WO1999046293A1 - Proteine a doigt de zinc derivee de cellules hematopoietiques - Google Patents

Proteine a doigt de zinc derivee de cellules hematopoietiques Download PDF

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Publication number
WO1999046293A1
WO1999046293A1 PCT/CN1998/000035 CN9800035W WO9946293A1 WO 1999046293 A1 WO1999046293 A1 WO 1999046293A1 CN 9800035 W CN9800035 W CN 9800035W WO 9946293 A1 WO9946293 A1 WO 9946293A1
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Prior art keywords
polypeptide
cbmacd04
seq
polynucleotide
nucleotide sequence
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PCT/CN1998/000035
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English (en)
Inventor
Jisheng Wu
Gang Fu
Qinghua Zhang
Yaxin Wang
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Shanghai Second Medical University
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Priority to PCT/CN1998/000035 priority Critical patent/WO1999046293A1/fr
Publication of WO1999046293A1 publication Critical patent/WO1999046293A1/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by 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 zinc finger protein gene family, hereinafter referred to as CBMACD04 The invention also relates to inhibiting or activating the action of such polynucleotides and polypeptides
  • Proteins containing zinc finger motifs are often transcription factors or transcription repressors involved in cell division and proliferation
  • a typical zinc finger motif is the C2H2 motif, ⁇ hich consists of repeated domains of two invariant cysteines and two histidines, with a loop of variable length, but containing an invariant leucine and either a tyrosine or phenylalanine located within each loop Zinc finger proteins can bind directly to the DNA double helix
  • the transcription factor TFIIIA is one example of a C2H2 zinc finger protein, and the first identified C2H2 protein
  • the C2H2 zinc finger motif has also been identified in a number of other transcnption factors, presumed transcription factors, and steroid receptors This indicates that the zinc finger protein gene family has an established, proven history as therapeutic targets Clearly there is a need for identification and charactenzation of further members of the zinc finger protein gene family which can play a role m preventing, ameliorating or correcting dysfunctions or diseases,
  • the invention relates to CBMACD04 polypeptides and recombinant mate ⁇ als and methods for their production
  • Another aspect of the invention relates to methods for using such CBMACD04 polypeptides and polynucleotides
  • Such uses include the treatment of cancer and AIDS, among others
  • the invention relates to methods to identify agonists and antagonists using the materials provided by the invention, and treating conditions associated with CBMACD04 imbalance with the identified compounds
  • Yet another aspect of the invention relates to diagnostic assays for detecting diseases associated with inappropriate CBMACD04 activity or levels DESCRIPTION OF THE INVENTION Definitions
  • CBMACD04 refers, among others, generally to a polypeptide having the ammo acid sequence set forth in SEQ ID NO 2 or an allehc variant thereof
  • CBMACD04 activity or CBMACD04 polypeptide activity or “biological activity of the CBMACD04 or CBMACD04 polypeptide” refers to the metabolic or physiologic function of said CBMACD04 including similar activities or improved activities or these activities with decreased undesirable side-effects Also included are antigenic and immunogenic activities of said CBMACD04
  • CBMACD04 gene refers to a polynucleotide having the nucleotide sequence set forth in SEQ ID NO 1 or allehc 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 immunoglobulin 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 mate ⁇ als 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, 1 e , peptide isosteres
  • Polypeptide refers to both short chains, commonly referred to as peptides, oligopeptides or ohgomers, and to longer chains, generally referred to as proteins Polypeptides may contain ammo acids other than the 20 gene-encoded ammo 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 m a voluminous research literature Modifications can occur anywhere in a polypeptide, including the peptide backbone, the ammo 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
  • 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 m nucleotide sequence from another, reference polynucleotide
  • a typical variant of a polypeptide differs in ammo acid sequence from another, reference polypeptide Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical
  • a vanant 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 allehc variant, or it may be a variant that is not known to occur naturally Non-naturally occur ⁇ ng variants of polynucleo
  • 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.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J. , et al. , Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F et al , J. Molec. Biol. 215- 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al.
  • a program useful with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI.
  • the aforementioned parameters are the default parameters for polynucleotide comparisons.
