AU2008202039A1 - Gene encoding labyrinthin, a marker for cancer - Google Patents

Description

S&F Ref: 521740D2

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: The following statement is a full performing it known to me/us: ImmvaRx, Inc., of 7135 Sentinel Rd, Rockford, Illinois, 61107, United States of America James A. Radosevich Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Gene encoding labyrinthin, a marker for cancer description of this invention, including the best method of 5845c(1228072_1) 00 ~The invention el~ to a gene encoding a protein and peptid= dhcrefrOM that includes an epitope, a C a~r assiated antigen, useWu as a marker thAt is not restrcted to previously def tned histological classes of canter. Antigernic peptides are useful as a vaccine foLt ent end preveation of cancer, and for the prepat~ion of CI ~new, specific, xnonoclor4liatibodime Antiscense mnoleculges are useful in 00 pharm~acetical composin. and are useful for diagnosis and teatment N BACKGOROUJND OF 4 I WDNTION Cancer ('cenccr is, 'a malignan turnor," wherein 4 "tumor" is an abnorrmal mass of tissue, that need not be 4=4nnt "neoplasmo" is a form, of new growth) is a leading of denth in mena azdwonten throutghout the world. In the Uaftod Sta~es alone, over I million new cases are c~agased each year, and over 0.5 million deaths are reported annually (Landislet al., 1998). Historically, tumnors are grouped and treazed, based in part by the tissutes in which they arise for example, breast cancer, colon cancer, and lung cancer, oihd the like. Yet. within lung cancer, for example, it is wel r Iecognized thAt these t=&rs wrc a very heterogeneous group of neoplarns. This is 413o true for tumors arising in other tissues. la part, because of this heterogeneity, there are complex and inosistent classification schernes which are used for hurran tumo~s. Preyiou 4±terpito treat cancer have been harnpered by: the arbitrary classification of tumors anrisig within given tissues, and by using microscopic methods based on how the~e tumors look (histological classification). Although exi~sting classifications for various fl~unr types have some propos~c va[lve, almost all the classifications Wai to predict responsiveness to treatments and likelihood of cure or disease course. Improved classi~cation schemes based on the biological constitution.

of thes neoplasmas is req4red to 3ignificantiy alter the suvival statstics of humans who have cancer. Oe approach to solving these probce is to loczte molecules specifr to tmors, preferably antigens in molecules tbat are markers for cancer cells.

(A "marlcer" is de~ned he-Ire=' as any property which can be used to distinguish cancer fr normal tissues and 6=o other disease states.) The markers' presence is then a bm~is for cliaflcan 00-- Monroclonal antibodies (MCAs) prepared by somatic cell hybidzaon tecb~niqucs, usually irt mice, are useful molecular probes for the detection and discrmiation of cellular antigens, and therefore have grent potential for detecting cancer associated anigens. These antibodies bind to specific antigens and the binding Sis detectable by woll known tnetbods. When binding occurs, the inference is made that a specific 4adgea is present. 'Those cancer associated antigens which are exposed to the cell nurace or found in the cancer tna s, are molecuLr targets for the Cmmn N systems (including host antibodies) of the host Recent findings suggest that cancer 00 patients who have antibodies against their- umors, do better than those who do not maount ths type of imzrunc response (Livingston et al., 1994). Therefore, nattoral, N induced, or admninistered antibodies are a prmising therapeutic approach.

The hunzzation of aoa-huzrnan MCA-s (the process by which non-hartan MCA reactive sites are shuttled int 'o cloned humnan antibodies and expressed) results in reduced immunogenicity of the foreign antibodies without the loss of their specific is binding in in vivo and in ex vivo applications. MCAs can be used as in vivo imaging agents, diagnnotic tests, and for thcrany (Ra4dosevich er al., 1988, 1990; Rosen. er al., 1988).

Vaccine theapy is a well established approach directed at inducing an immune response without exposure to the causative agent of a disease or condition. Many vaccines are available, for exAnipIc, to utimulmt a respouns in a host to bacterial a=d viral agents. The use of unior associated antigens (markers) in a vaccine could prevent priary cancer occurtence, and could also provide a means to prevent recw~ence of the disease.

Gene therapy is a means by which the genetic make-up of cells is modified to express the gene of iaterest. There are n~y forms of Rene therapy including: ge replacement, andsense suppression therapy, and surrogate gene expression.

Discovering genes encoding cancer-associated, preferably cancer.'specjflc antigens (markers) opens the door to genetic intervention against cancer cell proliferation. The accurate and consistont use of a cancer marker to diff~erentiate cancerus from normnal tissue, not only baw diagnostic poteatial, but is also desirable for teatment and prognosis. Therefore, such markers have been sought.

Recent studies have shown thazt the enzyme encoding hun aspartyl betahydroxylase (HAAII) is overexpressed in some hurnan-adencarcjnorza cell lines, and 00 in primary hepatocellular cancers, therefore could be a muarker. ThCe gone maid to encode H-AAII has been cloned and seqmenced (Gronke et al., 1989, 1990; Wang er al., 1991; Tia etal., 1992, 1994; Korioth eta!., 1 9 9 4; Lavaissiereetal, 1996).

1-lowover, Little is knwn about 1{AAH eKcprssion in human timors in geana (Lavaissiere et al., 1996).

The study of the HAAI- enzymne grew out of the study of its bovine counterpar (Gronke et al., 1989, 1990; Wang oftal, 1991: JRa efta., 1992). Bovine N aspartyl beta-hydroxtylase is an hnitr~ellular, glycosylated protein, localized in the N0 rough endoplsmic reticulum. The protein has been reported to have three major species of =noecules; a 95 klodaltoa formt, and two activ forms with molecular ci ~weights of 56 and 52 kilodaltonw respectively (Lavaisgler et al., 1996).

Using standard biochernical methods, bovine asparryl beta-hyrdroxvlase (bAAII) has been purified and characterized (Gronke er 1990; Wang et al., 199 1).

The activity of the enzyre has been shown to be correlated with the 52 and 56 ldlodalton species which were purified.

Immunologically, a related higher mnolecular weight form (85-90 kilodalton) was also obsered. As part of the purification, bAAH is bound to Con A sepharose, wbich is consiptent with the conclusion that the enzyme is glycosylad. (Subsequent reports an the DNA sequence show three possible glycosylation sites, with one site being very close to the Imown active enzyrme domain.) The protein is very acidic in nature, and a detergcni 1is not required to solubilize the active ftauciion- The active enzyme site is dependetr from the biochemically isolated bovine protein (bAAH) on the presence of bistidinc at position 675 (hfa oral,, 1994).

A partial amino acid sequence was obtained for H'AA.H. DNA probes DNA probc is 4 molecule having a nudleotide seuence that is capable of binding to a specified nucleoodde sequence uadcr certain conditions) deduced from this amino acid sequence was used to screen a bovire cDNA librury (yin. er al., 1992). (A cDNA library coulaip.s the sections of DNA that encode for gene products for example, peptides as opposed to genontic DNA). Several overlapping cDNA sequences in the library contaimed as 764 amino acid open reading fr-ame (ORF) sequenc which will be expected to encode an g5 kilodalton protaiL Also present in, this ORF sequence were two other possible Ptart codons, that is, locations at which encoding begins. The most 3st=r codon was preceded by a. ribosome binding site. Translation of the clone 00 4 having this sequarice resulted in a prolein that was about 8:5 lcilodaltoym, AMtisenia was raised to the membrane fraction of human MG-63 calls and was used to irniunoscre a cONA library made from MG-63 cells. Data on one cr was reported which could encode a 757 amino acid protein, and, by seTuece analysis, was found to have strong N-terminal homology with bAAHI (Korioth cr al., 1994). When this clone was used in an in vitr~o t-aslation systemn (an artificial cocktail of nnnl cell cytoplasm used to conivert rnRNA into protein), EL 36 kilodalton protin was Nproduced. It was suggested that th~is was due to post:-=atiadonal cleaage.

ri The H-AAHi enzyme is responsible for the mnodification of speciflc aspartic 00 010 acid residu~es within the epidermal! growth factor-like domains of proteins. It has5 been hypothesized that these modified aspar~c acid residues allow the epidermal growti factor-like domains to become calcium binding domains. (Groake et al, 1989, 1990; Wang of al., 1991; 11a et 1992, 1994; Korioth etal., 1994; Lavaissiere et a., 1996).

