WO2000008134A2 - HUMAN HtrA SERINE PROTEASE - Google Patents

Abstract

The subject invention concerns novel materials and methods for the detection of human osteoarthritis and cancer. Specifically, a human HtrA gene and gene product are disclosed which are differentially expressed when comparing osteoarthritic tissue to non-osteoarthritic tissue. A further aspect of the invention concerns compounds which antagonize the biological activity of HtrA protein and methods for identifying these compounds. Another aspect of the present invention concerns pharmaceutical compositions comprising such compounds.

Description

Human HtrA Serine Protease

BACKGROUND

Osteoarthritis (OA), the most prevalent form of degenerative joint disease, involves chondrocyte loss and breakdown of extracellular matrix components, leading to degeneration of articular cartilage and eventual deterioration of joint function (1 ). OA is characterized by degeneration and loss of articular cartilage and alterations of subchondral bone. Although it is the most common of the rheumatic diseases, its pathogenesis is not well understood (6). The disorder may be secondary to other diseases that cause joint deformity or to repeated joint trauma, but in many patients, no such associated factor is present. The incidence of osteoarthritis increases with age, but the disease is not caused solely by aging of articular tissues. The pathology differs from that of the inflammatory rheumatic diseases, such as rheumatoid arthritis, in that osteoarthritis is associated with only minor degrees of inflammation.

Epidemiologic evidence provides support for several different theories of disease causation, including the separate contribution of metabolic and mechanical factors and the importance of heredity and a generalized predisposition to disease. Some factors have an important impact on disease in one joint but not in others. Therefore, risk factors for OA in all joints are probably not the same, even though the biology of the disease may be the same. For example, joint injury such as a ligament tear may be a prominent cause of OA in the knee, which is especially susceptible to injury, but may not be a significant factor in causing OA in the hip, in which ligamentous injuries are unusual.

From a therapeutic perspective, it is important to understand the molecular events triggering the onset of OA and the biochemical pathways responsible for the disease progression which appear to be influenced by a complexity of environmental and genetic factors (2-4). Chondrocytes, the exclusive cell-type in cartilage, maintain the integrity of the collagen/proteoglycan network by responding to a variety of stresses, including normal mechanical load as well as abnormal trauma and injury (5). The cellular response to stress stimuli occurs through the regulation of a myriad of signal transduction pathways leading to alterations in gene expression.

In bacteria, HtrA is a critical component of the universal cellular response to stress, characterized by the induction of a set of so-called "heat shock" proteins (19). In addition to temperature elevation, heat shock proteins are induced by oxidative stress (20), viral (phage) infection (21), and intracellular expression of aberrant proteins (22). A functional htrA (high temperature requirement) gene is indispensable for the bacterial cell to survive heat shock (11 ). HtrA is identical to DegP (23), a serine endoprotease originally named "Do" as one of several proteolytic activities purified from E. coli (24). Mutation of the active site serine- 236 to alanine in HtrA results in loss of protease activity in vitro and loss of function, as determined by the inability to suppress the thermosensitivity of htrA null mutants (11). Lipinska et al. (11) suggested that the role of HtrA is proteolytic cleavage of toxic denatured proteins in the periplasmic space. Additional experimental evidence supporting this hypothesis has been recently reported by other investigators (25-26).

Due to the extreme debilitating effects of osteoarthritis, there is a need for a process to diagnose the onset and progression of osteoarthritis in order to assess appropriate therapeutic measures and their effectiveness.

There is also a need to detect and measure the differential expression of genes and gene products which are altered in this disease state, such that this differential expression can be determined diagnostically to predict the onset of the disease state.

A further need exists for identifying additional factor(s) and others which interact with and regulate the biological function of the gene products which show differential expression in OA, so that they may be administered to patients in need of such treatment.

There also exists a need for pharmaceuticals comprising the factor(s) which interact with the differentially expressed gene product such that they may be administered to a patient in need thereof for the treatment and/or prevention of diseases in which this gene product is differentially expressed. The nucleotide sequence of ORF480 is identical to a recently described "transformation-sensitive" cDNA expressed in human fibroblasts (8). ORF480 codes for a protein with distinct domains of homology to human mac25 (9) and to the bacterial serine protease (HtrA) that is critical for the cellular response to thermal and oxidative stress (10-11). Accordingly, the gene and gene product of ORF480 is referred to herein as "HtrA", "HtrA cDNA", "HtrA mRNA", "human HtrA", "human HtrA homologue", "HtrA protein" and "HtrA polypeptide".

SUMMARY

Toward these ends, and others, an aspect of the present invention encompasses assay techniques for detecting arthritic conditions by measuring human HtrA expression levels in bodily samples, preferably body tissue and fluid samples. A preferred embodiment of the assay aspect of the invention provides assays for measuring human HtrA mRNA expression in body tissue samples derived from a patient.

Another embodiment of the assay aspect of the invention provides assays for measuring human HtrA polypeptide levels comprising incubating a body tissue or fluid sample, which has been obtained from a patient, with an anti-human HtrA antibody and measuring the level of bound anti-human HtrA antibody in the body tissue or fluid sample.

In accordance with yet another aspect of the present invention, there are provided human HtrA antagonists (inhibitors) and methods for identifying such antagonists, wherein such antagonists reduce or prevent the effect of human HtrA polypeptide. Among preferred antagonists are those which mimic human HtrA so as to bind to human HtrA receptor or binding molecules but do not elicit a human HtrA- induced response or more than one human HtrA-induced response. In another embodiment of this aspect of the present invention there are provided antagonists which are small molecules and antibodies and the like which bind to human HtrA polypeptide and regulate its biological activity. Also among preferred antagonists are molecules that bind to or interact with human HtrA so as to inhibit an effect of human HtrA or more than one effect of human HtrA or which prevent expression of human HtrA. In accordance with this aspect of the invention there are provided assays for detecting antagonists to human HtrA which regulate human HtrA expression and/or activity. In accordance with one embodiment of this aspect of the invention there is provided anti-sense polynucleotides which regulate transcription of the human HtrA gene.

It also is an object of the invention to provide human HtrA polypeptides, particularly human HtrA polypeptides, that are differentially expressed in arthritic conditions and therefore, when detected via assay, allows a diagnosis of arthritic conditions. In a preferred embodiment the polypeptide comprises the sequence shown in Fig. 1 (SEQ ID NO:2). In accordance with this aspect of the invention there are provided novel polypeptides of human origin as well as biologically, diagnostically or therapeutically useful fragments, variants and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing.

It is another object of the invention to provide a process for producing the aforementioned polypeptides, polypeptide fragments, variants and derivatives, fragments of the variants and derivatives, and analogs of the foregoing. In a preferred embodiment of this aspect of the invention there are provided methods for producing the aforementioned human HtrA polypeptides comprising culturing host cells having expressibly incorporated therein a vector containing an exogenously- derived human HtrA-encoding polynucleotide under conditions for expression of human HtrA polypeptides in the host and then recovering the expressed polypeptide.

In accordance with another object the invention there are provided products, compositions, processes and methods that utilize the aforementioned polypeptides and polynucleotides for research, biological, clinical and therapeutic purposes, inter alia.

In certain additional preferred embodiments of this aspect of the invention there are provided antibodies against human HtrA polypeptides and methods for their production. In certain particularly preferred embodiments in this regard, the antibodies are highly selective for human HtrA polypeptides or portions of human HtrA polypeptides.

In still another embodiment of the present invention there are provided methods of treating conditions resulting from expression of human HtrA comprising administering antagonists to human HtrA in pharmaceutically acceptable amounts to treat conditions resulting from the activity of human HtrA.

In yet another aspect of the present invention there are provided kits comprising the components necessary for detecting an above-normal expression of human HtrA polynucleotides or polypeptides in body tissue samples derived from a patient.

Other objects, features, advantages and aspects of the present invention will become apparent to those of skill from the following description. It should be understood, however, that the following description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following description and from reading the other parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. They are illustrative only and do not limit the invention otherwise disclosed herein.

Figure 1. Identification of differential display clones 49A50 and 58A5. Magnified phosphor screen images of two separate differential display gels show the PCR products, indicated by the arrows, corresponding to 49A50 (left panel) and 58A5 (right panel) amplified by RT-PCR from mRNA isolated from osteoarthritic cartilage (OA, individual patients indicated by numbers) or non- arthritic cartilage (NA, individual samples indicated by letters).

Figure 2. Schematic illustration of ORF480 cDNA clones and protein coding domains. Genbank entry D87258, illustrated as a shaded bar, is a 2036 bp cDNA from human osteoblasts (SEQ ID NO:1) containing a 480 codon open reading frame (lighter shaded area) encoded by nucleotide 49 to nucleotide 1491 of SEQ ID NO:1. Overlapping differential display PCR products (49A50 and 58A5), EST sequences (accession numbers W47107 and W67176), and cDNA clones from human cartilage (C05,C13) and normal dermal fibroblasts (pcDNA3/ORF480) relative to D87258 are depicted as horizontal lines drawn to scale. The mac25 and HtrA homology domains within ORF480 are indicated as open rectangles, preceded by the secretory signal sequence (S). The Kazal-type inhibitor motif (Kl) within the mac25 domain and the PDZ-related sequence within the HtrA domain are depicted as open triangles.

