WO1994028161A1

WO1994028161A1 - Antibodies specific for dcc gene product

Info

Publication number: WO1994028161A1
Application number: PCT/US1994/005277
Authority: WO
Inventors: Arthur Bruskin; David E. Jarosz; Karen Johnson; Kenneth W. Kinzler; Bert Vogelstein; James R. Zabrecky
Original assignee: The Johns Hopkins University
Priority date: 1993-05-26
Filing date: 1994-05-18
Publication date: 1994-12-08
Also published as: EP0700446A1; JPH09501045A; CA2163422A1

Abstract

Antibodies are disclosed which are able to detect the DCC protein in biological samples, despite the extremely low level of expression of DCC. Immunological methods for detecting DCC alterations are also disclosed. These reagents and methods allow detection of DCC mutations without the need to analyze this very large gene in individual test samples.

Description

ANTIBODIES SPECIFIC FOR DCC GENE PRODUCT

This invention was made using U.S. Government funds (NIH CA-43460). The government retains certain rights in this invention.

TECHNICAL FEELP OF THE INVENTION

This invention relates to the area of cancer diagnostics. In particular, it relates to immunological methods and tools for determining expression of the tumor suppressor gene DCC.

BACKGROUND OF THE INVENTION

The development of human cancer has been proposed to be a multi-step process (Foulds, Cancer Res. 17:355-356 (1957); Nowell, Cancer Res. 46:2203 (1985)). Colorectal tumorigenesis provides an excellent model system to test this multi-step hypothesis at the molecular level (Vogelstein, et al., New Eng. J. Med. 319:525 (1988)). Colorectal tumors progress through a continuum of histopathologically identifiable states, proceeding from normal epithelium, through benign tumors called adenomas, to the malignant stage known as carcinomas (Sugarbaker, Colorectal cancer: Principles and practices of Oncology (ed. V.T. Devita, S. Hellman, and S.A. Rosenberg) 2nd edition pg. 800. J.B. Lippincott, Philadelphia). Several genes have now been identified which are altered during this progression. They include an oncogene (K-ras) (Bos, et al., Nature 327:293 (1987); Forrester, et al. , Nature 327:29% (1987)) and three tumor suppressor genes (p53, DCC and APC) (Baker, et al., Science 244:211 (1989); Fearon, et al., Science 247:49 (1990); Kinzler, et al., Science 253:661 (1991); Groden, et al., Cell 66:589 (1991)) which are mutated in a preferred order during tumor progression. The tumor suppressor gene DCC (deleted in colorectal carcinoma), located on the long arm of chromosome 18, encodes a cell surface protein.

Frequent and consistent loss of heterozygosity (LOH) of specific chromosomes in human tumors has been associated with the presence of tumor suppressor genes contained within the region of loss (Knudson, Cancer Res. 45:1437 (1985); Friend, et al., Nature 323:643 (1986); Baker, et al., Science 244:211 (1989)). Based on this association, a search for potential tumor suppressor genes important in the development of colorectal cancer was pursued by analyzing all the non-acrocentric chromosomal arms in colorectal tumor DNA samples for loss of heterozygosity. This study revealed loss of heterozygosity on several chromosomal arms in a significant number of the colorectal tumors. One, the long arm of chromosome 18, was lost in approximately 45% of late adenomas and 75% of carcinomas (Vogelstein, et al., New Eng. J. Med. 319:525 (1988)). To delineate further the critical region of loss, eight carcinomas, which had lost part but not all of 18q, were examined extensively with numerous probes spanning the length of the long arm of chromosome 18. One common region of deletion was centered at 18q21.3. Probes from genes and anonymous DNA segments which had been previously shown to map to this region were used to screen colorectal carcinoma DNA samples for possible alterations. One anonymous probe, pl5-65, detected a homozygous deletion in one colorectal carcinoma. Homozygous deletions are rare and have been noted in tumors at loci encoding tumor suppressor genes (Wolf and Rotter, Proc. Nat'l. Acad. Sci. U.S.A. 82:190 (1985)). These results strongly suggested that p15-65 was within, or at least very near, a potential tumor suppressor gene.

Consequently, pl5-65 was used as a starting point for a bidirectional chromosomal walk which eventually encompassed 370 kb of contiguous genomic DNA. The walk generated 117 Eco RI fragments which were used as probes in cross-species Southern blot analysis. Twenty-four of the fragments hybridized to at least one of the species tested. Rat and human homologues from seven of the evolutionarily conserved fragments were cloned and sequenced. The sequences were examined for the presence of open reading frames as well as for 5' and 3' splice donor and acceptor sites, which would indicate possible exons. A sensitive expression assay for mRNA was developed using the polymerase chain reaction to amplify cDNA. These studies revealed expression of two of the candidate exons in most normal tissues, including colonic mucosa. In contrast, 15 to 17 colorectal cancers failed to express the exons. The gene containing these exons was named DCC (for deleted in colorectal carcinoma), and the results were consistent with the possibility that DCC represented a target tumor suppressor gene on chromosome 18q (Fearon, et al., Science 247:49 (1990)). The cDNA was cloned and the nucleic acid sequence of DCC was found to predict a 1447 amino acid transmembrane protein with a unique cytoplasmic domain and an extracellular portion with significant homology to neural cell adhesion molecules and other cell surface glycoproteins (Figure 1). This was an intriguing finding, as numerous studies of human cancers have revealed abnormalities in cell-cell relationships, including cell-cell and cell-extracellular matrix adhesion, loss of inhibition of growth by cell contact (Todaro and Green, J. Cell Biol. 17:299 (1963)), and disorganized tissue architecture (Cotran, et al., Robbins Pathologic Basis of Disease 4th Edition pp. 244, W.B. Saunders Company, Philadelphia (1989)).

Somatic mutations of putative tumor suppressor genes in human tumors have been used as evidence for their importance in the development of the tumors analyzed (Baker, et al., Science 244:211 (1989)). The DCC gene has been shown to be somatically altered in colorectal carcinomas by a variety of mechanisms, including deletions, insertions and point mutations (Fearon, et al., Science 247:49 (1990)). The search for additional mutations is ongoing. There is a need in the art for additional tools and methods for assessing somatic alterations of the DCC gene.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a preparation of antibodies for detecting somatic alterations of the DCC gene.

It is another object of the invention to provide an immunological method for determining mutations in a human DCC gene. It is yet another object of the invention to provide solid supports for use in performing enzyme-linked immunosorbent assays.

It is still another object of the invention to provide hybridomas which produce antibodies which are useful in the determining the somatic alterations of the DCC gene.

These and other objects of the invention are provided by one or more embodiments described below. In one embodiment of the invention an antibody preparation is provided which consists essentially of antibodies which: (1) are specifically immunoreactive with an epitope on the extracellular domain or on the cytoplasmic domain of DCC protein and (2) do not cross-react with neural cell adhesion molecules.

In another embodiment of the invention a method for detecting mutations in a human DCC gene is provided. The method comprises the steps of: extracting proteins from a sample selected from the group consisting of a tissue and a body fluid; separating said extracted proteins on a polyacrylamide gel; blotting said separated proteins onto a filter; contacting said filter with antibodies which: (1) are specifically immunoreactive with DCC protein and (2) do not cross-react with neural cell adhesion molecules, to bind said antibodies to proteins blotted on said filter; detecting the antibodies which bind to said proteins, wherein the absence of binding of antibodies to a protein having the size of DCC indicates the presence of a DCC mutation in the sample.

In one more embodiment of the invention a method for determining the presence in a human of DCC mutations is provided. The method comprises the steps of: contacting a body sample of a human with a first antibody which: (1) is specifically immunoreactive with an epitope of DCC and (2) does not cross-react with neural cell adhesion molecules to bind components of said body sample to said antibody; determining the amount of antibody which is bound to said body sample, wherein an absence of binding of said antibody to said body sample indicates the presence of a DCC mutation in the human.

In yet another embodiment of the invention a solid support for use in performing enzyme-linked immunosorbent assays (ELISA) is provided. The solid supports are coated with an antibody: (1) which is specifically immunoreactive with an epitope contained within the extracellular domain of DCC and (2) does not cross-react with neural cell adhesion molecules.

In still another embodiment of the invention a hybridoma cell is provided. The hybridoma cell secretes an antibody: (1) which is specifically immunoreactive with an epitope contained within the extracellular or the intracellular domain of DCC and (2) does not cross-react with neural cell adhesion molecules.

The present invention thus provides the art with immunological methods and reagents for assessing the mutational status of DCC in human tissues.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present invention that antibodies can be isolated which are immunologically specific for DCC protein. DCC was known to have substantial homology to NCAMs (neural cell adhesion molecules); nonetheless antibodies have been found which react with DCC but not with NCAMs. Such antibodies can be used diagnostically. It has been determined that DCC is expressed in one type of differentiated cells in normal colonic epithelium, the goblet cells. However, DCC expression is lost during the progression of a subset of colorectal tumors. (The non-mucinous carcinomas and late stage adenomas lose DCC expression.) The ability of antibodies to detect DCC expression in normal colonic cells is unexpected, since no DCC specific mRNA could be detected in such cells by Northern blotting. The antibodies of the present invention can be used to test colonic samples for expression of DCC. Lack of DCC expression is associated with a poorer prognosis, as well as being indicative of the stage of the colorectal tumor.

