation required the presence of the 91 kD protein; IFN-7 did not enhance transcription in U3A cells transfected with the reporter vector alone or a vector lacking the GAS site.
  • Cells transfected with the reporter vector and the Tyr 7 " 1 mutant did not respond to IFN-7, suggesting a requirement for phosphorylation for gene activation.
  • EXAMPLE 5 THE ARG 6 " 2 RESIDUE IN THE 91KD SH2 DOMAIN IS REQUIRED FOR TYROSINE PHOSPHORYLATION
  • the 91 kD protein has a sequence from Try 572 to Pro 67 " that resembles SH2 domains (38), amino acid regions known bind tightly to tyrosine phosphates (39). Since ligand activated kinases often present a phosphotyrosine to a substrate, we tested the requirement for the SH2 domain in the 91 kD protein in ligand-mediated phosphorylation.
  • the Arg 155 residue in the v-src SH2 domain is crucial for direct interaction between a phosphotyrosine residue in the SH2 domain (40, 41) and Arg 602 of the kD protein is in a comparable position within the SH2 homology (38).
  • the above represents evidence that the 91kD protein is the tyrosine kinase target when IFN7 is the ligand.
  • the 91kD protein is the tyrosine kinase target when IFN7 is the ligand.
  • two different ligands acting through two different receptors both use these family members.
  • this family of proteins With only a modest number of family members and combinatorial use in response to different ligands, this family of proteins becomes an even more likely possibility to represent a general link between ligand-occupied receptors and transcriptional control of specific genes in the nucleus.
  • the 91 kD protein is an IFN-7 dependent tyrosine kinase substrate as indeed it had earlier proved to be in response to IFN- ⁇ (15). 5) The 91 kD protein but not the 113 kD protein moved to the nucleus in response to IFN-7 treatment. These experiments prove but do strongly suggest that the same 91 kD protein acts differently in different DNA binding complexes that are triggered by either IFN- ⁇ or IFN-7.
  • the trk protein which has an intracellular tyrosine kinase domain, associates with the NGF receptor when that receptor is occupied (23).
  • the lck protein a member of the src family of tyrosine kinases, is co-precipitated with the T cell receptor (24). It is possible to predict that signal transduction to the nucleus through these two receptors could involve latent cytoplasmic substrates that form part of activated transcription factors. In any event, it seems possible that there are kinases like trk or lck associated with the IFN-7 receptor or with IFN- ⁇ receptor.
  • the 91 kD protein is specifically translocated the nucleus in the wake of IFN-7 stimulation. While the present work strongly implicates the 91 kD protein as important in the immediate IFN-7 transcriptional response of the GBP gene, two points should also be clear. First, it is not known whether the 91 kD protein acts on its own to activate transcription. Second, it is not known how widely used the 91 kD protein is in the immediate IFN-7 transcriptional response. Only a few genes have been studied that are activated immediately by IFN-7 without new protein synthesis. It is at present uncertain whether activation of these genes operates through the 91 kD binding site.
  • the present examples demonstrate that phosphorylation of Tyr 701 on the 91 kD protein induces nuclear translocation and DNA binding of the protein.
  • the phosphorylated 91 kD protein directly or indirectly activates transcription in response to IFN-8. This function of the phospho-91 kD protein has been indirectly confirmed by the inability of a non-phosphorylated mutant 91 kD protein to induce transcription.
  • the 84 kD protein acts in parallel with the 91 kD protein up to the point of gene activation: the 84 kD protein can be phosphorylated and translocated and binds to DNA. However, only the 91 kD protein acts by itself as a direct DNA binding protein capable of transcriptional activation. These results suggest that the 38 COOH-terminal amino acids of the 91 kD are essential for activation of transcription through a GAS site. It is possible that the 84 kD protein functions to regular activity of the 94 kD protein.
  • Stat91 (a 91 kD protein that acts as a signal transducer and activator of transcription) is inactive in the cytoplasm of untreated cells but is activated by phosphorylation on tyrosine in response to a number of polypeptide ligands- including IFN- ⁇ and IFN-7.
  • This example reports that inactive Stat91 in the cytoplasm of untreated cells is a monomer and upon IFN-7 induced phosphorylation it forms a stable homodimer.
  • the dimer is capable of binding to a specific DNA sequence directing transcription.
  • Dissociation and reassociation assays show that dimerization of Stat91 is mediated through SH2-phosphotyrosyl peptide interactions.
  • Dimerization involving SH2 recognition of specific phosphotyrosyl peptides may well provide a prototype for interactions among family members of STAT proteins to form different transcription complexes and Jak2 for the IFN-7 pathway (42, 43, 44). These kinases themselves become tyrosine phosphorylated to carry out specific signaling events.
  • Expression construct MNC-84 was made by insertion of the cDNA into the Not I-Bam HI cloning site of an expression vector PMNC (45, 35).
  • MNC-91L was made by insertion of the Stat91 cDNA into the Not I -Bam HI cloning sites of pMNC without the stop codon at the end, resulting the production of a long form of Stat91 with a C-terminal tag of 34 amino acids encoded by PMNC vector.
