KR101535717B1 - Composition for pancreatic cancer diagnosis comprising complememt factor i-specific binding polypeptide or antibody - Google Patents

Abstract

The present invention relates to a method for diagnosing pancreatic cancer by analyzing levels of complement factor I protein in a sample of a subject as a biomarker for diagnosing pancreatic cancer, and a method for diagnosing pancreatic cancer comprising a polypeptide or an antibody specifically binding to a complement factor I protein ≪ / RTI > According to the present invention, a composition for diagnosing pancreatic cancer comprising a polypeptide or an antibody that specifically binds to a complement factor I protein can be used to diagnose pancreatic cancer early by analyzing the expression amount of complement factor I protein in plasma.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing pancreatic cancer, which comprises a polypeptide or an antibody that specifically binds to a complement factor I protein. The present invention also relates to a composition for diagnosing pancreatic cancer,

The present invention relates to a composition for diagnosing pancreatic cancer comprising a polypeptide or an antibody that specifically binds to a complement factor I protein, and a method for providing information for diagnosing pancreatic cancer using the composition.

Pancreatic cancer is the ninth most common major cancer in the world, with the fourth highest mortality rate [Non-Patent Document 1]. In most countries, pancreatic cancer accounts for only 2-3% of all malignant tumors, but the proportion of deaths accounts for as much as 6% of all tumor-related deaths. The reason for this is that the 2-year survival rate of pancreatic cancer is so low as to be only about 10% and the 5-year survival rate does not exceed 5%. [Non-patent document 2].

Since pancreatic cancer does not show symptoms until it has progressed considerably, once the pancreatic cancer is diagnosed, it is very difficult to perform the operation because it is already advanced. In addition, even if surgery is possible, 80 to 90% of patients who have undergone surgery will recur and die. In addition to surgery, radiotherapy and chemotherapy are performed to treat pancreatic cancer, but most of them have a limited effect on the survival of patients. Therefore, early diagnosis of pancreatic cancer is very important.

Diagnostic methods for pancreatic cancer include biopsy using computed tomography (CT) or magnetic resonance imaging (MRI). Recently, pancreatic cancer is a clinical sample that can be used to diagnose pancreatic cancer. And the presence or absence of pancreatic cancer. Although carbohydrate antigen 19-9 (CA 19-9) is the most commonly used tumor marker in relation to pancreatic cancer, it is known that it is elevated in the plasma of cancer patients of digestive system including biliary tract in addition to pancreatic cancer. It is also known that it is elevated in the presence of cholangitis and biliary obstruction without malignant tumors (Source: National Health Information Portal). In early cancer, the blood level of CA 19-9 markers is normal, so it can not be used for early diagnosis. It is used only for the prognosis of pancreatic cancer and follow-up examination after treatment.

Complement factor I is a protein called CFI which is also called C3b inactivator and C3b / C4b inactivator (C3b / C4b inactivator). It does not work on C3 but works with complement factor H on C3b produced by C3 convertase to decompose C3b into C3c and C3b and acts to inhibit hemolytic activity, immune adherence, and immune phagocytosis in C3b. In the alternative pathway, complement factor I degrades the complement protein to prevent complement activation. Complement factor I is known as a secreted protein, and there are six glycan sites as glycoproteins.

According to the present reports, complement factor I is known to be secreted from serum of various patients such as gastric cancer [Non-Patent Document 3] and senile macular degeneration [Non-Patent Document 4], but the incidence of complement factor I and pancreatic cancer in blood Is not known.

Lowenfels, A. B., et al., Best Pract Res Clin Gastroenterol, 197-209, 2006 Lee, C. N., et al., Pancreas, 94-94, 2012 Liu W., et al., Clin Chim Acta., 337, 119-126, 2007 Ennis S., et al., Eur J Hum Genet., 18, 15-16, 2010

Accordingly, the present applicant has discovered a complement factor II protein as a biomarker for diagnosis of a more specific and sensitive new pancreatic cancer, and a method of providing information for diagnosis of pancreatic cancer using the protein; And a polypeptide or antibody that specifically binds to a complement factor I protein.

The inventors of the present invention selected the complement factor I protein as a candidate for biomarkers for diagnosing pancreatic cancer in order to investigate early diagnosis of pancreatic cancer marker and method of diagnosing pancreatic cancer, and found that the presence of complement factor I protein in normal and pancreatic cancer patients And observed changes. As a result of the study, it was confirmed that the level of complement factor I protein was significantly higher in plasma of pancreatic cancer patients than that of the normal control group, and the present invention was completed.

Accordingly, the present invention provides a method for providing information for diagnosis of pancreatic cancer comprising quantifying the level of the complement factor I protein in a sample of an individual.

