Preparation of deoxycytidine kinase phosphorylation antibody and its application in efficacy prediction of gemcitabine

Abstract

The present application relates to the biomedical field, specifically is the reagent for detecting the phosphorylation level of deoxycytidine kinase (DCK) S74 site in tumor tissue in the preparation of gemcitabine efficacy evaluation reagent and companion diagnostic kit, and the preparation of DCK S74 site phosphorylation polyclonal and monoclonal antibody. The application of specific antibody in tumor cells and tissues can effectively evaluate the catalytic activity of DCK and the efficacy of gemcitabine. The present application prepares the antibody for specifically recognizing the phosphorylation modification of DCK S74 site, which has good immunoblotting and immunohistochemical detection efficiency. The present application provides polyclonal and monoclonal antibodies for detecting the phosphorylation of DCK S74 site, which can be used for predicting the efficacy of gemcitabine, and is easy to use in basic research and clinical promotion.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the design and preparation of polyclonal and monoclonal antibodies against deoxycytidine kinase (DCK) phosphorylation at the S74 site, and the application of these antibodies to evaluate the antitumor efficacy of gemcitabine by immunoblotting or immunohistochemical detection. Background Technology

[0002] Gemcitabine is a deoxycytidine-like chemotherapy drug widely used as first-line or off-label treatment for various solid tumors, including pancreatic ductal adenocarcinoma, biliary tract tumors, breast cancer, ovarian cancer, and non-small cell lung cancer (Binenbaum et al., 2015). Despite its significant clinical success, over 70% of patients with advanced cholangiocarcinoma and over 90% of patients with pancreatic cancer do not respond well to gemcitabine. Predicting the efficacy of gemcitabine-based first-line chemotherapy regimens and promptly initiating second-line or targeted therapy remains a challenge in the treatment of advanced cholangiocarcinoma and pancreatic cancer. Developing biomarkers that can predict gemcitabine efficacy could prevent delays in treatment due to ineffective first-line regimens and improve the treatment outcomes for advanced tumors.

[0003] Gemcitabine exerts its antitumor effect through three steps: The first step is uptake. Gemcitabine first needs to enter the cell via human nucleoside transporters (hNTs) on the cell membrane surface. Studies have shown that gemcitabine is mainly taken up into the cell via hENT1, and secondly via hENT2, hCNT1, and hCNT3 (Ritzel, MW, Ng, AM, Yao, SY, Graham, K., Loewen, SK, Smith, KM, Hyde, RJ, Karpinski, E., Cass, CE, Baldwin, SA, et al. (2001). Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). Mol MembrBiol 18, 65-72.). The second step is activation. After gemcitabine enters the cell, it undergoes a series of phosphorylations to eventually form the activated metabolite, nucleoside triphosphate (dFdCTP). The first step, phosphorylation by deoxycytidine kinase (DCK), is the key rate-limiting step in the entire metabolic process (Ohhashi, S., Ohuchida, K., Mizumoto, K., Fujita, H., Egami, T., Yu, J., Toma, H., Sadatomi, S., Nagai, E., and Tanaka, M. (2008). Down-regulation of deoxycytidine kinase enhances acquired resistance to gemcitabine in pancreatic cancer. Anticancer Res 28, 2205-2212.).The third step involves DNA incorporation. As a deoxycytidine analog, gemcitabine competitively binds to DNA with intracellular deoxycytidine (dC), inhibiting DNA synthesis and causing double-strand breaks, thereby leading to cell death (de Sousa Cavalcante, L., and Monteiro, G. (2014). Gemcitabine: metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur J Pharmacol 741, 8-16.). Since DCK plays a central activating role in gemcitabine drug metabolism, detecting DCK activity can predict the clinical efficacy of gemcitabine.

