WO1999046295A1 - Gene homologue au gene b(2)gene de drosophile et proteine ypr015c de levure putative 26,5kd - Google Patents

Gene homologue au gene b(2)gene de drosophile et proteine ypr015c de levure putative 26,5kd Download PDF

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Publication number
WO1999046295A1
WO1999046295A1 PCT/CN1998/000037 CN9800037W WO9946295A1 WO 1999046295 A1 WO1999046295 A1 WO 1999046295A1 CN 9800037 W CN9800037 W CN 9800037W WO 9946295 A1 WO9946295 A1 WO 9946295A1
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Prior art keywords
polypeptide
cbcajcio
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000037
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English (en)
Inventor
Qinghua Zhang
Gang Fu
Juan Zhou
Mao Mao
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Shanghai Second Medical University
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Priority to PCT/CN1998/000037 priority Critical patent/WO1999046295A1/fr
Publication of WO1999046295A1 publication Critical patent/WO1999046295A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded b them and to the use of such polynucleotides and polypeptides, and to their production More particularly, the polynucleotides and polypeptides of the present invention relate to the Drosophila b(2)gcn Gene family, hereinafter referred to as CBCAJCIO The invention also relates to inhibiting or actuating the action of such polynucleotides and polypeptides
  • the Drosophila b(2)gcn gene encodes a polypeptide found in benign gonial cell neoplasms
  • the b(2)gcn gene product is expressed early in development and is thought to be involved in cell specific differentiation as a tumor suppressor This indicates that the Drosophila b(2)gcn Gene familj has an established, proven history as therapeutic targets Clearly there is a need for identification and characte ⁇ zation of further members of the Drosophila b(2)gcn Gene family which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, cancer, leukemia, lymphoma, arte ⁇ osclerosis, and autoimmune diseases
  • the invention relates to CBCAJCIO polypeptides and recombinant matenals and methods for their production
  • Another aspect of the invention relates to methods for using such CBCAJCIO polypeptides and polynucleotides
  • Such uses include the treatment of cancer, leukemia, lymphoma, artenosclerosis, and autoimmune diseases, among others
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBCAJC 10 imbalance with the identified compounds
  • CBCAJCIO refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allelic variant thereof
  • CBCAJCIO activity or CBCAJCIO polypeptide activity refers to the metabolic or physiologic function of said CBCAJCIO including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and lmmunogenic activities of said CBCAJCIO
  • CBCAJCIO gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allelic variants thereof and/or their complements
  • Antibodies as used herein includes polyclonal and monoclonal antibodies, chime ⁇ c, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other lmmunoglobulin expression library
  • Isolated means altered “by the hand of man” from the natural state If an "isolated” composition or substance occurs in nature, it has been changed or removed from its original environment, or both For example, a polynucleotide or a polypeptide naturally present in a living animal 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
  • Polynucleotide generally refers to any poly ⁇ bonucleotide or polydeox ⁇ bonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA
  • Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- 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 refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA
  • polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons "Modified" bases include, for example, t ⁇ tylated
  • Polypeptide refers to any peptide or protein comprising two or more ammo acids joined to each other by peptide bonds or modified peptide bonds, I e , peptide isosteres "Polypeptide” refers to both short chains, commonly referred to as peptides, o gopeptides or ohgomers, and to
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids
  • Polypeptides include ammo acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Polypeptides may be branched as a result of ubiquitmation, and they may be cyclic, with or without branching Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods Modifications include acetylation, acyl, acyl-N-linked
  • Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties
  • a typical variant of a polynucleotide differs in nucleotide sequence from another reference polynucleotide Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide Nucleotide changes may result in amino acid substitutions, additions, deletions fusions and truncations in the polypeptide encoded b% the reference sequence, as discussed below
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide Generall) . differences are limited so that the
  • a variant and reference polypeptide are closely similar overall and, in many regions, identical
  • a variant and reference polypeptide may differ in ammo acid sequence by one or more substitutions, additions, deletions in any combination
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally
  • Non-naturally occurring vanants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis
  • Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences
  • Identity and similarity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Bwcomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data, Part I, Griffin, A M , and Griffin, H G , eds , Humana Press, New Jersey, 1994, Sequence Analysis in Molecular Biology , von Heinje, G , Academic Press, 1987, and Sequence Analysis Primer, G ⁇ bskov, M and
  • Preferred parameters for polypeptide sequence comparison include the following
  • Preferred polynucleotide embodiments further include an isolated polynucleotide comprising a polynucleotide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polynucleotide reference sequence of SEQ ID NO 1 , wherein said reference sequence may be identical to the sequence of SEQ ID NO 1 or may include up to a certain integer number of nucleotide alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one nucleotide deletion substitution, including transition and transversion, or insertion, and wherein said alterations may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among the nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of nucleotide alterations is determined by multiplying the total number of nucleotides in SEQ ID NO 1 by the numerical percent of the
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO 1
  • y is 0 50 for 50% , 0 60 for 60% , 0 70 for 70% , 0 80 for 80% , 0 85 for 85 % , 0 90 for 90% , 0 95 for 95 % , 0 97 for 97% or 1 00 for 100%
  • am non integer product of x n and y is rounded down to the nearest integer prior to subtracting it from x n .
