CA2201865C - Laminin chains: diagnostic and therapeutic use - Google Patents

Abstract

The instant invention provides for the identification diagnosis, monitoring, and treatment of invasive cells using the laminin 5 gamma-2 chain protein or nucleic acid sequence, or antibodies thereto.</SDOA B>

Description

76909-228(S) Laminin Chains: Diagnostic and Therapeutic Use Background of the Invention Laminins are a family of basement membrane proteins which function in cell differentiation, adhesion, and migration, in addition to being true structural components (Tryggvason K, Curr. Opn. Cell Biol., 1993, 5:877-882). The laminin molecule is a cross-shaped heterotiimer consisting of one heavy a chain (-400 kd) and two light chains, B and y (130-200 kd) (nomenclature according to Burgeson et al., Matrix Bio1, 1994, 14:209-211). Laminin exists in numerous isoforms that are formed by different combinations of laminin chain vanients which currently amount to at least nine.
Kalinin/laminin 5 (most likely also identical to the adhesion molecule nicein) is a recently identified laminin isoform which is a functional adhesion component for epithelial cells (Tryggvason. 1993, supra. ; Burgeson et al..
1994, supra.; Rousselle et al., J. Cell Bio., 1991, 114:567-576; Kallunki et al., J.
Cell Biol. , 1992, 119:679-693; Marinkovich et al., J. Biol. Chem., 1992, 267:17900-1?906; Vailly et al.. Eur. J. Biachen>., 1994, 219:209-218). Kalinin/laminin 5 contains unique laminin varient chains, one of which, the ~1. chain, has recently been cloned and sequenced (Kallunki et al., 1992, supra. , previously named B2t).
The Y1 chain has a mass of -130 kd and is thus smaller than the "classical" -kd Q1 and y1 light chains of laminin. The domain structure of the ~2 chain also differs from that of the Y 1 chain in that it lacks the amino-terminal globular domain (domain V1) bc;lieved to function in intermolecular cross-linking of laminin molecules to form networks (Yurcheno and O'Rear, in Molecular and Cellular A~~s of Basement Membranes, 1993, (ed. Rohrbach and Timpl.
Academic Press, San Diego, pp. 20-47). In addition, domains III, IV, and V
(containing EGF-like repeats) in Y2 are shorter than in the ~yl chain (Kallunki et a1..1992, supra. ).
By in situ hybridization the y2 chain was found to be expressed in epithelial cells of many embryonic tissues such as those of skin, lung, and kidney (Kallunki et al.. 1992, supra. ), and antibodies to kalinin/laminin 5, react with basement membranes of the same tissues (Rousselle et al., 1991, supra.;
Verr,mdo et al.. Lab. Invest.. 1991. 64:85-92).
The different laminin chains have been shown to have quite varying tissue distribution as determined by immunohistological studies, Northern, and in situ hybridization analyses. For example, the A and M chains on the one hand, and the R'O 96/10646 PCT/EP95/03918 B1 (!31) and S (B2) chains on the other, have been shown to be mutually exclusive (see for example Vuolteenaho et al., J. Cell Biol., 1994, 124:381-394). In vitro studies have indicated that laminin mediates a variety of biological functions such as stimulation of cell proliferation, cell adhesion, differentiation, and neurite outgrowth. The cellular activities are thought to be mediated by cell memebrane receptors, many of which are members of the integrin family (Ruoslahti, E. J.
Clin. Invest., 1991, 87:1-5; Mecham, R.P. FASEB J., 1991, 5:2538-2546; Hynes, R. Cell, 1992, 69:11-25).
Recently a new nomenclature for describing laminins has been agreed to as in the following Table 1 (after Burgeson et al., 1994, supra. ) Table 1 laminin chainsenes heterotrimers and of laminin New Previous Gene New Chains Previous al A, Ae LAMAl laminin-1al(31y1 EHS laminin a2 M, Am LAMA2 laminin-2a2(3lyl merosin a3 200kDa LAMA3 laminin-3al~i2y1 s-laminin (31 B 1, B 1e LAMB 1 laminin-4a2(32y1 s-merosin (32 S, B is LAMB2 laminin-5a3(33y~2kalinin/nicein (33 140 kDa LAMB3 laminin-6a3~ilyl k-laminin 'y1 B2, B2e LAMC1 Iaminin-7a3~32y1 ks-laminin B2t LAMC2 8ummary of the Invention The instant invention provides for methods of detecting kalinin/Iaminin 5 expression in tissue comprising detecting a signal from assayed tissue, such signal resulting from specifically hybridizing tissue with an effective amount of a nucleic acid probe, which probe contains a sense or antisense portion of kalinin/laminin 5 gamma-2 nucleic acid sequence (Kallunki et al., 1992, sc~pra.).
In particular, where the nucleic acid probe is DNA, RNA, radiolabelled, enzyme labelled, chemiluminescent labelled, avidin or biotin labelled, derived from human kalinin/laminin 5 gamma-2 nucleic acid sequence, incorporated into an extrachromasomal self-replicating vector, a viral vector, is linear, circularized, or contiains modified nucleotides. In the preferred embodiment the probes are linearized specific regions of the 'y2 gene.
The instant invention also provides for methods for detecting the presence of invasive cells in tissue comprising detecting a signal from assayed tissue, such 76909-228(S) signal resulting from contacting tissue with an effective amount of a nucleic acid probe, which probe contains a sense or antisense portion of kalinin/laminin 5 gamma-2 nucleic acid sequence (Kallunki et al., 1992, supra. ). In particular, where the nucleic acid probe is DNA, RNA, radiolabelled, enzyme labelled, chemiluminescent labelled, avidin or biotin labelled, derived from human kalinin/laminin 5 gamma-2 nucleic acid sequence, incorporated into an extrachromasomal self-replicating vector, a viral vector, is linear, circularized, or contiains modified nucleotides. In the preferred embodiment the probes are linearized specific regions of the y2 gene. The instant method also provides for the diagnosis of the absence of n chain expression, useful for the monitoring of therapies, and the progress of malignant cell transformation leading to accurate determination of the extent of invasive cell activity.
The instant invention father provides for a method for detecting kalinin/laminin 5 expression in tissue comprising detecting a signal from assayed tissue, such signal resulting from contacting tissue with an effective amount of a labeled probe, which probe contains an antibody immunoreactive with a portion of kalinin/laminin 5 gamma-2 protein.
Further provided is a method for detecting invasive cells in tissue comprising detecting a signal from assayed tissue, such signal resulting from contacting tissue with an effective amount of a labeled probe, which probe contains an antibody immunoreactive with a portion of kalinin/laminin 5 gamma-2 protein. Also provided is a method for detecting kalinin/laminin 5-in tissue comprising detecting a signal from assayed tissue. such signal resulting from contacting tissue with an effective amount of a labeled probe, which probe contains an antibody immunoreactive with a portion of kalinin/laminin 5 gamma-2 protein. Thus the method of the instant invention provides for the absence of .
such signal as diagnostic for the absence of invasive cells.