[0004] DCK is the rate-limiting enzyme of the deoxynucleoside rescue pathway in mammalian cells and plays a key role in the activation of many nucleoside-like antitumor and antiviral drugs, such as gemcitabine, fludarabine, cladribine, zalcitabine, and lamivudine (De Clercq, E. (2004). Antiviral drugs in current clinical use. J Clin Virol 30, 115-133. Galmarini, CM, Mackey, JR, and Dumontet, C. (2002). Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 3, 415-424. Pettitt, AR (2003). Mechanism of action of purine analogues in chronic lymphocyticleukaemia. Br J Haematol 121, 692-702.). However, DCK rarely mutates, is deleted, or is dysregulated in tumors, so simply detecting DCK protein expression levels to predict the efficacy of gemcitabine may not be satisfactory.It is noteworthy that the catalytic activity of DCK is regulated by its own phosphorylation modification, including sites such as T3, S11, S15, and S74; among them, S74 is the main phosphorylation site. Phosphorylation at this site significantly enhances the affinity of DCK for various substrates, including gemcitabine, thereby promoting the catalytic activity of DCK and the efficacy of gemcitabine. In addition, the DCK S74 site can undergo phosphorylation modification in response to stimulation by various drugs, including gemcitabine (Smal, C., Vertommen, D., Bertrand, L., Ntamashimikiro, S., Rider, MH, Van Den Neste, E., and Bontemps, F. (2006). Identification of in vivo phosphorylation sites on human deoxycytidine kinase. Role of Ser-74 in the control of enzyme activity. J Biol Chem. 281,4887-4893. Zhong, R., Xin, R., Chen, Z., Liang, N., Liu, Y., Ma, S., and Liu, X. (2016). The Role of Deoxycytidine Kinase (dCK) in Radiation-Induced Cell Death. Int J Mol Sci 17.); Therefore, developing specific antibodies to detect the phosphorylation level of DCK S74 site using immunoblotting or immunohistochemistry can conveniently and effectively assess the catalytic activity of DCK, thereby predicting the efficacy of gemcitabine. Currently, there are no commercially available antibodies that identify DCK S74 site phosphorylation, and there is also a lack of reagents or kits for determining gemcitabine efficacy by detecting DCK S74 site phosphorylation. In addition, the correlation between DCK S74 site phosphorylation and the efficacy of gemcitabine chemotherapy in oncology clinical cohorts has not been experimentally verified. Summary of the Invention

[0005] The purpose of this invention is to provide novel antibodies that specifically recognize DCK S74 phosphorylation modification. This series of antibodies exhibits good immunoblotting and immunohistochemical detection efficiency. This invention also provides a novel method for predicting the antitumor efficacy of gemcitabine. Specifically, by applying this specific antibody to detect the phosphorylation level of DCK S74 in tumor tissue via immunohistochemistry, the efficacy of gemcitabine can be effectively predicted. The novel antibodies and efficacy evaluation methods provided by this invention can be used in the development of reagents or companion diagnostic kits for predicting gemcitabine efficacy, which is beneficial for clinical application.

[0006] This invention synthesizes two peptides containing phosphorylation modification at the DCK S74 site, conjugates them with hemocyanin (KLH) as an antigen, and immunizes New Zealand rabbits with the mixture. The resulting purified peptides then specifically recognize the phosphorylated peptides and DCK S74 phosphorylated protein (p-DCK S74 polyclonal antibody). This invention demonstrates that the obtained rabbit polyclonal antibody can effectively detect DCK S74 phosphorylation levels in cells and tissues. In a preferred embodiment of this invention, this specific antibody was used to score 98 patients with cholangiocarcinoma treated with gemcitabine chemotherapy using immunohistochemical methods, dividing them into two groups: those with high DCK S74 phosphorylation expression and those with low expression. Combined with imaging pathological evaluation and prognostic survival statistical analysis, it was confirmed that patients in the high-expression group had a better response to gemcitabine treatment and longer progression-free survival and overall survival.

[0007] Furthermore, this invention synthesizes two polypeptides containing phosphorylation modification at the S74 site, conjugates them with hemocyanin (KLH) as an antigen, and immunizes mice with the mixture. After verification by serum titer and immunoblotting, hybridoma cell fusion, establishment of positive monoclonal cell lines and antibody purification, a mouse monoclonal antibody (p-DCK S74 monoclonal antibody) meeting the requirements is obtained, with the clone number 52-D4-A2.

[0008] In summary, this invention provides polyclonal and monoclonal antibodies that specifically recognize phosphorylation modification at the DCK S74 site, exhibiting good immunoblotting and immunohistochemical detection efficiency. These antibodies can be used to predict the efficacy of gemcitabine and are easy to apply in basic research and clinical practice.

[0009] In a first aspect, the present invention provides the use of a reagent for detecting the phosphorylation level of DCK S74 site in tumor tissue in the preparation of gemcitabine efficacy evaluation reagents and companion diagnostic kits.

[0010] Furthermore, the reagent used to detect the phosphorylation level of DCK S74 site in tumor tissue is a specific antibody that recognizes phosphorylation modification at the DCK S74 site.

[0011] Furthermore, the specific antibody is a polyclonal antibody, monoclonal antibody, or nanobody that specifically recognizes the phosphorylation modification at the DCK S74 site.

