WO1992003466A1 - Lymphokine 154 - Google Patents

Lymphokine 154 Download PDF

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Publication number
WO1992003466A1
WO1992003466A1 PCT/US1991/005749 US9105749W WO9203466A1 WO 1992003466 A1 WO1992003466 A1 WO 1992003466A1 US 9105749 W US9105749 W US 9105749W WO 9203466 A1 WO9203466 A1 WO 9203466A1
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WO
WIPO (PCT)
Prior art keywords
protein
present
lymphokine
sequence given
dna segment
Prior art date
Application number
PCT/US1991/005749
Other languages
English (en)
Inventor
Kathleen Kelly
Ulrich Siebenlist
Kevin S. Smith
Original Assignee
The United States Of America, Represented By The Secretary, United States Department Of Commerce
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 The United States Of America, Represented By The Secretary, United States Department Of Commerce filed Critical The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority to CA002089258A priority Critical patent/CA2089258A1/fr
Priority to JP91514525A priority patent/JPH05508778A/ja
Publication of WO1992003466A1 publication Critical patent/WO1992003466A1/fr

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • the present invention relates to secreted proteins (lymphokines) produced by activated T lymphocytes.
  • the present invention relates to lymphokine 154. Background Information
  • Quiescent non-dividing T lymphocytes respond to antigen and mitogen stimulation by the activation of cellular genes which are thought to control events that lead to cell proliferation and the expression of a differ ⁇ entiated phenotype.
  • Treatment of human peripheral blood T cells with the mitogenic combination of phytohemmagglutin (PHA) and phorbol 12-myristate 13-acetate (PMA) appears to mimic the effects of antigen plus antigen presenting cells in vivo. These two agents are known to induce the expres ⁇ sion of genes which are important for progression through the cell cycle (Reed et al., Proc. Natl. Acad. Sci. USA. 83:3982, 1986).
  • T lymphocyte activation further induces genes producing secreted factors that direct the growth and differentiation of many cell types, predominantly, but not exclusively, those of the immune system (Dumonde et al., Nature. 224:38 f 1969; Mosmann, T.R. "Helper T cells and their lymphokines.” T cells. Feld ann, Lamb and Owen ed. 1988 John Wiley and Sons, New York; Rocklin et al. Adv. Immunol. 29:55, 1980). Culture supernatants from activat ⁇ ed T cells display multiple biological activities includ- ing T cell growth (Taniguchi et al., Nature. 302:305, 1983), B cell growth and differentiation (Namen et al., Nature.
  • lymphokines and cytokines including IL-2 through IL-8 (Campbell et al., Proc. Natl. Acad. Sci. USA. 84:6629, 1987; Hirano et al., Nature. 324:73, 1986; Namen et al. , Nature. 333:571, 1988; Taniguchi et al. , Nature. 302:305, 1983; Yang et al., Cell. 47:3, 1986; Yokota et al. , Proc. Natl. Acad. Sci. USA. 83:5894, 1986; Yoshimura et al., Proc. Natl. Acad. Sci. USA.
  • IFN- ⁇ (Gray et al., Nature. 295:503, 1982), GM-CSF (Wong et al., Science. 228:810, 1985), TNF (Cuturi et al., J. Exp. Med. 165:1581, 1987), LT (Cuturi et al., J. Exp. Med. 165:1581, 1987) and others have been identified, purified and the genes encoding them have subsequently been molecu ⁇ larly cloned. The cloned genes have proven to be immense- ly useful for the generation of pure recombinant proteins which can be tested for biological functions in the absence of other secreted products.
  • lymphokines that have been de ⁇ scribed, each has its own unique spectrum of biological properties. Although many lymphokines are being developed for clinical utility, most lymphokines act on such complex biological systems as to effect a part of the desired result. Thus, new lymphokines not only present the potential for entirely new spectrums of biological activities, but also could act synergistically with previously described products to effect the overall efficacy of the treatment.
  • the present invention relates to a DNA segment encoding protein 154. In another embodiment, the present invention relates to protein 154 substantially free of proteins with which it is normally associated.
  • the present invention relates to a recombinantly produced or chemically synthe ⁇ sized protein having all, or a unique portion of the amino acid sequence given in Figure 2.
  • the present invention relates to a recombinant DNA construct and to host cells transformed therewith.
  • the DNA construct comprises a DNA segment of the present invention encoding protein 154 and a vector.
  • the present invention relates to a method of producing recombinant protein 154 which comprises culturing host cells of the present invention, in a manner allowing expression of protein 154 encoded therein and isolating the protein.
