Multiplexed biomarkers for cancer diagnosis and their uses

Abstract

This paper relates to a cancer diagnostic multiple biomarker and its uses, and to a cancer diagnostic composition comprising a preparation for measuring the expression levels of IGFI, KLK2, PKCα, and TRPM8 proteins or their mRNA. Using actual serum samples from cancer patients, it has been confirmed that detecting the specific marker combination of IGFI, KLK2, PKCα, and TRPM8 not only enables rapid and accurate cancer diagnosis, but also allows for simultaneous multiplex detection of one or more cancers. Therefore, it is expected that the combination of IGFI, KLK2, PKCα, and TRPM8 will enable rapid and accurate diagnosis of various cancers, including prostate cancer.

Description

[Technical Field]

[0001] The present invention relates to multiple biomarkers for cancer diagnosis and uses thereof, and more particularly to a cancer diagnostic composition comprising a preparation for measuring the expression levels of PKCα, IGFI, KLK2, and / or TRPM8 protein or its mRNA.

[0002] This application claims priority based on Korean Patent Application No. 10-2022-0101571, filed on August 12, 2022, the entire contents of which are incorporated herein by reference in their entirety in the specification and drawings. [Background technology]

[0003] Angiogenesis disorders, such as cancer, are one of the most deadly threats to human health. Approximately 1.3 million new cancer cases occur annually in the United States alone, making it the second leading cause of death after cardiovascular disease. While significant advances have been made in medical treatment for certain cancers, the overall five-year survival rate for all cancers has improved by only about 10% over the past 20 years. Because cancer, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, it is extremely difficult to detect and treat them in time.

[0004] Therefore, the importance of early cancer diagnosis is increasing worldwide. Accordingly, research into early cancer diagnosis methods is increasing. However, current cancer diagnosis methods are performed using invasive methods such as tissue sampling and endoscopic examination. In particular, tissue examination is performed by removing a portion of the suspected disease site and observing it under a microscope. Therefore, when using a needle, punch, endoscope, or laparoscope to collect a tissue sample, the body must be incised, which not only causes considerable inconvenience to the patient but also leaves scars and requires a long recovery time.

[0005] Meanwhile, prostate cancer (PCa) is one of the most common causes of cancer-related deaths among American men. This is statistically proven, with prostate cancer, respiratory cancer, and colorectal cancer accounting for 46% of cancer-related deaths in 2016. Prostate cancer, in particular, has been reported to be associated with factors related to economic development, and it is a growing problem in Asia, where many developing countries are located. In South Korea, the prevalence of prostate cancer tripled between 2007 and 2013, and its mortality rate is rapidly increasing among young men (under 70 years old).

[0006] Traditional prostate cancer screening methods involve measuring serum prostate-specific antigen (PSA) levels and then performing a diagnostic biopsy after a transrectal palpation. Currently, prostate cancer is diagnosed when a serum PSA level exceeds 4, along with a doctor's judgment. While this method can identify most malignant tumors, the efficacy of standard PSA testing has recently been questioned. Only approximately 25% of men with elevated PSA levels (>4.0 ng / mL) are diagnosed with prostate cancer after a biopsy, with many false-negative results. Furthermore, biopsies, which can be potentially dangerous to patients, are not always accurate due to the heterogeneity of cancers, requiring multiple repeated tests. Furthermore, the final recommendations of the US Preventive Services Task Force (USPSTF) do not recommend PSA-based testing for prostate cancer diagnosis.

[0007] Therefore, the present inventors attempted to establish a new biomarker combination and diagnostic method that can reinforce the diagnosis of prostate cancer along with confirmation of PSA values. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the inventors investigated various markers to improve the accuracy and speed of cancer diagnosis and confirmed that the combination of KLK2, IGFI, and PKCα has excellent diagnostic effects for various cancers, including prostate cancer and breast cancer, and thus completed the invention.

[0009] Therefore, an object of the present invention is to provide a composition for diagnosing cancer, which comprises a preparation for measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGFI, KLK2, and TRPM8, or their mRNA.

[0010] Another object of the present invention is to provide a cancer diagnosis kit comprising the preparation.

[0011] It is yet another object of the present invention to provide a lateral flow assay strip for cancer diagnosis, which comprises the formulation.

[0012] It is yet another object of the present invention to provide a method for providing information for cancer diagnosis.

[0013] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description. [Means for solving the problem]

[0014] To achieve the above object, the present invention provides a cancer diagnostic composition comprising a preparation for measuring the expression levels of IGFI, KLK2, PKCα, and / or TRPM8 proteins or their mRNAs.

[0015] The present invention also provides a cancer diagnostic use of a preparation for measuring the expression levels of IGFI, KLK2, PKCα, and / or TRPM8 proteins or their mRNAs.

[0016] The present invention also provides use of a preparation for measuring the expression levels of IGFI, KLK2, PKCα, and / or TRPM8 proteins or their mRNAs for the manufacture of a cancer diagnostic agent.

[0017] The present invention also provides a cancer diagnostic kit comprising the composition.

[0018] The present invention also provides a method for providing information for cancer diagnosis, comprising the steps of:

[0019] (a) measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGFI, KLK2, and TRPM8, or their mRNAs, in a biological sample isolated from a subject; and

[0020] (b) determining that the subject has cancer or is at risk of developing cancer if the expression level of the measured protein or its mRNA is increased compared to a control group;

[0021] The present invention also provides a method for diagnosing cancer, comprising the steps of:

[0022] (a) measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGFI, KLK2, and TRPM8, or their mRNAs, in a biological sample isolated from a subject; and

[0023] (b) determining that the subject has cancer or is at risk of developing cancer if the expression level of the measured protein or its mRNA is increased compared to a control group;

[0024] In one embodiment of the present invention, the diagnostic composition or kit can simultaneously detect one or more cancers, but is not limited thereto.

[0025] In one embodiment of the present invention, the cancer may be one or more selected from the group consisting of colon cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, malignant melanoma, lymphoma, aplastic anemia, and blood cancer, but is not limited to this.

[0026] In another embodiment of the present invention, the composition may be used with one or more samples selected from the group consisting of, but not limited to, blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, peritoneal fluid, cervical or vaginal secretions, urine, and cerebrospinal fluid as the sample to be analyzed.

[0027] In yet another embodiment of the present invention, the expression level of the protein may be measured using, but is not limited to, an antibody or aptamer specific to the protein.

[0028] In yet another embodiment of the present invention, the expression level of the mRNA may be measured using, but is not limited to, a probe or primer that specifically binds to the mRNA.

[0029] In yet another embodiment of the present invention, the composition may further comprise, but is not limited to, an agent for measuring the expression level of prostate-specific antigen (PSA) protein or its mRNA.

[0030] In yet another embodiment of the present invention, the kit may include, but is not limited to, a lateral flow assay strip.

[0031] In yet another embodiment of the present invention, the lateral flow assay strip may include, but is not limited to, a sample pad, a conjugation pad, a membrane, and an absorbent pad.

[0032] In yet another embodiment of the present invention, the lateral flow assay strip may comprise gold nanoparticles onto which one or more antibodies selected from the group consisting of anti-PKCα antibody, anti-IGFI antibody, anti-KLK2 antibody, and anti-TRPM8 antibody are immobilized, but is not limited thereto.

[0033] In yet another embodiment of the present invention, the expression level of the protein may be measured using, but is not limited to, enzyme-linked immunosorbent assay (ELISA) or lateral flow assay. [Effects of the Invention]

[0034] The present inventors have confirmed that detecting the specific marker combination of the present invention, IGFI, KLK2, PKCα, and / or TRPM8, using serum samples from actual cancer patients not only enables rapid and accurate cancer diagnosis, but also allows for simultaneous detection of one or more cancers. Therefore, it is expected that the combination of IGFI, KLK2, PKCα, and / or TRPM8 will enable rapid and accurate diagnosis of various cancers, including prostate cancer and breast cancer. In particular, the lateral flow assay strip of the present invention contains gold nanoparticles to which biomarker-specific antibodies are immobilized, and has been confirmed to simultaneously detect multiple biomarkers, enabling more accurate cancer diagnosis than when detecting a single biomarker. Therefore, it is expected that the lateral flow assay strip of the present invention will be useful for the simultaneous diagnosis of multiple cancers. [Brief explanation of the drawings]