  • Alterations of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2 may create nonsense, missense or frameshift mutations in this coding sequence and thereby alter the polypeptide encoded by the polynucleotide following such alterations.
  • Preferred polypeptide embodiments further include an isolated polypeptide comprising a polypeptide having at least a 50,60, 70, 80, 85, 90, 95, 97 or 100% identity to a polypeptide reference sequence of SEQ ID NO:2, wherein said reference sequence may be identical to the sequence of SEQ ID NO: 2 or may include up to a certain integer number of amino acid alterations as compared to the reference sequence, wherein said alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence, and wherein said number of amino acid alterations is determined by multiplying the total number of amino acids in SEQ ID NO:2 by the numerical percent of the respective percent identity and subtracting that product from said total number of amino acids in SEQ ID NO:2, or
  • n a is the number of amino acid alterations
  • x a is the total number of amino acids in SEQ ID NO:2
  • y is 0.50 for 50% , 0.60 for 60% , 0.70 for 70% , 0.80 for 80% , 0.85 for 85 % , 0.90 for 90% , 0.95 for 95 % , 0.97 for 97% or 1.00 for 100% , and wherein any non-integer product of x a and y is rounded down to the nearest integer prior to subtracting it from x-,
  • the present invention relates to CBCAJCIO polypeptides (or CBCAJCIO proteins)
  • the CBCAJCIO polypeptides include the polypeptide of SEQ ID NO 2, as well as polypeptides comprising the amino acid sequence of SEQ ID NO 2, and polypeptides comprising the amino acid sequence which have at least 80% identity to that of SEQ ID NO 2 over its entire length, and still more preferably at least 90% identity, and even still more preferably at least 95% identity to SEQ ID NO 2 Furthermore, those with at least 97-99% are highly preferred Also included within CBCAJCIO polypeptides are polypeptides having the amino acid sequence which
  • CBCAJCIO polypeptide exhibit at least one biological activity of CBCAJCIO
  • the CBCAJCIO polypeptides may be in the form of the "mature" protein or may be a part of a larger protein such as a fusion protein It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification such as multiple histidine residues, or an additional sequence for stability during recombinant production Fragments of the CBCAJCIO polypeptides are also included in the invention
  • a fragment is a polypeptide having an ammo acid sequence that entirely is the same as part, but not all, of the ammo acid sequence of the aforementioned CBCAJC 10 polypeptide
  • Preferred fragments m include, for example, truncation polypeptides having the ammo acid sequence of CBCAJC 10 polypeptides, except for deletion of a contmuous se ⁇ es of residues that mcludes the amino terminus, or a contmuous senes of residues that mcludes the carboxyl terminus or deletion of two contmuous senes of residues, one including the ammo terminus and one including the carboxyl terminus
  • fragments charactenzed by structural or functional attnbutes such as fragments that compnse alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic mdex regions
  • Other preferred fragments are biologically active fragments Biologically active fragments are those that
  • CBCAJC 10 polynucleotides CBCAJC 10 polynucleotides mclude isolated polynucleotides which encode the CBCAJCIO polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBCAJC 10 polynucleotide of the mvention mclude a polynucleotide compnsmg the nucleotide sequence contained m SEQ ID NO 1 encodmg a CBCAJC 10 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBCAJCIO polynucleotides further mclude a polynucleotide compnsmg a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBCAJCIO polypeptide of SEQ ID NO 2, and a polynucleotide comprising a
  • CBCAJCIO of the mvention is structurally related to other proteins of the Drosophila b(2)gcn Gene family, as shown by the results of sequencmg the cDNA of Table 1 (SEQ ID NO 1) encodmg human CBCAJCIO
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 71 to 805) encodmg a polypeptide of 245 ammo acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 72 2% identity (using FASTA) in 245 am o acid residues with yeast hypothetica 1 26 5 kd protein yprO 15c (P Hieter et al , Cell 42 913-921 , 1985 and R Yano et al , Mol Cell Biol 1 1 754-764,1991)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) nas about 72 3% identity (using F
  • CBCAJCIO SEQ ID NO 2 is 77% identical over 35 ammo acid residues to pig unknown protem fragment (AK,W ⁇ nteroe,et al , Mamm Genom 7 509- 517,1996)