  • 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: l, 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:
  • n n is the number of nucleotide alterations
  • x n is the total number of nucleotides in SEQ ID NO: l
  • 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%
  • any 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 a
  • the present invention relates to CBMACD04 polypeptides (or CBMACD04 proteins)
  • the CBMACD04 polypeptides include the polypeptide of SEQ ID NO.2, as well as
  • CBMACD04 polypeptides comprising the ammo acid sequence of SEQ ID NO 2
  • polypeptides comprising the ammo 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
  • those with at least 97-99% are highly preferred
  • those with at least 97-99% are highly preferred
  • CBMACD04 polypeptide exhibit at least one biological activity of CBMACD04
  • the CBMACD04 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 am
  • Preferred fragments include, for example, truncation polypeptides having the amino acid sequence of CBMACD04 polypeptides, except for deletion of a continuous se ⁇ es of residues that includes the ammo terminus, or a continuous se ⁇ es of residues that includes the carboxyl terminus or deletion of two continuous senes of residues, one including the amino terminus and one including the carboxyl terminus
  • fragments charactenzed by structural or functional attributes 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, hydrophi c regions, hydrophobe regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface- forming regions, substrate binding region, and high antigenic index regions
  • Other preferred fragments are biologically active fragments Biologically active fragments are those that mediate CBMACD04 activity, including those with a similar activity or an improved activity
  • the CBMACD04 polypeptides of the invention can be prepared in any suitable manner
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods Means for preparing such polypeptides are well understood in the art
  • CBMACD04 polynucleotides include isolated polynucleotides which encode the CBMACD04 polypeptides and fragments, and polynucleotides closely related thereto More specifically, CBMACD04 polynucleotide of the invention mclude a polynucleotide comp ⁇ sing the nucleotide sequence contained in SEQ ID NO 1 encoding a CBMACD04 polypeptide of SEQ ID NO 2, and polynucleotide having the particular sequence of SEQ ID NO 1 CBMACD04 polynucleotides further include a polynucleotide comp ⁇ sing a nucleotide sequence that has at least 80% identity over its entire length to a nucleotide sequence encoding the CBMACD04 polypeptide of SEQ ID NO 2, and a polynucleotide comp ⁇ smg a nucleotide sequence that is at least
  • CBMACD04 of the invention is structurally related to other proteins of the zinc finger protein gene family, as shown by the results of sequencing the cDNA of Table 1 (SEQ ID NO 1) encoding human CBMACD04
  • the cDNA sequence of SEQ ID NO 1 contains an open reading frame (nucleotide number 135 to 1193) encoding a polypeptide of 353 amino acids of SEQ ID NO 2
  • the ammo acid sequence of Table 2 (SEQ ID NO 2) has about 792% identity (using FASTA) in 351 amino acid residues with Human Kruppel related zmc finger protein HTF 10 (J Bellefroid et al , Proc Natl Acad Sci U S A 88 (9), 3608-3612,1991)
  • the nucleotide sequence of Table 1 (SEQ ID NO 1) has about 69% identity (using FASTA) in 1619 nucleotide residues with Human Kruppel related zinc finger protein HTF10 (E J Bellefroid et al , Proc Nat
  • a nucleotide sequence of a human CBMACD04 (SEQ ID NO 1)
  • One polynucleotide of the present invention encoding CBMACD04 may be obtained usmg standard cloning and screening, from a cDNA library de ⁇ ved from mRNA m cells of human cord blood using the expressed sequence tag (EST) analysis (Adams, M D , et al Science (19 1) 252 1651- 1656, Adams, M D et al , Nature, (1992) 355 632-634, Adams, M D , et al , Nature (1995) 377 Supp 3-174)
  • EST expressed sequence tag
  • 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
  • the nucleotide sequence encoding CBMACD04 polypeptide of SEQ ID NO 2 may be identical to the polypeptide encoding sequence contained in Table 1 (nucleotide number 135 to 1193 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 in reading frame with other coding sequences, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence, or other fusion peptide portions
  • a marker sequence which facilitates pu ⁇ fication of the fused polypeptide can be encoded
  • the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc ) and desc ⁇ bed in Gentz et al , Proc Natl Acad Sci USA ( 1989) 86 821 -
  • polynucleotide may also contain non-coding 5 ' and 3 ' sequences, such as transc ⁇ bed, non-translated sequences, splicing and polyadenylation signals, ⁇ bosome binding sites and sequences that stabilize mRNA
  • polynucleotides encoding CBMACD04 va ⁇ ants compnse the amino acid sequence CBMACD04 polypeptide of Table 2 (SEQ ID NO 2) in which several, 5-10, 1-5, 1-3, 1-2 or 1 amino acid residues are substituted, deleted or added, m any combination
  • the present invention further relates to polynucleotides that hyb ⁇ dize to the herein above- desc ⁇ bed sequences
