KR101098186B1 - Pauf-specific human monoclonal antibody, pharmaceutical composition for treating cancer comprising the same and method for detecting cancer using the same - Google Patents

Abstract

The present invention relates to human monoclonal antibodies that specifically bind to PAUF proteins, PAUF proteins associated with cancer, and PAUF nucleic acid molecules encoding them, and their use. It is about. More specifically, a cancer diagnostic marker comprising a human monoclonal antibody, a PAUF protein, or a PAUF nucleic acid molecule encoding the same, specifically binding to PAUF protein. A method for diagnosing cancer and a method for diagnosing cancer, the method for diagnosing cancer, and a method for diagnosing cancer, the method for screening a composition for treating or preventing cancer by measuring the level, activity and the like of the marker and the level of the marker The present invention relates to a composition for treating or preventing cancer, which reduces activity. The PAUF protein and PAUF nucleic acid molecules encoding the same according to the present invention are very specific for the development of cancer, and are particularly markers or therapeutic targets for pancreatic cancer. In addition, anti-PUF antibodies or functional fragments thereof that specifically bind to PAUF proteins may be expressed in PAUF proteins, including pancreatic cancer and gastric cancer. By specifically binding to the cancer can be diagnosed early, and inhibits the activity of PAUF protein inhibits the growth, infiltration and migration of cancer cells.

Description

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

The present invention relates to a human monoclonal antibody specifically binding to a PAUF protein, a cancer-related PAUF protein, and a PAUF nucleic acid molecule encoding the same, and its use. About. More specifically, a marker for cancer diagnosis comprising a human monoclonal antibody specifically binding to a PAUF protein, a PAUF protein, or a PAUF nucleic acid molecule encoding the same , A composition for diagnosis of cancer comprising an antibody that specifically binds to the marker, and a method for diagnosing cancer, and a method for screening a composition for treating or preventing cancer by measuring the level and activity of the marker, and the level of the marker , It relates to a composition for the treatment or prevention of cancer to reduce the activity.

The global cancer incidence rate is increasing by more than 5% every year due to the increase in the elderly population and the environmental deterioration caused by the rapid development of the industry, and according to the WHO report, the cancer incidence population will increase to 30 million in the next 25 years, of which 20 million. It is estimated that they will die of cancer (Ahn Soon-Gil (2003) Anticancer drug research and development trend, Health industry technology trend 2003 summer: 10-18). According to the results of deaths and causes of death in 2006 by the National Statistical Office, cancer ranks the highest among the three leading causes of death, and it was investigated as the cause of death with the largest increase in mortality. In addition, the research results that the cost of socioeconomic losses due to cancer amounts to about 11 trillion won per year suggests that the importance of cancer prevention and treatment and related research is high (Faint et al. (2004) Pipeline Insight: Cancer Overview). , Datamonitor DMHC2025).

Pancreatic cancer is not very high, accounting for about 2% of all cancer patients, but the mortality rate is known to be very high as the 4th place in all cancer-related deaths. This is because pancreatic cancer does not show any signs at the beginning, making early diagnosis difficult, and the metastasis rate is high.Therefore, at the time of detection, it has already progressed to a broadly metastasized terminal stage where surgical resection is not possible, and crucially, there is no effective treatment. (Faint et al. (2004) Datamonitor DMHC2045; Garcea et al. (2005) Pancreatology 5:514-529).

In addition, there is currently no method for diagnosing pancreatic cancer from human-derived body fluids containing blood. Currently, MRI, CT, etc. can be used for examination, but if the examination is possible with these methods, it is already in the terminal stage. In addition, the CA19-9 assay, which is used as a blood test for pancreatic cancer, has a very low specificity and cannot be used for diagnostic purposes. It is used only as a method of monitoring the treatment of a disease. (Method of cancer screening announced by the Ministry of Health and Welfare). For advanced pancreatic cancer, the proportion of patients who can undergo surgical resection due to the presence of distant metastases is about 10-15% of all pancreatic cancer patients, and the average survival time prolongation is 15 even when intensive treatment with resection and chemotherapy are received. It is on the very low side, about ~18 months. In the case of patients who cannot be resected, gemcitabine, an intravenous 2-deoxycitatin nucleoside analogue that can induce apoptosis of human pancreatic cancer cells and inhibit tumor growth and progression, is used as a basic prescription. However, it is being evaluated as having poor effect and many side effects that lower the quality of life of patients. Therefore, there is a strong need for the development of an effective and new therapeutic agent for the 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).

Anticancer drugs currently used clinically can be classified as chemotherapeutic drugs and biotherapy drugs. Chemotherapeutic agents, which have been actively developed in the past, are drugs that exhibit toxicity to cancer cells, and show limitations in that there are problems that show toxicity to normal cells as well as cancer cells and drug resistance as a kind of defense mechanism within cells. Accordingly, in recent years, biotherapeutics of the concept that can inhibit the progression of cancer cells by weakening the activity of cancer cells by restoring or increasing the immune function of the human body have emerged as an alternative and are being actively developed. Biotherapy agents currently used or being developed include recombinant antibodies such as cytokines and monoclonal antibodies, nucleic acid molecule therapeutics, and angiogenesis inhibitors (Kim Yeolhong et al. (2004) Cancer genome research and development of new anticancer drugs, health industry technology. Trend 2004-Summer 55~61; Ahn Soon-Gil (2003) Anticancer drug R&D trend, Health industry technology trend 2003-Summer 10~18).

Among these, therapeutic monoclonal antibodies are known to have lower side effects than conventional chemotherapeutic agents with cytotoxicity because they act only on disease-specific targets because of their high response specificity as a representative biotherapy agent. Antibodies exhibit therapeutic effects through various mechanisms. They can specifically bind to the antigen to inhibit signal transduction or induce cell death by cross-linking, and also activate the immune system in vivo to have its effect. Can also represent. Therefore, monoclonal antibodies as anticancer agents have the advantage of not only suppressing the function of the target by specifically tracking cancer cells, but also by causing an immune response to effectively remove cancer cells, and are gradually becoming the mainstream of cancer treatment. Of the monoclonal antibodies already used as antibody therapeutics, more than 20 types of monoclonal antibodies as of 2005 were approved for diagnosis and treatment, and more than half of them were developed for diagnosis and treatment of various cancers.

Accordingly, the present inventors discovered that the PAUF protein and the PAUF nucleic acid molecule encoding the same are very specific for the onset of cancer, and in particular, become markers or therapeutic targets in pancreatic cancer, By developing an anti-PAUF antibody that specifically binds to the PAUF protein, the antibody is applied to the PAUF protein expressed in various cancer cells including pancreatic cancer and gastric cancer. The present invention was completed by confirming that cancer can be diagnosed early by binding specifically, and inhibiting the growth, invasion and migration of cancer cells by inhibiting the activity of PAUF protein.

An object of the present invention is to provide a human monoclonal antibody or functional fragment thereof that specifically binds to PAUF.

Another object of the present invention is to provide a nucleic acid molecule encoding the antibody.

Another object of the present invention is to provide a marker for cancer diagnosis comprising a PAUF protein or a PAUF nucleic acid molecule encoding the same.

Another object of the present invention is to provide a composition for diagnosis of cancer comprising at least one selected from the group consisting of proteins, peptides, aptamers, and antibodies that specifically bind to PAUF proteins or immunogenic fragments thereof. It is to do.

Another object of the present invention is to provide a cancer diagnostic kit comprising at least one selected from the group consisting of proteins, peptides, aptamers, and antibodies that specifically bind to PAUF proteins or immunogenic fragments thereof. It is to do.

Another object of the present invention includes the step of measuring the level of the PAUF protein, the level of the PAUF nucleic acid molecule encoding it, or the activity of the PAUF protein. It is to provide a method of diagnosing cancer.

