GB2369363A - Matrix metalloproteases - Google Patents

Abstract

An isolated matrix metalloprotease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or (ii) a variant thereof which comprises a catalytic domain capable of binding a zinc residue or (iii) a fragment of (i) or (ii) which comprises a catalytic domain capable of binding a zinc residue.

Description

PROTEIN

Field of the Invention

The present invention relates to matrix metalloprotease polypeptides.

Background of the Invention

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteases responsible for controlled destruction of extracelluar matrix (ECM) components during normal physiological processes such as fetal development, 10 wound healing, cellular migration and angiogenesis. Matrix degradation is a tightly regulated process, controlled by the relative levels of active MMPs and the naturally occurring tissue metalloproteinase inhibitors (TIMPs) which keep them in check.

Disregulated MMP activity results in runaway ECM destruction, implicated in a wide variety of pathological conditions including arthritis, atherosclerosis, 15 emphysema, ulceration, neurodegeneration and cancer metastasis. MMPs are multidomain proteins, typically containing an Nterminal prodomain, catalytic domain and C-terminal hemopexin or extracellular matrix binding domains. A subclass of the MMP family, the membrane type MMPs (MT-MMPs) possess a transmembrane domain near the Cterminus. The prodomain maintains the enzyme 20 in an inactive state in the mature zymogen by binding the active site zinc residue via a conserved cysteine residue (the cysteine switch), normally positioned near the prodomain N-terminus. Active MMPs typically lack the prodomain, which is displaced from the active site and cleaved off during the activation process. The catalytic domain contains a conserved zinc binding sequence (HEATH).

Summary of the Invention

A novel matrix metalloprotease, referred to herein as HIPHUM35, is now provided. HIPHUM35 is shown to be primarily expressed in adipose tissue, cerebellum, jejunum, lung, myometrium, omentum, prostate, small intestine and 30 testis. Expression is upregulated in parasupranuclear palsy (PSP) brain, in chronic obstructive pulmonary disease (COPD) lung, VEGF treated endothelial- cells and peripheral blood mononuclear cells (PBMCs). Expression is downregulated in colon tumour, breast tumour and lung carcinoma. The novel matrix metalloprotease is a

screening target for the identification and development of novel pharmaceutical agents, including modulators of matrix metalloprotease activity. These agents may be used in the treatment and/or prophylaxis of CNS diseases such as parasupranuclear palsy (PSP), respiratory diseases such as chronic obstructive pulmonary disease 5 (COPD), inflammatory respiratory diseases such as fibrotic diseases of the lung and cancers such as lung, colon, breast and endometrial carcinomas (DECK; Accordingly, the present invention provides an isolated matrix metalloprotease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2; 10 (ii) a variant thereof which comprises a catalytic domain capable of binding a zinc residue; or (iii) a fragment of (i) or (ii) which comprises a catalytic domain capable of binding a zinc residue.

According to another aspect of the invention there is provided a 15 polynucleotide encoding a polypeptide of the invention which polynucleotide includes a sequence comprising: (a) the nucleic acid sequence of SEQ ID NO: 1 and/or a sequence complementary thereto, (b) a sequence which hybridises under stringent conditions to a sequence 20 as defined in (a); (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 65% identity to a sequence as defined in (a), (b) or (c).

25 The invention also provides: - an expression vector which comprises a polynucleotide of the invention and which is capable of expressing a polypeptide of the invention; - a host cell comprising an expression vector of the invention; - a method of producing a polypeptide of the invention which method 30 comprises maintaining a host cell of the invention under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide; - an antibody specific for a polypeptide of the invention;

- a method for identification of a substance that modulates matrix metalloprotease activity and/or expression, which method comprises contacting a polypeptide, polynucleotide, expression vector or host cell of the invention with a test substance and determining the effect of the test 5 substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by the said polynucleotide, thereby to determine whether the test substance modulates matrix metalloprotease activity and/or expression; - a compound which modulates matrix metalloprotease activity and which is 10 identifiable by the method referred to above; - a method of treating a subject having a disorder that is responsive to matrix metalloprotease stimulation or modulation, which method comprises administering to said subject an effective amount of substance of the invention; and 15 - use of a substance that stimulates or modulates matrix metalloprotease activity in the manufacture of a medicament for the treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of matrix metalloprotease activity.

Preferably the disorder is selected from CNS diseases such as 20 parasupranuclear palsy (PSP), respiratory diseases such as chronic obstructive pulmonary disease (COPD), inflammatory respiratory diseases such as fibrotic diseases of the lung and cancers such as lung, colon, breast and endometrial carcinomas (DEC).

25 Brief Description of the Sequences

SEQ ID NO: 1 shows the nucleotide and amino acid sequences of human protein HIPHUM35.

SEQ ID NO: 2 is the amino acid sequence alone of HIPHUM35.

30 Brief Description of the Figures

Figure 1 shows amacroglobulin capture by Hiphum 35, demonstrating that Hiphum - 35 has protease activity.

Detailed Description of the Invention

Throughout the present specification and the accompanying claims the words

"comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are 5 intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows., The present invention relates to a human matrix metalloprotease, referred to herein as HIPHUM35, and variants thereof. Sequence information for HIPHUM35 is provided in SEQ ID NO: 1 (nucleotide and amino acid) and in SEQ ID NO: 2. A 10 polypeptide of the invention thus consists essentially of the amino acid sequence of SEQ ID NO: 2 or of a variant of that sequence, or of a fragment of either thereof.

Polypeptides of the invention may be in a substantially isolated form. It will be understood that the polypeptide may be mixed with carriers or diluents which will not interfere with the intended purpose of the polypeptide and still be regarded as 15 substantially isolated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 50%, e.g. more than 80%, 90%, 95% or 99%, by weight of the polypeptide in the preparation is a polypeptide of the invention.

Routine methods, can be employed to purify andlor synthesise the proteins according 20 to the invention. Such methods are well understood by persons skilled in the art, and include techniques such as those disclosed in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2n Edition, CSH Laboratory Press, 1989, the disclosure of

which is included herein in its entirety by way of reference.

The term "variant" refers to a polypeptide which has a same essential 25 character or basic biological functionality as HIPHUM35. The essential character of HIPHUM35 can be defined as follows: HIPHUM35 is a matrix metalloprotease.

Preferably a variant polypeptide is one which binds to the same extracellular matrix component as HIPHUM35. Preferably the polypeptide comprises a catalytic domain capable of binding a zinc residue. Typically the zinc residue binds to the consensus 30 zinc binding sequence HEXXH, preferably HEIGH. Typically the catalytic domain is capable of cleaving a polypeptide when the metalloprotease is in an active state.

The metalloprotease is in an active state when the polypeptide does not comprise a prodomain. Therefore, a polypeptide of the invention may comprise a prodomain

and be in an inactive state or may lack a prodomain and be in an active state. A polypeptide comprising a prodomain may be cleaved to remove the prodomain and activate protease activity. Cleavage of the prodomain to activate the metalloprotease may occur as a result of the enzymatic activity of any suitable protease which may be 5 a matrix metalloprotease or a protease of another family. The protease may comprise a mutated prodomain that does not function to maintain the polypeptide in an inactive state, for example because of mutation of the cysteine switch motif (PRCGVP) in the prodomain. The cysteine switch motif contains a cysteine residue which interacts with the zinc residue in the active site of the catalytic domain.

