JP2003527094A - Novel human organic anion transporter-like protein and polynucleotide encoding the same - Google Patents

Abstract

  (57) [Summary] Disclosed are novel human polynucleotide and polypeptide sequences that can be used in therapeutic, diagnostic and pharmacogenetic applications.

Description

Detailed Description of the Invention

[0001] Introduction The present invention is the discovery of novel human polynucleotide sequences encoding a protein with sequence similarity with animal organic ion transporter protein, it relates to the identification and characterization. The invention provides polynucleotides, host cell expression systems, encoded proteins, fusion proteins, polypeptides and peptides, antibodies directed against the encoded proteins and peptides, and lacking or excess of the disclosed sequences. Expressing genetically engineered animals, antagonists and agonists of the described proteins, and modulating expression or activity of proteins encoded by the disclosed sequences that can be used in diagnostics, drug screening, clinical trial monitoring and treatment of physiological disorders. Including compounds of BACKGROUND ART Transporter proteins mediate transmembrane transmembrane influx and efflux of compounds and / or ions. Because of the physiological importance of such a given function, the transporter protein is a subject to be scrutinized and a drug candidate. SUMMARY OF THE INVENTION The present invention relates to the discovery, identification and characterization of nucleotides encoding novel human proteins, and the corresponding amino acid sequences of those proteins. Novel human proteins (NHPs) are animal organic anion transporter proteins,
More specifically, it was first described herein to have structural similarities to the prostaglandin transporter protein. Thus, NHPs represent a new family of proteins with homologies and similarities across strains and species.

It has been described herein that the novel human nucleic acid sequences encode the open reading frames (ORFs) of protein / 536 or 535 amino acids (see SEQ ID NO: 2).

The present invention includes the following. Small molecules, large molecules, mutant NHPs, or agonists and antagonists of said NHPs, including peptides and antibodies that compete with naturally occurring NHPs, and peptides for inhibiting the expression of said NHPs (eg, antisense and ribozyme molecules, And gene or regulatory sequence replacement constructs), or to enhance expression of the NHP polynucleotides (eg, expression constructs that place the sequences under the control of a strong promoter system). The present invention also includes both transgenic animals that express the NHP transgene and NHP "knockouts" that do not express functional NHP (which may be conditional).

Furthermore, the present invention provides a compound, ie an agonist or an antagonist, which regulates NHP expression and / or NHP activity utilizing the purified product of the NHP and / or NHP product or cells expressing the same. A method of identifying a compound that acts. Such compounds may be used as therapeutic agents to treat any of a variety of conditions associated with biological or imbalance disorders. SEQUENCE LISTING AND EXPLANATION OF DRAWING The sequence listing shows the transporter-like ORF sequences encoding the NHP amino acid sequences. DETAILED DESCRIPTION OF THE INVENTION The NHPs described first herein are, inter alia, human cell lines and human brain, pituitary gland, spinal cord, lymph nodes, trachea, testis, heart, fat, skin, pericardium, hypothalamic cells. Is a novel protein expressed in. The sequences are gene trapped cDNA and human lymph node cDNA library (Edge Biosystems, G
aithersburg, MD).

The present invention includes the following. Nucleotides set forth in the Sequence Listing, host cells expressing those nucleotides, and expression products of those nucleotides, and: (a) nucleotides and NHPs encoding mammalian homologues of the gene products, particularly including the NHPs. Product; (b) NHP corresponding to functional domain
Nucleotides encoding one or more portions of, and polypeptide products identified by their nucleotide sequences, including but not limited to novel regions of either active domain (s); (c ) An isolated nucleotide encoding a genetically engineered or naturally occurring variant of said NHP, wherein all or part of at least one domain has been deleted or altered, and their nucleotide sequences. Specified polypeptide products (including soluble proteins and peptides lacking all or part of the signal sequence,
(D) all or part of the coding region of NHP, or one of its domains fused to another peptide or polypeptide (eg, receptor or ligand binding domain, accessory protein / self binding domain, etc.) Or a nucleotide encoding a chimeric fusion protein comprising: (e) an oligonucleotide, an antisense polynucleotide, a ribozyme, a double-stranded RNA
Or a therapeutic or diagnostic derivative of said polynucleotide, such as a gene therapy construct containing the novel sequences disclosed in the Sequence Listing.

