WO1997032993A1 - Chemotactic cytokine iii - Google Patents

Chemotactic cytokine iii Download PDF

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Publication number
WO1997032993A1
WO1997032993A1 PCT/US1996/002985 US9602985W WO9732993A1 WO 1997032993 A1 WO1997032993 A1 WO 1997032993A1 US 9602985 W US9602985 W US 9602985W WO 9732993 A1 WO9732993 A1 WO 9732993A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
cciii
polypeptides
dna
Prior art date
Application number
PCT/US1996/002985
Other languages
French (fr)
Inventor
Jian Ni
Guo-Liang Yu
Reiner L. Gentz
Original Assignee
Human Genome Sciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Human Genome Sciences, Inc. filed Critical Human Genome Sciences, Inc.
Priority to PCT/US1996/002985 priority Critical patent/WO1997032993A1/en
Priority to AU55234/96A priority patent/AU5523496A/en
Publication of WO1997032993A1 publication Critical patent/WO1997032993A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • This invention relates, in part, to newly identified is polynucleotides and polypeptides; variants and derivatives of the
  • the invention relates to polynucleotides and polypeptides 2 of human chemotactic cytokine III, hereinafter referred to as 3 "CCIII". 4 5 BACKGROUND OF THE INVENTION 6
  • cytokines and in particular chemotactic 9 cytokines or chemokines, are an example.
  • Chemokines also referred to as intercrine cytokines, are a i subfamily of structurally and functionally related chemotactic 2 cytokines.
  • chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two 5 disulfide bonds. Based on the arrangement of the first two 6 cysteine residues, chemokines have been classified subfamilies, 7 according to the arrangement of the cysteine residues with respect 8 to the nearest cysteine residue. In the alpha subfamily, for 9 example, the first two cysteines are separated by one amino acid
  • the two cysteines are in an adjacent position and are,
  • the intercrine cytokines exhibit a wide variety of functions.
  • a hallmark feature is their ability to elicit chemotactic migration
  • chemokines have ⁇ proinflammatory activity and are involved in multiple steps during 9 an inflammatory reaction. These activities include stimulation of 0 histamine release, lysosomal enzyme and leukotriene release, i increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities.
  • macrophage inflammatory protein 1 is able to suppress hematopoietic stem cell proliferation
  • platelet factor-4 is a potent inhibitor of endothelial cell growth
  • Interleukin-3 IL-8 promotes proliferation of keratinocytes
  • GRO is an autocrine growth factor for melanoma cells.
  • chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis.
  • C-C branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophage ⁇ which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation.
  • members of one branch may exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches.
  • members of the C-C branch act predominantly on mononuclear cells and members of the C-X-C branch act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline.
  • Some chemokines from one family show characteristics of the other. 1
  • chemokines from one family show characteristics of the other. 1 Clearly, there is a need for identification and
  • 3 cells particularly cells of the immune system, and which can play a role in preventing, ameliorating or correcting dysfunctions or
  • polypeptide of the present invention has the conserved
  • polypeptides inter alia, that have been 3 identified as novel CCIII polypeptides by homology between the 4 amino acid sequence set out in Figure 1 (SEQ ID NO:2) and known s amino acid sequences of other proteins such as murine cytokine- 6 induced neutrophil chemoattractant 2 set out in Figure 2 and (SEQ 7 ID NO:9) .
  • SEQ ID NO:2 4 amino acid sequence set out in Figure 1
  • SEQ 7 ID NO:9 known s amino acid sequences of other proteins
  • the polynucleotide comprises the region encoding human 4 CCIII in the sequence set out in Figure l (SEQ ID NO:l) . 5
  • an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97408.
  • isolated nucleic acid molecules encoding human CCIII including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives.
  • biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives are provided.
  • allelic variants of human CCIII are naturally occurring allelic variants of human CCIII.
  • CCIII polypeptides particularly human CCIII polypeptides
  • CCIII novel polypeptides of human origin referred to herein as CCIII as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.
  • this aspect of the invention are variants of human CCIII encoded by naturally occurring alleles of the human CCIII gene. It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing.
  • methods for producing the aforementioned CCIII polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human CCIII-encoding polynucleotide under conditions for expression of human CCIII in the host and then recovering the expressed polypeptide.
  • products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia .
  • products, compositions and methods for, among other things: assessing CCIII expression in cells by determining CCIII polypeptides or CCIII- encoding mRNA; to treat tumors, chronic infections, leukemia, T- cell mediated auto-immune diseases, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate growth factor activity, to inhibit angiogenesis and to promote wound healing in vi tro, ex vivo or in vivo by exposing cells to CCIII polypeptides or polynucleotides as disclosed herein,- assaying 1 genetic variation and aberrations, such as defects, in CCIII genes;
  • the antibodies are highly selective for human CCIII.
  • CCIII agonists there are provided CCIII agonists.
  • preferred agonists are
  • CCIII antagonists are those which mimic CCIII so as to bind to CCIII 3 receptor(s) or binding molecules but not elicit a CCIII-induced 4 response or more than one CCIII-induced response.
  • CCIII-induced 4 response or more than one CCIII-induced response.
  • molecules that bind to or interact with 6 CCIII so as to inhibit an effect of CCIII or more than one effect 7 of CCIII or which prevent expression of CCIII .
  • the antagonists may 8 be employed to treat and/or prevent, for example, 9 glomerulonephritis, inflammation, cerebral ischemia, HTLV-1 related 0 diseases, arthritis, infectious diseases, auto-immune diseases, i hyper-eosinophilic syndrome, endotoxic shock, ahterosclerosis, 2 allergies, bone marrow failure and asthma.
  • compositions comprising a CCIII polynucleotide or a CCIII 5 polypeptide for administration to cells in vi tro, to cells ex vivo 6 and to cells in vivo, or to a multicellular organism.
  • compositions comprise a CCIII polynucleotide for expression of 9 a CCIII polypeptide in a host organism for treatment of disease.
  • a CCIII polypeptide in a host organism for treatment of disease.
  • Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of CCIII.
  • FIG. 1 shows the nucleotide and deduced amino acid sequence of human CCIII, wherein the underlined portion represents a putative leader sequence.
  • Figure 2 shows the regions of similarity between amino acid sequences of CCIII and murine cytokine-induced neutrophil chemoattractant 2 polypeptides.
  • Figure 3 shows structural and functional features of CCIII deduced by the indicated techniques, as a function of amino acid sequence.
  • DIGESTION of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA.
  • the various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled artisan.
  • 1 ⁇ g of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 ⁇ l 1 of reaction buffer.
  • GENETIC ELEMENT generally means a polynucleotide comprising 5 a region that encodes a polypeptide or a region that regulates 6 transcription or translation or other processes important to 7 expression of the polypeptide in a host cell, or a polynucleotide 8 comprising both a region that encodes a polypeptide and a region 9 operably linked thereto that regulates expression.
  • Genetic elements may be comprised within a vector that i replicates as an episomal element; that is, as a molecule 2 physically independent of the host cell genome.
  • chromosomes may be 3 comprised within mini-chromosomes, such as those that arise during 4 amplification of transfected DNA by methotrexate selection in 5 eukaryotic cells.
  • Genetic elements also may be comprised within 6 a host cell genome; not in their natural state but, rather, 7 following manipulation such as isolation, cloning and introduction 8 into a host cell in the form of purified DNA or in a vector, among 9 others.
  • 0 ISOLATED means altered "by the hand of man” from its natural i state; i.e., that, if it occurs in nature, it has been changed or 2 removed from its original environment, or both.
  • a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not “isolated, " but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein.
  • the term isolated means that it is separated from the chromosome and cell in which it naturally occurs.
  • such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance.
  • the isolated polynucleotides alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein.
  • LIGATION refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs.
  • OLIGONUCLEOTIDE refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others.
  • Oligonucleotide ⁇ such as single-stranded DNA probe oligonucleotide ⁇ , often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers.
  • oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms. Initially, chemically synthesized DNAs typically are obtained without a 5' phosphate. The 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules.
  • a phosphate can be added by standard techniques, such as those that employ a kinase and ATP.
  • the 3 ' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide.
  • a ligase such as T4 DNA ligase
  • this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotide ( ⁇ ) prior to ligation.
  • PLASMIDS generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art.
  • Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures.
  • Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art.
  • those of skill readily may construct any number of other plasmids suitable for use in the invention.
  • POLYNUCLEOTIDE generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, ⁇ ingle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules .
  • One of the molecules of a triple-helical region often is an oligonucleotide.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
  • DNAs or RNAs comprising unusual base ⁇ , such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • POLYPEPTIDES as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art .
  • amino acids including the terminal amino acids
  • chemical modification techniques which are well known to the art .
  • polypeptides are not always entirely linear.
  • 4 polypeptides may be branched as a result of ubiquitination, and 5 they may be circular, with or without branching, generally as a 6 result of posttranslation events, including natural processing 7 event and events brought about by human manipulation which do not 8 occur naturally.
  • Circular, branched and branched circular i polypeptides may be synthesized by non-translation natural process
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or
  • the modifications that occur in a polypeptide often will be 3 a function of how it is made.
  • polypeptides made by expressing 4 a cloned gene in a host for instance, the nature and extent of the s modifications in large part will be determined by the host cell 6 posttranslational modification capacity and the modification 7 signals present in the polypeptide amino acid sequence.
  • glycosylation often does not occur in 9 bacterial hosts such as E. coli.
  • glycosylation 0 when glycosylation 0 is desired, a polypeptide should be expressed in a glycosylating i host, generally a eukaryotic cell. Insect cell often carry out the 2 same posttranslational glycosylations as mammalian cells and, for 3 this reason, insect cell expression systems have been developed to 4 express efficiently mammalian proteins having native patterns of 5 glycosylation, inter alia. Similar considerations apply to other 6 modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications.
  • polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthe ⁇ ized by expressing a polynucleotide in a host cell.
  • VARIANT(S) of polynucleotides or polypeptides are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail. 1 (1) A polynucleotide that differs in nucleotide sequence from
  • variants 7 variant may be silent. That is, they may not alter the amino acids ⁇ encoded by the polynucleotide. Where alterations are limited to 9 silent changes of this type a variant will encode a polypeptide 0 with the same amino acid sequence as the reference. Also as noted i below, changes in the nucleotide sequence of the variant may alter 2 the amino acid sequence of a polypeptide encoded by the reference 3 polynucleotide. Such nucleotide changes may result in amino acid 4 substitutions, additions, deletions, fusions and truncations in the 5 polypeptide encoded by the reference sequence, as discussed below. 6 7 (2) A polypeptide that differs in amino acid sequence from 8 another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid 2 sequence by one or more substitutions, additions, deletions, 3 fusions and truncations, which may be present in any combination.
  • RECEPTOR MOLECULE refers to molecules which 6 bind or interact specifically with CCIII polypeptides of the present invention, including not only classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which also may be referred to as “binding molecules” and “interaction molecules,” respectively and as “CCIII binding molecules” and “CCIII interaction molecules.” Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it 1 may be specific to several groups of proteins at least one of which
  • Receptors also may be non-naturally occurring, such as
  • the present invention relates to novel CCIII polypeptides and
  • polypeptides and ii polynucleotides of a novel human CCIII which is related by amino
  • the invention relates especially to CCIII
  • the deposited clone or as the "cDNA of the ie deposited clone.” It will be appreciated that the nucleotide and
  • Figure 1 (SEQ ID N0:1) includes reference to the sequence of the 4 human cDNA of the deposited clone. 5 6 Polynucleotides 7 In accordance with one aspect of the present invention, there 8 are provided isolated polynucleotides which encode the CCIII 9 polypeptide having the deduced amino acid sequence of Figure l (SEQ 0 ID NO:2) or the CCIII polypeptide encoded by the cDNA in the i deposited clone.
  • a 4 polynucleotide of the present invention encoding human CCIII 5 polypeptide may be obtained using standard cloning and screening 6 procedures, such as those for cloning cDNAs using mRNA from cells 7 of a human adrenal gland tumor, cerebellum, fetal heart and fetal 8 lung as starting material.
  • standard cloning and screening 6 procedures such as those for cloning cDNAs using mRNA from cells 7 of a human adrenal gland tumor, cerebellum, fetal heart and fetal 8 lung as starting material.
  • the 1 polynucleotide set out in Figure 1 (SEQ ID N0:1) was discovered in
  • Human CCIII of the invention is structurally related to other proteins of the chemokine family, as shown by the results of
  • CCIII has a deduced molecular weight of about ii 8.5682 kDa, has a Molar extinction coefficient of 14660 ⁇ 5%, an
  • the protein exhibits greatest homology to murine cytokine-
  • Polynucleotides of the present invention may be in the form
  • RNA such as mRNA
  • DNA including, for ie instance, cDNA and genomic DNA obtained by cloning or produced by
  • Single-stranded DNA may 2i be the coding strand, also known as the sense strand, or it may be 2 the non-coding strand, also referred to as the anti-sense strand.
  • the coding sequence which encodes the polypeptide may be 5 identical to the coding sequence of the polynucleotide shown in 6 Figure 1 (SEQ ID NO:l) or that of the deposited clone.
  • Polynucleotides of the present invention which encode the 2 polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptide encoded 3 by the deposited cDNA may include, but are not limited to the 4 coding sequence for the mature polypeptide, by itself; the coding 5 sequence for the mature polypeptide and additional coding 6 sequences, such as those encoding a leader or secretory sequence, 7 such as a pre-, or pro- or prepro- protein sequence; the coding 8 sequence of the mature polypeptide, with or without the 9 aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example -ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
  • the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector, among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984), for instance.
  • polynucleotide encoding a polypeptide encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly the human CCIII having the amino acid sequence set out in Figure 1 (SEQ ID NO:2) or the amino acid sequence of the human CCIII encoded by the cDNA of the deposited clone.
  • the term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by introns) together with additional regions, that also may contain coding and/or non-coding sequences.
  • the present invention further relates to variants of the herein above-described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone.
  • a variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis 1 techniques, including those applied to polynucleotides, cells or
  • variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions,
  • the variants may be altered
  • polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding the CCIII polypeptide of the cDNA of the deposited clone and polynucleotides complementary thereto.
  • polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred.
  • those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
  • polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 (SEQ ID N0:1) or the human cDNA of the deposited clone.
  • the present invention further relates to polynucleotides that hybridize to the herein above-described sequences.
  • the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polynucleotides of the invention may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding CCIII and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the human CCIII gene.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 30 bases and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases and will have 50 bases or less.
  • the coding region of the CCIII gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia.
  • the polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) .
  • Such sequences may play a role in processing of a protein from precursor to a mature form, may facilitate protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things.
  • the additional amino acids may be processed away from the mature protein by cellular enzymes.
  • a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein) , a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • a leader sequence which may be referred to as a preprotein
  • a preproprotein which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
  • a deposit containing a human CCIII cDNA has been deposited with the American Type Culture Collection, as noted above. Also as noted above, the cDNA deposit is referred to herein as "the deposited clone” or as "the cDNA of the deposited clone.” The deposited clone was deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on January 2, 1996 and assigned ATCC Deposit No. 97408. The deposited material is a pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) that contains the full length CCIII cDNA.
  • the deposit has been made under the terms of the Budapest Treaty on the international recognition of the deposit of micro- organisms for purposes of patent procedure.
  • the strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
  • the deposit is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 112.
  • the present invention relates to the sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are 1 controlling in the event of any conflict with any description of
  • a license may be required to make, use or sell the deposited
  • the present invention further relates to a human CCIII
  • polypeptides of the present invention include o the polypeptide of SEQ ID NO:2 (in particular the mature i polypeptide) as well as polypeptides which have at least 70% 2 similarity (preferably at least 70% identity) to the polypeptide 3 of SEQ ID NO:2 and more preferably at least 90% similarity (more 4 preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 s and still more preferably at least 95% similarity (still more 6 preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 7 and also include portions of such polypeptides with such portion 8 of the polypeptide generally containing at least 30 amino acids and 9 more preferably at least 50 amino acids.
  • fragments As known in the art "similarity" between two polypeptides is i determined by comparing the amino acid sequence and its conserved 2 amino acid substitutes of one polypeptide to the sequence of a 3 second polypeptide. 4
  • the invention also relates to fragments, analogs and 5 derivatives of these polypeptides.
  • fragment 6 "derivative” and “analog” when referring to the polypeptide of 7 Figure 1 (SEQ ID NO:2) or that encoded by the deposited human cDNA, ⁇ means a polypeptide which retains essentially the same biological 9 function or activity as such polypeptide.
  • an analog includes 0 a proprotein which can be activated by cleavage of the proprotein 1 portion to produce an active mature polypeptide.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of 6 Figure 1 may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be 1 one encoded by the genetic code, or (ii) one in which one or more
  • substitutions are those that vary from a reference 3 by conservative amino acid substitutions .
  • Such substitutions are 4 those that substitute a given amino acid in a polypeptide by 5 another amino acid of like characteristics.
  • 6 conservative substitutions are the replacements, one for another, 7 among the aliphatic amino acids Ala, Val, Leu and lie; interchange 8 of the hydroxyl residues Ser and Thr, exchange of the acidic 9 residues Asp and Glu, substitution between the amide residues Asn 0 and Gin, exchange of the basic residues Lys and Arg and 1 replacements among the aromatic residues Phe, Tyr.
  • variants, 3 analogs, derivatives and fragments, and variants, analogs and 4 derivatives of the fragments having the amino acid sequence of the 5 CCIII polypeptide of Figure l (SEQ ID NO:2) or of the cDNA in the 6 deposited clone, in which several, a few, 5 to 10, l to 5, l to 3, 7 2, 1 or no amino acid residues are substituted, deleted or added, 8 in any combination.
  • silent 9 substitutions, additions and deletions which do not alter the properties and activities of the CCIII .
  • conservative substitutions are also especially preferred in this regard.
  • polypeptides having the amino acid sequence of Figure 1 SEQ ID NO:2 or that encoded by the deposited clone without substitutions.
  • the polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • fragments comprising fragments of CCIII, most particularly fragments of the CCIII having the amino acid set out in Figure 1 (SEQ ID NO:2) , and fragments of variants and derivatives of the CCIII of Figure 1 (SEQ ID NO:2) .
  • a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned CCIII polypeptides and variants or derivatives thereof.
  • Such fragments may be "free-standing,” i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region.