[0012] Furthermore, the application involves detecting the phosphorylation level of the DCKS74 site in pathological specimens from tumor patients using immunohistochemistry and evaluating the staining results, i.e., the immunohistochemical staining score. Based on this score, tumor patients are divided into high and low expression groups of DCKS74 phosphorylation. Patients in the high expression group of DCKS74 phosphorylation respond well to gemcitabine treatment, while patients in the low expression group respond poorly to gemcitabine treatment.

[0013] In a second aspect, the present invention provides a polyclonal antibody that specifically recognizes phosphorylated modification at the DCK S74 site. The method for preparing the polyclonal antibody involves synthesizing peptide 1: Cys+EELTMS(p)QKNGG and peptide 2: TMS(p)QKNGGNVLQMMY+Cys, conjugating them with hemocyanin as an antigen, and then immunizing New Zealand rabbits with the mixture. The purified antibody is required to have a titer greater than 1:64000 and to specifically recognize the phosphorylated peptide and the DCK S74 phosphorylated protein.

[0014] Immunoblot analysis revealed that the prepared p-DCK S74 polyclonal antibody effectively recognized phosphorylated DCK S74 peptide (p-Seq1), but could not detect unphosphorylated p-DCK S74 peptide (Seq1); after treatment with λPPase, the phosphorylated DCK S74 peptide (p-Seq1) could not be recognized by the p-DCK S74 polyclonal antibody. Figure 1 D); similarly, p-DCK S74 antibodies can recognize wild-type DCK but not the unphosphorylated DCK S74A mutant (D); Figure 1 E). The above results demonstrate that the p-DCK S74 antibody prepared in this invention has high specificity and sensitivity, and can only recognize phosphorylation at the wild-type DCK S74 site, and can be used for subsequent DCK S74 phosphorylation detection.

[0015] In a third aspect, the present invention provides a mouse monoclonal antibody that specifically recognizes phosphorylation modification at the DCK S74 site, clone number 52-D4-A2; the preparation method of the monoclonal antibody is as follows: peptide 1: Cys+EELTMS(p)QKNGG and peptide 2: TMS(p)QKNGGNVLQMMY+Cys are synthesized and conjugated with hemocyanin as an antigen, and mice are immunized with the mixture. After verification by serum titer and immunoblotting, the spleens of positive mice are selected for cell fusion; positive hybridoma cells are obtained by screening in HAT medium, and the positive hybridoma cells are subcloned to obtain monoclonal cell lines by limiting dilution. During each subcloning period, 2-3 rounds of indirect ELISA screening are performed to obtain positive monoclonal cell lines that meet the requirements, and the p-DCK S74 monoclonal antibody is purified to obtain clone number 52-D4-A2.

[0016] This invention confirms that the obtained mouse monoclonal antibody can effectively detect DCK S74 phosphorylation levels in cells and tissues; Western blot analysis shows ( Figure 4 The obtained mouse monoclonal antibody showed higher specificity than the aforementioned rabbit polyclonal antibody, with clear target bands and fewer non-specific bands; furthermore, immunohistochemical detection showed ( Figure 5The obtained mouse monoclonal antibody showed less non-specific staining in the cytoplasm and less background interference compared to the aforementioned rabbit polyclonal antibody; the above experimental data show that the obtained mouse monoclonal antibody is superior to the aforementioned rabbit polyclonal antibody.

[0017] Furthermore, the antibody that specifically recognizes DCK S74 phosphorylation modification provided by this invention can be optimized, modified, and combined with other adjuvants, and the level of DCK S74 phosphorylation in tumor cells and tissues can be detected in various ways. In a preferred embodiment of this invention, detection is preferentially performed by immunoblotting and immunohistochemistry; the effective detection dose of the antibody described in this invention can be a dilution ratio of 1:10 to 1:100000, more preferably a dilution ratio of 1:100 to 1:1000.

[0018] The method for specifically identifying phosphorylated antibodies at the DCK S74 site and predicting the antitumor efficacy of citalbine provided by this invention can be used for companion diagnostic identification of tumor drugs, preferably for cholangiocarcinoma, pancreatic ductal adenocarcinoma, cervical cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

[0019] In a fourth aspect, the present invention provides a kit for predicting the antitumor efficacy of gemcitabine, the kit comprising a specific antibody as described above that recognizes phosphorylation modification at the DCK S74 site. Specifically, by applying the aforementioned specific antibody to detect the phosphorylation level of the DCK S74 site in tumor tissue via immunohistochemistry, the efficacy of gemcitabine can be effectively predicted. This is achieved by combining DCK S74 site phosphorylation immunohistochemical staining scores with clinical data analysis. In a preferred embodiment, the present invention demonstrates that cholangiocarcinoma patients with high DCK S74 phosphorylation expression showed better responsiveness to gemcitabine treatment, longer progression-free survival, and longer overall survival.