An enzyme related to HAAH, 4spartyl beta-bydroxylase (AAa), was first studied because it spci~cvlly modified select aspartic acid or asparagine residues in a group of biologically important proteins including the vitamin K-dependent coagulation factors VII, EX, and X Other proteins hle C, S, and Z also have this modi~cation (Groake et 1989, 1990; Wang et al., 1991 Tia ct al.. 1992, 1994; Koiloth er al., 1994; Lavissiere ef al., 1996), Aspartic adid and asparagime residues have be.-n shown to be modified by HAAH in proteins containing epidermal growth factor-like domains, The function of the beta-hydraxyasparric and betahydroyaspaugire meidues is unknown, howcver, Ii as been speulated that this modification is required for calcium binding in the epidennal growth factor EGF .lie donminse of selected proteins.

Antibodies were raised to human hepatocellular carcinoma FOCUS cells (Lavaisaiere et al., 1990). Ons IMCA reacted with an antigen that wAs highly expressed in hepatacellular carcinomas (Lavaissiere st al., 1996). Immnoscreeag using this antibody and a lambda gt1 I I HepG2 library resulted in the isolation of pardal cD1NA, which was subsequeatly used to isolate a larger clone.

A human adence~inom a cell Lime designated A549 was reported as having very high levels of HtAAHI activity (Lavaissiere et 1996). A mouse monoclonal antibody designated MCA-44-3A6 Parent No. 4,816,402) was produced agai=s 00-5 the human aderocarcinoma cell line A549 (ATCC accession number CCL 185) (Rdsvich et al., 1985). The Antibody recogr~izd a cell surface, aon-alycosylated anfigenic protein having an estimated apparent m~olecular weight of 40 k.Da).

The antigen. was excpressed by A-549 cells, and was found to be v. good marker; that is, it was, frequcntly expresmd by cancers which looked like adenocarcizoomas when examined histologic~Ily (Radosevich. ea al., 1990a; Lee ef al, 1985). MCA 44-3A6 is uniqu~e in that it is the first monoclonal antibody which has this binding specificity. The results from an Internationa.1 Workshop for Lung Cancer confluncd other related published 5ndings on MCA 44-~3A6 (Stahel, 1994).

0010 The antibody designaed MCA 44-3A6 bas clinical tuility because it differentiatrs antigens emsociarc4 '~th adanocarcinomas. The normal and fatal tisme distribution of the antigez is restricted to some glandular tissues (Radosevich et ci., 1991). Detection can occur on formalin fixed-paraffibi embedded tissue (Radosevich at 1985, 1988, 1990a. 1990b; Lee et al., 1985, 1986; Pichi. etal., 1988; Combs et al., 1988b, 1988Sc; BanneT r al 1.98:5). The antibody has a restricted binding pattern withiahuman pulmonary tumors (Lee e al., 1985; Banner er al., 1985; Radosevich e, al., 1990a, 1990b).

In a study of over rwo hundred pulmonary cancers, MCA 44-3A6 was found to react with all of the -Adenocarainomas tested, many of the large cell carcinomas, as well as with subsets of intermediate neuroendocrina small cell lung cancers, well-.

differentiated newoendocrine sm4l cell carcinonta, carcinaids, but not mresctlleliornas. MCA 44-3A6 does not react with squamous cell carcinoma, bronchioloalveolpr carcinoma, or smal cell carcinoma (Lee er al, 1985). MCA 44- 3A6 is useful in distinguishing adenocarcinonms that are metastatic to the pleura from mesothelima. (Lee et al., 1986). The antibody has selected reactivity among aclenocarcinornas and in large call carcinomas (Piebi er al., 1988; Radasevich et a., 1990b).

In a study of over 40 cases of luzg cancer comparing cytological and histological findin&3, MCA 441-3A6 was found to be useful in cytological diagnosis and was consistent with the histological fiading (Banner at al., 1985). Histology is the study of tissues (which are made of cells). Cytology is the study of cells which have been removed from tam org nnization;.l context which is common referred io as tissue.

Cells removed fromn tissues do aot always behave tu., same as if they were in-the tissue 00 from which thty were derived. Fortunately, the antigen delected by .MCA 44-3A6 expressed in adenocarcinoma cells in tissue behaves in the same ways as adonocarcinornaz cells removed frorn tissues. This is a very div.postically important chcteitic. Siniiar correlations using cytologically prepared cell blocks of puiraona~y cnouas. as well as AC3 presenting in body fluids from other sites throu~ghout tht body were demoristrated (Lee et al,, 1985; Spagnolo et al., 1991; Combs or al,, 1 998c), Also, MCA 44-3A6 binds to adenocaxcinomas from, sites other tbim lung cancer, The expresson of tlhe antigen in primary and metastatic lesions was ri also reported (Combs et al., 1988a). The utility of the MCA antibody in 00 010 diffrentiating cancer from benigni lesions in human bremst tissue was also noted (Duda el' al., 199 1).

The cellular localization of the antigen detected by MCA 44-3A6 was determined. By using live cell radioimmunoasays (a radioactive antibody test directed at drteining binding of the antibody to live cells), imuncoluorescence, and live cell fluorescance activated cell sorter (FAGS) analysis, the antigen detected by MACA 44-3A6, was shown to be on the outside surface of the cell (Radosevich er al., 1985). Additional studies using immunmogold-electron microscopy and FACS analysis bave demonstrated that this antigen is non-modulated (that is not interalized by the cancer cell when bound by in antibody), is expressed on. the extracellular surface of the pla1n nebrane, and is not cell cycle specific that is, the cell makes prnteizn all the tie it is going through the process of cell replication, and also whten it is not dividing CRadosevich et 199 The entigen is not found in the serum of normal or tumor bearing patients, =n4 is not shed into the ctzlture inedia by positive coll lines (that is, cancer cells are known to blebofftportionis of their cell membranes 2S and release them into the surrounding fluid) (RAdosevich er al,, 1985). Recently 3 of 27 randomly tesed adcnocarcinoma, patents were found to have nrall&y occurring antibodies to the antigen. In additon, radiolabeled MCA 44-3A6 was used to localize A549 tuors growing in nude m.ice. A duoxorubicin immunoconjugate MCA 44-346 is selectively oxic in, vitro (Sinicule et al., 199 1).

Detrtmination of the nucleotide and amino acid sequences of the antigen detected by MCA 44-3A6 would enhance the usefulness of thiis anigen in cu~cer diagnosis, treatment and prevention.

BRIEF SUMMARY OF THE INVDNTON 00 0 The antigen detected by the antibody MCA 44-3A6 as described in the Background is now designated as "Labyrinthin." A gene (designated labyrinthin; abbreviated lab) characterized by a unique nucleotide sequence that encodes the antigen detected by MCA 44-3A6 was isolated and characterized. The notation lab signifies the nucleic DNA/RNA forms; "Lab" notation refers to the protein which is encoded by the lab DNA/RNA.

The invention described herein used the antibody MCA 44-3A6 as a tool to clone Sthe gene encoding Lab. In addition, an epitope (the necessary binding site for an antibody found on the antigen) for MCA 44-3A6 was identified on the Lab protein expressed by the clone to be PTGEPQ (standard abbreviations for amino acids). The epitope represents 00 an important immunodominant sequence; that is, when injected into animals, the animals readily produce antibodies to this sequence.

