WO2001023553A2 - Metastasis-associated antigen c4.4a - Google Patents

Abstract

The present invention relates to gene expression in normal cells and cells of metastasizing tumors and particularly to a novel metastasis-associated antigen, C4.4A, and isolated nucleic acid molecules encoding C4.4A. Also provided are vectors, host cells, antibodies, and recombinant methods for producing this human protein. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating proliferative disorders associated with a metastasizing tumor.

Description

Novel metastasis-associated antigen, C4.4A

FIELD OF THE INVENTION

The present invention relates to gene expression in normal cells and cells of metastasizing tumors and particularly to a novel metastasis-associated antigen, C4.4A, and the gene encoding C4.4A.

BACKGROUND OF THE TECHNOLOGY

Tumor progression is a complex process, which involves detachment from the primary tumor, migration through the extracellular matrix, penetration through the basal membrane, adaptation to the circulation pressure, attachment to the endothelia of the vessel wall and settlement and growth in distant organs. According to these distinct requirements, metastatic cells frequently display a whole array of qualitatively or quantitatively altered gene products. These include, as the most frequent ones, cell-cell and cell-matrix adhesion molecules as well as matrix-degrading enzymes, their activators, inhibitors and receptors.

Furthermore, it has long been hypothesized that the complex process of tumor progression is pursuit by master or regulatory gene products which initiate the performance of a cellular program. Those programs are by no means supposed to be tumor-specific. Rather, the metastasizing tumor cell adapts or reactivates expression of regulatory gene products used during embryogenesis , organogenesis , stem cell differentiation and lymphocyte activation, to name the most frequent programmatic changes of the physiology of multicellular organisms .

Recently a subline of a pancreatic adenocarcinoma of the rat has been described, which metastasizes via the lymphatic system and expresses a variety of surface molecules, which are also expressed on non-related, metastasizing tumor cells, but never an non-metastasizing tumors. Despite this restriction to the metastatic phenotype, all of these molecules were detected under physiological conditions, whereby the expression pattern varied between widespread distribution and restriction to few selected tissue layers. All of these molecules were expressed or upregulated during implantation and placentation. Some, though not all were differentially expressed during organogenesis. One of these molecules has been identified as a variant isoform of the adhesion molecule CD44. The cDNA of three additional molecules has been meanwhile cloned: D6.1A is a tetraspanin molecule and the homologue of the human tumor antigen CO-029. The molecule is widely expressed and there is evidence that its metastasis-associated function is brought about by its linkage to additional surface structures, particularly integrins . A molecule, D5.7A, is the homologue to the human panepithelial antigen EGP314, whose function in tumor progression is currently under investigation.

Matrix degrading enzymes like matrix metalloproteinases (MMP) and urokinase (uPA) are known to play an important role in metastasis formation and much effort has been put into trials to prevent activation of such enzymes as a therapeutic regimen. Unfortunately, these enzymes can also be found in normal tissue. Thus, they are not useful as markers specific for metastasizing tumors and, even more importantly, the inhibition of the activity or expression of these enzymes for tumor therapy might be accompanied by severe side effects.

These limitations and failings of the prior art to provide meaningful specific markers which correlate with the presence of metastasizing tumors has created a need for markers which can be used diagnostically, prognostically and therapeutically over the course of this disease. The present invention fullfills such a need by the provision of C4.4A and the gene encoding C4.4A: Only metastasizing tumor cells exhibit significant expression of C4.4A, whereas normal adult tissue shows no or only a very restricted expression of C4.4A.

SUMMARY OF THE INVENTION

The present invention is based on the isolation of a gene encoding a novel metastasis-associated antigen, C4.4A, particularly the rat and human C4.4A. Both antigens are expressed under physiological conditions only in the gravid uterus and on epithelial cells of the upper gastrointestinal tract. The rat cDNA of the antigen (1.637 bp) codes for a 352 amino acid long glycosylphosphatidyl-inositol (GPI) anchored molecule, whose molecular weight varies in different cells between 94 - 98 kDa according to the degree of N- and 0- glycosylation. The length of the human C4.4A is 347 amino acids. Data base searches have revealed a low degree of homology to the receptor for the plasminogen activator (uPAR) . After intrafootpad and intravenous application of rat C4.4A transfected and mocktransfected tumor cells, an increased number of lung nodules was detected with the former, whereby the individual metastatic nodules amalgamated without any encapsulation of the tumor tissue. Furthermore, C4.4A is involved in adhesion to laminin and, although transfection of a non-metastasizing tumor line with the molecule was not sufficient, constitutively C4.4A-positive tumor cells penetrated through matrigel . This process could be completely prevented by C4.4A. The observed influence of C4.4A on metastasis formation and matrix penetration indicates that C4.4A exerts functional activity similar to uPAR, i.e. via activation of matrix degrading enzymes. In the adult organism there is only a very restricted expression of C4.4A observed. Furthermore, there is an upregulation of expression during implantation and the reactivation exclusively on metastasizing tumor cells. Additionally, an upregulation of C4.4A expression is also seen in primary malignant melanoma and lymph nodes as well as skin metastases of malignant melanoma. From the above results it is apparent that the modulation of C4.4A, e.g. the inhibition of its expression or activity, is of therapeutic interest, e.g. for the prevention of tumor progression. Moreover, the C4.4A molecule is an important target for screening, e.g. for diagnostic purposes.

The present invention, thus, provides a C4.4A protein as well as a nucleic acid molecule encoding the protein and, moreover, an antisense RNA, a ribozyme and an inhibitor, which allow to inhibit the expression or the acitivity of C4.4A.

In one embodiment, the present invention provides a diagnostic method for detecting a metastasizing tumor associated with C4.4A in a tissue of a subject, comprising contacting a sample containing C4.4A or C4.4A encoding mRNA with a reagent which detects C4.4A or the corresponding mRNA.

In another embodiment, the present invention provides a method of treating a metastasizing tumor associated with C4.4A, comprising administering to a subject with such an disorder a therapeutically effect amount of a reagent which modulates, e.g. inhibits, C4.4A expression or the activity of the protein, e.g. the above described compounds.

Finally, the present invention provides a method of gene therapy comprising introducing into cells of a subject an expression vector comprising a nucleoitde sequence encoding the above mentioned antisense RNA or ribozyme, in operable linkage with a promotor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Nucleic acid sequence and deduced amino acid sequence of the rat C4.4A

Figure 2 : Nucleic acid sequence and deduced amino acid sequence of the human C4.4A

Figure 3 : Molecular weight of the rat C4.4A protein and mRNA

(a) Lysates of BSp73AS (AS) cells (non-metastasizing) , BSp73AS cells transfected with CD44v4-v7 (AS-14) (metastasizing due to CD44v4-v7 expression) , BSp73ASML (ASML) (metastasizing) , Regressor (metastasizing) , BSp3A (Sp3A; non-metastasizing pheochromoblastoma) , mock- transfected C0S-7 cells, COS-7 cells transfected with

C4.4A cDNA (COS-7 C4.4) and COS-7 cells transfected with the cDNA of an additional metastasis-associated antigen isolated from the RG library, D6.1A (COS-7 D6.1) were blotted and stained with C4.4 (first antibody) and goat anti-mouse IgGl-HRPO (second antibody). The C4.4A molecule is expressed in BSp73ASML, Regressor and C4.4A cDNA transfected COS-7 cells. The molecular weight varies between BSp73ASML (98 kDa) and RG as well as COS-7 C4.4

(94 kDa) . (b) PCR amplification of C4.4 cDNA after reverse transcription of mRNA of the cell lines described in (a) and a C4.4A cDNA transfected BSp73AS clone (AS-lBl) confirmed the presence of C4.4A mRNA in the BSp73ASML, Regresssor and C4.4A cDNA transfected BSp73AS cells. In all three lines the molecular sine of the mRNA-derived cDNA was identical, i.e. 1100 bp.

Figure 4: Glycosylation of the rat C4.4A molecule

BSp73ASML (ASML) and two C .4A cDNA transfected BSp73AS clones

(AS-lBl and AS-2A2) were tunicamycin treated. Cell lysates were blotted and stained with C4.4 (first anibody) and goat anti-mouse IgGl-HRPO (second antibody) . Lysates of tunicamycin-treated BSp73AS cells (AS) were included as negative control, lysates of the metastatic colon carcinoma line Progressor were included as positive control. By inhibition of N-glycosylation the molecular weight of the C4.4A molecule as detected on BSp73AS cells was reduced to 66 kDa, the molecular weight of the tunicamycin-treated C4.4A cDNA transfected BS73AS cells was in the range of 63 kDa. The molecular weight of C4.4A cDNA transfected AS cells was not changed by inhibiting O-glycosylation, the molecular weight of C4.4A on BSp73ASML was reduced to 94 kDa corresponding to the molecular weight of C4.4A on PROG.

Figure 5: Histological appearance of C4.4A transfected tumor cells

Paraffin sections through lung metastases of BSp73AS-mock (a) , BSp73AS-lBl (b) , BSp73AS-2A2 (c) , BSp73AS-lBl (higher magnification) (d) and BSp6S-C4.4A (e) are shown. While the metastasis formed by the mock transfected tumor line is encapsulated by fibrous tissue (open arrow head) , (a) , lung metastases of rat C4.4A transfected tumors are not encapsulated (closed arrow head) (b, c and e) and degrade the bronchus epithelium (closed arrow) (e) and the vessel endothelium (open arrow) (d) .

