WO2010036031A2 - Pauf-specific human monoclonal antibody, pharmaceutical composition for treating cancer comprising the same and method for detecting cancer using the same - Google Patents

Abstract

Disclosed are a PAUF protein associated with the onset of cancer, a human monoclonal antibody specific to the PAUF protein, and a nucleic acid molecule coding for the PAUF protein, and uses thereof. Closely correlated with the onset of cancer, the PAUF protein or the PAUF nucleic acid molecule can be used as a diagnostic marker or a therapeutic target. In addition, having the ability to bind specifically to PAUF presented on the surface of various cancer cells including pancreatic cancer cells, the anti-PAUF antibody or its functional fragment allows the diagnosis of cancer in the early stages thereof and can inhibit the growth, invasion or migration of cancer cells by inducing the cells to undergo apoptosis, and thus are useful in the diagnosis and treatment of cancer.

Description

PAUF-SPECIFIC HUMAN MONOCLONAL ANTIBODY, PHARMACEUTICAL COMPOSITION FOR TREATING CANCER COMPRISING THE SAME AND METHOD FOR DETECTING CANCER USING THE SAME

The present invention relates to a PAUF protein associated with the onset of cancer, a human monoclonal antibody specific to the PAUF protein, and a nucleic acid molecule coding for the PAUF protein, and uses thereof. More particularly, the present invention relates to a human monoclonal antibody binding specifically to a PAUF protein, a diagnostic marker for cancer comprising a PAUF protein or PAUF nucleic acid, a diagnositic composition for cancer comprising an antibody specific to the marker, a method for the diagnosis of cancer using the composition, a method of screening a material therapeutic or preventive of cancer by determining the expression level of the marker, and a composition for the treatment or prevention of cancer featuring the inhibitory activity against the marker.

With the rapid growth of the elderly population and environmental degradation attributed to rapid industrial development, the global incidence of cancer is increasing at a rate of 5% each year. A WHO report expects that the population diagnosed with cancer will increase to 30 million within 25 years and that 20 million people will die from cancer in the world (Ahn, Soon Kil (2003), Trends in Research and Development of Anticancer Agents, Overview of Health Industry Technology 2003 Summer:10-18). According to statistics related to death and the causes of death in 2006, published by the Korea National Statistical Office, cancer ranks first among the three leading causes of death, with the greatest annual increment of mortality. Also, a research result reported that the social and economic loss attributed to cancer amounts to about 11.3 billion dollars every year, indicating that research into the prophylaxis and treatment of cancer is very important (Faint et al. (2004), Pipeline Insight: Cancer Overview, Datamonitor DMHC2025).

Pancreatic cancer accounts for about 2% of all instances of cancer. In spite of the low rate of incidence, pancreatic cancer has one of the highest mortality rates of all cancers and is fourth in the list of cancers which result in death in Korea. Patients diagnosed with pancreatic cancer typically have a poor prognosis partly because the cancer usually exhibits no symptoms early on, leading to locally advanced or metastatic disease at the time of diagnosis. In many cases, pancreatic cancer has developed into the final stages and has spread over too wide of a region for surgical resection to be viable. Importantly, there are no effective curatives (Faint et al. (2004) Datamonitor DMHC2045; Garcea et al. (2005) Pancreatology 5:514-529).

Further, there have been no methods used to diagnose pancreatic cancer from human bodily fluids including blood. Currently, MRI or CT is able to diagnose pancreatic cancer, but only after it enters the later stage. CA19-9 assay is too low in specificity to be applied to the diagnosis of pancreatic cancer, and is used only for monitoring the treatment of diseases (Notification of Ministry for health, welfare and family affairs: Method for diagnosis of Cancer). The only chance patients with pancreatic cancer have for a cure is complete surgical resection of the tumor. At the time of diagnosis, however, only 10 ~ 15% of progressive pancreatic cancer cases are typically found to be potentially operable due to the presence of distant metastasis. However, despite maximal medical or surgical management, results of the treatment of patients with pancreatic cancer are dismal. Patients with this disease have a median survival of 15 ~ 18 months following diagnosis. Currently, the most common strategy in the treatment of advanced pancreatic cancer which is unresectable is treatment with gemcitabine, an i.v.-administered 2-deoxycytidine nucleoside analogue, which induces apoptosis of human pancreatic cancer cells and can inhibit tumor growth and progression. However, this chemotherapy is not highly effective and lowers the quality of life of the patients due to significant side effects. Clearly, new, effective treatment strategies are required to combat this deadly disease (Faint et al. (2004), Datamonitor DMHC2045; Garcea et al. (2005) Pancreatology 5:514-529; Kern et al. (2002) Cancer Biol Therapy 1:607-613; Laheru and Jaffee (2005) Nature Rev Cancer 5:459-467).

Current anti-cancer agents in clinical use may be divided into chemotherapeutic agents and biotherapeutic agents. Chemotherapy, in its most general sense, refers to treatment of disease by chemicals that kill cancer cells. Virtually all chemotherapeutic regimens can cause significant side effects such as inducing cytotoxicity in normal cells and inducing chemotherapy resistance as a result of the defense mechanism of tumor cells. Arising as alternatives to chemotherapy, biotherapeutic agents have recently been actively developed because they can inhibit the progression of cancer by enhancing the immune system or by suppressing the activity of cancer cells. Examples of currently used or developed chemotherapeutic agents include cytokines, recombinant antibodies such as monoclonal antibodies, gene therapy agents, and angiogenesis inhibitors (Kim, Yeol Hong et al., (2004), Study on Cancer Gene and Development of Novel Anticancer Agents, Overview of Health Industry Technology 2003 Summer 10~18).

Therapeutic monoclonal antibodies, representative of biotherapeutic agents, act only on disease-specific targets thanks to the high reaction specificity thereof and thus are known to show much lower side effects than do conventional cytotoxic chemicals. The therapeutic effects of antibodies are obtained through various mechanisms. For example, antibodies may bind specifically to corresponding antigens to inhibit signaling or may induce apoptosis by cross-linking. Also, the therapeutic effects may be extracted by activating the immune system with antibodies. As anticancer agents, monoclonal antibodies can be responsible for the functions of tracing cancer cells, suppressing the functions of targets, eliciting immune responses and effectively eliminating cancer cells. With these advantages, monoclonal antibodies are playing an increasingly major role in cancer therapy. 20 kinds of monoclonal antibodies were already approved for use in diagnosis and treatment in 2005. More than half of them were applied to the diagnosis and treatment of various cancers.

Leading to the present invention, intensive and thorough research into the treatment of cancer, conducted by the present inventors, resulted in the finding that both the PAUF protein and the PAUF nucleic acid molecule are very specific for the onset of cancer and become markers or targets for the treatment particularly of pancreatic cancer and that antibodies specific to PAUF proteins can be used for the diagnosis of caner at an early stage and are inhibitory of the growth, invasion and migration of various cancers including pancreatic cancer.

It is therefore an object of the present invention to provide a human monoclonal antibody specific to PAUF and a functional fragment thereof.

It is another object of the present invention to provide a nucleic acid molecule coding for the antibody.

It is a further object of the present invention to provide a diagnostic marker for cancer, comprising a PAUF protein or a PAUF nucleic acid molecule coding for the PAUF protein.

It is a still further object of the present invention to provide a composition for diagnosing cancer, comprising at least one selected from a group consisting of a protein, a peptide, an aptamer and an antibody, all of which specifically bind to a PAUF protein or an immunogenic fragment thereof.

It is still another object of the present invention to provide a diagnostic kit for caner, comprising at least one selected from a group consisting of a protein, a peptide, an aptamer and an antibody, all of which specifically bind to a PAUF protein or an immunogenic fragment thereof.