According to a first embodiment of the invention there is provided the use of a DNA molecule with a nucleotide sequence as shown if FIG. 1 or a sequence showing at least about 70% homology to the nucleotide sequence in FIG. 1 and having the biological Is properties of labyrinthin, for the diagnosis of cancer.

According to a second embodiment the present invention provides a cDNA molecule with a nucleotide sequence as shown in FIG. 1 and a cDNA molecule with a nucleotide sequence showing about 70% homology to the nucleotide sequence in FIG. 1 and having the biological properties of labyrinthin.

In a third embodiment of the invention there is provided a segment of the cDNA molecule of the second embodiment, wherein the segment extends from the start codon (ATG) to the stop codon TAA in the nucleotide sequence and includes 765 base pairs.

In another embodiment of the invention there is provided an amino acid sequence encoded by the cDNA, or segment thereof, defined above.

In another embodiment of the invention there is provided a method to diagnose cancer cells in a sample of cells, said method comprising: contacting the sample of cells with a labeled probe that is capable of hybridizing to a cDNA molecule with a nucleotide sequence as shown in FIG. 1 or a fragment thereof, under stringent conditions; determining whether the probe has hybridized with nucleotide sequences in the sample; and inferring the presence of the sequence if the probe has hybridized, said presence being diagnostic of cancer.

In another embodiment of the invention there is provided use of a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of 1225080 IJIN 00 FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof, for the preparation of a vaccine for immunising a subject against cancer.

In another embodiment of the invention there is provided a peptide selected from Os the group consisting of all sequences that are between 5 and 20 amino acids in length, aligned as are the amino acids in FIG. 2, wherein the position of the first amino acid of Sthe sequence of n amino acids is selected from the group consisting of position 1 to n-5 to SIn a further embodiment of the invention there is provided an antibody directed to a 00 0 10 peptide having an amino acid sequence selected from the sequences defined above, excluding monoclonal antibody 44-3A6.

In another embodiment of the invention there is provided a method to attenuate the effects of expression of the cDNA molecule, or segment thereof, as defined above, said method comprising: obtaining an antisense molecule to the cDNA molecule or its expression product and hybridising the antisense molecule to the cDNA molecule or its expression product.

The present invention also provides a vaccine for immunising a subject against cancer, the vaccine comprising a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG.

2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof.

The present invention also provides a vaccine comprising a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG.

1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof when used in immunising a subject against cancer.

The present invention also provides a method of immunising a subject against cancer, said method comprising administering to said subject a vaccine comprising a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof.

1225080 I.JIN 00 0 The present invention further provides the use of a molecule having an amino acid N sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof, for the manufacture of a medicament for the treatment or prevention of cancer in a subject.

Also disclosed herein is the use of lab DNA in the sense (where the normal C€ transcription of a DNA sequence which proceeds from the 3 to the 5 end to produce a ,IC mRNA strand from the sense strand of DNA, the mRNA being complementary to the DNA) expression made for; the marking of human tumors by nucleotide probes; (2) 00 0to the detection of DNA and mRNA expression of lab in cells and tissues; the transformation of cells into a glandular-like cell type; the production of Lab antigen in vivo for immunization; the ex vivo expression of Lab for immunization to produce antibodies; and production of Lab in vitro. Use of an antisense molecule, e.g. for example, by production of a mRNA or DNA strand in the reverse orientation to a sense molecule, to suppress the growth of labyrinthin-expressing (cancerous) cells in another aspect of the invention.

Also disclosed herein is a vector comprising a DNA molecule with a nucleotide sequence encoding at least an epitope of the Lab antigen, and suitable regulatory sequences to allow expression in a host cell.

Also disclosed herein is an amino acid sequence deduced from the protein coding region of the lab gene. Selected regions of the sequence were found via immunological methods, to produce effects corresponding to effects from both naturally occurring (from cancer cells), chemically produced (synthetically produced peptides), and expression products of the cloned lab gene.

Also disclosed herein is the use of the entire deduced amino acid sequence of Lab, peptides derived from Lab, or chemically produced (synthetic) Lab 1225080 I.JIN 00 peptides, or Any combination Of thles! Molecules, f~or use in the prepaxation of vaccines to Prevent human c~nc ers and/or to treat humans vth cancer. For purposes of the Preent invenion, "bumnans with cancer" are those persons who have the Lab anti gen detected In their cells. These preparations may also be used to prevent patients fromn ever having these tumors prior to their &3rt occurrcnce, Monoclonal, antibodies directed to the Lab protein 1 or aztigeuic components or derivatives of Lab proteins, are useful for detection of Lab and for other purposes.

N ~Monoclonal antibodies which are made in species other than those which react with N- the Lab antigen can be modified by a number of molecular cloning methods such that 00 they retain their binding with the Labyrinthin peptides, yet are not immaunog.-nic in N humans (Sastry e al, 1989; Sambrook et al., 1990). Ia brief, this is dome by replacing the binding site sequence of a cloned human antibody gene, with the binding site sequence of the non-human monoclonal vitibody of interest. These humanized" MVC As ame used a~s therapeutic and diagnostic =egents, in vivo, ax vivao, amd in vitro.

The use of the Lob protein or antigenic peptides derived therefr~om in diagnostic assays for cancer is a way to monitor patients for the presene and arnount of antibody that they have in their blood or other body fluids or tissre. This detection is not limited to cancer of a class or classes previously defined, but is useful for cancer cells that have the Lab marker entigen The degree off scroconversion, Ms measured by icichniques known to those of akll1 in the art (for example, ELISA (Engrl pnd Perlhrann, 1971)] may be used to monitor treatmnent effects.

00 9 Treatmient with antisonse molecules to lob or antibodies to Lab in a phamnaeudtcal composition, ii an approach to treat patients who have Lab in or on, their cancer cells.

BRIEF DESCRIPTION OF THE DRAWiNGS FIG. 1 is the nuclelc acid sequence of the lab &mne (SEQ 1ID NO. I).

FIG. 2 Is the amino acid sequenc: for Lab, deduced from the lab geze (SEQ ID NQ:2).

IFICY. 3 is an ilustration of the lab gene and how it is related to the HAAI{ 00 010 DETAILEBD DESCRION OF THE IVNTION' Molecular Biolog oLabyinthin: To demtonstrate Tha± the epitope.MCA 44-3A6 is encode-d by a protein sequence, high molecular weight DNA from tht cell line A.549 was isolat-ed. This DNA was co-precipitated (via calcium.) with a plasmaid (.pSneo), and used lo tansfect a mouse cell l"a designated B78H1 celLs (Albino el is at., 1985), This rnouse cell line is negative for the expression of the epitope and weas reported to have a high frequency of' incorporaton and expression of any human DNA seqt4ences. If a given B78H1 cell was in a state to take vp DNA, it would be exjpected to have taken up both humnan DNA and the plasntid DNA. Thc plasmid DNA makes the cell rrsistant to 04 18 (a n ormally toxic drug). Therefore, if a cel normally sensitive to G418 growth inhibitor grows in 0419, it had to have taken up the plosntid, ad may also have taken up one orr more A549 DNA sequences, After 0418 selection (a way of choosing only cells which have resistance to growth in C0418 by the uptakc/exprcssion of the lNco gene oA PSVnCO plasmid, and therefore rprmsening c~eils that were in a state to uptakce other DNA at the sam~e timne), approximately 15 of W1x0 clones were deed using irmunoselection with MCA 44-3A6. This findli~g is consistent with conclusion Thut hu~man A549 cells have DNA that encodes Lab and possess,=s the regularity sequences accessary f'or the expression of Lab.

comparisoft of HAAR- and Lahv thn: Bmuse the DNA sequence of lab was deterniined as an aspect of the present inventiort, HAAR and lab could be compared. ?{AAH and the lab nucleotide seqiqences have some internal fragm=n similarities, but are different on either side of the fizgment, and are related to different products, This conclusion ij based in p=x by the analysis and homnology of the DNA sequences reported for these two genes. Specifically, the Wa -T region has no 00 hOMOLOgY With HAAH. The protein coding region of tab has about a 96 homology with an internal segment of the proposed protein coding region for HAA~i.

