WO1995002053A1 - Composants purifies de complexes de regulation de transcription de mammiferes et leurs analogues - Google Patents

Composants purifies de complexes de regulation de transcription de mammiferes et leurs analogues Download PDF

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WO1995002053A1
WO1995002053A1 PCT/US1994/007297 US9407297W WO9502053A1 WO 1995002053 A1 WO1995002053 A1 WO 1995002053A1 US 9407297 W US9407297 W US 9407297W WO 9502053 A1 WO9502053 A1 WO 9502053A1
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ser
pro
leu
protein
gly
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PCT/US1994/007297
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English (en)
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Naoko Arai
Esteban S. Masuda
Hiroshi Tokumitsu
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Schering Corporation
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Priority to AU73188/94A priority Critical patent/AU7318894A/en
Publication of WO1995002053A1 publication Critical patent/WO1995002053A1/fr

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention provides compositions related to proteins which function in controlling development and differentiation of mammalian cells, e.g., cells of a mammalian immune system.
  • mammalian cells e.g., cells of a mammalian immune system.
  • proteins and mimetics which regulate development, differentiation, and expression of various genes, including various cytokines.
  • cytokine production including interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) by the stimulation of antigen-specific receptors of T lymphocytes
  • IL-2 interleukin-2
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • CsA cyclosporin A
  • FK506 inhibit the stimulation elicited by A23187 and PMA.
  • NF-CLEO ⁇ nuclear Factor of Activated I cells
  • the transcription regulation is mediated by quaternary protein complexes comprising a number of proteins.
  • the AP-1 subcomponent of an NF-AT comple is made up of two proteins, a representative of the jun family, and a representative of the fos family; see Jain et al. (1992) Nature 356:801-804.
  • the lack of complete characterization of the other components of the NF-AT complex has led to the inability to understand the important components and interactions which regulate cytokine transcription and expression.
  • NF-AT is a lymphoid-specific transcription factor involved in regulation of the IL-2 gene and is considered to be an important regulator in early T cell activation. Recent studies have shown that NF-AT is a multimeric protein complex composed of the previously identified transcription factor AP-1 and a cytoplasmic component. See: Jain et al. M992) Nature 356:801-804: Northrop et al. (1993) J. Biol. Chem. 268:2917-2923; and McCaffrey et al. (1993) J. Biol. Chem. 268:3747-3752.
  • the present invention is based, in part, upon the isolation of various components of a complex of proteins which interact to function as a transcription regulatory entity.
  • the purified component designated NF-AT120, interacts with AP-1 proteins (comprising a member of each of the jun and fos protein families) to form a multiprotein complex which binds to an NF-AT recognition sequence.
  • the invention identifies a common factor that regulates transcriptional activation of the GM-CSF and IL-2 genes upon T cell activation.
  • the invention embraces purified natural forms as well as analogues and homologues, e.g., mutations (muteins) of the natural sequence, fusion proteins, chemical mimetics, antibodies, and other structural or functional analogues. It is also directed to isolated genes encoding proteins of the invention. Various uses of these different protein or nucleic acid compositions are also provided.
  • the present invention provides a substantially pure component of an NF-AT protein complex, or peptide thereof, or a fusion protein comprising NF-AT120 protein sequence; an antibody specific for binding to an NF-AT120 protein; and a nucleic acid encoding an NF-AT120 protein or fragment thereof.
  • the protein or peptide can be from a warm blooded animal selected from the group of birds and mammals, including a mouse, rat, or human; can comprise at least one polypeptide segment of SEQ ID NO: 1 through 5, 35, 37, 39 and 41 ; can exhibit a post-translational modification pattern distinct from natural NF-AT120 protein; can exhibit at least one of the features disclosed in Table 1 ; or can induce transcription of a cytokine. (All SEQ IDs are given together immediately before the Claims.)
  • a further embodiment is a composition comprising such a protein and a pharmaceutically acceptable carrier.
  • the antigen can be a mammalian protein, e.g., from a mouse, rat, or human; the antibody can be raised against a peptide sequence of SEQ ID NO: 1 through 5, 35, 37, 39 and 41; the antibody can be a monoclonal antibody; or the antibody can be labeled.
  • nucleic acid in nucleic acid embodiments, can comprise a sequence of SEQ ID NO: 6 through 24, 34, 36, 38 and 40.
  • the invention also embraces a kit comprising a substantially pure NF-AT120 protein or fragment, e.g., as a positive control; an antibody which specifically binds an NF-AT120 protein; or a nucleic acid encoding an NF-AT120 protein or peptide.
  • a kit comprising a substantially pure NF-AT120 protein or fragment, e.g., as a positive control; an antibody which specifically binds an NF-AT120 protein; or a nucleic acid encoding an NF-AT120 protein or peptide.
  • the availability of these reagents also provides methods of modulating physiology or development of a cell comprising contacting said cell with an NF-AT120 protein or with an analogue or homologue thereof, e.g., by introducing said NF-AT120 protein or analogue or homologue thereof into a cell.
  • an inhibitor might be an antibody against a mammalian NF-AT120 protein or the cell may be a hematopoi
  • Figure 1 shows a mobility shift assay of affinity-purified NF-AT.
  • Nuclear extracts of PMA/A23187-stimulated Jurkat cells (NE) were assayed in the presence of 250 ng/ ⁇ l poly-dldC (lanes 1 and 5).
  • Mobility shift assays of affinity- purified NF-AT (30 ng) were carried out without poly-dldC (lanes 2-4, and 6-11 ) using 32 P-labeled NF-AT and CLEO oligonucleotide as probes. The probes are listed at the top with the competitors identified below each probe.
  • Figure 2 shows a purification of the 120 kDa component of NF-AT (NF-AT120) by Mono Q chromatography.
  • Affinity-purified NF-AT was applied in the presence of 6 M urea and eluted by KCI gradient (0.05 - 0.8 M) from a Mono Q column.
  • Mobility shift assays were carried out in a solution containing 1 ⁇ l of each fraction, 100 ng/ ⁇ l polydldC in the absence (Figure 2A) or presence (Figure 2B) of 10 ng of affinity-purified Jurkat AP-1.
  • the right lane in Figure 2B shows NF-AT binding of 10 ng of affinity-purified Jurkat AP-1.
  • Figure 2C shows analysis by SDS-7.5% PAGE (polyacrylamide gel electrophoresis) of each fraction from Mono Q chromatography.
  • the left lane (M) indicates molecular weight standards (kDa). Fraction numbers are listed below each figure.
  • Figure 3 shows that recombinant cJun/cFos heterodimer reconstitutes NF-AT DNA-binding with the NF-AT120 protein.
  • Mobility shift assays were carried out using NF-AT DNA probe in a solution containing 10 ng of affinity- purified Jurkat AP-1 (lane 2) or various combinations of recombinant cJun (0.2 ⁇ M) and cFos (0.2 ⁇ M) in either the presence (+) or absence (-) of the Mono Q-purified 120 kDa protein (1 ⁇ l). The proteins are listed on top.
  • Figure 4 shows that NF-AT120 reconstitutes the DNA-binding activity to NF-AT and CLEO elements with AP-1.
  • Figure 4A shows analysis by SDS-7.5 % PAGE of purified NF-AT120 (lane 1 ).
  • the left lane (M) indicates molecular weight standards (kDa).
  • Figure 4B shows mobility shift assays carried out using NF-AT DNA probe and CLEO DNA probe.
  • the reaction solutions contain the 120 kDa protein from Mono Q fraction ((Q), lanes 2, 3, 7, 8,) or renatured NF-AT120 protein from gel slice ((G), lanes 4, 5, 9, 10) in either the presence (+ AP-1 ) or absence of 10 ng of affinity-purified Jurkat AP-1.
  • NF-AT DNA binding of affinity-purified NF-AT (30 ng) is indicated in lane 1.
  • FIG. 5 is a diagrammatic representation showing how proteins of the present invention are currently believed to bind to dsDNA. However, the validity of the claims of the present invention is not dependent upon the correctness of this belief.
  • PKC represents protein kinase C
  • CHX represents cyclohexamide
  • A is AP-1 as defined herein
  • CsA is cyclosporin A
  • B represents the C family of NF-AT, which is further defined in the Sequence Listing.