10 Preferably a polypeptide of the invention comprises a hemopexin domain.

Preferably a polypeptide of the invention binds to an extracellular matrix component.

More preferably binding of a polypeptide of the invention to an extracellular matrix component occurs via a hemopexin domain.

Typically, a polypeptide of the invention is capable of cleaving a polypeptide 15 such as a component of the extracellular matrix or a metalloprotease when activated.

A polypeptide of the invention may be capable of binding a tissue inhibitor of metalloproteases (TIMP). Binding of a polypeptide of the invention to a TIMP typically inactivates metalloprotease activity. A polypeptide of the invention may be capable of activating a matrix metalloprotease by cleaving a prodomain.

20 A polypeptide having a same essential character as HIPHUM35 may be identified by monitoring for a function of the matrix metalloprotease selected from cleavage of extracellular matrix proteins such as collagen (including fibrillar collagen), elastin, laminin, vitronectin, fibronectin, enamel matrix, aggrecan, other matrix and basement membrane proteins and proteoglycans, cleavage of 25 plasminogen and/or amyloid precursor protein (APP), cleavage of an artificial substrate such as a fluorescent substrate or colourimetric substrate and activation of MMPs or other proteases, either alone or in conjunction with other components (ea.

TIMPs). In another aspect of the invention, a variant is one which does not show the 30 same activity as HIPHUM35 but is one which inhibits a basic function of HIPHUM35. For example, a variant polypeptide is one which inhibits protease activity of HIPHUM35, for example by binding to extracellular matrix component to prevent extracellular matrix component binding to HIPHUM35.

A typical assay for determining whether a polypeptide possesses HIPHUM35 protease activity comprises monitoring fluorescence or colourimetric changes of a fluorescent or colourimetric substrate of HIPHUM35 following incubation of the polypeptide with a said substrate.

5 Typically, polypeptides with more than about 70% identity preferably at least 80% or at least 90% and particularly preferably at least 95% at least 97 % or at least 99% identity, with the amino acid sequences of SEQ ID NO: 2, are considered as variants of the proteins. Such variants may include allelic variants and the deletion, modification or addition of single amino acids or groups of amino acids within the 10 protein sequence, as long as the peptide maintains a basic biological functionality of the HIPHUM35 receptor.

Amino acid substitutions may be made, for example from 1, 2 or 3 to 10, 20 or 30 substitutions. The modified polypeptide generally retains activity as a matrix metalloprotease. Conservative substitutions may be made, for example according to 15 the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other.

ALIPHATIC Non-polar A ILV l Polar-uncharged C S T M Polar-charged D E AROMATIC 11 F W Y

Shorter polypeptide sequences are within the scope of the invention. For 20 example, a peptide of at least 20 amino acids or up to 50, 60, 70, 80, 100, 150, 200, 300 or 400 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates a basic biological functionality of HIPHUM35.

In particular, but not exclusively, this aspect of the invention encompasses the

situation where the protein is a fragment of the complete protein sequence and may represent a catalytic domain or substrate binding domain. Preferred fragments include fragments comprising the cysteine switch domain found at amino acid residues 115 to 122 of full length HIPHUM35 and/or one or more of the haemopexin 5 domain repeat elements found at amino acid residues 333 to 391, 394 to 449, 441 to 498 and 506 to 549 of full length HIPHUM35. Such fragments can be used to construct chimeric proteases preferably with another protease, more preferably with another member of the family of matrix metalloproteases. Such chimeric proteases may comprise different domains from different metalloproteases. For example, a 1 0 fragment comprising the zinc protease domain of a polypeptide of the invention may be fused to a fragment of a different metalloprotease comprising one or more haemopexin repeat elements. The haemopexin domain of a chimeric protease may comprise one or more repeat elements from a polypeptide of the invention and one or more repeat elements from another metalloprotease.

1 5 Fragments of HIPHUM35 or a variant thereof can also be used to raise anti HIPHUM35 antibodies. In this embodiment the fragment may comprise an epitope of the HIPHUM35 polypeptide and may otherwise not demonstrate the catalytic, substrate binding or other properties of HIPHUM35.

Polypeptides of the invention may be chemically modified, e.g. post 20 translationally modified. For example, they may be glycosylated or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote insertion into the cell membrane. Such modified polypeptides fall within the scope of the term "polypeptide" of the invention.

25 The invention also includes nucleodde sequences that encode for HIPHUM35 or a variant thereof as well as nucleotide sequences which are complementary thereto. The nucleotide sequence may be RNA or DNA including genomic DNA, synthetic DNA or cDNA. Preferably the nucleotide sequence is a DNA sequence and most preferably, a cDNA sequence. Nucleotide sequence information is provided in 30 SEQ ID NO: 1. Such nucleotides can be isolated from human cells or synthesised according to methods well known in the art, as described by way of example in Sambrook et al, 1989.

Typically a polynucleotide of the invention comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 1.

A polynucleotide of the invention can hydridize to the coding sequence or the 5 complement of the coding sequence of SEQ ID NO: 1 at a level significantly above background. Background hybridization may occur, for example, because of other

cDNAs present in a cDNA library. The signal level generated by the interaction between a polynucleotide of the invention and the coding sequence or complement of the coding sequence of SEQ ID NO: 1 is typically at least 10 fold, preferably at least 10 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 1. The intensity of interaction may be measured, for example, by radiolabelling the probe, e.g. with 32p. Selective hybridization may typically be achieved using conditions of medium to high stringency. However, such hybridisation may be carried out under any suitable conditions known in the art (see 15 Sambrook et al, 1989. For example, if high stringency is required suitable conditions include from 0.1 to 0.2 x SSC at 60 C up to 65 C. If lower stringency is required suitable conditions include 2 x SSC at 60 C.

The coding sequence of SEQ ID NO: 1 may be modified by nucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or 100 substitutions. The 20 polynucleotide of SEQ ID NO: 1 may alternatively or additionally be modified by one or more insertions andlor deletions and/or by an extension at either or both ends.

A polynucleotide may include one or more introns, for example may comprise genomic DNA. Additional sequences such as signal sequences which may assist in insertion of the polypeptide in a cell membrane may also be included. The modified 25 polynucleotide generally encodes a polypeptide which has a HIPHUM35 activity.

Alternatively, a polynucleotide encodes a catalytic or substrate-binding portion of a polypeptide or a polypeptide which inhibits HIPHUM35 activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for 30 example as shown in the Table above.

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO: 1 will generally have at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at

least 99% sequence identity to the coding sequence of SEQ ID NO: 1 over a region of at least 20, preferably at least 30, for instance at least 40, at least 60, more preferably at least 100 contiguous nucleotides or most preferably over the full length of SEQ ID NO: 1.

5 For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (typically on their default settings), for example as described in Altschul (1993) J. Mol. Evol.

1 0 36:290-300; Altschul et al (1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http://www.ncbi.nim.nih. gov/).

