WO1997041221A1 - Metastasis-associated antigen and antibodies thereto - Google Patents

Abstract

There are provided membrane-associated polypeptides having the sequence shown in SEQ ID Nos. 1 and 9. Also provided are immunogenic determinants derived from said polypeptides and antibodies raised thereto. The polypeptides, their derived antigenic determinants and the antibodies are useful for the diagnosis and treatment of metastatic potential in tumors.

Description

Metastasis-Associated Antigen and Antibodies thereto

The present invention relates to a novel antigen which is closely associated with metastatic behaviour of tumour cells in vitro and in vivo, as well as to antibodies recognising said antigen and uses thereof in diagnosis and treatment of malignancies.

Metastasis is a multi-stage process by which tumour cells leave the site of a primary tumour, enter blood and lymph vessels, migrate to distant parts of the body and form novel foci of tumour growth. In keeping with the apparently complex nature of metastasis, experimental data suggest that no one factor is exclusively responsible for the determination of metastatic potential. Rather, a cascade of events, each of which incrementally increases the liability of a tumour to metastasis, leads to metastasis and tumour spread. Each of the events of the metastatic cascade is not believed to play a role of identical importance in each metastatic situation. A given metastatic cell is likely to possess some less developed metastatic properties, but still possess high metastatic potential because other properties are highly pronounced.

For this reason, a reliable marker for metastatic potential is difficult to determine. There are indications, however, that the cell surface is closely involved with many aspects of the metastatic cascade. The detachment of tumour cells, migration trough tissues and final attachment to form a new focus of growth are events which all involve the behaviour of the cell surface. ,

It is known to attempt to raise antibodies directed to antigens preferentially expressed by tumour cells which show metastatic potential. For example, Vollmers and Birchmeier, (1983) PNAS 80, 3739-3733 & 6863-6867, demonstrated that certain antibodies raised to mouse melanoma cell line surface protein were able to inhibit cell adhesion and block metastasis. The antibodies appeared to target antigens of between 40 and 50 kDa. By raising antisera to membrane associated proteins and assessing cross-reactivity with transformed cells, we have now been able to identify novel 150kDa cell-surface proteins which correlate extremely closely with metastatic potential both in vitro and in vivo.

Summary of the Invention

According to the present invention, we provide membrane-associated polypeptides having the sequence shown in SEQ ID NOS. 2 and 9, respectively. Also provided are immuno¬ genic determinants derived from said polypeptides and antibodies raised thereto. The polypeptides, their derived antigenic determinants and the antibodies are useful for the diagnosis and treatment of metastatic potential in tumours.

Brief Description of the Figures

Figure 1 shows the deduced amino acid sequence of the metastasis-related antigen p150 isolated from mouse. The sequence data derived by amino acid microsequencing is underlined.

Figure 2 shows a sequence homology comparison between p150 from mouse and related sequences.

Detailed Description of the Invention

By raising monoclonal antibodies to membrane fractions derived from human placenta and subsequently screening the antibodies for cell surface staining in transformed versus non- transformed cells, an antibody which identifies an antigen preferentially expressed on transformed cells can be identified. This antigen, referred to herein as p150 from mouse, has an apparent molecular weight on SDS-PAGE of ca. 150kDa and the sequence shown in SEQ ID No. 2. The human homologue is represented in SEQ ID No. 9.

The antigens are expressed in tumours, tumour cell lines and primary cells derived from tumours, and appear at much lower levels in normal human tissue. The levels of expression of the antigens are not consistent between tumours and cell lines derived therefrom, including primary cell lines. However, within each separate system, we have determined that the antigens closely correlate with metastatic potential. The level of expression of the antigens appears to be dependent on the differentiation state of the tumour cell. Thus, relatively undifferentiated tumour cells, which have a high metastatic potential, express relatively high levels of p150, while more differentiated cells express a lower amount of these antigens. Tumour tissue derived from actual metastases appears to express the most elevated levels of p150.

The present invention includes the p150 proteins both from mouse and human and all derivatives thereof which are useful for diagnosing the metastatic potential of a tumour. Preferably, unless otherwise indicated or implied, such derivatives are encompassed by the term "p150". The high degree of correlation of p150 with metastatic potential makes the antigens useful for determining the correct treatment to be used, as it is known that a high metastatic potential correlates with a poor prognosis. Accordingly, the derivatives of p150 comprised in the invention include all those which retain characteristics of p150 which allow them to be identified in diagnostic assays. The preferred characteristic retained by the p150 derivatives of the invention is a common antigenic determinant shared with p150.

"Common antigenic determinant" means that the derivative in question at least one antigenic function of p150. Antigenic functions includes possession of an epitope or antigenic site that is capable of cross-reacting with antibodies raised against a naturally occurring or denatured p150 polypeptide or fragment thereof. Thus p150 as provided by the present invention includes splice variants encoded by mRNA generated by alternative splicing of a primary transcript, amino acid mutants, glycosylation variants and other covalent derivatives of p150 which retain the physiological and/or physical properties of p150. Exemplary derivatives include molecules wherein the protein of the invention is covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid. Such a moiety may be a detectable moiety such as an enzyme or a radioisotope. Further included are naturally occurring variants of p150 found with a particular species, preferably a mammal. Such a variant may be encoded by a related gene of the same gene family, by an allelic variant of a particular gene, or represent an alternative splicing variant of the p150 gene.

Derivatives which retain common antigenic determinants can be fragments of p150. Fragments of p150 comprise individual domains thereof, as well as smaller polypeptides derived from the domains. Preferably, smaller polypeptides derived from p150 according to the invention define a single epitope which is characteristic of p150. Fragments may in theory be almost any size, as long as they retain one characteristic of p150. Preferably, fragments will be between 5 and 500 amino acids in length. Longer fragments are regarded as truncations of the full-length p150 and generally encompassed by the term "p150".

Derivatives of p150 also comprise mutants thereof, which may contain amino acid deletions, additions or substitutions, subject to the requirement to maintain at least one feature characteristic of p150. Thus, conservative amino acid substitutions may be made substantially without altering the nature of p150, as may truncations from the 5' or 3' ends. Deletions and substitutions may moreover be made to the fragments of p150 comprised by the invention. p150 mutants may be produced from a DNA encoding p150 which has been subjected to in vitro mutagenesis resulting e.g. in an addition, exchange and/or deletion of one or more amino acids. For example, substitutional, deletional or insertional variants of p150 can be prepared by recombinant methods and screened for immuno-crossreactivity with the native forms of p150.

The fragments, mutants and other derivative of p150 preferably retain substantial homology with p150. As used herein, "homology" means that the two entities share sufficient characteristics for the skilled person to determine that they are similar in origin and function. Preferably, homology is used to refer to sequence identity. Thus, the derivatives of p150 preferably retain substantial sequence identity with the sequences of SEQ ID Nos. 2 and 9, respectively.

"Substantial homology", where homology indicates sequence identity, means more than 50% sequence identity, preferably more than 75% sequence identity and most preferably a sequence identity of 90% or more.

Preferably, the proteins or derivatives thereof of the invention are provided in isolated form. "Isolated" means that the respective protein or derivative thereof has been identified and is free of one or more components of its natural environment. Isolated pi 50 includes p150 in a recombinant cell culture. P150 present in an organism expressing a recombinant p150 gene, whether the p150 protein is "isolated" or otherwise, is included within the scope of the present invention.

The polypeptides according to the invention are closely associated with the metastatic process in tumour cells. Accordingly, the invention provides a composition comprising a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.

According to a further aspect of the present invention, there are provided nucleic acids encoding p150. In addition to being useful for the production of recombinant p150 protein, these nucleic acids are also useful as probes, thus readily enabling those skilled in the art to identify and/or isolate nucleic acid encoding p150. The nucleic acid may be unlabelled or labelled with a detectable moiety. Furthermore, nucleic acids according to the invention are useful e.g. in a method determining the presence of p150-specific nucleic acid, said method comprising hybridising the DNA (or RNA) encoding (or complementary to) p150 to test sample nucleic acid and determining the presence of p150. In another aspect, the invention provides nucleic acid sequences that are complementary to, or hybridise under stringent conditions to, a nucleic acid sequence encoding p150.

The invention also provides a method for amplifying a nucleic acid test sample comprising priming a nucleic acid polymerase (chain) reaction with nucleic acid (DNA or RNA) encoding (or complementary to) p150.

In still another aspect of the invention, the nucleic acid is DNA and further comprises a replicable vector comprising the nucleic acid encoding p150 operably linked to control sequences recognised by a host transformed by the vector. Furthermore the invention provides host cells transformed with such a vector and a method of using a nucleic acid encoding p150 to effect the production of p150, comprising expressing p150 nucleic acid in a culture of the transformed host cells and, if desired, recovering p150 from the host cell culture.

Furthermore, the present invention relates to isolated p150 proteins and derivatives thereof encoded by the above-described nucleic acids.

Isolated p150 nucleic acid includes a nucleic acid that is free from at least one contaminant nucleic acid with which it is ordinarily associated in the natural source of p150 nucleic acid or in crude nucleic acid preparations, such as DNA libraries and the like. Isolated nucleic acid thus is present in other than in the form or setting in which it is found in nature. However, isolated p150 encoding nucleic acid includes p150 nucleic acid in ordinarily p150- expressing cells where the nucleic acid is in a chromosomal location different from that of natural cells or is otherwise flanked by a different DNA sequence than that found in nature.

In accordance with the present invention, there are provided isolated nucleic acids, e.g. DNAs or RNAs, encoding p150, particularly mammalian p150, e.g. human p150, or fragments thereof. In particular, the invention provides DNA molecules encoding p150, or a fragment thereof. By definition, such a DNA comprises a coding single stranded DNA, a double stranded DNA of said coding DNA and complementary DNA thereto, or this complementary (single stranded) DNA itself. Exemplary nucleic acids encoding p150 are represented in SEQ ID Nos. 1 and 8.

The preferred sequences encoding p150 are those having substantially the same nucleotide sequence as the coding sequences in SEQ ID Nos. 1 and 8, with the nucleic acids having the same sequence as the coding sequences in SEQ ID Nos. 1 and 8 being most preferred. As used herein, nucleotide sequences which are substantially the same share at least about 90 % identity. However, in the case of splice variants having e.g. an additional exon sequence homology may be lower.

The nucleic acids of the invention, whether used as probes or otherwise, are preferably substantially homologous to the sequences of p150 as shown in SEQ ID Nos. 1 and 8. The terms "substantially" and "homologous" are used as hereinbefore defined with reference to the p150 polypeptide(s).

Preferably, nucleic acids according to the invention are fragments of the p150-encoding sequences, or derivatives thereof as hereinbefore defined in relation to polypeptides. Fragments of the nucleic acid sequences of a few nucleotides in length, preferably 5 to 150 nucleotides in length, are especially useful as probes.

Exemplary nucleic acids can alternatively be characterised as those nucleotide sequences which encode a p150 protein and hybridise to the DNA sequences set forth SEQ ID Nos. 1 and 8, respectively, or a selected fragment of said DNA sequences. Preferred are such sequences encoding p150 which hybridise under high-stringency conditions to the sequences of SEQ ID Nos. 1 and 8, respectively. Stringency of hybridisation refers to conditions under which polynucleic acids hybrids are stable. Such conditions are evident to those of ordinary skill in the field. As known to those of skill in the art, the stability of hybrids is reflected in the melting temperature (T_m) of the hybrid which decreases approximately 1 to 1.5°C with every 1% decrease in sequence homology. In general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridisation reaction is performed under conditions of higher stringency, followed by washes of varying stringency.

As used herein, high stringency refers to conditions that permit hybridisation of only those nucleic acid sequences that form stable hybrids in 1 M Na⁺ at 65-68 °C. High stringency conditions can be provided, for example, by hybridisation in an aqueous solution containing 6x SSC, 5x Denhardt's, 1 % SDS (sodium dodecyl sulphate), 0.1 Na⁺ pyrophosphate and 0.1 mg/ml denatured salmon sperm DNA as non specific competitor. Following hybridisation, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridisation temperature in 0.2 - 0.1x SSC, 0.1 % SDS.

Moderate stringency refers to conditions equivalent to hybridisation in the above described solution but at about 60-62°C. In that case the final wash is performed at the hybridisation temperature in 1x SSC, 0.1 % SDS.

Low stringency refers to conditions equivalent to hybridisation in the above described solution at about 50-52°C. In that case, the final wash is performed at the hybridisation temperature in 2x SSC, 0.1 % SDS.

It is understood that these conditions may be adapted and duplicated using a variety of buffers, e.g. formamide-based buffers, and temperatures. Denhardt's solution and SSC are well known to those of skill in the art as are other suitable hybridisation buffers (see, e.g. Sambrook, et al., eds. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York or Ausubel, et al., eds. (1990) Current Protocols in Molecular Biology, John Wiley & Sons, Inc.). Optimal hybridisation conditions have to be determined empirically, as the length and the GC content of the probe also play a role.

Given the guidance provided herein, the nucleic acids of the invention are obtainable according to methods well known in the art. For example, a DNA of the invention is obtainable by chemical synthesis, using polymerase chain reaction (PCR) or by screening a genomic library or a suitable cDNA library prepared from a source believed to possess p150 and to express it at a detectable level.

Chemical methods for synthesis of a nucleic acid of interest are known in the art and include triester, phosphite, phosphoramidite and H-phosphonate methods, PCR and other autoprimer methods as well as oligonucleotide synthesis on solid supports. These methods may be used if the entire nucleic acid sequence of the nucleic acid is known, or the sequence of the nucleic acid complementary to the coding strand is available. Alternatively, if the target amino acid sequence is known, one may infer potential nucleic acid sequences using known and preferred coding residues for each amino acid residue.

An alternative means to isolate the gene encoding p150 is to use PCR technology as described e.g. in section 14 of Sambrook et al., 1989. This method requires the use of oligonucleotide probes that will hybridise to p150 nucleic acid. Strategies for selection of oligonucleotides are described below.

Libraries are screened with probes or analytical tools designed to identify the gene of interest or the protein encoded by it. For cDNA expression libraries suitable means include monoclonal or polyclonal antibodies that recognise and specifically bind to p150; oligonucleotides of about 20 to 80 bases in length that encode known or suspected p150 cDNA from the same or different species; and/or complementary or homologous cDNAs or fragments thereof that encode the same or a hybridising gene. Appropriate probes for screening genomic DNA libraries include, but are not limited to oligonucleotides, cDNAs or fragments thereof that encode the same or hybridising DNA; and/or homologous genomic DNAs or fragments thereof.

A nucleic acid encoding p150 may be isolated by screening suitable cDNA or genomic libraries under suitable hybridisation conditions with a probe, i.e. a nucleic acid disclosed herein including oligonucleotides derivable from the sequences set forth in SEQ ID Nos. 1 and 8. Suitable libraries are commercially available or can be prepared e.g. from cell lines, tissue samples, and the like. As used herein, a probe is e.g. a single-stranded DNA or RNA that has a sequence of nucleotides that includes between 10 and 50, preferably between 15 and 30 and most preferably at least about 20 contiguous bases that are the same as (or the complement of) an equivalent or greater number of contiguous bases set forth in SEQ ID Nos. 1 and 8, respectively. The nucleic acid sequences selected as probes should be of sufficient length and sufficiently unambiguous so that false positive results are minimised. The nucleotide sequences are usually based on conserved or highly homologous nucleotide sequences or regions of p150. The nucleic acids used as probes may be degenerate at one or more positions. The use of degenerate oligonucleotides may be of particular importance where a library is screened from a species in which preferential codon usage in that species is not known.

Preferred regions from which to construct probes include 5' and/or 3' coding sequences, sequences predicted to encode ligand binding sites, and the like. For example, either the full-length cDNA clone disclosed herein or fragments thereof can be used as probes. Preferably, nucleic acid probes of the invention are labelled with suitable label means for ready detection upon hybridisation. For example, a suitable label means is a radiolabel. The preferred method of labelling a DNA fragment is by incorporating a^32P dATP with the Klenow fragment of DNA polymerase in a random priming reaction, as is well known in the art. Oligonucleotides are usually end-labelled with g^32P-labelled ATP and polynucleotide kinase. However, other methods (e.g. non-radioactive) may also be used to label the fragment or oligonucleotide, including e.g. enzyme labelling, fluorescent labelling with suitable fluorophores and biotinylation.

After screening the library, e.g. with a portion of DNA including substantially the entire p150- encoding sequence or a suitable oligonucleotide based on a portion of said DNA, positive clones are identified by detecting a hybridisation signal; the identified clones are characterised by restriction enzyme mapping and/or DNA sequence analysis, and then examined, e.g. by comparison with the sequences set forth herein, to ascertain whether they include DNA encoding a complete p150 (i.e., if they include translation initiation and termination codons). If the selected clones are incomplete, they may be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones may include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, complete clones may be identified by comparison with the DNAs and deduced amino acid sequences provided herein.

In order to detect any abnormality of endogenous p150, genetic screening may be carried out using the nucleotide sequences of the invention as hybridisation probes. Such probes can e.g. be used to screen specific tissues and body fluids like e.g. blood, urine, spinal- fluid, ascites and serum. Also, based on the nucleic acid sequences provided herein antisense-type therapeutic agents may be designed.

It is envisaged that the nucleic acids of the invention can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion or inversion of a nucleotide stretch, and any combination thereof. Such mutants can be used e.g. to produce a p150 mutant that has an amino acid sequence differing from the p150 sequences as found in nature. Mutagenesis may be predetermined (site-specific) or random. A mutation which is not a silent mutation must not place sequences out of reading frames and preferably will not create complementary regions that could hybridise to produce secondary mRNA structure such as loops or hairpins.

The cDNA or genomic DNA encoding native or mutant p150 can be incorporated into vectors for further manipulation. As used herein, vector (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan. Many vectors are available, and selection of appropriate vector will depend on the intended use of the vector, i.e. whether it is to be used for DNA amplification or for DNA expression, the size of the DNA to be inserted into the vector, and the host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell for which it is compatible. The vector components generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence and a signal sequence.

Both expression and cloning vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors, these sequences are those which enable the vector to replicate independently of the host chromosomal DNA, and include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (e.g. SV 40, polyoma, adenovirus) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors unless these are used in mammalian cells competent for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, i.e. they are capable of replication in at least one class of organisms but can be transfected into another organism for expression. For example, a vector is cloned in E. coli and then the same vector is transfected into yeast or mammalian cells even though it is not capable of replicating independently of the host cell chromosome. DNA may also be replicated by insertion into the host genome. However, the recovery of genomic DNA encoding p150 is more complex than that of exogenously replicated vector because restriction enzyme digestion is required to excise p150 DNA. DNA can be amplified by PCR and be directly transfected into the host cells without any replication component.

Advantageously, an expression and cloning vector may contain a selection gene also referred to as selectable marker. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available from complex media.

As to a selective gene marker appropriate for yeast, any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene. Suitable markers for yeast are, for example, those conferring resistance to antibiotics G418, hygromycin or bleomycin, or provide for prototrophy in an auxotrophic yeast mutant, for example the URA3. LEU2, LYS2. TRP1. or HIS3 gene. Since the replication of vectors is conveniently done in E. coli, an E. coli genetic marker and an E. coli origin of replication are advantageously included. These can be obtained from ]Ξ. coli plasmids, such as pBR322, Bluescript^® vector or a pUC plasmid, e.g. pUClδ or pUC19, which contain both E. coli replication origin and E. coli genetic marker conferring resistance to antibiotics, such as ampicillin.

Suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up p150 nucleic acid, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes conferring resistance to G418 or hygromycin. The mammalian cell transformants are placed under selection pressure which only those transformants which have taken up and are expressing the marker are uniquely adapted to survive. In the case of a DHFR or glutamine synthase (GS) marker, selection pressure can be imposed by culturing the transformants under conditions in which the pressure is progressively increased, thereby leading to amplification (at its chromosomal integration site) of both the selection gene and the linked DNA that encodes p150. Amplification is the process by which genes in greater demand for the production of a protein critical for growth, together with closely associated genes which may encode a desired protein, are reiterated in tandem within the chromosomes of recombinant cells. Increased quantities of desired protein are usually synthesised from thus amplified DNA.

Expression and cloning vectors usually contain a promoter that is recognised by the host organism and is operably linked to p150 nucleic acid. Such a promoter may be inducible or constitutive. The promoters are operably linked to DNA encoding p150 by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector. Both the native p150 promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of p150 DNA.

Promoters suitable for use with prokaryotic hosts include, for example, the b-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (trp) promoter system and hybrid promoters such as the tac promoter. Their nucleotide sequences have been published, thereby enabling the skilled worker operably to ligate them to DNA encoding p150, using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems will also generally contain a Shine-Delgarno sequence operably linked to the DNA encoding p150.

Moreover, the p150 genes according to the invention preferably include a secretion sequence in order to facilitate secretion of the corresponding polypeptide from bacterial hosts, such that it will be produced as a soluble native peptide rather than in an inclusion body. The peptide may be recovered from the bacterial periplasmic space, or the culture medium, as appropriate.

Suitable promoting sequences for use with yeast hosts may be regulated or constitutive and are preferably derived from a highly expressed yeast gene, especially a Saccharomyces cerevisiae gene. Thus, the promoter of the TRP1 gene, the ADHI or ADH 11 gene, the acid phosphatase (PH05) gene, a promoter of the yeast mating pheromone genes coding for the a- or a-factor or a promoter derived from a gene encoding a glycolytic enzyme such as the promoter of the enolase, glyceraldehyde-3-phosphate dehydrogenase (GAP), 3-phospho glycerate kinase (PGK). hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, those phosphate isomerase, phosphoglucose isomerase or glucokinase genes, or a promoter from the TATA binding protein (TBP) gene can be used. Furthermore, it is possible to use hybrid promoters comprising upstream activation sequences (UAS) of one yeast gene and downstream promoter elements including a functional TATA box of another yeast gene, for example a hybrid promoter including the UAS(s) of the yeast PH05 gene and downstream promoter elements including a functional TATA box of the yeast GAP gene (PH05-GAP hybrid promoter). A suitable constitutive PHO5 promoter is e.g. a shortened acid phosphatase PH05 promoter devoid of the upstream regulatory elements (UAS) such as the PH05 (-173) promoter element starting at nucleotide -173 and ending at nucleotide -9 of the PH05 gene.

p150 gene transcription from vectors in mammalian hosts may be controlled by promoters derived from the genomes of viruses such as polyoma virus, adenovirus, fowlpox virus, bovine papilloma virus, avian sarcoma virus, cytomegalovirus (CMV), a retrovirus and Simian Virus 40 (SV40), from heterologous mammalian promoters such as the actin promoter or a very strong promoter, e.g. a ribosomal protein promoter, and from the promoter normally associated with p150 sequence, provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding p150 by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are relatively orientation and position independent. Many enhancer sequences are known from mammalian genes (e.g. elastase and globin). However, typically one will employ an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270) and the CMV early promoter enhancer. The enhancer may be spliced into the vector at a position 5' or 3' to p150 DNA, but is preferably located at a site 5' from the promoter.

Advantageously, a eukaryotic expression vector encoding p150 may comprise a locus control region (LCR). LCRs are capable of directing high-level integration site independent expression of transgenes integrated into host cell chromatin, which is of importance especially where the p150 gene is to be expressed in the context of a permanently- transfected eukaryotic cell line in which chromosomal integration of the vector has occurred, in vectors designed for gene therapy applications or in transgenic animals.

Suitable eukaryotic host cells for expression of p150 include yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms will also contain sequences necessary for the termination of transcription and for stabilising the mRNA. Such sequences are commonly available from the 5' and 3' untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNAs encoding p150.

An expression vector includes any vector capable of expressing p150 nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of expression of such DNAs. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector, that upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those with ordinary skill in the art and include those that are replicable in eukaryotic and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. For example, DNAs encoding p150 may be inserted into a vector suitable for expression of cDNAs in mammalian cells, e.g. a CMV enhancer-based vector such as pEVRF (Matthias, et al., (1989) NAR 17, 6418).

Particularly useful for practising the present invention are expression vectors that provide for the transient expression of DNA encoding p150 in mammalian cells. Transient expression usually involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector, and, in turn, synthesises high levels of p150. For the purposes of the present invention, transient expression systems are useful e.g. for identifying p150 mutants, to identify potential phosphorylation sites, or to characterise functional domains of the protein.

Construction of vectors according to the invention employs conventional ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to generate the plasmids required. If desired, analysis to confirm correct sequences in the constructed plasmids is performed in a known fashion. Suitable methods for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyses for assessing p150 expression and function are known to those skilled in the art. Gene presence, amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA, dot blotting (DNA or RNA analysis), or in situ hybridisation, using an appropriately labelled probe based on a sequence provided herein. Those skilled in the art will readily envisage how these methods may be modified, if desired.

In accordance with another embodiment of the present invention, there are provided cells containing the above-described nucleic acids. Such host cells such as prokaryote, yeast and higher eukaryote cells may be used for replicating DNA and producing p150. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive organisms, such as E. coli, e.g. E. coli K-12 strains, DH5a and HB101 , or Bacilli. Further hosts suitable for p150 encoding vectors include eukaryotic microbes such as filamentous fungi or yeast, e.g. Saccharomyces cerevisiae. Higher eukaryotic cells include insect and vertebrate cells, particularly mammalian cells. In recent years propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are epithelial or fibroblastic cell lines such as Chinese hamster ovary (CHO) cells, NIH 3T3 cells, HeLa cells or 293T cells. The host cells referred to in this disclosure comprise cells in in vitro culture as well as cells that are within a host animal.

DNA may be stably incorporated into cells or may be transiently expressed using methods known in the art. Stably transfected mammalian cells may be prepared by transfecting cells with an expression vector having a selectable marker gene, and growing the transfected cells under conditions selective for cells expressing the marker gene. To prepare transient transfectants, mammalian cells are transfected with a reporter gene to monitor transfection efficiency.

To produce such stably or transiently transfected cells, the cells should be transfected with a sufficient amount of p150-encoding nucleic acid to form p150. The precise amounts of DNA encoding p150 may be empirically determined and optimised for a particular cell and assay.

Host cells are transfected or, preferably, transformed with the above-captioned expression or cloning vectors of this invention and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Heterologous DNA may be introduced into host cells by any method known in the art, such as transfection with a vector encoding a heterologous DNA by the calcium phosphate coprecipitation technique or by electroporation. Numerous methods of transfection are known to the skilled worker in the field. Successful transfection is generally recognised when any indication of the operation of this vector occurs in the host cell. Transformation is achieved using standard techniques appropriate to the particular host cells used.

Incorporation of cloned DNA into a suitable expression vector, transfection of eukaryotic cells with a plasmid vector or a combination of plasmid vectors, each encoding one or more distinct genes or with linear DNA, and selection of transfected cells are well known in the art (see, e.g. Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press).

Transfected or transformed cells are cultured using media and culturing methods known in the art, preferably under conditions, whereby p150 encoded by the DNA is expressed. The composition of suitable media is known to those in the art, so that they can be readily prepared. Suitable culturing media are also commercially available.

While the DNAs provided herein may be expressed in any suitable host cell, e.g. those referred to above, preferred for expression of DNA encoding functional p150 are eukaryotic expression systems such as baculovirus-based systems and, particularly, mammalian expression systems, including commercially available systems and other systems known to those of skill in the art.

In preferred embodiments, p150 encoding DNA is ligated into a vector, and introduced into suitable host cells to produce transformed cell lines that express p150. The resulting cell lines can then be produced in quantity for reproducible qualitative and/or quantitative analysis of the effect(s) of potential drugs affecting p150 function. Thus p150 expressing cells may be employed for the identification of compounds, particularly small antagonising molecules interfering with p150 function are useful. An alternative to achieve an antagonistic effect is to rely on over-expression of antisense p150 RNA. Thus host cells expressing p150 are useful for drug screening and it is a further object of the present invention to provide a method for identifying compounds which modulate and preferable reduce the activity of p150, said method comprising exposing cells containing heterologous DNA encoding p150, wherein said cells produce functional p150, to at least one compound or signal whose ability to modulate the activity of said p150 is sought to be determined, and thereafter monitoring said cells for changes caused by said modulation. Such an assay enables the identification of agonists, antagonists and allosteric modulators of p150.

Cell-based screening assays can be designed e.g. by constructing cell lines in which the expression of a reporter protein, i.e. an easily assayable protein, such as b galactosidase, chloramphenicol acetyltransferase (CAT) or luciferase, is dependent on p150. Such an assay enables the detection of compounds that directly modulate p150 function, e.g. compounds that antagonise p150, or compounds that inhibit other cellular functions required for the activity of p150. An in vitro assay for p150 requires that it may be produced in large amounts in a functional form using recombinant DNA methods. An assay is then designed to measure a functional property of the p150. It has been found that p150 is expressed mostly in cells of high metastatic potential. Thus the present invention also provides a method to exogenously affect p150 dependent processes occurring in such cells. Recombinant p150 producing host cells, e.g. mammalian cells, can be contacted with a test compound, and the modulating effect(s) thereof can then be evaluated by comparing the p150-mediated response in the presence and absence of test compound, or relating the p150-mediated response of test cells, or control cells (i.e., cells that do not express p150), to the presence of the compound.

As used herein, a compound or signal that modulates the activity of p150 refers to a compound that alters the activity of p150 in such a way that the activity of p150 is different in the presence of the compound or signal (as compared to the absence of said compound or signal).

The invention also provides a transgenic non-human mammal which has been modified to modulate the expression of endogenous p150. Preferably, the transgenic non-human mammal is a transgenic mouse. For example, therefore, a transgenic mouse may be designed in which p150 production is greatly reduced or eliminated Alternatively, the transgenic mouse of the invention may express elevated levels of p150, or may be subject to regulation of p150 expression in a developmentally or tissue-specific manner, or via control by exogenous agents. Study of such an animal provides insights into the importance of p150 in vivo.

Moreover, since the polypeptides according to the invention are closely associated with the metastatic process in tumour cells, the invention provides a composition comprising a nucleic acid encoding a polypeptide according to the invention or an antagonist thereto for use as a medicament in the treatment or diagnosis of tumours.

In a preferred embodiment, there is provided a transcription unit encoding a polypeptide according to the invention or an antagonist thereto for use in a method of treatment of a tumour or other condition, for example a condition involving aberrant p150 gene expression, by gene therapy techniques. The transcription unit provided according to the present aspect of the invention comprises regulatable control regions which include a promoter, together with one or more enhancers and/or LCRs. The transcription unit may be delivered to the subject by any suitable means, including viral vectors, especially retroviral vectors, adeno- and adeno associated viral vectors, non-viral delivery systems, including liposomal and antibody targeted delivery systems, and direct uptake of naked DNA. The target tissue is advantageously a tumour tissue.

In accordance with yet another embodiment of the present invention, there are provided antibodies specifically recognising and binding to p150. For example, such antibodies may be generated against the p150 having the amino acid sequences set forth in SEQ ID Nos. 2 and 9, respectively. Alternatively, p150 or p150 fragments (which may also be synthesised by in vitro methods) are fused (by recombinant expression or an in vitro peptidyl bond) to an immunogenic polypeptide and this fusion polypeptide, in turn, is used to raise antibodies against a p150 epitope.

Anti-p150 antibodies are recovered from the serum of immunised animals. Alternatively, monoclonal antibodies are prepared from cells in vitro or from in vivo immunised animals in conventional manner. Preferred antibodies identified by routine screening specifically identify p150 on tumour cells with a high metastatic potential.

The antibodies of the invention are useful for studying p150 tissue localisation, screening of an expression library to identify nucleic acids encoding p150 or the structure of functional domains, as well as for the purification of p150, and the like. Preferably, however, they are useful in the diagnosis and treatment of tumours and especially tumour metastasis.

Antibodies according to the invention may be whole antibodies of natural classes, such as IgE and IgM antibodies, but are preferably IgG antibodies. Moreover, the invention includes antibody fragments, such as Fab, F(ab')₂, Fv and ScFv. Small fragments, such Fv and ScFv, possess advantageous properties for diagnostic and therapeutic applications on account of their small size and consequent superior tissue distribution.

The antibodies according to the invention are especially indicated for the diagnosis and therapy of cancer. Accordingly, they may be altered antibodies comprising an effector protein such as a toxin or a label. Especially preferred are labels which allow the imaging of the distribution of the antibody in a tumour in vivo. Such labels may be radioactive labels or radioopaque labels, such as metal particles, which are readily visualisable within the body of a patient. Moreover, the may be fluorescent labels or other labels which are visualisable on tissue samples removed from patients.

Recombinant DNA technology may be used to improve the antibodies of the invention. Thus, chimeric antibodies may be constructed in order to decrease the immunogenicity thereof in diagnostic or therapeutic applications. Moreover, immunogenicity may be minimised by humanising the antibodies by CDR grafting [see European Patent Application 0 239 400 (Winter)] and, optionally, framework modification [see international patent application WO 90/07861 (Protein Design Labs)].

Antibodies according to the invention may be obtained from animal serum, or, in the case of monoclonal antibodies or fragments thereof, produced in cell culture. Preferably, monoclonal antibodies according to the invention are antibody 6G7 (DSM 2256) and antibody 6G10 (DSM 2257). Recombinant DNA technology may be used to produce the antibodies according to established procedure, in bacterial or preferable in mammalian cell culture. The selected cell culture system preferably secretes the antibody product.

Therefore, the present invention includes a process for the production of an antibody according to the invention comprising culturing a host, e.g. E. coli or a mammalian cell, which has been transformed with a hybrid vector comprising an expression cassette comprising a promoter operably linked to a first DNA sequence encoding a signal peptide linked in the proper reading frame to a second DNA sequence encoding said protein, and isolating said protein.

Multiplication of hybridoma cells or mammalian host cells in vitro is carried out in suitable culture media, which are the customary standard culture media, for example Dulbecco's Modified Eagle Medium (DMEM) or RPM1 1640 medium, optionally replenished by a mammalian serum, e.g. fetal calf serum, or trace elements and growth sustaining supplements, e.g. feeder cells such as normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages, 2-aminoethanol, insulin, transferrin, low density lipoprotein, oleic acid, or the like. Multiplication of host cells which are bacterial cells or yeast cells is likewise carried out in suitable culture media known in the art, for example for bacteria in medium LB, N2CYM, NZYM, NZM, Terrific Broth, SOB, SOC, 2 x YT, or M9 Minimal Medium, and for yeast in medium YPD, YEPD, Minimal Medium, or Complete Minimal Dropout Medium.

In vitro production provides relatively pure antibody preparations and allows scale-up to give large amounts of the desired antibodies. Techniques for bacterial cell, yeast or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g. in an airlift reactor or in a continuous stirrer reactor, or immobilised or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads or ceramic cartridges.

Large quantities of the desired antibodies can also be obtained by multiplying mammalian cells in vivo. For this purpose, hybridoma cells producing the desired antibodies are injected into histocompatible mammals to cause growth of antibody-producing tumours. Optionally, the animals are primed with a hydrocarbon, especially mineral oils such as pristane (tetramethyl-pentadecane), prior to the injection. After one to three weeks, the antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells obtained by fusion of suitable myeloma cells with antibody-producing spleen cells from Balb/c mice, or transfected cells derived from hybridoma cell line Sp2/0 that produce the desired antibodies are injected intraperitoneally into Balb/c mice optionally pre-treated with pristane, and, after one to two weeks, ascitic fluid is taken from the animals.

The cell culture supernatants are screened for the desired antibodies, preferentially by immunofluorescent staining of cells expressing p150, by immunoblotting, by an enzyme immunoassay, e.g. a sandwich assay or a dot-assay, or a radioimmunoassay.

For isolation of the antibodies, the immunoglobulins in the culture supernatants or in the ascitic fluid may be concentrated, e.g. by precipitation with ammonium sulphate, dialysis against hygroscopic material such as polyethylene glycol, filtration through selective membranes, or the like. If necessary and/or desired, the antibodies are purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose and/or (immuno)-affinity chromatography, e.g. affinity chromatography with p150 protein or with Protein-A.

The invention further concerns hybridoma cells secreting the monoclonal antibodies of the invention. The preferred hybridoma cells of the invention are genetically stable, secrete monoclonal antibodies of the invention of the desired specificity and can be activated from deep-frozen cultures by thawing and recloning.

The invention also concerns a process for the preparation of a hybridoma cell line secreting monoclonal antibodies directed to p150, characterised in that a suitable mammal, for example a Balb/c mouse, is immunised with purified p150 protein, an antigenic carrier containing purified p150 or with cells bearing p150, antibody-producing cells of the immunised mammal are fused with cells of a suitable myeloma cell line, the hybrid cells obtained in the fusion are cloned, and cell clones secreting the desired antibodies are selected. For example spleen cells of Balb/c mice immunised with cells bearing p150 are fused with cells of the myeloma cell line PAI or the myeloma cell line Sp2/0-Ag14, the obtained hybrid cells are screened for secretion of the desired antibodies, and positive hybridoma cells are cloned.

Preferred is a process for the preparation of a hybridoma cell line, characterised in that Balb/c mice are immunised by injecting subcutaneously and/or intraperitoneally between 10 and 10⁷ and 10^s cells of human tumour origin which express p150 containing a suitable adjuvant several times, e.g. four to six times, over several months, e.g. between two and four months, and spleen cells from the immunised mice are taken two to four days after the last injection and fused with cells of the myeloma cell line PAI in the presence of a fusion promoter, preferably polyethylene glycol. Preferably the myeloma cells are fused with a three- to twentyfold excess of spleen cells from the immunised mice in a solution containing about 30 % to about 50 % polyethylene glycol of a molecular weight around 4000. After the fusion the cells are expanded in suitable culture media as described hereinbefore, supplemented with a selection medium, for example HAT medium, at regular intervals in order to prevent normal myeloma cells from overgrowing the desired hybridoma cells.

The invention also concerns recombinant DNAs comprising an insert coding for a heavy chain variable domain and/or for a light chain variable domain of antibodies directed to the extracellular domain of p150 as described hereinbefore. By definition such DNAs comprise coding single stranded DNAs, double stranded DNAs consisting of said coding DNAs and of complementary DNAs thereto, or these complementary (single stranded) DNAs themselves. Furthermore, DNA encoding a heavy chain variable domain and/or for a light chain variable domain of antibodies directed p150 can be enzymatically or chemically synthesised DNA having the authentic DNA sequence coding for a heavy chain variable domain and/or for the light chain variable domain, or a mutant thereof. A mutant of the authentic DNA is a DNA encoding a heavy chain variable domain and/or a light chain variable domain of the above-mentioned antibodies in which one or more amino acids are deleted or exchanged with one or more other amino acids. Preferably said modification(s) are outside the CDRs of the heavy chain variable domain and/or of the light chain variable domain of the antibody. Such a mutant DNA is also intended to be a silent mutant wherein one or more nucleotides are replaced by other nucleotides with the new codons coding for the same amino acid(s). Such a mutant sequence is also a degenerated sequence. Degenerated sequences are degenerated within the meaning of the genetic code in that an unlimited number of nucleotides are replaced by other nucleotides without resulting in a change of the amino acid sequence originally encoded. Such degenerated sequences may be useful due to their different restriction sites and/or frequency of particular codons which are preferred by the specific host, particularly E. coli, to obtain an optimal expression of the heavy chain murine variable domain and/or a light chain murine variable domain.