[0035] [Figure 1A] Schematic diagram of the multiplexed lateral fluid analysis method for early diagnosis of prostate cancer using a multi-biomarker array, showing (a) an LFA strip without sample, (b) the strip showing positive results after adding serum and urine samples from cancer patients, and (c) negative results for healthy individual specimens. [Figure 1B] This figure shows UV-Vis spectra of gold nanoparticles at various pH levels, and is the result of an experiment to prevent aggregation of gold nanoparticles and select an appropriate pH. [Figure 1C] This graph shows the change in absorbance of gold nanoparticles at different pH levels. It shows that pH 9 is the optimal condition, with the least amount of change in absorbance. [Figure 1D] This graph shows the color change of gold nanoparticles at various pH levels over time, and is the result of an experiment on the stability of gold nanoparticles. The result at pH 9 shows that the color change is minimal even after 7 days. [Figure 1E] This figure shows spectra at different reaction times for immobilizing anti-PKCα antibody (top row) and anti-TRPM8 antibody (bottom row) on gold nanoparticles, and the extent of spectral change was confirmed at 10-minute intervals up to 60 minutes. [Figure 1F] 1 is a graph showing the results of optimizing the reaction time for immobilizing anti-PKCα antibody and anti-TRPM8 antibody on gold nanoparticles, showing that a large amount of antibody is immobilized within 10 to 30 minutes. [Figure 1G] This figure shows the results of visual / color observation of gold nanoparticle-antibody conjugates at various antibody concentrations to determine the optimal antibody concentration for immobilization of gold nanoparticles and antibodies. The red dotted lines indicate the optimal antibody concentration for immobilization (anti-KLK2, 3 μg / mL; anti-IGFI, 3 μg / mL; anti-TRPM8, 2 μg / mL; and anti-PKCα, 4 μg / mL). [Figure 1H] The spectra of gold nanoparticle-antibody conjugates were observed at different antibody concentrations to determine the optimal antibody concentration for immobilizing gold nanoparticles and antibodies. The optimal antibody concentration for each antibody is indicated by a red square. [Figure 1I] The size of the synthesized gold nanoparticles (top row) and the results of surface zeta potential analysis (bottom row) are shown. [Figure 1J] 1 shows the UV-Vis spectrum of the synthesized gold nanoparticles. [Figure 1K] Electron micrographs of gold nanoparticles ((a) gold nanoparticles with a diameter of approximately 40 nm, (b) gold nanoparticles bound to antibodies, (c-d) electron micrographs of gold nanoparticles bound to antibodies on an absorbent pad after treatment). [Figure 1L]FIG. 1 shows FTIR spectra of gold nanoparticles bound to KLK2, IGFI, TRPM8, or PKC-α antibodies. [Figure 2] FIG. 1 shows cell growth curves of each cell line to confirm the growth stage of cancer cells. [Figure 3] FIG. 1 shows the results of Western blot analysis of cell extracts (lysates) from four different stages of the cell growth cycle. [Figure 4] This figure shows the results of validating KLK2, IGFI, and PKCα biomarkers for cancer diagnosis using the indirect ELISA method, comparing the absorbance (levels of each biomarker) in normal samples, prostate cancer samples, and breast cancer samples. [Figure 5] FIG. 1 shows a plasmid map for cloning human genes (KLK2, IGFI, PKCα, TRPM8) into the vector pET-22b(+). [Figure 6] FIG. 10 shows the results of agarose gel electrophoresis, confirming the cloning of an expression vector using restriction enzymes. [Figure 7] FIG. 1 shows the results of immuno-Western blot electrophoresis of the insoluble fraction of recombinant proteins. [Figure 8] FIG. 1 shows the results of immuno-Western blot electrophoresis to confirm the level of expression at different IPTG (Isopropyl β-D-1-thiogalactopyranoside) concentrations (0, 0.25, 0.5, 0.75, 1 mM). [Figure 9] FIG. 1 shows the results of immuno-Western blot electrophoresis to confirm the expression level of each biomarker at each expression temperature (2: 12° C., 3: 25° C., 4: 37° C.). [Figure 10A] This figure shows the results of indirect ELISA analysis for cancer diagnosis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 10B] This figure shows the results of indirect ELISA analysis for cancer diagnosis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 10C]This figure shows the results of indirect ELISA analysis for cancer diagnosis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 10D] Figure 1 shows the results of cancer diagnostic sandwich ELISA analysis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 10E] Figure 1 shows the results of cancer diagnostic sandwich ELISA analysis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 10F] Figure 1 shows the results of cancer diagnostic sandwich ELISA analysis using purified PKCα, IGFI, and KLK2 biomarker proteins. [Figure 11A] Figure 1 shows the results of lateral fluid analysis for IGFI antigen (test / control band intensity changes and images of LFA strips (inset) for IGFI antigen at various concentrations (1 ng / mL to 8000 ng / mL)). [Figure 11B] FIG. 11 shows the results of lateral fluid analysis for IGFI antigen (log-corrected results for the IGFI band in FIG. 11A above). [Figure 11C] Figure 1 shows the results of lateral fluid analysis for IGFI antigen (test / control band intensity changes and images of LFA strips (inset) for the target antigen IGFI in serum spiked samples at various concentrations (1 ng / mL to 2000 ng / mL)). [Figure 11D] FIG. 1 shows the results of lateral fluid analysis for IGFI antigen (corrected results for various concentrations of the target antigen IGFI in serum spiked samples). [Figure 12A] This figure shows the results of lateral fluid analysis for KLK2 antigen (test / control band intensity changes and images of LFA strips (inset) for KLK2 antigen at various concentrations (1 ng / mL to 8000 ng / mL)). [Figure 12B] FIG. 12 shows the results of lateral fluid analysis for KLK2 antigen (log-corrected results for the KLK2 band in FIG. 12A). [Figure 12C]Figure 1 shows the results of lateral flow analysis for KLK2 antigen (test / control band intensity changes and images of LFA strips (inset) for the target antigen KLK2 in serum spike samples of various concentrations (1 ng / mL to 1000 ng / mL)). [Figure 12D] FIG. 1 shows the results of lateral fluid analysis for KLK2 antigen (corrected results for various concentrations of the target antigen KLK2 in serum spike samples). [Figure 13A] FIG. 1 shows the results of lateral fluid analysis for PKCα antigen (test / control band intensity changes and images of LFA strips (inset) for PKCα antigen at various concentrations (1 ng / mL to 4000 ng / mL)). [Figure 13B] FIG. 13 shows the results of lateral fluid analysis for PKCα antigen (log-corrected results for the PKCα band in FIG. 13A). [Figure 13C] Figure 1 shows the results of lateral flow assay for PKCα antigen (test / control band intensity changes and images of LFA strips (inset) for the target antigen PKCα in serum spike samples of various concentrations (1 ng / mL to 4000 ng / mL)). [Figure 13D] FIG. 1 shows the results of lateral fluid analysis for PKCα antigen (corrected results for various concentrations of the target antigen PKCα in serum spiked samples). [Figure 14] FIG. 1 shows the results of diagnosing IGFI biomarkers from serum samples of normal subjects and cancer patients (liver cancer, lung cancer, colon cancer, breast cancer, and prostate cancer) using a lateral flow analysis strip. [Figure 15] FIG. 1 shows the results of diagnosing the KLK2 biomarker from serum samples of normal subjects and cancer patients (liver cancer, lung cancer, colon cancer, breast cancer, and prostate cancer) using a lateral flow analysis strip. [Figure 16] FIG. 1 shows the results of diagnosing the PKCα biomarker in serum samples from normal subjects and cancer patients (liver cancer, lung cancer, colon cancer, breast cancer, and prostate cancer) using a lateral flow assay strip. [Figure 17]Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results on the top and a TL band intensity graph on the bottom (detection results for serum spiked samples using 500 ng / mL of biomarkers). [Figure 18] Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper panel and a TL band intensity graph in the lower panel (detection results for normal serum samples). [Figure 19] Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper row and a TL band intensity graph in the lower row (detection results for serum samples from liver cancer patients). [Figure 20] Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper panel and a TL band intensity graph in the lower panel (detection results for serum samples from lung cancer patients). [Figure 21] Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper panel and a TL band intensity graph in the lower panel (detection results for serum samples from colorectal cancer patients). [Figure 22] Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper panel and a TL band intensity graph in the lower panel (detection results for serum samples from breast cancer patients). [Figure 23]Figure 1 shows the results of simultaneous detection of PKCα, IGFI, and KLK2 biomarkers from each sample using a lateral flow assay strip for multiplex biomarker detection. Each figure shows a photograph of the LFA multiplex detection results in the upper panel and a TL band intensity graph in the lower panel (detection results for serum samples from prostate cancer patients). DETAILED DESCRIPTION OF THE INVENTION

[0036] In one embodiment of the present invention, prostate cancer-related cell lines were selected to select prostate cancer biomarkers, and it was confirmed that KLK2, TMEFF2, TRPM8, and KLK4 were highly expressed in prostate cancer cell lines (see Example 1).

[0037] In another example of the present invention, the usefulness of KLK2, IGFI, PKCα, and TRPM8 as cancer diagnostic markers was confirmed (see Example 2).

[0038] In yet another example of the present invention, the antigen recognition efficiency of anti-IGFI, anti-KLK2, and anti-PKCα antibodies was measured using indirect ELISA and sandwich ELISA, and it was confirmed that these antibodies effectively detected the recombinant antigen proteins KLK2, IGFI, and PKCα (see Example 4).

[0039] In yet another example of the present invention, in order to confirm the efficacy of the cancer diagnostic biomarkers according to the present invention, lateral fluid analysis was performed on standard samples for each biomarker. As a result, each biomarker was detected from the LFA strip, and it was confirmed that KLK2, IGFI, and PKCα all showed good linearity and were well detected in a reaction time of less than 10 minutes (see Example 6).

[0040] In yet another example of the present invention, various clinical samples from cancers such as breast cancer, lung cancer, liver cancer, colon cancer, and prostate cancer were processed into LFA strips and lateral flow analysis was performed for each single biomarker. As a result, it was confirmed that KLK2, IGFI, and PKCα all had high activity in cancer patient samples and had excellent diagnostic efficacy with sensitivity, selectivity, and accuracy of 90-100% (see Example 7).

[0041] In yet another example of the present invention, lateral flow analysis of clinical samples was performed using an LFA strip for detecting multiple biomarkers. As a result, it was confirmed that the biomarkers according to the present invention were simultaneously detected when cancer patient samples were processed, thereby enabling more accurate diagnosis of cancer (see Example 8).

[0042] Therefore, the present inventors have confirmed that cancer can be diagnosed accurately and quickly using KLK2, IGFI, PKCα, and TRPM8, and have completed the diagnostic composition of the present invention.

[0043] The present invention will be described in detail below.

[0044] One aspect of the present invention relates to a cancer diagnostic composition comprising a preparation for measuring the expression levels of PKCα, IGFI, KLK2, and / or TRPM8 proteins or their mRNAs. The present invention also provides a cancer diagnostic kit comprising the preparation.

[0045] As used herein, "polynucleotide" or "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single- or double-stranded form. Unless otherwise specified, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included. Generally, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), while RNA contains uracil (U) instead of thymine. In a double-stranded nucleic acid, A forms hydrogen bonds with T or U, and C forms hydrogen bonds with G; this relationship between bases is called "complementary."

[0046] On the other hand, mRNA (messenger RNA) is an RNA that transmits the genetic information of a specific gene's base sequence to ribosomes during the protein synthesis process and acts as a blueprint for polypeptide synthesis (protein translation). Single-stranded mRNA is synthesized through the transcription process using a gene as a template.

[0047] As used herein, "protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues, such as those commonly found in proteins in their natural state.

[0048] The single-letter (three-letter) amino acids used herein refer to the following amino acids according to the standard abbreviation conventions in the field of biochemistry: A (Ala): alanine; C (Cys): cysteine; D (Asp): aspartic acid; E (Glu): glutamic acid; F (Phe): phenylalanine; G (Gly): glycine; H (His): histidine; I (IIe): isoleucine; K (Lys): lysine; L (Leu): leucine; M (Met): methionine; N (Asn): asparagine; O (Ply): pyrrolysine; P (Pro): proline; Q (Gln): glutamine; R (Arg): arginine; S (Ser): serine; T (Thr): threonine; U (Sec): selenocysteine; V (Val): valine; W (Trp): tryptophan; Y (Tyr): tyrosine.

[0049] The term "complementary" as used herein means that a targeting moiety in a nucleic acid molecule is sufficiently complementary to selectively hybridize to a target (e.g., a SNP of the present invention) under predetermined hybridization or annealing conditions, specifically, physiological conditions (in a cell), and may have one or more mismatched base sequences. The term encompasses both substantially complementary and perfectly complementary, and more specifically, perfectly complementary.