Figure 3. ORF480 contains a Kazal-type inhibitor motif within the mac25 homology domain. A) Amino acid sequence alignment of ORF480 (amino acids 12-90 and 107 to 158of SEQ ID NO:2) and mac25 (SEQ ID NO:3) is shown with identical ( I ) and similar ( : ) residues indicated. The Kazal-type inhibitor motif is outlined by a rectangle. B) Multiple sequence alignment of the conserved Kazal- type inhibitor motif within ORF480 (amino acids 117-140 of SEQ ID NO:2) (shaded), human (amino acid 87 to 110 of SEQ ID NO:3) and murine mac25 (SEQ ID NO:4) (9,16), follistatin (SEQ ID NO:5) (31 ), agrin (SEQ ID NO:6) (32), and several known serine protease inhibitors: elastase inhibitor from Anemonia sulcata (SEQ ID NO:7) (33), rhodiin (SEQ ID NO:8) (34), a representative sequence of the ovomucoid third domain (SEQ ID NO:9) (35), tryptase inhibitor from Hirudo medicinalis (SEQ ID NO:10) (36), protease inhibitor from crayfish (SEQ ID NO:11 ) (37), and human trypsin inhibitor C (SEQ ID NO:12) (38).

Figure 4. Elevated levels of ORF480-encoded HtrA mRNA and protein in human OA cartilage. A) Comparison of mRNA expression in osteoarthritic (OA) versus non-arthritic (NA) cartilage by semi-quantitative RT-PCR. A composite image is shown from a representative experiment; PCR products were resolved by polyacrylamide gel electrophoresis followed by staining with SYBR™-Green (Molecular Probes). Band intensities were quantified using a Phosphorlmager™ and ImageQuant software (Molecular Dynamics). ^* The mean ratio of expression, normalized to actin, in OA cartilage to that in NA cartilage from multiple (n) experiments using at least 3 samples from each is indicated. COL3A and COL2A are abbreviations for pro-alpha1 (III) and pro-alpha1 (II) collagens, respectively. B) Detection of ORF480-encoded HtrA protein in high-salt extracts of human OA and NA cartilage by immunoblot analysis (see Experiment 5). The anti-HtrA antiserum exhibits low cross-reactivity with serum albumin. As a positive control (+), a supernatant sample from cultured 293 cells transfected with pcDNA3/ORF480 was included in the immunoblot analysis. Figure 5. Expression of ORF480 cDNA and functional analysis of its encoded protein, HtrA. A) In vitro transcription/translation of ORF480 and ORF480-Ser328Ala cDNAs yields the expected -50 kDa translation product (indicated by the arrow), resolved by SDS-polyacrylamide gel electrophoresis. Note the presence of three lower molecular weight protein bands in lanes 1 and 2 (duplicate samples of ORF480) which are absent in lanes 3 and 4 (duplicate samples of the Ser328Ala mutant). B) Culture supernatants from 293 cells transfected with pcDNA3/ORF480 were analyzed by immunoblot analysis (upper panel) and proteolytic activity against β-casein (lower panel). Samples 1 and 2 are negative controls; samples 3-7 are from independent clones expressing decreasing levels of HtrA protein. Specific HtrA-generated cleavage products of β -casein are indicated by the arrows. C) Human HtrA protein produced in baculovirus-infected Sf9 cells exhibits autoproteolytic activity (upper panel, immunoblot analysis) with enhanced degradative activity against β-casein (lower panel) upon prolonged culture incubation. Post-infection incubation times in hrs for each sample are indicated. A group of immunoreactive degradation products are framed by the bracket. Specific HtrA-generated cleavage products of casein are indicated by the arrows. D) The high-molecular weight protein band (marked with * in panels B, C, and D), detected by immunoblot analysis with anti-HtrA antibody, is a stable complex of HtrA and α-1 -antitrypsin (hereinafter "AAT"). Transfected 293 cells expressing ORF480-encoded HtrA (or the Ser328Ala mutant) cultured in the presence of fetal bovine serum (FBS), serum-free medium (SFM), or SFM with purified AAT added. The culture incubation times (hr) after the addition of fresh media are indicated. Molecular weight standards (BioRad) are shown (kDa).

Figure 6. Mammalian HtrA is highly conserved. A) Human ORF480 cDNA hybridizes to genomic DNA of several species. A ZOO BLOT (Clontech), containing of EcoR l-digested genomic (4 μg DNA per lane) from different vertebrate species (indicated above each lane), was hybridized with the HtrA- related segment of ORF480 as described in Example 8. B) Amino acid sequence alignment of human HtrA with selected mammalian homologues, as well as E. coli HtrA (SEQ ID NO:28). The translated amino acid sequence of ORF480 (single- letter code) is shown with residue numbers indicated to the right. Identical amino acid sequences between ORF480 and human mac25 (see Fig. 3) are indicated ( ^* ) above the ORF480 residues. The 16 cysteine residues in the amino-terminal domain of ORF480 are underlined. The conserved serine protease active site sequence (39) is outlined by a rectangle, with the serine residue of the active site shaded. Identical residues between human, bovine (SEQ ID NO:29), rabbit (SEQ ID NO:30), and guinea pig (SEQ ID NO:31) HtrA homologues are denoted with a hyphen, and the actual amino acid substitutions in each species are shown.

DEFINITIONS

The following illustrative explanations are provided to facilitate understanding of certain terms used frequently herein, particularly in the examples. The explanations are provided as a convenience and are not limitative of the invention.

"Identity" as the term is used herein, refers to a polynucleotide or polypeptide sequence which comprises a percentage of the same bases as a reference polynucleotide or polypeptide (SEQ ID NO:1 or SEQ ID NO:2). For example, a polynucleotide or polypeptide which is at least 90% identical to a reference polynucleotide or polypeptide, has polynucleotide bases or amino acid residues which are identical in 90% of the bases or residues which make up the reference polynucleotide or polypeptide and may have different bases or residues in 10% of the bases or residues which comprise that polynucleotide or polypeptide sequence. Exemplary algorithms for determining "identity" are the BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information) and FAST program. "Identity" may be determined by procedures which are well-known in the art.

"Plasmids" generally are designated herein by a lower case p preceded and/or followed by capital letters and/or numbers, in accordance with standard naming conventions that are familiar to those of skill in the art. Starting plasmids disclosed herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention. The properties, construction and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill from the present disclosure.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The term "polypeptide" is used interchangeably herein with the terms "compound(s)", "polypeptides" and "protein(s)".

DESCRIPTION

Human HtrA Proteins

The present invention relates to human HtrA polypeptides, among other things, as described in greater detail below. Human HtrA exhibits autocatalytic cleavage and endoproteolytic activity against an exogenous substrate, for example, β-casein. HtrA protein, when incubated in the presence of serum, binds to and forms a stable complex with AAT. In addition, the sequence of the HtrA- related domain of ORF480 is highly conserved among mammalian species as shown in Fig. 6.

Levels of human HrtA mRNA and protein are significantly elevated in osteoarthritic cartilage. Human HrtA cDNA encodes a protein with two distinct domains of homology. The amino terminal domain is homologous to mac25, a recently characterized gene product related to IGF-BP (9) and follistatin (16). The second domain, comprising the majority of the amino acid sequence, is greater than 40% identical to bacterial HtrA serine protease. Based on the conservation of HrtA polynucleotide sequence at the DNA level in mammals and at the amino acid level in bacteria, and on the biological activity of human HtrA, it is believed that function is also conserved. Identification of physiologically relevant substrates for human HtrA include other proteases, extracellular matrix proteins, growth factors, and proteins that modulate growth factors. The evidence presented herein that AAT binds to and forms a stable complex with HtrA is evidence that this serine protease inhibitor plays a role in its regulation in vivo; levels of protease inhibitors are known to be altered in osteoarthritic cartilage (27).

The high degree of conservation among mammalian HtrA and the addition of a new functional domain during evolution, namely mac25, suggests a biological role for the ORF480-encoded protein beyond that of processing denatured proteins. Although mac25 was initially described as a member of the IGF- binding protein family (9), Kato et al. (16) noted that mac25 is more closely related to follistatin, an activin-binding protein. They demonstrated that its expression in transfected osteosarcoma cells results in clonal growth inhibition. A similar phenomenon was observed during the process of isolating clones of human 293 cells transfected with ORF480 cDNA (7). While Applicants will not be held to any particular theory or scope of activity for the polypeptides of the present invention, it is possible that human HtrA is involved in cell growth regulation, perhaps via modulation of growth factor systems other than IGF, e.g. the activin/inhibin system (28).

The Kazal-type inhibitor motif in ORF480-encoded protein, which is conserved in a diverse group of serine protease inhibitors, also occurs within mac25, follistatin, and agrin. Agrin and agrin-related proteins appear to function as extracellular components that bind to and regulate the activity of growth factors (29). Recombinant agrin has been shown to inhibit serine proteases of the trypsin class, but not the thrombin class (30). The presence of the protease inhibitor motif in ORF480 suggests that the human HtrA serine protease is a self- regulating enzyme which also regulates other serine proteases. Identification, cloning, and sequence analysis of ORF480 cDNA

In the course of screening for differences in gene expression between osteoarthritic (OA) and non-arthritic (NA) cartilage by mRNA differential display, two PCR fragments, designated 49A50 and 58A5, were identified using different arbitrary primers (Fig.1 ). Sequence analysis of 49A50 and 58A5 demonstrated that the two cloned PCR products correspond to the 3' end of the same mRNA. Using the 5' RACE technique (13), a ~1.4 kb PCR fragment corresponding to 49A50/58A5 was isolated and cloned from OA cartilage-derived cDNA. The DNA sequences of two independent clones derived from the RACE products, C05 and C13, revealed overlapping open reading frames of 337 and 328 codons, respectively; however, no ATG initiation codon was evident (Fig. 2). Together with Northern blot data showing a transcript size of over 2 kb, these results suggested that C05 and C13 were partial cDNA clones. The initial BLASTN (14) search of the Genbank and EST databases identified two 3' EST sequences (accession numbers W47107 and W67176) and a 2036 bp cDNA (SEQ ID NO:1) entry (accession number D87258; (12)). The translated sequence of Genbank entry D87258 contains an open reading frame of 480 amino acids ((ORF480) SEQ ID NO:2). The DNA sequence of clone C05 was identical to bases 477- 2036 of SEQ ID NO:1 (D87258), except for a 1 bp difference (most probably due to a PCR-generated error). Thus, the cDNA clones isolated from OA cartilage and the D87258 sequence cloned from osteoblasts represent the same gene product. RT-PCR analysis indicated that the mRNA for ORF480 is expressed in human placenta and in normal human dermal fibroblasts. Overlapping PCR- generated fragments corresponding to the entire ORF480 were isolated from cDNA derived from fibroblast RNA. The DNA sequence of the fibroblast-derived ORF480 cDNA was determined to be identical to Genbank entry D87258.

