WO1995031468A1 - Macrophage migration inhibitory factor-3 - Google Patents

Macrophage migration inhibitory factor-3 Download PDF

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Publication number
WO1995031468A1
WO1995031468A1 PCT/US1994/005385 US9405385W WO9531468A1 WO 1995031468 A1 WO1995031468 A1 WO 1995031468A1 US 9405385 W US9405385 W US 9405385W WO 9531468 A1 WO9531468 A1 WO 9531468A1
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WIPO (PCT)
Prior art keywords
polypeptide
mif
polynucleotide
dna
sequence
Prior art date
Application number
PCT/US1994/005385
Other languages
French (fr)
Inventor
Haodong Li
Lisa M. Fitzgerald
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to EP94923875A priority Critical patent/EP0770086A4/en
Priority to PCT/US1994/005385 priority patent/WO1995031468A1/en
Priority to AU73942/94A priority patent/AU7394294A/en
Priority to JP7529593A priority patent/JPH10500301A/en
Publication of WO1995031468A1 publication Critical patent/WO1995031468A1/en
Priority to US08/903,224 priority patent/US5986060A/en
Priority to US10/178,731 priority patent/US20030166863A1/en

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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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptide ⁇ encoded by such polynucleotides, the use of such polynucleotides and polypeptide ⁇ . as well as the production of such polynucleotides and polypeptide ⁇ . More particularly, the polypeptide of the present invention is Macrophage Migration Inhibitory Factor-3 (MIF-3). The invention also relates to inhibiting the action of such polypeptides.
  • MIF-3 Macrophage Migration Inhibitory Factor-3
  • lympho ines that play an important role in immunoregulation, inflammation and effector mechanisms of cellular immunity.
  • MIF migration inhibitory factor
  • MIF activity is correlated with a variety of inflammatory responses including delayed hypersensitivity and cellular immunity (Rocklin, R.E. et al.. New Engl. J. Med., 282:1340-1343 (1970); allograft rejection (Al-Askari, S. et al., Nature, 205:916-917 (1965); and rheumatoid polyarthritic synovialis (Odink et al., Nature, 330:80-82 (1987).
  • MIF is a lymphokine known to be produced by activated T cells. MIF is a major secreted protein released by the anterior pituitary cells. A large number of publications have reported the isolation and identification of putative MIF molecules. For example, MIF-1 was purified to homogeneity from the serum-free culture supernatant of a human T cell hybridoma clone called F5. Oki, S., Lymphokine Cytokine Res., 10:273-80 (1991). Also, an MIF-2, which is more hydrophobic than MIF-1, was purified to homogeneity from the same clone, (Hirose, S., et al., M, Microbiol. Immunol., 35:235-45 (1991)). The polypeptide of the present invention, MIF-3, is structurally related to the MIF family.
  • polypeptide of the present invention is of human origin.
  • polynucleotides (DNA or RNA) which encode such polypeptide ⁇ .
  • antagonist/inhibitor ⁇ to such polypeptides which may be used to inhibit the action of such polypeptides, for example, in the treatment of septic shock, lethal endotoxaemia and ocular inflammation.
  • Figure 1 depict ⁇ the polynucleotide sequence and corresponding deduced amino acid sequence of MIF-3.
  • the polypeptide encoded by the amino sequence shown is the preprotien form of the polypeptide, and the standard one letter abbreviation for amino acids is used.
  • nucleic acid which encode ⁇ for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited a ⁇ ATCC Deposit No. 75712 on March 18, 1994.
  • the polynucleotide of this invention wa ⁇ di ⁇ covered from a cDNA library derived from human T cell ⁇ . It is structurally related to the human MIF family. It contains an open reading frame encoding a protein of approximately 118 amino acid residues. The protein exhibits the highest degree of homology to human MIF with 34 % identity and 78 % similarity over the entire amino acid sequence.
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sen ⁇ e) strand.
  • the coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein ⁇ equence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding ⁇ equence 5' and/or 3 ' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variants of the hereinabove de ⁇ cribed polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotide ⁇ encoding the ⁇ ame mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variant ⁇ of such polynucleotide ⁇ which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variants include deletion variants, substitution variants and addition or in ⁇ ertion variant ⁇ .
  • the polynucleotide may have a coding ⁇ equence which i ⁇ a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a sub ⁇ titution, deletion or addition of one or more nucleotide ⁇ , which doe ⁇ not ⁇ ub ⁇ tantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • the marker sequence may be a hexa- hi ⁇ tidine tag supplied by a pQE-9 vector to provide for p ..fication of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, i ⁇ u ⁇ ed.
  • the HA tag correspond ⁇ to an epitope derived from the influenza hemagglutinin protein (Wil ⁇ on, I., et al.. Cell, 37:767 (1984)).
  • the present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there i ⁇ at least 50% and preferably 70% identity between the sequences.
  • the present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides .
  • ⁇ tringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptide ⁇ which retain substantially the same biological function or activity a ⁇ the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
  • the deposit(s) referred to herein will be maintained under the terms of the Budape ⁇ t Treaty on the International Recognition of the Deposit of Micro-organis ⁇ for purposes of Patent Procedure. These deposits are provided merely a ⁇ convenience to those of ⁇ kill in the art and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein.
  • a license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
  • the present invention further relates to a MIF-3 polypeptide which has the deduced amino acid ⁇ equence of Figure 1 or which ha ⁇ the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
  • fragment when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the ⁇ ame biological function or activity as such polypeptide.
  • an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a ⁇ ynthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the depo ⁇ ited cDNA may be (i) one in which one or more of the amino acid re ⁇ idue ⁇ are substituted with a conserved or non-conserved amino acid re ⁇ idue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residue ⁇ include ⁇ a ⁇ ub ⁇ tituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acid ⁇ are fused to the mature polypeptide, such a ⁇ a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • Such fragments, derivatives and analogs are deemed
  • polypeptide ⁇ and polynucleotide ⁇ of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material i ⁇ removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptide ⁇ could be part of a compo ⁇ ition, and ⁇ till be i ⁇ olated in that such vector or composition is not part of its natural environment.
  • the present invention also relates to vectors which include polynucleotide ⁇ of the pre ⁇ ent invention, ho ⁇ t cell ⁇ which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Ho ⁇ t cells are genetically engineered (transduced or transformed or transfected) with the vector ⁇ of this invention which may be, for example, a cloning vector or an expre ⁇ ion vector.
  • the vector may be, for example, in the
  • SUBSTITUTE SHEET (RULE 261 form of a plasmid, a viral particle, a phage, etc.
  • the engineered ho ⁇ t cell ⁇ can be cultured in conventional nutrient media modified a ⁇ appropriate for activating promoter ⁇ , ⁇ electing transformants or amplifying the MIF genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the ho ⁇ t cell ⁇ elected for expre ⁇ ion, and will be apparent to the ordinarily ⁇ killed artisan.
  • the polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques.
  • the polynucleotide may be included in any one of a variety of expression vectors for expres ⁇ ing a polypeptide.
  • Such vectors include chromosomal, nonchromo ⁇ omal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmid ⁇ ; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenoviru ⁇ , fowl pox viru ⁇ , and pseudorabie ⁇ .
  • any other vector may be used as long a ⁇ it i ⁇ replicable and viable in the ho ⁇ t.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures.
  • the DNA sequence is inserted into an appropriate re ⁇ triction endonuclease site( ⁇ ) by procedures known in the art. Such procedure ⁇ and other ⁇ are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence( ⁇ ) (promoter) to direct mRNA synthe ⁇ i ⁇ .
  • promoter promoter
  • a ⁇ repre ⁇ entative example ⁇ of such promoter ⁇ there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contain ⁇ a ribo ⁇ ome binding ⁇ ite for tran ⁇ lation initiation and a tran ⁇ cription terminator.
  • the vector may also i: .lude appropriate sequences for amplifying expre ⁇ ion.
  • the exp spion vector ⁇ preferably contain one or more selectable mar; er genes to provide a phenotypic trait for selection of transformed ho ⁇ t cells such as dihydrofolate reductase or neomycin resi ⁇ tance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well a ⁇ an appropriate promoter or control ⁇ equence, may be employed to tran ⁇ form an appropriate ho ⁇ t to permit the ho ⁇ t to expre ⁇ the protein.