This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy 1 5 some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be 20 increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.

The BLAST algorithm parameters W. T and X determine the sensitivity and speed of 25 the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff end Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) aligrunents (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity 30 between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-57X7. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences

would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about O.01, and most preferably less than about 0.001.

5 Any combination of the above mentioned degrees of sequence identity and minimum sizes may be used to define polynucleotides of the invention, with the more stringent combinations (i.e. hugher sequence identity over longer lengths) being preferred. Thus, for example a polynucleotide which has at least 90% sequence identity over 25, preferably over 30 nucleotides forms one aspect of the invention, as 10 does a polynucleotide which has at least 95% sequence identity over 40 nucleotides.

The nucleotides according to the invention have utility in production of the proteins according to the invention, which may take place in vitro, in vivo or ex vivo.

The nucleotides may be involved in recombinant protein synthesis or indeed as therapeutic agents in their own right, utilised in gene therapy techniques. Nucleotides 15 complementary to those encoding HIPHUM35, or antisense sequences, may also be used in gene therapy.

Polynucleotides of the invention may be used as a primer, e.g. a PCR primer, a primer for an alternative amplification reaction, a probe e.g. labelled with a revealing label by conventional means using radioactive or non-radioactive labels, or 20 the polynucleotides may be cloned into vectors.

Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in 25 length, for example up to 200, 300,-400, 500, 600, 700, 800, 900, 1000, 1250 or 1500 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of the coding sequence of SEQ ID NO: 1.

The present invention also includes expression vectors that comprise nucleotide sequences encoding the proteins or variants thereof of the invention. Such 30 expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to

allow for protein expression. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. 1989.

Polynucleotides according to the invention may also be inserted into the 5 vectors described above in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense polynucleotides may also be produced by synthetic means. Such antisense polyoucleotides may be used as test compounds in the assays of the invention or may be useful in a method of treatment of the human or animal body by therapy.

10 Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

15 A regulatory sequence, such as a promoter, "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with a origin of replication, optionally a promoter for the expression of the said 20 polyoucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also 25 be adapted to be used in viva, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be 30 induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as p- actin promoters, may be used. Tissue-

specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human 5 cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic 10 genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention

into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the 15 polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to 20 the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

The invention also includes cells that have been modif ed to express the HIPHUM35 polypeptide or a variant thereof. Such cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect 25 cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells such as bacterial cells. Particular examples of cells which may be modif ed by insertion of vectors encoding for a polypeptide according to the invention include mammalian HEK293T, CHO, HeLa, BHK, 3T3 and COS cells. Preferably the cell line selected will be one which is not only stable, but also 30 allows for mature glycosylation of a polypeptide. Expression may be achieved in transformed oocytes. A polypeptide of the invention may be expressed in cells of a transgenic non-human animal, preferably a mouse. A transgenic non-human animal expressing a polypeptide of the invention is included within the scope of the

invention. A polypeptide of the invention may also be expressed in Xenopus Levis oocytes. A polypeptide for use in an assay of the invention may be overexpressed in bacterial cells, such as E.Coli, and isolated from the bacterial culture.

5 According to another aspect, the present invention also relates to antibodies, specific for a polypeptide of the invention. Such antibodiesare for example useful in purification, isolation or screening methods involving irnmunoprecipitation techniques or, indeed, as therapeutic agents in their own right.

Antibodies may be raised against specific epitopes of the polypeptides 10 according to the invention. Such antibodies may be used to block substrate binding to the receptor. An antibody, or other compound, "specifically binds" to a protein when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other proteins.

A variety of protocols for competitive binding or immunoradiometric assays to 15 determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 1 58, 1 211 -1226, 1 993). Such immuneassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

Antibodies of the invention may be antibodies to human polypeptides or 20 fragments thereof. For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR- grafted antibodies or humanised antibodies.

25 Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample, which method comprises: I providing an antibody of the invention; II incubating a biological sample with said antibody under conditions which allow for the formation of an antibodyantigen complex; and 30 III determining whether antibody-antigen complex comprising said antibody is formed. A sample may be for example a tissue extract, blood, serum and saliva.

Antibodies of the invention may be bound to a solid support and/or packaged into

kits in a suitable container along with suitable reagents, controls, instructions, etc. Antibodies may be linked to a revealing label and thus may be suitable for use in methods of in viva HIPHUM35 imaging.

Antibodies of the invention can be produced by any suitable method. Means 5 for preparing and characterizing antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the 1 0 "immunogen".

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and 15 the IgG fraction purified.

A method for producing a monoclonal antibody comprises immortalizing cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

20 An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunization of human lymphocytes, followed by transformation of the 25 lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat or mouse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier.

30 The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

An important aspect of the present invention is the use of polypeptides according to the invention in screening methods. The screening methods may be

used to identify substances that bind to matrix metalloprotease and in particular which bind to HIPHUM35 such as a substrate for the enzyme. Screening methods may also be used to identify agonists or antagonists which may modulate matrix metalloprotease activity, inhibitors or activators of HIPHUM35 activity, and/or 5 agents which up-regulate or down-regulate HIPHUM35 expression.

Any suitable format may be used for the assay. In general terms such screening methods may involve contacting a polypeptide of the invention with a test substance and monitoring for binding of the test substance to the polypeptide or measuring protease activity. A polypeptide of the invention may be incubated with a 10 test substance. Modulation of matrix metalloprotease activity may be determined. In a preferred aspect, the assay is a cell-based assay. Preferably the assay may be carried out in a single well of a microtitre plate. Assay formats which allow high throughput screening are preferred.

A typical assay for determining whether a test substance acts as an inhibitor 15 or activator of HIPHUM35 activity comprises contacting a fluorescent or colouri netric substrate with a polypeptide of the invention and a test substance and monitoring protease activity by monitoring any change in the fluorescence or light emission. Any changes in the fluorescence of a substrate as a result of its proteolytic degradation by a polypeptide of the invention may be detected using a fluorescence 20 plate reader. Colourimetic changes may be measured using a spectrophotometer.

The inhibitory or stimulatory activity of a test substance may be determined by comparing any fluorescent or colourimetric changes observed in the presence of a test substance to any changes observed in the absence of a test substance and/or in the presence of a known inhibitor of HIPHUM35 activity.

25 Modulator activity can be determined by contacting cells expressing a polypeptide of the invention with a substance under investigation and by monitoring an effect mediated by the polypeptide. The cells expressing the polypeptide may be in vitro or in vivo. The polypeptide of the invention may be naturally or recombinantly expressed. Preferably, the assay is carried out in vitro using cells 30 expressing recombinant polypeptide. Preferably, control experiments are carried out on cells which do not express the polypeptide of the invention to establish whether the observed responses are the result of activation of the polypeptide.

The binding of a test substance to a polypeptide of the invention can be determined directly. For example, a radiolabelled test substance can be incubated with the polypeptide of the invention and binding of the test substance to the polypeptide can be monitored. Typically, the radiolabelled test substance can be 5 incubated with cell membranes containing the polypeptide until equilibrium is reached. The membranes can then be separated from a non-bound test substance and dissolved in scintillation fluid to allow the radioactive content to be determined by scintillation counting. Non-specific binding of the test substance may also be determined by carrying out a competitive binding assay.

