JP2004500051A - Novel human melastatin-like protein and polynucleotide encoding the protein - Google Patents

Abstract

Disclosed are novel human polynucleotide and polypeptide sequences that can be used in therapy, diagnosis, and pharmacogenomic applications.

Description

[0001]
This application claims priority to US Provisional Application No. 60 / 162,678, filed November 1, 1999, and incorporated herein by reference in its entirety.
1. Introduction
The present invention relates to the discovery, identification, and characterization of novel human polynucleotides that encode proteins that share sequence similarity with mammalian tumor progression inhibitor proteins. The present invention relates to the described polynucleotides, host cell expression systems, encoded proteins, fusion proteins, polypeptides and peptides, antibodies to the encoded proteins and peptides, and genetics that lack or overexpress the disclosed genes. Engineered animals, antagonists and agonists of the protein, and other that modulate the expression or activity of a protein encoded by the disclosed genes, which can be used in diagnosis, drug screening, clinical trial monitoring and treatment of physiological disorders Including compounds.
2. Background of the Invention
Tumor progression inhibitors (TPIs) include genes whose expression is inversely correlated with tumor virulence. Given the therapeutic significance of tumor virulence, TPI proteins such as melastatin have been linked to tumor growth, spread, and metastasis.
3. Summary of the Invention
The present invention relates to the discovery, identification, and characterization of nucleotides that encode novel human proteins, and the corresponding amino acid sequences of these proteins. The novel human protein (NHP) described herein for the first time shares structural similarity with the mammalian melastatin protein.
[0002]
The novel human nucleic acid sequences described herein have lengths of 803, 823, 948, 1,134, 1,197, 1,579, 778, 798, 923, 1,109, 1,172, 1,554, 793, 813, 938, 1,124, 1,187, 1,569, 768, 788, 913, 1,099, 1,162, 1,544, 366, 386, 511, 697, 760, 1,142, 356, 376, 501, 687, 750, 1,132, 89, 127, 108, 12, 74, and 25 amino acids (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, respectively) 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 4,76,78,80,82, and 84 see) encoding the alternative protein / open reading frame (ORF).
[0003]
The invention also includes the agonists and antagonists of the described NHPs, including small molecules, large molecules, mutant NHPs, or portions thereof that compete with native NHPs, peptides, and antibodies, and the expression of the described NHPs (Eg, antisense and ribozyme molecules, and gene or regulatory sequence replacement constructs) or nucleotide sequences that can be used to enhance expression of the described NHP sequences (eg, a strong promoter system). Expression constructs under the control of NHPs), as well as transgenic animals expressing the NHP transgene, or "knockouts" that do not express a functional NHP (which may be conditional). Knockout ES cell lines were generated that contained a gene trap mutation in the murine ortholog of the described gene.
[0004]
In addition, the present invention also utilizes purified preparations of the described NHPs and / or NHP products, or compounds that modulate NHP expression and / or NHP activity utilizing cells that express NHPs, ie, as agonists or antagonists. It also relates to a method for identifying a compound that acts. Such compounds can be used as therapeutics for the treatment of any of a wide variety of conditions associated with biological disorders or imbalances.
4. Sequence listing and figure description
The sequence listing provides the sequence of the described NHP @ ORF encoding the described NHP amino acid sequence. SEQ ID NO: 85 describes the adjacent 5 'and 3' sequences along with the full length NHP'ORF.
5. DETAILED DESCRIPTION OF THE INVENTION
The NHPs described herein for the first time are novel proteins expressed, inter alia, in human cell lines and in adult brain, pituitary, prostate, thymus, thyroid, testis, kidney, and gene-trapped human cell lines. .