  • GST fusion protein expression plasmids were constructed by the using the pGEX- 2T vector (Pharmacia).
  • GST-91SH2 encodes amino acids 573 to 672 of Stat91;
  • GST-91mSH2 encodes amino acids 573 to 672 of Stat91 with an Arg-602- > Leu- 602 mutation:
  • GST-91SH3 encodes amino acids 506 to 564 of Stat91.
  • DNA Transfection was carried by the calcium phosphate method, and stable cell lines were selected in Dulbecco's modified Eagle's medium containing G418 (0.5 mg/ml, Gibco), as described (45).
  • Preparation of Cell Extracts Crude whole cell extracts were prepared as described (31). Cytoplasmic and nuclear extracts were prepared essentially as described (46).
  • Affinity Purification Affinity purification with a biotinylated oligonucleotide was described (31). The sequence of the biotinylated GAS oligonucleotide was from the Ly6E gene promoter (34).
  • Nondenaturing Polyacrylamide Gel Analysis A nondenatured protein molecular weight marker kit with a range of molecular weights from 14 to 545 kD was obtained from Sigma. Determining molecular weights using nondenaturing polyacrylamide gel was carried out following the manufacturer's procedure, which is a modification of the methods of Bryan and Davis (47, 48). Phosphorylated and unphosphorylated Stat91 samples obtained from affinity purification using a biotinylated GAS oligonucleotide (31) were resuspended in a buffer containing 10 mM Tris (pH 6.7), 16% glycerol, 0.04% bromphenol blue (BPB). The mixtures were analyzed on 4.5%.
  • Phosphorylated and unphosphorylated Stat91 samples obtained from affinity purification using a biotinylated GAS oligonucleotide (31) were resuspended in a buffer containing 10 mM Tris (pH 6.7), 16% glycerol
  • Extracts were incubated with various concentrations of peptides or fusion proteins, and 32 P-labeled GAS oligonucleotide probe in gel shift buffer was then added to promote the formation of protein- DNA complex followed by mobility shift analysis. This assay did not involve guanidium hydrochloride treatment.
  • Fusion Proteins Bacterially expressed GST fusion proteins were purified using standard techniques, as described in Birge et al., 1992. Fusion proteins were quantified by O.D. absorbance at 280nm. Aliquotes were frozen at -70°C. Results
  • Stat91 Binds DNA as a Dimer. Long or short versions of DNA binding protein can produce, respectively, a slower or a faster migrating band during gel retardation assays. Finding intermediate gel shift bands produced by mixing two different sized species provides evidence of dimerization of the DNA binding proteins. Since Stat91 requires specific tyrosine phosphorylation in ligand-treated cells for its DNA binding, we sought evidence of formation of such heterodimers, first in transfected cells. An expression vector (MNC911) encoding Stat91L, a recombinant form of Stat91 containing an additional 34 amino acid carboxyl terminal tag was generated.
  • a Stat84 expression vector (MNC84) was also available (45). From somatic cell genetic experiments, mutant human cell lines (U3) are known that lack the Stat91/84 mRNA and proteins (29,30). The U3 cells were therefore separately transfected with vectors encoding Stat84 (MNC84) or Stat91L (MNC91L) or a mixture of both vectors. Permanent transfectants expressing Stat84 (C84), Stat91L (C91L) or both proteins (Cmx) were isolated ( Figure 11 A).
  • the middle band formed by extracts of the Cmx cells is clearly identified as a heterodimer of Stat84 and Stat91L.
  • both Stat91 and Stat84 bind DNA as homodimers and, if present in the same cell, will form heterodimers.
  • any reassociated or remaining dimers can be assayed.
  • addition of DNA to form the stable protein-DNA complex should lead to the detection of homodimers as well as heterodimers.
  • subunits- of the dimer may not be able to re-form and no DNA-protein complexes would be detected (Figure 13).
  • the Stat91 sequence contains an SH2 domain (amino acids 569 to 700, see discussion below), and we knew that Tyr-701 was the single phosphorylated tyrosine residue required for DNA binding activity (supra, 45).
  • Activated Stat84 or Stat91L was obtained from IFN-7-treated C84 or C91L cells and mixed in the presence of various concentrations of the peptides followed by gel mobility shift analysis.
  • the non-phosphorylated peptide had no effect on the presence of the two gel shift bands characteristic of Stat84 or Stat91L homodimers ( Figure 14, lane 2-4).
  • the phosphorylated peptide (91Y-p) at the concentration of 4 ⁇ M clearly promoted the exchange between the subunits of Stat84 dimers and Stat91L dimers to form heterodimers ( Figure 14. lane 5).
  • peptide 91Y-p but not the unphosphorylated peptide dissociated the dimers and blocked the formation of DNA protein complexes ( Figure 14, lane 7).
  • the apparent stability of Stat91 dimer may be due to a high association rate coupled with a high dissociation rate of SH2-phosphotyrosyl peptide interactions as suggested (Felder et al. , 1993, Mol. Cell Biol. 13: 1449-1455) coupled with interactions between other domains of Stat91 that may contribute stability to the Stat91 dimer. Interference by homologous phosphopeptides with the -SH2- phosphotyrosine interaction would then lower stability sufficiently to allow complete dissociation and heterodimerization.