Zumbrunn and Trueb (8) reported the isolation of the same cDNA from human fibroblast, the expression of which was repressed in SV40-transformed cells. Their analysis of the sequence encoded by the cDNA revealed a putative signal sequence (amino acids 1 -22 of SEQ ID NO:2), an IGF-binding protein-3 homology domain (amino acids 1 -140 of SEQ ID NO:2), a Kazal-type inhibitor motif (amino acids 97-155 of SEQ ID NO:2), and the major domain of homology to E. coli HtrA (amino acids 140-480 of SEQ ID NO:2). In addition, a recent survey of proteins containing so-called PDZ domains (15) includes bacterial HtrA, as well as the human HtrA homologue. The location of the PDZ domain within ORF480 (amino acids 372 to 466 of SEQ ID NO:2), is indicated schematically in Fig. 2.

A BLASTP search of the Genbank protein sequence database, revealed that human mac25, a presumed member of the IGF-binding protein family (9), was found to have the highest degree of homology to the amino-terminal domain of ORF480 (Fig. 3A). For instance, the p value for the mac25 alignment with ORF480, residues 107-158, is 1.4 x 10^'23, compared to p=3.8 x 10^"8 for IGF-BP-3. Interestingly, unlike IGF-binding proteins, the sequence of mac25 within the region homologous to ORF480 contains a conserved Kazal-type serine protease inhibitor motif (Fig. 3). Moreover as pointed out by others (16), mac25 is more closely related to follistatin than to IGF-BP, and the sequence of follistatin also contains the Kazal-type inhibitor motif (Fig. 3).

Differential expression of ORF480 mRNA and HtrA protein in OA cartilage To verify the initial differential display results and quantify the relative difference in mRNA levels of human HtrA in OA compared to non-arthritic cartilage, oligonucleotide primer pairs specific for a 3' segment of ORF480 cDNA were used for semi-quantitative RT-PCR (Fig. 4A ). The results of this analysis, using expression levels of β-actin and heat shock protein with a molecular weight of about 60,000 daltons (Hsp60) for comparison, indicate that ORF480 mRNA is present at levels ~7 fold higher in OA than in non-arthritic controls. Also included in Fig. 4A are data for both type II (COL2A) and type III (COL3A) collagen mRNA, also identified in the differential display screening, show that levels of these transcripts are significantly elevated in the OA-derived samples used, a finding consistent with previous reports of induced collagen synthesis in remodeling OA cartilage (17).

To confirm differential expression of human HtrA protein in OA cartilage, high-salt extracts of OA and control cartilage samples were tested by immunoblot analysis using an antiserum prepared against the HtrA domain expressed in E. coli (Fig. 4B). Applicants found that this data reveals an unexpected difference in the levels of HtrA protein detected in OA versus non-arthritic cartilage extracts, results which, while consistent with the analysis of mRNA levels, suggest some degree of post-transcriptional regulation.

Expression and proteolytic activity of ORF480-encoded protein

A cDNA segment containing the entire coding region of ORF480 was inserted into expression vector pcDNA3 (see Experiment 7 and Fig. 2). This construct directed the expression of the expected size protein (-50 kDa) in an in vitro transcription/translation system (Fig. 5A, lanes 1 and 2). Lower molecular weight products (-40-45 kDa) were also evident in the gel electrophoresis pattern, a pattern remarkably similar to that observed for E. co// HtrA protein (11 ,18). Converting the conserved active-site serine-328 in ORF480 to alanine by site-directed mutagenesis resulted in the elimination of the lower molecular weight proteins (Fig. 5A, lanes 3 and 4). This result, which was previously shown for E. co// HtrA (18), demonstrates that the translation product of ORF480, the human HtrA protein, is a serine protease with autocatalytic activity.

Human 293 cells transfected with expression vector pcDNA3/ORF480 synthesize and secrete a 50 kDa protein as detected by immunoblot analysis (Fig.5B) using HtrA-specific antiserum. The media from different clones derived from transfected 293 cells contain an endoprotease that cleaves β-casein, resulting in at least 4 distinct fragments of the substrate that can be resolved by SDS-PAGE. The level of proteolytic activity in each sample, shown in lower panel of Fig. 5B, correlates with the relative amount of immunoreactive HtrA protein in the immunoblot shown in the upper panel. The Ser328Ala mutant of ORF480 does not digest β-casein.

The autocatalytic activity of ORF480-encoded protein observed in the cell- free expression system described above is also evident when the protein is produced in cell culture over an extended time period. When ORF480 is expressed in baculovirus infected Sf9 cells (Fig. 5C), the primary translation product (-50 kDa), which is comprised of amino acids 30 to 480 of SEQ ID NO:2, appears in the culture medium between 24 and 41 hr post-infection. In samples taken after 65 and 73 hr, a set of lower molecular weight proteins become apparent by immunoblot analysis. These proteins are a 42 kDa protein which is amino acids 99 to 480 of SEQ ID NO:2; a 38 kDa protein which is amino acids 30 to 373 of SEQ ID NO:2; and a 30 kDa protein which is amino acids 99 to 373 of SEQ ID NO:2. The endoproteolytic activity against β-casein, shown in the lower panel of Fig. 5C, is notably elevated in samples from later incubation times. These results suggests that autodegradation results in a processed form of ORF480-encoded protein with enhanced activity against an exogenous substrate.

High-MW complex of HtrA with AAT

In immunoblots of ORF480 protein expressed in both 293 cells and in baculovirus-infected Sf9 cells, high MW bands (-140 kDa) were evident (indicated with asterisks in Figs. 5B and 5C). Amino acid sequence analysis (Edman degradation) of the protein isolated from this fraction indicated a 1 :1 ratio of ORF480 sequence (XAPLAAGXPDRXEPA (amino acids 30-44 of SEQ ID NO:2,) and the amino-terminal sequence of the mature form of AAT (XVLQGHAVXE (SEQ ID NO:13)). The formation of the complex requires the presence of serum or the addition of purified AAT in serum-free medium, and an active site serine-328 (Fig. 5D)

The mammalian HtrA gene is evolutionarily conserved

Figure 6A shows a genomic "zoo blot" analysis, where total EcoR I -digested genomic DNA isolated from various mammalian and one avian species, was hybridized with ³²P-labeled human cDNA under relatively high stringency as shown in Example 8. The results, which show specific hybridization to a limited number of genomic DNA fragments, show that the gene sequence coding for the HtrA-related domain of ORF480 is conserved among vertebrate species. This interpretation was confirmed by analysis of the nucleotide sequences of partial ORF480 cDNA clones isolated from bovine, guinea pig, and rabbit, which are 91%, 89%, and 88% identical to the human sequence, respectively. Moreover, the amino acid sequences derived from the respective cDNA sequences of the three mammalian species are 98% identical to the human sequence (Fig 6B). Assays

The present invention relates to assays for diagnosing disease conditions by quantifying the level of expression of human HtrA in a body tissue sample derived from a patient. Disease states include but are not limited to, arthritis such as osteoarthrits (OA), and cancer. In accordance with a preferred embodiment of the invention there is provided a method of diagnosing OA by quantifying human HtrA mRNA expression in a body tissue sample derived from a patient. Examples of suitable body tissues include but are not limited to cartilage, blood, serum, saliva, urine, synovial fluid, etc. In an aspect of the invention, cartilage is removed from a patient and reverse transcriptase-polymerase chain reaction (RT- PCR) is used to quantify the level of human HtrA mRNA expression in the cartilage sample. The level of human HtrA mRNA thus quantified is then compared to the level of human HtrA mRNA expression from a host known not to have osteoarthritic conditions and referred to herein as a control. If the level of human HtrA from the patient being diagnosed is substantially higher than that of the control this will indicate to the attending medical professional the onset of OA. A substantially higher level of expression in this context refers to about three times or greater expression in the patient as compared to the control. In a preferred aspect of the invention the patient is a human and the human HtrA is human HtrA mRNA.

Other suitable exemplary methods for determining the level of expression of mRNA include Northern blot analysis, as described by, among others, J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory) which is hereby incorporated by reference in its entirety. A representative assay includes isolating total cellular RNA samples with RNAzol B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). An acceptable quantity of total RNA is isolated from tissue samples. The RNA is size resolved by electrophoresis through a 1% agarose gel under strongly denaturing conditions. RNA is blotted from the gel onto a nylon filter, and the filter then is prepared for hybridization to a detectably labeled polynucleotide probe.

As a probe to detect mRNA that encodes human HtrA, the antisense strand of the coding region of the human HtrA cDNA is labeled to a high specific activity. The cDNA is labeled by primer extension, using the Prime-It kit, available from Stratagene. The reaction is carried out using cDNA, following the standard reaction protocol as recommended by the supplier. The labeled polynucleotide is purified away from other labeled reaction components by column chromatography using a Select-G-50 column, obtained from 5-Prime - 3-Prime, Inc. of 5603 Arapahoe Road, Boulder, CO 80303.

The labeled probe is hybridized to the filter, at a concentration of 1 ,000,000 cpm/ml, in a small volume of 7% SDS, 0.5 M NaP04, pH 7.4 at 65°C, overnight. Thereafter the probe solution is drained and the filter is washed twice at room temperature and twice at 60°C with 0.5 x SSC, 0.1% SDS. The filter then is dried and exposed to film at -70°C overnight with an intensifying screen. Autoradiography shows the level of human HtrA mRNA.