  • the present invention provides the amino acid sequences of and DNA sequences encoding various mammalian proteins which exhibit properties of regulating transcription of immunologically relevant proteins, e.g., cytokines.
  • cytokines immunologically relevant proteins
  • proteins are designated components of a Nuclear Factor of Activated T cells (NF-AT) because they were initially characterized as proteins which associate in a complex and regulate transcription of various cytokines, e.g., IL-2 and GM-CSF.
  • the proteins are one component, referred to herein as NF-AT120, and exhibit features characteristic of a transcriptional regulatory factor component, e.g., specific association with particular genetic regulatory sequences when associated with other components, i.e., AP-1, of the complex.
  • the present invention provides substantially purified NF-AT120 proteins, derived from either natural sources or recombinant sources.
  • the proteins exhibit properties as described, both physicochemical and biological. See Table 1: Table 1 : Physical properties of human NF-AT120 protein.
  • NF-AT complex Affinity binding of NF-AT complex to a CLEO DNA sequence or NF-AT DNA binding sequence, but competed by oligonucleotides containing NF-AT DNA binding and AP-1 DNA binding sites.
  • NF-AT DNA binding affinity chromatography in 0.2% NP-40, washed with 1 M urea, elutes in 0.3 M KCI without urea.
  • Lys C protease fragments of a purified human NF-AT120 protein amino acid sequence are presented as SEQ ID NO: 1 through 5, reading from the amino to the carboxy end.
  • Other peptide sequences are provided by sequences from cloned nucleic acids encoding the proteins; see SEQ ID NO: 35, 37, 39, and 41. These amino acid sequences are important in providing partial sequence information in the protein, to enable the protein to be distinguished from other proteins.
  • the peptide sequences allow preparation of peptides to generate antibodies that recognize such segments, and/or allow preparation of oligonucleotide probes, both of which are strategies for isolation, e.g., cloning, of genes encoding such sequences.
  • another peptide- sequencing experiment showed two peptides, whose overlapped sequence reads, by amino acid cycle:
  • human NF-AT120 protein shall encompass, when used in a protein context, a protein containing at least some of the amino acid sequences of SEQ ID NO: 1 through 5, 35, 37, 39, or 41 , or a significant fragment of such a protein, or protein sequences encoded by isolated NF-AT120 genes or transcripts.
  • SEQ ID NO: 1 corresponds to residues 389-395 of SEQ ID NO: 36;
  • SEQ ID NO: 3 corresponds to residues 279-287; residues 2-15 of SEQ ID NO: 4 correspond to residues 415-428; and residues 1-5 of SEQ ID NO: 5 correspond to residues 677-681.
  • SEQ ID NO: 5 may be encoded by an alternative splicing variant of a message.
  • the term also refers to a human-derived polypeptide which exhibits similar biological function or interacts with NF-AT120 protein-specific binding components, including AP-1 proteins.
  • the specific binding components which include antibodies, typically bind to an NF-AT120 protein with high affinity, e.g., at better than about 100 nM, usually at better than about 30 nM or about 10 nM, and more preferably at better than about 3 nM. Under certain circumstances, the binding may require additional physiologically relevant cofactors or components.
  • Homologous proteins would be found in mammalian species other than human, e.g., in other primates, in rats, and in mice. Non-mammalian species should also possess structurally or functionally related genes and proteins.
  • polypeptide as used herein includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least 12 or 16 amino acids, preferably at least 20 or 24 amino acids, and, in particularly preferred embodiments, at least 28 or even 30 or more amino acids.
  • binding agent or “binding composition” refers to molecules that bind with specificity to NF-AT120.
  • One embodiment includes antibodies that bind specifically to NF-AT120.
  • a second embodiment includes proteins, like protein complex AP-1 , that complex specifically with NF-AT120 protein or specifically associate with it, as in a natural physiologically relevant protein- protein interaction.
  • the binding agent or binding composition may be a polymer or a chemical reagent.
  • a functional analog thereto may be a protein with structural modifications, or may be a wholly unrelated molecule, e.g., one which has a molecular shape which interacts with the appropriate binding determinants, preferably exhibiting similar biological properties.
  • Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Various soluble fragments of the NF-AT120 are also included. Many variables affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent.
  • the temperature at which the polypeptide is used ranges from about 4°C to about 65°C. Usually the temperature at use is higher than about 18°C and more usually higher than about 22°C.
  • the temperature will usually be about room temperature or higher, but less than the denaturation temperature of components in the assay.
  • the temperature will usually be body temperature, typically about 37°C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the electrolytes will usually approximate in situ physiological conditions, but may be modified to higher or lower ionic strength where advantageous.
  • the actual ions may be modified, e.g., to conform to standard buffers used in physiological or analytical contexts.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to form an NF-AT complex, or to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the NF-AT120 typically associates with AP-1 proteins to confer many of its natural biological functions.
  • the solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent.
  • the solvent will have a pH near neutrality, typically between about 5 and 10, and preferably about 7.5.
  • a detergent will be added, typically a mild non-denaturing one, e.g., CHS or CHAPS, in a concentration low enough to avoid significant disruption of structural or physiological properties of the protein.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions.
  • the determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge; see: Freifelder (1982) Physical Biochemistry (2d edJ. W.H. Freeman; and Cantor and Schimmel (1980) Biophysical Chemistry, parts 1-3, W.H. Freeman & Co., San Francisco; each of which is hereby incorporated herein by reference.
  • a sample containing a putatively soluble polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 minutes, and soluble molecules will remain in the supernatant.
  • a soluble particle or polypeptide will typically be less than about 30S, usually less than about 15S or even 10S, preferably less than about 6S, and, in particular preferred embodiments, less than about 4S or even 3S.
  • the NF-AT120 protein exhibits important structural and physical characteristics which confer an important biological function.
  • the first is an ability to associate with other components, e.g., AP-1 proteins, to form a complex having affinity for specific DNA sequences. This specificity of binding provides a biological function in regulation of both IL-2 and GM-CSF cytokine expression.
  • This invention also encompasses proteins or peptides having substantial amino acid sequence homology with the amino acid sequences of the NF-AT120 protein.
  • the variants include species or allelic variants.
  • Amino acid sequence homology, or sequence identity is determined by optimizing residue matches, if necessary introducing gaps as required. This changes when conservative substitutions are considered to be matches. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Homologous amino acid sequences are typically intended to include natural allelic and interspecies variations in each respective protein sequence.
  • Typical homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced) to 50-100% homology (if conservative substitutions are included) with the amino acid sequence of the NF-AT120 protein.
  • Homology measures will be at least about 35%, generally at least 45%, often at least 55%, typically at least 65%, usually at least 75%, preferably at least 80%, and in particularly preferred embodiments, at least 85% or more. See also: Needleham et al. (1970) J. Mol. Biol.. 48:443-453; Sankoff et al. (1983) Chapter One in Time Warns. String Edits, and Macromolecules: The Theory and Practice of Seouence Comparison. Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA, and from the University of Wisconsin Genetics Computer Group, Madison, Wl.
  • the isolated NF-AT120 protein DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, or antigenic activity. These modified sequences can be used to produce mutant antigens or to enhance expression. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant NF-AT120 protein derivatives include predetermined or site-specific mutations of the respective protein or its fragments.
  • “Mutant NF-AT120 protein” encompasses a polypeptide otherwise falling within the homology definition of the human NF-AT120 protein as set forth above, but having an amino acid sequence which differs from that of NF-AT120 protein as found in nature, whether by way of deletion, substitution, or insertion.
  • site specific mutant NF-AT120 protein generally includes proteins having significant homology with a protein having sequences of SEQ ID NO: 1 through 5, 35, 37, 39 or 41, and sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and preferred embodiments thereof contain most of the disclosed sequences. Similar concepts apply to different NF-AT120 proteins, particularly those found in various warm blooded animals, e.g., mammals and birds.
  • NF-AT120 protein mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy-terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR) techniques; see also Sambrook et al. (1989) and Ausubel et al. (1987 and Supplements).
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins.
  • a heterologous fusion protein is a fusion of proteins or segments which naturally are not normally fused in the same manner.
  • the fusion product of an immunoglobulin with an NF-AT120 protein polypeptide is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar concept applies to heterologous nucleic acid sequences.
  • new constructs may be made by combining similar functional domains from other proteins.