As described above, the present invention provides (a) a human DNA sequence (and a vector containing the same) shown in the sequence listing and a complementary sequence of the DNA sequence shown in the sequence listing under high stringency conditions [ For example, 0.5M NaHPO ₄ , 7% sodium dodecyl sulfate (SDS
) Hybridization to filter-bound DNA in 1 mM EDTA at 65 ° C. and washing in 0.1 × SSC / 0.1% SDS at 68 ° C. (Aussub)
el, F.F. M. et al. Hen, 1989, Current Protoco
ls in Molecular Biology, Vol. I, Green
Publishing Associates, Inc., and John Willy
& Sons, New York, p. 2.10.3)] Any nucleotide sequence encoding a contiguous NHP open reading frame (ORF) that hybridizes and encodes a functionally equivalent gene product is considered. Furthermore, it hybridizes to the complementary sequence of the DNA sequence encoding and expressing the amino acid sequence shown in the sequence listing under moderate stringent conditions [eg 0.2 × SSC / 0.1% S
Wash in DS at 42 ° C. (Ausubel, et al., 1989, supra)],
Also, any nucleotide sequence that encodes a functionally equivalent NHP gene product is contemplated. Functional equivalents of NHP include natural NHPs present in other species,
And native or genetically engineered mutated NHPs (site-directed mutagenesis, gene shuffling, for example by the specific evolution described in USP 5,837,458). The present invention also includes degenerate nucleic acid variants of the disclosed NHP polynucleotide sequences.

Further considered are polynucleotides encoding NHP ORFs, or about 99%, 95%, 90% or about 85% homology to the corresponding region of Sequence Listing 1 (eg, GCG sequence analysis package. Are measured by BLAST sequence comparison analysis using standard default parameters) and their functional equivalents encoded by the polynucleotide sequences.

The invention also includes a nucleic acid molecule, preferably a DNA molecule, which hybridizes to the NHP gene nucleotide sequence and is therefore the complementary sequence of the NHP gene. Such hybridization conditions may be highly stringent, as described above, or less stringent. When the nucleic acid molecule is a deoxyoligonucleotide ("DNA oligobody"), the molecule is generally about 16 to about 10
0 bases, about 20 to about 80 bases, or about 34 to about 45 bases in length, or any variation or combination of sizes shown therein, including contiguous regions of the sequences first disclosed in the Sequence Listing of the present invention. is there. Such oligonucleotides can be used in connection with the polymerase chain reaction (PCR) to screen libraries, isolate clones, prepare templates for cloning and sequencing, etc.

Alternatively, such NHP oligonucleotides can be used as hybridization probes for library screening and evaluation of gene expression patterns (especially using microarray or high throughput "chip" formats). In addition, a series of the NHP oligonucleotide sequences or their complements can be used to express all or part of the NHP sequence. Generally, oligonucleotides of from about 16 to about 40 (or any natural number in the range) nucleotides in length may partially overlap with each other and / or NH with oligonucleotides that do not overlap.
The P sequence can be expressed. Accordingly, the NHP polynucleotide sequences should generally include at least about 2 or 3 distinct oligonucleotides each of which is first disclosed in the Sequence Listing herein and is at least about 18, preferably at least about 25 nucleotides in length. is there. Such an oligonucleotide sequence begins at any nucleotide present in the sequence in the sequence listing and proceeds in either the sense (5'- to -3 ') or antisense orientation to said sequence. May be.

Regarding the oligonucleotide probe, the high stringency condition is, for example, 6 × SSC.
/0.05% sodium pyrophosphate at 37 ° C. (for 14 base oligos),
48 ° C (for 17-base oligo), 55 ° C (for 20-base oligo),
And wash at 60 ° C. (for 23 base oligos). These nucleic acid molecules can encode or act as NHP gene antisense molecules useful, for example, in NHP gene regulation (to obtain antisense primers in amplification reactions of NHP gene nucleic acid sequences, and / Or as an antisense primer). For NHP gene regulation, those methods can be used to regulate biological function. Furthermore, such an array can also be converted to N
It can be used as part of a ribozyme and / or triple helix sequence useful for HP gene regulation.

The inhibitory antisense or double-stranded oligonucleotide may comprise at least one modified base moiety selected from the group including but not limited to: 5-fluorouracil, 5-bromo. Uracil, 5-chlorouracil,
5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5
-(Carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine , 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5
-Methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylcueocin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil , 2-methylthio-N6-isopentenyladenine, uracil-5-
Oxyacetic acid (v), Wibutoxin, Pseudouracil, Cueosin, 2-
Thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,
6-diaminopurine.

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.