It is yet another object of the present invention to provide a method for the diagnosis of cancer, comprising determining the level of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

It is yet a further object of the present invention to provide a method for detecting PAUF protein, comprising bringing the antibody into contact with a biological specimen to examine whether the level of PAUF protein is increased or decreased.

It is yet a still another object of the present invention to provide a method for screening a material preventive or curative of cancer, comprising determining the level of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

It is an additional object of the present invention to provide a composition for inhibiting the growth and metastasis of cancer, comprising as an active ingredient a material inhibitory of the expression of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

It is an additional object of the present invention to provide a composition for the treatment or prevention of cancer, comprising as an active ingredient a material degrading the expression of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

In an embodiment for achieving the object thereof, the present invention pertains to a human monoclonal antibody binding specifically to PAUF or a functional fragment thereof.

PAUF is overexpressed in cancer, particularly in pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer. The present inventors found that PAUF is overexpressed in pancreatic cancer at the early stage, compared to normal cells. Moreover, overexpression of PAUF protein is observed in patients who have pancreatic cancer. In addition, it was observed that the growth, metastasis and invasion of cancer are likely to occur in cell lines expressing PAUF.

For use in determining the presence or level of PAUF or inhibiting the growth, metastasis and invasion of cancer, an antibody specific for PAUF was developed by the present inventors.

In a preferred embodiment, the present invention provides a human monoclonal antibody or functional fragment thereof specially binding to PAUF (Pancreatic adenocarcinoma upregulating factor), comprising a light chain variable region of which an amino acid sequence is selected from the group consisting of: (a) a light chain variable region comprising CDR1 as defined by SEQ ID NO.25, CDR2 as defined by SEQ ID NO. 26, and CDR3 as defined by SEQ ID NO.27; (b) a light chain variable region comprising CDR1 as defined by SEQ ID NO.31, CDR2 as defined by SEQ ID NO.32, and CDR3 as defined by SEQ ID NO.33; (c) a light chain variable region comprising CDR1 as defined by SEQ ID NO.37, CDR2 as defined by SEQ ID NO.38, and CDR3 as defined by SEQ ID NO.39; and (d) a light chain variable region comprising CDR1 as defined by SEQ ID NO.43, CDR2 as defined by SEQ ID NO.44, and CDR3 as defined by SEQ ID NO.45.

In another preferred embodiment, the present invention provides a human monoclonal antibody or functional fragment thereof specially binding to PAUF (Pancreatic adenocarcinoma upregulating factor) comprising a heavy chain variable region of which an amino acid sequence is selected from the group consisting of: (a) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.28, CDR2 as defined by SEQ ID NO.29, and CDR3 as defined by SEQ ID NO.30; (b) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.34, CDR2 as defined by SEQ ID NO.35, and CDR3 as defined by SEQ ID NO.36; (c) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.40, CDR2 as defined by SEQ ID NO.41, and CDR3 as defined by SEQ ID NO.42; and (d) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.46, CDR2 as defined by SEQ ID NO.47, and CDR3 as defined by SEQ ID NO.48.

The term 'PAUF (Pancreatic Adenocarcinoma Upregulating Factor)', as used herein, refers to a novel protein as defined by SEQ ID NO. 50, found and named by the present inventors.

The above PAUF (Pancreatic Adenocarcinoma Upregulating Factor) comprises isolated proteins, allelic variants of the proteins, and conservative amino acid substitutions of the proteins. As used herein, the 'protein' or 'polypeptide' refers, in part, to a protein that has the human amino acid sequence depicted in SEQ ID NO. 50. The terms also comprise naturally occurring allelic variants and proteins that have a slightly different amino acid sequence than that specifically recited above. Allelic variants, though possessing a slightly different amino acid sequence than those recited above, will still have the same or similar biological functions associated with these proteins.

As used herein, the family of proteins related to the human amino acid sequence of SEQ ID NO. 50 refers to proteins that have been isolated from organisms in addition to humans.

The above PAUF protein is preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the protein from cellular constituents that are normally associated with the protein. A skilled artisan can readily employ standard purification methods to obtain an isolated protein.

The above PAUF protein further includes insertion, deletion or conservative amino acid substitution variants of SEQ ID NO. 50. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the protein. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of the protein, in certain instances, may be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to render the peptide more hydrophobic or hydrophilic, without adversely affecting the biological activities of the protein.

Ordinarily, the allelic variants, the conservative substitution variants, and the members of the protein family, will have an amino acid sequence having at least about 50%, 60%, 70% or 75% amino acid sequence identity with the sequence set forth in SEQ ID NO. 50, more preferably at least about 80-90%, even more preferably at least about 92-94%, and most preferably at least about 95%, 98% or 99% sequence identity. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with SEQ ID NO. 50, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity (see section B for the relevant parameters). Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into the peptide sequence shall not be construed as affecting homology.

Thus, The above PAUF protein includes molecules having the amino acid sequence disclosed in SEQ ID NO. 50; amino acid sequence variants wherein one or more amino acid residues has been inserted N- or C-terminal to, or within, the disclosed coding sequence; and amino acid sequence variants of the disclosed sequence, or their fragments as defined above, that have been substituted by at least one residue. Such fragments, also referred to as peptides or polypeptides, may contain antigenic regions, functional regions of the protein identified as regions of the amino acid sequence which correspond to known protein domains, as well as regions of pronounced hydrophilicity. The regions are all easily identifiable by using commonly available protein sequence analysis software such as MacVector (Oxford Molecular).

Contemplated variants further include those containing predetermined mutations by, e.g., homologous recombination, site-directed or PCR mutagenesis, and the corresponding proteins of other animal species, including but not limited to rabbit, mouse, rat, porcine, bovine, ovine, equine and non-human primate species, and the alleles or other naturally occurring variants of the family of proteins; and derivatives wherein the protein has been covalently modified by substitution, chemical, enzymatic, or other appropriate means with a moiety other than a naturally occurring amino acid (for example a detectable moiety such as an enzyme or radioisotope).

Sequences of PAUF protein and nucleic acid molecule are indicated by GenBank (Accession number EF067313). Preferably, the PAUF nucleic acid molecule has a sequence as defined by SEQ ID NO. 49.

The antibody specially binding to PAUF in accordance with the present invention comprises specific CDRs (Complementarity Determining Region) for binding to an antigen, but is not limited thereto.

Preferably, the antibody specific for PAUF protein in accordance with the present invention may comprise CDRs (Complementarity Determining Region) which includes a specific sequence. More preferably, the monoclonal antibody comprises a CDR in the light chain or a CDR in the heavy chain, respectively, or all CDRs in the light and heavy chains. It will be understood by persons skilled in the art that the monoclonal antibody of the present invention may be generated by grafting CDRs onto framework regions (FR) of a known therapeutic antibody.

The antibody specific to PAUF in accordance with the present invention may be polyclonal or monoclonal with preference for monoclonal antibody. Most preferably, the antibody is a human monoclonal antibody or a functional fragment thereof as described above.

The term 'monoclonal antibody', as used herein, is recognized in the art and refers to a highly specific antibody directed by a single antigenic determinant (epitope). Typically, polyclonal antibodies are a mixture of immunoglobulin molecules secreted against a specific antigen by different B cell lines, each recognizing a different epitope. By contrast, monoclonal antibodies are derived from a single cell line. A polyclonal antibody is lower in specificity than a monoclonal antibody because it has cross-reactivity to different proteins. Further, antisera are different in antibody titer and specificity according to lots. For these reasons, drugs utilizing polyclonal antibodies are difficult to control in quality upon production. In contrast, monoclonal antibodies can improve diagnostic assay which is based on antigen-antibody binding by improving selectivity and specificity. Also advantageously, monoclonal antibodies are not contaminated with other immunoglobulins because they are produced by culturing hybridoma.