  • a ⁇ repre ⁇ entative example ⁇ of appropriate ho ⁇ ts there may be mentioned: bacterial cell ⁇ , such as E. coli. Strepto vces. Salmonella typhimuriu : fungal cells, such a ⁇ yeast; insect cells such as Drosophila and Sf9 animal cell ⁇ ⁇ uch a ⁇ CHO, COS or Bowe ⁇ melanoma; plant cell ⁇ , etc.
  • bacterial cell ⁇ such as E. coli. Strepto vces. Salmonella typhimuriu
  • fungal cells such a ⁇ yeast
  • insect cells such as Drosophila and Sf9 animal cell ⁇ ⁇ uch a ⁇ CHO, COS or Bowe ⁇ melanoma
  • plant cell ⁇ etc.
  • the selection of an appropriate host i ⁇ deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequence ⁇ a ⁇ broadly de ⁇ cribed above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a ⁇ equence of the invention ha ⁇ been in ⁇ erted, in a forward or rever ⁇ e orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vector ⁇ and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example.
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter region ⁇ can be ⁇ elected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are PKK232-8 and PCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P R , P L and trp.
  • Eukaryotic promoter ⁇ include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter i ⁇ well within the level of ordinary ⁇ kill in the art.
  • the pre ⁇ ent invention relate ⁇ to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such a ⁇ a yea ⁇ t cell, or the ho ⁇ t cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cell ⁇ under the control of appropriate promoter ⁇ .
  • Cell-free tran ⁇ lation ⁇ y ⁇ tem ⁇ can also be employed to produce ⁇ uch protein ⁇ using RNAs derived from the DNA con ⁇ truct ⁇ of the pre ⁇ ent invention.
  • Appropriate cloning and expre ⁇ ion vector ⁇ for u ⁇ e with prokaryotic and eukaryotic ho ⁇ t ⁇ are de ⁇ cribed by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
  • Enhancers are ci ⁇ -acting element ⁇ of DNA, ually about from 10 to 300 bp that act on a promoter to increase it ⁇ tran ⁇ cription.
  • Example ⁇ including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyo a enhancer on the late side of the replication origin, and adenovirus enhancer ⁇ .
  • recombinant expre ⁇ ion vector ⁇ will include origin ⁇ of replication and ⁇ electable marker ⁇ permitting transformation of the ho ⁇ t cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expre ⁇ ed gene to direct transcription of a down ⁇ tream ⁇ tructural ⁇ equence.
  • a promoter derived from a highly-expre ⁇ ed gene to direct transcription of a down ⁇ tream ⁇ tructural ⁇ equence can be derived from operon ⁇ encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), ⁇ -factor, acid pho ⁇ phatase, or heat shock pr ins, among other ⁇ .
  • PGK 3-phosphoglycerate kinase
  • ⁇ -factor acid pho ⁇ phatase
  • heat shock pr ins among other ⁇ .
  • heterologou ⁇ ⁇ tructural ⁇ equence i ⁇ assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterxal use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination ⁇ ignal ⁇ in operable reading phase with a functional promoter.
  • the vector will compri ⁇ e one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the ho ⁇ t.
  • Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimuriu and variou ⁇ species within the genera Pseudo ona ⁇ , Streptomyce ⁇ , and Staphylococcu ⁇ , although others may also be employed a ⁇ a matter of choice.
  • u ⁇ eful expre ⁇ ion vector ⁇ for bacterial u ⁇ e can compri ⁇ e a ⁇ electable marker and bacterial origin of replication derived from commercially available pla ⁇ mids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017).
  • cloning vector pBR322 ATCC 37017
  • Such commercial vectors include, for example, PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, I, USA).
  • the selected promoter i ⁇ induced by appropriate mean ⁇ e.g., temperature ⁇ hift or chemical induction
  • cell ⁇ are cultured for an additional period.
  • Cell ⁇ are typically harve ⁇ ted by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expres ⁇ ion of protein ⁇ can be di ⁇ rupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell ly ⁇ ing agent ⁇ , ⁇ uch method ⁇ are well know to tho ⁇ e skilled in the art.
  • mammalian cell culture sy ⁇ tem ⁇ can al ⁇ o be employed to expre ⁇ recombinant protein.
  • Example ⁇ of mammalian expre ⁇ ion ⁇ ystems include the COS-7 lines of monkey kidney fibroblasts, de ⁇ cribed by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expre ⁇ sing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expres ⁇ ion vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding ⁇ ite ⁇ , polyadenylation ⁇ ite, ⁇ plice donor and acceptor ⁇ ite ⁇ , transcriptional termination sequence ⁇ , and 5' flanking nontranscribed sequences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the MIF-3 polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, pho ⁇ phocellulo ⁇ e chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion pre ⁇ ent during purification. (Price et al., J. Biol. Chem., 244:917 (1969)). Protein refolding ⁇ tep ⁇ can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification step ⁇ .
  • HPLC high performance liquid chromatography
  • the polypeptide ⁇ of the pre ⁇ ent invention may be a naturally purified product, or a product of chemical ⁇ ynthetic procedure ⁇ , or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, in ⁇ ect and mammalian cell ⁇ in culture).
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, in ⁇ ect and mammalian cell ⁇ in culture.
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may also include an initial methionine amino acid residue.
  • MIF-3 proteins of the pre ⁇ ent invetion have displayed biological activitie ⁇ which indicate it ⁇ role as a general activator or several different acrophage functions. Further, MIF-3 both inhbiti ⁇ the migration of human macrophage ⁇ and ⁇ timulate ⁇ the activity of macrophages.
  • the MIF-3 polypeptides of the present invention may be employed as an anti-tumor agent. Activated macrophages alone or in combination with specific anti-tumor monoclonal antibodies have considerable tumoricidal capacity. Similarly, the ability of MIF-3 to promote macrophage- mediated killing of certain pathogens indicates the use of this molecule in treating various infections, including tuberculosis, Hunsen di ⁇ ea ⁇ e and Candida.
  • MIF-3 may be exploited in a therapeutic agent for treating wounds.
  • Local application of MIF-3 at the site of injury may result in increased numbers of activated macrophages concentrated within the wound, thereby increasing the rate of healing of the wound.
  • MIF-3 may be used as a general immune stimulus to increase the immunity generated against specific vaccines.
  • MIF proteins have the ability to enhance macrophages to present antigens to T cells. Therefore, MIF-3 may be emplyed to potentiate the immune response to different antigens. This is extremely important in cases such as AIDS or AIDS related complex.
  • MIF-3 may also be employed to enhance the detoxification function of the liver.
  • a protein having MIF activity in the rat liver links the chemical and immunological detoxification system ⁇ . This protein actuate ⁇ both glutothione S-tran ⁇ fera ⁇ e (GSTs) and MIF activity.
  • GSTs glutothione S-tran ⁇ fera ⁇ e
  • the MIF polypeptide ⁇ may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA polynucleotide
  • cell ⁇ may be engineered by procedure ⁇ known in the art by u ⁇ e of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cell ⁇ may be engineered in vivo for expre ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the pre ⁇ ent invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo .
  • the expression vehicle for engineering cells may be other than a retroviru ⁇ , for example, an adenoviru ⁇ which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • the polypeptides of the present invention may be employed in combination with a ⁇ uitable pharmaceutical carrier.
  • a ⁇ uitable pharmaceutical carrier include ⁇ but i ⁇ not limited to ⁇ aline, buffered ⁇ aline, dextro ⁇ e, water, glycerol, ethanol, and combination ⁇ thereof.
  • a carrier include ⁇ but i ⁇ not limited to ⁇ aline, buffered ⁇ aline, dextro ⁇ e, water, glycerol, ethanol, and combination ⁇ thereof.
  • the formulation should ⁇ uit the mode of administration.
  • the invention also provide ⁇ a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a ⁇ ociated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or ⁇ ale of pharmaceutical ⁇ or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the polypeptides of the present invention may be employed in conjunction with other therapeutic compound ⁇ .
  • the pharmaceutical compo ⁇ ition ⁇ may be admini ⁇ tered in a convenient manner ⁇ uch a ⁇ by the topical, intravenou ⁇ , intraperitoneal, intramu ⁇ cular, subcutaneous, intranasal or intradermal routes.
  • the amounts and dosage regimens of MIF and administered to a subject will depend on a number of factors ⁇ uch as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician.