  • fragments When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of a CCIII polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the amino terminus of the CCIII fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from CCIII.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full- length polypeptide by peptide synthesis,- therefore, the fragments may be employed as intermediates for producing the full-length polypeptides . Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • polypeptide fragments of the invention there may be mentioned those which have from about 15 to about 81 amino acids. In this context about includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes.
  • about 30-81 amino acids in this context means a polypeptide fragment of 30 or minus several, a few, 5, 4, 3, 2 or 1 amino acids to 81 plus or minus several a few, 5, 4, 3, 2 or l amino acid residues, i.e., ranges as broad as 30 minus several amino acids to 81 plus several amino acids to as narrow as 30 plus several amino acids to 81 minu ⁇ several amino acids.
  • Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes.
  • Particularly highly preferred are the recited ranges plus or minus as many as 3 amino acids at either or at both the recited extremes.
  • ranges plus or minus 1 amino acid at either or at both extremes or the recited ranges with no additions or deletions are preferred.
  • fragments from about 15 to about 54 amino acids are especially preferred fragments of the invention.
  • Truncation mutants include CCIII polypeptides having the amino acid sequence of Figure 1 (SEQ ID NO:2) , or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus.
  • Fragments having the size ranges set out about are preferred embodiments of truncation fragments, which are especially preferred among fragments generally.
  • fragments characterized by structural or functional attributes of CCIII are also preferred.
  • Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions”) , beta-sheet and beta-sheet-forming regions (“beta-regions”) , turn and turn-forming regions (“turn-regions”) , 1 coil and coil-forming regions (“coil-regions”) , hydrophilic
  • preferred regions include Gamier-Robson alpha-regions, beta- 0 regions, turn-regions and coil-regions, Chou-Fasman alpha-regions, i beta-regions and turn-regions, Kyte-Doolittle hydrophilic regions 2 and hydrophilic regions, Eisenberg alpha and beta amphipathic 3 regions, Karplus-Schulz flexible regions, Emini surface-forming 4 regions and Jameson-Wolf high antigenic index regions.
  • highly preferred fragments in this regard are those that 6 comprise regions of CCIII that combine several structural features, 7 such as several of the features set out above.
  • the s regions defined by the residues about 15 to about 80 residues of 9 Figure 1 which all are characterized by amino acid 0 compositions highly characteristic of turn-regions, hydrophilic i regions, flexible-regions, surface-forming regions, and high 2 antigenic inde -regions, are especially highly preferred regions.
  • Such regions may be comprised within a larger polypeptide or may 4 be by themselves a preferred fragment of the present invention, as 5 discussed above. It will be appreciated that the term "about” as 6 used in this paragraph has the meaning set out above regarding 7 fragments in general. 8 Further preferred regions are those that mediate activities 9 of CCIII.
  • fragments that 0 have a chemical, biological or other activity of CCIII, including i those with a similar activity or an improved activity, or with a 2 decreased undesirable activity.
  • Highly preferred in this regard 3 are fragments that contain regions that are homologs in sequence, 4 or in position, or in both sequence and to active regions of 5 related polypeptides, such as the related polypeptide set out in 6 Figure 2 (SEQ ID NO:9) .
  • Particular preferred fragments in 7 these regards are truncation mutants, as discussed above. 8
  • the invention also relates to, 9 among others, polynucleotides encoding the aforementioned i fragments, polynucleotides that hybridize to polynucleotides
  • preferred polynucleotides are those that correspondent to
  • the present invention also relates to vectors which include 10 polynucleotides of the present invention, host cells which are ii genetically engineered with vectors of the invention and the
  • Host cells can be genetically engineered to incorporate
  • polynucleotides may be introduced into host cells
  • polynucleotides may be introduced independently, co-introduced or
  • polynucleotides of the invention may be 3 transfected into host cells with another, separate, polynucleotide 4 encoding a selectable marker, using standard techniques for co- 5 transfection and selection in, for instance, mammalian cells.
  • the polynucleotides generally will be stably incorporated 7 into the host cell genome.
  • the polynucleotides may be joined to a vector 9 containing a selectable marker for propagation in a host.
  • the 0 vector construct may be introduced into host cells by the i aforementioned techniques.
  • a plasmid vector is 2 introduced as DNA in a precipitate, such as a calcium phosphate 3 precipitate, or in a complex with a charged lipid.
  • Electroporation 4 also may be used to introduce polynucleotides into a host.
  • the 5 vector is a virus, it may be packaged in vitro or introduced into 6 a packaging cell and the packaged virus may be transduced into 7 cells.
  • a wide variety of techniques suitable for making 8 polynucleotides and for introducing polynucleotides into cells in 9 accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length in Sambrook et al.
  • the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
  • the vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction.
  • Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
  • vectors are those for expression of polynucleotides and polypeptides of the present invention.
  • such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression.
  • Such specific expression may be inducible expres ⁇ ion or expres ⁇ ion only in certain types of cells or both inducible and cell-specific.
  • inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • a variety of vectors suitable to this aspect of the invention including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
  • the engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the l present invention as will be apparent to those of skill in the art.
  • Such vectors include chromosomal,
  • yeast chromosomal elements from viruses such as baculoviruses,
  • papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl
  • any vector suitable 4 to maintain, propagate or express polynucleotides to express a s polypeptide in a host may be used for expression in this regard.
  • 6 7 The appropriate DNA sequence may be inserted into the vector 8 by any of a variety of well-known and routine techniques.
  • a DNA sequence for expression is joined to an expression 0 vector by cleaving the DNA sequence and the expression vector with i one or more restriction endonucleases and then joining the 2 restriction fragments together using T4 DNA ligase.
  • Procedures for 3 restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expres ⁇ ion vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein.
  • the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription.
  • promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein. 1 In general, expression construct ⁇ will contain sites for
  • construct ⁇ may contain control region ⁇ that
  • Vectors for propagation and expression generally will include 4 selectable markers. Such markers also may be suitable for s amplification or the vectors may contain additional markers for 6 this purpo ⁇ e.
  • the expression vectors preferably 7 contain one or more selectable marker genes to provide a phenotypic s trait for selection of transformed host cells.
  • Preferred markers 9 include dihydrofolate reductase or neomycin resistance for 0 eukaryotic cell culture, and tetracycline or ampicillin resistance i gene ⁇ for culturing E.
  • the vector containing the appropriate DNA sequence as 3 described elsewhere herein, as well as an appropriate promoter, and 4 other appropriate control sequences, may be introduced into an 5 appropriate host using a variety of well known techniques suitable 6 to expres ⁇ ion therein of a de ⁇ ired polypeptide.
  • appropriate hosts include bacterial cells, such as E. 8 coli, Streptomyces and Salmonella typhimurium cells; fungal cells, 9 such as yeast cells; insect cells such as Drosophila S2 and 0 Spodoptera Sf9 cell ⁇ ; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
  • the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequence ⁇ de ⁇ cribed above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been in ⁇ erted.
  • the ⁇ equence may be inserted in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • vectors suitable for use in bacteria are known to tho ⁇ e of skill in the art, and there are many commercially available vectors suitable for use in the present invention.
  • the following vectors, which are commercially available, are provided by way of example.
  • vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen,- pBS vectors, Phagescript vectors, Bluescript vector ⁇ , pNH ⁇ A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • eukaryotic vector ⁇ are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are li ⁇ ted solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with thi ⁇ aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
  • Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter.
  • a reporter transcription unit lacking a promoter region such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter.
  • CAT activity which can be detected by standard CAT assays.
  • Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7.
  • promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.
  • E. coli lad and lacZ and promoters are the E. coli lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the
  • RSV metallothionein 9
  • metallothionein promoters such as the mouse 0 metallothionein-I promoter.
  • the ho ⁇ t cell can 8 be a higher eukaryotic cell, ⁇ uch a ⁇ a mammalian cell, or a lower 9 eukaryotic cell, such a ⁇ a yea ⁇ t cell, or the host cell can be a 0 prokaryotic cell, such as a bacterial cell.
  • 1 Introduction of the construct into the host cell can be 2 effected by calcium phosphate transfection, DEAE-dextran mediated 3 transfection, cationic lipid-mediated transfection, 4 electroporation, transduction, infection or other method ⁇ .
  • Such 5 method ⁇ are de ⁇ cribed in many standard laboratory manuals, such as 6 Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986) .
  • Constructs in host cell ⁇ can be used in a conventional manner 8 to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be 0 synthetically produced by conventional peptide synthe ⁇ izers.
  • i Mature proteins can be expressed in mammalian cells, yeast, 2 bacteria, or other cells under the control of appropriate 3 promoter ⁇ .
  • Cell-free tran ⁇ lation systems can also be employed to 4 produce such proteins using RNAs derived from the DNA constructs of the present invention.
  • recombinant expres ⁇ ion vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.
  • promoters are those derived from the genes that encode glycolytic enzymes such as 3- phosphoglycerate kinase ("PGK”), a-factor, acid phosphatase, and heat shock proteins, among others.
  • PPG 3- phosphoglycerate kinase
  • Selectable markers include the ampicillin resistance gene of E. coli and the trpl gene of S. cerevisiae. Tran ⁇ cription of the DNA encoding the polypeptide ⁇ of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector.
  • Enhancers are cis- acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques ⁇ o that it is operably linked to the promoter for expression.
  • the polynucleotide will be positioned so that the transcription start site is located appropriately 5 ' to a ribosome binding site.
  • the ribosome binding site will be 5 ' to the AUG that initiate ⁇ translation of the polypeptide to be expressed.
  • initiation codon usually AUG
  • translation stop codon at the end of the polypeptide
  • polyadenylation signal at the end of the polypeptide
  • transcription termination signal appropriately disposed at the 3' end of the transcribed region.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signal ⁇ .
  • the polypeptide may be expressed in a modified form, such as
  • 2 a fusion protein, and may include not only secretion signals but
  • 5 amino acid ⁇ may be added to the N-terminus of the polypeptide to
  • the invention include Escherischia coli, Bacillus subtilis and
  • 22 expres ⁇ ion vectors for bacterial use can comprise a selectable
  • 26 vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
  • Microbial cells employed in expression of proteins can be J9 disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.
  • Variou ⁇ mammalian cell culture ⁇ ystems can be employed for expression, as well.
  • Examples of mammalian expres ⁇ ion systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 (1981) .
  • Other cell lines capable of expres ⁇ ing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell line ⁇ .
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any nece ⁇ ary ribo ⁇ ome binding ⁇ ite ⁇ , polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expres ⁇ ion.
  • DNA sequences derived from the SV40 splice ⁇ ite ⁇ , and the SV40 polyadenylation sites are used for required non-transcribed genetic elements of these type ⁇ .
  • the CCIII polypeptide can be recovered and purified from recombinant cell culture ⁇ by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. CCIII polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties CCIII.
  • Polynucleotide a ⁇ ay ⁇ Thi ⁇ invention is also related to the use of the CCIII polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of CCIII associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disea ⁇ e which results from under-expression over-expression or altered expression of CCIII, for example, neoplasia such as cancers and tumors.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such a ⁇ from blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysi ⁇ .
  • PCR Saiki et al., Nature, 324: 163-166 (1986)
  • RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding CCIII can be used to identify and analyze CCIII expression and mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled CCIII RNA or alternatively, radiolabeled CCIII antisense DNA ⁇ equence ⁇ . Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segment ⁇ . The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double- stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is 1 performed by conventional procedures with radiolabeled nucleotide
  • the detection of a specific DNA sequence may be achieved 8 by methods such as hybridization, RNase protection, chemical 9 cleavage, direct DNA sequencing or the use of restriction enzymes, 0 (e.g., restriction fragment length polymorphisms ("RFLP") and i Southern blotting of genomic DNA.
  • restriction enzymes 0 (e.g., restriction fragment length polymorphisms ("RFLP") and i Southern blotting of genomic DNA. 2
  • mutation ⁇ al ⁇ o can be detected by in ⁇ itu analy ⁇ is.
  • Chromosome assays The sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disea ⁇ e.
  • the cDNA herein disclosed is used to clone genomic DNA of a CCIII gene.
  • genomic DNA is used for in situ chromosome mapping using well known techniques for this purpose.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA.
  • Computer analy ⁇ is of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification proces ⁇ .
  • the ⁇ e primer ⁇ are then u ⁇ ed for PCR ⁇ creening of somatic cell hybrid ⁇ containing individual human chromo ⁇ ome ⁇ . Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosome ⁇ or pool ⁇ of large genomic clone ⁇ in an analogous manner.
  • mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization FISH
  • FISH Fluorescence in situ hybridization
  • This technique can be used with cDNA as short as 50 or 60. For a review of this technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988) .
  • a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disea ⁇ e.
  • a cDNA precisely localized to a chromosomal region associated with the disea ⁇ e could be one of between 50 and 500 potential cau ⁇ ative gene ⁇ . (Thi ⁇ assumes l megabase mapping resolution and one gene per 20 kb) .
  • Polypeptide as ⁇ ays also relates to a diagnostic assay ⁇ ⁇ uch as quantitative and diagnostic assays for detecting levels of CCIII protein in cells and tissues, including determination of normal and abnormal levels.
  • a diagnostic assay in accordance with the invention for detecting over-expression of CCIII protein compared to normal control tissue samples may be used to detect the presence of a neoplasia, for example.
  • Assay techniques that can be used to determine levels of a protein, such as an CCIII protein of the present invention, in a sample derived from a host are well-known to those of skill in the art.
  • An ELISA as ⁇ ay initially comprises preparing an antibody specific to CCIII, preferably a monoclonal antibody.
  • a reporter antibody generally is prepared which binds to the monoclonal antibody.
  • the reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.
  • a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.
  • a polystyrene dish that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin.
  • the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any CCIII proteins attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer.
  • the reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to CCIII. Unattached reporter antibody 1 is then washed out.
  • Reagents for peroxidase activity including
  • the polypeptides, their fragments or other derivatives, or 6 analogs thereof, or cells expressing them can be used as an 7 immunogen to produce antibodies thereto. These antibodies can be, 8 for example, polyclonal or monoclonal antibodies.
  • the present 9 invention also includes chimeric, single chain, and humanized 0 antibodies, as well as Fab fragments, or the product of an Fab i expression library. Various procedures known in the art may be 2 used for the production of such antibodies and fragments.
  • Antibodies generated again ⁇ t the polypeptides corresponding 4 to a sequence of the present invention can be obtained by direct 5 injection of the polypeptides into an animal or by administering 6 the polypeptides to an animal, preferably a nonhuman. The antibody 7 so obtained will then bind the polypeptides itself. In this 8 manner, even a sequence encoding only a fragment of the 9 polypeptides can be used to generate antibodies binding the whole 0 native polypeptides. Such antibodies can then be used to isolate i the polypeptide from tissue expressing that polypeptide.
  • the antibodies may be employed to isolate or to identify 3 clones expressing the polypeptide or purify a polypeptide of the present invention by attachment to a solid support for isolation and/or purification by affinity chromatography.
  • any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C. , Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., pg.
  • CCIII may also be employed to inhibit epidermal keratinocyte proliferation for treatment of psoria ⁇ is, which is characterized by keratinocyte hyper-proliferation.
  • CCIII may also be employed to treat neoplasia, such as cancers and tumors, by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophage ⁇ and by inhibiting the angiogenesis of tumor ⁇ . They may also be employed to enhance host defenses against resistant chronic and acute infection ⁇ , for example, mycobacterial infection ⁇ via the attraction and activation of microbicidal leukocytes.
  • CCIII may also be employed to inhibit T cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-immune disease ⁇ and lymphocytic leukemia ⁇ .
  • CCIII may also be employed to stimulate wound healing, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells and also via its control of excessive TGF/3-mediated fibrosis.
  • CCIII may also be employed to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis.
  • CCIII also increases the presence of eosinophils which have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis.
  • This invention also provides a method for identification of
  • expression cloning may be employed for this
  • 16 library is divided into pools and the pools are transfected
  • CCIII can be
  • Sub-pools are prepared from these positives, transfected 6 into host cells and screened as described above. Using an iterative 7 sub-pooling and re-screening process, one or more single clones 8 that encode the putative binding molecule, such as a receptor 9 molecule, can be isolated.
  • a labeled ligand can be photoaffinity linked to i a cell extract, such as a membrane or a membrane extract, prepared 2 from cells that express a molecule that it binds, such as a 3 receptor molecule.
  • Cross-linked material is resolved by 4 polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray 5 film.
  • the labeled complex containing the ligand-receptor can be 6 excised, resolved into peptide fragments, and subjected to protein 7 microsequencing.
  • the amino acid sequence obtained from 8 microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule.
  • Polypeptides of the invention also can be used to asse ⁇ CCIII binding capacity of CCIII binding molecule ⁇ , ⁇ uch a ⁇ receptor molecules, in cell ⁇ or in cell-free preparation ⁇ .
  • Agoni ⁇ ts and antagonist ⁇ - assays and molecules The invention also provides a method of screening compounds to identify those which enhance or block the action of CCIII on cells, such as its interaction with CCIII-binding molecules ⁇ uch a ⁇ receptor molecule ⁇ .
  • An agonist is a compound which increases the natural biological functions of CCIII or which functions in a manner similar to CCIII, while antagonists decrease or eliminate such functions.
  • a cellular compartment such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds CCIII, such as a molecule of a signaling or regulatory pathway modulated by CCIII.
  • the preparation is incubated with labeled CCIII in the absence or the presence of a candidate molecule which may be a CCIII agoni ⁇ t or antagonist.
  • a candidate molecule which may be a CCIII agoni ⁇ t or antagonist.
  • the ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand.
  • Molecules which bind gratuitously, i.e., without inducing the effects of CCIII on binding the CCIII binding molecule are most likely to be good antagonist ⁇ .
  • Molecule ⁇ that bind well and elicit effect ⁇ that are the ⁇ ame as or closely related to CCIII are agonists.
  • CCIII-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of CCIII or molecules that elicit the same effect ⁇ as CCIII.
  • Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
  • an assay for CCIII antagonists is a competitive assay that combines CCIII and a potential antagonist with membrane-bound CCIII receptor molecules or recombinant CCIII 1 receptor molecule ⁇ under appropriate condition ⁇ for a competitive
  • CCIII can be labeled, such as by radioactivity
  • Potential antagonists include small organic molecules,
  • Potential antagonists also may be small organic molecules, a ⁇ o peptide, a polypeptide ⁇ uch as a closely related protein or ii antibody that binds the same site ⁇ on a binding molecule, such as
  • Potential antagonists include a small molecule which binds to
  • 19 small molecule ⁇ include but are not limited to ⁇ mall organic 0 molecules, peptides or peptide-like molecules. i Other potential antagonists include antisense molecule ⁇ . 2 Antisen ⁇ e technology can be u ⁇ ed to control gene expre ⁇ ion through 3 anti ⁇ en ⁇ e DNA or RNA or through triple-helix formation. Antisen ⁇ e 4 techniques are discussed, for example, in - Okano, J. Neurochem. 5 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF 6 GENE EXPRESSION, CRC Press, Boca Raton, FL (1988) .
  • Triple helix 7 formation is di ⁇ cussed in, for instance Lee et al., Nucleic Acids 8 Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); 9 and Dervan et al., Science 251: 1360 (1991) .
  • the methods are based 0 on binding of a polynucleotide to a complementary DNA or RNA.
  • the 5' coding portion of a polynucleotide that encodes the 2 mature polypeptide of the present invention may be used to design 3 an antisen ⁇ e RNA oligonucleotide of from about 10 to 40 base pairs 4 in length.
  • a DNA oligonucleotide is designed to be complementary 5 to a region of the gene involved in transcription thereby 6 preventing transcription and the production of CCIII.
  • the 7 antisen ⁇ e RNA oligonucleotide hybridize ⁇ to the mRNA in vivo and 8 blocks translation of the mRNA molecule into CCIII polypeptide.
  • the 9 oligonucleotides described above can also be delivered to cells 1 such that the antisense RNA or DNA may be expressed in vivo to
  • the antagonists may be employed in a composition with a
  • the antagonists may be employed for instance to inhibit the
  • auto-immune disea ⁇ e ⁇ examples include multiple 2 sclerosis, and insulin-dependent diabetes.
  • the antagonists may also be employed to treat infectious 4 disea ⁇ e ⁇ including ⁇ ilicosis, ⁇ arcoidosis, idiopathic pulmonary 5 fibrosis by preventing the recruitment and activation of 6 mononuclear phagocytes. They may also be employed to treat 7 idiopathic hyper-eo ⁇ inophilic ⁇ yndrome by preventing eo ⁇ inophil 8 production and migration.
  • Endotoxic ⁇ hock may also be treated by 9 the antagonists by preventing the migration of macrophages and 0 their production of the human chemokine polypeptides of the present i invention.
  • the antagonist ⁇ may al ⁇ o be employed for treating 3 atherosclerosis, by preventing monocyte infiltration in the artery 4 wall.
  • the antagonists may also be employed to treat histamine- 6 mediated allergic reactions and immunological disorders including 7 late phase allergic reactions, chronic urticaria, and atopic 8 dermatitis by inhibiting chemokine-induced mast cell and basophil 9 degranulation and release of histamine.
  • IgE-mediated allergic 0 reactions such as allergic asthma, rhinitis, and eczema may also i be treated.
  • the antagonists may also be employed to treat chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with seque ⁇ tration of mononuclear phagocytes in the lung. Antagonist ⁇ may also be employed to treat rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies. The antagonists may be employed to interfere with the deleterious cascade ⁇ attributed primarily to IL-1 and TNF, which prevent ⁇ the bio ⁇ ynthesis of other inflammatory cytokines.
  • the antagonist ⁇ may be employed to prevent inflammation.
  • the antagoni ⁇ ts may also be employed to inhibit prostaglandin- independent fever induced by chemokines.
  • the antagonists may also be employed to treat ca ⁇ e ⁇ of bone marrow failure, for example, apla ⁇ tic anemia and myelody ⁇ pla ⁇ tic syndrome.
  • the antagonists may also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung.
  • the antagoni ⁇ ts may also be employed to treat subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung.
  • the antagonists may also be employed to treat glomerulonephritis, cerebral ischemia and HTLV-1 related diseases.
  • the antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
  • compositions comprising the polynucleotide or the polypeptides discus ⁇ ed above or the agonists or antagonists.
  • the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject.
  • Such compo ⁇ itions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combination ⁇ thereof.
  • the formulation ⁇ hould ⁇ uit the mode of admini ⁇ tration.
  • the invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned composition ⁇ of the invention.
  • a ⁇ sociated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
  • compositions and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
  • the pharmaceutical composition ⁇ may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.
  • the pharmaceutical composition ⁇ generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications. In general, the composition ⁇ are administered in an amount of at lea ⁇ t about 10 ⁇ g/kg body weight. In mo ⁇ t cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day.
  • dose is from about 10 ug/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.
  • CCIII polynucleotides, polypeptides, agonists and antagonist ⁇ that are polypeptide ⁇ may be employed in accordance with the present invention by expre ⁇ ion of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy.”
  • cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide.
  • cells may be 1 engineered ex vivo by the use of a retroviral plasmid vector
  • cells may be engineered in vivo for expression of
  • polynucleotide of the invention may be engineered for expression
  • the retroviral expression construct then may be isolated and 10 introduced into a packaging cell is transduced with a retroviral ii plasmid vector containing RNA encoding a polypeptide of the present
  • 14 cells may be administered to a patient for engineering cells in is vivo and expres ⁇ ion of the polypeptide in vivo.
  • the retroviral pla ⁇ mid vector is derived 6 from Moloney Murine Leukemia Virus. 7 Such vectors well include one or more promoters for expressing 8 the polypeptide.
  • Suitable promoters which may be employed include, 9 but are not limited to, the retroviral LTR; the SV40 promoter; and 0 the human cytomegalovirus (CMV) promoter described in Miller et i al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., 2 cellular promoters such as eukaryotic cellular promoters including, 3 but not limited to, the histone, RNA polymerase III, and ⁇ -actin 4 promoters) .
  • Other viral promoters which may be employed include, 5 but are not limited to, adenovirus promoters, thymidine kina ⁇ e (TK) 6 promoters, and B19 parvovirus promoter ⁇ .
  • the ⁇ election of a 7 ⁇ uitable promoter will be apparent to tho ⁇ e skilled in the art from 8 the teachings contained herein.
  • the nucleic acid sequence encoding the polypeptide of the present invention will be placed under the control of a suitable promoter.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial viru ⁇ (RSV) promoter; inducible promoter ⁇ , ⁇ uch a ⁇ the MMT promoter, the metallothionein promoter; heat ⁇ hock promoter ⁇ ; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described) ; the ⁇ -actin promoter; and human growth hormone promoters.
  • CMV cytomegalovirus
  • RSV respiratory syncytial viru ⁇
  • inducible promoter ⁇ ⁇ uch a ⁇ the
  • the promoter also may be the native promoter which controls the gene encoding the polypeptide.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • Example ⁇ of packaging cell ⁇ which may be tran ⁇ fected include, but are not limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17- H2, YCRE, YCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14 (1990) .
  • the vector may be transduced into the packaging cells through any means known in the art.
  • retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then admini ⁇ tered to a ho ⁇ t.
  • the producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequence (s) encoding the polypeptides.
  • retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cell ⁇ , hepatocyte ⁇ , fibrobla ⁇ t ⁇ , myobla ⁇ ts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • ligations were accomplished using standard buffer ⁇ , incubation temperatures and times, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of DNA.
  • ligase T4 DNA ligase
  • the DNA sequence encoding human CCIII in the deposited polynucleotide was amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the human CCIII protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer had the sequence 5' CGCCCA TGGTGGCCGCCGCGCAGG 3' (SEQ ID NO:3) containing a Neo I restriction site, which encodes a start AUG, followed by 16 nucleotides of the human CCIII coding sequence set out in Figure 1.
  • the 3' primer had the ⁇ equence 5' CGCAAGCTTGCAGAG CTCAATTTA 3' (SEQ ID NO:4) containing the underlined BamHI restriction site followed by 15 nucleotides complementary to CCIII non-coding sequence set out in Figure 1, including the stop codon.
  • the restriction ⁇ ⁇ ites were convenient to restriction enzyme sites in the bacterial expres ⁇ ion vector ⁇ pQE-7 which were u ⁇ ed for bacterial expre ⁇ ion in the ⁇ e example ⁇ . (Qiagen, Inc. Chat ⁇ worth, CA) .
  • pQE-7 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori”) , an IPTG inducible promoter, a ribosome binding site (“RBS”) , a 6-His tag and restriction enzyme sites.
  • the amplified human CCIII DNA and the vector pQE-7 both were digested with Neo I and Hind III and the digested DNAs then were ligated together. Insertion of the CCIII DNA into the Neo I/Hind III restricted vector placed the CCIII coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in- frame with an initiating AUG appropriately positioned for translation of CCIII.
  • the ligation mixture was transformed into competent E. coli cells using standard procedures.
  • E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresse ⁇ lac repre ⁇ or and confers kanamycin resistance (“Kanr"), was used in carrying out the illustrative example described here.
  • This strain which i ⁇ only one of many that are ⁇ uitable for expre ⁇ sing CCIII, is available commercially from Qiagen. Transformant ⁇ were identified by their ability to grow on LB plates in the presence of ampicillin.
  • Plasmid DNA was isolated from resi ⁇ tant colonie ⁇ and the identity of the cloned DNA wa ⁇ confirmed by re ⁇ triction analy ⁇ i ⁇ .
  • Clones containing the de ⁇ ired constructs were grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ug/ml) and kanamycin (25 ug/ml) .
  • the 0/N culture was used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250.
  • the cells were grown to an optical density at 600nm ("OD600”) of between 0.4 and 0.6.
  • IPTG Isopropyl-B-D-thiogalactopyrano ⁇ ide
  • the 8M urea solution containing the solubilized protein was passed over a PD-10 column in 2X phosphate buffered saline ("PBS") , thereby removing the urea, exchanging the buffer and refolding the protein.
  • the protein was purified by a further step of chromatography to remove endotoxin. Then, it was sterile filtered.
  • the sterile filtered protein preparation was stored in 2X PBS at a concentration of 95 micrograms per mL. Analy ⁇ i ⁇ of the preparation by standard method ⁇ of polyacrylamide gel electrophore ⁇ i ⁇ revealed that the preparation contained about 90% monomer CCIII having the expected molecular weight of, approximately, 8.5 kDa.
  • the cDNA sequence encoding the full length human CCIII protein, in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
  • the 5' primer has the ⁇ equence 5' CGCGGATCCGCCATCATG GCGCCCGGAGTG 3' (SEQ ID NO:5) containing the underlined BamHI re ⁇ triction enzyme ⁇ ite followed by Kozak ⁇ equence and 15 ba ⁇ es of the sequence of CCIII of Figure 1. Inserted into an expres ⁇ ion vector, a ⁇ de ⁇ cribed below, the 5' end of the amplified fragment encoding human CCIII provides an efficient signal peptide.
  • the 3' primer has the sequence 5' CGCGGTACCGCAGAG CTCAATTTA 3' (SEQ ID NO:6) containing the underlined Asp718 restriction followed by nucleotides complementary to the last 15 nucleotides of the CCIII non-coding sequence set out in Figure 1, including the stop codon.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) .
  • the fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel.
  • This fragment is designated herein F2.
  • the vector pA2 is used to expres ⁇ the CCIII protein in the baculovirus expression system, using ⁇ tandard methods, such as those described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) .
  • Thi ⁇ expres ⁇ ion vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites.
  • the signal peptide of AcMNPV gp67 is located just upstream of a BamHI site.
  • the polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation.
  • SV40 simian virus 40
  • the beta-galacto ⁇ ida ⁇ e gene from E.coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
  • the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expres ⁇ the cloned polynucleotide.
  • baculoviru ⁇ vector ⁇ could be used in place of pA2- GP, ⁇ uch a ⁇ pAc373, pVL94l and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signal ⁇ for tran ⁇ cription, tran ⁇ lation, trafficking and the like, such a ⁇ an in-frame AUG and a ⁇ ignal peptide, a ⁇ required.
  • Such vectors are described in Luckow et al., Virology 170: 31-39, among others.
  • the plasmid is digested with the restriction enzymes Asp 718 band BamHI and then is dephosphorylated u ⁇ ing calf inte ⁇ tinal phosphata ⁇ e, u ⁇ ing routine procedure ⁇ known in the art.
  • the DNA i ⁇ then isolated from a 1% agarose gel using a commercially 1 available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This
  • V2 vector DNA
  • pBacCCIII 9 ⁇ equence of the cloned fragment is confirmed by DNA sequencing.
  • pBacCCIII i 5 ⁇ g of the plasmid pBacCCIII is co-transfected with 1.0 ⁇ g 2 of a commercially available linearized baculoviru ⁇ DNA 3 ("BaculoGoldTM baculoviru ⁇ DNA", Pharmingen, San Diego, CA.), u ⁇ ing 4 the lipofection method described by Feigner ⁇ t al. , Proc. Natl. 5 Acad. Sci. USA 84: 7413-7417 (1987) .
  • the plate is rocked back and forth to mix the newly added 4 solution.
  • the plate is then incubated for 5 hours at 27'C.
  • the transfection solution is removed from the plate and l 6 ml of Grace's insect medium supplemented with 10% fetal calf serum 7 is added.
  • the plate is put back into an incubator and cultivation 8 is continued at 27'C for four days.
  • 9 After four days the supernatant is collected and a plaque 0 assay is performed, as described by Summers and Smith, cited above.
  • An agarose gel with "Blue Gal" (Life Technologies Inc., 2 Gaithersburg) is used to allow easy identification and isolation 3 of gal-expres ⁇ ing clone ⁇ , which produce blue- ⁇ tained plaque ⁇ .
  • the agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatant ⁇ of the ⁇ e culture dishes are harvested and then they are stored at 4'C. A clone containing properly inserted CCIII is identified by DNA analysi ⁇ including re ⁇ triction mapping and ⁇ equencing. This is designated herein as V-CCIII. Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
  • the cells are infected with the recombinant baculovirus V-CCIII at a multiplicity of infection ("MOI") of about 2 (about l to about 3) .
  • MOI multiplicity of infection
  • the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg) .
  • 5 ⁇ Ci of 35S-methionine and 5 ⁇ Ci 35S cysteine available from Amersham
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled protein ⁇ are vi ⁇ ualized by SDS-PAGE and autoradiography.
  • the expres ⁇ ion plasmid, CCIII HA, i ⁇ made by cloning a cDNA encoding CCIII into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.) .
  • the expression vector pcDNAI/amp contains: (l) an E.coli origin of replication effective for propagation in E.
  • coli and other prokaryotic cell (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells,- (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expre ⁇ sion control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by mean ⁇ of restriction sites in the polylinker.
  • a DNA fragment encoding the entire CCIII precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al. , Cell 37: 767 (1984) .
  • the fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the pla ⁇ mid construction strategy is as follows.
  • the CCIII cDNA of the deposit clone is amplified using primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expres ⁇ ion of CCIII in E. coli and S. furgiperda.
  • primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expres ⁇ ion of CCIII in E. coli and S. furgiperda.
  • one of the primer ⁇ contain ⁇ a heamaglutinin tag ("HA tag") a ⁇ described above.
  • Suitable primers include that following, which are used in thi ⁇ example.
  • the 5' primer containing the underlined Bam HI ⁇ ite, an AUG start codon and 15 codons thereafter, has the following sequence,- 5' CGCGGATCCACCATGGCGCCCGGAGTGGCC 3' (SEQ ID NO:7).
  • the 3' primer containing the underlined Xba I site, ⁇ top codon, HA tag and 15 bp of 3' coding ⁇ equence (at the 3' end) has the following sequence 5 ' CGGTCTAGATCAAGCGTAGTCTGG GACGTCGTATGGGTACAAAGGGAAAGCCGG 3 ' (SEQ ID NO : 8 ) .
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Pla ⁇ mid DNA i ⁇ i ⁇ olated from re ⁇ i ⁇ tant colonie ⁇ and examined by restriction analysi ⁇ and gel ⁇ izing for the pre ⁇ ence of the CCIII-encoding fragment.
  • COS cell ⁇ are transfected with an expression vector, as de ⁇ cribed above, using DEAE-DEXTRAN, as de ⁇ cribed, for in ⁇ tance, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Pre ⁇ , Cold Spring Harbor, New York (1989) .
  • Cells are incubated under conditions for expres ⁇ ion of CCIII by the vector. 1 Expression of the CCIII HA fusion protein is detected by
  • Total cellular RNA 2 sample ⁇ are i ⁇ olated with RNAzolTM B ⁇ y ⁇ tem (Biotecx Laboratorie ⁇ , 3 Inc. 6023 South Loop Ea ⁇ t, Houston, TX 77033) . 4 About 10 ⁇ g of Total RNA is isolated from tissue samples.
  • RNA is size resolved by electrophoresis through a 1% agarose gel 6 under strongly denaturing conditions. RNA is blotted from the gel 7 onto a nylon filter, and the filter then is prepared for 8 hybridization to a detectably labeled polynucleotide probe. 9 As a probe to detect mRNA that encodes CCIII, the antisense 0 strand of the coding region of the cDNA insert in the depo ⁇ ited i clone i ⁇ labeled to a high specific activity.
  • the cDNA is labeled 2 by primer extension, using the Prime-It kit, available from 3 Stratagene. The reaction is carried out using 50 ng of the cDNA, 4 following the standard reaction protocol as recommended by the supplier.
  • the labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303. 1
  • the labeled probe is hybridized to the filter, at a
  • the flask is inverted - the chunks of tissue 9 remain fixed to the bottom of the flask - and fresh media is added 0 (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) .
  • fresh media 0 (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) .
  • the tissue is then incubated at 37'C for approximately one week. 2 At this time, fresh media is added and subsequently changed every 3 several days. After an additional two weeks in culture, a 4 monolayer of fibroblast ⁇ emerge ⁇ . The monolayer is trypsinized and 5 scaled into larger flasks. 6
  • a vector for gene therapy is digested with re ⁇ triction enzymes for cloning a fragment to be expres ⁇ ed.
  • the dige ⁇ ted vector is treated with calf intestinal phosphata ⁇ e to prevent ⁇ elf-ligation.
  • the dephosphorylated, linear vector is fractionated on an agarose gel and purified. Cytostatin cDNA capable of expressing active CCIII, is isolated. The ends of the fragment are modified, if neces ⁇ ary, for cloning into the vector. For instance, 5" overhanging may be treated with DNA polymerase to create blunt ends. 3 ' overhanging ends may be removed using Si nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase. Equal quantities of the Moloney murine leukemia virus linear backbone and the CCIII fragment are mixed together and joined using T4 DNA ligase.
