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WO1998011227A1 - Variante de la proteine-2 chimiotactique de granulocytes - Google Patents

Variante de la proteine-2 chimiotactique de granulocytes Download PDF

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Publication number
WO1998011227A1
WO1998011227A1 PCT/US1997/016034 US9716034W WO9811227A1 WO 1998011227 A1 WO1998011227 A1 WO 1998011227A1 US 9716034 W US9716034 W US 9716034W WO 9811227 A1 WO9811227 A1 WO 9811227A1
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WIPO (PCT)
Prior art keywords
ngcp
sequence
cys
gly
seq
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PCT/US1997/016034
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English (en)
Inventor
Olga Bandman
Roger Coleman
Lynn E. Murry
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Incyte Pharmaceuticals, Inc.
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Publication date
Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to CA002265455A priority Critical patent/CA2265455A1/fr
Priority to AU42661/97A priority patent/AU4266197A/en
Priority to EP97941010A priority patent/EP0958365A1/fr
Priority to JP10513829A priority patent/JP2001500019A/ja
Publication of WO1998011227A1 publication Critical patent/WO1998011227A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to nucleic acid and amino acid sequences of a novel granulocyte chemotactic protein 2 variant which shares features with alpha intercrines and to the use of these sequences in the diagnosis, study, prevention and treatment of disease.
  • Chemokines are small polypeptides, generally about 70-100 amino acids in length, 6-1 1 kD in molecular weight, and active over a 1-100 ng/ l concentration range. Initially, they were isolated and purified from inflamed tissues and characterized relative to their bioactivity. More recently, chemokines have been discovered and produced through molecular cloning techniques and characterized by structural as well as functional analysis.
  • Chemokines participate in gradients of chemoattractant factors which are involved in leukocyte trafficking in different inflammatory situations. They mediate the expression of particular adhesion molecules on endothelial cells, stimulate proliferation of specific cell types. and regulate the activation of cells which bear specific receptors.
  • CXC chemokines are related through cysteine motifs and generally fall into three families, CXC chemokines ( ⁇ ), CC chemokines ( ⁇ ) and C chemokines (g; Graves DT and Y Jiang (1995) Crit Rev Oral Biol Med 6:109-118 and Kelner G et al (1994) Science 266: 1395-99).
  • CXC chemokines include the well described platelet factor-4 as well as the nonplatelet chemokines epithelial-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2).
  • Chemokines are generally expressed and secreted in response to proinflammatory cytokines, such as interleukin-lB and tumor necrosis factor, endotoxin, mitogens, particulates. bacteria or viruses.
  • ENA-78 consists of 76 amino acids and has a molecular weight of 8,357 (Walz A et al (1991) J Exp Med 174:1355-62). When endothelial cells express and secrete ENA-78, it stimulates neutrophils, chemotaxis, increased intraceliular calcium, and exocytosis.
  • the genomic DNA for ENA-78 was cloned by Corbett et al (1994; Biochem Biophys Res Commun 205:612- 17) from human chromosome 4 as a 2.2 kb fragment containing four exons and three introns.
  • the open reading frame of 342 nucleotides encodes a protein of 1 14 amino acids.
  • the upstream flanking region is contains a promoter binding site for nuclear factor kappa B (Chang M et al (1994) J Biol Chem 269:41 :25277-82).
  • GCP-2 is a 6 kD protein isolated from the supernatants of human MG63 osteosarcoma cells. Proost et al (1993) J Immunol 150:1000-10) identified this cxc chemokine by amino acid sequencing and report that using HPLC separation, GCP-2 appears to exists in four different N- terminal forms. At concentrations of 3-10 nM, GCP-2 attracts and activates neutrophils in vitro, causes granulocyte accumulation in vivo, and has no effect on monocytes. Chemokines and their chemoattractant activities are being studied to provide a better understanding of and the means for intervening at the molecular level in various pathogenic processes.
  • Hirose et al (1995; Br J Cancer 72:708-714) showed that the transfection of chemokine genes into tumor cells of nude mice reduced tumorigenicity. This anti-tumor activity was attributed to the recruitment and activity of neutrophilic granulocytes to the site of the cancer.
  • chemokine expression and secretion may be useful in preventing pathogenesis in myocardial tissues.
  • Seino Y et al (1995; Cytokine 7:301-304) used PCR to show the expression of leukocyte CC and CXC chemotactic cytokines in endomyocardial biopsies from patients with idiopathic dilated cardiomyopathy. This suggests that cytotoxic action of leukocytes recruited to the site might be contributing to inflammatory heart muscle disorders.
  • the present invention discloses a novel granulocyte chemotactic protein 2 variant, hereinafter referred to as NGCP, which shares features with other alpha intercrines involved in the chemoattraction and activation of leukocytes, particularly neutrophils and granulocytes. Accordingly, the invention features a substantially purified NGCP, as shown in the amino acid sequence of SEQ ID NO: 1.
  • polynucleotide which encode NGCP.
  • polynucleotide is the nucleotide sequence of
  • the invention features polynucleotide sequences that hybridize under stringent conditions to SEQ ID NO:2.
  • the invention further relates to the nucleic acid sequence encoding NGCP, oligonucleotides, peptide nucleic acids, fragments, portions or antisense molecules thereof.
  • the present invention relates, in part, to the inclusion of the nucleic acid sequence encoding NGCP in an expression vector which can be used to transform host cells or organisms.
  • the invention also provides therapeutic transformation of cells or tissues involved in the cancers, particularly melanomas and cytokine sensitive tumors, immune deficiencies, or excessive immune responses.
  • the present invention also relates to a method for producing NGCP or a fragment thereof. It contemplates the delivery of purified NGCP, alone or in a pharmaceutically acceptable excipient, to cancerous cells or tissues. It also encompasses antibodies which bind specifically to NGCP and can be used to monitor testing of cytokine sensitive tumors and immune system disorders.
  • Figures 1A and IB shows the amino acid sequence (SEQ ID NO:l ) and nucleic acid sequence (SEQ ID NO:2) of the novel granulocyte chemotactic protein 2 variant.