Antibodies to PAUF may be produced using typical methods known in the art, for example, the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U. S. Patent No. 4,816,56) or the phage antibody library method (Clackson et al, Nature, 352:624-628(1991) and Marks et al, J. Mol. Biol., 222:58, 1-597(1991)). As concerns details about general items of concern related to antibody production, reference may be made to: Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and, Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley/Greene, NY, 1991. For example, hybridoma cells producing monoclonal antibodies may be obtained by fusing immortal cell lines to antibody-producing lymphocytes the technologies for which are well known to those skilled in the art and can be easily carried out. Polyclonal antibodies may be obtained by injecting a PAFU protein antigen into an appropriate animal, collecting antisera from the animal, and isolating antibodies from the antisera by an affinity technique.

In the present invention, human monoclonal antibodies specific to PAUF can be selected using well-known combinatorial antibody library and phage display technologies (see Example 1).

One of the most successful applications of phage display technology to biological studies is to select various recombinant antibody fragments (scFv or Fab) from antibody libraries. When utilizing the phage display technology which enables large libraries of proteins to be screened and amplified in an in vitro selection process, a number of human antibodies which are available as agents for in vivo diagnosis and treatment can be obtained from phage display antibody libraries through antigen-specific selection.

In principle, an antibody display library is constructed by cloning antibody genes, whether naive, immunized, or semi-synthesized, into a phagemid vector at the 5'end site of gIII and introducing the phagemids into E. coli. The phagemid clones with antibody genes of interest inserted thereinto express antibody-pIII fusion proteins in the host cells. All of the structural proteins necessary for the assembly of recombinant phage virions, such as wild-type (wt) pIII, etc., are supplied by helper phages such as M13K07 or VCSM13. Through a process called phage rescue, recombinant phage virions are produced. These recombinant phages display wt pIII and antibody-pIII fusion proteins at the same time on their surface. The phagemid DNA is preferentially packaged inside the recombinant phage, resulting in the formation of genotype-phenotype linkage. Theoretically, this phage packaging process enforces oligovalent display of antibody fragments on the surface of the recombinant phage particles. As a result, the phage display technology allows the separation of target molecule-specific antibody clones from the background of target molecule-non-specific clones by panning.

When produced, the human monoclonal antibody of the present invention can be used without purification or with purification by, for example, affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, etc.

In an embodiment of the present invention, cell lines which can stably produce the antibodies according to the present invention are prepared. To this end, first, the light chains and the heavy chains of the antibodies are inserted into an expression vector which is in turn transformed into a DHFR-deficient CHO cell line (CHO-DG44). For the amplification of the transformed genes, the transformed cells are cultured in selection media containing MTX (methotrexate), a DHFR inhibitor, at increasing concentrations. Cell lines which express the antibody with high efficiency are selected and cultured on a mass scale (see Example 3).

As long as it has the binding property of recognizing specifically PAUF, any antibody may be used in the present invention. For example, not only complete forms having two full-length heavy chains and two full-length light chains, but also functional fragments of antibody molecules fall within the scope of the present invention. The term 'functional fragment of an antibody', as used herein, refers to a fragment which retains at least an antigen-binding function, and may include Fab, F(ab'), F(ab')₂, and Fv. The antibody according to the present invention or a functional fragment thereof, which can bind specifically to PAUF, comprises (a) a light chain variable region defined as a polypeptide represented by one selected from a group consisting of amino acid sequences of SEQ ID Nos. 1 to 4, and/or (b) a heavy chain variable region defined as a polypeptide represented by one selected from a group consisting of amino acid sequences of SEQ ID Nos. 5 to 8.

Preferably, the human monoclonal antibody according to the present invention comprises light chain amino acid sequence as defined by one selected from among SEQ ID NOS. 9 to 12; or/and a heavy chain amino acid sequence as defined by one selectd from among SEQ ID NOS. 13 to 16.

In accordance with another aspect thereof, the present invention provides a nucleic acid molecule encoding the PAUF-specific monoclonal antibody or its fragment. In a preferred embodiment, the nucleic acid molecule of the present invention comprises a base sequence selected from among base sequences of SEQ ID Nos. 17 to 20 coding respectively for the amino acid sequences of SEQ ID Nos. 1 to 4 for the light chain variable region, and/or a base sequence selected from among base sequences of SEQ ID Nos. 21 to 24 coding respectively for the amino acid sequences of SEQ ID Nos. 5 to 8 for the heavy chain variable region.

The human monoclonal antibody or its functional fragment in accordance with the present invention specifically binds to PAUF in various cancer cells to suppress the growth, migration and/or metastasis of cancer, leading to the death of tumor cells. Preferably, the cancer is pancreatic cancer, stomach cancer, ovarian cancer or colorectal cancer.

Also, the present invention pertains to novel uses of PAUF protein or PAUF nucleic acid molecule as diagnostic markers or therapeutic targets in relation with cancer, based on the finding that PAUF is overexpressed in cancer and the growth, metastasis and invasion of cancer cells are likely to occur in the cell line where PAUF is overexpressed.

In accordance with an embodiment thereof, the present invention provides a diagnostic marker for cancer, comprising a PAUF protein or a PAUF nucleic acid molecule coding for the same.

The diagnostic marker according to the present invention is selective for pancreatic cancer, stomach cancer and colorectal cancer and particularly specific for pancreatic cancer.

While normal persons were observed to be negative, PAUF protein was detected in patients with pancreatic cancer, with a total diagnosis specificity of 100%. Among 13 pancreatic cancer specimens, eight were decided to be positive, with a selectivity of 61.5%, and the other five negative specimens were observed to have slightly higher PAUF levels compared to normal specimens (FIG. 7), indicating that the PAUF protein can be useful as a diagnostic marker for cancer (see Example 8).

In accordance with another embodiment thereof, the present invention provides a diagnostic composition and kit for cancer, comprising at least one selected from a group consisting of a protein, a peptide, an aptamer and an antibody all of which bind specifically to a PAUF protein or an immunogenic fragment thereof.

Preferably, the antibody is the human monoclonal antibody according to the present invention or a functional fragment thereof.

In accordance with still another embodiment thereof, the present invention provides a method for detecting a PAUF protein using the human monoclonal antibody or the functional fragment thereof. The detection of PAUF protein can be accomplished by bringing the human monoclonal antibody of the present invention or the functional fragment thereof into contact with a biological specimen to examine whether the level of PAUF protein increases or decreases.

The human monoclonal antibody or its functional fragment in accordance with the present invention specifically binds to PAUF in various cancer cells where PAUF protein is expressed, to suppress the growth, migration and/or metastasis of cancer, leading to the death of tumor cells. Preferably, the cancer is pancreatic cancer, stomach cancer, ovarian cancer or colorectal cancer.

In addition to the ingredients described above, the composition or the kit in accordance with the present invention may further comprise another ingredient. For example, the composition or kit may further include a reagent capable of detecting an antigen-antibody complex, e.g., a labeled secondary antibody, a chromophore, an enzyme (i.e., conjugated with an antibody) and its substrate or other substances capable of binding to the antibody (FIG. 7). The composition or kit may be in the form of multiple separate packages or compartments.

The diagnostic composition and kit in accordance with the present invention can be used to analyze the onset, progression and alleviation of cancer. Therefore, the term 'diagnosis', as used herein, is intended to include the meaning of determining the onset, progression and alleviation of diseases as well as determining the presence of diseases.

The composition and kit of the present invention is useful in the diagnosis of the cancer whose onset and progression are associated with the overexpression of PAUF, that is, pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer, with preference for pancreatic cancer.

Advantageously, the diagnostic composition of the present invention is used to diagnose the conditions of cancer with high efficiency. Most advantageously, the diagnostic composition is applicable to the diagnosis of cancer when it is in its early stages.

