WO2001016354A1 - Modulation of he4 in inflammatory and renal diseases - Google Patents

Abstract

The invention relates generally to the changes in gene expression in inflammatory diseases and renal diseases such as IgAN and NCGN. The invention relates specifically to a novel HE4 sequence which was identified in IgAN kidney tissue. Methods are described for diagnosing inflammatory and renal diseases, such as immunoglobulin A nephropathy by measuring the levels of HE4 in nucleic acid or cell samples from patients. Also described are methods of screening modulators of HE4.

Description

Modulation of HE4 in Inflammatory and Renal Diseases Inventors William E. Munger, Hong-Wei Sun and Ronald J. Falk

FIELD OF INVENTION

This invention relates to methods of correlating gene expression with inflammatory diseases and renal disorders in healthy and disease samples and methods for drug screening using identified gene product candidates as targets. Specifically, this invention relates to methods which monitor the expression of HE4, including an HE4 which has been identified in kidney tissue from a patient diagnosed with immunoglobulin A nephropathy (IgAN). Such monitoring can serve 1) as a predictor of glomerulonephritis, including mesangial proliferation; inflammation; necrotizing crescentic glomerulonephritis (NCGN); minimal change disease and sclerosis, 2) as a means of identifying agents that modulate HE4 expression in mesangial and other tissues and 3) broadly as a general marker for renal diseases and inflammatory diseases including noninfectious systemic inflammatory responses (SIRS). This application claims the benefit of U.S. Provisional Application 60/152,123, which is herein incorporated by reference.

BACKGROUND

Kidney Disease

Kidney disease affects many millions of people in the United States and worldwide. In 1990 the US had greater than 200,000 patients in end stage renal disease (ESRD) programs, largely as a result of renal involvement by glomerular diseases. One form of glomerular disease alone, diabetic glomerulonephropathy, affects millions with health costs in the billions of dollars.

The mechanisms of glomerular diseases are quite varied. Although certain common mechanisms may underlie blood or protein in the urine (e.g., loss of the glomerular charge barrier), the nature of the processes initiating renal injury differ. For example, immune- mediated renal injury is a major pathogenic mechanism of glomerular damage. In diabetic nephropathy, other mechanism are clearly at work. At this time kidney disease usually cannot be cured. Once the glomeruli (filtering units) are damaged, they cannot be repaired. Therefore, treatment focuses on slowing the progression of the disease and preventing complications.

Diagnosis and Renal Biopsy While diagnosis of the renal patient may involve urinanalysis, microscopic examination and radiologic studies, when these procedures fail to give a diagnosis or are not indicated for clinical reasons (e.g., patient cannot cooperate, has bleeding abnormalities; only a solitary kidney exists or blood pressure is too high), the use of kidney biopsy may be required (Cecil Textbook of Medicine 20th edition, 1996, (Bennett et al. eds.) p. 517, W.B. Saunders Company, Philadelphia, PA). There are two biopsy types, open and percutaneous, the former being reserved for those who present specific clinical risks (see above) or where visual control of biopsy is critical and wherein the expectation of obtaining therapeutically meaningful information is high (Cecil Textbook of Medicine 20th edition, 1996 and The Merck Manual 16th edition, 1992, (Berkow et al. eds) p. 1660, Merck Research Laboratories, Rahway, NJ). Renal biopsy is performed to: (1) help establish a histologic diagnosis; (2) help estimate prognosis and potential reversibility or progression of the renal lesion; (3) estimate the value of therapeutic modalities; and (4) determine the natural history of renal diseases (The Merck Manual 16th edition, 1992).

IgA Nephropathy IgA nephropathy is the most common form of glomerulonephritis world wide

(Rychlik et al, Ann Med Interne (Paris) (1999) 150(2):117-126; Galla JH., Kidney Int (1995) 47(2):377-387; Tractman et al, Pediatr Res (1996) 40(4):620-626). It is characterized by recurrent heavy blood and/or protein in the urine (i.e., hematuria and proteinuria, respectively) caused by deposits of immunoglobulin A(IgA) inside the glomeruli, particularly diffuse IgA deposits in mesangial cells. While IgAN causes little to no pain, about 30% of patients will develop ESRD after 20 years, particularly those who present with hypertension, heavy proteinuria or renal insufficiency (Galla JH., 1995). IgAN can also cause a vasculitis which can lead to necrotizing crescentic glomerulonephritis (NCGN), the latter ultimately resulting in renal failure and end-stage renal disease.

Clinical predictors of progressive disease are elevated serum creatinine concentration at presentation, increased systemic blood pressure and persistent protein excretion (Rychlik et al, 1999). No blood or urine test is sufficiently reliable for diagnosis, depending mainly upon biopsy for diagnostic information (Galla JH., 1995). The diagnosis therefore, continues to rely on the findings of the dominant or codominant mesengial deposition of IgA on immunohistologic examination of the kidney and includes glomemlosclerosis, tubular atrophy/interstitial fibrosis, extension of immune deposits to the perivascular space and epithelial cell proliferation. At present, therapy is disappointing, and many physicians choose to treat only those patients at highest risk for progression to renal failure. A significant percentage of patients who must receive transplants have morphogenic recurrence in the allograft, but graft loss is uncommon and patients show excellent allograft survival.

Cellular Events and IgAN

Glomerular disorders such as IgAN that progress to renal failure are characterized by mesangial cell proliferation and accumulation of mesangial matrix. Mesangial cell are cells found within the glomerular lobules of kidneys, where they serve as structural supports, regulate blood flow, are phagocytic and may act as accessory cells presenting antigen in immune responses. Factors that control mesangial cell function include cytokines and growth mediators, matrix components including integrins, and interactions with other cells such as the endothelial and epithelial cells. When exposed to an injurious stimulus, the mesangial cell responds by cellular proliferation and matrix synthesis leading to an increase in mesangial cells and matrix accumulation that ultimately progresses to the development of glomemlosclerosis (Lee GS., Ann Acad Med Singapore (1995) 24(6):851-855). Thus, there is a current interest in the discovery of new agents that interrupt the pathways of mesangial proliferation to sclerosis. Genes and IgAN

IgAN is thought to involve abnormalities in the production and/or catabolism of IgA. Some polymorphism evidence suggests that genetic factors determine the susceptibility to and rate of progression of IgAN. For example, genes involving blood pressure regulation, especially a polymorphism for angiotensin converting enzyme, have been found to be associated with disease progression (Schmidt et al, Ann Med Interne (Paris) (1999) 150(2):86-89 and Chen et al, Chin MedJ(Engl) (1997) 110(7):526-529).

While some studies have focused on genetic factors, others have attempted to characterize IgAN pathogenesis by examining gene expression using RT-PCR and/or Northern blotting. For example, as cytokines have been found to play a relevant role in the pathogenesis of IgAN, gene expression of proinflammatory cytokines, immuno-regulatory cytokines and growth factors have been analyzed (Yano et al, J Clin Immunol (1997) 17(5):396-403). In renal tissue, mRNA for IL-lα and PDGF-B have both been shown to be increased in biopsies from IgA patients. Further, expression of TGF-βl (Yang et al, Nephron (1997) 77(3):290-297), γ, δ-T cell receptor V regions (Olive et al, Kidney Int

(1997) 52(4): 1047-1053), PDGF B-chain (Terada et al, J Am Soc Nephrol (1997) 8(5):817- 819 and Nakamura et al, Clin Immunol Immunopathol (1992) 63(2):173-181), decay accelerating factor (DAF, Abe et al, Kidney Int (1998) 54(1):120-130), inducible NO synthase (Kashem et al, Kidney Int (1996) 50(2): 392-399) and monocyte chemo tactic peptide-1 (Grandaliano et al, Transplantation (1997) 63(3):414-420) have all been shown to be modulated as a function of IgAN. However, global examination of expression profiles, including differential display analysis, has not been employed to characterize IgAN.