  • DNA-binding or other segments may be "swapped" between different new fusion polypeptides or fragments; see, e.g.: Cunningham et al. (1989) Science. 243:1330-1336; and O'Dowd et al. (1988) J. Biol. Chem.. 263:15985-15992, each of which is incorporated herein by reference.
  • new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of DNA-binding specificities or protein-binding specificities and other functional domains.
  • a double-stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., by PCR techniques.
  • NF-AT120 proteins The blocking of physiological response to NF-AT120 proteins may result from the inhibition of binding of the protein to either DNA or other components of the NF-AT complex, likely through competitive inhibition.
  • in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated NF-AT120 protein, soluble fragments comprising DNA or AP-1 protein binding segments of these proteins, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either DNA- or protein-binding segment mutations and modifications, or compounds which can disrupt or facilitate such interactions.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to NF-AT120 or AP-1 protein fragments compete with a test compound for binding to the protein.
  • the antibodies can be used to detect the presence of any polypeptide which shares one or more antigenic binding sites of the NF-AT120 and can also be used to occupy binding sites on the protein that might otherwise interact with another component of an NF-AT complex.
  • neutralizing antibodies against the NF-AT120 protein and soluble fragments of the protein which contain a high affinity binding site to specific targets can be used to inhibit transcriptional function in tissues, e.g., tissues experiencing abnormal physiology.
  • “Derivatives" of the NF-AT120 protein antigens include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the NF-AT120 protein amino acid side chains or at the N- or C-termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.
  • Acyl groups are preferably selected from the group of alkanoyl moieties including C2 to C18 normal alkanoyl. Covalent attachment to carrier proteins may be important when immunogenic moieties are haptens.
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation may be effected by appropriate enzymes. Also embraced are versions of the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • a major group of derivatives comprises covalent conjugates of the NF-AT120 protein or fragments thereof with other proteins or polypeptides.
  • These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups.
  • Preferred protein derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.
  • Fusion polypeptides between the NF-AT120 proteins and other homologous or heterologous proteins are also provided.
  • Homologous polypeptides may be fusions between different nuclear proteins, resulting in, e.g., a hybrid protein exhibiting DNA binding specificity or nuclear factor binding specificity.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a DNA-binding segment, so that the presence or location of the fusion protein may be easily determined; see, e.g., Dull et al., U.S. Patent No.
  • genes fusion partners include bacterial ⁇ -galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor; see, e.g., Godowski et al. (1988) Science 241:812-816.
  • Some polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands, or fusions allowing for simple purification and processing.
  • Fusion proteins will typically be made either by recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally in, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold Spring Harbor Laboratory. Techniques for synthesis of polypeptides are described in, for example: Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986) Science 232:341-347; and Atherton et al. (1989) Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford.
  • This invention also contemplates the use of derivatives of the NF-AT120 proteins other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties. These derivatives generally fall into the three classes: (1) salts, (2) side-chain covalent modifications and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.
  • covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as affinity purification of binding partners.
  • an NF-AT120 protein antigen can be immobilized by covalent bonding to a solid support such as cyanogenbromide- activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-NF-AT120 protein antibodies or its binding partners.
  • the NF-AT120 proteins can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays. Purification of NF-AT120 protein may be effected by immobilized antibodies or binding partners.
  • a solubilized NF-AT120 protein or fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for the protein or any fragments thereof.
  • the purified proteins can be used to screen monoclonal antibodies or antigen-binding fragments prepared by immunization with various forms of impure preparations containing the protein.
  • the term "antibodies” also encompasses antigen-binding fragments of natural antibodies.
  • the purified NF-AT120 proteins can also be used as a reagent to detect any antibodies generated in response to the presence of elevated levels of the protein, protein complex, or cell fragments containing the protein, each of which may be diagnostic of an abnormal or specific physiological or disease condition, e.g., autoimmunity.
  • protein fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below.
  • this invention contemplates antibodies raised against amino acid sequences shown in Table 1, or fragments of proteins containing them.
  • the present invention contemplates the isolation of additional closely related species variants. Southern, Northern, and Western blot analysis should establish that similar genetic entities exist in other mammals. It is likely that the NF-AT120 proteins are widespread in various orders and species, e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.
  • the invention also provides means to isolate a group of related antigens displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects and mechanism of action of the proteins will be greatly accelerated by the isolation and characterization of distinct species variants of the proteins.
  • the present invention provides useful probes for identifying additional homologous genetic entities in different species.
  • the isolated genes will allow transformation of cells lacking expression of a corresponding NF-AT120 protein, e.g., either species types or cells which lack corresponding proteins and exhibit negative background activity. Expression of transformed genes will allow isolation of antigenically pure cell lines, with defined or single species variants. This approach will allow for more sensitive detection and discrimination of the physiological effects of proteins regulating nuclear transcription. Subcellular fragments, e.g., cytoplasts or membrane fragments, can be isolated and used in appropriate assays.
  • DNA-binding segments or AP-1 binding segments can be substituted between species variants to determine what structural features are important in both DNA binding affinity and specificity, as well as transcriptional activation.
  • An array of different NF-AT120 variants will be used to screen for factors exhibiting combined properties of interaction with different DNA or AP-1 species variants.
  • NF-AT120 protein Various functions would probably involve segments of the factor which are normally accessible to DNA or protein binding. Dissection of what domains of the protein are involved in DNA binding or AP-1 protein interaction can be performed, along with elucidation of other NF-AT components. The specific segments of interaction of NF-AT120 protein with other components may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of activation or suppression of transcription will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants.
  • NF-AT120 protein may exhibit differential developmental, tissue-specific, or other expression patterns.
  • Upstream or downstream genetic regions e.g., control elements
  • Structural studies of the factors will lead to design of new factors, particularly analogues exhibiting constitutive suppression of activation of transcription. This can be combined with previously described screening methods to isolate proteins exhibiting desired spectra of activities.
  • the present invention provides important reagents related to a physiological NF-AT120 interaction with AP-1 and/or regulatory DNA segments.
  • a physiological NF-AT120 interaction with AP-1 and/or regulatory DNA segments.
  • the foregoing description has focused primarily upon a human NF-AT120 protein, those of skill in the art will immediately recognize that the invention encompasses other species variants, e.g., rat and other mammalian species or allelic variants, as well as other variants thereof.
  • Antibodies can be raised to the various NF-AT120 proteins, including species or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to NF-AT120 proteins either in their active forms or in their inactive forms. Anti- idiotypic antibodies are also contemplated.
  • Antibodies including binding fragments, e.g., Fab, Fab 2 , Fv, etc., and single-chain versions, against predetermined fragments of the proteins can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins. Monoclonal antibodies are isolated from cells secreting the desired antibody. These antibodies can be screened for binding to normal or mutant NF-AT120 proteins, or screened for suppressive or helper activity.
  • These monoclonal antibodies will usually bind with a KQ of at most about 1 mM but more usually with stronger binding, e.g., with a KQ of at most about 300 ⁇ M, typically at most about 10 ⁇ M, more typically at most about 30 ⁇ M, preferably at most about 10 ⁇ M, and more preferably at most about 3 ⁇ M or better.
  • the antibodies including antigen-binding fragments, e.g., Fab, Fab 2 , Fv, etc., of this invention can have significant diagnostic or therapeutic value. They can be potent inhibitors that bind to the protein or complex and inhibit a biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides so that, when the antibody binds to the protein, which can also be expressed on a cell surface or secreted into the proximal environment, the cell or nearby cells are killed. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting or cell labeling. The antibodies of this invention can also be useful in diagnostic applications.
  • capture or non-neutralizing antibodies they can be screened for ability to bind to the proteins without inhibiting biological function.
  • neutralizing antibodies they can be useful in competitive binding assays. They will also be useful in detecting or quantifying NF-AT120 protein or its complexes. See, e.g., Chan (ed.) (1987) Immunoassav: A Practical Guide. Academic Press, Orlando, FL; Price and Newman (eds.) (1991) Principles and Practice of Immunoassav. Stockton Press, N.Y.; and Ngo (ed.) (1988) Nonisotooic Immunoassav. Plenum Press, N.Y.
  • Protein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.
  • a protein or its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See: Microbiology. Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serolo ⁇ ical Reactions. Dover Publications, New York, and Williams et al. (1967) Methods in Immunology and Immunochemistry. Vol. 1, Academic Press, New York, for descriptions of methods of preparing polyclonal antisera.