The 31 region has no homology with the H~AAH reported sequence. Virtually all of the other data comparing IiAAE and labyrin~thin vre different, for example; molecular S weights of the proteins, cellular localization, chromosome localization, (4) histologial presentation in nornuil tissues and tmtors, northern blot expression, imnmunological findings.

N- Altboigh th.: protein coding region of lab is identical to an inzearma region. of 0- N0 the sequence reported for 1{AAKI the 5' untramslated region of HAAH is different, and part of tbt 5' nanslated protein codi.ng region of HAAR~ is missing fromt that found in c-I the lab clone, From both IiAA and lab clones, the deduced protein would be expected to be very acidic in mature and therefore would run anomalously in SDS gels. As predicted, the Lab protenrimigrates anomolously in SDS gets. What was cloned and disclosed, in. the present invention migrates identically to the rative protein IS founmd in several cell lines. ConvincimS evidence that the correct gene fragment eacoding the antigen detectd by MCA 44-3A6 has been cloned (m.RNA) is that when the recombinant protein is mnade, that recombinant protein should aci (in. thi s case, have, an apparent molcular weight) the sarn as an independent biologically derived source of that protein. Lab provided from clones has thz characteristics of Lab from The deduced amino acid sequence encoded by FtAAH requires the use of an open reading frame which would produce a protein that is 85-90 kilodaltons, and does not uke into account that there ane several start codons and other shorter open readidS rames. The deduced IAAH protein (biochemically) is glycosylated and the reported sequenceW halycosyton sites Korioth et aL, 1994; Lavaissiere er al, 1996). To the coatrary, Lab is not glycosylated, nor does it have predicted glycosy~ered sites.

The deduced 1{AAH amino acid sequence contains a region shared bry the Lab amino acid sequence wh~ich is predicted to be very hydrkophobic. Lab requires strong d.-tergents in order to be soluble; HAAH does not. The increased expression of HAAR{ (by enzyme =ivity measurements) in the saecell line (A549) which was used to clone and study tab extensively, suggi=~ that both of these gene products may be importani to the AC phnotype and that at least A549 cells make both functional {A.H and. Lab, SuccessfU trarisLecti of the antisease to lab into A549 resulted in 00 .1 a marked decrease in expression of lab and in the growth rate of the cells. The expression of a sense lab construct in NII4-)T3 cells (norms.! mouse fibrob~asts) resulted in a marked chainge in phenotype, a phenotype consistent with that of ACs.

Therefore, lab expression is associated withi conversion of normal1 cells to czncerous Scells. Lab and HAM-I have powtial calciumn binding domains in common.

eDNA Library Constuttion end Clonin A ODNA lambd4 gtl I phage library was constructed 4sing nRNA which was isolated from actively growing A549 cells (Sarnbrook erta., 1990). This oligo(4T)-pimc-d cDNA was cloned into the Eco RI 00 site using Ero R1 linkers. The library has about 83% clear (containing an insrt) plaques wihaztiter of 1.2 xc I 10 /ini representing a minimum of 1.4 6 x N ~independent plaques whch by PolyzmeT=~ Chain R~eaction, have, insert sizes rarlg~ng from 0.6 to 5 kIlobazs. Since Lab is a 40 kilodalton intera protein, (a protein. which is embedded in the plasmna membrane) the theoretical Mul length mP NA encoding this protein, including a potenial leader sequence is e.stima~ed to be about 1.1 kilobases.

is This library was immunoscreened ujsing the antibody MCA 44.3A6. Eight independently derived phage stocks (identical phage which are from the same plaque) were isolated. These have all been plaque purified by repeated cycles of immiunoscreening/isolation. Upon Eco RI di-tstion of these eight isolates, inserts of about 2kb were seen. The largest insert was isolated (2AWA) and the Eco RI Eragmeit was cloned into the pOEM-3Z plasmid.

Seauencing and SecgvenceAnalysis: The DNA fragment designated 2AIA1 was Foundto have a~n insert of 2442 base pairs in length (FIG. containing a untwnslated region, a ribosome binding site, and a scart codon which would be ex pete4 to encod: a 2155 unino acid protein (FIG. The 3' untranlated region is mrkable ia that it contains only four~ instaibility sequences; ATITA ()Cu or a., 1997). In addidlon there are seclaences found in the very 3' end of mRNA's which result in adenylation of the mRnlNA (Sambrook er al., 1990). The lab sequence contains both a s'vb-optrna (ATTAAA-) and optimal (AATAAA) poly-adenylation site, These =r sequences found in the very 31 end of znRNA's which result in, adenylation of the mRN'A. This finding provides molecular data which supports the cellular and biochemical dama that has been outlined herein. (The HAAH clone has a poly A signal, brut the whole 3' region has not been sequenced.) 00 -12.

N A calcium binding site motif is noted in the Lab amino and sequence (FIG, 2), however, it is out of the knowzn required s uctural context wo be a binding site. In this case, the ca.lcium l1imiting~ sequence is There, but it is not in a proteinl sequence context that is known to make it work a-s a binding site. Homology was noted with (ab and an EST clone (designated #05501) which represented only a portion of the 3' unt-anslated region amd independintly cmifirmed this portion of the sequence. Some internal1 fragment homology is also noted with H{AAR4 but the 5'unawslated and part of the 51 translated region is diffaient (58 amino acids), es well a~s a major portion of the 3' 00 coding region is missing in lab (FIG. 3).

010 Qenon~ic DN4 C~iigadAayi:Using a ?CR fragment representing the protein coding region of lab U. 4 probe, a genornic lambda FIX U libary made from the human loulmonary fibroblast cell line WI-3 8 was screcne&d Tea primary plaques were isolated out of approximately -xI 0' screened plaques. Using sevn of these as target DNA, Polymerase Chain Reac~tion conditions were estabtished with primers for is the protein coding region, producing a 765 base pair fragnemt the expecttd protein coding region for lab. On Northern blots (a method used to qualitatively assess mIZNA) lab only detectA bad noted at 2.7 kIlobases. The recombinant protein made from the lab clone, when tested on Western blots (a method used to qualitatively define proteins) using MCA 44-3A6, has the s~me relative mobility as the Leb protein when. made by A549 cells.

Lab and HAA14 genes give differcut results in the proteins they encode.

HAAH consistently gives two bands on Northern blot analysis (2.6 and 4.3 kilobases) suggesting that the 2.6 kilobase band is due to altematiye splicing that is, the cell cuts and splices the raRNA, Also, if 10b and HAAH art the same gene, HAAIH should be detcted in all tissues and cancer cell lines in which Lab is found. However, Lab is not seen on Norrhe' blots of cell lines EMT6 or QV,-DB, nor is there irmaunor-activity in these cells; indicating that Lab is not made, and that Lab protein is not produced in these calls. Lab protein, is rarely ecpressed in normal cells, where both the HAAHJ mR2'IA and HAA}I proten have been reported to be expressed ia almost evezy tissue studied.

mRNLA Anriaysis: North=r blot analysis of the DNA ftagment from the A549 cell line using lab cDNA as a probe identified a single band of about 2.7 kilobases.

'This is expected based on the r.DNA (2442 base pairs) and a poly.A tail of about 300 00 -13.

base pairs. Northerna blot analysils of the mouse Cell line, EINT6, and of th~e hunta large cell carcinomz cell line, QU-DB, confirm that no tanscxipt for lab is produced by these cells, This is consistent with irim~o assays which are negative for lab expression on these cells.