Figure 6: Influence of C4.4A on cell proliferation and laminin binding

The tumor lines PROG, 73ASML, 73AS-mock and 73AS-1B1 were labeled with ⁵¹Cr and were seeded (5 x 10⁴ cells/well) on BSA or laminin coated plates. Where indicated, the medium contained 10 μg/ml control IgGl and C4.4 , respectively. Cells were incubated for 30 - 120 min. Non-adherent cells were washed off and adherent cells were detached by trypsin treatment. Radioactivity was determined in a γ-counter. Mean c.p.m. +/- s.d. of triplicate cultures are shown, (a) Cells were incubated in RPMI/5% FCS for 30 min. and 120 min. at 37°C on BSA or laminin coated plates, the medium containing either

10 μg/ml control IgGl or C4.4 ; (b) Cells were incubated for 30 min. at 4°C or at 37°C; (c) Cells were seeded in RPMI/0,5%

BSA, or in RPMI/0,5% BSA, 1 inM EDTA and were incubated for 30 min. at 37°C; (d) Cells were seeded in RPMI/5% FCS or in

RPMI/5% FCS containing 100 μg/ml aprotinin and were incubated for 30 min. and 120 min. at 37°C.

Figure 7: Alignment of human C4.4A and human uPAR

Alignment was done with the "GAP" program (GCG Wisconsin) Asterisks indicate potential N-glycosylation sites and the GPI anchoring site is underlined. The three domains of the human C4.4A and the uPAR are indicated, homologies are mainly restricted to domains 1 and 2.

Figure 8: Chromosomal localization of human C4.4A

The fluorescence in situ hybridization (FISH) analysis of the human C4.4A gene was done with clones LLNLP704, F09964Q3 and LLNLP704 D03144Q19. The hybridization was performed with a metaphase preparation of immortalized lymphocytes. A G-banded chromosome 19 is shown on the top of the figure and the same metaphase spread is shown in the FISH on the bottom. Human C4.4A maps to chromosomal band 19ql3. l-ql3.2 (arrow).

Figure 9: Expression of C4.4A on non-transformed human tissue

a. A Northern blot was probed with C4.4A as described in Example 1. Each lane represents 2 μg of poly (A) -RNA of brain, placenta, lung, liver, skeletal muscle, kidney and pancreas. The lower panel shows the GAPDH control, b. Expression of C4.4A on non-transformed tissues analyzed by RT-PCR. RT-PCR probes derived from poly A RNA were separated on a 1% agarose gel. First and last lane: 100 bp ladder, lane 2: negative control (H₂0) , lane 3-14: tissue samples, lane 15-17: three positive controls with the depicted copy numbers of hC4.4A derived from a plasmid. The lower panel shows the GAPDH control .

Figure 10: Expression of C4.4A on human tumor cell lines

a. Expression of C4.4A on melanoma lines analyzed by RT-PCR: RT-PCR probes derived from polyA RNA were separated on a 1 % agarose gel. Frist lane: 100 bp ladder, lane 2-6; melanoma lines, last lane: negative control (H₂0) . The lower panel shows the GAPDH control, b. Expression of C4.4A on naevi , primary melanoma and metastasis of malignant melanoma analyzed by RT- PCR: RT-PCR probes derived from polyA RNA were separated on a 1% agarose gel. First lane: 100 bp ladder, lane 2: lymph node metastasis, lane 3: skin metastasis, lane 4 and 5: primary malignant melanoma, lane 6: naevus .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated nucleic acid molecule encoding the metastasis-associated antigen C4.4A, particularly the rat and human C4.4A, or a protein exhibiting biological properties of the metastasis-associated antigen

C4.4A and being selected from the group consisting of

(a) a nucleic acid molecule encoding a protein that comprises the amino acid sequence depicted in Figure 1 and 2, respectively;

(b) a nucleic acid molecule comprising the nucleotide sequence depicted in Figure 1 and 2, respectively;

(c) a nucleic acid molecule included in DSMZ Deposit No: 13013 and 13014, respectively; (d) a nucleic acid molecule which hybridizes to a nucleic acid molecule specified in (a) to (c) ;

(e) a nucleic acid molecule the nucleic acid sequence of which deviates from the nucleic sequences specified in (a) to (d) due to the degeneration of the genetic code; and

(f) a nucleic acid molecule which represents a fragment, derivative or allelic variant of a nucleic acid sequence specified in (a) to (e) .

As used herein, a protein exhibiting biological properties of metastasis-associated antigen C4.4A is understood to be a protein having at least one of the acitivities of C4.4A as illustrated in the Examples, below.

As used herein, the term "isolated nucleic acid molecule" includes nucleic acid molecules substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which it is naturally associated. In a first embodiment, the invention provides an isolated nucleic acid molecule encoding the metastasis-associated antigen C4.4A comprising the amino acid sequence depicted in Figure 1 and 2, respectively. The present innvention also provides a nucleic acid molecule comprising the nucleotide sequence depicted in Figure 1 and 2, respectively.

The present invention provides not only the generated nucleotide sequence identified in Figure 1 and 2, respectively and the predicted translated amino acid sequence shown in Figure 1 and 2, respectively, but also plasmid DNA containing a DNA C4.4A (rat) deposited with the DSMZ on 18.08.1999 under DSM 13013 and a DNA C4.4A (human) deposited with the DSMZ on 18.08.1999 under DSM 13014, respectively. The nucleotide sequence of each deposited C4.4A clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted C4.4A amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by each deposited clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited C4. A encoding DNA; collecting the protein, and determining its sequence.

The nucleic acid molecules of the invention can be both DNA and RNA molecules. Suitable DNA molecules are, for example, genomic or cDNA molecules. It is understood that all nucleic acid molecules encoding all or a portion of C4.4A are also included, as long as they encode a polypeptide with C4.4A activity. The nucleic acid molecules of the invention can be isolated from natural sources or can be synthesized according to known methods .

The present invention also provides nucleic acid molecules which hybridize to the above nucleic acid molecules. As used herein, the term "hybridize" has the meaning of hybridization under conventional hybridization conditions, preferably under stringent conditions as described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Also contemplated are nucleic acid molecules that hybridize to the C4.4A nucleic acid molecules at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency) ; salt conditions, or temperature. For example, lower stringency conditions include an overnight incubation at 37°C in a solution comprising 6X SSPE (20X SSPE = 3M NaCl; 0.2M NaH₂P04 ; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA, followed by washes at 50°C with 1 X SSPE, 0.1% SDS. In addition, to achieve even lower stringency, washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC) . Variations in the above conditions may be accomplished through the inclusion and/or substitution of alternate blocking reagents used to suppress background in hybridization experiments. The inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.

Nucleic acid molecules that hybridize to the molecules of the invention can be isolated, e.g., from genomic or cDNA libraries that were produced from rat or human cell lines or tissues. In order to identify and isolate such nucleic acid molecules the molecules of the invention or parts of these molecules or the reverse complements of these molecules can be used, for example by means of hybridization according to conventional methods (see, e.g., Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, 2^nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) . As a hybridization probe nucleic acid molecules can be used, for example, that have exactly or basically the nucleotide sequence depicted in Figure 1 and 2, respectively, or parts of these sequences. The fragments used as hybridization probe can be synthetic fragments that were produced by means of conventional synthesis methods and the sequence of which basically corresponds to the sequence of a nucleic acid molecule of the invention.

The nucleic acid molecules of the present invention also include molecules with sequences that are degenerate as a result of the genetic code.

In a further embodiment, the present invention provides nucleic acid molecules which comprise fragments, derivatives and allelic variants of the nucleic acid molecules described above encoding a protein of the invention. "Fragments" are understood to be parts of the nucleic acid molecules that are long enough to encode one of the described proteins. The term "derivative" in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above at one or several positions but have a high level of homology to these sequences . Homology hereby means a sequence identity of at least 40 %, in particular an identity of at least 60 %, preferably of more than 80 % and particularly preferred of more than 90 %. These proteins encoded by the nucleic acid molecules have a sequence identity to the amino acid sequence depicted in Figure 1 and 2, respectively, of at least 80 %, preferably of 85 % and particularly preferred of more than 90 %, 95 %, 97 % and 99 %. The deviations to the above-described nucleic acid molecules may have been produced by deletion, substitution, insertion or recombination .

The nucleic acid molecules that are homologous to the above- described molecules and that represent derivatives of these molecules usually are variations of these molecules that represent modifications having the same biological function. They can be naturally occurring variations, for example sequences from other organisms, or mutations that can either occur naturally or that have been introduced by specific mutagenesis. Furthermore, the variations can be synthetically produced sequences. The allelic variants can be either naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA processes.

Generally, by means of conventional molecular biological processes it is possible (see, e.g., Sambrook et al . , 1989, Molecular Cloning, A Laboratory Manual, 2^nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) to introduce different mutations into the nucleic acid molecules of the invention. As a result C4.4A proteins or C4.4A related proteins with possibly modified biological properties are synthesized. One possibility is the production of deletion mutants in which nucleic acid molecules are produced by continuous deletions from the 5 ' - or 3 ' -terminal of the coding DNA sequence and that lead to the synthesis of proteins that are shortened accordingly. Another possibility is the introduction of single-point mutation at positions where a modification of the amino acid sequence influences, e.g., the enzyme activity or the regulation of the enzyme. By this method muteins can be produced, for example, that possess a modified K,.-value or that are no longer subject to the regulation mechanisms that normally exist in the cell, e.g. with regard to allosteric regulation or covalent modification. Such muteins might also be valuable as therapeutically useful antagonists of C4.4A.