  • a typical method involves hyperimmunization of an animal with an antigen.
  • the blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.
  • the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to selection of libraries of antibodies in phage or similar vectors. See: Huse et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward et al. (1989) Nature 341:544-546.
  • the polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies.
  • the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include: U.S. Patents Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced; see Cabilly, U.S. Patent No. 4,816,567.
  • the antibodies of this invention can also be used for affinity chromato ⁇ graphy in isolating proteins.
  • Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, SEPHADEX, or the like, where a cell lysate may be passed through the column, and the column is washed and then eluted with increasing concentrations of a mild denaturant, whereby the purified NF-AT120 protein or NF-AT will be released.
  • the antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • Antibodies raised against an NF-AT120 protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • the described peptide sequences and the related reagents are useful in isolating a DNA clone encoding NF-AT120 protein, e.g., from a natural source. Typically, it will be useful in isolating a gene from human, and similar procedures will be applied to isolate genes from other species, e.g., warm blooded animals, such as birds and mammals. Cross hybridization will allow isolation of analogous factors from other species. A number of different approaches should be available to successfully isolate a suitable nucleic acid clone.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above.
  • Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g.: Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.
  • the AP-1 complex can be used as a specific binding reagent, and advantage can be taken of its specificity of binding, very much as an antibody would be used.
  • a specific binding composition will be useful for screening an expression library made from a cell line which expresses an NF-AT120 protein.
  • the screening can be standard staining of surface-expressed protein, or panning. Screening of intracellular expression can also be performed by various staining or immunofluorescence procedures.
  • the binding compositions could be used to affinity purify or sort out cells expressing the protein.
  • the peptide segments can also be used to predict appropriate oligonucleotides to screen a library.
  • the genetic code can be used to select appropriate oligonucleotides useful as probes for screening; see, e.g., SEQ ID NO: 6 through 24.
  • oligonucleotides were useful in selecting correct clones from a library, e.g., inserts with sequences described in SEQ ID NO: 34, 36, 38 or 40.
  • Complementary sequences will also be used as probes, primers, or antisense molecules. It is recognized that minimally degenerate primers will typically be preferred, so long as the primers are of sufficient length, e.g., preferably at least about 18 nucleotides.
  • This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding NF-AT120 protein polypeptide.
  • this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide which is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact factor, or fragment, and have an amino acid sequence as disclosed in SEQ ID NO: 1 through 5, 35, 37, 39 or 41.
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins that are homologous to an NF-AT120 protein or which was isolated using cDNA encoding an NF-AT120 protein as a probe, e.g., other members of the subfamilies of NF-AT120.
  • the isolated DNA can have the respective regulatory sequences in the 5'- and 3'-flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • a double-stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., by PCR techniques.
  • an "isolated" nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components that naturally accompany a native sequence, e.g., ribosomes, polymerases and flanking genomic sequences from the originating species.
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure molecule includes isolated forms of the molecule.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • a "recombinant" nucleic acid is defined either by its method of production or by its structure.
  • the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production, and generally using some in vitro steps.
  • the structure can be a nucleic acid made by fusion of two fragments which are not naturally contiguous to each other, but it is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any unnaturally occurring vector are encompassed, as are nucleic acids comprising sequences derived using any synthetic oligonucleotide process.
  • Such a process is often used to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence-recognition site.
  • nucleic acid segments of desired functions are joined together to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms.
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site-specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features, may be incorporated by design.
  • site-specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features, may be incorporated by design.
  • a similar concept is intended for a recombinant polypeptide, e.g., a fusion polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides nucleotides, generally at least 23 or 29 nucleotides, often at least 35 or 41 nucleotides, preferably at least 47 or 53 nucleotides, and in particularly preferred embodiments will be of 56 or more nucleotides.
  • a DNA which codes for an NF-AT120 protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or homologous proteins, e.g., the members of the C, P and X subfamilies, as well as DNAs which code for homologous proteins from different species. There are likely homologues in other orders and species, including primates and rodents. Various NF-AT120 proteins should be homologous and are encompassed herein. However, even proteins that have a more distant evolutionary relationship to the NF-AT120 protein can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate NF-AT120 proteins are of particular interest.
  • This invention further covers recombinant DNA molecules and fragments having a DNA sequence identical to or highly homologous to the isolated DNAs set forth herein.
  • the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication.
  • recombinant clones derived from the genomic sequences e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g.: Goodnow (1992) "Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn et al.
  • homologous nucleic acid sequences when compared, exhibit significant similarity.
  • the standards for homology in nucleic acids either are measures for homology generally used in the art by sequence comparison or are based upon hybridization conditions. The hybridization conditions are described in greater detail below.
  • Substantial homology in the context of comparing nucleic acid sequences means that the segments are identical when optimally aligned, allowing for appropriate nucleotide insertions or deletions, in at least about 50% or even 59% of the nucleotides, generally at least 65% or even 71%, usually at least about 77% or even 85%, preferably at least about 95 to 98% or more, and, in particular embodiments, as many as about 99% or more of the nucleotides. Since dsDNA uses specific nucleotide pairing, the comparison can also be made on the basis of a complementary strand.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions to a strand or its complement, typically using a sequence derived from SEQ ID NO: 6 through 24, 34, 36, 38 and 40.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 40 nucleotides, preferably at least about 75% over a stretch of at least about 25 nucleotides, and most preferably at least about 90%. See Kanehisa (1984) Nucl. Acids Res.. 12:203-213.
  • the length of homology comparison may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 24 nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides.
  • Stringent conditions in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other variables, typically those controlled in hybridization reactions.
  • Stringent temperature conditions will usually include temperatures in excess of about 30°C, usually in excess of about 37°C, typically in excess of about 55°C, and preferably in excess of about 70°C.
  • Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 400 mM, typically less than about 300 mM, and preferably less than about 150 mM.
  • the combination of variables is much more important than the measure of any single variable: see, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
  • NF-AT120 protein from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. Homology may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody which exhibits less species specificity may be useful in expression cloning approaches.
  • DNA which encodes an NF-AT120 protein or fragments thereof can be obtained by chemical synthesis, by screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples.
  • This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length protein or fragments which can in turn, for example, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure/function studies.
  • Each antigen or its fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially purified to be free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent.
  • the antigen, or portions thereof, may be expressed as fusions with other proteins.
  • Expression vectors are typically self- replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used.
  • the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • the vectors of this invention contain DNA which encodes an NF-AT120 protein, or a fragment thereof, typically encoding a biologically active polypeptide.
  • the DNA can be under the control of a viral promoter and can encode a selection marker.
  • This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNA coding for an NF-AT120 protein in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the protein is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.
  • expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell: e.g., it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain an origin of replication that is recognized by the host cell. It is also possible to use vectors that cause integration of an NF-AT120 protein gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Vectors as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector, but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual. Elsevier, N.Y., and Rodriquez et al. (1988) (eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses. Buttersworth, Boston, MA.
  • Transformed cells include cells, preferably mammalian, that have been transformed or transfected with vectors that contain the NF-AT120 protein gene and have been constructed using recombinant DNA techniques.
  • Transformed host cells usually express the protein or its fragments; however, for purposes of cloning, amplifying, and manipulating its DNA, they do not need to express the protein.
  • This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the protein to accumulate in the culture.
  • the protein can be recovered, either from the culture or from the culture medium.
  • DNA sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.
  • "operably linked” means contiguous and in reading frame; however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e.g., £ coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictyostelium.
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host- vector systems include a wide variety of vectors for many different species.
  • a representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the NF-AT120 proteins or its fragments include, but are not limited to, such vectors as: those containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the plN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al.
  • Lower eukaryotes e.g., yeasts and Dictyostelium
  • yeasts and Dictyostelium may be transformed with NF-AT120 protein sequence containing vectors.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used to generically represent lower eukaryotes, although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless they are of the integrating type), a selection gene, a promoter, DNA encoding the desired protein or its fragments, and sequences for termination of translation, polyadenylation, and termination of transcription.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase-2 promoter or metallothionine promoter: see, e.g., Stearns et al. (1990) Methods in Enzymoloov 185:280-297; and chapter 29 in Wu et al. (eds.) (1989) Recombinant DNA Methodology. Academic Press, San Diego. Higher eukaryotic tissue culture cells are the preferred host cells for expression of the functionally active NF-AT120 protein.