10 Substances that inhibit the interaction of a polypeptide of the invention with a HIPHUM35 substrate or with another protease may also be identified through a yeast 2-hybrid assay or other protein interaction assay such as a co-immunoprecipitation or an ELISA based technique.

Assays may be carried out using cells expressing HIPHUM35, and incubating 15 such cells with the test substance optionally in the presence of a HIPHUM35 substrate. The results of the assay are compared to the results obtained using the same assay in the absence of the test substance. Cells expressing HIPHUM35 constitutively may be provided for use in assays for HIPHUM35 function.

Additional test substances may be introduced in any assay to look for inhibitors or 20 activators of substrate binding or inhibitors or activators of protease activity.

Assays may also be carried out to identify substances which modify HIPHUM35 expression, for example substances which up- or down- regulate expression. Such assays may be carried out for example by using antibodies for HIPHUM35 to monitor levels of HIPHUM35 expression. Other assays which can be 25 used to monitor the effect of a test substance on HIPHUM35 expression include using a reporter gene construct driven by the HIPHUM35 regulatory sequences as the promoter sequence and monitoring for expression of the reporter polypeptide.

Additional control experiments may be carried out.

Suitable test substances which can be tested in the above assays include 30 combinatorial libraries, defined chemical entities and compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display (e.g. phage display libraries) and antibody products.

Typically, organic molecules will be screened, preferably small organic

molecules which have a molecular weight of from 50 to 2500 daltons.

Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of 5 synthetic or natural compounds. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or 10 activation tested individually. Test substances may be used at a concentration of from lnM to 1 OmM, preferably from 1 1lm to l mM or from 1 IBM to 1 OO,uM, more preferably from 1,uM to 1 AIM. Preferably, the activity of a test substance is compared to the activity shown by a known activator or inhibitor. A test substance which acts as an inhibitor may produce a 50% inhibition of activity of the receptor.

15 Alternatively a test substance which acts as an activator may produce 50% of the maximal activity produced using a known activator.

Another aspect of the present invention is the use of polynucleotides encoding the HIPHUM35 polypeptides of the invention to identify mutations in HIPHUM35 genes which may be implicated in human disorders. Identification of 20 such mutations may be used to assist in diagnosis or susceptibility to such disorders and in assessing the physiology of such disorders. Polynucleotides may also be used in hybridization studies to monitor for up- or down-regulation of HIPHUM35 expression. Polynucleotides such as SEQ ID NO: 1 or fragments thereof may be used to identify allelic variants, genomic DNA and species variants.

25 The present invention provides a method for detecting variation in the expressed products encoded by HIPHUM35 genes. This may comprise determining the level of an HIPHUM35 expressed in cells or determining specific alterations in the expressed product. Sequences of interest for diagnostic purposes include, but are not limited to, the conserved portions as identified by sequence similarity and 30 conservation of intron/exon structure. The diagnosis may be performed in conjunction with kindred studies to determine whether a mutation of interest co-

segregates with disease phenotype in a family.

Diagnostic procedures may be performed on polynucleotides isolated from an individual or alternatively, may be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary. Appropriate 5 procedures are described in, for example, Nuovo, G.J., 1992, "PCR In Situ Hybridization: Protocols And Applications", Raven Press, NY). Such analysis techniques include, DNA or RNA blotting analyses, single stranded conformational polymorphism analyses, in situ hybridization assays, and polymerase chain reaction analyses. Such analyses may reveal both quantitative aspects of the expression 1 0 pattern of a HIPHUM35, and qualitative aspects of HIPHUM35 expression and/or composition. Alternative diagnostic methods for the detection of HIPHUM35 nucleic acid molecules may involve their amplification, e.g. by PCR (the experimental embodiment set forth in U.S. Patent No. 4,683,202), ligase chain reaction (Barany, 1 5 1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. 15 USA 86:1173 1177), Q- Beta Replicase (Lizard) et al., 1988, Bio/Technology 6:1197) or any other nucleic acid amplification method, followed by the detection of the amplified 20 molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

Particularly suitable diagnostic methods are chip-based DNA technologies such as those described by Hacia et al., 1996, Nature Genetics 14:441-447 and 25 Shoemaker et al., 1996, Nature Genetics 14:450-456. Briefly, these techniques involve quantitative methods for analyzing large numbers of nucleic acid sequence targets rapidly and accurately. By tagging with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization.

30 Following detection, the results seen in a given patient may be compared with a statistically significant reference group of normal patients and patients that have HIPHUM35 related pathologies. In this way, it is possible to correlate the amount or

kind of HIPHUM35 encoded product detected with various clinical states or predisposition to clinical states.

Another aspect of the present invention is the use of the substances that have been identified by screening techniques referred to above in the treatment of disease 5 states, which are responsive to regulation of matrix metalloprotease activity. The treatment may be therapeutic or prophylactic. The condition of a patient suffering from such a disease state can thus be improved.

In particular, such substances may be used in the treatment of CNS diseases such as parasupranuclear palsy (PSP), respiratory diseases such as chronic 10 obstructive pulmonary disease (COPD), inflammatory respiratory diseases such as fibrotic diseases of the lung and cancers such as lung, colon, breast and endometrial carcinomas (DEC).

Substances that act as inhibitors of HIPHUM35 activity may be used in the treatment of disease states in which HIPHUM35 expression is up-regulated such as 15 PSP or COPD. Substances that act as activators of HIPHUM35 activity may be used in the treatment of disease states in which expression of HIPHUM35 is down regulated such as lung, colon or breast cancer.

Additional disease states that may be treated include atherosclerosis and multiple sclerosis (MS).

20 Substances identified according to the screening methods outlined above may be formulated with standard pharmaceutically acceptable carriers and/or excipients as is routine in the pharmaceutical art. For example, a suitable substance may be dissolved in physiological saline or water for injections. The exact nature of a formulation will depend upon several factors including the particular substance to be 25 administered and the desired route of administration. Suitable types of formulation are fully described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Eastern Pennsylvania, 1 7th Ed. 1985, the disclosure of which is included

herein of its entirety by way of reference.

The substances may be administered by enteral or parenteral routes such as 30 via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, topical or other appropriate administration routes.

A therapeutically effective amount of a modulator is administered to a patient. The dose of a modulator may be determined according to various

parameters, especially according to the substance used, the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 5 mg per kg of body weight, according to the activity of the specific modulator, the age, weight and conditions of the subject to be treated, the type and severity of the degeneration and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Nucleic acid encoding HIPHUM35 or a variant thereof which inhibits 10 HIPHUM35 activity may be administered to the mammal. In particular, a nucleic acid encoding a polypeptide with HIPHUM35 activity may be administered to a subject suffering from a condition in which HIPHUM35 expression is down-

regulated, such as lung, colon or breast cancer. A nucleic acid encoding a variant of HIPHUM35 that inhibits HIPHUM35 activity may be administered to a patient 15 suffering from a condition in which HIPHUM35 expression is up-regulated such as PSP or COPD. Nucleic acid, such as RNA or DNA, and preferably, DNA, is provided in the form of a vector, such as the polynucleotides described above, which may be expressed in the cells of the mammal.