76909-228(S) According to another aspect of the present invention, there is provided a method for detecting the presence of invasive cells in tis:~ue comprising detecting a signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains (i) a sense or ' antisense portion of kalinin/laminin 5 gamma-2 chain nucleic acid set forth in Figure 4A, or (._i) a nucleic acid that specifically hybridizes to the nucleic acid of (i).
According to still another aspect of the present invention, there is provided a method for monitoring the presence of invasive cells in tis:~ue comprising detecting a signal or absence of signal from t:he tissue assayed, such signal resulting from specifica115r hybridizing the tissue with an effective amount of a nuc7_eic acid probe, which probe contains (i) a sense or antisense portion of kalinin/laminin 5 gamma-2 chain nucleic acid set forth in Figure 4A, or (ii) a nucleic acid that specifically hybridizes to the nucleic acid of (i).
According to yet anothex- aspect of the present invention, there is provided a method for detecting the presence of invasive cells in tissue comprising detecting a signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains a sense or antisense portion of a nucleic acid encoding kalinin/laminin 5 gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A.
According to a further aspect of the present invention, there is provided a method for monitoring the presence of invasive cells in tis~;ue comprising detecting a 3a 76909-228(S) signal or absence of signal from 1=he tissue assayed, such signal resulting from specificall~r hybridizing the tissue with an effective amount of a nuc=Leic acid probe, which probe contains a sense or antisen;~e portion of a nucleic acid encoding kalinin/laminin 5 gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A.
According to a further aspect of the present invention, there is provided a method for detecting invasive cells in tissue comprising detecting a signal from assayed tissue, such signal resulting frorl contacting tissue with an effective amount of a labelled probe, which probe contains an antibody specifically immunore~ictive with a portion of kalinin/laminin 5 gamma-2 chain px-otein, wherein the kalinin/laminin 5 gamma-2 chain px-otein is set forth in Figure 4A.
According to a further inspect, there is provided a method for monitoring invasive ce7.ls in malignant tissue comprising detecting a signal from assayed, malignant tissue, such signal resulting from contacting tissue with an effective amount of a labelled probe, which contains an antibody specifically immunoreacti.ve with a portion of kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.
According to a further aspect, there is provided use, for one or more of inhibiting invasive growth of a malignant cell, inhibiting tumor cell invasion of non-malignant tissue, inhibiting scudding of tumor masses, and inhibiting tumor cell interaction with basement 3b 76909-228(S) membrane, of a nucleic acid which contains an antisense portion of a nucleic acid encoding kalinin/laminin 5 gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A, and wherein the antisense portion effectively inh__bits the translation of endogenous kalinin/laminin 5 gamma-2 chain encoding mRNA.
According to a further aspect, there is provided use, in manufacture of a medicament for one or more of inhibiting invasive growth of mal~_gnant cells, inhibiting tumor cell invasion of non-malignant tissue, inhibiting budding of tumor masses, inhibiting tumor cell interaction with basement membrane, and evaluating extent of invasive cell activity in a patient, of an effective inhibiting amount of an antibody specificall~T immunoreactive with a portion of a kalinin/laminin 5 ganuna-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.
According to a further aspect, there is provided use, for one or more of inhibiting invasive growth of malignant cells, inhibiting tumor cell invasion of non-malignant tissue, inhibiting budding of tumor masses, inhibiting tumor cell interaction with basement membrane, and evaluating extent of invasive cell activity in a patient, of an effective inhibitir.:g amount of an antibody specifically immunoreactive with a. portion of a kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.
3c 76909-228(S) Brief Description of the Drawings Figure 1 shows In situ hybridization of a specimen of colon adenocarcinoma for y2 chain mRNA using a S-35 labelled anti-sense RNA probe der='med from plasmid pbb2r-02.
Magnification: 1A x 100; 1B-1D x Ei40.
Figure 2 shows In situ Hybridization for y2 chain mRNA on sections of ductal mammary carcinoma (2A), malignant melanoma (2B), squamous cell carc__noma 3d WO 96/10646 _ '~ ~ PGT/EP95/03918~
of the skin (2C-2D), and squamous cell carcinoma of the vulva (2E-2G).
Magnification: 2C x 100, all others x 640.
Figure 3 is incisionally wounded mouse skin (72 hours after wounding) showing signal for ~2 chain in keratinocytes at the leading edge of the migrating epithelium (curved arrow). Magnification: x 640.
Figure 4 shows the nucelic acid sequence for the y2 chain cDNA and the derived amino acid sequence. Figure 4A is the full cDNA for the 5,200 base pair sequence, availible from EMB/GenBank/DDBJ under the accession number 215008. Figure 4B is the nucleotide and derived amino acid sequence of the alternative 3' end sequence from cDNA clones providing a sequence of 4,316 base pairs, availible from EMB/GenBank/DDBJ under the accession number 215009. (Kallunki et al., 1992, sacpra.).
Detailed Description of the Invention Epidermolysis bullosa (EB) is a group of mechano-bullous disorders characterized by fragility of the skin and mucous membranes (see Lin & Carter eds., Epidermolysis bullosa Basic and clinical a pects, 1992, Springer Verlag, N.Y.; Fine et al., J. Any. Acad Dern~atol., 1991, 24:119-135). The functional forms of EB (JEB) are characterized by tissue separation at the level of the lamina lucida within the dermal-epidermal basement membrane, and no specific mutation had yet to be reported. Recently it has been proposed that the genes for a lamina lucida protien kalinin/nicein/epiligin may be a candidate in some forms of JEB
(Verrando et al., 1991, supra.). Several lines of evidence suggest that anchoring filament proteins could be defective in some forms of JEB. First, attenuation or absence of immunoreactivity with anti-kalinin(epiligrin) antibodies has been noted in the skin of patients with the most severe (Herlitz) type of JEB. The immunofluorescence staining patterns may be of prognostic value in classifying JEB, and these immunoreagents have been used for prenatal diagnosis of JEB
using fetal skin biopsy specimins. Second, the kalinin/laminin 5 ~2 chain is expressed in epithelial cells of the skin, trachea and kidneys, tissues which are frequently affected by JEB.
Since the majority of cases are of the generalized (Herlitz) phenotype (H-3~ JEB), JEB patients have been classified into Herlitz and non-Herlitz types.
Clinical features of H-JEB include mechanical fragility of the skin, with WO 96/10646 ~ ~ ~ ~ ~ ~ ~ PCT/EP95103918 widespread blistering and erosions, rapid deteuoration and neonatal death, often from sepsis. Longtern survival is rare.
Efforts to identify the basic defect in JEB began with the observation that a monoclonal antibody that binds to the lamina lucida of the epidermal basement membrane zone of normal skin, fails to react with the lamina lucida of H-JEB
skin ' (Verrando et al., 1991, stcpra.). The antigen recognized by~this antibody was purified from keratinocyte culture medium and termed BM600/nicein.
Keratinocytes cultured from the skin of H-JEB patients attach poorly to substrate and fail to accumulate immunologically detectable nicein. Further experiments with antibodies specific for the a3 chain of nicein, demonstrated that they were capable of inducing the rounding and detachment of adherent keratinocytes without affecting fibroblasts (Rousselle et al., 1991, sacpra.). Thus the correlation in vivo and i~z vitro of the dermoepidermal separation with deficient nicein/kalinin/laminin 5 immunoreactivity and the separation induced by anti-nicein antibody have made the genes encoding this protein strong candidates for the site of H-JEB mutations.
The importance of the y2 chain of nicein/kalinin/laminin 5 in JEB, and epithelial tissues prompted the investigation into the role such adhesion contacts between epithelial cells may play in abberant cells. Of primary interest was the role ~2 chain of nicein/kalinin/laminin 5 abberant expression may play in cancer tissue, and a possible role in cancer dissemination.
It has been recently shown that in colon adenocarcinoma, a significant positive correlation between the degree of tumor budding and the recurrence of tumors following curative surgery exists, and that this fact is likely to reflect a higher invasive potential of budding cancer cells as compared with cancer cells located deeper in the tumor (Hase et al., Dis. Colon Rectaenz, 1993, 36:627-635).
Therefore, as demonstrated in Example 3 below, the instant invention allows for the useful prognostic determination of success of surgery, means for monitoring progression of tumor budding and subsequent prognosis.
The identification of the role of 'y2 chain allows for the novel use of kalinin/laminin 5 y2 chain and its ligand, as diagnostic probes of the tumor cell/basement membrane adhesion interface that is crucial for the invasion of non-malignant tissues, and identifies invasive cells.
Thus the identification of the role of ~2 chain allows for the novel therapeutic intervention of binding of kalinin/laminin 5 to its ligand, and thereby reducing the tumor cell/basement membrane adhesion that is crucial for the invasion of non-malignant tissues, and method for inhibiting the budding of tumor 76909-228(S) masses, and a means for determing the level of ~2 chain expression as a measure of budding activity of a given tumor. .
As demonstrated in Example 3 below, the Y1 chain of kalinin/laminin 5 is preferentially expressed by invasively growing malignant cells in human carcinomas. Furthermore, migrating keratinocytes in wound healing also expressed this gene, pointing to a role of ~2 chain in epithelial cell migration both in malignant and in nonmalignant pathological conditions. The consistent expression of the Y1 chain, gene in invading cancer cells reflects a functional importance of this molecule in vivo in establishing contacts between the invading malignant cells and a provisional matrix in the immediate surroundings of the cancer cells. The instant invention provides methods for the identification of, and diagnosis of invasive cells and tissues, and for the monitoring of the progress of therapeutic treatments.
In a preferred embodiment of this aspect of the instant invention the nucleic acid probe comprises a specifically hybridizing fragment of the'y2 chain cDNA nucleic acid sequenee. In this embodiment, the nucleic acid sequence comprises all or a specifically hybridizing fragment of an open reading frame of the nucelic acid sequence for the YI chain (figure 4) encoding the amino acid sequence of the y1 chain (Figure 4). It will be understood that the term "specfically hybridizing" when used to describe a fragment of nucleic acid encoding a human laminin ~2 chain gene is intended to mean that. nucleic acid hybridization of such a fragment is stable under high stringency conditions of hybridization and washing as the term "high stringency" would be understood by those having skill in the molecular biological arts.
Further, the instant invention provides for the therapeutic treatment of such invasive tissues by using YI chain or biologically active fragments thereof to interfere with the interactions between abberant y2 chain and surrounding tissues.
The instant invention also provides for the intervention of y2 chain interaction with surrounding tissues by using specific anti-n chain antibodies (monoclonal or polyclonal) to inhibit the YI chain biological activity.
The instant disclosure also allows one to ablate the invasive cell phenotypic ~L chain expression by using genetic manipulation to "knock-out"
the functional expression of the Y1 chain gene in cancer cells, or to completely "knock-out" the functional Y1 chain gene in the genome of cancer cells. Such knock-outs can be accomplished by using genetic molecular biological techniques for inserting homologous recombination into genomic DNA, targeted transposon insertion, or random insenionldeletion mutations in the genomic DNA.