~Antiseno pnd Sense cDNA Expressicn: The plasmid (pB](-CMVW) (Samnbrook et at,, 1990) maay carry either the sense or antisense fMl length cDNA lab into A549 and NIH MT cells, An antisenwe molecule can be, for example, a complementary sequence to a sense molecule that hybridizes with the sene molecule, preventing its 00 expression. Using the MTT assay (Siddique et al., 1992) to ass=s the growth rate of A549 cells expressing antisense to lab, a marked reduction. in growth rate wa~s aoted.

c-i The antiscnse transfected A549 ceIs appear to have a greater degree of contact inhibition. A dctectbje amount of Lab is reduced in these antisense fransfected cells.

NIH--3T3 cells convert from a fibroblws-tike cell type morphology (large, thin spindle shaped) to a& large, adcnocarcinoina appe~rig cells (very round, plump) when sense expression~ occurs.

Chram2sgame Loalizaion: The chromosome localization for lab, using ful length cDNA as a probe via I .itu hybridization (Sambrook et al., 1990) is tentatively on chromosome 2q 12-14, with possibly some icactivity to chromosomes 4 and 8.

Using the sarne probe (the full length cDNA sequence of lab) and FACS sorted chromosomes (Lebo er al., 1985) staining was also noted on chromosome 2, with weak staining on 4 and none on S. The use of genomic clones will be of particular value in resolving these da because higher stringency hybridization conditions than that allowable for the cDNA, can be used, thereby reducing background signals, This is yet another proof that the correct gene was cloned and that the results are not due to 23 a Inetltod utifact, There rnay be mutations in the genomnic, DNA of tumors and for the present invenion, DANA was, cloned from ttunor cells (A549). Therefore, mutated gene could have b=e cloned. However, that is not the case because the genontic DNA front a normal cell (DNYA) produced the same sequence as what was cloned as desribed hereint. Therefore, A normal gene was cloned from A549 cells, The weak sigtaJs on. clhrosomes 4 and 8 are consistent with a pseudogene or a relazed gene.

For example, HAA14 has been reported to be on chromosome 8q12 by in situ hybriclizaton, so this result on chromosome 8 could reflect the RAM-I and lab sequence homology.

00 -14- (71 Poten Moecuar harateizatonofabv~ntin:Previous work us.ing Western blot apalysis qualitative assay to assess antieens) has shown that the Lab antigen is a 40 kilodalton (by relative rnobility) protein detectable in A549 cells (Radosevich et al,, 1985). The epiiope does not appear to be modulated or blocked by lectins. and is selectively expressed on the cell surface primarily localized to the plasma membrane. (Radosevich et al, 1985, 19~9 Lab is sensitive to proteases, but not lipid or carbohydrate altering reactionsi (Radosevich et aL, 1985). The N biochemical properties of Lab are consistoat with Lab being an integral membrane 00 protein, Having a deduced amino acid sequence from the lab gene of the present tavention, aows Ranther characterization of the Lab~ protein. Extensive computer analysis of Lab has identified a eukaryotic leadier-Uke sequence and theoretical cleavAge site, 3 myristylation sequence sites, a weak membrane anchoring domain (MAD and a strong membrane anchoring domain (M4AD If) (FIG, [(In the I{AAHi sequence, there =r 58 (thcorertical) wnino azids followed by 4 sequence homology In the Lab protein ooding seque=c and an additional 445 a=!no acid 3'to the lab sequence.)) Wben Lab is expressed as a fuzsioa protein In a bacterial GST fusion expression symtm (pGEMEX-2T) (Amereham ?barmacia Biotech, Inc,, Piscataway, New Jersey, 08854, USA), and subjected to Wetern blot analysis using the antibody MCA 44-3A6, the resulting blots demonstrate t~t the expressed cleaved fusion protein has the sarne relative mnobility as the protein detected in. A549 cclls. The deduced molecular weight for Lab is 28.8 ItilodalToris and on. Western blots it has a relative mobility identical to the form expr~sed by A549 cells (apparent relative mobility =40 kilodaltoas). The 55 glutmlic and 27 aspartic acid residues a (82 residues combined ame almost uniformly distributed throughout the protein amino acidjs total; 228 n= leader sequence), except for the leader sequence and the sn'vngest membrane anchoring domain (MAD 11). These d=t suggest that Lab migrates anomlom~ly in SDS gels. Cell lines othei than A549 (for example, adanocarcinomas DU- 145, ATCCO H TB-8l, ZR-75-1, ATCC CRL-1 504, and so forth) have an antigen detected with the samte molecular weight antigen as Lab.

Neither a 85-90 kilodalTon molecular weight species, nor a 52 and 56 killodalton molectlar wigh: species is noted when-priobing Western. blocs for Lab.

00 EpiOne MIpiQ U;Sing-te Antibody MCA 443Aad Vcce CC* b1tvp lab: Using Polyrnerse Chain Reaction and the CST fusion protein systemi, subclones of the protein codinig region were made 1 and epitopes mapped the binding of IMCA 44- 3A6 to six amino acids (PTGEPQ) represeating amino acids #I 17-122 of Lab (PP" peptide). In order to 4lcrnae this epitope, the entire codinS region was divided into regions, Polyrnerse Chain Reaction primensw=r designed to amplify each region, and the subsequent expressioa of Polymerase Chain Reaction products were cloned N and testd by Western blot aniaysis using the antibody MCA 44-3A6.

NI The DNA frpgrnent representing the positve Western blot result was then 00 010 ftirther subdivided. Polymerase Chain Reaction products were geaerated and cloned, CI expressed, and tested via Wester blot, Consucts were made in this way both from the 5' end and the 3' end and the intervals of the number of' analno acids were reduced upon each round. This reaulted in the last round represeating acone* amizo acid difference from the preavious round (in both directions), such that one could deducce the exact binding site of the MCA 44-3A6. This demonstrates that at least these six amio acids are excposed to the external cell surface. To further prove the point, the DNA encoding only these six amnino acids have been cloned and the fusion protein is positive by Western blot analysis. Synthetically pre-pared 71P peptide can be specifically detected by MCA 44-3A6, and the synthetic pep tide was iramlunogcnic in 5 of 5 mice tested. Computer analysis/modelig also predicted that this epitope would be very immunogenic using computer assisted analysis (GCG programs) (Genetics Computer Group, Madison, WT 53703), Vaccn reraton: A vaccine is a preparation of antigens), which when given. to a host, results in the host producing antibodies against the antigen(s). The host response results in the host being irmune to the disame to which the vaccine was directed. Vaccine teaunt therefQre, prevents the clinical presentation of a disease, without the host being exposed to the dismae causing agents. Lab has anl the 00 -6 clrateristcs of a. preferred cancer vaccine. The lab gene is frequently expressed by tumors which look like adenocarcinomas, is exPressed an the outside of the cells, is expressed by all of the cells within a given cancer, is expressed az all times by these cancer cells, and is infrequently expressed by normal cells. Lab protein (peptides) can be produced by any number of miethods using molecular cloning techniques, and c= be produced in large qlaantities, tu making it a practical antigen to use as a vaccine.

After the Lab protein has been purnified so that it is suitable for injectioa into humans, it is administered to n4vidu~ls inrrsdctmally, subcutaneously, or by other routes, so as to challenDge the immune system to produce antibodies against this protein 00 010 (peptides).