The 5 '-end of the open reading frame (ORF) of DCC has an initial hydrophobic sequence of 25 amino acids, suggestive of a signal sequence found in membrane-bound proteins, followed by 725 amino acids with significant homology to neural cell adhesion molecules (NCAMs) and other related cell surface glycoproteins. The DCC protein also contains an 1100 amino acid extracellular domain of four immunoglobulin-like C2 domains and six fibronectin type III domains, and an intracellular (cytoplasmic) domain of 324 amino acids. The cytoplasmic domain has no significant homology to any sequence in the

Genbank data base.

Polyclonal antibodies have been generated against bacterial fusion proteins containing various parts of the DCC cDNA sequence. Iodination of cell surface proteins by the lactoperoxidase method, followed by immunoprecipitation with anti-DCC antibodies has documented cell surface labeling of DCC. Immunocytochemistry of transfected cell lines expressing high levels of DCC demonstrates a diffuse membrane staining pattern with areas of cell-cell contact showing intense staining. This suggests that higher protein concentrations exist locally at these cellular contact sites.

According to one aspect of the invention antibodies are provided. Two types of antibodies have been prepared which are useful diagnostically. One type is specifically immunoreactive with an epitope on the extracellular domain of DCC protein, i.e., amino acids 26-1126 (SEQ ID NO:2), but does not cross-react with neural cell adhesion molecules. The other type is specifically immunoreactive with an epitope contained within the intracellular domain of DCC, i.e., amino acids 1123-1447 (SEQ ID NO:2) and also does not cross-react with neural cell adhesion molecules. One portion of the DCC gene which encodes part of the extracellular domain consists of base pairs 1195-2854 (SEQ ID NO:1). Another portion comprises base pairs 1-3189 (SEQ ID NO:1). Chimeric genes can be constructed which contain these or other portions of DCC. The fusion proteins can be expressed and purified according to known means. The fusion proteins can be used to immunize animals to raise either polyclonal or monoclonal antibodies. Two monoclonal antibodies according to the present invention are made by the hybridoma cell lines AF5 and AF1, which have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 (ATCC) as HB 11289 and 11299, respectively. Other cell lines may be isolated which secrete antibodies which have the same epitopic specificity as these cell lines. These may be antibodies which are isotype switch variants, anti-anti-idiotypic antibodies, or independently derived antibodies. See, e.g., Spira et al., (1985) "The Generation of Better Monoclonal Antibodies Through Somatic Mutations", Hybridoma Technology in the Biosciences and Medicine (ed. Springer), pp. 77-78, Plenum Press, N.Y.

According to the present invention a number of different immunological assays can be performed. One such assay is a Western Blot in which proteins are extracted from a test sample. The test sample may be either a tissue, cancerous or normal, or a body fluid, such as serum, blood, urine, sputum, saliva, feces etc. Any antibody which is specifically immunoreactive with DCC protein can be used. If a protein having the size of DCC is not present on the Western blot, then a DCC mutation is indicated in the human sample donor. Although DCC has a predicted molecular weight of 153 kd, a variety of larger bands have been observed, such as a 180 kd and a 190 kd protein. This variation in size is attributed to N-glycosylation.

Other immunological assay formats can also be used. These include immunohistochemistry, ELISA, immunoprecipitation. A body sample is tested for immunoreactivity with an antibody which is specifically immunoreactive with an epitope of DCC and which does not cross-react with neural cell adhesion molecules. The body sample can comprise a fixed cell sample, such as peripheral blood mononuclear cells, a fixed or frozen tissue section, an extract of cells or tissues, a body fluid, such as serum, blood, urine, sputum, saliva, feces etc. The body sample and the antibody are contacted under conditions where antibodyantigen complexes form and are stable. Such conditions are known in the art. Under some assay conditions it may be desirable to use an antibody which binds to an epitope which is contained within the extracellular domain of DCC. For other assay conditions, it may be desirable to use an antibody which binds to an epitope which is contained within the intracellular domain of DCC. It appears that the latter type of antibody is preferred for immunohistochemistry on tissue sections. It may also be desirable that adjacent, normal tissue to the tissue sample to be tested be collected and tested. Thus the absence of binding in the test tissue sample can be confirmed as representing a DCC mutation, if the adjacent normal tissue can be shown to bind to anti-DCC antibodies. In the case of ELISA assays, either the antibody or the body sample may be attached to the solid support on which the assay is performed. According to one aspect of the invention, solid supports, which are typically microtiter dishes or dipsticks, are coated with an antibody which is specifically immunoreactive with DCC. The solid support may be any material which is typically used in immunological assays, including plastics, glass, or other polymeric substances. The form of the support may be sheets, wells, tubes, beads, fabrics, etc.

EXAMPLES EXAMPLE 1

This example describes the preparation of polyclonal anti-DCC antibodies.

Two rabbit polyclonal antibodies were raised against bacterial fusion proteins containing non-overlapping epitopes of DCC, using selected portions of the DCC cDNA. One antibody was raised to a 1.65 kb portion of the extracellular domain (nucleotides 591-1239, SEQ ID NO:1) and the other was raised to the entire cytoplasmic domain (nucleotides 3309-4453, SEQ ID NO: 1). The bacterial fusion proteins were constructed in pATH vectors (Kinzler, et al., Molecular and Cellular Biology, 10: 649-642, 1990) with the non-overlapping portions of the DCC cDNA. The fusion proteins were isolated from SDS-polyacrylamide gels and injected subcutaneously in rabbits. The antisera were affinity purified using bacterial fusion proteins from constructs in pGEMEX expression vectors.

EXAMPLE 2

This example describes the screening of cell lines and tissues for those which express detectable levels of the DCC gene product and also describes the determination of its cellular localization.

Tissue culture cell lines were metabolically labelled with ³⁵S-methionine for four hours at 37°C. The cells were lysed in RIPA buffer (10 mM Tris(pH 7.5) and 0.5% SDS) under denaturing conditions. Approximately 200×10⁶ counts were immunoprecipitated with affinity purified anti-DCC antibodies and Protein A sepharose beads. The samples were analyzed by SDS-PAGE.

For Western blots, cell lines and tissues were solubilized in Laemmli's sample buffer and 100-200 μg of protein were loaded per lane in SDS- polyacrylamide gels. The proteins were transferred to nitrocellulose with a semidry electroblot transfer apparatus. The filters were blocked with 10% goat serum and 10% non-fat dried milk in Tris-buffered saline and incubated with affinity purified anti-DCC antibodies for 120 minutes at room temperature. The Amersham enhanced chemiluminescence system was employed for detection.

Frozen tissue was cryostat sectioned at 7μm thickness for immunohistochemistry. The sections were fixed for 30 minutes at room temperature in 10% buffered formalin. Endogenous peroxidase activity was blocked at incubation in 0.3% H₂O₂ and signal intensity was increased using the antigen retrieval system from Biogenex. The sections were blocked with goat serum followed by incubation with 3 ng/μl of anti-DCC antibody. A secondary goat anti-rabbit biotinylated antibody, in conjunction with the Vectastain Elite reagent from Vector Laboratories, was used for detection.

In an initial screen of thirty-two cell lines by immunoprecipitation, DCC expression was detected in only one cell line, 577MF, a non-seminomatous testicular germ cell tumor. Both antibodies precipitated a protein of approximately 180 kd from metabolically labelled 577MF cells, providing strong evidence that it represents the DCC gene product. The sequence of the full length DCC cDNA predicts an unmodified transmembrane molecule of approximately 153 kd. The sequence also suggests that DCC could be N-glycosylated (Fearon, et al., Science 247:49 (1990)), possibly explaining the larger apparent molecular weight observed in 577MF. All of the colorectal carcinoma cell lines tested by immunoprecipitation were negative, correlating with previous RNA expression assays in which DCC RNA was not detectable by RNase protection, but was detected by reverse-transcription polymerase chain reaction.

Immunoprecipitation of 577MF cells labelled by cell surface iodination demonstrated that the DCC protein was present on the cell surface. To investigate further the cellular location of the DCC product, CHO cells transfected with a constitutive expression vector containing the full length DCC cDNA were stained with DCC antibodies. The pattern of staining was consistent with a diffuse membrane localization of DCC. In addition, the signal showed increased intensity at cell-cell contact sites. This pattern of staining has been reported in cell lines expressing molecules known to be involved in cell adhesion. These studies indicate that DCC is a cell surface molecule and suggest that it may be concentrated at sites of cell-cell contact.

Tissue distribution of DCC expression was examined by Western blot analysis. Expression studies on normal tissues by reverse transcription polymerase chain reaction revealed that the highest level of expression was in brain. By Western blot analysis, a faint, but detectable, band was present at approximately 180 kd in human brain tissue lysates; however, DCC was not detected in any other tissues. We sought to increase the sensitivity of our expression assay by employing immunohistochemistry.