[0005]
The present invention relates to nucleotides presented in the Sequence Listing, host cells expressing such nucleotides, expression products of such nucleotides, and: (a) mammalian homologues of the described sequences (specifically described NHPs and NHP-related products) (B) nucleotides encoding one or more portions corresponding to a functional domain of NHP, including but not limited to a novel region of the active domain, and their nucleotide sequence (C) isolated nucleotides encoding genetically engineered or naturally occurring variants in which all or part of at least one domain of the described NHPs has been deleted or altered, and A polypeptide product identified by the nucleotide sequence of (D) all or part of the coding region of NHP, or one of its domains, fused to another peptide or polypeptide; A nucleotide encoding a chimeric fusion protein comprising one (eg, a receptor or ligand binding domain, an accessory protein / self-binding domain, etc.); or (e) a therapeutic or diagnostic derivative of the described polynucleotide, eg, for the first time in a sequence listing. Includes oligonucleotides, antisense polynucleotides, ribozymes, dsRNA, or gene therapy constructs comprising the disclosed sequences.
[0006]
As discussed above, the present invention includes: (a) the human DNA sequence presented in the sequence listing (and the vector containing the sequence), and further comprising the complement of the DNA sequence presented in the sequence listing: Under very stringent conditions, for example, 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), hybridization to filter-bound DNA in 1 mM EDTA at 65 ° C., and washing at 68 ° C. in 0.1 × SSC / 0.1% SDS (Ausubel F. et al. M. et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, 0.3, and High Dye at New Wiley & Sons, Inc .; Any nucleotide sequence that encodes an adjacent NHP open reading frame (ORF) that encodes a similarly equivalent gene product is contemplated. It is further contemplated that the complement of the DNA sequence encoding and expressing the amino acid sequence presented in the Sequence Listing may be added under moderately stringent conditions, for example, in 0.2 × SSC / 0.1% SDS. , 42 ° C. (Ausubel et al., 1989, supra), yet may be any nucleotide sequence that encodes a functionally equivalent NHP product. Functional equivalents of NHPs include naturally occurring NHPs found in other species and naturally occurring or engineered (site-directed mutagenesis, gene shuffling, eg, US Pat. No. 5,837,458). Or any mutated NHPs, as directed by the directed evolution. The invention also includes degenerate nucleic acid variants of the disclosed NHP polynucleotide sequences.
[0007]
Also contemplated is a polynucleotide encoding the NHP ORF, or a polynucleotide sequence that is about 99, 95, 90 or about 85 percent similar or identical to the corresponding region of the nucleotide sequence in the Sequence Listing (eg, standard default settings). (As measured by BLAST sequence comparison analysis using a GCG sequence analysis package using).
[0008]
The invention also includes nucleic acid molecules, preferably DNA molecules, which hybridize to the described NHP nucleotide sequence and are therefore the complements thereof. Such hybridization conditions may be very stringent, or less stringent, as described above. When the nucleic acid molecules are deoxyoligonucleotides ("DNA oligos"), those molecules comprise contiguous regions of the sequence disclosed for the first time in the Sequence Listing herein, generally from about 16 to about 100 bases, from about 20 to about 100 bases. 80 or about 34 to about 45 bases in length, or any variation or combination of the sizes indicated there. These oligonucleotides can be used in combination with the polymerase chain reaction (PCR) to screen libraries, isolate clones, and prepare cloning and sequencing templates.
[0009]
Alternatively, such NHP oligonucleotides can be used as hybridization probes to screen libraries and evaluate gene expression patterns (especially using microarray or high-throughput "chip" formats). In addition, a series of described NHP oligonucleotide sequences, or their complements, can be used to present all or a portion of the described NHP sequences. Typically, oligonucleotides between about 16 and about 40 (or any integer within this range) nucleotides in length may partially overlap one another and / or the NHP sequences may May be presented using non-oligonucleotides. Thus, the described NHP polynucleotide sequences will typically comprise at least about 2 or 3 separate, at least about 18, and preferably about 25 nucleotides, each disclosed for the first time in the Sequence Listing herein. Should be included. Such an oligonucleotide sequence may begin with any nucleotide present within the sequence in the Sequence Listing and proceed in either the sense (5 'to 3') or antisense direction relative to the described sequence. .