  • the ligation mixture is used to transform E. Coli and the bacteria are then plated onto agar-containing kanamycin. Kanamycin phenotype and restriction analysis confirm that the vector has the properly inserted gene.
  • Packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • the vector containing the CCIII gene is introduced into the packaging cells by standard techniques. Infectious viral particles containing the CCIII gene are collected from the packaging cells, which now are called producer cells. Fresh media is added to the producer cells, and after an appropriate incubation period media is harvested from the plates of confluent producer cells.
  • the media containing the infectious viral particles, is filtered through a Millipore filter to remove detached producer cells.
  • the filtered media then is used to infect fibroblast cells.
  • Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the filtered media.
  • Polybrene (Aldrich) may be included in the media to facilitate transduction. After appropriate incubation, the media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblast ⁇ will be infected and no ⁇ election is required. If the titer is low, then it is necessary to use a retroviral vector that has a ⁇ electable marker, such as neo or his, to select out transduced cells for expansion.
  • Engineered fibroblasts then may be injected into rats, either alone or after having been grown to confluence on microcarrier beads, such as cytodex 3 beads.
  • the injected fibroblast ⁇ produce CCIII product, and the biological actions of the protein are conveyed to the ho ⁇ t.
  • the invention may be practiced otherwi ⁇ e than a ⁇ particularly de ⁇ cribed in the foregoing de ⁇ cription and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

Abstract

Human chemotactic cytokine III polypeptides and DNA (RNA) encoding such chemotactic cytokines and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such chemotactic cytokines for the treatment of leukemia, tumors, chronic infections, auto-immune disease, fibrotic disorders, wound healing and psoriasis. Antagonists against such chemotactic cytokines and their use as a therapeutic to treat rheumatoid arthritis, auto-immune and chronic and acute inflammatory and infective diseases, allergic reactions, prostaglandin-independent fever, cerebral ischemia, glomerulonephritis, HTLV-1 related diseases and bone marrow failure are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to mutations in the nucleic acid sequences and altered concentrations of the polypeptides. Also disclosed are diagnostic assays for detecting mutations in the polynucleotides encoding chemotactic cytokine III and for detecting altered levels of the polypeptide in a host.

Description

2
3
4
5
6
7 β
9 10 11
12 CHEMOTACTIC CYTOKINE III
13
14 This invention relates, in part, to newly identified is polynucleotides and polypeptides; variants and derivatives of the
16 polynucleotides and polypeptides; processes for making the
17 polynucleotides and the polypeptides, and their variants and ie derivatives; agonists and antagonists of the polypeptides; and uses
19 of the polynucleotides, polypeptides, variants, derivatives,
20 agonists and antagonists. In particular, in these and in other i regards, the invention relates to polynucleotides and polypeptides 2 of human chemotactic cytokine III, hereinafter referred to as 3 "CCIII". 4 5 BACKGROUND OF THE INVENTION 6 The ability to control the migration and "trafficking" of 7 various cell types is controlled by a subset of factors, or 8 proteins, among which cytokines, and in particular chemotactic 9 cytokines or chemokines, are an example. 0 Chemokines, also referred to as intercrine cytokines, are a i subfamily of structurally and functionally related chemotactic 2 cytokines. These molecules are 8-10 kd in size. In general, 3 chemokines exhibit 20% to 75% homology at the amino acid level and are characterized by four conserved cysteine residues that form two 5 disulfide bonds. Based on the arrangement of the first two 6 cysteine residues, chemokines have been classified subfamilies, 7 according to the arrangement of the cysteine residues with respect 8 to the nearest cysteine residue. In the alpha subfamily, for 9 example, the first two cysteines are separated by one amino acid
-1- ι and hence are referred to as the "C-X-C" subfamily. in the beta
2 subfamily, the two cysteines are in an adjacent position and are,
3 therefore, referred to as the "C-C" subfamily.
4 The intercrine cytokines exhibit a wide variety of functions.
5 A hallmark feature is their ability to elicit chemotactic migration
6 of distinct cell types, including monocytes, neutrophils, T
7 lymphocytes, basophils and fibroblasts. Many chemokines have β proinflammatory activity and are involved in multiple steps during 9 an inflammatory reaction. These activities include stimulation of 0 histamine release, lysosomal enzyme and leukotriene release, i increased adherence of target immune cells to endothelial cells, enhanced binding of complement proteins, induced expression of granulocyte adhesion molecules and complement receptors, and respiratory burst. In addition to their involvement in inflammation, certain chemokines have been shown to exhibit other activities. For example, macrophage inflammatory protein 1 (MIP-l) is able to suppress hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent inhibitor of endothelial cell growth, Interleukin-3 (IL-8) promotes proliferation of keratinocytes, and GRO is an autocrine growth factor for melanoma cells. In light of the diverse biological activities, it is not surprising that chemokines have been implicated in a number of physiological and disease conditions, including lymphocyte trafficking, wound healing, hematopoietic regulation and immunological disorders such as allergy, asthma and arthritis. Members of the "C-C" branch exert their effects on the following cells: eosinophils which destroy parasites to lessen parasitic infection and cause chronic inflammation in the airways of the respiratory system; macrophageε which suppress tumor formation in vertebrates; and basophils which release histamine which plays a role in allergic inflammation. However, members of one branch may exert an effect on cells which are normally responsive to the other branch of chemokines and, therefore, no precise role can be attached to the members of the branches. While members of the C-C branch act predominantly on mononuclear cells and members of the C-X-C branch act predominantly on neutrophils a distinct chemoattractant property cannot be assigned to a chemokine based on this guideline. Some chemokines from one family show characteristics of the other. 1 Clearly, there is a need for identification and
2 characterization of such factors that regulate the migration of
3 cells, particularly cells of the immune system, and which can play a role in preventing, ameliorating or correcting dysfunctions or
5 diseases.
6 The polypeptide of the present invention has the conserved
7 cysteine residues of the "C-X-X-C" region, and has amino acid β sequence homology to known chemokines.
9 0 SUMMARY OF THE INVENTION i Toward these ends, and others, it is an object of the present 2 invention to provide polypeptides, inter alia, that have been 3 identified as novel CCIII polypeptides by homology between the 4 amino acid sequence set out in Figure 1 (SEQ ID NO:2) and known s amino acid sequences of other proteins such as murine cytokine- 6 induced neutrophil chemoattractant 2 set out in Figure 2 and (SEQ 7 ID NO:9) . 8 It is a further object of the invention, moreover, to provide 9 polynucleotides that encode polypeptides having chemotactic 0 activity, particularly polynucleotides that encode the polypeptide i herein designated CCIII. 2 In a particularly preferred embodiment of this aspect of the 3 invention the polynucleotide comprises the region encoding human 4 CCIII in the sequence set out in Figure l (SEQ ID NO:l) . 5 In accordance with this aspect of the present invention there is provided an isolated nucleic acid molecule encoding a mature polypeptide expressed by the human cDNA contained in ATCC Deposit No. 97408. In accordance with this aspect of the invention there are provided isolated nucleic acid molecules encoding human CCIII, including mRNAs, cDNAs, genomic DNAs and, in further embodiments of this aspect of the invention, biologically, diagnostically, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of the variants, analogs and derivatives. Among the particularly preferred embodiments of this aspect of the invention are naturally occurring allelic variants of human CCIII. It also is an object of the invention to provide CCIII polypeptides, particularly human CCIII polypeptides, that may be employed for therapeutic purposes, for example, to treat tumors, chronic infections, leukemia, T-cell mediated auto-immune diseases, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate growth factor activity, to inhibit angiogenesis and to promote wound healing. In accordance with this aspect of the invention there are provided novel polypeptides of human origin referred to herein as CCIII as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing. Among the particularly preferred embodiments of this aspect of the invention are variants of human CCIII encoded by naturally occurring alleles of the human CCIII gene. It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned CCIII polypeptides comprising culturing host cells having expressibly incorporated therein an exogenously-derived human CCIII-encoding polynucleotide under conditions for expression of human CCIII in the host and then recovering the expressed polypeptide. In accordance with another object the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia . In accordance with certain preferred embodiments of this aspect of the invention, there are provided products, compositions and methods, inter alia , for, among other things: assessing CCIII expression in cells by determining CCIII polypeptides or CCIII- encoding mRNA; to treat tumors, chronic infections, leukemia, T- cell mediated auto-immune diseases, parasitic infections, psoriasis, asthma, allergy, to regulate hematopoiesis, to stimulate growth factor activity, to inhibit angiogenesis and to promote wound healing in vi tro, ex vivo or in vivo by exposing cells to CCIII polypeptides or polynucleotides as disclosed herein,- assaying 1 genetic variation and aberrations, such as defects, in CCIII genes;
2 and administering a CCIII polypeptide or polynucleotide to an
3 organism to augment CCIII function or remediate CCIII dysfunction.
4 In accordance with certain preferred embodiments of this and
5 other aspects of the invention there are provided probes that
6 hybridize to human CCIII sequences.
7 In certain additional preferred embodiments of this aspect of
8 the invention there are provided antibodies against CCIII
9 polypeptides. In certain particularly preferred embodiments in 10 this regard, the antibodies are highly selective for human CCIII.
11
12 In accordance with another aspect of the present invention,
13 there are provided CCIII agonists. Among preferred agonists are
14 molecules that mimic CCIII, that bind to CCIII-binding molecules is or receptor molecules, and that elicit or augment CCIII-induced
16 responses. Also among preferred agonists are molecules that
17 interact with CCIII or CCIII polypeptides, or with other modulators ie of CCIII activities, and thereby potentiate or augment an effect
19 of CCIII or more than one effect of CCIII.
20 In accordance with yet another aspect of the present 2i invention, there are provided CCIII antagonists. Among preferred 2 antagonists are those which mimic CCIII so as to bind to CCIII 3 receptor(s) or binding molecules but not elicit a CCIII-induced 4 response or more than one CCIII-induced response. Also among 5 preferred antagonists are molecules that bind to or interact with 6 CCIII so as to inhibit an effect of CCIII or more than one effect 7 of CCIII or which prevent expression of CCIII . The antagonists may 8 be employed to treat and/or prevent, for example, 9 glomerulonephritis, inflammation, cerebral ischemia, HTLV-1 related 0 diseases, arthritis, infectious diseases, auto-immune diseases, i hyper-eosinophilic syndrome, endotoxic shock, ahterosclerosis, 2 allergies, bone marrow failure and asthma. 3 In further aspect of the invention there are provided 4 compositions comprising a CCIII polynucleotide or a CCIII 5 polypeptide for administration to cells in vi tro, to cells ex vivo 6 and to cells in vivo, or to a multicellular organism. In certain 7 particularly preferred embodiments of this aspect of the invention, 8 the compositions comprise a CCIII polynucleotide for expression of 9 a CCIII polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of CCIII. Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS The following drawings depict certain embodiments of the invention. They are illustrative only and do not limit the invention otherwise disclosed herein. Figure 1 shows the nucleotide and deduced amino acid sequence of human CCIII, wherein the underlined portion represents a putative leader sequence. Figure 2 shows the regions of similarity between amino acid sequences of CCIII and murine cytokine-induced neutrophil chemoattractant 2 polypeptides. Figure 3 shows structural and functional features of CCIII deduced by the indicated techniques, as a function of amino acid sequence. GLOSSARY The following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the examples. The explanations are provided as a convenience and are not limitative of the invention. DIGESTION of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled artisan. For analytical purposes, typically, 1 μg of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 μl 1 of reaction buffer. For the purpose of isolating DNA fragments for
2 plasmid construction, typically 5 to 50 μg of DNA are digested with
3 20 to 250 units of enzyme in proportionately larger volumes.
4 Appropriate buffers and substrate amounts for particular
5 restriction enzymes are described in standard laboratory manuals,
6 such as those referenced below, and they are specified by
7 commercial suppliers.
8 Incubation times of about 1 hour at 37'C are ordinarily used,
9 but conditions may vary in accordance with standard procedures, the 10 supplier's instructions and the particulars of the reaction. After ii digestion, reactions may be analyzed, and fragments may be purified
12 by electrophoresis through an agarose or polyacrylamide gel, using 3 well known methods that are routine for those skilled in the art. GENETIC ELEMENT generally means a polynucleotide comprising 5 a region that encodes a polypeptide or a region that regulates 6 transcription or translation or other processes important to 7 expression of the polypeptide in a host cell, or a polynucleotide 8 comprising both a region that encodes a polypeptide and a region 9 operably linked thereto that regulates expression. 0 Genetic elements may be comprised within a vector that i replicates as an episomal element; that is, as a molecule 2 physically independent of the host cell genome. They may be 3 comprised within mini-chromosomes, such as those that arise during 4 amplification of transfected DNA by methotrexate selection in 5 eukaryotic cells. Genetic elements also may be comprised within 6 a host cell genome; not in their natural state but, rather, 7 following manipulation such as isolation, cloning and introduction 8 into a host cell in the form of purified DNA or in a vector, among 9 others. 0 ISOLATED means altered "by the hand of man" from its natural i state; i.e., that, if it occurs in nature, it has been changed or 2 removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide naturally present in a living animal in its natural state is not "isolated, " but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. For example, with respect to polynucleotides, the term isolated means that it is separated from the chromosome and cell in which it naturally occurs. As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNAs, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or in whole organisms. Introduced into host cells in culture or in whole organisms, such DNAs still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleotides and polypeptides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides or polypeptides, for example, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides or polypeptides within the meaning of that term as it is employed herein. LIGATION refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double stranded DNAs. Techniques for ligation are well known to the art and protocols for ligation are described in standard laboratory manuals and references, such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed. ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) and Maniatis et al., pg. 146, as cited below. OLIGONUCLEOTIDE (S) refers to relatively short polynucleotides. Often the term refers to single-stranded deoxyribonucleotides, but it can refer as well to single-or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others. Oligonucleotideε, such as single-stranded DNA probe oligonucleotideε, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms. Initially, chemically synthesized DNAs typically are obtained without a 5' phosphate. The 5' ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules. Where ligation of such oligonucleotides is desired, a phosphate can be added by standard techniques, such as those that employ a kinase and ATP. The 3 ' end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5' phosphate of another polynucleotide, such as another oligonucleotide. As is well known, this reaction can be prevented selectively, where desired, by removing the 5' phosphates of the other polynucleotide (ε) prior to ligation. PLASMIDS generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure. POLYNUCLEOTIDE (S) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, εingle- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules . One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual baseε, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. POLYPEPTIDES, as used herein, includes all polypeptides as described below. The basic structure of polypeptides is well known and has been described in innumerable textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art . Even the common modifications that occur naturally in polypeptides are too numerouε to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those i of skill in the art. Among the known modifications which may
2 be present in polypeptides of the present are, to name an
3 illustrative few, acetylation, acylation, ADP-ribosylation,
4 amidation, covalent attachment of flavin, covalent attachment of
5 a heme moiety, covalent attachment of a nucleotide or nucleotide
6 derivative, covalent attachment of a lipid or lipid derivative,
7 covalent attachment of phosphotidylinositol, cross-linking,
8 cyclization, disulfide bond formation, demethylation, formation of
9 covalent cross-links, formation of cystine, formation of 10 pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI ii anchor formation, hydroxylation, iodination, methylation,
12 myristoylation, oxidation, proteolytic processing, phosphorylation,
13 prenylation, racemization, selenoylation, sulfation, transfer-RNA
14 mediated addition of amino acids to proteins such as arginylation, is and ubiquitination. ie Such modifications are well known to those of skill and have
17 been described in great detail in the scientific literature.
18 Several particularly common modifications, glycosylation, lipid
19 attachment, sulfation, gamma-carboxylation of glutamic acid
20 residueε, hydroxylation and ADP-ribosylation, for instance, are i described in most basic texts, such as, for instance PROTEINS - 2 STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton, W. 3 H. Freeman and Company, New York (1993) . Many detailed reviews are 4 available on this subject, such as, for example, those provided by 5 Wold, F., Posttranslational Protein Modifications.- Perspectives and 6 Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF 7 PROTEINS, B. C. Johnson, Ed., Academic Press, New York (1983); 8 Seif er et al. , Analysis for protein modifications and nonprotein 9 cofactors, Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., 0 Protein Synthesis: Posttranslational Modifications and Aging, Ann. 1 N.Y. Acad. Sci. 663: 48-62 (1992). 2 It will be appreciated, as is well known and as noted above, 3 that polypeptides are not always entirely linear. For instance, 4 polypeptides may be branched as a result of ubiquitination, and 5 they may be circular, with or without branching, generally as a 6 result of posttranslation events, including natural processing 7 event and events brought about by human manipulation which do not 8 occur naturally. Circular, branched and branched circular i polypeptides may be synthesized by non-translation natural process
2 and by entirely synthetic methods, as well.
3 Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or
5 carboxyl termini. In fact, blockage of the amino or carboxyl group
6 in a polypeptide, or both, by a covalent modification, is common
7 in naturally occurring and synthetic polypeptides and such
8 modifications may be present in polypeptides of the present
9 invention, as well. For instance, the amino terminal residue of 0 polypeptides made in E. coli, prior to proteolytic processing, i almost invariably will be N-formylmethionine. 2 The modifications that occur in a polypeptide often will be 3 a function of how it is made. For polypeptides made by expressing 4 a cloned gene in a host, for instance, the nature and extent of the s modifications in large part will be determined by the host cell 6 posttranslational modification capacity and the modification 7 signals present in the polypeptide amino acid sequence. For s instance, as is well known, glycosylation often does not occur in 9 bacterial hosts such as E. coli. Accordingly, when glycosylation 0 is desired, a polypeptide should be expressed in a glycosylating i host, generally a eukaryotic cell. Insect cell often carry out the 2 same posttranslational glycosylations as mammalian cells and, for 3 this reason, insect cell expression systems have been developed to 4 express efficiently mammalian proteins having native patterns of 5 glycosylation, inter alia. Similar considerations apply to other 6 modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. In general, as used herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides syntheεized by expressing a polynucleotide in a host cell. VARIANT(S) of polynucleotides or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail. 1 (1) A polynucleotide that differs in nucleotide sequence from
2 another, reference, polynucleotide. Generally, differences are
3 limited so that the nucleotide sequences of the reference and the
4 variant are closely similar overall and, in many regions,
5 identical.