Another object of the present invention is to provide a method for detecting a PAUF protein comprising the step of measuring an increase or decrease in the level of a PAUF protein by contacting the antibody of claim 1 with a biological sample. will be.

Another object of the present invention includes the step of measuring the level of the PAUF protein, the level of the PAUF nucleic acid molecule encoding it, or the activity of the PAUF protein. It is to provide a method for screening a substance for treating or preventing cancer.

Another object of the present invention is to effectively reduce the expression of the PAUF protein, the expression of the PAUF nucleic acid molecule encoding it, or the activity of the PAUF protein. It is to provide a composition for inhibiting cancer growth and metastasis, including as a component.

Another object of the present invention is to effectively reduce the expression of the PAUF protein, the expression of the PAUF nucleic acid molecule encoding it, or the activity of the PAUF protein. It is to provide a composition for the treatment or prevention of cancer containing as an ingredient.

Hereinafter, the present invention will be described in detail.

As one aspect for achieving the above object, the present invention relates to a human monoclonal antibody or a functional fragment thereof that specifically binds to PAUF.

PYUF is overexpressed in cancer, particularly in pancreatic cancer, gastric cancer, ovarian cancer and colon cancer.

The present inventors confirmed that the PAF is overexpressed in the early stage of pancreatic cancer, and that it is overexpressed in pancreatic cancer cell lines as well as pancreatic cancer patients compared to normal cell lines. In addition, it was also confirmed that cancer cell growth, metastasis, and invasion occur well in cell lines overexpressing PAFF.

Accordingly, the present inventors have developed an antibody that specifically binds to the PRF to measure the presence and level of the PAF, or to use it to inhibit the growth, metastasis, and penetration of cancer.

More specifically, in one embodiment, the human monoclonal antibody or functional fragment thereof is (a) a light chain variable region CDR1 defined by SEQ ID NO: 25, a light chain variable region CDR2 defined by SEQ ID NO: 26, and SEQ ID NO: 27. A light chain variable region including the defined light chain variable region CDR3; (b) a light chain variable region comprising a light chain variable region CDR1 defined by SEQ ID NO: 31, a light chain variable region CDR2 defined by SEQ ID NO: 32, and a light chain variable region CDR3 defined by SEQ ID NO: 33; (c) a light chain variable region comprising a light chain variable region CDR1 defined by SEQ ID NO: 37, a light chain variable region CDR2 defined by SEQ ID NO: 38, and a light chain variable region CDR3 defined by SEQ ID NO: 39; And (d) one selected from the group consisting of a light chain variable region CDR1 defined by SEQ ID NO: 43, a light chain variable region CDR2 defined by SEQ ID NO: 44, and a light chain variable region CDR3 defined by SEQ ID NO: 45. It may include a light chain variable region.

In addition, in another embodiment, the human monoclonal antibody or functional fragment thereof is (a) a heavy chain variable region CDR1 defined by SEQ ID NO: 28, a heavy chain variable region CDR2 defined by SEQ ID NO: 29, and a heavy chain variable defined by SEQ ID NO: 30. A heavy chain variable region comprising region CDR3; (b) a heavy chain variable region comprising a heavy chain variable region CDR1 defined by SEQ ID NO: 34, a heavy chain variable region CDR2 defined by SEQ ID NO: 35, and a heavy chain variable region CDR3 defined by SEQ ID NO: 36; (c) a heavy chain variable region comprising a heavy chain variable region CDR1 defined by SEQ ID NO: 40, a heavy chain variable region CDR2 defined by SEQ ID NO: 41, and a heavy chain variable region CDR3 defined by SEQ ID NO: 42; And (d) one selected from the group consisting of heavy chain variable region CDR1 defined by SEQ ID NO: 46, heavy chain variable region CDR2 defined by SEQ ID NO: 47, and heavy chain variable region CDR3 defined by SEQ ID NO: 48 It may include a heavy chain variable region.

The term'PAUF (Pancreatic Adenocarcinoma Upregulating Factor)' of the present invention was named by the present inventor as a novel protein. It is preferably defined by SEQ ID NO: 50.

The PAF protein includes an isolated protein, an allelic modification of the protein, and a conservative amino acid substitution form of the protein. As described herein, in part'protein'or'polypeptide' refers to a protein having the human amino acid sequence shown in SEQ ID NO: 50. The term also includes naturally occurring allelic modifications and specifically proteins with amino acid sequences slightly different from those described above. Even if they contain amino acid sequences that differ slightly from those described above, alleles will still have the same or similar biological functions associated with these proteins.

In addition, as described herein, the protein family associated with the human amino acid sequence of SEQ ID NO: 50 refers to proteins that have been isolated from organisms in addition to humans.

The PAYUF protein is preferably in an isolated form. As used herein, proteins are said to be isolated when physical, mechanical, or chemical methods are used to remove proteins from cellular components that are generally associated with proteins. One of skill in the art can readily use standard purification methods to obtain an isolated protein.

The PAF protein further includes an insertion, deletion or conservative amino acid substitution modification of SEQ ID NO: 50. As used herein, conservative modification refers to an alteration in the amino acid sequence that does not adversely affect the biological function of the protein. Substitutions, insertions, or deletions are said to act inversely on a protein when the altered sequence disrupts or disrupts the function associated with the protein. For example, the overall charge, structure or hydrophobic/hydrophilic properties of a protein can be altered without adversely affecting its biological activity. Thus, for example, the amino acid sequence can be altered so that it does not adversely affect the biological activity of the protein, and the peptide becomes more hydrophobic or hydrophilic.

Typically, allelic modifications, conservative substitutional modifications, and some protein families are at least about 50%, 60%, 70% or 75%, more preferably at least about 80-90% of the sequence set forth in SEQ ID NO: 50, More preferably at least about 92-94%, and most preferably at least about 95%, 98% or 99% amino acid sequence identity. After aligning the sequences and introducing gaps to have maximum percent homology if necessary, such sequences as if the percent of amino acid sequence residues of the sequence candidate were identical to SEQ ID NO: 50, without considering conservative substitutions as part of the sequence identity. Identity or homology to is defined herein. Fusion proteins, or N-terminal, C-terminal or internal extensions, deletions, or insertions into peptide sequences are not contemplated as acting on homology. Thus, the protein is a molecule having the amino acid sequence disclosed in SEQ ID NO: 50; Amino acid sequence variants in which one or more amino acid residues are inserted into or within the disclosed coding sequence; Amino acid sequence variants of the disclosed sequences in which at least one residue has been substituted, or fragments thereof as defined above. In addition, such fragments, called peptides or polypeptides, may contain not only an obvious hydrophobic site, but also an antigenic site or a functional site of a protein identified as a site of an amino acid sequence corresponding to an existing protein domain. These sites can be easily identified by using commonly available protein sequencing software such as MacVector (Oxford Molecular).

Expected variants include, for example, homologous rearrangements, site-specific or PCR mutagenesis, and equivalents of other animal species including, but not limited to, rabbits, mice, pigs, cattle, sheep, horses and non-human primates. Proteins, and variants of alleles or other naturally occurring protein families; And mutations intended by derivatives covalently modified by chemical, enzymatic or other suitable means, substituted with functional groups other than naturally occurring amino acids (e.g., detectable functional groups such as enzymes or radioactive isotopes). It includes more to do.

In addition, the sequence of the protein and nucleic acid molecule of the PAUF is registered in GeneBank (Accession number EF067313), and preferably, the PAUF nucleic acid molecule is defined as SEQ ID NO: 49.

Antibodies used to specifically bind to the PFAF protein include, but are not limited to, specific CDRs (Complementarity Determining Region) for antigen binding.