The expression studies indicate that expression of DCC in cell lines and in tissues is extremely low. However, the previous analyses were performed with total cellular mRNA or total cellular protein. If DCC expression were locally expressed at high concentrations at either intra- or inter-cellular locations it might be more readily detected by in situ methods. We tested this hypothesis on tissue from the central nervous system, based on the earlier mRNA expression data. Staining of frozen human spinal cord sections demonstrated strong staining in axons at several levels of the spinal cord. DCC expression appeared to be present in both myelinated and non-myelinated nerve cells. Subsequent examination of brain revealed a similar specificity, with pronounced staining limited to nerve tracks predominantly in the white matter. Some nerve cell bodies were also stained, particularly Puriάnje cells in the cerebellum. The expression of DCC in Purkinje cells was verified by in situ hybridization using DCC antisense RNA probes. This technique was then applied to tissues. Although faint staining of occasional cells was observed in several tissues (including bladder and endometrium) the most pronounced staining was found in colon and skin. In the skin, only the more differentiated keritinocytes stained.

In the colon, immunohistochemical staining localized detectable DCC expression to a small subset of colonic epithelial cells, specifically the goblet cell population. Normal colonic mucosa is composed of epithelial gland like structures called crypts, surrounded by the supporting lamina propria which contains fibroblasts and inflammatory cells. The colonic crypts contain two major differentiated cell types: absorptive cells, called enterocytes, responsible for fluid and electrolyte transport; and secretory cells, known as goblet cells, which produce and secrete mucin. The goblet cells are the most conspicuous cell type because of their large apical intracellular mucin accumulation. However, they only account for approximately 20-30% of the conic crypt epithelium (Hafez, Cell Growth and Differentiation 1:611 (1990)).

The expression pattern of DCC within the goblet cells is specific. The signal is localized to the basolateral aspect of the cell and in the supranuclear cytoplasmic space, just below the apical mucin (data not shown). It was not possible to determine with certainty whether DCC was localized to the cell membrane in these sections, but the expression pattern is consistent with that possibility. In addition, the supranuclear staining may represent nascent protein in the golgi apparatus where nascent protein is known to be localized (Bloom and Fawcett, A Textbook of Histology, pg. 662, W.B. Saunders, Philadelphia (1975)).

Pre-immune serum did not stain any of the reactive cells described above. As a further control for specificity, the analyses were performed in the presence of soluble fusion proteins as competitors. DCC fusion protein at the same concentration as the DCC antibody completely blocked the signal detected by the antibody in the goblet cells, whereas an irrelevant fusion protein, even at five-fold the antibody concentration, failed to reduce the signal.

The restriction of DCC expression to a subset of differential epithelial cells in the colon raises many questions. From a standpoint of colorectal tumorigenesis the pattern is interesting because colorectal tumors commonly lose cells with the goblet morphology and lose normal mucin production as they progress (Boland, Proc. Nat'l. Acad. Sci. U.S.A. 79:2051 (1982).

EXAMPLE 3

This example demonstrates the status of expression of the DCC gene product in tissue sections from colorectal tumors at different stages during its progression. Immunohistochemistry was then used to investigate DCC protein expression in various tumors of the colorectum. We first examined hyperplastic polyps. These tumors, as their name implies, consist of hyperproliferating cells that are not neoplastic. They retain a relatively normal architectural pattern and features indicative of cellular differentiation. Although epidemiological studies have suggested an association of hyperplastic polyps with colorectal cancer, there is little evidence that they are precursors to cancer. Relatively intense staining was observed in these polyps with the anti-DCC antibody in all of the epithelial cells within these polyps. In addition, the staining within each cell had a different pattern than that observed in the goblet cells of the normal colonic epithelium. In the cells of the hyperplastic polyp, the staining was not confined to the basolateral regions but appeared to be dispersed over the entire cell. Normal epithelial cells adjacent to the polyps showed the expected distribution of DCC staining confined to the goblet cells. Thus, DCC expression was increased rather than decreased in these non-neoplastic but proliferative and differentiating cells.

We then examined adenomatous polyps, generally considered to be the direct precursors of colorectal cancers. Early and intermediate stage adenomas retained expression of DCC in a largely normal pattern, limited to goblet cells. Two late adenomas with significant dysplasia revealed undetectable levels of DCC protein, with complete absence of staining in the neoplastic epithelium. Six invasive colorectal carcinomas were next examined. Four of the carcinomas completely lacked detectable DCC staining. However, two of the carcinomas stained intensely for DCC.

The relationship between DCC expression and mucin differentiation in the various colonic tumors was striking. Alcian Blue (AB), a histochemical stain which specifically stains mucins, was used to counterstain several of the tissue sections described above. Colocalization of AB staining cells and DCC reactivity was obvious in the normal colon. In hyperplastic polyps, increased AB staining was associated with elevated DCC expression. In adenomatous polyps and cancers, DCC reactivity precisely correlated with the AB staining. Most colorectal cancers were poorly differentiated, exhibited low or absent AB staining, and no DCC antibody reactivity. Early and intermediate adenomas co-ordinately stained with AB and DCC antibodies.

The two DCC positive carcinomas were found with AB staining to represent "mucinous carcinomas", an uncommon type of CRC which produces abundant mucus. The results, in toto, suggested that most colorectal cancers lose DCC expression concomitant with loss of differentiation towards the mucin producing pathway, but occasionally this loss did not occur. To further evaluate this issue, we retrospectively analyzed 49 cases of colorectal cancers for loss of DCC alleles and histologic phenotype. Seven mucinous tumors were identified in these 50 cases, and in only 1 of the 7 was there evidence of DCC loss. In contrast, in the 43 non-mucin tumors, DCC LOH (loss of heterozygosity) was observed in 34 (79%). These results suggest that there are at least two colorectal tumorigenesis pathways: one which is associated with DCC loss and the failure to differentiate towards mucin-producing cells, and the less common mucinous type unassociated with DCC alteration.

EXAMPLE 4

This example demonstrates the production of monoclonal antibodies which are specifically immunoreactive with the extracellular portion of DCC.

An expression construct was generated by first introducing a translation termination codon into a full length DCC cDNA contained in a plasmid vector for in vivo recombination into vaccinia virus, such that the open reading frame was interrupted at codon 1063. In vivo recombination with this plasmid resulted in a recombinant virus (vAbT 492) which expresses amino acids 1-1063 (the entire extracellular domain except for the C-terminal 34 amino acids) of DCC with the addition of a single serine residue at the carboxy terminus. Lacking its membrane anchor but still possessing the signal sequence, the truncated DCC gene product is secreted.

Briefly, a 4.5 kb Xho I - Eco RI fragment, containing the entire DCC coding region, was cloned into a HindIII M insertion vector for in vivo recombination into vaccinia virus. A universal translational termination oligonucleotide (Pharmacia) was inserted into the Bal I site at nucleotide 3211. Insertion into the correct Bal I site was confirmed by restriction analysis with Ase I. The resulting plasmid encodes the sequence of amino acids 1-1063 of DCC whose expression is regulated by the vaccinia virus 40k promoter.

In order to verify expression and secretion of the extracellular domain of the DCC protein, Vero cells were infected with vAbT 492 at a MOI of 10 in serum-free media. After 24 hr, the media was collected, clarified by centrifugation and analyzed by SDS-PAGE. Vero cells infected with NYCBH (Lyons et al., Infect, and Immun. 58:4089-4098 (1990); ATCC VR-325) served as the control. Comparison of the conditioned media revealed the presence of an additional protein of apparent molecular mass of 150 kDa in the media from vAbT 492 infected cells. The difference between the calculated molecular mass of the amino acid backbone (115 kDa) and the observed apparent molecular mass is most likely due to glycosylation. The extracellular domain of DCC protein (gp150^DCC) was expressed as a major component of the conditioned media at levels suitable for proceeding with purification.

Since a biochemical function of the DCC protein is still unknown, it was not possible to develop a quantitative assay for gp150^DCC. Therefore, throughout purification, the presence of the protein at 150 kDa was monitored by SDS-PAGE and immunoblot.

Lectin affinity chromatography was attempted as a first step in purification of gp150^DCC because the structurally related glycoprotein, NCAM, has been shown to bind to lectins. Con A and wheat germ lectins were tested for their ability to bind gp150^DCC. Although gp150^DCC bound to both affinity matrices, binding was more efficient and specific to Con A.

gp150^DCC from 100-fold-concentrated, conditioned media from vAbT 492 infected Vero cells was efficiently bound to and eluted from a Con A agarose column. Binding of gp150^DCC appeared to be complete after the first pass through the column. Elution with 0.5 M methyl-D-mannopyranoside resulted in substantial enrichment of gp150^DCC with only a few contaminating proteins of lower apparent molecular weight. Since gp150^DCC eluted slowly over several fractions the elution was followed with a wash of one column volume of 8 M urea to determine if any gp150^DCC remained bound to the column. By SDS-PAGE, no additional gp150^DCC was detected in the urea wash. However, a protein of ~ 30 kDa which presumably represented Con A monomer that had been stripped from the column was detected. This indicated that all of the bound gp150^DCC had been effectively eluted by the methyl-D-mannopyranoside solution.

In order to separate gp150^DCC from the major contaminants of lower apparent molecular weight, individual fractions from the Con A column were further purified by HPLC size exclusion chromatography. SDS-PAGE demonstrated that the peak between 15 and 17 min represented purified gp150^DCC and that the lower molecular weight contaminants were well resolved. The gp150^DCC preparation was substantially purified and appeared to contain only a single minor contaminating protein.