In accordance with a further embodiment thereof, the present invention provides a method of screening a material therapeutic or preventive of cancer, comprising:

(a) bringing a test sample into contact with a cell containing a PAUF protein or nucleic acid molecule;

(b) measuring the level of the PAUF protein or nucleic acid molecule or the activity of the PAUF protein; and

(c) judging the test sample as being therapeutic or preventive of cancer when the level or the activity is determined to be down-regulated.

Herein, the term 'prevention of cancer' is intended to refer to any action resulting in the suppression of carcinogenesis or the delay of the growth of cancer through the administration of a material obtained by the screening method of the present invention, or a composition comprising a material suppressive of the expression of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein, for example, at least one selected from a group consisting of a protein, an aptamer, a peptide and an antibody, all of which bind specifically to PAUF.

As used herein, the term 'treatment' is intended to refer to any action resulting in an improvement in the symptoms of the disease or which advantageously alters a disease state after a material has been administered, the material having been obtained by the screening method of the present invention or is composed of the composition thereof.

To begin with, according to the screening method of the present invention, a test material to be analyzed is brought into contact with a cell containing a PAUF protein or a PAUF nucleic acid molecule.

As used herein, the term 'test material' is intended to mean an unknown material which is used in performing a screening method which is carried out for purposes of examining whether it has influence on the expression level of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein or not. Examples of the test material useful in the present invention include chemicals, nucleotides, antisense-RNA, siRNA (small interferenceRNA), peptides, proteins, aptamers, and natural extracts, but are not limited thereto.

Subsequently, the cell treated with the test material is measured for the level of its PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein. Down-regulation in the level of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein leads to the judgement that the test sample is therapeutic or preventive of cancer.

As used herein, the term 'measurement of the level of protein or nucleic acid' is intended to refer to a process of examining whether and how much of the PAUF protein or the PAUF nucleic acid molecule is expressed in a biological sample.

The measurement of the level of PAUF nucleic acid molecule can be carried out using various well-known methods. For example, RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3^rd ed. Cold Spring Harbor Press(2001)), Nothern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRCpress), hybridization on cDNA microarray (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3^rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3^rd ed. Cold Spring Harbor Press (2001)) may be used for the measurement.

When employing an RT-PCR protocol, first, total RNA is isolated from the treated cells, and a single-stranded cDNA is prepared with oligo dT primers in the presence of reverse transcriptase. PCR is performed using a gene-specific primer set with the cDNA serving as a template. After the electrophoresis of the PCR product, the bands thus formed are analyzed to determine the levels of PAUF nucleic acids in the cells.

As for the measurement of the level of PAUF protein, it can be obtained using various immunoassays known in the art. For example, a change in PAUF protein level can be detected using radioimmunoassay, radioimmunoprecipitation assay, immunoprecipitation, ELISA (enzyme-linked immunosorbentassay), capture-ELISA, suppressive or competitive ELISA, or sandwich ELISA.

Concerning the immunoassay or immunostaining, reference may be made to Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton,Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology,Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; and, Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In addition, the protein level may be determined using a protein, an aptamer or a peptide, all of which bind specifically to PAUF protein, with preference for an antibody to PAUF protein.

Methods of measuring protein levels using antibodies include Western blotting, ELISA (enzyme linked immuNosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistostaining assay, immunoprecipitation assay, complement fixation assay, FACS, and protein chips, but is not limited thereto.

When making assays to perform the analysis, a quantitative comparison can be made between the antigen-antibody complexes in a normal control and a patient suspected of having cancer. Based on this comparison, a significant increase in the level of the PAUF nucleic acid molecule or the PAUF protein can be determined, thus giving information necessary for the diagnosis of cancer.

As used herein, the term 'antigen-antibody complex' is intended to refer to binding products of the PAUF protein to an antibody specific thereto. The antigen-antibody complex thus formed may be quantitatively determined by measuring the size of the signal generated by the detection label.

Such a detection label may be selected from a group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, but the present invention is not limited to these examples.

In accordance with still a further embodiment thereof, the present invention provides a pharmaceutical composition for the inhibition of cancer growth and metastasis, comprising as an active ingredient a material regulative of the expression of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

In accordance with yet a further embodiment thereof, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer, comprising as an active ingredient a material regulative of the expression of PAUF protein or PAUF nucleic acid molecule or the activity of PAUF protein.

In the composition, at least one selected from a group consisting of a protein, an aptamer, a peptide and an antibody, all of which bind specifically to PAUF, may be contained.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Examples of the pharmaceutically acceptable carrier suitable for oral administration include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, dye and a fragrant. For injection formulation, a buffer, a preservative, a pain reducer, a solubilizer, an isotonic agent, and a stabilizer may be used in combination. For local administration, the composition may further comprise a base, an excipient, a lubricant and/or a preservative. The pharmaceutical composition of the present invention may be formulated, along with a pharmaceutically acceptable carrier, into various dosage forms. For example, the composition may be in the oral dosage form of tablets, troches, capsules, elixirs, suspensions, syrups or wafers. For injections, the composition may be formulated into unit dose ampule forms or multiple dose ampule forms. Also, the composition may be applied to solutions, suspensions, tablets, pills, capsules, and suspended release forms. Examples of carriers, excipients or diluents suitable for the formulation of the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, methyl cellulose, amorphous cellulose, polypyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. A filler, an anticoagulant, a lubricant, a wetting agent, a fragrant and/or a preservative may be additionally used. The effective dosage of the pharmaceutical composition in accordance with the present invention depends on various factors, including kinds of cancer, route of administration, patient's age, gender and weight, and severity of diseases, etc.

In a preferred embodiment, the composition of the present invention may further comprise a therapeutic agent which can be conjugated with the human monoclonal antibody of the present invention. This therapeutic agent is selected from among radionuclides, drugs, lymphokines, toxins, and bispecific antibodies. Examples of the drugs or toxins include Etoposide, teniposide, adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, cis-platinum and cis-platinum analogues, bleomycins, esperamicins, 5-fluorouracil, melphalan, and nitrogen mustard, but are not limited thereto.

In the composition and kit for the inhibition of cancer growth and metastasis or for the treatment or prevention of cancer, the PAUF-specific human monoclonal antibody or its functional fragment is used to inhibit the functions of PAUF in various cancers to suppress the growth and metastasis of the cancers, thereby killing the tumor cells. Preferably,the treatment or prevention of cancer is achieved by regulating the proliferation, migration or invasion of cancer. Preferably, the cancer is pancreatic cancer, stomach cancer, ovarian cancer, or colorectal cancer.

In greater detail, the method of the present invention comprises administering the pharmaceutical composition to the body in a pharmaceutically effective dosage. The pharmaceutical composition may be administered parenterally, subcutaneously, intrapulmonarily or intranasally. For local immunosuppressive therapy, the composition may, if desired, be administered using a suitable method including intralesional administration. Parenteral injections include intramuscular, intravenous, intraarterial, intraperitoneal and subcutaneous routes. Preferred administration modes include intravenous, subcutaneous, intradermal, intramuscular and drip injections.

In an embodiment of the present invention, the pancreatic cancer cell line Panc-1 was treated with the monoclonal antibody of the present invention and examined for proliferation to determine the effect of the monoclonal antibody on the growth of cancer cells. The cells were found to proliferate at lower rates upon treatment with the antibody than with a control antibody (FIG. 3). In other words, the monoclonal antibody according to the present invention inhibited the growth and/or survival of cancer cells. The effect of the monoclonal antibody of the present invention on the migration of cancer cells was also examined. In this regard, after being treated with the monoclonal antibody, the pancreatic cell lines CFPAC-1 and Panc-1 were assayed for migration ability. As a result, the cells treated with the antibody of the present invention were observed to decrease in migration ability as compared to those treated with a control antibody (FIG. 4). Likewise, the cancer cell lines treated with the antibody of the present invention were found to decease in invasion ability, compared to those treated with a control antibody, as measured by invasion assay (FIG. 5). Taken together, these data demonstrate that the PAUF-specific antibody according to the present invention can function to inhibit the growth, migration, invasion and/or metastasis of cancer cells.