6 As noted below, changes in the nucleotide sequence of the
7 variant may be silent. That is, they may not alter the amino acids β encoded by the polynucleotide. Where alterations are limited to 9 silent changes of this type a variant will encode a polypeptide 0 with the same amino acid sequence as the reference. Also as noted i below, changes in the nucleotide sequence of the variant may alter 2 the amino acid sequence of a polypeptide encoded by the reference 3 polynucleotide. Such nucleotide changes may result in amino acid 4 substitutions, additions, deletions, fusions and truncations in the 5 polypeptide encoded by the reference sequence, as discussed below. 6 7 (2) A polypeptide that differs in amino acid sequence from 8 another, reference polypeptide. Generally, differences are limited 9 so that the sequences of the reference and the variant are closely 0 similar overall and, in many region, identical. i A variant and reference polypeptide may differ in amino acid 2 sequence by one or more substitutions, additions, deletions, 3 fusions and truncations, which may be present in any combination. 4 5 RECEPTOR MOLECULE, as used herein, refers to molecules which 6 bind or interact specifically with CCIII polypeptides of the present invention, including not only classic receptors, which are preferred, but also other molecules that specifically bind to or interact with polypeptides of the invention (which also may be referred to as "binding molecules" and "interaction molecules," respectively and as "CCIII binding molecules" and "CCIII interaction molecules." Binding between polypeptides of the invention and such molecules, including receptor or binding or interaction molecules may be exclusive to polypeptides of the invention, which is very highly preferred, or it may be highly specific for polypeptides of the invention, which is highly preferred, or it may be highly specific to a group of proteins that includes polypeptides of the invention, which is preferred, or it 1 may be specific to several groups of proteins at least one of which
2 includes polypeptides of the invention.
3 Receptors also may be non-naturally occurring, such as
4 antibodies and antibody-derived reagents that bind specifically to
5 polypeptides of the invention.
6
7 DESCRIPTION OF THE INVENTION
8 The present invention relates to novel CCIII polypeptides and
9 polynucleotides, among other things, as described in greater detail 10 below. In particular, the invention relates to polypeptides and ii polynucleotides of a novel human CCIII, which is related by amino
12 acid sequence homology to murine cytokine-induced neutrophil
13 chemoattractant-2. The invention relates especially to CCIII
14 having the nucleotide and amino acid sequences set out in Figure
15 1 (SEQ ID NOS:l and 2) , and to the CCIII nucleotide and amino acid
16 sequences of the human cDNA in ATCC Deposit No. 97408, which is
17 herein referred to as "the deposited clone" or as the "cDNA of the ie deposited clone." It will be appreciated that the nucleotide and
19 amino acid sequences set out in Figure 1 (SEQ ID N0S:l and 2) were
20 obtained by sequencing the cDNA of the deposited clone. Hence, the 2i sequence of the deposited clone is controlling as to any
22 discrepancies between the two and any reference to the sequence of
23 Figure 1 (SEQ ID N0:1) includes reference to the sequence of the 4 human cDNA of the deposited clone. 5 6 Polynucleotides 7 In accordance with one aspect of the present invention, there 8 are provided isolated polynucleotides which encode the CCIII 9 polypeptide having the deduced amino acid sequence of Figure l (SEQ 0 ID NO:2) or the CCIII polypeptide encoded by the cDNA in the i deposited clone. 2 Using the information provided herein, such as the 3 polynucleotide sequence set out in Figure 1 (SEQ ID N0:l) , a 4 polynucleotide of the present invention encoding human CCIII 5 polypeptide may be obtained using standard cloning and screening 6 procedures, such as those for cloning cDNAs using mRNA from cells 7 of a human adrenal gland tumor, cerebellum, fetal heart and fetal 8 lung as starting material. Illustrative of the invention, the 1 polynucleotide set out in Figure 1 (SEQ ID N0:1) was discovered in
2 a cDNA library derived from human microvascular endothelial cells.
3 Human CCIII of the invention is structurally related to other proteins of the chemokine family, as shown by the results of
5 sequencing the human cDNA encoding CCIII in the deposited clone.
6 The cDNA sequence thus obtained is set out in Figure l (SEQ ID
7 N0:1). It contains an open reading frame encoding a protein of
8 about 81 amino acid residues which contains a putative leader
9 sequence of 28 amino acids such that the mature protein comprises 10 53 amino acids. CCIII has a deduced molecular weight of about ii 8.5682 kDa, has a Molar extinction coefficient of 14660±5%, an
12 isoelectric point of 8.0 and a 1.73 charge at pH of 7.0.
13 The protein exhibits greatest homology to murine cytokine-
14 induced neutrophil chemoattractant 2 among known proteins, wherein
15 there is about 32.353 % identity and about 45.588 % similarity.
16 Polynucleotides of the present invention may be in the form
17 of RNA, such as mRNA, or in the form of DNA, including, for ie instance, cDNA and genomic DNA obtained by cloning or produced by
19 chemical synthetic techniques or by a combination thereof. The DNA
20 may be double-stranded or single-stranded. Single-stranded DNA may 2i be the coding strand, also known as the sense strand, or it may be 2 the non-coding strand, also referred to as the anti-sense strand. 3 4 The coding sequence which encodes the polypeptide may be 5 identical to the coding sequence of the polynucleotide shown in 6 Figure 1 (SEQ ID NO:l) or that of the deposited clone. It also may 7 be a polynucleotide with a different sequence, which, as a result 8 of the redundancy (degeneracy) of the genetic code, encodes the 9 polypeptide of the DNA of Figure 1 (SEQ ID NO:l) or of the 0 deposited cDNA. 1 Polynucleotides of the present invention which encode the 2 polypeptide of Figure 1 (SEQ ID NO:2) or the polypeptide encoded 3 by the deposited cDNA may include, but are not limited to the 4 coding sequence for the mature polypeptide, by itself; the coding 5 sequence for the mature polypeptide and additional coding 6 sequences, such as those encoding a leader or secretory sequence, 7 such as a pre-, or pro- or prepro- protein sequence; the coding 8 sequence of the mature polypeptide, with or without the 9 aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example -ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, for instance, the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector, among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci., USA 86: 821- 824 (1989) , for instance, hexa-histidine provides for convenient purification of the fusion protein. The HA tag corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984), for instance. In accordance with the foregoing, the term "polynucleotide encoding a polypeptide" as used herein encompasses polynucleotides which include a sequence encoding a polypeptide of the present invention, particularly the human CCIII having the amino acid sequence set out in Figure 1 (SEQ ID NO:2) or the amino acid sequence of the human CCIII encoded by the cDNA of the deposited clone. The term encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by introns) together with additional regions, that also may contain coding and/or non-coding sequences. The present invention further relates to variants of the herein above-described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the deposited clone. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis 1 techniques, including those applied to polynucleotides, cells or
2 organisms.
3 Among variants in this regard are variants that differ from the aforementioned polynucleotides by nucleotide substitutions,
5 deletions or additions. The substitutions, deletions or additions
6 may involve one or more nucleotides. The variants may be altered
7 in coding or non-coding regions or both. Alterations in the coding
8 regions may produce conservative or non-conservative amino acid
9 substitutions, deletions or additions.
10 Among the particularly preferred embodiments of the invention ii in this regard are polynucleotides encoding polypeptides having the
12 amino acid sequence of CCIII set out in Figure 1 (SEQ ID NO:2) or
13 the amino acid sequence of CCIII of the cDNA of the deposited 4 clone; variants, analogs, derivatives and fragments thereof, and 5 fragments of the variants, analogs and derivatives. 6 Further particularly preferred in this regard are 7 polynucleotides encoding CCIII variants, analogs, derivatives and 8 fragments, and variants, analogs and derivatives of the fragments, 9 which have the amino acid sequence of the CCIII polypeptide of 0 Figure 1 (SEQ ID NO:2) or of the deposit in which several, a few, 1 5 to 10, 1 to 5, 1 to 3, 2, l or no amino acid residues are 2 substituted, deleted or added, in any combination. Especially 3 preferred among these are silent substitutions, additions and 4 deletions, which do not alter the properties and activities of the 5 CCIII. Also especially preferred in this regard are conservative 6 substitutions . Most highly preferred are polynucleotides encoding 7 polypeptides having the amino acid sequence of Figure l (SEQ ID 8 NO:2) or of the deposit, without substitutions. 9 Further preferred embodiments of the invention are 0 polynucleotides that are at least 70% identical to a polynucleotide i encoding the CCIII polypeptide having the amino acid sequence set 2 out in Figure l (SEQ ID NO:2) , and polynucleotides which are 3 complementary to such polynucleotides. Alternatively, highly preferred are polynucleotides that comprise a region that is at least 80% identical to a polynucleotide encoding the CCIII polypeptide of the cDNA of the deposited clone and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred. Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred. Particularly preferred embodiments in this respect, moreover, are polynucleotides which encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 (SEQ ID N0:1) or the human cDNA of the deposited clone. The present invention further relates to polynucleotides that hybridize to the herein above-described sequences. In this regard, the present invention especially relates to polynucleotides which hybridize under stringent conditions to the herein above-described polynucleotides. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. As discussed additionally herein regarding polynucleotide assays of the invention, for instance, polynucleotides of the invention as discussed above, may be used as a hybridization probe for cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding CCIII and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the human CCIII gene. Such probes generally will comprise at least 15 bases. Preferably, such probes will have at least 30 bases and may have at least 50 bases. Particularly preferred probes will have at least 30 bases and will have 50 bases or less. For example, the coding region of the CCIII gene may be isolated by screening using the known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to. The polynucleotides and polypeptides of the present invention may be employed as research reagents and materials for discovery of treatments and diagnostics to human disease, as further discussed herein relating to polynucleotide assays, inter alia. The polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) . Such sequences may play a role in processing of a protein from precursor to a mature form, may facilitate protein trafficking, may prolong or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be processed away from the mature protein by cellular enzymes. In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein) , a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
Deposited materials A deposit containing a human CCIII cDNA has been deposited with the American Type Culture Collection, as noted above. Also as noted above, the cDNA deposit is referred to herein as "the deposited clone" or as "the cDNA of the deposited clone." The deposited clone was deposited with the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Maryland 20852, USA, on January 2, 1996 and assigned ATCC Deposit No. 97408. The deposited material is a pBluescript SK (-) plasmid (Stratagene, La Jolla, CA) that contains the full length CCIII cDNA. The deposit has been made under the terms of the Budapest Treaty on the international recognition of the deposit of micro- organisms for purposes of patent procedure. The strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent. The deposit is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. §112. Thus, the present invention relates to the sequence of the polynucleotides contained in the deposited material, as well as the amino acid sequence of the polypeptide encoded thereby, are 1 controlling in the event of any conflict with any description of
2 sequences herein.
3 A license may be required to make, use or sell the deposited
4 materials, and no such license is hereby granted.
5
6 Polypeptides
7 The present invention further relates to a human CCIII
8 polypeptide which has the deduced amino acid sequence of Figure 1
9 (SEQ ID NO:2) . The polypeptides of the present invention include o the polypeptide of SEQ ID NO:2 (in particular the mature i polypeptide) as well as polypeptides which have at least 70% 2 similarity (preferably at least 70% identity) to the polypeptide 3 of SEQ ID NO:2 and more preferably at least 90% similarity (more 4 preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 s and still more preferably at least 95% similarity (still more 6 preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 7 and also include portions of such polypeptides with such portion 8 of the polypeptide generally containing at least 30 amino acids and 9 more preferably at least 50 amino acids. 0 As known in the art "similarity" between two polypeptides is i determined by comparing the amino acid sequence and its conserved 2 amino acid substitutes of one polypeptide to the sequence of a 3 second polypeptide. 4 The invention also relates to fragments, analogs and 5 derivatives of these polypeptides. The terms "fragment," 6 "derivative" and "analog" when referring to the polypeptide of 7 Figure 1 (SEQ ID NO:2) or that encoded by the deposited human cDNA, β means a polypeptide which retains essentially the same biological 9 function or activity as such polypeptide. Thus, an analog includes 0 a proprotein which can be activated by cleavage of the proprotein 1 portion to produce an active mature polypeptide. 2 The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide. In certain preferred embodiments it is a recombinant polypeptide. The fragment, derivative or analog of the polypeptide of 6 Figure 1 (SEQ ID NO:2) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be 1 one encoded by the genetic code, or (ii) one in which one or more
2 of the amino acid residues includes a substituent group, or (iii)
3 one in which the mature polypeptide is fused with another compound,
4 such as a compound to increase the half-life of the polypeptide
5 (for example, polyethylene glycol) , or (iv) one in which the
6 additional amino acids are fused to the mature polypeptide, such
7 as a leader or secretory sequence or a sequence which is employed
8 for purification of the mature polypeptide or a proprotein
9 sequence. Such fragments, derivatives and analogs are deemed to lo be within the scope of those skilled in the art from the teachings ii herein.
12 Among the particularly preferred embodiments of the invention
13 in this regard are polypeptides having the amino acid sequence of
14 CCIII set out in Figure 1 (SEQ ID NO:2), variants, analogs, is derivatives and fragments thereof, and variants, analogs and
16 derivatives of the fragments. Alternatively, particularly
17 preferred embodiments of the invention in this regard are ie polypeptides having the amino acid sequence of the CCIII of the
19 cDNA in the deposited clone, variants, analogs, derivatives and
20 fragments thereof, and variants, analogs and derivatives of the i fragments. 2 Among preferred variants are those that vary from a reference 3 by conservative amino acid substitutions . Such substitutions are 4 those that substitute a given amino acid in a polypeptide by 5 another amino acid of like characteristics. Typically seen as 6 conservative substitutions are the replacements, one for another, 7 among the aliphatic amino acids Ala, Val, Leu and lie; interchange 8 of the hydroxyl residues Ser and Thr, exchange of the acidic 9 residues Asp and Glu, substitution between the amide residues Asn 0 and Gin, exchange of the basic residues Lys and Arg and 1 replacements among the aromatic residues Phe, Tyr. 2 Further particularly preferred in this regard are variants, 3 analogs, derivatives and fragments, and variants, analogs and 4 derivatives of the fragments, having the amino acid sequence of the 5 CCIII polypeptide of Figure l (SEQ ID NO:2) or of the cDNA in the 6 deposited clone, in which several, a few, 5 to 10, l to 5, l to 3, 7 2, 1 or no amino acid residues are substituted, deleted or added, 8 in any combination. Especially preferred among these are silent 9 substitutions, additions and deletions, which do not alter the properties and activities of the CCIII . Also especially preferred in this regard are conservative substitutions. Most highly preferred are polypeptides having the amino acid sequence of Figure 1 (SEQ ID NO:2) or that encoded by the deposited clone without substitutions. The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
Fragments Also among preferred embodiments of this aspect of the present invention are polypeptides comprising fragments of CCIII, most particularly fragments of the CCIII having the amino acid set out in Figure 1 (SEQ ID NO:2) , and fragments of variants and derivatives of the CCIII of Figure 1 (SEQ ID NO:2) . In this regard a fragment is a polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned CCIII polypeptides and variants or derivatives thereof. Such fragments may be "free-standing," i.e., not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region. When comprised within a larger polypeptide, the presently discussed fragments most preferably form a single continuous region. However, several fragments may be comprised within a single larger polypeptide. For instance, certain preferred embodiments relate to a fragment of a CCIII polypeptide of the present comprised within a precursor polypeptide designed for expression in a host and having heterologous pre and pro- polypeptide regions fused to the amino terminus of the CCIII fragment and an additional region fused to the carboxyl terminus of the fragment. Therefore, fragments in one aspect of the meaning intended herein, refers to the portion or portions of a fusion polypeptide or fusion protein derived from CCIII. Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full- length polypeptide by peptide synthesis,- therefore, the fragments may be employed as intermediates for producing the full-length polypeptides . Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention. As representative examples of polypeptide fragments of the invention, there may be mentioned those which have from about 15 to about 81 amino acids. In this context about includes the particularly recited range and ranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acid at either extreme or at both extremes. For instance, about 30-81 amino acids in this context means a polypeptide fragment of 30 or minus several, a few, 5, 4, 3, 2 or 1 amino acids to 81 plus or minus several a few, 5, 4, 3, 2 or l amino acid residues, i.e., ranges as broad as 30 minus several amino acids to 81 plus several amino acids to as narrow as 30 plus several amino acids to 81 minuε several amino acids. Highly preferred in this regard are the recited ranges plus or minus as many as 5 amino acids at either or at both extremes. Particularly highly preferred are the recited ranges plus or minus as many as 3 amino acids at either or at both the recited extremes. Especially particularly highly preferred are ranges plus or minus 1 amino acid at either or at both extremes or the recited ranges with no additions or deletions. Most highly preferred of all in this regard are fragments from about 15 to about 54 amino acids. Among especially preferred fragments of the invention are truncation mutants of CCIII. Truncation mutants include CCIII polypeptides having the amino acid sequence of Figure 1 (SEQ ID NO:2) , or of variants or derivatives thereof, except for deletion of a continuous series of residues (that is, a continuous region, part or portion) that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus or, as in double truncation mutants, deletion of two continuous series of residues, one including the amino terminus and one including the carboxyl terminus. Fragments having the size ranges set out about also are preferred embodiments of truncation fragments, which are especially preferred among fragments generally. Also preferred in this aspect of the invention are fragments characterized by structural or functional attributes of CCIII. Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions") , beta-sheet and beta-sheet-forming regions ("beta-regions") , turn and turn-forming regions ("turn-regions") , 1 coil and coil-forming regions ("coil-regions") , hydrophilic
2 regions, hydrophobic regions, alpha amphipathic regions, beta
3 amphipathic regions, flexible regions, surface-forming regions and
4 high antigenic index regions of CCIII.