  • the alignment was produced using MacDNAsisTM software (Hitachi Software Engineering Co Ltd, San Bruno CA).
  • Figure 2 shows the amino acid sequence alignments among granulocyte chemotactic protein 2 variant (SEQ ID NO: 1 ),ENA-78 (GI 607031 ; SEQ ID NO:3 ; Walz et al, supra), GCP-2 (GI 462170; SEQ ID NO:4; Proost et al, supra) and GCP-2 (GI 415589; SEQ ID NO:5; Proost et al (1993) Biochemistry 32:10170-77). These alignments were produced using the multisequence alignment program of DNAStarTM software (DNAStar Inc, Madison WI).
  • Figure 3 shows the hydrophobicity plot for granulocyte chemotactic protein 2 variant, SEQ ID NO: 1 (MacDNAsis software); the X axis reflects amino acid position, and the negative Y axis, hydrophobicity.
  • Figure 4 shows the hydrophobicity plot for ENA-78, SEQ ID NO:3.
  • Figure 5 shows the isoelectric plot for granulocyte chemotactic protein 2 variant, SEQ ID NO:l (MacDNAsis software).
  • Figure 6 shows the isoelectric plot for ENA-78, SEQ ID NO:4.
  • Figures 7A and 7B shows the alignment between the nucleic acid sequences of granulocyte chemotactic protein 2 variant and a human EST annotated as 5" similar to granulocyte chemotactic protein-2 (GI 973875; Hillier L et al (1995) Unpublished; DNAStarTM software).
  • Nucleic acid sequence refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
  • amino acid sequence refers to peptide or protein sequence.
  • Consensus as used herein may refer to a nucleic acid sequence 1 ) which has been resequenced to resolve uncalled bases, 2) which has been extended using XL-PCR (Perkin Elmer) in the 5' or the 3' direction and resequenced, 3) which has been assembled from the overlapping sequences of more than one Incyte clone GCG Fragment Assembly System, (GCG, Madison WI), or 4) which has been both extended and assembled.
  • XL-PCR Perkin Elmer
  • Protein nucleic acid refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary (template) strand of nucleic acid (Nielsen PE et al (1993) Anticancer Drug Des 8:53-63).
  • a “deletion” is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • An "insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring NGCP.
  • substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
  • NGCP refers to the amino acid sequence of substantially purified NGCP obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic or recombinant.
  • a '"variant" of NGCP is defined as an amino acid sequence differs by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, eg, replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, eg, replacement of a glycine with a tryptophan.
  • Similar minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNAStar software.
  • biologically active refers to NGCP having structural, regulatory or biochemical functions of a naturally occurring NGCP.
  • immunologically active defines the capability of the natural, recombinant or synthetic NGCP, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • nucleic acid derivative refers to the chemical modification of a nucleic acid encoding NGCP or the encoded NGCP. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.
  • a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of natural NGCP.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
  • hybridization shall include "any process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994)
  • Amplification is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach CW and GS Dveksler (1995) PCR Primer, a . Laboratory Manual. Cold Spring Harbor Press, Plainview NY).
  • "Stringency” typically occurs in a range from about Tm-5°C (5°C below the Tm of the probe)to about 20°C to 25°C below Tm.
  • a stringency hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences.
  • the consensus nucleotide sequence encodes a novel granulocyte chemotactic protein-2 variant of 114 amino acid residues (SEQ ID NO: 1 ).
  • the consensus sequence is based on the extension and assembly of Incyte clones 949299 (PANCNOT05) and 1321776 (BLADNOT04).
  • FIG. 1A and IB The nucleotide and amino acid alignments of NGCP are shown in Figures 1A and IB.
  • Figure 2 presents the alignment and shows the conserved cysteine residues, C 19 , C 49 , C 5I , C 75 , and C 9 aware among NGCP and the related chemokines, ENA-78 (GI 607031 ; SEQ ID NO:3); and GCP-2 (GI 462170; SEQ ID NO:4 and GI 415589; SEQ ID NO:5).
  • the hydrophobicity plots of NGCP ( Figure 3) and ENA-78 ( Figure 4) are similar; however as shown in Figure 2, there are significant differences between the actual residues that comprise the leader and mature protein sequences of these molecules.
  • FIG. 7A and 7B shows the alignment and 70% identity between the nucleic acid sequences of NGCP and the human EST (GI 973875; SEQ ID NO:6) annotated as 5" similar to granulocyte chemotactic protein-2.
  • a three frame translation of the first 180 nucleotides of this sequence reveals several frame shifts and predicts the exact leader sequence for the published ENA-78 peptide.
  • NGCP displays the general has no obvious N-glycosylation sites.
  • the NGCP Coding Sequences The nucleic acid and deduced amino acid sequences of NGCP are shown in Figures 1 A and IB.
  • any nucleic acid sequence which encodes NGCP can be used to generate recombinant molecules which express NGCP.
  • a partial sequence encoding NGCP was first isolated as Incyte Clone 949299 from a pancreas cDNA library (PANCNOT05). It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of NGCP-encoding nucleotide sequences, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence encoding naturally occurring NGCP, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode NGCP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring sequence under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding NGCP or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of Figure 1 under various conditions of stringency.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques. Methods in Enzymology. Vol 152, Academic Press, San Diego CA) incorporated herein by reference, and may be used at a defined "stringency”.
  • Altered nucleic acid sequences encoding NGCP which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent NGCP.
  • the protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent NGCP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of NGCP is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine phenylalanine, and tyrosine.
  • alleles encoding NGCP include alleles encoding NGCP.