R'O 96/10646 ~ n ~ ~ ~ PCT/EP95/03918 1I_ The instant disclosure also allows for the therapeutic treatment of invasive cell phenotype by the inhibition of functional y2 chain expression in targeted cells by using anti-sense technology, such methods for anti-sense production, stabilization, delivery, and therapeutic approaches are reviewed in Uhlmann et al., 1990, Chenz. Reviews 90:543-584).
Thus the instant invention provides for a method of detection, diagnosis, prognosis, monitoring, and therapeutic treatment of invasive cell phenotypes.
The examples below are meant by way of illustration, and are not meant to be limiting as to the scope of the instant disclosure.
lExamnle 1~ Mutation in thex2 Chain Gene LAMC2 is critical in ~nme cases ofof .TEB
A unique scanning strategy using RT-PCR amplification of LAMC2 sequences was devised to detect truncated forms of ~2 chain gene transcripts (Pulkkinen et al., Natccre Genetics, 1994, 6:293-298). The 3.6 kilobase coding sequence of the LAMC2 mRNA, was reverse transcribed and amplified with eight pairs of primers, producing overlapping PCR amplimers designated A-H. The PCR products were then examined by agarose gel electrophoresis, followed by MDE heteroduplex analysis. If bands with altered mobility were detected, the PCR products were sequenced, and compared with normal sequences from unaffected family members or unrelated individuals. Intron/exon borders were identified by PCR analysis of genomic DNA, deduced by comparison with cDNA
sequences.
A point mutation~roduceS exon Skipnine When a panel of five unrelated JEB patients were analysed, the primers used to amplify segment C (nt 1046-1537) produced markedly shortened band of 273 base pairs, as compared with the normal 491 base pairs. No evidence of the normal sized band was noted, suggesting that the patient was homozygous for this allele. Direct sequencing revealed that the shortened product resulted from the deletion of 219 base pairs corresponding to nucleotides 1184-1402 in the cDNA, thus exon 9 was deleted. The remaining nuclotide sequences within this and other PCR products did not reveal any additional mutations upon MDE analysis.
Subsequent examination of the genomic DNA revealed that the sequences for exons 8, 9 and 10 were present, however a homozygous G for A substitution at the 3' acceptor splice site at the junction of intron 8 and exon 9, abolished the obligatory splice site sequence (AG).