Nucleic acid encoding the polypeptide may be administered by any available 20 technique. For example, the nucleic acid may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the nucleic acid may be delivered directly across the skin using a nucleic acid delivery device such as particle-mediated gene delivery. The nucleic acid may be administered topically to the skin, or to mucosal surfaces for example by intranasal, 25 oral, intravaginal or intrarectal administration.

Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents.

Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The 30 dosage of the nucleic acid to be administered can be altered. Typically the nucleic acid is administered in the range of lpg to lmg, preferably to lpg to 1 Ollg nucleic acid for particle mediated gene delivery and 1 0pg to 1 mg for other routes.

The following Examples illustrate the invention.

Example 1: Character sation of the sequence A matrix metalloprotease, designated as HIPHUM35 has been identified. The nucleotide and amino acid sequences of the receptor have been determined. These are set out below in SEQ ID NOs: 1 and 2. Suitable primers and probes were 5 designed and used to analyse tissue expression. HIPHUM35 was found to be primarily expressed in adipose tissue, cerebellum, jejunum, lung, myometrium, omentum, prostate, small intestine and testis. Expression is upregulated in parasupranuclear palsy (PSP) brain, in chronic obstructive pulmonary disease (COPD) lung, VEGF treated endothelial cells and peripheral blood mononuclear 10 cells (PBMCs). Expression is downregulated in colon tumour, breast tumour and lung carcinoma.

Original screens on normal and disease Taqman plates revealed significant expression profiles to link HIPHUM 35 with diseases such as CNS diseases, for example parasupranuclear palsy (PSP), respiratory diseases such as chronic 15 obstructive pulmonary disease (COPD), inflammatory respiratory diseases such as fibrotic diseases of the lung and cancers such as lung, colon, breast.

HIPHUM 35 was found to be localised to chromosome 10q25-q26. This locus has been associated with the occurrence of cancers. One report details the presence of 20 genes on 1 Oq25-qter that are associated with proliferation that may be involved in the early steps of tumorigenesis of the breast (Ojopi EP. Rogatto SR. Caldeira JR.

Barbieri-Neto J. Squire JA. Comparative genomic hybridization detects novel amplifications in fibroadenomas of the breast. Genes, Chromosomes & Cancer.

30(1):25-31, 2001 Jan.). It has also been suggest that tumour suppresser genes are 25 localised on lOq25-q26, a locus that has been associated with patients suffering from endometrial carcinoma (Palmieri G. Manca A. Cossu A. Ruiu G. Pisano M. Cherchi P. Dessole S. Pintus A. Massarelli G. Tanda F. Pirastu M. Microsatellite analysis at 10q25-q26 in Sardinian patients with sporadic endometrial carcinoma: identification of specification patterns of genetic alteration. Cancer. 89(8):1773-82, 2000 Oct 15.).

30 Furthermore, frequent loss of a specific chromosomic region in cancers is often associated with the inactivation of a tumor-suppressor gene. The long arm of chromosome 10 (lOq) is deleted in several types oftumour including squamous-cell carcinomas present in the respiratory tract (Gasparotto D. Vukosavljevic T. Piccinin

S. Barzan L. Sulfaro S. Armellin M. Boiocchi M. Maestro R. Loss of heterozygosity at 1 0q in tumors of the upper respiratory tract is associated with poor prognosis. International Journal of Cancer. 84(4) :432-6, 1999 Aug 20.). A final report also details deletions on chromosome 10q25-q26 were highly significant for 5 metastasising carcinoma's (Bockmuhl U. Petersen S. Schmidt S. Wolf G. Jahnke V. Dietel M. Petersen I. Patterns of chromosomal alterations in metastasizing and nonmetastasizing primary head and neck carcinomas. Cancer Research.

57(23):5213-6, 1997 Dec 1.).

10 As Taqman screening of the disease plate showed significant levels of expression in patients suffering from lung, breast and colon cancers, secondary plates were screened to analyse this further. The data collected from breast-, lung- and colon-

tumour plates corroborated significant fold changes in HIPHUM 35 expression in tumour versus normal (or non tumour but taken from same patient) samples taken 15 from cancer patients. The strongest correlation is measured from the breast tumour plate with expression levels for 1 1 out of 12 of the tumour samples being significantly different from normal (non-tumour) tissue (ie., significantly higher or lower). Similarly out of the 20 lung tumour samples, 10 contained significantly different levels of HIPHUM 35 from the normal samples and from the 19 colon 20 tumour samples, signif cantly different levels of HIPHUM 35 were detected in 7 tumour samples in comparison to the equivalent normal samples.

As Taqman screening of the disease plate had showed significant levels of expression in lung, secondary plates were screened to analyse this further. The data 25 collected from lung- and leukocyte-plates corroborated significant expression in cell types central to respiratory diseases as well as other inflammatory conditions.

Hiphuin35 is expressed in many haematopoietic cells including monocytes, macrophages, dendritic cells, B cells and differentiated T cells. As such Hiphum35 is implicated in the function of these cells, which are involved in many pathological 30 responses in respiratory diseases such as asthma, COPD and allergic rhinitis, as well as a number of diseases including haemato-proliferative disorders such as lymphomas and leukemias; and conditions with an inflammatory component such as autoimmune diseases (for example rheumatoid arthritis, Crohn's, multiple sclerosis;

lupus), and inflammatory bowel disease or osteoarthritis.

E m le 2 Screenin for substances which exhibit rotein modulating activity xa p. p Preparations of a purified polypeptide of the invention are generated for 5 screening purposes. 96 and 384 well plate, high throughput screens (HTS) are employed using fluorescence or colourimetric indicator molecules, including but not limited to metalloprotease substrates such as GI 142612A (DNP-Pro-Cha-Gly-

Cys(Me)-His-Als-Lys(NMA)-NH2) and TES substrate (Ac-Pro-Leu-Gly-

SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2Hs) (Bachem). Also, amacroglobulin 1 0 trapping could be used as described in example 4 below. Secondary screening involves the same technology. Tertiary screens involve the study of modulators in rat, mouse and guinea-pig models of disease relevant to the target.

A brief screening assay protocol is as follows: A polypeptide of the invention is expressed in E. Coli, purified and refolded 15 by direct dilution in assay buffer (200mM NaCl, 50mM Tris, 5mM CaCl2, 10pM ZnSO4, 0. 01% Brij 35, pH 7.5). Test substances are provided in pools of 10 at 5mM for each test substance for high throughput screening or are serially diluted in dose response assays. The screening assay is run on an automated system incorporating an OCRA rail to move the plates, a Tecan liquid handler; a Multimek liquid handler 20 and a Titertek liquid handler. (The assay may also be run on manually or in combination with any suitable liquid handling equipment.) 60 pl of serially diluted test substance is added to 96 well plates (4 control wells with no inhibitor), 60 Ill of a solution of a polypeptide of the invention (0.5 M to 150 I1M) is then added to each well and 50 mM EDTA (20 'al of 0.5M EDTA) is 25 added to 4 inhibited control wells. The plates are then incubated at room temperature for 20 minutes before initiating with substrate (60 Ill GI 142612A for a fluorescence assay or 10 1 of DNTB (5,5'-dithio-bis(2nitrobenzoic acid) at 5mM in 150mM Tris base) followed by 10 p1 TES substrate for a colourimetric assay). In a fluorescence assay, the plates are read in a Fluostar (SLT) fluorescence plate reader or equivalent 30 at an excitation of 343 nm and an emission of 450 nm. In a colourimetric assay the plates are read continuously in a SLT spectrophotometer at 405 nm for 3 minutes.