  • any higher eukaryotic tissue culture cell line is workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source: see, e.g., Miller (1988) Ann. Rev. Microbiol. 42:177-99.
  • mammalian cells are generally preferred for their protein processing patterns, both cotranslational and posttranslational. Transformation or transfection and propagation of such cells is described in, e.g., Ausubel, et al. (eds.) (1987 and Supplements) Current Protocols in Molecular Biology. Greene/Wiley, New York.
  • useful cell lines include HeLa cells, Chinese hamster ovary
  • CHO baby rat kidney
  • COS monkey
  • Expression vectors for such cell lines usually include an origin of replication, a promoter, a site for initiation of translation, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a site for termination of transcription. These vectors also usually contain a selection gene or amplification gene.
  • Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus.
  • suitable expression vectors include pCDNAI ; pCD [see Okayama et al. (1985) Mol. Cell Biol.. 5: 1136-1142]; pMCIneo Poly-A [see Thomas et al. (1987) Cell. 51:503-512]; and a baculovirus vector such as pAC 373 or pAC 610.
  • NF-AT120 protein polypeptide in a system which provides a specific or defined glycosylation pattern.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the NF-AT120 protein gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable or approximated in prokaryote or other cells.
  • the NF-AT120 protein, or a fragment thereof, may be engineered to be linked by phosphatidylinositol (PI) to a cell membrane, but can be removed from membranes by treatment with a phosphatidylinositol-cleaving enzyme, e.g., phosphatidylinositol phospholipase-C.
  • PI phosphatidylinositol
  • a phosphatidylinositol-cleaving enzyme e.g., phosphatidylinositol phospholipase-C.
  • NF-AT120 proteins have been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described by: Stewart and Young in Solid Phase Peptide Synthesis (1984), Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky, The Practice of Peptide Synthesis. (1984), Springer- Verlag, New York; and Bodanszky, The Principles of Peotide Synthesis. (1984), Springer- Verlag, New York.
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, 4-nitrophenyl ester, N-hydroxy- succinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide/additive process, can be used.
  • Solid-phase and solution-phase syntheses are both applicable to the foregoing processes.
  • the NF-AT120 protein, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction are typically protected to prevent coupling at an incorrect location.
  • the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group.
  • the insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group.
  • examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonyl-hydrazide resins, and the like.
  • An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step-by-step. After the complete sequence has been synthesized , the peptide is split off from the insoluble carrier to produce the peptide.
  • This solid-phase approach is generally described by Merrifield et al. (1963) in J. Am. Chem. Soc. 85:2149-2156.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like.
  • the NF-AT120 proteins of this invention can be obtained in varying degrees of purity depending upon their desired use. Purification can be accomplished by use of the protein purification techniques disclosed herein or by the use of the antibodies herein described in immunoabsorbant affinity chromatography. This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the protein, or lysates or supernatants of cells producing the NF-AT120 protein as a result of DNA techniques; see below.
  • the present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in the general description for developmental abnormalities, or below in the description of kits for diagnosis.
  • This invention also provides reagents with significant therapeutic value.
  • NF-AT120 proteins naturally occurring or recombinant
  • fragments thereof and antibodies thereto should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions.
  • abnormal proliferation e.g., cancerous conditions, or degenerative conditions.
  • modulation of expression of cytokine genes is possible, but an NF-AT120 may be a component in regulatory proteins affecting other genes.
  • Abnormal proliferation .
  • a disease or disorder associated with abnormal transcriptional regulation by an NF-AT120 protein should be a likely target for an inhibitor or stimulator.
  • the factor probably plays a role in regulation or development of hematopoietic cells, e.g., lymphoid cells, which affect immunological responses, e.g., autoimmune or immunodeficiency disorders.
  • Antibodies raised to recombinant NF-AT120 protein can be prepared and purified and then administered to a patient. These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients. These combinations can be filtered sterile and placed into dosage forms by iyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also contemplates use of antibodies or binding fragments thereof, including forms which do not bind complement.
  • Drug screening using NF-AT120 or fragments thereof can be performed to identify compounds having binding affinity to NF-AT120 protein, including isolating associated components. Similar screening for compounds which interact with AP-1 will identify proteins which modulate NF-AT function. Subsequent biological assays can then be utilized to determine if the compound has intrinsic modulatory activity and is therefore useful in that it blocks the activity of the protein. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of NF-AT120 protein. This invention further contemplates the therapeutic use of antibodies to NF-AT120 protein as transcription modulators. This approach should be particularly useful with other NF-AT120 protein species variants.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Penn.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in The Merck Index. Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow-release formulations or a slow-release apparatus will often be utilized for continuous administration; see also Langer, (1990) Science 249:1527-1533.
  • NF-AT120 protein, fragments thereof, and antibodies to it or its fragments may be administered directly to the host to be treated. Alternatively, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in many conventional dosage formulations. Whereas it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typical ⁇ ly comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral administration (including subcutaneous, intramuscular, intravenous and intradermal administration).
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g.: Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications. Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets. Dekker, New York; and Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems. Dekker, New York.
  • the therapy Of this invention may be combined with or used in association with other chemotherapeutic or chemopreventive agents.
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins, or the AP-1 complex or DNA.
  • methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor et al., (1991 ) Science 251 :767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate. Structural diversity libraries can be used.
  • suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble NF-AT120 protein as provided by this invention.
  • modulators can normally be found once the protein or complex has been structurally defined. Testing of potential protein analogues is now possible with the development of highly automated assay methods using the information provided herein. In particular, new agonists and antagonists will be discovered by using screening techniques described herein. Of particular importance are compounds found to have a combined binding affinity for multiple AP-1 types, e.g., compounds which can serve as modulators for species variants of NF-AT120 protein.
  • This invention is particularly useful for screening compounds by using recombinant NFAT120 in a variety of drug-screening techniques.
  • the advantages of using a recombinant protein in screening for specific proteins include: (a) improved renewable source of the protein from a specific source; (b) potentially greater number of NF-AT120 proteins per cell giving better signal- to-noise ratio in assays; and (c) species variant specificity (theoretically giving greater biological and disease specificity).
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the protein.
  • Cells may be isolated which express the protein in isolation from others.
  • Such cells either in viable or fixed form, can be used for standard binding assays. See also: Parce et al. (1989) Science. 246:243-247: and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA. 87:4007-4011 ; which describe sensitive methods to detect cellular responses.
  • Viable cells could also be used to screen for the effects of drugs on NF-AT120 protein mediated functions: e.g., second messenger levels, i.e., Ca 2+ ; cell proliferation; inositol phosphate pool changes; and others.
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.
  • Calcium sensitive dyes will be useful for detecting Ca 2+ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
  • Another method utilizes membranes from transformed eukaryotic or prokaryotic host cells as the source of the NF-AT120 protein. These cells are stably transformed with DNA vectors directing the expression of an NF-AT120 protein, e.g., an engineered membrane bound form. Essentially, the membranes would be prepared from the cells and used in a binding assay such as the competitive assay set forth above.
  • Still another approach is to use solubilized and unpurified NF-AT120 protein or solubilized and purified NF-AT120 protein from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to NF-AT120 and is described in detail International Patent Application no. WO 84/03564 (Commonwealth Serum Labs.), published on September 13 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface; see Fodor et al. (1991).
  • all the pins are reacted with solubilized and unpurified NF-AT120, or with solubilized and purified NF-AT120, and washed.
  • the next step involves detecting bound NF-AT120.
  • Rational drug design may also be based upon structural studies of the molecular shapes of the NF-AT120 protein and other binding partners. These may be other proteins which mediate other functions in response to NF-AT120 binding, or other proteins which normally interact with the factor, or even DNA segments.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or two-dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions.
  • x-ray crystallography or two-dimensional NMR techniques.
  • Purified NF-AT120 protein can be coated directly onto plates for use in the aforementioned drug-screening techniques. However, non-neutralizing antibodies to these factors can be used as capture antibodies to immobilize the respective factor on the solid phase. Kits
  • kits and methods for detecting the presence of an NF-AT120 will have a compartment containing either a defined NF-AT120 protein peptide or gene segment or a reagent which recognizes one or the other, e.g., antibodies or fragments thereof.