In addition, the monoclonal antibody of the present invention was examined for in vivo therapeutic activity against caner. To this end, first, the pancreatic cancer cell line CFPAC-1 was transplanted into immunodeficient mice. The antibody was injected into the mice via the tail vein. As compared to those injected with a control, the mice treated with the antibody of the present invention were observed to decrease in tumor size (FIG. 6). Accordingly, the antibody of the present invention functioned to inhibit the progression of tumors in vivo.

Sequence lists are given in Table, below. They include amino acid sequences and corresponding base sequences for light chain variable regions and heavy chain variable regions of four anti-PAUF antibodies according to the present invention.

Table 1

8F3 Light Chain Variable Region a.a. Sequence	SEQ ID NO.1	DIQMTQSPSSLSASVGDRVTITCRASQSISRYLDWYQQKPGKAPRLLIYSTSTLQRGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPFAFGPGTKVDIKR
3A4 Light Chain Variable Region a.a. Sequence	SEQ ID NO.2	DIQMTQSPSSLAVSLGERATIDCRSSQSLLHSSNNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCHQYYSTPLTFGGGTKVDIKR
36C9 Light Chain Variable Region a.a. Sequence	SEQ ID NO.3	QLVLTQPPSASGPPGQRVTISCSGSRSNIGSNTVNWYQHLPGTAPKLLIHTNNQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEGDYYCAAWDDSLNGHFVFGTGTKVTV
6C4 Light Chain Variable Region a.a. Sequence	SEQ ID NO.4	DIVMTQSPLSLSASVGDRLTITCRASQSILTYLNWYQQKPGKAPKLLVYAASSLQPGVPSRFSGRGSGTDFTLTISGLQPDDFALYYCQQSYSSPYSFGPGTKVDIKR
8F3 Heavy Chain Variable Region a.a. Sequence	SEQ ID NO.5	MAQVQLVQSGGGLIQPGGSLRLSCAASGFTVSSHYMNWVRQAPGKGLEWVSIIYSGGSTYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCARDIPRTVSPRTRAMDVWGQGTSVTVSS
3A4 Heavy Chain Variable Region a.a. Sequence	SEQ ID NO.6	MAQVQLVQSGGGLVQPGGSLRVSCAASGFPFTTYAMTWVRQAPGRGLEWVAVISGSGRTTHYADSVKGRFTISRDASKNTLYLQMNSLRAEDTAVYYCAKTSHPRQLAVAGPFQHWGQGTLITVSS
36C9 Heavy Chain Variable Region a.a. Sequence	SEQ ID NO.7	MAQVQLVESGGGVVLPGGSLRLSCAASGFPFGSYAVSWVRQAPGKGLEWVSAISPRNRYIYYADSVKGRFTISRDNAKNSLYLEMNSLGAEDTAVYYCAKDRLLRGAIVSALGHWGQGTLVTVSS
6C4 Heavy Chain Variable Region a.a. Sequence	SEQ ID NO.8	MAQVQLVESGGGLVQPGGSLRLSCAASGFTFDRYWMTWVRQAPGKGLEWVANIKHDGSEKDYVDSVKGRFIISRDNAKKSLYLQMNSLRAEDTAVYYCARRRSYGRSTYYLDSWGQGTQITVSS
8F3 Light Chain a.a. Sequence	SEQ ID NO.9	MGWSYIILFLVATATDVHSSGVGSDIQMTQSPSSLSASVGDRVTITCRASQSISRYLDWYQQKPGKAPRLLIYSTSTLQRGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPFAFGPGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
3A4 Light Chain a.a. Sequence	SEQ ID NO.10	MGWSYIILFLVATATDVHSSGVGSDIQMTQSPSSLAVSLGERATIDCRSSQSLLHSSNNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCHQYYSTPLTFGGGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
36C9 Light Chain a.a. Sequence	SEQ ID NO.11	MGWSYIILFLVATATDVHSSGVGSQLVLTQPPSASGPPGQRVTISCSGSRSNIGSNTVNWYQHLPGTAPKLLIHTNNQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEGDYYCAAWDDSLNGHFVFGTGTKVTVXRWRTKVEIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
6C4 Light Chain a.a. Sequence	SEQ ID NO.12	MGWSYIILFLVATATDVHSSGVGSDIVMTQSPLSLSASVGDRLTITCRASQSILTYLNWYQQKPGKAPKLLVYAASSLQPGVPSRFSGRGSGTDFTLTISGLQPDDFALYYCQQSYSSPYSFGPGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
8F3 Heavy Chain a.a. Sequence	SEQ ID NO.13	MGWSYIILFLVATATDVHSAQPAMAQVQLVQSGGGLIQPGGSLRLSCAASGFTVSSHYMNWVRQAPGKGLEWVSIIYSGGSTYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCARDIPRTVSPRTRAMDVWGQGTSVTVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
3A4 Heavy Chain a.a. Sequence	SEQ ID NO.14	MGWSYIILFLVATATDVHSAQPAMAQVQLVQSGGGLVQPGGSLRVSCAASGFPFTTYAMTWVRQAPGRGLEWVAVISGSGRTTHYADSVKGRFTISRDASKNTLYLQMNSLRAEDTAVYYCAKTSHPRQLAVAGPFQHWGQGTLITVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
36C9 Heavy Chain a.a. Sequence	SEQ ID NO.15	MGWSYIILFLVATATDVHSAQPAMAQVQLVESGGGVVLPGGSLRLSCAASGFPFGSYAVSWVRQAPGKGLEWVSAISPRNRYIYYADSVKGRFTISRDNAKNSLYLEMNSLGAEDTAVYYCAKDRLLRGAIVSALGHWGQGTLVTVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
6C4 Heavy Chain a.a. Sequence	SEQ ID NO.16	MGWSYIILFLVATATDVHSAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTFDRYWMTWVRQAPGKGLEWVANIKHDGSEKDYVDSVKGRFIISRDNAKKSLYLQMNSLRAEDTAVYYCARRRSYGRSTYYLDSWGQGTQITVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Table 2

