EP1002104A1 - Novel metalloprotease - Google Patents

Abstract

Novel proteins ADAM 16a and ADAM 16b have been identified. They may be used, for example, as contraceptive vaccines or in screening.

Description

Novel Metalloprotease

The present invention relates inter alia to novel molecules and uses thereof.

Recently a new family of proteins, called ADAMs, was identified whose members are type I integral membrane proteins characterized by a disintegrin and metalloprotease domain (see Wolfsberg et al., 1995a; Wolfsberg et al., 1995b; Huovila et al., 1996 for recent reviews). The ADAMs are related to the crotalid snake venom disintegrin metalloproteases (SVMPs) and matrix metalloprotease (MMP) families, whose members lack transmembrane domains. Full-length ADAM cDNAs all encode a signal peptide followed by proprotein, Zn ⁺-metalloprotease, disintegrin, transmembrane region and cytoplasmic tail. Members of this family have been found in many animal species including mammals, Xenopus (Alfandari et al., 1997), Drosophila (Fambrough et al., 1996; Rooke et al., 1996) and nematodes (Podbilewicz, 1996; GenBank H89394), but none have as yet been found in unicellular eukaryotes or plants. Nor are ADAM genes found in the sequenced genomes of bacteria or S. cerevisiae.

In spite of the ADAM'S conservation of structural domains, they appear to play rather diverse roles in development.

ADAM 12 (meltrin-α) was shown to be involved in myoblast, and perhaps also osteoclast fusion (Yagami-Hiromasa et al., 1995). ADAM14, or Kuzbanian, is involved in axonal extension in Drosophila (Fambrough et a/., 1996; Rooke et al., 1996). The ADAM 11 encoding gene is rearranged in primary breast tumors (Emi ef a/., 1993).

Another ADAM, TACE (TNF-α convertase), is required for cleavage of TNF-α from its membrane-bound precursor (Black et al., 1997; Moss et al., 1997a; Moss et al., 1997b). ADAM 10 (MADAM) can degrade myelin basic protein, although it is not clear whether this is its physiological target (Howard et al., 1996).

ADAM 1 and -2, also named fertilin-α/β or PH30- /β are expressed as heterodimers on the posterior head of mammalian spermatocytes and play a role in oocyte adhesion and fusion (Wolfsberg et al., 1993; Myles et al., 1994; Carroll et al., 1995; Wolfsberg and White, 1996; Myles and Primakoff, 1997). In mouse, it has been shown that fertilin β binds the α6/β1 integrin on oocytes, and that this interaction is essential for sperm-egg binding (Almeida et al., 1995). This subunit encodes a non-functional metalloprotease domain, which is absent from the mature protein (Blobel et al., 1990). The α subunit encodes an active protease domain and encodes a putative fusion peptide postulated to be involved in cell-cell fusion (Blobel et al., 1992; White, 1992; Muga et al., 1994). Fertilins α and β have been found in rat, rabbit, mouse, macaque and two species of guinea pig. In humans only the β subunit has been cloned (Gupta et al., 1996; Vidaeus et al., 1997). Surprisingly, the only human fertilin α gene is non-functional (Jury et al., 1997).

The present invention, is based upon the identification of two polypeptides, which are members of the ADAM family and which are referred to herein as ADAM 16a and ADAM 16b. These polypeptides were identified unexpectedly during an attempt to find a completely different protein, known as a lymphocyte sheddase. Previous studies involving the direct screening of a human testis cDNA library with monkey fertilin-α has failed to yield any functional human, testic-specific ADAMs (Jury et al., 1997). The identification of ADAM 16a and ADAM 16b by the present inventor represents a major advance in the field of human fertility, the applications of which will be discussed later.

The putative amino acid sequences of ADAM 16a and ADAM 16b are provided in Figures 3A and 3B respectively, with the underlined portion of Figure 3A representing a signal sequence that is not present in the mature polypeptide .

The present invention includes within its scope not only ADAM 16a and ADAM 16b, but also derivatives thereof.

Thus, according to the present invention there is provided a polypeptide, which :

a) comprises the non-underlined amino acid sequence shown in Figure 3A or Figure 3B b) has one or more amino acid deletions, insertions or substitutions relative to the non-underlined amino acid sequence shown in Figure 3A or Figure 3B, but has at least 40% amino acid sequence identity with said sequence; or c) is a fragment of a polypeptide as defined in a) or b) above, which is at least 10 amino acids long.

[The term "polypeptide" is used herein in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds. It therefore includes within its scope substances, which may sometimes be referred to in the literature as peptides, polypeptides or proteins.]

ADAM16a and 16b were identified by the present inventor from cDNA studies and were shown to be closely related members of the ADAM family of membrane metalloproteases. Comparison of their predicted translation products with known ADAMs shows they are related to fertilin-α, fertilin-β and meltrin-γ (ADAM 1 , 2 and 9). Meltrin-γ mRNA is ubiquitously expressed (Yagami- Hiromasa et al., 1995) and not particularly up-regulated in testis (data not shown). ADAM 16a and ADAM 16b's exclusive expression in human testis and sequence similarity with fertilin-α and -β suggests they too are expressed on sperm cells and involved in sperm maturation and/or fertilization. In addition to fertilins, various other testis-specific ADAMs have been found in other species: ADAM 4 and 5 and cyritestin in mouse (Wolfsberg et al., 1995b), and tMDC l-IV in monkey (Perry et al., 1994; Perry et al., 1995b). ADAM 16a and b are less similar to these than to the fertilins, which suggests they represent a novel type of ADAMs, restricted to humans. Analysis of their domains suggest that ADAM 16a and b could be involved in adhesion to egg cells or play a role in sperm-egg fusion or (ADAM 16a) proteolytic processing of other fertilins. Since the only human fertilin α gene is non-functional, it is possible that ADAM 16a or ADAM 16b functionally replace this subunit. This is supported by the observations that, like fertilin-α, ADAM 16a cDNA encodes a functional protease domain and that both ADAM 16a and ADAM 16b encode potential fusion peptides, and are testis-specifically transcribed. In four different virus families that replicate via cell-fusion, as well as in the pore that mediates intra-Golgi transport, the basic pore-forming subunit is a trimer (see White, 1992 for a review). It is likewise possible that multiple proteins are involved in sperm-egg fusion, and that both ADAM 16a and -b are part of this complex.