The term mutant is intended to include a DNA mutant obtained by in vitro mutagenesis of the authentic DNA according to methods known in the art.

For the assembly of complete tetrameric immunoglobulin molecules and the expression of chimeric antibodies, the recombinant DNA inserts coding for heavy and light chain variable domains are fused with the corresponding DNAs coding for heavy and light chain constant domains, then transferred into appropriate host cells, for example after incorporation into hybrid vectors.

The invention therefore also concerns recombinant DNAs comprising an insert coding for a heavy chain murine variable domain of an antibody directed p150 fused to a human constant domain g, for example g1 , g2, g3 or g4, preferably g1 or g4. Likewise the invention concerns recombinant DNAs comprising an insert coding for a light chain murine variable domain of an antibody directed to p150 fused to a human constant domain k or I, preferably k. ln another embodiment the invention pertains to recombinant DNAs coding for a recombinant antibody wherein the heavy chain variable domain and the light chain variable domain are linked by way of a DNA insert coding for a spacer group, optionally comprising a signal sequence facilitating the processing of the antibody in the host cell and/or a DNA coding for a peptide facilitating the purification of the antibody and/or a DNA coding for a cleavage site and/or a DNA coding for a peptide spacer and/or a DNA coding for an effector molecule.

The DNA coding for an effector molecule is intended to be a DNA coding for the effector molecules useful in diagnostic or therapeutic applications. Thus, effector molecules which are toxins or enzymes, especially enzymes capable of catalysing the activation of prodrugs, are particularly indicated. The DNA encoding such an effector molecule has the sequence of a naturally occurring enzyme or toxin encoding DNA, or a mutant thereof, and can be prepared by methods well known in the art.

Antibodies and antibody fragments according to the invention are useful in the diagnosis and therapy of tumours. Accordingly, the invention provides a composition for the therapy or diagnosis of tumours comprising an antibody according to the invention.

In the case of a diagnostic composition, the antibody is preferably provided together with means for detecting the antibody, which may be enzymatic, fluorescent, radioisotopic or other means. These means can e.g. be used to screen specific tissues and body fluids like e.g. blood, serum, ascites, spinal-fluid and urine. The antibody and the detection means may be provided for simultaneous, simultaneous separate or sequential use, in a diagnostic kit intended for the diagnosis of tumour conditions.

The invention further provides a method for determining the metastatic potential of a tumour cell, comprising assessing the level of p150 expression in said cell, a higher level of p150 expression being indicative of a lower state of differentiation and consequently a higher metastatic potential.

Moreover, the invention provides a method for the therapy of tumours comprising administering to a patient a therapeutically effective dose of an antibody, a polypeptide or a nucleic acid according to the invention. The invention is further described, for the purposes of illustration only, in the following examples.

Example 1

Metastasis-specific antibody

Due to the interest in the molecular components playing a role in the metastatic cascade monoclonal antibodies (Mabs) are raised against fractions containing membrane proteins derived from human placenta in order to identify such molecules possibly involved in this complex event.

Membrane-associated proteins are isolated from human placental tissue. The rationale for the selection of placental tissue is that since the trophoblast invades surrounding tissue, it is likely that invasion-associated proteins will be expressed therein.

200 g fresh human placenta is used as a starting material. The placenta is cut into small pieces and then washed several times in ice cold PBS in order to remove blood. The small pieces are transferred into a beaker containing 200 ml lysis buffer (100 mM Octyl-beta-D- glucoside, 1 mM PMSF, 5 mg/ ml Leupeptin and 1 mM Mn²⁺ dissolved in PBS ), homogenised with a Warren blender, stirred for 4 h at 4 °C and then once again homogenised with a Polytron. Solid matter is then removed by centrifugation at 25000g for 60 minutes.

The supernatant is applied to a preequilibrated 10 ml column charged with Sepharose^® conjugated with the hexapeptide GRGDSP. The column is then extensively washed with lysis buffer. Bound material is eluted in lysis buffer containing 10 mM EDTA. 1 ml eluate fractions are analysed by SDS-PAGE under reducing conditions.

Balb/c mice are then used for raising Mabs against putative RGD-binding proteins. Each animal receives about 50-100 mg of partially purified membrane proteins derived from the Sepharose^®-GRGDSP column, plus 0.5 ml Freunds Adjuvants injected ip. As booster applications, the animals twice receive 50-100 mg of protein plus 0.5 ml Freunds incomplete Adjuvants every 3-4 weeks. Finally, 2 days before the fusion of the splenocytes with the myeloma cells ( PAI cells), the animals receive another 20-30 mg of protein intravenously. The resulting Mabs are screened for cell surface staining and expression levels of the newly defined antigens in transformed versus non transformed cells. Comparison is made between 3T3 cells in a non-transformed state, and 3T3 cells transformed with src or ras, as well as CEF68 cells grown at permissive or non-permissive temperatures.

Cell surface staining is analysed by immunofluorescence, as follows. Cells are grown on glass coverslips or on glass slides, washed in PBS or in DME and fixed for 30 min. in formaldehyde 3.7% in PBS or in DME at room temperature (20°C). Cells are then permeabilised in 0.5 % TX-100 ( in PBS or DME ) for 1-3 min at room temperature. Subsequently, cells are washed 3-4 times in PBS or DME at room temperature. Liquid is drained off and 0.03ml of a first antibody raised against the membrane-associated protein fractions diluted in DME / 10% FCS is added. The cells are incubated for 60 minutes at 37°C in a humid incubator.

After incubation, the cells are washed 3-4 times in PBS or DME at room temperature. 0.03 ml of a second, anti-mouse antibody labelled with FITC or TRITC diluted in DME / 10% FCS is added, and the cells incubated for 60 minutes at 37°C in a humid incubator. Subsequently, they are washed 3-4 times in PBS or DME and once in water. They are then embedded in an appropriate mounting medium and stored in the dark. Fluorescence can then be assessed by viewing the embedded cells using suitable fluorescence microscopy equipment.

An interesting MAb, H11VII, is selected and further tested in the B16 F1/ F10 mouse melanoma system. For this purpose melanoma cells are incubated with the MAb prior to tail vein injection into mice. After 3 weeks the animals are sacrificed and the lung colonies counted. A 80-85% inhibition of the lung colonisation ability can be observed.

In order to reconfirm the results obtained with the original MAb (H11 VII) a new set of Mabs against mouse p150 is raised. The outcome of the in vivo lung colonisation assay performed with the new anti-p150 Mabs is shown in Table 1. Table 1

B16F1 cells

treatment number of lung colonies mean std %inhibition control 25,16,2,1,14,20,25,24 16 10 H11VII 1,0,5,9,0,4,0,0,5,9 3 4 80

B16F10 cells

treatment number of lung colonies mean std %inhibition untreated 36,45,23,20,52,64,>300(64), 46

>300(64) control 27,22,38,22,20,28,37,64,93, 44 29

>300(93) H11VII 0,0,0,1,3,4,10,11,12,16 85

B16 F1 cells H11VII induced

treatment number of lung colonies mean std fold indue. control 2,6,5,5,13,0,3,1,2,35 4 4

H11VII ind Oh 28,97,92,37,61,7,43,17,5,47 43 32 10 H11Vllind40h 41,45,47,74,21,26,51,127,101,131 66 40 16

Table 1 (cont)

B16 F1 cells

treatment number of lung coionl mean std %inhibition

4H9 0,3,>50,>50,22,>50,20 ? ? promotion?

6G7 1 ,9,4,9,2,1 ,4,3,8,0 4 3 80

H11VII 1 1 ,1 ,13,11 ,0,7,16 8 5 50 control 22,33,24,7,19,30,4,34 22 11

1 B10 25,1 ,13,13,0,7,8,16,10 10 8 50

6G10 9,6,3,4,7,2,2,7,23,0 6 6 70

A general problem by using antibodies in an in vivo system is the possible stimulation of the host immune system. Especially the cytotoxic as well as the natural killer activities towards cells tagged with antibodies can be influenced. Due to the fact that peripheral cellular blood components do not express p150 in detectable amounts, it is possible to reduce the influence of the host immune system by changing the treatment. Instead of incubation of the cancer cells with Mabs prior to injection we pretreat the host animals 24 hrs before they received F1 cells. The results of this experiment, in which the original MAb (H11 VII) as well as a new MAb raised against mouse p150 (6G7) are used, are outlined in Table 2.

Table 2

B16 F1 cells injected into pretreated animals

pretreatment number of lung colonies mean std %inhibition control 1 ,4,13,31 ,40,44,63,>99(63),>99(63),>99(63 39 25 n/a

)

H11VII 1 ,1 ,5,7,10,15,17,25,41 14 13 65 6G7 0,1 ,2,5,6,1 1 ,19,20,22 10 9 75 Mice are pretreated with the monoclonal antibodies for 24hours before injection of B16 cells. O.5mg of antibody is injected i.p. Antibodies are prepared by precipitation in 28g/100ml (NH₄)₂S0₄ and ConA chromatography.

Example 2

Metastasis-specific antigen

Using western blotting according to standard protocols, antibody H11 VII is seen to recognise bands on SDS-PAGE migrating at approximately 28, 35, 70 and 150 KDa. The bands are purified from the gel and rabbit polyclonal antisera raised thereto. The antiserum raised against the 28KDa band cross-reacts with the 150KDa band on western blots.

The antigen recognised by MAb H1 1VII can be identified as a 150 kDa membrane associated protein (p150). P150 is expressed in B16F1 cells in large amounts. Therefore, these cells are used as starting material for the purification of p150.

A crude membrane fraction is isolated from B16F1 cells by centrifugation at 100,000g and lysis in 1%NP40, 2mM Tris-HCl pH 7.5, containing PMSF and aprotinin protease inhibitors. The supernatant is loaded on a Sepharose^® Q column, run off, transferred to a Sepharose^® S column, again run off, and separated by reducing SDS-PAGE on a 5% gel. The 150KDa band is electrocuted.

Example 3

Analysis of p150 and cDNA cloning

P150 is microsequenced by digestion with trypsin, V8 and Lys-C followed by sequencing using Edman degradation on an Applied Biosystems sequencer. An oligonucleotide deduced from the sequence used to probe a mouse melanoma B16F1 library.

Sequencing the clones picked out of the B16F1 mouse melanoma c-DNA library identifies an ORF of 3.6 kb encoding the 142 kDa carboxy terminal part of p150. The missing 5'part was obtained by applying race technique using a kit from Clontech. Taking all the sequence information together, we can establish an ORF of 4.03 kb encoding a polypeptide of 1343 aa. This ORF, referred to as p150 is shown in SEQ ID No. 1. The deduced protein sequence is shown in SEQ ID No. 2 and Figure 1. The peptide sequences obtained from protein microsequencing are underlined in Figure 1. The cDNA and derived amino acid sequences of the human homologue, also referred to as p150 are shown in SEQ ID Nos. 8 and 9, respectively.

Striking homologies are found between p150 and two mouse proteins called centrosomin A (280 amino acids from the sequence shown in SEQ ID No. 3) and B (430 amino acids from the sequence shown in SEQ ID No. 4) respectively. These two proteins are most likely splice variants. They are found to be associated with centrosomes. Unfortunately no functional studies have been carried out on centrosomins, but, due to the cellular localisation, they are thought to take part in the organisation of the spindle apparatus and therefore could be candidates for possible tubulin binding.

Sequence alignment between p150, centrosomin A and centrosomin B shows that the centrosomins share close homology with p150 throughout their lengths, but p150 extends both 5' and 3' of both centrosomins. A sequence comparison is given in Figure 2.

Another 3 ORFs showing significant homologies to p150 exist in the Swiss Prot databank. A yeast ORF (YBR079; SEQ ID No. 5) showing 51% similarity and 29% identity, a tobacco ORF (NTPNL35; SEQ ID No. 6) with 59% similarity and 37% identity and an ORF found in nematode C.elegans (EGL-45; SEQ ID No. 7) showing 56% similarity and 36% identity. Unfortunately functional data are available only for EGL-45; the two other ORFs are of unknown function. Mutational studies performed on EGL-45 suggest an involvement of this protein in the regulatory steps of the hermaphrodite specific neuronal differentiation. Expression of EGL-45 is found in most cells of the worm. Null mutants give rise to a sterile or even a lethal phenotype.

All four sequences show a common overall organisation: namely a stretch of 300 aa at the amino terminus with significant identities between 30-45%, followed by a stretch of about 200aa of little homology which is followed by a highly charged stretch of about 400aa predicted to form coiled-coil domains (according to Lupas et al., Science 252, 1162, 1991).

The sequence alignments of 6 proteins described hereinabove (including the centrosomins) are shown in Figure 2. Example 4

Metastasis-specific expression of p150

It is reasoned that p150 on B16F1/F10 cells must be expressed on the cell surface, otherwise it would not be possible to influence the metastatic cascade by applying Mabs to the cell surface. In order to verify the localisation of p150, FACS analysis is carried out as follows:

Starting with a confluent 6cm tissue culture dish with B16 F1 or F10 cells (in DME-medium), antibody (dilution 1/10 - 1/100) is added to adherent cells or detached cells (EDTA, PBS w/o Ca²⁺ / Mg²⁺, wash once in PBS /1% BSA, spin down and add the antibody).

Cells are incubated either on the tissue culture plate ( at 20 or 37°C ) or in an Eppendorf tube for 1-2 h. Subsequently, cells are washed with PBS /1% BSA on the plate and detached, or washed by spinning down in the Eppendorf tube and adding PBS /1 % BSA. The washing step is repeated 3-4 times.

Cells which were labelled on the plate are then taken up in 1 ml PBS /1 % BSA.

A second antibody conjugated with FITC or TRITC is added to the cells in PBS /1% BSA and incubated for 1 h at 20°C while slowly shaking. The cells are washed 2-3 times with PBS /1% BSA. A FACscan may then be carried out according to standard protocols.

FACS analysis demonstrates that P150 is clearly expressed at the cell surface of B16 F1 mouse melanoma cells. The labelling pattern as well as the intensity of the staining is independent of the fact whether the cells were labelled in suspension or they were attached to tissue culture dishes.

Results are confirmed by cell surface biotiπylation on living F1 cells, as follows:

A confluent 15 cm dish of B16 F1 or HeLa cells is washed 3-4 times with cold PBS. 10 ml PBS containing 0.05 ml Biotinamidocaproate-N- Hydroxysuccimide Ester (Sigma B 2643), which was dissolved in DMSO at 100mg/ml, is then added and the cells incubated for 40 - 60 minutes in the cold room (4°C). The cells are then washed 2-3 times with cold PBS and lysed in cold lysis buffer (see p150 purification) for 30 minutes on ice. Undissolved material is spun down at 13000g for 10 minutes and the cleared cell lysate incubated with Avidin Agarose beads for 30 minutes on a shaker in the cold room.

The Avidin Agarose beads are washed with lysis buffer 3-4 times, once with cold ddH₂0 and boiled in SDS-PAGE sample buffer. SDS-PAGE is run and western blotting carried out according to standard protocols.

B16 F1 cells are seen to express p150 on the cell surface as determined by the above cell surface biotinylation method. However only a very small proportion of total cellular p150 is exposed at the cell surface. This seems to be involved in cell-cell and/or in cell- extracellular matrix interactions as already indicated by the results obtained from the in vivo colonisation experiments. Contrary to this finding in B16 cells, in HeLa cells no cell surface biotinylation of p150 could be observed.

According to its amino acid sequence, p150 contains a hydrophobic stretch of 70 aa (1050- 1120), which could serve as a transmembrane domain. However, most of the cellular p150 is found in the cytoplasmic compartment associated with endogenous membranes.

On HeLa cells, in contrary to the B16F1 cells, p150 could not be detected on the cell surface by biotinylation technique. Therefore, it might be possible, that the distribution of p150 depends on the cell type and on its status.

B16 cells show a strong transformed phenotype; e.g. they do not spread out very well in cell culture. As a consequence, the cytoskeletal elements are not as well separated as in cells which show a more spread out phenotype. Therefore we chose HeLa cells for our localisation studies. Using confocal microscopy, a strong perinuclear staining is observed, while throughout the cytoplasm staining is associated with vescicular structures. In cells undergoing mitosis, staining is observed to be associated with the midbody, in a pattern similar to that of tubulin.

Assessed by western blotting according to standard procedures, p150 is normally expressed in several mouse organs. We find a high expression level in pancreas, as well as liver, although signal in liver might be artefact due to high protein concentration in that region, and a medium level in brain, thymus, and testis. Low expression of p150 occurs in lung, spleen and kidney. P150 expression in testis could reflect the fact that there we can find a lot of undifferentiated, proliferating cells. In testis, p150 and b-tubulin codistribute on structures resembling sperm tails.

In order to analyse the connection between transformation and p150 expression, we analyse various cell types in different states of transformation using western blotting and find that a positive correlation between the state of transformation and the expression level of p150 can be observed. Thus, we observe higher levels of p150 in 3T3 cells transformed with ras than in non-transformed cells, and much higher levels in 3T3 cells transformed with v-src. CEF cells transformed with a temperature sensitive transforming gene product, NY68, show higher levels of p150 expression at the permissive (37°C) than the non- permissive (41 °C) temperature.

When the expression of p150 assessed by western blotting is correlated to differentiation state, an inverse relationship between the grade of differentiation and the expression level of p150 is observed in all cell lines so far investigated, including ES cells differentiated according to standard procedures, stably differentiated F9 cells and B16F1 cells treated with retinoic acid (0.01 and 0.1 M).

The expression of p150 correlates inversely with malignancy in different B16 variants. The two most malignant variants, F10 and LS9, have a lower level of p150 expression than F1 cells. This seems to contradict the relationship between transformation state and p150 expression levels. However, it is known that in the B16 system the metastatic ability in most cases correlates directly with differentiation state. Thus, in B16 cells, the more differentiated cells are more malignant. This is in contradistinction to what is found in other model systems and in clinics where greater malignancy and poor prognosis correlates inversely with differentiation state.

In order to examine the expression of p150 in other systems, several human tumour cell lines are tested for p150 expression. Some breast cancer cell lines (HBL 100, BT 20, T47D, SKBR3, MB231 and MB453), a liver cancer cell line (Hep G2) as well as a ovarian cancer cell line (SKO V3) are investigated. The p150 levels are different in these lines, but p150 appears to be universally present.

Example 5

Expression of p150 in human tumours

Methods

Tumor and normal tissue samples

From 22 fresh mastectomy specimen, cancer tissue and non-cancerous normal tissue is chosen macroscopically by an experienced pathologist for transfer to the laboratory. The tissue arrives in the laboratory about 5 hours after mastectomy at latest, is frozen and stored at -80°C. Corresponding cancer and normal tissue are fixed in 10% buffered formalin and embedded in paraffin. 5 micron sections are cut for hematoxylin/eosin staining. For immunohistochemistry the 5 micron sections are mounted on cementit (Merz und Benteli

SA, Niederwangen, Switzerland) covered super frost slides. Presence of invasive cancer and of normal tissue is confirmed by histology in each case.

