JP2017520763A - Specific biomarker sets for noninvasive diagnosis of liver cancer - Google Patents

Abstract

Cells within the liver tumor mass contain a unique protein / tumor antigen set when compared to normal liver tissue epithelial cells in close proximity to the tumor. The presence of tumor antigens is consistent with the production of autoantibodies against these tumor antigens. The present invention relates to identification and elucidation of a protein set that can function as a novel marker set for liver cancer diagnosis and prognosis determination. In particular, the present invention relates to a kit that enables diagnostic and prognostic measurement of autoantibodies in the serum of patients with liver cancer. The present invention is a verified liver cancer protein / tumor antigen set (Bmi1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, midkine, IL-17A and IL26. Provides a non-invasive, specific, sensitive and cost-effective detection and quantification method that complements conventional diagnostic methods.

Description

(Copyright indication / permission)
Part of the content of this patent document contains information that is subject to copyright protection. The copyright holder will not challenge any copy by any person of the Patent and Trademark Office application document or the recorded patent document or patent specification, but reserves all other copyrights for anything else. The following notice applies to the following processes, experiments and data and the drawings attached to them: Copyright 2014, Vision Global Holdings Limited, Unauthorized Duplication (Related Application) This application is a US non-provisional patent application 14 Claims priority to US / 321,867 (filed July 2, 2014) and US non-provisional patent application 14 / 321,870 (filed July 2, 2014), the contents of which are hereby incorporated by reference in their entirety. included.
(Technical field)
The present invention discloses a method for detecting and quantifying a list of specific and novel hepatocellular carcinoma (HCC) tumor biomarkers by measuring corresponding autoantibodies in the serum of liver cancer patients. The biomarker set includes Bmi1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, midkine, IL-17A, and IL26. More specifically, the present invention further discloses a high-throughput and sensitive test kit design. The kit facilitates the collection of peripheral serum from patients to detect liver cancer early and in a non-invasive manner by measuring autoantibodies against at least one biomarker selected from the biomarker set. Available. The present invention further enables the identification of signature biomarker patterns for staging along with the detection of recurrence during the monitoring period after chemotherapy treatment. The present invention will support automatic data analysis.

Hepatocellular carcinoma (HCC) is the second most common cancer in China, accounting for 5.7% of the total population [1]. Most HCC patients show rapid tumor progression resulting in high mortality. Early diagnosis of the disease is essential to improve overall survival. So far, the most common HCC detection method is a blood test that measures the level of an HCC tumor marker (eg, alpha fetoprotein (AFP)). AFP is a plasma protein that is produced in the yolk sac and liver during fetal development and functions like serum albumin. Under normal conditions, AFP levels gradually decline after birth and remain low in adults. An increase in tumor marker levels indicates the possibility of liver cancer. However, the main challenge of AFP testing is excessive false positives. The reason is that HCC is not the only cause of elevated AFP levels, alcoholic hepatitis, chronic hepatitis or cirrhosis is also associated with increased AFP.
Despite the general advocacy of AFP testing for the diagnosis of liver cancer, the results are inconclusive. Suspected patients will need to perform an ultrasound imaging, CT scan or contrast MRI scan for further confirmation. A liver biopsy will be performed to identify whether the tumor is benign or malignant. However, normal detection of HCC has some limitations. (A) About 20% of liver cancers do not cause elevated levels of commonly used HCC tumor markers [2]. (B) Viral cirrhosis produces false positive results on blood tests [3]. (C) Ultrasound cannot detect small tumors [4]. (D) CT scans require high radiation doses and are less sensitive for tumors smaller than 1 cm [5]. (E) MRI scans are expensive and the processing procedure takes a lot of time. Because of these limitations, it is desirable to develop new biomarker screens with high sensitivity and specificity for the early diagnosis of HCC and / or prognosis of HCC and to complement conventional methods.
HCC tumor cells tend to produce a unique set of proteins when compared to normal liver epithelial cells alongside the tumor. Validation of validated tumor biomarkers has a high potential and facilitates the diagnosis of HCC. However, not all biomarkers themselves can be found with simple diagnostic serum or urine. Alternatively, autoantibodies specific for the biomarker provide an opportunity to assess the expression of the biomarker. The presence of tumor biomarkers has been shown in many cancers to be consistent with the production of autoantibodies against these tumor antigens [6-8]. Detection of autoantibodies in patient sera will allow us to more efficiently investigate the presence of biomarkers. Ideally, examining autoantibodies in peripheral blood would be evidence of detection of liver cancer in an early and non-invasive manner. One common obstacle that hinders clinical use of biomarkers is that they have not yet been verified after discovery. However, once verified, such a test would be cost effective and accurate. Prototype design also facilitates high-throughput screening. This can reduce the cost required for normal liver cancer diagnosis.