ADAM 16a and -b's chromosomal location have been determined. It was suspected that their genes might be clustered with other ADAMs. However, the finding that both ADAM 16's map to chromosome 14 does not support this idea. Thus, human TACE maps to chromosome 2. ADAM 11 is on chromosome 17 (Emi et al., 1993) and fertilin β on chromosome 8 (Vidaeus et al., 1997). Finally, the genes encoding the two mouse fertilins α and β, ADAM 4 and 5 and cyritestin were mapped to locations that are syntenic with human chromosomes 12, 8, 6, 8 and 8, respectively (Lemaire et al., 1994; Cho et al., 1996). It is therefore not likely that ADAM 16a and -b share direct common ancestry with these five testis-specific murine ADAMs. The fact that both ADAM 16's map to the same locus on chromosome 14 strongly suggests that they arose by gene duplication.

Two observations suggest that the human fertilin α gene was inactivated only very recently in human evolution. First, it has been found as a functional gene in five other mammalian species, including another primate. Second, unlike most pseudogenes, human fertilin α RNA is still transcribed, in spite of its "coding region" carrying multiple out-of-frame mutations (Jury et al., 1997).

On the other hand, ADAM 16a and -b seem to have appeared recently in human evolution, since they do not have orthologs even in the well-studied macaque (Perry et al., 1994; Perry et al., 1995a; Perry et al., 1995b). The fact that both these events must have occurred after the branching of what would evolve into macaques and humans lends further weight to the idea that ADAM 16a and -b evolved to become human fertilin-α analogues.

The profusion of testis-specific ADAMs in mammals and their poor evolutionary conservation are surprising and are difficult to explain in terms of sperm fitness per se only. This situation contrasts sharply with the high conservation of, e.g., transcription factors (Li et al., 1992), but is more similar to genes associated with rapidly co-evolving host-pathogen systems, such as those involved in the immune response. A possible explanation is that sperm-associated ADAMs also play a role in direct intraspecies sperm competition through their protease or perhaps integrin-binding activities. Such a role would especially have been relevant before humans evolved concealed oestrus.

Whatever the reason for this evolutionary drift, a consequence of testis ADAMs' diversity is that non-steroid-based contraceptives or immunocontraceptive antigens may be based on the polypeptides of the present invention rather than animal models. For drug development it may be an asset that testis-specific ADAMs are composed of distinct functional domains with clearly identifiable functions.

Various aspects of the present invention will now be discussed in order that its full scope can be appreciated.

Preferred polypeptides of the present invention are those which include one or more of the following functional regions:

a) a metalloprotease region (This is thought to be involved in autocatylytic cleavage or in processing of fertilin β) b) a disintegrin region. (This is thought to be involved in recognising oocytes) c) a fusion peptide region. (This is thought to be involved in sperm-egg fusion)

Other regions may also be present, e.g. one or more of the following regions may be present:

d) a signal peptide region e) a proprotein region f) a disintegrin region g) a cysteine-rich region h) an EGF-like repeat region i) a transmembrane region j) a cytoplasmic region

A skilled person is able to determine whether or not any of the regions a) to j) listed above are present in a given polypeptide. These regions are easiest determined by sequence comparisons. Sequences are aligned using appropriate software such as BLAST, BestFit or CLUSTAL as is, for instance, integrated into the Wisconsin Sequence Analysis Package GCG 8.0, or in "Align Plus" from Educational and Scientific Software. A query sequence can be compared to a sequence database, for instance the GenBank of EMBL public databases. An example alignment of fertilins is given in Wolfsberg et al., 1995b.

It will be appreciated that the presence of all of the above regions is not essential in order for polypeptides of the present invention to be of utility. For example, signal peptide regions and proprotein region are not present in mature, active polypeptides; a transmembrane region is not required where it is desired to provide a soluble rather than a membrane-bound polypeptide; a cytoplasmic region is not needed if cytoplasmic activity does not occur or is not required for a given function; etc.

Polypeptides of the present invention may be produced by techniques known to those skilled in the art. For example gene-cloning techniques may be used to provide a nucleic acid sequence encoding such a polypeptide. Such techniques are discussed in greater detail later in relation to nucleic acid molecules of the present invention.

Alternatively, chemical synthesis techniques may be used to produce polypeptides of the present invention. Such techniques generally utilise solid phase synthesis. Chemical synthesis techniques that allow polypeptides having particular sequences to be produced have now been automated. Apparatuses capable of chemically synthesising long polypeptides are available, for example, from Applied Biosystems. If desired however, short polypeptides can be synthesised initially and can then be ligated to produce longer polypeptides. A polypeptide of the present invention may be provided in substantially pure form. Thus it may be provided in a composition in which it is the predominant polypeptide component present (i.e. where it is present at a level, when determined on a weight/weight basis, of more than 50% of the total polypeptide present in the composition; preferably at a level of more than 75%, of more than 90%, or of more than 95% of the total polypeptide present in the composition).

As explained previously, a polypeptide of the present invention either:

a) comprises the non-underlined amino acid sequence shown in Figure 3A or

Figure 3B b) has one or more amino acid deletions, insertions or substitutions relative to the non-underlined amino acid sequence shown in Figure 3A or Figure 3B, but has at least 40% amino acid sequence identity with said sequence; or c) is a fragment of a polypeptide as defined in a) or b) above, which is at least 10 amino acids long.

In order to appreciate the present invention more fully, polypeptides within the scope of each of a), b) and c) above will now be discussed in greater detail.

Polypeptides within the scope of a)

A polypeptide within the scope of a) may consist of the non-underlined amino acid sequence shown in Figure 3A or Figure 3B, or may have an additional N-terminal and/or an additional C-terminal amino acid sequence.

Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene-cloning techniques to ligate together nucleic acid molecules encoding polypeptides or parts thereof, followed by expressing a polypeptide encoded by the nucleic acid molecule produced by ligation.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al., Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification, transportation or purification.

For example, a signal sequence may be present to direct the transport of the polypeptide to a particular location within a cell or to export the polypeptide from the cell (e.g. the underlined part of Figure 3A may be present). Different signal sequences can be used for different expression systems.

Another example of the provision of an additional sequence is where a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments that bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer.

Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a substance of the present invention and need not provide any particular advantageous characteristic.

Polypeptides within the scope ofb)

Turning now to the polypeptides defined in b) above, it will be appreciated by the person skilled in the art that these are variants of the polypeptides given in a) above.

The skilled person will appreciate that various changes can sometimes be made to the amino acid sequence of a polypeptide which has a particular activity to produce variants (often known as "muteins") which still have said activity. Such variants of the polypeptides described in a) above are within the scope of the present invention and are discussed in greater detail below in sections (1) to (iii). They include allelic and non-allelic variants. (i) Substitutions

An example of a variant of the present invention is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids with one or more other amino acids.

The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a polypeptide can often be substituted by one or more other such amino acids without eliminating a desired property of that polypeptide.

For example, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic).

Other amino acids that can often be substituted for one another include:

phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).

Substitutions of this nature are often referred to as "conservative" or "semi- conservative" amino acid substitutions.