Preferably, the monoclonal antibody of the present invention comprises a specific sequence, more preferably each of a light chain CDR, a heavy chain CDR, or a light chain CDR and a heavy chain CDR. It is known to those skilled in the art that the monoclonal antibody of the present invention can be obtained by grafting CDRs into the framework regions (FRs) of known therapeutic antibodies.

The specific antibody against PFA used in the present invention is a polyclonal antibody or a monoclonal antibody, preferably a monoclonal antibody, and most preferably the human monoclonal antibody described above or a functional fragment thereof.

The term "monoclonal antibody" of the present invention refers to a highly specific antibody directed against a single antigenic site (epitope) as a term known in the art. Typically, unlike polyclonal antibodies, which contain different antibodies directed against different epitopes, monoclonal antibodies are directed against a single epitope on the antigen. Polyclonal antibodies generally have a cross-reaction, so their specificity is low, and the antibody titer and specificity fluctuate for each lot of antisera.Therefore, the manufacture of diagnostic drugs using polyclonal antibodies has a problem that it is inherently difficult to control quality. , Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays using antigen-antibody binding, and also have another advantage of not being contaminated by other immunoglobulins because they are produced by cultivation of hybridomas. Have.

Antibodies against PFA are commonly practiced in the art, for example, fusion methods (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), recombinant DNA methods (US Patent 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)). Can be manufactured by General procedures for antibody production are described in 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, the production of hybridoma cells producing monoclonal antibodies is accomplished by fusion of an immortalized cell line with antibody-producing lymphocytes, and techniques required for this process are well known to those skilled in the art and can be easily performed. Polyclonal antibodies can be obtained by injecting a PFAF protein antigen into a suitable animal, collecting antiserum from the animal, and then isolating the antibody from the antiserum using a known affinity technique.

In the present invention, a human monoclonal antibody that specifically binds to PAUF can be selected using a known combinatorial antibody library and phage display technology (see Example 1).

The most successful application of phage display technology in biology-related research is the selection of various genetically recombinant antibody fragments (scFv or Fab form) from antibody libraries, and if this technology is used, affinity with antigens from phage display antibody libraries. Through the selection process, numerous human antibodies that can be used in a short time as an in vivo diagnosis and treatment can be obtained.

In general, antibody display libraries are produced by cloning an antibody gene obtained from naive, immunized, or semi-synthetic origin at the 5'terminal site of gIII already prepared in a phagemid vector and then introducing it into E. coli. Phagemid clones into which the antibody gene is inserted express the antibody-pIII fusion protein in E. coli, and all structural proteins such as wild type (wt) pIII required for the formation of genetically recombined phage are helpers such as M13K07 or VCSM13. It is provided by phage, and recombinant phage is produced in E. coli through a process called phage rescue. The recombinant phage generated through this process simultaneously displays the wt pIII and antibody-pIII fusion proteins on the surface, and the phagemid DNA is preferentially packaged in the recombinant phage, resulting in genotype-phenotype linkage. Is formed. This phage packaging process theoretically makes it possible to display the oligovalent antibody fragments on the surface of the recombinant phage, and thus, the powerful selection ability of the phage display technology obtained as a result of this process has resulted in a number of target molecules-non-specific. Among the background clones, only target molecule-specific functional antibody clones can be isolated by panning.

The human monoclonal antibody of the present invention may be used without purification, and may be purified using various conventional methods such as affinity chromatography, size exclusion chromatography, and ion exchange chromatography.

As a specific example, the present inventors recombined the light and heavy chains of the selected antibody into an expression vector for antibody production in order to develop a cell line stably expressing the antibody of the present invention. The expression vector was transformed into a DHFR-deficient CHO cell line (CHO-DG44). In order to amplify the transformed gene, methotrexate (MTX), an inhibitor of DHFR, was added to the selective medium and the concentration was increased stepwise to obtain a cell line with a high level of antibody expression, and the cell line was cultured in large quantities (Example 3).

As long as the antibody of the present invention has the characteristic of binding to specifically recognize PAUF, it is not only a complete form having the total length of two heavy chains and two light chains, but also as a functional fragment of an antibody molecule. Can be used. The functional fragment of an antibody molecule means a fragment having at least an antigen-binding function, and may include Fab, F(ab'), F(ab') _2, Fv, and the like. The antibody or antibody fragment according to the present invention is preferably selected from (a) SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 in order to retain the binding function to the PAUF. Light chain variable region defined by sequence; And/or (b) may include a heavy chain variable region defined by a sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

More preferably, the light chain amino acid sequence of the human monoclonal antibody according to the present invention may be selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, and the heavy chain amino acid sequence is SEQ ID NO: 13, SEQ ID NO: 14 , SEQ ID NO: 15 and SEQ ID NO: 16.

In yet another aspect, the present invention relates to a nucleic acid molecule encoding the PAUF-specific antibody or antibody fragment. Preferably, the nucleic acid molecule of the present invention is a light chain variable region, the base sequence defined by SEQ ID NO: 17 encoding SEQ ID NO: 1, the base sequence of SEQ ID NO: 18 encoding SEQ ID NO: 2, and SEQ ID NO: 19 encoding SEQ ID NO: 3 It may include a nucleotide sequence selected from the nucleotide sequence of SEQ ID NO: 20 encoding SEQ ID NO: 4 and the nucleotide sequence defined by SEQ ID NO: 21 encoding SEQ ID NO: 5 as a heavy chain variable region, and SEQ ID NO: 6 It may include a nucleotide sequence selected from the nucleotide sequence of SEQ ID NO: 22 encoding, the nucleotide sequence of SEQ ID NO: 23 encoding SEQ ID NO: 7 and the nucleotide sequence of SEQ ID NO: 24 encoding SEQ ID NO: 8.

The human monoclonal antibody of the present invention or a functional antibody fragment thereof specifically binds to PAUF in various cancer cells expressing the PAUF protein to prevent the growth, migration, and/or metastasis of the cancer. Inhibits it so that cancer can be cured. Preferably, the cancer is pancreatic cancer, gastric cancer, ovarian cancer and colon cancer.

The present invention relates to a novel use of a PAUF protein and a nucleic acid molecule encoding the same, and to a novel use of PAUF as a diagnostic marker or therapeutic target in relation to cancer. This novel use of the present invention is based on the findings of the present inventors that PAUF is overexpressed in cancer and that cancer cell growth, metastasis, and invasion occur well in cell lines that overexpress it.

In one aspect, the present invention relates to a marker for cancer diagnosis comprising a PAUF protein or a PAUF nucleic acid molecule encoding the same.

The marker of the present invention is specific for pancreatic cancer, gastric cancer, ovarian cancer and colon cancer, and in particular, is specific for pancreatic cancer.

The present inventors confirmed that PAUF protein was measured in pancreatic cancer patients, and in particular, all normal subjects were negative, showing a total diagnostic specificity of 100%, and 8 of 13 pancreatic cancer positive samples were positive. It was determined that the sensitivity was 61.5%, and it was confirmed that the sample showed a slightly higher value compared to other samples (Fig. 7). The results of this experiment show that the PAUF protein is useful as a cancer diagnostic marker (see Example 8).

In another aspect, the present invention is a composition and kit for cancer diagnosis comprising at least one selected from the group consisting of proteins, peptides, aptamers, and antibodies that specifically bind to a PAUF protein or an immunogenic fragment thereof. Provides.

The antibody is preferably a human monoclonal antibody according to the present invention or a functional fragment thereof.

In another aspect, the present invention provides a method for detecting PAUF protein using a human monoclonal antibody or a functional antibody fragment thereof. When the human monoclonal antibody of the present invention or a functional antibody fragment thereof is contacted with a biological sample to measure the increase or decrease in the level of the PAUF protein, the PAUF protein can be detected.