Preparation of tissue extracts

Frozen human tumor and normal tissue is cut into small pieces, suspended in STE buffer (0.1 M NaCl, 0.01 M Tris-HCl pH 7.6, 1 mM EDTA) with protease inhibitors aprotinin 2 μg/ml, leupeptin 1 μg/ml and 1 mM PMSF and homogenized with a Polytron homogenizer. The homogenates are centrifuged at 1000 g for 15 minutes and the supernatants are mixed with one volume of 2x sample buffer consisting of 0.1 M Tris-HCl pH 6.8, 4% SDS and 20 % glycerol. This mixture is incubated for 30 minutes at 56°C and recentrifuged at 15,000 g for 10 minutes. The protein concentration of the supernatants is determined with a BCA protein assay kit (Pierce, Rockford, IL).

Preparation of NP-40 extracts of cell lines

Cells are washed twice with PBS and lysed in ice-cold lysis buffer (0.01 mM Tris-HCl pH 7.5, 0.5 % Nonidet P-40 and 1 mM PMSF) in the dish for 30 minutes at 4°C. The lysed cells are centrifuged at 15,000 g at 4°C. The supernatants are mixed with sample buffer as described above. The samples are heated for 2 minutes at 95°C and the protein concentrations determined. Immunoblotting

Extracts of cells or surgical specimens containing 20 or 40 μg of protein are subjected to electrophoresis in the presence of 0.1% SDS in 5% SDS-PAGE minigels. After electrophoresis, the proteins are transferred electrophoretically at 1 mA per cm² for 90 minutes onto a PVDF membrane. The membranes are blocked in blocking solution (0.1 M Tris-HCl pH 7.4, 0.1 M MgCI₂, 0.5% Tween 20, 1% Triton X-100, 1 % BSA, 5% FCS) for 30 minutes at room temperature (RT), decorated with a chicken Ab against p150 (Sophie 1 :2000) or with Mab 6G10 (supernatant 1 :20) overnight at 4°C, washed with blocking solution and then incubated for 1 hour at RT with a goat anti-chicken peroxidase-coupled second antibody (1 :2000, Southern Biotechnology Ass. Inc., Birmingham, AL) or rabbit anti- mouse and swine anti-rabbit (both peroxidase-coupled, Dako Diagnostics AG, Zug, Switzerland) at dilutions of 1 :2000. Then the membranes are washed with blocking solution, PBS and water and the protein-antibody complexes are visualized by ECL (Amersham International, UK) according to the manufacturer's protocol. Quantification of the Western blots are done by using a ¹²⁵lodine-labeled second antibody (Amersham International, UK). The blots are scanned with a Phosphorimager unit (Molecular Dynamics, Sunnyvale, CA) and the relative amounts of p150 are determined by measuring the radioactivity present in the appropriate bands.

Immunohistochemistry

Representative tissue blocks of normal and carcinoma tissue are taken from three cases for immunohistological examination. The 5 micron paraffin sections are deparaffinized, rehydrated and washed twice in Tris-NaCI buffer (30 mM Tris (pH 7.5), 0.9% NaCl). Internal peroxidases are blocked with blocking solution (25 mM β-D-glucose, 25 mM sodium azide and 0.6 units/ml glucose oxidase (Sigma, Switzerland) in Tris-NaCI buffer) for 1 hour at 37°C. After washes in Tris-NaCI buffer, the sections are heated in 500 ml citrate buffer (0.1 mM, pH 6.0) in the microwave oven operated at 750 W for 10 minutes. The sections are allowed to cool down and washed again in Tris-NaCI buffer before blocking with 1 % BSA in Tris-NaCI buffer for 15 minutes. The chicken antibody against p150 (Sophie 1/1000) is applied for 60 minutes at RT in the same buffer, whereas the Mab 6G7, the monospecific rabbit anti- NH₂ terminal peptide (225 amino acids) antibody and the Mab against Ki-67 (MIB1 , Dianova GmbH, Germany) are incubated with the sections overnight at 6°C. Subsequently the sections are washed twice, incubated with second antibodies (goat anti- chicken, goat anti-mouse or goat anti-rabbit; all peroxidase coupled, diluted 1 :2000 , Southern Biotechnology Ass. Inc., Birmingham, AL) for half an hour, rinsed with Tris-NaCI- buffer and incubated with the peroxidase substrate 3'-3'-diaminobenzidine (Sigma, Switzerland) 2% in 50 mM Tris pH 7.6 for 5 to 10 minutes at RT. The paraffin sections are counterstained with stabilized hematoxylin (Shandon, Astmoore, GB), dehydrated, mounted in Depex (BDH, Gurr, UK) and photographed.

A. Screening of 22 mammary carcinoma

Expression of p150 in human mammary tumors

Based on the fact that transformation and dedifferentiation are main features of cancer cells primary human tumors are screened for p150 expression using the panel of antibodies developed in the early stage of these studies.

The mean age of our 22 patients with primary breast cancer when operated was 64 years (range 37-90 y) and the mean tumor size was 48 mm (range 20-140 mm). 55% of the tumors were poorly differentiated (Gill according to the Elston and Ellis grading system (12)) while 14% were well differentiated. The predominant histological type (91%) was ductal invasive, the others were lobular invasive tumors. Metastasis to axillary lymph nodes was detected in 59%, in 27% the lymph nodes showed no metastasis and from all other patients no lymph nodes were available. In 21 of the 22 patients Western blotting have revealed significantly higher p150 expression in tumors than in the normal tissue counterparts of the same patients. With patient #14139 we have been able to investigate lymphnode metastasis; p150 expression in this context is even higher than in the primary carcinoma. One case (#10594) displayed an aberrant result. This patient, however, was the only one who had received preoperative chemotherapy and did not show any p150 overexpression in the cancer tissue.

Immunohistochemistrv

A distinct pattern of p150 expression is observed in breast cancer tissue versus the control tissue. Invasive and in situ ductal cancer react equally positive. Necrotic tissue is found to be negative for p150 expression. Normal non proliferating tissue shows faint focal cytoplasmic staining of luminal epithelial cells as do myoepithelial cells. A few nuclei in proliferating epithelial cells also present positive staining. Stromal tissue is negative for p150 in all cases.

Proliferative and precancerous alterations in breast tissue such as epitheliosis, apocrine metaplasia and adenosis show staining for p150. No correlation between Ki-67 staining and p150 expression in normal breast tissue is observed.

B. Screening of additional 31 mammary carcinoma

The mean age of the 31 patients with primary breast cancer when operated was 61 years (range 36-85 years) and the mean tumor size was 28 mm (range 9-90 mm). Fifty-five percent of the cancers were poorly differentiated (Gill according to the Elston and Ellis grading system), 32% were moderately differentiated and 13% were benign. The predominant histological type (67%) was ductal invasive, the others were lobular invasive (16%), fibroadenomas (10%), one squamous cell carcinoma, one a mucinous carcinoma and one was a papilloma.

Metastasis to lymphnode was detected in 42%, in 29% no lymph nodes were involved and in 29% of the patients lymph nodes were not available. Thirty of 31 patients show significantly higher p150 expression in tumors than in normal tissue counterparts in Western blot analysis.

One patient (#18976) shows no p150 expression neither in the normal breast tissue nor in the carcinoma. By histology this tumor is determined to be a rare squamous cell carcinoma of the breast. Interestingly, squamous cell carcinomas of the cervix show almost a loss of p150 expression as soon as the tumor starts to invade. Therefore, it is suggested, that the behavior in p150 expression is strongly dependent on the histological type and origin of the particular tumor.

Remarkably, p150 expression in the benign fibroadenomas and papilloma is much lower than in malignant mammary carcinoma. C. Screening of cervical cancer

Extensive studies have been carried out on squamous cell carcinoma of the cervix. Specimens of 165 patients have been investigated by immunohistochemistry. According to histological grading they have been divided into 3 groups containing 77 cases with precancerous lesions, 68 cases with invasive tumors and 20 healthy controls. The precancerous lesions significantly present the highest level of p150 expression whereas invasive stages show expression levels comparable to controls. Furthermore 120 month survival is significantly increased (p < 0.01) in those patients who show highest expression levels of p150.

In contrast to mammary carcinoma, malignancy in cervical squamous cell carcinoma inversely correlates with p150 expression levels. Apparently, the behavior of neoplastic tissue in terms of p150 expression strongly depends on the histological origin. Therefore, it is not a general issue for p150, that expression levels increase with enhanced proliferative ability. Thus it appears that p150 can be used both as a dedifferentiation and as a proliferation marker.

P. Screening of colon cancer

Nine cases of colon cancer are investigated by Western blot analysis. p150 is clearly upregulated in all nine cases in comparison with the control tissue. An additional band of about 120 kD which is considered to be a degradation product, appears exclusively in the carcinoma. This additional band is not recognized by an antibody against the NH₂-terminal peptide of p150. This implies that in colon carcinoma the amino-terminal part of p150 might be specifically degraded by high protease activity.

Immunohistochemistry, which is done on 120 cases including the 9 cases presented in Western blot analysis, clearly reconfirms our findings. Furthermore, the results suggest that p150 plays a role in the dedifferentiation process in colorectal carcinoma. E. Screening of lung and thyroid cancer

Several cases of lung and thyroide cancer are investigated as well. The data suggest an upregulation of p150 in these malignancies too.

F. Screening of oesophagal cancer

Several cases of oesophagal cancer are investigated as well. From 70 cases 30 are screened by Western blot analysis and 40 by immunohistochemistry. All tissue samples screened with either method show positive results.

Deposit Data:

Antibodies according to the invention have been deposited in accordance with the

Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen,

Maschenroder Weg 1b, D-3300 Braunschweig, Germany, under the following accession numbers:

6G7 DSM 2256 11th April 1996 6G10DSM 2257 11th April 1996.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVARTIS AG

(B) STREET: SCHWARZWALDALLEE

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP): 4002

(G) TELEPHONE: +41 61 696 11 11 (H) TELEFAX: +41 61 696 7976 (I) TELEX: 962 991

(ii) TITLE OF INVENTION: Metastasis-Associated Antigen and Antibodies thereto

(iii) NUMBER OF SEQUENCES: 9

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5137 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:128..4157

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCCGGCTGGG CGCGGGCATC TGCTGGCGAG GCGCGTGGAA GCTCGCGCTA GTTCCCCTCC 60

GCCTTCTCTC CCGGTCCAGG CCACTAGGGA GTTCGCTGAC GCCGGGTGAA CTGAGCGTAC 120

CGTCAAG ATG CCG GCC TAT TTC CAG AGG CCG GAA AAT GCC CTC AAG CGC 169 Met Pro Ala Tyr Phe Gin Arg Pro Glu Asn Ala Leu Lys Arg 1 5 10

GCC AAC GAA TTT CTT GAG GTT GGC AAA AAG CAG CCT GCC CTG GAT GTT 217 Ala Asn Glu Phe Leu Glu Val Gly Lys Lys Gin Pro Ala Leu Asp Val 15 20 25 30

CTT TAT GAT GTA ATG AAA AGT AAG AAG CAC AGA ACA TGG CAG AAG ATA 265 Leu Tyr Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gin Lys lie 35 40 45

CAT GAG CCA ATT ATG CTG AAA TAC TTG GAA CTG TGT GTG GAT CTT CGT 313 His Glu Pro lie Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg 50 55 60

AAG AGC CAC TTG GCT AAG GAA GGG TTA TAT CAA TAT AAG AAC ATA TGC 361 Lys Ser His Leu Ala Lys Glu Gly Leu Tyr Gin Tyr Lys Asn lie Cys 65 70 75

CAA CAG GTA AAC ATT AAA TCT TTA GAA GAT GTT GTT AGG GCA TAT TTG 409 Gin Gin Val Asn lie Lys Ser Leu Glu Asp Val Val Arg Ala Tyr Leu 80 85 90 AAA TTA GCA GAG GAA AAA ACA GAA GCT GCT AAA GAA GAG TCC CAA CAA 457 Lys Leu Ala Glu Glu Lys Thr Glu Ala Ala Lys Glu Glu Ser Gin Gin 95 100 105 110

ATG GTG TTA GAT ATA GAA GAT CTG GAT AAT ATT CAG ACT CCT GAG AGT 505 Met Val Leu Asp lie Glu Asp Leu Asp Asn lie Gin Thr Pro Glu Ser 115 120 125

GTT CTC TTA AGT GCA GTA AGT GGG GAA GAT ACT CAG GAT CGT ACT GAC 553 Val Leu Leu Ser Ala Val Ser Gly Glu Asp Thr Gin Asp Arg Thr Asp 130 135 140

AGA TTG CTA CTG ACT CCC TGG GTC AAG TTC CTG TGG GAT CAT ACA GGC 601 Arg Leu Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Asp His Thr Gly 145 150 155

AGT GTT TTG GAC CTT CTT CGA AAC AAT TCT AGA GTA GAG CGC CTT TAC 649 Ser Val Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr 160 165 170

CAT GAT ATC GCC CAA CAA GCT TTC AAA TTC TGC CTC CAG TAT ACT CGG 697 His Asp lie Ala Gin Gin Ala Phe Lys Phe Cys Leu Gin Tyr Thr Arg 175 180 185 190

AAG GCT GAG TTC CGC AAG CTA TGT GAC AAC TTG CGA ATC GAC TTA TCC 745 Lys Ala Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg lie Asp Leu Ser 195 200 205

CAG ATT CAG CGC CAC CAT AAC CAA AGC ACA GCA ATT AAT CTT AAT AAT 793 Gin lie Gin Arg His His Asn Gin Ser Thr Ala lie Asn Leu Asn Asn 210 215 220

CCA GAG AGC CAG TCT ATG CAT TTG GAA ACC AGA CTT GTT CAG TTG GAC 841 Pro Glu Ser Gin Ser Met His Leu Glu Thr Arg Leu Val Gin Leu Asp 225 230 235 AGT GCT ATC AGC ATG GAA TTA TGG CAG GAA GCC TTC AAA GCT GTG GAA 889 Ser Ala lie Ser Met Glu Leu Trp Gln Glu Ala Phe Lys Ala Val Glu 240 245 250

GAT ATT CAT GGA CTA TTT TCC TTG TCT AAG AAA CCA CCT AAG CCT CAG 937 Asp lie His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gin 255 260 265 270

TTG ATG GCA AAT TAC TAT AAC AAA GTT TCA ACA GTG TTT TGG AAA TCT 985 Leu Met Ala Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser 275 280 285

GGA AAT GCT TTG TTC CAT GCA TCT ACA CTT CAT CGT CTT TAT CAT CTG 1033 Gly Asn Ala Leu Phe His Ala Ser Thr Leu His Arg Leu Tyr His Leu 290 295 300

TCT AGA GAA ATG AGA AAG AAT CTT ACA CAA GAA GAG ATG CAA AGA ATG 1081 Ser Arg Glu Met Arg Lys Asn Leu Thr Gin Glu Glu Met Gin Arg Met 305 310 315

TCT ACT AGA GTC CTT TTG GCG ACT CTT TCC ATT CCT ATT ACT CCT GAG 1129 Ser Thr Arg Val Leu Leu Ala Thr Leu Ser lie Pro lie Thr Pro Glu 320 325 330

CGA ACT GAT ATT GCT CGA CTT TTG GAT ATG GAT GGT ATT ATA GTT GAG 1177 Arg Thr Asp lie Ala Arg Leu Leu Asp Met Asp Gly lie lie Val Glu 335 340 345 350

AAG CAG CGA CGC CTT GCC ACA TTG CTA GGT CTT CAA GCC CCA CCC ACA 1225 Lys Gin Arg Arg Leu Ala Thr Leu Leu Gly Leu Gin Ala Pro Pro Thr 355 360 365

CGA ATC GGC CTA ATT AAT GAT ATG GTC AGG TTC AGT GTG CTA CAG TAT 1273 Arg lie Gly Leu lie Asn Asp Met Val Arg Phe Ser Val Leu Gin Tyr 370 375 380 GTT GTC CCA GAA GTG AAA GAC CTT TAC AAC TGG TTG GAG GTG GAA TTC 1321 Val Val Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe 385 390 395

AAC CCA CTA AAA CTC TGT GAG AGA GTT ACA AAG GTA TTA AAT TGG GTT 1369 Asn Pro Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val 400 405 410

AGG GAA CAA CCT GAA AAA GAA CCA GAA TTG CAA CAA TAT GTA CCA CAA 1417 Arg Glu Gin Pro Glu Lys Glu Pro Glu Leu Gin Gin Tyr Val Pro Gin 415 420 425 430

CTC CAG AAC AAT ACC ATA CTC CGC CTT CTG CAA CAG GTG GCA CAG ATT 1465 Leu Gin Asn Asn Thr lie Leu Arg Leu Leu Gin Gin Val Ala Gin lie 435 440 445

TAT CAG AGC ATT GAG TTT TCT CGT TTG ACT TCT CTG GTT CCT TTT GTT 1513 Tyr Gin Ser lie Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val 450 455 460

GAT GCT TTC CAA CTG GAA CGG GCC ATA GTA GAT GCA GCC AGG CAC TGT 1561 Asp Ala Phe Gin Leu Glu Arg Ala lie Val Asp Ala Ala Arg His Cys 465 470 475

GAC CTG CAG GTA CGT ATT GAC CAT ACT TCC CGG ACT CTG AGT TTT GGA 1609 Asp Leu Gin Val Arg lie Asp His Thr Ser Arg Thr Leu Ser Phe Gly 480 485 490

TCA GAT TTG AAT TAT GCA ACT CGA GAA GAT GCC CCA GTT GGC CCT CAT 1657 Ser Asp Leu Asn Tyr Ala Thr Arg Glu Asp Ala Pro Val Gly Pro His 495 500 505 510

CTG CAG AGC ATG CCT TCA GAG CAG ATA AGA AAC CAG CTC ACG GCC ATG 1705 Leu Gin Ser Met Pro Ser Glu Gin lie Arg Asn Gin Leu Thr Ala Met 515 520 525 TCC TCA GTG CTT GCC AAA GCA CTT GAA GTC ATC AGA CCT GCT CAC ATT 1753 Ser Ser Val Leu Ala Lys Ala Leu Glu Val lie Arg Pro Ala His lie 530 535 540

CTG CAA GAG AAA GAA GAA CAG CAT CAA TTG GCA GTT AAT GCA TAT CTT 1801 Leu Gin Glu Lys Glu Glu Gin His Gin Leu Ala Val Asn Ala Tyr Leu 545 550 555

AAA AAT TCA AGA AAA GAG CAC CAG AGG ATC CTG GCT CGG AGA CAG ACA 1849 Lys Asn Ser Arg Lys Glu His Gin Arg lie Leu Ala Arg Arg Gin Thr 560 565 570

ATT GAG GAA AGA AAA GAG CGT CTT GAG AGT CTG AAT ATT CAA CGT GAG 1897 lie Glu Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn lie Gin Arg Glu 575 580 585 590

AAG GAA GAA CTC GAG CAG AGG GAA GCT GAA CTC CAG AAG TAC GAA AGG 1945 Lys Glu Glu Leu Glu Gin Arg Glu Ala Glu Leu Gin Lys Tyr Glu Arg 595 600 605

CTG AAG AAG AAA GGC TGC CAA GAG GCA AAG GAG CGA GAG AAG GAA CGA 1993 Leu Lys Lys Lys Gly Cys Gin Glu Ala Lys Glu Arg Glu Lys Glu Arg 610 615 620