  • the do ⁇ age is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptom ⁇ , etc.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphism ⁇ ) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes as ⁇ ociated with di ⁇ ea ⁇ e.
  • ⁇ equence ⁇ can be mapped to chromo ⁇ ome ⁇ by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is u ⁇ ed to rapidly select primer ⁇ that do not ⁇ pan more than one exon in the genomic DNA, thu ⁇ complicating the amplification proce ⁇ . These primers are then used for PCR ⁇ creening of ⁇ omatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategie ⁇ that can ⁇ imilarly be u ⁇ ed to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosome ⁇ and preselection by hybridization to construct chromo ⁇ ome ⁇ pecific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clones to a metapha ⁇ e chromo ⁇ omal ⁇ pread can be u ⁇ ed to provide a precise chromosomal location in one step.
  • This technique can be u ⁇ ed with cDNA as short as 500 or 600 bases; however, clones larger t.*"an 2,000 bp have a higher likelihood of binding to a unique cnromo ⁇ omal location with ⁇ ufficient ⁇ ignal intensity for simple detection.
  • FISH requires use of the clones from which the EST was derived, and the longer the better.
  • a cDNA preci ⁇ ely localized to a chromo ⁇ omal region a ⁇ ociated with the di ⁇ ea ⁇ e could be one of between 50 and 500 potential causative genes. (This as ⁇ umes 1 megabase mapping re ⁇ olution and one gene per 20 kb).
  • Compari ⁇ on of affected and unaffected individual ⁇ generally involve ⁇ first looking for structural alteration ⁇ in the chromo ⁇ ome ⁇ , ⁇ uch as deletions or tran ⁇ locations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individual ⁇ i ⁇ required to confirm the presence of a mutation and to di ⁇ tingui ⁇ h mutation ⁇ from polymorphi ⁇ m ⁇ .
  • the polypeptide ⁇ , their fragments or other derivatives, or analogs thereof, or cells expres ⁇ ing them can be u ⁇ ed as an immunogen to produce antibodie ⁇ thereto.
  • the ⁇ e antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also include ⁇ chimeric, ⁇ ingle chain, and humanized antibodies, as well as Fab fragment ⁇ , or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated again ⁇ t the polypeptide ⁇ corre ⁇ ponding to a ⁇ equence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptide ⁇ itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodie ⁇ can then be u ⁇ ed to i ⁇ olate the polypeptide from tissue expressing that polypeptide.
  • Example ⁇ include the hybridoma technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodie ⁇ (Cole, et al., 1985, in Monoclonal Antibodie ⁇ and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Thi ⁇ invention if further related to a method for identification of MIF-3 receptor ⁇ .
  • the gene encoding a receptor can be identified by expre ⁇ ion cloning, which comprises preparing polyadenylated RNA from a cell responsive to MIF-3 and a cDNA library created from this RNA i ⁇ divided into pools and used to tran ⁇ fect COS cells or other cells that are not respon ⁇ ive to MIF-3. Tran ⁇ fected cell ⁇ which are grown on glas ⁇ ⁇ lide ⁇ are expo ⁇ ed to labeled MIF-3.
  • MIF- 3 can be labeled by a variety of mean ⁇ including iodidation or inclu ⁇ ion of a recognition ⁇ ite for a ⁇ ite- ⁇ pecific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retran ⁇ fected using an iterative sub-pooling and rescreening proce ⁇ , eventually yielding a ⁇ ingle clone that encode ⁇ the putative receptor.
  • labeled MIF-3 can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cros ⁇ -linked material is resolved by PAGE and exposed to x-ray film.
  • the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein micro ⁇ equencing.
  • the amino acid ⁇ equence obtained from microsequencing would be used to design a set of generate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
  • This invention also provides a method of screening drugs to identify those which enhance (agonists) or block (antagonist ⁇ ) interaction of MIF-3 to it ⁇ receptor.
  • An agonist is a compound which increases the natural biological functions of MIF-3, while an antagoni ⁇ t eliminates ⁇ uch function ⁇ .
  • a mammalian cell or membrane preparation expre ⁇ ing an MIF-3 receptor would be incubated with labeled MIF-3 in the presence of drug. The ability of drug to enhance or block this interaction could then be measured.
  • the response of a known second messenger sy ⁇ tem following interaction of ligand and receptor would be mea ⁇ ured compared in the pre ⁇ ence or absence of drug.
  • Such second messenger sy ⁇ tems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydroly ⁇ i ⁇ .
  • the pre ⁇ ent invention i ⁇ al ⁇ o directed to antagoni ⁇ t/inhibitor molecule ⁇ of the polypeptides of the present invention and their use to inhibit or eliminate the function of the polypeptide.
  • an antagonist is an antibody against the MIF-3 polypeptide or, in some ca ⁇ es, an oligonucleotide which bind ⁇ to the polypeptide.
  • An antagonist may also be be a peptide derivate of MIF-3 which recognizes and binds to MIF-3 receptor sites but has no biological function thereby effectivfely blocking the receptor ⁇ .
  • An example of an inhibitor is an anti ⁇ en ⁇ e construct prepared using antisense technology. Anti ⁇ en ⁇ e technology can be u ⁇ ed to control gene expre ⁇ ion through triple-helix formation or anti ⁇ en ⁇ e DNA or RNA, both of which method ⁇ are ba ⁇ ed on binding of a polynucleotide to DNA or RNA.
  • the 5' coding portion of the polynucleotide ⁇ equence, which encode ⁇ for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the gene involved in tran ⁇ cription (triple helix - ⁇ ee Lee et al., Nucl. Acid ⁇ Re ⁇ ., 3:173 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al.. Science, 251: 1360 (1991)), thereby preventing transcription and the production of MIF-3.
  • the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks tran ⁇ lation of the mRNA molecule into the MIF-3 (antisen ⁇ e - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotide ⁇ a ⁇ Anti ⁇ en ⁇ e Inhibitor ⁇ of Gene Expression, CRC Press, Boca Raton, FL (1988)).
  • the oligonucleotide ⁇ described above can also be delivered to cells ⁇ uch that the anti ⁇ en ⁇ e RNA or DNA may be expre ⁇ sed in vivo to inhibit production of MIF-3.
  • the antagonist/inhibitors may be employed to protect again ⁇ t lethal endotoxaemia and septic shock.
  • Cytokines including Macrophage Migration Inhibitory Proteins, are critical in the often fatal cascade of events that causes septic shock.
  • An endotoxin is a lipopolysaccharide (LPS) moiety of gram-negative bacillary cell walls. This endotoxin cause ⁇ vaso-constriction of small arteries and veins, which leads to increa ⁇ ed peripheral vascular re ⁇ i ⁇ tance and decreased cardiac output. These are the symptom ⁇ of lethal endotoxaemia which lead ⁇ to ⁇ eptic shock.
  • Anterior pituitary cells specifically release MIF proteins in response to the presence of LPS. These pituitary-derived MIF proteins contribute to circulating MIF proteins which are already pre ⁇ ent in the po ⁇ t-acute phase of endotoxaemia.
  • the antagonist/inhibitors may also be used to treat ocular inflammations since partial sequencing of ⁇ mall len ⁇ proteins has identified an MIF protein in the calf lens. Accordingly, MIF proteins may act as intercellular mes ⁇ enger ⁇ or part of the machinery of cellular differentiation, whereby over-expre ⁇ ion of MIF protein ⁇ may lead to ocular inflammation.
  • the antibodie ⁇ to MIF-3 may be employed for diagno ⁇ ing disease progression and efficacy of therapeutic intervention since the level of MIF-3 in circulation may correlate with the ⁇ tate of a disease.
  • the antagonist/inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.
  • the present invention also relates to an assay for identifying potential antagonist/inhibitors specific to MIF- 3.
  • An example of such an assay combines MIF-3 and a potential antagonist/inhibitor with membrane-bound MIF-3 receptors or recombinant MIF-3-receptors under appropriate conditions for a competitive inhibition as ⁇ ay.
  • MIF-3 can be labeled, ⁇ uch a ⁇ by radioactivity, ⁇ uch that the number of MIF-3 molecule ⁇ bound to the receptor can determine the effectivene ⁇ of the potential antagoni ⁇ t/inhibitor.
  • “Pla ⁇ mid ⁇ ” are designated by a lower case p preceded and/or followed by capital letters and/or numbers.