  • the present invention relates to an antibody specific for protein 154.
  • Figures 1A, IB and 1C show the nucleotide sequence of the pAT154 cDNA.
  • Figure 2 shows the putative amino acid sequence of the longest open reading frame of the pAT154 cDNA.
  • the classical hydrophobic leader region contained in the protein is underlined.
  • Figure 3 shows the hydrophobicity plot of the protein 154.
  • Figure 4 demonstrates in vitro transcription of a full-length cDNA clone followed by in vitro translation and SDS polyacrylamide gel electrophoresis (SDS PAGE) .
  • the first two lanes are low molecular weight and high molecular weight standards, respectively.
  • the arrow points to the 154 translated protein with a molecular weight of approximately 15 kd.
  • One approach to characterizing additional biologi ⁇ cal activities in supernatants of activated T cells, which activities may be mediated by unidentified proteins, is to directly identify genes that are expressed in the acti ⁇ vated T cells, the protein products of which have struc ⁇ tural features suggesting they are secretory peptides.
  • the present inventors have isolated more than 60 genes that are induced in human peripheral blood T cells by mitogens (Zipfel et al. , Mol. Cell. Biol. 9:1034-1040, 1989) . Of these clones, the inventors have fully characterized the 154 gene encoding a lymphokine.
  • the present invention relates to a DNA segment encoding all, or a unique portion, of protein 154 (lymphokine 154) and to the protein (or polypeptides) encoded therein, or allelic variations thereof.
  • the present invention relates, in particular, to the human protein 154 and to the equivalent protein in other spe- cies.
  • the mouse protein 154 which has been cloned and partially sequenced by the present inventors.
  • a "unique portion" as used herein is defined as consisting of at least five (or six) amino acids, or correspondingly, at least 15 (or 18) nucleotides) .
  • the present invention relates to a DNA segment that encodes the entire amino acid sequence given in Figure 2 (such as, for example, the DNA segment pAT154 given in Figures 1A, IB and 1C) , or any unique portion thereof.
  • DNA segments to which the invention relates also include those encoding substantially the same protein as that of Figure 2, which includes, for example, allelic forms thereof or the mouse lymphokine 154.
  • DNA segments of the present invention can be used as probes for detect ⁇ ing the presence of DNA or RNA encoding lymphokine 154 in a sample.
  • the present invention also relates to nucleotide sequences complementary to the segments referenced above.
  • the present invention further relates to protein 154 substantially free of proteins with which it is normally associated.
  • the lymphokine 154 can be purified by one skilled in the art using protocols known in the art for protein purification.
  • the present invention also relates to the lymphokine 154 and to peptide fragments thereof which are chemically synthesized or recombinantly produced.
  • the protein and polypeptides the present invention can be used as antigens, in protocols known in the art, to produce antibodies thereto, both monoclonal and poly- clonal.
  • a polypsptide having an amino acid sequence corresponding to an epitope of the sequence given in Figure 2 (or an allelic variation thereof) can be used to generate antibodies specific for lymphokine 154.
  • Such antibodies can be used in bioassays to detect the presence of protein 154 in samples, such as biological samples.
  • the present invention further relates to a recom ⁇ binant DNA construct and to a host cell transformed therewith.
  • a recombinant DNA construct comprising the above-described DNA segment encoding all, or a unique portion of lymphokine 154 and a vector can be constructed without undue experimentation.
  • Suitable vectors for use in the present invention include, but are not limited to, the bacterial vector pET-3 (Studier et al.
  • the DNA segment can be isolated for activated T-cells or take the form of a cDNA clone.
  • the DNA segment can be present in the vector operably linked to regulatory elements, includ ⁇ ing, for example a promoter.
  • the host cell can be pro- karyotic (such as bacterial) , lower eukaryotic (such as fungal, including yeast) or higher eukaryotic (such as mammalian) .
  • Host cells of the present invention can be used to generate recombinantly produced lymphokine 154.
  • the host cells can be cultured under conditions such that protein 154 (or a unique polypeptide thereof) encoded in the construct is expressed.