The present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of human HtrA polypeptide in cells and tissues, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for detecting over-expression of human HtrA polypeptide compared to normal control tissue samples may be used to detect the presence of arthritis, including but not limited to osteoarthritis and rheumatoid arthritis, and other diseases such as cancer, for example. Assay techniques that can be used to determine levels of a polypeptide, such as a human HtrA polypeptide of the present invention, in a sample derived from a host, are well- known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays. Among these ELISAs frequently are preferred. An ELISA assay initially requires preparing an antibody specific to the antigen, human HtrA, which can be monoclonal or polyclonal. In addition a reporter antibody is prepared which binds to the specific antibody, or directly to the antigen itself. To the reporter antibody is attached a detectable entity such as a radioactive, fluorescent or enzymatic reagent, in this example horseradish peroxidase enzyme.

To carry out an ELISA the specific antibody to HtrA is first incubated on a solid support, e.g. a polystyrene well, that binds the antibody permanently. Any free binding sites on the wells are then covered by incubating with an unrelated protein such as bovine serum albumin (hereinafter "BSA"). Next, sample removed from a host is incubated on the antibody bound to the wells; standards with known amounts of antigen may also be included to provide a quantitative measure. After the wells are washed with buffer, a second specific antibody to HtrA, which either is a monoclonal or polyclonal antibody different from the first antibody used to coat the well, or is a specific antibody already conjugated to a detection reagent, is incubated in the wells. During this time the second antibody attaches to any human HtrA polypeptides attached to the first antibody coated on the polystyrene well. Unbound antibody is washed out with buffer. If a reporter conjugated antibody has not already been used, a third antibody linked to a reporter molecule, e.g. horseradish peroxidase, is placed in the dish which can bind to the second antibody without binding to the first antibody used to coat the plate. Unattached reporter antibody is then washed out. A reagent to detect peroxidase activity, for example a colorimetric substrate, is then added to the well. Immobilized peroxidase, linked to human HtrA polypeptide through the antibodies, produces a colored reaction product. The amount of color developed in a given time period indicates the amount of human HtrA polypeptide present in the sample. Quantitative results typically are obtained by reference to a standard curve.

Also, antibodies may be used to quantify the amount of a protein present in a sample wherein antibodies specific to human HtrA polypeptide are attached to a solid support and a sample derived from the host is passed over the solid support. The antibody is then eluted and the amount of bound HtrA polypeptide is quantified. The amount of HtrA polypeptide detected can be correlated to a quantity of human HtrA polypeptide in the sample. The preferred body tissues employed for the above- described assays are cartilage extracts, synovial fluid, blood serum and urine with synovial fluid and serum being most preferred.

If the level of human HtrA from the patient being diagnosed is substantially higher than that of the control this will indicate to the attending medical professional the onset of OA. A substantially higher level of expression in this context refers to three times or greater expression in the patient as compared to the control. In a preferred aspect of the invention the patient is a human and the human HtrA is human HtrA protein. The present invention further relates to polypeptides which have the deduced amino acid sequence of Fig. 6 (SEQ ID NO:2), as well as fragments, derivatives and analogs of such polypeptide. The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Fig. 6 (SEQ ID NO:2) means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active polypeptide. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragments, derivatives or analogs of the polypeptide of Fig. 6 (SEQ ID NO:2) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2 as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) and even more preferably at least 97% similarity (still more preferably at least 97% identity) and most preferably at least 99% similarity (still more preferably at least 99% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As known in the art, "similarity" or "identity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similarity or identity may be determined by procedures which are well-known in the art, for example, a BLAST program (Basic Local Alignment Search Tool at the National Center for Biological Information).

A variant, i.e. a "fragment", "analog" or "derivative" polypeptide, and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. However, all variants of the amino acid sequence of SEQ ID NO:2 will contain the minimum protease domain which is a fragment containing amino acid 161 to amino acid 373 of SEQ ID NO:2. This was determined by expressing the amino acid 191 to amino acid 480 fragment and determining that it lacked protease activity. The 161 to 373 amino acid fragment of SEQ ID NO:2 was then expressed and which was determined to be active, thereby defining the minimum protease domain.

Among preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like character. Conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu, substitution between the amide residues Asn and Gin, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.

Host cells can be genetically engineered to incorporate polynucleotides and express polypeptides of the present invention. For instance, polynucleotides may be introduced into host cells using well known techniques of infection, transduction, transfection, transvection and transformation. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.

Thus, for instance, polynucleotides may be transfected into host cells with another, separate, polynucleotide encoding a selectable marker, using standard techniques for co-transfection and selection in, for instance, mammalian cells. In this case the polynucleotides generally will be stably incoφorated into the host cell genome.

Alternatively, the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. The vector construct may be introduced into host cells by the aforementioned techniques. Generally, a plasmid vector is introduced as DNA in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. Electroporation also may be used to introduce polynucleotides into a host. If the vector is a virus, it may be packaged in vitro or introduced into a packaging cell and the packaged virus may be transduced into cells. A wide variety of techniques suitable for making polynucleotides and for introducing polynucleotides into cells in accordance with this aspect of the invention are well known and routine to those of skill in the art. Such techniques are reviewed at length in Sambrook et al. cited above, which is illustrative of the many laboratory manuals that detail these techniques.

In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double- stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells. The vectors, in the case of phage and viral vectors also may be and preferably are introduced into cells as packaged or encapsidated virus by well known techniques for infection and transduction. Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expression of polynucleotides and polypeptides of the present invention. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrient media, which may be modified as appropriate for, inter alia, activating promoters, selecting transformants or amplifying genes. Culture conditions, such as temperature, pH and the like, previously used with the host cell selected for expression generally will be suitable for expression of polypeptides of the present invention as will be apparent to those of skill in the art.

A great variety of expression vectors can be used to express a polypeptide of the invention. Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques, in general, a DNA sequence for expression is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase. Procedures for restriction and ligation that can be used to this end are well known and routine to those of skill. Suitable procedures in this regard, and for constructing expression vectors using alternative techniques, which also are well known and routine to those skill, are set forth in great detail in Sambrook et al. cited elsewhere herein. The DNA sequence in the expression vector may be operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include the phage lambda PL promoter, the E. coli lac, tip and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. It will be understood that numerous promoters not mentioned are suitable for use in this aspect of the invention are well known and readily may be employed by those of skill in the manner illustrated by the discussion and the examples herein.

In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate as well as engender expression. Generally, in accordance with many commonly practiced procedures, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.

Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline, theomycin, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.

The vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention.

Various mammalian cell culture systems can be employed for expression, as well. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblast, described in Gluzman et al., Cell 23: 175 (1981). Other cell lines capable of expressing a compatible vector include for example, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines.

More particularly, the present invention also includes recombinant constructs, such as expression constructs, comprising one or more of the sequences described above. The constructs comprise a vector, such as a plasmid or viral vector, into which such a sequence of the invention has been inserted. The sequence may be inserted in a forward or reverse orientation. In certain preferred embodiments in this regard, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and there are many commercially available vectors suitable for use in the present invention.

The following vectors, which are commercially available, are provided by way of example. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pcDNA3 available from Invitrogen; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.

Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("cat") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter- containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT assays. Vectors suitable to this end are well known and readily available. Two such vectors are pKK232-8 and pCM7. Thus, promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene.

Among known bacterial promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lad and lacZ promoters, the T3 and T7 promoters, the T5 tac promoter, the lambda PR, PL promoters and the trp promoter. Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-l promoter.

Selection of appropriate vectors and promoters for expression in a host cell is a well known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host perse are routine skills in the art.

Generally, recombinant expression vectors will include origins of replication, a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence, and a selectable marker to permit isolation of vector containing cells after exposure to the vector.

The present invention also relates to host cells containing the above-described constructs discussed above. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.

Constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers. Proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The invention also provides a method of identifying antagonists which reduce or block the action of human HtrA protein, such as its interaction with human HtrA- binding molecules such as receptor molecules or with HtrA itself. For example, a cellular compartment, such as a membrane or a preparation thereof, such as a membrane-preparation, may be prepared from a cell that expresses a molecule that binds human HtrA protein, such as a molecule of a signaling or regulatory pathway modulated by human HtrA protein. The preparation is incubated with labeled human HtrA protein in me absence or the presence of a candidate molecule which may be a human HtrA antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand. Molecules which bind gratuitously, i.e., without inducing the effects of human HtrA on binding the human HtrA binding molecule, are most likely to be good antagonists.

Human HtrA-like effects of potential antagonists may by measured, for instance, by determining activity of a second messenger system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of human HtrA or molecules that elicit the same effects as human HtrA. Second messenger systems that may be useful in this regard include but are not limited to AMP guanylate cyclase, ion channel or phosphoinositide hydrolysis second messenger systems.

Another example of an assay for identifying human HtrA antagonists is a competitive assay that combines human HtrA and a potential antagonist with a known HtrA substrate, for example, the insulin chain β (Sigma) as set forth in Example 11. Appropriate conditions for a competitive inhibition assay including optimal kinetic parameters are first determined with HtrA and insulin chain β. Human HtrA activity is determined by measuring the disappearance of the substrate using HPLC. The same assay is then performed in the presence of a potential inhibitor or antagonist and the rate of disappearance of the substrate is again measured to determine the effectiveness of the candidate antagonist.

Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a receptor molecule, without inducing human HtrA-induced activities, thereby preventing the action of human HtrA by excluding human HtrA from binding.

Other potential antagonists include antisense molecules for preventing expression of the HtrA gene. Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in - Okano, J. Neurochem. 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073 (1979); Cooney et al., Science 241 : 456 (1988); and Dervan et al., Science 251 : 1360 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA. For example, the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene (or promotor) involved in transcription thereby preventing transcription and the production of human HtrA. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into human HtrA polypeptide. The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of human HtrA.