In yet another embodiment, the antisense oligonucleotides are phosphorothioates, phosphorodithioates, phosphoramidothioates, phosphoramidates, phosphordiamidates, methylphosphonates, alkyl phosphotriesters and formacetals, or It comprises at least one modified phosphate backbone selected from the group consisting of its analogs.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. Unlike normal β-units, α-anomeric oligonucleotides are complementary to RNs that complement specific double-stranded hybrids in which the strands run parallel to each other.
A. (Gautier et al., 1987, Nucl. Acid
s Res. , 15: 6625-6641). This oligonucleotide is 2'-
O-methyl ribonucleotide (Inoue et al., 1987, Nucl)
． Acids Res. , 15: 6131-6148), or chimeric RNA-
DNA analogues (Inoue et al., 1987, FEBS Lett. 2).
15: 327-330). Alternatively, double-stranded RNA can be used to disrupt expression and action of the target NHP. Oligonucleotides of the invention may be prepared according to standard methods known in the art, for example in an automated DNA synthesizer (eg Biose).
(commercially available from Arch, Applied Biosystems, etc.)). As an example, phosphorothioate oligonucleotides are described by Stein et al. (1988, Nucl. Acids Res., 16:32.
09) and methylphosphonate oligonucleotides can be prepared using a controlled pore glass polymer support (Sarin et al., 1988,
Proc. Natl. Acad. Sci. U. S. A. , 85: 7448-74
51) etc.

Low stringency conditions are well known to those of skill in the art and will depend on the library and the individual organism from which the label sequence is derived, but will be predictable. For guidance on such conditions see, for example: Sambrook et al. ，
1989, Molecular Cloning, A Laboratory.
Manual (and its regular revisions), Cold Springs Har
bor Press, New York; and Ausubel et al. , 1
989, Current Protocols in Molecular B
Iology, Green Publishing Associates and Wiley interscience, New York.

Alternatively, an appropriately labeled NHP nucleotide probe can be used to screen a human genomic library by employing appropriate stringency conditions or by PCR. Identification and elucidation of human genomic clones includes, but is not limited to, confirmation of polymorphisms, including but not limited to nucleotide repeats, microsatellite alleles, single nucleotide polymorphisms, or coding single nucleotide polymorphisms. Useful for determining loci / alleles genomic structure and designing diagnostic tests. Used in amplification assays to detect mutations in exons, introns, splice sites (eg, splice acceptor and / or donor sites), etc., using sequences from regions flanking the intron / exon boundaries of human genes, for example Primers can be designed and used in diagnostic and pharmacogenomics.

In addition, PCR is performed using two degenerate or “wobble” oligonucleotide primer pools designed based on the amino acid sequences within the NHP products described herein to allow the nucleic acid of the organism of interest to be identified. Can be used to isolate NHP homologous sequences. The reaction template is known or suspected to express the NHP gene / allele, reverse transcription of mRNA prepared from human or non-human cell lines or tissues, such as prostate, rectum, colon or adrenal gland. It may be total RNA, mRNA and / or cDNA obtained by Amplification sequence is the target NH
The PCR product can be subcloned and sequenced to confirm that it is the sequence of the P gene. The PCR fragment can then be used to isolate a full-length cDNA clone in a variety of ways. For example, the amplified fragment 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 by screening a genomic library.

The full-length cDNA sequence can also be isolated using the PCR method. For example, standard methods can isolate RNA from a suitable cellular or tissue source (ie, those known or suspected to express NHP sequences, such as testis tissue). For this RNA, an oligonucleotide primer specific for the most 5'-end amplified fragment was used for priming for first strand synthesis and reverse transcription (RT
) The reaction can be carried out. The resulting RNA / DNA hybrid can then be "tailed" by standard terminal transferase reactions, the hybrid can be digested with RNase H and then primed for second strand synthesis with complementary primers. In this way, the cDNA sequence upstream of the amplified fragment can be isolated. For an overview of cloning methods you can use, see Sambrook
k et al. , 1989 (supra).

The cDNA encoding the mutant NHP gene can be isolated using, for example, PCR. In this case, the first cDNA strand is hybridized with the oligo-dT oligonucleotide to mRNA isolated from tissue known or suspected to be expressed in an individual suspected of carrying the mutant NHP allele, It can be synthesized by extending a new chain with reverse transcriptase. 5'of normal gene
A second cDNA was prepared using an oligonucleotide that specifically hybridizes to the end.
Synthesize chains. The product is then amplified by PCR with these two primers, cloned into an appropriate vector if desired and D
Perform NA sequence analysis. By comparing the DNA sequence of the mutant NHP allele to that of the normal NHP allele, the mutation (one or more) responsible for the loss or alteration of function of the mutant NHP gene product can be identified.

Alternatively, DNA obtained from an individual suspected or known to carry the mutant NHP allele (eg, a human exhibiting an NHP-related phenotype such as immunodeficiency, obesity, hypertension, etc.) Genomic libraries can be used to construct, or cDNA from tissues known or suspected to express mutant NHP alleles can be used to construct cDNA libraries. The normal NHP gene, or any suitable fragment thereof, can then be labeled and used as a probe to identify the corresponding mutant NHP allele in such a library. Clones containing the mutant NHP gene sequence can then be purified and sequenced by established methods.