In addition, the human monoclonal antibody of the present invention or a functional antibody fragment thereof specifically binds to PAUF in various cancer cells expressing the PAF protein to grow, migrate, invade, and/or Or it can cure cancer by inhibiting metastasis. Preferably, the cancer is pancreatic cancer, gastric cancer, ovarian cancer and colon cancer.

In addition to the above components, the compositions and kits of the present invention may additionally include other components, and in addition, reagents capable of detecting antigen-antibody conjugates, for example, antigens or antibodies to solid bodies, chips, or beads. Methods of immobilization, labeled secondary antibodies, chromophores, enzymes (eg, conjugated with an antibody) and substrates thereof or other substances capable of binding to an antibody may be included (see FIG. 7 ). In addition, it may be manufactured as a plurality of separate packaging or compartment kits containing the above components.

The diagnostic composition and kit of the present invention can analyze the onset, development, and alleviation of cancer. Therefore, the term'diagnosis' used in the present specification has a meaning including not only determining the presence or absence of a disease, but also determining the onset, development, and alleviation of the disease.

The compositions and kits of the present invention are particularly useful for diagnosing cancers developed and developed by overexpression of PAUF, preferably pancreatic cancer, gastric cancer, ovarian cancer and colon cancer, and most preferably pancreatic cancer.

The diagnostic composition of the present invention has the advantage of being able to diagnose cancer conditions, especially pancreatic cancer with excellent efficiency, and furthermore, can discriminately diagnose cancer at an early stage.

In another aspect, the present invention provides a method for screening a substance for treating or preventing cancer comprising the following steps:

(a) contacting a test substance with a cell containing a PAUF protein or a nucleic acid molecule;

(b) measuring the level of the PAUF protein, the level of a PAUF nucleic acid molecule encoding it, or the activity of the PAUF protein; And

(c) when the measured value is determined to be down-regulated, determining that the test substance is a substance for treating or preventing cancer.

The term'prevention' as used herein refers to the expression of a substance obtained by a screening method according to the present invention or a PAUF protein according to the present invention, and expression of a PAUF nucleic acid molecule encoding the substance. , Or a substance that reduces the activity of a PAUF protein, for example, at least one selected from the group consisting of a protein, aptamer, peptide, and antibody that specifically binds to PAUF It refers to any action of inhibiting or delaying the onset of the disease by administration of a composition comprising a.

The term'treatment' as used herein refers to any action in which symptoms of the disease are improved or beneficially changed by administration of a substance obtained by the screening method according to the present invention or the composition.

According to the method of the present invention, first, a test substance to be analyzed is brought into contact with a cell containing a PAF protein or a nucleic acid molecule encoding the same.

As used herein, the term'test substance' is an unknown used in screening to examine whether it affects the level of the PAF protein, the level of the PAF nucleic acid molecule, or the activity of the PAF protein. Means the substance of. The sample includes, but is not limited to, a chemical substance, a nucleotide, an antisense-RNA, a small interference RNA (siRNA), a peptide, a protein, an aptamer, and a natural product extract.

Subsequently, the level of the PAF protein, the level of the PAF nucleic acid molecule, or the activity of the PAF protein are measured in the cells treated with the test substance. As a result of the measurement, if the level of the PAF protein, the level of the PAF nucleic acid molecule, or the activity of the PAF protein is reduced-downregulated, the test substance can be determined as a substance for the treatment or prevention of cancer. have.

In the present invention, "measurement of the level of a nucleic acid molecule or protein" refers to a process of confirming the presence and level of expression of a PAUF protein expressed in a biological sample or a nucleic acid molecule encoding the same.

Measurement of the change in the level of the PFAF nucleic acid molecule can be carried out through various methods known in the art. For example, RTPCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern Blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102 -108, CRCpress), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) or in situ hybridization reaction (Sambrook et al., Molecular Cloning A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)) can be used.

In the case of performing according to the RT-PCR protocol, first, total RNA is isolated from the cells treated with the sample, and then first-stranded cDNA is prepared using an oligo dT primer and a reverse transcriptase. Subsequently, the first-stranded cDNA is used as a template, and a PCR reaction is performed using a PAF nucleic acid molecule-specific primer set. Then, the PCR amplification product is subjected to electrophoresis, and the formed band is analyzed to measure the level change of the PFAF nucleic acid molecule.

Measurement of changes in the level of the PAF protein can be performed through various immunoassay methods known in the art. For example, changes in the amount of PAF protein include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbentassay (ELISA), capture-ELISA, inhibition or hardwood analysis, and sandwich analysis. It is not limited.

The immunoassay or immunostaining method is described in Enzyme Immunoassay, ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, Using Antibodies: ALAboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

In addition, the level of the protein can be confirmed with proteins, aptamers, peptides, etc. that specifically bind to the PFA protein, but is not limited thereto, and preferably an antibody that specifically binds to the protein is used. .

Analysis methods for measuring protein levels using antibodies include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunity. Diffusion method, rocket immunoelectrophoresis, tissue immunostaining, Immunoprecipitation Assay, Complement Fixation Assay, FACS, protein chip, etc., but are not limited thereto.

Through the above analysis methods, the amount of antigen-antibody complex formation in the normal control group and the amount of antigen-antibody complex formation in cancer suspected patients can be compared. By determining whether the level is increased, it is possible to diagnose whether the suspected cancer patient is actually a cancer patient.

The term'antigen-antibody complex' as used in the present invention means a combination of PAUF and an antibody specific thereto, and the amount of formation of the antigen-antibody complex is determined by the signal of the detection label. It can be measured quantitatively through size.

The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto.

In another aspect, the present invention is effective in reducing the expression of the PAUF protein, the expression of the PAUF nucleic acid molecule encoding the same, or the activity of the PAUF protein. It relates to a composition for inhibiting cancer growth and metastasis comprising as a component.

In addition, in another aspect, the present invention provides a substance that reduces the expression of a PAUF protein, the expression of a PAUF nucleic acid molecule encoding it, or the activity of a PAUF protein. It relates to a composition for treating or preventing cancer, including as an active ingredient.

The composition may contain one or more selected from the group consisting of proteins, aptamers, peptides, and antibodies that specifically bind to PAUF.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colors and fragrances, etc. for oral administration, and buffers, preservatives, and painlessness in the case of injections. An agent, a solubilizing agent, an isotonic agent, and a stabilizer may be mixed and used, and in the case of topical administration, a base agent, an excipient, a lubricant and a preservative may be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups and wafers, and in the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, and sustained-release preparations. On the other hand, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring and preservatives, and the like may additionally be included. In addition, the dosage of the pharmaceutical composition of the present invention is determined according to various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease.

In another specific embodiment, the composition of the present invention is a therapeutic agent capable of binding to the above-described antibody, and includes a radionuclide, a drug, a lymphokine, a toxin, a bispecific antibody, and the like. The above-described drugs and toxins include etoposide, teniposide, adriamycin, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, cis-platinum analogues, bleomycins, esperamicin. Liu, 5-fluorouracil, melphalan and other nitrogen mustards, and the like, but are not limited thereto.

The composition for inhibiting the growth and metastasis of cancer or the composition for treating or preventing cancer according to the present invention is a PAUF-specific human monoclonal antibody or a functional fragment thereof in various cancer cells. It is intended to cure cancer by inhibiting the function of (PAUF), inhibiting the growth and metastasis of cancer. The treatment or prevention of the cancer is preferably carried out by inhibiting the proliferation, migration or invasion of the cancer. The cancer is preferably pancreatic cancer, gastric cancer, ovarian cancer or colon cancer.