[0010]
For oligonucleotide probes, very stringent conditions are, for example, 37 ° C. (14 bases oligo), 48 ° C. (17 bases oligo), 55 ° C. (20 bases) in 6 × SSC / 0.05% sodium pyrophosphate. Oligo), and washing at 60 ° C. (23 base oligo). These nucleic acid molecules may encode, or act as antisense molecules for, NHP gene antisense molecules, for example, useful for NHP gene regulation (to obtain antisense primers in amplification reactions of NHP nucleic acid sequences, And / or as antisense primers). With respect to NHP gene regulation, such techniques can be used to regulate biological functions. In addition, such sequences can be used as part of a ribozyme and / or triple helix sequence that is also useful for NHP gene regulation.
[0011]
Inhibitory antisense or double-stranded oligonucleotides further include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin (queosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminome Ruuracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid ( v), wybutoxine, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5- Selected from the group comprising oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine At least one modified base moiety It can be included.
[0012]
The antisense oligonucleotide can also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0013]
In yet another embodiment, the antisense oligonucleotide is a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphorodiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or a similar thereof. At least one modified phosphate backbone selected from the group consisting of analogs.
[0014]
In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. The α-anomeric oligonucleotide forms a specific double-stranded hybrid with the complementary RNA, in which the chains run parallel to each other, in contrast to the normal β-unit (Gautier et al., 1987, Nucl. Acids Res. 15: 6625-6641). Oligonucleotides can be 2'-O-methyl ribonucleotides (Inoue et al., 1987, Nucl. Acids Res. 15: 6131-148), or chimeric RNA-DNA analogs (Inoue et al., 1987, FEBS Lett. 215: 327-). 330). Alternatively, double-stranded RNA can be used to disrupt target NHP expression and function.
[0015]
Oligonucleotides of the invention can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (e.g., commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16: 3209), and methylphosphonate oligonucleotides can be synthesized using controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85: 7448-7451).
[0016]
Low stringency conditions will be known to those of skill in the art, and will depend on the particular organism from which the tag and sequence are derived, as is predictable. Guidance on such conditions can be found, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual (and periodic revisions thereof), Cold Spring Harbor Press, New York; and Ausubel et al., 1989, Current Protocols, Digital Catalog See Publishing Associates and Wiley Interscience, New York.
[0017]
Alternatively, a human genomic library can be screened using appropriately labeled NHP nucleotide probes, using appropriately stringent conditions, or by PCR. The identification and characterization of human genomic clones identifies polymorphisms, including but not limited to nucleotide repeats, microsatellite alleles, single nucleotide polymorphisms, or coding single nucleotide polymorphisms, and / Help determine the genomic structure of the allele and design diagnostic tests. For example, internal to exons, introns, splice sites (eg, splicing acceptor and / or donor sites) that can be used in diagnostics and pharmacogenomics using sequences from regions adjacent to the intron / exon boundaries of the human gene. Primers can be designed for use in amplification assays to detect mutations.
[0018]
In addition, by performing PCR using two degenerate or "wobble" oligonucleotide primer pools designed based on the amino acid sequences within the NHP products disclosed herein, the organism of interest can be obtained. NHP gene homologs can be isolated from the nucleic acid of origin. The template for the reaction is the total RNA, mRNA, and / or mRNA prepared from a human or non-human cell line or tissue that is known or suspected to express an allele of the NHP gene. It may be cDNA obtained by reverse transcription.
[0019]
The PCR product can be subcloned and sequenced to ensure that the amplified sequence corresponds to the sequence of the desired NHP gene. Thereafter, a full-length cDNA clone can be isolated by various methods using the PCR fragment. For example, the amplified fragments can be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, labeled fragments can be used to isolate genomic clones through genomic library screening.
[0020]
PCR technology can also be used to isolate full-length cDNA sequences. For example, RNA can be isolated from a suitable cell or tissue source (ie, one known to, or suspected of expressing, the NHP gene) according to standard methods. The reverse transcription (RT) reaction can be performed on RNA using an oligonucleotide primer specific for the 5 'end of the amplified fragment for priming first strand synthesis. The resulting RNA / DNA hybrid can then be "tailed" using a standard terminal transferase reaction, digesting the hybrid with RNase H, and then priming the second strand synthesis with a complementary primer. it can. Thus, it is possible to isolate the cDNA sequence upstream of the amplified fragment. For a review of cloning strategies that can be used, see, for example, Sambrook et al., 1989, supra.