Percentage inhibition is calculated for each concentration (unknown values = U) in the dose response based on the range determined using the value of the

difference of the control (no test substance) (mean of 4 = Cl) and the EDTA treated wells (mean of 4 = C2) using the equation 100*(1-(U-C2)/(C1C2).

Example 3: Construction of Fraements of Hiphum 35 5 Full Length Hiphum 35 H = Hinge region The signal domain runs from amino acid O to amino acid 39. The propetide domain 10 runs from amino acid 40 to amino acid 142. The catalytic domain runs from amino acid 143 to amino acid 324. There is a polymorphism at position 191 of the catalytic domain. In Seq ID No: 1 this is shown as a valine, however in some individuals, this amino acid may be an alanine. The Hinge domain runs from amino acid 325 to amino acid 339. The Hemopexin domain runs from amino acid 340 to amino acid 15 569 the end of the polypeptide.

Construct 1: ( nucleotide sequence 1256 to 1918 and peptide sequence 140 to 360) -( 1256bp - 1918bp) HipHum 35 construct no. 1 was designed to include primarily the catalytic domain.

There is some extension beyond the catalytic domain into the hinge region and the construct terminates at the start of the hemopexin domain. Sole expression of the catalytic domain for other MMPs, such as MMP12, has resulted in active 25 recombinant protein and the design of construct 1 is based on this work. Two versions have been prepared, one of which has a six histidine tag fused at the amino terminal to aid purification and the other is a native construct with no tag at all. Both have been designed to be expressed in a bacterial system.

Construct 2: ( nucleotide sequence 1256 to 1816 and peptide sequence 140 to 326) (1256bp- 1816bp)

HipHum 35 construct no.2 is a modification of construct no.1. It was seenthat construct 1 included a second cysteine residue located in the hinge region, the first cysteine being within the catalytic domain. There was a suggestion that this second 5 cysteine residue could form di-sulphide bonds and prevent the correct re-folding of Hiphum 35 and cause the functionality of the recombinant protein to be affected.

Construct 2 has been shortened by 102 nucleotides at the c terminal end in order to remove the cysteine and now terminates two residues into the hinge region. A six histidine tag has been fused to the amino terminal and as before the construct has 1 0 been designed to be expressed in a bacterial system.

Construct 3: (nucleotide sequence 956 to 1918) | Pro _ The closest human family member to Hiphum 35 is MMP 19 and this protease has been successfully expressed and characterised by constructing a recombinant protein comprising the propeptide and catalytic domain. In order to enhance correct protein folding and activity construct 3 is based on MMP19 work and is being designed to 20 include both the pro and catalytic domains.

Example 4: Evidence for Protease Activity Protease activity can be confirmed by a2Macroglobulin trapping, see Loechel, F et al. (1999) J. Biol.Chem. 274, 13427 - 13433. Human a2Macroglobulin is a plasma 25 glycoprotein with a molecular mass of 725kDa and consists of four identical subunits of about 180kDa. It binds to and inhibits most endopeptidases regardless of their substrate specificity.

The protease cleaves a 'bait' region of cc2M located near the middle of the subunit.

30 This event triggers a large conformational change in a2M and entraps the protease.

The protein gel shown in figure 1 shows an additional band of >200kDa in the HH35/a2M sample, this could represent HH35 entrapped within an a2M subunit. The

recombinant HH35 protein used in this assay included the catalytic domain only, ie construct 2.

SEQUENCE LISTING

<110> GLAXO GROUP LTO

5 <120> PROTEIN

<130> QG 10210

<140> 10 <141>

<160> 2

<170> PatentIn Ver. 2.1 <210> 1

<211> 1707

<212i DNA <213> Homo sapiens <220> <221> CDS

<222> (1) (1707).

25 <400> 1.

atg ctc gcc gcc tcc ale ttc cgt ccg ace ctg ctg ctc tgc tgg ctg 48 Met Leu Ala Ala Ser Ile Phe Arg Pro Thr Leu Leu Leu Cys Trp Leu 1 5 10 15

30 gct gct ccc tgg ccc acc cag ccc gag agt ctc ttc cac agc cgg gac 96 Ala Ala Pro Trp Pro Thr Gln Pro Glu Ser Leu Phe His Ser Arg Asp 20 25 30

cgc leg gac ctg gag ccg tcc cca ctg cgc cag gcc aag ccc att gcc 144 35 Arg Ser Asp Leu Glu Pro Ser Pro Leu Arg Gln Ala Lys Pro Ile Ala - 35 40 45

gac ctc cac gct gct cag cgg ttc ctg tcc age tac ggc tgg lea 999 192 Asp Leu His Ala Ala Gln Arg Phe Leu Ser Arg Tyr Gly Trp Ser Gly 40 50 55 60

gtg tgg gcg gcc tgg 999 ccc agt ccc gag 999 ccg ccg gag acc ccc 240 Val Trp Ala Ala Trp Gly Pro Ser Pro Glu Gly Pro Pro Glu Thr Pro 65 70 75 80

aag ggc gcc gcc ctg gcc gag gcg gtg cgc agg ttc cag cgg gcg aac 288 Lys Gly Ala Ala Leu Ala Glu Ala Val Arg Arg Phe Gln Arg Ala Asn 85 90 95

50 gcg ctg ccg gcc agc 999 gag ctg gac gcg gcc acc cta gcg gcc atg 336 Ala Leu Pro Ala Ser Gly Glu Leu Asp Ala Ala Thr Leu Ala Ala Met 100 105 110

aac cgg ccg cgc tgc 999 gtc ccg gac atg cgc cca ccg ccc ccc tcc 384 55 Asn Arg Pro Arg Cys Gly Val Pro Asp Met Arg Pro Pro Pro Pro Ser 115 120 125

gcc ccg cct leg ccc ccg ggc ccg ccc ccc age gcc cgc tcc agg cgc 432 Ala Pro Pro Ser Pro Pro Gly Pro Pro Pro Arg Ala Arg Ser Arg Arg 130 135 140

tcc ccg cgg gcg ccg ctg tcc ttg tcc cgg cgg ggt tgg cag ccc cgg 480 Ser Pro Arg Ala Pro Leu Ser Leu Ser Arg Arg Gly Trp Gln Pro Arg 145 150 155 160