For the manipulation in prokaryotic cells by means of genetic engineering the nucleic acid molecules of the invention or parts of these molecules can be introduced into plasmids allowing a mutagenesis or a modification of a sequence by recombination of DNA sequences . By means of conventional methods (cf. Sambrook et al . , 1989, Molecular Cloning: A Laboratory Manu l, 2^nd edition, Cold Spring Harbor Laboratory Press, NY, USA) bases can be exchanged and natural or synthetic sequences can be added. In order to link the DNA fragments with each other adapters or linkers can be added to the fragments. Furthermore, manipulations can be performed that provide suitable cleavage sites or that remove superfluous DNA or cleavage sites. If insertions, deletions or substitutions are possible, in vitro mutagenesis, primer repair, restriction or ligation can be performed. As analysis method usually sequence analysis, restriction analysis and other biochemical or molecular biological methods are used.

The proteins encoded by the various variants of the nucleic acid molecules of the invention show certain common characteristics, such as enzyme activity, molecular weight, immunological reactivity or conformation or physical properties like the e 1 e c t o rphor e t i c a 1 mobility, chromatographic behavior, sedimenta ion coefficients, solubility, spectroscopic properties, stability; pH optimum, temperature optimum.

The invention furthermore relates to vectors containing the nucleic acid molecules of the invention. Preferably, they are plasmids, cosmids, viruses, bacteriophages and other vectors usually used in the field of genetic engineering. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria, the pMSXND expression vector for expression in mammalian cells and baculovirus-derived vectors for expression in insect cells. Preferably, the nucleic acid molecule of the invention is operatively linked to the regulatory elements in the recombinant vector of the invention that guarantee the transcription and synthesis of an RNA in prokaryotic and/or eukaryotic cells that can be translated. The nucleotide sequence to be transcribed can be operably linked to a promotor like a T7 , metallothionein I or polyhedrin promotor.

In a further embodiment, the present invention relates to recombinant host cells transiently or stably containing the nucleic acid molecules or vectors of the invention. A host cell is understood to be an organism that is capable to take up in vi tro recombinant DNA and, if the case may be, to synthesize the proteins encoded by the nucleic acid molecules of the invention. Preferably, these cells are prokaryotic or eukaryotic cells, for example mammalian cells, bacterial cells, insect cells or yeast cells. The host cells of the invention are preferably characterized by the fact that the introduced nucleic acid molecule of the invention either is heterologous with regard to the transformed cell, i.e. that it does not naturally occur in these cells, or is localized at a place in the genome different from that of the corresponding naturally occurring sequence.

A further embodiment of the invention relates to isolated proteins exhibiting biological properties of the metastasis- associated antigen C4.4A and being encoded by the nucleic acid molecules of the invention, as well as to methods for their production, whereby, e.g, a host cell of the invention is cultivated under conditions allowing the synthesis of the protein and the protein is subsequently isolated from the cultivated cells and/or the culture medium. Isolation and purification of the recombinantly produced proteins may be carried out by conventional means including preparative chromatography and affinity and immunological separations involving affinity chromatography with monoclonal or polyclonal antibodies, e.g. with the antibody C4.4.

As used herein, the term "isolated protein " includes proteins substantially free of other proteins, nucleic acids, lipids, carbohydrates or other materials with which it is naturally associated. Such proteins however not only comprise recombinantly produced proteins but include isolated naturally occurring proteins, synthetically produced proteins, or proteins produced by a combination of these methods. Means for preparing such proteins are well understood in the art. The C4.4A proteins are preferably in a substantially purified form. A recombinantly produced version of C4.4A protein, including the secreted protein, can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988) .

In another preferred embodiment the invention relates to nucleic acid molecules specifically hybridizing to transcripts of the nucleic acid molecules of the invention. These nucleic acid molecules can be used, for example, as probes in the diagnostic assay and/or kit described below and, preferably, are oligonucleotides having a length of at least 10, in particular of at least 15 and particularly preferred of at least 50 nucleotides. The nucleic acid molecules and oligonucleotides of the invention can also be used, for example, as primers for a PCR reaction.

In a further preferred embodiment, the present invention relates to an antisense RNA sequence characterized in that it is complementary to an mRNA transcribed from the nucleic acid molecule of the present invention or a part thereof and can selectively bind to said mRNA, said sequence being capable of inhibiting the synthesis of the protein encoded by said nucleic acid molecules, and a ribozyme characterized in that it is complementary to an mRNA transcribed from the nucleic acid molecule of the present invention or a part thereof and can selectively bind to and cleave said mRNA, thus inhibiting the synthesis of the proteins encoded by said nucleic acid molecules. Ribozymes which are composed of a single RNA chain are RNA enzymes, i.e. catalytic RNAs , which can intermolecularly cleave a target RNA, for example the mRNA transcribed from the C4.4A gene. It is now possible to construct ribozymes which are able to cleave the target RNA at a specific site by following the strategies described in the literature, (see, e.g., Tanner et al . , in: Antisense Research and Applications, CRC Press Inc. (1993), 415-426). The two main requirements for such ribozymes are the catalytic domain and regions which are complementary to the target RNA and which allow them to bind to its substrate, which is a prerequisite for cleavage. Said complementary sequences, i.e., the antisense RNA or ribozyme, are useful for repression of C4.4A expression, e.g. in the case of the treatment of a cell proliferative disorder associated with metastasizing tumors. Preferably, the antisense RNA and ribozyme of the invention are complementary to the coding region of the C4.4A mRNA, e.g. to the 5' part of the coding region. The person skilled in the art provided with the sequences of the nucleic acid molecules of the present invention will be in a position to produce and utilize the above described antisense RNAs or ribozymes. The region of the antisense RNA and ribozyme, respectively, which shows complementarity to the mRNA transcribed from the nucleic acid molecules of the present invention preferably has a length of at least 10, in particular of at least 15 and particularly preferred of at least 50 nucleotides.

In still a further embodiment, the present invention relates to C4.4A inhibitors which fulfill a similar purpose as the antisense RNAs or ribozymes mentioned above, i.e. reduction or elimination of biologically active C4.4A molecules. Such inhibitors can be, for instance, structural analogues of the C4.4A protein that act as antagonists. In addition, such inhibitors comprise molecules identified by the use of the recombinantly produced C4.4A, e.g. the recombinantly produced C4.4A can be used to screen for and identify C4.4A inhibitors, for example, by exploiting the capability of potential inhibitors to bind to C4.4A under appropriate conditions. The inhibitors can, for example, be identified by preparing a test mixture wherein the inhibtor candidate is incubated with C4.4A under appropriate conditions that allow C4.4A to be in a native conformation. Such an in vitro test system can be established according to methods well known in the art. Inhibitors of C4.4A can be identified, for example, by first screening for either synthetic or naturally occuring molecules that bind to the recombinantly produced C4.4A and then, in a second step, by testing those selected molecules in cellular assays for inhibition of CIITA, as reflected by inhibition of at least one of the biological activities of C4.4A as described in the Examples, below. Such screening for molecules that bind C4.4A could easily performed on a large scale, e.g. by screening candidate molecules from libraries of synthetic and/or natural molecules. Such an inhibitor is, e.g., a synthetic organic chemical, a natural fermentation product, a substance extracted from a microorganism, plant or animal, or a peptid .

The present invention also provides a method for detecting a cell proliferative disorder associated with a metastasizing tumor which comprises contacting a sample suspected to contain C4.4A or the C4.4A encoding mRNA with a reagent which reacts with C4.4A or the C4.4A encoding mRNA and detecting C4.4A or the C4.4A encoding mRNA. When the target is mRNA, the reagent is typically a nucleic acid probe or a primer for PCR. The person skilled in the art is in a position to design suitable nucleic acids probes based on the information as regards the nucleotide sequence of C4.4A as depicted in Figure 1 and 2, respectively. When the target is a C4.4A protein, the reagent is typically an antibody probe. The term "antibody", preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monclonal antibody preparations . Monoclonal antibodies are made from an antigen containing fragments of the C4.4A protein of the invention by methods well known to those skilled in the art (see, e.g., Kδhler et al . , Nature 256 (1975), 495). As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al . , J. Nucl . Med. 24:316-325 (1983).) Thus, these fragments are preferred, as well as the products of a FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies. The target cellular component, i.e. C4.4A or a C4.4A encoding mRNA, e.g., in biological fluids or tissues, may be detected directly in situ or it may be isolated from other cell components by common methods known to those skilled in the art before contacting with a probe. Detection methods include Northern blot analysis, RNase protection, in situ methods, PCR, LCR, immunoassays and other detection assays that are known to those skilled in the art.

Useful tissue samples include tissue of heart, renal, brain, colon, breast, urogenital, uterine, hematopoietic, prostate, thymus, lung, testis, pancreas and ovarian. A preferred tissue sample of the present invention is pancreas.

The probes can be detectably labeled, for example, with a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme .

C4.4A expression in tissues can be studied with classical immunohistological methods (Jalkanen et al . , J. Cell. Biol . 101 (1985), 976-985; Jalkanen et al . , J. Cell. Biol. 105 (1987), 3087-3096). Other antibody based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA) . Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine (¹²⁵I, ¹²¹I) , carbon

(¹⁴C) , sulfur (³⁵S) , tritium (³H) , indium (¹¹²In) , and technetium

("mTcj , and fluorescent labels, such as fluorescein and rhodamine, and biotin. In addition to assaying C4.4A levels in a biological sample, C4.4A can also be detected in vivo by imaging. Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, NMR or ESR. For X-radiography, suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject. Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma. A protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety, such as a radioisotope (for example, ¹³¹I, ¹¹²In, ⁹⁹mTc) , a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally , subcutaneously, or intraperitoneally) into the mammal. It will be understood in the art that the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of "mTc . The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al. , " Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments." (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds . , Masson Publishing Inc. (1982)).