  • the present invention especially relates to polynucleotides which hyb ⁇ dize under stringent conditions to the herein above- esc ⁇ 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 invention which are identical or sufficiently identical to a nucleotide sequence contained in SEQ ID NO 1 or a fragment thereof, may be used as hyb ⁇ di ⁇ ation probes for cDNA and genomic DNA, to isolate full-length cDNAs and genomic clones encoding CBMACD04 polypeptide and to isolate cDNA and genomic clones of other genes (including genes encoding homologs and orthologs from species other than human) that have a high sequence simila ⁇ ty to the CBMACD04 gene
  • Such hyb ⁇ dization techniques are known to those of skill in the art Typically these nucleotide sequences are 80% identical, preferably 90% identical, more preferably 95% identical to that of the referent
  • the probes generally will comp ⁇ se at least 15 nucleotides Preferably, such probes will have at least 30 nucleotides and may have at least 50 nucleotides Particularly preferred probes will range between 30 and 50 nucleotides
  • CBMACD04 polynucleotides of the present invention further mclude a nucleotide sequence comp ⁇ sing a nucleotide sequence that hyb ⁇ dize under stnngent condition to a nucleotide sequence having SEQ ID NO 1 or a fragment thereof
  • poKpeptide comp ⁇ smg amino acid sequence encoded by nucleotide sequence obtamed by the above hyb ⁇ dization condition Such hyb ⁇ dization techniques
  • polynucleotides and polypeptides of the present invention may be employed as research reagents and mate ⁇ als for discovery of treatments and diagnostics to animal and human disease
  • the present invention also relates to vectors which comp ⁇ se a polynucleotide or polynucleotides of the present invention, 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 using RNAs de ⁇ ved 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 invention
  • Introduction of polynucleotides into host cells can be effected by methods desc ⁇ bed in 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 (1 89) such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection
  • Representative examples of approp ⁇ ate hosts include bacte ⁇ al cells, such as streptococci, staphylococci, E coh, Streptomyces and Bacillus subt hs cells, fungal cells, such as yeast
  • a great vanety of expression systems can be used Such systems mclude, among others, chromosomal, episomal and virus-denved systems, e g , vectors de ⁇ ved from bactenal plasmids, from bactenophage, 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 bactenophage 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 approp ⁇ ate nucleot
  • appropnate secretion signals may be incorporated mto the desired polypeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
  • the polypeptide be produced at the surface of the cell
  • the cells may be harvested prior to use in the screening assay
  • CBMACD04 polypeptide is secreted into the medium
  • the medium can be recovered in order to recover and purify the polypeptide, if produced intracellularly, the cells must first be lysed before the polypeptide is recovered
  • CBMACD04 polypeptides can be recovered and pu ⁇ fied from recombinant cell cultures 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 lectm chromatography
  • high performance liquid chromatography is employed for punfication
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or
  • This invention also relates to the use of CBMACD04 polynucleotides for use as diagnostic reagents Detection of a mutated form of CBMACD04 gene associated with a dysfunction will provide a 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 CBMACD04 Individuals carrying mutations in the CBMACD04 gene may be detected at the DNA level by a vanety of techniques
  • Nucleic acids for diagnosis may be obtamed from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy mate ⁇ al
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using 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 in compa ⁇ son to the normal genotype
  • Point mutations can be identified b ⁇ hybndizing amplified DNA to labeled CBMACD04 nucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences m melting temperatures
  • DNA sequence differences may also be detected by alterations in electrophoretic mobility
  • cancer and AIDS can be diagnosed by methods comprising determining from a sample derived from a subject an abnormally decreased or increased level of CBMACD04 polypeptide or CBMACD04 mRNA Decreased or increased expression can be measured at the RNA level using any of the methods well known m 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 protein, such as an CBMACD04 polypeptide, in a sample de ⁇ ved from a host are well-known to those of skill m the art Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays
  • the present invention relates to a diagonostic kit for a disease or suspectabi ty to a disease, particularly cancer and AIDS, which comprises (a) a CBMACD04 polynucleotide, preferably the nucleotide sequence of SEQ ID NO 1, or a fragment thereof, (b) a nucleotide sequence complementary to that of (a),
  • a CBMACD04 polypeptide preferably the polypeptide of SEQ ID NO 2, or a fragment thereof, or
  • nucleotide sequences of the present mvention are also valuable for chromosome identification
  • sequence is specifically targeted to and can hybndize with a particular location on an
  • mapping of relevant sequences to chromosomes is an important first step m correlating those sequences with gene associated disease
  • m correlating those sequences with gene associated disease
  • 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