8F3 Light Chain Variable Region Base Sequence	SEQ ID NO.17	GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGGTATTTAGATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAGACTCCTGATCTATTCTACTTCCACTTTGCAAAGAGGGGTCCCATCAAGATTCAGTGGCGGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCGCATTCGGCCCTGGGACCAAAGTGGATATCAAACGT
3A4 Light Chain Variable Region Base Sequence	SEQ ID NO.18	GACATCCAGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCGACTGCAGGTCCAGCCAGAGTCTTTTACACAGCTCCAACAATAAGAACTACTTAGCTTGGTTCCAGCAGAAACCAGGACAGCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATGTGGCAGTCTATTACTGTCACCAATATTATAGTACTCCCCTCACTTTCGGCGGAGGGACCAAGGTGGATATCAAACGT
36C9 Light Chain Variable Region Base Sequence	SEQ ID NO.19	CAGCTCGTGCTGACTCAGCCACCCTCAGCGTCTGGGCCCCCCGGGCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGGTCCAACATCGGAAGTAATACTGTTAACTGGTATCAGCACCTCCCAGGAACGGCCCCCAAGCTCCTCATCCATACTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCGTCAGTGGGCTCCAGTCTGAGGATGAGGGTGATTATTATTGTGCAGCATGGGATGACAGCCTGAATGGCCATTTTGTCTTCGGAACTGGGACCAAGGTCACCGTC
6C4 Light Chain Variable Region Base Sequence	SEQ ID NO.20	GATATTGTGATGACCCAGTCTCCACTCTCCCTGTCTGCATCTGTAGGCGACAGACTCACTATCACTTGCCGGGCAAGTCAGAGCATTCTTACCTATTTAAATTGGTATCAACAAAAACCAGGGAAAGCCCCGAAGCTCCTAGTCTATGCTGCATCCAGTTTGCAACCTGGGGTCCCATCAAGGTTCAGTGGCCGCGGCTCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGACGATTTTGCACTTTACTACTGTCAACAGAGTTACAGTAGTCCGTACTCTTTCGGGCCTGGGACCAAAGTGGATATCAAACGT
8F3 Heavy Chain Variable Region Base Sequence	SEQ ID NO.21	ATGGCCCAGGTGCAGCTGGTACAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGTCATTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGCGGTGGTAGTACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACTCTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATATCCCTCGTACGGTGTCCCCACGTACCAGAGCTATGGACGTCTGGGGCCAAGGGACTTCGGTCACCGTCTCCTCA
3A4 Heavy Chain Variable Region Base Sequence	SEQ ID NO.22	ATGGCCCAGGTGCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCCCGTTTACCACTTATGCCATGACTTGGGTCCGCCAGGCTCCAGGGAGGGGACTGGAGTGGGTCGCAGTTATAAGTGGTAGTGGAAGAACCACACACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACGCTTCCAAGAACACACTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAACAAGCCACCCCCGACAACTAGCAGTGGCTGGCCCCTTCCAGCACTGGGGCCAGGGCACCCTGATCACCGTCTCCTCA
36C9 Heavy Chain Variable Region Base Sequence	SEQ ID NO.23	ATGGCCCAGGTGCAGCTGGTAGAGTCTGGGGGAGGCGTGGTCCTGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCCCCTTTGGCAGCTATGCCGTGAGCTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTCCTAGGAATCGTTACATATACTACGCAGACTCAGTGAAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAACTCACTTTATCTGGAAATGAACAGTCTGGGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATCGCCTACTTAGGGGGGCGATAGTGAGTGCCCTTGGGCACTGGGGCCAGGGAACTCTGGTCACCGTCTCCTCA
6C4 Heavy Chain Variable Region Base Sequence	SEQ ID NO.24	ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTCCAACCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTTACTTTTGATAGATATTGGATGACCTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCATGATGGAAGTGAGAAAGACTATGTGGACTCTGTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAAGTCGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGACGGCGCTCATATGGTCGGAGTACTTACTATTTGGACTCCTGGGGCCAGGGAACCCAGATCACCGTCTCCTCA

As described above, the PAUF protein and PAUF nucleic acid molecule according to the present invention is closely correlated with the onset of cancer and can be a marker or a therapeutic target particularly for pancreatic cancer. Also, the anti-PAUF antibody or its functional fragment according to the present invention binds specifically to PAUF on various cancer cells including pancreatic cancer and stomach cancer, to allow the diagnosis of cancer when it is in the early stages, to inhibit the growth, invasion or migration of cancer cells, and to induce the cancer cells to undergo apoptosis, so that it is useful in the diagnosis and treatment of cancer.

FIG. 1 shows purified monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 on acrylamide gel.

FIG. 2 is a titer curve showing the binding of the antibodies 8F3, 3A4, 36C9 and 6C4 to PAUF protein.

FIG. 3 shows the inhibitory activity of the antibodies 8F3, 3A4, 36C9 and 6C4 against the growth of a pancreatic cancer cell line (Panc-1).

FIG. 4 shows the inhibitory activity of the antibodies 8F3, 3A4, 36C9 and 6C4 against the migration of two pancreatic cancer cell lines (Panc-1 and CFPAC-1).

FIG. 5 shows the inhibitory activity of the antibodies 8F3, 3A4, 36C9 and 6C4 against the invasion of two pancreatic cancer cell lines (Panc-1 and CFPAC-1).

FIG. 6 shows the inhibitory activity of the antibodies 8F3 and 36C9 against the growth of the pancreatic cancer cell line transplanted into mice.

FIG. 7 shows the ability of the humanized antibody 8F3 and polyclonal antibodies to PAUF to detect PAUF in blood and the detection of PAUF with the antibody specifically in sera from pancreatic cancer patients.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Screening of PAUF-Specific Monoclonal Antibody

The PAUF protein was immobilized onto a plastic tube and buffered for 2 hours in a buffer (2% skim milk, PBS). After the removal of the buffer, a human antibody display phage library in a buffer was added to the plastic tube and incubated at room temperature for 2 hours. The tube was washed 10 times with a washing buffer (0.1% Tween 20, PBS) and then 10 times with PBS. Thereafter, the phages were eluted by incubation at room temperature for 10 min with 100mM TEA (triethylamine). To the phage eluate was added 1M Tris (pH 7.5), followed by incubation at room temperature for 5 min for neutralization. Then, an E. coli culture was added to the phage elutate. The E. coli thus infected with the phage was recovered as a cell pellet by centrifugation at 3,000 xg for 5 min and spread over 2xYT plates. E. coli colonies formed after incubation at 37℃ for 16 hours were suspended in 2xYT media and cultured. The infected E. coli, after being recovered from the suspension, was inoculated into fresh 2xYT media and supplemented with helper phage to primarily screen the phage. With this primarily screened phage library, this screening process was further conducted four times. After five rounds of the screening, clones were randomly selected from 2xYT plates and cultured in 2xYT media. To the clone culture was added helper phage to rescue phages. The phages thus produced were examined for specificity for PAUF by PAUF ELISA (Example 3). The phages which were found to show PAUF specificity were selected and subjected to base sequencing. Through these processes, 8F3, 3A4, 36C9 and 6C4 antibodies, which bind specifically to PAUF were excavated.

EXAMPLE 2: Purification of PAUF-Specific Human Monoclonal Antibody

CHO-DG44 cell lines which can express PAUF-specific human monoclonal antibodies in a large quantity were established. To this end, first, light chain variable regions of the 10 monoclonal antibodies excavated by use of the method described in Example 1 were inserted into the BstXI site of pIgGLD while the heavy chain variable regions were inserted into the SfiI site of pIgGHD, so that they were converted to IgG forms. pIgGLD and pIgGHD are expression vectors for antibody production which contain constant regions of light and heavy chains of IgG, respectively. The expression plasmids for IgG were transfected into the DHFR-deficient CHO cell line CHO-DG44 with the aid of Lipofectamin Plus (Invitrogen, CA) according to the protocol provided by the manufacturer. The stably transfected cells were selected in the presence of 550 ㎍/㎖ of G418 (Invitogen, CA) over 2 weeks. The stable transfectants were adapted to MTX (Sigma, MO) while the concentration was increased from 10 nM at intervals of two weeks. The monoclonal antibodies secreted to the media from the adapted cell lines were quantitatively analyzed using ELISA. High-expression cell lines were selected and applied to the mass production of the monoclonal antibodies.

The selected cell lines were grown in SF CHO Media (JBI, KR) in tissue culture flasks to express the antibodies. The antibodies of the present invention may be purified using various methods including affinity chromatography and ion exchange chromatography. All of the four antibodies 8F3, 3A4, 36C9 and 6C4 shown in FIG. 5 were purified as follows. The human monoclonal antibodies secreted into the SF CHO Media were first subjected to affinity chromatography using Protein A columns which can bind to Fc regions of antibodies. A culture medium containing the expressed antibodies was forced to pass once through a 0.45 ㎛ membrane in a filter and loaded on Protein A columns which were pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.0). A 20 mM sodium phosphate buffer (pH 7.0) was dropwise added to the Protein A column to wash off unbound proteins. The monoclonal antibodies were eluted with 100 mM sodium citrate buffer (pH3). The pH of the resulting eluted fractions was adjusted into 7 ~ 7.5 with 1 M Tris buffer (pH 9.0). Then, the eluted monoclonal antibody fractions were pooled and concentrated using a 3K MWCO spin column with a buffer exchange with 20 mM sodium phosphate (pH 7.0). This sample was loaded on a cation exchange chromatography column which was previously equilibrated with 20 mM sodium phosphate buffer (pH7.0). Unbound materials were washed off with the same buffer, followed by eluting monoclonal antibodies of interest with 20mM sodium phosphate, 1 M NaCl (pH 7.0). As a result, the monoclonal antibodies were purified, and endotoxin content was minimized. FIG. 1 shows purified monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 on acrylamide gel.