For evaluating whether the concentration of C4.4A or the

C4.4A encoding mRNA is normal or increased, thus indicative for the presence of a metastasizing tumor, the measured concentration is compared with the concentration in a normal tissue .

The present invention also relates to a method for preventing, treating, or ameliorating a cell proliferative disorder associated with a metastasizing tumor which comprises administering to a mammalian subject a therapeutically effective amount of a reagent which decreases or inhibits C4.4A expression or the activity of C4.4A. Examples of such reagents are the above described antisense RNAs, ribozymes or inhibitors, e.g. C4.4A specific antibodies. For example, administration of an antibody directed to C4.4A can bind and reduce overproduction of the protein.

For administration these reagents are preferably combined with suitable pharmaceutical carriers. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emuslions, various types of wetting agents, sterile solutions etc.. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitabel dose. Administration of the suitable compositions may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intrader al administration. The route of administration, of course, depends on the nature of the metastasizing tumor and the kind of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind of the metastasizing tumor, general health and other drugs being administered concurrently.

The delivery of the antisense RNAs or ribozymes of the invention can be achieved by direct application or, preferably, by using a recombinant expression vector such as a chimeric virus containing these compounds or a colloidal dispersion system. By delivering these nucleic acids to the desired target, the intracellular expression of C4.4A and, thus, the level of C4.4A can be decreased resulting in the inhibition of the negative effects of C4.4A as regards the metastasis formation discussed above.

The above nucleic acids can be administered directly to the target site, e.g., by ballistic delivery, as a colloidal dispersion system or by catheter to a site in artery. The colloidal dispersion systems which can be used for delivery of the above nucleic acids include macromolecule complexes, nanocapsules, microspheres , beads and lipid-based systems including oil-in-water emulsions, (mixed) micelles, liposomes and lipoplexes. The preferred colloidal system is a liposome. The composition of the liposome is usually a combination of phospholipids and steroids, especially cholesterol. The skilled person is in a position to select such liposomes which are suitable for the delivery of the desired nucleic acid molecule. Organ-specific or cell-specific liposomes can be used in order to achieve delivery only to the desired metastasizing tumor. The targeting of liposomes can be carried out by the person skilled in the art by applying commonly known methods. This targeting includes passive targeting (utilizing the natural tendency of the liposomes to distribute to cells of the RES in organs which contain sinusoidal capillaries) or active targeting (for example by coupling the liposome to a specific ligand, e.g., an antibody, a receptor, sugar, glycolipid, protein etc., by well known methods). In the present invention monoclonal antibodies are preferably used to target liposomes to specific tumors via specific cell- surface ligands.

Preferred recombinant vectors useful for gene therapy are viral vectors, e.g. adenovirus, herpes virus, vaccinia, or, more preferably, an RNA virus such as a retrovirus. Even more preferably, the retroviral vector is a derivative of a murine or avian retrovirus . Examples of such retroviral vectors which can be used in the present invention are: Moloney murine leukemia virus (MoMuLV) , Harvey murine sarcoma virus (HaMuSV) , murine mammary tumor virus (MuMTV) and Rous sarcoma virus

(RSV) . Most preferably, a non-human primate retroviral vector is employed, such as the gibbon ape leukemia virus (GaLV) , providing a broader host range compared to murine vectors. Since recombinant retroviruses are defective, assistance is required in order to produce infectious particles. Such assistance can be provided, e.g., by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. Suitable helper cell lines are well known to those skilled in the art. Said vectors can additionally contain a gene encoding a selectable marker so that the transduced cells can be identified. Moreover, the retroviral vectors can be modified in such a way that they become target specific. This can be achieved, e.g., by inserting a polynucleotid encoding a sugar, a glycolipid, or a protein, preferably an antibody. Those skilled in the art know additional methods for generating target specific vectors. Further suitable vectors and methods for in vitro- or in vivo- gene therapy are described in the literature and are known to the persons skilled in the art; see, e.g., WO 94/29469 or WO 97/00957.

In order to achieve expression only in the target organ, i.e. the metastasizing tumor to be treated, the nucleic acids encoding, e.g. an antisense RNA or ribozyme can also be operably linked to a tissue specific promotor and used for gene therapy. Such promotors are well known to those skilled in the art (see e.g. Zimmermann et al , 1994, Neuron 12, 11-24; Vidal et al . 1990, EMBO J. 9, 833-840; Mayford et al . , 1995, Cell 81, 891-904; Pinkert et al . , 1987, Genes & Dev. 1, 268- 76) .

For use in the diagnostic research discussed above, kits are also provided by the present invention. Such kits are useful for the detection of a target cellular component, which is C4.4A or C4.4A encoding mRNA, wherein the presence or an increased concentration of C4.4A or C4.4A encoding mRNA is indicative for a cell proliferative disorder associated with a metastasizing tumor, said kit comprising a probe for detection of C4.4A or C4.4A encoding mRNA. The probe can be detectably labeled. Such probe may be an antibody or oligonucleotide specific for C4.4A or C4.4A encoding mRNA. In a preferred embodiment, said kit contains a C4.4A specific antibody and allows said diagnosis by ELISA and contains the antibody bound to a solid support, for example, a polystyrene microtiter dish or nitrocellulose paper, using techniques known in the art. Alternatively, said kits are based on a RIA and contain said antibody marked with a radioactive isotope. In a preferred embodiment of the kit of the invention the C4.4A-antibody is labelled with enzymes, fluorescent compounds, luminescent compounds, ferromagnetic probes or radioactive compounds. The kit of the invention may comprise one or more containers filled with, for example, one or more probes of the invention. Associated with container (s) of the kit can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

EXAMPLES

The following Examples are intented to illustrate, but not to limit the invention. While such Examples are typical of those that might be used, other methods known to those skilled in the art may alternatively be utilized.

EXAMPLE 1 MATERIALS AND METHODS

(a) rat C4.4A Animals and tumors

BDX rats were obtained from Charles River, Sulzfeld, Germany. They were kept under SPF conditions, fed with conventional diet and water at libitum. They were used for experiments at the age of 8 to 10 weeks.

The following tumor lines were used: BSP73ASML

(metastasizing pancreatic adenocarcinoma) and BSp73AS (low- metastasizing pancreatic adenocarcinoma) (Matzku et al . , Invasion.Metast. (1983), 109-123), BSp6S (subline of a non- metastasizing fibrosarcoma) and BSp3A (subline of a non- metastasizing pheochromoblastoma) (Zδller et al . , Br.J. Cancer 37 (1978), 61-66). BSp73AS-C4.4A (BSp73A transfected with cDNA coding for the C4.4A molecules) and BSp73AS-mock (BS73AS transfected with the pCDNA3-vector) are described below. All these tumor lines were derived from the BDX rat strain. The tumor lines regressor (RG) and progressor (PROG) , a metastasizing colon carcinoma of the BDIX strain has been kindly provided by F.Martin (Reisser et al . , Int. J. Cancer 53. (1993), 651-656). The adherently growing tumor lines were cultured in RPMI 1640 supplemented with 10% fetal calf serum (FCS) . Confluent cultures were either trypsinized (BSp73ASML, PROG and RG) or treated with EDTA (BSp73AS, BSp6S, BSp3A) and split. COS-7 cells were obtained from the DSMZ. They were cultured in DMEM supplemented with 10% fetal calf serum.

Antibodies The mAB C4.4 (mouse IgGl) and an isotype matched control antibody, 3-9, binding a Ga-chelate complex have been described previously (Matzku et al . , Cancer Res. 49_. (1989), 1294-1299; Zoller et al . , J.Nucl.Med. 33 (1992), 1366-1372). A rabbit anti-rat uPAR was obtained from American Diagnostics. Polyclonal rabbit anti-mouse IgGl and fluoresecence dye labeled derivatives as well as an FITC labelled anti-rat IgG were obtained from Southern Biotechnology (Birmingham, AL) . For FACS analyses cells were harvested, washed and resuspended at 5 x 10⁵ cells in 50 μl PBS containing 2% FCS. Cells were incubated with the first antibody (10 μg/ml) for 30 min. at 4°C and, after washing, with the second, dye-labeled antibody (30 min. at 4°C) at appropriate dilutions. After washing again fluorescence staining was evaluated using an EPICS XL (Coulter Hialeah, FL) .

cDNA library and selection of clones coding for the rat surface molecule C4.4A mRNA was prepared from the colon carcinoma cell line RG and oligo(dT) . Unprimed cDNA was prepared using the Librarian Kit (Invitrogen, San Diego) . The cDNA was inserted unidirectional into the mammalian expression vector pcDNA3 after ligation of BstXI adaptors and cleavage with Notl . The ligated DNA was transformed into E.coli TOP10P. After cultivation, isolation of the plasmid DNA and plating of aliquots, the size of the library was estimated to about 2 x 10⁷ clones.

For the isolation of clones coding for the metastasis- associated surface molecule C4.4A 15 μg of the cDNA were transfected into COS-7 cells by electroporation. After 3 days cells were stained with the mAB C4.4 and a PE-coupled goat anti-mouse IgG. Strongly stained cells were isolated by floruescence activated cell sorting using a FACStar (Becton Dickinson, Mountain Viwe, CA) . Plasmid DNA of the stained cells was isolated by lysis according to the method described by Hirt, J. Mol. Biol. 26 (1967), 365-369. Genomic DNA and proteins were separated by centrifugation. The plasmid DNA/RNA mixture was digested with RNase A (20 μg/ml) and Proteinase K (50 μg/ml), followed by a phenol-chloroform extraction to remove residual proteins. The remaining plasmid DNA was precipitated and used for the transformation of E.coli by electroporation. The procedure was repeated three times. Thereafter the plasmid DNA was isolated from single bacterial colonies and was used for DEAE transfection of COS-7 cells. After 3 days cells were analysed for antigen expression by FACS. The cDNA of positive clones was sequenced according to the method of Sanger.