[0021]
The cDNA encoding the mutant NHP gene can be isolated, for example, by using PCR. In this case, the first cDNA strand is ligated to oligo dT in mRNA isolated from tissues known or suspected to be expressed in individuals having the supposedly mutated NHP allele. It can be synthesized by hybridizing the oligonucleotide and extending the new strand with reverse transcriptase. Thereafter, the second strand of the cDNA is synthesized using an oligonucleotide that specifically hybridizes to the 5 'end of the normal gene. Using these two primers, the product is then amplified via PCR, optionally cloned into a suitable vector, and subjected to DNA sequence analysis, through methods known to those skilled in the art. By comparing the DNA sequence of a mutant NHP allele with that of a corresponding normal NHP allele, one or more mutations involved in loss of function or alteration of the mutant NHP gene product can be ascertained It is.
[0022]
Alternatively, obtained from an individual presumed or known to have a mutant NHP allele (eg, an individual who develops an NHP-related phenotype, such as obesity, hypertension, connective tissue disorders, infertility, etc.). DNA can be used to construct a genomic library, or cDNA libraries can be constructed using RNA from tissues known or suspected of expressing mutant NHP alleles. be able to. Thereafter, the normal NHP sequence, or any suitable fragment thereof, may be labeled and used in such libraries as a probe to identify the corresponding mutant NHP allele. Thereafter, the clone containing the mutant NHP sequence can be purified and subjected to sequence analysis according to methods known to those skilled in the art.
[0023]
In addition, RNA isolated from, for example, an individual suspected of having, or known to have, a mutant NHP allele, from a tissue known or suspected of expressing such a mutant allele. An expression library can be constructed using cDNA synthesized from the above. In this format, the gene product created by the putative mutant tissue is expressed and, as described below, using standard antibody screening techniques in combination with antibodies raised against the normal NHP product, Screening is possible (for screening techniques see, for example, Harlow E. and Lane, supervision, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor).
[0024]
Furthermore, screening can be accomplished by screening with a labeled NHP fusion protein, such as, for example, an alkaline phosphatase-NHP fusion protein or an NHP-alkaline phosphatase fusion protein. If an NHP mutation results in an expressed gene product of altered function (eg, as a result of a missense or frameshift mutation), a polyclonal antibody against NHP may cross-react with the corresponding mutated NHP gene product. . Library clones detected via reaction with such labeled antibodies can be purified and subjected to sequence analysis according to methods known in the art.
[0025]
The invention also relates to (a) a DNA vector containing any of the aforementioned NHP coding sequences and / or their complements (ie, antisense); and (b) a control element that directs the expression of the coding sequence. A DNA expression vector (eg, a baculovirus as described in US Pat. No. 5,869,336, incorporated herein by reference) comprising any of the aforementioned NHP coding sequences related to (c) in the host cell. A genetically engineered host cell comprising any of the foregoing NHP coding sequences operably associated with a control element that directs the expression of the coding sequence; and (d) an exogenously introduced control. It also includes genetically engineered host cells that express the endogenous NHP gene under the control of elements (ie, gene activation). As used herein, control elements include, but are not limited to, inducible and non-inducible promoters, enhancers, operators, and other elements known to those of skill in the art to drive and control expression. included. Such control elements include, but are not limited to, the human cytomegalovirus (hCMV) immediate early gene, controllable viral elements (particularly the retroviral LTR promoter), the early or late promoter of the SV40 adenovirus, the lac system, trp Major operators and promoter regions of the phage lambda system, the TAC system, the TRC system, the regulatory region of the fd coat protein, the promoter of 3-phosphoglycerate kinase (PGK), the promoter of acid phosphatase, and the promoter of the yeast α-mating factor. included.