The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described. The antagonists may be employed for instance to treat and/or prevent arthritis, including but not limited to osteoarthritis, rheumatoid arthritis, and cancer. The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides or a fragment thereof into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide. The antibodies may also be used to bind a soluble form of the polypeptide and therefore render it ineffective to perform its intended biological function. See Example 10.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler, G. and Milstein, C, Nature 256: 495-497 (1975), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4: 72 (1983) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., pg. 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985).

Techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The above-described antibodies may be employed to isolate or to identify clones expressing the polypeptide or purify the polypeptide of the present invention by attachment of the antibody to a solid support for isolation and/or purification by affinity chromatography.

A specific example of the use of the polypeptide, or a portion thereof, of the present invention to prepare an antibody specific therefore is set forth in Example 10. In Example 10, HtrASP-1 (amino acid 191 to 480 of SEQ ID NO:2) was used to immunogize two rabbits from which the antibody was collected and used to immunoprecipitate HtrA.

The present invention also includes a kit for performing the assay aspect of the invention. Such a kit includes vials or vessels for incubating a body tissue sample, the components necessary for quantifying human HtrA polynucleotides, for example, via RT-PCR. A kit for quantifying human HtrA polypeptide may contain anti-HtrA antibodies, for example, the antibodies may be prepared via the procedure set forth in Example 10.

The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the antagonists or inhibitors of the invention. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

Administration

The antagonist or inhibitor compounds of the present invention may be administered as pharmaceutical compositions either alone or in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others.

The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a specific indication or indications. In general, the compositions are administered in an amount of at least about 10 μg/kg body weight. In most cases they will be administered in an amount not in excess of about 8 mg/kg body weight per day. Preferably, in most cases, dose is from about 10 μg/kg to about 1 mg/kg body weight, daily. It will be appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like.

Gene therapy

The HtrA polynucleotides, polypeptides and antagonists that are polypeptides may be employed in accordance with the present invention by expression of such polypeptides in vivo, in treatment modalities often referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with a polynucleotide, such as a DNA or RNA, encoding a polypeptide ex vivo, and the engineered cells then can be provided to a patient to be treated with the polypeptide. For example, cells may be engineered ex vivo by the use of a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention. Such methods are well-known in the art and their use in the present invention will be apparent from the teachings herein.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by procedures known in the art. For example, a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector, as discussed above. The retroviral expression construct then may be isolated and introduced into a packaging cell is transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein above mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors well include one or more promoters for expressing the polypeptide. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller et al., Biotechniques 7: 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, RNA polymerase III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention will be placed under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs herein above described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501 , PA317, Y-2, Y-AM, PA12, T19-14X, VT-19- 17-H2, YCRE, YCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, A., Human Gene Therapy 1 : 5-14 (1990). The vector may be transduced into the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vector particles, which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplification's, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

EXAMPLES

All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), herein referred to as "Sambrook."

All parts or amounts set out in the following examples are by weight, unless otherwise specified.

Example 1 mRNA differential display and RT-PCR product identification

Total RNA from OA and non-arthritic human cartilage was isolated according to the method of Amin, A.R., et al. (1997) J. Clin. Invest 99, 1231-1237 and supplied to our laboratories by Drs. I. Patel and A. Amin (Hospital for Joint Diseases, New York University Medical School). Independent biochemical analyses of the isolated cartilage (R. Goldberg, personal communication), as well as the differential mRNA expression of type II and type III collagens were consistent with the indicated pathological state of the samples used in these examples.

First strand cDNA was synthesized from 0.2 μg of total RNA with each of the 3 anchored oligo-dT primers from GenHunter Corporation. The reaction (20 μl) was carried out at 37°C for 60 min. For PCR amplification, 1μl of the cDNA served as template in a 10 μl reaction mix containing 10 mM Tris-HCI (pH 8.4), 1.5 mM MgCI₂, 50 mM KCI, 0.001 % gelatin, 2 μM dNTPs, 0.2 μM of 5' arbitrary primer (AP-49 or AP-58 from the RNAimage kits obtained from GenHunter Corporation), 2 μM of the same anchored primer used in the cDNA synthesis, 5 μ Ci of α-[³³P]dATP (2,000 Ci/mmole, Dupont-New England Nuclear) and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer). Samples were subjected to 40 cycles of amplification under the following conditions: denaturing at 94°C, 30 sec, annealing at 40°C, 2 min, extension at 72°C, 30 sec, and a final extension at 72° C, 5 min. The resulting PCR products were resolved on a denaturing polyacrylamide gel and visualized by autoradiography of the dried gel. PCR products of interest were excised from the gel, and the DNA was eluted and re- amplified by PCR using the same primers and conditions described above, excluding the radio-labeled nucleotide. PCR products were analyzed on a 1.5% agarose gel and ligated into the cloning vector PCR II 2.1 (TA cloning Kit, Invitrogen). Clones of the PCR-generated fragments were obtained by transformation of E. coli strain DH5α (Gibco/BRL). DNA sequences were determined for εt least 3 independent clones of each fragment using Dye Terminator Cycle Sequencing on an ABI PRISM 377 DNA sequencing system (Perkin-Elmer).

Example 2 Quantification of PCR Products First strand cDNA was synthesized from total RNA isolated from OA and non- arthritic cartilage. 200 ng of total RNA and 10 pmoles of primer T₃₀VN (where V=A,C,G and N=A,C,G,T) were mixed in a 6 μl volume, heated to 72°C for 3 min and quenched on ice for 3 min. Buffer and MMLV reverse transcriptase were added to final concentrations of 50 mM Tris-HCI (pH 8.3), 6 mM MgCI₂, 75 mM KCI, 1 mM dNTPs, and 10 units MMLV reverse transcriptase in 10 μl. This mixture was incubated at 42°C for 1.5 hr, 94°C for 5 min, and quenched on ice. Serial dilutions of cDNA from different individuals were used for PCR amplification with a primer set for actin (forward:

GGAGTCCTGTGGCATCCACGAAACTAC (SEQ ID NO: 14) and reverse: CACATCTGCTGGAAGGTGGACAGCG (SEQ ID NO:15)) under the following conditions: 25 μl reaction volume with 10 mM Tris-HCI (pH 8.3) , 50 mM KCI, 1.5 mM MgCI₂, 0.001% gelatin, 20 μM of dNTPs, 1.25 units AmpliTaq Gold Polymerase (Perkin Elmer); 94°C, 8.5 min; 32 cycles of 94°C, 30 sec; 63°C, 30 sec; 72°C, 2 min, and a final incubation at 72°C, 7 min. Nine μl of the reaction mix was run on a 10% polyacrylamide gel, stained with SYBR™ Green I (Molecular Probes), and quantified using fluorescence imaging and ImageQuant software (Molecular Dynamics). Concentrations were chosen to ensure that the reaction was within the log phase of amplification. These conditions were used to carried out PCR reactions to quantify relative levels of expression for 49A50/58A5 (ORF480), Hsp60, type II collagen, and type III collagen. Levels of expression were normalized to the levels of actin for each sample. A negative control reaction with no template was carried out for each primer set to verify the absence of contamination.

Example 3 5' RACE cloning of ORF480 from OA cartilage

Separate first strand cDNA syntheses were carried out with 500 ng of total RNA isolated from 4 OA cartilage samples. Aliquots were combined for the synthesis of double stranded cDNA with anchors ligated at both ends according to the Marathon™ cDNA system (Clontech). An antisense primer derived from the sequence of clones 58A5/49A50 (TGTGCATTGACCTTTGGGTGCTGAC (SEQ ID NO: 16) and an anchor-specific primer were used for 5' RACE stepdown PCR: 15 mM KOAc, 3.5 mM MgOAc, 75 μg/ml BSA, 0.2 mM dNTPs, KlenTaq-1 DNA polymerase mix (Clontech), 94°C, 5min; 5 cycles of 94°C, 30 sec, 72°C, 2 min; 5 cycles of 94°C, 30 sec, 70°C, 2 min; 25 cycles of 94°C, 30 sec, 68°C, 2 min, and a final incubation at 72°C, 7 min. Reaction products were run on a 1.4% agarose gel. DNA fragments between 1.5-3 Kb and between 1-1.5 Kb were isolated from the gel and cloned using the TA cloning kit (Invitrogen). Colonies were screened by PCR with 2 ORF480 specific primers. The clones with the longest inserts were identified by PCR using T7 and M13 (reverse) primers. Plasmid DNA from clones were prepared and sequenced.

Example 4 Isolation of ORF480 from MRHF human fibroblasts

The cDNA for ORF480 was generated by PCR using as template cDNA derived from MRHF fibroblast RNA. Initially, PCR primers AAACGGATCCACCATGCAGATCCCGCGCGCC (SEQ ID NO:17) and AAACGAATTCCTATGGGTCAATTTCTTCGGG (SEQ ID NO;18), corresponding to 5' end and 3' ends of the coding region of Genbank entry D87258, were used. PCR was performed with Pfu polymerase for 25 cycles (94 °C, 1 min; 58 °C, 1 min; 72 °C, 3 min). However, due to the high GC content within a region 5' of the unique Hind III site, PCR with this pair of primers generated a cDNA fragment with an internal deletion of 380 bp within the GC-rich region. The sequence of the DNA fragment 3' of the unique Hind III site was correct. Therefore, primers AAACGGATCCAGAGTCGCCATGCAGATC (SEQ ID NO:19) and TTGTCACGATCAGTCCATCT (SEQ ID NO:20) were used to generate a DNA fragment 5' of the unique Hind III site. PCR conditions were modified to include 10% DMSO, which allowed the generation of the correctly amplified fragment. The resulting two DNA fragments were joined together at the Hind III site and subcloned into Bam Hl/Eco Rl sites of pcDNA3 (Invitrogen). The final cDNA fragment of ORF480 corresponds to nucleotides 38 to 1491 of SEQ ID NO:1. Example 5 Immunoblot analysis of HtrA-related protein in cartilage extracts

Frozen milled human cartilage (50 mg) was extracted with 100 ul of 50 mM Tris- HCI buffer containing 1 M NaCI and a cocktail of protease inhibitors (Boehringer Mannheim) at 4 °C. Samples were prepared for SDS gel electrophoresis and immunoblot analysis using the ECL detection system (Amersham). Rabbit antiserum was generated against the human HtrA protein, amino acid 161 to 480 of SEQ ID NO:2, expressed in E. coli (Hu and Peppard, unpublished).