In one embodiment of the invention, the method may further comprise comparing and comparing levels of complement factor I protein in a sample of a pancreatic cancer candidate patient and a normal control sample. If the two samples are compared and the level of complement factor I protein in the sample of the pancreatic cancer candidate patient is higher than that of the normal control sample, the candidate patient can be classified as a pancreatic cancer patient. For example, a candidate patient may be classified as a pancreatic cancer patient when the level of complement factor I protein in a patient with pancreatic cancer candidate is more than two times the level of complement factor I protein in a normal control sample.

In one embodiment of the present invention, the complement factor I is a secreted protein secreted from macrophages. Therefore, in order to analyze proteins, normal plasma and plasma of pancreatic cancer patients can be used as samples. Plasma Because of the presence of well-known high abundant proteins, these proteins were removed using an immune antibody-attached column called Hu-6 (Agilent) and plasma proteins present in rare concentrations were subjected to multiple amine specific Type stable isotope (iTRAQ) experiment.

Methods for quantifying the levels of the complement factor I protein can utilize multiplexed amine-specific stable isotope assays, ELISA, radioimmunoassay, radiated immunodiffusion, immunoelectrophoresis, or mass spectrometry. For example, multiple - amine - specific stable isotope studies confirm that complement factor I is increased in plasma from normal plasma in pancreatic cancer patients. Thus, the present inventors have demonstrated the difference in the presence of complement factor I in blood and the expression level between normal human and pancreatic cancer patients using the proteomics technique. Western blots can be used to quantify and compare levels of complement factor I protein in samples of pancreatic cancer candidate patients and normal controls. Western blot is a widely used method for detecting proteins, and is a method of detecting a sample using an antigen and an antibody reaction. Specifically, a sample is developed through electrophoresis, and then transferred to a membrane and then developed to detect and analyze protein expression. As shown in FIG. 6, the levels of complement factor I protein in normal subjects and pancreatic cancer patients can be compared using Western blotting. Whether the proven protein is a complement factor I protein can be confirmed using antibody immunoprecipitation and mass spectrometry.

The present invention also provides a composition for diagnosing pancreatic cancer comprising a polypeptide or an antibody that specifically binds to a complement factor I protein and a diagnostic kit. The complement factor I protein may be one having the amino acid sequence of SEQ ID NO: 1.

SEQ ID NO: 1:

MKLLHVFLLFLCFHLRFCKVTYTSQEDLVEKKCLAKKYTHLSCDKVFCQPWQRCIEGTCVCKLPYQCPKNGTAVCATNRRSFPTYCQQKSLECLHPGTKFLNNGTCTAEGKFSVSLKHGNTDSEGIVEVKLVDQDKTMFICKSSWSMREANVACLDLGFQQGADTQRRFKLSDLSINSTECLHVHCRGLETSLAECTFTKRRTMGYQDFADVVCYTQKADSPMDDFFQCVNGKYISQMKACDGINDCGDQSDELCCKACQGKGFHCKSGVCIPSQYQCNGEVDCITGEDEVGCAGFASVTQEETEILTADMDAERRRIKSLLPKLSCGVKNRMHIRRKRIVGGKRAQLGDLPWQVAIKDASGITCGGIYIGGCWILTAAHCLRASKTHRYQIWTTVVDWIHPDLKRIVIEYVDRIIFHENYNAGTYQNDIALIEMKKDGNKKDCELPRSIPACVPWSPYLFQPNDTCIVSGWGREKDNERVFSLQWGEVKLISNCSKFYGNRFYEKEMECAGTYDGSIDACKGDSGGPLVCMDANNVTYVWGVVSWGENCGKPEFPGVYTKVANYFDWISYHVGRPFISQYNV

As used herein, a polypeptide refers to a polymer of amino acids that is not of a specific length and includes peptides, oligopeptides, and protein fragments. In one embodiment of the invention, the polypeptide that specifically binds to the complement factor I protein may be a polypeptide having the binding domain of the complement factor I protein. Polypeptides that specifically bind to the complement factor I protein can be obtained by conventional methods known in the art, for example, the phage display method (Smith GP, "Filamentous fusion phage: Smith ", Petrenko VA, "Phage display ". Chem. Rev. 97 (2): 391410 (1997)).

The antibody refers to an immunoglobulin gene or immunoglobulin gene capable of specifically binding to an antigen or epitope, or a peptide or polypeptide substantially encoded thereby derived from or fragments thereof. Antibodies herein include various forms of antibody constructs including, but not limited to, whole antibodies and antibody fragments. The antibody fragment comprises a portion of the full length antibody, the variable domain of the antibody, or at least the antigen binding site of the antibody. Examples of antibody fragments include multispecific antibodies formed from diabodies, single-chain antibody molecules and antibody fragments.