[0026]
The invention also relates to antibodies and anti-idiotype antibodies (including Fab fragments), NHP antagonists and agonists, as well as compounds or nucleotide constructs that inhibit the expression of the NHP gene (transcription factor inhibitors, antisense and ribozyme molecules, or genes). Or a control sequence replacement construct), or a compound or nucleotide construct that promotes expression of NHPs (eg, an expression construct in which the NHP coding sequence is operably associated with an expression control element such as a promoter, promoter / enhancer). Including.
[0027]
NHPs or NHP peptides, NHP fusion proteins, NHP nucleotide sequences, antibodies, antagonists and agonists may be useful to detect mutated or inappropriately expressed NHPs for diagnosis of disease. NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists, and genetically engineered cells and animals are symptomatically or in vivo disrupting the normal functioning of NHPs. It can be used to screen for agents that are effective in treating phenotypic expression (or high-throughput screening of combinatorial libraries). The use of engineered host cells and / or animals is advantageous in that not only can such systems identify compounds that bind to the endogenous receptor for NHPs, but also compounds that elicit NHP-mediated activities or pathways. is there.
[0028]
Finally, NHP products can be used as therapeutic agents. For example, soluble derivatives such as NHP peptides / domains corresponding to NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins, ie, fusion of NHPs to IgFc, or domains of NHPs), NHP antibodies and anti-idiotypes Antibodies (including Fab fragments), antagonists or agonists (including compounds that modulate or act on a downstream target in the NHP-mediated pathway) can be used to directly treat a disease or disorder. For example, administration of an effective amount of soluble NHP, or an NHP-IgFc fusion protein or anti-idiotypic antibody that mimics NHP (or its Fab) activates or effectively antagonizes the endogenous NHP receptor. be able to. Using nucleotide constructs encoding such NHP products, host cells can be genetically engineered to express such products in vivo; these genetically engineered cells can be engineered in vivo into "bioreactors". And delivers the NHP, NHP peptide, or NHP fusion protein to the body in a continuous supply. Nucleotide constructs encoding antisense and ribozyme molecules, as well as functional NHPs and mutant NHPs, can also be used in "gene therapy" approaches for the regulation of NHP expression. Accordingly, the present invention also includes drug formulations and methods for treating biological disorders.
[0029]
Various aspects of the invention are described in further detail in the following subsections.
5.1. NHP sequence
The cDNA sequences of the described NHPs and the corresponding deduced amino acid sequences are shown in the sequence listing. NHP nucleotides were obtained from the cluster human gene trap sequence, EST, and kidney cDNA library (Edge @ Biosystems, Geysersburg, MD). Suitable uses and applications for the described sequences, as well as similar sequences, are described in US Pat. No. 5,674,739, which is incorporated herein in its entirety.
[0030]
5.2. NHP and NHP polypeptide
NHPs, polypeptides, peptide fragments, mutations, truncated, or truncated forms of NHPs, and / or NHP fusion proteins can be prepared for a variety of uses. These uses include, but are not limited to, production of antibodies, use as reagents in diagnostic assays, use for identification of other cellular gene products associated with NHP, mental, biological, or Included is the use of the assay as a reagent to screen for compounds that can be used as pharmaceutical reagents useful in the therapeutic treatment of medical disorders and diseases.
[0031]
The sequence listing discloses the amino acid sequence encoded by the described NHP gene. The NHP typically has an initiator methionine in a DNA sequence background that coincides with the translation start site.
[0032]
The NHP amino acid sequences of the present invention include the amino acid sequences presented in the Sequence Listing, as well as analogs and derivatives thereof. In addition, corresponding NHP homologs from other species are included in the present invention. Indeed, any NHP protein encoded by the NHP nucleotide sequence described above is within the scope of the invention, as is any novel polynucleotide sequence that encodes all or any novel portion of the amino acid sequence presented in the Sequence Listing. . The degeneracy of the genetic code is known, and therefore, each amino acid provided in the sequence listing is a known nucleic acid "triplet" codon capable of encoding the amino acid, or, in many cases, the general form of the codons. This is a typical example. As such, as contemplated herein, the amino acid sequences presented in the Sequence Listing may be considered together with the genetic code (eg, “Molecular Cell Biology”, 1986, incorporated herein). , J. ｎDarnell et al.,, Scientific American Books, New York, NY, pp. 109, Table 4-1), among all of the various variations and combinations of nucleic acid sequences capable of encoding such amino acid sequences. Is a typical representative example.