(ii) Deletions Amino acid deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining a desired property. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example if the polypeptide is to be used in medicine, dosage levels can be reduced. As explained above the polypeptides shown in Figures 3A and 3B have various distinct regions and, for various applications of the present invention, many of these regions will not be needed.

(Hi) Insertions

Amino acid insertions relative to a polypeptide as defined in a) above can also be made. This may be done to alter the properties of the polypeptide (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).

Polypeptides incorporating amino acid changes (whether substitutions, deletions or insertions) relative to the sequence of a polypeptide as defined in a) above can be provided using any suitable techniques. For example, a nucleic acid sequence incorporating a desired sequence change can be provided by site directed mutagenesis. This can then be used to allow the expression of a polypeptide having a corresponding change in its amino acid sequence.

Whatever amino acid changes may be made, preferred polypeptides of the present invention have at least 50% amino acid sequence identity with the non- underlined amino acid sequence shown in Figure 3A or Figure 3B, more preferably the degree of sequence identity is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred. Where high degrees of sequence identity are present there may be relatively few differences in amino acid sequence. Thus for example there may be less than 20, less than 10, or even less than 5 differences.

For the purposes of the present invention sequence identity can be determined by optimally aligning two sequences, using for instance "BestFit" in the Wisconsin Sequence Analysis Package GCG 8.0.

Polypeptides within the scope of )

As discussed supra, it is often advantageous to reduce the length of a polypeptide. Feature c) of the present invention therefore covers fragments of the polypeptides a) or b) above which are at least 10 amino acids long. Desirably these fragments are at least 20, at least 50 or at least 100 amino acids long.

Preferred Polypeptides of the Present Invention

Preferred polypeptides within the scope of the present invention include the polypeptides ADAM 16a and ADAM 16b consisting of the amino acid sequences shown in Figures 3 A and 3B respectively; larger polypeptides comprising these sequences; fragments of ADAM 16a and ADAM 16b; and chimaeras comprising all or part of ADAM 16a fused to all or part of ADAM 16b.

Uses of Polypeptides of the Present Invention

A) Medical Uses Polypeptides of the present invention may be used in medicine. Preferred treatments are human treatments, although veterinary treatments are not excluded. The treatment may be prophylactic or may be in respect of an existing condition. Examples include:

Contraceptive Treatment

Polypeptides within the scope of the present invention are believed to be associated with important functions of sperm cells. For example, polypeptides comprising a disintegrin region may be involved in recognising oocytes; polypeptides comprising a fusion peptide region may be involved in sperm-egg fusion; and polypeptides comprising a metalloprotease region may be involved in autocatalytic cleavage or in the processing of fertilin β.

Polypeptides of the present invention can be used to provide contraceptive vaccines, which may be effective in males and/or females by promoting an immune response that may block or at least reduce the effectivity of one or more of the functions discussed above. Polypeptides used in a vaccine will comprise one or more epitopes. One or more polypeptides of the present invention may be present in a single vaccine. Thus multi-component vaccines may be provided (e.g. all or part of ADAM16a may be present together with all or part of ADAM16b).

A polypeptide comprising one or more epitopes may be fused to another polypeptide that need not contain any epitopes present on ADAM 16a or ADAM 16b to provide a fusion polypeptide with improved antigenicity.

Hybrid molecules comprising all or part of ADAM 16a fused to all or part of ADAM16b may be provided.

Fertility Treatment

Polypeptides of the present invention may be used to assist in in vitro fertilization. Thus they may be provided in combination with spermatozoa and/or oocytes.

A polypeptide of the present invention may be used in the manufacture of a medicament for any of the treatments mentioned above.

The medicament will usually be supplied as part of a pharmaceutical composition, which may include a pharmaceutically acceptable carrier. This pharmaceutical composition will generally be provided in a sterile form in a sealed container. It may be provided in unit dosage form, will generally be provided in a sealed container, and can be provided as part of a kit. Such a kit is within the scope of the present invention. It would normally (although not necessarily) include instructions for use. A plurality of unit dosage forms may be provided.

Pharmaceutical compositions within the scope of the present invention may include one or more of the following: preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odourants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants. They may also contain therapeutically active agents in addition to polypeptides of the present invention. They may be provided in controlled release form, e.g. so as to be effective over a period of at least a week, or, more preferably, of at least a month. Preferred pharmaceutical compositions of the present invention include contraceptive vaccines. Desirably a vaccine of the present invention comprises an adjuvant, e.g. an aluminum-salt based adjuvant.

Routes of Administration

The pharmaceutical compositions within the scope of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such a composition may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a suitable carrier under sterile conditions.

Dosages

Dosages of a polypeptide of the present invention can vary between wide limits, depending upon the nature of the treatment, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. However, without being bound by any particular dosages, for oral administration a daily dosage of a polypeptide of the present invention of from 1μg to 1 mg/kg body weight may be suitable. The dosage may be repeated as often as appropriate. If side effects develop then the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.

B) Diagnostics Uses

In addition to the medical uses discussed above, polypeptides of the present invention can be used in diagnosis. For example they may be useful in diagnosing infertility. Some infertile men and women are known to carry (auto-) antibodies against certain gamete antigens. The polypeptides of the present invention could be used in identifying such antibodies.

C) Screening Uses Polypeptides of the present invention can also be used in screening. For example, they can be used to screen for contraceptive agents that block sperm fertility.

A polypeptide comprising a protease region (e.g. a metalloprotease region, such as a zinc protease) can be expressed in a micro-organism and suitable targets selected using a phage display assay. (This technique involves a library of phages each carrying a random peptide sequence in an external "spike"). These tools can be used to screen for compounds that inhibit the protease. (See e.g. Smith, M.M., Shi, L. and Navre, M., (1995) Rapid identification of highly active and selective substrates for stromelysin and matήlysin using bacteriophage peptide display libraries. J. Biol. Chem. 270 (12) 6440-6449; Matthews, D.J. and Wells, J.A. (1993) Substrate phage: selection of protease substrates by monovalent phage display. Science 260 (5111) 1113-1117; or Matthews, D.J., Goodman, L.J., Gorman, CM. and Wells, J.A. (1994) A survey of furin substrate specificty using substrate phage display. Protein Sci. 3(8) 1197-1205). Once such a target has been found, it is possible to fix a dye covalently linked with the target peptide to a microtiter plate and incubate this with ADAM 16 protease. If a chemical inhibits the protease, the dye remains bound to the ELISA plate, otherwise it can be washed out. This type of screen is commonplace in the pharmaceutical industry to randomly screen automatically hundreds of thousands of different compounds.)