Further, for example, synthesized from RNA isolated from tissue of an individual suspected or known to carry the mutant allele, known or suspected to express the mutant NHP allele. An expression library can be constructed using the cDNA. In this way, the gene product formed by the putative mutant tissue can be expressed and screened using standard antibody screening methods in combination with antibodies raised against normal NHP products (for screening methods see, eg, Harlow, E. and Lane, 1988, "Antibody.
s: A Laboratory Manual ", Cold Spring H
Arbor Press, Cold Spring Harbor, see).

Furthermore, labeled NHP fusion proteins, such as alkaline phosphatase-NH
Screening can be performed with P or NHP-alkaline phosphatase fusion proteins. If the NHP fusion expresses a gene product with altered function (eg, as a result of missense or frameshift mutations), polyclonal antibodies against NHP are likely to cross-react with the corresponding mutated NHP sequence product. Library clones detected by reaction of them with such labeled antibodies can be purified and sequenced by methods well known to those skilled in the art.

A further application of the novel human polynucleotide sequences described is in the molecular mutagenesis / evolution of proteins at least partially encoded by the novel sequences described, for example using polynucleotide shuffling or related methodologies. The use of them. Such approaches are described in US Pat. Nos. 5,830,721 and 5,837,458, which are hereby incorporated by reference in their entireties.

The present invention also encompasses the following: (a) a DNA vector containing any of the above NHP coding sequences and / or their complementary sequences (ie antisense); (b) directing the expression of the coding sequences. A DNA expression vector comprising any of the NHP coding sequences operably linked to a regulatory element (eg, the baculovirus described in US Pat. No. 5,869,336, incorporated herein by reference); (C) a genetically engineered host cell comprising any of the aforementioned NHP coding sequences operably linked to regulatory elements that direct the expression of the coding sequence in the host cell; and (d) an endogenous NHP gene. A genetically engineered host cell that is expressed under the control of an exogenously introduced regulatory element (ie, gene activation). Regulatory elements, as used herein, include inducible and non-inducible promoters, enhancers, operators, and other elements known to those of skill in the art to drive and regulate expression. Not limited. These regulatory elements include, but are not limited to, the regulatable viral elements of the cytomegalovirus (hCMV) immediate early gene (particularly the retroviral LTR promoter), the early or late SV40 adenovirus. Promoter, lac system, trp system, TAC system, TRC system, main operator and promoter region of phage lambda, fd coat protein control region, 3-phosphoglycerate kinase (PGK) promoter, acid phosphatase promoter, and yeast α -A mating factor promoter.

As in this example, some of the NHP peptides or polypeptides described are believed to be membrane bound, but by devising the expression system, soluble derivatives of NHP (which can be used in therapeutic applications ( NHP extracellular and / or intracellular domains, or corresponding truncated polypeptides lacking one or more transmembrane domains), and / or NHP fusion protein products (particularly to NHP-Ig fusion proteins, ie IgFc). NHP domain, eg, fusion of ECD, ΔTM), NHP antibody and anti-idiotype antibody (Fa
b fragment) can be produced. The expression system described above is preferably engineered to recover the desired peptide or polypeptide from the culture. A sequence similar to the sequence existing near the 3'region of the described OFR has been reported as a human secretory protein in gene 28 clone HLHSH36, so that one or more regions or domains of the described protein are It should be noted that it may circulate as a soluble molecule of.

The invention also includes the following: antibodies and anti-idiotypic antibodies (Fabs).
Fragments), compounds and nucleotide constructs that inhibit the expression of NHP coding sequences (transcription factor inhibitors, antisense and ribozyme molecules, or gene or regulatory sequence replacement constructs), or A compound or nucleotide that promotes the expression of NHP (eg, an expression construct in which the coding sequence of NHP is operably linked to an expression control element such as a promoter or a promoter / enhancer).

NHPs or NHP peptides, NHP fusion proteins, and NHP nucleotide sequences, antibodies, antagonists and agonists can be useful in detecting mutant or misexpressed NHPs for the diagnosis of disease. The NHP proteins or peptides, NHP fusion proteins, NHP nucleotide sequences, host cell expression systems, antibodies, antagonists, agonists, and genetically engineered cells and animals are symptomatic for the perturbation of normal NHP function in the body or It can also be used in drug screening (or high throughput screening of combinatorial libraries) to identify candidates that are effective in treating phenotypic expression. The use of genetically engineered host cells and / or animals is
These systems are advantageous in that not only can they identify compounds that can bind to the endogenous receptor / ligand for NHP, but they can also identify compounds that trigger NHP-mediated activity or pathways.

