JP2001520015A - Human mammoglobin homolog - Google Patents

Abstract

(57) Abstract The present invention provides polynucleotides that identify and encode human mammoglobin homologs (HMH) and HMH. The present invention also provides expression vectors, host cells, agonists, antibodies, and antagonists. The present invention also provides a method for treating and preventing a disease associated with the expression of HMH.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Technical Field The present invention is human breast globin (Mammoglobin) homologs of the nucleic acid and amino acid sequences, and diagnosis of neoplastic disease and endometriosis, preventing those relates to the use of these sequences in the treatment It is.

[0002] function of cells (Background of the Invention) is regulated by the timing and amount of expression attributable to several thousand genes. Such regulation of expression is metabolically important in that it prevents intermediates from being synthesized and stored when proteins are not needed and conserves energy. Disregulation of gene expression is also known to result in disease development and progression, and has been linked to neoplastic diseases. Such dysregulation can alter the location, timing, or amount of gene expression, and these changes can be monitored and used for diagnosis. For example, different types of breast cancer (invasive, non-invasive, hereditary, ductal, lymphatic, metastatic, etc.) are distinguished by monitoring the expression of genes associated with the type and stage of the neoplasm can do. Nucleic acids are particularly useful in predicting a predisposition to breast cancer, and antibodies to marker proteins can be used in tests to determine the presence or stage of a disease state. Changes in the expression pattern of the BRCA marker
It has been associated with metastatic breast cancer and has been linked to familial susceptibility to the development of breast cancer. BRCA1, a well-known marker, is mapped to 17q21 (Hall, JM et al.
(1990) Science 250: 1584-9), a recently characterized marker BR
CA2 has been mapped to 13q 12-13 (Fischer, SG et al. (1996) Proc. Natl.
Acad. Sci. 93: 690-694).

[0003] Other genes whose expression patterns change upon the onset of disease include those of various steroid binding proteins. One example is uteroglobi
n) There is mammoglobin, a breast-specific steroid binding protein of the gene family. The cDNA encoding mammoglobin was cloned from primary human breast adenocarcinoma and its expression was reported to be restricted to adult breast tissue. Analysis of breast cancer biopsies has shown that mRNA levels are 10-fold higher in tumors compared to normal breast tissue (Watson, MA et al. (1996)
) Cancer Res. 56: 860-65).

[0004] Another disease state that has been found to be associated with steroid-stimulated cell proliferation is endometriosis. Endometrial tissue growth is part of the normal menstrual cycle, but endometriosis occurs when endometrial cells detach from the uterus and migrate to the abdomen and proliferate. This condition may not be detected for months or years and may include fibrosis throughout the pelvic cavity, infertility due to obstruction of the fallopian tubes, and bleeding within the mass with menstruation that varies in volume and severity. Causes considerable systemic discomfort. Treatment with a gonadotropin inhibitor, such as danazol, is effective in mild or elderly women, but often before the pharmacological treatment is applied, the peritoneal or fallopian tube / Surgery to remove endometrial cells from around the ovaries is needed.

[0005] Given the histopathological, etiological, and genetic heterogeneity of breast cancer and the definitive diagnosis of endometriosis, early treatment is possible because of the novel humans.
The discovery of mammoglobin homologs and polynucleotides encoding them fulfills the need in the art by providing new compositions useful for diagnosing, preventing, and treating neoplastic diseases and endometriosis. .

[0006] SUMMARY OF THE INVENTION The present invention, SEQ ID NO: 1 (SEQ ID NO: 1) or a substantially purified polypeptide having the amino acid sequence of the fragment, human breast globin homolog (HMH) I will provide a.

The invention further provides an isolated and substantially purified polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, and a composition comprising the polynucleotide sequence. I do. The present invention also provides a polynucleotide sequence that hybridizes under stringent conditions to a polynucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or a fragment of the polynucleotide sequence. The invention further provides a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 1, or a polynucleotide sequence that includes a sequence that is complementary to a fragment or variant of said polynucleotide sequence.

[0008] The present invention also provides an isolated and purified polynucleotide sequence having the sequence of SEQ ID NO: 2 or a mutant sequence thereof. Further, the present invention provides the
Provided are polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequence of NO: 2. The present invention also provides a polynucleotide sequence having a sequence complementary to the sequence of SEQ ID NO: 2, or a fragment or mutant sequence thereof.

[0009] The present invention further provides an expression vector comprising at least a fragment of any of the polynucleotide sequences described in the claims. In yet another embodiment, an expression vector comprising the polynucleotide sequence is included in a host cell.

The present invention also provides a method for producing a polypeptide having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, wherein (a) a polypeptide encoding HMH under conditions suitable for expression of the polypeptide. Amino acid sequence of SEQ ID NO: 1 or a fragment thereof, comprising: culturing a host cell containing an expression vector containing at least a fragment of a nucleotide sequence; and (b) recovering the polypeptide from a medium of the host cell. A method for producing a polypeptide having the formula:

[0011] The present invention also provides a pharmaceutical composition comprising substantially purified HMH having the amino acid sequence of SEQ ID NO: 1 together with a suitable pharmaceutical carrier.

[0012] The invention also provides a purified antagonist of the polypeptide of SEQ ID NO: 1.

[0013] The present invention also relates to a method for diagnosing endometriosis, comprising the steps of:
dominal fluid), (b) binding an antibody against HMH and peritoneal fluid under conditions suitable for antibody binding, and (c) detecting a human mammoglobin homolog. The process wherein the presence of the bound molecule indicates the presence of endometriosis.

[0014] The invention further provides a purified agonist of the polypeptide of SEQ ID NO: 1.

The present invention also provides a method for treating or preventing a neoplastic disease, which comprises the step of administering an effective amount of an HMH antagonist to a patient in need of such treatment. Provide a way.

The present invention also provides a method for treating or preventing endometriosis, comprising the step of administering an effective amount of an antagonist of HMH to a patient in need of such treatment. Provide preventive measures.

The present invention also relates to a method for detecting a polynucleotide encoding HMH in a biological sample, comprising: (a) a sequence complementary to the polynucleotide sequence encoding SEQ ID NO: 1; And (b) detecting the hybridization complex, wherein the presence of the complex determines HMH in the biological sample. Having a correlation with the presence of the encoding polynucleotide,
And a method for detecting a polynucleotide encoding HMH in a biological sample. In some embodiments, prior to hybridization,
The nucleic acid material of the biological sample is amplified by a polymerase chain reaction.

[0018] (Embodiment of the invention) protein of the present invention, nucleic acid sequences, and before describing how the present invention,
It is to be understood that it is not limited to the particular methodology, protocols, cell lines, vectors, and reagents disclosed herein, which may vary. Further, the terminology used in this specification is used for the purpose of describing only specific examples, and is intended to limit the scope of the present invention which is limited only by the description of the claims. It should be understood that it is not.

In this specification and the appended claims, “a” and “the” are used to mean a singular number.
It should be noted that what is described as has a plural meaning unless the context clearly indicates otherwise. Thus, for example, reference to "a certain host cell" includes a plurality of such host cells, and "a certain antibody" refers to one or more types of antibodies and those skilled in the art. Well-known equivalents are also shown.

[0020] Unless defined otherwise, all technical and scientific terms used herein are
It has the same meaning as commonly understood by a person of ordinary skill in the art to which this invention belongs. Although any methods or materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, vectors, and methodologies reported in the literature that may be used in the context of the present invention, and are incorporated herein by reference. Part of the book. In addition, any disclosure in this specification should not be construed as an admission that such disclosure in the present invention does not precede the prior art.

Definitions As used herein, HMH may be derived from any species, particularly mammals such as cows, sheep, pigs, mice, horses, and preferably humans, and may be natural, synthetic, 1 is the amino acid sequence of substantially purified HMH obtained from either semi-synthetic or recombinant.

As used herein, the term “agonist” is a molecule that, when bound to HMH, enhances the effect of HMH or prolongs the duration of the effect. Agonists include
Proteins, nucleic acids, carbohydrates, and any other molecules that bind to and modulate the effects of HMH can be included.

As used herein, “allele” or “allele sequence” is an allelic form of a gene encoding HMH. An allele results from mutation of at least one position in a nucleic acid sequence, resulting in a mutated mRNA or polypeptide.
Alternatively, the structure or function of the polypeptide may or may not change.
A given intact gene or recombinant gene may have no, one, or multiple allelic forms. Generally, allelic mutations result from natural deletions, additions and substitutions of nucleotides. Each of these types of mutations can occur alone or simultaneously with other mutations, once or more than once in a given sequence.

As used herein, a “mutated” nucleic acid sequence that encodes HMH includes substitution, insertion or deletion of different nucleotide residues, and consequently encodes the same or functionally equivalent HMH. It is a polynucleotide. This definition includes HMH
Polymorphisms having loci other than the normal chromosomal loci of polynucleotide sequences encoding, polymorphisms caused by inappropriate or unexpected hybridization with alleles, and specific oligonucleotides of polynucleotides encoding HMH It contains polymorphisms that are easily detectable or difficult to detect using a probe. The encoded protein may also be "mutated", including deletions, insertions and substitutions of amino acid residues which produce a silent mutation and result in a functionally equivalent HMH. Intentional amino acid substitutions can be made based on similarities in residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and also amphipathicity, as long as the biological activity of HMH is retained. it can. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids having an uncharged polar head group with near hydrophilicity include leucine. , Isoleucine, valine, glycine,
Includes alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

As used herein, “amino acid sequence” is an oligopeptide, peptide, polypeptide, or protein sequence and fragments thereof, which are naturally occurring or synthetic molecules. Fragments of HMH preferably have a length of about 5 to about 15 amino acids and retain the biological or immunological activity of HMH. Here, when "amino acid sequence" refers to the amino acid sequence of a naturally-occurring protein molecule, "amino acid sequence" or similar terms refer to the complete amino acid sequence associated with the protein molecule described herein. It is not used to limit to the original amino acid sequence.

As used herein, “amplification” refers to the generation of additional copies of a nucleic acid sequence, usually performed using the polymerase chain reaction (PCR) method well known to those skilled in the art (Dieffenbach, CW). And GS Dveksler (1995) PCR Primer. A Laboratory Manual , Cold Spring Harbor Press, Plainview, NY).

As used herein, the term “antagonist” refers to a molecule that, when bound to HMH, reduces the magnitude of the biological or immunological effect of HMH or shortens the duration of the effect. It is. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, or other molecules that reduce the effects of HMH.

As used herein, the term “antibody” refers to an entire antibody molecule, eg, Fa, F (a
b ′) ₂ and fragments thereof that can bind to an antigenic determinant such as Fv. HM
Antibodies that bind H polypeptides can be generated by using the entire polypeptide or a fragment thereof containing the small peptide of interest as the antigen for immunization. The polypeptide or peptide used to immunize the animal is R
It can be made by translation or chemical synthesis of NA and can be coupled to a carrier protein if necessary. Commonly used carriers that chemically bind to the peptide include bovine serum albumin and thyroglobulin. An animal (eg, mouse, rat, or rabbit) is immunized with the conjugated peptide.

As used herein, the term “antigenic determinant” is a fragment of a molecule (ie, an epitope) that makes contact with a particular antibody. When a host animal is immunized with a portion, or fragment, of a protein, various regions of the protein can trigger the production of antibodies that specifically bind to a given region or three-dimensional structure on the protein. Such a region or structure is called an antigenic determinant. Antigenic determinants can compete with the original antigen (ie, the immunogen used to elicit the immune response) for binding to the antibody.

As used herein, the term “antisense” is a composition that includes a nucleotide sequence that is complementary to a specific DNA or RNA sequence. The term "antisense strand"
Used in the sense of a nucleic acid strand complementary to the "sense" strand. Antisense molecules include peptide nucleic acids, and antisense molecules can be created by any method, including synthesis and transcription. Once introduced into the cell, the complementary nucleotide combines with the natural sequence created by the cell to form a duplex, which inhibits further transcription and translation. The expression "minus (-)" is used in the sense of the antisense strand,
“Plus (+)” is sometimes used to mean the sense strand.