[0020] In a fifth aspect, the present invention provides a novel method for predicting the efficacy of gemcitabine in treating tumors, namely, by using the aforementioned specific antibody to detect the phosphorylation level of the DCK S74 site in tumor tissue via immunohistochemistry, the efficacy of gemcitabine can be effectively predicted.

[0021] Furthermore, the key technology of the new method for predicting the antitumor efficacy of gemcitabine provided by the present invention is to detect the phosphorylation level of DCK S74 site in tumor cells and tissues, including but not limited to detection by immunohistochemistry, immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay, quantitative mass spectrometry and other methods.

[0022] The advantages of this invention are:

[0023] 1. Gemcitabine is a deoxycytidine-like prodrug that requires activation by DCK phosphorylation. The catalytic activity of DCK is affected by its own S74 site phosphorylation modification. Detecting the phosphorylation level of DCK at the S74 site in tumor cells and tissues using specific antibodies can effectively assess the catalytic activity of DCK and the efficacy of gemcitabine.

[0024] 2. This invention provides antibodies for detecting DCK S74 phosphorylation, which can be used to predict the efficacy of gemcitabine treatment and are easy to apply in basic research and clinical practice. This invention prepares polyclonal and monoclonal antibodies that specifically recognize DCK S74 phosphorylation modification by designing two DCK phosphorylated peptides for immunization, exhibiting good immunoblotting and immunohistochemical detection efficiency.

[0025] 3. This invention provides antibodies that specifically recognize phosphorylation modification at the DCK S74 site and kits and methods for predicting gemcitabine efficacy. These can be used to develop reagents for evaluating gemcitabine efficacy or companion diagnostic kits, avoiding delays in treatment due to ineffective first-line chemotherapy based on gemcitabine, allowing cancer patients to receive timely second-line treatment, improving the treatment level for patients with advanced cancer, and benefiting clinical application. Preservation information for biomaterial samples:

[0026] Depository Institution: China Center for Type Culture Collection (CCTCC)

[0027] Address: Wuhan University, Wuhan, China

[0028] Deposit date: October 11, 2023

[0029] Accession number: CCTCC NO: C2023306

[0030] Classification and nomenclature: Hybridoma cell line pDCK Attached Figure Description

[0031] Figure 1This section shows the preparation and validation of a rabbit polyclonal antibody (p-DCK S74) phosphorylated at the DCK S74 site. A shows the synthesis and use of DCK phosphorylated peptide 1 (p-Seq1:Cys+EELTMS(p)QKNGG) and peptide 2 (p-Seq2:TMS(p)QKNGGNVLQMMY+Cys), along with their corresponding non-phosphorylated peptides (Seq1 and Seq2), for antigen immunization. B shows the flowchart of immunizing two New Zealand white rabbits with a mixture of the two phosphorylated peptide antigens, validating the antiserum titer, purifying the antibody, and screening to obtain a qualified p-DCK S74 polyclonal antibody. C shows the ELISA detection of the titer of the prepared p-DCK S74 antibody binding to the DCK phosphorylated peptide p-Seq1 and the non-phosphorylated peptide Seq1. D shows the immunoblotting detection of the recognition ability of the prepared p-DCK S74 antibody for Seq1, p-Seq1, and p-Seq1 treated with λPPase. E shows the ability of the p-DCK S74 antibody prepared by immunoblotting to recognize wild-type DCK (DCK WT) and DCK with S74 site mutation (DCK S74A) transfected in HEK-293T cells.

[0032] Figure 2 The immunoblotting and immunohistochemical assays of the prepared p-DCK S74 rabbit polyclonal antibody are shown. A shows the immunoblotting detection of DCK phosphorylation levels in HCCC-9810, RBE, SK-CHA-1, and CCLP1 cholangiocarcinoma cells treated with gemcitabine (0, 5, 20 μM, 24 h) using the prepared p-DCK S74 rabbit polyclonal antibody. B shows the immunohistochemical detection of DCK phosphorylation levels in gemcitabine-treated and control cholangiocarcinoma PDX xenograft tissues (CC1, CC2, CC13, CC17, CC21) using the prepared p-DCK S74 rabbit polyclonal antibody; gemcitabine was administered intraperitoneally at 10 mg / kg, three times a week for 5 weeks. Scale bar: 50 μm.