Example 6 Generation of Ser328Ala mutation in ORF480

The Ser328Ala mutation was generated using the QuikChange Site-Directed mutagenesis kit (Stratagene). To avoid difficulty in primer extension with Pfu polymerase through the GC rich region located 5' of the unique Hind \\\ site of ORF480 cDNA, we cloned a DNA fragment, corresponding to amino acid residue 161 to the end of the coding region, into pcDNA3 and used it as a template. Primers used for the mutagenesis reaction were

CATCAACTATGGAAACGCGGGAGGCCCGTTAG (SEQ ID NO:21 )and CTAACGGGCCTCCCGCGTTTCCATAGTTGATG (SEQ ID NO:22). After confirming by sequence analysis that the correct mutation had been generated, the full length ORF480 was reassembled into pcDNA3. The resulting plasmid was designated pcDNA3-ORF480-Ser328Ala.

Example 7 Translation and Expression of ORF480

The translation product of ORF480 was synthesized in vitro using pcDNA3- ORF480 and pcDNA3-ORF480-Ser328Ala as templates in the TNT T7 Coupled Reticulocyte Lysate System (Promega), incorporating ³⁵S-methionine. The reaction products were separated on a 10% SDS-polyacrylamide gel. At the end of the run, the gel was dried under vacuum at 80 °C and analyzed on a Phosphorlmager™ (Molecular Dynamics). Heterologous expression of ORF480- Human embryonic kidney cells, 293, (ATCC) were grown in Minimal Essential Medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 1X antibiotic-antimycotic solution (Gibco) at 37°C in a humidified C0₂ incubator. Cells were stably transfected with the pcDNA3/ORF480 expression vector using the ProFection Mammalian Transf ection System (Promega). Clones were then selected by incubation with G418 (Gibco) at a concentration of 400 mg/ml and confirmed by immunoblot analysis of serum-free media. Sf-9 insect cells were maintained as suspension cultures at 28°C in Sf-900ll SF medium. Recombinant baculovirus stocks carrying the ORF480 cDNA were generated utilizing the pFASTBAC1/ORF-480 donor plasmid and the BAC-TO-BAC Baculovirus Expression System (Gibco). Optimal infection conditions were determined by varying the multiplicity of infection and conducting time course assays. Expression of the secreted ORF480 protein was confirmed by immunoblot blot analysis. In order to monitor protease activity of ORF480 protein, 30 μl of culture medium were incubated with 10 mg of β-casein (Sigma) in 50 mM Tris-HCI (pH 7.5) for 1 hr at 37 °C, and the reaction products were resolved by SDS- polyacrylamide gel electrophoresis.

Expression of Lys191 to Pro480 of human HtrA serine protease in E. coli An expression vector pET3d-HtrASP-1 containing the nucleotide sequence encoding human HtrA serine protease from Lys191 to Pro480 of SEQ ID NO:2 was constructed as follows. The cDNA for this expression construct was generated by first removing the Hind III fragment, containing the sequence for Met1 to Arg190 of HtrA serine protease, from pcDNA3/ORF480. This was followed by insertion of a linker that introduced a Nco I site and Met-Ala codons into the construct. The two primers for generating the linker are AGCTAAGAATTCAGGAAACAAAACCATGGCAA (SEQ ID NO:23) and AGCTTTGCCATGGTTTTGTTTCCTGAATTCTT (SEQ ID NO:24). The resulting construct was digested with Ncol/Notl and subcloned into the Ncol/Notl restriction sites of the expression vector pET3d(Not I) to create the expression vector pET3d-HtrASP-1. This expression construct generates a translation product, HtrASP-1 , that begins with Met-Ala followed by Lys191 to Pro480 of HtrA serine protease.

For expression of the recombinant protein, pET3d-HtrASP-1 was transformed into E. coli strain BL21 (DE3)pLysS. Cells were grown at 37 °C in LB medium containing 150 mg/ml of ampicillin and 68 mg/ml of chloramphenicol with constant shaking. When the A₆oo of the culture reached 0.5, isopropyl-b-D- thiogalactopyranoside was added to 0.6 mM. Cells were pelleted 4 hours later by centrifugation and stored at -80 °C.

For preparation of gel slices containing the expressed recombinant HtrASP-1 , cell pellets obtained from 200 ml of culture were resuspended in 40 ml of 50 mM Tris- HCI pH 7.5. After sonication, the inclusion body fraction was collected by centrifugation and solubilized in 5 ml of 8 M urea in 50 mM Tris-HCI pH 7.5. The solubilized inclusion body fraction was then separated on 10% preparative SDS- PAGE. Protein band corresponding to the recombinant HtrASP-1 , identified by staining with Commassie blue R-250, was excised from the gel and used as an antigen for raising antibodies. The estimate yield of the recombinant HtrASP-1 was 6-8 mg.

Expression of the protease domain of human HtrA serine protease (Asp161 to Pro480) in E. coli

An expression vector pET3d-HtrASP-2 containing the nucleotide sequence encoding human HtrA serine protease from Asp161 to Pro480 was constructed as follows. First, a DNA fragment corresponding to Asp161 to Arg190 of HtrA serine protease cDNA was generated by a PCR reaction using primer pair A/B (A, sense: AACAAGCTTGAATTCACCATGGATCCCAACAGTTTGCGCCA (SEQ ID NO:25); B, antisense: TTGTCACGATCAGTCCATCT (SEQ ID NO:26)) with pcDNA/HtrASP as a template. Primer A, which also includes a Nco I restriction site and a methionine translation initiation codon, corresponds to nucleotides 481 to 500 of the protein coding region of the HtrA serine protease cDNA. Primer B corresponds to nucleotides 633 to 652 of the protein coding region of the HtrA serine protease cDNA in the antisense orientation. PCR amplification was performed with Pfu polymerase for 25 cycles (94 °C, 45 sec; 58 °C, 45 sec; 72 °C, 45 sec). Next, the resulting PCR fragment was digested with Nco I/Hind III and ligated with Hind Ill/Not I fragment of pcDNA/HtrASP (Lys191 to Pro480 of HtrA serine protease) and Nco I/Not I digested pET3d(Not I) to create the expression vector pET3d-HtrASP-2.

For expression of the recombinant protein, pET3d-HtrASP-2 was transformed into E. coli strain BL21 (DE3)pLysS as set forth above.

Example 8 Genomic blot hybridization

A ZOO-BLOT (Clontech) membrane filter, containing EcoR l-digested genomic DNA from various species, was prehybridized for 30 min at 65°C, then hybridized with a random-primed ³²P-labeled BamH I- EcoR I (-900 bp, HtrA-related domain) fragment of ORF480 in rapid hybridization buffer (Amersham) at 65°C for 90 min. The hybridized filter was washed with 2X SSC (sodium chloride/sodium citrate) / 0.1 % SDS for 20 min at room temperature, twice for 10 min at 65°C, and once with 0.1X SSC, 0.1 % SDS at 65°C for 10 min. The results were visualized using a Phosphorlmager™ (Molecular Dynamics).

Example 9 Cloning mammalian homologues of HtrA

Bovine HtrA was isolated from a lung cDNA phage library (Clontech) by hybridization screening using the human cDNA as a probe. HtrA cDNA fragments were isolated by PCR from rabbit and guinea pig liver cDNA using primers designed from the human coding sequence corresponding to regions of maximum amino acid sequence identity with E. coli HtrA. The DNA sequences of the derived clones were determined using Dye Terminator Cycle Sequencing on an ABI PRISM 377 DNA sequencing system (Perkin-Elmer). Example 10 Antibody to HtrA serine protease domain - preparation and use

a. Antiserum preparation

1. Anti-gel band antisera - Rabbits 87 & 88

Gel bands containing HtrASP-1 from Experiment 7 were kept frozen at -20°C. One gel band in a 15ml polystyrene tube was chopped into small pieces with a metal spatula and 0.5ml phosphate buffered saline (pH 7.2) (PBS) and 0.5ml Freund's Complete Adjuvant (Sigma, St Louis, MO) were added. The mixture was homogenized to a thin paste and taken up via an 18 gauge needle into a syringe. Two rabbits (#s 87 & 88) were immunized subcutaneously at two sites in the scapular region, with equal volumes of the mixture. After 24 days this process was repeated with a freshly homogenized gel band. At 19 and 29 days later, blood was collected from the ear veins of the animals and the serum prepared.

Initially these sera were tested for activity in Western blotting. While both antisera were found to react with HtrA, Rabbit 88 serum contained the greater activity. Both antisera revealed bands corresponding to intact HtrA and smaller breakdown products; also, both reacted with a high molecular weight protein band corresponding to HtrA bound to AAT from fetal bovine serum in the growth medium. Rabbit 88 antiserum also reacted weakly with BSA. This reactivity was removed by absorption on immobilized BSA.