As used herein, the term “biologically active” is a protein that has a structural, regulatory, or biochemical function of a naturally occurring molecule. Similarly, "immunologically active" refers to the action of natural, recombinant, or synthetic HMH, or an oligopeptide thereof, on a particular animal or cell in a particular immune response and binding to a particular antibody. Ability.

As used herein, the term “complementary” or “complementarity” refers to the spontaneous binding of polynucleotides by forming base pairs under acceptable salt concentrations and temperature conditions. For example, the sequence "AGT" binds to the complementary sequence "TCA". 2
Complementarity between single-stranded molecules of a book is "partial", in which only some nucleic acids bind, or completely complementary if there is complete complementarity between the single-stranded molecules It can be something. The degree of complementarity between nucleic acid strands significantly affects the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions that depend on the binding between nucleic acid strands and in the design and use of PNA molecules.

As used herein, the term “composition containing a predetermined polynucleotide sequence” refers to any substance containing a predetermined polynucleotide sequence. The composition may be in the form of a powder formulation or an aqueous solution. A composition comprising a polynucleotide sequence encoding HMH (SEQ ID NO: 1) or a fragment thereof (eg, SEQ ID NO: 2 and a fragment thereof) can be used as a hybridization probe.
The probe can be stored in a lyophilized state and can be conjugated to a stabilizing agent such as a carbohydrate. In hybridization, the probe can be dispersed in an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS) and other substances (eg, Denhardt's solution, milk powder, salmon sperm DNA, etc.).

As used herein, “consensus” refers to a nucleic acid sequence obtained by resequencing and separating unnecessary bases, or using a XL-PCR ^™ (Perkin Elmer, Norwalk, Conn.) In the 5 ′ direction and / or 3 ′. A computer program (eg, GELVIEW ^™ Fragment Assembl) for combining nucleic acid sequences or fragments that have been extended and resequenced in
y system, GCG, Madison WI), and is a nucleic acid sequence derived by combining overlapping sequences of two or more types of Incyte clones. Consensus sequences may be created by both extension and combination.

As used herein, the expression “correlated with the expression of a polynucleotide” means that the presence of a ribonucleic acid having a similarity to SEQ ID NO: 2 is detected by Northern analysis. In the presence of mRNA encoding HMH, and thus correlate with the expression of transcripts from the gene encoding the protein.

As used herein, a “deletion” is a change in the nucleotide or amino acid sequence such that one or more nucleotides or amino acid residues are missing.

As used herein, the term “derivative” refers to a nucleic acid encoding HMH or a nucleic acid complementary thereto or a chemically modified HMH encoded. Examples of such modifications include hydrogen to alkyl, acyl, or amino groups. The nucleic acid derivative encodes a polypeptide that retains the biological or immunological function of the unmodified HMH. A derivative polypeptide retains the biological or immunological function of the original polypeptide and has been modified by glycosylation, polyethylene glycolation (PEGylation), or some other process.

As used herein, the term “homology” refers to a degree of complementarity. It may be partially homologous or completely homologous (ie, identical). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid, and is designated by the functional expression "substantially homologous." Inhibition of hybridization between the perfectly complementary sequence and the target sequence can be determined using hybridization assays (Southern or Northern blot, solution hybridization, etc.) under low stringency conditions.
A substantially homologous sequence or probe will compete for, and inhibit, binding (ie, hybridization) with a sequence or probe that is completely homologous to the target sequence under conditions of low stringency. This does not mean that low stringency conditions allow for non-specific binding. Low stringency conditions require that the binding of the two sequences to each other be a specific (ie, selective) interaction. The absence of non-specific binding can be determined by using a second target sequence that also has no partial degree of complementarity (ie, less than about 30% identity). In the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.

The human artificial chromosome (HAC) is a linear, small chromosome containing a DNA sequence of 10K to 10M in size and containing all the elements required for stable division and maintenance of split chromosomes (Harrington, JJ). (1997) Nat Genet. 15: 345-355).

As used herein, the term “humanized antibody” is an antibody molecule in which amino acids have been substituted in the non-antigen binding region in order to make the antibody more accessible to a human antibody while retaining the original binding ability.

As used herein, the term “hybridization (hybridization)” is a process in which a nucleic acid strand binds to a complementary strand by forming base pairs.

As used herein, the term “hybridization complex” refers to the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases.
A complex formed by two nucleic acid sequences. These hydrogen bonds can be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond to form an antiparallel structure. Hybridization complexes may be formed in solution (eg, in the case of C ₀ t or R ₀ t analysis), or nucleic acids may be combined with one nucleic acid present in solution with a solid support (eg, a cell or its nucleic acid). (An immobilized paper, membrane, filter, pin, or glass slide or other suitable substrate).

As used herein, “insertion” or “addition” refers to one or more compared to a naturally occurring molecule.
A change in nucleotide sequence or amino acid sequence in which one or more nucleotides or amino acid residues are added.

A “microarray” is an array of individual synthesized polynucleotides or oligonucleotides at high density on a substrate. The substrate may be, for example, paper, nylon or other types of membranes, filter paper, chips, glass slides, or any other suitable solid support.

As used herein, the term “modulation” is a change in the activity of HMH. For example, modulation may increase or decrease the activity of a protein, alter binding characteristics, or otherwise alter HMH.
Results in changes in the biological, functional, and immunological properties of

As used herein, “nucleic acid sequence” refers to single-stranded or double-stranded, and sense or antisense strands of genomic or synthetic DNA, RNA, oligonucleotides, nucleotides, or polynucleotides. Nucleotides and fragments thereof. "
A "fragment" is a nucleic acid sequence having a length of at least 60 nucleotides, most preferably a length of at least 100 nucleotides or at least 1000 nucleotides,
And a fragment of 10,000 nucleotides or more.

The term “oligonucleotide” is a nucleic acid sequence that can be used in PCR amplification or hybridization assays, or microarrays, and is about 6 or more nucleotides in length, up to about 60 nucleotides, preferably 15 to 30 nucleotides. , More preferably 20 to 25 nucleotides. Oligonucleotides herein are referred to by the term "as defined generally in the art.
Substantially synonymous with “Ambrimer”, “Primer”, “Oligomer”, and “Probe”.

As used herein, “peptide nucleic acid” PNA refers to an antisense molecule, ie, an antigenic agent, comprising an oligonucleotide 5 or more nucleotides in length attached to a peptide backbone of amino acid residues terminating in lysine. Terminal lysine confers solubility to this material. Polyethylene glycolation of PNA can extend its lifespan in cells. In such cells, PNA preferentially binds to complementary single-stranded DNA or RNA, stopping transcript elongation. (Niel
sen, PE et al. (1993) Anticancer Drug Des. 8: 53-63).

[0049] As used herein, the term "portion" with respect to a protein (as used for "portion (minute) of a given protein") is a fragment of that protein. The size of this fragment ranges from 5 amino acid residues to (the total number of amino acids in the sequence-1) amino acids. Thus, a protein “comprising at least a portion (min) of the amino acid sequence of SEQ ID NO: 1” includes full-length human HMH and fragments thereof.

As used herein, the term “sample” is used in its broadest sense. Biological samples suspected of containing a nucleic acid encoding HMH or a fragment thereof or HMH itself include body fluids, extracts from chromosomes, organelles or cell membranes isolated from cells, cells, ( Genomic DNA, RNA, or cDNA (in solution or bound to a solid support), tissue, or tissue print
) Others are included.

As used herein, the term “specific binding” or “specifically binds” is the interaction of a protein or peptide with agonists, antibodies, and antagonists. This interaction depends on the presence of specific structures (ie, antigenic determinants, or epitopes) on the protein that are recognized by the binding molecule. For example, if the antibody is specific for epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a protein containing epitope A (i.e., unbound, unlabeled A) is present. , The amount of labeled A bound to the antibody is reduced.

As used herein, the term “stringent conditions” or “stringency” refers to conditions for hybridization as defined by nucleic acids, salts, and temperature. These conditions are well known in the art and can be varied depending on whether the purpose is to identify or detect the same polynucleotide sequence or to identify or detect closely related polynucleotide sequences. . Nominal equivalent conditions, either low stringency conditions or high stringency conditions, include, for example, sequence length and properties (DNA, R
NA, base composition), nature of target (DNA, RNA, base composition), environment (whether present or fixed in solution, etc.), concentration of salts and other components (eg, formamide, dextran sulfate, and / or Or the presence or absence of polyethylene glycol) and the reaction temperature (from 5 ° C below the melting temperature (Tm) of the probe to about 20 to 25 ° C of Tm)
To the bottom). By altering one or more elements, conditions of either low or high stringency that are different but equivalent to the conditions listed above can be created.

As used herein, the term “substantially purified” refers to a substance that has been removed from its natural environment and isolated or separated so that it has more than 60% of its other constituents naturally associated with it. Preferably, it is a nucleic acid or amino acid sequence from which 75% or more, most preferably 90% or more, has been removed.

As used herein, “substitution” means replacing one or more nucleotides or amino acids with different nucleotides or amino acids.

As used herein, “transformation” refers to the process by which foreign DNA enters and changes a recipient cell. This process can occur under natural or artificial conditions using a variety of well-known methods. Transformation can be performed by known methods for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. This method is selected based on the type of host cell to be transformed and includes, but is not limited to, a method of infecting a virus, a method using electroporation, lipofection, and a method using a particle gun. There can be.
Such "transformed" cells include plasmids in which the inserted DNA is either autonomously replicating or stably transformed cells capable of replicating as part of the host chromosome. is there. Also, some of these cells undergo transient expression of the inserted DNA or RNA for a limited time.

As used herein, a “variant” of HMH is an amino acid sequence in which one or more amino acids have been changed. The variant may contain "conservative" changes, wherein the substituted amino acid has similar structural and chemical properties, for example, when leucine is replaced by isoleucine. In rare cases, a variant may change "non-conservatively," for example, replacing glycine with tryptophan. Similar minor changes include amino acid deletions or insertions, or both. Any amino acid that can be substituted, inserted, or deleted without impairing biological or immunological activity can be determined using a well-known computer program such as, for example, DNASTAR software. .

[0057] This invention is, (hereinafter referred to as "HMH") novel human breast globin homolog
, HMH, and the discovery of the use of these compositions for the diagnosis, prevention, or treatment of neoplastic diseases and endometriosis.

The nucleic acid encoding HMH of the present invention may be a breast cDNA library (BRASTNOT
05) was originally identified using computer search for amino acid sequence alignments in Insights clone No. 2295453. The consensus sequence SEQ ID NO: 2 has the following duplicated and / or extended nucleic acid sequences: Incyte Clone No. 2295453 (origin BRASTNOT05), 2381443 (origin ISLTNOT01), and 2600564 (origin UTRSNOT10) ).

In one embodiment, the invention encompasses a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 shown in FIGS. 1A and 1B. HMH has a length of 95 amino acids, has one N-glycosylation site at N (68) QSH, S (38) IPE, S (70) HR, T ( No. 73) LK and T (No. 83) VYD have four phosphorylatable sites. HMH also has a D (No. 51) or D (No. 53) that acts as a "cap" for calcium binding of uteroglobin. As shown in FIG. 2, HMH is composed of human mammoglobin (GI 1199596).
Has chemical and structural homology to SEQ ID NO: 3); Specifically, H
MH shares 57% sequence identity with human mammoglobin. From the results of Northern analysis of HMH (FIG. 3), the expression of this sequence in various libraries can be seen. More than 86% of such libraries are derived from breast tissue and more than 52% It was related to breast cancer. Of particular note is the expression of HMH in endometrial cells.

The present invention also includes HMH variants. Preferred HMH variants have at least 80%, more preferably at least 9%, the HMH amino acid sequence (SEQ ID NO: 1).
It has 0% amino acid sequence identity and retains at least one of the biological, immunological, or other functional properties or activities of HMH. The most preferred HMH variants are those having at least 95% amino acid sequence identity to the sequence of SEQ ID NO: 1.

The present invention also includes a polynucleotide encoding HMH. Therefore HM
Any of the nucleic acid sequences encoding the H amino acid sequence can be used to create a recombinant molecule that expresses HMH. In certain embodiments, the invention encompasses a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 2 shown in FIGS. 1A and 1B.