ATC CTT CAA GAA CAC GAG CAA ATC AAG AAG AAA ACT GTT CGG GAG CGG 2041 lie Leu Gin Glu His Glu Gin lie Lys Lys Lys Thr Val Arg Glu Arg 625 630 635

TTA GAG CAG ATC AAG AAG ACA GAG CTG GGC GCC AAA GCA TTT AAA GAT 2089 Leu Glu Gin lie Lys Lys Thr Glu Leu Gly Ala Lys Ala Phe Lys Asp 640 645 650

ATT GAC ATT GAA GAC CTT GAA GAA CTG GAT CCA GAT TTT ATT ATG GCC 2137 lie Asp lie Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe lie Met Ala 655 660 665 670 AAA CAG GTT GAA CAA CTG GAG AAA GAA AAG AAG GAA CTT CAG GAA CGC 2185 Lys Gin Val Glu Gin Leu Glu Lys Glu Lys Lys Glu Leu Gin Glu Arg 675 680 685

CTG AAG AAT CAA GAA AAG AAG ATT GAC TAT TTT GAG AGA GCT AAG CGT 2233 Leu Lys Asn Gin Glu Lys Lys lie Asp Tyr Phe Glu Arg Ala Lys Arg 690 695 700

TTG GAA GAA ATT CCT TTA ATA AAG AGT GCT TAT GAG GAA CAA AGG GTT 2281 Leu Glu Glu lie Pro Leu lie Lys Ser Ala Tyr Glu Glu Gin Arg Val 705 710 715

AAA GAC ATG GAC CTG TGG GAA CAG CAA GAA GAA GAA AGA ATC ACT ACA 2329 Lys Asp Met Asp Leu Trp Glu Gin Gin Glu Glu Glu Arg lie Thr Thr 720 725 730

ATG CAG CTA GAA CGA GAA AAA GCT CTG GAG CAT AAG AAT AGG ATG TCA 2377 Met Gin Leu Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser 735 740 745 750

CGA ATG TTG GAA GAC AGA GAT CTA TTT GTG ATG CGC CTC AAA GCT GCC 2425 Arg Met Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ala Ala 755 760 765

CGG CAG TCT GTC TAC GAG GAA AAA CTG AAA CAG TTT GAA GAG CGC TTA 2473 Arg Gin Ser Val Tyr Glu Glu Lys Leu Lys Gin Phe Glu Glu Arg Leu 770 775 780

GCA GAA GAA AGG CAT AGT CGC CTA GAA GAT CGG AAA AGG CAG CGG AAA 2521 Ala Glu Glu Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gin Arg Lys 785 790 795

GAA GAA CGC AAA ATA ACT TAT TAC AGA GAA AGG AAG AAG AAG AGC AGA 2569 Glu Glu Arg Lys lie Thr Tyr Tyr Arg Glu Arg Lys Lys Lys Ser Arg 800 805 810 GGA GGG CAG AGG AGC AGA TGC TCA AAG ACG GAG AAG AAA GAG AAC GTG 2617 Gly Gly Gin Arg Ser Arg Cys Ser Lys Thr Glu Lys Lys Glu Asn Val 815 820 825 830

CTG AGA GAG CAA AAC GCG GAG GAA GAA CTT AGA GAG TAT CAG GAG CGA 2665 Leu Arg Glu Gin Asn Ala Glu Glu Glu Leu Arg Glu Tyr Gin Glu Arg 835 840 845

GTC AAG AAA CTA GAA GAA GTA GAA AGG AAA AAG CGG CAA AGA GAG CTG 2713 Val Lys Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gin Arg Glu Leu 850 855 860

GAA ATT GAA GAA AGG GAA AGG CGC AGA GAG GAA GAA AGG AGA CTT GGT 2761 Glu lie Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly 865 870 875

GAT GAT CCA CTT TCT AGG AAG GAC TCT CGG TGG GGA GAT AGA GAT TCA 2809 Asp Asp Pro Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser 880 885 890

GAA GGC ACC TGG AGG AAA GGA CCA GAA GCT GAC TCT GAG TGG AGA AGA 2857 Glu Gly Thr Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg 895 900 905 910

GGC CCA CCA GAA AAG GAG TGG AGA CGA GAA ACT CGG GAT GAT GAG AGG 2905 Gly Pro Pro Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg 915 920 925

CCT CAC AGG AGA GAT GAG GAT CGG CTG AGG CGC TTG GGG GGT GAC GAT 2953 Pro His Arg Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp 930 935 940

GAA GAG AGA GAG TCT TCT CTT AGA CCG GAT GAT GAT CGT ATC CCC AGG 3001 Glu Glu Arg Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg lie Pro Arg 945 950 955 CGT GGC CTG GAT GAT GAC AGA GGG CCT AGA CGT GGT CCC GAT GAA GAT 3049 Arg Gly Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Asp Glu Asp 960 965 970

AGA TTT TCT CGT AGA GGA ACA GAT GAT GAC CGT CCT TCC TGG CGT AAT 3097 Arg Phe Ser Arg Arg Gly Thr Asp Asp Asp Arg Pro Ser Trp Arg Asn 975 980 985 990

GCA GAT GAT GAT AGG CCT CCC AGG CGA ATT GGT GAT GAT GAC AGG GGA 3145 Ala Asp Asp Asp Arg Pro Pro Arg Arg lie Gly Asp Asp Asp Arg Gly 995 1000 1005

AGC TGG CGT CAC ACA GAT GAC GAC AGA CCA CCC AGA CGG GGA CTG GAT 3193 Ser Trp Arg His Thr Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp 1010 1015 1020

GAT GAG AGA GGA AGC TGG CGT ACA GCA GAT GAG GAC AGA GGA CCT AGA 3241 Asp Glu Arg Gly Ser Trp Arg Thr Ala Asp Glu Asp Arg Gly Pro Arg 1025 1030 1035

CGA GGG ATG GAT GAT GAC CGG GGG CCA AGA CGA GGA GGT GCT GAT GAT 3289 Arg Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ala Asp Asp 1040 1045 1050

GAA CGG TCA TCT GGC GGA ATG CTG ATG ATG ATA GGG GTC CCA GGA GAG 3337 Glu Arg Ser Ser Gly Gly Met Leu Met Met lie Gly Val Pro Gly Glu 1055 1060 1065 1070

GCA TGG ATG ATG ATA GGG GTC CCA GGC GAG GGC TGG ATG ATG ACC GAG 3385 Ala Trp Met Met lie Gly Val Pro Gly Glu Gly Trp Met Met Thr Glu 1075 1080 1085

GAC CTT GGA GGA ATG CTG CTG AAG ATA GGA TTT CCA GGA GAG GTG CAG 3433 Asp Leu Gly Gly Met Leu Leu Lys lie Gly Phe Pro Gly Glu Val Gin 1090 1095 1100 ATG ATG ACC GAG GGC CTT GGA GAA ATA TGG ATG ATG ATA GGG TTC CTA 3481 Met Met Thr Glu Gly Leu Gly Glu lie Trp Met Met lie Gly Phe Leu 1105 1110 1115

GAA GGG GTG ATG ATG CAA GAC CTG GTC CTT GGA GAC CAT TTG TCA AGC 3529 Glu Gly Val Met Met Gin Asp Leu Val Leu Gly Asp His Leu Ser Ser 1120 1125 1130

CAG GTG GAT GGA GAG AGA AAG AAA AGG CTA GAG AAG AGA GTT GGG GTC 3577 Gin Val Asp Gly Glu Arg Lys Lys Arg Leu Glu Lys Arg Val Gly Val 1135 1140 1145 1150

CAC CTC GAG AAT CAA GAC CAT CAG AAG. AAC GTG AAT GGG ATA GAG ACA 3625 His Leu Glu Asn Gin Asp His Gin Lys Asn Val Asn Gly lie Glu Thr 1155 1160 1165

AAG AGA AGG ACA GAG ATA ATC AAG ATC GAG AGG AGA ATG ACA AAG ACC 3673 Lys Arg Arg Thr Glu lie lie Lys lie Glu Arg Arg Met Thr Lys Thr 1170 1175 1180

TTG AAC GAG ATA GGG ACA GAG AGA GAG ATG GGG ACC GGG AGG ATT CGA 3721 Leu Asn Glu lie Gly Thr Glu Arg Glu Met Gly Thr Gly Arg lie Arg 1185 1190 1195

TTC AGA AGA CCC AGG GAT GAA GGT GGC TGG AGA AGA GGA CCA GCA GAA 3769 Phe Arg Arg Pro Arg Asp Glu Gly Gly Trp Arg Arg Gly Pro Ala Glu 1200 1205 1210

GAA TCT TCA AGC TGG AGA GAT TCA AGT CGC CGT GAT GAT AGG GAC AGG 3817 Glu Ser Ser Ser Trp Arg Asp Ser Ser Arg Arg Asp Asp Arg Asp Arg 1215 1220 1225 1230

GAA GAC CGT CGA CGG GAT AGA GAT GAT CGT CGT GAT CTG AGA GAC CTG 3865 Glu Asp Arg Arg Arg Asp Arg Asp Asp Arg Arg Asp Leu Arg Asp Leu 1235 1240 1245 AGA GAC AGA AGG GAT TTA AGA GAT GAT AGA GAT CGG AGA GGA CCT CCC 3913 Arg Asp Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg Gly Pro Pro 1250 1255 1260

CTC AGA TCA GAG CGA GAA GAA GCA AGC TCT TGG AGA CGC ACT GAT GAC 3961 Leu Arg Ser Glu Arg Glu Glu Ala Ser Ser Trp Arg Arg Thr Asp Asp 1265 1270 1275

AGG AAA GAT GAC CGG ACT GAA GAG AGG GAT CCA CCT CGT CGT GTT CCT 4009 Arg Lys Asp Asp Arg Thr Glu Glu Arg Asp Pro Pro Arg Arg Val Pro 1280 1285 1290

CCC CCA GCT CTT TCA AGA GAT CGA GAA AGA GAG CGA GAA CGA GAA GGT 4057 Pro Pro Ala Leu Ser Arg Asp Arg Glu Arg Glu Arg Glu Arg Glu Gly 1295 1300 1305 1310

GAG AAA GAG AAA GCA TCC TGG AGA GCT GAG AAA GAT AGG GAG TCC CTT 4105 Glu Lys Glu Lys Ala Ser Trp Arg Ala Glu Lys Asp Arg Glu Ser Leu 1315 1320 1325

CGT CGT ACT AAG AAT GAA ACT GAT GAA GAT GGA TGG ACC ACA GTA CGA 4153 Arg Arg Thr Lys Asn Glu Thr Asp Glu Asp Gly Trp Thr Thr Val Arg 1330 1335 1340

CGT T AAGTCCCAAG ATGATGGAGT CAAACTTGTG GCTTACATAG GTTTGATCAC 4207 Arg

ATTCAAGGAT TATTATACTT GTGCTTCAAC CAGTCTAAAT TGAATTCTTT AATGTTGTCT 4267

CACCATAACA CAAAAAGCAT GAACTTGTAT TAATCATATA TAATAGATTG ATCATGCACT 4327

GTATTCACAG GAGGTTGGAA AACCATGCCA TTTTCTGGAA CTTAAGGTGT TGCATTATTT 4387

CATCAATCAT TTGTTAAAAA AAAAAAACTA AAAAATAAAA ATGTGAACCC TTCAGTGTAA 4447

ACACCTTATC TTGGTATACA ATGATCTTTT TCTTTTGTTT TGAAGTATCA GATATTAATT 4507 TGGAATAAGG TAAGGTTCTC TTGAAACATT TGAAAACCCT TTAAGCCAAC TGATCTGACA 4567

GCTTTCCCAT CAGTAGAAGT GGGAACATAC CTTCTTAGGT ATTTACTATT AACTACATGT 4627

AGGCAGTTTA TAGCTTCTGA TCAGTTAGTA GACATTACAA ACACTGGTTG TAATGGGGTT 4687

TTCTGTAGAC TTTACTTGAG AGGTGAGTAT AAAGCATTTT TTAGTCATCA TCATGACGAT 4747

GCTGCTCAAG TGCAGATCCA GAACAGTACA GCGTTGGGTT CCTAGAGCAT TTGGTAAACT 4807

GTTGTGGGTT TTTTCTTTCT GTTGCCAAAA CTGCTTTTCC ACTAATTCAT GCCTTTCAAG 4867

CATTTTAAAT ATGACAATAT TTATAAATGT GTGGTTTGGA GGAATTGTTT AAATTCTTTT 4927

TCCTAATTTT CTTTTTTCAG GATAGATTAT TTCAACAAGT AATTTGTAGT GATGACTGTG 4987

TTGACTTCAA TTTTGGAGTG TAGTAGCTGT GTTTAAAAAA AAGAGTAATA ACCATTTGGT 5047

CTTATTGAAG CCAACACGGA ATTTGCTGCT GTGTTTTTTT CTTTGGTGAT AAATAAAATA 5107

CTTACATAAT TGGAAAAAAA AAAAAAAAAA 5137

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1343 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Pro Ala Tyr Phe Gin Arg Pro Glu Asn Ala Leu Lys Arg Ala Asn 1 5 10 15 Glu Phe Leu Glu Val Gly Lys Lys Gin Pro Ala Leu Asp Val Leu Tyr 20 25 30

Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gin Lys lie His Glu 35 40 45

Pro lie Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg Lys Ser

50 55 60

His Leu Ala Lys Glu Gly Leu Tyr Gin Tyr Lys Asn lie Cys Gin Gin 65 70 75 80

Val Asn lie Lys Ser Leu Glu Asp Val Val Arg Ala Tyr Leu Lys Leu 85 90 95

Ala Glu Glu Lys Thr Glu Ala Ala Lys Glu Glu Ser Gin Gin Met Val 100 105 110

Leu Asp lie Glu Asp Leu Asp Asn lie Gin Thr Pro Glu Ser Val Leu 115 120 125

Leu Ser Ala Val Ser Gly Glu Asp Thr Gin Asp Arg Thr Asp Arg Leu 130 135 140

Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Asp His Thr Gly Ser Val 145 150 155 160

Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr His Asp 165 170 175

lie Ala Gin Gin Ala Phe Lys Phe Cys Leu Gin Tyr Thr Arg Lys Ala 180 185 190

Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg lie Asp Leu Ser Gin lie 195 200 205 Gln Arg His His Asn Gin Ser Thr Ala lie Asn Leu Asn Asn Pro Glu 210 215 220

Ser Gin Ser Met His Leu Glu Thr Arg Leu Val Gin Leu Asp Ser Ala 225 230 235 240

lie Ser Met Glu Leu Trp Gin Glu Ala Phe Lys Ala Val Glu Asp lie 245 250 255

His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gin Leu Met 260 265 270

Ala Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser Gly Asn 275 280 285

Ala Leu Phe His Ala Ser Thr Leu His Arg Leu Tyr His Leu Ser Arg 290 295 300

Glu Met Arg Lys Asn Leu Thr Gin Glu Glu Met Gin Arg Met Ser Thr 305 310 315 320

Arg Val Leu Leu Ala Thr Leu Ser lie Pro lie Thr Pro Glu Arg Thr 325 330 335

Asp lie Ala Arg Leu Leu Asp Met Asp Gly lie lie Val Glu Lys Gin 340 345 350

Arg Arg Leu Ala Thr Leu Leu Gly Leu Gin Ala Pro Pro Thr Arg lie 355 360 365

Gly Leu lie Asn Asp Met Val Arg Phe Ser Val Leu Gin Tyr Val Val 370 375 380

Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe Asn Pro 385 390 395 400 Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val Arg Glu 405 410 415

Gin Pro Glu Lys Glu Pro Glu Leu Gin Gin Tyr Val Pro Gin Leu Gin 420 425 430

Asn Asn Thr lie Leu Arg Leu Leu Gin Gin Val Ala Gin lie Tyr Gin 435 440 445

Ser lie Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val Asp Ala 450 455 460

Phe Gin Leu Glu Arg Ala lie Val Asp Ala Ala Arg His Cys Asp Leu 465 470 475 480

Gin Val Arg lie Asp His Thr Ser Arg Thr Leu Ser Phe Gly Ser Asp 485 490 495

Leu Asn Tyr Ala Thr Arg Glu Asp Ala Pro Val Gly Pro His Leu Gin 500 505 510

Ser Met Pro Ser Glu Gin lie Arg Asn Gin Leu Thr Ala Met Ser Ser 515 520 525

Val Leu Ala Lys Ala Leu Glu Val lie Arg Pro Ala His lie Leu Gin 530 535 540

Glu Lys Glu Glu Gin His Gin Leu Ala Val Asn Ala Tyr Leu Lys Asn 545 550 555 560

Ser Arg Lys Glu His Gin Arg lie Leu Ala Arg Arg Gin Thr lie Glu 565 570 575

Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn lie Gin Arg Glu Lys Glu 580 585 590 Glu Leu Glu Gin Arg Glu Ala Glu Leu Gin Lys Tyr Glu Arg Leu Lys 595 600 605

Lys Lys Gly Cys Gin Glu Ala Lys Glu Arg Glu Lys Glu Arg lie Leu 610 615 620

Gin Glu His Glu Gin lie Lys Lys Lys Thr Val Arg Glu Arg Leu Glu 625 630 635 640

Gin lie Lys Lys Thr Glu Leu Gly Ala Lys Ala Phe Lys Asp lie Asp 645 650 655

lie Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe lie Met Ala Lys Gin 660 665 670

Val Glu Gin Leu Glu Lys Glu Lys Lys Glu Leu Gin Glu Arg Leu Lys 675 680 685

Asn Gin Glu Lys Lys lie Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu 690 695 700

Glu lie Pro Leu lie Lys Ser Ala Tyr Glu Glu Gin Arg Val Lys Asp 705 710 715 720

Met Asp Leu Trp Glu Gin Gin Glu Glu Glu Arg lie Thr Thr Met Gin 725 730 735

Leu Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met 740 745 750

Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ala Ala Arg Gin 755 760 765

Ser Val Tyr Glu Glu Lys Leu Lys Gin Phe Glu Glu Arg Leu Ala Glu 770 775 780 Glu Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gin Arg Lys Glu Glu 785 790 795 800

Arg Lys lie Thr Tyr Tyr Arg Glu Arg Lys Lys Lys Ser Arg Gly Gly 805 810 815

Gin Arg Ser Arg Cys Ser Lys Thr Glu Lys Lys Glu Asn Val Leu Arg 820 825 830

Glu Gin Asn Ala Glu Glu Glu Leu Arg Glu Tyr Gin Glu Arg Val Lys 835 840 845

Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gin Arg Glu Leu Glu lie 850 855 860

Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp Asp 865 870 875 880

Pro Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu Gly 885 890 895

Thr Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg Gly Pro 900 905 910

Pro Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg Pro His 915 920 925

Arg Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp Glu Glu 930 935 940

Arg Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg lie Pro Arg Arg Gly 945 950 955 960

Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Asp Glu Asp Arg Phe 965 970 975 Ser Arg Arg Gly Thr Asp Asp Asp Arg Pro Ser Trp Arg Asn Ala Asp 980 ^"985 990

Asp Asp Arg Pro Pro Arg Arg lie Gly Asp Asp Asp Arg Gly Ser Trp 995 1000 1005

Arg His Thr Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp Asp Glu 1010 1015 1020

Arg Gly Ser Trp Arg Thr Ala Asp Glu Asp Arg Gly Pro Arg Arg Gly 1025 1030 1035 1040

Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ala Asp Asp Glu Arg 1045 1050 1055