The purified protein was subjected to N-terminal sequence analysis to verify that it was the extracellular domain of the DCC gene product. The sequence generated from the first 15 cycles exactly match the sequence predicted from the cDNA starting at Phe-32. This specifies the N-terminus of the processed DCC gene product in Vero cells and confirms the presence of a signal sequence of 31 amino acids. The result is in agreement with the predicted N-terminus based on hydrophobicity analysis and genomic structure.

Data from N-terminal sequence analysis was used to estimate total protein based on the yield from the initial cycle. Extrapolating from the proportion of material processed, the yield of purified gp150^DCC from a 40 roller bottle prep was estimated to be greater than 250 ug. This provided sufficient quantities for use as an antigen for monoclonal antibody development.

Mice were immunized with the purified, secreted DCC protein fragment and their spleen cells fused with myeloma cells, according to known techniques.

The initial hybridomas were screened by ELISA on the Con A-purified DCC. All positives were transferred to 48-well plates, re-grown, and re-screened by ELISA on Con A purified DCC that had been further purified by HPLC size exclusion chromatography. All positives were single cell cloned (primary cloning) and single clones were screened on HPLC-purified DCC. Positive primary clones went through a secondary cloning and screening. Three such clones were AF5,

AE6, and AF1.

Monoclonal antibodies were characterized by western blot and immunoprecipitation on lysates of SW 480 cells transfected with full length or extracellular DCC, CHO Kl cells transfected with extracellular DCC, and BSC-40 cells infected with recombinant vaccinia virus expressing full length DCC.

For the Western Blots, 100 μg each of the SW 480 lysates, along with 60 μg of the recombinant vaccinia lysate were separated by SDS-PAGE and transferred to nitrocellulose. The blots were probed with undiluted hybridoma supematants. For immunoprecipitation, the CHO Kl cells and the BSC-40 cells infected with recombinant vaccinia virus were metabolically labeled with ³⁵S-methionine. The lysate was then precipitated using 5 μg of each purified antibody.

577 MF is a human cell line which expresses a 190 kD, endogenous form of DCC. A lysate of these cells was immunoprecipitated with 10 μg of each purified antibody. The precipitations were then separated by SDS-PAGE and transferred to nitrocellulose. The blot was probed with 10 ug/ml of purified antibody. In this format, all 3 monoclonal antibodies are able to precipitate the 190 kD DCC produced by 577 MF.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Bruskin, Arthur

Jarosz, David E.

Johnson, Karen

Kinzler, Kenneth W.

Vogelstein, Bert

Zabrecky, James R.

(ii) TITLE OF INVENTION: Antibodies Specific for DCC Gene Product

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Banner, Birch, McKie & Beckett

(B) STREET: 1001 G Street, N.W.

(C) CITY: Washington

(D) STATE: D.C

(E) COUNTRY: USA

(F) ZIP: 20001

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kagan, Sarah A.

(B) REGISTRATION NUMBER: 32,141

(C) REFERENCE/DOCKET NUMBER: 01107.42709

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202.508.9100

(B) TELEFAX: 202.508.9299

(C) TELEX: 197430 BBMB UT

(2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4608 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(viii) POSITION IN GENOME:

(A) CHROMOSOME/SEGMENT: 18q21 (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..4342

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

ATG GAG AAT AGT CTT AGA TGT GTT TGG GTA CCC AAG CTG GCT TTT GTA 48 Met Glu Asn Ser Leu Arg Cys Val Trp Val Pro Lys Leu Ala Phe Val

1 5 10 15

CTC TTC GGA GCT TCC TTG CTC AGC GCG CAT CTT CAA GTA ACC GGT TTT 96 Leu Phe Gly Ala Ser Leu Leu Ser Ala His Leu Gln Val Thr Gly Phe

20 25 30

CAA ATT AAA GCT TTC ACA GCA CTG CGC TTC CTC TCA GAA CCT TCT GAT 144 Gln Ile Lys Ala Phe Thr Ala Leu Arg Phe Leu Ser Glu Pro Ser Asp

35 40 45

GCC GTC ACA ATG CGG GGA GGA AAT GTC CTC CTC GAC TGC TCC GCG GAG 192 Ala Val Thr Met Arg Gly Gly Asn Val Leu Leu Asp Cys Ser Ala Glu

50 55 60

TCC GAC CGA GGA GTT CCA GTG ATC AAG TGG AAG AAA GAT GGC ATT CAT 240 Ser Asp Arg Gly Val Pro Val Ile Lys Trp Lys Lys Asp Gly Ile His

65 70 75 80

CTG GCC TTG GGA ATG GAT GAA AGG AAG CAG CAA CTT TCA AAT GGG TCT 288 Leu Ala Leu Gly Met Asp Glu Arg Lys Gln Gln Leu Ser Asn Gly Ser

85 90 95

CTG CTG ATA CAA AAC ATA CTT CAT TCC AGA CAC CAC AAG CCA GAT GAG 336 Leu Leu Ile Gln Asn Ile Leu His Ser Arg His His Lys Pro Asp Glu

100 105 110

GGA CTT TAC CAA TGT GAG GCA TCT TTA GGA GAT TCT GGC TCA ATT ATT 384 Gly Leu Tyr Gln Cys Glu Ala Ser Leu Gly Asp Ser Gly Ser Ile Ile

115 120 125

AGT CGG ACA GCA AAA GTT GCA GTA GCA GGA CCA CTG AGG TTC CTT TCA 432 Ser Arg Thr Ala Lys Val Ala Val Ala Gly Pro Leu Arg Phe Leu Ser

130 135 140

CAG ACA GAA TCT GTC ACA GCC TTC ATG GGA GAC ACA GTG CTA CTC AAG 480 Gln Thr Glu Ser Val Thr Ala Phe Met Gly Asp Thr Val Leu Leu Lys

145 150 155 160

TGT GAA GTC ATT GGG GAG CCC ATG CCA ACA ATC CAC TGG CAG AAG AAC 528 Cys Glu Val Ile Gly Glu Pro Met Pro Thr Ile His Trp Gln Lys Asn

165 170 175

CAA CAA GAC CTG ACT CCA ATC CCA GGT GAC TCC CGA GTG GTG GTC TTG 576 Gln Gln Asp Leu Thr Pro Ile Pro Gly Asp Ser Arg Val Val Val Leu

180 185 190

CCC TCT GGA GCA TTG CAG ATC AGC CGA CTC CAA CCG GGG GAC ATT GGA 624 Pro Ser Gly Ala Leu Gln Ile Ser Arg Leu Gln Pro Gly Asp Ile Gly

195 200 205

ATT TAC CGA TGC TCA GCT CGA AAT CCA GCC AGC TCA AGA ACA GGA AAT 672 Ile Tyr Arg Cys Ser Ala Arg Asn Pro Ala Ser Ser Arg Thr Gly Asn

210 215 220

GAA GCA GAA GTC AGA ATT TTA TCA GAT CCA GGA CTG CAT AGA CAG CTG 720 Glu Ala Glu Val Arg Ile Leu Ser Asp Pro Gly Leu His Arg Gln Leu 225 230 235 240

TAT TTT CTG CAA AGA CCA TCC AAT GTA GTA GCC ATT GAA GGA AAA GAT 768 Tyr Phe Leu Gln Arg Pro Ser Asn Val Val Ala Ile Glu Gly Lys Asp

245 250 255

GCT GTC CTG GAA TGT TGT GTT TCT GGC TAT CCT CCA CCA AGT TTT ACC 816 Ala Val Leu Glu Cys Cys Val Ser Gly Tyr Pro Pro Pro Ser Phe Thr

260 265 270

TGG TTA CGA GGC GAG GAA GTC ATC CAA CTC AGG TCT AAA AAG TAT TCT 864 Trp Leu Arg Gly Glu Glu Val Ile Gln Leu Arg Ser Lys Lys Tyr Ser

275 280 285

TTA TTG GGT GGA AGC AAC TTG CTT ATC TCC AAT GTG ACA GAT GAT GAC 912 Leu Leu Gly Gly Ser Asn Leu Leu Ile Ser Asn Val Thr Asp Asp Asp

290 295 300

AGT GGA ATG TAT ACC TGT GTT GTC ACA TAT AAA AAT GAG AAT ATT AGT 960 Ser Gly Met Tyr Thr Cys Val Val Thr Tyr Lys Asn Glu Asn Ile Ser

305 310 315 320

GCC TCT GCA GAG CTC ACA GTC TTG GTT CCG CCA TGG TTT TTA AAT CAT 1008 Ala Ser Ala Glu Leu Thr Val Leu Val Pro Pro Trp Phe Leu Asn His

325 330 335

CCT TCC AAC CTG TAT GCC TAT GAA AGC ATG GAT ATT GAG TTT GAA TGT 1056 Pro Ser Asn Leu Tyr Ala Tyr Glu Ser Met Asp Ile Glu Phe Glu Cys