~The 4se of molecular modeling and computer assisted analysis OCO programs (Genetics Crystal Group, Madison, W1 $3703) allows the identification of smaflll portions of a molecule, slightly larger than an cpiiope (six to seven amino acids for proteins), which are expected to be on the stwface of a protein molecule. In addition, the degree of hydrophobicity or hydtophilicity of a given sequence, and how immunogenic the sequence wouald be int animals, can be detenmined (Genetics Crystal Grroup, Madison, WI 53703)- After defiriing which sequences meet thesc criteria, the peptidecs are synthetically mode, or produced by a number of standard methods, One or more of these peptides can then be formnulated to be used as a vaccime, and adm i "stered to the ho at au outline d above, a3 a vaccine, A vaccine comprisiag a molecule having an amino acid sequenc- selected from the group of sequences encoded by the cDNA of FIG. 1, sequences of FIG. 2, encoded by the cDNA, the peptidecs APPEDNPVED (SEQ ID NO:6), E-EQQEVPPDT (SEQ ID NO:7), DGPTGEPQQE (SEQ ID NO:S), and EQENPDSSEPV (SEQ M) 23 NO:9), and any fragnicnta or omibinations thereof.

A given vaccine may be administered once to a host, or may be administered many times. In order for some Datients to recognize a given vaccine, am adjuvant may also need to be adrnirstered with the peptides. Adjuvants are nonspecific immcune stimulators which heighten the LIMUne readiness and aid in the conversion of the host from not having detectable serum antibodies to having very high titerr serum antibodies. It is this high level (titer) of antibodies, whic13 effectively protects the host from the diseases or coaditions to which the aztibodies ar.- directed.

00 -7 Functional Studies: Studies directed at understanding the cellular ftinctions of Lab are extensions of cell local aton/characterizationa studies (S iddique et al., 1992).

Changes in leyels of L~ab in response to extracellulax exposure to vaious response to extnacellulaz exposure to va~rious cations (CA, NMg', Cu~, aad Fe") were undertaken. Lab expression in A549 cells was only modulated by Ca". Using the highly specif c: fluorc~cenl Fur4-2/AM Ca1 method of moaiu.ring cytosoio (Molecular Probes rac., Eugenc, OR 97402) it was demonstatd that: the interal Ca"" concentr~tion is higher in A549 cells than in QTJ-DB cells; and that the A,549 cell Line respoads to various external Ca7' levels (Siddique et aL, 1992). Since pli 00 can modalate intracellular free Ca" levels, external p1-i manipulations should result in changes in the expression levels of Lab. Etraceilular pH- changes (In the prTence of normal Ca concenrutions) result in: a parallel change in intracellular pH us measured by SNARF-1 AMJFACS, (Molecular Probes Inc., Eugene, OR 97402) (2) t-ascript levels increas~e for Lab (when compared to GAPDH expression via Northern is blot); and that Lab protein. also incTeascs (using Western/Slot blot analysis). The intracellular changes in pHi (due to external changes) for A549 cells are identical to those reported for normal cells. The incrased expression of lab is also not due to cell death (as measured by N=I assays) (Siddique it al., 1992). In additoon, incubation of recombinant Lab at various pHI sohidons does not alter immunoreacdNvity.

Peinmnary data suggest that when these experimnts are conducted on A549 cells grown in reduced Ca-, the induced expression of lab is blunted.

Methodg of Piggosing Cancer Cells in a Samoile of Cel-]: Biological samples from a subject are used to detenuine whether cancer =e11s, are present in the subject.

Examples of suitable samnples include blood and biopsy mnaterial. Ono method of ditgnosis is to expose DNA form cells in thoi samaple to a labeled probe that is capable of hybridizing to the lab gene, or a fragment thereof, under stringent conditions.- for example, 6x ssc; 0.05x blotto; 50% for~mIde; 42TC (Sambrook et al,, 1990). Of cournse, the hybridizing conitions arm altered to acbieve opdm~ Sensitivity and specificity depending on the nature of the biological sample, type of cancer, method of probe preparAtin, and method of tissue preparution.

After contacting the sample with the probe, the rext step is determing whether the probe has hybridized with nucleotide sequences of the DNA fromn thfe 00 sample, from which the presence of the lab gene is infened said presence being diagnostc of canc er.

Another diagnostic method is to obtain mnonoclonal antibodies preferably 0 labled. either antibodies already existing, or new ones directed to the aatigenic peptides th= are aspects of thc present invention, anid contat a sample with these to detect the Lab antigeo. Thnese mnonoclonal antibodies axe usefta in the development of VM~ specific assays for the detection of Lab antigen, and allow the tests to b~e carried out in many different formats; resulting in a& broader application in science and 00 medicine.

The current invention is usefuli in that it* describes a new gene which is expressed on the surface of tumors, which was not previously reported. This gene is not tissue specific, and therefore will allow the detection of tumors regardless of the organ, in which They arise, Likewise, the use of this gene to produce a vaccine for these tumors, will have a very bropd application. Dianostic tests will also have this broad tissue. use, nacidng the detection of Lab/lab a. "Pan-rnarke9' for can=e, in particular for what have been designated previously. adenocarcinomas.

00 -19- DOCUMENTS CITED Albino, AP', Ca, LH{, Kontor, RRS, el~ al. DNA-mediated transf'er of human tuelanora. cell surface glycaprotein gp13O: Identification of transfectants by erytbrocytc rosetting. Mol. Cell. Biol. 5!692-697, 1985, Banner B3F, Gould YE, Radosevich JA, et al. Application of monoclonal antibody 44-3A6 in the cytodiagnosis and classification of palmonary caicinomas.

Diag. Cytopathol. 1:300-307, 1985.

c-i Brown, DT and Moore, M. Mooclonal antibodies against two human lung c-i carcinoma cell link. Br. I. Can. 46:794-801, 19 00 Combs SG, Hidvegi D)F, Ma Y, at al. Plernorphic. Carcinom of the c-i Pancreas; A rare case report of combined histological features of pleorrorphik adenccarcinoma and giant cell trmor ofthe pancreas. Diag. Cytopathal. 4-316-322, 1988a, Combs S0, Radosevich JA, Ma Y, et al. Expression of the Antigenic is Determinant Recognized by the Monoclonal Antibody 44-3A6 on SeLect Humnan Adenocarcinojna and iNormal HumanTissues, Tumor Blol. 9:1 16-122, 1988b.

Combs SG, Radosevicli JA, and ST Rosen. Cytological expression of the adenoacinoma antigen marker in human body fluids, Tumor Biol. 9i:116-122, 1989c, Dudtt RB, August CZ, Radoscvich TA and ST Rosen. Moinoclonal Antibody 4-4-3A6 as a Marker for Differentiation of Breast Cancer. Tumor Biol. 12-.254-260, 1992.

Engvall, E and Perlnn, P. Enzyme linked inumosorbent assay (ELISA): Quantitative assay of ISG. iznmijochernisrry. 8:87-874, 1971.

Gronkc VanDusen WI, Garsky VM, Jacobs JW, Sardana MK, Stem AM.L and PA Friedman. Aspartyl bet bydrwyiase: In vitro hydroxylation of a syntb--tic peptide based on the struct=n of the first growth factoT-like domain of' bumn fctor IX PNAS. 86:3609-3613, 1989.

Gronice nS, Welsch DT, VanDuscrn W, Garsky VIK Sardana Stern AIM, and PA Friedman. Partial purification and chazacterization of bovine liver aspartyl beta hydroxylase, J. Biol. Chem., 365-8558-8565, 1990.

00 lia S. Van-Dusen, WI, Diehl1 RE, Kohn NF, Dixon RAF, El~lison KG, S tern AM, =44 PA Friedman. cD'NA cloniing and expression of bovine E1spanyl (asparageinyI) b eta.'hydroxy Lase, J. Blod. Chem,26:42132,9.

Jia S. McGinns K, Van Dusen WI, Burke CJ, Kuo A, Crriflfn PR, Sardana, MK, Ellison KO, St=r AM, and PA.Friedmn A fully active caalytic domain of bovine aspartyl. (asparaginyl) beta-hydroxylage expressed in Escbericbia coti, Characterization and evidence for the identification. of an active-sita region in~ vertebrate elph-keroglutarate-dependent dioxygenases. PNAS 91:7227-7231, 1994.