  • the dimer formation between phospho Stat91 is the first case in eukaryotes where dimer formation is regulated by phosphorylation, and the only one thus far dependent on tyrosine phosphorylation.
  • Dimerization with the STAT protein family will be important. It seems likely that in cells treated with IFN- ⁇ , there is Statl 13-Stat91 interaction (15). This may well be mediated through SH2 and phosphotyrosyl peptide interactions as described above, leading to a complex (a probable dimer of Stat91 -Statl 13) which joins with a 48 kD DNA binding protein (a member of another family of DNA binding factors) to make a complex capable of binding to a different DNA site.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • TCA AAA TTC CTG GAG CAG GTT CAC CAG CTT TAT GAT GAC AGT TTT CCC 277 Ser Lys Phe Leu Glu Gin Val His Gin Leu Tyr Asp Asp Ser Phe Pro 15 20 25 ATG GAA ATC AGA CAG TAC CTG GCA CAG TGG TTA GAA AAG CAA GAC TGG 325 Met Glu He Arg Gin Tyr Leu Ala Gin Trp Leu Glu Lys Gin Asp Trp 30 35 40
  • TCT GTC ACC AAA AGA GGT CTC AAT GTG GAC CAG CTG AAC ATG TTG GGA 1765 Ser Val Thr Lys Arg Gly Leu Asn Val Asp Gin Leu Asn Met Leu Gly 510 515 520
  • ACTTTTTCCA GACACTTTTT TGAGTGGATG ATGTTTCGTG AAGTATACTG TATTTTTACC 3566
  • GAG AGT CTG CAG CAA GTT CGG CAG CAG CTT AAA
  • TCT GTC ACC AAA AGA GGT CTC AAT GTG GAC CAG CTG AAC ATG TTG GGA 1765 Ser Val Thr Lys Arg Gly Leu Asn Val Asp Gin Leu Asn Met Leu Gly 510 515 520
  • CAGAAGAGTG ACATGTTTAC AAACCTCAAG CCAGCCTTGC TCCTGGCTGG GGCCTGTTGA 2402
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • GAA AGG AAG ATT TTG GAA AAT GCC CAA AGA TTT AAT CAG GCC CAG GAG 385 Glu Arg Lys He Leu Glu Asn Ala Gin Arg Phe Asn Gin Ala Gin Glu 115 120 125
  • GGT ACG CAC ACA AAA GTG ATG AAC ATG GAA GAA TCC ACC AAC GGA AGT 1201 Gly Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr Asn Gly Ser 385 390 395
  • AAG GAA AAT ATT AAT GAT AAA AAT TTC TCC TTC TGG CCT TGG ATT GAC 1681 Lys Glu Asn He Asn Asp Lys Asn Phe Ser Phe Trp Pro Trp He Asp 545 550 555
  • GCT CAA AGA GCA CAC CTC CTG GAA
  • AAA CTG AGA TTA CTA ATA AAA TTG CCG GAA CTA AAC TAT CAG GTG AAA 1110 Lys Leu Arg Leu Leu He Lys Leu Pro Glu Leu Asn Tyr Gin Val Lys 345 350 355

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Abstract

Il existe des facteurs de reconnaissance de récepteurs qui reconnaissent le récepteur cellulaire spécifique auquel un ligand spécifique a été fixé, et qui peuvent par conséquent signaler et/ou amorcer la fixation du facteur de transcription au site d'ADN. Le facteur de reconnaissance de récepteurs est dans un cas, une partie d'un facteur de transcription, et peut également avoir une interaction avec d'autres facteurs de transcription afin de provoquer leur activation et leur déplacement jusqu'au noyau pour fixer l'ADN. Le facteur de reconnaissance de récepteurs semble avoir une activité indépendante du second messager, puisque des perturbations manifestes dans des concentrations de second messager n'ont aucun effet. Le concept de l'invention est illustré par les résultats d'études menées avec une transcription de gènes stimulés par interféron (IFN), et notamment, l'activation provoquée par les interférons à la fois IFNα et IFNη. L'invention concerne également de l'ADN ainsi que des séquences d'acides aminés spécifiques à divers facteurs de reconnaissance des récepteurs humains et murins, ainsi que des fragments polypeptidiques de deux des gènes ISGF-3, des anticorps ayant également été préparés et testés. Les polypeptides confirment une implication directe de la tyrosine kinase dans la transmission du message intracellulaire. De plus l'invention concerne de nombreuses matières et applications diagnostiques et thérapeutiques.
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US08/212,185 US6605442B1 (en) 1992-03-19 1994-03-11 Methods of testing drugs or agents that modulate the activity of receptor recognition factors
US212185 1994-03-11
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US6030780A (en) * 1996-10-15 2000-02-29 The Rockefeller University Purified Stat proteins and methods of purifying thereof
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US6087478A (en) * 1998-01-23 2000-07-11 The Rockefeller University Crystal of the N-terminal domain of a STAT protein and methods of use thereof
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