  • an "allele” or “allelic sequence” is an alternative form of the nucleic acid sequence encoding NGCP. Alleles result from a mutation, ie. a change in the nucleic acid sequence, and generally produce altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • Methods for DNA sequencing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland OH)), Taq polymerase (Perkin Elmer, Norwalk CT), thermostable T7 polymerase (Amersham, Chicago IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg MD).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the ABI 377 DNA sequencers (Perkin Elmer). Extending the Polynucleotide Sequence
  • the polynucleotide sequence encoding NGCP may be extended utilizing partial nucleotide sequence and various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • Gobinda et al (1993; PCR Methods Applic 2:318-22) disclose "restriction-site" polymerase chain reaction (PCR) as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia T et al (1988) Nucleic Acids Res 16:8186).
  • the primers may be designed using OLIGO® 4.06 Primer Analysis Software (1992; National Biosciences Inc, Plymouth MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Capture PCR (Lagerstrom M et al (1991 ) PCR Methods Applic 1 : 11 1-19) is a method for
  • Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before PCR.
  • PCR Parker JD et al (1991) Nucleic Acids Res 19:3055-60
  • PCR nested primers
  • PromoterFinderTM Click-FinderTM
  • PromoterFinder libraries can be used to walk in genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs.
  • random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
  • a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • Capillary electrophoresis may be used to analyze either the size or confirm the nucleotide sequence in sequencing or PCR products.
  • Systems for rapid sequencing are available from Perkin Elmer, Beckman Instruments (Fullerton CA), and other companies.
  • Capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
  • Output/light intensity is converted to electrical signal using appropriate software (eg. GenotyperTM and Sequence NavigatorTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled.
  • Capillary electrophoresis is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
  • the reproducible sequencing of up to 350 bp of Ml 3 phage DNA in 30 min has been reported (Ruiz-Martinez MC et al (1993) Anal Chem 65:2851-8). Expression of the Nucleotide Sequence
  • polynucleotide sequences which encode NGCP, fragments of the polypeptide, fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of NGCP in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence, may be used to clone and express NGCP. As will be understood by those of skill in the art, it may be advantageous to produce NGCP-encoding nucleotide sequences possessing non-naturally occurring codons.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of NGCP expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • the nucleotide sequences of the present invention can be engineered in order to alter
  • NGCP-encoding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
  • a natural, modified or recombinant NGCP- encoding sequence may be ligated to a heterologous sequence to encode a fusion protein.
  • a heterologous sequence For example, for screening of peptide libraries for inhibitors of NGCP activity, it may be useful to encode a chimeric NGCP protein that is recognized by a commercially available antibody.
  • a fusion protein may also be engineered to contain a cleavage site located between NGCP and the heterologous protein sequence, so that the NGCP may be cleaved and substantially purified away from the heterologous moiety.
  • sequence encoding NGCP may be synthesized, whole or in part, using chemical methods well known in the art (see Caruthers MH et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al(1980) Nuc Acids Res Symp Ser 225-32, etc).
  • the protein itself could be produced using chemical methods to synthesize an amino acid sequence for NGCP, whole or in part.
  • peptide synthesis can be performed using various solid-phase techniques (Roberge JY et al (1995) Science 269:202-204) and automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (eg, Creighton (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co, New York NY).
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (eg, the Edman degradation procedure; Creighton, supra). Additionally the amino acid sequence of NGCP, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
  • nucleotide sequence encoding NGCP or its functional equivalent is inserted into an appropriate expression vector, ie, a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express a sequence encoding NGCP. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg, Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (eg, baculovirus); plant cell systems transfected with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vector
  • control elements or “regulatory sequences” of these systems vary in their strength and specificities and are those nontranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla CA) or pSportl (Gibco BRL) and ptrp-lac hybrids and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (eg, heat shock, RUBISCO; and storage protein genes) or from plant viruses (eg, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding NGCP, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • Promoters or enhancers derived from the genomes of plant cells eg, heat shock, RUBISCO; and storage protein genes
  • plant viruses eg, viral promoters or leader sequences
  • a number of expression vectors may be selected depending upon the use intended for NGCP. For example, when large quantities of NGCP are needed for the 5 induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding NGCP may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems are designed to
  • NGCP may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al (1984) Nature 310:511-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al (1987) EMBO J 3:1311).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al
  • NGCP is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the sequence encoding NGCP may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the sequence encoding NGCP will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
  • a number of viral-based expression systems may be utilized.
  • a sequence encoding NGCP may be ligated into an adenovirus transcription/ translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing in infected host cells (Logan and Shenk (1984) Proc Natl Acad Sci 81 :3655-59).
  • transcription enhancers such as the rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of a sequence encoding NGCP. These signals include the ATG initiation codon and adjacent sequences. In cases where the sequence encoding NGCP, its initiation codon and upstream sequences are inserted into the most appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic.