76909-228(S) Examination of another patient revealed that PCR product F (nt 2248-2777).corresponding to domains I and II of the n chain, was a band with altered mobility. Sequencing the abnormal product revealed a 20 by deletion, followed by a single base pair (G) insertion in the coding region corresponding to exon 16.
This mutation causes a frameshift which results in a premature stop codon 51 base pairs downstream from the deletion-insertion, predicting a'truncated kalinin/laminin 5 y2 chain terminating at residue 830.
RT-PCR and MDE analyses RNA isolated from Fbroblast cell cultures of JEB patients was used as template for RT-PCR of the LAMC2 mRNA. (Epidermal keratinocytes can also be used). cDNA was prepared from 50 ug of total RNA in a volume of l001tL
according to manufacturer's reccomendations (BRL), and oligonucleotide primers were synthesized on the basis of the cDNA sequence (Figure 4; Kallunki et al.;
1992, supra.), to generate -500 base pair products, which spanned the entire coding region.
For PCR amplification, 1 ~tl. of cDNA was used as template and amplification conditions were 94 C for 5 min followed by 95 C for 45 sec, 60 C
for 45 sec and 72 C for 45 sec for 35 cycles in an OmniGene thermal cycler (Marsh Scientific). Amplification was performed in a total volume of 25 ~tL
containing 1.5 mM MgCl2, and 2 U Taq polymerise (Boehringer Mannheim).
Aliquots of 5 ~tL were anilysed on 2 % agarose gels and MDE hetei~oduplex analysis was performed according to the manufacturer's reccomendation (AT
Biochemicals). Heteroduplexes were visualized by staining with ethidium bromide. If a band of altered mobility was detected in heteroduplex analysis, the PCR product was subcloncd into the TA vector (Invitrogen), and sequenced by standard techniques.
DNA isolated either from fibroblast cultures or from specimens obtained from buccal smears, was used as template for amplification of genomic sequences. For amplification of introns 8 and 16, -500 ng of genomic DNA was used as template and the following oligomer primers were utilized.
5' GGCTCACCAAGACTTACACA 3' (SEQ ID NO.:1);
5' GAATCACTGAGCAGCTGAAC 3' (SEQ ID N0.:2);
5' CAGTACCAGAACCGAGTTCG 3' (SEQ ID N0.:3);
5' CTGGTTACCAGGCTTGAGAG 3' (SEQ ID N0.:4);
~' TTACTGCGGAATCTCACAGC 3' (SEQ ID NO.:S);
5' TACACTGTTCAACCCAGGGT 3' (SEQ ID N0.:6);
*Trade-mark ", WO 96!10646 ~ ~ ~ ~ ~ PCT/EP95I03918 5' AAACAAGCCCTCTCACTGGT 3' (SEQ ID N0.:7);
. - 5' GCGGAGACTGTGCTGATAAG 3' (SEQ ID N0.:8);
5' CATACCTCTCTACATGGCAT 3' (SEQ ID N0.:9);
' S' AGTCTCGCTGAATCTCTCTT 3' (SEQ ID NO.:10);
5' TTACAACTAGCATGGTGCCC 3' (SEQ ID NO.:11).
' Amplification conditions were 94 C for 7 min followed by 95 C for 1.5 min, 56 C (intron 8) or 58 C (intron 16) for 1 min and 72 C for 1.5 min for 35 cycles in an OmniGene thermal cycles (Marsh Scientific). Amplification was performed in a total volume of 25 ~tL containing 1.5 mM MgCl2, and 2 U Taq polymerise (Boehringer Mannheim). The PCR products were subcloned and sequenced as above.
Verification of Mutations The putative mutations detected in the PCR products were verified at the genomic level in both cases. For this purpose, a search for a potential change in restriction endonuclease sites as a result of the mutation was peuormed.
Amplification conditions were 94 C for 7 min followed by 94 C for 1 min, 58 C for 45 sec and 72 C for 45 sec for 35 cycles in an OmniGene thermal cycles (Marsh Scientific). PCR products were analysed on 2.5% agarose gels.
The methods described allow for the screening of patients for mutations in the ~2 chain which will cowelate with JEB. As demonstrated, the results have identified a homozygous point mutation resulting in exon skipping, and a heterozygous deletion-insertion mutation. This demonstrating the effective screening for, and identillcation of, y2 chain mutations which correlate with JEB.
The methods are thus useful for diagnosis, prenatal screening, early screening and detection, as well as detailed examination of JEB. Further, the results show that the functional role of ~2 chain expression in epithelial cells is important in determining proper intercellular connectivity, relating to the integrity of tissues and cell interactions.
Example 2~ Mutation in the ~2 Chain Gene LAM 2 is Critical inH-.TEB
The correlation both i~z vivo and in viwo of the dermo-epidermal separation in H-JEB, with deficient immunoreactivity of anti-nicein/kalinin/laminin 5 antibodies, and the separation induced by anti-nicein/kalinin/laminin 5 antibodies have made the genes encoding this protein strong candidates for the site of H-JEB
mutations. In this example, it is demonstrated that the molecular defect which causes H-JEB is linked to the gene encoding nicein/kalinin/laminin 5 ~2 chain.
In 76909-228 (S) particular, the occurence of a homozygous premature termination codon mutation is the specific cause in an examined case of H-JEB (Aberdam et al.; Nature Genetics, 1994, 6:299-304).
Expression of mRNA encoding the three nicein subunits by northern analysis of RNA isolated from primary keratinocyte culture of a H-JEB patient was determined as the initial screen. Hybridization with probes for the oc3 and Q3 subunits was normal, but no hybridization with a cDNA encoding the ~2 subunit was detected. Examination of the genomic DNA for gross abnormalities, such as large deletions, insertions or rearrangements, in LAMC2 (the YI subunit gene) by Southern blot analysis turned up no abnormalities when the genomic DNA was digested with BamHI, BgII, HindIII, PstI or PvuII and probed with full length LAMC2 cDNA.
Possible mutations in the y2 subunit were sought by using cDNA reverse transcribed from total RNA purified from cultured keratinocytes of the H-JEB
patient, and subjected to PCR amplification. The size of the ampl~ed products was checked by electrophoresis on 2% agarose gels and compared with that obtained from healthy controls.
No major differences were detected in the agarose gels, and the PCR
products were examined by heteroduplex analysis (MDE). Heteroduplex analysis of the most 5' PCR product (nt 35-726) revealed the presence of a homoduplex in the proband (pateint) and the controls. However, when the amplified PCR
products from the patient and control were mixed together, an additional band with altered mobility, representing heteroduplexes, was detected, suggesting a homozygous mutation in the patient's LAMC2 cDNA . This amplified fragment corresponded to domain V of the ~2 protein (Vailly et al., Eur. J.
Biochem.,1994, 219:209-218). Sequencing detected a C to T transition at position +283, leading to a nonsense mutation in which a termination codon TGA replaces an arginine (CGA), perhaps arising as a result of the hypermutability of 5-methyl-cytosine to thymine at CpG nucleotides. This mutation, 895X, leads to wncation of the 'y2 subunit polypeptide at amino acid 95 and loss of a TaqI restriction site (TOGA). Digestion of cDNA with TaqI confirmed the presence of a homozygous mutation in the DNA of the H-JEB patient. No other mutations were detected.
To confirm the cosegregation of the mutation with the loss of the TaqI
restriction site, eight genotyped individuals of the family of the patient were screened. In each case, a 120 base pair fragment was amplified by PCR using genomic DNA templates and primers flanking the restriction site. Upon digestion of the wild type amplification product, two clevage fragments of 80 and 40 base 76909-228(S) pairs are generated. Consistent with the presence of a heterozygous mutation in carriers of this genotype. DNA fragments of 120, 80 and 40_base pairs, indicative of a wild type genotype, were found in the paternal grandmother and two other relatives.
Cell Culture Epidermis was separated from detmis by dispase treatment at 37 C.
Keratinocytes were dissociated in 0.25% trypsin at 37 C and plated onto a feeder layer of irradiated mouse :1T3 cells (ICN) (Rheinwald & Green. Cell. 175, 6:331-334). Keratinocytes were grown in a 1:1 mixture of DMEM and Ham's F12 (BRL) containing 10% Fetal Calf Serum (FCS), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 ug/mL of penicillin and strptomycin, 10 ng/mL transferrin, 180 ~tM
adenine and 20 pM T3 (Simon & Green, Cell, 1985. 40:677-683). H-JEB
keratinocytes were expanded after gentle dissociation in 0.05 trypsin, 0.02°k EDTA.
Northern Blot Analysis Total RNA was prepared from H-JEB and normal cultured keratinocytes according to standard methods (Chomzynski & Sacchi, Anal. Biochem. , 1987, 162:156-159). RNA was electrophoresed in 1.2% denaturing agarose gels *
containing 1.2 M formaldehyde and transferred onto Hybond N membrane (Amersham). Membranes were hybridized at high stringency with P-32 labeled cDNA probes corresponding to the different chains of nicein, and then exposed on Hyperfilm MP (Amersham) with intensifying screens. Radiolabeled cDNA probes NA1, ItAL-5.5C, and PCR 1.3 (Vailly et al., 1994, supra.), were used to detect the mRNAs for nicein chains a3, (33 and y2, respectively.
RT-PCR and heteroduplex analysis (MDE) 50 ~tg of total RNA isolated from cultured keratinocytes from JEB patient, and unrelated healthy controls were reverse transcribed in a volume of l001tL
as recommended by the manufacturer (BRL). 1 ~tL of the reaction product was used to amplify overlapping regions of the cDNA that spanned the open reading frame.
Primer pair used to identify the mutation R95X: (L) 5'-GAGCGCAGAGTGAGAACCAC-3', (R) 5'-ACTGTATTCTGCAGAGCTGC-3'. PCR cycling conditions were: 94 C, 5 min, followed by 94 C, 45 sec; 60 C.