340 345 350

ACA GTC TCT GGA AAG CCT GTG CCC ACT GTG AAT TGG ATG AAG AAT GGA 1104 Thr Val Ser Gly Lys Pro Val Pro Thr Val Asn Trp Met Lys Asn Gly

355 360 365

GAT GTG GTC ATT CCT AGT GAT TAT TTT CAG ATA GTG GGA GGA AGC AAC 1152 Asp Val Val Ile Pro Ser Asp Tyr Phe Gln Ile Val Gly Gly Ser Asn

370 375 380

TTA CGG ATA CTT GGG GTG GTG AAG TCA GAT GAA GGC TTT TAT CAA TGT 1200 Leu Arg Ile Leu Gly Val Val Lys Ser Asp Glu Gly Phe Tyr Gln Cys

385 390 395 400

GTG GCT GAA AAT GAG GCT GGA AAT GCC CAG ACC AGT GCA CAG CTC ATT 1248 Val Ala Glu Asn Glu Ala Gly Asn Ala Gln Thr Ser Ala Gln Leu Ile

405 410 415

GTC CCT AAG CCT GCA ATC CCA AGC TCC AGT GTC CTC CCT TCG GCT CCC 1296 Val Pro Lys Pro Ala Ile Pro Ser Ser Ser Val Leu Pro Ser Ala Pro

420 425 430

AGA GAT GTG GTC CCT GTC TTG GTT TCC AGC CGA TTT GTC CGT CTC AGC 1344 Arg Asp Val Val Pro Val Leu Val Ser Ser Arg Phe Val Arg Leu Ser

435 440 445

TGG CGC CCA CCT GCA GAA GCG AAA GGG AAC ATT CAA ACT TTC ACG GTC 1392 Trp Arg Pro Pro Ala Glu Ala Lys Gly Asn Ile Gln Thr Phe Thr Val

450 455 460

TTT TTC TCC AGA GAA GGT GAC AAC AGG GAA CGA GCA TTG AAT ACA ACA 1440 Phe Phe Ser Arg Glu Gly Asp Asn Arg Glu Arg Ala Leu Asn Thr Thr

465 470 475 480 CAG CCT GGG TCC CTT CAG CTC ACT GTG GGA AAC CTG AAG CCA GAA GCC 1488 Gln Pro Gly Ser Leu Gln Leu Thr Val Gly Asn Leu Lys Pro Glu Ala

485 490 495

ATG TAC ACC TTT CGA GTT GTG GCT TAC AAT GAA TGG GGA CCG GGA GAG 1536 Met Tyr Thr Phe Arg Val Val Ala Tyr Asn Glu Trp Gly Pro Gly Glu

500 505 510

AGT TCT CAA CCC ATC AAG GTG GCC ACA CAG CCT GAG TTG CAA GTT CCA 1584 Ser Ser Gln Pro Ile Lys Val Ala Thr Gln Pro Glu Leu Gln Val Pro

515 520 525

GGG CCA GTA GAA AAC CTG CAA GCT GTA TCT ACC TCA CCT ACC TCA ATT 1632 Gly Pro Val Glu Asn Leu Gln Ala Val Ser Thr Ser Pro Thr Ser Ile

530 535 540

CTT ATT ACC TGG GAA CCC CCT GCC TAT GCA AAC GGT CCA GTC CAA GGT 1680 Leu Ile Thr Trp Glu Pro Pro Ala Tyr Ala Asn Gly Pro Val Gln Gly

545 550 555 560

TAC AGA TTG TTC TGC ACT GAG GTG TCC ACA GGA AAA GAA CAG AAT ATA 1728 Tyr Arg Leu Phe Cys Thr Glu Val Ser Thr Gly Lys Glu Gln Asn Ile

565 570 575

GAG GTT GAT GGA CTA TCT TAT AAA CTG GAA GGC CTG AAA AAA TTC ACC 1776 Glu Val Asp Gly Leu Ser Tyr Lys Leu Glu Gly Leu Lys Lys Phe Thr

580 585 590

GAA TAT AGT CTT CGA TTC TTA GCT TAT AAT CGC TAT GGT CCG GGC GTC 1824 Glu Tyr Ser Leu Arg Phe Leu Ala Tyr Asn Arg Tyr Gly Pro Gly Val

595 600 605

TCT ACT GAT GAT ATA ACA GTG GTT ACA CTT TCT GAC GTG CCA AGT GCC 1872 Ser Thr Asp Asp Ile Thr Val Val Thr Leu Ser Asp Val Pro Ser Ala

610 615 620

CCG CCT CAG AAC GTC TCC CTG GAA GTG GTC AAT TCA AGA AGT ATC AAA 1920 Pro Pro Gln Asn Val Ser Leu Glu Val Val Asn Ser Arg Ser Ile Lys

625 630 635 640

GTT AGC TGG CTG CCT CCT CCA TCA GGA ACA CAA AAT GGA TTT ATT ACC 1968 Val Ser Trp Leu Pro Pro Pro Ser Gly Thr Gln Asn Gly Phe Ile Thr

645 650 655

GGC TAT AAA ATT CGA CAC AGA AAG ACG ACC CGC AGG GGT GAG ATG GAA 2016 Gly Tyr Lys Ile Arg His Arg Lys Thr Thr Arg Arg Gly Glu Met Glu

660 665 670

ACA CTG GAG CCA AAC AAC CTC TGG TAC CTA TTC ACA GGA CTG GAG AAA 2064 Thr Leu Glu Pro Asn Asn Leu Trp Tyr Leu Phe Thr Gly Leu Glu Lys

675 680 685

GGA AGT CAG TAC AGT TTC CAG GTG TCA GCC ATG ACA GTC AAT GGT ACT 2112 Gly Ser Gln Tyr Ser Phe Gln Val Ser Ala Met Thr Val Asn Gly Thr

690 695 700

GGA CCA CCT TCC AAC TGG TAT ACT GCA GAG ACT CCA GAG AAT GAT CTA 2160 Gly Pro Pro Ser Asn Trp Tyr Thr Ala Glu Thr Pro Glu Asn Asp Leu

705 710 715 720

GAT GAA TCT CAA GTT CCT GAT CAA CCA AGC TCT CTT CAT GTG AGG CCC 2208 Asp Glu Ser Gln Val Pro Asp Gln Pro Ser Ser Leu His Val Arg Pro

725 730 735 CAG ACT AAC TGC ATC ATC ATG AGT TGG ACT CCT CCC TTG AAC CCA AAC 2256 Gln Thr Asn Cys Ile Ile Met Ser Trp Thr Pro Pro Leu Asn Pro Asn

740 745 750

ATC GTG GTG CGA GGT TAT ATT ATC GGT TAT GGC GTT GGG AGC CCT TAC 2304 Ile Val Val Arg Gly Tyr Ile Ile Gly Tyr Gly Val Gly Ser Pro Tyr

755 760 765

GCT GAG ACA GTG CGT GTG GAC AGC AAG CAG CGA TAT TAT TCC ATT GAG 2352 Ala Glu Thr Val Arg Val Asp Ser Lys Gln Arg Tyr Tyr Ser Ile Glu

770 775 780

AGG TTA GAG TCA AGT TCC CAT TAT GTA ATC TCC CTA AAA GCT TTT AAC 2400 Arg Leu Glu Ser Ser Ser His Tyr Val Ile Ser Leu Lys Ala Phe Asn

785 790 795 800

AAT GCC GGA GAA GGA GTT CCT CTT TAT GAA AGT GCC ACC ACC AGG TCT 2448 Asn Ala Gly Glu Gly Val Pro Leu Tyr Glu Ser Ala Thr Thr Arg Ser

805 810 815

ATA ACC GAT CCC ACT GAC CCA GTT GAT TAT TAT CCT TTG CTT GAT GAT 2496 Ile Thr Asp Pro Thr Asp Pro Val Asp Tyr Tyr Pro Leu Leu Asp Asp

820 825 830

TTC CCC ACC TCG GTC CCA GAT CTC TCC ACC CCC ATG CTC CCA CCA GTA 2544 Phe Pro Thr Ser Val Pro Asp Leu Ser Thr Pro Met Leu Pro Pro Val

835 840 845

GGT GTA CAG GCT GTG GCT CTT ACC CAT GAT GCT GTG AGG GTC AGC TGG 2592 Gly Val Gln Ala Val Ala Leu Thr His Asp Ala Val Arg Val Ser Trp

850 855 860

GCA GAC AAC TCT GTC CCT AAG AAC CAA AAG ACG TCT GAG GTG CGA CTT 2640 Ala Asp Asn Ser Val Pro Lys Asn Gln Lys Thr Ser Glu Val Arg Leu

865 870 875 880

TAC ACC GTC CGG TGG AGA ACC AGC TTT TCT GCA AGT GCA AAA TAC AAG 2688 Tyr Thr Val Arg Trp Arg Thr Ser Phe Ser Ala Ser Ala Lys Tyr Lys

885 890 895

TCA GAA GAC ACA ACA TCT CTA AGT TAC ACA GCA ACA GGC CTC AAA CCA 2736 Ser Glu Asp Thr Thr Ser Leu Ser Tyr Thr Ala Thr Gly Leu Lys Pro