  • the ⁇ tarting plasmids herein are either commercially available. publicly available on an unrestricted basi ⁇ , or can be con ⁇ tructed from available pla ⁇ mids in accord with published procedures.
  • equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
  • “Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan.
  • For analytical purpose ⁇ typically 1 ⁇ g of pla ⁇ mid or DNA fragment is used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
  • For the purpose of isolating DNA fragments for plasmid con ⁇ truction typically 5 to 50 ⁇ g of DNA are dige ⁇ ted with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 * C are ordinarily used, but may vary in accordance with the supplier's instruction . After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
  • Oligonucleotides refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. “Ligation” refers to the process of forming phosphodie ⁇ ter bond ⁇ between two double stranded nucleic acid fragments (Maniatis, T., et al..
  • ligation may be accomplished u ⁇ ing known buffers and conditions with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
  • ligase T4 DNA ligase
  • Example 1 Bacterial Expres ⁇ ion and Purification of MIF-3
  • the 5' oligonucleotide primer has the sequence CCCGCATGCCGTTCCTGGAGCTGG contains an Sph I restriction enzyme site and 19 nucleotide ⁇ of MIF-3 coding ⁇ equence starting from the initiation codon.
  • the 3' sequence CCCAGATCTTAAAAAAGTCATGACCGT contains complementary sequence ⁇ to a Bgl II site and i ⁇ followed by 18 nucleotide ⁇ preceeding the termination codon of MIF-3.
  • the re ⁇ triction enzyme sites correspond to the Sph I and Bam HI restriction enzyme sites on the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311).
  • pQE-70 encode ⁇ antibiotic resistance (Amp r ), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), and puts the His tag to the 3' end of the gene.
  • pQE-70 was then digested with Sph I and Bam HI.
  • the amplified sequences were ligated into pQE-70 and were inserted in frame with the sequence encoding for the histidine tag.
  • Figure 2 shows a schematic representation of this arrangement.
  • the ligation mixture wa ⁇ then u ⁇ ed to tran ⁇ form E. coli ⁇ train M15/rep 4 available from Qiagen under the trademark M15/rep 4 by the procedure de ⁇ cribed in Sambrook, J. et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989).
  • M15/rep4 contains multiple copies of the plasmid pREP4, which expres ⁇ es the lad repressor and also confers kanamycin resistance (Kan r ).
  • Transformant ⁇ are identified by their ability to grow on LB plates and ampicillin/kanamycin re ⁇ istant colonies were selected. Plasmid DNA wa ⁇ isolated and confirmed by restriction analysi ⁇ . Clone ⁇ containing the desired construct ⁇ were grown overnight (0/N) in liquid culture in LB media ⁇ upplemented with both Amp (100 ug/ l) and Kan (25 ug/ml) . The O/N culture i ⁇ u ⁇ ed to inoculate a large culture at a ratio of 1:100 to 1:250.
  • the cells were grown to an optical density 600 (O.D. 600 ) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D-thiogalacto pyranoside
  • IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression.
  • Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HC1.
  • ⁇ olubilized MIF-3 wa ⁇ purified from this solution by chromatography on a Nickel-Chelate column under condition ⁇ that allow for tight binding by protein ⁇ containing the 6-Hi ⁇ tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIF-3 (95 % pure) was eluted from the column in 6 molar guanidine HC1 pH 5.0.
  • Protein renaturation out of GnHCl can be accomplished by several protocols. (Jaenicke, R. and Rudolph, R. , Protein Structure - A Practical Approach, IRL Press, New York (1990)). Initially, step dialysis is utilized to remove the GnHCL. Alternatively, the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decreasing linear GnHCL gradient is run. The protein is allowed to renature while bound to the column and i ⁇ ⁇ ub ⁇ equently eluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tri ⁇ -HCl pH 7.5 and 10% Glycerol. Finally, ⁇ oluble protein is dialyzed against a storage buffer containing 5 mM Ammonium Bicarbonate. The purified protein was analyzed by SDS-PAGE. See Figure 3.
  • plasmid, MIF-3 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resi ⁇ tance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire MIF-3 precur ⁇ or and a HA tag fu ⁇ ed in frame to it ⁇ 3' end i ⁇ cloned into the polylinker region of the vector, therefore, the recombinant protein expre ⁇ ion i ⁇ directed under the CMV promoter.
  • the HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously de ⁇ cribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767).
  • the infusion of HA tag to the target protein allows ea ⁇ y detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is described as follows:
  • the DNA sequence encoding for MIF-3, ATCC # 75712, is constructed by PCR on the original EST cloned u ⁇ ing two primers: the 5' primer CCCAAGCTTATGCCGTTCCTGGAACTG contains a Hind III site followed by 18 nucleotides of MIF-3 coding sequence starting from the initiation codon; the 3' sequence CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATAAAAAAGTCATGACCGTC contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 19 nucleotides of the MIF-3 coding sequence (not including the stop codon).
  • the PCR product contains a Hind III site, MIF-3 coding ⁇ equence followed by HA tag fu ⁇ ed in frame, a translation termination stop codon next to the HA tag, and an Xba I site.
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hind III and Xba I restriction enzymes and ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning System ⁇ , 11099 North Torrey Pine ⁇ Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected.
  • Cells are labelled for 8 hours with 3S S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell ly ⁇ ate and culture media are precipitated with a HA ⁇ pecific monoclonal antibody. Protein ⁇ precipitated are analyzed on 15% SDS-PAGE gels.
  • RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • MOLECULE TYPE PROTEIN
  • Ser lie Ser Ser lie Gly Val Val Gly Thr Ala Glu A ⁇ p A ⁇ n Arg
  • Trp Gin lie Gly Lys lie Gly Thr Val Met Thr Phe Leu

Abstract

The present invention relates to a human MIF-3 and DNA (RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques and antibodies and antagonists/inhibitors against such polypeptide. Also provided are methods of using the polypeptide therapeutically for treating cancer, infections, acceleration of wound healing, stimulating the immune system, as an anti-inflammatory. Methods of using antibodies and antagonists/inhibitors for therapeutic purposes, is also disclosed, for example, for treating lethal endotoxaemia, ocular inflammation and diagnosing immune diseases.

Description

MACROPHAGE MIGRATION INHIBITORY FACTOR-3
This invention relates to newly identified polynucleotides, polypeptideε encoded by such polynucleotides, the use of such polynucleotides and polypeptideε. as well as the production of such polynucleotides and polypeptideε. More particularly, the polypeptide of the present invention is Macrophage Migration Inhibitory Factor-3 (MIF-3). The invention also relates to inhibiting the action of such polypeptides.
In response to antigenic or mitogenic stimulation- lymphocytes secrete protein mediators called lympho ines that play an important role in immunoregulation, inflammation and effector mechanisms of cellular immunity, (Miyajima, A., et al., FASEB J., 38:2462-2473 (1988)). The first reported lymphokine activity was observed in culture supernatants of antigenically sensitized and activated guinea pig lymphocytes. This activity was named migration inhibitory factor (MIF) for its ability to prevent the migration of guinea pig macrophages out of capillary tubes in vitro, (Bloom, B.R., et al.. Science, 153:80-82 (1966)).
The detection of MIF activity is correlated with a variety of inflammatory responses including delayed hypersensitivity and cellular immunity (Rocklin, R.E. et al.. New Engl. J. Med., 282:1340-1343 (1970); allograft rejection (Al-Askari, S. et al., Nature, 205:916-917 (1965); and rheumatoid polyarthritic synovialis (Odink et al., Nature, 330:80-82 (1987).
MIF is a lymphokine known to be produced by activated T cells. MIF is a major secreted protein released by the anterior pituitary cells. A large number of publications have reported the isolation and identification of putative MIF molecules. For example, MIF-1 was purified to homogeneity from the serum-free culture supernatant of a human T cell hybridoma clone called F5. Oki, S., Lymphokine Cytokine Res., 10:273-80 (1991). Also, an MIF-2, which is more hydrophobic than MIF-1, was purified to homogeneity from the same clone, (Hirose, S., et al., M, Microbiol. Immunol., 35:235-45 (1991)). The polypeptide of the present invention, MIF-3, is structurally related to the MIF family.
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is MIF-3, as well as fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided polynucleotides (DNA or RNA) which encode such polypeptideε.