  • 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 CBMACD04 polypeptides
  • 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 CBMACD04 polypeptides can be obtamed by administering the polypeptides or epitope-bea ⁇ ng fragments, analogs or cells to an animal, preferably a nonhuman, usmg routme protocols
  • any technique which provides antibodies produced by continuous cell lme cultures can be used Examples include the hybndoma technique (Kohler, G and Milstein, C , Nature (1975) 256 495-497), the t ⁇ oma technique, the human B-cell hybndoma technique (Kozbor et
  • the above-descnbed antibodies may be employed to isolate or to identify clones expressmg the polypeptide or to pu ⁇ fy the polypeptides by affinity chromatography
  • Antibodies against CBMACD04 polypeptides may also be employed to treat cancer and AIDS, among others Vaccines
  • Another aspect of the invention relates to a method for inducing an lmmunological response in a mammal which comprises inoculating the mammal with CBMACD04 polypeptide, or a fragment thereof, adequate to produce antibody and/or T cell immune response to protect said animal from cancer and AIDS, among others
  • Yet another aspect of the invention relates to a method of inducing lmmunological response in a mammal which comp ⁇ ses, dehve ⁇ ng CBMACD04 polypeptide via a vector directing expression of CBMACD04 polynucleotide in vivo m 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 m that mammal to a CBMACD04 polypeptide wherein the composition comp ⁇ ses a CBMACD04 polypeptide or CBMACD04 gene
  • the vaccine formulation may further comprise a suitable carrier Since CBMACD04 polypeptide may be broken down in the stomach, it is preferably administered parenterally (including subcutaneous, intramuscular, intravenous, mtradermal etc injection)
  • parenterally including subcutaneous, intramuscular, intravenous, mtradermal 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 lnstomc with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
  • the formulations may be presented in unit-dose or multi
  • the CBMACD04 polypeptide of the present invention may be employed in a screening process for compounds which activate (agonists) or inhibit activation of (antagonists, or otherwise called inhibitors) the CBMACD04 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 bra ⁇ es, and natural product mixtures
  • agonists or antagonists may be natural or modified substrates, gands, receptors, enzymes, etc , as the case may be, of the polypeptide of the
  • CBMACD04 polypeptides are responsible for many biological functions, including many pathologies Accordingly, it is desirous to find compounds and drugs which stimulate CBMACD04 polypeptide on the one hand and which can inhibit the function of CBMACD04 polypeptide on the other hand
  • agonists are employed for therapeutic and prophylactic purposes for such conditions as cancer and AIDS Antagonists may be employed for a vanety of therapeutic and prophylactic purposes for such conditions as cancer and AIDS
  • such screening procedures may involve using appropnate cells which express the CBMACD04 polypeptide or respond to CBMACD04 polypeptide of the present mvention
  • Such cells mclude cells from mammals, yeast, Drosophila or E cob Cells which express the CBMACD04 polypeptide (or cell membrane containing the expressed polypeptide) or respond to CBMACD04 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 CBMACD04 activity
  • the assays may simply test binding of a candidate compound wherein adherence to the cells bearing the CBMACD04 polypeptide is detected by means of a label directly or indirectly associated with the candidate compound or in an assay involving competition with a labeled competitor Further, these assays may test whether the candidate compound results in a signal generated by activation of the CBMACD04 polypeptide, using detection systems approp ⁇ ate to the cells bearing the CBMACD04 polypeptide Inhibitors of activation are generally assayed m 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 CBMACD04 polypeptide to form a mixture, measuring CBMACD04 activity in the mixture, and comparing the CBMACD04 activity of the mixture to a standard
  • the CBMACD04 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 CBMACD04 mRNA and protein in cells
  • an ELISA may be constructed for measuring secreted or cell associated levels of CBMACD04 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of CBMACD04 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues
  • the CBMACD04 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 binding and crosshnking assays in which the CBMACD04 is labeled with a radioactive isotope (eg 1251), chemically modified (eg biotmylated), 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 cloning of the receptor, these binding assays can be used to identify agonists and antagonists of CBMACD04 which compete with the binding of CBMACD04 to its receptors, if any Standard methods for conducting screening assays are well understood in the art Examples of potential CBMACD04 polypeptide antagonists include antibodies or,
  • the present invention relates to a screening kit for identifying agonists, antagonists, hgands, receptors, substrates, enzymes, etc for CBMACD04 polypeptides, or compounds which decrease or enhance the production of CBMACD04 polypeptides, which comprises (a) a CBMACD04 polypeptide, preferably that of SEQ ID NO 2.