[0033]
The invention also relates to, but is not limited to, the ability to bind to and cleave NHP substrates, or to achieve the same or a complementary downstream pathway, or cellular metabolism (eg, proteolytic activity, ionic Also included are proteins that are functionally equivalent to the NHPs encoded by the nucleotide sequences described herein, as judged by any of a number of criteria, including changes in flux, tyrosine phosphorylation, and the like. Such functionally equivalent NHP proteins are, but are not limited to, additions or substitutions of amino acid residues within the amino acid sequence encoded by the NHP nucleotide sequence described above, but which produce a silent change and therefore And those that produce functionally equivalent gene products. Amino acid substitutions can be made based on similarities in the polar, charged, soluble, hydrophobic, hydrophilic, and / or amphiphilic properties of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine. And glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
[0034]
A variety of host expression vector systems can be used to express the NHP nucleotide sequences of the present invention. If, as in the example of the present invention, the NHP peptide or polypeptide is considered to be a soluble or secreted molecule, the peptide or polypeptide can be recovered from the culture medium. Such expression systems also include engineered host cells that express NHPs, or functional equivalents, in situ. Purification or enrichment of NHPs from such expression systems can be accomplished using appropriate detergents and lipid micelles, and methods known to those skilled in the art. However, such engineered host cells not only retain the structural and functional properties of NHPs, but can be used in situations where it is important to assess biological activity, such as drug screening assays. .
[0035]
Expression systems that can be used for the purposes of the present invention include, but are not limited to, bacteria (eg, E. coli) transformed with a recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vector containing an NHP nucleotide sequence. Microorganisms such as E. coli and B. subtilis; yeast transformed with a recombinant yeast expression vector containing an NHP nucleotide sequence (eg, Saccharomyces, Pichia); NHP sequences An insect cell line infected with a recombinant virus expression vector (eg, a baculovirus) containing a recombinant virus expression vector (eg, a cauliflower mosaic virus) containing an NHP nucleotide sequence; aMV; a plant cell line infected with tobacco mosaic virus (TMbac), TMV), or transformed with a recombinant plasmid expression vector (eg, Ti plasmid); or a promoter derived from the genome of a mammalian cell (eg, a metallothionein promoter) ) Or a mammalian cell line harboring a recombinant expression construct comprising a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter) (eg, COS, CHO, BHK, 293, 3T3).
[0036]
In bacterial systems, a number of expression vectors may be suitably selected depending upon the use intended for the NHP product being expressed. For example, when attempting to produce large amounts of such proteins, either for the production of NHPs or for pharmaceutical compositions comprising NHPs, or to generate antibodies to NHPs, high levels of fusion protein products that are readily purified Vectors that direct expression may be desirable. Such vectors include, but are not limited to, E. coli in which the NHP coding sequence can be individually ligated in frame with the lacZ coding region, such that a fusion protein is produced. Expression vector pUR278 (Ruther et al., 1983, EMBO J. 2: 1791); pIN vector (Inouye & Inouye, ８５1985, Nucleic Acids Res. 13: 3101-3109; Van Heake & Schom. : 5503-5509); and their equivalents. The pGEX vector (Pharmacia or American Type Culture Collection) can also be used to express a foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads, followed by elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site to allow the cloned target gene product to be released from the GST moiety.
[0037]
In an insect system, Autographa californica nuclear polypolyhydrosis virus (AcNPV) is used as a vector to express foreign sequences. The virus grows in Spodoptera frugiperda cells. The NHP gene coding sequence can be individually cloned into a nonessential region of the virus (eg, the polyhedrin gene) and placed under the control of an AcNPV promoter (eg, the polyhedrin promoter). Successful insertion of the NHP gene coding sequence will result in inactivation of the polyhedrin gene and production of a non-occlusive recombinant virus (ie, a virus that lacks the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (eg, Smith et al., 1983, J. Virol. 46: 584; Smith, U.S. Pat. No. 4,215,051).