  • Enhancers appropriate to the cell system in use (Scharf D et al (1994) Results Probl Cell Differ 20:125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, W138. etc have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express NGCP may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following the introduction of the vector, cells may be allowed to grow for 1 -2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) genes which can be employed in tk- or aprt- cells, respectively.
  • antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin F et al (1981) J Mol Biol 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
  • marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • a marker gene can be placed in tandem with the sequence encoding NGCP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem sequence as well.
  • host cells which contain the sequence encoding NGCP and expressing NGCP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein.
  • the presence of the polynucleotide sequence encoding NGCP can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of the sequence encoding NGCP.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the nucleic acid sequence to detect transformants containing DNA or RNA encoding NGCP.
  • oligonucleotides or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides which can be used as a probe or amplimer.
  • Means for producing labeled hybridization or PCR probes for detecting related sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • the NGCP-encoding sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
  • Patents teaching the use of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.
  • recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 incorporated herein by reference.
  • NGCP Purification of NGCP
  • Host cells transformed with a nucleotide sequence encoding NGCP may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing sequence encoding NGCP can be designed with signal sequences which direct secretion of NGCP through a prokaryotic or eukaryotic cell membrane.
  • NGCP may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
  • the inclusion of a cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and NGCP is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein comprising the sequence encoding NGCP and nucleic acid sequence encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site.
  • the histidine residues facilitate purification while the enterokinase cleavage site provides a means for purifying NGCP from the fusion protein.
  • fragments of NGCP may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al (1969) Solid-Phase Peptide Synthesis. WH Freeman Co, San Francisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154). in vitro protein synthesis may be performed using manual techniques or by automation.
  • NGCP neurotrophic factor
  • NGCP or its derivatives may be used therapeutical ly in the treatment of disease states such as leukemia and other cancers, immune deficiencies, or excessive immune responses.
  • Transfection of minute amounts of NGCP into tumor cells may reduce tumorigenicity.
  • NGCP could be administered to induce proteolytic activity of neutrophilic granulocytes resulting in tumor regression. Since it appears that the expression of NGCP in mammals (Broxmeyer, supra) is controlled by a feedback mechanism, NGCP can be administered to a patient for its myelosuppressive effect on bone marrow progenitor cell populations. NGCP, with or without preincubation in acetonitril, would be expected to suppress granulocyte-macrophage, erythroid and multipotential progenitor cells; therefore, daily chemokine treatment may be useful for patients with acute or chronic leukemia.
  • a therapeutic composition comprising NGCP may have application in the prevention and treatment of individuals subject to disease conditions which compromise the immune system.
  • Such conditions include, but are not limited to, HIV infection, Job-Buckley syndrome, lazy leukocyte syndrome, acquired agranulocytosis, and Chediak-Higashi syndrome. Although the pathologies of these diseases differ, common features include impairment of the immune system with subsequent predisposition to infections. Administration of minute amounts of NGCP might be expected to attract and activate leukocytes to the site for systemic defense.
  • NGCP Antibodies may be useful in preventing pathogenesis. In those situations, it is useful to prevent the chemoattraction and activity of excessive leukocytes which cause cellular destruction. Such conditions include idiopathic dilated cardiomyopathy, emphysema, lupus, myasthenia gravis, pancreatitis, and rheumatoid arthritis.
  • NGCP Antibodies include idiopathic dilated cardiomyopathy, emphysema, lupus, myasthenia gravis, pancreatitis, and rheumatoid arthritis.
  • NGCP-specific antibodies are useful for the diagnosis and treatment of conditions and diseases associated with expression of NGCP.
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Neutralizing antibodies, ie, those which inhibit dimer formation, are especially preferred for diagnostics and therapeutics.
  • NGCP for antibody induction does not require biological activity; however, the protein fragment, or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids. Preferably, they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of NGCP amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to NGCP.
  • various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with NGCP or any portion, fragment or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to NGCP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (1975 Nature 256:495-497), the human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today 4:72; Cote et al (1983) Proc Natl Acad Sci 80:2026-2030) and the EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy. Alan R Liss Inc, New York NY, pp 77-96).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991 ; Nature 349:293-299).
  • Antibody fragments which contain specific binding sites for NGCP may also be generated.
  • fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse WD et al (1989) Science 256:1275-1281).
  • NGCP antibodies are useful for the diagnosis of conditions or diseases characterized by expression of NGCP or in assays to monitor patients being treated with NGCP, its fragments, agonists or inhibitors.
  • Diagnostic assays for NGCP include methods utilizing the antibody and a label to detect NGCP in human body fluids or extracts of cells or tissues.
  • the polypeptides and antibodies of the present invention may be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
  • reporter molecules A wide variety of reporter molecules are known, several of which were described above.