Specifically, the treatment method of the present invention includes administering the pharmaceutical composition in a pharmaceutically effective amount into the human body. The pharmaceutical composition may be administered parenterally, subcutaneously, intraperitoneally, intrapulmonally, and intranasally, and for local immunosuppressive treatment, if necessary, by a suitable method including intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraperitoneal or subcutaneous administration. Preferred modes of administration are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and instillation.

As a specific example, in order to investigate the effect of the human monoclonal antibody of the present invention on the growth of cancer cells, cell proliferation was investigated after the pancreatic cancer cell line Panc-1 was treated with the monoclonal antibody. As a result, it was confirmed that the cell proliferation was reduced compared to the cell line treated with the control antibody when the antibody was treated (see FIG. 3). That is, it was observed that inhibition of the growth and/or survival of cancer cells was induced by the antibody. Thereafter, in order to investigate the effect of the monoclonal antibody on the mobility of cancer cells, the pancreatic cancer cell line CFPAC-1 cell line and the Panc-1 cell line were treated with the monoclonal antibody and then a mobility assay was performed. As a result, it was confirmed that the cancer cell migration ability decreased when the antibody was treated compared to the control group (see FIG. 4). In a similar manner, when the cancer cell penetration assay was performed, a decrease in the cancer cell penetration ability by the antibody was observed compared to the control group (see FIG. 5). Through this, it was found that PAUF-specific antibodies can inhibit the growth, migration, penetration and/or metastasis of cancer cells.

In addition, in order to investigate the effect of the monoclonal antibody on cancer treatment in vivo, the pancreatic cancer cell line CFPAC-1 cell line was xenografted into immunodeficient mice. The antibody was injected into the tail vein and compared with the control antibody, it was confirmed that the tumor size was reduced (see FIG. 6). That is, it was observed that tumor progression in vivo was decreased by the antibody.

Hereinafter, the following table is a sequence listing, showing a list of amino acid sequences and nucleotide sequences corresponding to the light chain variable region, heavy chain variable region, and light chain CDR and heavy chain CDR of the four anti-PAUF antibodies of the present invention.

8F3 light chain variable region amino acid sequence SEQ ID NO: 1 DIQMTQSPSSLSASVGDRVTITCRASQSISRYLDWYQQKPGKAPRLLIYSTSTLQRGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPFAFGPGTKVDIKR
3A4 light chain variable region amino acid sequence SEQ ID NO:2 DIQMTQSPSSLAVSLGERATIDCRSSQSLLHSSNNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCHQYYSTPLTFGGGTKVDIKR
36C9 light chain variable region amino acid sequence SEQ ID NO:3 QLVLTQPPSASGPPGQRVTISCSGSRSNIGSNTVNWYQHLPGTAPKLLIHTNNQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEGDYYCAAWDDSLNGHFVFGTGTKVTV
6C4 light chain variable region amino acid sequence SEQ ID NO:4 DIVMTQSPLSLSASVGDRLTITCRASQSILTYLNWYQQKPGKAPKLLVYAASSLQPGVPSRFSGRGSGTDFTLTISGLQPDDFALYYCQQSYSSPYSFGPGTKVDIKR
8F3 heavy chain variable region amino acid sequence SEQ ID NO: 5 MAQVQLVQSGGGLIQPGGSLRLSCAASGFTVSSHYMNWVRQAPGKGLEWVSIIYSGGSTYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCARDIPRTVSPRTRAMDVWGQGTSVTVSS 3A4 heavy chain variable region amino acid sequence SEQ ID NO: 6 MAQVQLVQSGGGLVQPGGSLRVSCAASGFPFTTYAMTWVRQAPGRGLEWVAVISGSGRTTHYADSVKGRFTISRDASKNTLYLQMNSLRAEDTAVYYCAKTSHPRQLAVAGPFQHWGQGTLITVSS 36C9 heavy chain variable region amino acid sequence SEQ ID NO: 7 MAQVQLVESGGGVVLPGGSLRLSCAASGFPFGSYAVSWVRQAPGKGLEWVSAISPRNRYIYYADSVKGRFTISRDNAKNSLYLEMNSLGAEDTAVYYCAKDRLLRGAIVSALGHWGQGTLVTVSS 6C4 heavy chain variable region amino acid sequence SEQ ID NO: 8 MAQVQLVESGGGLVQPGGSLRLSCAASGFTFDRYWMTWVRQAPGKGLEWVANIKHDGSEKDYVDSVKGRFIISRDNAKKSLYLQMNSLRAEDTAVYYCARRRSYGRSTYYLDSWGQGTQITVSS 8F3 light chain amino acid sequence SEQ ID NO: 9 MGWSYIILFLVATATDVHSSGVGSDIQMTQSPSSLSASVGDRVTITCRASQSISRYLDWYQQKPGKAPRLLIYSTSTLQRGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTPFAFGPGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 3A4 light chain amino acid sequence Sequence number
10 MGWSYIILFLVATATDVHSSGVGSDIQMTQSPSSLAVSLGERATIDCRSSQSLLHSSNNKNYLAWFQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDVAVYYCHQYYSTPLTFGGGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 36C9 light chain amino acid sequence Sequence number
11 MGWSYIILFLVATATDVHSSGVGSQLVLTQPPSASGPPGQRVTISCSGSRSNIGSNTVNWYQHLPGTAPKLLIHTNNQRPSGVPDRFSGSKSGTSASLAVSGLQSEDEGDYYCAAWDDSLNGHFVFGTGTKVTVXRWRTKVEIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 6C4 light chain amino acid sequence Sequence number
12 MGWSYIILFLVATATDVHSSGVGSDIVMTQSPLSLSASVGDRLTITCRASQSILTYLNWYQQKPGKAPKLLVYAASSLQPGVPSRFSGRGSGTDFTLTISGLQPDDFALYYCQQSYSSPYSFGPGTKVDIKRGGASLVERSVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 8F3 heavy chain amino acid sequence Sequence number
13 MGWSYIILFLVATATDVHSAQPAMAQVQLVQSGGGLIQPGGSLRLSCAASGFTVSSHYMNWVRQAPGKGLEWVSIIYSGGSTYYADSVKGRFTISRDKSKNTLYLQMNSLRAEDTAVYYCARDIPRTVSPRTRAMDVWGQGTSVTVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3A4 heavy chain amino acid sequence Sequence number
14 MGWSYIILFLVATATDVHSAQPAMAQVQLVQSGGGLVQPGGSLRVSCAASGFPFTTYAMTWVRQAPGRGLEWVAVISGSGRTTHYADSVKGRFTISRDASKNTLYLQMNSLRAEDTAVYYCAKTSHPRQLAVAGPFQHWGQGTLITVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 36C9 heavy chain amino acid sequence Sequence number
15 MGWSYIILFLVATATDVHSAQPAMAQVQLVESGGGVVLPGGSLRLSCAASGFPFGSYAVSWVRQAPGKGLEWVSAISPRNRYIYYADSVKGRFTISRDNAKNSLYLEMNSLGAEDTAVYYCAKDRLLRGAIVSALGHWGQGTLVTVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 6C4 heavy chain amino acid sequence Sequence number
16 MGWSYIILFLVATATDVHSAQPAMAQVQLVESGGGLVQPGGSLRLSCAASGFTFDRYWMTWVRQAPGKGLEWVANIKHDGSEKDYVDSVKGRFIISRDNAKKSLYLQMNSLRAEDTAVYYCARRRSYGRSTYYLDSWGQGTQITVSSGLGGLASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