Noninfectious Inflammation and End Stage Illness

The syndrome previously known as "sepsis" is now recognized as a generalized response to a number of insults that cause whole body activation of inflammatory mediators. Systemic inflammatory response syndrome (SIRS) is one example of the organism's inflammatory responses that can be triggered by infections, however, noninfectious disorders such as trauma (Cecil Textbook of Medicine, 1992), over-exertion (Shephard et al, Int J Sports Med (1998) 19(3):159-171), respiratory distress syndrome (ARDS) (Bass et al, Chest Surg Clin N Am (1997) 7(2):429-442), bypass surgery leading to pulmonary disfunction (Hensel et al, Anesthesiology (1998) 89(1):93-104), preterm labor syndrome (Dudley., Am J Obstet Gynecol (1999) 180:(1 Pt 3):S251-256), intraperitoneal hyperthermic perfusion (Sumida et al, Anest Analg (1999) 88(4):771-776), pancreatitis (Cecil Textbook of Medicine, 1992) and renal failure (Cecil Textbook of Medicine, 1992) can also lead to systemic inflammation. In fact, chronic activation of the systemic inflammatory response is a common and unifying factor in many end stage illnesses, particularly end stage renal disease (Bistrian BR., Am J Kidney Dis (1998) 32(6 Suppl 4):S113-117). As with IgAN, SIRS is correlated with the expression of proinflammatory cytokines, especially TNF-α, IL-6, IL-8, including the activation of such inflammatory cytokines by transcription factor nuclear factor KB (see Kovacich et al, JThorac Cardiovasc Surg (1999) 118( 1 ) : 154- 162). Thus, elucidation of both renal disease specific gene expression and common pathways involving proinflammatory modulators such as cytokines might be useful in monitoring inflammatory responses.

HE4

Human epididymal proteins represent a near ubiquitous mammalian family of proteins which have been localized in epididymal epithelium, within the lumen of the epididymal duct and vas deferens, and also on the surface of spermatozoa (Ellerbrock et al, Int JAndrol (1994) 17(6):314-323; Uhlenbruck et al, Int JAndrol (1993) 16(1):53-61; Pera et al, IntJ Androl (1994) 17(6):324-330; Xu et al, Arch Androl (1996) 37(2):135-141). As is the case in other species, a link to a specific function for human epididymal proteins such a maturation or storage has not be conclusively demonstrated.

Northern blot and in situ transcript hybridization specifically localize HE4 (Human epididymis gene product) mRNA to the epithelial cells of the epididymal duct predominantly within the distal sections. It is postulated that the gene product functions in sperm maturation as indicated by amino acid similarities to extracellular proteinase inhibitors in genital tract mucous secretion (Kirchoff et al, Biol Repro (1991) 45(2):350-357). SUMMARY OF THE INVENTION

In the present invention, a novel HE4 sequence has been identified in nucleic acid samples from diseased kidney biopsies. The diagnosis of many renal diseases, such as IgAN, continue to rely on highly invasive techniques (i.e., renal biopsy) to establish histo logical involvement as there is no sufficiently reliable blood or urine test for diagnosis. Because the expression of HE4 has been correlated with various glomerulonephrotic disorders, demonstration of such expression in nucleic acid samples obtained from living patients can serve as a diagnostic for detection of IgAN, necrotizing crescentic glomerulonephritis (NCGN) and minimal change disease, potentially replacing present invasive modalities. Moreover, cells in which HE4 can be induced can be used to screen compounds which modulate the expression of this gene.

Progressive renal disease commonly involves inflammatory and tissue remodeling processes in which proteases play a significant role. As a putative proteinase inhibitor, the novel HE4 sequence may be a protective response and therefore its regulation therapeutically could ameliorate damage to the kidneys. More broadly, because many progressive disease responses reflect an unhealthy physiologic and biochemical imbalance as opposed to a necessary state of homeostasis for health, either lowered or elevated HE4 production in general or lowered or elevated HE4 production outside a range that is homeostatically healthy could be deleterious. Therefore, measuring the level of HE4 could provide important diagnostic and prognostic information. Relatedly, modulating HE4 therapeutically up or down could provide a significant means of treating diseases in which HE4 plays a role. The novel HE4 sequence is also expressed in salivary gland, tests, lung, utems, trachea, prostate, thyroid, heart, pancreas, colon, and other tissues (see Figure 4). Therefor, it might play a significant role in disease processes associated with these tissues, particularly disease processes that involve inflammation and scarring.

The invention includes an isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:2; (b) an isolated nucleic acid molecule comprising SEQ ID NO:l; and (c) an isolated nucleic acid molecule which encodes an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4. The invention also includes vectors containing the nucleic acid molecules, host cells transformed with these nucleic acid molecules and recombinant methods of expressing the encoded proteins.

The invention also includes an isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 and an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4. In another embodiment, the invention includes methods of detecting HE4 in patient fluid or tissue samples as well as methods for screening for agents which modulate the expression or function of HE4.

This and other aspects of the present invention will become clearly understood by reference to the following figures, examples and description of the invention.

BRIEF DESCRIPTIONS OF THE DRAWINGS

Figure 1. Autoradiographic image of the distribution of HE4 (JT22962) expression in various human tissues determined by Northern blot analysis.

Figure 2. Graphic representation of HE4 expression in renal biopsy samples. RT-PCR is performed on nucleic acids from the biopsied renal tissues listed. Relative expression is determined by comparing the amount of HE4 PCR product in any given sample to that of GAP-DH expression.

Figure 3. Expression profile of HE4 sequence in renal disease using Q-RT-PCR. RT- PCR is performed on nucleic acids from minimum change disease; mild, moderate and severe forms of IgAN; and NCGN. Relative expression is determined by comparing the amount of HE4 PCR product in any given sample to that of GAP-DH expression.

Figure 4. Graphic representation of HE4 expression in various tissues. RT-PCR is performed on nucleic acids from the tissues listed. Relative expression is determined by comparing the amount of the HE4 PCR product in any given sample to that of GAP-DH expression.

Figure 5. READS analysis showing co-fractionating bands corresponding to HE4 from normal and diseased human renal biopsy samples.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based in part on identifying genes that are differentially regulated or expressed in human diseased kidney tissue compared to normal kidney tissue. Specifically, a novel HE4 sequence has been identified that is differentially regulated in inflammatory and renal diseases and whose expression can be correlated, for example, with minimal change disease, IgAN (including correlation with degrees of severity of the disease), and NCGN. Further, monitoring of expression is considered to be indicative of treatment efficacy as well as predictive of end stage renal disease development. The gene, as well as the peptides they encode, can serve as targets for agents that can be used to modulate the activity of HE4. For example, agents may be identified which modulate biological processes associated with injury to the kidney such as mesangial cell injury by cytokine/macrophage infiltration. Agents may also be identified which modulate the biological processes associated with recovery from IgA injury to the kidney.

Definitions "Sclerosis" herein refers to a thickening or hardening of a body part. Sclerosis also refers to a disease characterized by this thickening or hardening.

"IgA Nephropathy or Berger's Disease" refers to a group of disorders featuring recurrent episodes of macroscopic hematuria (blood in urine), mild proteinuria (protein in urine), glomerular changes, with or without progression to renal failure.

"Minimal Change Disease" refers to a disorder of the kidneys which largely affects the glomerulus, the blood filtering structure. This disorder is one common cause of nephrotic syndrome in children affecting 2 to 3 children per 100,000 population under age 16 in the U.S. Minimal change disease is also seen rarely in adults. The cause is unknown but may be related to an autoimmune illness. Presently, minimal change disease can only be diagnosed by renal biopsy.

"Necrotizing Crescentic Glomerulonephropathy" refers to a relatively uncommon form of acute glomerulonephritis that results in damage within the glomerulus of the kidney. There is rapid loss of kidney function with the formation of crescents on microscopic analysis (kidney biopsy). This disorder may result in acute glomerulonephritis or nephrotic syndrome and ultimately results in renal failure and end-stage renal disease.

"Mesangial cells" refer to cells found within the glomerular lobules of mammalian kidney, where they serve as structural supports, may regulate blood flow, are phagocytic and may act as accessory cells, presenting antigen in immune responses. "Ischemia" herein refers to a decrease in blood supply to a body organ, tissue or part caused by constriction or obstmction of blood vessels.