[0050] In the present invention, the cancer may be selected from the group consisting of colon cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, malignant melanoma, lymphoma, aplastic anemia, and blood cancer, and may be preferably, but not limited to, prostate cancer, breast cancer, liver cancer, colorectal cancer, and / or lung cancer.

[0051] In the present invention, the IGFI (Insulin-like growth factor I) gene may comprise or consist of the nucleotide sequence of NCBI Reference Sequence: NM_000618, NM_001111283.3, NM_001111284.2, or NM_001111285.3, and the IGFI protein may comprise or consist of the amino acid sequence of NCBI Reference Sequence: NP_000609.1, NP_001104753.1, NP_001104754.1, or NP_001104755.1, but is not limited thereto. In the present invention, the term "IGFI" may be used interchangeably with "IGF1."

[0052] In the present invention, the KLK2 (kallikrein related peptidase 2) gene may comprise or consist of the nucleotide sequence of NCBI Reference Sequence: NM_001002231.3, NM_001256080.2, or NM_005551.5, and the KLK2 protein may comprise or consist of the amino acid sequence of NCBI Reference Sequence: NP_001002231.1, NP_001243009.1, or NP_005542.1, but is not limited to these.

[0053] In the present invention, the PKCα (PKC alpha, PRKCA, AAG6, PRKACA) gene may comprise or consist of the nucleotide sequence of NCBI Reference Sequence: NM_002737.3, and the PKCα protein may comprise or consist of the amino acid sequence of NCBI Reference Sequence: NP_002728.2, but is not limited to these.

[0054] In the present invention, the TPRM8 (transient receptor potential cation channel subfamily M member 8) gene may comprise or consist of the nucleotide sequence of NCBI Reference Sequence: FJ895300.1, M_024080.5, or XM_024453133.1, and the TPRM8 protein may comprise or consist of the amino acid sequence of NCBI Reference Sequence: ACQ66098.1, NP_076985.4, or XP_024308901.1, but is not limited to these.

[0055] In the present invention, a "primer" refers to a short single-stranded oligonucleotide that serves as a starting point for DNA synthesis. A primer specifically binds to a template polynucleotide under appropriate buffer and temperature conditions, and DNA is synthesized by DNA polymerase adding a nucleotide triphosphate containing a base complementary to the template DNA to the primer. A primer generally consists of a sequence of 15 to 30 bases, and the temperature at which it binds to the template strand (melting temperature, Tm) varies depending on its base composition and length. The primer sequence does not need to be completely complementary to a portion of the template base sequence; it only needs to have a length and complementarity suitable for the purpose of amplifying a specific region of mRNA or cDNA by DNA synthesis and measuring the amount of mRNA. Therefore, in the present invention, primer pairs can be easily designed by referring to the base sequence of the gene or its mRNA, cDNA, or genomic DNA. The primers for the amplification reaction consist of a set (pair) of primers that complementarily bind to the template (sense) and the opposite (antisense) ends of a specific section of the mRNA to be amplified.

[0056] In the present invention, the term "probe" refers to a polynucleotide fragment, such as RNA or DNA, having a length ranging from as short as a few base pairs to as long as several hundred base pairs, which can specifically bind to mRNA, cDNA (complementary DNA), DNA, etc. of a specific gene. Because it is labeled, the presence or absence, expression level, etc., of the target mRNA or cDNA to which it binds can be confirmed. The selection of probe and hybridization conditions can be appropriately determined using techniques known in the art. The probe may be used in diagnostic methods for detecting alleles. Diagnostic methods include detection methods based on nucleic acid hybridization, such as Southern blotting, and in methods using DNA chips, the probe may be provided in a form pre-bound to the substrate of the DNA chip.

[0057] In the present invention, primers or probes can be chemically synthesized using phosphoramidite solid support synthesis or other widely known methods. Furthermore, primers or probes can be modified in various ways by methods known in the art, as long as they do not interfere with hybridization with the target polynucleotide to be detected. Examples of such modifications include methylation, gapping, substitution of one or more natural nucleotides with analogs, and internucleotide modifications, such as uncharged linkers (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) or charged linkers (e.g., phosphorothioates, phosphorodithioates, etc.), and attachment of fluorescent or enzymatic labeling materials.

[0058] In the present invention, the primer or probe is not limited to a specific sequence, as long as it can detect the mRNA of the target gene.

[0059] In the present invention, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. Aptamers for various desired target substances (proteins, sugars, dyes, DNA, metal ions, cells, etc.) can be developed using a method called SELEX (Systematic Evolution of Ligands of Exponential Enrichment). The aptamer is not limited to a specific type as long as it can detect the target protein of the present invention.

[0060] In the present invention, the term "antibody" refers to a protein molecule that specifically binds to an antigenic site. Antibodies may be produced by methods commonly used in the art, such as fusion methods, recombinant DNA methods, or phage antibody library methods. In some embodiments, antibodies or antibody fragments may be derived from different organisms, including humans, mice, rats, hamsters, rabbits, or camels, and may be, for example, monoclonal or polyclonal antibodies, immunologically active fragments, antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single-chain Fv molecules, or chimeric antibodies. The antibody fragment may be any fragment that contains the antigen-binding site of the antibody. For example, the fragment may be selected from the group consisting of scFv, (scFv)2, Fab, Fab', and F(ab')2 of the antibody, but is not limited thereto.

[0061] In the present invention, the composition may further comprise, but is not limited to, a preparation for measuring the expression level of prostate-specific antigen (PSA) protein or its mRNA.

[0062] In the present invention, a "kit" refers to a tool that enables cancer diagnosis by including a preparation for measuring the expression levels of PKCα, IGFI, KLK2, and / or TRPM8 proteins or their mRNAs. In addition to the substance for detecting the expression levels of the gene, mRNA, or protein, the kit of the present invention may also include other components, compositions, solutions, devices, etc. generally required for these detection methods. In this regard, the substance for detecting the expression levels of the gene, mRNA, or protein may be applied one or more times, with no limit on the number of times. There is no limit on the order in which each substance is applied, and each substance may be applied simultaneously or at different times.

[0063] In addition to the preparation for measuring the biomarker protein or its mRNA level of the present invention, the kit of the present invention may further comprise tools and / or reagents known in the art for use in immunological analysis.

[0064] In the present invention, the kit may include, but is not limited to, a lateral flow assay (LFA) strip. That is, the present invention provides a lateral flow assay strip capable of detecting the biomarkers (PKCα, IGFI, KLK2, and / or TRPM8) according to the present invention. The lateral flow assay may also be referred to as a lateral flow assay or a lateral flow immunoassay. A lateral flow immunoassay strip may be composed of, but is not limited to, a sample pad, a conjugation pad, a membrane, and an absorbent pad. In the lateral flow assay according to the present invention, target proteins in a sample injected into the sample pad of the LFA strip bind to gold nanoparticle-antibody conjugates immobilized on the conjugate pad and flow along the membrane (detection pad) by capillary action. At this time, the gold nanoparticles serving as the detection indicator (probe) develop color upon binding to a secondary antibody immobilized in the detection region, allowing the results to be confirmed. Lateral flow immunoassays evaluate detection factors with the naked eye by detecting the color development of gold nanoparticles that form immune complexes with target substances. For precise diagnosis and detection of target substances, improved analytical techniques are required to implement highly sensitive analysis and develop a reading system capable of quantitative analysis. In one embodiment, the present invention uses gold nanoparticles as a substance for labeling the binding between a target substance and an antibody. However, this is merely a preferred example, and any type of label known in the art that can be used to detect immune complexes with LFAs may be used without limitation.

[0065] Therefore, the LFA strip of the present invention may include a sample pad, a conjugate pad, a membrane, and an absorbent pad, and the conjugate pad may have a conjugate of gold nanoparticles and an antibody (i.e., an antibody immobilized on gold nanoparticles) immobilized thereon. The antibody may be, but is not limited to, one or more of the cancer diagnostic biomarkers of the present invention, i.e., a PKCα-specific antibody, an IGFI-specific antibody, a KLK2-specific antibody, and a TRPM8-specific antibody.

[0066] The total length of the LFA strip according to the present invention may be, but is not limited to, 20 to 100 mm, 20 to 80 mm, 20 to 60 mm, 20 to 55 mm, 30 to 100 mm, 40 to 100 mm, 30 to 80 mm, or 30 to 60 mm, and the diameter of the LFA strip may be, but is not limited to, 1 to 20 mm, 1 to 15 mm, 1 to 10 mm, or 1 to 5 mm.

[0067] Preferably, the diameter of the gold nanoparticles may be, but is not limited to, 10 to 100 nm, 10 to 80 nm, 10 to 60 nm, 10 to 50 nm, 20 to 50 nm, or 30 to 50 nm.Furthermore, the surface charge of the gold nanoparticles may be, but is not limited to, -100 to 0 mV, -80 to 0 mV, -60 to 0 mV, -80 to -10 mV, -80 to -20 mV, or -60 to -20 mV.

[0068] In another embodiment of the present invention, the kit may be, but is not limited to, an RT-PCR kit or a microarray chip kit.

[0069] The RT-PCR kit may include a pair of primers capable of amplifying a nucleic acid containing the SNP site, as well as a test tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, an RNAse inhibitor, DEPC-water, sterile water, etc. The kit may also include a pair of primers specific to a gene used as a quantitative control.

[0070] The microarray chip kit may include a microarray having a substrate on which the nucleic acid containing the SNP site is immobilized. The microarray may be a conventional microarray except that it contains the polynucleotide, primer, or probe of the present invention. Nucleic acid hybridization and detection of hybridization results on a microarray are well known in the art. For example, the detection can be performed by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal, including fluorescent substances such as Cy3 and Cy5, hybridizing the sample on the microarray, and detecting the signal generated from the labeling substance to detect the hybridization results.

[0071] The kit of the present invention (particularly, the LFA strip) is characterized by its ability to rapidly detect biomarkers and thereby diagnose cancer. For example, the kit of the present invention may have a reaction time for biomarker detection of less than 30 minutes, less than 20 minutes, less than 15 minutes, or less than 10 minutes, but is not limited thereto. The lower limit of the reaction time may be 10 seconds, 20 seconds, 30 seconds, or 1 minute, but those skilled in the art will clearly understand that the lower limit is not an essential element within the range.

[0072] The composition and cancer are as described above. The cancer diagnostic kit may further include a composition, solution, or device having one or more other components suitable for the analysis method.

[0073] In the present invention, the kit may include a container; instructions; and a preparation for measuring the expression levels of the PKCα, IGFI, KLK2, and / or TRPM8 protein or its mRNA. The container may serve to package, store, and fix the substance. The container may be made of, for example, but is not limited to, plastic or a glass bottle. The instructions may include information regarding the characteristics and manufacturing method of the composition or kit of the present invention.