In accordance with yet a further aspect of the present invention, there - is provided a process for producing such polypeptides by recombinant techniques.
In accordance with another aspect of the present invention there are provided antibodies against such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptideε for therapeutic purposes, for example, for treating cancer, infections, accelerating wound healing and stimulating the immune syεtem. In accordance with yet another aspect of the present invention, there are provided antagonist/inhibitorε to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of septic shock, lethal endotoxaemia and ocular inflammation.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
The following drawings are illustrative of embodiments of the invention and are not meant to limit the εcope of the invention as encompassed by the claims.
Figure 1 depictε the polynucleotide sequence and corresponding deduced amino acid sequence of MIF-3. The polypeptide encoded by the amino sequence shown is the preprotien form of the polypeptide, and the standard one letter abbreviation for amino acids is used.
In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodeε for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited aε ATCC Deposit No. 75712 on March 18, 1994.
The polynucleotide of this invention waε diεcovered from a cDNA library derived from human T cellε. It is structurally related to the human MIF family. It contains an open reading frame encoding a protein of approximately 118 amino acid residues. The protein exhibits the highest degree of homology to human MIF with 34 % identity and 78 % similarity over the entire amino acid sequence.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-senεe) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same, mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein εequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding εequence 5' and/or 3 ' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove deεcribed polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotideε encoding the εame mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variantε of such polynucleotideε which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or inεertion variantε.
Aε hereinabove indicated, the polynucleotide may have a coding εequence which iε a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a subεtitution, deletion or addition of one or more nucleotideε, which doeε not εubεtantially alter the function of the encoded polypeptide.
The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence
Figure imgf000007_0001
which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- hiβtidine tag supplied by a pQE-9 vector to provide for p ..fication of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, iε uεed. The HA tag correspondε to an epitope derived from the influenza hemagglutinin protein (Wilεon, I., et al.. Cell, 37:767 (1984)).
The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there iε at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . Aε herein uεed, the term "εtringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotideε which hybridize to the hereinabove described polynucleotideε in a preferred embodiment encode polypeptideε which retain substantially the same biological function or activity aε the mature polypeptide encoded by the cDNA of Figure 1 or the deposited cDNA.
The deposit(s) referred to herein will be maintained under the terms of the Budapeεt Treaty on the International Recognition of the Deposit of Micro-organis ε for purposes of Patent Procedure. These deposits are provided merely aε convenience to those of εkill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
The present invention further relates to a MIF-3 polypeptide which has the deduced amino acid εequence of Figure 1 or which haε the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the εame biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a εynthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the depoεited cDNA may be (i) one in which one or more of the amino acid reεidueε are substituted with a conserved or non-conserved amino acid reεidue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residueε includeε a εubεtituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acidε are fused to the mature polypeptide, such aε a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the εcope of those skilled in the art from the teachingε herein.
The polypeptideε and polynucleotideε of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material iε removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptideε could be part of a compoεition, and εtill be iεolated in that such vector or composition is not part of its natural environment.
The present invention also relates to vectors which include polynucleotideε of the preεent invention, hoεt cellε which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
Hoεt cells are genetically engineered (transduced or transformed or transfected) with the vectorε of this invention which may be, for example, a cloning vector or an expreεεion vector. The vector may be, for example, in the
-7-
SUBSTITUTE SHEET (RULE 261 form of a plasmid, a viral particle, a phage, etc. The engineered hoεt cellε can be cultured in conventional nutrient media modified aε appropriate for activating promoterε, εelecting transformants or amplifying the MIF genes. The culture conditions, such as temperature, pH and the like, are those previously used with the hoεt cell εelected for expreεεion, and will be apparent to the ordinarily εkilled artisan.
The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expresεing a polypeptide. Such vectors include chromosomal, nonchromoεomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmidε; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenoviruε, fowl pox viruε, and pseudorabieε. However, any other vector may be used as long aε it iε replicable and viable in the hoεt.
The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate reεtriction endonuclease site(ε) by procedures known in the art. Such procedureε and otherε are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(ε) (promoter) to direct mRNA syntheεiε. Aε repreεentative exampleε of such promoterε, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also containε a riboεome binding εite for tranεlation initiation and a tranεcription terminator. The vector may also i: .lude appropriate sequences for amplifying expreεεion.
In addition, the exp spion vectorε preferably contain one or more selectable mar; er genes to provide a phenotypic trait for selection of transformed hoεt cells such as dihydrofolate reductase or neomycin resiεtance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
The vector containing the appropriate DNA sequence as hereinabove described, as well aε an appropriate promoter or control εequence, may be employed to tranεform an appropriate hoεt to permit the hoεt to expreεε the protein.
Aε repreεentative exampleε of appropriate hoεts, there may be mentioned: bacterial cellε, such as E. coli. Strepto vces. Salmonella typhimuriu : fungal cells, such aε yeast; insect cells such as Drosophila and Sf9 animal cellε εuch aε CHO, COS or Boweε melanoma; plant cellε, etc. The selection of an appropriate host iε deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs comprising one or more of the sequenceε aε broadly deεcribed above. The constructs comprise a vector, such as a plasmid or viral vector, into which a εequence of the invention haε been inεerted, in a forward or reverεe orientation. In a preferred aεpect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectorε and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pbε, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, NH4:A (Stratagene); ptrc99a, pKK223- 3, pKK233-3, pDR540, p..IT5 -.Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
Promoter regionε can be εelected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter iε well within the level of ordinary εkill in the art.
In a further embodiment, the preεent invention relateε to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such aε a yeaεt cell, or the hoεt cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L., Dibner, M. , Battey, I., Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cellε under the control of appropriate promoterε. Cell-free tranεlation εyεtemε can also be employed to produce εuch proteinε using RNAs derived from the DNA conεtructε of the preεent invention. Appropriate cloning and expreεεion vectorε for uεe with prokaryotic and eukaryotic hoεtε are deεcribed by Sambrook, et al.. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are ciε-acting elementε of DNA, ually about from 10 to 300 bp that act on a promoter to increase itε tranεcription. Exampleε including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyo a enhancer on the late side of the replication origin, and adenovirus enhancerε.
Generally, recombinant expreεεion vectorε will include originε of replication and εelectable markerε permitting transformation of the hoεt cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expreεεed gene to direct transcription of a downεtream εtructural εequence. Such promoterε can be derived from operonε encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phoεphatase, or heat shock pr ins, among otherε. The heterologouε εtructural εequence iε assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterxal use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination εignalε in operable reading phase with a functional promoter. The vector will compriεe one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the hoεt. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella typhimuriu and variouε species within the genera Pseudo onaε, Streptomyceε, and Staphylococcuε, although others may also be employed aε a matter of choice.
Aε a repreεentative but nonlimiting example, uεeful expreεεion vectorε for bacterial uεe can compriεe a εelectable marker and bacterial origin of replication derived from commercially available plaεmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, PKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, I, USA). These pBR322 "backbone" sectionε are combined with an appropriate promoter and the εtructural εequence to be expreεεed.
Following tranεformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter iε induced by appropriate meanε (e.g., temperature εhift or chemical induction) and cellε are cultured for an additional period.
Cellε are typically harveεted by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cells employed in expresεion of proteinε can be diεrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lyεing agentε, εuch methodε are well know to thoεe skilled in the art.
Various mammalian cell culture syεtemε can alεo be employed to expreεε recombinant protein. Exampleε of mammalian expreεεion εystems include the COS-7 lines of monkey kidney fibroblasts, deεcribed by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expreεsing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expresεion vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding εiteε, polyadenylation εite, εplice donor and acceptor εiteε, transcriptional termination sequenceε, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The MIF-3 polypeptide can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phoεphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography hydroxylapatite chromatography and lectin chromatography. It is preferred to have low concentrations (approximately 0.15-5 mM) of calcium ion preεent during purification. (Price et al., J. Biol. Chem., 244:917 (1969)). Protein refolding εtepε can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification stepε.
The polypeptideε of the preεent invention may be a naturally purified product, or a product of chemical εynthetic procedureε, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, inεect and mammalian cellε in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
The MIF-3 proteins of the preεent invetion have displayed biological activitieε which indicate itε role as a general activator or several different acrophage functions. Further, MIF-3 both inhbitiε the migration of human macrophageε and εtimulateε the activity of macrophages.