5 Certain preferred regions in these regards are set out in
6 Figure 3, and include, but are not limited to, regions of the
7 aforementioned typeε identified by analysis of the amino acid
8 sequence set out in Figure 1. As set out in Figure 3, such
9 preferred regions include Gamier-Robson alpha-regions, beta- 0 regions, turn-regions and coil-regions, Chou-Fasman alpha-regions, i beta-regions and turn-regions, Kyte-Doolittle hydrophilic regions 2 and hydrophilic regions, Eisenberg alpha and beta amphipathic 3 regions, Karplus-Schulz flexible regions, Emini surface-forming 4 regions and Jameson-Wolf high antigenic index regions. s Among highly preferred fragments in this regard are those that 6 comprise regions of CCIII that combine several structural features, 7 such as several of the features set out above. In this regard, the s regions defined by the residues about 15 to about 80 residues of 9 Figure 1 (SEQ ID NO:2) , which all are characterized by amino acid 0 compositions highly characteristic of turn-regions, hydrophilic i regions, flexible-regions, surface-forming regions, and high 2 antigenic inde -regions, are especially highly preferred regions. 3 Such regions may be comprised within a larger polypeptide or may 4 be by themselves a preferred fragment of the present invention, as 5 discussed above. It will be appreciated that the term "about" as 6 used in this paragraph has the meaning set out above regarding 7 fragments in general. 8 Further preferred regions are those that mediate activities 9 of CCIII. Most highly preferred in this regard are fragments that 0 have a chemical, biological or other activity of CCIII, including i those with a similar activity or an improved activity, or with a 2 decreased undesirable activity. Highly preferred in this regard 3 are fragments that contain regions that are homologs in sequence, 4 or in position, or in both sequence and to active regions of 5 related polypeptides, such as the related polypeptide set out in 6 Figure 2 (SEQ ID NO:9) . Among particularly preferred fragments in 7 these regards are truncation mutants, as discussed above. 8 It will be appreciated that the invention also relates to, 9 among others, polynucleotides encoding the aforementioned i fragments, polynucleotides that hybridize to polynucleotides
2 encoding the fragments, particularly those that hybridize under
3 stringent conditions, and polynucleotides, such as PCR primers, for
4 amplifying polynucleotides that encode the fragments. In these
5 regards, preferred polynucleotides are those that correspondent to
6 the preferred fragments, as discussed above.
7
8 Vectors, host cells, expression
9 The present invention also relates to vectors which include 10 polynucleotides of the present invention, host cells which are ii genetically engineered with vectors of the invention and the
12 production of polypeptides of the invention by recombinant
13 techniques.
14 Host cells can be genetically engineered to incorporate
15 polynucleotides and expresε polypeptides of the present invention.
16 For instance, polynucleotides may be introduced into host cells
17 using well known techniques of infection, transduction, ie transfection, transvection and transformation. The polynucleotides
19 may be introduced alone or with other polynucleotides. Such other
20 polynucleotides may be introduced independently, co-introduced or
21 introduced joined to the polynucleotides of the invention.
22 Thus, for instance, polynucleotides of the invention may be 3 transfected into host cells with another, separate, polynucleotide 4 encoding a selectable marker, using standard techniques for co- 5 transfection and selection in, for instance, mammalian cells. In 6 this case the polynucleotides generally will be stably incorporated 7 into the host cell genome. 8 Alternatively, the polynucleotides may be joined to a vector 9 containing a selectable marker for propagation in a host. The 0 vector construct may be introduced into host cells by the i aforementioned techniques. Generally, a plasmid vector is 2 introduced as DNA in a precipitate, such as a calcium phosphate 3 precipitate, or in a complex with a charged lipid. Electroporation 4 also may be used to introduce polynucleotides into a host. If the 5 vector is a virus, it may be packaged in vitro or introduced into 6 a packaging cell and the packaged virus may be transduced into 7 cells. A wide variety of techniques suitable for making 8 polynucleotides and for introducing polynucleotides into cells in 9 accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length in Sambrook et al. cited above, which is illustrative of the many laboratory manuals that detail these techniques. In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells. Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host. In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expresεion or expresεion only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art. The engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the l present invention as will be apparent to those of skill in the art.
2
3 A great variety of expression vectors can be used to express
4 a polypeptide of the invention. Such vectors include chromosomal,
5 episomal and virus-derived vectors e.g., vectors derived from
6 bacterial plasmids, from bacteriophage, from yeast episomes, from
7 yeast chromosomal elements, from viruses such as baculoviruses,
8 papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
9 pox viruses, pseudorabies viruses and retroviruses, and vectors 0 derived from combinations thereof, such as those derived from i plasmid and bacteriophage genetic elements, such as cosmidε and 2 phagemids, all may be used for expresεion in accordance with thiε 3 aspect of the present invention. Generally, any vector suitable 4 to maintain, propagate or express polynucleotides to express a s polypeptide in a host may be used for expression in this regard. 6 7 The appropriate DNA sequence may be inserted into the vector 8 by any of a variety of well-known and routine techniques. In 9 general, a DNA sequence for expression is joined to an expression 0 vector by cleaving the DNA sequence and the expression vector with i one or more restriction endonucleases and then joining the 2 restriction fragments together using T4 DNA ligase. Procedures for 3 restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expresεion vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein. The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein. 1 In general, expression constructε will contain sites for
2 transcription initiation and termination, and, in the transcribed
3 region, a ribosome binding site for translation. The coding
4 portion of the mature transcripts expresεed by the constructs will s include a translation initiating AUG at the beginning and a
6 termination codon appropriately positioned at the end of the
7 polypeptide to be translated.
8 In addition, the constructε may contain control regionε that
9 regulate as well as engender expression. Generally, in accordance 0 with many commonly practiced procedures, such regions will operate i by controlling transcription, such as repressor binding sites and 2 enhancers, among others. 3 Vectors for propagation and expression generally will include 4 selectable markers. Such markers also may be suitable for s amplification or the vectors may contain additional markers for 6 this purpoεe. In thiε regard, the expression vectors preferably 7 contain one or more selectable marker genes to provide a phenotypic s trait for selection of transformed host cells. Preferred markers 9 include dihydrofolate reductase or neomycin resistance for 0 eukaryotic cell culture, and tetracycline or ampicillin resistance i geneε for culturing E. coli and other bacteria. 2 The vector containing the appropriate DNA sequence as 3 described elsewhere herein, as well as an appropriate promoter, and 4 other appropriate control sequences, may be introduced into an 5 appropriate host using a variety of well known techniques suitable 6 to expresεion therein of a deεired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. 8 coli, Streptomyces and Salmonella typhimurium cells; fungal cells, 9 such as yeast cells; insect cells such as Drosophila S2 and 0 Spodoptera Sf9 cellε; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention. More particularly, the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequenceε deεcribed above. The constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inεerted. The εequence may be inserted in a forward or reverse orientation. In certain preferred embodiments in this regard, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to thoεe of skill in the art, and there are many commercially available vectors suitable for use in the present invention. The following vectors, which are commercially available, are provided by way of example. Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen,- pBS vectors, Phagescript vectors, Bluescript vectorε, pNHΘA, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectorε are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are liεted solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with thiε aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention. Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene. ι Among known bacterial promoters suitable for expression of
2 polynucleotides and polypeptides in accordance with the present
3 invention are the E. coli lad and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters and the
5 trp promoter. Among known eukaryotic promoters suitable in
6 this regard are the CMV immediate early promoter, the HSV thymidine
7 kinase promoter, the early and late SV40 promoters, the promoters
8 of retroviral LTRs, such as those of the Rous sarcoma virus
9 ("RSV") , and metallothionein promoters, such as the mouse 0 metallothionein-I promoter. i Selection of appropriate vectors and promoters for expresεion 2 in a host cell is a well known procedure and the requisite 3 techniques for expression vector construction, introduction of the 4 vector into the host and expression in the host are routine skills 5 in the art. 6 The present invention also relates to host cells containing 7 the above-described constructε discusεed above. The hoεt cell can 8 be a higher eukaryotic cell, εuch aε a mammalian cell, or a lower 9 eukaryotic cell, such aε a yeaεt cell, or the host cell can be a 0 prokaryotic cell, such as a bacterial cell. 1 Introduction of the construct into the host cell can be 2 effected by calcium phosphate transfection, DEAE-dextran mediated 3 transfection, cationic lipid-mediated transfection, 4 electroporation, transduction, infection or other methodε. Such 5 methodε are deεcribed in many standard laboratory manuals, such as 6 Davis et al. BASIC METHODS IN MOLECULAR BIOLOGY, (1986) . 7 Constructs in host cellε can be used in a conventional manner 8 to produce the gene product encoded by the recombinant sequence. 9 Alternatively, the polypeptides of the invention can be 0 synthetically produced by conventional peptide syntheεizers. i Mature proteins can be expressed in mammalian cells, yeast, 2 bacteria, or other cells under the control of appropriate 3 promoterε . Cell-free tranεlation systems can also be employed to 4 produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) . Generally, recombinant expresεion vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector. Among suitable promoters are those derived from the genes that encode glycolytic enzymes such as 3- phosphoglycerate kinase ("PGK"), a-factor, acid phosphatase, and heat shock proteins, among others. Selectable markers include the ampicillin resistance gene of E. coli and the trpl gene of S. cerevisiae. Tranεcription of the DNA encoding the polypeptideε of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis- acting elements of DNA, usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type. Examples of enhancers include the SV40 enhancer, which is located on the late side of the replication origin at bp 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Polynucleotides of the invention, encoding the heterologous structural sequence of a polypeptide of the invention generally will be inserted into the vector using standard techniques εo that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5 ' to a ribosome binding site. The ribosome binding site will be 5 ' to the AUG that initiateε translation of the polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiating AUG. Also, generally, there will be a translation stop codon at the end of the polypeptide and there will be a polyadenylation signal and a transcription termination signal appropriately disposed at the 3' end of the transcribed region. For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic εpace or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polypeptide. The signals may be endogenous to the polypeptide or they may be heterologous signalε. ι The polypeptide may be expressed in a modified form, such as
2 a fusion protein, and may include not only secretion signals but
3 also additional heterologous functional regions. Thus, for
4 inεtance, a region of additional amino acids, particularly charged
5 amino acidε, may be added to the N-terminus of the polypeptide to
6 improve stability and persistence in the host cell, during
7 purification or during subεequent handling and storage. Also,
8 region also may be added to the polypeptide to facilitate
9 purification. Such regions may be removed prior to final 10 preparation of the polypeptide. The addition of peptide moieties ii to polypeptides to engender secretion or excretion, to improve
12 stability and to facilitate purification, among others, are
13 familiar and routine techniques in the art.
14 Suitable prokaryotic hosts for propagation, maintenance or
15 expression of polynucleotides and polypeptides in accordance with
16 the invention include Escherischia coli, Bacillus subtilis and
17 Salmonella typhimurium. Various specieε of Pseudomonas, ie Streptomyces, and Staphylococcus are suitable hosts in this regard.
19 Moreover, many other hosts also known to those of skill may be
20 employed in thiε regard.
21 As a representative but non-limiting example, useful
22 expresεion vectors for bacterial use can comprise a selectable
23 marker and bacterial origin of replication derived from
24 commercially available plasmids comprising genetic elements of the
25 well known cloning vector pBR322 (ATCC 37017) . Such commercial
26 vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
27 Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
28 These pBR322 "backbone" sections are combined with an appropriate
29 promoter and the structural sequence to be expressed.
30 Following transformation of a suitable host strain and growth
31 of the host strain to an appropriate cell density, where the
32 selected promoter is inducible it is induced by appropriate means
33 (e.g., temperature shift or exposure to chemical inducer) and cells
34 are cultured for an additional period.
35 Cells typically then are harvested by centrifugation,
36 disrupted by physical or chemical means, and the resulting crude
37 extract retained for further purification.
38 Microbial cells employed in expression of proteins can be J9 disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art. Variouε mammalian cell culture εystems can be employed for expression, as well. Examples of mammalian expresεion systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 (1981) . Other cell lines capable of expresεing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lineε. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any neceεεary riboεome binding εiteε, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences that are necessary for expresεion. In certain preferred embodimentε in this regard DNA sequences derived from the SV40 splice εiteε, and the SV40 polyadenylation sites are used for required non-transcribed genetic elements of these typeε. The CCIII polypeptide can be recovered and purified from recombinant cell cultureε by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes. CCIII polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties CCIII. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are illustrated further by the following discusεion. Polynucleotide aεεayε Thiε invention is also related to the use of the CCIII polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of CCIII associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a diseaεe which results from under-expression over-expression or altered expression of CCIII, for example, neoplasia such as cancers and tumors. Individuals carrying mutations in the human CCIII gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such aε from blood, urine, saliva, tissue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysiε. PCR (Saiki et al., Nature, 324: 163-166 (1986)) . RNA or cDNA may also be used in the same ways. As an example, PCR primers complementary to the nucleic acid encoding CCIII can be used to identify and analyze CCIII expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled CCIII RNA or alternatively, radiolabeled CCIII antisense DNA εequenceε. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segmentε. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method. For example, a sequencing primer is used with double- stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is 1 performed by conventional procedures with radiolabeled nucleotide
2 or by automatic sequencing procedures with fluorescent-tags.
3 Genetic testing based on DNA sequence differences may be
4 achieved by detection of alteration in electrophoretic mobility of
5 DNA fragments in gels, with or without denaturing agents. Small
6 sequence deletions and insertions can be visualized by high
7 resolution gel electrophoresis. DNA fragments of different
8 sequences may be distinguished on denaturing formamide gradient
9 gels in which the mobilities of different DNA fragments are 0 retarded in the gel at different positionε according to their i specific melting or partial melting temperatureε (εee, e.g., Myerε 2 et al., Science, 230: 1242 (1985)). 3 Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or s the chemical cleavage method (e.g., Cotton et al., Proc. Natl. 6 Acad. Sci., USA, 85: 4397-4401 (1985)) . 7 Thus, the detection of a specific DNA sequence may be achieved 8 by methods such as hybridization, RNase protection, chemical 9 cleavage, direct DNA sequencing or the use of restriction enzymes, 0 (e.g., restriction fragment length polymorphisms ("RFLP") and i Southern blotting of genomic DNA. 2 In addition to more conventional gel-electrophoresiε and DNA 3 εequencing, mutationε alεo can be detected by in εitu analyεis.
Chromosome assays The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with diseaεe. In certain preferred embodiments in this regard, the cDNA herein disclosed is used to clone genomic DNA of a CCIII gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA the is used for in situ chromosome mapping using well known techniques for this purpose. Typically, in accordance with routine procedures for chromosome mapping, some trial and error may be necessary to identify a genomic probe that gives a good in situ hybridization signal. In some cases, in addition, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analyεis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification procesε. Theεe primerε are then uεed for PCR εcreening of somatic cell hybridε containing individual human chromoεomeε. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomeε or poolε of large genomic cloneε in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries. Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphaεe chromoεomal εpread can be uεed to provide a preciεe chromoεomal location in one step. This technique can be used with cDNA as short as 50 or 60. For a review of this technique, see Verma et al., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988) . Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, MENDELIAN INHERITANCE IN MAN, available on line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseaseε that have been mapped to the εame chromoεomal region are then identified through linkage analysiε (coinheritance of phyεically adjacent genes) . Next, it is necesεary to determine the differenceε in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the diseaεe. With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the diseaεe could be one of between 50 and 500 potential cauεative geneε. (Thiε assumes l megabase mapping resolution and one gene per 20 kb) .
Polypeptide asεays The present invention also relates to a diagnostic assayε εuch as quantitative and diagnostic assays for detecting levels of CCIII protein in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of CCIII protein compared to normal control tissue samples may be used to detect the presence of a neoplasia, for example. Assay techniques that can be used to determine levels of a protein, such as an CCIII protein of the present invention, in a sample derived from a host are well-known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysiε and ELISA asεays. Among theεe ELlSAs frequently are preferred. An ELISA asεay initially comprises preparing an antibody specific to CCIII, preferably a monoclonal antibody. In addition a reporter antibody generally is prepared which binds to the monoclonal antibody. The reporter antibody is attached a detectable reagent such as radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme. To carry out an ELISA a sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein such as bovine serum albumin. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any CCIII proteins attached to the polystyrene dish. Unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to CCIII. Unattached reporter antibody 1 is then washed out. Reagents for peroxidase activity, including
2 a colorimetric substrate are then added to the dish. Immobilized
3 peroxidase, linked to CCIII through the primary and secondary
4 antibodies, produces a colored reaction product. The amount of
5 color developed in a given time period indicates the amount of
6 CCIII protein present in the sample. Quantitative results
7 typically are obtained by reference to a standard curve. β A competition assay may be employed wherein antibodies
9 specific to CCIII attached to a solid support and labeled CCIII and o a sample derived from the host are passed over the solid support i and the amount of label detected attached to the solid support can 2 be correlated to a quantity of CCIII in the sample. 3 4 Antibodies 5 The polypeptides, their fragments or other derivatives, or 6 analogs thereof, or cells expressing them can be used as an 7 immunogen to produce antibodies thereto. These antibodies can be, 8 for example, polyclonal or monoclonal antibodies. The present 9 invention also includes chimeric, single chain, and humanized 0 antibodies, as well as Fab fragments, or the product of an Fab i expression library. Various procedures known in the art may be 2 used for the production of such antibodies and fragments. 3 Antibodies generated againεt the polypeptides corresponding 4 to a sequence of the present invention can be obtained by direct 5 injection of the polypeptides into an animal or by administering 6 the polypeptides to an animal, preferably a nonhuman. The antibody 7 so obtained will then bind the polypeptides itself. In this 8 manner, even a sequence encoding only a fragment of the 9 polypeptides can be used to generate antibodies binding the whole 0 native polypeptides. Such antibodies can then be used to isolate i the polypeptide from tissue expressing that polypeptide. 2 The antibodies may be employed to isolate or to identify 3 clones expressing the polypeptide or purify a polypeptide of the present invention by attachment to a solid support for isolation and/or purification by affinity chromatography. 6 For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C. , Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Lisε, Inc. (1985) . Techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, tranεgenic mice, or other organiεmε εuch as other mammalε, may be uεed to expreεs humanized antibodies to immunogenic polypeptide products of this invention. Thus, among others, CCIII may be employed to inhibit bone marrow stem cell colony formation as adjunct protective treatment during cancer chemotherapy and for leukemia. CCIII may also be employed to inhibit epidermal keratinocyte proliferation for treatment of psoriaεis, which is characterized by keratinocyte hyper-proliferation. CCIII may also be employed to treat neoplasia, such as cancers and tumors, by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophageε and by inhibiting the angiogenesis of tumorε. They may also be employed to enhance host defenses against resistant chronic and acute infectionε, for example, mycobacterial infectionε via the attraction and activation of microbicidal leukocytes. CCIII may also be employed to inhibit T cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-immune diseaseε and lymphocytic leukemiaε. CCIII may also be employed to stimulate wound healing, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells and also via its control of excessive TGF/3-mediated fibrosis. In this same manner, CCIII may also be employed to treat other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis. CCIII also increases the presence of eosinophils which have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis.