The following is a list of references that are sometimes cited herein. The contents of each of these references are hereby incorporated by reference in their entirety.
(1) Chen JG, Zhang SW. Liver cancer epidemic in China: past, present and future. Semin Cancer Biol. 2011; 21 (1): 59-69
(2) Okuda K, Peters RL. Human alpha-1 fetoprotein. Hepatocellular Carcinoma. 1976: 353-67
(3) Lok AS, Lai CL. Alpha-fetoprotein monitoring in Chinese patients with chronic hepatitis E virus infection: role in the early detection of hepatocellular carcinoma. Hepatolog.y 1989; 9: 110-115
(4) Colombo M, de Franchis R, Del Ninno E, Sangiovanni A, De Fazio C, Tommasini M, Donato MF, Piva A, Di Carlo V, Dioguardi N. Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl J Med. 1991; 325: 675-80
(5) Sahani DV, Kalva SP. Imaging the Liver. The Oncologist. 2004; 9 (4): 385-397
(6) Masutomi K, Kaneko S, Yasukawa M, Arai K, Murakami S, Kobayashi K. Identification of serum anti-human telomerase reverse transcriptase (hTERT) auto-antibodies during progression to hepatocellular carcinoma. Oncogene. 2002 Aug 29; 21 ( 38): 5946-50.
(7) Karanikas V, Khalil S, Kerenidi T, Gourgoulianis KI, Germenis AE. Anti-survivin antibody responses in lung cancer. Cancer Lett. 2009 Sep 18; 282 (2): 159-66.
(8) Wang YQ, Zhang HH, Liu CL, Xia Q, Wu H, Yu XH, Kong W. Correlation between auto-antibodies to survivin and MUC1 variable number tandem repeats in colorectal cancer. Asian Pac J Cancer Prev. 2012; 13 (11): 5557-62.

The present invention provides a method for detecting and quantifying autoantibodies against a list of specific tumor biomarkers for the purpose of cancer diagnosis and staging. Compared to normal liver epithelial cells, HCC tumor cells tend to produce a unique set of proteins. Evaluation of the unique protein set (multibiomarkers) will complement conventional diagnostic methods and facilitate early detection of cancer.
Liver cancer biomarker sets (Bmi1, VCC1, SUMO-4, RhoA, TXN, ET) derived from paired patient biopsies (tumor biopsy versus adjacent normal tissue) by using a method based on 2D / mass spectrometry -1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, midkine, IL-17A and IL26) are identified in the present invention.
The specificity and accuracy of this liver cancer biomarker set is subsequently verified and combined for diagnosis of liver cancer. In the present invention, this list of biomarker proteins is expressed from cDNA clones, purified, and bound to fluorescent microsphere beads having various emission wavelengths. Autoantibodies against the protein of interest in the patient's serum bind immunologically to the protein-bead conjugate. This autoantibody is subsequently interacted with a PE-conjugated secondary antibody. The specific fluorescent signal of the microsphere beads serves as a discriminator for the bound biomarker. By measuring the fluorescence intensity provided by the PE-conjugated secondary antibody of the complex, autoantibodies can be detected and quantified. Since autoantibodies are produced in the patient's serum in proportion to the abundance of HCC tumor cell biomarkers, the higher the fluorescence intensity resulting from high concentrations of autoantibodies, the higher the expression of the corresponding biomarker Is shown. The minimum detection limit for each biomarker for whole serum autoantibodies is about 0.15 ng / mL.
Compared to the serum of healthy subjects, the level of autoantibodies against the target biomarker is higher in cancer patients. Furthermore, different sera from patients with liver cancer at different stages can be compared to create a signature pattern for staging. Thus, the present invention allows non-invasive evaluation of targeted liver cancer biomarkers. This allows detection of recurrence in the monitoring period after chemotherapy treatment, as well as identification for detection of HCC in the early stage and staging of the signature biomarker pattern.
Embodiments of the invention are described in more detail below with reference to the drawings. The drawings are as follows:

Differences in protein expression patterns between tumor biopsies and adjacent normal tissues are shown by two-dimensional / mass spectrometry (the analysis results in the identification of 15 specific biomarkers that are up-regulated in liver cancer). The arrow indicates the position of the spot identified in the mass spectrometry two-dimensional gel. Figure 2 shows a set of validation performed 15 liver cancer biomarkers and their corresponding molecular weights as measured by targeting in the present invention. The workflow for expressing biomarkers from cDNA clones is shown. Figure 2 shows a workflow for purifying biomarkers expressed from E. coli. The workflow for measuring autoantibodies with the BioPlex system is shown. The binding of biomarker and bioplex is shown. FIG. 2 shows an illustration of a biomarker-bioplex bead conjugate complex that is immunoreactive with a primary antibody and a PE-conjugated secondary antibody. Figure 2 shows gel electrophoresis of DNA insert released from plasmid cleaved by restriction enzymes HindIII and BamH1. Shown are Coomassie blue stained SDS-PAGE demonstrating IPTG induction of the following biomarkers: (a) Bmi1, (b) SOD1, (c) IL-17A, (d) TXN, and (e) midkine. The elution profile of Bmi1 in AKTA is shown. The elution profile of SOD-1 in AKTA is shown. The elution profile of IL-17A in AKTA is shown. Shown is Coomassie blue stained SDS-PAGE demonstrating purification of His-tagged Bmi1 biomarker. Fraction C is a bacterial lysate. Shown is Coomassie blue stained SDS-PAGE demonstrating purification of His-tagged SOD-1 biomarker. Fraction B is the bacteria that induced IPTG, and fraction C is the bacterial lysate. Shown is Coomassie Blue stained SDS-PAGE demonstrating purification of a His-tagged IL-17A biomarker. Fraction A is a bacterium that has not undergone IPTG induction, Fraction B is a bacterium that has undergone IPTG induction, and Fraction C is a bacterial lysate. A standard curve showing fluorescence intensity against anti-Bmi1 antibody concentration is shown. It is a schematic diagram which shows the design of the said test | inspection. Patient sera containing autoantibodies are mixed in wells containing 15 types of beads corresponding to 15 biomarkers of the biomarker set, followed by the addition of PE-conjugated secondary antibody.

Definitions The term “biomarker” refers to a protein that is uniquely expressed or upregulated in a tumor compared to normal epithelial cells.
The term “biomarker set” refers to a specific combination of biomarkers identified from a paired patient biopsy (tumor biopsy vs. adjacent normal tissue) and is the target of the measurement of the present invention.
The term “autoantibody” refers to an antibody produced by the patient's body that pairs with the expression of the tumor biomarker, which is present in the circulation and can be collected in peripheral serum.
Bmi1 (polycomb ring finger) is the protein component of the polycomb group (PcG) multiprotein PRC1-like complex. This is necessary to maintain the transcriptional repression status of many genes (including the Hox gene) throughout development. The regulation is via monoubiquitin addition of histone H2A'Lys-119 ', which provides expression (modifies histones and reconstitutes chromatin).
VCC1 or CXCL17 (chemokine (CXC motif) ligand 17) has an essential role in angiogenesis, and possibly in tumor development. Furthermore, it has been proposed to be a housekeeping chemokine that regulates the recruitment of non-activated blood monocytes and immature dendritic cells to tissues. It can also play a role in innate defense against infection. VCC1 dysfunction is closely related to duodenal inflammation and cholera.
SUMO-4 (small ubiquitin-like modifier 4) belongs to the family of small ubiquitin-related modifiers and is located in the cytoplasm. It adheres covalently to a target protein, IKBA, and controls its intracellular localization, stability or activity. This ultimately results in negative regulation of NF-kappa-B-dependent transcription of the IL12B gene.

RhoA (Ras homologue family member A) regulates the signaling pathway that links plasma membrane receptors to the assembly of adhesions and actin stress fibers. It is also involved in microtubule-dependent signaling essential during cytokinesis in the cell cycle, as well as other signaling pathways required for microtubule stability and cell migration and adhesion.
TXN (thioredoxin) forms a homodimer and is required for the redox reaction in the reversible oxidation of its active center dithiol to disulfide and catalyzes the dithiol-disulfide exchange reaction. Thioredoxin has been reported to be associated with breast mucinous carcinoma.
ET-1 (endothelin 1) is a potent vasoconstrictor produced by vascular endothelial cells. It binds to endothelin receptors that are widely expressed in all tissues, including non-vascular structures such as epithelial cells, glia, and neurons. Apart from its main role in maintaining vascular tone, ET-1 has also been proposed to have mitotic co-help activity and enhance the action of other growth factors.
UBE2C (ubiquitin conjugating enzyme E2C) belongs to the E2 ubiquitin conjugating enzyme family. The above is one of three enzymes required for ubiquitin addition, an important cellular mechanism that targets abnormal proteins for degradation. More specifically, UBE2C is required for degradation and cell cycle progression as a target for mitotic cyclins. Thus, it is believed that this protein may also be involved in cancer progression.
HDGF2 is called hepatoma-derived growth factor 2. This protein, which is highly expressed in a variety of tumors, has been reported to play a central role in the development and progression of several tumors. Although the mechanism has not been identified so far, HDGF2 has been proposed to have mitogenic activity, angiogenic activity, neurotrophic activity, and anti-apoptotic activity.
EGF21 (fibroblast growth factor 21) is a family member of the EGF family. The EGF family is involved in a variety of biological processes including embryonic development, cell proliferation, morphogenesis, tissue repair, tumor growth and invasion. More specifically, EGF21 stimulates glucose updates in various adipocytes through induction of glucose transport factor SLC2A1 / GLUT1 expression. EGF21 has been reported to be associated with fatty liver disease.