Similarly, a polypeptide of the present invention can be used to screen for compounds that inhibit binding to a ligand. The polypeptide may comprise a disintegrin domain, although this is not essential. For instance, a polypeptide comprising a disintegrin domain or other region of an ADAM 16 is linked with a dye, and added to a microtiter plate which is coated with the oocyte structure that serves as its ligand. One can then search for compounds that inhibit the staining of wells with the dye. This need not necessarily involve the "disintegrin" domain, as recent work has showed that fertilin α polypeptides lacking this domain are still capable of binding oocytes. (Evans, J.P., Schutz, R.M., and

Kopf, G.S., (1997) Characterisation of the binding of recombinant mouse sperm fertilin α subunit to mouse eggs: Evidence for function as a cell adhesion molecule in sperm-egg binding. Dev. Biol. 187 94-106.) Also, a polypeptide comprising a fusion peptide region can be used to induce fusion of artificial liposomes containing a fluorescent dye. Similar experiments have already been done for fertilin α from other species. (See e.g. Martin, I. And Ruysschaert, J.M., (1997) Comparison of lipid vesicle fusion induced by the putative fusion peptide of fertilin (a protein active in sperm-egg fusion) and the NH2-terminal domain of the HIV2 gp41. FEBS Lett. 405(3) 351-355.) Such a model is relatively easy to convert into a drug screen. Such a screen could involve lipid vesicles containing fluorescent compounds, such as described by Martin et al (1997). It has been shown that mouse fertilin α peptides can induce vesicle fusion, an event that can be measured by a change in fluorescence. A similar model using the human ADAM 16 fusogenic peptide could be set up for the industrial screening of large numbers of compounds that inhibit this fusion. Most of the manipulation in this type of work is now done by robots, using 256- well microtiter plates.

Preferred screening procedures of the present invention are automated.

D) Uses in Raising or Selecting Antibodies

One further use of the polypeptides of the present invention is in raising or selecting antibodies.

The present invention therefore includes antibodies that bind to a polypeptide of the present invention. Preferred antibodies bind specifically to polypeptides of the present invention.

Antibodies within the scope of the present invention may be monoclonal or polyclonal.

Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when the substance of the present invention is injected into the animal. If necessary, an adjuvant may be administered together with the substance of the present invention. The antibodies can then be purified by virtue of their binding to a substance of the present invention.

Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing myeloma cells and spleen cells which produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique {Nature 256 52-55 (1975)) or variations upon this technique can be used.

Techniques for producing monoclonal and polyclonal antibodies which bind to a particular polypeptide are now well developed in the art. They are discussed in standard immunology textbooks, for example in Roitt et al, Immunology second edition (1989), Churchill Livingstone, London.

In addition to whole antibodies, the present invention includes derivatives thereof which are capable of binding to polypeptides of the present invention. Thus the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12 372-379 (September 1994).

Antibody fragments include, for example, Fab, F(ab')₂ and Fv fragments (these are discussed in Roitt et al [supra], for example). Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V_h and V, regions, which contribute to the stability of the molecule. Other synthetic constructs which can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains.

Synthetic constructs include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Synthetic constructs also include molecules comprising an additional moiety which provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label). Alternatively, it may be a pharmaceutically active agent.

The antibodies or derivatives thereof of the present invention have a wide variety of uses in addition to their use in purification of polypeptides discussed above.

They can be used in therapy. For example they may be used :

a) to provide passive contraceptive immunisation (cf the contraceptive vaccine discussed above using proteins of the present invention to stimulate an immune response) or b) in fertility treatment to select and/or concentrate competent sperm cells ( e.g. by FACS)

They can be used in diagnosis. For example they may be used :

to diagnose infertility by being used to determine whether or not polypeptides within the scope of the present invention (especially ADAM 16a or ADAM 16b or a part thereof) are present in an individual. If such polypeptides are not present in functional form then the individual may be infertile or of low fertility.

Preferred antibodies or derivatives thereof bind to ADAM 16a or ADAM 16b or to a part thereof.

Nucleic Acid Molecules of the Present Invention and Uses Thereof

Nucleic Acids

The present invention also includes nucleic acid molecules within its scope.

Such nucleic acid molecules:

a) code for a polypeptide according to the present invention; or b) are complementary to molecules as defined in a) above; or c) hybridise to molecules as defined in a) or b) above.

These nucleic acid molecules and their uses will now be discussed in greater detail below:

The polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well-known degeneracy of the genetic code. All of these coding nucleic acid molecules are within the scope of the present invention. They may be administered to an individual and used to express polypeptides of the present invention. Thus they may be used for the same treatments as the polypeptides of the present invention. The nucleic acid molecules may be used directly, e.g. they may be injected into muscle (optionally after being first incorporated into a lipid-based carrier, such as a liposome). Alternatively they may be inserted into vectors. Vectors for use in treatments include replication-deficient adenoviruses, retroviruses or adeno-associated viruses.

Vectors may be used in cloning. They may be introduced into host cells to enable the expression of polypeptides of the present invention using techniques known to the person skilled in the art. Alternatively, cell free expression systems may be used. By using an appropriate expression system the polypeptides can be produced in a desired form. For example, the polypeptides can be produced by micro-organisms such as bacteria or yeast, by baculovirus-infected, cultured insect cells; mammalian cells (such as Chinese hamster cells) or transgenic animals that, for instance, secrete the proteins in milk. Where glycosylation is desired, eukaryotic (preferably mammalian) expression systems are preferred.

Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide will lack this residue. Polypeptides may initially be expressed to include signal sequences. Different signal sequences may be provided for different expression systems. Alternatively, signal sequences may be absent . Techniques for cloning, expressing and purifying polypeptides are well known to the skilled person. Various such techniques are disclosed in standard text-books, such as in Sambrook et al [Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989)]; in Old & Primrose [Principles of Gene Manipulation 5th Edition, Blackwell Scientific Publications (1994); and in Stryer [Biochemistry 4th Edition, W H Freeman and Company (1995)].

Preferred techniques for cloning, expressing and purifying a polypeptide of the present invention are summarised below:

cDNA encoding ADAM 16a/b protein can be subcloned into "pFastBac" plasmids, which can be purchased from GIBCO-BRL, InVitroGen and other companies, preferably in such a way that the encoded polypeptide has a "tag" consisting of six histidine residues. The recombinant plasmid can be used to make recombinant baculovirus (following the manufacturer's instructions) and expressed in cultured insect cells. The polypeptide can then be purified on affinity columns containing either nickel ions, to which the histidines bind, or using an anti-His₆ antibody. There are also other peptide "tags" for which monoclonal antibodies exist. Some commercially available "FastBac" plasmids already have DNA encoding His₆, and a signal peptide that causes secretion by insect cells.