sec; 72 C, 45 sec; for 35 cycles, and extension at 72 C for 5 min. 5 ~tL
aliquots *Trade-mark 76909-228 (S) were run in 2% agarose gels. Heteroduplex analysis was performed as recommended by the manufacturer (MDE, AT Biochemicals). Heteroduplexes were visualized under UV light in the presence of ethidium bromide and photographed. Amplified cDNA fragments with altered mobility were subcloned into the TA vector according to the manufacturer's recommendations (Invitrogen).
Sequence analysis were then performed using standard techniques.
PCR reactions on genomic DNA (50 ~tg) were carried out using -the upstream primer 5'-TTCCTTTCCCCTACCTTGTG-3' and the downstream primer 5'-TGTGGAAGCCTGGCAGACAT-3', which are located in the intron 2 and exon 3 of LAMC2 respectively. PCR conditions were: 95 C, 5 min, followed by 94 C. 45 sec; 56 C, 45 sec; 72 C. 45 sec; for 35 cycles, and extension at for 5 min. PCIt products were used for restriction analysis. 20 ~tL of PCR
product obtained from genomic DNA was digested with TaqI for 2 hours (Boehringer Mannheim). Clevage products were electrophoresed (2.4°Xo agarose) stained and visualized under UV light.
Thus the methods allow for the screening of patients for mutations in the 'y2 chain which correlate with H-JEB. As demonstrated, the results have identified a nonsense mutation resulting in a wncated ~2 chain, leading to severe H-JEB.
This was futher confirmed by specific amplification and restriction enzyme analysis of both the patient and relatives. Thus demonstrating the effective screening for, and identification of, y2 chain mutations which correlate with H-1EB. The methods are thus useful for diagnosis, prenatal screening, early screening and detection, as well as detailed examination of H-JEB.
Furthermore.
the results demonstrate the significance of the yZ chain in forming proper cellular contacts:
.xa rle 3: y~Chain as Diagnostic for Invasive Tissues In this example, in siru hybridization is used to demonstrate the expression of the kalinin/laminin 5 ~(1 chain in a variety of human cancer tissues and in skin wound healing in mice (Pyke et al., Amer. J. Pathol., Oct 1994, 145(4):1-10).
Thirty-six routinely processed, formalin-fixed and paraffin wax-embedded 35- specimens from cancer surgery performed from 1991 to 1993 were drawn from pathology department files at Herlev Hospital (Copenhagen. Denmark). The specimens were evaluated according to standard criteria and included 16 cases of 76909-228(S) moderately or well-differentiated colon adenocarcinomas. 7 cases of ductal mammary carcinomas, 4 squamous cell carcinomas (2 skin, 1 cervix, 1 vulva), 3 malignant melanomas, and 6 sarcomas (3 leiomyosarcomas, 2 malingnant fibrous histiocytomas, 1 neurofibrosarcoma).
All samples were selected upon histological examination of a hematoxylin and eosin-stained section to ensure that they showed a well preserved morphology throughout and contained representative areas of both cancerous tissue and surrounding apparently normal, unaffected tissue. The broad zone separating these two tissue compartments is referred to as the invasive front in the following.
No estimation of the effect of variations in fixation conditions was attempted, but in. a previous study of plasminogen activating system components using specimens of colon adenocarcinomas collected using the same procedures, very little variation in relative mRNA levels was found (Pyke, C. PhD. Thesis, 1993, University of Copenhagen, Denmark). In addition, tissue from incisionally wounded mouse skin prepared as described by Romer et al. (J. Invest. Dermatol. , 1994, 102:519 522), was fixed and paraftin-embedded the same way as the hum:u~ cancer specimens.
For preparation of total RNA from six samples of colon adenocarcinomas.
tissues were snap-frozen in liquid nitrogen immediately following resection and RNA was prepared.
Fragments of the cDNA for the y2 chain of human kalinin/laminin 5 was inserted into RNA transcription vectors by restriction enzyme cutting of clone L 15 covering base pairs 2995 to 3840 (Figure 4; Kallunki et al.. 1992, supra.
). In brief, plasmids phb2t-O1 and phb2t-02 were prepared by insertion of the complete L15 ~1 chain cDNA in sense and anti-sense orientation into the polylinker of plasmid vectors SP64 and SP65 (both Promega. Madison, WI), respectively. In addition, two non-overlapping fragments of clone L15 were bluntend cloned into the EcoRV-site of pKS(Bluescript)II(+) (Stratagene, La Jolla, CA) transcription vector and the resulting plasmids were verified by dideoxy sequencing according to Sanger et al ( PNAS( USA), 1977, 74:5463-5471 ). Plasmid phb2t-03 cover bases 3003-3239 and phb2t-OS cover bases 3239 to 3839, numbers referring to cDNA
sequence 215008 in the EMBLJGenBank/DDBJ database as reported by Kallunki et al., ( 1992, sups a. ; Figure 4).
Similarly, cDNA fragments of other human laminin chains were prepared in RNA transcription vectors, yielding the following plasmid constructs (numbers *Trade-mark 76909-228(S) in brackets refer to base pair numbers in the EMBL/GenBank/DDBJ sequence database by the listed accession numbers); chain al: plasmid phae-O1 (3244-(accession No. X58531, Nissinen et al., Biochem. J., 1991, 276:369-379) in pKS(Bluescript)II(+)); chain X31: plasmid phble-O1 (3460-4366 (accession No.
J02778, Pikkarainen et al., J. Bio1 Chem., 1987, 262:10454-10462) in pKS(Bluescript)II(+)); chain y1: plasmids A1PSP64 and A1PSP65 (919-1535 (accession No. M55210, Pikkarainen et al., J. Biol. Chem., 1988, 263:6751-6758) in SP64 and SP65 repectively (sense and anti-sense orientation)).
All plasmids were linearized for transcription using restriction endonucleases and 5 ltg of the linearized plasmids was extracted with phenol and with choloroform/isoamyl alcohol (25:1), precipitated with ethanol, and redissolved in water. Each transcription reaction contained 1 ~1g linearized DNA
template, and transcriptions were performed essentially as recommended by the manufacturer of the polymerises. The RNA was hydrolyzed in 0.1 mol/L sodium carbonate buffer, pH 10.2, containing 1U mmol/L dithiothreitol (DTT) to an average size of 100 bases. RNA probes transcribed from opposite strands of the same plasmid template, yielding sense and anti-sense transcripts, were adjusted to 1x106 cpm/~tL and stored at -20 C until used. Probes were applied to tissue sections.
In situ Hyhridi~ation:
In situ Hybridization was performed as described by Pyke et al., (Am. J.
PathoL , 1991. 38:1059-1067) with S35 labeled RNA probes prepared as described above. In brief, paraffin sections were cut, placed on gelatinized slides, heated to 60 C for 30 minutes, deparaffinized in xylene, and rehydrated through graded alcohols to PBS (0.01 mol/L, sodium phosphate buffer, pH 7.4, containing 0.14 mol/L NaCI). The slides were then washed twice in PBS, incubated with S ~tg/mL
proteinase K in 50 mmol/L Tris/HCI, pH 8.0, with 5 mmol/L EDTA for 7.5 minutes, washed in PBS (2 minutes), dehydrated in graded ethanols, and air-dried before the RNA probe (-80 pg/pL) was applied. The hybridization solution consisted of deionirxd formamide (50%), dextrin sulfate (10%), tRNA (1 ~tg/~tL), Ficoll 400 (0.02% (w/v)), polyvinylpyrrolidone (0.02% (w/v)), BSA fraction V
(0.02% (w/v)), 10 mmol/L DTT, 0.3 M NaCI. 0.5 mmol/L EDTA. 10 mmol/L
Tris-HCI, and 10 mmol/L NaP04 (pH 6.8). Sections were covered by alcohol-washed, autoclaved coverslips and hybridized at 47 C overnight (16 to I8 hours) in a chamber humidified with 10 ml of a mixture similar to the hybridization solution, except for the omission of probe, dextrin sulfate. DTT, and tRNA
*Trade-mark ~WO 96/10646 a ~ ~ ~ ~ ~ ~ ~ PCTIEP95/03918 (washing mixture). After hybridization, slides were washed in washing mixture for 2 x 1 hour at 50 C, followed by 0:5 mol/L NaCI, 1 mmol/L EDTA, 10 mmol/L
Tris-HCl (pH 7.2) (NTE) with 10 mmollL DTT at 37 C for 15 minutes. After ' treatment with RNAse A (20 ~tg/mL) in NTE at 37 C for 30 minutes, the sections were washed in NTE at 37 C (2 x 30 minutes), and in 2 L of 15 mmol/L sodium ' chloride, 1.5 mmol/L sodium citrate, pH 7.0, with 1 mmol/L DTT for 30 minutes at room temperature with stirring. Sections were then dehydrated and air-dried.
Finally, autoradiographic emulsion was applied according to the manufacturer's reccomendations, and sections were stored in black airtight boxes at 4 C until they were developed after 1 to 2 weeks of exposure.
Results : Laminin oc 1 R1 y1 andw2 chains All rounds of in sitccr~ hybridization include both sense and anti-sense RNA
probes for each of the genes studied. As negative controls, sense RNA probes are applied to adjacent sections and these probes consistently are negative. As a positive control of the ~2 chain hybridizations, two anti-sense probes derived from non-overlapping'y2 chain cDNA clones are used on a number of sections. To summarizes the y2 chain expression found; all carcinomas were positive except for one case of mammary duct carcinoma, and all three cases of leiomyosarcomas, both cases of malignant fibrous histiocytoma, and the only case of neurofibrosarcoma. The positive controls always give similar staining on adjacent sections (see Figure 2, E and G). Fifteen of the malignant cases and all mouse tissue blocks were hybridized on two or more separate occasions giving the same hybridization pattern. All cell types other than those described below were negative in all cases.
Colon Adenocarcinoma Sixteen specimens of colon adenocarcinoma were investigated by in situ hybridization for expression of the ~2 chain (Figure 1 ). In all of these cases, mRNA for yZ chain was present exclusively in cancer cells and in most of the cases, staining was confined to a distinct subpopulation of cancer cells at the invasive front (Figure 1, A-D). A characteristic feature of y2 chain containing cancer cells at the invasive front was that they appeared to represent cells in the process of branching or dissociating from larger well differentiated epithelial glands, a phenomenon referred to in the literature as tumor budding or tumor-cell dissociation.