Transfection of COS-7 cells and C4.4A negative tumor lines with C4.4-CDNA

The tumor lines BSp73AS, BSp6S and BSp3A were harvested during growth in log phase. Cells (1 x 10⁷/ml) were resuspended in RPMI 1640 containing 10% FCS and were mixed with 15 μg of the pcDNA3-C4.4A plasmid in a steril cuvette. Electroporation was performed after 15 min. incubation at room temperature at 260 V and 1050 μF using a Bio-Rad Genepulser with capacitance extender. Transfected cells were selected by growth in RPMI 1640 containing 10% FCS and 500 μg/ml G418. G418 resistant cells were analysed for C4.4A expression by flowcytometry and were cloned.

PCR

Total RNA was extracted by the guanidine isothiocyanate/acid phenol method of Chomczynski and Sacchi (Analytical Biochem. 162 (1987), 156-159). cDNA was synthesized and subjected to PCR amplification using 2 μg total RNA and 1 μg oligo(dT)₁₅ for synthesis of the first strand and C4.4A-specific primers for the amplification. The following C4.4A specific oligonucleotides were used: 5 ' TGCTACAGCTGCGTGCAA and 3 ' TTGGAACTGCGGATGCTG . Amplification was performed at 59°C with 5' and 3' oligonucleotides for 35 cycles. Amplification of GAPDH was performed accordingly at 53°C. PCR products were analysed on a 1% agarose gel.

SDS-PAGE and Western blot

SDS-PAGE was performed under non-reducing conditions using 8 or 10% gels according to the method of Laemmli (Nature 227 (1970) , 680-685) . Gels were transferred to PVDF membranes (Millipore) by the method of Towbin et al . (Proc.Natl.Acad.Sci. USA 76 (1979), 4350-4354). Membranes were blocked by incubation in PBS containing 5% (w/v) non-fat dry milk powder and 0,1% (v/v) Tween 20® for 1 h at room temperature. After incubation for 1 h with the appropriate first antibody, membranes were washed four times with PBS/0, 1% Tween 20®, incubated for 1 h with goat anti-mouse IgGl- horseradish peroxidase (HRPO) (1:5000 in PBS/0,1% Tween 20®) and washed four times with PBS/0, 1% Tween 20®. Detection was performed by the enhanced chemiluminescence assay according to the manufacturer's protocol (Amersham, Braunschweig, Germany) .

Anchorage, glycosyla ion and solubility N- and O-glycosylation were inhibited by plating 2 x 10⁵ cells (six well plate) and growing the cells in RPMI 1640, 10% FCS containing either 2,5 μg/ml Tunicamycin or 2 mM phenyl-alpha- GalNac. Thereafter cells were harvested, suspended in 300 μl SDS sample buffer and analysed by Western blot. To determine detergent solubility cells growing in log phase were harvested, washed with PBS and resuspended in 100 μl of extraction buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 0,5 mM PMSF) containing the following detergents: 0,5% and 1% Triton X-100, NP-40, CHAPS and 60 mM octyl-glucoside . Insoluble material was collected by centrifugation for 2 min. at 12 000 r.p.m. in a Heraeus Biofuge 13R at 4°C . The supernatant containing the detergent- soluble proteins was removed and mixed with 1/6 volume of 6 x sample buffer. The pelleted material was washed once with 500 μg sample buffer. DNA was shared by sonification. Membrane anchorage was determined by GPI-PLC treatment (Ferguson and Williams, Ann. Rev. Biochem. 57 (1988), 285-320). 5 x 10⁶C4.4A- transfected and untransfected AS cells were harvested, washed twice with PBS and resuspended in 100 μl PBS containing 1 mM MgCl₂. After incubation for 1 h with 0.25 U Phosphatidyl- inositol (GPI) -specific phospholipase C (Boehringer Mannheim, Germany) at 37°C, cells were analysed by Western blot and flow cytometry.

Adhesion, migration and proliferation assays

In adhesion studies cells were labeled with ⁵¹Cr and were seeded either on a monolayer of adherent cells or on plates coated with components of the extracellular matrix, i . e .hyaluronic acid (HA), collagen type I, type III and type IV, laminin (LA) , fibronectin and vitronectin. The components of the extracellular matrix, except for HA wer coated at 10 μg/ml; HA was coated at a concentration of 100 μg/ml. To differentiate between binding and spreading, the assay was run at 4°C and 37°C. The protease inhibitor aprotinin (100 μg/ml) was added to exclude laminin degradation. Where indicated, anti-C4.4 (10 μg/ml) was added to the culture medium. Cells were incubated for 30 min. to 2 h and were washed repeatedly. The adherent cells were lysed by 2% SDS, transferred into counting vials and counted in a γ-counter. The percentage of adherent cells is shown.

The migratory activity of C4.4A⁺ cells was evaluated in a matrigel assay: Membranes with a pore size of 8 μm were coated with 50 μl matrigel (Sigma, St. Louis, MO, USA). The membranes were inserted into 24 well plates which contained medium without supplements. Tumor cells (1 x 10⁴) were suspended in RPMI 1640 supplemented with 10% FCS and, where indicated, with 10 μg/ml C4.4 and were seeded on the matrigel. Plates were incubated for 48 h at 37°C. Thereafter cells at the bottom of the chamber were counted using an inverted microscope.

Proliferation of C4.4A transfected cells was tested by [³H] thymidine incorporation. Cells were seeded on control IgG or C4.4 coated plates. The supernatant contained either a control antibody or C4.4. [³H] thymidine was added immediately or after 24 to 72 h. After 8 h of incubation with [³H] thymidine cells were trypsinized and transferred to a β-counter to evaluate thymidine incorporation.

Metastasis assay

BDX rats received 5 x 10⁵ tumor cells, i.v. or i.f.p.. In the latter case the tumor and the draining lymph node were excised by amputation in the knee as soon as the primary tumor reached a diameter of 0.5 cm. Animals were observed for local tumor growth, weight loss, anemia and shortage of breathing. They were sacrified before reaching a moribund stage. Metastasis formation was controlled macroscopically and by histology.

Statistics

Significance of differences was calculated by the two tailed Student's t-test.

(b) human C4.4A

1. Isolation and expression of human C4.4A

Human tissue and cell lines

Shock frozen normal human tissue of placenta, skin, thymus, colon, tonsils, kidney, liver and stomach was kindly provided by P.Mδller, Dep. of Pathology, University Ulm, Germany. The tumor lines COLO-205, COLO-320DM, HAT-29, LoVo, SW480, SW707, SW948, WIDR (colon carcinoma), A-375, BLM, BT474, Bult, COLO, COLO-4, COLO-8, F-mex, Fo-1, Mel-Juso. Mewo, ML68, MML1, MM412, MZ2, SK-Mel-25, SK-Mel-28, TGIG, 530C1 (malignant melanoma), MDA-MB-415, MDA-MB-435S, MDA-MB-436, MML2B, TD470

(mammary carcinoma), BJAB, HL60, Joki , Jurkat, OCI

(hematopoietic malignancies), Bδ, BxPC-3, 8.18 (carcinoma of the pancreatic gland), KTCM-1M, KTCM-129, KTCM-140 (renal cell carcinoma) , MKN (stomach carcinoma) , Igrov (carcinoma of the ovary) , JAR (choriocarcinoma) , COLO-680 (esophagus carcinoma) ,

Calu-6 (lung carcinoma) , HeLa (cervix carcinoma) , HAT 1080

(fibrocarcinoma) , Hep G2 and Chang liver (liver carcinoma) were either obtained from the American Type Culture Collection or from the Central Tumor Bank of the German Cancer Research Center. W+ is a newly established melanoma line, kindly provided by S. Seiter, Department of Dermatology, Medical University of the Saarland. cDNA library and selection of clones coding for the human C4.4A (hC4.4A)

Image filter No. 10 containing Soares 2NbHP8-9W normal 8 to 9 week placental library was screened with a ³²P labeled C4.4A cDNA probe of the rat. One of the labeled clones (IMAGEp9 8 Ao5562Q6) was sequenced using the chain termination method and compared with the rat sequence.

Fluorescence in situ hybridization (FISH)

For isolation of chromosomal clones of the hC4.4A gene a PAC (Pl-derived artificial chromosome) library was screened with the hC4.4A-cDNA ( ICRF library No704, Resource Center Primary Data Base, German Human Genome Porject, Max Planck Institute for Molecular Genetics, Berlin) . Two positive clones (LLNLP704 F09964Q3 and LLNLP704 D03144Q19) were isolated and used for FISH analysis. PAC-DNA was labeled with DIG-11-dUTP (Boehringer, Mannheim) by nick translation. Suppression of repetitive sequences, denaturation, hybridization and fluorescence detection were performed according to routine procedures. Anti-DIG mouse IgG_K (Boehringer, Mannheim) and Cy3-conjugated sheep anti-mouse IgG (Dianova) were used to detect Digoxigenin labeled probes. Chromosomes were counterstained with DAPI . Analysis was performed using a Zeiss Axiophot microscope. Images were collected and merged using a cooled CCD camera (KAF 1400, Photometries) and IPLab Spectrum software .