  • a kit for determining the binding affinity of a test compound to an NF-AT120 protein would typically comprise: a test compound; a labeled compound, for example an antibody having known binding affinity for the protein; a source of NF-AT120 protein (naturally occurring or recombinant); and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the protein.
  • a test compound for example an antibody having known binding affinity for the protein
  • a source of NF-AT120 protein naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the protein.
  • a preferred kit for determining the concentration of, for example, an NF-AT120 protein in a sample would typically comprise a labeled compound, e.g., AP-1 or antibody, having known binding affinity for the protein, a source of NF-AT120 (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the NF-AT120 protein. Compartments containing reagents, and instructions, will normally be provided. Antibodies, including antigen-binding fragments, specific for the
  • NF-AT120 protein or fragments are useful in diagnostic applications to detect the presence of elevated levels of NF-AT120 protein and/or its fragments.
  • diagnostic assays can employ lysates, live cells, fixed cells, immuno ⁇ fluorescence, cell cultures, and body fluids, and further can involve the detection of antigens related to the NF-AT120 in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and bound complex) or heterogeneous (with a separation step).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • EMIT enzyme-multiplied immunoassay technique
  • SFIA substrate-labeled fluorescent immunoassay
  • unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to an NF-AT120 protein or to a particular fragment thereof.
  • Similar assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual. CSH.
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against an NF-AT120 protein, since the latter antibodies may be diagnostic of various abnormal states, e.g., autoimmune conditions.
  • NF-AT120 protein may result in production of various immunologicai reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal differentiation.
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or unlabeled antibody or labeled NF-AT120 protein is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, and may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non- covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the test compound, NF-AT120 protein, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125 I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
  • the NF-AT120 protein can be immobilized on various matrixes followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the NF-AT120 protein to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin- avidin.
  • the last step in this approach generally involves the precipitation of protein/binding partner or protein/antibody complex by various methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • Suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
  • Methods for linking proteins or their fragments to the various labels have been extensively reported in the literature. Many of the techniques involve the use of activated carboxyl groups either through the use of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in these applications.
  • oligonucleotide or polynucleotide sequences taken from the sequence of an NF-AT120 protein. These sequences can be used as probes for detecting levels of the message in samples from patients suspected of having an abnormal condition, e.g., cancer or developmental problem.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has been described above.
  • an oligonucleotide probe should have at least about 14 nucleotides and usually at least about 18 nucleo- tides, and the polynucleotide probes may be up to several kilobases.
  • Various labels may be employed, most commonly radionuclides, particularly 32 P.
  • biotin modified nucleotides for introduction into a polynucleotide.
  • the biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like.
  • antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out in any conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT), and hybrid arrested translation (HART). This also includes amplification techniques such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers; see, e.g., Viallet et al. (1989) Progress in Growth Factor Res.. 1:89-97.
  • Methods for protein purification include such methods as precipitation with ammonium sulfate, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g.: Ausubel et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification” in Methods in Enzvmologv. vol. 182, and other volumes in this series; and manufacturers' literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA.
  • Combination with recombinant techniques allows fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused through a protease-removable sequence.
  • appropriate segments e.g., to a FLAG sequence or an equivalent which can be fused through a protease-removable sequence.
  • the pellet was dissolved with 7-8 ml of Buffer A (20 mM HEPES (pH 7.9), 0.1 M KCI, 1 mM EDTA, 1 mM EGTA, 5% glycerol, 1 mM DTT, 10 ⁇ g/ml leupeptin, and 0.1 mM PMSF), and 2 ml of the sample was applied to a SUPEROSE 6 gel filtration column (HR 16/50, Pharmacia Co.) on an FPLC system at a flow rate of 0.3 ml/min. Each fraction was subjected to mobility shift assay using 32 P-labeled NF-AT oligonucleotide as a probe. NP-40 and polydldC were added to the fractions containing NF-AT binding at 0.2 % and 13 ⁇ g/ml, respectively.
  • Buffer A 20 mM HEPES (pH 7.9), 0.1 M KCI, 1 mM EDTA, 1 mM EGTA, 5% g
  • oligonucleotides used for competition and mobility shift assays contained the following sequences (only one strand is shown; sequence overhangs are in lower-case letters):
  • CLEO element 5'-gatcGTCACCA ⁇ AATCATTTCCTCTAACTGT-3'; see Miyatake et al. (1991) Mol. Cell. Biol.. 11:5894-901, and SEQ ID NO: 25;
  • AP-1 site 5'-tcgaGCTATGACTCATCCG-3'; see Nakabeppu et al. (1988) ⁇ , 55:907-15, and SEQ ID NO: 26;
  • NF-AT site 5'-gatcGGAGGAAAAACTGTTTCATACAGAAGGCGT-3'; see Emmel et al. (1989) Science. 246:1617-20, and SEQ ID NO: 27.
  • the DNA binding reactions were performed at room temperature for 30 min in a solution (10 ⁇ l) containing 10 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 10% glycerol, KCI adjusted to 100 mM, 0.5 ng of 32 P-labeled probe (50,000 cpm), and protein sample as indicated in the figure legend, with or without 100 ng/ ⁇ l of polydldC.
  • Samples were analyzed on 4% native polyacrylamide gels (Tris-glycine-EDTA buffer) at 120 v, which were dried and exposed to Kodak X-OMAT film. Denaturation and renaturation of the 120 kDa component of NF-AT
  • the gel piece corresponding to NF-AT120 was excised and eluted overnight with 200 ⁇ l of 20 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM EGTA, 2 mM DTT, 5% glycerol, 0.1% SDS and 0.1 mg/ml BSA.
  • the eluted protein was precipitated with 5 volumes of acetone at -20°C and then washed with cold methanol.
  • the protein precipitate was dissolved with 3 ⁇ l of 8 M urea, diluted with buffer B, and left overnight at 4°C to renature.
  • Reconstituted NF-AT binding was assayed by mixing 1 ⁇ l of renatured 120 kDa protein with 10 ng of affinity-purified Jurkat AP-1 proteins. Mobility shift conditions were as described above in the presence of 100 ng/ ⁇ l of polydldC.
  • NF-AT oligonucleotide-coupled SEPHAROSE 4B (50 ⁇ g of DNA per ml of resin) was prepared by the method of Kadonaga and Tjian (1986) Proc. Natl. Acad. Sci. U.S.A.. 83:5889-5893 using CNBr-activated SEPHAROSE 4B (Pharmacia Co.). SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (1970) Nature (London), 227:680-685. Protein concentration was determined by the method of Bradford (1976) Anal. Biochem.. 72:248-254, using bovine serum albumin as a standard. All other chemicals were reagent grade or better. A combination of both phorbol ester (PMA) and calcium ionophore
  • PMA phorbol ester
  • cytokines that include IL-2 and GM-CSF. Production of these cytokines is inhibited by the immunosuppressive drug CsA.
  • CsA immunosuppressive drug
  • the CLEO element (position -54 to -40) was shown to be essential for induction of transcription at the GM-CSF promoter in a stimulation- dependent manner.
  • the 3'-half of the CLEO sequence is identical to that of the NF-AT element (Table 2), which is important for NF-AT binding.
  • One of the CLEO binding nuclear factors (NF-CLEO ⁇ ) is inducible with PMA/A23187 and sensitive to cycloheximide and CsA, suggesting that this factor is related to NF-AT.
  • Table 3 provides further data on the specificity of binding of NF-AT to the CLEO binding sequence.
  • NF-AT Element Crabtree, Science (1989), 243:355-361, Ullman et al., Ann. Rev. Immunol. (1990), 8:421-452, and Jain et al., Nature (1992), 356:801-804; CLEO Element: Arai et al., Pharmac. Ther. (1992), 55:303-318, and
  • affinity- purified NF-AT were prepared from nuclear extracts of Jurkat cells stimulated with PMA and A23187. After precipitation with ammonium sulfate and gel filtration with SUPEROSE 6, the fractions containing NF-AT-binding activity were applied to the NF-AT oligonucleotide coupled to a SEPHAROSE 4B column. The column was washed, and elution was carried out with 0.3 M KCI-containing buffer. An elution profile of the affinity purified NF-AT, when compared to the protein concentration of each fraction, yielded a single peak.