WO 96/10646 PGT/EP95/03918~
In normal-looking colon mucosa distal from the invasive carcinoma, moderate signals for y2 chain mRNA were observed in two specimens in the epithelial cells of a few mucosal glands that showed clear morphological signs of glandular disintegration and phagocytic cell infiltration. Apart from this, a weak signal was seen in luminal epithelial cells in normal looking colon mucosa in most specimens.
Weak signals for laminin chains a1,131, and y1 mRNAs were detected in cancerous areas of the 6 colon cancers studied for the expression of these genes.
The expression of each of the three genes showed a similar distribution.
Expression in stromal cells with a fibroblast-like morphology as well as in endothelial cells of smaller vessels was consistently found . In marked contrast to the ~2, chain expression in the same samples, expression of a 1,131, or y1 was never found in cancer cells and no correlation between expression of a1,131, and y1 chains with sites of invasion was found. Adjacent normal-looking parts of the samples were negative or only weakly positive for these laminin chains.
Figure 1 shows In situ hybuidization of a specimen of colon adenocarcinoma for ~yf? chain mRNA using a S-35 labeled anti-sense RNA probe derived from plasmid pbb2r-02. Figure 1A is a cluster of heavily labeled cancer cells at the invasive front (open an-ow) in close proximity to a well-differentiated glandular structure (straight arrow). Figure 1B shows a high-magnification view of the area at the open arrow in 1 A. Note that the isolated cancer cells show prominent labeling, whereas many coherent cancer cells of an adjacent glandular structure are negative (straight arrow). Figure 1C shows the same pattern at an invasive focus in another part of the same specimen. Figure 1D shows strong ~2 chain expression in-cancer cells engaged in a bifurcation process (curved arrows).
The malignant glandular epithelium from which the ~L chain-positive cancer cells are branching is negative (straight arrow). Magnification:lA x 100; 1B-1D x 640.
Ductal Mammarv Carcinomas Six of the seven cases showed a prominent signal for y2 chain in a small subpopulation of cells intimately associated with invasively growing malignant glandular structures. The most prominent signal was seen in cells located at the border between malignant and surrounding sti~omal tissue in glandular structures that exhibited clear histological signs of active invasion (Figure 2A). On careful examination it was concluded that the majority of the positive cells were cancer cells but it was not possible to determine if the cells of myoepithelial origin were WO 96/10646 ~ ~ ~ ~ ~ ~ PCT/EP9~/03918 also positive in some cases. One case was totally negative. Normal-appearing glandular tissue was negative in all cases.
Weak signals for laminin chains a1,131, and y1 mRNAs were detected in fibroblast-like stromal cells throughout cancerous areas in one of the cases..
Malignant Melanoma In all three cases strong hybridization of y2 chain was found in a population of cancer cells.in the radial growth phase (Figure 2B). Laminin chains a1,131, and y1 were weakly expressed in the endothelium of small vessels and in fibroblast-like stromal cells throughout the affected areas in the two cases studied for these components. In addition, a weak signal for these chains was seen in sebaceous glands of adjacent normal skin.
~Squamous Cell Carcinoma In all four squamous cell carcinomas investigated, the same pattern of ~2 chain expression was found as in other carcinomas. The signals were found only in cancer cells, and only in areas with signs of ongoing invasion (Figure 2, C-G).
The four cases were also studied for mRNA of a1,131, and y1 chains. In the two skin cancers, it was found that a very weak signal occured in malignant cells, and that the weak signal was in all cancer cells and of an equal intensity.
This is in clear contrast to the pattern of expression of the y2 chain. As seen in melanomas, epithelial cells of sebaceous glands present in adjacent unaffected skin were weakly positive for these laminin chains. In the other two cases (cervix and vulva) weak expression of a1,131, and y1 chains were seen only in endothelial and libroblast-like stromal ells throughout the cancerous areas (Figure 2F).
Figure 2 shows In situ hybridization for ~2 chain mRNA on sections of duetal mammary carcinoma (2A), malignant melanoma (2B), squamous cell carcinoma of the skin (2C-2D), and squamous cell carcinoma of the vulva (2E-2G). In 2A, cancer shows prominent signal for ~2 chain mRNA in cells bordering the zone between malignant glandular tissue and surrounding mesenchyme (curved avows). Cancer cells located more centrally in individual malignant glandular structures are negative for ~2 chain mRNA (straight arrows). Note the wedge shaped form of the invading glandular tissue. (All images marked X' are darkfield images of the respective sections). Figure 2B shows 'y2 chain mRNA
signal in a subpopulation of cancer cells of radially growing malignant epithelium (curved arrows). Adjacent malignant epithelium showing a different growth pattern is devoid signal (straight arrow). Figure 2C shows y2 chain mRNA