[0033] Figure 3The application of p-DCK S74 rabbit polyclonal antibody to detect DCK phosphorylation levels in a cohort of cholangiocarcinoma patients confirms that patients with higher DCK phosphorylation levels respond better to gemcitabine treatment. A waterfall plot in Figure A shows the change in tumor burden from start to best objective response in 98 patients with advanced cholangiocarcinoma after receiving gemcitabine-based first-line chemotherapy; a representative image of p-DCK S74 staining is shown below, scale bar: 50 μm; patients were divided into a low p-DCK S74 expression group (≤1 point) and a high p-DCK S74 expression group (≥1 point) based on the intensity of p-DCK S74 staining; the dashed line above the X-axis represents a 20% increase in the sum of the diameters of target lesions from the minimum, and the dashed line below the X-axis represents a 30% decrease in the sum of the diameters of target lesions from the baseline; PD: disease progression, SD: stable disease, PR: partial remission. Tables and bar charts in section B summarize the efficacy evaluation results and proportions of cholangiocarcinoma patients with high or low p-DCK S74 expression; p-values ​​were obtained using the chi-square test. Sections C and D use Kaplan-Meier survival analysis to determine progression-free survival (PFS) and overall survival (OS) in cholangiocarcinoma patients; cholangiocarcinoma patients receiving gemcitabine chemotherapy were grouped according to their p-DCK S74 staining scores; p-values ​​were obtained using the log-rank test.

[0034] Figure 4 This demonstrates the detection efficacy of the prepared p-DCK S74 phosphorylation mouse monoclonal antibody (p-DCK S74 monoclonal antibody) in Western blotting. A shows the ability of the prepared p-DCK S74 mouse monoclonal antibody (52-D4-A2) to recognize wild-type DCK (DCK WT) and S74-mutated DCK (DCK S74A) transfected in HEK-293T cells using Western blotting. B shows the detection of DCK phosphorylation levels in RBE cholangiocarcinoma cells after treatment with gemcitabine (5 μM, 6 h) using both the prepared p-DCK S74 rabbit polyclonal antibody and mouse monoclonal antibody.

[0035] Figure 5 This shows the immunohistochemical staining of the prepared DCK S74 mouse monoclonal antibody and rabbit polyclonal antibody in cholangiocarcinoma tumor tissue. Detailed Implementation

[0036] The specific embodiments of the present invention will be described in detail below with reference to the examples. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0037] The DCK S74 phosphorylated modified peptides 1 (p-Seq1:Cys+EELTMS(p)QKNGG) and 2 (p-Seq2:TMS(p)QKNGGNVLQMMY+Cys), as well as their corresponding unphosphorylated peptides Seq1 (CEELTMSQKNGG, SEQ ID NO:1) and Seq2 (TMSQKNGGNVLQMMYC, SEQ ID NO:2), were synthesized by Wuhan Pujian Biotechnology Co., Ltd.; the wild-type DCK plasmid (DCK-WT) was purchased from Beijing Yiqiao Shenzhou Co., Ltd., and the DCK S74A mutant plasmid was constructed by mutating the wild-type plasmid and verified by sequencing; β-actin (AC026) and GAPDH (AC002) antibodies were purchased from ABclonal, and the DCK antibody was purchased from Proteintech Group (17758-1-Ig); p-DCK S74 rabbit polyclonal antibody and mouse monoclonal antibody (52-D4-A2) were prepared by the inventors and subjected to specificity testing; λ phosphatase (λPPase) was purchased from NEB; gemcitabine (GEM) was purchased from Selleck; a mixture of protease inhibitors and phosphatase inhibitors was purchased from Topscience; human cholangiocarcinoma cells RBE, HCCC-9810, CCLP1, and human embryonic renal epithelial cells HEK-293T were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; 6-week-old BALB / c (nu / nu) female nude mice (SPF grade) were purchased from Shanghai Bikai Biotechnology; human cholangiocarcinoma cells SK-CHA-1; and a human cholangiocarcinoma tumor xenograft model (Patient-derived tumor) were also used. (For cell line culture and the establishment of human cholangiocarcinoma PDX models, please refer to the literature Jiang, TY, Pan, YF, Wan, ZH, Lin, YK, Zhu, B., Yuan, ZG, Ma, YH, Shi, YY, Zeng, TM, Dong, LW, et al. (2020). PTEN status determines chemosensitivity to proteasome inhibition in cholangiocarcinoma. Sci Transl Med 12.). Other reagents or instruments used, unless otherwise specified, are commercially available products.