[0074] The immunological analysis can be any method capable of measuring the binding of an antigen and an antibody, such as immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme-linked immunoabsorbent assay (ELISA), immunoblotting, Farr assay, immunoprecipitation, latex agglutination, hemagglutination, nephelometry, immunodiffusion, counter-current electrophoresis, single-radical immunodiffusion, protein chips, and immunofluorescence.

[0075] The tools and / or reagents used in immunological analysis include a suitable carrier or support, a label capable of generating a detectable signal, a solubilizing agent, and a detergent. Furthermore, if the labeling substance is an enzyme, a substrate capable of measuring the enzyme activity and a reaction quencher may also be included. As described above, the present invention is an embodiment in which gold nanoparticles are used as a substance for labeling the binding between a target substance and an antibody. However, this is merely a preferred example, and any type of labeling substance known in the art that can be used for the purpose of detecting immune complexes with LFA may be included without limitation.

[0076] The antigens included in the kits of the present invention may be immobilized on a suitable carrier or support using various methods, as described in the literature (Antibodies: A Laboratory Manual, Harlow & Lane; Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sepatex, Sepharose, liposomes, carboxymethylcellulose, polyacrylamide, polystyrene, gabbro, filter paper, ion exchange resins, plastic films, plastic tubes, glass, polyamine-methyl vinyl ether-maleic acid copolymers, amino acid copolymers, ethylene maleic acid copolymers, nylon, cups, flat packs, etc. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, such as a sphere (beads), a cylinder (the inner surface of a test tube or well), or a flat surface (sheets, test strips).

[0077] The inventors have confirmed the optimum conditions for manufacturing the LFA strip according to the present invention through specific examples.

[0078] For example, in producing an LFA strip according to the present invention, the optimal antibody concentration for immobilizing biomarker-specific antibodies and gold nanoparticles may be, but is not limited to, 1 to 10 μg / mL, 1 to 9 μg / mL, 1 to 8 μg / mL, 1 to 7 μg / mL, 1 to 6 μg / mL, 1 to 5 μg / mL, 1 to 4 μg / mL, 1 to 3 μg / mL, 1 to 2 μg / mL, 2 to 5 μg / mL, or 2 to 4 μg / mL based on 500 μL of gold nanoparticle dispersion.

[0079] To promote the immobilization of the antibody to the gold nanoparticles, NaCl may be added at a concentration of 5 to 30%, 5 to 20%, 5 to 15%, or 5 to 10%, and in a volume of 10 to 100 μl, 10 to 80 μl, 10 to 60 μl, 20 to 100 μl, 40 to 80 μl, or 40 to 60 μl, but is not limited thereto.

[0080] Furthermore, the antibody may be immobilized on the gold nanoparticles at a pH of 4 to 10, pH 5 to 10, pH 6 to 10, pH 7 to 10, pH 8 to 10, pH 8 to 9.5, or pH 8.5 to 9.5, but is not limited thereto.

[0081] Furthermore, the antibody may be immobilized on the gold nanoparticles for, but is not limited to, 5 to 60 minutes, 5 to 50 minutes, 5 to 40 minutes, 5 to 35 minutes, or 10 to 60 minutes.

[0082] In yet another aspect of the present invention, there is provided a method for providing information for cancer diagnosis or a method for diagnosing cancer, comprising the steps of:

[0083] (a) measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGFI, KLK2, and TRPM8, or their mRNAs, in a biological sample isolated from a subject; and

[0084] (b) determining that the subject has cancer or is at risk of developing cancer if the expression level of the measured protein or its mRNA is increased compared to a control group;

[0085] In the present invention, "expression" refers to the process by which a polypeptide is produced from a structural gene, said process including transcription of the gene into mRNA and translation of such mRNA into the polypeptide.

[0086] In the present invention, the "control group" may be normal individuals, but is not limited thereto.

[0087] In the present invention, the mRNA may be detected by a method known in the art, such as, but not limited to, a method selected from the group consisting of polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time PCR (quantitative PCR, quantitative real-time PCR), RNase protection assay (RPA), Southern blotting, Northern blotting, and DNA chips.

[0088] In the present invention, the expression level of the protein may be detected by a method known in the art, such as, but not limited to, Western blotting, lateral flow analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radial immunodiffusion, Ouchterlony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorter (FACS), and protein chip. Preferably, the protein expression level is detected using enzyme-linked immunosorbent assay (ELISA) or lateral flow analysis. More preferably, the diagnostic or information-providing method may be performed using the LFA strip of the present invention.

[0089] Enzyme-linked immunosorbent assays (ELISAs) include various ELISA methods, such as direct ELISA, which uses a labeled antibody that recognizes an antigen attached to a solid support; indirect ELISA, which uses a labeled secondary antibody that recognizes a capture antibody in a complex of an antibody that recognizes the antigen attached to a solid support; direct sandwich ELISA, which uses a separate labeled antibody that recognizes the antigen in an antibody-antigen complex attached to a solid support, or an antibody attached to a solid recognizes the antigen in an antigen-antibody complex and uses a labeled secondary antibody that recognizes this antibody; and indirect sandwich ELISA, which uses a labeled secondary antibody that recognizes this antibody after reacting it with a separate antibody that recognizes the antigen in an antibody-antigen complex attached to a solid support.

[0090] In the present invention, the term "subject" refers to a subject for diagnosing cancer, and more specifically refers to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0091] In the present invention, the term "sample" refers to a specimen to be analyzed to confirm the presence or expression level of PKCα, IGFI, KLK2, and / or TRPM8 protein or mRNA for the diagnosis of cancer in an individual. The sample may be any sample collected from a subject to be diagnosed for cancer, including, but not limited to, cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, various secretions, urine, and feces. In the present invention, the sample may be one or more selected from the group consisting of bronchial biopsy tissue, bronchial epithelium, nasal tissue, and lung tissue, but is not limited thereto. In the present invention, the sample may be pretreated before use in detection. For example, pretreatment may include homogenization, filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc.

[0092] The present invention also provides a method for treating cancer, comprising the steps of:

[0093] (a) measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGFI, KLK2, and TRPM8, or their mRNAs, in a biological sample isolated from a subject;

[0094] (b) diagnosing cancer when the expression level of the measured protein or its mRNA is increased compared to a control group; and

[0095] (c) treating cancer in the individual diagnosed with cancer in step (b).

[0096] In the present invention, the term "treatment" refers to any action that improves or beneficially alters the target disease and its associated metabolic abnormalities, and methods such as chemotherapy, radiotherapy, surgery, biological therapy, or antibiotic administration can be used.

[0097] In the present invention, chemotherapy refers to the use of chemical substances to treat a specific disease, as well as the entire body of drugs used in that process.

[0098] In the present invention, the drug may be, for example, paclitaxel, doxorubicin, 5-fluorouracil, cisplatin, imatinib, carboplatin, oxaliplatin, tegafur, irinotecan, docetaxel, cyclophosphamide, gemcitabine, ifosfamide, mitomycin C, or the like. C), vincristine, etoposide, methotrexate, topotecan, tamoxifen, vinorelbine, camptothecin, daunorubicin, chlorambucil, bryostatin-1, calicheamicin, maytansine, levamisole, DNA recombinant interferon alfa-2a, mitoxantrone, nimustine, interferon alfa-2a alfa-2a, doxifluridine, formestane, leuprolide acetate, megestrol acetateacetate, carmofur, teniposide, bleomycin, carmustine, heptaplatin, exemestane, anastrozole, estramustine, capecitabine, goserelin acetate, potassium polysaccharide, medroxyprogesterone acetate, epirubicin, letrozole, pirarubicin, topotecan, altretamine, toremifene citrate, BCNU, taxotere, actinomycin D D), and synthetic analogs thereof, and modified or identical substances that exhibit the same therapeutic effect.

[0099] In the present invention, radiation therapy refers to the irradiation of a patient with high-energy radiation, including, but not limited to, x-rays, gamma rays, and neutrons. Such types of therapy include, but are not limited to, external beam radiation therapy, internal radiation therapy, implantable radiation therapy, brachytherapy, and systemic radiation therapy.

[0100] In the present invention, the term "surgery" includes any therapeutic or diagnostic procedure involving the procedural action of a hand or a hand in combination with an instrument on an individual's body to achieve a curative, therapeutic or diagnostic effect.

[0101] In the present invention, "individual" means a subject in need of treatment or diagnosis of a disease, and more specifically means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, or cow.

[0102] In the present invention, the biological therapy refers to a treatment method that directly or indirectly utilizes the immune system of the human body using a biological preparation containing a substance derived from or produced by an organism, and the biological preparation includes vaccines, allergens, antigens, hormones, cytokines, enzymes, blood and plasma, immune serum, monoclonal antibodies, fermentation products, antitoxins, and laboratory diagnostic agents, etc., whose potency and stability cannot be evaluated by physical or chemical tests alone.

[0103] In the present invention, the biological agent may be, for example, adalimumab, alemtuzumab, bevacizumab, cetuximab, daratumumab, panitumumab, rituximab, trastuzumab, pertuzumab, ipilimumab, or the like. b), nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, tocilizumab, sarilumab, satralizumab, and siltuximab, but are not limited to these.

[0104] In the present invention, when the term "comprise" is used, it means that other components may be further included rather than excluding other components, unless otherwise specified. The terms "(a) step of" or "a step of" used throughout the present invention do not mean "a step for".

[0105] Since the present invention can be modified in various ways and can have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to the specific embodiments, but includes all modifications, equivalents, or alternatives within the spirit and technical scope of the present invention. In describing the present invention, if it is determined that a detailed description of related publicly known technology may obscure the gist of the present invention, such detailed description will be omitted.

[0106] Preferred examples are presented below to aid in understanding the present invention, but the following examples are provided merely to facilitate understanding of the present invention and are not intended to limit the scope of the present invention.

[0107] [Experimental Method] 1. Reagents and Experimental Materials Silver nitrate and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (Louis, MO, USA). Dialysis membranes (MWCO 1 kDa) were procured from Spectrum Labs Inc. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and sodium phosphate monobasic monohydrophate were purchased from Alfaisal (Ward Hill, MA, USA). Dulbecco's Modified Eagle's Medium (DMEM) and RPMI 1640 medium supplemented with 10% (v / v) heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin were purchased from GenDEPOT (Katy, TX, USA). The human Hep2B hepatoma cell line, HEK293 human embryonic kidney 293 nontumor cell line, RAW264.7 murine macrophage cell line, and SCC7 murine carcinoma cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Sodium phosphate heptahydrate was purchased from Kanto Chemical (Tokyo, Japan). Tris base was obtained from Bio-Rad (Hercules, CA, USA). Deionized water (18.2 mΩ / cm) was filtered using a Milli-Q system (Milli-Q, Billerica, MA, USA).