Ser Ser Gly Gly Met Leu Met Met He Gly Val Pro Gly Glu Ala Trp 1060 1065 1070

Met Met He Gly Val Pro Gly Glu Gly Trp Met Met Thr Glu Asp Leu 1075 1080 1085

Gly Gly Met Leu Leu Lys He Gly Phe Pro Gly Glu Val Gin Met Met 1090 1095 1100

Thr Glu Gly Leu Gly Glu He Trp Met Met He Gly Phe Leu Glu Gly 1105 1110 1115 1120

Val Met Met Gin Asp Leu Val Leu Gly Asp His Leu Ser Ser Gin Val 1125 1130 1135

Asp Gly Glu Arg Lys Lys Arg Leu Glu Lys Arg Val Gly Val His Leu 1140 1145 1150

Glu Asn Gin Asp His Gin Lys Asn Val Asn Gly He Glu Thr Lys Arg 1155 1160 1165 Arg Thr Glu He He Lys He Glu Arg Arg Met Thr Lys Thr Leu Asn 1170 1175 1180

Glu He Gly Thr Glu Arg Glu Met Gly Thr Gly Arg He Arg Phe Arg 1185 1190 1195 1200

Arg Pro Arg Asp Glu Gly Gly Trp Arg Arg Gly Pro Ala Glu Glu Ser 1205 1210 1215

Ser Ser Trp Arg Asp Ser Ser Arg Arg Asp Asp Arg Asp Arg Glu Asp 1220 1225 1230

Arg Arg Arg Asp Arg Asp Asp Arg Arg Asp Leu Arg Asp Leu Arg Asp 1235 1240 1245

Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg Gly Pro Pro Leu Arg 1250 1255 1260

Ser Glu Arg Glu Glu Ala Ser Ser Trp Arg Arg Thr Asp Asp Arg Lys 1265 1270 1275 1280

Asp Asp Arg Thr Glu Glu Arg Asp Pro Pro Arg Arg Val Pro Pro Pro 1285 1290 1295

Ala Leu Ser Arg Asp Arg Glu Arg Glu Arg Glu Arg Glu Gly Glu Lys 1300 1305 1310

Glu Lys Ala Ser Trp Arg Ala Glu Lys Asp Arg Glu Ser Leu Arg Arg 1315 1320 1325

Thr Lys Asn Glu Thr Asp Glu Asp Gly Trp Thr Thr Val Arg Arg 1330 1335 1340 (2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 276 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Met Pro Ser Glu Gin He Arg Asn Gin Leu Thr Ala Met Ser Ser Val

1 5 10 15

Leu Ala Lys Ala He Glu Val He Arg Pro Ala His He Leu Gin Glu 20 25 30

Lys Glu Glu Gin His Gin Leu Ala Val Asn Ala Tyr Leu Lys Asn Ser 35 40 45

Arg Lys Glu His Gin Arg He Leu Ala Arg Arg Gin Thr He Glu Glu 50 55 60

Arg Lys Glu Arg Leu Glu Ser Leu Asn He Gin Arg Glu Lys Glu Glu 65 70 75 80

Leu Glu Gin Arg Glu Ala Glu Leu Gin Lys Val Arg Lys Ala Glu Glu 85 90 95

Glu Arg Leu Pro Arg Gly Lys Gly Ala Arg Glu Gly Thr Asn Pro Ser 100 105 110 Arg Thr Arg Ala Asn Gin Glu Glu Asn Cys Ser Gly Ala Val Arg Ala 115 120 125

Asp Gin Glu Asp Arg Leu Gly Ala Lys Ala Phe Lys Asp He Asp He 130 135 140

Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe He Met Ala Lys Gin Val 145 150 155 160

Glu Gin Leu Glu Lys Glu Lys Lys Asp Tyr Gin Glu Arg Leu Lys Asn 165 170 175

Gin Glu Lys Lys He Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu Glu 180 185 190

He Pro Leu He Lys Ser Ala Tyr Glu Glu His Arg Val Lys Asp Met 195 200 205

Asp Leu Trp Glu Gin Gin Glu Glu Glu Arg He Thr Thr Met Gin Leu 210 215 220

Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met Leu 225 230 235 240

Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Val Ala Arg Gin Ser 245 250 255

Val Tyr Glu Glu Lys Leu Lys Gin Phe Glu Glu Arg Leu Ala Glu Glu 260 265 270

Ser He Val Ala 275 (2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 447 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Pro Ser Glu Gin He Arg Asn Gin Leu Thr Ala Met Ser Ser Val 1 5 10 15

Leu Ala Lys Ala He Glu Val He Arg Pro Ala His He Leu Gin Glu 20 25 30

Lys Glu Glu Gin His Gin Leu Ala Val Asn Ala Tyr Leu Lys Asn Ser 35 40 45

Arg Lys Glu His Gin Arg He Leu Ala Arg Arg Gin Thr He Glu Glu 50 55 60

Arg Lys Glu Arg Leu Glu Ser Leu Asn He Gin Arg Glu Lys Glu Glu 65 70 75 80

Leu Glu Gin Arg Glu Ala Glu Leu Gin Lys Val Arg Lys Ala Glu Glu 85 90 95

Glu Arg Leu Pro Arg Gly Lys Gly Ala Arg Glu Gly Thr Asn Pro Ser 100 105 110 Arg Thr Arg Ala Asn Gin Glu Glu Asn Cys Ser Gly Ala Val Arg Ala 115 120 125

Asp Gin Glu Asp Arg Leu Gly Ala Lys Ala Phe Lys Asp He Asp He 130 135 140

Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe He Met Ala Lys Gin Val 145 150 155 160

Glu Gin Leu Glu Lys Glu Lys Lys Asp Tyr Gin Glu Arg Leu Lys Asn 165 170 175

Gin Glu Lys Lys He Asp Tyr Phe Glu Arg Ala Lys Arg Leu Glu Glu 180 185 190

He Pro Leu He Lys Ser Ala Tyr Glu Glu His Arg Val Lys Asp Met 195 200 205

Asp Leu Trp Glu Gin Gin Glu Glu Glu Arg He Thr Thr Met Gin Leu 210 215 220

Glu Arg Glu Lys Ala Leu Glu His Lys Asn Arg Met Ser Arg Met Leu 225 230 235 240

Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Val Ala Arg Gin Ser 245 250 255

Val Tyr Glu Glu Lys Leu Lys Gin Phe Glu Glu Arg Leu Ala Glu Glu 260 265 270

Arg His Ser Arg Leu Glu Asp Arg Lys Arg Gin Arg Lys Glu Glu Arg 275 280 285

Lys He Thr Tyr Tyr Arg Glu Lys Glu Glu Glu Glu Gin Arg Arg Ala 290 295 300 Glu Glu Gin Met Leu Lys Glu Arg Glu Glu Arg Glu Arg Ala Glu Arg 305 310 315 320

Ala Lys Arg Glu Glu Glu Leu Arg Glu Tyr Gin Glu Arg Val Lys Lys 325 330 335

Leu Glu Glu Val Glu Arg Lys Lys Arg Gin Arg Glu Leu Glu He Glu 340 345 350

Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp Asp Pro 355 360 365

Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu Gly Thr 370 375 380

Trp Arg Lys Gly Pro Glu Ala Asp Ser Glu Trp Arg Arg Gly Pro Pro 385 390 395 400

Glu Lys Glu Trp Arg Arg Glu Thr Arg Asp Asp Glu Arg Pro His Arg 405 410 415

Arg Asp Glu Asp Arg Leu Arg Arg Leu Gly Gly Asp Asp Glu Glu Arg

420 425 430

Glu Ser Ser Leu Arg Pro Asp Asp Asp Arg He Pro Arg Arg Gly 435 440 445

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 964 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear ( ii) MOLECULE TYPE : peptide

( iii) HYPOTHETICAL: NO

( iv) ANTI-SENSE : NO

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 5 :

Met Ma Pro Pro Pro Phe Arg Pro Glu Asn Ma He Lys Arg Ma Asp

1 5 10 15

Glu Leu He Ser Val Gly Glu Lys Gin Ma Ma Leu Gin Ser Leu His 20 25 30

Asp Phe He Thr Ma Arg Arg He Arg Trp Ma Thr Pro Ser Thr Val 35 40 45

Glu Pro Val Val Phe Lys Phe Leu Glu He Gly Val Glu Leu Lys Lys 50 55 60

Gly Lys Leu Leu Lys Asp Gly Leu His Gin Tyr Lys Lys Leu He Gin 65 70 75 80

Gly Ser Thr Glu Gly Leu Val Ser Val Gly Ma Val Ma Arg Lys Phe 85 90 95

He Asp Leu Val Glu Ser Lys He Ma Ser Glu Gin Thr Arg Ma Asp 100 105 110

Glu Leu Gin Lys Gin Glu He Asp Asp Asp Leu Glu Gly Gly Val Thr 115 120 125

Pro Glu Asn Leu Leu He Ser Val Tyr Glu Ser Asp Gin Ser Val Ma 130 135 140 Gly Phe Asn Asp Glu Ma He Thr Ser Trp Leu Arg Phe Thr Trp Glu 145 150 155 160

Ser Tyr Arg Ma Val Leu Asp Leu Leu Arg Asn Asn Ma Leu Leu Glu 165 170 175

He Thr Tyr Ser Gly Val Val Lys Lys Thr Met His Phe Cys Leu Lys 180 185 190

Tyr Gin Arg Lys Asn Glu Phe Lys Arg Leu Ma Glu Met Leu Arg Gin 195 200 205

His Leu Asp Ma Ma Asn Tyr Gin Gin Ser Lys Ser Gly Asn Asn Leu 210 215 220

Val Asp Leu Ser Asp Ma Asp Thr Leu Gin Arg Tyr Leu Asp Gin Arg 225 230 235 240

Phe Gin Gin Val Asp Val Ser Val Lys Leu Glu Leu Trp His Glu Ma 245 250 255

Tyr Arg Ser He Glu Asp Val Phe His Leu Met Lys He Ser Lys Arg 260 265 270

Ma Pro Lys Pro Ser Thr Leu Ma Asn Tyr Tyr Glu Asn Leu Val Lys 275 280 285

Val Phe Phe Val Ser Gly Asp Pro Leu Leu His Thr Thr Ma Trp Lys 290 295 300

Lys Phe Tyr Lys Leu Tyr Ser Thr Asn Pro Arg Ma Thr Glu Glu Glu 305 310 315 320

Phe Lys Thr Tyr Ser Ser Thr He Phe Leu Ser Ma He Ser Thr Gin 325 330 335 Leu Asp Glu He Pro Ser He Gly Tyr Asp Pro His Leu Arg Met Tyr 340 345 350

Arg Leu Leu Asn Leu Asp Ma Lys Pro Thr Arg Lys Glu Met Leu Gin 355 360 365

Ser He He Glu Asp Glu Ser He Tyr Gly Lys Val Asp Glu Glu Leu 370 375 380

Lys Glu Leu Tyr Asp He He Glu Val Asn Phe Asp Val Asp Thr Val 385 390 395 400

Lys Gin Gin Leu Glu Asn Leu Leu Val Lys Leu Ser Ser Lys Thr Tyr 405 410 415

Phe Ser Gin Tyr He Ma Pro Leu Arg Asp Val He Met Arg Arg Val 420 425 430

Phe Val Ma Ma Ser Gin Lys Phe Thr Thr Val Ser Gin Ser Glu Leu 435 440 445

Tyr Lys Leu Ma Thr Leu Pro Ma Pro Leu Asp Leu Ser Ma Trp Asp 450 455 460

He Glu Lys Ser Leu Leu Gin Ma Ma Val Glu Asp Tyr Val Ser He 465 470 475 480

Thr He Asp His Glu Ser Ma Lys Val Thr Phe Ma Lys Asp Pro Phe 485 490 495

Asp He Phe Ma Ser Thr Ma Ser Lys Glu Val Ser Glu Glu Glu Asn 500 505 510

Thr Glu Pro Glu Val Gin Glu Glu Lys Glu Glu Thr Asp Glu Ma Leu 515 520 525 Gly Pro Gin Glu Thr Glu Asp Gly Glu Glu Lys Glu Glu Glu Ser Asp 530 535 540

Pro Val He He Arg Asn Ser Tyr He His Asn Lys Leu Leu Glu Leu 545 550 555 560

Ser Asn Val Leu His Asp Val Asp Ser Phe Asn Asn Ma Ser Tyr Met 565 570 575

Glu Lys Val Arg He Ma Arg Glu Thr Leu He Lys Lys Asn Lys Asp 580 585 590

Asp Leu Glu Lys He Ser Lys He Val Asp Glu Arg Val Lys Arg Ser 595 600 605

Gin Glu Gin Lys Gin Lys His Met Glu His Ma Ma Leu His Ma Glu 610 615 620

Gin Asp Ma Glu Val Arg Gin Gin Arg He Leu Glu Glu Lys Ma Ma 625 630 635 640

He Glu Ma Lys Leu Glu Glu Glu Ma His Arg Arg Leu He Glu Lys 645 650 655

Lys Lys Arg Glu Phe Glu Ma He Lys Glu Arg Glu He Thr Lys Met 660 665 670

He Thr Glu Val Asn Ma Lys Gly His Val Tyr He Asp Pro Asn Glu 675 680 685

Ma Lys Ser Leu Asp Leu Asp Thr He Lys Gin Val He He Ma Glu 690 695 700

Val Ser Lys Asn Lys Ser Glu Leu Glu Ser Arg Met Glu Tyr Ma Met 705 710 715 720 Lys Lys Leu Asp His Thr Glu Arg Ma Leu Arg Lys Val Glu Leu Pro 725 730 735

Leu Leu Gin Lys Glu Val Asp Lys Leu Gin Glu Thr Asp Thr Ma Asn 740 745 750

Tyr Glu Ma Met Lys Lys Lys He Val Asp Ma Ma Lys Ma Glu Tyr 755 760 765

Glu Ma Arg Met Ma Asp Arg Lys Asn Leu Val Met Val Tyr Asp Asp 770 775 780

Tyr Leu Lys Phe Lys Glu His Val Ser Gly Thr Lys Glu Ser Glu Leu 785 790 795 800

Ma Ma He Arg Asn Gin Lys Lys Ma Glu Leu Glu Ma Ma Lys Lys 805 810 815

Ma Arg He Glu Glu Val Arg Lys Arg Arg Tyr Glu Glu Ma He Ma 820 825 830

Arg Arg Lys Glu Glu He Ma Asn Ma Glu Arg Gin Lys Arg Ma Gin 835 840 845

Glu Leu Ma Glu Ma Thr Arg Lys Gin Arg Glu He Glu Glu Ma Ma 850 855 860

Ma Lys Lys Ser Thr Pro Tyr Ser Phe Arg Ma Gly Asn Arg Glu Pro 865 870 875 880

Pro Ser Thr Pro Ser Thr Leu Pro Lys Ma Thr Val Ser Pro Asp Lys 885 890 895

Ma Lys Leu Asp Met He Ma Gin Lys Gin Arg Glu Met Glu Glu Ma 900 905 910 Ile Glu Gin Arg Leu Ma Gly Arg Thr Ma Gly Gly Ser Ser Pro Ma 915 920 925

Thr Pro Ma Thr Pro Ma Thr Pro Ma Thr Pro Thr Pro Ser Ser Gly 930 935 940

Pro Lys Lys Met Thr Met Ma Glu Lys Leu Arg Ma Lys Arg Leu Ma 945 950 955 960

Lys Gly Gly Arg

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 958 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Ma Thr Phe Ma Lys Pro Glu Asn Ma Leu Lys Arg Ma Glu Glu 1 5 10 15

Leu He Thr Val Gly Gin Lys Gin Glu Ma Leu Gin Ma Leu His Asp 20 25 30 Leu He Thr Ser Arg Arg Tyr Arg Ma Trp Gin Lys Thr Leu Glu Arg

35 ^" 40 45

He Met Phe Lys Tyr Val Glu Leu Cys Val Asp Met Arg Arg Gly Arg 50 55 60

Phe Ma Lys Asp Gly Leu He Gin Tyr Arg He Val Cys Gin Gin Val 65 70 75 80

Asn He Asn Ser Leu Glu Glu Val He Lys His Phe Met His Leu Ma 85 90 95

Thr Glu Arg Ma Glu Leu Ma Arg Asn Gin Ma Gin Ma Leu Glu Glu 100 105 110

Ma Leu Asp Val Glu Asp Leu Glu Ma Asp Lys Arg Pro Glu Asp Leu 115 120 125

Met Leu Ser Tyr Val Ser Gly Glu Lys Gly Lys Asp Arg Ser Asp Arg 130 135 140

Glu Leu Val Thr Pro Trp Phe Lys Phe Leu Trp Glu Thr Tyr Arg Thr 145 150 155 160

Val Leu Glu He Leu Arg Asn Asn Ser Arg Leu Glu Ma Leu Tyr Ma 165 170 175

Met Thr Ma His Arg Ma Phe Gin Phe Cys Lys Gin Tyr Lys Arg Thr 180 185 190

Thr Glu Phe Arg Arg Leu Cys Glu He He Arg Asn His Leu Ma Asn 195 200 205

Leu Asn Lys Tyr Arg Asp Gin Arg Asp Arg Pro Asp Leu Ser Ma Pro 210 215 220 Glu Ser Leu Gin Leu Tyr Leu Asp Thr Arg Phe Glu Gin Leu Lys Val 225 230 235 240

Ma Thr Glu Leu Gly Leu Trp Gin Glu Ma Phe Arg Ser He Glu Asp 245 250 255

He Tyr Gly Leu Met Cys Met Val Lys Lys Thr Pro Lys Ma Ser Leu 260 265 270

Met Val Val Tyr Tyr Gly Lys Leu Thr Glu He Phe Trp Met Ser Ser 275 280 285

Asn His Leu Tyr His Ma Tyr Ma Trp Leu Lys Leu Phe Ser Leu Gin 290 295 300

Lys Ser Phe Asn Lys Asn Leu Ser Gin Lys Asp Leu Gin Leu He Ma 305 310 315 320

Ser Ser Val Val Leu Ma Ma Leu Ser Val Pro Pro Tyr Asp Gin Ser 325 330 335

Tyr Gly Ma Ser His Leu Glu Leu Glu Asn Glu Lys Glu Arg Ser Leu 340 345 350

Arg Val Ma Asn Leu He Gly Phe Glu Val Glu Pro Lys Ma Glu Asn 355 360 365

Arg Val Ma Leu Ser Arg Ser Ser Leu Leu Ser Glu Leu Val Ser Lys 370 375 380

Gly Val Met Ser Cys Val Thr Gin Glu Val Lys Asp Leu Tyr His Leu 385 390 395 400

Leu Glu Asn Glu Phe Leu Pro Leu Asp Leu Ma Leu Lys Val Gin Pro 405 410 415 Val Leu Ser Lys He Ser Lys Leu Gly Gly Lys Leu Ser Ser Val Ser 420 425 430