  • CBCAJCIO polypeptides and polynucleotides of the present mvention are expected to have, mter alia, similar biological functions/properties to their homologous polypeptides and polynucleotides, and their utility is obvious to anyone skilled m the art
  • a nucleotide sequence of a human CBCAJCIO (SEQ ID NO: 1).
  • One polynucleotide of the present invention encodmg CBCAJCIO may be obtained usmg standard clonmg and screenmg, from a cDNA library denved from mRNA in cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (1991) 252 165 1 - 1656, Adams, M D et al , Nature, (1992) 355 632-634; Adams, M O . et al . Nature ( 1995) 377 Supp 3-174)
  • Polynucleotides of the invention can also be obtained from natural sources such as genomic DNA libraries or can be synthesized using well known and commercially available techniques
  • nucleotide sequence encoding CBCAJCIO polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 71 to 805 of SEQ ID NO 1), or it may be a sequence, which as a result of the redundancy (degeneracy) of the genetic code, also encodes the polypeptide of SEQ ID NO 2 When the polynucleotides of the invention are used for the recombinant production of
  • the polynucleotide may include the coding sequence for the mature polypeptide or a fragment thereof, by itself, the coding sequence for the mature polypeptide or fragment m reading frame with other coding sequences, such as those encodmg a leader or secretory sequence, a pre-, or pro- or prepro- protem sequence, or other fusion peptide portions
  • a marker sequence which facilitates punfication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidtne peptide, as provided m the pQE vector (Qiagen, Inc ) and descnbed m Gentz et al , Proc Natl Acad Sci USA ( 1989) 86 821- 824, or is an HA tag
  • the polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transcnbed,
  • polynucleotides encodmg CBCAJCIO vanants compnse the ammo acid sequence CBCAJCIO polypeptide of Table 2 (SEQ ID NO 2) m which several, 5-10. 1-5. 1-3, 1-2 or 1 ammo acid residues are substituted, deleted or added, m any combination
  • the present mvention further relates to polynucleotides that hybndize to the herem above- desc ⁇ bed sequences
  • the present mvention especially relates to polynucleotides which hybndize under stringent conditions to the herem above-desc ⁇ bed polynucleotides
  • stringent conditions means hyb ⁇ dization will occur only if there is at least 80%, and preferably at least 90%, and more preferably at least 95%, yet even more preferably 97-99% identity between the sequences
  • Polynucleotides of the mvention which are identical or sufficiently identical to a nucleotide sequence contained m SEQ ID NO 1 or a fragment thereof, may be used as hybndization probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encodmg CBCAJC 10 polypeptide and to isolate cDNA and genomic clones of other genes (including genes enco
  • CBCAJCIO polynucleotides of the present mvention further mclude a nucleotide sequence compnsmg a nucleotide sequence that hybndize under stringent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof
  • polypeptide compnsmg ammo acid sequence encoded by nucleotide sequence obtained by the above hybndization condition Such h
  • the present mvention also relates to vectors which compnse a polynucleotide or polynucleotides of the present mvention, and host cells which are genetically engineered with vectors of the mvention and to the production of polypeptides of the mvention by recombinant techniques Cell -free translation systems can also be employed to produce such proteins usmg RNAs denved from the DNA constructs of the present mvention
  • host cells can be genetically engmeered to incorporate expression systems or portions thereof for polynucleotides of the present mvention
  • Introduction of polynucleotides mto host cells can be effected by methods desc ⁇ bed m many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY (1986) and Sambrook et al MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y (1989) such as calcium phosphate transfection, DEAE-dextran mediated transfection transvection, microi ⁇ jection, cationic lipid-mediated transfection, electroporation transduction scrape loading, ballistic mtroduction or infection
  • approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci staphylococci, E coll, Streptomyces and Bacillus subtih cells, fungal cells, such as yeast cells and
  • Asperg llus cells insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells, and plant cells
  • a great va ⁇ ety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those de ⁇ ved from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
  • the expression systems may contain control regions that regulate as well as engender expression Generally, any system or vector suitable to maintain, propagate or express polynucleotides to produce a polypeptide m a host may be used The