[0108] 2. Immunological screening of biomarkers using cancer cell line cultures Normal prostate cells (RWPE1), prostate cancer cell lines (PC3, DU145, 22RV1, LNCap), and hepatoma cell line (Hep3B) were purchased from the American Type Culture Collection (ATCC). The purchased cell lines were cultured at 37°C in DMEM or RPMI medium supplemented with 10% heat-inactivated FBS and 1% antibiotics. The culture experiments were carried out under a 95% air and 5.0% CO2 atmosphere. All cell lines were cultured in Petri dishes with the appropriate medium and kept on ice until lysed. The cells were washed twice with ice-cold PBS. The cells were lysed using RIPA buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS], together with the protease inhibitors 0.1 M PMSF and 10 M sodium fluoride. The cell lysate was centrifuged at 12,000 xg at 4°C for 10 minutes.

[0109] Protein concentration was quantified using a bicinchoninic acid (BCA) kit (Sigma). 50 g of sample was resuspended in SDS buffer and heated at 95°C for 5 minutes, followed by separation using pre-cast SDS gels (Bio-Rad). Anti-TRPM8 antibody (1:2000 dilution; Abcam #Ab109308), anti-IGFI antibody (1:2000 dilution; Abcam #Ab133542), anti-PKCα antibody (1:2000 dilution; Abcam #Ab11423), and anti-KLK2 antibody (1:400 dilution; Abcam #Ab11423) were incubated overnight on polyvinylidene fluoride membranes. After appropriate blocking, the membranes were incubated overnight with anti-TRPM8 antibody, washed with TBS buffer containing 0.1% Tween-20 (Abcam), and then reacted with the required HRP-conjugated antibody. Chemiluminescence analysis was performed using a chemiluminescent substrate (ChemiDoc Imaging System, Bio-Rad).

[0110] 3. Expression and purification of KLK2, IGFI, PKCα, and TRPM8 antigens The protein amino acid sequences of all target biomarkers were confirmed using the National Center for Biotechnology Information (NCBI) protein BLAST. Information on the gene sequences, sizes, and gene IDs of these biomarkers was reconfirmed using NCBI BLAST-n and the HUGO gene nomenclature committee. KLK2, IGFI, PKCα, and TRP8, encoded with a C-terminal His-tag, were cloned into the pET-22b(+) vector and transformed into Escherichia coli BL21(DE3) for expression. 100 μL of E. coli BL21(DE3) cells containing the recombinant plasmids were inoculated into Luria-Bertani medium containing 100 μg / mL ampicillin at 37°C and 25°C. The samples were incubated until the absorbance at 600 nm reached 0.35–0.4, and then 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce fusion protein expression. After incubation at various temperatures, cells were harvested by centrifugation at 4,254 × g for 5 minutes, and the supernatant was removed. Cells were disrupted in cell disruption buffer by sonication (30 seconds on, 30 seconds off, 10 minutes) and then centrifuged at 15,815 × g at 4°C for 20 minutes to obtain soluble and insoluble protein fractions (KLK2, IGFI, PKCα, and TRPM8). Optimal conditions for antigen expression of KLK2 (30.6 kDa), IGFI (18.6 kDa), PKCα (76.2 kDa), and TRPM8 (123.4 kDa) were confirmed using polyacrylamide gel electrophoresis (SDS-PAGE). The collected fusion proteins were purified using an AKTA Purifier equipped with a protein Ni column (GE Healthcare Bio-Science, Piscataway, NJ, USA) and stored at -80°C. Protein concentrations were measured using a Smart micro-BCA protein quantification kit (Intron Biotechnology, Seongnam, Korea).

[0111] 4. Serum Sample Preparation Forty serum samples from cancer patients, including breast cancer (n=10), colon cancer (n=10), and prostate cancer (n=10), as well as standard serum samples (n=10) were analyzed. Because the serum samples used for the PKA autoantibody ELISA were not intended to differentiate the stage or location of cancer, they were collected from patients with various active malignancies and analyzed in comparison with a control group (normal individual sample antigen). The serum samples were stored at -80°C in 200 μL volumes, but were thawed only once before use; diluted serum samples were discarded.

[0112] 5.ELISA Anti-ECPKA IgG autoantibodies were measured by solid-phase sandwich ELISA. 100 μL of purified human PKA antibody (e.g., 3G2, concentration: 1 μg / mL) was coated onto the plate and incubated overnight at 4°C. After washing three times with wash buffer [PBST, 0.05% (w / v) Tween-20], the plate was incubated for 2 hours with 1% (w / v) BSA at 37°C, washed twice with 0.05% (w / v) Tween-20 in PBS (200 μL / well), and then washed three times with wash buffer. Then, 100 μL of 1 / 8,000 diluted PKA antigen (end product) was added, and the plate was incubated for 2 hours at 37°C in dilution buffer [PBS (pH 7.4), 1% (w / v) BSA (fatty acid-free fraction V), 0.05% (w / v) Tween-20]. After washing with washing buffer, 100 μL of 1 / 20–1 / 30 diluted serum sample [dilution buffer: PBS (pH 7.4), 1% (w / v) BSA (fatty acid-free fraction V), 0.05% (w / v) Tween-20] was added and the plate was incubated at 37°C for 2 hours. After washing with washing buffer, 100 μL of 1 / 150,000 diluted anti-human IgG-HRP conjugated antibody in PBS, 1% (w / v) BSA, and 0.05% (w / v) Tween-20 was added and incubated at 37°C for 2 hours. After washing three times with washing buffer, 100 μL of TMB substrate solution was added and incubated at 37°C for 15 minutes. The reaction was then stopped by adding 100 μL of 0.5 mol / L H2SO4 solution. Absorbance was measured at 450 nm and analyzed using an ELISA reader (Synergy microplate reader, BioTek).

[0113] 6. Gold Nanoparticle Synthesis and Antibody Immobilization 6-1. Synthesis of gold nanoparticles Gold nanoparticles were synthesized by the citrate reduction method of HAuCl4. Briefly, a solution of 0.01% HAuCl4 (50 mL) and 1% citrate (0.5 mL) was prepared. The HAuCl4 solution was heated to a maximum of 95°C, and then 0.5 mL of sodium citrate was added. After approximately 25 seconds, the solution turned a faint blue color, confirming the formation of primary gold nanoparticles, and after approximately 80 seconds, it turned a bright red color, indicating the formation of dispersed spherical nanoparticles. HAuCl4(Au 3+ From Au 0 Complete reduction of the antibody (in the PBS solution) was achieved after 5–10 minutes of initial nanoparticle nucleation, which allowed nanoparticle formation. 10 μL of all antibodies (100 μg / mL) in PBS was added to 1.5 mL of gold nanoparticle solution and left at room temperature for 30 minutes. BSA solution (0.1% w / v, 100 μL) was added to block the gold nanoparticle surface and incubated for 20 minutes. The solution was then centrifuged at 9,358 × g for 20 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 10% sucrose-containing PBS (1 mM, pH 7.4).

[0114] Zeta potential analysis was performed to confirm the change in surface charge of the gold nanoparticles, and showed a pattern in which the charge decreased with increasing surface area (-51.46 mV, -36.83 mV, -29.43 mV), as shown in Figure 1I. The absorption spectra of all colloidal gold solutions, shown in Figure 1J, showed that each gold nanoparticle had surface absorption at 520 nm, 525 nm, and 529 nm, and the size distribution was large.

[0115] To improve the sensitivity of the LFA biosensor, we attempted to obtain gold nanoparticles with a diameter of approximately 20-40 nm. The size of the gold nanoparticles produced by the above process was analyzed using dynamic light scattering, confirming a diameter of 37.69 ± 3.71 nm. After antibody immobilization, the nanoparticle size was observed to be 45.18 ± 3.25 nm (Figure 1K (a) and (b)). Furthermore, after processing the gold nanoparticles with antibody bound to them on the absorbent pad of the LFA strip, electron microscopy revealed that the gold nanoparticles and antibody were still bound (Figure 1K (c) and (d)).

[0116] Furthermore, to confirm the successful binding of gold nanoparticles and antibodies, FT-IR spectroscopy was performed to examine the spectrum, and the successful binding of gold nanoparticles and antibodies was confirmed. Specifically, the peak at 3350 cm -1 A broad peak of (-OH) and ~2950 cm -1 The (-CH) peak at 1378 cm indicates the presence of hydroxyl and alkane groups on each surface (Figure 1L). -1 and 1425 cm -1 The naked eye showed that there was a stretching vibration of the -C=O group of the citrate molecule absorbed at approximately 1644 cm -1 The vibrations at approximately 2950, 1640, 1430, and 1380 cm indicated the presence of –C=C groups on the gold nanoparticle surface. After binding with the antibody, the vibrations at approximately 2950, 1640, 1430, and 1380 cm were observed. -1 The characteristic features of the protein were attributed to the vibration and stretching of -NH, -CH, and -CO groups, and the bending vibration of the CN group of the protein was observed at 11001 cm -1 Located at about 1050cm -1 The peaks indicate the presence of -CO / -COC groups, respectively (Fig. 1L).

[0117] The antibodies immobilized on gold nanoparticles in the present invention are shown in the table below.

[0118] [List of target biomarker antigens and their corresponding antibodies used in the present invention]

[0119] [Table 1-1]

[0120] 6-2. Optimization of pH conditions for immobilization of gold nanoparticles and antibodies Optimization experiments were conducted under various pH conditions (4.0–10.0) for the immobilization of gold nanoparticles and antibodies. To maximize the safety of the prepared gold nanoparticles, storage and reaction must be induced under moderate pH conditions. Gold colloid solutions were stable without visible precipitates at pH 8.0 or higher, whereas rapid accumulation and precipitation occurred at pH below 5.0. The electrolyte concentration of the gold colloid solution was also a crucial factor for stability. The optimal pH was selected based on minimal decrease in absorbance and no clear color change from red to blue at other pH values (6.0–10.0). The UV-Vis spectrum (Figure 1B) and observable color change (Figure 1C and Figure 2D, inset) confirmed that the optimal pH was 9.0.

[0121] 6-3. Optimization of reaction time for gold nanoparticle and antibody immobilization Reaction experiments were performed to determine the optimal concentration and time for the binding of gold nanoparticles and antibodies. Because it depends on the physical properties (amino acid nature, size) and chemical properties (charge, PI) of the antibody, different concentrations of antibodies take different times to bind at the optimal pH.