900 905 910

AAC ACA ATG TAT GAA TTC TCG GTC ATG GTA ACA AAA AAC AGA AGG TCC 2784 Asn Thr Met Tyr Glu Phe Ser Val Met Val Thr Lys Asn Arg Arg Ser

915 920 925

AGT ACT TGG AGC ATG ACT GCA CAT GCC ACC ACG TAT GAA GCA GCC CCC 2832 Ser Thr Trp Ser Met Thr Ala His Ala Thr Thr Tyr Glu Ala Ala Pro

930 935 940

ACC TCT GCT CCC AAG GAC TTT ACA GTC ATT ACT AGG GAA GGG AAG CCT 2880 Thr Ser Ala Pro Lys Asp Phe Thr Val Ile Thr Arg Glu Gly Lys Pro

945 950 955 960

CGT GCC GTC ATT GTG AGT TGG CAG CCT CCC TTG GAA GCC AAT GGG AAA 2928 Arg Ala Val Ile Val Ser Trp Gln Pro Pro Leu Glu Ala Asn Gly Lys

965 970 975

ATT ACT GCT TAC ATC TTA TTT TAT ACC TTG GAC AAG AAC ATC CCA ATT 2976 Ile Thr Ala Tyr Ile Leu Phe Tyr Thr Leu Asp Lys Asn Ile Pro Ile

980 985 990 GAT GAC TGG ATT ATG GAA ACA ATC AGT GGT GAT AGG CTT ACT CAT CAA 3024 Asp Asp Trp Ile Met Glu Thr Ile Ser Gly Asp Arg Leu Thr His Gln

995 1000 1005

ATC ATG GAT CTC AAC CTT GAT ACT ATG TAT TAC TTT CGA ATT CAA GCA 3072 Ile Met Asp Leu Asn Leu Asp Thr Met Tyr Tyr Phe Arg Ile Gln Ala

1010 1015 1020

CGA AAT TCA AAA GGA GTG GGG CCA CTC TCT GAT CCC ATC CTC TTC AGG 3120 Arg Asn Ser Lys Gly Val Gly Pro Leu Ser Asp Pro Ile Leu Phe Arg

1025 1030 1035 1040

ACT CTG AAA GTG GAA CAC CCT GAC AAA ATG GCT AAT GAC CAA GGT CGT 3168 Thr Leu Lys Val Glu His Pro Asp Lys Met Ala Asn Asp Gln Gly Arg

1045 1050 1055

CAT GGA GAT GGA GGT TAT TGG CCA GTT GAT ACT AAT TTG ATT GAT AGA 3216 His Gly Asp Gly Gly Tyr Trp Pro Val Asp Thr Asn Leu Ile Asp Arg

1060 1065 1070

AGC ACC CTA AAT GAG CCG CCA ATT GGA CAA ATG CAC CCC CCG CAT GGC 3264 Ser Thr Leu Asn Glu Pro Pro Ile Gly Gln Met His Pro Pro His Gly

1075 1080 1085

AGT GTC ACT CCT CAG AAG AAC AGC AAC CTG CTT GTG ATC ATT GTG GTC 3312 Ser Val Thr Pro Gln Lys Asn Ser Asn Leu Leu Val Ile Ile Val Val

1090 1095 1100

ACC GTT GGT GTC ATC ACA GTG CTG GTA GTG GTC ATC GTG GCT GTG ATT 3360 Thr Val Gly Val Ile Thr Val Leu Val Val Val Ile Val Ala Val Ile

1105 1110 1115 1120

TGC ACC CGA CGC TCT TCA GCC CAG CAG AGA AAG AAA CGG GCC ACC CAC 3408 Cys Thr Arg Arg Ser Ser Ala Gln Gln Arg Lys Lys Arg Ala Thr His

1125 1130 1135

AGT GCT GGC AAA AGG AAG GGC AGC CAG AAG GAC CTC CGA CCC CCT GAT 3456 Ser Ala Gly Lys Arg Lys Gly Ser Gln Lys Asp Leu Arg Pro Pro Asp

1140 1145 1150

CTT TGG ATC CAT CAT GAA GAA ATG GAG ATG AAA AAT ATT GAA AAG CCA 3504 Leu Trp Ile His His Glu Glu Met Glu Met Lys Asn Ile Glu Lys Pro

1155 1160 1165

TCT GGC ACT GAC CCT GCA GGA AGG GAC TCT CCC ATC CAA AGT TGC CAA 3552 Ser Gly Thr Asp Pro Ala Gly Arg Asp Ser Pro Ile Gln Ser Cys Gln

1170 1175 1180

GAC CTC ACA CCA GTC AGC CAC AGC CAG TCA GAA ACC CAA CTG GGA AGC 3600 Asp Leu Thr Pro Val Ser His Ser Gln Ser Glu Thr Gln Leu Gly Ser

1185 1190 1195 1200

AAA AGC ACC TCT CAT TCA GGT CAA GAC ACT GAG GAA GCA GGG AGC TCT 3648 Lye Ser Thr Ser Hie Ser Gly Gln Asp Thr Glu Glu Ala Gly Ser Ser

1205 1210 1215

ATG TCC ACT CTG GAG AGG TCG CTG GCT GCA CGC CGA GCC CCC CGG GCC 3696 Met Ser Thr Leu Glu Arg Ser Leu Ala Ala Arg Arg Ala Pro Arg Ala

1220 1225 1230

AAG CTC ATG ATT CCC ATG GAT GCC CAG TCC AAC AAT CCT GCT GTC GTG 3744 Lys Leu Met Ile Pro Met Asp Ala Gln Ser Asn Asn Pro Ala Val Val

1235 1240 1245 AGC GCC ATC CCG GTG CCA ACG CTA GAA AGT GCC CAG TAC CCA GGA ATC 3792 Ser Ala Ile Pro Val Pro Thr Leu Glu Ser Ala Gln Tyr Pro Gly Ile

1250 1255 1260

CTC CCG TCT CCC ACC TGT GGA TAT CCC CAC CCG CAG TTC ACT CTC CGG 3840 Leu Pro Ser Pro Thr Cys Gly Tyr Pro His Pro Gln Phe Thr Leu Arg

1265 1270 1275 1280

CCT GTG CCA TTC CCA ACA CTC TCA GTG GAC CGA GGT TTC GGA GCA GGA 3888 Pro Val Pro Phe Pro Thr Leu Ser Val Asp Arg Gly Phe Gly Ala Gly

1285 1290 1295

AGA AGT CAG TCA GTG AGT GAA GGA CCA ACT ACC CAA CAA CCA CCT ATG 3936 Arg Ser Gln Ser Val Ser Glu Gly Pro Thr Thr Gln Gln Pro Pro Met

1300 1305 1310

CTG CCC CCA TCT CAG CCT GAG CAT TCT AGC AGC GAG GAG GCA CCA AGC 3984 Leu Pro Pro Ser Gln Pro Glu His Ser Ser Ser Glu Glu Ala Pro Ser

1315 1320 1325

AGA ACC ATC CCC ACA GCT TGT GTT CGA CCA ACT CAC CCA CTC CGC AGC 4032 Arg Thr Ile Pro Thr Ala Cys Val Arg Pro Thr His Pro Leu Arg Ser

1330 1335 1340

TTT GCT AAT CCT TTG CTA CCT CCA CCA ATG AGT GCA ATA GAA CCG AAA 4080 Phe Ala Asn Pro Leu Leu Pro Pro Pro Met Ser Ala Ile Glu Pro Lys

1345 1350 1355 1360

GTC CCT TAC ACA CCA CTT TTG TCT CAG CCA GGG CCC ACT CTT CCT AAG 4128 Val Pro Tyr Thr Pro Leu Leu Ser Gln Pro Gly Pro Thr Leu Pro Lys

1365 1370 1375

ACC CAT GTG AAA ACA GCC TCC CTT GGG TTG GCT GGA AAA GCA AGA TCC 4176 Thr His Val Lys Thr Ala Ser Leu Gly Leu Ala Gly Lys Ala Arg Ser

1380 1385 1390

CCT TTG CTT CCT GTG TCT GTG CCA ACA GCC CCT GAA GTG TCT GAG GAG 4224 Pro Leu Leu Pro Val Ser Val Pro Thr Ala Pro Glu Val Ser Glu Glu

1395 1400 1405

AGC CAC AAA CCA ACA GAG GAT TCA GCC AAT GTG TAT GAA CAG GAT GAT 4272 Ser His Lys Pro Thr Glu Asp Ser Ala Asn Val Tyr Glu Gln Asp Asp

1410 1415 1420

CTG AGT GAA CAA ATG GCA AGT TTG GAA GGA CTC ATG AAG CAG CTT AAT 4320 Leu Ser Glu Gln Met Ala Ser Leu Glu Gly Leu Met Lys Gln Leu Asn

1425 1430 1435 1440

GCC ATC ACA GGC TCA GCC TTT T AACATGTATT TCTGAATGGA TGAGGTGAAT 4372 Ala Ile Thr Gly Ser Ala Phe

1445

TTTCCGGGAA CTTTGCAGCA TACCAATTAC CCATAAACAG CACACCTGTG TCCAAGAACT 4432

CTAACCAGTG TACAGGTCAC CCATCAGGAC CACTCAGTTA AGGAAGATCC TGAAGCAGTT 4492

CAGAAGGAAT AAGCATTCCT TCTTTCACAG GCATCAGGAA TTGTCAAATG ATGATTATGA 4552

GTTCCCTAAA CAAAAGCAAA GATGCATTTT CACTGCAATG TCAAAGTTTA GCTGCT 4608

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1447 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Glu Asn Ser Leu Arg Cys Val Trp Val Pro Lys Leu Ala Phe Val 1 5 10 15