Finally, the NHP product can be used as a treatment. For example, soluble derivatives such as NHP peptides / domains corresponding to NHP, NHP fusion protein products (particularly NHP-Ig fusion proteins, ie fusions or domains of NHP to IgFc), NHP antibodies and anti-idiotypic antibodies (Fab fragments). ,) And antagonists or agonists (including compounds that modulate or act on downstream targets in the NHP-mediated pathway) can be used to directly treat the disease or disorder. For example, an effective amount of soluble NHP, or N
Administration of an HPP-mimicking NHP-IgFc fusion protein or anti-idiotypic antibody (or Fab fragment thereof) could activate or effectively neutralize endogenous NHP or a protein that interacts with it.
Nucleotide constructs encoding such NHP products can be used to engineer host cells to express such products in vivo; these engineered cells are engineered. Acts as a "bioreactor" in the body, continuously supplying NHP, NHP peptides, or NHP fusion proteins to the body. Nucleotide constructs encoding functional NHPs, mutant NHPs, and antisense and ribozyme molecules can also be used in "gene therapy" modalities that regulate NHP expression. Accordingly, the present invention also includes pharmaceutical formulations and methods for treating biological disorders.

Various aspects of the invention are described in further detail in the following subsections. 5.1 NHP Sequence The cDNA sequence of the NHP and the corresponding deduced amino acid sequence are shown in the sequence listing.
The NHP coding sequence was obtained from a human cDNA library using probes and / or primers obtained from human gene trapped sequence tags.
The ORF has a predicted hydrophobic leader sequence characteristic of membrane proteins.

Expression analysis showed that the NHPs were expressible in human tissues as well as gene trap human cells. In addition to the aforementioned organic ion transporter proteins, the NHPs have considerable similarities with prostaglandin class transporters of various species. Due to the physiological importance of prostaglandins, these transporters have been the subject of scrutiny, for example, as shown in US Pat. No. 5,792,851, which is hereby incorporated by reference in its entirety.

The sequence was found to be such that the ORF initiates translation at the tandem ATG codon, or the transcript selection is SEQ ID NO: 3 with sequences 103-151 deleted (upstream from the tandem start codon). Can take various forms, including the fact that Two
When translation is initiated at the second of the two start codons, the resulting NHP is SEQ ID NO: 2.
It will be one amino acid shorter (at the 5'end) than the NHP product shown in.

5.2 NHPs and NHP Polypeptides NHPs, polypeptides, peptide fragments, mutated, truncated or deleted forms of NHPs, and / or NHP fusion proteins can be prepared for a variety of uses. It may be used as a reagent in antibody production, as a reagent in diagnostic assays, as a medicament useful in the identification of other cellular gene products associated with NHP, in the therapeutic treatment of mental, biological or medical disorders and diseases. It includes, but is not limited to, use as a reagent in an assay for screening compounds.

The Sequence Listing discloses the amino acid sequences encoded by the NHP coding sequences. NHPs have an initiator methionine in the DNA sequence context that corresponds to the translation start site.

The NHP amino acid sequences of the present invention include the amino acid sequences shown in the sequence listing, as well as their analogs and derivatives. In addition, the corresponding NHP homologous sequences from other species are also included in the invention. In fact, any NHP protein encoded by the above NHP nucleotide sequences is included within the scope of the invention, as are novel polynucleotide sequences encoding all or any novel portion of the amino acid sequences set forth in the Sequence Listing. Included in the present invention. The degeneracy of the genetic code is well known, and therefore each amino acid shown in the sequence listing is a general representative of the well known nucleic acid "triplet" codons (or often codons) that can encode that amino acid. Therefore, as intended herein, the amino acid sequences shown in the Sequence Listing are based on the genetic code (eg, “Molecular Cell Biology”, 1986, J.
． Edited by Darnell et al., P. 109, see Table 4-1, Scientific A
(Merican Books, New York, NY; incorporated herein by reference), is a general representative of all the various variations and combinations of nucleic acid sequences that can encode such amino acid sequences.

The present invention also includes proteins functionally equivalent to the NHP encoded by the nucleotide sequences described herein, as judged by any of a number of criteria. These criteria include, but are not limited to: the ability of NHPs to bind or dissociate with substrates; the ability to affect equivalent or complementary downstream pathways or to alter cellular metabolism (eg, , Proteolytic activity, ion flux, tyrosine phosphorylation, etc.). Such functionally equivalent NHP proteins include additions or substitutions of amino acid residues within the amino acid sequence encoded by the above-described NHP nucleotide sequence, but which results in silent changes and is thus functionally equivalent. These include, but are not limited to, those that produce a gene product. Amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic similarity of the residues involved. For example non-polar (hydrophobic)
Amino acid residues 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 The (basic) amino acids include arginine, lysine and histidine; the negatively charged (acidic) amino acids include arginic acid and glutamic acid.