The MIF-3 polypeptides of the present invention may be employed as an anti-tumor agent. Activated macrophages alone or in combination with specific anti-tumor monoclonal antibodies have considerable tumoricidal capacity. Similarly, the ability of MIF-3 to promote macrophage- mediated killing of certain pathogens indicates the use of this molecule in treating various infections, including tuberculosis, Hunsen diεeaεe and Candida.
In addition, the ability of MIF-3 to prevent the migration of macrophages may be exploited in a therapeutic agent for treating wounds. Local application of MIF-3 at the site of injury may result in increased numbers of activated macrophages concentrated within the wound, thereby increasing the rate of healing of the wound.
In addition, MIF-3 may be used as a general immune stimulus to increase the immunity generated against specific vaccines. MIF proteins have the ability to enhance macrophages to present antigens to T cells. Therefore, MIF-3 may be emplyed to potentiate the immune response to different antigens. This is extremely important in cases such as AIDS or AIDS related complex.
MIF-3 may also be employed to enhance the detoxification function of the liver. There is evidence that a protein having MIF activity in the rat liver links the chemical and immunological detoxification systemε. This protein actuateε both glutothione S-tranεferaεe (GSTs) and MIF activity. Primary εtructure compariεonε reveal significant similarity between GSTs and MIF.
The MIF polypeptideε may also be employed in accordance with the present invention by expression of such polypeptides in vivo, which is often referred to as "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo , with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art. For example, cellε may be engineered by procedureε known in the art by uεe of a retroviral particle containing RNA encoding a polypeptide of the present invention.
Similarly, cellε may be engineered in vivo for expreεεion of a polypeptide in vivo by, for example, procedureε known in the art. Aε known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the preεent invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo . These and other methods for administering a polypeptide of the present invention by such method εhould be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retroviruε, for example, an adenoviruε which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
The polypeptides of the present invention may be employed in combination with a εuitable pharmaceutical carrier. Such compoεitionε compriεe a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includeε but iε not limited to εaline, buffered εaline, dextroεe, water, glycerol, ethanol, and combinationε thereof. The formulation should εuit the mode of administration.
The invention also provideε a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Aεεociated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or εale of pharmaceuticalε or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compoundε.
The pharmaceutical compoεitionε may be adminiεtered in a convenient manner εuch aε by the topical, intravenouε, intraperitoneal, intramuεcular, subcutaneous, intranasal or intradermal routes. The amounts and dosage regimens of MIF and administered to a subject will depend on a number of factors εuch as the mode of administration, the nature of the condition being treated and the judgment of the prescribing physician. Generally speaking, they are given, for example, in therapeutically effective doseε of at least about 10 μg/kg body weight and in most cases they will be administered in an amount not in excesε of about 8 mg/Kg body weight per day and preferably the doεage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptomε, etc.
The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular siteε on the chromoεome. Few chromosome marking reagents based on actual sequence data (repeat polymorphismε) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes asεociated with diεeaεe.
Briefly, εequenceε can be mapped to chromoεomeε by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is uεed to rapidly select primerε that do not εpan more than one exon in the genomic DNA, thuε complicating the amplification proceεε. These primers are then used for PCR εcreening of εomatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategieε that can εimilarly be uεed to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomeε and preselection by hybridization to construct chromoεome εpecific-cDNA libraries.
Fluorescence in situ hybridization (FISH) of a cDNA clones to a metaphaεe chromoεomal εpread can be uεed to provide a precise chromosomal location in one step. This technique can be uεed with cDNA as short as 500 or 600 bases; however, clones larger t.*"an 2,000 bp have a higher likelihood of binding to a unique cnromoεomal location with εufficient εignal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp is good, 4,000 iε better, and more than 4,000 iε probably not necesεary to get good reεultε a reaεonable percentage of the time. For a review of thiε technique, see Verma et al.. Human Chromoεomeε: a Manual of Basic Techniques, Pergamon Press, New York (1988).
Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johnε Hopkinε University Welch Medical Library) . The relationship between geneε and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
Next, it is necesεary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in εome or all of the affected individualε but not in any normal individualε, then the mutation iε likely to be the cauεative agent of the diεeaεe.
With current reεolution of phyεical mapping and genetic mapping techniques, a cDNA preciεely localized to a chromoεomal region aεεociated with the diεeaεe could be one of between 50 and 500 potential causative genes. (This asεumes 1 megabase mapping reεolution and one gene per 20 kb).
Compariεon of affected and unaffected individualε generally involveε first looking for structural alterationε in the chromoεomeε, εuch as deletions or tranεlocations that are visible from chromosome spreads or detectable using PCR based on that cDNA sequence. Ultimately, complete sequencing of genes from several individualε iε required to confirm the presence of a mutation and to diεtinguiεh mutationε from polymorphiεmε.
The polypeptideε, their fragments or other derivatives, or analogs thereof, or cells expresεing them can be uεed as an immunogen to produce antibodieε thereto. Theεe antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includeε chimeric, εingle chain, and humanized antibodies, as well as Fab fragmentε, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated againεt the polypeptideε correεponding to a εequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptideε itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodieε can then be uεed to iεolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be uεed. Exampleε include the hybridoma technique (Kohler and Milεtein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodieε (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
Thiε invention if further related to a method for identification of MIF-3 receptorε. The gene encoding a receptor can be identified by expreεεion cloning, which comprises preparing polyadenylated RNA from a cell responsive to MIF-3 and a cDNA library created from this RNA iε divided into pools and used to tranεfect COS cells or other cells that are not responεive to MIF-3. Tranεfected cellε which are grown on glasε εlideε are expoεed to labeled MIF-3. MIF- 3 can be labeled by a variety of meanε including iodidation or incluεion of a recognition εite for a εite-εpecific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retranεfected using an iterative sub-pooling and rescreening proceεε, eventually yielding a εingle clone that encodeε the putative receptor. As an alternative approach for receptor identification, labeled MIF-3 can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Crosε-linked material is resolved by PAGE and exposed to x-ray film. The labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microεequencing. The amino acid εequence obtained from microsequencing would be used to design a set of generate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
This invention also provides a method of screening drugs to identify those which enhance (agonists) or block (antagonistε) interaction of MIF-3 to itε receptor. An agonist is a compound which increases the natural biological functions of MIF-3, while an antagoniεt eliminates εuch functionε. Aε an example, a mammalian cell or membrane preparation expreεεing an MIF-3 receptor would be incubated with labeled MIF-3 in the presence of drug. The ability of drug to enhance or block this interaction could then be measured. Alternatively, the response of a known second messenger syεtem following interaction of ligand and receptor would be meaεured compared in the preεence or absence of drug. Such second messenger syεtems include but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolyεiε.
The preεent invention iε alεo directed to antagoniεt/inhibitor moleculeε of the polypeptides of the present invention and their use to inhibit or eliminate the function of the polypeptide.
An example of an antagonist is an antibody against the MIF-3 polypeptide or, in some caεes, an oligonucleotide which bindε to the polypeptide. An antagonist may also be be a peptide derivate of MIF-3 which recognizes and binds to MIF-3 receptor sites but has no biological function thereby effectivfely blocking the receptorε. An example of an inhibitor is an antiεenεe construct prepared using antisense technology. Antiεenεe technology can be uεed to control gene expreεεion through triple-helix formation or antiεenεe DNA or RNA, both of which methodε are baεed on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide εequence, which encodeε for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in tranεcription (triple helix - εee Lee et al., Nucl. Acidε Reε., 6:3073 (1979); Cooney et al. Science, 241:456 (1988); and Dervan et al.. Science, 251: 1360 (1991)), thereby preventing transcription and the production of MIF-3. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks tranεlation of the mRNA molecule into the MIF-3 (antisenεe - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotideε aε Antiεenεe Inhibitorε of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotideε described above can also be delivered to cells εuch that the antiεenεe RNA or DNA may be expreεsed in vivo to inhibit production of MIF-3.
The antagonist/inhibitors may be employed to protect againεt lethal endotoxaemia and septic shock. Cytokines, including Macrophage Migration Inhibitory Proteins, are critical in the often fatal cascade of events that causes septic shock. An endotoxin is a lipopolysaccharide (LPS) moiety of gram-negative bacillary cell walls. This endotoxin causeε vaso-constriction of small arteries and veins, which leads to increaεed peripheral vascular reεiεtance and decreased cardiac output. These are the symptomε of lethal endotoxaemia which leadε to εeptic shock. Anterior pituitary cells specifically release MIF proteins in response to the presence of LPS. These pituitary-derived MIF proteins contribute to circulating MIF proteins which are already preεent in the poεt-acute phase of endotoxaemia.