[0122] The antibody concentration (1-20 μg / mL) can be observed using a spectrophotometer from the spectrum at 530 nm or 400-800 nm, and the decrease in absorbance at these wavelengths was measured to determine whether or not the antibody bound. Anti-TRPM8 and anti-PKCα antibodies showed an increase in absorbance, indicating that they bound within 10 and 30 minutes, respectively (Figures 1E and 1F). The presence of 10% (w / w) sucrose in the antibody-immobilized gold nanoparticles resulted in a larger change in absorbance.

[0123] 6-4. Optimization of concentrations for gold nanoparticle and antibody immobilization Adding an appropriate amount of salt (NaCl) can induce rapid and efficient aggregation of gold nanoparticles even at basic pH conditions, allowing the selection of an appropriate antibody concentration for conjugation with the colloid. The remaining unbound colloidal gold nanoparticles reacted with salt [10% (w / w) NaCl], inducing aggregate formation along with a color change and optical absorbance. Figure 1G shows colloidal gold nanoparticles (A) and aggregated gold nanoparticles (B) after the addition of salt.

[0124] The concentrations of antibodies that completely bind to gold nanoparticles were determined by observing the color change caused by the reaction with the colloid, and optimal concentrations of 3, 3, 2, and 4 μg / mL were selected for anti-KLK2, anti-IGFI, anti-TRPM8, and anti-PKCα antibodies (Figure 1G). The optimal concentrations were selected from the spectral changes observed in the reaction at different antibody concentrations (Figure 1H).

[0125] The optimized conditions for immobilizing the biomarker-specific antibody according to the present invention onto gold nanoparticles are shown in Tables 1 to 4 below.

[0126] [Table 1]

[0127] [Table 2]

[0128] [Table 3]

[0129] [Table 4]

[0130] 7. Lateral Fluidic Strip Fabrication and Biomarker Detection A standard lateral flow analysis strip consists of a sample plate, nitrocellulose (NC) membrane, absorbent pad, and adhesive backing, with the NC membrane being the most important component. To immobilize the test and control areas of the NC membrane, KLK2 (1 mg / mL), IGFI (1 mg / mL), PKCα (1 mg / mL), TRPM8 (1 mg / mL), and anti-mouse / rabbit IgG (HRP) antibodies were inserted at a volume ratio of 1:10. The NC membrane was then loaded with the test and control areas attached to the adhesive backing, and the sample pad and absorbent pad were then placed on top of it in succession. As shown in Figure 1(a), the sample and absorbent pads covered the front and edge surfaces of the NC membrane by approximately 2 mm, allowing for smooth flow through the entire strip via optimal capillary chromatography. Finally, the assembled pre-assembled strip was cut into 4 mm widths and stored in the dark. In this study, different concentrations of anti-KLK2, anti-TRPM8, anti-IGFI, and anti-PKCα antibodies were conjugated to gold nanoparticles and incubated at 37°C for 3 hours. Then, 10 μL of a sample from a cancer patient or a normal individual was applied to the sample pad. 10 μL of PBST (PBS buffer containing 0.05% (w / v) Tween-20) was passed through the entire strip to wash away nonspecific binding from the test line, and the specific binding of the antigen to the antibody was confirmed after 15 minutes. As shown in Figure 1A(b), the signal gradually appeared, and the finally filled strip was placed in the identification channel of a strip reader for quantitative analysis. In addition, one or both uniform line bands may appear on the sample-treated strip. The colorimetric intensity of the test and control lines was recorded after analysis using a smartphone and a ChemiDoc® MP Imaging System (Bio-Rad Laboratories).

[0131] 8. Other analytical methods The size of the prepared gold nanoparticles and gold nanoparticle-anti-KLK2 antibody, gold nanoparticle-anti-IGFI antibody, gold nanoparticle-anti-PKCα antibody, and gold nanoparticle-anti-TRPM8 antibody complexes was confirmed by dynamic light scattering (DLS). DLS and zeta potential were measured using an Otsuka ELSZ-1000 (Otsuka Electronics, Osaka, Japan). UV-Vis spectra were measured using a spectrophotometer (V-670, Jusco, Tokyo, Japan). The surface chemistry of the gold nanoparticles and the successful conjugation of the gold nanoparticles with anti-KLK2, anti-IGFI, anti-PKα, and anti-TRPM8 antibodies were analyzed using UV-Vis spectrophotometer and Fourier transform infrared (FT-IR) spectroscopy. Samples were attached to aluminum holders with carbon tape and then covered with a gold (Au) film at 4 mA current for 80 seconds for scanning electron microscopy (SEM).

[0132] [Example] Example 1. Prostate cancer biomarker discovery Six cell lines were selected to screen for prostate cancer biomarkers. Four of these cell lines, including DU145, 22RV1, PC3, and LNCaP cells, were used as standard prostate cancer cells. The other two, normal prostate cells (RWPE1) and hepatoma cells (Hep3B), were selected as positive and negative controls, respectively. Prostate-specific antigen (PSA) has traditionally been the most widely used biomarker for detecting prostate cancer. Unlike PSA, hK2 is known to be more strongly expressed in benign epithelial cells, especially malignant tumor cells. Clinical trials have shown that serum hK2 can help distinguish between malignant and benign causes of elevated PSA, and other prostate cancer protein biomarkers have been investigated. Additional research is needed before these or other potential new prostate cancer biomarkers can be used to replace PSA testing. Any potential biomarkers should demonstrate complementary results that can diagnose clinically meaningful cancers, rather than surpassing PSA.

[0133] Although various cancer biomarkers are known, it is important to select strong biomarkers such as PSP, TMEFF2, KLK4, KLK2, and TRPM8, along with PSA, to develop diagnostic methods. Cell extracts were collected by cell lysis, and the cell growth cycle of all cell lines was confirmed in this study to understand the growth stage of cancer cell lines (Figure 2).

[0134] The expression of all cancer biomarkers was verified using SDS-PAGE and immunological Western blotting analysis, and blot observation confirmed that proteins of their native size were detected. Although proteins showed higher levels of expression in cell culture medium precipitate samples, it was confirmed that expression of individual proteins varied in some cell lines. In other words, repeated experiments using cell culture medium and culture medium precipitates showed no reproducibility, so cell extracts were used for accurate biomarker expression analysis.

[0135] As shown in Figure 3, the four biomarkers KLK2, TMEFF2, TRPM8, and KLK4 were detected in all four cell line growth stages (early log phase, late log phase, arrest phase, and death phase), and it was observed that the concentrations of the biomarkers varied depending on the cell growth cycle stage. Biomarker expression was analyzed separately for different cell lines at each stage, and all biomarkers were well expressed, as indicated by the band sizes per sample.

[0136] Example 2. Prostate cancer biomarker screening The expression levels of all biomarkers were monitored by collecting the same number of cells for all cell extract experiments. Biomarkers with consistently high expression levels, such as PSA, KLK2, TRPM8, and TMEFF2, were selected as positive controls. It has been reported that PSA can produce false-positive results in non-prostate cancer patients and false-negative results in metastatic prostate cancer patients. KLK2 and TRPM8 were particularly suitable biomarkers because they were highly expressed at all stages, even in the early log phase, and were highly consistent across all cancer cell lines. IGFI and PKCα were also selected as additional biomarkers.

[0137] Insulin-like growth factor-I (IGFI) is a useful circulating marker for diagnosing and tracking growth hormone (GH) deficiency and excess in clinical practice, and has been proposed as a more broadly applicable marker for predicting cancer vulnerability and non-cancer growth characteristics (Brabant, Eur. J. Endocrinol., 148:S15-S20, 2003; Glynn and Agha, Inter. J. Endocrinol., 2012:972-627, 2012). Numerous preclinical and experimental studies have associated higher circulating levels of IGFI in adults with an increased risk of many other cancers and low-grade diseases, including breast, prostate, ovarian, endometrial, colon, and lung cancer, although a lower risk of ovarian cancer has been reported (Yu et al., J. Natl. Cancer Inst., 91:151-156, 1999; Li et al., Cell. Phys. Biochem., 38:589-597, 2016).

[0138] IGF-1 can also promote normal epithelial breast cell proliferation. Animal studies have implicated the IGF cascade in breast tumorigenesis and, consequently, it has been extensively investigated in relation to breast cancer pathogenesis (LeRoith and Roberts, Cancer Lett., 195:127-137, 2003).

[0139] Recently, Chan et al. published a study that found a close correlation between plasma insulin-like growth factor 1 (IGFI) levels and prostate cancer risk (Chan et al., Science, 279:563-566, 1998). Various studies have demonstrated a signaling role for the IGFI receptor (IGFI) in neoplastic transformation (Pollak et al., Nat. Rev. Cancer, 4:505-518, 2004; Vella et al., Biochim. Biophys. Acta, Mol. Cell Res., 1866:118-522).

[0140] Furthermore, one of the changes that occurs as prostate adenocarcinoma progresses is the release of IGFI by epithelial cells (Wang et al., Oncogene, 38:6338-6353, 20190). Furthermore, serum IGFI levels have been reported to be associated with prostate cancer (Sreenivasulu et al., 21:138-144, 2018; Janiczek et al., J. Immunol. Res., 2020:4910595, 2020). Studies using transgenic mice expressing IGFI directly in primary prostate epithelial cells have provided molecular evidence for a link between IGFI expression and prostate carcinogenesis (Ahearn et al., Carcinogenesis, 39:1431-1437, 2018).

[0141] The results of these extensive studies to date have been somewhat consistent, and further research has shown a steadily increasing correlation between IGFI levels and the risk of prostate cancer. These reports support the theory that elevated serum IGFI levels in young men can lead to prostate cancer several years later. In other words, prolonged exposure of prostate epithelial cells to large amounts of IGFI increases the risk of hyperplasia in the cytoplasm of prostate intraepithelial neoplasia, ultimately increasing the risk of developing prostate adenocarcinoma.

[0142] The formation, persistence, and death of cells are mediated by various signaling chains, with phosphorylation mechanisms playing a key role. Normal cells often mutate into cancer cells through altered signaling pathways that result in the overactivation of kinases and their successors, ultimately resulting in uncontrolled proliferation or enhanced survival advantages. One essential group of kinases that regulates these activities is known as protein kinase C (PKC), which has been extensively studied as an effector of seven transmembrane G protein-coupled receptors and tyrosine kinases. PKC contains at least 10 distinct isoenzymes, grouped into three modules based on their biochemical and structural properties. The lipid secondary transporter diacylglycerol (DAG) produced in the membrane by the C1 domain binds to PKC, triggering the carboxyl ester molecule adjacent to the DAG-binding site (C1 domain) (Kolczynska et al., Lipids Health Dis., 19:1-15, 2020; Griner and Kazanietz, Nat. Rev. Cancer, 7:281-294, 2007; Hanauske et al., Curr. Pharm. Des., 10:1923-1936, 2004). Cells express numerous PKC isoenzymes, which may have distinct overlapping or distinct biological functions.