Alternatively the cDNA can be expressed in bacteria using, for instance, an inducible plasmid such as pBAD (Guzman, L.M., Belin, D., Carson, M.J. and Beckwith, J. (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J. Bacteriol. 177 (14) 4121- 4130). If the encoded product is toxic for bacteria (as is often the case for proteases), the bacteria can be grown in absence of recombinant protein production and then induced by adding arabinose to induce the pBAD promoter.

In addition to nucleic acid molecules coding for polypeptides of the present invention (referred to herein as "coding" nucleic acid molecules), the present invention also includes nucleic acid molecules complementary thereto. Thus, for example, both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another). Also included are mRNA molecules and complementary DNA molecules (e.g. cDNA molecules).

Nucleic acid molecules which can hybridise to one or more of the nucleic acid molecules discussed above are also covered by the present invention. Such nucleic acid molecules are referred to herein as "hybridising" nucleic acid molecules. Hybridising nucleic acid molecules can be useful as probes or primers, for example. Probes can be used to purify and/or to identify nucleic acids. They may be used in diagnosis. For example probes may be used to determine whether or not an individual has a gene encoding functional ADAM 16a or ADAM16b. The absence of a functional ADAM 16a or ADAM 16b gene may be indicative of infertility. Primers are useful in amplifying nucleic acids or parts thereof, e.g. by PCR techniques (including reverse PCR).

Desirably such hybridising molecules are at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length.

Preferred hybridising molecules hybridise under stringent hybridisation conditions. One example of stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution which is about 0.9 molar. However, the skilled person will be able to vary such parameters as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc.

Most preferably, hybridising nucleic acid molecules of the present invention hybridise to a DNA molecule having the sequence shown in Figure 3A or 3B, to an RNA equivalent thereof, or to a complementary sequence to any of the aforesaid molecules.

It is important to note that nucleic acid molecules for use in the present invention include those with classical DNA or RNA structures, but also variants with modified (non-phosphodiester) backbones. A large number of derivatives are now available in which the phosphodiester linkage has been replaced but the deoxyribose retained. Two successful attempts to replace the entire deoxyribose phosphate backbone have been reported, the morpholino derivatives and the peptide nucleic acids (PNA), which contain an N-(2-aminoethyl)glycine-based pseudopeptide backbone. (See Nielsen, P.E., Annual Review of Biophysics & Biomolecular Structure, 24 167-83 (1995)).

In addition to being used as probes or primers, hybridising nucleic acid molecules of the present invention can be used as antisense molecules to alter the expression of polypeptides of the present invention by binding to complementary nucleic acid molecules. (Generally this can be achieved by providing nucleic acid molecules (e.g. RNA molecules) that bind to RNA molecules that would normally be translated, thereby preventing translation due to the formation of duplexes.) This technique can be used in antisense therapy. For example, antisense RNA expressing constructs could be used to inhibit ADAM 16a or ADAM 16b production (e.g. using recombinant viruses as expression vectors) and are therefore of utility in contraception. Antisense molecules may be in the form of DNA or RNA or variants thereof (e.g. PNA).

Hybridising molecules may also be provided as ribozymes. Ribozymes can be used to regulate expression by binding to and cleaving RNA molecules which include particular target sequences.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of a) or b) above (e.g. at least 50%, at least 75% or at least 90% sequence identity). As will be appreciated by the skilled person, the higher the sequence identity a given single stranded nucleic acid molecule has with another nucleic acid molecule, the greater the likelihood that it will hybridise to a nucleic acid molecule which is complementary to that other nucleic acid molecule under appropriate conditions.

In view of the foregoing description, the skilled person will appreciate that a large number of nucleic acids are within the scope of the present invention. Therefore, unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1) they may be DNA or RNA or a variant thereof (e.g. PNA); 2) they may be single or double stranded;

3) they may be provided in recombinant form i.e. covalently linked to a heterologous 5' and/or a 3' flanking sequence to provide a chimaeric molecule (e.g. a vector) which does not occur in nature; 4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature;

5) they may be provided in substantially pure form, e.g. by using probes to isolate cloned molecules having a desired target sequence or by using chemical synthesis techniques. (Thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids);

6) they may be provided with introns (e.g. as a full-length gene) or without introns (e.g. as cDNA).

Description of Figures

The present invention will now be described, by way of example only, with reference to the accompanying drawing; wherein:

Figure 1 shows the frequency distribution of B-cell cDNA fragments obtained by

RT-PCR and degenerate oligonucleotides for metalloprotease- and disintegrin domains. Meltrin-γ corresponds to GenBank (Benson et al., 1997) U41766 and D14665; TACE: TNF-α convertase, GenBank (HSU86755, HSU69611 and HSU69612). ORF#6 is a novel cDNA (see part 1 of the "Results" section). In addition, 14 fragments were found that had no open reading frame.

Figure 2 shows the tissue distribution of ADAM16a (panel A) and ADAM16b (panel B) mRNA. The probes for the Northern blots comprised 2.2 or 1.8 kbp of cDNA sequence, respectively. Exposure was overnight (A) or 5 h (B). Sk. Muscle is skeletal muscle; p.b.: peripheral blood. Size markers are in 10³ nucleotides.

Figure 3 shows the sequence of ADAM 16a (A) and ADAM 16b (B) and their predicted encoded products. The signal peptide of ADAM 16a is underlined. Figure 4 shows a comparison of the predicted products encoded by ADAM 16a and -b. Amino acids that are shared by both proteins are indicated with an "*" in ADAM 16b; gaps are indicated with dashes. The predicted signal peptide in ADAM 16a is boxed; the extent of the different domains is indicated by the dotted lines.

Figure 5 shows a comparison of ADAM 16 and -b with previously characterized genes. (A) Best GenBank matches for ADAM 16a, as determined by the BLAST (Madden er a/., 1996) algorithm, fert: fertilin. (B) Phylogenetic tree of ADAM 16a and -b and closely related ADAMs. The tree was generated using the CLUSTAL algorithm (Higgins et al., 1996). Hs: human; Mf. macacque; Rn: rat; Mm: mouse; Oc: rabbit; Cc: guinea pig; fert: fertilin. ADAM 9 is meltrin-γ (U41766).

Figure 6 shows a helical wheel-representation of the putative fusogenic peptides encoded by ADAM 16a (A) and ADAM 16b (B). Hydrophobic residues are shaded.

Figure 7 shows the genomic mapping of ADAM 16a. Agarose gels of PCR fragments from hybrid cell lines carrying multiple, short fragments of human genomic DNA, using oligonucleotides NMPraseN3 and -C3 (A) or -N and -C (B). Size markers: "1 kb ladder" (Gibco-BRL). Numbers of positive hybridomas, and the products of 359 bp (A) or 123 bp (B) are indicated by arrows. ADAM 16b was positive for the same set of hybrids, plus #4 (data not shown).