  • the eluted fractions were then subjected to a mobility shift assay using 32 P-labeled NF-AT oligonucleotides as a probe, which showed that affinity- purified NF-AT shifts the probe as a single band in either the absence ( Figure 1 , lanes 2 and 10) or the presence of polydldC, and the mobility of the band is the same as that of Jurkat nuclear extracts stimulated with PMA/A23187 ( Figure 1 , lane 1 ).
  • NF-CLEO ⁇ CLE0- binding protein
  • FIG. 1 affinity-purified NF-AT can bind the CLEO probe strongly and shifts the probe to a position that corresponds to NF-CLEO ⁇ .
  • This binding is a specific interaction between NF-AT120 proteins and CLEO sequence, because it was blocked with NF-AT and AP-1 oligonucleotides ( Figure 1 , lanes 8 and 9), but not with the Sp1 sequence.
  • NF-AT was purified to near homogeneity to determine whether the NF-CLEO ⁇ shared the same component with NF-AT.
  • Affinity-purified NF-AT was dialyzed against a buffer containing 6 M urea, 20 mM HEPES (pH 7.9), 1 mM EGTA, and 1 mM EDTA to dissociate the AP-1 components from the other component of NF-AT, and was then subjected to Mono-Q chromatography in the presence of 6 M urea. After elution by a KCI gradient (0.05 - 0.8 M), each fraction was assayed by mobility shift in either the absence or the presence of affinity-purified AP-1 proteins from activated Jurkat cells. As shown in Figure 2A and Figure 2B, the Mono Q fractions at a position of 0.2 M KCI contained a protein, which reconstituted NF-AT DNA binding activity with AP-1 proteins (fractions 13 - 19).
  • NF-AT120 The DNA-binding component of NF-AT (NF-AT120) was detected in a cytoplasmic fraction of Jurkat cells by elution and renaturation after SDS- PAGE; it has a molecular mass between 94 and 116 kDa. It was also detected by the same method in nuclear extracts of an activated murine T-cell clone, Ar-5, whose apparent molecular mass was between 90 and 125 kDa (NF-ATp).
  • the protein band corresponding to 120 kDa on SDS- PAGE after the Mono Q fraction was excised.
  • the protein was eluted from the gel slice, denatured with 8 M urea and renatured by dilution.
  • the 120 kDa protein, eluted and renatured from the gel reconstituted both NF-AT and CLEO DNA-binding activity with AP-1 proteins as well as the protein fraction from Mono Q chromatography ( Figure 4B).
  • NF-AT binds directly to the CLEO element on the GM-CSF promoter region and that both NF-CLEO ⁇ and NF-AT share the same nuclear component, NF-AT120, which can reconstitute the DNA-binding in vitro with AP-1 proteins, including recombinant cJun/cFos heterodimer.
  • NF-AT120 which can reconstitute the DNA-binding in vitro with AP-1 proteins, including recombinant cJun/cFos heterodimer.
  • NF-AT may contribute to coordinate regulation of the expression of both IL-2 and GM-CSF genes in T-cells.
  • the procedure for isolation of the 120 kDa component of NF-AT described here is helpful in elucidating the regulation of cytokine gene expression upon T cell activation.
  • the prep gel slice containing protein was briefly rinsed with water and acetonitrile to remove excess SDS, smashed into tiny fragments, taken to dryness under vacuum on a SpeedVac (Savant), and then solubilized in 0.2 ml 25 mM Tris buffer (pH 7.5) containing 0.7 ⁇ g LysC.
  • the cleavage reaction was carried out at 37°C for 24 hours.
  • the reaction mix was spun at 13,000 rpm and loaded onto a 2.1 x 100 mm AQUAPORE RP-300 reversed phase column, and peptides eluted with a linear 4-44% acetonitrile gradient (with constant 0.1% TFA). Eluting peptides were monitored at 214 nm and were collected by hand.
  • Peptide sequences were determined using an Applied Biosystems 477A Sequencer. Fragments provided peptide sequences of SEQ ID NOs: 1 through 5.
  • a 437 bp DNA fragment was amplified from Jurkat cDNA by PCR using degenerate oligonucleotides.
  • the two oligonucleotides of SEQ ID NOs: 32 and 33 were used.
  • the amplification was performed using the "step-cycle" program on a PCR THERMOCYCLER machine set to denature at 94°C for 1 min, anneal at 40°C for 1 min, and extend at 72°C for 10 sec, for a total of 40 cycles.
  • the 437 bp fragment was purified and cloned into the pCRTM ⁇ plasmid vector obtained from Invitrogen Corp. and sequenced by the chain-termination sequencing method.
  • SEQ ID NO: 34 represents the NF-AT120 nucleotide sequence and SEQ ID NO: 35 is the encoded amino acid sequence.
  • This nucleotide segment can be used to screen for related NF-AT proteins by hybridization.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g.: Coligan (1991 and Supplements) Current Protocols in Immunology. Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual. Cold Spring Harbor Press.
  • AP-1 is used as a specific binding reagent, and advantage can be taken of its specificity of binding, much as an antibody would be used.
  • the binding reagent is either labeled as described above, e.g., for fluorescence or otherwise, or immobilized to a substrate for panning methods.
  • the binding composition is used for screening of an expression library made from a cell line which expresses an NF-AT120 protein. Standard staining techniques are used to detect or sort intracellular or surface-expressed protein, or surface-expressing transformed cells are screened by panning.
  • ELISA solution e.g., VECTOR ELITE ABC horseradish peroxidase solution
  • use e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solution and incubate for 30 min. Wash cells once with HBSS, and then a second time for 2 min, to close the cells.
  • DAB VECTOR diaminobenzoic acid
  • the binding compositions are used to affinity purify or sort out cells expressing the protein; see, e.g., Sambrook et al. or Ausubel et al.
  • the peptide segments are used to predict appropriate oligonucleotides to screen a library.
  • the genetic code is used to select appropriate oligonucleotides useful as probes for directly screening a library; see, e.g., SEQ ID NO: 1 through 24.
  • polymerase chain reaction (PCR) techniques will be applied. Synthetic oligonucleotides in appropriate orientations are used as primers to select correct clones from a library.
  • Various combinations of upstream/downstream sense/antisense combinations are tested until an appropriate clone is amplified and detected. 3'- or 5'-anchor PCR techniques can also be applied.
  • Another strategy is to screen for a membrane-bound expression product by panning.
  • the cDNA library is constructed in an expression vector which attaches the product to the cell membrane.
  • the soluble binding partner or antibodies raised against the defined peptide fragments can be immobilized and used to immobilize expressing cells, immobilization may be achieved by use of appropriate antibodies which recognize, e.g., the soluble receptor construct, or by use of antibodies raised against the first antibodies. Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of appropriate expressing clones.
  • Phage expression libraries can be screened by soluble AP-1 or anti- fragment antibodies. Appropriate label techniques, e.g., antibodies, will allow specific labeling of appropriate clones.
  • Species variants are isolated using cross-species hybridization techniques based upon isolation of a full-length isolate or fragment from one species as a probe. Alternatively, similar assays can be developed, e.g., in mouse cells for isolation of a corresponding NF-AT120 protein.
  • SEQ ID NO: 1 is NF-AT120 peptide 1 sequence.
  • SEQ ID NO: 2 is NF-AT120 peptide 2 sequence.
  • SEQ ID NO: 3 is NF-AT120 peptide 3 sequence.
  • SEQ ID NO: 4 is NF-AT120 peptide 4 sequence.
  • SEQ ID NO: 5 is NF-AT120 peptide 5 sequence.
  • SEQ ID NO: 6 is nucleotide sequence for NF-AT peptide 1.
  • SEQ ID NO: 7 is nucleotide sequence for NF-AT peptide 2.
  • SEQ ID NO: 8 is nucleotide sequence for NF-AT peptide 2.
  • SEQ ID NO: 9 is nucleotide sequence for NF-AT peptide 3.
  • SEQ ID NO: 10 is nucleotide sequence for NF-AT peptide 3.
  • SEQ ID NO: 11 is nucleotide sequence for NF-AT peptide 3.
  • SEQ ID NO: 12 is nucleotide sequence for NF-AT peptide 3.
  • SEQ ID NO: 13 is nucleotide sequence for NF-AT peptide 4.