2. Anti-HtrA serine protease domain, amino acid 161 to 480 of SEQ ID NO:2 - Rabbit 45 & 46

The serine protease domain of HtrA amino acid 161 to 480 of SEQ ID NO:2 was cloned, expressed and purified. The purified protein was used to immunize two rabbits (#s 45 and 46) as follows: 0.5ml of a 1 mg/ml protein solution was homogenized with an equal volume of Freund's complete adjuvant and the mix injected (0.25ml) subcutaneously at two sites per rabbit. Three weeks later this process was repeated; then rabbit serum was obtained three and four weeks later. At this time the rabbits were re-immunized as before but with Freund's incomplete adjuvant. Serum was obtained after a further three weeks.

All sera were tested by Western blotting and reacted exclusively with HtrA, both free and bound to AAT; similar to Rabbits 87 and 88 sera, fragments of HtrA were also detectable.

b. Specific purification of anti-HtrA antibodies

Once the serine protease domain had been expressed and purified, an affinity column was prepared to enable specific purification of antibody from antisera. Two mg of purified protein in 0.1 M Na₂C0₃/NaHC0₃ buffer (pH 8.6) was covalently coupled to ω-Aminohexyl-Agarose (Sigma) which had been activated for 15 min with 1% gluteraldehyde (Sigma, EM grade) in the same buffer. After blocking vacant sites with glycylglycine, a brief rinse with 3M ammonium thiocyanate (AT) and a re-wash with PBS + 0.1% NaN₃ (PBS-A), the protein-gel conjugate was packed into a 0.7 x 5.0cm glass column (Bio-Rad). Rabbit antiserum was passed through the gel and then non-specific activity removed by washing in 0.5M NaCI buffered to pH 7.2 with 0.3M phosphate (3xPBS). The eluate was monitored at 280nm and when the absorbance returned to the baseline, AT was applied to remove the specific antibody. This was collected and immediately dialysed against PBS-A (3 x 500ml). Precipitated material was removed by centrifugation, the ODs at 280nm determined and the solution concentrated by ultrafiltration (Centricon-10 or -30, Amicon/Millipore) to 0.5- 1 mg/ml. This was stored at 4°C.

c. Immunoprecipitation

To 1 ml of supernatant from the HtrA-transfected 293 clone, or from untransfected 293 cells, 1 μg of specifically purified anti-HtrA antibody was added. The mixtures were incubated for 1 hr at 4°C, then 20μl of Protein A-Sepharose (25% gel bead suspension, Sigma) was added to each tube and the incubation continued for a further 1hr. The gel beads were centrifuged down and washed three times with 1 ml PBS. The supernatant was carefully removed and 20μl 2x SDS running buffer (Novex) containing 1 :20 2-mercaptoethanol was added. The mix was boiled for 4min and the gel beads spun down. The supernatant was removed and analyzed for the presence of HtrA by Western blotting using PVDF membranes (Novex, San Diego, CA), after 4-20% gradient reducing SDS-PAGE (Novex) using directly loaded supernatant medium from a clone of 293 cells stably transfected with the HtrA gene as a positive control. Blots were probed with the corresponding anti-HtrA rabbit antiserum at 1 :1 ,000 antiserum for 2hr at 20°C, followed by mouse monoclonal anti-rabbit IgG conjugated to alkaline phosphatase (Sigma, clone 96) at 1 :5,000 (2hr at 20°C). Activity was revealed using CDP-Star ™ (New England Biolabs, Beverly, MA) and a 5min exposure to X-Ray film (Amersham). Rabbit 88 affinity purified antibodies were able to immunoprecipitate HtrA and AAT-bound HtrA, confirming that they reacted with the protein in solution, not only in its denatured state.

d. Analysis of antibody/antigen binding by surface plasmon resonance using the BIAcore 2000™

Specifically purified Rabbit 88 antibody was covalently immobilized on a CM5 BIAcore™ (BIAcore, Inc., Piscataway, NJ) sensor "chip" using EDC conjugation with instructions supplied by the manufacturer. Purified HtrA was passed over the sensor surface (10μg/ml, 10μl/min flow rate) and the binding of the antigen to the immobilized antibody in real time was observed. The antigen in solution bound to the antibody, confirming the immunoprecipitation analysis that the antibody recognized the intact protein in solution, as well as denatured protein in SDS-PAGE. Similar observations were made with antibodies from rabbits 87, 45 and 46.

Example 11 HtrA Protease Assay

HtrA was assayed using oxidised insulin chain β (Sigma) as a substrate. The degradation products and the cleavage site within the substrate were identified by isolating and purifying the products of enzyme degradation by HPLC and peptide sequencing.

HtrA (0.56 μg) was added to 11 μM oxidised Insulin chain β and incubated in 50 mM HEPES, pH 7.5 containing 2M NaCI at 37°C for 80 min. in a total volume of 160 μl. The reaction was terminated by the addition of 32 μl 12% TFA (final 2% TFA). 8 μl of 50 mM HEPES containing 2% TFA was added and 170 μl was separated by reverse phase-HPLC (RP-HPLC) using a micro Bondapak C₁₈ column (Waters, Milford, MA). The column was equilibrated with 75% buffer A (5 mM NH40H-TFA in water) and 25% buffer B (5mM NH40H-TFA in acetonitrile). The gradient was 25 to 45% buffer B over 30 min at 1 ml/min. HtrA activity was determined by measuring the disappearance of the substrate. For the identification of the cleavage site fractions were collected and the two new peaks as well as the undigested insulin chain β isolated and sequenced.

HPLC was performed on a waters 840 System equipped with a model 712 WISP and two 590 HPLC pumps. A Lambda 481 spectrophotometer (Waters Milford, MA) was used to monitor UV absorbance at 214 nm.

The peptides isolated from the RP-HPLC were further analyzed for amino terminal analysis of new amino terminal generated as well as by mass spectrometry.

The structure of the insulin chain β is as follows

Phe-Ala-Asn-Gln-His-Leu-Cys[S03H]-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr- Leu-Val-Cys[S03H]-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala (SEQ ID NO:27). The cleavage site identified by amino terminal analysis and mass spectrometry using insulin chain β as a substrate was between Ala-Leu (bold) residues 14 and 15 of the 30 residue peptide.

Using the above HPLC method the following kinetic studies were carried out. The Km of Insulin chain β was determined to be 11μM, therefore the linearity of the rate of hydrolysis of the substrate was determined at 11 μM with incubation time. The results indicate that the rate of hydrolysis was linear up to 120 minutes using 11 μM of the substrate and 30 nM of the enzyme at 37°C. The linearity of the rate of hydrolysis of Insulin chain β with protein concentration of HtrA (10- 50 nM) was determined at 37°C for 80 minutes. The results indicate that the rates of hydrolysis was linear up to 50 nM of the enzyme.

Example 12 Gene therapeutic expression of human HtrA

Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted - the chunks of tissue remain fixed to the bottom of the flask - and fresh media is added (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin). The tissue is then incubated at 37°C for approximately one week. At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerges. The monolayer is trypsinized and scaled into larger flasks.

A vector for gene therapy is digested with restriction enzymes for cloning a fragment to be expressed. The digested vector is treated with calf intestinal phosphatase to prevent self-ligation. The dephosphorylated, linear vector is fractionated on an agarose gel and purified. HtrA cDNA capable of expressing active HtrA, is isolated. The ends of the fragment are modified, if necessary, for cloning into the vector. For instance, 5" overhanging may be treated with DNA polymerase to create blunt ends. 3' overhanging ends may be removed using S1 nuclease. Linkers may be ligated to blunt ends with T4 DNA ligase.

Equal quantities of the Moloney murine leukemia virus linear backbone and the HtrA fragment are mixed together and joined using T4 DNA ligase. The ligation mixture is used to transform E. Coli and the bacteria are then plated onto agar- containing kanamycin. Kanamycin phenotype and restriction analysis confirm that the vector has the properly inserted gene.

Packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The vector containing the HtrA gene is introduced into the packaging cells by standard techniques. Infectious viral particles containing the HtrA gene are collected from the packaging cells, which now are called producer cells.

Fresh media is added to the producer cells, and after an appropriate incubation period media is harvested from the plates of confluent producer cells. The media, containing the infectious viral particles, is filtered through a Millipore filter to remove detached producer cells. The filtered media then is used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the filtered media. Polybrene (Aldrich) may be included in the media to facilitate transduction. After appropriate incubation, the media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his, to select out transduced cells for expansion.

Engineered fibroblasts then may be injected into rats, either alone or after having been grown to confluence on microcarrier beads, such as cytodex 3 beads. The injected fibroblasts produce HtrA product, and the biological actions of the protein are conveyed to the host.

Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible without departing from the spirit and scope of the preferred versions contained herein.

All references referred to herein and set forth in the attached reference page, are hereby incorporated by reference in their entirety.

REFERENCES

1. Howell, D.S. (1984) in Osteoarthritis Diagnosis and Management (Moskowitz, R.W., Howell, D.S., Goldberg, V.M. and Mankin, H.J. eds.) pp.129-146, Sanders, Philadelphia.

2. Felson, D.T. (1990) Rheum. Dis. Clin. North Am. 16, 499-512

3. McAlindon, T. and Dieppe, P. (1990) Br. J. Rheumatol. 29, 471-473

4. Oddis, CN. (1996) Am. J. Med. 100,10s-15s

5. Muir, H. (1995) Bioessays 17, 1039-1048

6. Hammerman, D. (1989) N. Engl. J. Med. 320, 1322.

7. Klein and Hu, unpublished.

8. Zumbrunn, J., and Trueb, B. (1996) FEBS Letters 398, 187-192

9. Swisshelm, K., Ryan, K., Tsuchiya, K., and Sager, R. (1995) Proc. Natl. Acad. Sci. USA 92, 4472-4476

10. Lipinska, B., Sharma, S., and Georgopoulos, C. (1988) Nucleic Acids Res. 16, 10053-10067

11. Lipinska, B., Zylicz, M., and Georgopoulos, C. (1990) J. Bacteriol. 172, 1791 - 1797

12.0hno et al., unpublished

13. Frohman, M.A. (1993) Methods in Enzymol. 218, 340-358

H.AItschul, S. F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990) J.