10 ggc tac ccc gac ggc gga gct gcc cag gcc ttc tcc aag agg acg ctg 52 Gly Tyr Pro Asp Gly Gly Ala Ala Gln Ala Phe Ser Lys Arg Thr Leu 165 170 175

agc tgg cgg ctg ctg ggc gag gcc ctg apc agc caa ctg tcc gtg gcc 576 15 Ser Trp Arg Leu Leu Gly Glu Ala Leu Ser 5er Gln Leu Ser Val Ala 180 185 190

gac cag cgg cgc att gtg gcg ctg gcc ttc agg atg tgg agc gag gtg 624 Asp Gln Arg Arg Ile Val Ala Leu Ala Phe Arg Met Trp Ser Glu Val 20 195 200 205

acg ccg ctg gac ttc cgc gag gac ctg gcc gcc ccc ggg gcc gcg gtc 672 Thr Pro Leu Asp Phe Arg Glu Asp Leu Ala Ala Pro Gly Ala Ala Val 210 215 220

gac ale aag ctg ggc ttt ggg age ggc cgg cac ctg ggc tgt ccg cgg 720 Asp Ile Lys Leu Gly Phe Gly Arg Gly Arg His Leu Gly Cys Pro Arg 2?5 230 235 240

30 gcc ttc get ggg agc ggg cag gag ttt gca cac gcc tgg cgc cta ggt 768 Ala Phe Asp Gly Ser Gly Gln Glu Phe Ala His Ala Trp Arg Leu Gly 245. 250 255

gac att cac ttt gac gac gac gag cac ttc ace cct ccc acc agt gac 816 35 Asp Ile His Phe Asp Asp Asp Glu His Phe Thr Pro Pro Thr Ser Asp 260 265 270

acg ggc ale agc ctt ctc aag gtg gcc gtc cat gee att ggc cat gtc 864 Thr Gly Ile Ser Leu Leu Lys Val Ala Val His Glu Ile Gly His Val 40 275 280 285

ctg ggc ttg cct cac acc tac agg acg gga tcc ata atg caa cca aet 912 Leu Gly Leu Pro His Thr Tyr Arg Thr Gly Ser Ile Met Gln Pro Asn 290 295 300

tac att ccc cag gag cct gcc ttt gag ttg gac tgg lea gac agg aaa 960 Tyr Ile Pro Gln Glu Pro Ala Phe Glu Leu Asp Trp Ser Asp Arg Lys 305 310 315 320

5O gca att caa aag ctg tat ggc tcc tgt gag gga lea ttt get act gcg 1008 Ala Ile Gln Lys Leu Tyr Gly Ser Cys Glu Gly Ser Phe Asp Thr Ala 325 330 335

ttt gac tgg att cgc aaa gag age aac caa tat gga gag gtg atg gtg 1056 55 Phe Asp Trp Ile Arg Lys Glu Arg Asn Gln Tyr Gly Glu Val Met Val 340 345 350

age ttt agc ace tat ttc ttc cgt aac aDc tgg tac tgg ctt tat gaa 1104 Arg Phe Ser Thr Tyr Phe Phe Arg Asn Ser Trp Tyr Trp Leu Tyr Glu 355 360 365

5 eat cga aac eat agg ace cgc tat ggg gac cct ale caa ale ctc act 1152 Asn Arg Asn Asn Arg Thr Arg Tyr Gly Asp Pro Ile Gln Ile Leu Thr 370 375 380

ggc tgg cct gga ale cca ace cac aac ata get gcc ttt gtt cac ale 1200 10 Gly Trp Pro Gly Ile Pro Thr His Asn Ile Asp Ala Phe Val His Ile 385 390 395 400

tgg ace tgg aaa age get gaa cgt tat ttt ttt caa gga eat caa tac 1248 Trp Thr Trp Lys Arg Asp Glu Arg Tyr Phe Phe Gln Gly Asn Gln Tyr 15 405 410 415

tgg age tat gac agt gac aag get cag gcc ctc ace gaa get gaa caa 1296 Trp Arg Tyr Asp Ser Asp Lys Asp Gln Ala Leu Thr Glu Asp Glu Gln 420 425 430

gga aaa agc tat ccc aaa ttg att lea gaa gga ttt cct ggc ale cca 1344 Gly Lys Ser Tyr Pro Lys Leu Ile Ser Glu Gly Phe Pro Gly Ile Pro 435 440 445

25 agt ccc cta gac acg gcg ttt tat gac cga age cag aag tta att tac 1392 Ser Pro Leu Asp Thr Ala Phe Tyr Asp Arg Arg Gln Lys Leu Ile Tyr 450 455 460

ttc ttc aag gag tcc ctt gta ttt gca ttt get gtc aac age aet cga 1440 30 Phe Phe Lys Glu Ser Leu Val Phe Ala Phe Asp Val Asn Arg Asn Arg 465 470 475 480

gta ctt eat let tat cca aag agg att act gaa gtt ttt cca gca gta 1488 Val Leu Asn Ser Tyr Pro Lys Arg Ile Thr Glu Val Phe Pro Ala Val 35 485 490 495

- ata cca caa eat cat cct ttc age eat ata get tcc gct tat tac tcc 1536 Ile Pro Gln Asn His Pro Phe Arg Asn Ile Asp Ser Ala Tyr Tyr Ser 500 505 510

tat gca tac aac tcc att ttc ttt ttc aaa ggc eat gca tac tgg aag 1584 Tyr Ala Tyr Asn Ser Ile Phe Phe Phe Lys Gly Asn Ala Tyr Trp Lys 515 520 525

45 gta gtt eat gac aag gac aaa caa cag eat tcc tgg ctt cct gct eat 1632 Val Val Asn Asp Lys Asp Lys Gln Gln Asn Ser Trp Leu Pro Ala Asn 530 535 540

ggc tta ttt cca aaa aag ttt att lea gag aeg tgg ttt get gtt tgt 1680 50 Gly Leu Phe Pro Lys Lys Phe Ile Ser Glu Lys Trp Phe Asp Val Cys 545 550 555 560

gac gtc cat ale tcc ace ctg aac atg 1707 Asp Val His Ile Ser Thr Leu Asn Met 55 565

<210> 2

<211> 569

<212> PRT

<213> Homo sapiens <400> 2

Met Leu Ala Ala Ser Ile Phe Arg Pro Thr Leu Leu Leu Cys Trp Leu 1 5 10 15

10 Ala Ala Pro Trp Pro Thr Gln Pro Glu Ser Leu Phe His Ser Arg Asp 20 25 30

Arg Ser Asp Leu Glu Pro Ser Pro Leu Arg Gln Ala Lys Pro Ile Ala 35 40 45

Asp Leu His Ala Ala Gln Arg Phe Leu Ser Arg Tyr Gly Trp Ser Gly 50 55 60

Val Trp Ala Ala Trp Gly Pro Ser Pro Glu Gly Pro Pro Glu Thr Pro 20 65 70 75 80

Lys Gly Ala Ala Leu Ala Glu Ala Val Arg Arg Phe Gln Arg Ala Asn 85 90 95

25 Ala Leu Pro Ala Ser Gly Glu Leu Asp Ala Ala Thr Leu Ala Ala Met 100 105 110

Asn Arg Pro Arg Cys Gly Val Pro Asp Met Arg Pro Pro Pro Pro Ser 115 120 125

Ala Pro Pro Ser Pro Pro Gly Pro Pro Pro Arg Ala Arg Ser Arg Arg 130 135 140

Ser Pro Arg Ala Pro Leu Ser Leu Ser Arg Arg Gly Trp Gln Pro Arg 35 145 150 155 160