Materials and Methods

1 RT-PCR

OligodT-primed, ds cDNA was prepared (Marathon kit, ClonTech) from polyA- RNA isolated from tonsillar B-cells which had been purified by resetting on sheep red blood cells. Ten nanogram DNA was used as template in a PCR containing 30 mM Tricine pH 8.4, 2 mM MgCI₂, 5 mM β-mercaptoethanol, 0.1 mg/ml gelatin, 0.1% Thesit (Ponce and Micol, 1992), 0.2 mM of each of the four dNTP's (freshly prepared from buffered 100 mM solution; Pharmacia), 0.3 pmoles/μl of each oligonucleotides and 25 u/ml ?? Taq DNA polymerase, in 100 μl total volume. Buffer and enzyme were sealed off in 70 μl ?? from the other components using a wax bead (Perkin-Elmer) and the sample was cycled 40 times for 1 min at 94° C, 1 min 35° C and 20 sec at 72° C (no product was obtained at annealing temperatures of 45° C or 50° C). The oligonucleotides used were: ATA GAA TTC AYG ARI IIG GIC AY AAY TTY GG (Microsynth; l=inosine; encoding HEXGHNFG) and ATA GGA TCC III RTC RCA YTC YTC ICC encoding (GEECDXG). The material was phenol-extracted, precipitated, dissolved in water, digested with EcoRI and SamHI and fragments in the 200- 400 bp range purified from an agarose gel using glass-milk (Pharmacia). The fragments were subcloned into pBlueKSlT (BlueScript, Stratagene) plasmid and sequenced using an automated ABI-sequencer.

2 cDNA "walking"

Additional cDNA sequence was obtained by designing oligonucleotides corresponding to newly determined sequence. These oligonucleotides were then used in a PCR, using linker-ligated human placenta cDNA and nested AP-1 and AP-2 primers provided in the ClonTech "Marathon RT-PCR" kit. The products were subcloned into Bluescript plasmid and sequenced. This method had a tendency to yield relatively short (500-1 ,000 bp) fragments, and had to be applied iteratively.

3 Library screening

A human testis cDNA library (λgt11 , oligodT-primed, complexity 10⁶; ClonTech #HL1010b) was plated out on 1090Y E. coli cells, totalling 2x10⁶ plaques on 10 rectangular plates (25x25 cm each), and screened with the cDNA probe (position 20-2,190 in Fig. 3A).

Positive colonies were purified after repetitive cycles of plaque excision and re- hybridization. Finally, 2-10 plaques of the same cDNA clone were pooled in 0.5 ml SM plus 50 μl chlorophorm (Ausubel et al., 1989) eluted >4 h, and 10 μl was subjected to PCR, as described above, except that Pfu DNA polymerase (Stratagene) was used instead of Taq and that annealing was at 55° C and extension time at 72° C was 7.5 min (35 cycles). The λgtl 1 -specific primers were GAT TGG TGG CGA CGA CTC CT and CAA CTG GTA ATG GTA GCG AC. The products were phenol-extracted, digested with EcoRI, gel-purified, subcloned into pBlueKSII⁺ and sequenced using an automated ABI sequencer. Both strands of all sequences were read at least once.

4 Hybridizations

Gel-purified cDNA fragments were radiolabelled using α-[³²P]dCTP (Amersham) and HighPrime® (Boehringer Mannheim) and hybridized in buffer containing 50% distilled formamide (Gibco-BRL) and 0.5% SDS at 42° C using standard protocols (Ausubel et al., 1989). The filters were washed for several hours at room temperature in 0.2xSSC, then for one hour at 55° C. The Northern blots (ClonTech, cat#7760-1 , 7754-1 and 7759-1) contained 2 μg human polyA RNA from the tissues indicated. The blots have been tested with an actin probe for equivalent loadings (data not shown).

5 Genomic mapping

PCR-reactions were set up (50 μl) containing 0.5 μl (12.5 ng DNA) genomic DNA of each of the hamster/human hybrid cell lines from the Stanford G3 Radiation Hybrid Panel (RH01.05; Research Genetics). The samples were cycled 35 times with 1 min at 94° C, 1 min at 55° C and 30 s at 72° C. The oligonucleotides used for ADAM 16a were NMpraseN3 (TGC AAT TGT CAT ATA ATT TC) and -C3 (AGC TTC ATG CCT TGT GTG TC), or NMpraseN (CAG TGG TGT GTG TGC GAG CTA CAG TGG TGC) and NMpraseC (GAG GCG

GTT GAA TAC ATA ATC CAC TAC TGA). The oligonucleotides used for ADAM 16b were GTG ACA GTG CCT ACT GCT ATC A and CTC TCC CAC AAA AGA CAT CAG. Of the products 40 μl was concentrated in vacuo to 5-10 μl, separated on 1.5% agarose gels and viewed with a still video system.

Results

1 cDNA cloning of ADAM 16a and -16b The original aim of this study was to identify novel ADAMs that might be involved in shedding of adhesion molecules and cytokines, such as L-selectin, ICAM-3 and TNF-α (Hwang et al., 1993; del Pozo et al., 1994; Bjornberg et al., 1995; Kishimoto et al., 1995; Arribas et al., 1996; Feehan et al., 1996; Black et al., 1997; Moss et al., 1997a; Moss et al., 1997b). As there is evidence, and one concrete example (TACE) that disintegrin-metalloproteases, or ADAMs are involved in these processes, degenerate oligonucleotides were designed that encode the Zn²⁺-binding pocket (HEXGHNFG) consensus and conserved disintegrin domain (GEECDXG). An RT-PCR on B-cell RNA yielded three types of fragment carrying open reading frames (ORFs), namely meltrin-γ, TACE and one that had no database match ("ORF#6"; Fig. 1). In addition, a number of fragments were obtained that did not have an ORF (data not shown). The ORF#6 sequence could not be found in any of the public databases, but it showed good overall similarity with other ADAM proteins. Using 5'- and 3'-RACE protocols, a near-complete sequence of about 2.2 kbp was obtained for this cDNA from placenta (see Materials and Methods section 2).

2 Tissue distribution

The 2.2 kbp fragment obtained by RT-PCR was used as a probe on Northern Blots containing polyA RNA from various human tissues. A single signal was obtained for an mRNA of about 2,800 nts, and in testis only (Fig. 2A). Since the cDNA fragment was obtained from B-cell and placenta cDNA, this mRNA must also be expressed at very low levels in other tissues than testis. However, no signal was seen in these tissues on the Northern blots. All testis-specific ADAMs whose expression was closely examined (e.g. ADAM 1-5 in mouse, see Wolfsberg et al., 1995b) are expressed exclusively on spermatocytes. It is therefore very likely that ADAM 16a and -b are expressed in this tissue as well.