  • the CBCAJC 10 polypeptide is to be expressed for use m screenmg assays, generally, it is preferred that the polypeptide be produced at the surface of the cell In this event, the cells may be harvested prior to use m the screening assay If CBCAJCIO polypeptide is secreted into the medium, the medium can be recovered in order to recover and punfy the polypeptide, if produced mtracellularly, the cells must first be lysed before the polypeptide is recovered CBCAJC 10 polypeptides can be recovered and punfied from recombinant cell cultures by well-known methods mcludmg ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic mteraction chromatography, affinit.
  • This mvention also relates to the use of CBCAJCIO polynucleotides for use as diagnostic reagents Detection of a mutated form of CBCAJC 10 gene associated with a dysfunction will provide a
  • CBCAJCIO diagnostic tool that can add to or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression or altered expression of CBCAJC 10
  • Individuals carrying mutations m the CBCAJCIO gene may be detected at the DNA level by a va ⁇ ety of techniques
  • Nucleic acids for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy matenal
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by usmg PCR or other amplification techniques pnor to analysis RNA or cDNA may also be used in similar fashion
  • Deletions and insertions can be detected by a change m size of the amplified product m companson to the normal genotype
  • Pomt mutations can be identified by hyb ⁇ dizing amplified DNA to labeled CBCAJC 10 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched
  • the diagnostic assays offer a process for diagnosing or determining a susceptibility to cancer leukemia, lymphoma, arte ⁇ osclerosis, autoimmune diseases through detection of mutation m the CBCAJC 10 gene by the methods descnbed
  • cancer leukemia, lymphoma, artosclerosis, autoimmune diseases
  • methods comprising determining from a sample derived from a subject an abnormalK decreased or increased level of CBCAJCIO polypeptide or CBCAJCIO mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR, RT-PCR RNase protection Northern blotting and other hybridization methods
  • Assay techniques that can be used to determine levels of a protem, such as an CBCAJC 10 polypeptide, m a sample de ⁇ ved from a host are w ell-known to those of skill m the art
  • Such assay methods mclude radioimmunoassay s competitive-binding assa ⁇ s Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit for a disease or suspectabihty to a disease, particularly cancer, leukemia, lymphoma, arte ⁇ osclerosis, autoimmune diseases, which comprises
  • a CBCAJCIO polynucleotide preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof,
  • a CBCAJCIO polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • the nucleotide sequences of the present mvention are also valuable for chromosome identification
  • the sequence is specifically targeted to and can hybndize with a particular location on an individual human chromosome
  • the mapping of relevant sequences to chromosomes according to the present mvention is an important first step in correlating those sequences with gene associated disease 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, m V McKusick, Mendelian Inhentance m Man (available on lme through Johns Hopkins
  • the differences in the cDNA or genomic sequence between affected and unaffected individuals can also be determined 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 disease
  • Antibodies The polypeptides of the mvention or their fragments or analogs thereof, or cells expressing them can also be used as immunogens to produce antibodies lmmunospecific for the CBCAJC 10 pohpeptides
  • the term "lmmunospecific" means that the antibodies have substantiall greater affinity for the polypeptides of the mvention than their affinity for other related polypeptides in the p ⁇ or art
  • Antibodies generated against the CBCAJCIO polypeptides can be obtained by administering the polypeptides or epitope-beanng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routme protocols
  • any technique which provides antibodies produced by contmuous cell lme cultures can be used Examples mclude the hyb ⁇ doma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et al , Immunology Today (1983) 4 72) and the EBV-hybndoma technique (Cole et al , MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp 77-96, Alan R Liss, Inc , 1985)
  • the above-desc ⁇ bed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to punfy the polypeptides by affinity chromatography
  • Antibodies against CBCAJCIO polypeptides may also be employed to treat cancer, leukemia, lymphoma, artenosclerosis, and autoimmune diseases, among others