[0143] Over the past 20 years, extensive research on PKC has focused on PCa cells (LNCaP), which have emerged as one of the most widely studied models for PKC-α-stimulated apoptosis. PKC-α is expressed in most prostate cancer cell lines, and PKC levels are lower in hormone-responsive cell lines (LNCaP) than in hormone-unresponsive cell lines (PC-3, DU145, and PC-3M) (Griner and Kazanietz, Nat. Rev. Cancer, 7:281-294, 2007). PC3 prostate cell lines showed elevated PKC-α levels, which correlated with increased bcl2 expression and apoptosis (Hanauske et al., Curr. Pharm. Des., 10:1923-1936, 2004). Numerous studies have determined that inhibitor treatments activate cell suicide in LNCaP cells (Garzotto et al., Cancer Res., 59:5194-5201, 1999; Deveraux et al., EMBO J., 17:2215-2223, 1998). A key focus in this context has been the relative influence of the intrinsic and extrinsic cell suicide pathways on effectors and how PKC homology alters this amplification response. The intrinsic pathway is based on mitochondrial depolarization, while the extrinsic pathway involves the initiation of death receptors. Members of the Bcl-2 receptor family are key players in the intrinsic pathway through the regulation of cytochrome c and the subsequent initiation of caspase-9. The extrinsic pathway is triggered by the interaction of death ligands with plasma membrane receptors, leading to the recruitment of caspase-8 and adaptor molecules and subsequent stimulation of downstream caspases (Zimmermann and Green, J. Allergy Clin. Immunol., 108:S99-S103, 2001; Aggarwal, Nat. Rev. Immunol., 3:745-756, 2003). Modulation of the pathway by PKC isoenzymes is not fully understood.In androgen-sensitive prostate cancer cells, PKC is thought to disrupt the cell suicide cascade at various stages through isoenzyme-specific access (Isakov, N. Protein kinase C (PKC) isoforms in cancer, tumor promotion, and tumor suppression. In Seminars in Cancer Biology. 2018). Furthermore, immunoblotting studies of six human colon cancer cell lines at various stages of tumor growth also confirmed that PKC-α can be directly detected using specific antibodies in various carcinomas (Masur et al., Mol. Biol. Cell., 12:1973-1982, 2001).

[0144] The perfect tumor biomarker would allow a simple blood test to identify cancer. However, precision and sensitivity are reduced, and no single biomarker is identified as a true cancer marker. Currently available serum biomarkers focus on measuring cancer antigens (Garrett and Sell, Cellular cancer markers. Vol. 12, 2013: Springer Science & Business Media). For example, prostate-specific antigen (PSA) is used as a biomarker for prostate cancer (Gretzer and Partin, Urol. Clin. North Am., 30:677-686, 2003), carcinoembryonic antigen (CEA) is used to diagnose colorectal cancer, and the cancer antigen CA125 (Anderiesz and Quinn, Med. J. Aust., 178:655-656, 2003), the cancer antigen CA19-9 for gastrointestinal cancer, and the cancer antigen CA152 for breast cancer (Duffy et al., Tumor Biol., 40:1010428318776169, 2018). Other proteins, enzymes, and hormones have been used as indicators over the past 30 years, adding to these newer markers (Garrett and Sell, Cellular cancer markers. Vol. 12, 2013: Springer Science & Business Media). However, these markers are often lacking in precision and sensitivity, and their values increase in both positive and negative environments, which needs to be improved. The fact that the various biomarkers mentioned above are only detectable in limited carcinomas is why new biomarkers are needed, and the development of technology that can identify carcinomas using various biomarkers is also necessary.

[0145] For the reasons mentioned above, indirect ELISA analysis was performed using various actual cancer serum samples using specific IgG antibodies to estimate their specific binding ability to the cancer biomarkers (KLK2, IGFI, and PKCα) according to the present invention.

[0146] As shown in Figure 4, KLK2, IGFI, and PKCα all showed higher absorbance, i.e., higher expression levels, in patient-derived samples compared to normal samples, confirming that these biomarkers all have excellent diagnostic and prognostic capabilities for breast cancer and prostate cancer.

[0147] Example 3. Optimization of expression conditions for KLK2, IGFI, PKCα, and TRPM8 The target biomarker (TRPM8, KLK2, IGFI, PKCα) sequences were selected from Abcam's prostate biomarker sequences, and these sequences were also obtained and confirmed by blast searching the Protein Database Bank (PDB) and NCBI databases using search engines (NCBI, HGNC).

[0148] We attempted to obtain recombinant proteins in large quantities by transforming human gene fragments into E. coli in the form of plasmids and expressing them. The target vector was transformed into Escherichia coli Top10 and DH5α cells. The cloned recombinant plasmids were then purified and analyzed for accuracy of the cloned gene sequences.

[0149] Successful PCR amplification of the human gene fragment was confirmed and inserted into the pET-22b(+) vector for expression. E. coli harboring the pET-22b(+) plasmid was selected based on its reduced incubation time, lower cost, and ease of culturing for protein expression compared to yeast and mammalian vectors. To isolate recombinant proteins from E. coli, expression plasmids containing pET-KLK2, pET-IGFIA, pET-PKCα, and pET-TRPM8 under the control of a strong T7 promoter (Figure 5) were transformed into E. coli BL21(DE3). These recombinant plasmids were digested with appropriate restriction enzymes, and proper cloning was verified by gel electrophoresis (Figure 6). Recombinant protein expression was confirmed by Coomassie staining and immuno-Western blotting using an anti-6x His antibody (Figure 7).

[0150] Recombinant protein expression was achieved by treating the cells with IPTG as an inducer and then inducing the culture at 37°C for 6 hours. High expression was observed in the insoluble fraction, while weak expression was observed in the soluble fraction. Various concentrations of IPTG (0 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM) were then used to confirm the level of recombinant protein expression. This was done at 37°C for 6 hours, 25°C for 15 hours, and 12°C for 20 hours. The cells were then harvested, and the soluble and insoluble fractions were separated by sonication and centrifugation. After purification, the fractions were stored in small aliquots at -80°C.

[0151] Figure 5 shows a schematic diagram of the insertion of a recombinant gene into a selected vector using restriction enzyme sites (BamHI, SalI, SacI, NocI). The recombinant gene was inserted in frame with the pelB signal sequence, allowing for easy transcription using the T7 promoter and interrupted by the T7 terminator sequence. The addition of six histidines to the target gene facilitated protein purification.

[0152] Western blotting analysis of the recombinant proteins demonstrated that all proteins were highly expressed in the insoluble fraction (Figure 7). Therefore, the insoluble fractions were optimized for expression at different concentrations (0, 0.25, 0.5, 0.75, and 1 mM; Figure 8) and temperatures (12°C, 25°C, and 37°C; Figure 9).

[0153] Specifically, it was confirmed that expression of IGF1 was optimized at 37°C, KLK2 at 12°C, 25°C, and 37°C, PKCα at 25°C and 37°C, and TRPM8 at 25°C and 37°C.

[0154] Example 4. Detection by ELISA The antigen recognition efficiency of purified IGFI, KLK2, and PKCα antigens was observed using indirect ELISA (Figures 10A to 10C).

[0155] In addition, spiked samples of IGFI, KLK2, and PKCα antigens were analyzed using sandwich ELISA with anti-IGFI, anti-KLK2, and anti-PKCα antibodies for specific and accurate detection (Figures 10D-10F).

[0156] KLK2, IGFI, and PKCα recombinant antigens were measured at different concentrations (0.032 μg / mL, 0.16 μg / mL, 0.8 μg / mL, 4 μg / mL, 20 μg / mL, and 100 μg / mL) using their specific antibodies by solid-phase indirect ELISA and sandwich ELISA.

[0157] It was confirmed that by using the specific antibodies for all of the biomarkers through each ELISA analysis, the biomarkers could be used as detection targets for the purpose of diagnosing cancer from the serum of cancer patients.

[0158] Example 5. Preparation of LFA strips for lateral fluid analysis of KLK2, IGFI, PKCα, and TRPM8 To confirm the efficacy of cancer target antigen (IGFI, KLK2, PKCα, TRPM8) biomarkers, we attempted to perform lateral flow analysis using actual cancer patient serum samples. To do this, gold nanoparticles were conjugated to antibodies via electrostatic interactions, formulated, and stored at 4°C for subsequent experiments. First, all components of the LFA strip were prepared, and the control line (CL) and test line (TL) of the LFA strip were created using a dispersion system in which the reaction solution spreads via capillary action.

[0159] Conjugation pads were prepared by immersing glass fibers in the gold nanoparticle-antibody conjugate solution. The glass fibers were dried in an incubator at 37°C for 2 hours and then stored in a zipper-lock bag with dried pearls at 4°C. Sample pads were prepared by immersing cotton membranes in PBS buffer containing 0.05% (w / v) Tween-20 and 5% (w / v) BSA. The pads were dried in an oven at 37°C for 3–4 hours and then stored in a zipper bag with dried pearls at 4°C. Detection pads were fabricated by assembling a nitrocellulose membrane (NC membrane) onto a supporting adhesive card. Anti-KLK2, anti-IGFI, anti-PKCα, and anti-TRPM8 antibodies (1.0 mg / mL in PBS) were sprayed to form a test line (TL), and goat anti-rabbit IgG antibody (2.0 mg / mL in PBS) was sprayed 4 mm away from the TL to form a control line (CL). The detection pads were dried at room temperature for 8 hours and then stored at room temperature. All strip components were fabricated with a 2 mm overlap. The dimensions of the entire strip and all sections are listed in Table 5.

[0160] [Table 5]

[0161] Example 6. Antigen detection method using standard samples of KLK2, IGFI, and PKCα To measure the limit of detection (LOD), cancer target antigens (IGFI, KLK2, PKCα) were dissolved in PBS (pH 7.4) at various concentrations (1-8000 ng / mL) to prepare samples. A total volume of 100 μL of the sample solution was dropped onto the prepared LFA strip.

[0162] The results of IGFI detection are shown in Figures 11A to 11D, the results of KLK2 detection are shown in Figures 12A to 12D, and the results of PKCα analysis are shown in Figures 13A to 13D. As can be seen from these figures, IGFI, KLK2, and PKCα recombinant antigens were clearly detected in the T-line at various concentrations.