"Modulate" refers to the inhibition, induction, agonism and/or antagonism of the expression or function of an HE4 gene or gene product.

"Nucleic acid" includes DNA and RNA molecules and is used synonymously with the terms "nucleic acid sequence" and "polynucleotide."

"Transcriptional factors" refer to a class of proteins that bind to a promoter or to a nearby sequence of DNA to facilitate or prevent transcription initiation.

"Polypeptide" refers to an amino acid sequence including, but not limited to, proteins and protein fragments, naturally derived or synthetically produced. "Transcriptional profiling" refers to any assay method or technique which is capable of analyzing, quantitatively and/or qualitatively, one or more mRNA species found in a cell or a nucleic acid sample. For example, such assays include but are not limited to RT-PCR, quantitative PCR (Q-PCR), RNase protection assays, subtractive hybridization, READS and Northern blots.

"Whey Acidic Protein (WAP)" refers to a cysteine rich (four disulfide core), acidic protein, similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin. For example HE4 identified in IgAN renal tissue has two WAP domains:Gly-Phe-Thr-Leu-Val-Ser-Gly-Thr-Gly-Ala-Glu-Lys-Thr-Gly-Val-Cys-Pro-Glu- Leu-Gln-Ala-Asp-Gln-Asn-Cys-Thr-Gln-Glu-Cys-Val-Ser-Asp-Ser-Glu-Cys-Ala-Asp-Asn- Leu-Lys-Cys-Cys-Ser-Ala-Gly-Cys-Ala-Thr-Phe-Cys (WAP I) (amino acids 21 to 70 of SEQ ID NOs: 1 and 2); and Ser-Leu-Pro-Asn-Asp-Lys-Glu-Gly-Ser-Cys-Pro-Gln-Val-Asn-Ile- Asn-Phe-Pro-Gln-Leu-Gly-Leu-Cys-Arg-Asp-Gln-Cys-Gln-Val-Asp-Ser-Gln-Cys-Pro-Gly- Gln-Met-Lys-Cys-Cys-Arg-Asn-Gly-Cys-Gly-Lys-Val-Ser-Cys-Val-Thr-Pro-Asn-Phe (WAP II) (amino acids 71 to 124 of SEQ ID NOs: 1 and 2).

Preferred Embodiments

Epididymal proteins are known proteins whose function has not been firmly established. However they are thought to perform a maturation function in the epididymis of mammals and may be extracellular proteinase inhibitors based on sequence similarity of proteins having such a function in genital tract mucous secretions (Kirchoff et al, 1991). Attributes characteristic of HE4 and stmctural homologues of epididymal proteins in other species (e.g., CE4, see Ellerbrock et al, 1994) are: (i) they possess two whey acid protein domains; (ii) they belong to the "four disufide core" family of proteins; (iii) they can be found and isolated from epididymal epithelium, within the lumen of the epididymal duct and vas deferens, and also on the surface of spermatozoa; and (iv) they are glycosylated (Kirchoff et al, 1991).

HE4 and its stmctural homologues share extensive homology (See, e.g., Ellerbrock et al.1991 ; Uhlenbruck et al, 1993; Pera et al, 1994; XU et al, 1996). For example, probes from HE4 DNA can be used to screen Northern blots containing boar, bull and stallion mRNA to identify structurally homologous epididymal gene products. Further, antibodies recognizing HE4 proteins have been shown to react specifically in the epididymis of the boar and the bull (Uhlenbruck et al, 1993). As used herein, homology or identity can be determined by BLAST (Basic Local

Alignment Search Tool) analysis using the algorithm employed by the programs blastp, blastn, blastx, tblastn and tblastx (Karlin et al, Proc NatlAcad Sci USA (1990) 87: 2264- 2268 and Altschul, S. F. JMolEvol (1993) 36: 290-300, fully incorporated by reference) which are tailored for sequence similarity searching. The approach used by the BLAST program is to first consider similar segments between a query sequence and a database sequence, then to evaluate the statistical significance of all matches that are identified and finally to summarize only those matches which satisfy a preselected threshold of significance. For a discussion of basic issues in similarity searching of sequence databases, see Altschul et al. (Nature Genetics (1994) 6: 119-129) which is fully incorporated by reference. The search parameters for histogram, descriptions, alignments, expect (i.e., the statistical significance threshold for reporting matches against database sequences), cutoff, matrix and filter are at the default settings. The default scoring matrix used by blastp, blastx, tblastn, and tblastx is the BLOSUM62 matrix (Henikoff, et al, Proc Natl Acad Sci USA (1992) 89: 10915-10919, fully incorporated by reference). For blastn, the scoring matrix is set by the ratios of M (i.e., the reward score for a pair of matching residues) to N (i.e., the penalty score for mismatching residues), wherein the default values for M and N are 5 and -4, respectively.

In a preferred embodiment, HE4 DNA comprises the nucleic acid sequence as set forth in SEQ ID NO:l, wherein said DNA encodes the amino acid sequence as set forth in SEQ ID NO:2. As used herein, "HE4 DNA "is not limited to the sequences defined by SEQ ID NO: 1 , and includes those nucleic acid sequences which encode the present novel HE4 sequence which differs from the HE4 of Kirchoff et al. (SEQ ID NO: 3). For example, HE4 DNA encodes a polypeptide comprising 124 amino acids as set forth in SEQ ID NO:2 versus 125 amino acids as shown in Kirchoff et. al (1991) (SEQ ID NOs: 3 and 4). Further, HE4 DNA encodes a polypeptide comprising a Ser-71 (as set forth in SEQ ID NO:2) where Leu is present at the equivalent position of the encoded HE4 of Kirchoff et al (1991) (SEQ ID NOs: 3 and 4). Still further, between the equivalent Leu-72 and Pro-73 of SEQ ID NO:2, the HE4 of Kirchoff et al. has a cystinyl residue. Still further, the Ser-101 of the SEQ ID NO:2 is occupied by a threonyl residue in the HE4 of Kirchoff et al (1991) (SEQ ID NOs: 3 and 4). Any HE4 sequence, however, may be used in the methods of the invention, including natural variants, amino acid sequence variants, etc.

One means of diagnosing renal disease using the nucleic acids of the present invention involves obtaining kidney tissue from living subjects. Obtaining tissue samples from living sources is not problematic for tissues such as kidney as biopsy has become routine for certain procedures. For example, such procedures are used for dialysis and during transplant typing. (Yang et al, 1997).

In a preferred embodiment, diagnosing renal disease can be performed using body fluids. For example, HE4 protein or nucleic acid may be monitored in non-renal, accessible tissue (e.g., blood) and other body fluids which comprise renal filtrates (e.g., urine). For example, blood can be isolated and analyzed by transcriptional profiling to determine if HE4 expression has been modulated relative to a control. As IgAN can be accompanied by pyuria (i.e., the production of urine which contains white blood, see Ibels LS, et al, Medicine (Baltimore) (1994) 63(l):269-276), these cells may also be used to assay for HE4 levels. Such a determination may then be used to differentiate between urinary tract infection, tuberculosis, cancer and acute glomerulonephritis or severity of IgAN observed (e.g., mild, moderate, severe).