LECT2 (white blood cell-derived chemotaxin 1) is a secreted protein that acts as a chemotactic factor for neutrophils and stimulates the proliferation of chondrocytes and osteoblasts. This protein is implicated in acute liver failure.
SOD1 (superoxide dismutase 1) is a Cu / Zn-containing antioxidant enzyme and is required to decompose free superoxide radicals into molecular oxygen and hydrogen peroxide in the intermembrane space of cytosol, nucleus and mitochondria. This is important for maintaining cytosolic superoxide at low levels and thus protecting cells from oxidative stress and subsequent cell death.
STMN4 (Stathmin-like 4) is a small regulatory protein that relays integration of diverse intracellular signaling pathways and in turn controls cell proliferation, differentiation and function. This protein has also been shown to contribute to the control of microtubule dynamics by inhibiting microtubule polymerization and / or promoting microtubule depolymerization.
Midkine or NEGF2 (neurite growth promoting factor 2) is a secreted growth factor that binds to heparin and is responsive to retinoic acid. Midkine promotes cell proliferation, migration, and angiogenesis, especially during tumor development. Midkine has been previously shown to be associated with breast adenocarcinoma and soft tissue sarcoma.
IL-17A (interleukin 17A) is a pro-inflammatory cytokine produced by activated T cells. IL-17A regulates the activity of NF-kappa B and mitogen-activated protein kinase, stimulates expression of IL6 and cyclooxygenase 2, and further enhances nitric oxide production. Some chronic inflammation and sclerosis are usually associated with elevated IL-17A.
IL-26 (interleukin 26) belongs to the IL-10 cytokine family, is produced by activated T cells, and targets epithelial cells for signal transduction. IL-26 binds strongly to cell surface glycosaminoglycans (eg, heparin, heparan sulfate, and dermatan sulfate) (glycosaminoglycans are used to concentrate IL-26 on the surface of producing and target cells). Works like a co-receptor).

DETAILED DESCRIPTION OF THE INVENTION In the following description, a corresponding embodiment of a biomarker / multiple biomarker, a preferred example detection / verification / identification / quantification method is presented. Those skilled in the art will appreciate that modifications (including additions and / or substitutions) can be made without departing from the scope and spirit of the invention. Although specific details may be omitted so as not to obscure the present invention, the present disclosure is described to enable those skilled in the art to practice the teachings herein without undue experimentation. .
In the present invention, a liver tumor biomarker set for the detection and quantification of liver cancer is firstly used to elucidate differences in protein expression patterns between paired patient biopsies (tumor biopsy vs. adjacent normal tissue). They are identified by mass spectrometry (Figure 1). These biomarkers are verified by immunohistochemical staining with paraffin section HCC blocks and Western blot analysis of HCC patient sera. This provides a final list of 15 biomarkers that are evaluated in the present invention for purposes of liver cancer diagnosis (FIG. 2).
Based on the amino acid sequence of the targeted biomarker, a cDNA clone synthesized by a vendor was used for expression of the biomarker set (FIG. 3). Subsequently, the protein expressed from the cDNA clone was subjected to a series of purification steps (FIG. 4). Subsequently, the purified biomarker is bound to the bioplex beads via a stable amide bond. Bioplex is a type of fluorescent microsphere bead that is available as a panel that provides a unique fluorescent signal for identification in multiple components. The biomarker on the bead is recognized by a specific primary antibody, which is then bound to a PE-conjugated anti-human secondary antibody (FIG. 7). Thus, the Bioplex device measures two signals originating from the complex simultaneously. The fluorescence provided by the bioplex beads serves as a discriminator, while the signal generated from PE indicates that the biomarker is present in the complex. This also serves to distinguish biomarker bead conjugates with linked antibodies from biomarker beads due to non-immune reactions with antibodies.

In order to prove the significance of the biomarker of the present invention, the cDNA clone is confirmed by restriction enzyme digestion (FIG. 8). Transformed bacteria are induced by IPTG to express biomarker protein. Protein expression demonstrated by SDS-PAGE and Coomassie blue staining shows the protein band (Figure 9a-e), His-tagged Bmil, SOD1 and IL-17A proteins were purified by AKTA (Figure 10a-c), followed This is verified by SDS-PAGE and Coomassie blue staining (FIGS. 11a-c).
The sensitivity of the test is measured by spiking with a serial dilution of antibody. The lowest concentration of added antibody that can provide a signal indicates the sensitivity of the respective individual biomarker. On the other hand, a standard curve showing the fluorescence intensity of PE against serial dilutions of antibody is constructed (FIG. 12). A standard curve will be used to estimate the concentration of biomarker specific autoantibodies in patient sera by comparing PE intensities.
In the present invention, a multiplex mixture of 15 different Bioplex beads, each providing unique fluorescence, is bound to the biomarker set and preloaded into the wells of the plate (FIG. 13). Patient sera containing autoantibodies is loaded into the wells and allowed to interact with the biomarker conjugate. Subsequently, a PE-conjugated secondary antibody is added and allowed to bind to the autoantibody. With this device, excess secondary antibody is washed away and the complex comprising the biomarker bead conjugate and the linked antibody is measured individually. The inherent fluorescence of the bioplex beads identifies the biomarker, while the PE signal of the same complex indicates the presence of autoantibodies as the primary antibody (Figure 7). Taken together, this measurement will suggest the presence and relative concentration of autoantibodies in patient sera.