As will be appreciated by those skilled in the art, the degeneracy of the genetic code results in a wide variety of HMHs, including those that have minimal homology to the nucleotide sequence of any known naturally occurring gene. Can be created. Therefore, the present invention
It includes within its scope all possible nucleic acid sequence variations created by selecting combinations based on the possible codon choices. These combinations are made based on the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HMH, and all such variations are considered to be specifically disclosed herein. I want to be.

The nucleotide sequence encoding HMH and its variant sequences are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring sequence under appropriately selected stringency conditions, but have substantially different codons. It may be beneficial to create a nucleotide sequence that encodes the HMH or derivative thereof that has. In selecting codons, it can be chosen to increase the incidence of peptide expression in a particular prokaryotic or eukaryotic expression host, depending on the frequency with which particular codons are used by the host. Other reasons for altering the nucleotide sequence encoding HMH and its derivatives substantially without altering the encoded amino acid sequence include more desirable, eg, longer half-lives than transcripts made from naturally occurring sequences. Creating RNA transcripts with properties.

The scope of the present invention also includes the production of a DNA sequence encoding HMH or a derivative thereof or a fragment thereof by completely synthetic chemistry. Once made, the synthetic gene can be inserted into various available expression vectors and cell lines using well-known reagents. In addition, synthetic chemistry can be used to introduce mutations into the sequence encoding HMH or any fragment thereof.

Also included within the scope of the present invention are Wahl, GM and SLBerger (1987;
Methods Enzymol. 152: 399-407) and Kimmel, AR (1987; Methods in Enzymo).
l. 152: 507-511), there are polynucleotide sequences capable of hybridizing to the claimed nucleotide sequences, in particular the nucleotide sequence of SEQ ID NO: 2, under various stringency conditions.

Methods for DNA sequencing are well known and commonly available to those of skill in the art, and can be used to practice any of the embodiments of the present invention. In this method, for example, Sequenase which is a Klenow fragment of DNA polymerase I (US Biochemical
Corp. Cleveland OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago IL), or a calibration exo such as that found in the ELONGASE Amplification System available from Gibco BRL (Gaithersburg MD). Enzymes, such as a combination of a nuclease and a recombinant polymerase can be used. This process is based on the Hamilton Micro Lab2200 (Hamilton, Reno
, NV), Peltier Thermal Cycler (PTC200; MJ Reserch, Watertown MA) and
It is preferred to automate using equipment such as ABI Catalyst and ABI377 and 377 DNA sequencers (Perkin Elmer).

The HMH-encoding nucleic acid sequence can be extended, utilizing the partial nucleotide sequence, using a variety of well-known methods to detect upstream sequences, such as promoters and regulatory elements. For example, in one of the available methods, the "restriction site" PCR method, universal primers are used to obtain an unknown sequence adjacent to a known position (Sarkar, G. (1993) PCR Methods Applic 2: 318- 322). Specifically, genomic DNA is first amplified in the presence of a primer specific to a known region and a primer to a linker sequence. The amplified sequence is subjected to a second round of PCR using the same linker primer and another specific primer contained within the first primer. The product of each round of PCR is transcribed using the appropriate RNA polymerase and sequenced using reverse transcriptase.

The reverse PCR method can also be used to amplify or extend sequences using various primers based on known regions (Triglia, T. et al. (1988) Nucleic A
cids Res 16: 8186). The primers are OLIGO 4.06 Primer Analysis software (N
a commercially available software such as National Biosciences Inc., Plymouth MN) or other suitable program, 22-30 nucleotides in length and 50% GC content.
The above is designed so as to anneal to the target sequence at a temperature of about 68 to 72 ° C. In this method, several restriction enzymes are used to generate appropriate fragments of a known region of the gene. This fragment is then circularized by intramolecular ligation and used as a template for PCR.

Another method that can be used is the capture PCR method, in which DNA fragments adjacent to known sequences in human and yeast artificial chromosomal DNA are amplified by PCR (Lagerstrom, M. et al. (1991) PCR). Methods Applic 1: 111-119). In this method, the DNA is digested and ligated with a plurality of restriction enzymes before performing the PCR.
Recombinant double-stranded sequences can be incorporated into unknown fragments of the molecule.

Another method that can be used to obtain unknown sequences is Parker, JD et al. (1991; Nucleic Acids Res 19: 3055-3060). In addition, genomic DNA walking can be performed using PCR, nested primers, and PromoterFinder ^™ library (Clontech, Palo Alto CA). This process is useful for finding intron / exon junctions without having to screen the library.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Random primed libraries are also preferred in that they contain more sequences containing the 5 'region of the gene. The use of a randomly primed library is particularly preferable when an oligo d (T) library cannot obtain a full-length cDNA. Genomic libraries can also be useful for extending sequence to a non-transcribed 5 'regulatory region.

A commercially available capillary electrophoresis system can be used to size-analyze the nucleotide sequence of a product of sequencing or PCR and confirm its presence. In particular, capillary sequencing uses a flowable polymer for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) activated by a laser, and a wavelength emitted by a CCD camera. Is detected.
Output / light intensity is determined by appropriate software (eg, Genotyper ^™ and S from Perkin Elmer).
The signal is converted into an electric signal by using an equence Navigator ^™, and the entire process from sample loading to computer analysis and electronic data display is computer controlled. Capillary electrophoresis is particularly suitable for sequencing small pieces of DNA that are present in small amounts in a particular sample.

In another embodiment of the present invention, a polynucleotide sequence encoding HMH, or a fragment thereof, is incorporated into a recombinant DNA molecule to provide HM in a suitable host cell.
H, its fragments or functional equivalents can be induced. Due to the inherent degeneracy of the genetic code, other DNA sequences encoding substantially identical, ie, functionally equivalent, amino acid sequences can also be created, and these sequences can be used for cloning and expression of HMH. it can.

As will be appreciated by those skilled in the art, it may be beneficial to create HMH coding nucleotide sequences with non-naturally occurring codons. For example, selecting codons that are preferred in a particular prokaryotic or eukaryotic host to increase protein expression or have desirable properties such as a longer half-life than transcripts generated from naturally occurring sequences. Can be created.

The nucleotide sequences of the present invention can be recombined using known methods to modify the sequence encoding HMH for various purposes. The purpose of this sequence modification includes, but is not limited to, for example, cloning, processing and / or altering expression of the gene product. DNA rearrangement by random fragments and reassembly of gene fragments and synthetic oligonucleotides by PCR
Allows the nucleotide sequence to be recombined. For example, site-directed mutagenesis can result in the insertion of new restriction sites, altered glycosylation patterns, altered codon preferences, generation of splice variants, and introduction of mutations.

In another embodiment of the present invention, the original HMH coding sequence, modified HMH coding sequence or recombinant HMH coding sequence is linked to a heterologous sequence,
It can be a sequence encoding a fusion protein. For example, to screen for inhibitors of HMH activity from a peptide library, it may be useful to encode a chimeric HMH protein that is recognized by commercially available antibodies. The fusion protein can also be designed to have a cleavage site at a position between the HMH-encoding sequence and the heterologous protein sequence, so that the HMH can be cleaved and purified separately from the heterologous portion. Becomes

In another embodiment of the present invention, known chemical methods (eg, Caruthers. MH et al. (198)
0) Nucl. Acids Res. Symp. Ser. 7: 215-223; Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232) can be used to synthesize the whole or a part of the sequence encoding HMH. Alternatively, the protein itself can be produced using chemical methods to synthesize the amino acid sequence of HMH or a fragment thereof. For example, peptide synthesis can be performed by various solid-phase techniques (Roberge, JY et al. (1995) Science 269: 202-204), and the automation of the synthesis is achieved by using, for example, an ABI 431A peptide synthesizer (Perkin Elmer). can do.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (see, eg, Creighton T. (1983) Proteins Structure and Molecular Principles , WH Freeman and Co., NY). ). The composition of the synthesized peptide can be confirmed by amino acid analysis or sequencing (for example, Edman degradation method; Creighton, supra). Further, the HMH amino acid sequence or any part thereof may be modified by direct synthesis thereof and / or linked to a sequence derived from another protein or any part thereof using a chemical method. Thus, a mutant polypeptide can be created.

In order to express biologically active HMH, the nucleotide sequence encoding HMH or a functional equivalent thereof is converted to an appropriate expression vector, ie, the elements necessary for the transcription and translation of the inserted coding sequence. Insert into a vector containing

[0080] Methods known to those of skill in the art can be used to prepare expression vectors containing a sequence encoding HMH and appropriate transcriptional and translational regulatory regions. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination techniques. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Press, Planview NY and Ausubel,
FM etc.Current Protocol in Molecular Biology , John Wiley & Sons, New York,
It is listed in NY.

Various expression vector / host systems can be used to maintain and express the HMH coding sequence. Such things include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and viral expression. An insect cell line infected with a vector (eg, baculovirus), or a plant cell transformed with a virus expression vector (eg, cauliflower mosaic virus CaMV, tobacco mosaic virus TMV) or a bacterial expression vector (eg, Ti or pBR322 plasmid) And animal cell systems. The invention is not limited by the host cell used.

“Regulatory regions” or “control sequences” are the untranslated regions of a vector that interact with host cell proteins for transcription and translation, ie, enhancers, promoters, and 3 ′ untranslated regions. The strength and specificity of the action of such elements can vary. Depending on the vector system and host used, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning into bacterial systems, Bluescript phagemid (Stratagene, LaJolla CA) or pSportI ^™ plasmid (Gibco BRL
) Can be used, such as a hybrid lacZ promoter. In insect cells, the baculovirus polyhedrin promoter can be used. A promoter or enhancer derived from the genome of a plant cell (
For example, a heat shock RUBISCO and storage protein gene), or a promoter or enhancer from a plant virus (eg, a viral promoter or leader sequence) may be cloned into the vector. For mammalian cell lines, promoters from mammalian genes or from mammalian viruses are suitable.
If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HMH, vectors based on SV40 or EBV can be used with appropriate selectable markers.

[0083] In bacterial systems, a wide variety of expression vectors can be selected depending on the use of the HMH. For example, when a large amount of HMH is required for antibody induction, a vector that can express a fusion protein that can be easily purified at a high level can be used.
Such vectors include, but are not limited to, a multifunctional E. coli cloning and expression vector, Bluescript (Stratagene) (in this vector,
The sequence encoding HMH can be ligated in the frame of a vector with a sequence consisting of the amino terminal methionine and the subsequent seven residues of β-galactosidase to produce a hybrid protein, or a pIN vector (Van Heeke, G. and SM Sch
uster (1989) J. Biol. Chem. 264: 5503-5509). In addition, a pGEX vector (Promage, Madison WI) can 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 protein produced in such a system can be designed to include a heparin, thrombin, or factor Xa protease cleavage site so that the cloned polypeptide of interest can be released from the GST moiety at will.

For the yeast Saccharomyces cerevisiae , a variety of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH can be used. For an overview, see Ausubel et al. (Supra) and Grant et al. (1987) Methods Enzymol 153: 516-544.

When using plant expression vectors, expression of the sequence encoding HMH can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in TMV (Takamats
u, N. et al. (1987) EMBO J 6: 307-311). Alternatively, plant promoters such as the small subunit of RUBISCO, the heat shock promoter, may be used (Coruzzi, G. et al. (1984) EMBO J.
3: 1671-1680); Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105). These products can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in various publicly available documents (eg, McGraw Hill Yearbook of Science and Technology (1992) McGraw
See Hobbs, S. or Murry, LE, Hill NY, pp 191-196).

[0086] An insect system can also be used for expression of HMH. For example, one such system uses Au tographa californica nuclear polyhedrosis virus (AcNPV) as a vector for expressing foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae. The sequence encoding HMH can be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. H
Successful insertion of the MH coding sequence inactivates the polyhedrin gene and produces a mutant virus lacking the coat protein membrane. Next, the mutant virus was transferred to S. frugiperda cells or Trichoplusia .
Larvae can be infected and express HMH therein (Engelhard, EK et al. (1994) Proc. Nat. Acad. Sci. 91: 3224-3227).

[0087] In mammalian host cells, a variety of viral-based expression systems may be utilized. When an adenovirus is used as an expression vector, HMH
Can bind into an adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion into a non-essential E1 or E3 region in the viral genome results in a live virus capable of expressing HMH in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. . 81: 3655-3
659). In addition, transcription enhancers such as the Rous sarcoma virus (RSV) enhancer can be used to increase expression in mammalian host cells.