[0163] Specifically, a decrease in color intensity was observed in the test lines (TL) as the concentration of the target antigen samples was decreased from 10,000 ng / mL to 1 ng / mL, and the intensity of the red color increased as expected with increasing antigen concentration. All control lines (CL) appeared red, regardless of the presence or absence of cancer biomarkers. As the concentration of each antigen increased from 1 or 10 ng / mL to 10,000 ng / mL, all red test lines became thicker and darker. The LODs for the IGFI, KLK2, and PKCα antigens were measured by TL color intensity to be approximately 10 ng / mL, 1 ng / mL, and 50 ng / mL, respectively. The visual LODs of the TL for all target antigens were approximately 10 ng / mL.

[0164] Quantitative analysis of IGFI, KLK2, and PKCα was performed by monitoring the T / C band intensity in the test and control groups. The band intensity gradually increased with increasing concentrations of IGFI, KLK2, and PKCα. Based on these LFA results, calibration curves for IGFI, KLK2, and PKCα were determined.

[0165] As can be seen from the graphs in Figures 11A to 11D, IGFI showed good linearity between the TL band intensities (R2 = 0.911, 0.928), with low LOD values of 1.2 ng / mL and 0.9 ng / mL in the buffer and serum spiked samples, respectively.

[0166] As can be seen from the graphs in Figures 12A to 12D, KLK2 showed good linearity between the TL band intensities (R2 = 0.969, 0.977), and low LOD values of 2.1 ng / mL and 1.9 ng / mL were observed in the buffer and serum detection samples, respectively.

[0167] As can be seen from the graphs in Figures 13A to 13D, PKCα showed good linearity between the TL band intensities (R2 = 0.958, 0.933), and low LOD values of 1.5 ng / mL and 2.6 ng / mL were observed in the buffer and serum detection samples, respectively.

[0168] Next, 10 test strips were randomly selected and the reaction time for LFA activity and line color intensity was evaluated. The results of all reaction time measurements showed that the reaction time was approximately 30 seconds in PBS (pH 7.4), and the time to reach maximum color intensity was 9.26 ± 1.58 minutes. Therefore, the reaction time for this test was determined to be less than 10 minutes.

[0169] Example 7. Analysis of clinical samples for detection of IGFI, KLK2, and PKCα To evaluate the clinical efficacy of the LFA strip of the present invention, lateral flow analysis was performed using actual serum samples from cancer patients. Specifically, 20 clinical specimens (15 positive, 5 negative) were subjected to LFA based on IGFI, KLK2, and PKCα antibodies, and the results were analyzed qualitatively and quantitatively. The titer for the clinical specimens was measured using 20-fold diluted specimens. The clinical specimens included six samples each from normal subjects, liver cancer, lung cancer, colon cancer, breast cancer, and prostate cancer.

[0170] Sensitivity, specificity, and accuracy were calculated using the following equations:

[0171]

number

[0172] As a result, it was confirmed that the biomarkers IGF1, KLK2, and PKCα were clearly detected in LFA strips treated with patient samples of liver cancer, lung cancer, rectal cancer, breast cancer, or prostate cancer (FIGS. 14 to 16).

[0173] Specifically, as shown in Figure 14, IGFI was confirmed to be present to some extent in normal subjects, but the levels were lower than those in cancer patient samples. In particular, high color intensity of TL was observed in samples from lung cancer, breast cancer, and prostate cancer patients. These results indicate that IGFI can be used as a general-purpose biomarker. The sensitivity, selectivity, and accuracy of the IGFI biomarker were approximately 96%, 92%, and 87%, respectively.

[0174] As shown in Figure 15, the KLK2 biomarker was also analyzed using LFA strips in various cancer and normal specimens. KLK2 band intensity was generally higher in various cancer specimens than in normal specimens. In particular, TL color intensity was significantly higher in breast cancer and prostate cancer specimens than in other cancer specimens, confirming its high specificity for breast cancer diagnosis. The sensitivity, selectivity, and accuracy of the KLK2 biomarker were approximately 100%, 100%, and 90%, respectively.

[0175] As shown in Figure 16, the PKCα biomarker was also analyzed using the LFA strip in all five cancer samples and normal samples. Similar to the biomarkers mentioned above, PKCα also exhibited bands with generally higher intensity in cancer samples compared to normal samples. Furthermore, higher color intensities on the test line were observed in rectal cancer, breast cancer, and prostate cancer samples compared to other cancer samples, confirming its high relevance to breast cancer and rectal cancer. The sensitivity, selectivity, and accuracy of the PKCα biomarker were approximately 100%, 100%, and 80%, respectively.

[0176] Example 8. Lateral fluid analysis using LFA strips for multiple biomarker detection The above examples confirmed the usefulness of each of the cancer diagnostic biomarkers of the present invention as a diagnostic marker. Therefore, to further enhance the usefulness of these biomarkers as diagnostic markers, we investigated whether simultaneous detection of these biomarkers would enable more accurate cancer diagnosis. Therefore, we prepared an LFA strip on which PKCα, IGFI, and KLK2-specific antibodies were immobilized, enabling simultaneous detection of multiple biomarkers, and performed lateral flow analysis using this strip. Similarly, the antibodies were labeled with gold nanoparticles, and the sensitivity of the gold nanoparticle-based multiplexed detection LFA was evaluated by detecting and assessing serum containing IGFI, KLK2, and PKCα at a concentration of 500 ng / mL in PBS buffer.

[0177] Figures 17 to 23 show the results of biomarker detection in each sample using the gold nanoparticle-based multidetection LFA. Figures 17 and 18 show the analysis results of spiked samples and normal samples, respectively, while Figures 19 to 23 show the analysis results of cancer patient samples (liver cancer, lung cancer, colon cancer, breast cancer, and prostate cancer, respectively). Each figure shows photographs and quantitative color intensity graphs of TLs (TL1, PKCα; TL2, IGFI; TL3, KLK2) indicating the presence of three biomarkers (PKCα, IGFI, and KLK2). As can be seen from the figures, the normal serum samples exhibited lower color intensities than the TLs of the breast cancer, lung cancer, liver cancer, and colon cancer serum samples. The dark red color of gold nanoparticles was observed in the TLs of the multidetection LFA strips processed with each cancer patient sample, including the breast cancer patient sample, indicating the presence of high concentrations of the IGFI, KLK2, and PKCα biomarkers in each cancer patient sample.

[0178] Collectively, these results also suggest that the gold nanoparticle-based multidetection LFA allows simultaneous detection of multiple biomarkers according to the present invention, thereby enabling more accurate diagnosis of cancer.

[0179] Tables 6 and 7 show the results of tests using clinical specimens. The results show the cancer diagnosis results obtained by testing each biomarker from clinical specimens of cancer patients using enzyme-linked immunosorbent assay (ELISA) and lateral flow assay (LFA), with the positive rate indicated as + and the negative rate indicated as -. As can be seen from Table 6, when each biomarker is detected individually by ELISA, the negative rate is high and the diagnostic accuracy is relatively low. However, in Table 7, when biomarkers are simultaneously detected using the LFA strip according to the present invention, the positive rate for cancer diagnosis significantly increased, enabling more accurate cancer diagnosis.

[0180] [Table 6]

[0181] [Table 7]

[0182] These results indicate that the biomarkers used have high activity in samples from cancer patients and can be used to diagnose cancers such as breast cancer, prostate cancer, liver cancer, rectal cancer, lung cancer, colon cancer, and breast cancer, which are correlated with each other.

[0183] The above-described description of the present invention is for illustrative purposes only, and those skilled in the art can easily modify the present invention into other specific forms without changing the technical concept or essential features of the present invention. Therefore, the above-described embodiments are illustrative in all respects and are not limiting. [Industrial Applicability]

[0184] The present invention relates to a multiple biomarker for cancer diagnosis and its uses, and more specifically to a cancer diagnostic composition comprising a preparation for measuring the expression levels of IGFI, KLK2, PKCα, and TRPM8 proteins or their mRNAs. The present inventors have confirmed that by detecting the specific marker combination of the present invention, IGFI, KLK2, PKCα, and TRPM8, using serum samples from actual cancer patients, cancer diagnosis can be performed quickly and accurately, and one or more cancers can be simultaneously detected in multiplex. Therefore, it is expected that the combination of IGFI, KLK2, PKCα, and TRPM8 can be used to quickly and accurately diagnose various cancers, including prostate cancer, and therefore the industrial applicability of the present invention is recognized.

Claims

1. The present invention also includes a preparation for measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGF1, KLK2, and TRPM8, or their mRNA. A composition for cancer diagnosis, comprising:

2. The diagnostic composition is capable of simultaneously detecting one or more cancers. The composition of claim 1.

3. The cancer is at least one selected from the group consisting of colon cancer, colorectal cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, malignant melanoma, lymphoma, aplastic anemia, and blood cancer. The composition of claim 1.

4. The composition is for analyzing one or more samples selected from the group consisting of blood, plasma, serum, saliva, nasal fluid, sputum, joint capsule fluid, amniotic fluid, peritoneal fluid, cervical or vaginal secretions, urine, and cerebrospinal fluid. The composition of claim 1.

5. The expression level of the protein is measured with an antibody or aptamer specific to the protein. The composition of claim 1.

6. The expression level of the mRNA is measured using a probe or primer that specifically binds to the mRNA. The composition of claim 1.

7. The composition further comprises a formulation for measuring the expression level of prostate-specific antigen (PSA) protein or its mRNA. The composition of claim 1.

8. The composition of claim 1 A cancer diagnostic kit comprising:

9. The kit includes a lateral flow assay strip. The kit of claim 8.

10. The lateral flow assay strip includes a sample pad, a conjugation pad, a membrane, and an absorbent pad. The kit of claim 9.

11. The lateral flow assay strip comprises gold nanoparticles onto which one or more antibodies selected from the group consisting of an anti-PKCα antibody, an anti-IGF1 antibody, an anti-KLK2 antibody, and an anti-TRPM8 antibody are immobilized. The kit of claim 9.

12. (a) measuring the expression level of one or more proteins selected from the group consisting of PKCα, IGF1, KLK2, and TRPM8 or their mRNA in a biological sample isolated from a subject; and (b) determining that the subject has cancer or is at risk of developing cancer if the expression level of the measured protein or its mRNA is increased compared to a control group; 1. A method for providing information for cancer diagnosis, comprising:

13. The expression level of the protein is measured using enzyme-linked immunosorbent assay (ELISA) or lateral flow assay. The information providing method according to claim 12.

Images