  • the expressed protein can be isolated using standard protein isolation protocols.
  • the present invention also relates to a membrane bound form of the lymphokine 154 and host cells expressing such on their surfaces.
  • the membrane form of protein 154 can be generated with the addition of a transmembrane anchor encoding sequence on the DNA segment encoding the protein.
  • host cells transformed with a recombinant DNA construct encoding such a DNA segment will express protein 154 on their surface.
  • Lymphokines provide a wide array of activities relating to the immune system. Lymphokine activities range from recruiting cells, such as macrophages, to specific sites to mount an effective immune response, to fighting viral infections. Lymphokine 154 of the present invention may provide such activities and therefore could be used in various treatment regimens.
  • the protein or polypeptide of the present invention may exert cytotoxic effects on certain cells such as tumor cells and could therefore be used in the treatment of cancer.
  • the protein or polypep ⁇ tide of the present invention may have anti-viral and/or anti-parasitic activity (such as the activity of the lymphokine -interferon) , in which case the present invention could be used in the treatment of viral or parasitic infections.
  • Lymphokine 154 may also effect the growth and/or differentiation of cells, particularly, hematopoietic cells (an activity displayed by several of the interleukin lymphokines) . Such activity would be useful in the treatment of diseases which adversely affect and/or deplete cells of the immune system.
  • lymphokine 154 may have the ability to augment the immune system which would allow the protein or polypeptides of the present invention to be used, for example, to detect rejection of a transplanted organ by monitoring the presence of the protein or polypeptide in the blood or urine of the transplant patient. An increase in the amount of protein or polypeptide in the biological fluid of the patient could be an early indication that the immune system is rejecting the foreign organ.
  • CDNA clones of pAT154 were originally isolated from a previously described subtractive hybridization library (Zipfel et al., Mol. Cell. Biol. 9:1041-1048, 1989) . Additional clones were isolated from a ⁇ Zap library (Stratagene) (Short et al., Nuc. Acids. Res. 16:7583-7600, 1988), or from an oligo dT primed ⁇ gtlO library made following the Guebler-Hoffman method (Gubler et al., Gene. 25:263, 1983).
  • human peripheral blood T cells were isolated f om whole blood and stimulated for 4.5 hours in vitro using the mitogenic agents phytohemmagglutin (PHA) (1 ⁇ g/ml) and phorbol 12-myristate 13-acetate (PMA) (20 ng/ml) in the presence of cycloheximide (CX) (10 ⁇ g/ml) .
  • cDNA synthesized from the mRNA was cloned into ⁇ gtlO or ⁇ Zap and screened using a 453 bp probe of pAT154 original- ly isolated from the subtractive hybridization library. A total of seven clones covering the length of the mRNA were isolated, mapped and sequenced.
  • the nucleic acid sequence was determined on both st ands of pAT154 cDNA's by the Sanger dideoxy chain termination method (Sanger et al., Pro. Natl. Acad. Sci.
  • GenBank (V.63 + updates through 5/90) was utilized to search the nucleic acid and protein databanks for statistically relevant homologies in the published literature to the pAT154 cDNA and amino acid sequences. No sequences having significant homology with pAT154 were found.
  • IN VITRO TRANSCRIPTION OF 154 OPEN READING FRAME A total of 10 ⁇ g pAT154 cDNA containing the 154 open reading frame was linearized by a Cla I digestion of the pBlueScript's Multicloning site. The final digest was adjusted to 0.5 M NaCl and Proteinase K (1 mg/ml) treated for 30 min. at 37°C.
  • the cDNA was treated twice with 1:1 phenol/chloroform and ethanol precipitated and dried. The resulting pellet was resuspended at a final concentration of 0.5 ⁇ g/ ⁇ l using diethyl pyrocarbonate treated H 2 0.
  • T3RNA polymerase (Stratagene) was performed according to manu- facturer's instructions. A 1 ⁇ l aliquot of the mRNA was qualitatively tested by electrophoresis of a lxTBE 1% agarose gel for 10 min. IN VITRO TRANSLATION OF 154 PROTEIN.
  • the 154 mRNA was translated utilizing a rabbit reticulolysate system (Promega) .
  • Five microliters of 154 mRNA (representing 20% of the total mRNA transcribed) was mixed with rabbit reticulolysate, amino acids (minus cysteine) and cysteine, L-[ 35 S] (NEN) according to the Promega protocol. This mixture was incubated for 60 min. at 30°C. Free label was removed from the final translate by dialysis (EmproTech Prodialyzer) against 0.1 M NH 4 HC0 3 overnight at 4°C.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