The antagonist/inhibitors may also be used to treat ocular inflammations since partial sequencing of εmall lenε proteins has identified an MIF protein in the calf lens. Accordingly, MIF proteins may act as intercellular mesεengerε or part of the machinery of cellular differentiation, whereby over-expreεεion of MIF proteinε may lead to ocular inflammation.
The antibodieε to MIF-3 may be employed for diagnoεing disease progression and efficacy of therapeutic intervention since the level of MIF-3 in circulation may correlate with the εtate of a disease. The antagonist/inhibitors may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinabove described.
The present invention also relates to an assay for identifying potential antagonist/inhibitors specific to MIF- 3. An example of such an assay combines MIF-3 and a potential antagonist/inhibitor with membrane-bound MIF-3 receptors or recombinant MIF-3-receptors under appropriate conditions for a competitive inhibition asεay. MIF-3 can be labeled, εuch aε by radioactivity, εuch that the number of MIF-3 moleculeε bound to the receptor can determine the effectiveneεε of the potential antagoniεt/inhibitor.
The preεent invention will be further described with reference to the following examples; however, it iε to be underεtood that the preεent invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.
"Plaεmidε" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The εtarting plasmids herein are either commercially available. publicly available on an unrestricted basiε, or can be conεtructed from available plaεmids in accord with publiεhed procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposeε, typically 1 μg of plaεmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid conεtruction, typically 5 to 50 μg of DNA are digeεted with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37*C are ordinarily used, but may vary in accordance with the supplier's instruction . After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments iε performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al . , Nucleic Acidε Reε., 8:4057 (1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated. "Ligation" refers to the process of forming phosphodieεter bondε between two double stranded nucleic acid fragments (Maniatis, T., et al.. Id., p. 146). Unless otherwise provided, ligation may be accomplished uεing known buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.
Unleεε otherwiεe εtated, transformation was performed aε described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).
Example 1 Bacterial Expresεion and Purification of MIF-3 The DNA εequence encoding for MIF-3 ATCC # 75712 iε initially amplified using PCR oligonucleotide primers corresponding to the 5' terminus and sequences of the proceεsed MIF-3 protein and the vector sequences 3' to the MIF-3 gene. Additional nucleotides corresponding to MIF-3 were added to the 5 ' and 3' sequences respectively. The 5' oligonucleotide primer has the sequence CCCGCATGCCGTTCCTGGAGCTGG contains an Sph I restriction enzyme site and 19 nucleotideε of MIF-3 coding εequence starting from the initiation codon. The 3' sequence CCCAGATCTTAAAAAAGTCATGACCGT contains complementary sequenceε to a Bgl II site and iε followed by 18 nucleotideε preceeding the termination codon of MIF-3. The reεtriction enzyme sites correspond to the Sph I and Bam HI restriction enzyme sites on the bacterial expression vector pQE-70 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311). pQE-70 encodeε antibiotic resistance (Ampr), a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site (RBS), and puts the His tag to the 3' end of the gene. pQE-70 was then digested with Sph I and Bam HI. The amplified sequences were ligated into pQE-70 and were inserted in frame with the sequence encoding for the histidine tag. Figure 2 shows a schematic representation of this arrangement. The ligation mixture waε then uεed to tranεform E. coli εtrain M15/rep 4 available from Qiagen under the trademark M15/rep 4 by the procedure deεcribed in Sambrook, J. et al.. Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989). M15/rep4 contains multiple copies of the plasmid pREP4, which expresεes the lad repressor and also confers kanamycin resistance (Kanr). Transformantε are identified by their ability to grow on LB plates and ampicillin/kanamycin reεistant colonies were selected. Plasmid DNA waε isolated and confirmed by restriction analysiε. Cloneε containing the desired constructε were grown overnight (0/N) in liquid culture in LB media εupplemented with both Amp (100 ug/ l) and Kan (25 ug/ml) . The O/N culture iε uεed to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an optical density 600 (O.D.600) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") waε then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression. Cells were grown an extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell pellet was solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, εolubilized MIF-3 waε purified from this solution by chromatography on a Nickel-Chelate column under conditionε that allow for tight binding by proteinε containing the 6-Hiε tag. Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIF-3 (95 % pure) was eluted from the column in 6 molar guanidine HC1 pH 5.0.
Protein renaturation out of GnHCl can be accomplished by several protocols. (Jaenicke, R. and Rudolph, R. , Protein Structure - A Practical Approach, IRL Press, New York (1990)). Initially, step dialysis is utilized to remove the GnHCL. Alternatively, the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decreasing linear GnHCL gradient is run. The protein is allowed to renature while bound to the column and iε εubεequently eluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Triε-HCl pH 7.5 and 10% Glycerol. Finally, εoluble protein is dialyzed against a storage buffer containing 5 mM Ammonium Bicarbonate. The purified protein was analyzed by SDS-PAGE. See Figure 3.
Example 2 Expression of Recombinant MIF-3 in COS cells
The expression of plasmid, MIF-3 HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resiεtance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire MIF-3 precurεor and a HA tag fuεed in frame to itε 3' end iε cloned into the polylinker region of the vector, therefore, the recombinant protein expreεεion iε directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously deεcribed (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767). The infusion of HA tag to the target protein allows eaεy detection of the recombinant protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for MIF-3, ATCC # 75712, is constructed by PCR on the original EST cloned uεing two primers: the 5' primer CCCAAGCTTATGCCGTTCCTGGAACTG contains a Hind III site followed by 18 nucleotides of MIF-3 coding sequence starting from the initiation codon; the 3' sequence CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTATAAAAAAGTCATGACCGTC contains complementary sequences to an Xba I site, translation stop codon, HA tag and the last 19 nucleotides of the MIF-3 coding sequence (not including the stop codon). Therefore, the PCR product contains a Hind III site, MIF-3 coding εequence followed by HA tag fuεed in frame, a translation termination stop codon next to the HA tag, and an Xba I site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Hind III and Xba I restriction enzymes and ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systemε, 11099 North Torrey Pineε Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA iε iεolated from tranεformantε and examined by restriction analysis for the presence of the correct fragment. For expression of the recombinant MIF-3, COS cells are transfected with the expresεion vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritεch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expreεεion of the MIF-3 HA protein iε detected by radiolabelling and immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with 3SS-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5). (Wilson, I. et al.. Id. 37:767 (1984)). Both cell lyεate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gels.
Numerous modifications and variations of the present invention are posεible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwiεe than aε particularly described. SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: , ET AL.
(ii) TITLE OF INVENTION: Macrophage Migration Inhibitory
Factor - 3
(iii) NUMBER OF SEQUENCES: 2
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068
(V) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER:
(B) FILING DATE: (Viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: FERRARO, GREGORY D.
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-115
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 357 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: cDNA
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATGCCGTTCC TGGAGCTGGA CACGAATTTG CCCGCCAACC GAGTGCCCGC GGGGCTGGAG 60
AAACGACTCT GCGCCGCCGC TGCCTCCATC CTGGGCAAAC CTGCGGACCG CGTGAACTGT 120
ACGGTACGGC CGGGCCTGGC CATGGCGCTG AGCGGGTCCA CCGAGCCCTG CGCGCAGCTG 180
TCCATCTCCT CCATCGGCGT AGTGGGCACC GCCGAGGACA ACCGCAGCCA ACGCGCCCAC 240
TTCTTTGAGT TTCTCACCAA GGAGCTAGCC CTGGGCCAGG ACCGGATACT TATCCGCTTT 300
TTCCCCTTGG AGTCCTGGCA GATTGGCAAG ATAGGGACGG TCATGACTTT TTTATGA 357
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 118 AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Pro Pag Leu Glu Leu Asp Thr Asn Leu Pro Ala Aεn Arg Val
5 10 15
Pro Ala Gly Leu Glu Lys Arg Leu Cys Ala Ala Ala Ala Ser lie
20 25 30
Leu Gly Lyε Pro Ala Aεp Arg Val Aεn Val Thr Val Arg Pro Gly
35 40 45
Leu Ala Met Ala Leu Ser Gly Ser Thr Glu Pro Cyε Ala Gin Leu
50 55 60
Ser lie Ser Ser lie Gly Val Val Gly Thr Ala Glu Aεp Aεn Arg
65 70 75
Ser Hiε Ser Ala His Phe Phe Glu Phe Leu Thr Lys Glu Leu Ala
80 85 90
Leu Gly Gin Asp Arg lie Leu lie Arg Phe Phe Pro Leu Glu Ser
95 100 105 Trp Gin lie Gly Lys lie Gly Thr Val Met Thr Phe Leu
110 115

Claims

WHAT IS CLAIMED IS:
1. An isolated polynucleotide selected from the groups consisting of
(a) a polynucleotide encoding an MIF-3 polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of εaid polypeptide;
(b) a polynucleotide encoding an MIF-3 polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Depoεit No. 75712 or a fragment, analog or derivative of εaid polypeptide.