In one embodiment of the invention, the antibody may be a polyclonal antibody or a monoclonal antibody. Antibodies against the complement factor I protein can be produced using methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods 4,816,56) or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 . ≪ / RTI > General procedures for antibody preparation 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 preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing an immortalized cell line with an antibody-producing lymphocyte, and the techniques necessary for this process are well known and readily practicable by those skilled in the art . A polyclonal antibody can be obtained by injecting a complement factor I protein antigen into a suitable animal, collecting the antiserum from the animal, and then separating the antibody from the antiserum using a known affinity technique.

The present invention also provides a method for providing information for the diagnosis of pancreatic cancer comprising quantifying the level of expression of complement factor I. Specifically, the level of expression of complement factor I in pancreatic cells of normal pancreatic cancer cells and pancreatic cancer candidate cells can be quantified and compared to provide information for diagnosis of pancreatic cancer. As a result of comparison, if the expression of complement factor I in cells of a candidate pancreatic cancer patient is higher than that of normal pancreatic cell, the candidate patient can be classified as a pancreatic cancer patient. Methods for quantifying the expression level of complement factor I include, but are not limited to, RT-PCR or quantitative RT-PCR using a PCR primer for a gene encoding complement factor I, northern blot analysis using a probe for complement factor I Can be used.

As a biomarker for diagnosing pancreatic cancer according to the present invention, the level of the complement factor I protein in the individual sample can be analyzed to provide information for diagnosing pancreatic cancer, a pancreas comprising the polypeptide or antibody that specifically binds to the complement factor I protein Can be provided.

Figure 1 shows the result of confirming complement factor I peptides in plasma of normal and pancreatic cancer patients using a mass spectrometer.
FIG. 2 is a graph showing a ratio of complement factor I peptides that are specifically different in plasma from normal human and pancreatic cancer patients using the multiple amine-specific stable isotope assay (iTRAQ).
115, 118: normal plasma
117,121: Pancreatic cancer patient plasma
Figure 3 shows the results of one-dimensional electrophoresis after immunoprecipitation to demonstrate the presence of complement factor I in plasma.
1DE: One-dimensional electrophoresis
IP: Immunoprecipitation
FIG. 4 shows the results obtained by cutting out the gel band shown in FIG. 3, cutting out the protein into a peptide form, and identifying the complement factor I protein using a mass spectrometer.
FIG. 5 is a photograph showing the results of confirmation of the changes in complement factor I in representative plasma samples of five healthy persons and pancreatic cancer patients by immunoblotting. FIG.
N: Normal
C: Patient with pancreatic cancer
6 is a graph comparing the abundance of complement factor I in the plasma of a total of 26 healthy persons and pancreatic cancer patients.
Normal: Normal
PC: Pancreatic cancer patient

Hereinafter, the present invention will be described in detail with reference to examples. The following examples illustrate the invention and are not intended to limit the scope of the invention. These embodiments are provided so that the disclosure of the present invention is not limited thereto and that those skilled in the art will fully understand the scope of the present invention and that the present invention is not limited by the scope of the claims Only.

Example  1: Plasma gain

Plasma was obtained from Severance Hospital Gene Bank and Digestive Medicine Internal Medicine, and the entire process of obtaining samples was carried out with the approval of the Clinical Trials Committee (IRB). Plasma samples were dispensed in 200 μl aliquots and stored at -70 ° C until experiment.

Example  2: Multiple amine-specific stable isotope experiments

Plasma samples from 13 normal and 13 pancreatic cancer patients were treated with Hu-6 (Agilent) MARC (column) to remove plasma high abundant proteins, and plasma proteins were detected with amicones (millipore) ≪ / RTI > Proteins were then dissolved in 0.5 M TEAB (triethylammonium bicarbonate) and the proteins were denatured using 2% SDS. The disulfide bonds of the proteins were removed by adding 50 mM TCEP (tris- (2-carboxyethyl) phosphine) to the denatured proteins, and the cysteine group was blocked using 200 mM MMTS (Methyl methanethiosulfonate) . Subsequently, proteins were peptized using trypsin, labeled 115, 118 reagents for normal human plasma, and 117, 121 reagents for plasma of pancreatic cancer patients, respectively. After the labeled samples were combined, the saccharide was cleaved with a saccharide-digesting enzyme (PNGaseF), and the proteins were fractionated using a C18 column.