A variety of host expression vector systems may be utilized to express the NHP nucleotide sequences of the present invention. Examples are NHP peptides or polypeptides considered soluble or secreted molecules, NHP peptides or polypeptides that can be recovered from the medium. Such expression systems also include engineered host cells that express NHP or a functional equivalent thereof in situ. Purification or enrichment of NHPs from such expression systems can be done using appropriate detergents and lipid micelles and methods well known to those of skill in the art. But,
Such engineered host cells themselves can be used where it is important not only to maintain the structural and functional properties of the NHP but also to assess biological activity in, for example, drug screening assays.

Expression systems that can be used for the purposes of the present invention include, but are not limited to: microorganisms transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing NHP nucleotide sequences. , For example bacteria (eg E. coli, B. subtilis
)); Yeast transformed with a recombinant yeast expression vector containing an NHP nucleotide sequence (eg, Saccharomyces, Pichia).
hia)); insect cell lines infected with a recombinant viral expression vector containing NHP sequences (eg baculovirus); recombinant viral expression vectors containing NHP nucleotide sequences (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV). Plant cell lines infected with or transformed with recombinant plasmid expression vectors (eg Ti plasmid); or promoters derived from the genome of mammalian cells (eg metallothionein promoter)
Alternatively, a mammalian cell line (eg, COS, CHO, BHK, 239, 3T) harboring a recombinant expression construct containing a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter).
3).

In bacterial systems, a large number of expression vectors may be advantageously selected, depending on the intended use for the expressed NHP product. If one wishes to produce such a protein in large quantities, eg for preparing a pharmaceutical composition containing NHP, or for producing an antibody against NHP, it directs high level expression of a fusion protein product that is easily purified. It may be desirable to have a vector that does. Such vectors include, but are not limited to: E. coli expression vector pUR278.
(Ruther et al., 1983, EMBO J., 2: 1791),
In this case, the NHP coding sequences are individually labeled so that the fusion protein is produced.
The cZ coding region and the reading frame can be aligned and ligated into the vector; pIN vector (Inouye & Inouye, 1985, Nucleic
Acids Res. , 13: 3101-3109; Van Heeke &
Schuster, 1989, J. Am. Biol. Chem. , 264: 5503
-5509) and the like. The pGEX vector can also be used to express the foreign polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified by adsorption from lysed cells to glutathione-agarose beads followed by elution in the presence of free glutathione. The PGEX vector was designed to contain a thrombin or factor Xa protease cleavage site, which allows the cloned target gene product to be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhydrosis virus (A
cNPV) is used as a vector for expressing foreign genes. The virus is Spo
Proliferates in doptera frugiperda cells. The NHP gene coding sequences are individually cloned into nonessential regions of the virus (eg polyhedrin gene) and placed under control of the AcNPV promoter (eg polyhedrin promoter). Successful insertion of the NHP coding sequence inactivates the polyhedrin gene and produces a non-occluded recombinant virus (ie, a virus without the proteinaceous coat encoded by the polyhedrin gene). Will. These recombinant viruses are then Spodopte
used to infect Ra frugiperda cells, where the insert sequence is expressed (
For example, Smith et al. , 1983, J .; Virol. 46: 584
See Smith, USP 4,215,051).

In the case of mammalian host cells, a number of viral expression systems are available. When using adenovirus as an expression vector, the NHP gene nucleotide sequence of interest can be ligated to an adenovirus transcription / translation control complex, such as the late promoter and tripartite leader sequence.
This chimeric gene is then inserted in the adenovirus genome by in vitro or in vivo recombination. Nonessential regions of the viral genome (eg region E1 or E
3), a viable recombinant virus capable of expressing the NHP product in the infected host is obtained (eg Logan & Shenk, 1984, P.
roc. Natl. Acad. Sci. USA, 81: 3655-3659).
Specific initiation signals may also be required for efficient translation of inserted NHP gene nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. If the entire NHP coding sequence or cDNA, including its own initiation codon and flanking sequences, is inserted into a suitable expression vector, additional translational control signals may not be needed. However, if you insert only part of the NHP coding sequence,
An exogenous translational control signal (possibly including the ATG start codon) must be provided. Furthermore, the initiation codon must match the reading frame of the target coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons, whether natural or synthetic, can be of various origin. Expression efficiency can be increased by incorporating an appropriate transcription enhancer element, transcription terminator, or the like (Bittner et al.
, 1987, Methods in Enzymol. , 153: 516-54.
4).