[0038] All cholangiocarcinoma tissue samples used in this invention were obtained from the Third Affiliated Hospital of Naval Medical University; the use of samples and clinical information was reviewed and approved by the hospital's ethics committee, and informed consent was obtained from patients in writing. A specific implementation example in this invention includes a clinical cohort of cholangiocarcinoma patients treated with gemcitabine chemotherapy. The inclusion criteria were: (1) pathological or cytological diagnosis of cholangiocarcinoma; (2) measurable according to RECIST criteria; (3) age between 18 and 75 years; (4) WHO / ECOG performance status score of 0-2; (5) unresectable lesions or distant metastases at diagnosis, and no prior systemic treatment; (6) informed consent; (7) treatment with gemcitabine at a dose of 1000 mg / m². 2 The drug was administered intravenously on days 1 and 8. The primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), and safety. The chi-square test was used to assess the correlation between DCK phosphorylation protein expression and gemcitabine efficacy. Kaplan-Meier survival curves were plotted using GraphPad Prism (version 7.0), and differences in survival curves were compared using the log-rank method; p < 0.05 was considered statistically significant.

[0039] Example 1: Preparation and Validation of Rabbit Polyclonal Antibody Specifically Recognizing DCK S74 Phosphorylation Modification (p-DCK S74 Antibody)

[0040] The catalytic activity of DCK is primarily regulated by phosphorylation at the S74 site; however, readily available DCK S74 phosphorylation antibodies are lacking. This invention, based on the DCK protein sequence, predicts possible antigenic epitopes and designs two pairs of DCK S74 phosphorylated and non-phosphorylated peptides (Seq1 and p-Seq1, Seq2 and p-Seq2) for subsequent immunization. Figure 1 A). 20 mg each of p-Seq1 and p-Seq2 were synthesized with a purity of over 95%, of which 10 mg was conjugated with hemocyanin (KLH) as the antigen, and the remaining 10 mg of naked peptide was used for detection and purification. In this invention, two phosphorylated polypeptide antigens were mixed and used to immunize two New Zealand white rabbits; the antiserum titer was detected after the third and fourth immunizations using an indirect ELISA method; when the titer was greater than 1:32000, blood was collected to purify the antiserum for antigen-specific antibody purification, requiring a purified antibody titer greater than 1:64000. Figure 1B). ELISA assays confirmed that the antibody exhibited high binding titer to the phosphorylated peptide of DCK S74 (p-Seq1), but low binding titer to the non-phosphorylated peptide (Seq1), indicating that the antibody has high specificity. Figure 1 C). Subsequently, this invention, through immunoblotting detection, found that the prepared p-DCK S74 polyclonal antibody could effectively recognize the phosphorylated DCK S74 polypeptide (p-Seq1), but could not detect the non-phosphorylated p-DCK S74 polypeptide (Seq1); after treatment with λ phosphatase (λPPase) of the phosphorylated DCK S74 polypeptide (p-Seq1), it could not be recognized by the p-DCK S74 polyclonal antibody. Figure 1 D); similarly, p-DCK S74 antibodies can recognize wild-type DCK but not the unphosphorylated DCK S74A mutant (D); Figure 1 E). The above results demonstrate that the prepared p-DCK S74 antibody is highly specific and sensitive, and can only recognize phosphorylation at the wild-type DCK S74 site, making it suitable for subsequent DCK S74 phosphorylation detection.

[0041] Example 2: The prepared p-DCK S74 rabbit polyclonal antibody can be used to detect the phosphorylation level of DCK S74 site in tumor cells and tissues.

[0042] Phosphorylation of DCK at site S74 is subject to feedback activation by its substrates, such as deoxycytosine nucleotides and gemcitabine. Based on this, this invention validates the detection efficacy of p-DCK S74 rabbit polyclonal antibody against phosphorylated DCK in tumor cells and tissues using Western blotting and immunohistochemistry.

[0043] The immunoblotting detection was performed according to the following steps: (1) Preparation of protein samples: Collect cholangiocarcinoma cells treated with 20 μM gemcitabine and control cells, including HCCC-9810, RBE, SK-CHA-1 and CCLP14 cell lines; lyse with RIPA buffer (Beyotime), and add a mixture of protease inhibitor and phosphatase inhibitor (Topscience) to the lysate in advance; after sonication, centrifugation and BCA quantitative analysis, prepare protein samples of equal concentration in 1×SDS loading buffer; (2) After SDS-PAGE electrophoresis, transfer and membrane blocking of protein samples, incubate with specific primary antibody (4℃, overnight), and then incubate with fluorescein-conjugated secondary antibody (37℃, 60 minutes), wash and detect fluorescence signal using Odyssey fluorescence scanner (Li-Cor); (3) The dilution ratio of p-DCK S74 rabbit polyclonal antibody for immunoblotting is 1:1000, and the dilution ratio of secondary antibody is 1:5000. Experimental results showed that p-DCK S74 rabbit polyclonal antibody could detect gemcitabine-induced phosphorylated DCK protein in various cholangiocarcinoma cell lines, and this recognition was specific. Figure 2 A).