Gly Tyr Pro Asp Gly Gly Ala Ala Gln Ala Phe Ser Lys Arg Thr Leu 165 170 175

40 Ser Trp Arg Leu Leu Gly Glu Ala Leu Ser Ser Gln Leu Ser Val Ala 180 185 190

Asp Gln Arg Arg Ile Val Ala Leu Ala Phe Arg Met Trp Ser Glu Val 195 200 205

Thr Pro Leu Asp Phe Arg Glu Asp Leu Ala Ala Pro Gly Ala Ala Val 210 215 220

Asp Ile Lys Leu Gly Phe Gly Arg Gly Arg His Leu Gly Cys Pro Arg 50 225 230 235 240

Ala Phe Asp Gly Ser Gly Gln Glu Phe Ala His Ala Trp Arg Leu Gly 245 250 255

55 Asp Ile His Phe Asp Asp Asp Glu His Phe Thr Pro Pro Thr Ser Asp 260 265 270

t Thr Gly Ile Ser Leu Leu Lys Val Ala Val His Glu Ile Gly His Val 275 280 285

Leu Gly Leu Pro His Thr Tyr Arg Thr Gly Ser Ile Met Gln Pro Asn 5 290 295 300

Tyr Ile Pro Gln Glu Pro Ala Phe Glu Leu Asp Trp Ser Asp Arg Lys 305 310 315 320

10 Ala Ile Gln Lys Leu Tyr Gly Ser Cys Glu Gly Ser Phe Asp Thr Ala 325 330 335

Phe Asp Trp Ile Arg Lys Glu Arg Asn Gln Tyr Gly Glu Val Met Val 340 345 350

Arg Phe Ser Thr Tyr Phe Phe Arg Asn Ser Trp Tyr Trp Leu Tyr Glu 355 360 365

Asn Arg Asn Asn Arg Thr Arg Tyr Gly Asp Pro Ile Gln Ile Leu Thr 20 370 375 380

* Gly Trp Pro Gly Ile Pro Thr His Asn Ile Asp Ala Phe Val His Ile 385 390 395 400

25 Trp Thr Trp Lys Arg Asp Glu Arg Tyr Phe Phe Gln Gly Asn Gln Tyr 405 410 415

Trp Arg Tyr Asp Ser Asp Lys Asp Gln Ala Leu Thr Glu Asp Glu Gln 420 425 430

Gly Lys Ser Tyr Pro Lys Leu Ile Ser Glu Gly Phe Pro Gly Ile Pro 435 440 445

Ser Pro Leu Asp Thr Ala Phe Tyr Asp Arg Arg Gln Lys Leu Ile Tyr 35 450 455 460

Phe Phe Lys Glu Ser Leu Val Phe Ala Phe Asp Val Asn Arg Asn Arg 465 470 475 480

40 Val Leu Asn Ser Tyr Pro Lys Arg Ile Thr Glu Val Phe Pro Ala Val 485 490 495

Ile Pro Gln Asn His Pro Phe Arg Asn Ile Asp Ser Ala Tyr Tyr Ser 500 505 510

Tyr Ala Tyr Asn Ser Ile Phe Phe Phe Lys Gly Asn Ala Tyr Trp Lys 515 520 525

Val Val Asn Asp Lys Asp Lys Gln Gln Asn Ser Trp Leu Pro Ala Asn 50 530 535 540

Gly Leu Phe Pro Lys Lys Phe Ile Ser Glu Lys Trp Phe Asp Val Cys 545 550 555 560

55 Asp Val His Ile Ser Thr Leu Asn Met

Claims (16)

- CLAIMS

1. An isolated matrix metalloprotease polypeptide comprising (i) the amino acid sequence of SEQ ID NO: 2 or 5 (ii) a variant thereof which comprises a catalytic domain capable of binding a zinc residue or..

(iii) a fragment of (i) or (ii) which comprises a catalytic domain capable of binding a zinc residue.

2. A polypeptide according to claim 1 wherein the variant (ii) has at least 10 80% identity to the amino acid sequence of SEQ ID NO: 2.

3. A polynucleotide encoding a polypeptide according to claim 1 or 2.

4. A polynucleotide according to claim 3 which is a cDNA sequence.

5. A polynucleotide encoding a matrix metalloprotease polypeptide which comprises a catalytic domain capable of binding a zinc residue which 15 polynucleotide comprises: (a) the nucleic acid sequence of SEQ ID NO: 1 andlor a sequence complementary thereto; (b) a sequence which hybridises under stringent conditions to a sequence as defined in (a); 20 (c) a sequence that is degenerate as a result of the genetic code to a sequence as defined in (a) or (b); or (d) a sequence having at least 65% identity to a sequence as defined in (a), (b) or (c).

6. An expression vector comprising a polynucleotide according to any 25 one of claims 3 to 5.

7. A host cell comprising an expression vector according to claim 6.

8. An antibody specific for a polypeptide according to claim 1 or 2.

9. A method for the identification of a substance that modulates matrix metalloprotease activity and/or expression, which method comprises: 30 (i) contacting a test substance and a polypeptide according to claim 1 or 2, a polynucleotide according to any one of claims 3 to 5, an expression vector according to claim 6 or a host cell according to claim 7, and

(ii) determining the effect of the test substance on the activity and/or expression of the said polypeptide or the polypeptide encoded by said polynucleotide, thereby to determine whether the test substance modulates matrix metalloprotease activity and/or expression.

5

10. A method according to claim 9 wherein the polypeptide is in a substantially isolated form.

11. A substance which modulates matrix metalloprotease activity and which is identifiable by a method according to claim 9 or 10.

12. A method of treating a subject having a disorder that is responsive to 10 matrix metalloprotease modulation, which method comprises administering to said subject an effective amount of a substance according to claim 11.

13. A method according to claim 12 wherein the disorder is selected from CNS diseases such as parasupranuclear palsy (PSP), respiratory diseases such as chronic obstructive pulmonary disease (COPD), inflammatory respiratory diseases 15 such as fibrotic diseases of the lung and cancers such as lung, colon, breast and endometrial carcinomas (DEC).

14. Use of a substance as defined in claim 11 in the manufacture of a medicament for treatment or prophylaxis of a disorder that is responsive to stimulation or modulation of matrix metalloprotease activity.

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15. A use according to claim 14 wherein the disorder is selected from CNS diseases such as parasupranuclear palsy (PSP), respiratory diseases such as chronic obstructive pulmonary disease (COPD), inflammatory respiratory diseases such as fibrotic diseases of the lung and cancers such as lung, colon, breast and endometrial carcinomas (DEC).

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16. A method of producing a polypeptide according to claim 1 or 2, which method comprises maintaining a host cell as defined in claim 7 under conditions suitable for obtaining expression of the polypeptide and isolating the said polypeptide.