3 Library screening

In order to obtain a full-length sequence of the novel mRNA, a human testis- cDNA library was screened with the RT-PCR fragment. Among 2x10⁶ plaques screened, one group (20 clones) hybridized strongly, and a smaller group (7 clones) hybridized weakly. The first group comprised at least nine different clones which all corresponded to the original RT-PCR product, and was called "ADAM 16a". The second group (>six different clones) corresponded to a related, but distinct cDNA, that was named "ADAM 16b". The largest inserts from both groups were subcloned and their sequences determined (Fig. 3). The N-terminal sequence of the ADAM 16a protein was predicted to carry a signal sequence, upon analysis by the "SignalP V1.1" World Wide Webserver (Nielsen et al., 1997). The 5' sequence of the ADAM 16a clones displayed sequence divergence in their noncoding region, probably due to differential splicing (data not shown). The sequence shown in Fig. 3A was common to all ADAM 16a clones. While this work was in progress, an EST-sequence (HS1200890) from testis was reported whose sequence overlaps with the one shown in Fig. 3A. The 5' end of this EST sequence is non-coding however, and is likely to be the results of alternative splicing as well. No sequence heterogeneity was found in the coding portion of either ADAM 16a or 16b cDNAs.

The full-length ADAM 16b sequence was also used to probe the set of multiple tissue-Northern blots (Fig. 2B). The expression pattern of ADAM 16b was identical to what was found for ADAM 16a. Superimposition of the two films showed that ADAM 16b's mRNA was slightly shorter than ADAM 16a mRNA.

4 Sequence comparisons

Apart from the ESTs (HS1200890 and HS1200798, both ADAM 16a), no exact matches were found for ADAM 16a or -b in the public databases. A comparison of their predicted products (Fig. 4) showed that ADAM 16a and -b share 50% identity. Identities do not appear to cluster in particular domains. The same observation was made in a much wider comparison of ADAMs (Wolfsberg and White, 1996). The overall composition of domains is very similar to the one found in most ADAMs: a signal peptide, proprotein domain, metalloprotease domain with Zn²⁺ binding motif, disintegrin domain with disintegrin loop and "hairpin tip", cystein-rich domain, a putative fusion peptide, EGF-like repeat, transmembrane region and intracellular domain.

GenBank BLAST (Madden et al., 1996) database searches revealed a large number of significant similarities with previously identified ADAMs (Fig. 5A). For both ADAM 16a and -b, the best similarity was with meltrin-γ / ADAM9 (U41766), closely followed by fertilin-αl and -II from macaque (X79808 and X79809), the human fertilin-α pseudogene (Y09232) and fertilin-α from rat, mouse, rabbit and guinea pig, followed by the fertilin-β proteins. The most similar proteins in this list were compared as a group using the CLUSTAL (Higgins et al., 1996) algorithm and are represented in a tree in Fig. 5B. The tree shows two clusters of fertilin-β (left) and fertilin-α (bottom right). The two ADAMs 16 and ADAM 9 sprout between these clusters, the closest to the fertilin-α group.

5 Sequence analysis

5.1 The proprotein domain

Although ADAMs consist of clearly recognizable domains, it is not likely that all are capable of proteolysis, cell adhesion, fusion and intracellular signalling. In most ADAMs, the pro-protein domain is cleaved off, resulting in activation of the protease domain (when functional). This characteristic, shared with the soluble matrix metalloproteases (MMPs) and crotalid snake venom metalloproteases (SVMPs), is due to the presence of a so-called "cysteine switch" (van Wart and Birkedal-Hansen, 1990; Grams et al., 1993), a short motif with an un-paired cysteine that is thought to interact with Zn²⁺ in the catalytic domain. This switch can be found in ADAM 16a's proprotein domain, suggesting that this protein, too, requires proteolytic processing (Fig. 3A). In TNF-α convertase and ADAM 15, the pro-protein cleavage sites (RVKRR and RRRR) are readily identifiable as targets for the ubiquitous furin proteases (Matthews et al., 1994). Accordingly, TACE and ADAM 15 protein are mostly found in their processed form (Black et al., 1997; Herren et al., 1997). By contrast, ADAM 16a and b do not have an obvious cleavage site, which suggests that their proteolytic processing is regulated. This situation is similar to fertilin-α and fertilin-β, which are processed only during the later stages of spermatogenesis during epididymal passage, coincidental with the acquisition of fertilization competence (Blobel et al., 1990; Huovila er a/., 1996). Accordingly, full-length ADAM 16a was not properly processed in recombinant baculovirus-infected insect cells (data not shown). 5.2 The metalloprotease domains

The Zn²⁺-binding site in the ADAM 16a metalloprotease domain corresponds to the consensus sequence HEXGHNLGXXHD (Rawlings and Barrett, 1995). It is therefore likely that ADAM 16a is a functional protease, like fertilin α, TNF α convertase and ADAM 10. The Zn²⁺-binding site is followed by a "Met-turn", a structure that folds back and stabilizes the Zn ⁺-ligand, a characteristic of the adamalysin metzincin family of proteases (Stόcker and Bode, 1995). By contrast, the ADAM 16b sequence differs at two positions from the consensus Zn²⁺-binding site, one being a critical histidine residue thought to interact directly with the metal cation. This makes it unlikely that ADAM 16b can function as a protease.

A perfect consensus Zn²⁺-binding site is found in all SVMP's, but only in a minority of ADAMs. Among the five testis-specific mouse ADAM's, only fertilin-α is predicted to possess protease activity.

5.3 The disintegrin domains

The best-known disintegrin domains are those derived from SVMPs. Most of these carry the RGD tripeptide at the tip of a flexible disintegrin loop. Pll-type SVMPs interact with platelet integrin αllb/βllla, thus inhibiting blood clot formation and enhancing snake venom potency. Other SVMPs disrupt blood vessels, causing haemorrhage. By contrast, none of the ADAMs, with exception of ADAM 15 (metargidin; Kratzschmar et al., 1996; Herren et al., 1997) express RGD, and it is believed that, in general, ADAM disintegrin domains promote rather than disrupt cell-cell interaction (Wolfsberg and White, 1996). The disintegrin domain plays an important role in mouse fertilin β as ligand for egg integrin α6/β1 (Almeida et al., 1995). Similarly, guinea pig fertilin-β disintegrin peptides can block sperm-egg fusion (Myles et al., 1994). However, the disintegrin loop triplet shows poor conservation among fertilin βs from other species. It is not clear if this is so because other disintegrin subdomains are involved in integrin-ligand specificity or because sperm cells bind different egg ligands in different species. If, as has been argued, the third residue of the triplet must be acidic in functional disintegrins, then both ADAM 16a (VGE) and ADAM 16b (VNE) may encode active ligands.