Koricth F, Gieffers C, and I Frey. Cloning and characterizatioa of the human 0010 gene encoding aspa~y beta-hydroxylase. Gene 150:395-399, 1994.

Landis, SK~ Murray T, Bolden S, and PA Wingo. Cancer Staxistics, 1998, CA 44:6-9, Lavaissier L, ha S, NishiYanmn M dc la. Monte S, Stren AM, Wands JR, and PA Frieiman. Overcxpression of human aspartyl (asparaginyl) beta-hydroxylase in hepaxocellular carcinoma and cholanagiocaroinOruL I. Clin. Invest 98:1313-1323, 1996.

Lebo RV, Tolan DR, Bruce 51), Cheng MC, and YW Kam Spot blot analysis of sorted chromosomes assigns a fructose intolerance gene locus to chromosome 9.

Cytometry. 6:476-483, 1985, Let I. R.Adosevich IA, Rosen ST, et al, Immunohistochemi~try of lung carcfinas uiung monnclonal antibodty44-3A6, Can. Res. 45 -58 13-58 17, 1985, Lee 1, Radosevich JA, Chalfec 0, et Malignant Mesothc~omas: Improved Differential Diagnosi3 From Lung Adenocarzcinomas Using Monoclonal Andboides~ 44-3A6 and 624AI12. Amer. J. Path. 123:497-507, 198 6.

Livingston PO, Wang QYC, Adluri S, Tao Y. Padevan M, Parete R, Hanl on C, Calvea NV. Helling F, Rimye 0, Oeten HF, and LJ Old. Improved survival in AJCC stage MU melanma pateitns with GM2 antibodies: A randomized trial of adjuvant vaccination with GM2 ganglioside. J, Cliii. Oncol., 12:1036-1044, 1994.

Piehi MR, Gould YE, Radosevich 1A, et al. Irmnchitcejic identification of Exocrine atd Neuzoendocrine Subsets of Large Cell Lung Carinomas. Path. Res. and Frmc. 183:67S-682, 1988.

00 Radosevich JA, Ma y, Lee 1, et al. Monoclonal antibody 44-3A6 as a probe for a novel antigen found hur=a lung carcinomas with glandular differuntadon. Can.

Res. 45,5805- 5812, 1985.

Radosevich SA, Lee 1, Gould YE, and ST Rosen. Monoclonal antibody assays for lung cancer. In vitro diagnosis of human tumors using monoclonat antibodies.

Kupchick HZ and N Rose (Eds&) Marcel Dekker p 10 1- 119, 1988.

P-adosevich JA, Combs S0, a4nd ST Rnsen. I mnohisochrnical analysis of c-i lung cancer diffrentiation mwakems In: Lung Cancer Differcnaton, Lung Biology in Health and Disease. L'Enfant C, Bernal S, and B myliri S. (Eds.) Marcel Dekker, 0010 1990a.

Radosevicli JA, 1Nogwclt M, gosen ST. Y Sbimosato. rmunocytoch~aical analysis of human adenocarciaoomas and bronchioloalveolar carcinomas of the lung using the monoclonal antibody 44-3A6. Tumor Biology'. 1 1:1 1- 188, 1990b.

Radosevich JA, Combs SG, and ST Rosen. Expression of MCA 44-3A6 in human fetal davelopment. Tumor Biology 11.321 -329, 1991.U Radosevich IA, Siddique FS, Rosen ST, and W1 Kabat, Cell Cycle and EM Evaluation of the A enocarcizooma Antigen Recognized by the Monoc lonal Antibody 44-3A6. Br.J1. CarL 63:86-87, 1991.

Rosen ST, Mfuisbine .JL, Cuttitta F, and PG Abrams. Biology of Lung Cancer.

Marcl Dekkcer, Inc, New York, NY, VoL 37, 1988.

Samnbrook 1, Flitsch EF, and T Maniatis. Molecular cloning; a laboratory rnanual. 2nd Ed. Cold Sprin Harbor Lab. Press, 1990.

Sastry L, AIlng-Mzses K Huse WD, Short JM, Hay BN, Janda 1(0, Berikovis SJ, and Lerner. Cloning of the immnunological repertoire in Escherichia coli for 2S .generation of mionoclonal catalytic antibodies: Constrution, of a heavy chain vuxiable region-specific CONA Library. PNAS. 86;S728-5732, 1989.

Siddique FS, Tqbal Z, and JA R44ose-vich. Changes in the expression of the =uor-essociared Antigen recogized by monoclonal antibody 44-3A6 in AS49 cells due to calciurm. Tumor Blot, 13:142-151, 1992.

Sinkul.e J, Rosen ST, and IA Radosevich. MCA 44-3A6 Douxorubicin (Adriamnycin) hinmunconju gates: Comparative in In Vivo Anti-TurC Efftacy Of Differem Conjugatiion Methods. Tumor Bic]. 12,198-206, 1991, 00 -22- Spagnolo DV, Witak=e D, Carrelo S, el al. The 'um of monoclonal antibody 44-3A6 in cell blocks in the dieanosis of hIrg carcinozma, carcinomras Inetastatic to lun~g and pleurna, and pleumi rnalipant mesotheloma. Am, J. Clin. Path. 95:322.329, 1991.

Staht-I RA (Chairman). Tird latcmadonaI IASLC Workshop on Lung Tumor and Differenriation Antigens. later. J. Cancer Suppi. 8:6-26, 1994.

Wang Q, Vanflusen Wi', Petmald Ci', Qarky VM, Sterm AM, and PA Friedman.. Bovine liver aspa~ryl bm~-hydroxylase. J. Biol. CbenL 2,66:1400414010 c-I 1991.

00 10 Xu N, Chen C-Y A, and A.5 Shyu. Modulation of the fate of cytoplasmic mRI4A by AU-rich elenets: Key 3eqUenr.C fC~UreS controlling rnRNA deadezylation and decay. Mol. Cell. Biology. l17;461 1-4621, 1997.

Claims (6)