RT-PCR Total RNA was prepared by the guanidine isothiocyanate/acid phenol method. cDNA was synthesized and subjected to RT-PCR amplification using 9 μg of total RNA and 25 pmol of random hexanucleotides for the first strand synthesis and the following human C4.4A specific oligonucleotides for the amplification (32 cycles at 60° C) : GCC CCA GCA GCC CCA TAA TAA A and CAC CCA CCC CAC GCT CCA AAG T. For the amplification of uPAR (35 cycles at 55° C) the oligonucleotides AAC GAC ACC TTC CAC TTC and GCA CAG CCT CTT ACC ATA have been used. Amplification of GAPDH (oligonucleotides: ACC ACA GTC CAT GCC ATC AC and TCC ACC ACC CTG TTG CTG TA) was performed for 35 cycles at 56° C. RT-PCR products were analyzed on 1% or 2% agarose gels stained with ethidium bromide. The identity of the fragments was checked by sequencing.

Northern blots

10 μg of total RNA from human tissue or tumor cells were loaded per lane on a denaturing agarose gel . After gel electrophoresis the RNA was transferred to a positively charged nylon membrane by vacuum transfer. After UV- crosslinking of nucleic acid with a Stratal inker , hybridization was done with radioactively labeled RT-PCR fragments of human C4.4A, uPAR or GAPDH with stringent washing. A second filter with human poly (A) -RNA was obtained from Clontech (multiple tissue northern blot, MTN™) .

2. Upregulation of human C4.4A during progression of malignant melanoma Human tissue and cell lines:

Shock frozen normal human tissue of 7 nevi (5 congenital and 2 dermal nevi), 10 primary malignant melanoma (5 superficial spreading melanoma, 4 nodular melanoma 1 acrolentiginous melanoma) of different thickness according to the Breslow definition, 8 lymph node metastases and 7 skin metastases were collected at the Department of Dermatology, University of the Saarland, Homburg, Saar . Patients' consent had been obtained in all cases. Cultured melanocytes were obtained from PromoCell, Heidelberg, Germany, and were cultured as recommended for 4-6 passages. The melanoma lines BS1251, Colo38, W+ and MMLl have been obtained from the American Type Culture Collection or have been established at the Department. With the exception of MMLl, these lines have been derived from metastatic tissue.

Treatment of melanoma lines:

For the evaluation of activation dependent expression of hC4.4A, melanoma lines were starved overnight in RPMI 1640 medium not containing fetal calf serum. Thereafter they were cultured for 24 hours in medium containing 10 % heat inactivated fetal calf serum or 10 % ABO serum which was or was not heat-inactivated for 30 min at 56°C.

RNA in situ hybridization (SIH) :

In order to generate a human C4.4A-specific RNA-probe we first performed RT-PCR with 5μg RNA using the following primers: 5'- GCCCCAGCAGCCCCATAATAAA (hC4.4A 1037-1058) and 5'- CACCCACCCCACGCTCCAAAGT (hC4.4A 1483-1504). The resulting DNA product of 467 bp was gel-eluted and purified using the Qiaquick Gel Extraction Kit (Quiagen, Hilden, Germany) and subsequently cloned into the pCRII-Topo vector (Invitrogen, Groningen, The Netherlands). After sequence analysis, sense and antisense probes were generated by in vitro-transcription using T7 or Sp6 RNA-polymerase. The probe was digoxigenin

(DIG) -labelled using the Boehringer RNA DIG-labelling Kit

(Roche, Mannheim, Germany) . Cryosections (6μm) or cytospins

(10⁵ cells) were fixed in 4 % paraformaldehyde for 1 h and pretreated with lμg/ml pepsin in 0 , 1M HC1 for 5 min and with 0 , 1M triethanolamin/0, 25M acetic acid anhydrid for 10 min. Sections or cytospins were hybridized overnight with 400 ng/ml DIG-labelled sense or antisense hC4.4A-probe at 55°C. Subsequently, sections were incubated with an anti-DIG- alkaline phosphatase monoclonal antibody (Roche, Mannheim, Germany) for 2 h at room temperature. After an overnight incubation with nitroblue tetrazolium / 5-bromo-4-chloro-3- indolyl phosphate (Roche, Mannheim, Germany) sections were counterstained with methylgreen (Vector Laboratories, Burlingame, CA) and embedded in Kaiser's glycerin (Merck, Darmstadt, Germany) .

RT-PCR: mRNA was isolated with the Oligotex mRNA purification system (Quiagen, Hilden, Germany) using the manufacturers protocol. cDNA was synthesized using lμg of polyA⁺ RNA, MuMLV reverse transcriptase and oligo dT . First strand cDNA was subjected to RT-PCR amplification. Using polyA⁺ RNA-derived cDNA the following human C4.4A specific oligonucleotides have been used for the amplification (32 cycles at 55°C) : GCC CCA GCA GCC CCA TAA TAA A and CAC CCA CCC CAC GCT CCA AAG T. Amplification of GAPDH (oligonucleotides: GGT CGG AGT CAA CGG ATT TG and ATG AGC CCC AGC CTT CTC CAT) was performed for 35 cycles at 60°C. The 4.4A-specific and the GAPDH-specific primers amplify a PCR fragment of 460bp and 400 bp, respectively. RT-PCR products were analyzed on 1 % agarose gels stained with ethidium bromide. In real-time PCR, 2μl of cDNA was added to 18μl PCR mix (LightCycler Fast Start DNA Master SYBR Green I kit, Roche Diagnostics) . SYBR Green intercalates between double-stranded DNA and a fluorescence signal is generated through a laser beam. Fluorescence emission is measured and continuously monitored during PCR. The fluorescence signal is plotted versus cross points which mark the cycle number when fluorescence becomes significantly different from baseline signal .

EXAMPLE 2 CLONING OF THE RAT C4.4A cDNA

COS-7 cells were repeatedly transfected with plasmid DNA of a cDNA library derived from the metastatic rat colon carcinoma line RG. C4.4A positive cells were selected by fluorescence staining and FACS sorting. After three extractions according to the method of Hirt 12 bacterial colonies were selected and cDNA of individual colonies was transfected into COS-7 cells. One clone was isolated which gave positive FACS staining with C4.4 mAB. Western blot analysis confirmed that the cells expressed a C4.4-reactive molecule of an estimated molecular weight of about 94 kDa, corresponding to the molecule expressed on RG, where the cDNA was derived from. The C4.4A molecule of the metastatic ASML line had a slightly higher molecular weight. Sequencing of the C4.4 cDNA revealed a 1637 b long DNA with an open reading frame of 352 aa starting with the 5' ATG flanked by a sequence ACAGCTATGG similar to the consensus sequence GCC (A/G) CCATGG characteristic for translation initiation sites (Figure 1). The 5' untranslated region spans 80 nucleotides. Within the 500 bases of the 3' untranslated region there is a consensus sequence for poly (A) addition (Figure 1). The full length nucleotide sequence of the C4.4A revealed no strong homology to any known rat, mouse or human gene. However, there was a low level of homology to the uPAR of several species (Behrendt et al . , J.Biol.Chem. 266 (1991), 7842-7847) .

EXAMPLE 3

CLONING OF THE HUMAN C4.4A CDNA

A placental library was screened with a ³²P labeled C4.4A cDNA probe of the rat. One of the labeled clones was full length sequenced and compared with the rat sequence. The cDNA sequence and the deduced amino acid sequence are shown in Figure 2. The homology of human to rat C4.4A is 72.2% at the DNA level and 81.8% at the amino acid level (Figure 7). This corresponds to the homology between rat C4.4A and rat uPAR (44,9% at the cDNA level and 46,9% on the amino acid level).

EXAMPLE 4 CHARACTERIZATION OF THE RAT AND HUMAN C4.4A PROTEIN

The rat C4.4A molecule potentially spans 352 aa. The molecule has consensus sequences for seven potential N- glycosylation sites. Like the uPAR, it can be divided into three domains, where domains 1 and 2 show some homology to the uPAR of several species, while the third domain is unrelated. According to the aa sequence the C4.4A molecule has an isoelectric point of pH 7.04 and a theoretical MW of 36.96 kDa. Western blotting under non-reducing conditions revealed molecular weights of 98 kDa (ASML) and 94 kDa (PROG, AS-lBl, AS-2A2) (Figure 4) . To determine whether the higher molecular weight may be due to glycosylation, Western blots were performed after inhibition of N- or O-glycosylation . Tunicamycin treatment led to the appearence of an additional band of 66 kDa, which was the dominating band in lysates of the C4.4A transfected lines. Inhibition of O-glycosylation had no effect on the molecular weight of PROG and the transfected lines. The MW of BSp73ASML was slightly reduced corresponding to the MW of PROG. Thus, the rat C4.4A clearly is N- glycosylated. The apparanetly higher molecular weight as revealed by Western blotting after tunicamycin treatment may be a consequence of additional modifications of the GPI anchor (see below) , but ist not due to dimerization, because the MW of the molecule in immunoprecipitates after surface biotinylation is the same under reducing and non-reducing conditions. The C-terminal sequence of C4.4A suggests that the molecule may be phosphatidyl-inositol anchored. This suggestion was strengthened by the observation of a high degree of Triton X-100 insolubility of C4.4A, which is characteristic for GPI anchored molecules (Moller et al . , FEBS Lett. 301 (1992), 493-500). The GPI anchorage of C4.4A was finally proven by treatment with phosphatidyl-inositol phospholipase C. As shown by Western blotting and by fluorescence staining, C4.4 was removed from the cell membrane after phosphatidyl-inositol phospholipase C treatment. Like in the rat, the homolgy between human C4.4A and uPAR is restricted to domains 1 and 2, whereas domain 3 shows no homology. Human C4.4A has 6 N-glycosylation sites. Like the rat C4.4A as well as the rat and human uPAR it contains a potential GPI-anchoring sequence. Interestingly, the C4.4A gene, like the uPAR gene, is located on chromosome 19ql3.1- ql3.2 (Figure 8). In this context it should be pointed out that C4.4A and uPAR are the only members of the Ly-6 superfamily (Rock et al . , Immmunol . Rev. Ill (1989), 195-224) which are composed of 3 domains, while all other members consist of 1 domain (Ploug and Ellis, FEBS Lett. 349 (1994), 163-168) .