  • Another aspect of the invention relates to a method for inducing an lmmunological response in a mammal which comprises inoculating the mammal with CBCAJCIO polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer, leukemia, lymphoma, artenosclerosis, and autoimmune diseases, among others
  • a method of inducing lmmunological response in a mammal which comprises, delivering CBCAJCIO polypeptide via a vector directing expression of CBCAJC 10 polynucleotide in vivo in order to induce such an lmmunological response to produce antibody to protect said animal from diseases
  • composition which, when introduced into a mammalian host, induces an lmmunological response in that mammal to a CBCAJCIO polypeptide wherein the composition comprises a CBCAJCIO polypeptide or CBCAJCIO gene
  • the vaccine formulation may further comprise a suitable carrier Since CBCAJCIO polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, lntradermal etc injection)
  • parenterally including subcutaneous, intramuscular, intravenous, lntradermal etc injection
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation instonic with the blood of the recipient, and aqueous and non-aqueous sterile
  • the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid carrier immediately prior to use
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in water systems and other systems known in the art The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation
  • the CBCAJC 10 polypeptide of the present mvention may be employed m a screenmg process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBCAJCIO polypeptide of the present mvention
  • polypeptides of the mvention may also be used to assess identify agonist or antagonists from, for example, cells, cell-free preparations, chemical branes, and natural product mixtures
  • agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc , as the case may be, of the polypeptide of the present mvention, or may be structural or functional mimetics of the polypeptide of the present mvention See Coligan et al , Current Protocols in Immunology 1(2) Chapter 5 (19 1) CBCAJCIO polypeptides are responsible for many biological functions, mcludmg many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBCAJCIO polypeptide on the one
  • Antagonists are employed for therapeutic and prophylactic purposes for such conditions as cancer, leukemia, lymphoma, artosclerosis, and autoimmune diseases
  • Antagonists may be employed for a va ⁇ ety of therapeutic and prophylactic purposes for such conditions as cancer, leukemia, lymphoma. artenosclerosis, and autoimmune diseases
  • screenmg procedures may mvolve usmg appropnate cells which express the
  • CBCAJC 10 polypeptide or respond to CBCAJC 10 polypeptide of the present mvention Such cells mclude cells from mammals, yeast, Drosophila or E coli Cells which express the CBCAJC 10 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBCAJC 10 polypeptide are then contacted with a test compound to observe binding, or stimulation or inhibition of a functional response The ability of the cells which were contacted with the candidate compounds is compared with the same cells which were not contacted for CBCAJC 10 activity
  • these assays may test whether the candidate compound results in a signal generated by activation of the CBCAJCIO polypeptide, using detection systems appropriate to the cells bearing the CBCAJCIO polypeptide Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist by the presence of the candidate compound is observed
  • the assays may simply comprise the steps of mixing a candidate compound with a solution containing a CBCAJCIO polypeptide to form a mixture, measuring CBCAJCIO activity in the mixture, and comparing the CBCAJCIO activity of the mixture to a standard
  • the CBCAJCIO cDNA, protein and antibodies to the protein may also be used to configure assays for detecting the effect of added compounds on the production of CBCAJC 10 mRNA and protein in cells
  • an ELISA may be constructed for measunng secreted or cell associated levels of CBCAJCIO protein using monoclonal and polyclonal antibodies by standard methods known m the art, and this can be used to discover agents which may inhibit or enhance the production of CBCAJCIO (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBCAJCIO protein may be used to identify membrane bound or soluble receptors, if any, through standard receptor binding techniques known in the art These include, but are not limited to, hgand bmding and crosslinking assays in which the CBCAJCIO is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotinylated), or fused to a peptide sequence suitable for detection or purification, and incubated with a source of the putative receptor (cells, cell membranes, cell supernatants, tissue extracts, bodily fluids) Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy In addition to being used for purification and clonmg of the receptor, these binding assays can be used to identify agonists and antagonists of CBCAJCIO which compete with the binding of CBCAJCIO to its receptors, if any Standard methods for conducting screening assays are well understood in the art