[0044] Immunohistochemical detection was performed according to the following steps: (1) PDX tissues of human cholangiocarcinoma treated with gemcitabine and control were fixed in 4% neutral formaldehyde for 24 hours, then dehydrated and embedded in paraffin; the paraffin samples were cut into 4μm thick slices and adsorbed onto glass slides coated with polylysine; (2) Before the staining experiment, the tissue slices were baked in a 60℃ oven for 1 hour, and then routine immunohistochemical staining was performed, including dewaxing, peroxidase inactivation, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, hematoxylin staining, tap water blueing, and finally dehydration and mounting; (3) The dilution ratio of p-DCK S74 rabbit polyclonal antibody in immunohistochemistry was 1:200. The experimental results showed that the staining of p-DCK S74 rabbit polyclonal antibody was mainly concentrated in the cell nucleus, which was consistent with the subcellular localization of DCK. Figure 2 B); In addition, the staining of p-DCK S74 rabbit polyclonal antibody was significantly increased in PDX tumor tissue treated with gemcitabine, suggesting that gemcitabine can induce an increase in DCK phosphorylation level, further confirming the specificity and efficacy of p-DCK S74 rabbit polyclonal antibody at the tissue level.

[0045] Example 3: High expression of p-DCK S74 in patients with cholangiocarcinoma suggests better gemcitabine drug responsiveness

[0046] The aforementioned experiments confirmed that the prepared p-DCK S74 rabbit polyclonal antibody can effectively recognize phosphorylated DCK, thereby reflecting the catalytic activity of DCK and possessing potential clinical value in assessing gemcitabine drug sensitivity. Subsequently, we applied this antibody to verify the above hypothesis in 98 patients with advanced cholangiocarcinoma who received gemcitabine-based first-line chemotherapy. This invention assesses the expression of p-DCK S74 in cholangiocarcinoma samples using immunohistochemical staining. The expression was evaluated by two independent pathologists, and scores were assigned based on the intensity of p-DCK S74 staining. The scoring criteria were as follows: five fields of view were randomly selected from each slide of all specimens, considering both staining intensity and the frequency of positive cells; staining intensity was categorized as follows: 0 for no staining, 1 for light yellow granules, 2 for yellowish-brown granules, and 3 for dark brown granules; the frequency of positive cells was categorized as follows: 0 (less than 5%), 0.25 (5%–25%), 0.5 (6%–50%), 0.75 (51%–75%), and 1 (greater than 75%). When staining is uneven, each region is scored independently, and the results are summed. For example, a specimen containing 75% tumor cells stained to a score of 2 (2 × 0.75 = 1.5) and 25% tumor cells stained to a score of 1 (1 × 0.25 = 0.25) has a final score of 1.5 + 0.25 = 1.75. In statistical analysis, a p-DCK S74 staining score ≤ 1 indicates low expression, and a score > 1 indicates high expression.

[0047] Figure 3 Image A shows representative images of p-DCK S74 staining scores of 0, 1, 2, and 3. In this cholangiocarcinoma chemotherapy cohort, 60 patients showed low p-DCK S74 expression, and 38 patients showed high p-DCK S74 expression. The waterfall plot of tumor burden changes shows that patients in the p-DCK S74 high expression group were more likely to achieve stable disease (SD) and partial remission (PR) after receiving gemcitabine chemotherapy. Figure 3 A). The objective response rate in the p-DCK S74 high expression group was 42.1% (16 / 38), and the disease control rate was 86.8% (33 / 38), which were significantly higher than the 15.0% (9 / 60) and 65.0% (39 / 60) in the p-DCK S74 low expression group, respectively. Figure 3 (B) Furthermore, the PFS (p=0.006) and OS (p=0.034) of patients in the p-DCK S74 high expression group were significantly longer than those in the p-DCK S74 low expression group; these results confirm that patients in the p-DCK S74 high expression group have a better response to gemcitabine treatment. In summary, the p-DCK S74 rabbit polyclonal antibody prepared in this invention can be used in the development of companion diagnostic kits for predicting the clinical efficacy of gemcitabine.