In a standard randomized trial design, the average relative level of autoantibodies between healthy groups and patients diagnosed with liver cancer is compared. Student's t test is used to analyze variable significance. A significant difference indicates that the biomarker is specific for liver cancer. After the validation test, a biomarker-specific autoantibody concentration range is obtained for liver cancer positive and negative patients and serves as a reference point for future diagnosis. On the other hand, autoantibody expression patterns are also compared among liver cancer patients at various stages. The signature pattern of biomarker expression will indicate the stage of HCC.
Taking the relative autoantibody levels and biomarker expression patterns together, the present invention represents a variety of means to complement normal liver cancer diagnosis. The present invention further allows non-invasive detection of autoantibodies against verified targets in patient sera of the present invention, wherein said non-invasive detection identifies the extent and characteristics of the disease. Aside from the early detection of stage I liver cancer, the present invention also allows the generation of a signature pattern for staging and the detection of recurrence during the monitoring period after mastectomy or chemotherapy treatment.
EXAMPLES The following examples are provided by describing specific embodiments of the present invention without intent to limit the scope of the invention.

Example 1a: Protein extraction from patient biopsy
500 mg paired patient biopsies (tumor biopsy vs. adjacent normal tissue) are collected and washed with PBS. The tissue is frozen by submerging in liquid nitrogen and immediately homogenized with a mortar and pestle. To this homogenized sample, a lysis solution (8M urea, 4% CHAPS, 2% IPG buffer, 0.2 mg / mL PMSF) is added, followed by vortexing for at least 5 minutes until the tissue is completely dispersed. The lysate is subsequently clarified by centrifugation at 14,000 rpm for 10 minutes at 4 ° C. The supernatant is further cleaned with a 2D cleanup kit (Amersham) to remove salts and impurities. Resuspend pellet with minimum volume of rehydration solution (no DTT and IPG buffer added). The protein concentration is then measured with the Bio-Rad protein assay and an aliquot of 200 g / tube is stored at -70 ° C.
Example 1b: Protein separation by two-dimensional electrophoresis
To 1 mL of rehydrated stock solution, add 2.8 mg DTT, 5 μL Pharmalite or IPG buffer, and 2 μL bromophenol blue. 50-100 μg protein sample is added to a 13 cm Immobiline DryStrip (IPG strip) containing 250 μL of rehydration solution. After removing the protective cover, place this IPG strip on the strip holder appropriately with the gel side facing down and overlay the cover solution to prevent dehydration during electrophoresis. Subsequently, the strips are placed on Ettan IPGphor (Amersham) for isoelectric focusing (one-dimensional electrophoresis).
After one-dimensional electrophoresis, the IPG strips were equilibrated with 6M urea, 2% SDS, 50 mM Tris HCl (pH 6.8), 30% glycerol, 0.002% bromophenol blue, 100 mg DTT / 10 mL buffer, and 250 mg IAA / Equilibrate with 10 mL buffer) followed by 4-5 washes with 1 × SDS running buffer. The IPG strip is placed on a two-dimensional gel and overlaid with a sealing solution (0.5% cold-melted agarose, 0.002% bromophenol blue in 1 × SDS running buffer). Subsequently, two-dimensional electrophoresis is first performed at 30 mA for 15 minutes, followed by 60 mA for 3-4 hours.
Upon completion of two-dimensional electrophoresis, the gel is removed from the cassette, fixed and stained with silver nitrate. Identify 15 spots representing 15 up-regulated proteins (Figure 1). For protein identification (Figure 2), the silver-stained gel slice is destained and further trypsin digested to release the protein from the gel for MALDI-TOF analysis.