By using human artificial chromosomes (HACs), a DNA fragment larger than a DNA fragment that can be included in a plasmid and expressed can also be supplied. For therapeutic purposes, 6-10 M HACs can be constructed and supplied using conventional delivery methods (liposomes, polycationic amino polymers, or vesicles).

[0089] Specific initiation signals are also required for more efficient translation of the sequence encoding HMH. Such signals include the ATG initiation codon and adjacent sequences. If HMH and its initiation codon and upstream sequences are inserted into the appropriate expression vector, no other transcriptional or translational control signals are required. However, if only the coding sequence or a fragment thereof is inserted, an exogenous translational control signal must be provided, including the ATG initiation codon. In addition, the start codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be from a variety of sources, both natural and synthetic. For example, as described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162), the efficiency of expression can be improved by including appropriate enhancers in the particular cell line used. Can be increased.

In addition, a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences, or to process the expressed protein in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation (lipida
tion) and acylation. Post-translational processing that cuts off the "prepro" portion of the protein is also important for correct insertion, folding, and / or performance. A variety of host cells (eg, CHO, HeLa, MDCK, HEK293, and WI3) having specific cellular devices and characteristic mechanisms for such post-translational effects.
8) is available from the American Type Culture Collection (ATCC; Bethesda, MD) and can be selected from among them to ensure correct modification and processing of the foreign protein to be introduced.

[0091] Stable expression is desirable to ensure high-yield production of recombinant proteins over long periods of time. For example, a cell line that stably expresses HMH is transformed by using an expression vector containing the viral origin of replication and / or an endogenous expression element and a selectable marker gene on the same vector or on another vector. be able to. After the introduction of the vector, the cells are allowed to grow for 1-2 days in a concentrated medium before switching to a selective medium. The purpose of the selectable marker is to confer resistance for selection and allow cells expressing the introduced sequence to grow and recover based on its presence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.

[0092] Any number of selection systems can be used to recover transformed cell lines. The selection system is not limited to the following, but includes herpes simplex virus thymidine kinase (tk) (Wigler, M. et al. (1977) Cell 11: 223-32) and adenine phosphoribosyltransferase (aprt) (Lowy, I Others (1980) Cell 22: 817-23)
There are genes such as, respectively tk ^- used in a cell ^- or aprt. Also, resistance to antimetabolites, antibiotics or herbicides can be used as a basis for selection.
For example, dhfr confers resistance to methotrexate (Wigler, M. et al. (1980) Proc.
Natl. Acad. Sci. 77: 3567), npt confers resistance to the aminoglycoside neomycin and G-418 (Colberre-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14). )
, Als or pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase (Murry, supra). Other genes available for selection include trpB, which allows cells to use indole instead of tryptophan, and hisD, which allows cells to use histinol instead of histidine ( Hartman, SC and RC Mulligan (1988) Proc. Natl. Ac
ad. Sci. 85: 8047-51). Recently, not only to identify transformants, but also to quantify the amount of transient or stable protein expression by a particular vector system, such as anthocyanins, β-glucuronidase and its substrates.
Visible markers such as GUS and luciferase and its substrate luciferin have been used (Rhodes, CA et al. (1995) Methods Mol. Biol. 55:
121-131).

The presence / absence of marker gene expression also indicates the presence of the gene of interest,
It may be necessary to confirm its presence and expression. For example, when a sequence encoding HMH is inserted into a marker gene sequence, transformed cells containing the sequence encoding HMH can be identified as lacking the function of the marker gene. Alternatively, a marker gene can be placed in tandem with a sequence encoding HMH, both under the control of a single promoter. Expression of the marker gene in response to induction or selection will usually also indicate expression of the tandemly arranged sequence.

Alternatively, host cells containing a nucleic acid sequence encoding HMH and expressing HMH can be identified by various methods well known to those skilled in the art. Such methods include, but are not limited to, DNA-DNA or DNA-RNA hybridization, and techniques using membranes, solutions, or chips for detecting and / or quantifying nucleic acids and proteins. DNA-D using a probe, a part, or a fragment of a sequence encoding HMH, such as a protein bioassay or an immunoassay.
NA or DNA-RNA hybridization or amplification can detect the presence of a polynucleotide sequence encoding HMH. In assays utilizing nucleic acid amplification, oligonucleotides or oligomers based on a sequence encoding HMH are used to detect transformants containing DNA or RNA encoding HMH.

Various protocols are well known for detecting and measuring HMH expression, using either polyclonal or monoclonal antibodies specific for this protein. Examples of such protocols include enzyme linked immunoassays (ELISA).
, Radioimmunoassay (RIA) and fluorescence-activated cell sorter (FACS). A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the HMH polypeptide (
Two-site, monoclonal-based immunoassays are preferred, but competitive binding assays can also be used. These and other assays are described in other literature,
Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual , APS Press)
, St. Paul MN) and Maddox, DE et al. (1983, J. Exp. Med. 158: 1211-1216).

In addition, various labeling and conjugation techniques are well known and can be used in various nucleic acid and amino acid assays. As a means for producing a labeled hybridization probe or PCR probe for detecting closely related sequences,
Examples include oligo labeling, nick translation, terminal labeling, and PCR amplification using labeled nucleotides. Alternatively, the sequence encoding HMH or any fragment thereof is cloned into a vector for the production of an mRNA probe. Such vectors are well known and commercially available and can be used to T7, T3, or
RNA probes can be synthesized in vitro by adding a suitable RNA polymerase such as SP6 and labeled nucleotides. Such methods are described in various commercially available kits (Pharmacia & Upjohn (Kalamazoo, MI); Promega (
Madison WI); and US Biochemical Corp. (Cleveland OH)). Appropriate reporter molecules used to facilitate detection, ie, labels, include radionuclides, enzymes, fluorescers, chemiluminescent or dye agents, as well as substrates, cofactors, inhibitors, magnetic particles. And the like.

[0097] Host cells transformed with the nucleotide sequence encoding HMH can be cultured under conditions suitable for expressing and recovering the protein from the cell culture medium. Proteins produced by the transformed cells are either secreted or included intracellularly, depending on the sequence and / or the vector used. As will be appreciated by those skilled in the art, expression vectors containing a polynucleotide encoding HMH can be designed to include a signal sequence that directs secretion of HMH through the cell membrane of a prokaryotic or eukaryotic cell. Using other constructs,
The sequence encoding MH can be linked to a nucleotide sequence encoding a polypeptide domain that facilitates purification of the soluble protein. Domains that facilitate such purification include, but are not limited to, metal chelating peptides such as the histidine tryptophan module, which allow purification on immobilized metals, and purification on immobilized immunoglobulins And a domain used in a FLAGS extension / affinity purification system (Immunex Corp., Seattle WA). Between the purification domain and HMH, for example, a factor Xa or enterokinase specific sequence (Invitrogen, San Diego CA
Purification can be facilitated by including a cleavable linker sequence such as One such expression vector expresses a nucleic acid sequence encoding six histidine residues, along with a sequence encoding HMH, followed by a sequence encoding thioredoxin and an enterokinase cleavage site to create a fusion protein. . A histidine residue facilitates purification on IMIAC (described in Immobilized Metal Ion Affinity Chromatography, Porath, J. et al. (1992) Prot. Exp. Purif. 3: 263-281), as well as an enterokinase cleavage site. From fusion protein to HMH
Is a means for purifying. A description of vectors containing fusion proteins is provided in Kroll, DJ et al. (1993; DNA Cell Biol. 12: 441-453).

In addition to recombinant production, fragments of HMH can also be created by direct peptide synthesis using solid phase technology (Merrifield J. (1963) J. Am. Chem. Soc. 85:21).
49-2154). Protein synthesis can be done manually, but can also be automated. Automated synthesis can be performed, for example, using Applied Biosystem 431A Peptide Synthesizer (Pe
rkin Elmer). It is also possible to chemically synthesize various HMH fragments individually and combine them in a chemical manner to create full-length molecules.

There is chemical and structural homology between therapeutic HMH and human mammoglobin (Gl 1199596). In addition, HMH is expressed in breast cancer and endometrial cells and is thought to play a role in steroid responsive cell proliferation, especially in neoplastic disease and endometriosis.

Thus, in certain embodiments, an antagonist of HMH may be administered to a patient for the prevention or treatment of a neoplastic disease. Such diseases include, but are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and the like. Such cancers include, but are not limited to, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary , Pancreatic, parathyroid, penile, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer. In some embodiments, an antibody that specifically binds to HMH may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for delivering a drug to cells or tissues that express HMH. it can.

In another example, a vector expressing a sequence complementary to a polynucleotide encoding HMH is used for the treatment or prevention of a neoplastic disease, including but not limited to those listed above. It can be administered to a patient.

In another example, an antagonist of HMH may be administered to a patient for the prevention or treatment of endometriosis. In some embodiments, an antibody that specifically binds to HMH may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for delivering a drug to cells or tissues that express HMH. it can.

In another example, a vector expressing a sequence complementary to a polynucleotide encoding HMH may be administered to a patient for the treatment or prevention of endometriosis.

In another embodiment, a protein, antagonist, antibody, agonist,
Either the complementary sequence or the vector can be administered in combination with other suitable agents. One of skill in the art would be able to select an appropriate drug for use in combination therapy based on conventional pharmaceutical principles. By combining therapeutic agents, a synergistic effect that is effective in treating or preventing the various diseases described above can be obtained. By using this method, lower doses of each drug can increase the therapeutic effect and reduce the likelihood of side effects.

An antagonist of HMH can be produced using well-known methods. Specifically, purified HMH can be used to generate antibodies or to screen libraries of drugs to identify those that specifically bind to HMH.

[0106] Antibodies to HMH can be generated using known methods. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments made from Fab expression libraries. Neutralizing antibodies (ie, those that inhibit dimer formation) are particularly suitable for therapeutic use.

A variety of hosts, such as goats, rabbits, rats, mice, etc., can be immunized by injecting HMH or fragments or oligopeptides thereof having immunological properties to generate antibodies. Various adjuvants, depending on the species of the host, can be used to enhance the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, inorganic gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, key emulsions. Whole limpet hemocyanin and dinitrophenol. Among the adjuvants used in humans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are particularly preferred.

Oligopeptides, peptides used to elicit antibodies against HMH
Alternatively, the fragment thereof preferably has an amino acid sequence consisting of 5 or more amino acids, more preferably 10 or more amino acids. Also, these sequences are preferably identical to a portion of the amino acid sequence of the original protein, and include the entire amino acid sequence of the small, naturally occurring molecule. Short stretches of HMH amino acids can be fused with stretches of other proteins, such as keyhole limpet hemocyanin, to produce antibodies against the chimeric molecule.

[0109] Monoclonal antibodies to HMH can be obtained from a continuous cell line in culture.
ell line) using a technique for producing antibody molecules. Such techniques include, but are not limited to, hybridoma technology, human B cell hybridoma technology, EBV-hybridoma technology, and the like (Kohler, G. et al. (1975)
Nature 256: 495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81: 31-42; Co
te, RJ et al. (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole, SP et al. (1984)
Mol. Cell Biol. 62: 109-120).

In addition, “chimeric antibodies”, such as techniques for splicing mouse antibody genes to human antibody genes, to obtain molecules with appropriate antigen specificity and biological activity
(Morrison, SL et al. (1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger, MS et al. (1984) Nature 3).
12: 604-608; Takeda, S. et al. (1985) Nature 314: 452-454). Alternatively, well-known techniques for the production of single-chain antibodies can be applied to produce single-chain antibodies specific to HMH by well-known methods. Antibodies with related specificities but differing idiotypic configurations can be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton DR (1991) P
roc. Natl. Acad. Sci. 88: 11120-3).

In addition, literature (Orlandi, R. et al. (1989), Proc. Natl. Acad. Sci. 86: 3833-3837;
Winter, G. et al. (1991) Nature 349: 293-299), by screening a panel of highly specific binding reagents or recombinant immunoglobulin libraries, or in a lymphocyte population. Antibodies can also be produced by inducing the production of in vivo.