EXAMPLE 3: Assay of PAUF-Specific Human Monoclonal Antibody for Antigen-Binding Capacity

The PAUF protein was immobilized onto a plate and buffered for 2 hours in a buffer (2% skim milk, PBS). After the removal of the buffer, an human monoclonal antibody display phage eluate or purified human antibodies in buffer was added to the plate and incubated at 37℃ for 1 hour. Unbound antibodies were washed off five times with a washing solution (0.1% Tween 20, PBS). As a secondary antibody, anti-M13-HRP and anti-human IgG (Fc specific)-HRP were used for the phage elution or the human antibodies, respectively. A dilution (1:5000) of a suitable secondary antibody in buffer was added to the plate and incubated at 37℃ for 30 min. After washing five times with a washing solution (0.1% Tween 20, PBS), colors were developed with TMB and H₂O₂. Absorbance at 450nm was read to select PAUF-specific antibodies or measure the binding capacity of purified antibodies according to concentrations. FIG. 2 shows measurements of antigen-binding capacity of the purified antibodies 8F3, 3A4, 36C9 and 6C4 according to concentrations.

EXAMPLE 4: Proliferation Assay

Pancreatic cancer cells were plated at a density of 5x10⁴ cells/㎖ in 24-well plates and mixed with 3 ㎍/㎖ of the monoclonal antibody. The cells were incubated in a 37℃ CO₂ incubator while the medium was exchanged with a fresh one every two days. After incubation for 6 days, cells were recovered stained with 0.2% tryphan blue, and counted. Comparison was made between the numbers of living cells in antibody-treated wells and in a control, indicating that the four monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 could inhibit the growth of pancreatic cancer cells (FIG. 3).

EXAMPLE 5: Migration Assay

Pancreatic cells were isolated and washed twice with PBS. Various concentrations of the antibodies were added in such an amount as to adjust the number of cells to 1 x 10⁶ cells/㎖. In a Neuro Probe 48-well micro chamber from Neuroprobe Inc., the lower chamber was filled with 30 ㎕ of a DMEM medium supplemented with 10% FBS and covered with a 8 m pore size polycarbonate filter (25x80 mm, Neuroprobe). Then, the upper chamber was applied to the lower chamber and immobilized. A mixture of pancreatic cancer cells and antibody was added in an amount of 50 ㎕ per well to the immobilized upper chamber and incubated for 20 hours in a 37℃, CO₂ incubator. Afterwards, the cells which migrated to the lower chamber were fixed with 100% methanol and stained with a Giemsa solution. In at least three selected areas of a photograph taken, the stained cells were counted. This assay indicated that the four selected monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 could inhibit the migration of pancreatic cancer cells (FIG. 4).

EXAMPLE 6: Invasion Assay

Pancreatic cells were isolated and washed twice with PBS. Various concentrations of the antibodies were added in such an amount as to adjust the number of cells to 1 x 10⁶ cells/㎖. In a Neuro Probe 48-well micro chamber from Neuroprobe Inc., the lower chamber was filled with 30 ㎕ of a DMEM medium supplemented with 10% FBS and covered with a matrigel-coated, 8 m pore size polycarbonate filter (25x80 mm, Neuroprobe). Then, the upper chamber was applied to the lower chamber and immobilized. A mixture of pancreatic cancer cells and antibody was added in an amount of 50 ㎕ per well to the immobilized upper chamber and incubated for 20 hours in a 37℃ CO₂ incubator. Afterwards, the cells which migrated to the lower chamber were fixed with 100% methanol and stained with a Giemsa solution. In at least three selected areas of a photograph taken, the stained cells were counted. This assay indicated that the four selected monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 could inhibit the invasion of pancreatic cancer cells (FIG. 5).

EXAMPLE 7: Assay of PAUF-Specific Antibodies for Inhibitory Activity against Growth of Cancer Cell

CFPAC-1 cells were transplanted in an amount of 5 x 10⁶ cells into the hypodermis of 6~7-week-old Balb/c nu/nu nude mice. From 10 days after the transplantation, the monoclonal antibodies were injected at a concentration of 5 mg/kg twice a week for three weeks via the tail vein. Using a caliper, tumor volumes were measured twice a week. On the final day of the experiment, the nude mice were sacrificed with CO₂, and tumors were separated and weighed. The tumors were fixed, embedded in paraffin blocks, and stained with rabbit anti-PAUF polyclonal antibodies. The tumors of the mice injected with the PAUF-specific monoclonal antibodies were found to be lower in PAUF level than those of the mice injected with a control antibody, indicating that the two selected monoclonal antibodies 8F3 and 36C9 can inhibit the growth of the pancreatic cell line CFPAC-1 in mice (FIG. 6).

EXAMPLE 8: Measurement of Blood Level of PAUF with Anti-PAUF Antibody and Diagnosis of Pancreatic Cancer

The following experiment was conducted in order to determine whether pancreatic cancer can be diagnosed by detecting PAUF in blood. The anti-PAUF antibody 8F3 was diluted to a concentration of 1-10 μg/ml in PBS (pH 7.2) and the dilution was plated in an amount of 100 ㎕/well onto 96-well plates, followed by incubation of the sealed plates at 4℃ for 18 hours to sufficiently fix the antibody to the plates. After removal of unfixed antibodies, PBS containing 1% bovine serum albumin was added in an amount of 300 ㎕/well onto the plates and incubated at 37℃ for 2 hours. The solution was removed from the wells of the plates which were then dried, placed together with a desicant in a sealed vessel, and incubated at 4℃. Sera obtained from healthy persons or patients with pancreatic cancer, pancreatitis or other diseases were two-fold diluted in PBS containing 3% bovine serum albumin and plated in an amount of 100 ㎕/well onto the antibody-coated plates before incubation at 37℃ for 90 min. Each well was washed five times with 300 ㎕ of PBS containing 0.05% Tween 20, after which anti-PAUF polyclonal antibodies were plated at a concentration of 8 μg/ml/well, together with 100 ㎕ of PBS containing 5% horse serum and 100mM NaCl, onto the plates, followed by incubation at 37℃ for 90 min. The solution was aspirated from each well, and the plates were washed five times with PBS containing 5% Tween20. An HRP-conjugated anti-rabbit antibody was 1/2000 diluted in PBS containing 1% bovine serum and the dilution was plated in an amount of 100 ㎕/well onto the plates, followed by incubation at 37℃ for 30 min. The plates were washed five times with PBS containing 0.05% Tween20. A substrate solution containing 100 μg/ml tetrametyl benzidine, 0.006% hydrogen peroxide and citric acid in PBS (pH4.5) was added in an amount of 100 ㎕/well to the plates and allowed to develop a color for 30 min in a dark place. Color development was terminated by adding 50 ㎕ of a reaction stopper (1N sulfuric acid) to each well. Absorbance at 450 was read in a 96-well plate reader (Molecular Devices), with a reference wavelength at 650 nm. The colorant tetramethyl benzidine was degraded by the HRP conjugate with antibody to develop a color. Color development was thus analyzed by measuring the absorbance at 450 nm, to determine the presence of PAUF in the blood. The specimens used in this experiment were as follows.

(1) 26 serum samples from healthy persons.

(2) 13 pancreatic cancer-positive serum samples

(3) serum samples from patients with other types of cancer or disease: 15 sera of stomach cancer, one serum of colorectal cancer, one serum of breast cancer, three sera of hepatic cancer, and five sera of patients suffering from hepatitis.