EXAMPLE 5

C4.4A AND METASTASIS FORMATION

Rat C4.4A originally has been detected only on rat tumor lines, which metastasize via the lymphatic system. Thus, it was of special interest, whether C4.4A would be involved in the process of tumor progression. To evaluate a possible influence of C4.4A on tumorigenicity the two C4.4A transfected BSp73AS lines, AS-lBl and AS-2A2, were inoculated intravenously and survival time as well as metastasis formation were compared to mock-transfected cells. It became apparent that the survival time of all three groups was comparable and that all rats developed lung metastasis. However, while few and well encapsulated nodules were detected in rats receiving mock-transfected cells, distinct nodules where difficult to dissect in rats receiving AS-lBl and AS-2A2 and the lung tissue was largely replaced by the tumor load. Histological sections of the lung confirmed that AS-lBl and AS-2A2 were not encapsulated at all.

When mock-trans ected and C4.4A-transfected BSp73AS cells were inoculated intrafoodpad and the tumor together with the draining (popliteal) lymph node were excised at 2 weeks after inoculation, animals became moribund after about 2 months. Five out of six rats which had received mock-transfected BSp73AS cells had large tumor masses in the most proximal (inguinal) lymph node. Distant lymph nodes and the lung, with one exception, were free of tumor cells. Both C4.4A transfected lines grew more slowly and removing the primary tumor burden at 2 weeks after transfer was curative for two and three out of five rats, respectively. The remaining rats, where secondary tumor growth was noted, showed lung metastasis. However, only in two out of these five rats tumor cells could be recovered from the most proximal one and distant lymph nodes were free of tumor cells in all five rats. Instead, there was miliar dissemination of the tumor in the peritoneal cavity of three rats. To guarantee a higher rate of secondary tumor growth, the experiment was repeated and the primary tumor together with the draining lymph node was excised at 3 and 4 weeks after tumor cell application. When the primary tumor was excised at these later time points, eight out of nine and six out of six animals receiving mock- transfected cells developed lymph node metastases and only three animals remained free of lung metastases. Lung metastases (mean: 10.0 and 11.3, respectively) were small and well encapsulated (Figure 5a) . The number of lung metastases was slightly increased in rats receiving C4.4A transfected tumor cells (mean: 18.8 and 21.0, respectively). However, metastases in the lung were large and not encapsulated (Figure 5b-d) . Because this particular patterns of metastasis formation was also seen when C4.4A cDNA was transfected in a fibrosarcoma line (BSpβS) (Figure 5e) or in a pheochromoblastoma line (BSp3A) , it was concluded that the incapability of matrix formation and/or the strong degradation of the ECM were an autonomous feature of C4.4A and independent of the epithelial or mesoderm origin of the tumor cell. Thus, C4.4A did not suffice to initiate metastatic spread, but facilitated metastasis formation.

EXAMPLE 6 FUNCTIONAL ACTIVITY OF C4.4A IN SUBSTRATE ADHESION AND MATRIXGEL PENETRATION

Phosphatidyl-inositol anchored molecules display a variety of functions (Moller, Blood Coagul . Fibrinolysis 4

(1993), 293-303; Anderson, Semin. Immunol . 6 (1994), 89-95), which could be of importance in tumor progression, like the involvement in signal transduction, the capacity to support cell proliferation upon crosslinking and the participation in matrix degradation. Our studies did not reveal any linkage of C4.4A to phosphotyrosine kinases and cell proliferation was only influenced to a minor degree by C4.4A. But there is in vitro evidence that C4.4A is involved in matrix degradation. This observation is in line with the pattern of metastasis formation in vivo. This evidence is based on the results of two expermiental settings, adhesion to laminin and matrigel penetration. Expression of C4.4A, constitutively or via transfection, had no influence on binding to hyaluronic acid, fibronectin and vitronectin. Instead, C4.4A⁺ cells adhered strongly to laminin coated plates and binding was inhibited by C4.4 (Figure 6a). To differantiate between binding and spreading, the experiment was done in parallel at 4°C and 37°C. Particularly BSp73ASML cells, but also BSp73AS-lBl adhered more efficiently at 37°C (Figure 6b) . The interpretation of active adhesion via C4.4A was strengthened by the finding that C4.4A⁺ cells spread very rapidly on laminin coated plates, the phenomenon has not been observed on BSA or collagen coated plates. Since BSp73ASML as well as BSp73AS cells express α6βl and weakly α3 integrins it was next asked, whether adhesion to laminin may be integrin-dependent . The finding that in the presence of EDTA adhesion of C4.4A⁺ cells to laminin was weakened, but not abolished argues for laminin binding via

C4.4A to be at least partly an integrin-independent process

(Figure 6c) . Interestingly, as shown in Figure 6a, adhesion to laminin is transient, i.e. after 2 to 4 h less C4.4A⁺ cells adhered to laminin coated plates than after 30 min. In order to evaluate, whether the transient nature of adhesion may be brought about by degradation of laminin, the protease inhibitor aprotinin was added to the culture medium. In the presence of aprotinin, adhesion to laminin remained stable over the observation period of 2 h (Figure 6d) .

The transient adhesion of C4.4A⁺ cells to laminin strengthened the hypothesis that C4.4 may enable cells for degradation of elements of the extracellular matrix. Indeed when constitutively C4.4A⁺ cells BSp73ASML cells were layered on matrigel coated transwell plates, 66% migrated through the matrix within 24 h. In the presence of C4.4 (lOμg/ml) migration through the matrix was nearly completely inhibited. However, only a minority of cells transfected with C4.4A⁺ cDNA penetrated through the matrigel. One possible reason for the failure of C4.4A transfected BSp73AS cells to penetrate the matrigel could be the lack of C4.4A ligand expression, whereas on the consitutively C4.4A⁺ line BSp73ASML uPA or a corresponding molecule might bind to C4.4A, thus initiating the process of matrix degradation. Assuming that the transfected lines can recruit in vivo uPA or a homologous molecule from host cells, these experiments could provide a hint, why C4.4A⁺ tumor cells metastasize without capsule formation and by which mechanism C4.4A facilitates metastasis formation.

EXAMPLE 7 EXPRESSION OF hC4.4A IN HUMAN NONTRANSFORMED TISSUE AND TUMOR LINES

Expression of hC4.4A in normal tissue was evaluated by Northern blots using a filter containing poly (A) -RNA from brain, placenta, lung, liver, skeletal muscle, kidney and pancreas obtained by Clontech (Figure 9a) and by RT-PCR of human cDNA of skin, oesophagus, thymus , colon, spleen, kidney, lung, brain, liver, stomach and peripheral blood leukocytes

(Figure 9b). Expression of hC4.4A RNA was detected in skin, oesophagus and placenta derived RNA and very weakly on RNA of peripheral blood leukocytes. In a RT-PCR using uPAR primers, signals were detected in RNA derived from skin, thymus, colon, kidney and stomach (Figure 9b) . Thus, expression of hC4.4A appears to be similarly restricted as it was observed with rat C4.4A. Furthermore, there is no evidence that expression of hC4.4A and uPAR may be linked.

While the expression of hC4.4A on non-transformed tissue was very restricted, hC4.4A mRNA was found in 56% of tumor lines. This has been evaluated by RT-PCR (Figure 10) and confirmed by Northern blots using filters containing 10 μg of total RNA per lane. 9 colon carcinoma, 20 malignant melanoma, 5 mammary carcinoma, 5 malignancies of the hematopoietic system, 3 pancreatic adenocarcinoma, 3 renal cell carcinoma, 1 or 2 tumor cell lines of liver, chorion, stomach, esophagus, lung, cervix, ovary and 1 fibrosarcoma were tested. Human C4.4A was detected in 2 of 5 tested malignancies of the hematopoietic system. It was also found in 100% of malignant melanoma, 22% of colon carcinoma lines and 20% of mamma carcinoma lines

(Figure 10 and Table 1) .

When comparing the expression of hC4.4A with uPAR (Table 1), it became apparent that expression of the two molecules was independent. Yet, the overall frequency of expression of uPAR on tumors of different origin (70%) slightly exceeded expression of hC4.4A. Finally, it should be mentioned that a PCR analysis of a small number of tissue extracts of naevi , primary malignant melanoma and metastasis of malignant melanoma revealed expression of hC4.4A in 100 % of metastatic tissue, but only in a low percentage of primary tumors. Naevi did not express C4.4A.

SUMMARY OF EXAMPLES 2 - 7

In summary, the present invention identifies cDNAs encoding rat and human C4.4A, respectively. The rat C4.4A cDNA has been isolated from a metastasizing tumor line, i.e. is metastasis- associated expressed. The rat and human C4.4A is hardly expressed in normal tissues of the adult organism. By transfection of the rat C4.4A encoding cDNA into a low metastasizing tumor line it became apparent that C4.4A did not alter the metastasic potential of tumor cells, but influenced the manner of host tissue invasion: Metastasis formation of C4.4A transfected lines was either miliary or at least without any form of encapsulation. The GPI anchored C4.4A molecule displays structurally and functionally similarities to the uPAR and facilitates embedding of metastasizing tumor cells likely by degradation of the extracellular matrix.