Ser Val Pro Glu Val Gin Leu Ser Gin Tyr Val Pro Ma Leu Glu Lys 435 440 445

Leu Ma Thr Leu Arg Leu Leu Gin Gin Val Ser Gin Val Tyr Gin Thr 450 455 460

He Gin He Asp Asn He Ser Lys Met He Pro Phe Phe Asp Phe Thr 465 470 475 480

Val He Glu Lys He Ser Val Asp Ma Val Arg Arg Asn Phe Leu Ma 485 490 495

He Lys Val Asp His Met Lys Gly Leu Ser Ser Leu Val Asn Arg Val 500 505 510

Leu Arg Arg Lys Asp Ser Gly He He Cys Leu Phe Leu Ma Glu Ser 515 520 525

Leu Ser Lys Ma Arg Thr Met He Tyr Pro Pro Ma Lys Lys Ma Ma 530 535 540

Lys Leu Gly Glu Ma Leu Ser Asn Leu Ma Glu He Val Glu Lys Glu 545 550 555 560

His Lys Arg Leu Leu Ma Arg Lys Ser He He Glu Lys Arg Lys Glu 565 570 575

Glu Gin Glu Arg Leu Leu Leu Glu Met Glu Arg Val Glu Glu Thr Lys 580 585 590

Arg Arg Asp Val Gin Lys Met Thr Glu Glu Ma Glu Gin Lys Arg He 595 600 605 Ma Ma Glu Tyr Glu Gin Arg Arg Asn Gin Arg He Leu Lys Glu He 610 615 620

Glu Asp Arg Glu Leu Glu Glu Ma Gin Ma Leu Leu His Glu Ma Glu 625 630 635 640

Lys Arg Ser Lys Arg Lys Lys Lys Pro Val Leu Glu Gly Glu Lys Met 645 650 655

Thr Lys Lys Val He Met Glu Leu Ma Leu Asn Glu Gin Leu Arg Glu 660 665 670

Arg Gin Glu Met Glu Lys Lys Leu Leu Lys Phe Ma Lys Ser Met Asp 675 680 685

His Leu Glu Arg Ma Lys Arg Glu Glu Ma Ma Pro Leu He Glu Ser 690 695 700

Ma Phe Lys Gin Arg Leu Ma Glu Glu Ma Ma Leu His Glu Arg Glu 705 710 715 720

Gin Gin Gin Glu He Glu Leu Ser Arg Gin Arg His Ma Gly Asp Leu 725 730 735

Glu Glu Lys Arg Arg Leu Ma Arg Met Leu Glu Asn Lys Arg He Leu 740 745 750

Gin Glu Lys Val Val Ser Ser Arg Glu Ma Glu Phe Thr Arg Met Lys 755 760 765

Arg Glu Arg Gin Glu Arg He Ser Gin He He Gin Ser Arg Lys Gin 770 775 780

Glu Arg Glu Ma Arg Arg Lys Met He Phe Phe Leu Arg Ser Glu Glu 785 790 795 800 Glu Arg Gin Lys Arg Leu Gin Glu Glu Glu Glu Ma Arg Lys Arg Glu 805 810 815

Glu Ma Glu Arg Arg Lys Lys Glu Glu Ma Glu Arg Gin Ma Lys Leu 820 825 830

Asp Glu He Ma Glu Lys Gin Arg Arg Arg Met Leu Glu Leu Glu Glu 835 840 845

Lys Glu Lys Arg Glu Arg Glu Glu He Leu Arg Lys Ser Thr Ma Val 850 855 860

Leu Pro Lys Pro Ma Glu Pro Pro Thr Leu Gly Arg Pro Ma Glu Leu 865 870 875 880

Gly Gly Ma Ma Pro He Pro Ma Ma Ma Ma Thr Ma Pro Thr Pro 885 890 895

Gly Pro Gly Lys Tyr Val Pro Lys His Leu Arg Thr Lys Met Asp Gly 900 905 910

Ma Gly Gin Ma Pro Pro Pro Glu Thr Asp Lys Trp Gly Gly Gly Ser 915 920 925

Lys Pro Asp Asp Arg Pro Ser Trp Arg Asp Glu Arg Lys Pro Pro Ser 930 935 940

Phe Gly Ser Gly Ser Arg Thr Ser Trp Pro Ma Ser Arg Arg 945 950 955 (2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1076 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

Met Ma Pro Asn Tyr Phe Gin Lys Pro Glu Ma Ma Leu Lys Arg Ma 1 5 10 15

Glu Glu Leu He Gin Val Gly Lys Glu Ser Asp Ma Leu Asp Thr Leu 20 25 30

His Asp Thr He Lys Ma Arg Arg His Lys Gin Trp Thr Thr Val His 35 40 45

Glu Gin He Met He Lys His Met Glu Leu Cys Val Asp Leu Lys Lys 50 55 60

Gin His Leu Ma Lys Asp Ma Leu Phe Gin Tyr Lys Ma Leu Thr Gin 65 70 75 80

Gin He Asn Val Lys Ser Leu Glu Thr Val Val Val His Phe Leu Lys 85 90 95

Leu Ma Glu Gin Arg Thr Glu Asp Ma Gin Lys Gin Ser He Glu Lys 100 105 110 Val Glu Glu He Gly Asp Leu Asp Gin Gly Asp Val Pro Glu Arg Leu 115 120 125

Leu Leu Ma Val Val Ser Gly Ma Ma Ma Gin Asp Arg Met Asp Arg 130 135 140

Thr Val Leu Ma Pro Trp Leu Arg Phe Leu Trp Asp Ser Tyr Arg Asn 145 150 155 160

Cys Leu Glu Leu Leu Arg Asn Asn Ma Gin Val Glu Gin Leu Tyr His 165 170 175

Thr He Ser Arg His Ser Phe Thr Phe Cys Leu Arg Tyr Gin Arg Arg 180 185 190

Thr Glu Phe Arg Lys Leu Cys Asp Leu Leu Arg Met His Leu Asn Gin 195 200 205

He Gin Lys His Gin Tyr Ma Pro Asn Val Asn Ser Phe Arg Val Lys 210 215 220

Leu Thr Ser Pro Glu Ser Leu Gly Leu Met Gin Asp Thr Arg Leu He 225 230 235 240

Gin Leu Asp Thr Ma He Gin Met Glu Leu Trp Gin Glu Ma Tyr Lys 245 250 255

Ser Ma Glu Asp Val His Gly Met Met Gin Leu Ser Lys Asp Lys Asp 260 265 270

Lys Arg Thr Val Lys Pro Ma Ser Tyr Val Asn Tyr Tyr Asp Lys Leu 275 280 285

Ma Leu Val Phe Trp Lys Ma Gly Asn Ser Leu Phe His Ma Ma Ma 290 295 300 Leu Leu Gin Lys Phe He He Tyr Lys Asp Met Lys Lys Ser Phe Thr 305 310 315 320

Gin Asp Glu Ma Gin Glu Gin Ma Thr Arg Val Leu Leu Ma Thr Leu 325 330 335

Ser He Pro Glu Gly Ser Asp Ser Pro Ser Asp Leu Ser Arg Asn Leu 340 345 350

Asp He Glu Asp Gin His Val Ma Asn Met Arg Leu Leu Ser Asn Leu 355 360 365

Leu Arg Leu Pro He Ma Pro Thr Lys Asn Gly He Leu Lys Glu Ma 370 375 380

Ma Arg He Gly Val Pro Glu Ma Ma Gly Gin Thr Ma Lys Asp Leu 385 390 395 400

Tyr Lys Leu Leu Glu Ser Asn Phe Ser Pro Leu Lys Val Ma Lys Asp 405 410 415

Val Gin Ser Val Leu Asp Thr Val Thr Arg Pro Asp His Leu Gin Tyr 420 425 430

Val Glu Ser Leu Gin Ma Val Ma Ma Val Lys Ma Leu Lys Gin Val 435 440 445

Ser Val He Tyr Glu Ma He Ser Trp Glu Arg He Arg Lys He He 450 455 460

Pro Phe Tyr Ser Asp Leu Ma Leu Glu Arg Leu Val Val Glu Ma Ser 465 470 475 480

Lys His Arg He Val Lys Ma Gin Leu Asp His Arg Ma Asp Cys Val 485 490 495 Arg Phe Gly Ser Ser Asp Ma Thr Leu Ma Gly Gly Val Asp Glu Cys 500 505 510

Asp Asn Asn Glu Gly Phe Thr Gly Asp Asp Thr Gin Leu Gly Val Glu 515 520 525

Gly Val Arg Asn His Leu Glu Ma Met Tyr Thr Arg Leu Arg Gly Leu 530 535 540

Val Glu Gly Leu Asp Ma Glu Lys Arg Arg Lys Glu He Leu Lys Lys 545 550 555 560

He Glu Gly Gin Val Thr Ser Tyr Glu Lys Asn Arg Pro Thr Glu He 565 570 575

Glu Arg He His Arg Arg Lys Lys Met Leu Glu Asn Tyr Lys Glu Asn 580 585 590

Trp Glu Arg Val Lys Ma Glu Lys Thr Ma Ma Ma Ma Thr Glu Gin 595 600 605

Ma Lys Arg Glu Glu Ma Ma Arg Ma Glu Glu Met Lys Arg Leu Asp 610 615 620

Glu Gin Asn Lys Glu Ser Glu Arg Lys Arg Lys Gin Ma Glu Gin Asp 625 630 635 640

Glu He Gin Lys Lys He Lys Gin Asp Gin Leu Tyr Lys Met Gin Gin 645 650 655

Asn Ma He Tyr Gin Glu He He Lys Glu Lys Gly Leu Glu Gin Phe 660 665 670

Arg Asp Met Asp Pro Glu Gin Val Leu Arg Glu Gin Arg Glu Arg Leu 675 680 685 Asp Lys Glu Arg Ma Glu Thr Gin Arg Arg Leu Gin Gin Gin Glu Lys 690 695 700

Asn Phe Asp His His Val Arg Ma Leu His Leu Glu Glu Leu Asn Glu 705 710 715 720

Arg Arg Ma Val Met Asn Met Arg Leu Ser Glu Ma Pro Lys Leu His 725 730 735

Asp Leu Tyr Glu Glu Ma Arg He Ma Lys Glu He Ma Ma His Asp 740 745 750

Ser His Val Lys Leu Trp Gly Met Trp Asp Gin Val Arg Asp Ma Thr 755 760 765

Phe Asp Trp Val Glu Ser Val Lys He Asp Asn Gin Glu Thr Leu Glu 770 775 780

Lys Lys Leu Ser Asp Trp Gin Ma Lys Leu Glu Ma Val Arg Asn Asn 785 790 795 800

Arg Leu Ma Glu Arg Ma Glu Asp Arg Lys Lys Lys Arg Lys Glu Asp 805 810 815

Ma He Gin Ma Lys He Ma Glu Glu Arg Lys Lys Arg Glu Glu Glu 820 825 830

Glu Arg Ma Arg Leu Gin Val He Glu Gly Gin Arg Arg Gin His Asn 835 840 845

Asp Gly Arg Gly Arg Arg Glu Met Glu Asn Ser Val Ma Met Gin Asp 850 855 860

Asn Asp Trp Arg Arg Asn Pro Pro Arg Glu Ser Leu Pro Pro Arg Glu 865 870 875 880 Thr Arg Pro Met Arg Asp Gly Pro Thr Arg Glu Pro Arg Glu Phe Arg 885 890 895

Gly Asp Arg Asp Arg Glu Pro Arg Glu Pro Phe Arg Glu Val Pro Ser 900 905 910

Ser Lys Ma Asp Thr Asp Asn Ser Trp Arg Ser Ser Ma Gin Pro Thr 915 920 925

Arg Lys Pro Asp Asp Arg Arg Ser Asp Glu Phe Arg Arg Asn Asp Asp 930 935 940

Val Arg Arg Asn Asp Asp Val Arg Arg Asn Asp Pro Pro Arg Pro Ma 945 950 955 960

Ser Lys Ma Asp Thr Gly Asp Lys Trp Glu Arg Gly Val Lys Pro Val 965 970 975

Val Ser Pro Pro Lys Thr Asp Ma Pro Ser Val Ser Glu Pro Lys Ser 980 985 990

Glu Gly Pro Lys Arg Phe Val Pro Pro His Leu Arg Asn Arg Gin Gly 995 1000 1005

Gly Gly Gly Ma Gly Gly Ser Glu Glu Gin Ser Ma Pro Ma Arg Ser 1010 1015 1020

Gly Asn Ser Asn Val Thr Ser Pro Pro Asp Arg Ma Gin Gly Leu Arg 1025 1030 1035 1040

Gly Pro Pro Pro Thr Gly Arg Asn Ser Leu Pro Arg Arg Asp Gly Pro 1045 1050 1055

Pro Pro Arg Ser Asn Asn Thr Gly Asn Thr Gly Asn Ma Asp Ser Gly 1060 1065 1070 Ser Trp Arg Lys 1075

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5301 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION:127..4272

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

CCGGCTGGGC GCGGGCATCT GCTGGCGAGG CGCGTGGGAC CTTACGCTGG TTCCCCTTCG 60

TCTCCTCTCC CGGCCCGGGC CACTAGAGAG TTCGCTGACG CCGGGTGAGC TGAGCCTGCC 120

GCCAAGATGC CGGCCTATTT CCAGAGGCCG GAAAATGCCC TCAAACGCGC CAACGAATTT 180

CTTGAGGTTG GCAAAAAGCA GCCTGCTCTG GATCTTCTTT ATGATGTTAT GAAAAGTAAA 240

AAACATAGAA CATGGCAAAA GATACACGAA CCAATTATGT TGAAATACTT GGAACTTTGC 300

GTGGATCTTC GCAAGAGCCA CTTGGCAAAG GAGGGGTTAT ACCAGTATAA GAACATTTGT 360 CAACAGGTGA ACATAAAATC TCTGGAGGAT GTTGTTAGGG CATATTTGAA AATGGCAGAG 420

GAAAAAACTG AAGCTGCTAA AGAAGAATCT CAGCAGATGG TCTTAGATAT AGAGGATCTA 480

GATAATATTC AAACTCCTGA GAGTGTTCTC CTAAGTGCTG TAAGTGGTGA AGACACTCAG 540

GATCGTACTG ACAGATTACT TTTAACTCCA TGGGTTAAAT TCCTGTGGGA GTCTTACAGG 600

CAGTGTTTGG ACCTTCTTAG AAACAATTCT AGAGTAGAGC GCCTGTACCA TGATATTGCC 660

CAGCAAGCTT TCAAATTCTG CCTCCAATAC ACGCGTAAGG CTGAATTCCG TAAACTGTGT 720

GACAATTTGA GAATGCACTT ATCGCAGATT CAGCGCCACC ATAACCAAAG TACGGCAATC 780

AATCTTAATA ATCCAGAGAG CCAGTCCATG CATTTGGAAA CCAGACTTGT TCAGCTGGAC 840

AGTGCTATCA GCATGGAATT GTGGCAGGAA GCATTCAAAG CTGTGGAAGA TATTCACGGG 900

CTATTCTCCT TGTCTAAAAA ACCACCTAAA CCTCAGTTGA TGGCAAATTA CTATAACAAA 960

GTCTCAACTG TGTTTTGGAA ATCTGGAAAT GCTCTTTTTC ATGCATCTAC ACTCCATCGT 1020

CTTTACCATC TCTCTAGAGA AATGAGAAAG AATCTCACAC AAGACGAGAT GCAAAGAATG 1080

TCTACTAGAG TCCTTTTAGC CACTCTTTCC ATCCCTATTA CTCCTGAGCG TACGGATATT 1140

GCTCGACTTC TGGATACGGA TGGCATTATA GTTGAAAAAC AGCGTCGCCT TGCAACACTA 1200

CTAGGTCTTC AAGCCCCACC GACACGAATT GGCCTTATTA ATGATATGGT CAGATTTAAT 1260

GTACTACAAT ATGTTGTCCC AGAAGTGAAA GACCTTTACA ATTGGCTTGA AGTAGAATTT 1320

AACCCATTAA AACTCTGTGA GCGAGTCACA AAGGTTCTAA ATTGGGTTAG GGAACAACCT 1380

GAAAAGGAAC CGGAATTGCA GCAGTATGTG CCACAACTGC AAAACAACAC CATCCTCCAC 1440

CTTCTGCAGC AGGTGTCACA GATTTATCAG AGCATTGAGT TTTCTCGTTT GACTTCTTTG 1500 GTTCCTTTTG TTGATGCTTT CCAACTGGAA CGGGCCATAG TAGATGCAGC CAGGCATTGC 1560

GACTTGCAGG TTCGTATTGA TCACACTTCT CGGACCCTGA GTTTTGGATC TGATTTGAAT 1620

TATGCTACTC GAGAAGATGC TCCGATTGGT CCTCATTTGC AAAGCATGCC TTCAGAGCAG 1680

ATAAGAAACC AGCTGACAGC CATGTCCTCA GTACTTGCAA AAGCACTTGA AGTCATTAAA 1740

CCAGCTCATA TACTGCAAGA GAAAGAAGAA CAGCATCAGT TGGCTGCCAC TGCATACCTT 1800

AAAAATTCAC GAAAAGAGCA CCAGCGGATC CTGGCTCGCC GCCAGACAAT TGAGGAGAGA 1860

AAAGAGCGCC TTGAGAGTCT GAATATTCAG CGTGAGAAAG AAGAATTGGA ACAGAGGGAA 1920

GCTGAACTCC AGAAAGTGCG GAAGGCTGAG GAAGAGAGGC TGCGCCAGGA AGCAAAGGAG 1980

AGAGAGAAGG AGCGTATCTT ACAGGAACAT GAACAAATCA AAAAGAAAAC TGTCCGAGAG 2040

CGTTTGGAGC AGATCAAGAA AACAGAACTG GGTGCCAAAG CATTCAAAGA TATTGATATT 2100

GAAGACCTTG AGGAATTGGA TCCΛGATTTT ATCATGGCTA AACAGGTTGA ACAACTGGAG 2160

AAAGAAAAGA AAGAACTTCA AGAACGCCTA AAGAATCAAG AAAAGAAGAT TGACTATTTT 2220

GAAAGAGCCA AACGTTTGGA AGAAATTCCT TTGATAAAGA GCGCTTACGA GGAACAGAGA 2280

ATTAAAGACA TGGATCTGTG GGAGCAACAA GAGGAAGAAA GAATTACTAC AATGCAGCTA 2340

GAACGTGAAA AGGCTCTTGA ACATAAGAAT CGAATGTCAC GAATGCTTGA AGACAGAGAT 2400

TTATTCGTAA TGCGACTCAA AGCTGCACGG CAGTCTGTTT ATGAGGAAAA ACTTAAACAG 2460

TTTGAAGAGC GATTAGCAGA AGAAAGGCAT AATCGATTGG AAGAACGGAA AAGGCAGCGT 2520

AAAGAAGAAC GCAGGATAAC ATACTATAGA GAAAAAGAAG AGGAGGAGCA GAGAAGGGCA 2580

GGAGAACAAA TGCTACAAGA GCGGGAAGAG AGAGAGCGCG CCGAACGAGC AAAACGCGAG 2640 GAAGAGCTAC GAGAGTATCA GGAGCGGGTG AAGAAATTAG AAGAAGTGGA AAGGAAAAAA 2700