Example  3: Protein identification

Protein identification was analyzed by Q-STAR (Applied Biosystems) mass spectrometry (MS), and the peptide spectrum obtained by Q-STAR-MS [Fig. 1] was used to identify complement proteins using the MASCOT database. The proportion of complement factor I peptides that differed specifically in the plasma of normal and pancreatic cancer patients was determined (Figure 2).

Example  4: Plasma pancreatic cancer Marker  Verification of protein expression level - Immunoblotting

To examine the difference in protein concentration in pancreatic cancer plasma from pancreatic cancer marker protein, Western blot analysis was performed using the antibody from the same amount of protein in plasma of 13 normal and 13 pancreatic cancer patients. 10 μg of the plasma protein was separated on 10% SDS-PAGE, and the nitrocellulose (NC) membrane was placed on the gel to transfer the protein in the gel to the NC membrane. Then, TBS-T buffer containing 5% skimmed milk Solution [20 mM Tris, 137 mM sodium chloride, 0.1% Tween-20, pH 7.6] for 1 hour. Anti-complement factor I antibody (CFI, Abcam) was diluted 1: 1000 times in TBS-T buffer containing 5% skim milk and subjected to primary treatment and anti-mouse IgG-HRP (Santa Cruz) 10000 times the secondary treatment. The final NC membrane was reacted with ECL Plus Western Blotting reagent (GE Healthcare) for 1 minute, and then the intact complement protein was identified in each band using a Typhoon 9400 scanner to identify the target band present on the NC membrane [Figure 5] , And the amount of complement factor I present in normal and pancreatic cancer patients was compared with a graph (FIG. 6).

Example  5: Plasma Complement factor  I protein identification method

100 μg of plasma, 2 μg of anti-complement factor I antibody, and 10 μl of protein G agarose were immunoprecipitated in a 1 ml tube for 2 hours, then the complement factor I protein was isolated and normal and pancreatic cancer patient samples were dissolved The protein was denaturated in a buffer solution and separated by one-dimensional electrophoresis (FIG. 3). A gel band of about 75 kDa was cut out and dithiothrietol (DTT) / iodoacetic acid iodoacetic acid (IAA). After digesting with trypsin, the protein was cleaved with peptides, and the complement factor I protein was identified using a mass spectrometer (MALDI-TOF) (Fig. 4).