In a preferred embodiment, all assays will be carried-out with appropriate controls.

rDNA molecules Containing a Nucleic Acid Molecule The present invention further provides recombinant DNA molecules (rDNAs) that contain a coding sequence. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al, Molecular Cloning (1989). In the preferred rDNA molecules, a coding DNA sequence is operably linked to expression control sequences and/or vector sequences. The choice of vector and/or expression control sequences to which one of the protein family encoding sequences of the present invention is operably linked depends directly, as is well known in the art, on the functional properties desired, e.g., protein expression, and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the replication or insertion into the host chromosome, and preferably also expression, of the stmctural gene included in the rDNA molecule. Expression control elements that are used for regulating the expression of an operably linked protein encoding sequence are known in the art and include, but are not limited to, inducible promoters, constitutive promoters, secretion signals, and other regulatory elements. Preferably, the inducible promoter is readily controlled, such as being responsive to a nutrient in the host cell's medium. In one embodiment, the vector containing a coding nucleic acid molecule will include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons are well known in the art. In addition, vectors that include a prokaryotic replicon may also include a gene whose expression confers a detectable marker such as a dmg resistance. Typical bacterial dmg resistance genes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include a prokaryotic or bacteriophage promoter capable of directing the expression (transcription and translation) of the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention. Typical of such vector plasmids are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, N. J. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used to form a rDNA molecules that contains a coding sequence. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided cont-ώ-ing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-l/pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and the like eukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in an eukaryotic cell, preferably a drug resistance selection marker. A preferred drug resistance marker is the gene whose expression results in neomycin resistance, i.e., the neomycin phosphotransferase (neo) gene. (Southern et al, J. Mol. Anal. Genet. (1982) 1:327-341). Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the appropriate drug for the selectable marker.

Production of Recombinant Proteins using a rDNA Molecule

The present invention further provides methods for producing a protein of the invention using nucleic acid molecules herein described. In general terms, the production of a recombinant form of a protein typically involves the following steps:

First, a nucleic acid molecule is obtained that encodes a protein of the invention, such as the nucleic acid molecule having SEQ ID NO: 1 or nucleotides 23-397 of SEQ ID NO: 1. If the encoding sequence is unintermpted by introns, it is directly suitable for expression in any host.

The nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences, as described above, to form an expression unit containing the protein open reading frame. The expression unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of the recombinant protein. Optionally the recombinant protein is isolated from the medium or from the cells; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated. Each of the foregoing steps can be done in a variety of ways. For example, the desired coding sequences may be obtained from genomic fragments and used directly in appropriate hosts. The construction of expression vectors that are operable in a variety of hosts is accomplished using appropriate replicons and control sequences, as set forth above. The control sequences, expression vectors, and transformation methods are dependent on the type of host cell used to express the gene and were discussed in detail earlier. Suitable restriction sites can, if not normally available, be added to the ends of the coding sequence so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules of the invention to produce recombinant protein.

Methods to Identify Agents that Modulate the Expression of a Nucleic Acid

Encoding the IgAN Associated Protein.

Another embodiment of the present invention provides methods for identifying agents that modulate the expression of HE4. Such assays may utilize any available means of monitoring for changes in the expression level of HE4 mRNA. As used herein, an agent is said to modulate the expression of HE4, if it is capable of up- or down-regulating expression of an appropriate nucleic acid in a cell.

In one assay format, cell lines that contain reporter gene in-frame fusions between the HE4 promoter and any assayable fusion partner may be prepared. In another assay format, cell which express HE4 physiologically may be used. Additional assay formats may be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a protein of the invention, for example, a protein having SEQ ID NO:2. For instance, mRNA expression may be monitored directly by hybridization to the nucleic acids of the invention. Cell lines are exposed to the agent to be tested under appropriate conditions and time periods, after which total RNA or mRNA is isolated by standard procedures such those disclosed in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, 1989), and followed by the appropriate hybridization analysis for example Northern blots. Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells may be prepared from HE4 nucleic acids. It is preferable, but not necessary, to design probes which hybridize only with target nucleic acids under conditions of high stringency. Only highly complementary nucleic acid hybrids form under conditions of high stringency. Accordingly, the stringency of the assay conditions determines the amount of complementarity which should exist between two nucleic acid strands in order to form a hybrid. Stringency should be chosen to maximize the difference in stability between the probe:target hybrid and the potential probe:non-target hybrids.

Probes may be designed from HE4 nucleic acids through methods known in the art. For instance, the G+C content of the probe and the probe length can affect probe binding to its target sequence. Methods to optimize probe specificity are commonly known, such as those described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 1989) or Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995). "Stringent conditions" are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015M NaCl/0.0015M sodium citrate/0.1% SDS at 50°C, or (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine semm albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 rnM sodium citrate at 42°C. Another example is use of 50% formamide, 5 x SSC (0.75M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C. in 0.2 x SSC and 0.1% SDS. A skilled artisan can readily determine and vary the stringency conditions appropriately to obtain a clear and detectable hybridization signal. Hybridization conditions are modified using known methods, such as those described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 1989) and Ausubel et al. (Current Protocols in Molecular Biology, Greene Publishing Co., NY, 1995) as required for each probe. Hybridization of total cellular RNA or RNA enriched for polyA RNA can be accomplished in any available format. For instance, total cellular RNA or RNA enriched for polyA RNA can be affixed to a solid support and the solid support exposed to at least one probe comprising at least one, or part of one of the nucleic acids encoding HE4 under conditions in which the probe will specifically hybridize. Alternatively, nucleic acid fragments comprising at least one, or part of one of the nucleic acids encoding HE4 can be affixed to a solid support, such as a silicon wafer or a porous glass wafer. The wafer can then be exposed to total cellular RNA or polyA RNA from a sample under conditions in which the affixed sequences will specifically hybridize. Such glass wafers and hybridization methods are widely available, for example, those disclosed by Beattie (WO 95/11755). Silicon wafers and hybridization methods are also widely available, for example, those disclosed by Rava et al (U.S. Patent No: 5,874,219). By examining for differences between the ability of a given probe to specifically hybridize to an RNA sample from an untreated cell population versus a cell population exposed to an agent to be tested, agents which up or down regulate the expression HE4 is identified.

Hybridization for qualitative and quantitative analysis of mRNAs may also be carried out by using a RNase Protection Assay (i.e., RPA, see Ma et al, Methods (1996) 10:273-

238). Briefly, an expression vehicle comprising cDNA encoding the gene product and a phage specific DNA dependent RNA polymerase promoter (e.g., T7, T3 or SP6 RNA polymerase) is linearized at the 3' end of the cDNA molecule, downstream from the phage promoter, wherein such a linearized molecule is subsequently used as a template for synthesis of a labeled antisense transcript of the cDNA by in vitro transcription. The labeled transcript is then hybridized to a mixture of isolated RNA (i.e., total or fractionated mRNA) by incubation at 45 °C overnight in a buffer comprising 80% formamide, 40 mM Pipes, pH 6.4, 0.4 M NaCl and 1 mM EDTA. The resulting hybrids are then digested in a buffer comprising 40 μg/ml ribonuclease A and 2 μg/ml ribonuclease. After deactivation and extraction of extraneous proteins, the samples are loaded onto urea/polyacrylamide gels for analysis.

In another assay format for identification of agents which effect the expression of the instant gene products, cells or cell lines would first be identified which express said gene products physiologically. Cell and or cell lines so identified would be expected to comprise the necessary cellular machinery such that the fidelity of modulation of the transcriptional apparatus is maintained with regard to exogenous contact of agents with appropriate surface transduction mechanisms and or the cytosolic cascades. Further, such cells or cell lines may be transduced or transfected with an expression vehicle (e.g., a plasmid or viral vector) comprising an operable non-translated 5'-promoter containing end of the stmctural gene encoding the instant gene product fused to one or more antigenic fragments, which are peculiar to the instant gene products, wherein said fragments are under the transcriptional control of said promoter and are expressed as polypeptides whose molecular weight can be distinguished from the naturally occurring polypeptides or may further comprise an immunologically distinct tag. Such a process is well known in the art (see Sambrook et αl Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, 1989).

Cells or cell lines transduced or transfected as outlined above would then be contacted with agents under appropriate conditions; for example, the agent comprises a pharmaceutically acceptable excipient and is contacted with cells comprised in an aqueous physiological buffer such as phosphate buffered saline (PBS) at physiological pH, Eagles balanced salt solution (BSS) at physiological pH, PBS or BSS comprising semm or conditioned media comprising PBS or BSS and/or semm incubated at 37 °C . Said conditions may be modulated as deemed necessary by one of skill in the art. Subsequent to contacting the cells with the agent, said cells will be dismpted and the polypeptides of the dismptate are fractionated such that a polypeptide fraction is pooled and contacted with an antibody to be further processed by immunological assay (e.g., ELISA, immunoprecipitation or Western blot). The pool of proteins isolated from the "agent contacted" sample will be compared with a control sample where only the excipient is contacted with the cells and an increase or decrease in the immunologically generated signal from the "agent contacted" sample compared to the control will be used to distinguish the effectiveness of the agent. The agents identified by the above methods can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents.