  • a variety of protocols for measuring NGCP, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art.
  • Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • the amount of standard complex formation may be quantified by comparing various artificial membranes containing known quantities of NGCP with both control and disease samples from biopsied tissues. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • Drug Screening NGCP, its catalytic or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between NGCP and the agent being tested, may be measured.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding NGCP specifically compete with a test compound for binding NGCP. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with NGCP.
  • a polynucleotide sequence encoding NGCP or any part thereof may be used for diagnostic and/or therapeutic purposes.
  • the sequence encoding NGCP of this invention may be used to detect and quantitate gene expression in biopsied tissues in which NGCP may be expressed in response to oncogenes.
  • the diagnostic assay is useful to distinguish between absence, presence, and excess expression of NGCP and to monitor regulation of NGCP levels during therapeutic intervention.
  • Included in the scope of the invention are oligonucleotide sequences, antisense RNA and DNA molecules, and peptide nucleic acids, (PNA).
  • Another aspect of the subject invention is to provide for hybridization or PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding NGCP or closely related molecules.
  • the specificity of the probe whether it is made from a highly specific region, eg, 10 unique nucleotides in the 5' regulatory region, or a less specific region, eg, especially in the 3' region, and the stringency of the hybridization or amplification (maximal, high, intermediate or low) will determine whether the probe identifies only naturally occurring NGCP, alleles or related sequences.
  • Probes may also be used for the detection of related sequences and should preferably contain at least 50% of the nucleotides from any of these sequences encoding NGCP.
  • the hybridization probes of the subject invention may be derived from the nucleotide sequence of SEQ ID NO:2 or from genomic sequence including promoter, enhancer elements and introns of the naturally occurring sequence encoding NGCP.
  • Hybridization probes may be labeled by a variety of reporter groups, including radionuclides such as 32P or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • RNA probes for DNAs include the cloning of nucleic acid sequences encoding NGCP or NGCP derivatives into vectors for the production of mRNA probes.
  • vectors are known in the art and are commercially available and may be used to synthesize RNA probes i vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. Diagnostic Use
  • Polynucleotide sequences encoding NGCP may be used for the diagnosis of conditions or diseases with which the expression of NGCP is associated.
  • polynucleotide sequences encoding NGCP may be used in hybridization or PCR assays of fluids or tissues from biopsies to detect NGCP expression.
  • the form of such qualitative or quantitative methods may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin, chip and ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.
  • the NGCP-encoding nucleotide sequences disclosed herein provide the basis for assays that detect activation or induction associated with inflammation or disease.
  • the nucleotide sequence may be labeled by methods known in the art and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After an incubation period, the sample is washed with a compatible fluid which optionally contains a dye (or other label requiring a developer) if the nucleotide has been labeled with an enzyme. After the compatible fluid is rinsed off, the dye is quantitated and compared with a standard.
  • the nucleotide sequence has hybridized with nucleotide sequences in the sample, and the presence of elevated levels of nucleotide sequences encoding NGCP in the sample indicates the presence of the associated inflammation and/or disease.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regime in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for NGCP expression must be established. This is accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with NGCP, or a portion thereof, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained for normal subjects with a dilution series of NGCP run in the same experiment where a known amount of substantially purified NGCP is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients affected by NGCP-associated diseases. Deviation between standard and subject values establishes the presence of disease.
  • a therapeutic agent is administered and a treatment profile is generated. Such assays may be repeated on a regular basis to evaluate whether the values in the profile progress toward or return to the normal or standard pattern. Successive treatment profiles may be used to show the efficacy of treatment over a period of several days or several months.
  • PCR may be used as described in US Patent Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the sequence encoding NGCP. Such oligomers are generally chemically synthesized, but they may be generated enzymatically or produced from a recombinant source.
  • Oligomers generally comprise two nucleotide sequences, one with sense orientation (5'->3') and one with antisense (3' ⁇ -5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • methods which may be used to quantitate the expression of a particular molecule include radiolabeling (Melby PC et al 1993 J Immunol Methods 159:235-44) or biotinylating (Duplaa C et al 1993 Anal Biochem 229-36) nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated. Quantitation of multiple samples may be speeded up by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation. A definitive diagnosis of this type may allow health professionals to begin aggressive treatment and prevent further worsening of the condition.
  • nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known such as the triplet genetic code, specific base pair interactions, and the like. Therapeutic Use
  • the polynucleotide encoding NGCP disclosed herein may be useful in the treatment of immune deficiency diseases.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express antisense of the sequence encoding NGCP. See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra).
  • polynucleotides comprising full length cDNA sequence and/or its regulatory elements enable researchers to use the sequence encoding NGCP as an investigative tool in sense (Youssoufian H and HF Lodish 1993 Mol Cell Biol 13:98-104) or antisense (Eguchi et al (1991) Annu Rev Biochem 60:631-652) regulation of gene function.