  • This mvention provides methods of treating abnormal conditions such as, cancer and AIDS, related to both an excess of and insufficient amounts of CBMACD04 polypeptide activity If the activity of CBMACD04 polypeptide is in excess, several approaches are available One approach comp ⁇ ses administering to a subject an inhibitor compound (antagonist) as heremabove desc ⁇ bed along with a pharmaceutically acceptable earner in an amount effective to inhibit the function of the CBMACD04 polypeptide, such as, for example, by blocking the binding of hgands, substrates, receptors, enzymes, etc , or by inhibiting a second signal, and thereby alleviating the abnormal condition
  • soluble forms of CBMACD04 polypeptides still capable of binding the hgand, substrate, enzymes, receptors, etc m competition with endogenous CBMACD04 polypeptide may be administered Typical embodiments of such competitors comprise fragments of the CBMACD04 polypeptide
  • expression of the gene encoding endogenous CBMACD04 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 Ohgodeoxynucleotides as Antisense Inhibitois of Gene Expression, CRC Press, Boca Raton, FL (1988)
  • ohgonucleotides 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 ⁇ /
  • a polynucleotide of the mvention may be engmeered for expression in a replication defective retroviral vector, as discussed above
  • the retroviral expression construct may then be isolated and mtroduced mto a packagmg cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packagmg cell now produces infectious viral particles containing the gene of interest
  • These producer cells may be administered to a subject for engineering cells in vivo and
  • Peptides such as the soluble form of CBMACD04 polypeptides, and agonists and antagonist peptides or small molecules, may be formulated in combination with a suitable pharmaceutical earner
  • a suitable pharmaceutical earner Such formulations comp ⁇ se a therapeutically effective amount of the polypeptide or compound, and a pharmaceutically acceptable earner or excipient
  • earners mclude but are not limited to, saline,
  • the mvention further relates to pharmaceutical packs and kits comp ⁇ smg 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 in conjunction with other compounds, such as therapeutic compounds
  • systemic administration of the pharmaceutical compositions mclude injection, typically by mtravenous injection
  • Other injection routes such as subcutaneous, intramuscular, or mtrape ⁇ toneal
  • 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
  • Admimstration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like
  • the dosage range required depends on the choice of peptide, the route of admimstration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner Suitable dosages, however, are m the range of 0 1 - 100 ⁇ g/kg of subject Wide vanations m the needed dosage, however, are to be expected in view of the vanety of compounds available and the differing efficiencies of vanous routes of admimstration For example, oral administration would be expected to require higher dosages than admimstration by mtravenous injection Vanations m these dosage levels can be adjusted usmg standard empi ⁇ cal routmes for optimization, as is well understood m the 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 mtroduced mto the subject
  • CTCTGTGTCC TCTGCTCCTA GAGGCCCAGC CTCTGTGGCG CTGTTACCAG CAGTATTGGA 120

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Abstract

L'invention concerne des polypeptides et des polynucléotides CBMACD04 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 CBMACD04 dans la conception de protocoles pour le traitement de cancers et du SIDA, entre autres, et des doses diagnostiques pour ces pathologies.