In order to assay HE4 expression of the present invention in a physiologically relevant manner, tissues may be assayed under conditions which model IgAN, NCGN, minimal change disease, SIRS or another inflammatory disease, particularly inflamamatory diseases of the lungs. For example, some IgAN model systems include incubation of cultured cells with IL-1 and 11-6 (Lin et al, JLab Clin Med (1999) 133(l):55-63). Further, exposure of cells to ethanol (Smith et al, Alcohol (1993) 10(6):477-480) or gliadin (Amore et al, Am J Kidney Dis (1994) 23(2):290-301) have been shown to be adequate models for IgAN.

In a preferred embodiment, assays which incubate cells under conditions that simulate IgAN in vitro include, but are not limited to, for example, incubation of mesangial cells with cytokines, growth factors, antigens or primary alcohols. Assays which simulate IgAN in vivo include, but are not limited to oral immunization in animal models (Amore et al, 1994 and Trachtman et al, 1996), including oral administration of vomitoxin (Yan et al, Food Chem Toxicol (1998) 36(12):1095-1106) or gram-negative bacteria (or LPS, See Endo et al, Nephron (1993) 65(2): 196-205).

In a related aspect, NCGN can be simulated in vivo and in vitro by stimulation of rats or isolated mesengial cells with specific cytokines (e.g., INF-γ; see Coers et al.). Further, NCGN can be simulated by renal ischemia (see Brouwer et al, Kidney Int (1995) 47(4): 1121- 1129) or by immunization with myeloperoxidase (Brouwer et al, JExp Med (1993) 177(4): 905 -914). In another embodiment, SIRS is simulated by exercise (Iannoli et al, Adv Exp Med Biol (1997) 428:333-341) or by modulation of inflammatory cytokines similar to that seen for IgAN (e.g., IL-6, IL-8 and TGFα modulation). In another embodiment, minimal change disease can be simulated in vivo and in vitro by treatment of rats or rat podocytes with puromycin aminonucleoside (Bertram et al, Cell Tissue Res (1990) 260(3):555-563) or by removing α 2-6 linked sialic acids from glomerular filtration barriers (Gelberg et al, Lab Invest (1996) 74(5):907-920).

Candidate peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instmmentation and produced recombinantly using standard recombinant production systems (e.g., PCR and cloning). Peptide production using solid phase peptide synthesis is necessitated if non-gene-encoded amino acids are to be included in the candidate peptide product.

Methods to Identify Agents that Modulate at Least One Activity of HE4 Protein

Another embodiment of the present invention includes methods for identifying agents that modulate at least one activity of the HE4 protein. Such methods or assays may utilize any means of monitoring or detecting the desired protein(s).

In one format, the relative amounts of protein between a cell population exposed to an agent to be tested compared to an un-exposed control cell population may be assayed. In this format, probes such as specific antibodies are used to monitor the differential expression of the protein(s) in different cell populations. Cell lines or populations are exposed to the agent to be tested under appropriate conditions and time. Cellular lysates may be prepared from the exposed cell line or population and a control, unexposed cell line or population. The cellular lysates are then analyzed with the probe.

Antibodies directed against some HE4 polypeptides are known in the art (see Uhlenbruck et. al, 1993) and such antibodies may be used as probes. Further, antibodies against HE4 protein, for instance the protein of SEQ ID NO:2, may be prepared by immunizing suitable mammalian hosts using the peptides, polypeptides or proteins alone or conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier proteins are well known in the art. In some circumstances, direct conjugation using, for example, carbodiimide reagents may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation. Preferred peptides for preparing antibodies include sequences spanning amino acids 71, 73 or 101 of SEQ ID NO.:2 or 4.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, use of monoclonal preparations is preferred. Immortalized cell lines which secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications which effect immortalization of lymphocytes or spleen cells, as is generally known. The immortalized cell lines secreting the desired antibodies are screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera which contain the immunologically significant portion can be used as antagonists, as well as the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology, by recombinant means. Regions that bind specifically to the desired regions of the gene products can also be produced in the context of chimeras with multiple species origin.

Alternatively, the HE4 specific antibody can be humanized antibodies or human antibodies, as described in U. S. Patent No. 5,585,089 by Queen et al. See also Riechmann et al, Nature (1988) 332: 323-27.

Cell lines or populations are exposed under appropriate conditions to the agent to be tested. Agents which modulate the binding activity of the protein are identified by assaying the binding activity of the protein from the exposed cell line or population and a control, unexposed cell line or population, thereby identifying agents which modulate the binding activity of the protein. Binding assays to measure the ability of the agent to modulate the binding activity of proteins are widely available such as the assays disclosed by Morris et al, Oncogene (1991) 6(12):2339-48 and Cibelli et al, EurJBiochem (1996) 237(1):311-7.

Agents that are assayed in the above method can be randomly selected or rationally selected or designed. As used herein, an agent is said to be randomly selected when the agent is chosen randomly without considering the specific sequences involved in the association of the an HE4 protein alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism. As used herein, an agent is said to be rationally selected or designed when the agent is chosen on a nonrandom basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action. Another class of agents of the present invention are antibodies immunoreactive with critical positions of HE4 proteins. Antibody agents are obtained by immunization of suitable mammalian subjects with peptides, containing as antigenic regions, those portions of the protein intended to be targeted by the antibodies a sdescribed above.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be constmed as limiting in any way the remainder of the disclosure.

Uses for Agents that Modulate at Least One Activity of HE4.

As provided in the Examples, the proteins and nucleic acids of the invention, such as the protein having the amino acid sequence of SEQ ID NO: 2 are expressed in various tissues including differential expression in renal disease. Agents that modulate, up-or-down-regulate the expression of the protein or agents such as agonists or antagonists of at least one activity of the protein may be used to modulate biological and pathologic processes associated with the protein's function and activity. Further, because many progressive disease responses reflect an unhealthy physiologic and biochemical imbalance as opposed to a necessary state of homeostasis for health, either lowered or elevated HE4 production in general or lowered or elevated HE4 production outside a range that is homeostatically healthy could be deleterious. Therefore, modulating HE4 therapeutically up or down could provide a significant means of treating diseases in which HE4 plays a role.

The novel HE4 sequence is also expressed in salivary gland, tests, lung, uterus, trachea, prostate, thyroid, heart, pancreas, colon, and other tissues (see Figure 4). Therefor, it might play a significant role in disease processes associated with these tissues, particularly disease processes that involve inflammation and scarring. As used herein, a subject can be any mammal, so long as the mammal is in need of modulation of a pathological or biological process mediated by a protein of the invention. The term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects.

Pathological processes refer to a category of biological processes which produce a deleterious effect. For example, expression of a protein of the invention may be associated with kidney cell growth regeneration and/or recovery from kidney disease. As used herein, an agent is said to modulate a pathological process when the agent reduces the degree or severity of the process. For instance, kidney damage or ESRD may be prevented or disease progression modulated by the administration of agents which up-regulate or modulate in some way the expression or at least one activity of a protein of the invention.

The agents of the present invention can be provided alone, or in combination with other agents that modulate a particular pathological process. For example, an agent of the present invention can be administered in combination with other known drugs or may be combined with dialysis or anti-rejection drugs used during transplantation. As used herein, two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.

The agents of the present invention can be administered via parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

The present invention further provides compositions containing one or more agents which modulate expression or at least one activity of a protein of the invention. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages comprise 0.1 to 100 μg/kg body wt. The preferred dosages comprise 0.1 to 10 μg/kg body wt. The most preferred dosages comprise 0.1 to 1 μg/kg body wt.