  • sense or antisense oligomers, or larger fragments can be designed from various locations along the coding or control regions.
  • Genes encoding NGCP can be turned off by transfecting a cell or tissue with expression vectors which express high levels of a desired NGCP fragment. Such constructs can flood cells with untranslatable sense or antisense sequences. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector (Mettler I, personal communication) and even longer if appropriate replication elements are part of the vector system.
  • Methodtler I personal communication
  • antisense molecules DNA, RNA or PNA, to the control regions of the sequence encoding NGCP, ie, the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, eg, between -10 and +10 regions of the leader sequence, are preferred.
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of the sequence encoding NGCP.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding NGCP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues.
  • RNA molecules may be modified to increase intraceliular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • Methods for introducing vectors into cells or tissues include those methods discussed infra and which are equally suitable for in vivo, in vitro and ex vivo therapy.
  • vectors are introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient is presented in US Patent Nos. 5,399.493 and 5,437,994, disclosed herein by reference. Delivery by transfection and by liposome are quite well known in the art.
  • nucleotide sequences encoding NGCP disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including but not limited to such properties as the triplet genetic code and specific base pair interactions. Detection and Mapping of Related Polynucleotide Sequences
  • the nucleic acid sequence encoding NGCP can also be used to generate hybridization probes for mapping the naturally occurring genomic sequence.
  • the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques. These include in situ hybridization to chromosomal spreads, flow-sorted chromosomal preparations, or artificial chromosome constructions such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial Pl constructions or single chromosome cDNA libraries as reviewed in Price CM (1993; Blood Rev 7:127-34) and Trask BJ (1991; Trends Genet 7:149-54).
  • telangiectasia AT
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.
  • compositions are accomplished orally or parenterally.
  • Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Maack Publishing Co, Easton PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Dragee cores are provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, ie, dosage.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyi cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Manufacture and Storage
  • compositions of the present invention may be manufactured in a manner that known in the art, eg, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder in lmM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, eg, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors which ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, eg, ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • ED50 the dose therapeutically effective in 50% of the population
  • LD50 the dose lethal to 50% of the population.
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio,
  • LD50/ED50 Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, eg, tumor size and location; age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature. See US Patent Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • NGCP can be used as a therapeutic molecule to attract granulocytes to cancers for the purpose of destroying the cancerous cells or tissues.
  • the PANCNOT05 cDNA library was constructed from human pancreas tissue.
  • the donor was a 2 year old Hispanic male who died from anoxia.
  • the frozen tissue was homogenized and lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury NJ) in guanidinium isothiocyanate solution.
  • the lysate was centrifuged over a 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.
  • Qiagen Oligotex kit QIAGEN Inc; Chatsworth CA
  • mRNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Catalog #18248-013; Gibco/BRL). cDNAs were fractionated on a Sepharose CL4B column (Catalog #275105-01; Pharmacia), and those cDNAs exceeding 400 bp were ligated into pSport I. The plasmid pSport I was subsequently transformed into DH5aTM competent cells (Catalog #18258-012; Gibco BRL).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 Plasmid Kit for Rapid Extraction Alkaline Lysis Plasmid Minipreps (Catalog #26173; QIAGEN, Inc). This kit enables the simultaneous purification of 96 samples in a 96-well block using multichannel reagent dispensers.
  • the recommended protocol was employed except for the following changes: 1 ) the bacteria were cultured in 1 ml of sterile Terrific Broth (Catalog #2271 1 , LIFE TECHNOLOGIESTM) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures were incubated for 19 hours and at the end of incubation, the cells were lysed with 0.3 ml of lysis buffer; and 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the last step in the protocol, samples were transferred to a 96-well block for storage at 4° C. The cDN As were sequenced by the method of Sanger F and AR Coulson ( 1975 ; J Mol
  • the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith- Waterman alignments were used to display the results of the homology search. Peptide and protein sequence homologies were ascertained using the INHERIT TM 670
  • Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.
  • BLAST which stands for Basic Local Alignment Search Tool (Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al (1990) J Mol Biol 215:403-10), was used to search for local sequence alignments.
  • BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
  • the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
  • An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
  • the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
  • the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output. IV Northern Analysis
  • RNAs from a particular cell type or tissue have been bound (Sambrook et al. supra).
  • LIFESEQTM database (Incyte, Palo Alto CA). This analysis is much faster than multiple, membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
  • the basis of the search is the product score which is defined as: % sequence identity x % maximum BLAST score
  • homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