CGCCAAAGGG AGTTGGAAAT TGAAGAACGA GAACGGCGTA GAGAGGAAGA GAGAAGACTT 2760

GGCGATAGTT CCCTTTCTAG AAAGGACTCT CGTTGGGGAG ATAGAGATTC AGAAGGCACC 2820

TGGAGAAAAG GACCTGAAGC AGATTCTGAG TGGAGAAGAG GCCCGCCAGA GAAGGAGTGG 2880

AGACGTGGAG AAGGGCGAGA TGAGGACAGG TCTCATAGAA GAGATGAAGA GCGGCCCCGG 2940

CGTCTGGGGG ATGTTGAAGA TAGAGAGCCC TCTCTTAGAC CAGACGATGA TCGGGTTCCC 3000

CGGCGTGGCA TGGATGATGA CAGAGGCCCT AGACGTGGTC CTGAGGAAGA TAGGTTCTCT 3060

CGTCGTGGGG CAGACGATGA CCGGCCTTCC TGGCGTAACA CAGATGATGA CAGGCCTCCC 3120

AGACGAATTG CCGATGAAGA CAGGGGAAAC TGGCGTCATG CGGATGATGA CAGACCACCT 3180

AGACGAGGAC TGGATGAGGA CAGAGGAAGC TGGCGAACAG CTGATGAGGA CAGAGGACCA 3240

AGACGTGGGA TGGATGATGA CCGGGGGCCG AGGCGAGGAG GCGCTGATGA TGAGCGATCA 3300

TCCTGGCGTA ATGCTGATGA TGACCGGGGT CCCAGGCGAG GGTTGGATGA TGATCGGGGT 3360

CCCAGGCGAG GCATGGATGA TGACCGGGGT CCCAGGCGAG GCATGGATGA TGACCGGGGT 3420

CCCAGGCGAG GCATGGATGA TGACCGGGGT CCCAGGCGAG GGTTGGATGA TGATCGAGGA 3480

CCTTGGAGGA ACGCCGATGA TGACAGAATT CCCAGGCGTG GTGCAGAGGA TGACAGGGGC 3540

CCTTGGAGAA ACATGGATGA TGATCGCCTT TCAAGACGTG CTGATGATGA TCGGTTTCCC 3600

AGACGGGGTG ATGACTCAAG ACCTGGTCCT TGGAGACCAT TAGTCAAGCC AGGTGGATGG 3660

AGAGAGAAAG AAAAAGCCAG AGAGGAGAGC TGGGGTCCAC CTCGAGAATC AAGGCCATCA 3720

GAAGAACGTG AATGGGACAG AGAAAAAGAA AGGGACAGAG ATAATCAAGA TCGGGAGGAG 3780 AATGACAAGG ACCCTGAGAG AGAAAGGGAC AGAGAGAGAG ATGTGGATCG AGAGGATCGC 3840

TTCAGAAGAC CTAGGGATGA AGGTGGCTGG AGAAGAGGAC CAGCTGAGGA ATCTTCAAGC 3900

TGGAGAGACT CAAGTCGCCG GGACGATAGG GATAGGGATG ACCGTCGCCG TGAGAGGGAT 3960

GACCGGCGTG ATCTAAGAGA AAGACGAGAT CTAAGAGACG ACAGGGACCG AAGAGGACCT 4020

CCACTCAGAT CAGAACGTGA AGAAGTAAGT TCTTGGAGAC GTGCTGATGA CAGGAAAGAT 4080

GACCGGGTGG AAGAGCGGGA CCCTCCTCGT CGAGTTCCTC CCCCAGCTCT TTCAAGAGAC 4140

CGAGAAAGAG ACCGAGACCG AGAAAGAGAA GGTGAAAAAG AGAAGGCCTC ATGGAGAGCT 4200

GAGAAAGATA GGGAATCTCT CCGTCGTACT AAAAATGAGA CTGATGAAGA TGGATGGACC 4260

ACAGTACGAC GTTAACCCGG GCGCGTCTCA AGATAATGGA TTTAAACTGG TGTCTTAAAT 4320

AGGTTTGATC ACATTCAAGG ATTATTATAC TTGTGCTTCA ACCAATCTAA ATTGGATTCT 4380

TTAATGTTGT TTCACCATAA CACAAAAAGC ATGAACTTGT ATTAATCCTA TATAATAGAT 4440

TGATCATGCA CCATATCCAC AGGAGGTTGG AAAAACCATG CCATTTTCTG GAATTTAAGG 4500

GTGTTGCATT ATTTCATCAA TCATTTGTTG ACAAAAAAGA AAAACTAAAA AATAAATTTA 4560

AAATGTGAAC CTTCAGGTAT TGAGTAACAC CTTTATCTTG GTATAGAACT GATACTTTTT 4620

TTTGATTTTG AAATATCTGA TAATAATTTG GAATGAAGTA AGGTCCTGTT AAAATATATT 4680

TGAAGACCCT TTAAAGCAGT GAATCTGAAA CLAATTTTCAC ACCCTTAAGT GGTTGATACG 4740

TACCTATTTT AGGTATTTTG AGGTATTTAC CATAAACTAA ATTTAGAAAT TTTTTAGATT 4800

CACTTGAAGT AAACATTACA AACATTGGAT ACGGTGGGGT TTTCTTTAGA TTTTACTTGA 4860

GAGAAGGTGA GTACAAAGCA ATTTGCAGTT GTTGTAATGA CAAGATTACT GCGCAAGTGT 4920 GAATCCAAAC AGTATAGCTT TTAAATTTTA AAGCATTTGG TAAATTATCG CTGAGTTTTT 4980

TTCTGTTGCC AATAGCAAAC TGCTTTTCCA TTAATGGAGA ATTCATGCCT TTCAAGCATT 5040

TTAAATATGA CAATATTTAT AAATGTATGG TTTGGAGGAA TCGTTTAAAT TCTCTTTCCT 5100

AATTTTCTTT CTTTTGAAGA TAGATTCTTT CAACAAGTAA TTTGTAGTAA TGACTGTGTT 5160

GACTTCAATT TTGGAGCGCA GTAGCTATGT TAAAGATGAA CTATTTGGTC TCΛTTGAAGC 5220

CAACACAGAA CTTGCTGCTG TGTTTTTTCT TCAGTGATAA ATAAAATACT TACCAAAAAA 5280

AAAAAAAAAA AAAAAAAAAA A 5301

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1382 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Met Pro Ma Tyr Phe Gin Arg Pro Glu Asn Ma Leu Lys Arg Ma Asn 1 5 10 15

Glu Phe Leu Glu Val Gly Lys Lys Gin Pro Ma Leu Asp Val Leu Tyr 20 25 30

Asp Val Met Lys Ser Lys Lys His Arg Thr Trp Gin Lys He His Glu 35 40 45 Pro He Met Leu Lys Tyr Leu Glu Leu Cys Val Asp Leu Arg Lys Ser 50 55 60

His Leu Ma Lys Glu Gly Leu Tyr Gin Tyr Lys Asn He Cys Gin Gin 65 70 75 80

Val Asn He Lys Ser Leu Glu Asp Val Val Arg Ma Tyr Leu Lys Met 85 90 95

Ma Glu Glu Lys Thr Glu Ma Ma Lys Glu Glu Ser Gin Gin Met Val 100 105 110

Leu Asp He Glu Asp Leu Asp Asn He Gin Thr Pro Glu Ser Val Leu 115 120 125

Leu Ser Ma Val Ser Gly Glu Asp Thr Gin Asp Arg Thr Asp Arg Leu 130 135 140

Leu Leu Thr Pro Trp Val Lys Phe Leu Trp Glu Ser Tyr Arg Gin Cys

145 150 155 160

Leu Asp Leu Leu Arg Asn Asn Ser Arg Val Glu Arg Leu Tyr His Asp 165 170 175

He Ma Gin Gin Ma Phe Lys Phe Cys Leu Gin Tyr Thr Arg Lys Ma 180 185 190

Glu Phe Arg Lys Leu Cys Asp Asn Leu Arg Met His Leu Ser Gin He 195 200 205

Gin Arg His His Asn Gin Ser Thr Ma He Asn Leu Asn Asn Pro Glu 210 215 220

Ser Gin Ser Met His Leu Glu Thr Arg Leu Val Gin Leu Asp Ser Ma 225 230 235 240 Ile Ser Met Glu Leu Trp Gin Glu Ma Phe Lys Ma Val Glu Asp He 245 250 255

His Gly Leu Phe Ser Leu Ser Lys Lys Pro Pro Lys Pro Gin Leu Met 260 265 270

Ma Asn Tyr Tyr Asn Lys Val Ser Thr Val Phe Trp Lys Ser Gly Asn 275 280 285

Ma Leu Phe His Ma Ser Thr Leu His Arg Leu Tyr His Leu Ser Arg 290 295 300

Glu Met Arg Lys Asn Leu Thr Gin Asp Glu Met Gin Arg Met Ser Thr 305 310 315 320

Arg Val Leu Leu Ma Thr Leu Ser He Pro He Thr Pro Glu Arg Thr 325 330 335

Asp He Ma Arg Leu Leu Asp Thr Asp Gly He He Val Glu Lys Gin 340 345 350

Arg Arg Leu Ma Thr Leu Leu Gly Leu Gin Ma Pro Pro Thr Arg He 355 360 365

Gly Leu He Asn Asp Met Val Arg Phe Asn Val Leu Gin Tyr Val Val 370 375 380

Pro Glu Val Lys Asp Leu Tyr Asn Trp Leu Glu Val Glu Phe Asn Pro 385 390 395 400

Leu Lys Leu Cys Glu Arg Val Thr Lys Val Leu Asn Trp Val Arg Glu 405 410 415

Gin Pro Glu Lys Glu Pro Glu Leu Gin Gin Tyr Val Pro Gin Leu Gin 420 425 430 Asn Asn Thr He Leu His Leu Leu Gin Gin Val Ser Gin He Tyr Gin 435 440 445

Ser He Glu Phe Ser Arg Leu Thr Ser Leu Val Pro Phe Val Asp Ma 450 455 460

Phe Gin Leu Glu Arg Ma He Val Asp Ma Ma Arg His Cys Asp Leu 465 470 475 480

Gin Val Arg He Asp His Thr Ser Arg Thr Leu Ser Phe Gly Ser Asp 485 490 495

Leu Asn Tyr Ma Thr Arg Glu Asp Ma Pro He Gly Pro His Leu Gin 500 505 510

Ser Met Pro Ser Glu Gin He Arg Asn Gin Leu Thr Ma Met Ser Ser 515 520 525

Val Leu Ma Lys Ma Leu Glu Val He Lys Pro Ma His He Leu Gin 530 535 540

Glu Lys Glu Glu Gin His Gin Leu Ma Ma Thr Ma Tyr Leu Lys Asn 545 550 555 560

Ser Arg Lys Glu His Gin Arg He Leu Ma Arg Arg Gin Thr He Glu 565 570 575

Glu Arg Lys Glu Arg Leu Glu Ser Leu Asn He Gin Arg Glu Lys Glu 580 585 590

Glu Leu Glu Gin Arg Glu Ma Glu Leu Gin Lys Val Arg Lys Ma Glu 595 600 605

Glu Glu Arg Leu Arg Gin Glu Ma Lys Glu Arg Glu Lys Glu Arg He 610 615 620 Leu Gin Glu His Glu Gin He Lys Lys Lys Thr Val Arg Glu Arg Leu 625 630 635 640

Glu Gin He Lys Lys Thr Glu Leu Gly Ma Lys Ma Phe Lys Asp He 645 650 655

Asp He Glu Asp Leu Glu Glu Leu Asp Pro Asp Phe He Met Ma Lys 660 665 670

Gin Val Glu Gin Leu Glu Lys Glu Lys Lys Glu Leu Gin Glu Arg Leu 675 680 685

Lys Asn Gin Glu Lys Lys He Asp Tyr Phe Glu Arg Ma Lys Arg Leu 690 695 700

Glu Glu He Pro Leu He Lys Ser Ma Tyr Glu Glu Gin Arg He Lys 705 710 715 720

Asp Met Asp Leu Trp Glu Gin Gin Glu Glu Glu Arg He Thr Thr Met 725 730 735

Gin Leu Glu Arg Glu Lys Ma Leu Glu His Lys Asn Arg Met Ser Arg 740 745 750

Met Leu Glu Asp Arg Asp Leu Phe Val Met Arg Leu Lys Ma Ma Arg 755 760 765

Gin Ser Val Tyr Glu Glu Lys Leu Lys Gin Phe Glu Glu Arg Leu Ma 770 775 780

Glu Glu Arg His Asn Arg Leu Glu Glu Arg Lys Arg Gin Arg Lys Glu 785 790 795 800

Glu Arg Arg He Thr Tyr Tyr Arg Glu Lys Glu Glu Glu Glu Gin Arg 805 810 815 Arg Ma Gly Glu Gin Met Leu Gin Glu Arg Glu Glu Arg Glu Arg Ma 820 825 830

Glu Arg Ma Lys Arg Glu Glu Glu Leu Arg Glu Tyr Gin Glu Arg Val 835 840 845

Lys Lys Leu Glu Glu Val Glu Arg Lys Lys Arg Gin Arg Glu Leu Glu 850 855 860

He Glu Glu Arg Glu Arg Arg Arg Glu Glu Glu Arg Arg Leu Gly Asp 865 870 875 880

Ser Ser Leu Ser Arg Lys Asp Ser Arg Trp Gly Asp Arg Asp Ser Glu 885 890 895

Gly Thr Trp Arg Lys Gly Pro Glu Ma Asp Ser Glu Trp Arg Arg Gly 900 905 910

Pro Pro Glu Lys Glu Trp Arg Arg Gly Glu Gly Arg Asp Glu Asp Arg 915 920 925

Ser His Arg Arg Asp Glu Glu Arg Pro Arg Arg Leu Gly Asp Val Glu 930 935 940

Asp Arg Glu Pro Ser Leu Arg Pro Asp Asp Asp Arg Val Pro Arg Arg 945 950 955 960

Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Pro Glu Glu Asp Arg 965 970 975

Phe Ser Arg Arg Gly Ma Asp Asp Asp Arg Pro Ser Trp Arg Asn Thr 980 985 990

Asp Asp Asp Arg Pro Pro Arg Arg He Ma Asp Glu Asp Arg Gly Asn 995 1000 1005 Trp Arg His Ma Asp Asp Asp Arg Pro Pro Arg Arg Gly Leu Asp Glu 1010 1015 1020

Asp Arg Gly Ser Trp Arg Thr Ma Asp Glu Asp Arg Gly Pro Arg Arg 1025 1030 1035 1040

Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Gly Ma Asp Asp Glu 1045 1050 1055

Arg Ser Ser Trp Arg Asn Ma Asp Asp Asp Arg Gly Pro Arg Arg Gly 1060 1065 1070

Leu Asp Asp Asp Arg Gly Pro Arg Arg Gly Met Asp Asp Asp Arg Gly 1075 1080 1085

Pro Arg Arg Gly Met Asp Asp Asp Arg Gly Pro Arg Arg Gly Met Asp 1090 1095 1100

Asp Asp Arg Gly Pro Arg Arg Gly Leu Asp Asp Asp Arg Gly Pro Trp 1105 1110 1115 1120

Arg Asn Ma Asp Asp Asp Arg He Pro Arg Arg Gly Ma Glu Asp Asp 1125 1130 1135

Arg Gly Pro Trp Arg Asn Met Asp Asp Asp Arg Leu Ser Arg Arg Ma 1140 1145 1150

Asp Asp Asp Arg Phe Pro Arg Arg Gly Asp Asp Ser Arg Pro Gly Pro 1155 1160 1165

Trp Arg Pro Leu Val Lys Pro Gly Gly Trp Arg Glu Lys Glu Lys Ma 1170 1175 1180

Arg Glu Glu Ser Trp Gly Pro Pro Arg Glu Ser Arg Pro Ser Glu Glu 1185 1190 1195 1200 Arg Glu Trp Asp Arg Glu Lys Glu Arg Asp Arg Asp Asn Gin Asp Arg 1205 1210 1215

Glu Glu Asn Asp Lys Asp Pro Glu Arg Glu Arg Asp Arg Glu Arg Asp 1220 1225 1230

Val Asp Arg Glu Asp Arg Phe Arg Arg Pro Arg Asp Glu Gly Gly Trp 1235 1240 1245

Arg Arg Gly Pro Ma Glu Glu Ser Ser Ser Trp Arg Asp Ser Ser Arg 1250 1255 1260

Arg Asp Asp Arg Asp Arg Asp Asp Arg Arg Arg Glu Arg Asp Asp Arg 1265 1270 1275 1280

Arg Asp Leu Arg Glu Arg Arg Asp Leu Arg Asp Asp Arg Asp Arg Arg 1285 1290 1295

Gly Pro Pro Leu Arg Ser Glu Arg Glu Glu Val Ser Ser Trp Arg Arg 1300 1305 1310

Ma Asp Asp Arg Lys Asp Asp Arg Val Glu Glu Arg Asp Pro Pro Arg 1315 1320 1325

Arg Val Pro Pro Pro Ma Leu Ser Arg Asp Arg Glu Arg Asp Arg Asp 1330 1335 1340

Arg Glu Arg Glu Gly Glu Lys Glu Lys Ma Ser Trp Arg Ma Glu Lys 1345 1350 1355 1360

Asp Arg Glu Ser Leu Arg Arg Thr Lys Asn Glu Thr Asp Glu Asp Gly 1365 1370 1375

Trp Thr Thr Val Arg Arg 1380 INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bιs)

sheet j j

on an additional sheet Q

not for all designated Slates)

nature ofthe indications e.g., 'Accession

For receiving Office use only For International Bureau use only

This sheet was received with the international application | | This sheet was received by the International Bureau on:

Authorized officer Authorized officer fiføj ai_R. Pettier

Form PCT/RO/134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule I3bis)

A. The indications made below relate to the microorganism referred to in the description on page 39 , line 8-14

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

Deutsche Sanππlung von Mikroorganismen und Zellkulturen (DSHZ)

Address of depositary institution (including postal code and country)

Hascheroder Weg IB D-38124 Braunschweig Germany

Date of deposit Accession Number

11 April 1996 ( 11.04.96 ) DSM ACC2257

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

We request the Expert Solution where available

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the Intemationai Bureau later (specify the general nature of theindicalions e.g., 'Accession Number of Deposit")

For receiving Office use only For Intemationai Bureau use only

I XJ This sheet was received with the international application | | This sheet was received by the Intemationai Bureau on:

Authorized officer 'XM* Authorized officer

R.LR. Pettier

Form PCT/RO/134 (July 1992)

Claims

Claims

1. An antigenic determinant which cross-reacts with antibodies raised against a polypeptide shown in SEQ ID Nos. 2 and 9, respectively.

2. An antigenic determinant according to claim 1 which possesses substantial homology to a fragment of the polypeptide shown in SEQ ID Nos. 2 and 9, respectively.

3. The polypeptide shown in SEQ ID No.2 or SEQ ID No.9 , a fragment or a derivative thereof.

4. A nucleic acid substantially homologous to the nucleic acid shown in SEQ ID Nos. 1 and 8, respectively.

5. A fragment of a nucleic acid according to claim 4.

6. A nucleic acid or fragment thereof being able to hybridize to the nucleic acid or fragment according to claim 4 or 5.

7. An antibody or antibody fragment capable of specifically recognising an epitope of an antigenic determinant according to claim 1 or claim 2.

8. A composition for the therapy or diagnosis of tumours comprising an antigenic determinant according to claim 1 or claim 2, or a nucleic acid according to claim 4, 5 or 6.

9. A composition for the therapy or diagnosis of tumours comprising an antibody or antibody fragment according to claim 7.

10. A method for determining the metastatic potential of a tumour cell comprising assessing the level of p150 expression in said cell, a higher level of p150 expression being indicative of a lower state of differentiation and consequently a higher metastatic potential.

11. An antibody or antibody fragment according to claim 7 and means for detecting the antibody or antibody fragment for simultaneous, simultaneous separate or sequential use in the diagnosis or therapy of tumours.