[0112] Antibody fragments which contain specific binding sites for HMH can also be generated. Such fragments include, but are not limited to, reducing F (ab ') ₂ fragments and F (ab') ₂ fragments that can be generated by digestion of antibody molecules with pepsin, Fab fragments that can be produced by Alternatively, a monoclonal Fa having the desired specificity
A Fab expression library may be generated so that b-fragments can be identified quickly and easily (Huse, WD et al. (1989) Science 256: 1275-1281).

[0113] A variety of immunoassays can be used for screening to identify antibodies with the desired specificity. Various protocols for competitive binding assays or radioimmunoassays using either monoclonal or polyclonal antibodies with established specificity are well known in the art. In such an immunoassay, the amount of a complex formed between HMH and its specific antibody is measured. 2
Two sites monoclonal based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes
ssay) is preferred, but a competitive binding assay may be used (Maddox, supra).

In another embodiment of the present invention, a polynucleotide encoding HMH, or any fragment or complement thereof, can be used for therapeutic purposes. In some embodiments, in situations where it is desirable to inhibit transcription of mRNA, a complementary sequence to a polynucleotide encoding HMH can be used. Specifically, cells can be transformed with sequences complementary to the polynucleotide encoding HMH. Thus, complementary molecules or fragments can be used to modulate the activity of HMH, ie, to regulate gene function. Such techniques are now well known, and sense or antisense oligonucleotides, or larger fragments, are available from HMH
It can be designed from various positions of the coding region and the regulatory region of the coding sequence.

Expression vectors derived from retroviruses, adenoviruses, herpes or vaccinia viruses, or expression vectors derived from various bacterial plasmids, are used for delivery of nucleotide sequences to target organs, tissues, or cells. be able to. A vector expressing a nucleic acid sequence complementary to the polynucleotide of the gene encoding HMH can be prepared using methods well known to those skilled in the art. These techniques are described both in Sambrook et al. (Supra) and Ausubel et al. (Supra).

The gene encoding HMH can be blocked by transforming a cell or tissue with an expression vector that expresses a polynucleotide encoding HMH or a fragment thereof at a high level. Using such a construct, a non-translatable sense or antisense sequence can be introduced into cells. Such vectors, even when not incorporated into DNA, continue to transcribe the RNA molecule until the vector is destroyed by an endogenous nuclease. Such a transient expression,
Non-replicating vectors can last more than one month, and even longer if the appropriate replicating element is part of the vector system.

As described above, the sequence complementary to the control region, 5 ′ region, or regulatory region (signal sequence, promoter, enhancer, and intron) of the gene encoding HMH, that is, an antisense molecule (DNA, RNA or By designing a PNA), the manner of gene expression can be changed. Oligonucleotides derived from the transcription initiation site, e.g., a region approximately between +10 and -10 of the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base pairing. Triple helix pairing is useful because the double helix contains polymerase, transcription factors,
Alternatively, it inhibits the ability to unwind sufficiently for binding of the regulatory molecule. Recent therapeutic advances using triple helical DNA have been described in the literature (Huber, BE and BI Carr, Molecular and Immunologic Approaches ,
Gee, JE et al. (1994) at Futura Publishing Co, Mt Kisco NY). Complementary sequences, i.e., antisense molecules, can also be designed to prevent transcript binding to ribosomes and inhibit mRNA transcription.

[0118] Ribozymes, which are enzymatic RNA molecules, can also be used to catalyze the specific cleavage of RNA. In the mechanism of ribozyme action, a ribozyme molecule hybridizes to a complementary target RNA in a sequence-specific manner, followed by endonucleolytic cleavage. Examples of ribozymes that can be used include:
There are artificially synthesized hammerhead ribozyme molecules that can specifically and effectively catalyze the cleavage of HMH-encoding sequences by endonucleases.

[0119] Specific ribozyme cleavage sites in the RNA that can be targeted are identified by first scanning the ribozyme cleavage sites containing the sequences GUA, GUU and GUC in the target molecule. Once identified, it is possible to evaluate the characteristics of the secondary structure that stops the function of the oligonucleotide for a short RNA sequence consisting of 15 to 20 ribonucleotides corresponding to the region of the target gene including the cleavage site. . The suitability of the candidate target is also determined by the ribonuclease protection assay (ribonucl
This can be evaluated by measuring the accessibility for hybridization with a complementary oligonucleotide by an ease protection assay.

The complementary ribonucleic acid molecules and ribozymes of the present invention can be made by well-known methods for synthesizing nucleic acid molecules. These techniques include oligonucleotide chemical synthesis techniques such as solid phase phosphoramidite chemical synthesis. Or,
RNA molecules can be created by the transcription of DNA sequences encoding HMH in vivo and in vitro. Such a DNA sequence can be incorporated into various vectors having a suitable RNA polymerase promoter, such as T7 or SP6. Alternatively, a cDNA construct that synthesizes constitutive RNA can be transferred to a cell line,
It can be introduced into cells or tissues.

[0121] RNA molecules can be modified to increase their stability in cells or to increase their half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5 'end and / or 3' end of the molecule, and phosphorothioate (rather than phosphodiesterase linkages) within the backbone of the molecule. phosph
orothioate) or 2'O-methyl. This method (
concept) is originally used in the production of PNAs and can be extended to all of these molecules as follows. That is, inosine, queosine, as well as by modification of acetyl-, methyl-, thio-, and similar forms of adenine, guanine, cytidine, thymine, and uridine that are not readily recognized by endogenous endonucleases. And the inclusion of less commonly used bases such as wybutosine, the system can be extended to all of these molecules.

A number of methods are available for introducing vectors into cells or tissues,
The methods are equally suitable for in vivo , in vitro and ex vivo uses. In the case of ex vivo treatment, there is a method in which a vector is introduced into stem cells collected from a patient, propagated as a clone for autologous transplantation, and returned to the same patient. Delivery by transfection, liposome injection or delivery by polycation amino polymer (Goldman, CK et al. (1997) Nature Biotechnology 1
5: 462-66; incorporated herein by reference) can be performed using methods well known in the art.

[0123] Any of the above treatments can be applied to any suitable subject, including, for example, dogs, cats, cows, horses, rabbits, monkeys, and most preferably mammals, such as humans.

In yet another embodiment of the present invention, the pharmaceutical composition is administered with a pharmaceutically acceptable carrier to achieve the therapeutic effect described above. Such a pharmaceutical composition comprises HMH
, Antibodies to HMH, mimetics of HMH, agonists, antagonists, or inhibitors. The pharmaceutical compositions are administered alone or together with one or more other drugs, such as, for example, stabilizers, using a sterile, biocompatible pharmaceutical carrier. Such carriers include, but are not limited to, saline, buffered saline, glucose or water. Such compositions can be administered to the patient alone or in combination with other drugs or hormones.

The route of administration of the pharmaceutical composition used in the present invention is not limited to the following, but may be oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, Examples include intraventricular administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, enteral administration, local administration, sublingual administration, and rectal administration.

These pharmaceutical compositions contain, in addition to the principal component, suitable pharmaceutically acceptable excipients and additives such as excipients and additives that facilitate the processing of the active ingredient into pharmaceutically usable formulations. Carrier. Details of the formulation or administration techniques can be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co, Easton PA).

Pharmaceutical compositions for oral administration can be formulated in suitable dosage forms using pharmaceutically acceptable carriers well known in the art. With such a carrier, the pharmaceutical composition can be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for patients to take. it can.

Pharmaceutical preparations for oral administration are obtained by combining the main component with a solid excipient and then, if desired, grinding the resulting mixture to produce tablets or dragee cores. It is obtained by processing the mixture of granules after adding the appropriate additives, if necessary. Suitable additives include sugar or protein fillers such as sugars including lactose, sucrose, mannitol or sorbitol, starches such as corn, wheat, rice, potatoes, methylcellulose, hydroxypropylmethylcellulose or carboxyl. There are celluloses such as sodium methylcellulose, gums such as gum arabic or tragacanth, and proteins such as gelatin or collagen. If desired, disintegrating or solubilizing agents may be added, or the cross-linked polyvinyl pyrrolidone, agar, alginic acid such as sodium alginate, or a salt thereof such as sodium alginate.

[0129] Dragee cores are used by coating appropriate tablets with a concentrated sugar solution or the like. Solutions for making the tablets include gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or tablets for tablet identification or to indicate the amount or dosage of the principal component.

[0130] Orally administrable preparations include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a tablet, such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient in admixture with excipients or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, if desired, stabilizers. In soft capsules, the principal component is dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, liquid polyethylene glycol, with or without stabilizers.

Pharmaceutical preparations for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or carriers include fatty oils such as sesame oil, and synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Aminopolymers of non-lipid polycations can also be used for delivery. Appropriate agents or stabilizers may be added to the suspension, as desired, to increase solubility and allow for the preparation of highly concentrated solutions.

For topical or nasal mucosal administration, preparations will be formulated with an appropriate penetrant for the particular barrier to be permeated. Such penetrants are well-known in the art.

The pharmaceutical compositions of the present invention can be prepared by any known methods, eg, conventional mixing, dissolving, granulating,
Sugar coating, levigating, emulsifying, encapsulating, entrapping,
Alternatively, it is produced by freeze-drying.

The pharmaceutical compositions can also be provided as salts and can be formed with various acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like. Salts tend to be more soluble in aqueous or protonic solvents than the corresponding free base forms. In addition, preferred formulations include those in the range of 4.5-5.5, buffered before use, from 1 mM to 5 mM.
There are lyophilized powders that contain all or any of 0 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol.

Once prepared, the pharmaceutical compositions can be placed in an appropriate container and labeled for use in treatment of the indicated condition. In the case of HMH administration, such labels indicate the amount, frequency, and manner of administration.

[0136] Pharmaceutical compositions suitable for use in the present invention are those containing the principal component in an effective amount to achieve the desired purpose. Determination of an effective amount can be made well within the ability of those skilled in the art.

For any compound, the therapeutically effective amount can be estimated initially from assays of neoplastic cells or cell culture, usually in any animal model such as mouse, rabbit, dog, pig. This animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine effective doses and routes for administration in humans.

A therapeutically effective amount is an amount of an active ingredient that ameliorates a symptom or condition, for example, HMH or a fragment thereof, an antibody of HMH, an agonist, antagonist, or inhibitor of HMH. Therapeutic efficacy and toxicity of such compounds may be determined by standard pharmaceutical procedures in cell culture media or using laboratory animals, eg, ED50 (therapeutically effective amount in 50% of the population, 50% effective amount) And LD50 (50 of the population)
% Lethal dose). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that achieve the ED50 with little or no toxicity. The dosage will vary within this range depending upon the dosage form employed, the sensitivity of the patient, as well as the route of administration.

The exact dosage will be selected by the attending physician, in light of factors related to the patient that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient and to maintain the desired effect. Factors to consider include the severity of the disease state, the patient's general health, the patient's age, weight, and gender, diet, time and frequency of administration, concomitant medications, response sensitivity, and resistance / response to treatment. And the like. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the clearance rate of the particular formulation.

Typical dosages are in the range of 0.1-100,000 μg, with a total dosage of up to about 1 g, depending on the route of administration. Guidance as to particular dosages or delivery methods can be found in the literature commonly available to practitioners in the art. Those skilled in the art will employ different dosage forms for nucleotides than for proteins and inhibitors. Similarly, the mode of delivery of a polynucleotide or polypeptide will depend on the particular cell, condition, location, and the like.

Diagnostics In another embodiment, an antibody that specifically binds to HMH may be used to diagnose a condition or disease characterized by HMH expression, or may be treated with HMH or an agonist, antagonist, or inhibitor of HMH. Can be used in assays for monitoring patients who have Antibodies useful for diagnosis can be made in the same manner as for therapeutics described above. Diagnostic assays for HMH include the use of antibodies or labels to detect HMH in human body fluids, cell or tissue extracts. The antibody may be used with or without modification, and may be labeled by binding to the reporter molecule either covalently or non-covalently. A variety of reporter molecules are well known, some of which have been described above.