It may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various 1 hematopoietic progenitor cells, for example, to release mature
2 leukocytes from the bone marrow following chemotherapy.
3
4 CCIII binding molecules and assays
5 This invention also provides a method for identification of
6 molecules, such as receptor molecules, that bind CCIII. Genes
7 encoding proteins that bind CCIII, such as receptor proteins, can
8 be identified by numerous methods known to those of skill in the
9 art, for example, ligand panning and FACS sorting. Such methods 10 are described in many laboratory manuals such as, for instance, ii Coligan et al., Current Protocols in Immunology 1(2) : Chapter 5
12 (1991) .
13 For instance, expression cloning may be employed for this
14 purpose. To this end polyadenylated RNA iε prepared from a cell
15 responsive to CCIII, a cDNA library is created from this RNA, the
16 library is divided into pools and the pools are transfected
17 individually into cells that are not responεive to CCIII. The ie transfected cells then are exposed to labeled CCIII. (CCIII can
19 be labeled by a variety of well-known techniques including standard
20 methods of radio-iodination or inclusion of a recognition site for
21 a site-specific protein kinase.) Following exposure, the cells are
22 fixed and binding of cytostatin is determined. These procedures
23 conveniently are carried out on glass slideε.
24 Pools are identified of cDNA that produced CCIII-binding
25 cells. Sub-pools are prepared from these positives, transfected 6 into host cells and screened as described above. Using an iterative 7 sub-pooling and re-screening process, one or more single clones 8 that encode the putative binding molecule, such as a receptor 9 molecule, can be isolated. 0 Alternatively a labeled ligand can be photoaffinity linked to i a cell extract, such as a membrane or a membrane extract, prepared 2 from cells that express a molecule that it binds, such as a 3 receptor molecule. Cross-linked material is resolved by 4 polyacrylamide gel electrophoresis ("PAGE") and exposed to X-ray 5 film. The labeled complex containing the ligand-receptor can be 6 excised, resolved into peptide fragments, and subjected to protein 7 microsequencing. The amino acid sequence obtained from 8 microsequencing can be used to design unique or degenerate oligonucleotide probes to screen cDNA libraries to identify genes encoding the putative receptor molecule. Polypeptides of the invention also can be used to asseεε CCIII binding capacity of CCIII binding moleculeε, εuch aε receptor molecules, in cellε or in cell-free preparationε.
Agoniεts and antagonistε - assays and molecules The invention also provides a method of screening compounds to identify those which enhance or block the action of CCIII on cells, such as its interaction with CCIII-binding molecules εuch aε receptor moleculeε. An agonist is a compound which increases the natural biological functions of CCIII or which functions in a manner similar to CCIII, while antagonists decrease or eliminate such functions. For example, a cellular compartment, such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds CCIII, such as a molecule of a signaling or regulatory pathway modulated by CCIII. The preparation is incubated with labeled CCIII in the absence or the presence of a candidate molecule which may be a CCIII agoniεt or antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously, i.e., without inducing the effects of CCIII on binding the CCIII binding molecule, are most likely to be good antagonistε. Moleculeε that bind well and elicit effectε that are the εame as or closely related to CCIII are agonists. CCIII-like effects of potential agonists and antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of CCIII or molecules that elicit the same effectε as CCIII. Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.
Another example of an assay for CCIII antagonists is a competitive assay that combines CCIII and a potential antagonist with membrane-bound CCIII receptor molecules or recombinant CCIII 1 receptor moleculeε under appropriate conditionε for a competitive
2 inhibition aεsay. CCIII can be labeled, such as by radioactivity,
3 such that the number of CCIII molecules bound to a receptor
4 molecule can be determined accurately to assess the effectiveness
5 of the potential antagonist.
6 Potential antagonists include small organic molecules,
7 peptides, polypeptides and antibodies that bind to a polypeptide
8 of the invention and thereby inhibit or extinguish its activity.
9 Potential antagonists also may be small organic molecules, a ιo peptide, a polypeptide εuch as a closely related protein or ii antibody that binds the same siteε on a binding molecule, such as
12 a receptor molecule, without inducing CCIII-induced activities,
13 thereby preventing the action of CCIII by excluding CCIII from
14 binding. i s Potential antagonists include a small molecule which binds to
16 and occupies the binding site of the polypeptide thereby preventing
17 binding to cellular binding molecules, such as receptor molecules,
18 such that normal biological activity is prevented. Exampleε of
19 small moleculeε include but are not limited to εmall organic 0 molecules, peptides or peptide-like molecules. i Other potential antagonists include antisense moleculeε. 2 Antisenεe technology can be uεed to control gene expreεεion through 3 antiεenεe DNA or RNA or through triple-helix formation. Antisenεe 4 techniques are discussed, for example, in - Okano, J. Neurochem. 5 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF 6 GENE EXPRESSION, CRC Press, Boca Raton, FL (1988) . Triple helix 7 formation is diεcussed in, for instance Lee et al., Nucleic Acids 8 Research 6: 3073 (1979); Cooney et al., Science 241: 456 (1988); 9 and Dervan et al., Science 251: 1360 (1991) . The methods are based 0 on binding of a polynucleotide to a complementary DNA or RNA. For i example, the 5' coding portion of a polynucleotide that encodes the 2 mature polypeptide of the present invention may be used to design 3 an antisenεe RNA oligonucleotide of from about 10 to 40 base pairs 4 in length. A DNA oligonucleotide is designed to be complementary 5 to a region of the gene involved in transcription thereby 6 preventing transcription and the production of CCIII. The 7 antisenεe RNA oligonucleotide hybridizeε to the mRNA in vivo and 8 blocks translation of the mRNA molecule into CCIII polypeptide. The 9 oligonucleotides described above can also be delivered to cells 1 such that the antisense RNA or DNA may be expressed in vivo to
2 inhibit production of CCIII.
3 The antagonists may be employed in a composition with a
4 pharmaceutically acceptable carrier, e.g., as hereinafter
5 described.
6 The antagonists may be employed for instance to inhibit the
7 chemotaxiε and activation of macrophageε and their precursors, and
8 of neutrophils, basophils, B lymphocytes and some T cell subsets,
9 e.g., activated and CD8 cytotoxic T cells and natural killer cells, 10 in certain auto-immune and chronic inflammatory and infective ii diseases. Examples of auto-immune diseaεeε include multiple 2 sclerosis, and insulin-dependent diabetes. 3 The antagonists may also be employed to treat infectious 4 diseaεeε including εilicosis, εarcoidosis, idiopathic pulmonary 5 fibrosis by preventing the recruitment and activation of 6 mononuclear phagocytes. They may also be employed to treat 7 idiopathic hyper-eoεinophilic εyndrome by preventing eoεinophil 8 production and migration. Endotoxic εhock may also be treated by 9 the antagonists by preventing the migration of macrophages and 0 their production of the human chemokine polypeptides of the present i invention. 2 The antagonistε may alεo be employed for treating 3 atherosclerosis, by preventing monocyte infiltration in the artery 4 wall. 5 The antagonists may also be employed to treat histamine- 6 mediated allergic reactions and immunological disorders including 7 late phase allergic reactions, chronic urticaria, and atopic 8 dermatitis by inhibiting chemokine-induced mast cell and basophil 9 degranulation and release of histamine. IgE-mediated allergic 0 reactions such as allergic asthma, rhinitis, and eczema may also i be treated. 2 The antagonists may also be employed to treat chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequeεtration of mononuclear phagocytes in the lung. Antagonistε may also be employed to treat rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies. The antagonists may be employed to interfere with the deleterious cascadeε attributed primarily to IL-1 and TNF, which preventε the bioεynthesis of other inflammatory cytokines. In this way, the antagonistε may be employed to prevent inflammation. The antagoniεts may also be employed to inhibit prostaglandin- independent fever induced by chemokines. The antagonists may also be employed to treat caεeε of bone marrow failure, for example, aplaεtic anemia and myelodyεplaεtic syndrome. The antagonists may also be employed to treat asthma and allergy by preventing eosinophil accumulation in the lung. The antagoniεts may also be employed to treat subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung. The antagonists may also be employed to treat glomerulonephritis, cerebral ischemia and HTLV-1 related diseases. The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.
Compositions The invention also relateε to compoεitions comprising the polynucleotide or the polypeptides discusεed above or the agonists or antagonists. Thus, the polypeptides of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compoεitions comprise, for instance, a media additive or a therapeutically effective amount of a polypeptide of the invention and a pharmaceutically acceptable carrier or excipient. Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinationε thereof. The formulation εhould εuit the mode of adminiεtration. The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositionε of the invention. Aεsociated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
Administration Polypeptides and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds. The pharmaceutical compositionε may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others. The pharmaceutical compositionε generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications. In general, the compositionε are administered in an amount of at leaεt about 10 μg/kg body weight. In moεt cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10 ug/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.
Gene therapy The CCIII polynucleotides, polypeptides, agonists and antagonistε that are polypeptideε may be employed in accordance with the present invention by expreεεion of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy." Thus, for example, cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide. For example, cells may be 1 engineered ex vivo by the use of a retroviral plasmid vector
2 containing RNA encoding a polypeptide of the present invention.
3 Such methods are well-known in the art and their use in the present
4 invention will be apparent from the teachings herein.
5 Similarly, cells may be engineered in vivo for expression of
6 a polypeptide in vivo by procedures known in the art. For example,
7 a polynucleotide of the invention may be engineered for expression
8 in a replication defective retroviral vector, as discuεεed above.
9 The retroviral expression construct then may be isolated and 10 introduced into a packaging cell is transduced with a retroviral ii plasmid vector containing RNA encoding a polypeptide of the present
12 invention εuch that the packaging cell now produceε infectious
13 viral particles containing the gene of interest. These producer
14 cells may be administered to a patient for engineering cells in is vivo and expresεion of the polypeptide in vivo. Theεe and other
16 methods for administering a polypeptide of the present invention
17 by εuch method εhould be apparent to those skilled in the art from ie the teachings of the present invention.
19 Retroviruses from which the retroviral plasmid vectors herein
20 above mentioned may be derived include, but are not limited to,
21 Moloney Murine Leukemia Viruε, εpleen necrosis virus, retroviruses
22 such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis 3 virus, gibbon ape leukemia virus, human immunodeficiency virus, 4 adenovirus, Myeloproliferative Sarcoma Viruε, and mammary tumor 5 viruε. In one embodiment, the retroviral plaεmid vector is derived 6 from Moloney Murine Leukemia Virus. 7 Such vectors well include one or more promoters for expressing 8 the polypeptide. Suitable promoters which may be employed include, 9 but are not limited to, the retroviral LTR; the SV40 promoter; and 0 the human cytomegalovirus (CMV) promoter described in Miller et i al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., 2 cellular promoters such as eukaryotic cellular promoters including, 3 but not limited to, the histone, RNA polymerase III, and β-actin 4 promoters) . Other viral promoters which may be employed include, 5 but are not limited to, adenovirus promoters, thymidine kinaεe (TK) 6 promoters, and B19 parvovirus promoterε. The εelection of a 7 εuitable promoter will be apparent to thoεe skilled in the art from 8 the teachings contained herein. The nucleic acid sequence encoding the polypeptide of the present invention will be placed under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial viruε (RSV) promoter; inducible promoterε, εuch aε the MMT promoter, the metallothionein promoter; heat εhock promoterε; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described) ; the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide. The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Exampleε of packaging cellε which may be tranεfected include, but are not limited to, the PE501, PA317, Y-2, Y-AM, PA12, T19-14X, VT-19-17- H2, YCRE, YCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, A., Human Gene Therapy 1: 5-14 (1990) . The vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then adminiεtered to a hoεt. The producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequence (s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cellε, hepatocyteε, fibroblaεtε, myoblaεts, keratinocytes, endothelial cells, and bronchial epithelial cells.
EXAMPLES The present invention is further described by the following examples. The exampleε are provided solely to illuεtrate the invention by reference to εpecific embodimentε. Theεe exemplification's, while illustrating certain specific aspectε of the invention, do not portray the limitationε or circumscribe the scope of the discloεed invention. Certain termε used herein are explained in the foregoing glossary. All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniqueε of the following exampleε can be carried out aε described in εtandard laboratory manuals, such as Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. ; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) , herein referred to as "Sambrook." All parts or amounts εet out in the following examples are by weight, unleεε otherwiεe specified. Unleεs otherwise stated size separation of fragments in the examples below was carried out using standard techniques of agarose and polyacrylamide gel electrophoresiε ("PAGE") in Sambrook and numerous other referenceε εuch aε, for inεtance, by Goeddel et al., Nucleic Acidε Res. 8: 4057 (1980) . Unless described otherwise, ligations were accomplished using standard bufferε, incubation temperatures and times, approximately equimolar amounts of the DNA fragments to be ligated and approximately 10 units of T4 DNA ligase ("ligase") per 0.5 μg of DNA.
Example 1 Expression and purification of human CCIII using bacteria
The DNA sequence encoding human CCIII in the deposited polynucleotide was amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence of the human CCIII protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning were added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer had the sequence 5' CGCCCA TGGTGGCCGCCGCGCAGG 3' (SEQ ID NO:3) containing a Neo I restriction site, which encodes a start AUG, followed by 16 nucleotides of the human CCIII coding sequence set out in Figure 1. The 3' primer had the εequence 5' CGCAAGCTTGCAGAG CTCAATTTA 3' (SEQ ID NO:4) containing the underlined BamHI restriction site followed by 15 nucleotides complementary to CCIII non-coding sequence set out in Figure 1, including the stop codon. The restrictionε εites were convenient to restriction enzyme sites in the bacterial expresεion vectorε pQE-7 which were uεed for bacterial expreεεion in theεe exampleε. (Qiagen, Inc. Chatεworth, CA) . pQE-7 encodes ampicillin antibiotic resistance ("Ampr") and contains a bacterial origin of replication ("ori") , an IPTG inducible promoter, a ribosome binding site ("RBS") , a 6-His tag and restriction enzyme sites. The amplified human CCIII DNA and the vector pQE-7 both were digested with Neo I and Hind III and the digested DNAs then were ligated together. Insertion of the CCIII DNA into the Neo I/Hind III restricted vector placed the CCIII coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in- frame with an initiating AUG appropriately positioned for translation of CCIII. The ligation mixture was transformed into competent E. coli cells using standard procedures. Such procedures are described in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) . E. coli strain M15/rep4, containing multiple copies of the plasmid pREP4, which expresseε lac repreεεor and confers kanamycin resistance ("Kanr"), was used in carrying out the illustrative example described here. This strain, which iε only one of many that are εuitable for expreεsing CCIII, is available commercially from Qiagen. Transformantε were identified by their ability to grow on LB plates in the presence of ampicillin. Plasmid DNA was isolated from resiεtant colonieε and the identity of the cloned DNA waε confirmed by reεtriction analyεiε. Clones containing the deεired constructs were grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ug/ml) and kanamycin (25 ug/ml) . The 0/N culture was used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250. The cells were grown to an optical density at 600nm ("OD600") of between 0.4 and 0.6. Isopropyl-B-D-thiogalactopyranoεide ("IPTG") waε then added to a final concentration of 1 mM to induce tranεcription from lac repreεsor sensitive promoters, by inactivating the lad represεor. Cells subsequently were incubated further for 3 to 4 hours. Cells then were harvested by centrifugation and disrupted, by εtandard method . Inclusion bodies were purified from the disrupted cells using routine collection techniques, and protein waε εolubilized from the incluεion bodieε into 8M urea. The 8M urea solution containing the solubilized protein was passed over a PD-10 column in 2X phosphate buffered saline ("PBS") , thereby removing the urea, exchanging the buffer and refolding the protein. The protein was purified by a further step of chromatography to remove endotoxin. Then, it was sterile filtered. The sterile filtered protein preparation was stored in 2X PBS at a concentration of 95 micrograms per mL. Analyεiε of the preparation by standard methodε of polyacrylamide gel electrophoreεiε revealed that the preparation contained about 90% monomer CCIII having the expected molecular weight of, approximately, 8.5 kDa.
Example 2 Cloning and expreεεion of human CCIII in a baculoviruε expreεεion εystem
The cDNA sequence encoding the full length human CCIII protein, in the deposited clone is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene: The 5' primer has the εequence 5' CGCGGATCCGCCATCATG GCGCCCGGAGTG 3' (SEQ ID NO:5) containing the underlined BamHI reεtriction enzyme εite followed by Kozak εequence and 15 baεes of the sequence of CCIII of Figure 1. Inserted into an expresεion vector, aε deεcribed below, the 5' end of the amplified fragment encoding human CCIII provides an efficient signal peptide. An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M. , J. Mol. Biol. 196: 947-950 (1987) is appropriately located in the vector portion of the construct. The 3' primer has the sequence 5' CGCGGTACCGCAGAG CTCAATTTA 3' (SEQ ID NO:6) containing the underlined Asp718 restriction followed by nucleotides complementary to the last 15 nucleotides of the CCIII non-coding sequence set out in Figure 1, including the stop codon. The amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment then is digested with BamHI and Asp718 and again is purified on a 1% agarose gel. This fragment is designated herein F2. The vector pA2 is used to expresε the CCIII protein in the baculovirus expression system, using εtandard methods, such as those described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555 (1987) . Thiε expresεion vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV) followed by convenient restriction sites. The signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site. The polyadenylation site of the simian virus 40 ("SV40") is used for efficient polyadenylation. For an easy εelection of recombinant viruε the beta-galactoεidaεe gene from E.coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that expresε the cloned polynucleotide. Many other baculoviruε vectorε could be used in place of pA2- GP, εuch aε pAc373, pVL94l and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signalε for tranεcription, tranεlation, trafficking and the like, such aε an in-frame AUG and a εignal peptide, aε required. Such vectors are described in Luckow et al., Virology 170: 31-39, among others. The plasmid is digested with the restriction enzymes Asp 718 band BamHI and then is dephosphorylated uεing calf inteεtinal phosphataεe, uεing routine procedureε known in the art. The DNA iε then isolated from a 1% agarose gel using a commercially 1 available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.) . This
2 vector DNA is designated herein "V2".