In addition to the pharmacologically active agent, the compositions of the present invention may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts. In addition, suspensions of the active compounds as appropriate oily injection suspensions may be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers. Liposomes can also be used to encapsulate the agent for delivery into the cell. The pharmaceutical formulation for systemic administration according to the invention may be formulated for enteral, parenteral or topical administration. Indeed, all three types of formulations may be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, symps or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of this invention may be used alone or in combination, or in combination with other therapeutic or diagnostic agents. In certain preferred embodiments, the compounds of this invention may be coadministered along with other compounds typically prescribed for these conditions according to generally accepted medical practice. The compounds of this invention can be utilized in vivo, ordinarily in mammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, rats and mice, or in vitro.

Methods to Identify Binding Partners In order to identify proteases for which the HE4 polypeptides serves as substrates, another embodiment of the present invention provides methods for isolating and identifying binding partners of proteins of the invention. In general, a protein of the invention is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the protein of the invention. After mixing, peptides, polypeptides, proteins or other molecules that have become associated with a protein of the invention are separated from the mixture. The binding partner that bound to the protein of the invention can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein, for instance a protein comprising the entire amino acid sequence of SEQ ID NO: 2 can be used. Alternatively, a fragment of the protein can be used. Another embodiment includes protease inhibitor assays. For example, HE4 can be tested against a panel of proteases; e.g., ficin, papain, cathepsin H, cathepsin B (see Minakata et al, Biol Chem Hoppe Seyler (1985) 366(1):15-18 or elastase (Moriyama et al, Mol Hum Reprod (1998) 4(10):946-950). Briefly, the protease is incubated with its protease substrate in the presence and absence of HE4 protein or polypeptide. For example, when ficin is used, Nα-benzoyl-DL arginine-2-napthylamide (substrate) is incubated under appropriate conditions in the presence and absence of HE4. In this embodiment, relative inhibition is determined by absorbance at 530 nM (Minakata et al, 1985).

Identified proteinases will be screened to determine agents which modulate their enzymatic activity, including modulators of the expression of said proteinases. As used herein, a cellular extract refers to a preparation or fraction which is made from a lysed or dismpted cell. The preferred source of cellular extracts will be cells derived from human kidney tissue, for instance, renal biopsy tissue or tissue culture cells. Alternatively, cellular extracts may be prepared from normal human kidney tissue or available cell lines, particularly kidney derived cell lines.

A variety of methods can be used to obtain an extract of a cell. Cells can be disrupted using either physical or chemical dismption methods. Examples of physical dismption methods include, but are not limited to, sonication and mechanical shearing. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzyme lysis. A skilled artisan can readily adapt methods for preparing cellular extracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with the protein of the invention under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, the most preferred being conditions that closely resemble conditions found in the cytoplasm of a human cell. Features such as osmolarity, pH, temperature, and the concentration of cellular extract used, can be varied to optimize the association of the protein with the binding partner. After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be utilized to separate the mixture. For example, antibodies specific to a protein of the invention can be used to immunoprecipitate the binding partner complex. Alternatively, standard chemical separation techniques such as chromatography and density/sediment centrifugation can be used. After removal of non-associated cellular constituents found in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be accomplished by altering the salt concentration or pH of the mixture. To aid in separating associated binding partner pairs from the mixed extract, the protein of the invention can be immobilized on a solid support. For example, the protein can be attached to a nitrocellulose matrix or acrylic beads. Attachment of the protein to a solid support aids in separating peptide/binding partner pairs from other constituents found in the extract. The identified binding partners can be either a single protein or a complex made up of two or more proteins. Alternatively, binding partners may be identified using a Far- Western assay according to the procedures of Takayama et al, Methods Mol Biol (1997) 69:171-84 or Sauder et al, J Gen Virol (1996) 77(5):991-6 or identified through the use of epitope tagged proteins or GST fusion proteins.

Alternatively, the nucleic acid molecules of the invention can be used in a yeast two- hybrid system. The yeast two-hybrid system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules herein described.

Transgenic Animals

Transgenic animals containing mutant, knock-out or modified genes corresponding to HE4 or the cDNA sequence of SEQ ID NO:l are also included in the invention. Transgenic animals are genetically modified animals into which recombinant, exogenous or cloned genetic material has been experimentally transferred. Such genetic material is often referred to as a "transgene". The nucleic acid sequence of the transgene, in this case a form of SEQ ID NO: 1 , may be integrated either at a locus of a genome where that particular nucleic acid sequence is not otherwise normally found or at the normal locus for the transgene. The transgene may consist of nucleic acid sequences derived from the genome of the same species or of a different species than the species of the target animal.

The term "germ cell line transgenic animal" refers to a transgenic animal in which the genetic alteration or genetic information was introduced into a germ line cell, thereby conferring the ability of the transgenic animal to transfer the genetic information to offspring. If such offspring in fact possess some or all of that alteration or genetic information, then they too are transgenic animals.

The alteration or genetic information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or may be genetic information already possessed by the recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene. Transgenic animals can be produced by a variety of different methods including transfection, electroporation, microinjection, gene targeting in embryonic stem cells and recombinant viral and retroviral infection (see, e.g., U.S. Patent No. 4,736,866; U.S. Patent No. 5,602,307; Mullins et al, Hypertension (1993) 22(4): 630-633; Brenin et al, Surg Oncol (1997) 6(2)99-110; Tuan (ed.), Recombinant Gene Expression Protocols, Methods in Molecular Biology, 1997, No. 62, Humana Press).

A number of recombinant or transgenic mice have been produced, including those which express an activated oncogene sequence (U.S. Patent No. 4,736,866); express simian SV 40 T-antigen (U.S. Patent No. 5,728,915); lack the expression of interferon regulatory factor 1 (IRF-1) (U.S. Patent No. 5,731,490); exhibit dopaminergic dysfunction (U.S. Patent No. 5,723,719); express at least one human gene which participates in blood pressure control (U.S. Patent No. 5,731,489); display greater similarity to the conditions existing in naturally occurring Alzheimer's disease (U.S. Patent No. 5,720,936); have a reduced capacity to mediate cellular adhesion (U.S. Patent No. 5,602,307); possess a bovine growth hormone gene (Clutter et al, Genetics (1996) 143(4):1753-1760); or, are capable of generating a fully human antibody response (McCarthy The Lancet (1997) 349(9049):405).

While mice and rats remain the animals of choice for most transgenic experimentation, in some instances it is preferable or even necessary to use alternative animal species. Transgenic procedures have been successfully utilized in a variety of non-murine animals, including sheep, goats, pigs, dogs, cats, monkeys, chimpanzees, hamsters, rabbits, cows and guinea pigs (see, e.g., Kim et al, Mol ReprodDev (1997) 46(4):515-526; Houdebine Reprod Nutr Dev (1995) 35(6):609-617; Petters Reprod Fertil Dev (1994) 6(5):643-645; Schnieke et al, Science (1997) 278(5346):2130-2133; and Amoah J.4n.ma/ Science (1997) 75(2):578-585).

The method of introduction of nucleic acid fragments into recombination competent mammalian cells can be by any method which favors co-transformation of multiple nucleic acid molecules. Detailed procedures for producing transgenic animals are readily available to one skilled in the art, including the disclosures in U.S. Patent No. 5,489,743 and U.S. Patent No. 5,602,307.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

EXAMPLES

Example 1

Identification of Differentially Expressed IgAN mRNA Biopsy kidney tissue was obtained from subjects exhibiting IgA nephropathy and their age- and sex-matched controls by standard procedures (Yang et al, 1997).