For example, ELISA (enzyme linked immunoassay), RIA (radioimmunoassay)
As well as various protocols for measuring HMH, such as FACS (Fluorescent Display Cell Sorter), are well known in the art, and provide the basis for diagnosing changes or abnormalities in HMH expression. Normal values of HMH expression, ie, standard values, are the same as those of humor or cell extracts obtained from normal subjects, preferably mammals, and humans.
H antibody is established by conjugation under conditions suitable for complex formation. The standard complex formation can be quantified by various methods, preferably by using photometric means. The amount of HMH expressed in the affected tissue sample and the control sample of the subject's biopsy tissue is compared to a standard value. A parameter for diagnosing the disease is established from the deviation between the standard value and the value of the subject.

In another embodiment of the present invention, a polynucleotide encoding HMH can be used for diagnostic purposes. Polynucleotides that can be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNA. This polynucleotide is used to detect and quantify gene expression in biopsy tissues where HMH expression may be related to disease. Diagnostic assays are used to distinguish whether HMH is present, absent, or overexpressed, or to monitor regulation of HMH levels in therapeutic intervention. be able to.

In one embodiment, identifying a nucleic acid sequence encoding HMH using hybridization with a PCR probe capable of detecting a polynucleotide sequence, including genomic sequences, encoding HMH or closely related molecules. Can be. The specificity of the probe, ie, whether the probe is in a very highly specific region (eg, 10 unique nucleotides in the 5 ′ regulatory region) or a region of low specificity (eg, especially the 3 ′ coding region). Origin, and the stringency of hybridization or amplification (high, medium or low), whether the probe identifies only naturally occurring HMH, or allelic or closely related sequences Is determined.

Probes can also be used to detect closely related sequences, preferably
It should contain at least 50% nucleotides based on any sequence encoding HMH. The hybridization probe of the present invention has SEQ ID NO: 2.
Or from a genomic sequence including introns, promoters and enhancer elements of naturally occurring HMH.

As a means for preparing a hybridization probe specific to DNA encoding HMH, a method of cloning a nucleic acid sequence encoding HMH or an HMH derivative into a vector for producing an mRNA probe is used. is there. Such vectors are well known and commercially available and can be used for in vitro RNA probe synthesis by adding an appropriate RNA polymerase or an appropriate labeled nucleotide. Hybridization probes can be labeled with various reporter molecules. For example, it can be labeled with a radionuclide such as ³² P or ³⁵ S with an enzyme label such as alkaline phosphatase which binds to the probe via an avidin / biotin binding system.

The polynucleotide sequence encoding HMH can be used for diagnosis of a condition or disease associated with HMH expression. Examples of such conditions or diseases include cancers such as adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and the like. Such cancers include, but are not limited to, adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gallbladder, ganglion, digestive tract, heart, kidney, liver, lung, muscle, ovary , Pancreatic, parathyroid, penile, prostate, salivary gland, skin, spleen, testis, thymus, thyroid, and uterine cancer. There are also disorders of steroid-responsive cell proliferation, such as endometriosis. A polynucleotide sequence encoding HMH can be obtained by Southern blotting or Northern blotting using a patient's biopsy tissue or body fluid.
Detect changes in HMH expression by using dot blot or other membrane-based techniques, PCR techniques, dipstick assays (pin strips), pinning techniques, and ELISA assays, as well as microarrays. it can. Such qualitative or quantitative test methods are well known in the art.

In certain embodiments, nucleotide sequences encoding HMH may be useful in assays that detect activation or induction of various cancers, particularly those listed above.
The nucleotide sequence encoding HMH can be labeled by standard techniques and added to a patient's body fluid or tissue sample under conditions suitable for forming hybridization complexes. After a suitable incubation time, the sample is washed,
The signal is quantified and compared to a standard value. If the amount of signal in the biopsy or extracted sample is significantly different from the signal amount in the comparable control sample, the nucleotide sequence has hybridized to the nucleotide sequence of the sample and encodes HMH in the sample. The occurrence of a change in the level of the nucleotide sequence indicates the presence of the associated disease. Such assays can also be used in animal studies, clinical trials, or in monitoring the treatment of an individual patient to assess the effectiveness of a particular therapeutic treatment.

To establish a basis for the diagnosis of diseases associated with the expression of HMH, a normal, ie standard, expression profile is established. This standard profile is established by combining extracts of bodily fluids or cells taken from normal subjects, either animals or humans, with HMH-encoding sequences or fragments thereof under conditions suitable for hybridization or amplification. I can do it. The amount of standard hybridization can be quantified by comparing values obtained in experiments using known amounts of substantially purified HMH with values obtained in normal subjects. Standard values obtained from a normal sample can be compared to values obtained from a sample of a patient exhibiting symptoms of the disease. The presence of the disease is confirmed using the deviation between the standard value and the subject value.

[0150] Once the disease has been confirmed and the treatment protocol has begun, the hybridization assay is repeated periodically to determine whether the level of expression in the patient has begun to approach that observed in normal patients. Can be evaluated. The results from continuous assays can be used to determine the efficacy of treatment over a period of days or months.

For cancer, the relatively high abundance of the transcript in the patient's biopsy tissue indicates a predisposition to the development of the disease. In other words, it can be a means of detecting a disease before actual clinical symptoms appear. This type of definitive diagnosis allows healthcare professionals to take preventive measures or initiate aggressive treatment earlier and prevent the onset and further progression of cancer.

Another diagnostic use of oligonucleotides designed from sequences encoding HMH may be in PCR. Such oligomers can be chemically synthesized, made using enzymes, or made in vitro.
Oligomers are preferably two nucleotide sequences used under optimal conditions to identify a particular gene or state, namely the nucleotide sequence in the sense direction (5 '→ 3') and the antisense direction (3 '← 5 '). Also, a set of two identical oligomers or nested oligomers, or a degenerate pool of oligomers can be used under lower stringency conditions for the detection and / or quantification of closely related DNA or RNA sequences.

Methods that can be used to quantify HMH expression include radiolabeling (ra
use of diolabeling or biotin-labeled nucleotides, use of control nucleic acid coamplification, and use of standard graph curves drawn to complement experimental results (Melby, PC et al. (1993)). J. Immunol. Methods, 159: 235
-44; Duplaa, C. et al. (1993) Anal. Biochem. 229-236). For quantification of large numbers of samples, the oligomers of interest are present in multiple dilutions and can be further quantified by performing ELISA-type assays that can be quickly quantified using a spectrophotometer or colorimetrically. Speed can be increased.

In another example, oligonucleotides or larger fragments from any of the polynucleotide sequences disclosed herein can be used as targets in a microarray. By using microarrays, the expression levels of many genes can be monitored simultaneously (to generate transcript images), and gene variants, mutations, and polymorphisms can be identified. This information can be used to determine gene function, understand the genetic basis of disease, diagnose disease, and develop therapeutics and monitor their activity.

In some embodiments, PCT applications WO 95/11995 (Chee et al.), Lockhart, DJ et al.
96; Nat. Biotech. 14: 1675-1680) and Schena, M. et al. (1996; Proc. Natl. Aca).
d. Prepare and use a microarray according to the method described in Sci. 93: 10614-10619). The above references are incorporated herein by reference.

Preferably, the microarray is composed of a number of unique single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNA, fixed to a solid support. The length of the oligonucleotide is preferably a length of 6 to 60 nucleotides, more preferably a length of 15 to 30 nucleotides, and most preferably a length of 20 to 25 nucleotides. . For certain types of microarrays, it may be preferable to use oligonucleotides that are only 7 to 10 nucleotides in length. Microarrays contain oligonucleotides that cover a known 5 ′ or 3 ′ sequence, continuous oligonucleotides that cover a full-length sequence, or unique oligonucleotides selected from specific regions along the length of the sequence. Can have. The polynucleotide used in the microarray may be an oligonucleotide specific to a target gene or genes whose sequence fragment is known, or one or more species common to a particular cell type, development or disease state. May be an oligonucleotide specific to the unidentified cDNA.

To generate oligonucleotides against known sequences for microarrays, a computer algorithm is used to examine the gene of interest from the 5 ′ or more preferably the 3 ′ end of the nucleotide sequence. The algorithm identifies oligomers of defined length that have a GC content that is unique to the gene and that is within the range suitable for hybridization, and that have no putative secondary structure that could interfere with hybridization. In some cases, it may be appropriate to use pairs of oligonucleotides on a microarray. The plurality of "pairs" are preferably identical except for a single nucleotide located at the center of the sequence. The second oligonucleotide of the pair (only one nucleotide is mismatched) serves as control. The number of oligonucleotide pairs can range from 2 pairs to 1 million pairs. The oligomer is synthesized at a predetermined region on the substrate using a light irradiation chemical process. This substrate can be paper, nylon, or another type of membrane,
It can be a filter paper, chip, glass slide, or any other suitable solid support.

In another embodiment, the oligomer is obtained from PCT application WO 95/251116 (Baldeschweiler
Can be synthesized on the surface of the substrate by using a chemical bonding procedure and an ink-jet device as described in E. et al. The PCT application is incorporated herein by reference. Another embodiment uses a "lattice" array, similar to a dot blot (or slot blot), using a vacuum system, heat,
CDNA fragments or oligonucleotides can be placed and attached on the surface of the substrate using UV, mechanical or chemical coupling procedures. Arrays can be made by hand or commercially available devices (slot blot or dot blot devices),
8, 24, 96, which can be made by using materials (any suitable solid support) and machines (including robotic devices), and can effectively use commercially available instruments
384, 1536, or 6144 oligonucleotides, or 2 to 10
It may contain any number of oligonucleotides in the range of 10,000.

To perform analysis of a sample using a microarray, polynucleotides are extracted from a biological sample. The biological sample can be obtained from bodily fluids (eg, blood, urine, saliva, sputum, gastric juice, etc.), cultured cells, biopsy tissue, or other tissue preparations. To generate probes, polynucleotides extracted from a sample are used to create nucleic acid sequences that are complementary to nucleic acids on a microarray. If the microarray consists of cDNA, antisense RNA (aRNA) is a suitable probe. Thus, in one embodiment, mRNA is used to generate cDNA, and the cDNA is used in the presence of fluorescently labeled nucleotides to generate fragments, ie, oligonucleotide aRNA probes. These fluorescently labeled probes are incubated with the microarray such that the probe sequences hybridize to the microarray cDNA oligonucleotides. In another embodiment, a complementary nucleic acid sequence is used as a probe. This complementary nucleic acid sequence includes restriction enzymes, PCR technology, and Oligolav well known in the art of hybridization technology.
Also included are polynucleotides, fragments, and complementary or antisense sequences generated using eling or TransProbe kits (Pharmacia).

Incubation conditions are adjusted so that hybridization occurs with either exact complementarity or with various lower levels of complementarity.
After removing the hybridized probe, the level and pattern of fluorescence is determined using a scanner. The scanned image is examined to determine the degree of complementarity and relative amount of each oligonucleotide sequence on the microarray. By using a detection system, the absence, presence, and amount of hybridization can be measured simultaneously for all individual sequences. This data can be used for large-scale correlation studies for sequences, mutations, variants, or polymorphisms in a sample (Heller, RA et al. (1997) Proc. Natl. Ac
ad. Sci. 94: 2150-55).

In another embodiment of the present invention, nucleic acid sequences encoding HMH can be used to create useful hybridization probes for naturally occurring genomic sequence mapping. This sequence may map to a particular chromosome, a particular region of the chromosome, or an artificial chromosome construct. Examples of artificial chromosome products include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs).
), Bacterial P1 products or monochromosomal cDNA libraries, etc.
(1993) Blood Rev. 7: 127-134, and Trask, BJ (1991) Trends Genet. 7:14.
The outline is described in 9-154.

Fluorescence in situ hybridization (described in FISH, Verma et al. (1988) Human Chromosomes : A Manual of Basic Technique , Pergamon Press, New York, NY ”) is based on other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in various scientific journals and Online Mendelian Inheritance
in Man (OMIM). With the help of correlating the location of the HMH-encoding sequence on the physical chromosomal map with a particular disease (or predisposition to a particular disease), regions of DNA associated with a genetic disease can be determined. . The nucleotide sequences of the present invention can be used to detect differences in the gene sequence of normals, carriers, or patients.