  • the nucleic acid sequence of SEQ ID NO:2 is used to design oligo-nucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5'sequence from genomic libraries.
  • One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
  • Primers allow the extension of the know sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest (US Patent Application 08/487,112, filed June 7, 1995, specifically incorporated by reference).
  • the initial primers are designed from the cDNA using OLIGO ® 4.06 Primer Analysis Software (National Biosciences), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68° -72° C. Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations is avoided.
  • the original, selected cDNA libraries, or a human genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
  • PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA) and the following parameters:
  • Step 1 94 ° C for 1 min (initial denaturation)
  • Step 2 65 ° C for 1 min
  • Step 3 68° C for 6 min
  • Step 4 94° C for 15 sec
  • Step 6 68° C for 7 min
  • Step 7 Repeat step 4-6 for 15 additional cycles
  • Step 8 94° C for 15 sec
  • Step 1 Repeat step 8-10 for 12 cycles Step 12 72 ° C for 8 min
  • a 5-10 ⁇ l aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as QIAQuickTM (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
  • PCR amplification For PCR amplification, 18 ⁇ l of concentrated PCR reaction mix (3.3x) containing 4 units of rTth DNA polymerase, a vector primer and one or both of the gene specific primers used for the extension reaction are added to each well. Amplification is performed using the following conditions:
  • Step 1 94° C for 60 sec
  • Step 2 94° C for 20 sec
  • Step 3 55° C for 30 sec
  • Step 4 72° C for 90 sec
  • Step 5 Repeat steps 2-4 for an additional 29 cycles
  • Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06
  • the labeled oligonucleotides are substantially purified with Sephadex G-25 super fine resin column (Pharmacia). A portion containing 10 7 counts per minute of each of the sense and antisense oligonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPont NEN ® ). The DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 °C.
  • blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARTM film (Kodak, Rochester NY) is exposed to the blots in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale CA) for several hours, hybridization patterns are compared visually. VII Antisense Molecules
  • the sequence encoding NGCP is used to inhibit in vivo or in vitro expression of naturally occurring sequence. Although use of antisense oligonucleotides, comprising about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments.
  • An oligonucleotide complementary to the coding sequence of NGCP as shown in Figures 1 A and IB is used to inhibit expression of naturally occurring sequence.
  • the complementary oligonucleotide is designed from the most unique 5' sequence as shown in Figures 1 A and IB and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of a transcript encoding NGCP by preventing the ribosome from binding.
  • an effective antisense oligonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or early coding sequence of the polypeptide as shown in Figures 1 A and IB. VIII Expression of NGCP
  • NGCP is accomplished by subcloning the cDNAs into appropriate vectors and transfecting the vectors into host cells.
  • the cloning vector, pSport. previously used for the generation of the cDNA library is used to express NGCP in E. coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
  • Induction of an isolated, transfected bacterial strain with IPTG using standard methods produces a fusion protein which consists of the first seven residues of ⁇ -galactosidase, about 5 to 15 residues of linker, and the full length NGCP.
  • the signal sequence directs the secretion of NGCP into the bacterial growth media which can be used directly in the following assay for activity.
  • chemotactic activity of NGCP is measured under agarose according to the method of Nelson et al (1975; J Immunol 1 15:1650). Mononuclear cells or granulocytes are exposed to serial dilutions of control media or media in which cells expressing recombinant NGCP were grown. After 2 hours incubation at 37°C, cells are fixed and stained. Spontaneous migration toward the control sample is compared to migration toward the test sample for various cell types. The specificity of the chemoattraction is determined by performing the agarose assay on specific populations of cells.
  • Blood cells obtained from venipuncture are fractionated by density gradient centrifugation and the chemotactic activity of NGCP is tested on enriched populations of neutrophils, peripheral blood mononuclear cells, granulocytes, monocytes and lymphocytes.
  • enriched cell populations may be further fractionated using CD8 T and CD4 + specific antibodies for negative selection of CD4 + and CD8' enriched T-cell populations, respectively.
  • NGCP is substantially purified using PAGE electrophoresis (Sambrook, supra) is used to immunize rabbits and to produce antibodies using standard protocols.
  • the amino acid sequence translated from NGCP is analyzed using DNAStar software (DNAStar Inc) to determine regions of high immunogenicity and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art.
  • DNAStar Inc DNAStar Inc
  • Analysis to select appropriate epitopes, such as those near the C-terminus or in hydrophilic regions (shown in Figures 4 and 5) is described by Ausubel FM et al (supra).
  • the oligopeptides are 15 residues in length, synthesized using an Applied Biosystems Peptide Synthesizer Model 431 A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel FM et al, supra).
  • KLH keyhole limpet hemocyanin
  • MBS M-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant.
  • the resulting antisera are tested for antipeptide activity, for example, by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated, goat anti-rabbit IgG.
  • Naturally occurring or recombinant NGCP is substantially purified by immunoaffinity chromatography using antibodies specific for NGCP.
  • An immunoaffinity column is constructed by covalently coupling NGCP antibody to an activated chromatographic resin such as CnBr-activated Sepharose (Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • Membrane fractions from cells expressing NGCP are prepared by methods well known in the art. Alternatively, a recombinant NGCP fragment containing an appropriate signal sequence may be secreted in useful quantity into the medium in which transfected cells are grown.
  • the NGCP-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of NGCP (eg, high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody /NGCP binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and NGCP is collected.