[0048] Example 4: The prepared p-DCK S74 mouse monoclonal antibody (52-D4-A2) was superior to the previously obtained rabbit polyclonal antibody in Western blotting and immunohistochemical detection.

[0049] This invention synthesizes peptide 1: Cys+EELTMS(p)QKNGG and peptide 2: TMS(p)QKNGGNVLQMMY+Cys, and conjugates them with hemocyanin as an antigen. Mice are then immunized with the mixture. After verification by serum titer and immunoblotting, the spleens of positive mice are selected for cell fusion. Positive hybridoma cells are obtained by screening with HAT medium. The positive hybridoma cells are then subcloned using the limiting dilution method to obtain monoclonal cell lines. During each subcloning period, 2-3 rounds of indirect ELISA screening are performed to obtain positive monoclonal cell lines that meet the requirements. The p-DCK S74 monoclonal antibody is then purified to obtain clone number 52-D4-A2.

[0050] Subsequently, this invention, through immunoblotting detection, found that the prepared p-DCK S74 mouse monoclonal antibody could effectively recognize wild-type DCK, but could not recognize the non-phosphorylated DCK S74A mutant. Figure 4 A); Furthermore, compared to the aforementioned p-DCK S74 rabbit polyclonal antibody, the p-DCK S74 mouse monoclonal antibody showed clearer target bands and fewer non-specific bands when detecting DCK phosphorylation levels in gemcitabine-stimulated RBE cells. Figure 4B) suggests that p-DCK S74 mouse monoclonal antibody has better immunoblotting detection efficiency.

[0051] Furthermore, the present invention applied the prepared p-DCK S74 mouse monoclonal antibody and rabbit polyclonal antibody to perform immunohistochemical staining on cholangiocarcinoma tumor tissues; the results showed that the obtained p-DCK S74 mouse monoclonal antibody staining was mainly concentrated in the cell nucleus, consistent with the subcellular localization of DCK reported in the literature; compared with the aforementioned rabbit polyclonal antibody, the p-DCK S74 mouse monoclonal antibody showed less non-specific staining in the cytoplasm, reducing background interference. Figure 5 The above experimental data confirm that the obtained mouse monoclonal antibody has better immunohistochemical detection efficiency than the previously obtained rabbit polyclonal antibody.

[0052] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A monoclonal antibody that specifically recognizes phosphorylation modification at the DCK S74 site, characterized in that, The monoclonal antibody was secreted by a hybridoma cell line with accession number CCTCC NO:C2023306.

2. The monoclonal antibody according to claim 1, characterized in that, The method for preparing the monoclonal antibody is as follows: peptide 1: Cys+EELTMS(p)QKNGG and peptide 2: TMS(p)QKNGGNVLQMMY+Cys are synthesized and conjugated with hemocyanin as an antigen. Mice are immunized with the mixture, and after verification by serum titer and immunoblotting, the spleens of positive mice are selected for cell fusion. Positive hybridoma cells are obtained by screening in HAT medium. The positive hybridoma cells are subcloned to obtain monoclonal cell lines by limiting dilution. During each subcloning period, 2-3 rounds of indirect ELISA screening are performed to obtain positive monoclonal cell lines that meet the requirements. The p-DCK S74 monoclonal antibody is then purified to obtain clone number 52-D4-A2.

3. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a kit for predicting the antitumor efficacy of gemcitabine.

4. The application according to claim 3, characterized in that, The application involves detecting the phosphorylation level of DCK S74 in pathological specimens from tumor patients using immunohistochemistry and evaluating the staining results, i.e., the immunohistochemical staining score. Based on this score, tumor patients are divided into high and low expression groups of DCK S74 phosphorylation. Patients in the high expression group of DCK S74 phosphorylation respond well to gemcitabine treatment, while patients in the low expression group respond poorly to gemcitabine treatment.

5. The application according to claim 3, characterized in that, The tumors mentioned are bile duct cancer, pancreatic ductal adenocarcinoma, cervical cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

6. A kit for predicting the antitumor efficacy of gemcitabine, characterized in that, The kit contains the monoclonal antibody as described in claim 1 or 2.

7. The reagent kit according to claim 6, characterized in that, The kit described uses immunohistochemistry to detect the phosphorylation level of DCK S74 site in tumor tissue to predict the efficacy of gemcitabine. Tumor patients with high expression of DCK S74 site phosphorylation showed better response to gemcitabine treatment and longer progression-free survival and overall survival.

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