  • CBCAJCIO polypeptide antagonists examples include antibodies or, m some cases, oligonucleotides or protems which are closely related to the gands, substrates, receptors, enzymes, etc . as the case may be, of the CBCAJCIO polypeptide, e g , a fragment of the hgands, substrates, receptors. enzymes, etc , or small molecules which bmd to the polypetide of the present mvention but do not elicit a response, so that the activity of the polypeptide is prevented
  • the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for CBCAJCIO polypeptides. or compounds which decrease or enhance the production of CBCAJCIO polypeptides. which comprises
  • This mvention provides methods of treatmg abnormal conditions such as, cancer, leukemia, lymphoma, artenosclerosis, and autoimmune diseases, related to both an excess of and msufficient amounts of CBCAJCIO polypeptide activity
  • CBCAJCIO polypeptide If the activity of CBCAJCIO polypeptide is m excess, several approaches are available One approach compnses administering to a subject an inhibitor compound (antagonist) as hereinabove descnbed along with a pharmaceutically acceptable earner m an amount effective to inhibit the function of the CBCAJC 10 polypeptide, such as, for example, by blockmg the bmdmg of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition In another approach, soluble forms of CBCAJCIO polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc in competition with endogenous CBCAJCIO polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBCAJCIO polypeptide
  • expression of the gene encoding endogenous CBCAJCIO polypeptide can be inhibited using expression blocking techniques
  • Known such techniques involve the use of antisense sequences, either internally generated or separately administered See, for example, O'Connor, J Neurochem (1991) 56 560 in Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)
  • oligonucleotides which form triple helices with the gene can be supplied See, for example, Lee et al , Nucleic Acids Res (1979) 6 3073, Cooney et al , Science (1988) 241 456, Dervan et al , Science (1991) 251 1360
  • These ohgomers can be administered per se or the relevant ohgomers can be expressed in vivo
  • CBCAJC 10 For treatmg abnormal conditions related to an under-expression of CBCAJC 10 and its activity .
  • several approaches are also available.
  • One approach compnses administering to a subject a therapeutically effective amount of a compound which activates CBCAJCIO polypeptide, l e , an agonist as desc ⁇ bed above, m combination with a pharmaceutically acceptable earner, to thereby alleviate the abnormal condition
  • gene therapy may be employed to effect the endogenous production of CBCAJCIO by the relevant cells in the subject
  • a polynucleotide of the mvention may be employed to effect the endogenous production of CBCAJCIO by the relevant cells in the subject.
  • retro viral expression construct may then be isolated and introduced mto a packaging cell transduced with a retroviral plasmid vector containing RNA encodmg a polypeptide of the present mvention such that the packaging cell now produces infectious viral particles containing the gene of interest
  • retroviral plasmid vector containing RNA encodmg a polypeptide of the present mvention
  • These producer cells may be administered to a subject for engmeermg cells in vivo and expression of the polypeptide m vivo
  • gene therapy see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd (1996)
  • Another approach is to administer a therapeutic amount of CBCAJCIO polypeptides m combination with a suitable pharmaceutical earner
  • Peptides such as the soluble form of CBCAJCIO polypeptides, and agonists and antagonist peptides or small molecules, may be formulated m combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations compnse a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners mclude but are not limited to, salme, buffered salme, dextrose, water, glycerol, ethanol, and combinations thereof Formulation should suit the mode of administration, and is well within the skill of the art
  • the mvention further relates to pharmaceutical packs and kits compnsmg one or more containers filled with one or more of the ingredients of the aforementioned compositions of the mvention Polypeptides and other compounds of the present mvention may be employed alone or m conjunction with other compounds, such as therapeutic compounds