Leu Phe Gly Ala Ser Leu Leu Ser Ala His Leu Gln Val Thr Gly Phe

20 25 30

Gln Ile Lys Ala Phe Thr Ala Leu Arg Phe Leu Ser Glu Pro Ser Asp

35 40 45

Ala Val Thr Met Arg Gly Gly Asn Val Leu Leu Asp Cys Ser Ala Glu 50 55 60

Ser Asp Arg Gly Val Pro Val Ile Lys Trp Lys Lys Asp Gly Ile His 65 70 75 80

Leu Ala Leu Gly Met Asp Glu Arg Lys Gln Gln Leu Ser Asn Gly Ser

85 90 95

Leu Leu Ile Gln Asn Ile Leu His Ser Arg His His Lys Pro Asp Glu

100 105 110

Gly Leu Tyr Gln Cys Glu Ala Ser Leu Gly Asp Ser Gly Ser Ile Ile

115 120 125

Ser Arg Thr Ala Lys Val Ala Val Ala Gly Pro Leu Arg Phe Leu Ser 130 135 140

Gln Thr Glu Ser Val Thr Ala Phe Met Gly Asp Thr Val Leu Leu Lys 145 150 155 160

Cys Glu Val Ile Gly Glu Pro Met Pro Thr Ile His Trp Gln Lys Asn

165 170 175 Gln Gln Asp Leu Thr Pro Ile Pro Gly Asp Ser Arg Val Val Val Leu

180 185 190

Pro Ser Gly Ala Leu Gln Ile Ser Arg Leu Gln Pro Gly Asp Ile Gly

195 200 205

Ile Tyr Arg Cys Ser Ala Arg Asn Pro Ala Ser Ser Arg Thr Gly Asn 210 215 220

Glu Ala Glu Val Arg Ile Leu Ser Asp Pro Gly Leu His Arg Gln Leu 225 230 235 240

Tyr Phe Leu Gln Arg Pro Ser Asn Val Val Ala Ile Glu Gly Lys Asp

245 250 255

Ala Val Leu Glu Cys Cys Val Ser Gly Tyr Pro Pro Pro Ser Phe Thr

260 265 270

Trp Leu Arg Gly Glu Glu Val Ile Gln Leu Arg Ser Lys Lys Tyr Ser

275 280 285

Leu Leu Gly Gly Ser Asn Leu Leu Ile Ser Asn Val Thr Asp Asp Asp 290 295 300 Ser Gly Met Tyr Thr Cys Val Val Thr Tyr Lys Asn Glu Asn Ile Ser 305 310 315 320

Ala Ser Ala Glu Leu Thr Val Leu Val Pro Pro Trp Phe Leu Asn His

325 330 335

Pro Ser Asn Leu Tyr Ala Tyr Glu Ser Met Asp Ile Glu Phe Glu Cys

340 345 350

Thr Val Ser Gly Lys Pro Val Pro Thr Val Asn Trp Met Lys Asn Gly

355 360 365

Asp Val Val Ile Pro Ser Asp Tyr Phe Gln Ile Val Gly Gly Ser Asn 370 375 380

Leu Arg Ile Leu Gly Val Val Lys Ser Asp Glu Gly Phe Tyr Gln Cys 385 390 395 400

Val Ala Glu Asn Glu Ala Gly Asn Ala Gln Thr Ser Ala Gln Leu Ile

405 410 415

Val Pro Lys Pro Ala Ile Pro Ser Ser Ser Val Leu Pro Ser Ala Pro

420 425 430

Arg Asp Val Val Pro Val Leu Val Ser Ser Arg Phe Val Arg Leu Ser

435 440 445

Trp Arg Pro Pro Ala Glu Ala Lys Gly Asn Ile Gln Thr Phe Thr Val 450 455 460

Phe Phe Ser Arg Glu Gly Asp Asn Arg Glu Arg Ala Leu Asn Thr Thr 465 470 475 480 Gln Pro Gly Ser Leu Gln Leu Thr Val Gly Asn Leu Lys Pro Glu Ala

485 490 495

Met Tyr Thr Phe Arg Val Val Ala Tyr Asn Glu Trp Gly Pro Gly Glu

500 505 510

Ser Ser Gln Pro Ile Lys Val Ala Thr Gln Pro Glu Leu Gln Val Pro

515 520 525

Gly Pro Val Glu Asn Leu Gln Ala Val Ser Thr Ser Pro Thr Ser Ile 530 535 540

Leu Ile Thr Trp Glu Pro Pro Ala Tyr Ala Asn Gly Pro Val Gln Gly 545 550 555 560

Tyr Arg Leu Phe Cys Thr Glu Val Ser Thr Gly Lys Glu Gln Asn Ile

565 570 575

Glu Val Asp Gly Leu Ser Tyr Lys Leu Glu Gly Leu Lys Lys Phe Thr

580 585 590

Glu Tyr Ser Leu Arg Phe Leu Ala Tyr Asn Arg Tyr Gly Pro Gly Val

595 600 605

Ser Thr Asp Asp Ile Thr Val Val Thr Leu Ser Asp Val Pro Ser Ala 610 615 620

Pro Pro Gln Asn Val Ser Leu Glu Val Val Asn Ser Arg Ser Ile Lys 625 630 635 640

Val Ser Trp Leu Pro Pro Pro Ser Gly Thr Gln Asn Gly Phe Ile Thr

645 650 655 Gly Tyr Lys Ile Arg His Arg Lys Thr Thr Arg Arg Gly Glu Met Glu 660 665 670

Thr Leu Glu Pro Asn Asn Leu Trp Tyr Leu Phe Thr Gly Leu Glu Lys

675 680 685

Gly Ser Gln Tyr Ser Phe Gln Val Ser Ala Met Thr Val Asn Gly Thr 690 695 700

Gly Pro Pro Ser Asn Trp Tyr Thr Ala Glu Thr Pro Glu Asn Asp Leu 705 710 715 720

Asp Glu Ser Gln Val Pro Asp Gln Pro Ser Ser Leu His Val Arg Pro

725 730 735 Gln Thr Asn Cys Ile Ile Met Ser Trp Thr Pro Pro Leu Asn Pro Asn

740 745 750

Ile Val Val Arg Gly Tyr Ile Ile Gly Tyr Gly Val Gly Ser Pro Tyr

755 760 765

Ala Glu Thr Val Arg Val Asp Ser Lys Gln Arg Tyr Tyr Ser Ile Glu 770 775 780

Arg Leu Glu Ser Ser Ser His Tyr Val Ile Ser Leu Lys Ala Phe Asn 785 790 795 800

Asn Ala Gly Glu Gly Val Pro Leu Tyr Glu Ser Ala Thr Thr Arg Ser

805 810 815 Ile Thr Asp Pro Thr Asp Pro Val Asp Tyr Tyr Pro Leu Leu Asp Asp

820 825 830

Phe Pro Thr Ser Val Pro Asp Leu Ser Thr Pro Met Leu Pro Pro Val

835 840 845

Gly Val Gln Ala Val Ala Leu Thr His Asp Ala Val Arg Val Ser Trp 850 855 860

Ala Asp Asn Ser Val Pro Lys Asn Gln Lys Thr Ser Glu Val Arg Leu 865 870 875 880

Tyr Thr Val Arg Trp Arg Thr Ser Phe Ser Ala Ser Ala Lys Tyr Lys

885 890 895

Ser Glu Asp Thr Thr Ser Leu Ser Tyr Thr Ala Thr Gly Leu Lys Pro

900 905 910

Asn Thr Met Tyr Glu Phe Ser Val Met Val Thr Lys Asn Arg Arg Ser

915 920 925

Ser Thr Trp Ser Met Thr Ala His Ala Thr Thr Tyr Glu Ala Ala Pro 930 935 940

Thr Ser Ala Pro Lys Asp Phe Thr Val Ile Thr Arg Glu Gly Lys Pro 945 950 955 960

Arg Ala Val Ile Val Ser Trp Gln Pro Pro Leu Glu Ala Asn Gly Lys

965 970 975 Ile Thr Ala Tyr Ile Leu Phe Tyr Thr Leu Asp Lys Asn Ile Pro Ile

980 985 990

Asp Asp Trp Ile Met Glu Thr Ile Ser Gly Asp Arg Leu Thr His Gln

995 1000 1005 Ile Met Asp Leu Asn Leu Asp Thr Met Tyr Tyr Phe Arg Ile Gln Ala 1010 1015 1020

Arg Asn Ser Lys Gly Val Gly Pro Leu Ser Asp Pro Ile Leu Phe Arg 1025 1030 1035 1040