The results presented above suggest that an interference with metastasis formation by blockade of the C4.4A molecule is achievable. Finally, the C4.4A molecule could as well function as marker for metastasizing tumors, propably at an early stage of the disease, thus allowing a therapeutical treatment at an early stage and, as a consequence, increasing the prospects of effective curing. Moreover, the C4.4A molecule may also be useful for evaluating whether a metastasizing tumor is resistant to particular therapeutic regimens.

The foregoing is meant to illustrate, but not to limit, the scope of the invention. The person skilled in the art can readily envision and produce further embodiments, based on the above teachings, without undue experimentation.

EXAMPLE 8

Expression of hC4.4A on melanocytes and malignant melanoma

Expression of hC4.4A was evaluated by RT-PCR and ISH in melanocytes, unaltered skin, 7 nevi, 10 primary malignant melanoma, 8 lymph node and 7 skin metastasis of malignant melanoma (Table II) . As shown before, human skin weakly expressed hC4.4A, the expression being restricted to the stratum basale. Neither melanocytes nor nevi expressed hC4.4A. With primary malignant melanoma the picture was not uniform. By RT-PCR, no signal was obtained with 3 out of 10 tumors, weak signals were seen with 5 and strong signals with 2 samples. All skin and lymph node metastases expressed hC4.4A, the signals being in most instances stronger than those of primary tumors. Similar findings accounted for ISH. In primary malignant melanoma at least some cells were positive in all 10 tumors. A higher percentage of cells, though not all, were stained in sections of skin metastases . In lymph node metastases, the vast majority of tumor cells expressed hC4.4A mRNA.

Table II

These findings exclude that hC4.4A is a differentiation marker of cells of the melanocytic lineage. Furthermore, hC4.4A is not expressed constitutively on malignant melanoma cells, but its expression appears to be upregulated during tumor progession.

Activational state dependent expression of hC4.4A Because of the higher expression level of hC4.4 on metastases of malignant melanoma, it became tempting to speculate that the acivational state of tumor cells may have bearing on hC4.4A expression. To support the assumption (Table III), malignant melanoma cell lines were starved overnight, i.e. were cultured in the absence of fetal calf serum. Thereafter they were cultured in the presence of human serum, which had not been heat inactivated, and expression of hC4.4A was evaluated by quantitative PCR. Transcription of hC4.4A became upregulated by all 4 tested lines, the increase spanning a range from 2.3- to 8.2-fold. Importantly, when the cells were cultured in the presence of heat inactivated human serum, no such increase was observed, i.e. expression was in the same range or below the one of cells cultured in the presence of heat inactivated fetal calf serum.

Table III

Expression of rat C4.4A facilitates matrix degradation and interferes with adhesion to laminin. The finding that expression of hC4.4A is upregulated on metastasizing melanoma cells could well be in line with functional activity of hC4.4A in matrix degradation and is reminiscent of uPAR expression on colon carcinoma which correlates with the metastastic capacity. Activation of transcription is another phenomenon shared with the uPAR gene, transcription of which has been described to be regulated by nerve growth factor, epidermal growth factor, serum factor VII and Vila as well as by TGF- beta 1. Work is in progress to identify the heat-labile serum factor (s) responsible for the transcriptional regulation of hC4.4A. Our data indicate a very restricted expression in non- transformed cells and a high expression on metastases. Thus, hC4.4A will possibly be important as a prognostic indicator and a therapeutic target. Table I

Expression of hC4.4A and uPAR on human tumor lines (RT-PCR)

Tumor lines hC4.4A^a uPAR³ hC4.4A + uPAR

Malignant melanoma 20/20 8/13 8/13

Colon carcinoma 2/9 5/7 1/7

Mammary carcinoma 1/5 4/5 1/5

Leukemia, lymphoma 2/5 2/5 1/5

Pancreatic adenocarcinoma 2/3 1/1 1/1

Renal cell carcinoma 3/3 3/3 373

Hepatoma 1/2 1/1 1/1

Carcinoma of various tissues 2/6 3/4 1/4

Fibrosarcoma 0/1 1/1 0/1

Table 11 mRNA expression of hC4.4A: RT-PCR versus ISH

Tissue RT-PCR ISH

skin weak signal stratum basale (weak) freshly cultured melanocytes negative negative nevi negative (7/7) negative (7/7) primary malignant melanoma positive (7/10) positive (10/10) weak → strong few → most cells skin metastases positive (7/7) positive (7/7) medium → strong most cells lymph node metastases positive (8/8) positive (8/8) mostly strong most → all cells

Table I I I Modulation of hC4.4A expression on malignant melanoma lines by culture conditions

Melanoma Culture Signal intensity hC4.4A : GAPDH line condition GAPDH hC4.4A increase above baseline

BS1251 FCS 3.35 1523 1.00

ABO serum 1.95 5518 6.22

ABO serum inact. 1.63 514 0.69

Colo38 FCS 1.01 175 1.00

ABO serum 1.14 1628 8.16

ABO serum inact. 2.36 1069 2.62

W+ FCS 5.70 1079 1.00

ABO serum 4.24 3982 4.97

ABO serum inact. 5.40 1012 0.99

MML1 FCS 5.35 1717 1.00

ABO serum 4.06 3007 2.31

ABO serum inact. 4.77 1761 1.15

Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule encoding the metastasis-associated antigen C4.4A or a protein exhibiting biological properties of the metastasis- associated antigen C4.4A and being selected from the group consisting of

(a) a nucleic acid molecule encoding a protein that comprises the amino acid sequence depicted in Figure 1 and 2, respectively;

(b) a nucleic acid molecule comprising the nucleotide sequence depicted in Figure 1 and 2, respectively;

(c) a nucleic acid molecule included in DSMZ Deposit No: 13013 and 13014, respectively; (d) a nucleic acid molecule which hybridizes to a nucleic acid molecule specified in (a) to (c) ;

(e) a nucleic acid molecule the nucleic acid sequence of which deviates from the nucleic sequences specified in

(a) to (d) due to the degeneration of the genetic code ; and

(f) a nucleic acid molecule, which represents a fragment, derivative or allelic variation of a nucleic acid sequence specified in (a) to (e) .

2. A recombinant vector containing the nucleic acid molecule of claim 1.

3. The recombinant vector of claim 2 wherein the nucleic acid molecule is operatively linked to regulatory elements allowing transcription and synthesis of a translatable RNA in prokaryotic and/or eukaryotic host cells .

4. A recombinant host cell which contains the recombinant vector of claim 3.

5. The recombinant host cell of claim 4, which is a mammalian cell, a bacterial cell, an insect cell or a yeast cell .

6. An isolated protein exhibiting biological properties of the metastasis-associated antigen C4.4A which is encoded by a nucleic acid molecule of claim 1.

7. A recombinant host cell that expresses the isolated protein of claim 6.

8. A method of making an isolated protein exhibiting biological properties of the metastasis-associated antigen C4.4A comprising: (a) culturing the recombinant host cell of claim 7 under conditions such that said protein is expressed; and (b) recovering said protein.

9. The protein produced by the method of claim 8.

10. An antisense RNA sequence characterized in that it is complementary to an mRNA transcribed from a nucleic acid molecule of claim 1 or a part thereof and can selectively bind to said mRNA or part thereof, said sequence being capable of inhibiting the synthesis of the protein encoded by said nucleic acid molecule.

11. A ribozyme characterized in that it is complementary to an mRNA transcribed from a nucleic acid molecule of claim 1 or a part thereof and can selectively bind to and cleave said mRNA or part thereof, thus inhibiting the synthesis of the protein encoded by said nucleic acid molecule.

12. An inhibitor characterized in that it can suppress the activity of the protein of claim 9.

13. A method for detecting a cell proliferative disorder associated with a metastasizing tumor which comprises contacting a target sample suspected to contain C4.4A or the C4.4A encoding mRNA with a reagent which reacts with C4.4A or the C4.4A encoding mRNA and detecting C4.4A or the C4.4A encoding mRNA.

14. The method of claim 13, wherein the reagent is a nucleic acid.

15. The method of claim 13, wherein the reagent is an antibody.

16. The method of claim 13, wherein the reagent is detectably labeled.

17. The method of claim 16, wherein the label is selected from the group consisting of a radioisotope, a bioluminescent compound, a chemiluminescent compound, a fluorescent compound, a metal chelate, or an enzyme.

18. A method for preventing, treating, or ameliorating a cell proli f erative disorder associated with a metastasizing tumor which comprises administering to a mammalian subject a therapeutically effective amount of a reagent which decreases or inhibits C4.4A expression and/or the activity of C4.4A.

19. The method of claim 18, wherein the reagent is a nucleotide sequence comprising an antisense RNA.

20. The method of claim 18, wherein the reagent is a nucleotide sequence comprising a ribozyme.

21. The method of claim 18, wherein the reagent is an C4.4A inhibitor .

22. The method of claim 21, wherein the reagent is an C4.4 antibody or a fragment thereof .

23. A diagnostic kit useful for the detection of C4.4A or C4.4A encoding mRNA in a sample, wherein the presence or an increased concentration of C4.4A or C4.4A encoding mRNA is indicative for a cell proliferative disorder associated with a metastasizing tumor, said kit comprising a probe for detection of C4.4A or C4.4A encoding mRNA.

24. The kit of claim 23, wherein the target component to be detected is C4.4A and the probe is an antibody.