1. 2. A eDNA molecule witlh a nucleatido sequence L; show'n in FIG. 1. c-
2. 3. A 4CDNA moleculc with a ill4ocotide sequence showing about 70% homiology to the flucleotdc sequence in FIG. 1 00
3. 4. A semn of the CIDhA molecule otclaim 2, wherein the segment extends from~ the start codon (ATG) to the stop codo.1 TAX in the nucleotjde sequence and includcs 0 765 base pairs. S. A segment ofn 4 DNA su~cient to encode an epitope in a protein designated Lib detectable by tho antibody MCA
4. 44-3 A6, 6, An aMino acid sequence encoded by !be eDNA of either claim 2, 3, 4 or S. 7. A method to diagnc!; can= celLs in, a sample of cells, said method comprising- contacing t~e sample of cells with a labeled probe- tha is capable of hybridizing to a cDNA molecule with 4 plodde sequence as shown in FIG. I or a fragznen thereof, undcer stringsw conditou dete~iniaaa Whethtr the probel=a hybridized with nucleotide sequencea in the sample: and inforring the presence of the seqp1ence if the probe has hybridized, said presncz being diagnostic of cancer. S. Use of a mnolec'.4e having an amino acid sequence selected from the group of 4equenacs encoded by the CDZNA of FITG. 1, sequences encoded by the cONA of FIG. 2, the 00 24 peptides AJPPEDNPVED, B.EQQEVPPDT, DC-PTrGEPQQE, and EQEr-NPDSSEPV, and any fragmnt, or comibinations thereof, in a vacoine. 9. A molecule wvith az amino acid sequence selected from the group con~sisting of A-PPEDNPYED, EF-QQEVPT)T, DGPTGiEPQQE, aa4 E-QENPFDSSEPV, A molecule with the amino acid sequence PTGBPQ. ri11. A peptide selected from the group consisting of all sequces that are betw-en a,-4 20 am~ino acids in length, a~iped as are the: amio acids in FIG. 2, wherein the position 00 o h u ta i o ai ft es q e c faaan cd ssl ce r m t ri.c nitu 0 of posTion I to a-5 to 1o 12. An antibody directed to a peptide having an amino acid sequence selected frmthe sequence of claim 6, 8,9, 10 or 11, excludingmonoclonal antibody 44-.3A6, 13. The antibody of claim 12, Rier defined as a monoclonal antibody. 14. .An an~ibody produced in nm~mals against an amino acid sequence encoded by the DNA molecul2 o! claim 2, 3, 4, or 5 or a fragment thereof containing an epitope, I excluding monnoclonal antbody 44-3A6. A method to atten~te the effects of expression oftthe cDNA molecule of claim 2, 3, 4 or 5, said mnethod conaprisiag; obtaining an antisease molecule to the ODNA molecule or its expression product and b) hybridizing the aatise=s mol~ecule to the cDNA molecule or its expression product. 16, The mrethod of claimn 16, wherein the antisense molecule is at least 100 nucleotides in lengtlL 00 17. Use of a DNA molecule with a nucleotide sequence as shown in FIG. 1 or a sequence showing at least about 70% homology to the nucleotide sequence in FIG. 1 and having the biological properties of labyrinthin, for the diagnosis of cancer. 18. A cDNA molecule with a nucleotide sequence as shown in FIG. 1. 19. A cDNA molecule with a nucleotide sequence showing about 70% homology to the nucleotide sequence in FIG. 1 and having the biological properties of labyrinthin. 20. A segment of the cDNA molecule of claim 18, wherein the segment extends Sfrom the start codon (ATG) to the stop codon TAA in the nucleotide sequence and Sincludes 765 base pairs. 00 21. A segment of a cDNA sufficient to encode an epitope in a protein designated SLab encoded by the cDNA of claim 18 or 19. 22. An amino acid sequence encoded by the cDNA of any one of claims 18, 19, or 21. 23. A method to diagnose cancer cells in a sample of cells, said method Is comprising: contacting the sample of cells with a labeled probe that is capable of hybridizing to a cDNA molecule with a nucleotide sequence as shown in FIG. 1 or a fragment thereof, under stringent conditions; determining whether the probe has hybridized with nucleotide sequences in the sample; and inferring the presence of the sequence if the probe has hybridized, said presence being diagnostic of cancer. 24. Use of a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof, for the preparation of a vaccine for immunising a subject against cancer. A molecule with an amino acid sequence selected from the group consisting of APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV. 26. A molecule with the amino acid sequence PTGEPQ. 27. A peptide selected from the group consisting of all sequences that are between and 20 amino acids in length, aligned as are the amino acids in FIG. 2, wherein the position of the first amino acid of the sequence of n amino acids is selected from the group consisting of position 1 to n-5 to 1225080 I.JIN 00 O 0 28. An antibody directed to a peptide having an amino acid sequence selected from the sequence of any one of claims 22, 24, 25, 26 or 27, excluding monoclonal t antibody 44-3A6. 29. The antibody of claim 28, further defined as a monoclonal antibody. Ss 30. An antibody produced in mammals against an amino acid sequence encoded by the DNA molecule of any one of claims 18, 19, 20 or 21 or a fragment thereof Scontaining an epitope, excluding monoclonal antibody 44-3A6. 31. A method to attenuate the effects of expression of the cDNA molecule of any Sone of claims 18, 19, 20 or 21, said method comprising: 00 obtaining an antisense molecule to the cDNA molecule or its expression N product and hybridising the antisense molecule to the cDNA molecule or its expression product. 32. The method of claim 31, wherein the antisense molecule is at least 100 is nucleotides in length. 33. An amino acid sequence substantially as hereinbefore described with reference to Figure 2 of the accompanying drawings. 34. The use of claim 24, wherein the molecule has an amino acid sequence selected from APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof, for the manufacture of a vaccine for immunising a subject against cancer. The use of claim 24 or claim 34 wherein the subject is a human. 36. A vaccine for immunising a subject against cancer, the vaccine comprising a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof. 37. The vaccine of claim 36 wherein the molecule has an amino acid sequence selected from APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof. 38. The vaccine of claim 36 or 37 wherein the subject is a human. 39. A vaccine comprising a molecule having an amino acid sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and 1225080 I.JIN 00 0 EQENPDSSEPV, and any fragment, or combinations thereof, when used to immunise a subject against cancer. A vaccine when used according to claim 39, wherein the molecule has an amino acid sequence selected from APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, O s and EQENPDSSEPV, and any fragment, or combinations thereof. 41. A vaccine when used according to claim 39 or 40, wherein the subject is a human. .i 42. A method of immunising a subject against cancer, the method comprising 1 administering to said subject a vaccine comprising a molecule having an amino acid 00 S 0to sequence selected from the group of sequences encoded by the cDNA of FIG. 1, the N amino acid sequence of FIG. 2, the peptides APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof in an amount sufficient to elicit an immune response. 43. The method of claim 42 wherein the molecule has an amino acid sequence selected from APPEDNPVED, EEQQEVPPDT, DGPTGEPQQE, and EQENPDSSEPV, and any fragment, or combinations thereof. 44. The method of claim 42 or 43 wherein the vaccine is administered together with an appropriate adjuvant. The method of any one of claims 42 to 44 wherein the subject is a human. 46 A method for detecting a cDNA molecule selected from the group consisting of a nucleotide sequence as shown in FIG. 1, a nucleotide sequence showing about homology to the nucleotide sequence in FIG. a segment of the cDNA molecule wherein a segment extends from the start codon (ATG) to the stop codon TAA in the nucleotide sequence and includes 765 base pairs, and a segment of a cDNA sufficient to encode an epitope in a protein designated Lab detectable by the antibody MCA 44-3A6 in a sample of cells, said method comprising: contacting the sample of cells with a labelled probe that is capable of hybridizing to the cDNA or a fragment thereof, under stringent conditions, determining whether the probe has hybridized to the nucleotide sequence in that sample; and inferring the presence of the cDNA if the probe has hybridized.
5. 47. An isolated genomic DNA molecule from which a cDNA selected from the group consisting of a nucleotide sequence as shown in FIG. 1, a nucleotide sequence showing about 70% homology to the nucleotide sequence in FIG. 1, a segment of the cDNA molecule wherein a segment extends from the start codon (ATG) to the stop codon 1225080 I.JIN 00 STAA in the nucleotide sequence and includes 765 base pairs, and a segment of a cDNA C sufficient to encode an epitope in a protein designated Lab detectable by the antibody cMCA 44-3A6, is derived.
6. 48. A vector comprising the isolated cDNA molecule of a nucleotide sequence as shown in FIG. 1, a nucleotide sequence showing about 70% homology to the nucleotide sequence in FIG. 1, a segment of the cDNA molecule wherein a segment extends from the 0 start codon (ATG) to the stop codon TAA in the nucleotide sequence and includes 765 0base pairs, and a segment of a cDNA sufficient to encode an epitope in a protein designated Lab detectable by the antibody MCA 44-3A6. 00 o0 49. A vector comprising a cDNA molecule that encodes a molecule with an Samino acid sequence selected from the group of sequences encoded by the cDNA of C FIG.1, sequences encoded by the cDNA of FIG. 2, the peptides APPEDNPVED (SEQ ID NO:6), EEQQEVPPDT (SEQ ID NO:7), DGPTGEPQQE (SEQ ID NO:8), and EQENPDSSEPV (SEQ ID NO:9), and any fragment, or combination thereof. 1s Dated 6 May, 2008 ImmvaRx, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 1225080 I.JIN