[0163] Physical mapping techniques, such as in situ hybridization of chromosomal preparations and linkage analysis using established chromosomal markers, can be used to enlarge the genetic map. In many cases, even if the number or arm of a particular human chromosome is unknown, the gene arrangement on the chromosome of another mammal, such as a mouse, will reveal the relevant marker. New sequences can be assigned to chromosomal arms or parts thereof by physical mapping. This can provide valuable information to researchers investigating disease genes using positional cloning or other gene discovery techniques. Once a disease or syndrome has been coarsely localized to a particular genomic region, for example, ataxia telangiectasia (AT) was located at 11q22-23 (Gatti, RA et al. (1988) Nature 336: 577-580). If done, any sequence mapped to that region could represent a related gene, or a regulatory gene, for further study. The nucleotide sequence of the present invention, generated by translocation, inversion, etc.,
It can also be used to detect differences in chromosomal location between normals, carriers, and patients.

In another embodiment of the present invention, HMH, its catalytic or immunogenic fragments, oligopeptides can be used for screening compounds in various drug screening techniques. Fragments used in such screening may be in free form in solution, in a form attached to a solid support, in a form attached to a cell surface, or in a cell. Can be The binding complex formation between HMH and the drug under test can be measured.

As described in published PCT application WO 84/03564, alternative drug screening techniques can perform high-throughput screening for compounds with appropriate binding affinity for the protein of interest. In this method, when applied to HMH, a number of different miniature compounds to be tested are synthesized on a solid substrate such as a plastic pin or other surface. The compound to be tested is reacted with HMH or a fragment thereof and washed. The bound HMH is then detected by methods well known in the art. Also, purified HMH can be directly coated on a plate for use in the aforementioned drug screening techniques. Alternatively, the peptide can be captured using a non-neutralizing antibody and immobilized on a solid substrate.

In another example, a competitive drug screening assay can be used in which neutralizing antibodies capable of binding to HMH specifically compete with the compound under test for binding to HMH. In this method, antibodies can be used to detect the presence of any peptide that has one or more antigenic determinants of HMH in common.

In yet another embodiment, a molecular biology technique that has not been developed to date, but that the new technique has the properties of nucleotide sequences known to date (eg,
Any technique based on, but not limited to, the triplet genetic code and specific base pairing) can employ nucleotide sequences encoding HMH in that technique.

The following examples are merely illustrative of the present invention and are not intended to limit the present invention to these examples.

[0169]

【Example】

1. Preparation of BRUSTNOT05 cDNA Library A BRSTNOT05 cDNA library was obtained microscopically from a 58-year-old Caucasian female who underwent unilateral magnifying mastectomy after being diagnosed with multicentric invasive grade 4 lobular carcinoma of the breast. Prepared from normal breast tissue. Non-tumorous breast tissue, skin, papillae, and fascia were not involved. Also, no evidence of intravascular invasion was found. Eight lower central and two upper left axillary lymph node symptoms were negative. Prior to surgery, the patient had been prescribed tamoxifen, which inhibits the induction of breast cancer. In addition, Zantac® (Ranitidine hydrochloride; Gl
axo Wellcome, Inc., Research Triangle Park, NC), aspirin, powerful Tyleno
l® (acetaminophen; McNeil Consumer Products Company, For
t Washington, PA), and vitamin C. His history of this patient included skin cancer, cerebrovascular disease, atherosclerosis, rheumatic heart disease, and osteoarthritis. Family history included maternal breast cancer and sibling prostate cancer.

[0170] This frozen tissue was used as a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Inst.
ruments, Westbury, NY) and homogenized and dissolved in a guanidine isothiocyanate solution. This lysate is transferred to a Backman L8-70M ultracentrifuge (
The mixture was centrifuged by cesium chloride density gradient centrifugation using 5.7 M CsCl at a rotational speed of 25,000 revolutions per minute at ambient temperature for 18 hours using a Beckman SW28 rotor at Beckman Instruments. The RNA was extracted with acidic phenol pH 4.7, precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in RNase-free water and DNase treated at 37 ° C.
The process of extraction and precipitation of the RNA was repeated. Next, this mRNA is transferred to Qiagen Oligo
It was isolated using a tex kit (QIAGEN, Inc., Chatsworth, CA) and used to construct a cDNA library.

This mRNA was obtained from the SuperScript Plasmid System for cDNA Synthesis and Plasmid.
Handled according to the recommended protocol of mid Cloning (Part No. 18248-013, Gibco-BRL). This cDNA was fractionated on a Sepharose CL4B column (Part No. 275105-01; Pharmacia), and a cDNA having a size exceeding 400 bp was ligated to a pINCY1 vector. This plasmid pINCY1 was then transferred to DH5a ^™ competent cells (Part No. 18258-012; Gib
co-BRL).

2 Isolation and Sequencing of cDNA Clones Plasmid DNA was released from the cells, and this was purified using the REAL Prep 96 Plasmid Kit.
(Catalog # 26173, QIAGEN). This kit was capable of simultaneously amplifying 96 samples in a 96-well block using a multichannel reagent dispenser. The recommended protocol was used, but the following points were changed. (
1) Bacteria were cultured in 1 ml sterile Terrific Broth (article number 22711, Gibco / BRL) containing 25 mg / L carbenicillin and 0.4% glycerol. (2) After inoculation, the culture solution was incubated for 19 hours, and at the end of the incubation, the cells were lysed in 0.3 ml of lysis buffer. (3) After isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml of distilled water. After the final step of the protocol, samples were transferred to a 96-well block and stored at 4 ° C.

The sequencing of this cDNA was performed using Peltier Thermal Cyclers (MJ Research, Water
town, MA PTC200) and Applied Biosystems 377 DNA Sequencing System
Using Hamilton Micro Lab 2200 (Hamilton, Reno, NV) in combination with ms
The reading frame was determined by the method of Sanger et al. (1975, J. Mol. Biol. 94: 441f).

3 Using the nucleotide sequences of the homology search sequence listing of cDNA clones and their analogous proteins and the amino acid sequences inferred therefrom as query sequences, for example, GenBank, SwissProt, BLOCKS, and Pima II I searched the database. These databases contain annotated sequences that have already been identified, and a region having homology (similarity) was searched from this database using BLAST (for Basic Local Alignment Tool) (Al
tschul. SF (1993) J. Mol. Evol. 36: 290-300; Altschul et al. (1990) J. Mol. B
iol. 215: 403-10).

BLAST creates an alignment of both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of the alignment, BLAST is particularly useful in identifying exact matches, ie, homologs of prokaryotic (bacterial) or eukaryotic (animal, fungal, or plant) origin. When processing primary sequence patterns and gap penalties for secondary structure, Sm is incorporated herein by reference.
Other algorithms such as those described in ith RF and TF Smith (1992, Protein Engineering 5: 35-51) can be used. The sequences disclosed herein are at least 49 nucleotides in length and have no more than 12% of unnecessary bases (where N is recorded other than A, C, G, or T). .

The BLAST method is described by Karlin. S. and SF Altsch, which are hereby incorporated by reference.
As described in detail in ul (1993: Proc. Nat. Acad. Sci. 90: 5873-7), searching for a match between the query sequence and the sequence in the database, the statistical significance of any match found is Assess gender and report only matches that meet the user-selected significance threshold. Threshold in this application, 10 ^-25 nucleotides, 10 peptide ^- was set to ^14.

4 Northern Analysis Northern analysis is an experimental technique used to detect the presence of gene transcripts, and involves the labeling of a labeled nucleotide sequence and the RN of a particular cell type or tissue.
Hybridization with the membrane to which A is bound (Sambrook et al.,
Supra).

Using a similar computer technique using BLAST (Altschul, SF supra; Altschul, SF supra), use the GenBank or LIFESEQ ^™ database (Incyte Pharmaceuticals).
Search for identical or related molecules in databases such as ticals). This analysis can be performed in a much shorter time than hybridization using many membranes. In addition, the sensitivity of the computer search can be modified to determine whether a particular match is classified as an exact match or homologously.

The reference value of the search is a product score, which is defined by the following equation. (Sequence match (%) × Maximum BLAST score (%)) / 100 This product score takes into account both the degree of similarity between two sequences and the match in sequence length. For example, when the product score is 40, the match is accurate within an error of 1-2%, and when the score is 70, the match is exact. Homologous molecules are usually identified by selecting those that show a product score of 15-40, while those with lower scores are identified as closely related molecules.

The results of the Northern analysis are reported as a list of libraries in which transcripts encoding HMH are generated. A list of sequence abundance and percent abundance is also reported. Abundance directly reflects the number of detections of a particular transcript, and percent abundance is abundance divided by the total number of sequences detected in the cDNA library.

Extension of Sequence Encoding 5HMH Using the nucleic acid sequence of Incyte Clone No. 2295453, oligonucleotide primers were designed to extend the partial nucleotide sequence to full length.
One primer was synthesized to initiate extension in the antisense direction and the other primer was synthesized to extend the sequence in the sense direction. Using these primers, the known HMH sequence was extended "outside" to generate an amplicon containing a new, unknown nucleotide sequence of the region of interest. The first primer is OLIG
O 4.06 (National Biosciences) or other suitable program, is about 22 to about 30 nucleotides in length, has a GC content of 50% or more, and is about 68 It was designed to anneal to the target sequence at a temperature of 約 72 ° C. Stretches of any nucleotides that would result in hairpin structure and primer-primer dimerization were excluded.

This sequence was extended using a selected human cDNA library (Gibco / BRL). If more than one step extension is necessary or desirable, an additional set of primers is designed to further extend the known region.

High fidelity amplification is achieved by thorough mixing of the enzyme with the reaction mixture according to the instructions in the instructions for the XL-PCR kit (Perkin Elmer). When starting amplification with 40 pmol of each primer and all other components of the kit at the recommended concentration, the following parameters were used using a Peltier Thermal Cycler (PTC200; MJ Research, Watertown MA), ie, step 194: 1 minute at 65 ° C (initial denaturation) Step 2 1 minute at 65 ° C Step 3 6 minutes at 68 ° C Step 4 15 seconds at 94 ° C Step 5 1 minute at 65 ° C Step 6 7 minutes at 68 ° C Step 7 Steps 4-6 Repeat 15 more cycles Step 8 94 ° C for 15 seconds Step 9 65 ° C for 1 minute Step 10 68 ° C for 7 minutes 15 seconds Step 11 Repeat steps 8 to 12 for 12 cycles Step 12 72 ° C for 8 minutes Step 134 ° C (the (The temperature was maintained).

An aliquot of 5-10 μl of the reaction mixture was analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose minigel to determine whether the sequence extension reaction was successful. Were determined. Select the largest product or band, cut out of the gel,
Purification was performed using QIAQuick ^™ (QIAGEN Inc., Chatsworth, CA) and the single-stranded nucleotide overhang was excised using the Klenow enzyme to create blunt ends that facilitate recombination and cloning.

After ethanol precipitation, the product was redissolved in 13 μl ligation buffer and 1 μl
Of T4-DNA ligase (15 units) and 1 μl of T4 polynucleotide kinase were added, and the mixture was incubated for 2-3 hours at room temperature or overnight at 16 ° C. Competent E. coli cells (in 40 μl of appropriate culture medium)
The ligation mixture was transformed with 80 μl of SOC medium (Semb
rook et al., supra). After 1 hour incubation at 37 ° C, all transformation mixtures were plated on Luria Bertani (LB) agar (Sembrook et al., Supra) containing 2xCarb. At a later date, several colonies are randomly selected from each plate,
Cultivated in 150 μl of liquid LB / 2 × Carb medium in each well of a suitable commercially available sterile 96-well microtiter plate. Further at a later date, 5 μl each
Was transferred into a non-sterile 96-well plate and diluted 1:10 with water,
5 μl of each sample was transferred to a PCR array.

For PCR amplification, 18 μl of the enriched PCR reaction mixture (3.3 ×) containing 4 units of rTth DNA polymerase, vector primers, and / or primers specific to the gene used in the extension reaction were added to each well. Added. The amplification was carried out under the following conditions: Step 1 at 94 ° C. for 60 seconds Step 2 at 94 ° C. for 20 seconds Step 3 at 55 ° C. for 30 seconds Step 4 at 72 ° C. for 90 seconds Step 5 Steps 2 to 4 were further repeated 29 cycles Step 6 72 Performed at 74 ° C. (maintained) at 180 ° C. for 180 seconds.