Total cellular RNA was prepared from the kidney tissue described above as well as from control, non-IgAN kidney tissue using the procedure of Yang et al, 1997

Synthesis of cDNA was performed as previously described by Prashar et al. in WO 97/05286 and in Prashar et al, Proc NatlAcad Sci USA (1996) 93:659-663. Briefly, cDNA was synthesized according to the protocol described in the GIBCO/BRL kit for cDNA synthesis. The reaction mixture for first-strand synthesis included 6 μg of total RNA, and 200 ng of a mixture of anchored oligo(dT) primers degenerate at nl

(ACGTAATACGACTCACTATAGGGCGAATTGGGTCGACTTTTTTTTTTTTTTTTTnl wherein nl=A C or G) (SEQ ID NO: 5) along with other components for first-strand synthesis reaction except reverse transcriptase. This mixture was incubated at 65 °C for 5 mins, chilled on ice and the process repeated. Alternatively, the reaction mixture may include lOμg of total RNA, and 2 pmol of 1 of the 2 base-anchored oligo(dT) primers with a heel such as RP5.0 (CTCTCAAGGATCTTACCGCT(T)₁₈AT) (SEQ ID NO: 6), or

RP6.0 (TAATACCGCGCCACATAGCA(T)₁₈CG) (SEQ ID NO: 7), or RP9.2 (CAGGGTAGACGACGCTACGC(T)₁₈GA) (SEQ ID NO: 8) along with other components for first-strand synthesis reaction except reverse transcriptase. This mixture was then layered with mineral oil and incubated at 65 °C for 7 min followed by 50 °C for another 7 min. At this stage, 2 l of SUPERSCRIPT REVERSE TRANSCRIPTASE® (200 units/μl; GIBCO/BRL) was added quickly and mixed, and the reaction continued for 1 hr at 45-50°C. Second-strand synthesis was performed at 16°C for 2 hr. At the end of the reaction, the cDNAs were precipitated with ethanol and the yield of cDNA was calculated. In our experiments, =200 ng of cDNA was obtained from lOμg of total RNA. The adapter oligonucleotide sequences were

Al (TAGCGTCCGGCGCAGCGACGGCCAG) (SEQ ID NO: 9) and A2 (GATCCTGGCCGTCGGCTGTCTGTCGGCGC) (SEQ ID NO: 10). One microgram of oligonucleotide A2 was first phosphorylated at the 5 ' end using T4 polynucleotide kinase (PNK). After phosphorylation, PNK was heat denatured, and lμg of the oligonucleotide Al was added along with 10^χ annealing buffer (1 M NaCl/100 mM

Tris-HCl, pH8.0/10 mM EDTA, pH8.0) in a final vol of 20 μl. This mixture was then heated at 65 °C for 10 min followed by slow cooling to room temperature for 30 min, resulting in formation of the Y adapter at a final concentration of 100 ng/μl. About 20 ng of the cDNA was digested with 4 units oϊBgl II in a final vol of 10 μl for 30 min at 37°C. Two microliters (=4 ng of digested cDNA) of this reaction mixture was then used for ligation to 100 ng (= 50- fold) of the Y-shaped adapter in a final vol of 5μl for 16 hr at 15°C. After ligation, the reaction mixture was diluted with water to a final vol of 80 μl (adapter ligated cDNA concentration, =50 pg/μl) and heated at 65 °C for 10 min to denature T4 DNA ligase, and 2-μl aliquots (with = 100 pg of cDNA) were used for PCR. The following sets of primers were used for PCR amplification of the adapter ligated

3' -end cDNAs:

TGAAGCCGAGACGTCGGTCG(T)_lg nl, n2 (wherein nl, n2 = AA, AC, AG AT CA CC CG CT GA GC GG and GT) (SEQ ID NO: 11) as the 3' primer with Al as the 5' primer or alternatively RP 5.0, RP 6.0, or RP 9.2 used as 3' primers with primer Al.l serving as the 5' primer. To detect the PCR products on the display gel, 24 pmol of oligonucleotide Al or Al.l was 5' -end-labeled using 15 μl of [γ-³² P]ATP (Amersham; 3000 Ci/mmol) and PNK in a final volume of 20 μl for 30 min at 37°C. After heat denaturing PNK at 65 °C for 20 min, the labeled oligonucleotide was diluted to a final concentration of 2 μM in 80 μl with unlabeled oligonucleotide Al.l. The PCR mixture (20μl) consisted of 2 μl ( = 100 pg) of the template, 2μl of 10x PCR buffer (100 mM Tris-HCl, pH 8.3/500 mM KC1), 2 μl of 15 mM MgCl₂ to yield 1.5 mM final Mg²⁺ concentration optimum in the reaction mixture, 200 μM dNTPs, 200 nM each 5' and 3' PCR primers, and 1 unit of Amplitaq Gold®. Primers and dNTPs were added after preheating the reaction mixture containing the rest of the components at 85 °C. This "hot start" PCR was done to avoid artefactual amplification arising out of arbitrary annealing of PCR primers at lower temperatures during transition from room temperature to 94°C in the first PCR cycle. PCR consisted of 5 cycles of 94°C for 30 sec, 55°C for 2 min, and 72°C for 60 sec followed by 25 cycles of 94°C for 30 sec, 60°C for 2 min, and 72° C for 60 sec. A higher number of cycles resulted in smeary gel patterns. PCR products (2.5μl) were analyzed on 6% polyacrylamide sequencing gel. For double or multiple digestion following adapter ligation, 13.2 μl of the ligated cDNA sample was digested with a secondary restriction enzyme(s) in a final vol of 20 μl. From this solution, 3μl was used as a template for PCR. This template vol of 3 μl carried = 100 pg of the cDNA and 10 mM MgCl₂ (from the 10x enzyme buffer), which diluted to the optimum of 1.5 mM in the final PCR vol of 20 μl. Since Mg²⁺ comes from the restriction enzyme buffer, it was not included in the reaction mixture when amplifying secondarily cut cDNA. Individual cDNA fragments corresponding to mRNA species were separated by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. Bands were extracted from the display gels as described by Liang et al. (1995 Curr. Opin. Immunol. 7:274-280), reamplified using the 5 ' and 3 ' primers, and subcloned into pCR-Script with high efficiency using the PCR- Script cloning kit from Stratagene. Plasmids were sequenced by cycle sequencing on an ABI automated sequencer. Alternatively, bands were extracted (cored) from the display gels, PCR amplified and sequenced directly without subcloning (see Figure 5). SEQ. ID NO:l is the full-length cDNA sequence which corresponds to the differentially expressed band of Figure 5. SEQ ID NO:2 is the amino acid sequence of the encoded HE4 protein.

Example 2 Northern blot and PCR Expression Analysis Northern blots are prepared using a probe derived from SEQ ID NO:l with hybridization conditions as described by Sambrook et al. (1989), see Figure 1.

Quantitative PCR Analysis of Expression Levels Biopsy tissue is obtained from normal and diseased kidneys, wherein extracted tissues are lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. Real time PCR detection is accomplished by the use of the ABI PRISM 7700 Sequence Detection System. The 7700 measures the fluorescence intensity of the sample each cycle and is able to detect the presence of specific amplicons within the PCR reaction. Each sample is assayed for the level of GAPDH and clone JT22962 (HE4). GAPDH detection is performed using Perkin Elmer part#402869 according to the manufacturer's directions. Primers are designed for clone JT22962 (HE4) using Primer Express, a program developed by PE to efficiently find primers and probes for specific sequences. These primers are used in conjunction with SYBR green (Molecular Probes), a nonspecific double stranded DNA dye, to measure the expression level of clone JT22962 (HE4), which is normalized to the GAPDH level in each sample (see Figure 2 and Figure 3).

Tissue Distribution Tissues are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. PCR is carried out as described above. Quantative expression data was not normalized to GAPDH in each sample (see Figure 3).

Example 3 Method of Screening for Modulators of Kidney HE4 Expression Using IL-1 and IL-6 Using human cultured mesangial cells, IL-1 and 11-6 are administered to cells in culture according to the method of Lin et al. (1999). At specific time points pre- or at post- administration, candidate agents and diluent (i.e., carrier minus agent; control) are contacted with the human mesangial cells. Cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling means to determine whether the candidate agent modulates the expression of HE4. Agents which up- or down-regulate the expression of HE4 are then designated as modulators of the gene.