51 serum specimens from normal persons and patients with other diseases were judged as being negative, with a diagnotic specificity of 100%. Of 13 pancreatic cancer-positive specimens, eight were determined to be positive, with a sensitivity of 61.5 %. Further, the PAUF levels of the specimens which were determined to be negative were a little higher those of the sera independent of pancreatic cancer (FIG. 7). Thus, an upgraded analytical technique may afford higher sensitivity. Consequently, the method of the present invention allows the diagnosis of pancreatic cancer only with serum samples.

Closely correlated with the onset of cancer, the PAUF protein or the PAUF nucleic acid molecule in accordance with the present invention can be used as a diagnostic marker or a therapeutic target. In addition, having the ability to bind specifically to PAUF presented on the surface of various cancer cells including pancreatic cancer cells, as described hitherto, the anti-PAUF antibody or its functional fragment allows the diagnosis of cancer in the early stages thereof and can inhibit the growth, invasion or migration of cancer cells by inducing the cells to undergo apoptosis, and thus are useful in the diagnosis and treatment of cancer.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims (28)

1. A human monoclonal antibody or functional fragment thereof specially binding to PAUF (Pancreatic adenocarcinoma upregulating factor), comprising a light chain variable region of which an amino acid sequence is selected from the group consisting of:

   (a) a light chain variable region comprising CDR1 as defined by SEQ ID NO.25, CDR2 as defined by SEQ ID NO. 26, and CDR3 as defined by SEQ ID NO.27;

   (b) a light chain variable region comprising CDR1 as defined by SEQ ID NO.31, CDR2 as defined by SEQ ID NO.32, and CDR3 as defined by SEQ ID NO.33;

   (c) a light chain variable region comprising CDR1 as defined by SEQ ID NO.37, CDR2 as defined by SEQ ID NO.38, and CDR3 as defined by SEQ ID NO.39; and

   (d) a light chain variable region comprising CDR1 as defined by SEQ ID NO.43, CDR2 as defined by SEQ ID NO.44, and CDR3 as defined by SEQ ID NO.45.
2. A human monoclonal antibody or functional fragment thereof specially binding to PAUF (Pancreatic adenocarcinoma upregulating factor) comprising a heavy chain variable region of which an amino acid sequence is selected from the group consisting of:

   (a) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.28, CDR2 as defined by SEQ ID NO.29, and CDR3 as defined by SEQ ID NO.30;

   (b) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.34, CDR2 as defined by SEQ ID NO.35, and CDR3 as defined by SEQ ID NO.36;

   (c) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.40, CDR2 as defined by SEQ ID NO.41, and CDR3 as defined by SEQ ID NO.42; and

   (d) a heavy chain variable region comprising CDR1 as defined by SEQ ID NO.46, CDR2 as defined by SEQ ID NO.47, and CDR3 as defined by SEQ ID NO.48.
3. A human monoclonal antibody or a functional fragment thereof, comprising:

   (a) a light chain variable region defined as a polypeptide having an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOS. 1 to 4, or

   (b) a heavy chain variable region defined as a polypeptide having an amino acid sequence selected from a group consisting of amino acid sequences of SEQ ID NOS. 5 to 8; or

   (c) both (a) the light chain variable region and (b) the heavy chain variable region.
4. The human monoclonal antibody or the functional fragment thereof according to claim 3, wherein the antibody inhibits cancer cells from proliferating, migrating or invading.
5. The human monoclonal antibody or the functional fragment thereof according to claim 4, wherein the cancer cells are selected from a group consisting of pancreatic cancer cells, stomach cancer cells, ovarian cancer cells and colorectal cancer cells.
6. The human monoclonal antibody or the functional fragment thereof according to claim 3, wherein the functional fragment is in a form selected from a group consisting of Fab, F(ab'), F(ab')₂, and Fv.
7. The human monoclonal antibody or the functional fragment thereof according to claim 3, wherein the human monoclonal antibody comprises light chain amino acid sequences as defined by one selected from among SEQ ID NOS. 9 to 12; or a heavy chain amino acid sequences as defined by one selectd from among SEQ ID NOS. 13 to 16.
8. A nucleic acid molecule, encoding the human monoclonal antibody or the functional fragment of one of claims 1 to 3.
9. The nucleic acid molecule according to claim 8, wherein the nucleic acid molecule comprises a base sequence selected from a group consisting of:

   (a) a base sequence, encoding a light chain variable region, defined as one selected from among SEQ ID NOS. 17 to 20;

   (b) a base sequence, encoding a heavy chain variable region, defined as one selected from among SEQ ID NOS. 21 to 24; and

   (c) a combination of the base sequence of (a) and the base sequence of (b).
10. A marker for the diagnosis of cancer, comprising a PAUF protein having a sequence defined by SEQ ID NO. 50 or a nucleic acid molecule coding for the PAUF protein.
11. The marker according to claim 10, wherein the nucleic acid molecule has a nucleotide sequence defined by SEQ ID NO. 49.
12. The marker according to claim 10, wherein the cancer is selected from among pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer.
13. A composition for diagnosis of cancer, comprising at least one selected from a group consisting of a protein, a peptide, an aptamer and an antibody, all of which bind specifically to a PAUF protein or an immunogenic fragment thereof.
14. The composition according to claim 13, wherein the antibody is the human monoclonal antibody of one of claims 1 to 3 or the functional fragment thereof.
15. The composition according to claim 14, wherein the human monoclonal antibody or the functional fragment thereof is encoded by the nucleic acid molecule of claim 9.
16. The composition according to claim 13, wherein the cancer is selected from a group consisting of pancreatic cancer, stomach cancer, ovarian cancer, and colorectal cancer.
17. A kit for diagnosis of cancer, comprising at least one selected from a group consisting of a protein, a peptide, an aptamer and an antibody, all of which bind specifically to a PAUF protein or an immunogenic fragment thereof.
18. A method for diagnosing cancer, comprising measuring an expression level of a PAUF protein or a PAUF nucleic acid molecule or an activity of a PAUF protein.
19. The method according to claim 18, wherein the cancer is selected from a group consisting of pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer.
20. A method of detecting a PAUF protein, comprising:

    contacting a biological sample with an anti-PAUF antibody of one of claims 1 to 3; and

    measuring a level of the PAUF protein in the biological sample to determine whether the level is increased or decreased.

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21. A method of screening a material therapeutic or preventive of cancer, comprising:

    (a) bringing a test sample into contact with a cell containing a PAUF protein or nucleic acid molecule;

    (b) measuring a level of the PAUF protein or nucleic acid molecule or an activity of the PAUF protein; and

    (c) judging the test sample as being therapeutic or preventive of cancer when the level or the activity is determined to be down-regulated.
22. The method according to claim 21, wherein the cancer is selected from a group consisting of pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer.
23. A composition for inhibiting cancer growth and metastasis, comprising as an active ingredient a material suppressive of the expression of a PAUF protein or a PAUF nucleic acid molecule or the activity of a PAUF protein.
24. A composition for treatment or prevention of cancer, comprising as an active ingredient a material suppressive of the expression of a PAUF protein or a PAUF nucleic acid molecule or the activity of a PAUF protein.
25. The composition according to claim 24, comprising at least one selected from a group consisting of a protein, an aptamer, a peptide and an antibody, all of which bind specifically to PAUF.
26. The composition according to claim 25, wherein the antibody is the human monoclonal antibody of one of claims 1 to 3 or the functional fragment thereof.
27. The composition according to claim 26, wherein the human monoclonal antibody or the functional fragment thereof is encoded by the nucleic acid molecule of claim 9.
28. The composition according to claim 24, wherein the cancer is selected from a group consisting of pancreatic cancer, stomach cancer, ovarian cancer and colorectal cancer.

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