  • SEQ ID NO: 14 is nucleotide sequence for NF-AT peptide 4.
  • SEQ ID NO: 15 is nucleotide sequence for NF-AT peptide 4.
  • SEQ ID NO: 16 is nucleotide sequence for NF-AT peptide 4.
  • SEQ ID NO: 17 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 18 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 19 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 20 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 21 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 22 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 23 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 24 is nucleotide sequence for NF-AT peptide 5.
  • SEQ ID NO: 25 is CLE0 nucleotide sequence.
  • SEQ ID NO: 26 is AP-1 site.
  • SEQ ID NO: 27 is NF-AT site.
  • SEQ ID NO: 28 is huNF-AT element.
  • SEQ ID NO: 29 is huCLE ⁇ element.
  • SEQ ID NO: 30 is oNF-AT element.
  • SEQ ID NO: 31 is moCLE ⁇ element.
  • SEQ ID NO: 32 is L27-1S oligonucleotide.
  • SEQ ID NO: 33 is L33-4A oligonucleotide.
  • SEQ ID NO: 34 is huNF-AT120, P subfamily, nucleotide sequence
  • SEQ ID NO: 35 is huNF-AT120, P subfamily, amino acid sequence SEQ ID NO: 36 is huNF-AT120, C subfamily, nucleotide sequence
  • SEQ ID NO: 37 is huNF-AT120, C subfamily, amino acid sequence
  • SEQ ID NO: 38 is huNF-AT120, P subfamily, nucleotide sequence
  • SEQ ID NO: 39 is huNF-AT120, P subfamily, amino acid sequence
  • SEQ ID NO: 40 is huNF-AT120, X subfamily, nucleotide sequence SEQ ID NO: 41 is huNF-AT120, X subfamily, amino acid sequence (1) GENERAL INFORMATION:
  • MOLECULE TYPE peptide
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO:10: GTNTTRGARA THCCNCUNGA RCCNAAR 27
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA
  • CARGARCARA AYTTRGAYCA RACNTAYTTR CAYCARGTNA AYGARATHGT NAGRAAR 57
  • CARGARCARA AYCTNGAYCA RACNTAYTTR CAYCARGTNA AYGARATHGT NCGNAAR 57
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:22:
  • CARGARCARA AYCTNGAYCA RACNTAYTTR CAYCARGTNA AYGARATHGT NAGRAAR 57
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:30: TGAAACAAAT TTTCCTCC 18
  • MOLECULE TYPE cDNA
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:32:
  • MOLECULE TYPE cDNA
  • TCC TAC ATG AGC CCG ACC CTG CCC GCC CTG GAC TGG CAG CTG CCG TCC 1602 Ser Tyr Met Ser Pro Thr Leu Pro Ala Leu Asp Trp Gin Leu Pro Ser 410 415 420 CAC TCA GGC CCG TAT GAG CTT CGG ATT GAG GTG CAG CCC AAG TCC CAC 1650
  • GCC GGA ATC CTG AAA CTC AGA AAC TCC GAC ATT GAA CTT CGG AAA GGA 1986 Ala Gly He Leu Lys Leu Arg Asn Ser Asp He Glu Leu Arg Lys Gly 535 540 545
  • ATC CCA GTG ACT GCA TCC CTC CCT CCA CTT GAG TGG CCG CTG TCC
  • AGT 581 He Pro Val Thr Ala Ser Leu Pro Pro Leu Glu Trp Pro Leu Ser Ser 170 175 180 CAG TCA GGC TCT TAC GAG CTG CGG ATC GAG GTG CAG CCC AAG CCA CAT 629 Gin Ser Gly Ser Tyr Glu Leu Arg He Glu V ⁇ l Gin Pro Lys Pro His 185 190 195 200 CAC CGG GCC CAC TAT GAG ACA GAA GGC AGC CGA GGG GCT GTC AAA GCT 677 His Arg Ala His Tyr Glu Thr Glu Gly Ser Arg Gly Ala Val Lys Ala 205 210 215
  • CAC CCA GTC CCA GCC ATC AAG ACG GAG CCC ACG GAT GAA TAT GAC CCC 1445 His Pro V ⁇ l Pro Ala He Lys Thr Glu Pro Thr Asp Glu Tyr Asp Pro 460 465 470
  • AGC CCC AGG CCA GCC TCA GGA CCC TCA TCA AGG CCC ACA TCC CCC TGT 1116 Ser Pro Arg Pro Ala Ser Gly Pro Ser Ser Arg Pro Thr Ser Pro Cys
  • GGG AAA CGG AGG CAC TCC AGT GCT GAA GTT TGT TAT GCT GGG TCC CTT 1164 Gly Lys Arg Arg His Ser Ser Ala Glu Val Cys Tyr Ala Gly Ser Leu 275 280 285
  • ATTTGTACTG CTTTATGGTC ATTTGAAGGG CCCCTTAGTT TTTATGATAA TTTTTAAAAT 3928 AGGAACTTTT GATAAGACCT TCTAGAACCC CAAAAAAAAA AAAAAAAGAA AAAAAAAGAA 3988

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Abstract

Composants de facteurs de régulation de transcription de mammifères, et réactifs associés comprenant des protéines purifiées, des anticorps spécifiques, ainsi que les acides nucléiques codant pour ladite protéine. Sont également présentées des méthodes d'utilisation desdits réactifs et des trousses de diagnostic.
PCT/US1994/007297 1993-07-06 1994-07-05 Composants purifies de complexes de regulation de transcription de mammiferes et leurs analogues WO1995002053A1 (fr)

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US8848393A 1993-07-06 1993-07-06
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US11397193A 1993-08-30 1993-08-30
US08/113,971 1993-08-30
US14806193A 1993-11-05 1993-11-05
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0722452A1 (fr) * 1993-09-20 1996-07-24 The Board Of Trustees Of The Leland Stanford Junior University Polypeptides et polynucleotides nf-at
WO1996026959A1 (fr) * 1995-03-02 1996-09-06 Tularik, Inc. Facteurs nucleaires et determination par liaison
US5912168A (en) * 1996-08-30 1999-06-15 Genesis Research & Development Corporation Limited CD95 regulatory gene sequences
US6352830B1 (en) 1991-08-22 2002-03-05 The Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides and screening methods for immunosuppressive agents
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Cited By (16)

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Publication number Priority date Publication date Assignee Title
US6150099A (en) * 1991-08-22 2000-11-21 Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides
US6875571B2 (en) 1991-08-22 2005-04-05 The Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides and screening methods for immunosuppressive agents
US6352830B1 (en) 1991-08-22 2002-03-05 The Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides and screening methods for immunosuppressive agents
US6197925B1 (en) 1991-08-22 2001-03-06 Sara Lee Corporation NF-AT polypeptides and polynucleotides
EP1221483A1 (fr) * 1993-09-20 2002-07-10 The Board Of Trustees Of The Leland Stanford Junior University Polypeptides et polynucleotides nf-at
US6096515A (en) * 1993-09-20 2000-08-01 Board Of Trustees Of The Leland Stanford Junior University NF-AT polynucleotides
US6171781B1 (en) 1993-09-20 2001-01-09 The Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides
EP0722452A4 (fr) * 1993-09-20 1998-08-05 Univ Leland Stanford Junior Polypeptides et polynucleotides nf-at
US6312899B1 (en) 1993-09-20 2001-11-06 Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides
US6388052B1 (en) 1993-09-20 2002-05-14 Board Of Trustees Of The Leland Stanford Junior University NF-AT polypeptides and polynucleotides
EP0722452A1 (fr) * 1993-09-20 1996-07-24 The Board Of Trustees Of The Leland Stanford Junior University Polypeptides et polynucleotides nf-at
US5612455A (en) * 1994-07-05 1997-03-18 Tularik, Inc. Nuclear factors and binding assay
WO1996026959A1 (fr) * 1995-03-02 1996-09-06 Tularik, Inc. Facteurs nucleaires et determination par liaison
US5912168A (en) * 1996-08-30 1999-06-15 Genesis Research & Development Corporation Limited CD95 regulatory gene sequences
US6599741B1 (en) 1999-09-14 2003-07-29 Avontec Gmbh Modulating transcription of genes in vascular cells
US7186556B2 (en) 1999-09-14 2007-03-06 Avontec Gmbh Modulating transcription of genes in vascular cells

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