WD 96/10646 _ n ~ ~ PCT/EP95/03918 containing cancer cells at the invasive front (curved arrow). Note lack of signal in non-invasive areas of the tumor and in adjacent unaffected areas (straight anow).
Figure 2D is a higher magnification of area of curved arrow of 2C highlighting the prominent signal in invading cells (curved arrow). Adjacent cancer cells with tumor 'islets are negative (straight arrow). Figure 2E shows a strong signal for ~Z
chain mRNA is seen in invading cancer cells, using an anti-sense RNA probe derived from plasmid pb2t-03 (curved arrow). A postcapillary venule is negative (straight arrow). Figure 2F is a near adjacent section hybridized for laminin y1 chain. Note that the endothelial cells of the venule show signal (straight arrow) whereas the malignant epithelium is negative (curved awow). Figure 2G is another near-adjacent section which was hybridized for ~2 chain expression using an anti-sense RNA probe derived from a cDNA plasmid non-overlapping with that used for preparing the probe in 2E (phb2t-OS). Note that the hybridization patter is similar to that seen in 2E, with strong signal in invading cancer cells (curved arrow) and absence of signal in a vessel (straight arrow). Magnification: 2C x 100, all others x 640.
Sarcomas All six sarcomas tested in the study were totally negative for ~2 chain mRNA. The expression of other laminin chains was not done.
Mouse Wounded Skin .
To compare the gene expression of y2 chain in cancer tissue with a nonmalignant condition known to contain actively migrating epithelial cells showing a transient invasive phenotype, we hybridized sections of incisionally wounded mouse skin with ~2 chain sense and anti-sense RNA probes. Weak y2 chain expression was observed in the keratinocytes at the edge of 12-hour old wounds, and at later time points (1-5 days), strong signals for ~2 chain mRNA
was seen exclusively in the basal keratinocytes of the epidermal tongue moving under the wound clot (Figure 3). In adjacent normal-looking skin, keratinocytes were negative for y2 chain mRNA.
Figure 3 is incisionally wounded mouse skin (72 hours after wounding) showing signal for y2 chain in keratinocytes at the leading edge of the migrating epithelium (curved arrow). Whereas buccal keratinocytes located more distant to the site of injury show little or no signal (straight arrow). Note that the signal for ~L chain stops at the tip of invading keratinocytes (open arrow). A' is a dark field image of 2A. Magnification: x 640.

.~ . 2 2 ~ .~ $ 6 ~ PCT/EP95/03918 RNAse Protection Ass~Y
Plasmid phbt-03 was linearized with EcoRI and a radiolabeled RNA-anti-sense probe was prepared by transcription using P-32 UTP and T3 p~lymerase (Pyke et al., FEBS Letters, 1993, 326:69-75). RNAse protection assay, using 40 p.g ethanol-precipitated and DNAse I-treated total RNA from six samples of colon adenocarcinomas was performed as described in Pyke et al., (1993, sccpra.).
Protected mRNA regions were analyzed on a denaturing polyacrylamide gel and autoradiography.
The RNAse protection assay carried out on total RNA from the six samples confirmed the presence of genuine ~2 chain mRNA in all samples.
These results clearly demonstrate the important correlation of y2 chain expression and invasive cell phenotype in vivo, as detected In vrtro. Thus the instant methods present a novel and important method for the specific identification of invasive cell phenotypes in biopsied tissues. The knowledge of any information diagnostic for the presence or absence of invasive cells is useful for the monitoring and prognosis of continuing anti-carcinoma therapies.
Further the identification of the expression or non-expression of the ~2 chain provides important information as to the phenotypic nature of the tissue examined. Thus the instant example demonst~~ates the use of probes of y2 chain for detection of the presence, or absence, of invasive cells.
Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and all other equivalents are intended to be encompassed by the following claims.

WO 96/10646 ~ ~ ~ ~ ~ ~ ~ PCT/EP95/03918~
Ezamnle 4- inhibition of metASfisis via LRruinia v2 .-t,~«~
Background In this prospective example, a method for iz~fervcntion of mctasiisis and invasive cell activity via the Iaminin y2 chain is described_ Inhibition of fhe functional expression of fhe Iaminin y2 gene in cancerous cells is predicted to be an elfeciive means of inhibiting the invasive growth of such cells.
One approach for inlu-bition ofthe functional expression of the y2 gene urouId be to use antisense oligonuclcotides. The art of antisense I0 oligouucleotides is generally knouin (see gener.~y ~tisense Research and Applications_ ed_ Crooke & Lebleu, CRC Press, Ann Arbpr,Ml, T 993).
Since Zamccnik and Stephenson, Prac. lYatl cad Sci USR 75, 284-284 (1978), first demonstrated virus repltc~.tion inhibition by synthetic oligonucleotides, there has been much interest in the use of antisense IS oligonucleotides as agents for the selective modulation ofgene expression, f °tl' In YiL'o and in viva See, a ~g , A,grawal, ?-rends In Biotech I0, l 52 (/992); Chang and Pent, Prog BiopJrys: ~t~tole~ Biol. 58, 225 (1992); and UhImann and Peymann, Chem. Rev 90, 543 (1990). Antisense oliganucleotidcs arc constructed to be sufficiently complemen~y to a target ZO nucleic acid to hybridize with the target under the conditions of interest and inhibit expression of the target_ Antisense oligonucleotides may be designed to bind directly to DNA (the so-called uanti gene" approach) or to viral RNA or mRNA. Id Expression inhibifion is believed to occur by interfering With transcziption processing or translation, or inducement of target mRNA
25 cleavage by RNase FI»
Antisense oligonucleotides can be used as research toots in vitro to determine the biological function ofge~nes and proteins. They provide an easily used alternative to the Iabotious method of gene mutation (eg., deletion mutation) to selectively inhiibit gene expression. The importance of this method 30 is readily appreciated when one realizes that the elucidation of most known SUBSTITUTE SHEET (RULE 26) WO 96/10646 ~ 2 ~ '~ ~ ~ PCT/EP95~03918 biological processes has been determined by deletion mutation.
Antisense oligonucleotides also may be used to treat a variety of pathogenic diseases by inhibiting gene expression.of the pathogen in vivo.
Oligonucleotide phosphorotluoales (PS-oligos) have shown great therapeutic S potential as antisense-mediated inhibitors of gene expression (Stein and Cheng, Science 261, 1004 (1993) and references therein) as evidenced by a number of ongoing clinics! trials against AIDS and cancer. Agrawal and Tang, Aniisense Res cardDev 2, 261 (1992) and references therein, and Bayever et al., Antiserzre Res Dev 3, 383 (1993)_ Various methods have been developed for the synthesis of oligonucIeotides for such purposes. See getrerally, Methods in Molecular Biology. Yol. Z0: Protocols far Oligorrucleotides cmd Analogy: pp.
165-189 (S. Agrawal, .Ed., Humans Press, 1993j; Uligonucleotides cntd Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991 ); and Uhlmann and Peyman, supra. The phosporamiditc method (and variations thereon) is the most commonly used method of oligonucleotide synthesis. E.g:, Beaucage inMethods in Molecular Biology, Yol. 20, supra, pp. 33-6I; and Beaucage and Iyer, ~'eirahedron 48, 2223 (1992).
Animal Model S~rstem and Protocol In this application, antisense to the y2 gene will be administered to mice which have been inoculated with metastasizing cancer cells. In particular, it can be demonstrated that a mouse innoculated with a mouse colon cancer cell line which metastisizes to the lungs and liver exhibits expression of Iaminin y2, a_s detected by immano-histochemical staining of invasive tissues and metastisized tumors.
Nfice can be injected intraperitoneally (i_p.) or intramuscularlly (i.m_) with cultured marine colon cell line that has the ability to metastisize_ After a period of several weeks, the animal is sacraficed and the tissues examined for the expression of laminin y2.
As an initial study, such tumor cells can be transfected with plasmid SUBSTITUTE SHEET (RULE 26) R'O 96/10646 ~ ~ ~ ~ ~ PCT/EP95/03918~
containing an expression vector which generates anti-sense y2 messenger RNA, which can bind with any endogenously produced native y2 messenger RNA and thereby inhibit the translation and expression of y2 protein.
Examination of the ability of these transformed cells to metastasize, and the pattern of y2 expression will be examined.
In further experiments, other modes of delivery of stabilized and un-stabilized anti-sense y2 can be administered by many acceptable routes to demonstrate the efficacy of administering anti-sense y2 as a pharmaceutical for the inlu'bition of cancer cell metastasis. The teachings of the instant invention have clearly taught the means for identifying the effective target cancer phenotypes for such treatment.
To examine pertubation of the y2 protein, the tumor cells can be pre-treated with antibodies directed to the y2 protein, to inhibit the activity of the y2 chain protein in its functional role in tumor cell metastasis.
IS
Those skilled in the art will know, or be able to ascertair~ using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These and aI( other equivalents are intended to be encompassed by the following claims.