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Abstract

L'invention porte sur un polynucléotide qui identifie et code une nouvelle variante de la protéine-2 chimiotactique de granulocytes chez l'homme (NGCP). L'invention concerne des vecteurs d'expression et des cellules hôtes, obtenus par génie génétique, qui comprennent la séquence nucléotidique codant pour NGCP. L'invention se rapporte également à la production et à l'utilisation de NGCP sensiblement purifiée dans des compositions pharmaceutiques destinées à stopper la division cellulaire dans les cellules cancéreuses. L'invention concerne des méthodes diagnostiques dans lesquelles on utilise le polynucléotide à des fins d'hybridation avec les transcrits codant pour NGCP et pour les anticorps anti-NGCP se liant spécifiquement à NGCP.
PCT/US1997/016034 1996-09-12 1997-09-10 Variante de la proteine-2 chimiotactique de granulocytes WO1998011227A1 (fr)

Priority Applications (4)

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CA002265455A CA2265455A1 (fr) 1996-09-12 1997-09-10 Variante de la proteine-2 chimiotactique de granulocytes
AU42661/97A AU4266197A (en) 1996-09-12 1997-09-10 Granulocyte chemotactic protein 2 variant
EP97941010A EP0958365A1 (fr) 1996-09-12 1997-09-10 Variante de la proteine-2 chimiotactique de granulocytes
JP10513829A JP2001500019A (ja) 1996-09-12 1997-09-10 顆粒球走化性タンパク質２変異体

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US71328896A 1996-09-12 1996-09-12
US08/713,288 1996-09-12

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1312614A1 (fr) * 2001-11-16 2003-05-21 GenOdyssee Polynucleotides et polypeptides du gène GCP-2
US6908986B2 (en) 1996-03-18 2005-06-21 Human Genome Sciences, Inc. Chemokine alpha 3

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NO348172B1 (en) 2023-03-27 2024-09-16 Resitec As Lightly oxidized and deagglomerated silicon kerf particles as anode or anode precursor material in Li-ion batteries
NO20230344A1 (en) 2023-03-27 2024-09-30 Resitec As Drying of fine silicon powder in ambient air by empirically establishing powder reactivity

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994012537A2 (fr) * 1992-11-27 1994-06-09 Rega Institute Leuven University Proteine a chimiotaxie selective pour les granulocytes

Patent Citations (1)

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WO1994012537A2 (fr) * 1992-11-27 1994-06-09 Rega Institute Leuven University Proteine a chimiotaxie selective pour les granulocytes

Non-Patent Citations (3)

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CHANG M.-S. ET AL: "Cloning and characterization of the human neutrophil-activating peptide (ENA-78) gene", JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 269, 1994, MD US, pages 25277 - 25282, XP002049872 *
POWER C.A. ET AL: "Cloning of a full-length cDNA encoding the neutrophil-activating peptide ENA-78 from human platelets", GENE., vol. 151, 1994, AMSTERDAM NL, pages 333 - 334, XP002049873 *
PROOST P. ET AL: "Human and bovine granulocyte chemotactic protein-2: Complete amino acid sequence and fuctional characterization as chemokines", BIOCHEMISTRY., vol. 32, 1993, EASTON, PA US, pages 10170 - 10177, XP002049871 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6908986B2 (en) 1996-03-18 2005-06-21 Human Genome Sciences, Inc. Chemokine alpha 3
US7419662B2 (en) 1996-03-18 2008-09-02 Human Genome Sciences, Inc. Methods of making chemokine alpha 3 antibodies
EP1312614A1 (fr) * 2001-11-16 2003-05-21 GenOdyssee Polynucleotides et polypeptides du gène GCP-2

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