In addition, a host cell line 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 are important for the function of the protein. Different host cells have characteristic and specific mechanisms for 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 can be used that have the cellular machinery for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product. Such mammalian host cells include CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3.
, WI38, especially human cell lines, but is not limited to these.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express the NHP sequence can be engineered. Instead of using an expression vector containing replication of viral origin, host cells can be transformed with DNA that is controlled by the appropriate expression control elements (eg, promoters, enhancer sequences, transcription terminators, polyadenylation sites, etc.). Outpatient D
After the introduction of NA, engineered cells are allowed to grow for 1-2 days in enriched medium and then switched to selective medium. A selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cell to stably integrate the plasmid into the cell's chromosome,
Proliferate to form foci. It can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines expressing the desired NHP product. Such engineered cell lines are particularly useful for screening and evaluation of compounds that affect the intrinsic activity of NHP products.

A number of selection systems can be used, including but not limited to: Herpes simplex virus thymidine kinase (Wigler, et al.
, 1977, Cell, 11: 223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Szybalski, 19).
62, Proc. Natl. Acad. Sci. USA, 48: 2026), and adenine phosphoribosyl transferase (Lowy, et al., 1).
980, Cell, 22: 817) gene can be used in tk ⁻ , hgprt ⁻ or aprt ⁻ cells, respectively. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler, et al., 1980, Natl. Acad. Sci.
． USA, 77: 3567; O'Hare, et al. , 1981, Proc
． Natl. Acad. Sci. USA, 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 19).
81, Proc. Natl. Acad. Sci. USA, 78: 2072); n
eo, which confers resistance to the aminoglycoside G-418 (Colber
re-Garapin, et al. , 1981, J. Mol. Biol. , 1
50: 1); and hygro, which confers resistance to hygromycin (Santerre, et al., 1984, Gene, 30: 147).

Alternatively, any fusion protein can be readily purified by utilizing an antibody specific for the expressed fusion protein. For example, the system described by Janknecht et al. Can easily purify the native fusion protein expressed in human cell lines (Janknecht, et al., 1991, Proc. Natl. A.
cad. Sci. USA, 88: 8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translatable and fused to an amino-terminal tag consisting of 6 histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto a Ni2 ⁺ .nitriloacetic acid-agarose column and histidine-tagged proteins are selectively eluted with imidazole-containing buffer.

5.3 Antibodies to NHP Products One or more NHP epitopes, or epitopes of conserved variants of NHP,
Alternatively, an antibody that specifically recognizes a peptide fragment of NHP is also included in the present invention. Such antibodies include polyclonal antibodies, monoclonal antibodies (mA
b), humanized or chimeric antibody, single chain antibody, Fab fragment, F (ab ')
) ₂ fragments, fragments produced by a Fab expression library, anti-idiotypic (anti -Id) antibodies, and wherein any of the epitope-binding fragments of include, but are not limited thereto.

The antibodies of the invention can be used, for example, in the detection of NHP in biological samples and thus can be used as part of a diagnostic or prognostic method of examining a patient for abnormal amounts of NHP. Such antibodies in combination with compound screening methods, or N
It can also be used to assess the effect of test compounds on the expression and / or activity of HP coding region products. Moreover, such antibodies can be used in combination with gene therapy to assess, for example, normal and / or engineered NHP-expressing cells prior to introduction into a patient. Such an antibody can also be used for abnormal NH
It can be used as a method of inhibiting P activity. Thus, for example, such antibodies can be utilized as part of a therapeutic regimen.

For antibody production, NHPs, NHP peptides (eg, those corresponding to functional domains of NHPs), truncated NHP polypeptides (NHPs with one or more domains deleted), functional equivalents of NHPs, or Various host animals can be immunized by injecting mutant sequences of NHP. Such host animals include, but are not limited to, pigs, rabbits, mice, goats, and rats, to name but a few. Depending on the host species, various adjuvants may be used to enhance the immune response, including Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide or phosphate, detergents such as lysolecithin. , Pluronic polyols, polyanions, peptides, oil emulsions, and potentially useful human adjuvants such as BCG (Ba
pile Calmette-Guerin) and Corynebacte
Rium parvum, but is not limited to these. Alternatively, the immune response can be enhanced by combination and / or binding with molecules such as keyhole limpet hemocyanin, tetanus toxin, diphtheria toxin, ovalbumin, cholera toxin or fragments thereof. Polyclonal antibodies are a heterogeneous population of antibody molecules obtained from the sera of immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodies against a particular antigen, can be obtained by any method that produces antibody molecules in continuous culture cell lines. These include, but are not limited to, the following methods: Kohler and Milstein's hybridoma method (1975, Nature, 256:
495-497; and USP 4,376,110), human B cell hybridoma method (Kosbor et al., 1983, Immunology Tod.
ay, 4:72; Cole et al. , 1983, Proc. Natl. A
cad. Sci. USA, 80: 2026-2030), and the EBV-hybridoma method (Cole et al., 1985, Monoclonal An.
tibodies and Cancer Therapy, Alan R. et al. L
iss, pp. 77-96). Such antibodies include IgG, IgM, IgE,
It may be of any immunoglobulin class, including IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention can be cultivated in vitro or in vivo. This is the presently preferred method of production as it can produce high titers of mAb in vivo.