Example 2a (SEQ ID NO: 1)
Bmil amino acid sequence
MHRTTRIKITELNPHLMCVLCGGYFIDATTIIECLHSFCKTCIVRYLETSKYCPICDVQVHKTRPLLNIRSDKTLQDIVYKLVPGLFKNEMKRRRDFYAAHPSADAANGSNEDRGEVADEDKRIITDDEIISLSIEFFDQNRLDRKVNKDKEKSKEEVNDKRYLRCPAAMTVMHLRKFLRSKMDIPNTFQIDVMYEEEPLKDYYTLMDIAYIYTWRRNGPLPLKYRVRPTCKRMKISHQRDGLTNAGELESDSGSDKANSPAGGIPSTSSCLPSPSTPVQSPHPQFPHISSTMNGTSNSPSGNHQSSFANRPRKSSVNGSSATSSG
Example 2b (SEQ ID NO: 2)
VCC1 amino acid sequence
MKVLISSLLLLLPLMLMSMVSSSLNPGVARGHRDRGQASRRWLQEGGQECECKDWFLRAPRRKFMTVSGLPKKQCPCDHFKGNVKKTRHQRHHRKPNKHSRACQQFLKQCQLRSFALPL
Example 2c (SEQ ID NO: 3)
Amino acid sequence of SUMO-4
MANEKPTEEVKTENNNHINLKVAGQDGSVVQFKIKRQTPLSKLMKAYCEPRGLSVKQIRFRFGGQPISGTDKPAQLEMEDEDTIDVFQQPTGGVY
Example 2d (SEQ ID NO: 4)
RhoA amino acid sequence
MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAGQRG
Example 2e (SEQ ID NO: 5)
Amino acid sequence of TXN
MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEATINELV
Example 2f (SEQ ID NO: 6)
ET-1 amino acid sequence
MDYLLMIFSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRSKRALENLLPTKATDRENRCQCASQKDKKCWNFCQAGKELRAEDIMEKDWNNHKKGKDCSKLGKKCIYTHQRE
Example 2g (SEQ ID NO: 7)
Amino acid sequence of UBE2C
MASQNRDPAATSVAAARKGAEPSGGAARGPVGKRLQQELMTLMMSGDKGISAFPESDNLFKWVGTIHGAAGTVYEDLRYKLSLEFPSGYPYNAPTVKFLTPCYHPNVDTQGNICLDILKEKWSALYDVRTILLSIQSLLGEPNIDSPLNTHAAELWKEPTAFKKYVQYSK
Example 2h (SEQ ID NO: 8)
Amino acid sequence of HDGF2
MARPRPREYKAGDLVFAKMKGYPHWPARIDELPEGAVKPPANKYPIFFFGTHETAFLGPKDLFPYKEYKDKFGKSNKRKGFNEGLWEIENNPGVKFTGYQAIQQQSSSETEGEGGNTADASSEEEGDRVEEDGKGKRKNEKAGSKRKKSYTSKKDSSKQSRKSPGDEDDKDCKEEENTSSSEGGGND
Example 2i (SEQ ID NO: 9)
FGF21 amino acid sequence
MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLLSGPPG
Example 2j (SEQ ID NO: 10)
Amino acid sequence of LECT2
MFSTKALLLAGLISTALAGPWANICAGKSSNEIRTCDRHGCGQYSAQRSQRPHQGVDVLCSAGSTVYAPFTGMIVGQEKPYQNKNAINNGVRISGRGFCVKMFYIKPIKYKGPIKKGEKLGTLLPLQKVYPGIQSHVHIENCDSSDPTAYL
Example 2k (SEQ ID NO: 11)
Amino acid sequence of SOD1
MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ
Example 2l (SEQ ID NO: 12)
Amino acid sequence of STMN4
MTLAAYKEKMKELPLVSLFCSCFLADPLNKSSYKYEADTVDLNWCVISDMEVIELNKCTSGQSFEVILKPPSFDGVPEFNASLPRRRDPSLEEIQKKLEAAEERRKYQEAELLKHLAEKREHEREVIQKAIEENNNFIKMAKEKLAQKMESNKENREAHLAAMLERLQEKDKHAEEVRKEL
Example 2m (SEQ ID NO: 13)
Midkine amino acid sequence
MQHRGFLLLTLLALLALTSAVAKKKDKVKKGGPGSECAEWAWGPCTPSSKDCGVGFREGTCGAQTQRIRCRVPCNWKKEFGADCKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETIRVTKPCTPKTKAKAKAKKGKGKD
Example 2n (SEQ ID NO: 14)
IL-17A amino acid sequence
MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA
Example 2o (SEQ ID NO: 15)
IL-26 amino acid sequence
MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIYKAISELDILLSWIKKLLESSQ

Example 3a: Expression of a biomarker set DH5 competent cells are transformed with a His-tagged plasmid containing a cDNA insert encoding a biomarker set (301, FIG. 3). Single colonies are picked and grown in bacterial culture. Increase the number of plasmids and extract from bacteria by miniprep. This plasmid is further transformed into BL21DE3 or BL21DE3pLysS competent cells. Transformed bacteria are selected and grown in 2X 100 mL LB medium. When the bacterial culture reaches an optical density of 0.06, 200 μM IPTG is added to the 100 mL bacterial culture (303). Another 100 mL bacterial culture without IPTG is used as a negative control. These bacterial cultures are incubated at 30 ° C. with shaking. Save 500 μL of bacterial culture 3 hours after incubation and the next morning after overnight incubation and store at −20 ° C.
IPTG induced and uninduced bacterial cultures are mixed together in a 500 mL centrifuge bottle. Bacterial cells are collected by centrifugation at 9000 rpm for 20 minutes at 4 ° C. (304). Save 500 μL of the supernatant as another negative control and discard the remaining supernatant. Bacterial cultures collected at various time points and negative controls are run on SDS-PAGE to separate proteins (305). The gel is then stained overnight with Coomassie blue. After destaining the gel, protein induction can be confirmed by size check and comparison with a negative control.
Example 3b: Protein-purified bacterial cell pellet of biomarker set is resuspended in 10 mL of solubilization buffer at room temperature by vortex mixer. The resuspended cells are maintained on ice in a 50 mL centrifuge tube and the cells are completely lysed by sonication (70% amplitude, 10 rounds of 30 seconds, 30 second intervals) (401, FIG. 4). The lysed cells are centrifuged at 10,000 rpm for 1 hour at 4 ° C. (402). The supernatant is transferred to a dialysis tube, submerged in 1 L of unfiltered start buffer and stirred continuously at 4 ° C. for 4-6 hours (403). Continue dialysis overnight with another 1 L of starting buffer. The supernatant is further filtered through a 0.22 μm filter disc and syringe. The filtered sample is loaded (405) onto an AKTA apparatus (404) equipped with a HiTrap chelate column packed with 0.1 M nickel sulfate. A program is set in the AKTA apparatus so that the eluent is automatically collected (406). Various fractions of purified protein are checked by SDS-PAGE analysis (407).

Example 4a: Bioplex bead and protein binding Purified protein of the biomarker set is bound to bioplex beads (Bio-Rad) according to the manufacturer's manual (501). Briefly, unbound beads are vortexed for 30 seconds and then sonicated for 15 seconds. Centrifuge 100 μL of beads at maximum speed for 4 minutes and collect 1,250,000 beads in a reaction tube. After washing with 100 μL of bead wash buffer by centrifugation, the beads are resuspended in 80 μL of bead activation buffer. To the beads, freshly prepared 50 mg / mL S-NHS (10 μL) and freshly prepared 50 mg / mL EDAC (10 μL) are added followed by incubation for 20 minutes at room temperature in the dark (FIG. 6). Subsequently, the beads are washed twice with 150 μL of PBS.
Add 10 μg of protein to the washed beads, bring the total volume to 500 μL with PBS and incubate for 2 hours with shaking in the dark. After centrifugation at maximum speed for 4 minutes, the supernatant is removed. Add 250 μL of blocking buffer to the beads and shake for 30 minutes in the dark, then centrifuge at maximum speed for 4 minutes to remove the supernatant. The beads are washed gently and then resuspended in storage buffer and stored at 4 ° C. Count the number of beads with a hemocytometer.
Example 4b: Verification of protein-bead binding
To the HTS 96-well plate, 50 μL of conjugated Bioplex beads (100 beads / μL) is added to react the primary antibody followed by the secondary antibody (502). Serial dilutions of primary antibody for the biomarker set available on the market are prepared as 8,000, 4,000, 1,000, 250, 62.5, 15.625, 3.906, 0.977, 0.244 and 0.061 ng / mL. Add 50 μL of each dilution to each well. Two negative controls are performed by removing the primary antibody from the well and removing both the primary and secondary antibodies. The plate is then sealed with metal foil and kept on a shaker avoiding exposure to light at 350 rpm for 30 minutes.
After incubation, wash the beads 3 times with 150 μL PBS. 50 μL of PE-conjugated secondary antibody (8,000 ng / mL) is added to each well except the negative control. Seal the plate again and incubate for 30 minutes with shaking in the dark. Subsequently, the excess antibody is washed away with PBS. The scale of the Bioplex device is adjusted with a calibration kit and a verification kit. After loading the device with the HTS plate, the signals of both Bioplex beads and secondary antibody-bound PE (503) are measured (schematic diagram is shown in FIG. 7). The calibration curve is created by Logistic-5PL.
Example 4c: Collection of serum samples and determination of autoantibodies with the Bioplex system Whole blood samples are allowed to clot for 1 hour at 37 ° C. After centrifugation at 1000 g for 10 minutes at room temperature, supernatant serum containing autoantibodies is collected. Dilute serum with PBS when necessary. Load serum samples onto HTS plates pre-loaded with bioplex beads bound biomarker set and incubate for 30 minutes with shaking (Figure 13). Similar to the steps described in Example 4b, 50 μL of PE-conjugated secondary antibody (8000 ng / mL) is added to the PBS wash beads followed by shaking for an additional 30 minutes. After three rounds of washing, the plate is loaded into the Bioplex device and the fluorescent signal is measured (504). Subsequently, the concentration of the autoantibody is calculated from the standard curve.
The foregoing description of the invention has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations will be apparent to practitioners skilled in this art.
In order to best explain the principles of the invention and its practical application and thereby enable those skilled in the art to understand various embodiments and modifications suitable for the specific use intended, the present embodiments Is selected and described. It is intended that the scope of the invention be defined by the following claims and their equivalents.