[0038]
In mammalian host cells, a number of viral-based expression systems are available. When an adenovirus is used as an expression vector, the NHP nucleotide sequence of interest can be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence. The chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertions in non-essential regions of the viral genome (eg, regions E1 or E3) will result in a recombinant virus that is viable and capable of expressing NHP products in infected hosts (eg, Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81: 3655-3659). Certain initiation signals may also be required for efficient translation of the inserted NHP nucleotide sequence. These signals include the ATG start codon and adjacent sequences. If the entire NHP gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be required. However, if only a portion of the NHP coding sequence is inserted, an exogenous translational control signal, possibly including the ATG start codon, must be provided. In addition, the start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons may be of various origins, both natural and synthetic. Expression efficiency can be increased by including appropriate transcription enhancer elements, transcription terminators, and the like (see Bittner et al., 1987, Methods in Enzymol. 153: 516-544).
[0039]
In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and especially human cell lines.
[0040]
For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, a cell line that stably expresses the above-mentioned NHP sequence can be designed. Rather than using an expression vector containing a viral origin of replication, transforming the host cell with DNA regulated by appropriate expression control elements (eg, promoters, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Can be. Following the introduction of foreign DNA, engineered cells are grown for 1-2 days in enriched media and then transferred to selective media. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into the chromosome and grow to form a foci. The foci can then be cloned and developed into a cell line. This method can be suitably used to engineer cell lines expressing NHP products. Such engineered cell lines may be particularly useful for screening and evaluating compounds that affect the endogenous activity of NHP products.
[0041]
Several selection systems may be used and include, but are not limited to, herpes simplex virus thymidine kinase (Wigler et al., {1977, {Cell} 11: 223), hypoxanthine-guanine phosphoribosyltransferase ( The Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and the adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes were each tk⁻, Hgprt⁻Or apprt⁻Can be used on cells. In addition, the antimetabolite resistance is determined by dhfr (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt conferring resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neo (Colberre- conferring resistance to aminoglycoside G-418). Garpin et al., 1981, J. Mol. Biol. 150: 1); and hygro conferring resistance to hygromycin (Santere et al., 1984, Gene 30: 1). Can be used as the basis of selection for 7).
[0042]
Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the fusion protein to be expressed. For example, the system described in Janknecht et al. Allows for easy purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976). ). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid so that the open reading frame of the gene, when translated, is fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus were²⁺Load onto a nitriloacetic acid-agarose column and selectively elute histidine-tagged proteins with imidazole containing buffer.
[0043]
5.3. Antibodies to NHP products
Antibodies that specifically recognize one or more epitopes of NHP, or epitopes of conserved variants of NHP, or peptide fragments of NHP are also included in the invention. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ')₂Fragments, fragments produced by a Fab expression library, anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of any of the above.
[0044]
The antibodies of the present invention can be used, for example, in the detection of NHPs in biological samples, and thus can be used as part of a diagnostic or prognostic technique to examine patients for abnormal amounts of NHPs. Such antibodies can also be used to assess the effect of a test compound on the expression and / or activity of a NHP gene product, for example, in combination with a compound screening scheme. Further, such antibodies can be used in combination with gene therapy, for example, to evaluate normal and / or engineered NHP expressing cells prior to introduction into a patient. Such antibodies can further be used as a method for inhibition of abnormal NHP activity. Thus, such antibodies can be used as part of a therapy.