SUBSTITUTE SHEET (RULE 26)

Claims (33)

CLAIMS:

1. ~A method for detecting the presence of invasive cells in tissue comprising detecting a signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains (i) a sense or antisense portion of kalinin/laminin 5 gamma-2 chain nucleic acid set forth in Figure 4A, or (ii) a nucleic acid that specifically hybridizes to the nucleic acid of (i).

2. ~A method for monitoring the presence of invasive cells in tissue comprising detecting a signal or absence of signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains (i) a sense or antisense portion of kalinin/laminin 5 gamma-2 chain nucleic acid set forth in Figure 4A, or (ii) a nucleic acid that specifically hybridizes to the nucleic acid of (i).

3. ~A method for detecting t:he presence of invasive cells in tissue comprising detecting a signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains a sense or antisense portion of a nucleic acid encoding kalinin/laminin 5 gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A.

4. ~A method for monitoring the presence of invasive cells in tissue comprising detecting a signal or absence of signal from the tissue assayed, such signal resulting from specifically hybridizing the tissue with an effective amount of a nucleic acid probe, which probe contains a sense or antisense portion of a nucleic acid encoding kalinin/laminin gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A.

5. ~The method of any one of claims 1 to 4 where the nucleic acid probe is DNA.

6. ~The method of any one of claims 1 to 4 where the nucleic acid probe is RNA.

7. ~The method of any one of claims 1 to 6 where the nucleic acid probe is radiolabelled, enzyme labelled, chemiluminescent labelled, avidin or biotin labelled.

8. ~The method of any one of claims 1 to 7 where the nucleic acid probe is derived from human kalinin/laminin 5 gamma-2 chain nucleic acid sequence.

9. ~The method of any one of. claims 1 to 8 where the nucleic acid probe is incorporated into an extrachromosomal self-replicating vector.

10. ~The method of any one of claims 1 to 8 where the nucleic acid probe is incorporated into a viral vector.

11. ~The method of any one of claims 1 to 10 where the nucleic acid probe is linear.

12. ~The method of any one of claims 1 to 10 where the nucleic acid probe is circularized.

13. ~The method of any one of claims 1 to 12 where the nucleic acid probe contains modified nucleotides.

14. ~A method for detecting invasive cells in tissue comprising detecting a signal frog. assayed tissue, such signal resulting from contacting tissue with an effective amount of a labelled probe, which probe contains an antibody specifically immunoreactive with a portion of kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.

15. A method for monitoring invasive cells in malignant tissue comprising detecting a signal from assayed, malignant tissue, such signal resulting from contacting tissue with an effective amount of a labelled probe, which contains an antibody specifically immunoreactive with a portion of kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.

16. The method of any one of claims 1 to 15 wherein the invasive cells comprise carcinoma cells.

17. The method of claim 16 wherein the invasive cells comprise carcinoma cells selected from the group consisting of colon carcinoma, mammary carcinoma, squamous cell carcinoma, and melanoma cells.

18. The method of any one of claims 14 and 15 wherein the antibody is a polyclonal antibody.

19. The method of any one of claims 14 and 15 wherein the antibody is a monoclonal antibody.

20. Use, for one or more of inhibiting invasive growth of a malignant cell, inhibiting tumor cell invasion of non-malignant tissue, inhibiting budding of tumor masses, and inhibiting tumor cell interaction with basement membrane, of a nucleic acid which contains an antisense portion of a nucleic acid encoding kalinin/laminin 5 gamma-2 chain, wherein the kalinin/laminin 5 gamma-2 chain encoding nucleic acid is set forth in Figure 4A, and wherein the antisense portion effectively inhibits the translation of endogenous kalinin/laminin 5 gamma-2 chain encoding mRNA.

21. The use of claim 20, wherein the antisense nucleic acid is an oligonucleotide which binds with mRNA transcribed from kalinin/laminin 5 gamma-2 gene.

22. The use of claim 20 wherein the antisense nucleic acid is expressed from a plasmid construct.

23. The use of claim 20 wherein the antisense nucleic acid is an exogenously administered oligonucleotide.

24. Use, in manufacture of a medicament for one or more of inhibiting invasive growth of malignant cells, inhibiting tumor cell invasion of non-malignant tissue, inhibiting budding of tumor masses, inhibiting tumor cell interaction with basement membrane, and evaluating extent of invasive cell activity in a patient, of an effective inhibiting amount of an antibody specifically immunoreactive with a portion of a kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.

25. Use, for one or more of inhibiting invasive growth of malignant cells, inhibiting tumor cell invasion of non-malignant tissue, inhibiting budding of tumor masses, inhibiting tumor cell interaction with basement membrane, and evaluating extent of invasive cell activity in a patient, of an effective inhibiting amount of an antibody specifically immunoreactive with a portion of a kalinin/laminin 5 gamma-2 chain protein, wherein the kalinin/laminin 5 gamma-2 chain protein is set forth in Figure 4A.

26. The use of claim 24 or claim 25 wherein the use is for inhibiting invasive growth of malignant cells, and wherein the malignant cells comprise carcinoma cells.

27. The use of claim 24 or claim 25 wherein the use is for inhibiting tumor cell invasion of non-malignant tissue, and wherein the tumor cells comprise carcinoma cells.

28. The use of claim 24 or claim 25 wherein the use is for inhibiting budding of tumor messes, and wherein the tumor masses comprise carcinoma cells.

29. The use of claim 24 or claim 25 wherein the use is for inhibiting tumor cell interaction with basement membrane, and wherein the tumor cells comprise carcinoma cells.

30. The use of claim 24 or claim 25 wherein the use is for evaluating the extent of invasive cell activity in a patient, and wherein the invasive cells comprise carcinoma cells.

31. The use of any one of claims 26 to 30 wherein the carcinoma cells are selected from the group consisting of colon carcinoma, mammary carcinoma, squamous cell carcinoma, and melanoma cells.

32. The use of any one of claims 24 to 31, wherein the antibody is a polyclonal antibody.~

33. The use of any one of claims 24 to 31, wherein the antibody is a monoclonal antibody.