Mol.Biol. 215, 403-410 15. Ponting, C.P. (1997) Protein Science 6, 464-468 16.Kato, MN., Sato, H., Tsukada, T., Ikawa, Y., Aizawa, S., and Νagayoshi, M.

(1996) Oncogene ΛI, 1361-1364 17.Aigner, T., Bertling, W., Stoss, H., Weseloh, G., and von der Mark, K. (1993)

J. Clin. Invest. 91, 829-837 18.Skorko-Glonek, J., Wawrzynow, A., Krzewski, K., Kurpierz, K., and Lipinska,

B. (1995) Gene 163, 47-52 19. Νeidhardt, F.C., VanBogelen, R.A., and Vaughn, V. (1984) Annu. Rev. Genet.

18, 295-329 20.Christman, M. F., Morgan, R. W., Jacobson, F. S., and Ames, B. N. (1985)

Cell 41, 753-762 21. Bahl, H., Echols, H., Straus, D.B., Court, D., Crowl, R., and Georgopoulos,

C.P. (1987) Genes and Development \, 57-64 22.Goff, S.A. and Goldberg, A.L. (1985) Ce//41, 587-595 23. Strauch, K., Johnson, K., and Beckwith, J. (1989) J. Bacteriol. 171, 2689-2696 24. Goldberg, A.L., Swamy, K.H.S., Chung, C.H., and Larimore, F.S. (1982)

Methods in Enzymol. 80, 680-702 25.Laskowska, E., Kuczynska-Wisnik, D., Skorko-Glonek, J., and Taylor, A.

(1996) Molecular Microbiology 22, 555-571 26. Kolomar, H., Waller, P.R.H., and Sauer, R.T. (1996) J. Bacteriol. 178, 5925-

5929 27. Walton, E.A., Upfold, L.I., Stephens, R.W., Ghosh, P., and Taylor, T.K.F.

(1981) Semin. Arthritis Rheum. 1, 73-74 28. Petraglia, F. (1997) Placenta 18, 3-8

29. Patthy, L. and Nikolics, K. (1993) Trends in Neurosci. 16, 76-81 30. Biroc, S.L., Payan, D.G., and Fisher, J.M. (1993) Developmental Brain

Research 75, 119-129 31. Shimasaki, S., Koga, M., Esch, F., Cooksey, K., Mercado, M., Koba, A., Ueno,

N., Ying, S.-Y., Ling, N., and Guillemin, R. Proc. Natl. Acad. Sci. U.S.A. (1988)

85, 4218-4222 32. Rupp, F., Ozcelik, T., Linial, M., Peterson, K., Francke, U., and Scheller, R.J.

(1992) J. Neurosci. 12, 3535-3544 33.Tschesche, H., Kolkenbrock, H., and Bode, W. (1987) Biol. Chem. Hoppe-

Seyler 368, 1297-1304 34. Friedrich, P., Kroger, B., Bialojan, S., Lemaire, H.G., Hoffken, H.W.,

Reuschenbach, P., Otte, M., and Dodt, J. (1993) J. Biol. Chem. 268, 16216-

16222 35.Laskowski, M., Kato, I., Ardelt, W., Cook, J., Denton, A., Empie, M.W., Kohr,

W. J., Park, S. J., Parks, K., Schatzley, B. L., Schoenberger, O.L., Tashiro, M.,

Vichot, G., Whatley, H.E., Wieczorek, A., and Wieczorek, M. (1987)

Biochemistry 26 , 202-221 36.Sommerhoff, C.P., Sollner.C, Mentele, R., Piechottka, G.P., Auerswald, E.A., and Fritz, H. (1994) Biol. Chem. Hoppe-Seyler 375, 685-694

37. Johansson, M.W., Keyser, P., and Soderhall, K. (1994) Eur. J. Biochem. 223, 389-394

38. Kikuchi, N., Nagata, K., Yoshida, N., and Ogawa, M.J. (1985) J. Biochem. (Tokyo) 98, 687-694

39. Brenner, S. (1988) Nature 334, 528-530

Claims

WHAT IS CLAIMED IS:

1 . An isolated polypeptide comprising a member selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in SEQ ID NO:2; and (b) a polypeptide comprising at least 30 amino acids of the polypeptide of (a).

2. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 1 to amino acid 480 of SEQ ID NO:2.

3. The polypeptide of claim 1 which is human HtrA.

4. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 30 to amino acid 480 of SEQ ID NO:2.

5. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 99 to amino acid 480 of SEQ ID NO:2.

6. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 30 to amino acid 373 of SEQ ID NO:2.

7. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 99 to amino acid 373 of SEQ ID NO:2.

8. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 191 to amino acid 480 of SEQ ID NO:2.

9. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 161 to amino acid 480 of SEQ ID NO:2.

10. The polypeptide of claim 1 wherein the polypeptide comprises amino acid 161 to amino acid 373 of SEQ ID NO:2.

11.A process for producing a human HtrA polypeptide comprising: transforming a host cell with a vector comprising DNA which upon expression encodes the polypeptide of claim 1 ; culturing the host cell under conditions promoting expression of the polypeptide and recovering the expressed polypeptide.

12. The process of claim 1 1 wherein the DNA encodes the polypeptide of claim 2.

13. A human HtrA polypeptide prepared by the process of claim 1 1 .

14. The human HtrA polypeptide prepared by the process of claim 12.

15. A process for producing a cell which expresses a polypeptide comprising transforming the cell with a vector comprising DNA which upon expression encodes the polypeptide of claim 1.

16. The process of claim 15 wherein the DNA encodes the polypeptide of claim 2.

17. A cell prepared by the process of claim 15.

18. A cell prepared by the process of claim 16.

19. A host cell tranformed with a vector comprising DNA which upon expression encodes the polypeptide of claim 1.

20. The host cell of claim 16 wherein the vector comprises DNA which upon expression encodes the polypeptide of claim 2.

21.An antibody which binds to the polypeptide of claim 1.

22. An antibody which binds to the polypeptide of claim 2.

23. The antibody of claim 21 which is a monoclonal antibody.

24. The antibody of claim 21 which binds to amino acid 1 to amino acid 480 of SEQ ID NO:2.

25. A method of producing an antibody comprising: injecting HrtASP-1 into a mammal; purifying the antibody produced by the mammal; and recovering the antibody.

26. A method for measuring the presence and/or progression of a disease state in a mammal comprising:

(a) isolating an HtrA polynucleotide from a body tissue sample derived from the mammal;

(b) quantifying the level of expression of the HtrA polynucleotide; and (c) comparing the level of expression determined in step (b) to a control to detect the presence or absence of an altered level of expression of the polynucleotide.

27. The method of claim 26 wherein the polynucleotide is quantified via RT-PCR.

28. The method of claim 26 wherein the body tissue sample is selected from the group consisting of cartilage, urine, blood and synovial fluid.

29. The method of claim 26 wherein the disease state is selected from the group consisting of osteoarthritis and cancer.

30. The method of claim 26 wherein the mammal is a human.

31.The method of claim 26 wherein the altered level of expression is an over- expression.

32. The method of claim 26 wherein the polynucleotide is mRNA.

33. A method for measuring the presence and/or progression of a disease state in a mammal comprising:

(a) isolating an HtrA polypeptide from a body tissue sample derived from the mammal;

(b) quantifying the amount of HtrA polypeptide in the sample; and (c) comparing the amount determined in step (b) to a control to determine the presence or absence of an altered quantity of the polypeptide.

34. The method of claim 33 wherein the polypeptide is quantified via an ELISA assay.

35. The method of claim 33 wherein the body tissue sample is selected from the group consisting of cartilage, urine, blood and synovial fluid.

36. The method of claim 33 wherein the disease state is selected from the group consisting of osteoarthritis and cancer.

37. The method of claim 33 wherein the mammal is a human.

38. The method of claim 33 wherein the altered level of expression is an over- expression.

39. The method of claim 33 wherein the polypeptide is isolated using an antibody specific for HtrA polypeptide.

40. A method for identifying substances that antagonize or prevent the activity of HtrA protein comprising: (a) contacting HtrA protein with a substance; and

(b) determining the ability of the substance to antagonize or prevent the activity of

HtrA protein as compared to a control.

41.The method of claim 40 wherein the HtrA protein and substance are combined with a known substrate of the HtrA protein under conditions sufficient for the HtrA protein to act on the substrate and the ability of the substance to prevent

HtrA from acting on the substrate is determined by quantifying the amount of substrate cleaved by HtrA protein.

42. The method of claim 41 wherein the substrate is insulin chain β.

43. A substance identified by the method of claim 41.

44. A method for treating a disease associated with an over-expression of HtrA protein comprising administering a therapeutically effective amount of the substance of claim 43 to a subject in need thereof.

45. The method of claim 44 wherein the disease is osteoarthritis or cancer.

46. A pharmaceutical composition comprising the substance of claim 43 and a pharmaceutically acceptable carrier.

47. A diagnostic kit for detecting a disease state associated with an over- expression of HtrA protein comprising an anti-human HtrA antibody.

48. A diagnostic kit for detecting disease states associated with an over- expression of human HtrA mRNA.

49. A method of diagnosing conditions associated with altered levels of human HtrA polypeptide comprising: (a) incubating a body fluid sample with an anti-human HtrA antibody; and

(b) quantifying the level of bound anti-human HtrA antibody in the body fluid sample; and

(c) comparing the level determined in step (b) to a control to detect the presence or aosence of an altered level of expression of the polypeptide.

50. The method of claim 49 wherein quantifying the level of bound anti-human HtrA antibody in the body fluid sample is by ELISA.

51. The method of claim 49 wherein the body fluid sample is selected from the group consisting of blood, urine and synovial fluid.