In addition, “chimeric antibodies” (Morrison et al., 1984, Pr.
oc. Natl. Acad. Sci. , 81: 6851-6855; Neube.
rger et al. , 1984, Nature, 312: 604-608;
Takeda et al. , 1985, Nature, 314: 452-45.
The method by splicing the gene from the mouse antibody molecule with the appropriate antigen specificity developed for the production of 4) with the gene from the human antibody molecule with the appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, for example, a murine mAb-derived variable region and a human immunoglobulin constant region. Such techniques are described in US Pat. Nos. 6,075,181 and 5,877,397, each of which is incorporated by reference herein in its entirety.

Alternatively, the method described for the production of single chain antibodies (USP 4,946,7
78; Bird, 1988, Science, 242: 423-426; Hus.
ton et al. , 1988, Proc. Natl. Acad. Sci. U
SA, 85: 5879-5883; and Ward et al. , 1989,
Nature, 334: 544-546) can be adapted for the production of single chain antibodies to NHP coding sequence products. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region by amino acid bridges into a single chain polypeptide.

Antibody fragments that recognize specific epitopes can be formed by known methods.
For example, such fragments include F (ab ′) ₂ fragments that can be prepared by pepsin digestion of antibody molecules, and Fab fragments that can be prepared by reducing the disulfide bridges of F (ab ′) ₂ fragments. Not limited to. Alternatively, a Fab expression library was constructed (Huse e
t al. , 1989, Science, 245: 1275-1281), which allows rapid and easy identification of monoclonal Fab fragments with the desired specificity.

The antibody to NHP is then utilized and the NH 3 is analyzed using methods well known to those skilled in the art.
Anti-idiotypic antibodies that "mimic" P can be produced (eg G
reenspan & and Bona, 1993, FASEB J .; 7 (5
): 437-444; and Nissinoff, 1991, J. Am. Immuno
l. , 147 (8): 2429-2438). For example, an antibody that binds to the NHP domain and competitively inhibits NHP from binding to its cognate receptor is used to "mimic" NHP, thus binding, activating or moderately binding to NHP, NHP receptor or NHP ligand. Anti-idiotypic antibodies that are compatible can be produced. Such anti-idiotype antibodies or Fab fragments of such anti-idiotype antibodies can be used for therapy involving NHP-mediated pathways.

The scope of the invention is not limited by the specific embodiments described herein. These aspects are presented merely to illustrate individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those illustrated and described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are also included in the scope of the present invention. All cited documents, patents and patent applications are incorporated herein by reference in their entirety.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, MZ, SD, SL, SZ, TZ, UG , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, C A, CH, CN, CR, CU, CZ, DE, DK, DM , DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, K E, KG, KP, KR, KZ, LC, LK, LR, LS , LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, R U, SD, SE, SG, SI, SK, SL, TJ, TM , TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (72) Inventor Donoho, Gregory             Tau, Texas 77381, United States             Druns, Fallen Stone Dry             Bug 41 (72) Inventor Watler, Frank             Germany 82131 Stockdorf, Benno             Strasse 11 (72) Inventor Neels, Michael             Germany 82131 Stockdorf, Paul             -Keller-Strasse 6 (72) Inventor Fredrich, Glenn             Hugh, Texas 07004, USA             Ston, Herman Drive 2207, Bure             Land and Breland (72) Inventor Zambrowich, Bryan             The U., Texas 77382, USA             Druns, firezone play             18 (72) Inventor Sands, Arthur Tee             The U., Texas 77382, USA             Druns, Bristol Bend Sir             Curu 163 F term (reference) 2G045 AA40 DA12 DA13 DA36 FB02                       FB03                 4B024 AA01 AA11 CA01

Claims (3)

[Claims] 1. An isolated nucleic acid molecule comprising a nucleotide sequence of at least 24 contiguous bases in the novel NHP polynucleotide sequence set forth in SEQ ID NO: 1. 2. An isolated nucleic acid molecule comprising (a) an amino acid sequence shown in SEQ ID NO: 2; and (b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence. . 3. The isolated nucleic acid molecule of claim 1, wherein the NHP polynucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 2.