8F3 light chain variable region base sequence Sequence number
17 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGGTATTTAGATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAGACTCCTGATCTATTCTACTTCCACTTTGCAAAGAGGGGTCCCATCAAGATTCAGTGGCGGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCATTCGCATTCGGCCCTGGGACCAAAGTGGATATCAAACGT 3A4 light chain variable region base sequence Sequence number
18 GACATCCAGATGACCCAGTCTCCATCCTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCGACTGCAGGTCCAGCCAGAGTCTTTTACACAGCTCCAACAATAAGAACTACTTAGCTTGGTTCCAGCAGAAACCAGGACAGCCTCCTAAACTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATGTGGCAGTCTATTACTGTCACCAATATTATAGTACTCCCCTCACTTTCGGCGGAGGGACCAAGGTGGATATCAAACGT 36C9 light chain variable region base sequence Sequence number
19 CAGCTCGTGCTGACTCAGCCACCCTCAGCGTCTGGGCCCCCCGGGCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGGTCCAACATCGGAAGTAATACTGTTAACTGGTATCAGCACCTCCCAGGAACGGCCCCCAAGCTCCTCATCCATACTAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCGTCAGTGGGCTCCAGTCTGAGGATGAGGGTGATTATTATTGTGCAGCATGGGATGACAGCCTGAATGGCCATTTTGTCTTCGGAACTGGGACCAAGGTCACCGTC 6C4 light chain variable region base sequence Sequence number
20 GATATTGTGATGACCCAGTCTCCACTCTCCCTGTCTGCATCTGTAGGCGACAGACTCACTATCACTTGCCGGGCAAGTCAGAGCATTCTTACCTATTTAAATTGGTATCAACAAAAACCAGGGAAAGCCCCGAAGCTCCTAGTCTATGCTGCATCCAGTTTGCAACCTGGGGTCCCATCAAGGTTCAGTGGCCGCGGCTCTGGGACAGATTTCACTCTCACCATCAGCGGTCTGCAACCTGACGATTTTGCACTTTACTACTGTCAACAGAGTTACAGTAGTCCGTACTCTTTCGGGCCTGGGACCAAAGTGGATATCAAACGT 8F3 heavy chain variable region base sequence Sequence number
21 ATGGCCCAGGTGCAGCTGGTACAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGGTTCACCGTCAGTAGTCATTACATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGCGGTGGTAGTACATATTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAAGTCCAAGAACACTCTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGAGATATCCCTCGTACGGTGTCCCCACGTACCAGAGCTATGGACGTCTGGGGCCAAGGGACTTCGGTCACCGTCTCCTCA 3A4 heavy chain variable region base sequence Sequence number
22 ATGGCCCAGGTGCAGCTGGTACAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGAGTCTCCTGTGCAGCCTCTGGATTCCCGTTTACCACTTATGCCATGACTTGGGTCCGCCAGGCTCCAGGGAGGGGACTGGAGTGGGTCGCAGTTATAAGTGGTAGTGGAAGAACCACACACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACGCTTCCAAGAACACACTGTATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAACAAGCCACCCCCGACAACTAGCAGTGGCTGGCCCCTTCCAGCACTGGGGCCAGGGCACCCTGATCACCGTCTCCTCA 36C9 heavy chain variable region base sequence Sequence number
23 ATGGCCCAGGTGCAGCTGGTAGAGTCTGGGGGAGGCGTGGTCCTGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGCTTCCCCTTTGGCAGCTATGCCGTGAGCTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTCCTAGGAATCGTTACATATACTACGCAGACTCAGTGAAGGGCCGCTTCACCATCTCCAGAGACAACGCCAAGAACTCACTTTATCTGGAAATGAACAGTCTGGGAGCCGAGGACACGGCCGTGTATTACTGTGCGAAAGATCGCCTACTTAGGGGGGCGATAGTGAGTGCCCTTGGGCACTGGGGCCAGGGAACTCTGGTCACCGTCTCCTCA 6C4 heavy chain variable region base sequence Sequence number
24 ATGGCCCAGGTGCAGCTGGTGGAGTCGGGGGGAGGCTTGGTCCAACCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTTACTTTTGATAGATATTGGATGACCTGGGTCCGCCAGGCTCCGGGGAAGGGGCTGGAGTGGGTGGCCAACATAAAGCATGATGGAAGTGAGAAAGACTATGTGGACTCTGTGAAGGGCCGATTCATCATCTCCAGAGACAACGCCAAGAAGTCGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGACGGCGCTCATATGGTCGGAGTACTTACTATTTGGACTCCTGGGGCCAGGGAACCCAGATCACCGTCTCCTCA

8F3 light chain variable region CDR1 SEQ ID NO: 25 RASQSISRYLD 8F3 light chain variable region CDR2 SEQ ID NO: 26 STSTLQR 8F3 light chain variable region CDR3 SEQ ID NO: 27 QQSYSTPFAF 8F3 heavy chain variable region CDR1 SEQ ID NO: 28 SHYMN 8F3 heavy chain variable region CDR2 SEQ ID NO: 29 IIYSGGSTYYADSVKG 8F3 heavy chain variable region CDR3 SEQ ID NO: 30 DIPRTVSPRTRAMDV 3A4 light chain variable region CDR1 SEQ ID NO: 31 RSSQSLLHSSNNKNYLA 3A4 light chain variable region CDR2 SEQ ID NO: 32 WASTRESG 3A4 light chain variable region CDR3 SEQ ID NO: 33 HQYYSTPLTFG 3A4 heavy chain variable region CDR1 SEQ ID NO: 34 TYAMT 3A4 heavy chain variable region CDR2 SEQ ID NO: 35 VISGSGRTTHYADSVKG 3A4 heavy chain variable region CDR3 SEQ ID NO: 36 TSHPRQLAVAGPFQH 36C9 light chain variable region CDR1 SEQ ID NO: 37 SGSRSNIGSNTVN 36C9 light chain variable region CDR2 SEQ ID NO: 38 TNNQRPS 36C9 light chain variable region CDR3 SEQ ID NO: 39 AAWDDSLNGHFVF 36C9 heavy chain variable region CDR1 SEQ ID NO: 40 SYAVS 36C9 heavy chain variable region CDR2 SEQ ID NO: 41 AISPRNRYIYYADSVKG 36C9 heavy chain variable region CDR3 SEQ ID NO: 42 DRLLRGAIVSALGH 6C4 light chain variable region CDR1 SEQ ID NO: 43 RASQSILTYLN 6C4 light chain variable region CDR2 SEQ ID NO: 44 AASSLQPG 6C4 light chain variable region CDR3 SEQ ID NO: 45 QQSYSSPYSFG 6C4 heavy chain variable region CDR1 SEQ ID NO: 46 DRYWMT 6C4 heavy chain variable region CDR2 SEQ ID NO: 47 NIKHDGSEKDYVDSVKG 6C4 heavy chain variable region CDR3 SEQ ID NO: 48 RRSYGRSTYYLDS

Hereinafter, the present invention will be described in more detail by examples.

However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

As can be seen above, the PAUF protein according to the present invention and the PAUF nucleic acid molecule encoding the same are very specific for the onset of cancer, and in particular, a marker or treatment for pancreatic cancer. Become a target. In addition, the anti-PAUF antibody or a functional fragment thereof that specifically binds to the PAUF protein is a PAUF protein expressed in various cancer cells including pancreatic cancer and gastric cancer. It specifically binds to and enables early diagnosis of cancer, inhibits the growth, invasion or migration of cancer cells by inhibiting the activity of PAUF protein, and also induces the death of cancer cells for cancer treatment and diagnosis. Can be useful have.