2. The polynucleotide of Claim 1 wherein the polynucleotide iε DNA.
3. The polynucleotide of Claim 1 wherein the polynucleotide is RNA.
4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.
5. The polynucleotide of Claim 2 wherein εaid polynucleotide encodeε MIF-3 having the deduced amino acid εequence of Figure 1.
6. The polynucleotide of Claim 2 wherein said polynucleotide encodes the MIF-3 polypeptide encoded by the cDNA of ATCC Deposit No. 75712.
7. The polynucleotide of Claim 1 having the coding sequence for MIF-3 as εhown in Figure 1.
8. The polynucleotide of Claim 2 having the coding sequence for MIF-3 deposited as ATCC Deposit No. 75712.
9. A vector containing the DNA of Claim 2.
10. A host cell genetically engineered with the vector of Claim 9.
11. A process for producing a polypeptide comprising: expresεing from the hoεt cell of Claim 10 the polypeptide encoded by said DNA.
12. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 9.
13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having MIF-3 activity.
14. A polypeptide selected from the group consisting of (i) a MIF-3 polypeptide having the deduced amino acid sequence of Figure 1 and fragments, analogs and derivatives thereof and (ii) a MIF-3 polypeptide encoded by the cDNA of ATCC Deposit No. 75712 and fragments, analogs and derivatives of said polypeptide.
15. The polypeptide of Claim 14 wherein the polypeptide is MIF-3 having the deduced amino acid sequence of Figure 1.
16. An antibody againεt the polypeptide of claim 14.
17. An antagoniεt/inhibitor against the polypeptide of claim 14.
18. A method for the treatment of a patient having need of MIF-3 comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 14.
19. A method for the treatment of a patient having need to inhibit MIF-3 comprising: administering to the patient a therapeutically effective amount of the antagonist/inhibitor of Claim 17.
20. A pharmaceutical composition comprising the polypeptide of Claim 14 and a pharmaceutically acceptable carrier.
21. The method of Claim 18 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding said polypeptide and expresεing εaid polypeptide in vivo .
22. A method for identifying antagonist/inhibitors to MIF-3 comprising: combining labeled MIF-3 and a potential antagonist/inhibitor receptors for MIF-3 under appropriate conditions εuch that a competitive binding aεεay occurs; determining the amount of MIF-3 bound to the receptors; and identifying the effectiveness of the potential antagonist/inhibitor.
PCT/US1994/005385 1994-05-16 1994-05-16 Macrophage migration inhibitory factor-3 WO1995031468A1 (en)

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EP94923875A EP0770086A4 (en) 1994-05-16 1994-05-16 Macrophage migration inhibitory factor-3
PCT/US1994/005385 WO1995031468A1 (en) 1994-05-16 1994-05-16 Macrophage migration inhibitory factor-3
AU73942/94A AU7394294A (en) 1994-05-16 1994-05-16 Macrophage migration inhibitory factor-3
JP7529593A JPH10500301A (en) 1994-05-16 1994-05-16 Macrophage migration inhibitory factor-3
US08/903,224 US5986060A (en) 1994-05-16 1997-07-22 Macrophage Migration Inhibitory Factor-3
US10/178,731 US20030166863A1 (en) 1994-05-16 2002-06-25 Macrophage migration inhibitory factor-3

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US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease
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US6673344B1 (en) 1994-08-23 2004-01-06 Human Genome Sciences, Inc. Antibodies to human CKβ-10/MCP-4
US6391589B1 (en) 1994-08-23 2002-05-21 Human Genome Sciences, Inc. Human chemokine beta-10 mutant polypeptides
US6174995B1 (en) 1994-08-23 2001-01-16 Haodong Li Human chemokines, CKβ4 and CKβ10/MCP-4
US7183081B2 (en) 1994-08-23 2007-02-27 Human Genome Sciences, Inc. Human Ckβ-10 polynucleotides
US7138498B2 (en) 1994-08-23 2006-11-21 Human Genome Sciences, Inc. Antibodies to MCP-4
US6921645B2 (en) 1994-08-23 2005-07-26 Human Genome Sciences, Inc. Antibodies to chemokine β-4
US6458349B1 (en) 1995-06-02 2002-10-01 Human Genome Sciences, Inc. Chemokine β-4 polypeptides
EP0807439A3 (en) * 1996-05-14 1998-03-11 Smithkline Beecham Corporation Method of treating sepsis and adult respiratory distress syndrome
US6406688B1 (en) 1996-05-14 2002-06-18 Human Genome Sciences, Inc. Method of treating sepsis and ARDS with chemokine β-4
US6290948B1 (en) * 1996-05-14 2001-09-18 Smithkline Beecham Corporation Method of treating sepsis and ARDS using chamohine beta-10
EP0807439A2 (en) * 1996-05-14 1997-11-19 Smithkline Beecham Corporation Method of treating sepsis and adult respiratory distress syndrome
US8303951B2 (en) 1996-10-25 2012-11-06 Human Genome Sciences, Inc. Neutrokine-alpha antibodies and methods of use thereof
US8071092B1 (en) 1996-10-25 2011-12-06 Human Genome Sciences, Inc. Methods of inhibiting B lymphocytes using antibodies to Neutrokine-alpha
US8231873B2 (en) 1996-10-25 2012-07-31 Human Genome Sciences, Inc. Methods of treatment using antibodies to Neutrokine-alpha
US8173122B2 (en) 1996-10-25 2012-05-08 Human Genome Sciences, Inc. Methods of treatment using antibodies to neutrokine-alpha
US8212004B2 (en) 1999-03-02 2012-07-03 Human Genome Sciences, Inc. Neutrokine-alpha fusion proteins
US7879328B2 (en) 2000-06-16 2011-02-01 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator
US8101181B2 (en) 2000-06-16 2012-01-24 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US9187548B2 (en) 2000-06-16 2015-11-17 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US7605236B2 (en) 2000-06-16 2009-10-20 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US8062906B2 (en) 2000-08-18 2011-11-22 Human Genome Sciences, Inc. B-lymphocyte stimulator binding polypeptides and methods based thereon
WO2002020758A3 (en) * 2000-09-05 2003-06-12 Curagen Corp Novel proteins and nucleic acids encoding same
US7943741B2 (en) 2002-05-01 2011-05-17 Human Genome Sciences, Inc. Antibodies that specifically bind to chemokine β-4
US7375192B2 (en) 2002-05-01 2008-05-20 Human Genome Sciences, Inc. Antibodies that specifically bind to chemokine beta-4
US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease
US8211649B2 (en) 2006-03-31 2012-07-03 Human Genome Sciences, Inc. Methods of diagnosing and prognosing hodgkin's lymphoma
US9133273B2 (en) 2010-11-19 2015-09-15 Eisai R&D Management Co., Ltd. Nucleic acids encoding neutralizing anti-CCL20 antibodies
US9809647B2 (en) 2010-11-19 2017-11-07 Eisai R&D Management Co., Ltd. Neutralizing anti-CCL20 antibodies
US9958456B2 (en) 2011-10-07 2018-05-01 Baxalta Incorporated OxMIF as a diagnostic marker
WO2013184209A1 (en) * 2012-06-04 2013-12-12 Ludwig Institute For Cancer Research Ltd. Mif for use in methods of treating subjects with a neurodegenerative disorder

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AU7394294A (en) 1995-12-05
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EP0770086A4 (en) 1998-07-15

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