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> COMPOSITION FOR PANCREATIC CANCER DIAGNOSIS COMPRISING COMPLEMEMT          FACTOR I-SPECIFIC BINDING POLYPEPTIDE OR ANTIBODY <130> P130981 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 583 <212> PRT <213> Human <400> 1 Met Lys Leu Leu His Val Phe Leu Leu Phe Leu Cys Phe His Leu Arg   1 5 10 15 Phe Cys Lys Val Thr Tyr Thr Ser Gln Glu Asp Leu Val Glu Lys Lys              20 25 30 Cys Leu Ala Lys Lys Tyr Thr His Leu Ser Cys Asp Lys Val Phe Cys          35 40 45 Gln Pro Trp Gln Arg Cys Ile Glu Gly Thr Cys Val Cys Lys Leu Pro      50 55 60 Tyr Gln Cys Pro Lys Asn Gly Thr Ala Val Cys Ala Thr Asn Arg Arg  65 70 75 80 Ser Phe Pro Thr Tyr Cys Gln Gln Lys Ser Leu Glu Cys Leu His Pro                  85 90 95 Gly Thr Lys Phe Leu Asn Asn Gly Thr Cys Thr Ala Glu Gly Lys Phe             100 105 110 Ser Val Ser Leu Lys His Gly Asn Thr Asp Ser Glu Gly Ile Val Glu         115 120 125 Val Lys Leu Val Asp Gln Asp Lys Thr Met Phe Ile Cys Lys Ser Ser     130 135 140 Trp Ser Met Arg Glu Ala Asn Val Ala Cys Leu Asp Leu Gly Phe Gln 145 150 155 160 Gln Gly Ala Asp Thr Gln Arg Arg Phe Lys Leu Ser Asp Leu Ser Ile                 165 170 175 Asn Ser Thr Glu Cys Leu His Val His Cys Arg Gly Leu Glu Thr Ser             180 185 190 Leu Ala Glu Cys Thr Phe Thr Lys Arg Arg Thr Met Gly Tyr Gln Asp         195 200 205 Phe Ala Asp Val Val Cys Tyr Thr Gln Lys Ala Asp Ser Pro Met Asp     210 215 220 Asp Phe Phe Gln Cys Val Asn Gly Lys Tyr Ile Ser Gln Met Lys Ala 225 230 235 240 Cys Asp Gly Ile Asn Asp Cys Gly Asp Gln Ser Asp Glu Leu Cys Cys                 245 250 255 Lys Ala Cys Gln Gly Lys Gly Phe His Cys Lys Ser Gly Val Cys Ile             260 265 270 Pro Ser Gln Tyr Gln Cys Asn Gly Glu Val Asp Cys Ile Thr Gly Glu         275 280 285 Asp Glu Val Gly Cys Ala Gly Phe Ala Ser Val Thr Gln Glu Glu Thr     290 295 300 Glu Ile Leu Thr Ala Asp Met Asp Ala Glu Arg Arg Arg Ile Lys Ser 305 310 315 320 Leu Leu Pro Lys Leu Ser Cys Gly Val Lys Asn Arg Met His Ile Arg                 325 330 335 Arg Lys Arg Ile Val Gly Gly Lys Arg Ala Gln Leu Gly Asp Leu Pro             340 345 350 Trp Gln Val Ala Ile Lys Asp Ala Ser Gly Ile Thr Cys Gly Gly Ile         355 360 365 Tyr Ile Gly Gly Cys Trp Ile Leu Thr Ala Ala His Cys Leu Arg Ala     370 375 380 Ser Lys Thr His Arg Tyr Gln Ile Trp Thr Thr Val Val Asp Trp Ile 385 390 395 400 His Pro Asp Leu Lys Arg Ile Val Ile Glu Tyr Val Asp Arg Ile Ile                 405 410 415 Phe His Glu Asn Tyr Asn Ala Gly Thr Tyr Gln Asn Asp Ile Ala Leu             420 425 430 Ile Glu Met Lys Lys Asp Gly Asn Lys Lys Asp Cys Glu Leu Pro Arg         435 440 445 Ser Ile Pro Ala Cys Val Pro Trp Ser Pro Tyr Leu Phe Gln Pro Asn     450 455 460 Asp Thr Cys Ile Val Ser Gly Trp Gly Arg Glu Lys Asp Asn Glu Arg 465 470 475 480 Val Phe Ser Leu Gln Trp Gly Glu Val Lys Leu Ile Ser Asn Cys Ser                 485 490 495 Lys Phe Tyr Gly Asn Arg Phe Tyr Glu Lys Glu Met Glu Cys Ala Gly             500 505 510 Thr Tyr Asp Gly Ser Ile Asp Ala Cys Lys Gly Asp Ser Gly Gly Pro         515 520 525 Leu Val Cys Met Asp Ala Asn Asn Val Thr Tyr Val Trp Gly Val Val     530 535 540 Ser Trp Gly Glu Asn Cys Gly Lys Pro Glu Phe Pro Gly Val Tyr Thr 545 550 555 560 Lys Val Ala Asn Tyr Phe Asp Trp Ile Ser Tyr His Val Gly Arg Pro                 565 570 575 Phe Ile Ser Gln Tyr Asn Val             580

Claims (13)

And quantifying the level of the complement factor I protein in a sample of the individual.
The method according to claim 1,
Comparing the level of complement factor I protein in a sample of a candidate pancreatic cancer patient and a sample of a normal control.
3. The method of claim 2,
Further comprising classifying the candidate patient as a pancreatic cancer patient if the level of complement factor I protein in the patient of the candidate pancreatic cancer is higher than the level of the complement factor I protein in the normal control sample.
3. The method of claim 2,
Further comprising classifying the candidate patient as a pancreatic cancer patient when the level of complement factor I protein in the patient of the candidate pancreatic cancer is more than two times the level of the complement factor I protein in the normal control sample.
5. The method according to any one of claims 1 to 4,
Wherein the sample is blood.
The method according to claim 1,
Methods for quantifying the level of the complement factor I protein include, but are not limited to, multiplexed amine-specific stable isotope assays (iTRAQ), immunoblotting, ELISA, radioimmunoassay, Mass spectrometry.
A composition for diagnosing pancreatic cancer comprising a polypeptide or an antibody that specifically binds to a complement factor I protein.
8. The method of claim 7,
Wherein the complement factor I protein has the amino acid sequence of SEQ ID NO: 1.
8. The method of claim 7,
Wherein the polypeptide is a polypeptide comprising a complement factor I protein binding domain.
8. The method of claim 7,
Wherein the antibody is a polyclonal antibody or a monoclonal antibody.
A pancreatic cancer diagnostic kit comprising a polypeptide or an antibody that specifically binds to a complement factor I protein.
12. The method of claim 11,
Wherein the complement factor I protein has the amino acid sequence of SEQ ID NO: 1.
12. The method of claim 11,
Wherein the antibody is a polyclonal antibody or a monoclonal antibody.

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