1 shows a photograph of an acrylamide gel of purified 8F3, 3A4, 36C9, and 6C4 antibodies.
2A-2D show binding titration curves for binding of 8F3, 3A4, 36C9, and 6C4 antibodies to PAUF protein.
Figure 3 shows the growth inhibition of pancreatic cancer cell line (Panc-1) by 8F3, 3A4, 36C9, 6C4 antibodies.
4A-4D show the inhibition of migration ability of two pancreatic cancer cell lines (Panc-1, CFPAC-1) by 8F3, 3A4, 36C9, and 6C4 antibodies.
5A-5D show the inhibition of penetration ability of two pancreatic cancer cell lines (Panc-1, CFPAC-1) by 8F3, 3A4, 36C9, and 6C4 antibodies.
6A-6B show the therapeutic effect on the growth of CFPAC-1 pancreatic cancer xenografts in mice by 8F3 and 36C9 antibodies.
FIG. 7 shows that it is possible to measure PAF in blood using a humanized antibody 8F3 and a polyclonal antibody against PAF, and that PAF is specifically measured in blood of a pancreatic cancer patient.

< Example 1> PYUF ( PAUF ) Screening of specific monoclonal antibodies

After the PAUF protein is immobilized in a plastic tube, it is buffered for 2 hours using a buffer (2% skim milk, PBS). After removing the buffer, the human antibody display phage library mixed with the buffer was added and reacted at room temperature for 2 hours. Wash 10 times with a washing solution (0.1% Tween 20, PBS) and 10 times with PBS. After washing, 100 mM TEA (Triethylamine) is added and reacted at room temperature for 10 minutes to elute the phage. Add 1 M Tris (pH 7.5) to the eluted phage and react at room temperature for 5 minutes to neutralize. Infect the eluted phage solution by adding E. coli culture solution. Infected E. coli was centrifuged at 3,000 xg for 5 minutes, the supernatant was removed, inoculated on a 2xYT plate, and incubated at 37°C for 16 hours. An appropriate amount of 2xYT medium was added to suspend E. coli colonies to recover the formed colonies. The recovered E. coli is inoculated in 2xYT medium and supplementary phage is added to prepare a first screened phage library. Screening is repeated 4 more times using the screened phage library. After the 5th screening, clones are randomly selected from 2xYT plates and cultured in 2xYT medium. A phage is formed by adding auxiliary phage to the cultured clone. The formed phage was confirmed for specificity to PAUF using the PAUF ELISA method (see Example 3). The phage showing specificity is selected and the DNA sequence is analyzed. Through this method, monoclonal antibodies that specifically bind to PAUF, including four antibodies (8F3, 3A4, 36C9, 6C4) of Sequence Listings 1 to 8 were discovered.

< Example 2> PYUF ( PAUF ) Specific human monoclonal antibody purification

In order to establish a CHO-DG44 cell line capable of mass-expressing a specific human monoclonal antibody against PAUF, the light chain portion of 10 monoclonal antibodies discovered using the method of Example 1 was pIgGLD. Into the BstXI site of, the heavy chain portion was inserted into the SfiI site of pIgGHD and converted into an IgG form. pIgGLD and pIgGLD are expression vectors for antibody production that have nucleic acid molecules corresponding to the constant regions of the light and heavy chains of IgG, respectively. The expression plasmid of the IgG form of each monoclonal antibody was stably transfected into CHO-DG44, a DHFR-deficient CHO mutant cell line, according to the product instructions using a lipofectamin plus drug (Invitrogen, CA). . Stably transfected cells were selected in G418 G418 550 ug/ml (Invitogen, CA) for 2 weeks. The stable transfectant was further adapted by increasing the concentration in steps of 2 weeks from 10 nM concentration of MTX (Sigma, MO). The expression level of the monoclonal antibody secreted into the culture medium of the adapted cell line was calculated by ELISA, and the highly expressed cell line was selected and applied to mass purification of the monoclonal antibody.

The selected cell lines were grown in SF CHO culture medium (JBI, KR) in a tissue culture flask to express the antibody. Various methods including affinity chromatography and ion exchange chromatography can be used to purely purify the antibody of the present invention. All four antibodies 8F3, 3A4, 36C9 and 6C4 shown in FIG. 5 were purified under the following conditions. Human monoclonal antibodies expressed and secreted in CHO culture were first subjected to affinity chromatography using a Protein A column capable of recognizing and binding the Fc portion of the antibody. The culture medium containing the expressed antibody was passed once through a filter having a 0.45 μm membrane, washed with 20 mM sodium phosphate buffer (pH 7.0), and added dropwise to the equilibrated protein A column. To wash off unbound proteins, 20 mM sodium phosphate buffer (pH 7.0) was added dropwise to the Protein A column. Thereafter, the monoclonal antibody was eluted with 100 mM sodium citrate buffer (pH3), and the pH of the elution fraction was adjusted to 7-7.5 with 1 M Tris buffer (pH9.0). The elution fractionated monoclonal antibodies are combined to form a protein pool, and the buffer is exchanged with 20 mM sodium phosphate buffer (pH 7.0) using a spin column with a 3K MWCO standard membrane for application to cation exchange chromatography. And concentrated. This sample was added dropwise to cation exchange chromatography pre-equilibrated with 20 mM sodium phosphate buffer (pH 7.0), and the unbound material was washed with the buffer, and then 20 mM sodium phosphate, 1 A solution of M NaCl (pH7.0) was added dropwise to elute the target antibody. Thus, the monoclonal antibody was purified with minimal endotoxins inside. FIG. 1 shows photographs of acrylamide gels of monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 purified by the above method.

< Example 3> PYUF ( PAUF ) Antigen of specific human antibody Binding ability Measure ( PAUF ELISA )

After the PAUF protein is immobilized on a plate, it is buffered for 2 hours using a buffer (2% skim milk, PBS). After removing the buffer, the eluate or purified human antibody of the human antibody display phage mixed with the buffer is added to the plate and reacted at 37°C for 1 hour. Antibodies that do not bind to the antigen are removed by washing 5 times with a washing solution (0.1% Tween 20, PBS). Anti-M13-HRP is used when measuring the antigen-binding ability of the phage eluate with a secondary antibody, and anti-human IgG (Fc specific)-HRP is used when measuring the antigen-binding ability of a human antibody. After diluting the appropriate secondary antibody to a concentration of 1:5000 using a buffer solution, put it into the plate, and reacted at 37°C for 30 minutes. After washing 5 times with a washing solution (0.1% Tween 20, PBS), _{color development with TMB and H 2} O ₂ and measuring the absorbance at 450 nm to select an antibody specific to PAUF or by concentration of the purified antibody Check the binding ability. 2 is a result of measuring the binding capacity of the purified antibodies 8F3, 3A4, 36C9 and 6C4 by concentration.

< Example 4> growth Assay ( proliferation assay )

Pancreatic cancer cells were counted in 1 ml of medium by 5 x 10 ⁴ cells and cultured in a 24-well plate, and the monoclonal antibody was added at a concentration of 3 ug/ml, and the cells were cultured in a 37°C CO ₂ incubator for 2 days. The medium to which the antibody was added was changed once in each. After 6 days, the cells were recovered, stained in 0.2% Tryphan Blue solution, and the number of living cells was compared with the control group. Through this assay, it was confirmed that the selected four monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 can inhibit the growth rate of pancreatic cancer cell lines (see FIG. 3).

< Example 5> move Assay ( migration assay )

Pancreatic cancer cells are isolated and washed twice with PBS, and the antibodies are mixed by concentration to adjust the final cell count to 1 x 10 ⁶ cells/ml. Add 30 ul of DMEM medium containing 10% FBS in the lower chamber of Neuroprobe's Neuro Probe 48 well micro chamber, and put an 8 um pore polycarbonate filter (25x80 mm, Neuroprobe) on it. Put it. Place the upper chamber on it and fix it. 50 ul per well of the mixture of pancreatic cancer and antibody was added to the fixed upper chamber and incubated for 20 hours in a _{37°C CO 2 incubator.} After the cultivation was completed, the cells that had moved to the lower chamber were fixed with 100% methanol and stained with Kimja staining solution. Pictures of the stained cells were counted in at least three selected areas and then compared with the control group. Through this assay, it was confirmed that the selected four monoclonal antibodies 8F3, 3A4, 36C9, and 6C4 can inhibit the ability of pancreatic cancer cell lines to migrate (see FIG. 4).