An aliquot of the PCR reaction was run on an agarose gel with molecular weight markers. The size of the PCR product was compared to the original partial cDNA, the appropriate clone was selected, ligated to the plasmid and sequenced.

Similarly, using the nucleotide sequence of SEQ ID NO: 2 to obtain 5 ′ regulatory sequences using the procedures described above, obtaining oligonucleotides designed for 5 ′ extension, and It is possible to obtain an appropriate genomic library.

6 Labeling and Use of Hybridization Probes cDNA, mRNA and genomic DNA are screened using hybridization probes made from the sequence of SEQ ID NO: 2. About 20
The labeling of oligonucleotides consisting of base pairs of
The same procedure is generally used for DNA fragments. OLIGO4.06 (National Bi
Oligonucleotides were designed using state-of-the-art software such as Oscience) and 50 pmol of each oligomer, 250 μCi of [γ- ³² P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston
MA). Label the labeled oligonucleotide with S
Purify using ephadex G-25 ultrafine resin column (Pharmacia & Upjohn). An aliquot containing probes labeled per minute 10 ⁷ counts endonuclease AseI, Bgl II, EcoRI, Pst I, Xba1 or PvuII; common membrane of the digested human genomic DNA using one of DuPont NEN Used in the hybridization analysis used.

The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to a nylon membrane (Nytran Plus, Schleicher & Schuell, Durham NH).
Hybridization is performed at 40 ° C. for 16 hours. To remove non-specific signals, blots are washed sequentially at room temperature under increasingly stringent conditions of up to 0.1 × aqueous sodium citrate and 0.5% sodium dodecyl sulfate.
XOMAT AR ^TM film (Kodak, Rochester, NY) is loaded into a Phosphoimager cassette (Mol
Hybridization patterns are visually compared after exposure to the blots for several hours in ecular Dynamics, Sunnyvale, CA).

7 Microarrays To generate oligonucleotides for microarrays , SEQ ID NO:
: 2 is examined using a computer algorithm from the 3 'end of the nucleotide sequence. The algorithm identifies oligomers of defined length that are unique to the gene, have a GC content within the range suitable for hybridization, and are free of putative secondary structures that would prevent hybridization. . This algorithm identifies 20 sequence-specific oligonucleotides 20 nucleotides in length (20 mer). Only one central nucleotide in each sequence is changed to create a set of otherwise identical oligonucleotides. This process is repeated for each gene in the microarray, and two sets of 20 20-mers are synthesized and arranged on the surface of a silicon chip using a light-irradiated chemistry process (Chee, M. et al., PCT / WO95). / 11
995, which is hereby incorporated by reference).

Alternatively, oligomers are synthesized on the surface of a substrate using chemical conjugation procedures and ink jet equipment (Baldeschweiler, JD et al. PCT / WO95 / 25116, which is hereby incorporated by reference). Yet another method uses a "lattice-type" array similar to that used in dot blots (slot blots), using a vacuum system, thermal, UV, mechanical or chemical coupling procedures to remove cDNA fragments or The oligonucleotide is placed on the surface of the substrate and allowed to bind. Arrays can be made manually or by using commercially available materials and machines, and can have a grid of 8, 24, 94, 384, 1536, or 6144 dots. After hybridization, the microarray is washed to remove unhybridized probes, and the level and pattern of fluorescence are determined using a scanner. The scanned images are examined to determine the degree of complementarity and the relative amount / expression level of each oligonucleotide sequence on the microarray.

8 Complementary Polynucleotides A sequence that encodes HMH, or a sequence that is complementary to any portion thereof, is used to reduce, ie, inhibit, the expression of naturally occurring HMH. Although the use of oligonucleotides of about 15 to about 30 base pairs is specifically noted, essentially the same procedure can be used for smaller or larger sequence fragments. Olig
Design the appropriate oligonucleotides using o4.06 software and the coding sequence of HMH (SEQ ID NO: 1). To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5 'sequence and used to prevent promoter binding. To inhibit translation, complementary oligonucleotides are designed so that ribosomes do not bind to transcripts encoding HMH.

9 Expression of HMH Expression of HMH is achieved by subcloning the cDNA into a suitable vector and transfecting the vector into a host cell. in this case,
Cloning vectors are also used to express HMH in E. coli.
This vector has a β-galactosidase promoter upstream of the cloning site, followed by an amino-terminal Met, followed by seven residues of β-galactosidase. Following these eight residues are a bacteriophage promoter that facilitates transcription and a linker that contains many unique restriction sites.

The transformed strain was isolated, induced with IPTG in a standard manner, and fused with the original β-galactosidase at 7 residues, a linker of about 5-15 residues, and a full-length protein. Produce protein. This group of signal residues induces the secretion of HMH into the bacterial growth medium, which can be used directly in subsequent assays for activity.

Demonstration of 10 HMH activity The calcium binding activity of the human mammoglobin homolog was determined by Barnes et al.
Mol. Biol. 256: 392-404). In this assay, a ruthenium molecule between a mutant molecule created by replacing the active D (No. 51) or D (No. 53) "cap" residue of HMH with a nucleotide encoding N or K, and HMH. Compare red binding.

11 Production of HMH-Specific Antibodies To immunize rabbits and produce antibodies using standard protocols, PA
HMH substantially purified by GE electrophoresis (Sambrook, supra) is used. SE
The amino acid sequence deduced from Q ID NO: 2 was converted to DNASTAR software (DNASTAR
Inc.) to determine the region of high immunogenicity, synthesize the corresponding oligopeptide, and use it to produce antibodies using methods well known to those skilled in the art. The selection of suitable epitopes, such as epitopes near the C-terminus or in hydrophilic regions, is described in Ausubel et al., Supra, and other references.

Generally, oligopeptides having a length of 15 residues were synthesized by fmoc chemistry using a peptide synthesizer Model 431A from Applied Biosystems,
M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS:
It binds to keyhole limpet hemocyanin (KLH, Sigma, St. Louis, MO) by a reaction using Ausubel et al., Supra). Rabbits are immunized with the oligopeptide-KLH complex in Freund's complete adjuvant. To test the anti-peptide activity of the resulting antiserum, for example, the peptide can be bound to plastic and
Block with% BSA, react with rabbit antiserum, wash, and react with radioactive iodine-labeled goat anti-rabbit IgG.

12 Purification of Spontaneous HMH Using Specific Antibodies Spontaneously occurring HMH or recombinant HMH can be substantially purified by immunoaffinity chromatography using antibodies specific for HMH. The immunoaffinity column is CnBr-activated Sepharose (Pharmacia & Upjohn)
It is constructed by covalently binding an activated chromatography resin such as described above and an HMH antibody. After binding, the resin is blocked and washed according to the instructions for use.

The medium containing HMH was passed through an immunoaffinity column, and the column was subjected to HMH.
Is washed under conditions that allow for preferential adsorption (eg, with a high ionic strength buffer in the presence of a surfactant). The column is eluted under conditions that break the binding between the antibody and HMH (eg, a buffer of pH 2-3 or a high concentration of chaotropic ions such as urea or thiocyanate ions) to recover HMH.

13 Identification of Molecules That Interact with HMH HMH, or a biologically active fragment thereof, was prepared using ¹²⁵ I Bolton Hunter reagent (Bolto
n et al. (1973) Biochem. J. 133: 529). Candidate molecules pre-arranged in the wells of the multiwell plate are incubated with labeled HMH, washed, and the wells bearing the labeled HMH complex are assayed. Using data obtained using different concentrations of HMH, the association, affinity,
Calculate the numerical value of a number.

All publications and patent specifications mentioned above are incorporated herein by reference.
Various modifications of the method and system of the invention described herein which do not depart from the scope and spirit of the invention will be apparent to those skilled in the art. Although the invention has been described with reference to particularly preferred embodiments, it should be understood that the true scope of the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the claims.

[Sequence list]

[Brief description of the drawings]

FIG. 1A: Amino acid sequence of human mammoglobin homolog, HMH (SEQ ID NO:
1) and a diagram showing a nucleic acid sequence (SEQ ID NO: 2). Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Software Engineering Co., Ltd.,
San Bruno, CA).

FIG. 1B: Amino acid sequence of human mammoglobin homolog, HMH (SEQ ID NO:
1) and a diagram showing a nucleic acid sequence (SEQ ID NO: 2). Sequence alignment is performed using MacDNASIS PRO ^™ software (Hitachi Software Engineering Co., Ltd.,
San Bruno, CA).

FIG. 2. HMH (2295453; SEQ ID NO: 1) and human mammobrobin (GI 11
99596 shows an amino acid sequence alignment between SEQ ID NOs: 3). For sequence alignment, use DNASTAR ^™ software (DNASTAR Inc, Madison WI)
Using a multi-sequence alignment program.

FIG. 3 is a diagram showing the results of Northern analysis of HMH prepared electronically using a LIFESEQ ™ database (Incyte Pharmaceuticals Inc., Palo Alto, Calif.).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C12N 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/68 A 5/10 G01N 33/53 D C12P 21/02 33/566 C12Q 1/68 C12P 21/08 G01N 33/53 C12N 15/00 ZNAA 33/566 A61K 37/02 // C12P 21/08 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG , ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU , CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Shah, Parvi 94087 Sunnyvale, California, United States # 5 Queen Charlotte Drive 1608 (72) Inventor Marie, Lin E United States California 9402 8. Portola Valley Los Trancos Road 1124 [Continued summary]

Claims (20)

[Claims] 1. A substantially purified human mammoglobin homolog having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof. 2. A substantially purified, having at least 90% amino acid sequence identity to the sequence of SEQ ID NO: 1 and retaining at least one of the functional characteristics of a human mammoglobin homolog. Mutant of human mammoglobin homolog. 3. Encoding the human mammoglobin homolog of claim 1;
An isolated and purified polynucleotide sequence, or a fragment or variant sequence of said polynucleotide sequence. 4. A composition comprising the polynucleotide sequence of claim 3. 5. Hybridizing with the polynucleotide sequence of claim 3,
An isolated and purified polynucleotide sequence. 6. An isolated and purified polynucleotide sequence which is complementary to the polynucleotide sequence of claim 3 or a fragment or variant thereof. 7. An isolated and purified polynucleotide sequence comprising the sequence of SEQ ID NO: 2 or a fragment or variant thereof. 8. An isolated and purified polynucleotide sequence that is complementary to the polynucleotide sequence of claim 7. 9. An expression vector comprising at least a fragment of the polynucleotide sequence of claim 3. 10. A host cell comprising the expression vector according to claim 9. 11. A method for producing a polypeptide having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, comprising: (a) using the host cell of claim 10 under conditions suitable for expression of the polypeptide. A method for producing a polypeptide having the amino acid sequence of SEQ ID NO: 1 or a fragment thereof, comprising: culturing; and (b) recovering the polypeptide from the medium of the host cell. 12. A pharmaceutical composition comprising a substantially purified human mammoglobin homolog having the amino acid sequence of SEQ ID NO: 1 together with a suitable pharmaceutical carrier. 13. A purified antibody that specifically binds to the polypeptide of claim 1. 14. A purified agonist of the polypeptide of claim 1. 15. A purified antagonist of the polypeptide of claim 1. 16. A method for treating or preventing a neoplastic disease, comprising a step of administering an effective amount of the antagonist of claim 15 to a patient in need of such treatment. 17. A method for treating or preventing endometriosis, comprising the step of administering an effective amount of the antagonist of claim 15 to a patient in need of such treatment. Method. 18. A method for detecting a polynucleotide encoding a human mammoglobin homolog in a biological sample, comprising: (a) hybridizing the polynucleotide of claim 6 with a nucleic acid material of a biological sample. Forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the complex is a polynucleotide encoding a human mammoglobin homolog in the biological sample. A method for detecting a polynucleotide encoding a human mammoglobin homolog in a biological sample, the method comprising the steps of: 19. The method according to claim 18, wherein the nucleic acid material is amplified by polymerase chain reaction before hybridization. 20. A method for diagnosing endometriosis, comprising: (a) extracting abdominal fluid from a subject; and (b) conditions suitable for antibody binding. (C) detecting a human mammoglobin homolog, wherein the presence of the bound molecule indicates the presence of endometriosis. A method for diagnosing endometriosis.