[0045]
For the production of antibodies, a variety of host animals can be selected from NHPs, NHP peptides (eg, corresponding to functional domains of NHPs), truncated NHP polypeptides (NHPs in which one or more domains have been deleted), NHPs. Can be immunized by injection with a functional equivalent of or a mutant variant of NHP. Such host animals include, but are not limited to, pigs, rabbits, mice, goats, and rats, to name just a few. Depending on the host species, but not limited to, Freund's adjuvant (complete and incomplete), inorganic salts such as aluminum hydroxide or aluminum phosphate, lysolecithin, surfactants such as pluronic polyols, polyanions , Peptides, oil emulsions, and a variety of adjuvants, including potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and acne propionobacterium (Corynebacterium parvum). Can increase the immunological response. Alternatively, the immune response can be enhanced by combining and / or coupling with molecules such as keyhole limpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies are a heterogeneous population of antibody molecules from the serum of the immunized animal.
[0046]
Monoclonal antibodies, a homogeneous population of antibodies to a particular antigen, can be obtained by any technique that provides for the production of antibody molecules by continuously cultured cell lines. These include, but are not limited to, Kohler and Milstein's hybridoma technology (1975, Nature 256: 495-497; and U.S. Pat. No. 4,376,110), human B cell hybridoma technology (Kosbor et al., 1983). Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030), and EBV-Hybridoma technology (Cole et al., 1985, Monoclonal Ant.Rad.Ant. Inc., pp. 77-96). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of the present invention can be cultured in vitro or in vivo. This method is currently the preferred method of production because of the high titer production of mAbs in vivo.
[0047]
In addition, techniques developed for the production of "chimeric antibodies" by splicing genes from mouse antibody molecules of appropriate antigen specificity along with genes from human antibody molecules of appropriate biological activity (Morrison et al. Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature, 312: 604-608; Takeda et al., 1985, Nature, 314: 452-454). A chimeric antibody is a molecule in which different portions are derived from different animal species, for example, a molecule having a variable region derived from a murine mAb and a human immunoglobulin constant region. Such techniques are described in U.S. Patent Nos. 6,075,181 and 5,877,397, and their respective disclosures, which are incorporated herein by reference in their entirety.
[0048]
Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85) : 5879-5883; and Ward et al., 1989, Nature 334: 544-546) can be used to produce single chain antibodies to the NHP gene product. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
[0049]
Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, such fragments include, but are not limited to: F (ab '), which can be produced by pepsin digestion of an antibody molecule.₂Fragment, and F (ab ')₂Fab fragments that can be generated by reducing the disulfide bridges of the fragment are included. Alternatively, a Fab expression library can be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
[0050]
Antibodies to NHP can subsequently be used to generate anti-idiotypic antibodies that "mimic" a given NHP, using techniques known to those of skill in the art (eg, Greenspan & Bona, 1993, FASEB J7 (5) ): 437-444; and Nissinoff, 1991, J. Immunol. 147 (8): 2429-2438). For example, an antibody that binds to the NHP domain and competitively inhibits binding of NHP to its cognate receptor is used to “mimic” NHP, and thus bind to the receptor and Anti-idiotype antibodies that activate or neutralize the receptor can be generated. Such anti-idiotype antibodies or Fab fragments of such anti-idiotype antibodies can be used in therapeutic measures involving the NHP signaling pathway.
[0051]
The present invention is not limited to the scope according to the particular embodiments described herein, which are merely set forth by way of illustration of the individual embodiments of the present invention and are intended to be functionally equivalent. Methods and components are within the scope of the present invention. Indeed, various modifications of the invention will become apparent to those skilled in the art from the foregoing description, in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims. All cited publications, patents, and patent applications are incorporated herein by reference in their entirety.

Claims (4)

An isolated nucleic acid molecule comprising at least 24 contiguous bases of the nucleotide sequence disclosed for the first time in the NHP gene set forth in SEQ ID NO: 23. An isolated nucleic acid molecule which (a) encodes the amino acid sequence set forth in SEQ ID NO: 24; and (b) comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 23 or its complement. An isolated nucleic acid molecule which (a) encodes the amino acid sequence set forth in SEQ ID NO: 2; and (b) comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or its complement. An isolated nucleic acid molecule which (a) encodes the amino acid sequence set forth in SEQ ID NO: 48; and (b) comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 47 or its complement.