< Example 6> infiltration Assay ( invasion assay )

Pancreatic cancer cells are isolated and washed twice with PBS, and the antibodies are mixed by concentration to adjust the final number of cells to 1 x 10 ⁶ cells/ml. Add 30 ul of DMEM medium containing 10% FBS to the lower chamber of Neuroprobe's Neuro Probe 48 well micro chamber, and 8 um pore bicarbonate filter (polycarbonate) coated with matrigel on it. filter) (25x80mm, Neuroprobe). Place the upper chamber on it and fix it. 50 ul per well of the mixture of pancreatic cancer and antibody was added to the fixed upper chamber and incubated for 20 hours in a _{37° C. CO 2 incubator.} After the cultivation was completed, the cells moved to the lower chamber were fixed with 100% methanol and stained with Kimja stain solution. Pictures of the stained cells were counted in at least 3 selected areas and then compared with the control group. Through this assay, it was confirmed that the selected four monoclonal antibodies 8F3, 3A4, 36C9 and 6C4 can inhibit the invasion capacity of pancreatic cancer cell lines (see FIG. 5).

< Example 7> In the mouse model PYUF ( PAUF ) Cancer cell growth inhibition test of specific antibody

6-7 week old nude mice Balb/c nu/nu ^{were implanted with 5 x 10 6} CFPAC-1 cells subcutaneously. From the 10th day of transplantation, the monoclonal antibody was injected into the tail vein at a concentration of 5 mg/kg twice a week for 3 weeks. The tumor volume was measured twice a week using a Caliper. On the last day of the experiment, nude mice were _{sacrificed using CO 2} , and then tumors were separated and the weight was measured. The isolated tumor was fixed and a paraffin block was prepared, and tissue immunostaining for PAUF protein was performed using a polyclonal antibody against PAUF prepared in rabbits. As a result, it was confirmed that the amount of PAUF was small in the tumors of mice injected with PAUF-specific monoclonal antibodies compared to mice injected with the control antibody. Through this assay, it was confirmed that the two selected monoclonal antibodies 8F3 and 36C9 could inhibit the growth of the pancreatic cancer cell line, CFPAC-1, which was transplanted in a mouse model (see FIG. 6).

< Example Section 8> PYUF In the blood with antibodies PYUF Measurement and pancreatic cancer diagnosis experiment

The following experiment was performed to confirm the possibility of diagnosing pancreatic cancer through detection of PAF in blood according to the present invention. After diluting 8F3, an anti-PAF antibody at a concentration of 1-10 ug/ml, in PBS (pH 7.2), dispensing 100 ul into a 96-well plate, sealing the antibody dispensed into the well so that it is well fixed to the plate. It reacted at °C for 18 hours. After removing the solution remaining without adhering to the well plate, 300 ul of a phosphate buffer solution containing 1% bovine serum albumin was dispensed per well and reacted at 37° C. for 2 hours. The solution remaining in the well was removed, dried, and put in a sealed container together with a dehumidifying agent and stored in a refrigerator at 4°C. Serum of pancreatic cancer patients, normal people, pancreatitis and other diseases are diluted 1/2 in a phosphate buffer containing 3% bovine serum albumin in each well of the coated plate, and 100 ul of the solution is dispensed into each well at 37℃. It was allowed to react for 90 minutes. After the reaction, 300 ul of a phosphate buffer containing 0.05% Tween 20 was dispensed into each well, washed 5 times, and anti-PAF polyclonal antibody was added to each well at a concentration of 8 ug/ml 5%. 100 ul of a phosphate buffer solution containing horse serum and 100 mM NaCl was dispensed per well and reacted at 37° C. for 90 minutes. After the reaction was over, the remaining solution was discarded, and the well plate was washed 5 times using a phosphate buffer solution containing 5% Tween20. To confirm the specific reaction, the anti-rabbit antibody with horseradish enzyme (HRP) as a secondary antibody was diluted 1/2000 in a phosphate buffer containing 1% bovine serum, and 100 ul per well was dispensed. It was reacted at 37°C for 30 minutes. After the reaction, the well plate was washed 5 times using a phosphate buffer containing 0.05% Tween20, tetramethyl benzidine 100 ug/ml, hydrogen peroxide 0.006% en peroxide 0.006% And a substrate solution containing a citric acid phosphate buffer solution (pH4.5) was added each 100 to develop color in the dark for 30 minutes. 50 ul of the reaction stop solution (1N sulfuric acid solution) was added to each well to terminate the color development, and then absorbance was measured at 450 nm using a 96-well plate reader (Molecular Devices) with 650 nm as a reference wavelength. Since tetramethyl benzidine, a color developing agent in the substrate solution, is decomposed by the wasabi enzyme conjugate bound to the antibody to induce a color reaction, the degree of color development was measured by absorbance to detect the presence or absence of PAI in the blood of pancreatic cancer patients. The samples used in the experiment are as follows.

(1) 26 normal human serum samples

(2) 13 samples of pancreatic cancer positive serum

(3) Serum for patients with other cancers and other diseases: 15 gastric cancer, 1 rectal cancer, 1 breast cancer, 3 liver cancer, 5 samples of hepatitis

As a result of the experiment, a total of 51 samples of normal subjects and patients with other diseases were judged as negative, but the overall diagnostic specificity was 100%, and 8 of the 13 samples positive for pancreatic cancer showed a sensitivity of 61.5%, which was negative. The sample also exhibited a somewhat higher value compared to other samples (FIG. 7). The results of this experiment show that it is possible to secure a higher sensitivity according to the improvement of the analysis method, and it is possible to diagnose pancreatic cancer in the blood.

Attach electronic file of sequence list

Claims (6)

(a) a human monoclonal antibody comprising a light chain variable region as set out in SEQ ID NO: 1 and a heavy chain variable region as set out in SEQ ID NO: 5;
(b) a human monoclonal antibody comprising a light chain variable region as set out in SEQ ID NO: 2 and a heavy chain variable region as set out in SEQ ID NO: 6;
(c) a human monoclonal antibody comprising a light chain variable region as set out in SEQ ID NO: 3 and a heavy chain variable region as set out in SEQ ID NO: 7; And
(d) Human monoclonal antibody selected from the group consisting of a light chain variable region as set out in SEQ ID NO: 4 and a heavy chain variable region as set out in SEQ ID NO: 8.
The human monoclonal antibody of claim 1, wherein the antibody inhibits proliferation, migration or invasion of pancreatic cancer cells.
(a) a human monoclonal antibody comprising a light chain amino acid sequence as set out in SEQ ID NO: 9 and a heavy chain amino acid sequence as set out in SEQ ID NO: 13;
(b) a human monoclonal antibody comprising a light chain amino acid sequence as set out in SEQ ID NO: 10 and a heavy chain amino acid sequence as set out in SEQ ID NO: 14;
(c) a human monoclonal antibody comprising a light chain amino acid sequence as set out in SEQ ID NO: 11 and a heavy chain amino acid sequence as set out in SEQ ID NO: 15; And
(d) A human monoclonal antibody selected from human monoclonal antibodies comprising a light chain amino acid sequence as set out in SEQ ID NO: 12 and a heavy chain amino acid sequence as set out in SEQ ID NO: 16.
A pancreatic cancer diagnostic composition comprising the human monoclonal antibody of claim 1.
A kit for diagnosing pancreatic cancer comprising the human monoclonal antibody of claim 1.
A composition for treating pancreatic cancer comprising the human monoclonal antibody of claim 1.

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