PCT/CN1998/000035 1998-03-12 1998-03-12 Proteine a doigt de zinc derivee de cellules hematopoietiques WO1999046293A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021836A2 (fr) * 1999-09-23 2001-03-29 Incyte Genomics, Inc. Molecules pour le diagnostic et la therapeutique
WO2001023538A2 (fr) * 1999-09-28 2001-04-05 Incyte Genomics, Inc. Molecules de detection et de traitement de maladies
WO2001066580A1 (fr) * 2000-03-07 2001-09-13 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 13 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001066583A1 (fr) * 2000-03-10 2001-09-13 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 14 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001066582A1 (fr) * 2000-03-10 2001-09-13 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 27, et polynucleotide codant pour ce polypeptide
WO2001068690A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 15, et polynucleotide codant pour ce polypeptide
WO2001072803A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 15, et polynucleotide codant pour ce polypeptide
WO2002004500A1 (fr) * 2000-06-19 2002-01-17 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine a doigt de zinc fpm315-17, et polynucleotide codant ce polypeptide
WO2002004502A1 (fr) * 2000-06-19 2002-01-17 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine murine a doigt de zinc 16 (zfp-1), et polynucleotide codant ce polypeptide
WO2002010211A1 (fr) * 2000-06-30 2002-02-07 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 12.98, et polynucleotide codant ce polypeptide
WO2002020595A1 (fr) * 2000-06-12 2002-03-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide
WO2002020600A1 (fr) * 2000-06-28 2002-03-14 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 10.45, et polynucleotide codant ce polypeptide
WO2002040525A1 (fr) * 2000-06-30 2002-05-23 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 18.92, et polynucleotide codant ce polypeptide

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WO1996011267A1 (fr) * 1994-10-07 1996-04-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Adn et proteine a doigts de zinc et leur utilisation

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Publication number Priority date Publication date Assignee Title
WO1996011267A1 (fr) * 1994-10-07 1996-04-18 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Adn et proteine a doigts de zinc et leur utilisation

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Title
PROC. NATL. ACAD. SCI. U.S.A., 88(9), 1 May 1991, BELLEFROID E.J. et al., "The Evolutionarily Conserved Kruppel-Associated Box Domain Defines a Subfamily of Eukaryotic Multifingered Proteins", pages 3608-3612. *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001021836A3 (fr) * 1999-09-23 2002-01-31 Incyte Genomics Inc Molecules pour le diagnostic et la therapeutique
WO2001021836A2 (fr) * 1999-09-23 2001-03-29 Incyte Genomics, Inc. Molecules pour le diagnostic et la therapeutique
WO2001023538A2 (fr) * 1999-09-28 2001-04-05 Incyte Genomics, Inc. Molecules de detection et de traitement de maladies
WO2001023538A3 (fr) * 1999-09-28 2002-05-02 Incyte Genomics Inc Molecules de detection et de traitement de maladies
WO2001066580A1 (fr) * 2000-03-07 2001-09-13 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine 13 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001066583A1 (fr) * 2000-03-10 2001-09-13 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine 14 a doigt de zinc, et polynucleotide codant pour ce polypeptide
WO2001066582A1 (fr) * 2000-03-10 2001-09-13 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 27, et polynucleotide codant pour ce polypeptide
WO2001068690A1 (fr) * 2000-03-15 2001-09-20 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 15, et polynucleotide codant pour ce polypeptide
WO2001072803A1 (fr) * 2000-03-28 2001-10-04 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 15, et polynucleotide codant pour ce polypeptide
WO2002020595A1 (fr) * 2000-06-12 2002-03-14 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine a doigt de zinc humaine 18.04, et polynucleotide codant ce polypeptide
WO2002004500A1 (fr) * 2000-06-19 2002-01-17 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine humaine a doigt de zinc fpm315-17, et polynucleotide codant ce polypeptide
WO2002004502A1 (fr) * 2000-06-19 2002-01-17 Biowindow Gene Development Inc. Shanghai Nouveau polypeptide, proteine murine a doigt de zinc 16 (zfp-1), et polynucleotide codant ce polypeptide
WO2002020600A1 (fr) * 2000-06-28 2002-03-14 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 10.45, et polynucleotide codant ce polypeptide
WO2002010211A1 (fr) * 2000-06-30 2002-02-07 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 12.98, et polynucleotide codant ce polypeptide
WO2002040525A1 (fr) * 2000-06-30 2002-05-23 Shanghai Biowindow Gene Development Inc. Nouveau polypeptide, proteine humaine a doigt de zinc 18.92, et polynucleotide codant ce polypeptide

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