5.4 The potential fusion peptides

Putative fusion peptides have been identified in the cysteine-rich domains of fertilin-α and meltrin-α (Blobel et al., 1992; Muga et al., 1994; Huovila et al., 1996), which are involved in cell-cell fusion. Like those identified in fusogenic viruses, these peptides share as a common feature the ability to fold into an amphipathic α-helix, often interrupted with one or two central proline residues, with hydrophobic and hydrophilic faces; they are short (16-26 aminoacids), and are expressed in a membrane-anchored subunit (White, 1992). In both the ADAM 16a and -b cysteine-rich domains short hydrophobic stretches can be found that can be modelled into an amphipathic helix with a central proline residue (Fig. 6). This is compatible with a role for ADAM 16a and -b in mediating cell-cell fusion.

5.5 The cytoplasmic domains

Several ADAMs have relatively long cytoplasmic tails, and ADAMs 8-10, 12, 13 and 15 carry putative tyrosine kinase SH-3 binding domains (reviewed in Wolfsberg and White, 1996). However, the ADAM 16a and -b intracellular domains are very short, lack tyrosine residues and SH-3 consensus sequences and are unlikely to be involved in signalling.

6 Genomic mapping

The Stanford G3 Radiation Hybrid Panel was used to determine the genomic localization of ADAM 16a and -b. This panel consists of 83 hybrid cell lines which are derived from fusions between a hamster line and γ-irradiated human cells. As each hybridoma contains short, multiple fragments of the human genome, assignment of a given cDNA to a subset of these hybrids allows mapping with a precision that greatly exceeds in situ hybridization methods (Schuler et al., 1996). Two sets of oligonucleotides were used in a PCR with genomic DNA from these hybrids as template. Using this method, ADAM 16a cDNA was assigned to fourteen hybrids (Fig. 7). This result was decoded by the Radiation Hybrid Map Search Engine (version 2.0; Stanford Human Genome Center), which returned a very tight association (LOD score 12.93) with marker SHGC-36001 (GenBank H64417) on chromosome 14q24.1. ADAM 16b was mapped using the same method. The same set of hybrids was positive for this gene, plus hybrid 4 (data not shown). This pattern is identical to marker SHGC- 36001. Thus, both genes are tightly clustered and linked to marker SHGC- 36001 on the long arm of chromosome 14.

Abbreviations

ADAM, (protein encoding) A Disintegrin and Metalloprotease bp, base pair(s)

EGF, epithelial growth factor

EST, expressed sequence tag ICAM, intracellular adhesion molecule kbp, 1 ,000 bp

LOD, limit of detection

MMP, matrix metalloprotease

MDC, metalloprotease-like, cysteine-rich (protein) ORF, open reading frame

PCR, polymerase chain reaction

RACE, rapid (PCR) amplification of cDNA ends

RT-PCR, reverse transcription-PCR

SDS, sodium dodecyl sulphate SSC, 0.15 M NaCI, 15 mM Na citrate, pH 7.0

SVMP, snake venom metalloprotease

TACE, TNF-α convertase

TNF, Tumour necrosis factor

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Claims

1. A polypeptide, which:

a) comprises the non-underlined amino acid sequence shown in Figure 3A or Figure 3B b) has one or more amino acid deletions, insertions or substitutions relative to the non-underlined amino acid sequence shown in Figure 3A or Figure 3B, but has at least 40% amino acid sequence identity with said sequence; or c) is a fragment of a polypeptide as defined in a) or b) above, which is at least 10 amino acids long.

2. A polypeptide according to claim 1 , which has one or more of the following regions:

a) a metalloprotease region b) a disintegrin region c) a fusion peptide region.

3. A polypeptide according to claim 1 or claim 2, which consists of the amino acid sequence shown in Figure 3A or Figure 3B, or of a part thereof.

4. A polypeptide comprising a polypeptide according to claim 1 or claim 2, when covalently linked to another moiety.

5. A polypeptide according to any preceding claim, when in glycosylated form

6. A pharmaceutically acceptable composition comprising a polypeptide according to any preceding claim.

7. A pharmaceutically acceptable composition according to claim 6, which is a vaccine.

8. A pharmaceutically acceptable composition according to claim 7, which comprises an adjuvant.

9. A polypeptide according to any of claims 1 to 5 or a pharmaceutically acceptable composition according to any of claims 6 to 8, for use in medicine.

10. The use of a polypeptide according to any of claims 1 to 5 in the preparation of a contraceptive.

11. The use of a polypeptide according to any of claims 1 to 5 in the preparation of an agent for improving fertility.

12. A method for assessing fertility comprising determining whether or not a patient has antibodies that bind to a polypeptide according to any of claims 1 to 5.

13. The use of a polypeptide according to any of claims 1 to 5 in screening.

14. The use according to claim 13 in screening for a contraceptive agent.

15. The use according to claim 13 in screening for an agent capable of enhancing fertility.

16. The use of a polypeptide according to any of claims 1 to 5 in raising or selecting antibodies.

17. An antibody or a derivative thereof which binds to a polypeptide according to any claims 1 to 5.

18. A pharmaceutically acceptable composition comprising an antibody or a derivative thereof according to claim 17.

19. An antibody or a derivative thereof according to claim 17 or a pharmaceutically acceptable composition according to claim 18, for use in medicine

20. The use of an antibody or a derivative thereof according to claim 17 in the preparation of a contraceptive.

21. A method for assessing fertility comprising determining in vitro whether or not a patient has a polypeptide that binds to an antibody or a derivative thereof according to claim 17.

22. A nucleic acid molecule which:

a) codes for a polypeptide according to any of claims claim 1 to 5. b) is complementary to a molecule as defined in a) above, or c) hybridises to a molecule as defined in a) or b) above.

23. A vector comprising a nucleic acid molecule according to claim 22.

24. A host comprising a nucleic acid molecule according to claim 22 or a vector according to claim 23.

25. A method for obtaining a polypeptide according to any of claims 1 to 5, comprising incubating a host according to claim 24 under conditions causing expression of said polypeptide and then purifying said polypeptide.

26. A nucleic acid molecule, vector or host according to any of claims 22, 23, or 24 respectively, for use in medicine.

27. The use of a nucleic acid molecule, vector or host according to any of claims 22, 23, or 24 respectively in the preparation of a contraceptive.

28. The use of a nucleic acid molecule, vector or host according to any of claims 22, 23, or 24 respectively, in the preparation of an agent capable of enhancing fertility.

29. A method of assessing fertility comprising determining in vitro whether or not nucleic acid from a patient hybridises with a nucleic acid molecule according to claim 22.

30. The use of nucleic acid molecule according to claim 22 as a probe or as a primer.

31. An agent that has been identified by screening as described in any of claims 13 to 15.