  • systemic administration of the pharmaceutical compositions mclude injection typically by intravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or intrapentoneal
  • Alternative means for systemic administration mclude transmucosal and transdermal administration usmg penetrants such as bile salts or fusidic acids or other detergents
  • oral administration may also be possible
  • Administration of these compounds may also be topical and/or localized, m the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of administration the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are in the range of 0 1-100 ⁇ g/kg of subject Wide va ⁇ ations in the needed dosage, however, are to be expected m view of the va ⁇ ety of compounds available and the differing efficiencies of va ⁇ ous routes of administration For example, oral administration ould be expected to require higher dosages than administration by intravenous injection Va ⁇ ations in these
  • 20 dosage levels can be adjusted usmg standard empincal routmes for optimization, as is well understood m -he art
  • Polypeptides used m treatment can also be generated endogenously m the subject, m treatment modalities often referred to as "gene therapy" as descnbed above
  • m treatment modalities often referred to as "gene therapy” as descnbed above
  • cells from a subject may be engmeered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector The cells are then introduced mto the subject
  • CAAGCTCACC AACACCTACT GTCTGGTAGC GATCGGAGGC TCAGAGAACT TCTACAGTGT 180 GTTCGAGGGC GAGCTCTCCG ATACCATCCC CGTGGTGCAC GCGTCTATCG CCGGCTGCCG 240

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBCAJC10 et des procédés d'obtention de ces polypeptides par des techniques recombinantes. Font aussi l'objet de cette invention des procédés d'utilisation des polypeptides et des polynucléotides CBCAJC10 dans la conception de protocoles pour le traitement de cancers, de la leucémie, de lymphomes, d'artériosclérose et de maladies auto-immunes, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000037 1998-03-12 1998-03-12 Gene homologue au gene b(2)gene de drosophile et proteine ypr015c de levure putative 26,5kd WO1999046295A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002016411A2 (fr) * 2000-08-18 2002-02-28 Human Genome Sciences, Inc. Polypeptides de fixation et procedes associes
US8071092B1 (en) 1996-10-25 2011-12-06 Human Genome Sciences, Inc. Methods of inhibiting B lymphocytes using antibodies to Neutrokine-alpha
US8101181B2 (en) 2000-06-16 2012-01-24 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US8212004B2 (en) 1999-03-02 2012-07-03 Human Genome Sciences, Inc. Neutrokine-alpha fusion proteins
US8211649B2 (en) 2006-03-31 2012-07-03 Human Genome Sciences, Inc. Methods of diagnosing and prognosing hodgkin's lymphoma
US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
J. BIOL. CHEM., 272(48), (1997), BIFFO S. et al., "Isolation of a Novel beta4 Integrin-Binding Protein Úp27(BBP)¾ Highly Expressed in Epithelial Cell", pages 30314-30321. *
PROC. NATL. ACAD. SCI. U.S.A., 94(26), (1997), SI K. et al., "Molecular Cloning and Functional Expression of a Human cDNA Encoding Translation Initiation Factor 6", pages 14285-14290. *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8071092B1 (en) 1996-10-25 2011-12-06 Human Genome Sciences, Inc. Methods of inhibiting B lymphocytes using antibodies to Neutrokine-alpha
US8173122B2 (en) 1996-10-25 2012-05-08 Human Genome Sciences, Inc. Methods of treatment using antibodies to neutrokine-alpha
US8231873B2 (en) 1996-10-25 2012-07-31 Human Genome Sciences, Inc. Methods of treatment using antibodies to Neutrokine-alpha
US8303951B2 (en) 1996-10-25 2012-11-06 Human Genome Sciences, Inc. Neutrokine-alpha antibodies and methods of use thereof
US8212004B2 (en) 1999-03-02 2012-07-03 Human Genome Sciences, Inc. Neutrokine-alpha fusion proteins
US8101181B2 (en) 2000-06-16 2012-01-24 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US9187548B2 (en) 2000-06-16 2015-11-17 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
WO2002016411A2 (fr) * 2000-08-18 2002-02-28 Human Genome Sciences, Inc. Polypeptides de fixation et procedes associes
WO2002016411A3 (fr) * 2000-08-18 2003-09-25 Human Genome Sciences Inc Polypeptides de fixation et procedes associes
US8062906B2 (en) 2000-08-18 2011-11-22 Human Genome Sciences, Inc. B-lymphocyte stimulator binding polypeptides and methods based thereon
US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease
US8211649B2 (en) 2006-03-31 2012-07-03 Human Genome Sciences, Inc. Methods of diagnosing and prognosing hodgkin's lymphoma

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