On a identifié une nouvelle protéine sécrétée (la lymphokine) qui est appelée lymphokine 154. Cette invention concerne des segments d'ADN codant la protéine 154, ainsi que la protéine elle-même. En outre, l'invention concerne une structure d'ADN recombinant codant la protéine et des cellules transformées par ladite structure.
PCT/US1991/005749 1990-08-13 1991-08-13 Lymphokine 154 WO1992003466A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002089258A CA2089258A1 (fr) 1990-08-13 1991-08-13 Lymphokine 154
JP91514525A JPH05508778A (ja) 1990-08-13 1991-08-13 リンホカイン１５４

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US56610890A 1990-08-13 1990-08-13
US566,108 1990-08-13

Publications (1)

Publication Number Publication Date
WO1992003466A1 true WO1992003466A1 (fr) 1992-03-05

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Application Number Title Priority Date Filing Date
PCT/US1991/005749 WO1992003466A1 (fr) 1990-08-13 1991-08-13 Lymphokine 154

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EP (1) EP0544743A4 (fr)
JP (1) JPH05508778A (fr)
AU (1) AU8446891A (fr)
CA (1) CA2089258A1 (fr)
WO (1) WO1992003466A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025959A2 (fr) * 1996-12-11 1998-06-18 Chiron Corporation Proteines humaines secretees

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL92685A0 (en) * 1988-12-16 1990-09-17 Us Commerce Dna encoding lymphokine/cytokine like proteins,cells transformed thereby and production of said proteins and antibodies against them

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Molecular and Cellular Biology, Vol. 9, No. 3, issued March 1989, IRVING et al., "Mitogen-Induced Genes are subject To Multiple Pathways of Regulation In The Initial Stages Of T-Cell Activation", pages 1034-1040, see entire document. *
Molecular and Cellular Biology, Vol. 9, No. 3, issued March 1989, ZIPFEL et al., "Complexity of The Primary Genetic Response To Mitogenic Activation Of Human T Cells", pages 1041-1048, see entire document. *
Proceedings National Academy Of Sciences, Vol. 85, issued December 1988, LIPES et al., "Identification, cloning; and characterization of An Immune Activation Gene", pages 9704-9708, see entire document. *
See also references of EP0544743A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998025959A2 (fr) * 1996-12-11 1998-06-18 Chiron Corporation Proteines humaines secretees
WO1998025959A3 (fr) * 1996-12-11 1998-10-08 Chiron Corp Proteines humaines secretees

Also Published As

Publication number Publication date
EP0544743A4 (en) 1994-05-18
EP0544743A1 (fr) 1993-06-09
CA2089258A1 (fr) 1992-02-14
JPH05508778A (ja) 1993-12-09
AU8446891A (en) 1992-03-17

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