Thr Leu Lys Val Glu His Pro Asp Lys Met Ala Asn Asp Gln Gly Arg

1045 1050 1055

His Gly Asp Gly Gly Tyr Trp Pro Val Asp Thr Asn Leu Ile Asp Arg

1060 1065 1070

Ser Thr Leu Asn Glu Pro Pro Ile Gly Gln Met His Pro Pro His Gly

1075 1080 1085

Ser Val Thr Pro Gln Lys Asn Ser Asn Leu Leu Val Ile Ile Val Val 1090 1095 1100

Thr Val Gly Val Ile Thr Val Leu Val Val Val Ile Val Ala Val Ile 1105 1110 1115 1120

Cys Thr Arg Arg Ser Ser Ala Gln Gln Arg Lys Lys Arg Ala Thr His

1125 1130 1135

Ser Ala Gly Lys Arg Lys Gly Ser Gln Lys Asp Leu Arg Pro Pro Asp

1140 1145 1150

Leu Trp Ile His His Glu Glu Met Glu Met Lys Asn Ile Glu Lys Pro

1155 1160 1165

Ser Gly Thr Asp Pro Ala Gly Arg Asp Ser Pro Ile Gln Ser Cys Gln 1170 1175 1180

Asp Leu Thr Pro Val Ser His Ser Gln Ser Glu Thr Gln Leu Gly Ser 1185 1190 1195 1200

Lys Ser Thr Ser His Ser Gly Gln Asp Thr Glu Glu Ala Gly Ser Ser

1205 1210 1215

Met Ser Thr Leu Glu Arg Ser Leu Ala Ala Arg Arg Ala Pro Arg Ala

1220 1225 1230

Lys Leu Met Ile Pro Met Asp Ala Gln Ser Asn Asn Pro Ala Val Val

1235 1240 1245

Ser Ala Ile Pro Val Pro Thr Leu Glu Ser Ala Gln Tyr Pro Gly Ile 1250 1255 1260

Leu Pro Ser Pro Thr Cys Gly Tyr Pro His Pro Gln Phe Thr Leu Arg 1265 1270 1275 1280

Pro Val Pro Phe Pro Thr Leu Ser Val Asp Arg Gly Phe Gly Ala Gly

1285 1290 1295

Arg Ser Gln Ser Val Ser Glu Gly Pro Thr Thr Gln Gln Pro Pro Met

1300 1305 1310

Leu Pro Pro Ser Gln Pro Glu His Ser Ser Ser Glu Glu Ala Pro Ser

1315 1320 1325

Arg Thr Ile Pro Thr Ala Cys Val Arg Pro Thr His Pro Leu Arg Ser 1330 1335 1340

Phe Ala Asn Pro Leu Leu Pro Pro Pro Met Ser Ala Ile Glu Pro Lys 1345 1350 1355 1360 Val Pro Tyr Thr Pro Leu Leu Ser Gln Pro Gly Pro Thr Leu Pro Lye 1365 1370 1375

Thr His Val Lys Thr Ala Ser Leu Gly Leu Ala Gly Lys Ala Arg Ser

1380 1385 1390

Pro Leu Leu Pro Val Ser Val Pro Thr Ala Pro Glu Val Ser Glu Glu

1395 1400 1405

Ser His Lys Pro Thr Glu Asp Ser Ala Asn Val Tyr Glu Gln Asp Asp 1410 1415 1420

Leu Ser Glu Gln Met Ala Ser Leu Glu Gly Leu Met Lys Gln Leu Asn 1425 1430 1435 1440

Ala Ile Thr Gly Ser Ala Phe

1445

Claims

1. An antibody preparation consisting essentially of antibodies which: are specifically immunoreactive with an epitope on the extracellular domain of DCC protein and (2) do not cross-react with neural cell adhesion molecules.

2. An antibody preparation consisting essentially of antibodies which: are specifically immunoreactive with an epitope contained within the cytoplasmic domain of DCC and (2) do not cross-react with neural cell adhesion molecules.

3. The antibody preparation of claim 1 wherein the epitope is encoded by a segment of the DCC gene consisting of base pairs 1195-2854, as shown in SEQ ID NO: 1.

4. The antibody preparation of claim 1 wherein the epitope is contained within amino acids 1-1063 of DCC protein as shown in SEQ ID NO:2.

5. The antibody preparation of claim 1 wherein said antibodies are polyclonal.

6. The antibody preparation of claim 1 wherein said antibodies are monoclonal.

7. The antibody preparation of claim 2 wherein said antibodies are polyclonal.

8. The antibody preparation of claim 2 wherein said antibodies are monoclonal.

9. The antibody preparation of claim 1 wherein the antibodies are made by the cell line AF5 deposited at the ATCC as HB 11299.

10. The antibody preparation of claim 1 wherein the antibodies are made by the cell line AF1 deposited at the ATCC as HB 11289.

11. The antibody preparation of claim 2 wherein the epitope is contained within amino acids 3369-4341 of DCC protein as shown in SEQ ID NO:2.

12. A method for detecting mutations in a human DCC gene, comprising: extracting proteins from a sample selected from the group consisting of a tissue and a body fluid;

separating said extracted proteins on a polyacrylamide gel;

blotting said separated proteins onto a filter;

contacting said filter with antibodies which:

(1) are specifically immunoreactive with DCC protein and

(2) do not cross-react with neural cell adhesion molecules, to bind said antibodies to proteins blotted on said filter;

detecting the antibodies which bind to said proteins, wherein the absence of binding of antibodies to a protein having the size of DCC (approximately 115-190 kDa) indicates the presence of a DCC mutation in the sample.

13. The method of claim 12 wherein the antibody binds to an epitope located within the extracellular domain of DCC.

14. The method of claim 12 wherein the antibodies are made by the cell line AF5 deposited at the ATCC as HB 11299.

15. The method of claim 12 wherein the antibodies are made by the cell line AF1 deposited at the ATCC as HB 11289.

16. The method of claim 12 wherein the antibodies bind to an epitope located within the cytoplasmic domain of DCC.

17. The method of claim 12 wherein the sample is a tumor tissue.

18. The method of claim 12 wherein the sample is a preparation of peripheral blood mononuclear cells.

19. A method for determining the presence in a human of DCC mutations, comprising the steps of:

contacting a body sample of a human with a first antibody which: (1) is specifically immunoreactive with an epitope of DCC, and (2) does not crossreact with neural cell adhesion molecules, to bind components of said body sample to said antibody; determining the amount of antibody which is bound to said body sample, wherein failure of said body sample to bind to said antibody indicates the presence of a DCC mutation in the human.

20. The method of claim 19 wherein said epitope is contained within the extracellular domain.

21. The method of claim 19 wherein said epitope is contained within the cytoplasmic domain.

22. The method of claim 19 wherein said antibody is AF5, deposited at the ATCC as accession no. HB 11299.

23. The method of claim 19 wherein said antibody is AF1, deposited at the ATCC as accession no. HB 11289.

24. The method of claim 19 wherein the body sample is a tissue section.

25. The method of claim 19 wherein the body sample is a lysate of a tumor biopsy.

26. The method of claim 19 wherein the antibody is bound to a solid support.

27. The method of claim 19 wherein the body sample is bound to a solid support.

28. A solid support for use in performing enzyme-linked immunosorbent assays (ELISA), comprising:

a solid support which is coated with an antibody which is specifically immunoreactive with an epitope contained within the extracellular domain of DCC, and does not cross-react with neural cell adhesion molecules.

29. The solid support of claim 28 wherein the antibody is AF1, deposited at the ATCC as accession no. HB 11289.

30. The solid support of claim 28 wherein the antibody is AF5, deposited at the ATCC as accession no. HB 11299.

31. A solid support for use in performing enzyme-linked immunosorbent assays (ELISA), comprising: a solid support which is coated with an antibody which is specifically immunoreactive with an epitope contained within the intracellular domain of DCC, and does not cross-react with neural cell adhesion molecules.

32. A hybridoma cell which secretes an antibody which is specifically immunoreactive with an epitope contained within the extracellular domain of DCC, and does not cross-react with neural cell adhesion molecules.

33. A hybridoma cell which secretes an antibody which is specifically immunoreactive with an epitope contained within the intracellular domain of DCC, and does not cross-react with neural cell adhesion molecules.

34. The hybridoma cell of claim 33 which secretes an antibody which binds to the same epitope as does AF5 (ATCC deposit No. HB 11299).

35. The hybridoma cell of claim 33 which secretes an antibody which binds to the same epitope as does AF1 (ATCC deposit No. HB 11289).

36. A method for determining the presence in a human of DCC mutations, comprising the steps of:

contacting a first tissue section of a human with an antibody which (1) is specifically immunoreactive with an epitope contained within the intracytoplasmic domain of DCC, and (2) does not cross-react with neural cell adhesion molecules, to bind components of said tissue section to said antibody;

determining the amount of antibody which is bound to said tissue sample, wherein an absence of binding of said antibody to said tissue sample indicates the presence of a DCC mutation in the tissue sample.

37. The method of claim 36 further comprising:

contacting a normal tissue section of said human with said antibody to bind components of said normal tissue section to said solid support by means of said antibody;

determining the amount of antibody which is bound to said body sample, wherein the detection of binding of said antibody to said normal tissue sample confirms the presence of a DCC mutation in the first tissue section.