Industrial Application A kit comprising the claimed method and 15 identification biomarkers for identifying and quantifying the presence of autoantibodies in a patient's serum to detect and / or stage liver cancer It is also useful for developing drugs that target these markers for specific treatment of liver cancer.

Claims (16)

A method for detecting and quantifying autoantibodies against multiple tumor biomarkers for the purpose of diagnosing and staging liver cancer comprising:
Comparing tumor cells with normal cells and evaluating a set of proteins that are unique to the tumor cells but not unique to the normal cells as a plurality of biomarkers specific to the cancer;
Identifying the protein set of the paired patient biopsy sample by using two-dimensional or mass spectrometry technology;
Verifying the protein set for diagnosis of liver cancer;
Including
The protein set is expressed from a cDNA clone, purified, and further bound to fluorescent microsphere beads having various emission wavelengths to form protein-bead conjugates,
Autoantibodies to the protein set present in the patient's serum bind immunologically to the protein-bead conjugate,
Said method.   2. The method of claim 1, wherein the autoantibody is subsequently interacted with a PE-conjugated secondary antibody, and the fluorescent signal specific for the microsphere bead serves as a discriminator for the protein-bead conjugate.   3. The method of claim 2, wherein the fluorescence intensity provided by the complex PE-conjugated secondary antibody is measured to enable detection and quantification of autoantibodies.   The method of claim 1, further comprising the step of comparing serum between the patient and a healthy subject to determine the level of autoantibodies against the relevant biomarker.   2. The method of claim 1, further comprising comparing different sera obtained from different patients at different stages to create a signature pattern for staging.   The method of claim 1, wherein the protein set comprises at least one of the amino acid sequences of SEQ ID NOs: 1-15.   The plurality of tumor markers according to claim 1, comprising Bmi1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, midkine, IL-17A and IL26. the method of.   2. The method of claim 1, wherein the tumor cells comprise cells derived from hepatocellular carcinoma (HCC) tumor.   Each fluorescent microsphere bead has a unique fluorescence emission wavelength specific for each of the proteins corresponding to the presence of a specific tumor biomarker, while each of the proteins for that specific tumor biomarker The PE signal generated from the PE-conjugated secondary antibody bound to the protein-bead conjugate is an indicator of the presence of autoantibodies produced in the patient serum, and the fluorescence intensity of the PE signal is abundant in the specific tumor marker. The method of claim 1, which is proportional to   2. The method of claim 1, wherein each of the tumor biomarkers present in the autoantibodies is present at a low of about 0.15 ng / mL.   A kit for diagnosing and staging liver cancer by detecting and quantifying patient autoantibodies against multiple tumor biomarkers, expressed and purified from cDNA clones, and further fluorescent with various emission wavelengths Includes a protein set bound to microsphere beads to form a protein-bead conjugate, where the protein-bead conjugate binds immunologically with the autoantibody and is subsequently targeted by a PE-conjugated secondary antibody. The kit wherein the fluorescence signal and intensity are measured to determine the level of autoantibody in the patient compared to a normal subject.   The kit of claim 11, wherein the protein set comprises at least one of the amino acid sequences of SEQ ID NOs: 1-15.   The tumor biomarker according to claim 11, wherein Bmi1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, midkine, IL-17A and IL26. kit.   Fluorescent microsphere beads with different emission wavelengths are used for each of the cDNA clones to immunologically bind to autoantibodies and subsequently form protein-bead conjugates that are targeted by PE-conjugated secondary antibodies. 12. The kit of claim 11, comprising a His-tagged plasmid containing a cDNA insert corresponding to a protein expressed in competent cells that is bound.   Multiple mixtures of different fluorescent microspheres, each providing unique fluorescence, are combined with the expressed and purified protein set as previously described to form a protein-bead conjugate, and the protein-bead conjugate is Pre-load into container wells, load patient serum containing autoantibodies into each well, interact with the protein-bead conjugate, then add PE-conjugated secondary antibody to bind to the autoantibodies Followed by washing away any excess secondary antibody, and the complex containing the protein-bead conjugate and the linked antibody is individually measured and the specific fluorescence signal of the protein-bead conjugate is determined by the specific fluorescence signal. While the presence of the marker was revealed, the presence of autoantibodies and the presence of the tumor biomarker in patient sera by the PE signal from the secondary antibody of the complex 12. Kit according to claim 11, wherein relative concentrations are indicated.   12. The kit of claim 11, wherein each tumor biomarker present in the autoantibody is present at a low of about 0.15 ng / mL.