3 Fragment F2 and the dephosphorylated plaεmid V2 are ligated
4 together with T4 DNA ligaεe. E.coli HB101 cellε are transformed
5 with ligation mix and spread on culture plates. Bacteria are
6 identified that contain the plaεmid with the human CCIII gene by digesting DNA from individual colonieε using BamHI and Asp 718 and
8 then analyzing the digeεtion product by gel electrophoreεis. The
9 εequence of the cloned fragment is confirmed by DNA sequencing. o Thiε plasmid is designated herein pBacCCIII. i 5 μg of the plasmid pBacCCIII is co-transfected with 1.0 μg 2 of a commercially available linearized baculoviruε DNA 3 ("BaculoGold™ baculoviruε DNA", Pharmingen, San Diego, CA.), uεing 4 the lipofection method described by Feigner εt al. , Proc. Natl. 5 Acad. Sci. USA 84: 7413-7417 (1987) . lμg of BaculoGold™ virus DNA 6 and 5 μg of the plasmid pBacCCIII are mixed in a sterile well of 7 a microtiter plate containing 50 μl of serum free Grace's medium 8 (Life Technologies Inc., Gaithersburg, MD) . Afterwardε 10 μl 9 Lipofectin pluε 90 μl Grace's medium are added, mixed and incubated 0 for 15 minutes at room temperature. Then the transfection mixture i is added drop-wise to Sf9 inεect cellε (ATCC CRL 1711) εeeded in 2 a 35 mm tissue culture plate with 1 ml Grace's medium without 3 serum. The plate is rocked back and forth to mix the newly added 4 solution. The plate is then incubated for 5 hours at 27'C. After 5 5 hours the transfection solution is removed from the plate and l 6 ml of Grace's insect medium supplemented with 10% fetal calf serum 7 is added. The plate is put back into an incubator and cultivation 8 is continued at 27'C for four days. 9 After four days the supernatant is collected and a plaque 0 assay is performed, as described by Summers and Smith, cited above. i An agarose gel with "Blue Gal" (Life Technologies Inc., 2 Gaithersburg) is used to allow easy identification and isolation 3 of gal-expresεing cloneε, which produce blue-εtained plaqueε. (A detailed deεcription of a "plaque aεεay" of thiε type can alεo be found in the uεer' s guide for insect cell culture and baculovirology distributed by Life Technologies Inc. , Gaithersburg, page 9-10) . Four days after serial dilution, the virus is added to the cells. After appropriate incubation, blue stained plaqueε are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuεpended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatantε of theεe culture dishes are harvested and then they are stored at 4'C. A clone containing properly inserted CCIII is identified by DNA analysiε including reεtriction mapping and εequencing. This is designated herein as V-CCIII. Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-CCIII at a multiplicity of infection ("MOI") of about 2 (about l to about 3) . Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg) . 42 hours later, 5 μCi of 35S-methionine and 5 μCi 35S cysteine (available from Amersham) are added. The cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteinε are viεualized by SDS-PAGE and autoradiography.
Example 3 Expreεsion of CCIII in COS cells
The expresεion plasmid, CCIII HA, iε made by cloning a cDNA encoding CCIII into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.) . The expression vector pcDNAI/amp contains: (l) an E.coli origin of replication effective for propagation in E. coli and other prokaryotic cell; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells,- (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expreεsion control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by meanε of restriction sites in the polylinker. A DNA fragment encoding the entire CCIII precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al. , Cell 37: 767 (1984) . The fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope. The plaεmid construction strategy is as follows. The CCIII cDNA of the deposit clone is amplified using primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expresεion of CCIII in E. coli and S. furgiperda. To facilitate detection, purification and characterization of the expreεεed CCIII, one of the primerε containε a heamaglutinin tag ("HA tag") aε described above. Suitable primers include that following, which are used in thiε example. The 5' primer, containing the underlined Bam HI εite, an AUG start codon and 15 codons thereafter, has the following sequence,- 5' CGCGGATCCACCATGGCGCCCGGAGTGGCC 3' (SEQ ID NO:7). The 3' primer, containing the underlined Xba I site, εtop codon, HA tag and 15 bp of 3' coding εequence (at the 3' end) has the following sequence 5 ' CGGTCTAGATCAAGCGTAGTCTGG GACGTCGTATGGGTACAAAGGGAAAGCCGG 3 ' (SEQ ID NO : 8 ) . The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with and then ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plaεmid DNA iε iεolated from reεiεtant colonieε and examined by restriction analysiε and gel εizing for the preεence of the CCIII-encoding fragment. For expreεεion of recombinant CCIII, COS cellε are transfected with an expression vector, as deεcribed above, using DEAE-DEXTRAN, as deεcribed, for inεtance, in Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Laboratory Preεε, Cold Spring Harbor, New York (1989) . Cells are incubated under conditions for expresεion of CCIII by the vector. 1 Expression of the CCIII HA fusion protein is detected by
2 radiolabelling and immunoprecipitation, using methods described in,
3 for example Harlow et al., ANTIBODIES: A LABORATORY MANUAL, 2nd
4 Ed.,- Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
5 York (1988) . To this end, two days after transfection, the cells
6 are labeled by incubation in media containing 35S-cysteine for 8
7 hours. The cells and the media are collected, and the cells are
8 washed and the lysed with detergent-containing RIPA buffer: 150 mM
9 NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, 10 as described by Wilson et al. cited above. Proteins are ii precipitated from the cell lysate and from the culture media using
12 an HA-specific monoclonal antibody. The precipitated proteins then
13 are analyzed by SDS-PAGE gels and autoradiography. An expression
14 product of the expected size is seen in the cell lysate, which is 5 not seen in negative controls. 6 7 Example 4 Tissue distribution of CCIII expresεion 8 9 Northern blot analyεis is carried out to examine the levels 0 of expression of CCIII in human tisεueε, using methods described i by, among others, Sambrook et al, cited above. Total cellular RNA 2 sampleε are iεolated with RNAzol™ B εyεtem (Biotecx Laboratorieε, 3 Inc. 6023 South Loop Eaεt, Houston, TX 77033) . 4 About 10μg of Total RNA is isolated from tissue samples. The 5 RNA is size resolved by electrophoresis through a 1% agarose gel 6 under strongly denaturing conditions. RNA is blotted from the gel 7 onto a nylon filter, and the filter then is prepared for 8 hybridization to a detectably labeled polynucleotide probe. 9 As a probe to detect mRNA that encodes CCIII, the antisense 0 strand of the coding region of the cDNA insert in the depoεited i clone iε labeled to a high specific activity. The cDNA is labeled 2 by primer extension, using the Prime-It kit, available from 3 Stratagene. The reaction is carried out using 50 ng of the cDNA, 4 following the standard reaction protocol as recommended by the supplier. The labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303. 1 The labeled probe is hybridized to the filter, at a
2 concentration of 1,000,000 cpm/ml, in a small volume of 7% SDS, 0.5
3 M NaP04, pH 7.4 at 65 "C, overnight.
4 Thereafter the probe solution is drained and the filter is s washed twice at room temperature and twice at 60'C with 0.5 x SSC,
6 0.1% SDS. The filter then is dried and exposed to film at -70'C
7 overnight with an intensifying screen. 8
9 o Example 5 Gene therapeutic expresεion of human CCIII 1 2 Fibroblasts are obtained from a εubject by skin biopsy. The 3 resulting tisεue iε placed in tiεsue-culture medium and separated 4 into εmall pieceε. Small chunkε of the tiεεue are placed on a wet s εurface of a tiεεue culture flaεk, approximately ten pieceε are 6 placed in each flask. The flask is turned upside down, closed 7 tight and left at room temperature overnight. After 24 hours at s room temperature, the flask is inverted - the chunks of tissue 9 remain fixed to the bottom of the flask - and fresh media is added 0 (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) . i The tissue is then incubated at 37'C for approximately one week. 2 At this time, fresh media is added and subsequently changed every 3 several days. After an additional two weeks in culture, a 4 monolayer of fibroblastε emergeε. The monolayer is trypsinized and 5 scaled into larger flasks. 6 A vector for gene therapy is digested with reεtriction enzymes for cloning a fragment to be expresεed. The digeεted vector is treated with calf intestinal phosphataεe to prevent εelf-ligation. The dephosphorylated, linear vector is fractionated on an agarose gel and purified. Cytostatin cDNA capable of expressing active CCIII, is isolated. The ends of the fragment are modified, if necesεary, for cloning into the vector. For instance, 5" overhanging may be treated with DNA polymerase to create blunt ends. 3 ' overhanging ends may be removed using Si nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase. Equal quantities of the Moloney murine leukemia virus linear backbone and the CCIII fragment are mixed together and joined using T4 DNA ligase. The ligation mixture is used to transform E. Coli and the bacteria are then plated onto agar-containing kanamycin. Kanamycin phenotype and restriction analysis confirm that the vector has the properly inserted gene. Packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS) , penicillin and streptomycin. The vector containing the CCIII gene is introduced into the packaging cells by standard techniques. Infectious viral particles containing the CCIII gene are collected from the packaging cells, which now are called producer cells. Fresh media is added to the producer cells, and after an appropriate incubation period media is harvested from the plates of confluent producer cells. The media, containing the infectious viral particles, is filtered through a Millipore filter to remove detached producer cells. The filtered media then is used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the filtered media. Polybrene (Aldrich) may be included in the media to facilitate transduction. After appropriate incubation, the media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblastε will be infected and no εelection is required. If the titer is low, then it is necessary to use a retroviral vector that has a εelectable marker, such as neo or his, to select out transduced cells for expansion. Engineered fibroblasts then may be injected into rats, either alone or after having been grown to confluence on microcarrier beads, such as cytodex 3 beads. The injected fibroblastε produce CCIII product, and the biological actions of the protein are conveyed to the hoεt. It will be clear that the invention may be practiced otherwiεe than aε particularly deεcribed in the foregoing deεcription and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ni, Jian (ii) TITLE OF INVENTION: Chemotactic Cytokine III (iii) NUMBER OF SEQUENCES: 9
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,
STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068-1739
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNE /AGENT INFORMATION:
(A) NAME: Ferraro, Gregory D
(B) REGISTRATION NUMBER: 36,134
(C) REFERENCE/DOCKET NUMBER: 325800-522
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 371 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: sig_peptide
(B) LOCATION: 58..141
(ix) FEATURE:
(A) NAME/KEY: mat_peptide
(B) LOCATION: 142..298
(ix) FEATURE:
(A) NAME/KEY: CDS
<B) LOCATION: 58..300
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 : CTCGAGCTGG AGTCGGAGTT GTAACGCTCC ACTGACTGAT AGAGCGACCG GCCGACC 57
ATG GCG CCC GGA GTG GCC CGC GGG CCG ACG CCG TAC TGG AGG TTG CGC 105 Met Ala Pro Gly Val Ala Arg Gly Pro Thr Pro Tyr Trp Arg Leu Arg -28 -25 -20 -15
CTC GGT GGC GCC GCG CTG CTC CTG CTG CTC ATC CCG GTG GCC GCC GCG 153 Leu Gly Gly Ala Ala Leu Leu Leu Leu Leu He Pro Val Ala Ala Ala -10 -5 1
CAG GAG CCT CCC GGA GCT GCT TGT TCT CAG AAC ACA AAC AAA ACC TGT 201 Gin Glu Pro Pro Gly Ala Ala Cys Ser Gin Asn Thr Asn Lys Thr Cys 5 10 15 20
GAA GAG TGC CTG AAG AAC GTC TCC TGT CTT TGG TGC AAC ACT AAC AAG 249 Glu Glu Cys Leu Lys Asn Val Ser Cys Leu Trp Cys Asn Thr Asn Lys 25 30 35
GCT TGT CTG GAC TAC CCA GTT ACA AGC GTC TTG CCA CCG GCT TTC CCT 297 Ala Cys Leu Asp Tyr Pro Val Thr Ser Val Leu Pro Pro Ala Phe Pro 40 45 50
TTG TAAATTGAGC TCTGCACGCT GGGGAGTTTG TTGGGTGAAC TTTGAGGCGC 350
Leu
TGATCATCAC CATGTCGGTA G 371
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 81 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Pro Gly Val Ala Arg Gly Pro Thr Pro Tyr Trp Arg Leu Arg -28 -25 -20 -15
Leu Gly Gly Ala Ala Leu Leu Leu Leu Leu He Pro Val Ala Ala Ala -10 -5 l
Gin Glu Pro Pro Gly Ala Ala Cys Ser Gin Asn Thr Asn Lys Thr Cys 5 10 15 20
Glu Glu Cys Leu Lys Asn Val Ser Cys Leu Trp Cys Asn Thr Asn Lys 25 30 35
Ala Cys Leu Asp Tyr Pro Val Thr Ser Val Leu Pro Pro Ala Phe Pro 40 45 50
Leu
(2) INFORMATION FOR SEQ ID NO:3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: CGCCCATGGT GGCCGCCGCG CAGG 24
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: CGCAAGCTTG CAGAGCTCAA TTTA 24
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5: CGCGGATCCG CCATCATGGC GCCCGGAGTG 30
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGCGGTACCG CAGAGCTCAA TTTA 24
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid ; C) STRANDEDNESS: single
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CGCGGATCCA CCATGGCGCC CGGAGTGGCC 30 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: CGGTCTAGAT CAAGCGTAGT CTGGGACGTC GTATGGGTAC AAAGGGAAAG CCGG 54
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Met Ala Pro Pro Thr Arg Arg Leu Leu Asn Ala Ala Leu Leu Leu Leu 1 5 10 15
Leu Leu Leu Met Ala Thr Ser His Gin Pro Ser Gly Thr Val Val Ala 20 25 30
Arg Glu Leu Arg Cys Gin Cys Leu Lys Thr Leu Pro Arg Val Asp Phe 35 40 45
Glu Asn He Gin Ser Leu Thr Val Thr Pro Pro Gly Pro His Cys Thr 50 55 60
Gin Thr Glu Val He Ala Thr 65 70

Claims

1 What iε Claimed iε:
2 l. An isolated polynucleotide comprising a polynucleotide having
3 at leaεt 70% identity to a member εelected from the group
4 conεisting of:
5 (a) a polynucleotide encoding a polypeptide comprising an
6 amino acid sequence as set forth in SEQ ID NO:2;
7 (b) a polynucleotide encoding a polypeptide comprising amino
8 acid 1 to amino acid 53 of SEQ ID NO:2;
9 (c) a polynucleotide which is complementary to the o polynucleotide of (a) or (b) ; and i (d) a polynucleotide comprising at least 15 baseε of the
2 polynucleotide of (a) , (b) or (c) . 3 4 2. The polynucleotide of Claim 1 wherein the polynucleotide iε 5 DNA. 6 7 3. The polynucleotide of Claim 1 wherein the polynucleotide iε 8 RNA. 9 0 4. The polynucleotide of Claim 2 compriεing nucleotide 58 to 371 1 set forth in SEQ ID NO:l. 2
5. The polynucleotide of Claim 2 compriεing nucleotide 142 to 370 set forth in SEQ ID N0:1.
β 6. The polynucleotide of Claim 2 which encodes a polypeptide i comprising amino acid 1 to 53 of SEQ ID NO:2. 8 9
7. An isolated polynucleotide compriεing a polynucleotide having 0 at leaεt a 70% identity to a member selected from the group 1 consiεting of: 2 (a) a polynucleotide encoding the same mature polypeptide 3 expresεed by the human cDNA contained in ATCC Depoεit No. 97408; 4 (b) a polynucleotide complementary to the polynucleotide of 5 (a) ; and 6 (c) a polynucleotide compriεing at leaεt 15 baseε of the 7 polynucleotide of (a) or (b) . 8
8. A vector compriεing the DNA of Claim 2 l 9. A host cell comprising the vector of Claim 8.
2
3 10. A process for producing a polypeptide comprising: expressing
4 from the host cell of Claim 9 a polypeptide encoded by said human
5 cDNA. 6
7 11. A procesε for producing a cell which expreεses a polypeptide
8 comprising: transforming or transfecting the cell with the vector
9 of Claim 8.
10 ii 12. A polypeptide compriεing an amino acid εequence which iε at
12 leaεt 70% identical to a member εelected from the group consisting
13 of:
14 (a) a polypeptide comprising an amino acid sequence aε set
15 forth in SEQ ID NO:2;
16 (b) a polypeptide comprising amino acid 1 to 53 of SEQ ID
17 NO:2; ie (c) a polypeptide compriεing at least 15 amino acid residues
19 of the polypeptide of claim (a) or (b) .
20 i 13. A polypeptide comprising amino acid 1 to 53 of SEQ ID NO:2. 2 3 14. An agonist to the polypeptide of claim 12. 4 5 15. An antibody specific to the polypeptide of claim 12. 6 7 16. An antagoniεt which inhibitε the activity of the polypeptide 8 of claim 12. 9 0 17. A method for the treatment of a patient having need of CCIII 1 compriεing: adminiεtering to the patient a therapeutically 2 effective amount of the polypeptide of claim 12. 3 4 18. The method of Claim 17 wherein εaid therapeutically effective 5 amount of the polypeptide iε administered by providing to the 6 patient DNA encoding εaid polypeptide and expreεsing said 7 polypeptide in vivo. 8 1 19. A method for the treatment of a patient having need to inhibit
2 CCIII polypeptide comprising: administering to the patient a
3 therapeutically effective amount of the antagonist of Claim 16.
4
5 20. A process for diagnosing a diεeaεe or a susceptibility to a
6 disease related to expression of the polypeptide of claim 12
7 comprising: β determining a mutation in the nucleic acid sequence encoding
9 said polypeptide.
10 n 21. A diagnostic process comprising:
12 analyzing for the presence of the polypeptide of claim 12 in
13 a sample derived from a host.
14 is 22. A method for identifying compounds which bind to and activate
16 or inhibit a receptor for the polypeptide of claim 12 comprising: 7 contacting a cell expressing on the surface thereof a receptor s for the polypeptide, said receptor being associated with a second 9 component capable of providing a detectable signal in response to 0 the binding of a compound to said receptor, with a compound to be i screened under conditions to permit binding to the receptor; and 2 determining whether the compound binds to and activates or 3 inhibits the receptor by detecting the presence or absence of a 4 signal generated from the interaction of the compound with the 5 receptor.
PCT/US1996/002985 1996-03-05 1996-03-05 Chemotactic cytokine iii WO1997032993A1 (en)

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US7067117B1 (en) 1997-09-11 2006-06-27 Cambridge University Technical Services, Ltd. Compounds and methods to inhibit or augment an inflammatory response

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