Example 4

Method of Screening for Modulators of Kidney HE4 Expression Using Ethanol Ethanol is administered to cultured contractile mesangial cells in culture according to the method of Smith et al (1993). At specific time points pre or at post administration, candidate agents and diluent (i.e., carrier minus agent; control) are contacted with the cultured cells. Control and test cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down- regulate the expression of HE4 will then be designated as modulators of the gene.

Example 5

Method of Screening for Modulators of Kidney HE4 Expression Using Gliadin Gliadin is administered to cultured contractile mesangial cells in culture according to the method of Amore et al (1994). At specific time points pre or at post administration, candidate agents and diluent (i.e., carrier minus agent; control) are contacted with the cultured cells. Control and test cells are removed and lysed in an appropriate buffer for isolation of total and/or messenger RNA in a similar fashion as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down- regulate the expression of HE4 will then be designated as modulators of the gene.

Example 6 Method of Screening for Modulators of Kidney HE4 Expression Using an Animal

Model: Oral Immunization Oral immunization animal models for IgAN are well documented (Amore et al, 1994; Endo et al, 1993; Yan et al, 1998; and Tractman et al, 1996). For example, IgAN is induced in animals by oral administration of LPS (Endo et al, 1993), bovine gamma globulin (Trachtman et al, 1996), or vomitoxin (Yan et al, 1998). For rats, gliadin maybe used (Amore et al, 1994). Before or after antigen administration, agents are administered to the animals (including carrier-only for controls). At various time points and/or after administration of various concentrations of candidate agents using a single time point, the kidneys of the animals are removed for isolation of nucleic acids by standard methods as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down-regulate HE4 will then be designated as modulators of the gene.

Example 7

Use of ddY Mice A spontaneous model for IgAN exists in mice (e.g., ddY mice, see, Nakamura et al, 1992). Agents are administered to ddY mice at various time points or at various concentrations at a single time point. The kidneys of the animals are then removed for isolation of nucleic acids by standard methods as described in Sambrook et al. (1989). Isolated nucleic acids are then assayed by a transcriptional profiling assay to determine whether the candidate agent modulates the induction of HE4. Agents which up- or down- regulate HE4 will then be designated as modulators of the gene.

Although the present invention has been described in detail with reference to examples above, it is understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims. All cited patents and publications referred to in this application are herein incorporated by reference in their entirety.

Claims

We claim:

1. An isolated nucleic acid molecule selected from the group consisting of: (a) an isolated nucleic acid molecule that encodes the amino acid sequence of SEQ ID NO:2; (b) an isolated nucleic acid molecule comprising SEQ ID NO:l; and (c) an isolated nucleic acid molecule which encodes an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4.

2. The isolated nucleic acid molecule of claim 1 consisting of the sequence of SEQ ID NOT.

3. The isolated nucleic acid molecule of claim 2 comprising nucleotides 23-394 of SEQ ID NO: 1.

4. The isolated nucleic acid molecule of claim 1 comprising nucleotides 23 to 397 of SEQ ID NO: 1.

5. The isolated nucleic acid molecule of claim 1 wherein position 71 is a serine position 73 has been deleted and position 101 is a serine.

6. The isolated nucleic acid molecule of any one of claims 1-5, wherein said nucleic acid molecule is operably linked to one or more expression control elements.

7. A vector comprising an isolated nucleic acid molecule of any one of claims 1 -5.

8. A host cell transformed to contain the nucleic acid molecule of any one claims 1- 5.

9. A host cell comprising a vector of claim 7.

10. A host cell of claim 9, wherein said host is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

11. A method for producing a polypeptide comprising the step of culturing a host cell transformed with the nucleic acid molecule of any one of claims 1-5 under conditions in which the protein encoded by said nucleic acid molecule is expressed.

12. The method of claim 11, wherein said host cell is selected from the group consisting of prokaryotic hosts and eukaryotic hosts.

13. An isolated polypeptide produced by the method of claim 11.

14. An isolated polypeptide selected from the group consisting of an isolated polypeptide comprising the amino acid sequence of SEQ ID NO:2 and an HE4 protein containing a substitution or deletion of at least one amino acid corresponding to residue 71, 73 or 102 of sequence of SEQ ID NOs: 3 and 4.

15. An isolated antibody that specifically binds to a polypeptide of either claim 13 or 14.

16. The antibody of claim 14 wherein said antibody is a monoclonal or polyclonal antibody.

17. A method of diagnosing inflammation or renal disease in a patient comprising assaying for the expression of HE4 protein or nucleic acid in a sample, wherein the presence of HE4 is an indication of inflamation or renal disease.

18. The method of claim 17, wherein the sample is renal biopsy material obtained during dialysis.

19. The method claim 17, wherein the sample is renal biopsy material obtained during transplant typing.

20. The method of claim 17, wherein the assay comprises a transcriptional profiling (TP) assay.

21. The method of claim 20, wherein said TP assay is selected from the group consisting of RNAse protection assays, RT-PCR, Northern blot, READS and TAQMAN.

22. A method of screening for agents which modulate HE4 comprising the steps: a) incubating cells under conditions which simulate renal disease in the presence of an agent; b) determining whether HE4 is up- or down-regulated in the presence or absence of the agent, wherein up- or down-regulation of HE4 identifies the agent as a modulator.

23. The method of claim 22, wherein said renal disease is selected from the group consisting of IgAn, NCGN, minimal change disease and SIRS.

24. The method of claim 23, wherein said renal disease is IgAN.

25. The method of claim 24, wherein the incubation conditions comprise treating mesangial cells with IL-1 and IL-6.

26. The method of claim 24, wherein the incubation conditions comprise treating contractile mesangial cells with ethanol.

27. The method of claim 24, wherein the incubation conditions comprise treating mesangial cells with gliadin.

28. A method of screening for agents which modulate HE4 comprising the steps: a) administering an agent to a renal disease in vivo model system; and b) determining whether HE4 is modulated in the presence of the agent, wherein up or down regulation of HE4 identifies the agent as a modulator.

29. The method of claim 28, wherein said renal disease is selected from the group consisting of: IgAN, NCGN, minimal change disease and SIRS.

30. The method of claim 29, wherein said renal disease is IgAN.

31. The method of claim 29, wherein said in vivo model comprises intragastrically infusing ethanol in an animal.

32. The method of claim 29, wherein said in vivo model comprises orally immunizing an animal.

33. The method of claim 29, wherein said in vivo model comprises orally administering vomitoxin to an animal.

34. The method of claim 29, wherein said in vivo model comprises administering gram negative bacteria or lipopolysaccharide (LPS) to an animal.

35. The method of claim 29, wherein said in vivo model comprises administering an agent to ddY mice.

36. The method of claim 29, wherein said oral immunizing comprises administration of an antigen selected from the group consisting of gliadin and bovine gamma globulin.

37. A method of treatment of inflammation or a renal disease comprising administering to a patient in need thereof a therapeutically effective amount of an agent which modulates HE4.

38. The method of claim 37, wherein said renal disease is selected form the group consisting of: IgAN, NCGN, minimal change disease and SIRS.

39. The method of claim 38, wherein said renal disease is IgAN.

40. The method of claim 39, wherein the agent modulates mesangial cell proliferation, hypertension, glomemlosclerosis, hematuria and/or proteinuria.

41. An agent for the treatment of renal disease identified by the method of any one of claims 22-40.

42. The agent of claim 41, wherein said renal disease is selected from the group consisting of: IgAN, NCGN, minimal change disease and SIRS.

43. The agent of claim 42, wherein said renal disease is IgAN.

44. A pharmaceutical composition comprising at least one agent of claim 41 and a pharmaceutically acceptable carrier.

45. The pharmaceutical composition of claim 44 further comprising a second agent which modulates IgA production.

46. The pharmaceutical composition of claim 45, wherein said second agent is selected from the group consisting of cytokines, interleukins, and antibodies.

47. An method of treatment of IgA nephropathy comprising administering to a patient in need thereof a therapeutically effective amount of a composition which modulates HE4 and IgA production.

48. A non-human transgenic animal comprising a nucleic acid molecule of any of claims 1-5.

49. A non-human transgenic animal which does not express a nucleic acid molecule of any of claims 1-5.