FBW7 phosphorylation specific antibody and application thereof in evaluating prognosis of colon cancer

Abstract

The invention relates to the technical field of biological medicine, and discloses an FBW7 phosphorylation specific antibody and application thereof in evaluation of colon cancer prognosis. The FBW7 phosphorylation specific antibody is an antibody capable of specifically recognizing the 680th threonine phosphorylation site (pT680) of FBW7 protein, the antibody has high affinity (Kd is smaller than or equal to 1 nM) and high specificity, and endogenous FBW7 pT680 can be accurately detected through various methods such as Western Blot, immunofluorescence and immunohistochemistry. When the antibody is used for performing immunohistochemical staining on a colon cancer tissue chip, the obvious high expression of the FBW7 pT680 in the colon cancer is found for the first time, and the high expression level of the FBW7 pT680 is obviously related to the poor prognosis of a patient. The invention provides a key research tool for filling the blank in the field, and discloses a new application of the FBW7 pT680 as the prognosis biomarker of the colon cancer, and the FBW7 pT680 has important scientific research and clinical transformation values.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to FBW7 phosphorylation-specific antibodies and their application in assessing the prognosis of colorectal cancer. Background Technology

[0002] FBW7 (F-box and WD repeat domain-containing 7) is a key substrate recognition subunit of the SCF-type E3 ubiquitin ligase complex. As an important tumor suppressor, it plays a central role in regulating cell cycle, cell growth, differentiation, and apoptosis by targeting and degrading various oncoproteins (such as c-Myc, Cyclin E, Notch, and c-Jun). The activity and stability of FBW7 are precisely regulated by various post-translational modifications, among which phosphorylation is a crucial regulatory mechanism. Studies have shown that the phosphorylation state of threonine 680 (T680) in the FBW7 protein may directly affect its protein stability, subcellular localization, and substrate interaction, thereby influencing its tumor suppressor function. Inactivation or downregulation of FBW7 function has been observed in various malignant tumors (such as colorectal cancer, breast cancer, and lung cancer). Besides gene mutations, dysregulation of post-translational modifications caused by abnormal signaling pathways is also an important contributing factor.

[0003] Currently, research on the FBW7 protein primarily relies on antibodies that identify its total protein content. However, high-quality antibodies that specifically recognize specific phosphorylation sites of FBW7 (especially the T680 site) are extremely scarce globally, posing a significant bottleneck to research in this field. Existing commercially available FBW7 phosphorylation antibodies generally suffer from poor specificity, high background signal, and large batch-to-batch variability, severely impacting the reliability and reproducibility of research data. Because the phosphorylation level of FBW7 T680 cannot be accurately and specifically detected, researchers struggle to dynamically monitor this modification event under physiological or pathological conditions, and are unable to assess its association with disease development (especially cancer), treatment response, and prognosis.

[0004] Therefore, developing a high-quality antibody that can specifically, sensitively, and stably recognize FBW7 pT680 has become an urgent need in the fields of tumor biology, ubiquitination regulation, and translational medicine research. Summary of the Invention

[0005] To address the problems mentioned in the background section, the present invention is implemented through the following technical solution:

[0006] An FBW7 phosphorylation-specific antibody, wherein the FBW7 phosphorylation-specific antibody is an antibody or its antigen-binding fragment that specifically recognizes the phosphorylation site of threonine at position 680 of the FBW7 protein, and the antibody or its antigen-binding fragment satisfies at least one of the following characteristics:

[0007] (1) It can specifically bind to human FBW7 protein or its peptide containing phosphorylated threonine 680;

[0008] (2) No significant cross-reactivity with the unphosphorylated FBW7 T680 peptide;

[0009] (3) Identify an endogenous FBW7 protein band with a molecular weight of approximately 70 kDa in Western Blot;

[0010] (4) In immunofluorescence experiments, it is located in the cell nucleus;

[0011] (5) Specific labeling of cell nuclei in formalin-fixed and paraffin-embedded tissue sections during immunohistochemical staining;

[0012] (6) Its equilibrium dissociation constant Kd with the FBW7 pT680 peptide is ≤ 1 nM.

[0013] Furthermore, the antibody is a monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, or fully human antibody; the antigen-binding fragment is selected from Fab, F(ab')2, scFv, dsFv, nanobody, or single-domain antibody.

[0014] A nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.

[0015] A recombinant expression vector containing the nucleic acid molecule.

[0016] A host cell containing the recombinant expression vector.

[0017] A method for preparing the antibody or its antigen-binding fragment includes the following steps:

[0018] (1) Synthesize an immunogenic peptide containing phosphorylated threonine at position 680 of the FBW7 protein;

[0019] (2) Immunize mammals with the immunogenic peptides described above;

[0020] (3) Isolate B cells or serum from immunized animals;

[0021] (4) Screen for antibodies or antigen-binding fragments that can specifically recognize FBW7 pT680.

[0022] A detection kit comprising the antibody or its antigen-binding fragment, and auxiliary reagents for immunoassay.

[0023] Furthermore, the immunoassay is selected from Western blotting, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, immunoprecipitation, or flow cytometry.

[0024] Use of the antibody or its antigen-binding fragment, and the detection kit, in the preparation of a reagent for detecting the phosphorylation level of threonine at position 680 of the FBW7 protein.

[0025] The use of an FBW7 phosphorylation-specific antibody in assessing the prognosis of colorectal cancer includes the use of the FBW7 phosphorylation-specific antibody and the detection kit in the preparation of a product for assessing the prognosis of colorectal cancer.

[0026] Furthermore, the assessment of colorectal cancer prognosis involves detecting the expression level of FBW7 pT680 in colorectal cancer tissue samples using immunohistochemistry and then performing a correlation analysis between this level and the patient's disease-free survival or overall survival.

[0027] Furthermore, this includes a method for assessing the prognosis of colorectal cancer, comprising the following steps:

[0028] (1) Obtain colon cancer tissue samples from the subjects;

[0029] (2) The sample is subjected to immunohistochemical staining using the antibody or its antigen-binding fragment;

[0030] (3) Evaluate the staining results and determine the expression level of FBW7 pT680;

[0031] (4) High expression levels are associated with poor prognostic risk.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. This FBW7 phosphorylation-specific antibody and its application in assessing the prognosis of colorectal cancer completely overcomes the shortcomings of existing tools, such as poor specificity and low reliability. For the first time, researchers can accurately and reliably detect the phosphorylation dynamics of FBW7 T680 in various experimental systems (cells, tissues, body fluids). This provides an indispensable key reagent for in-depth research into the regulatory mechanisms of FBW7 post-translational modifications, its functional changes in physiological and pathological processes, and the cross-interactions of related signaling pathways.

[0034] 2. This invention relates to an FBW7 phosphorylation-specific antibody and its application in assessing the prognosis of colorectal cancer. For the first time, the developed antibody systematically evaluated the expression pattern of FBW7 pT680 and its clinical prognostic value in a large-scale colorectal cancer tissue microarray. The results clearly showed that FBW7 pT680 is specifically and highly expressed in colorectal cancer, and its high expression is significantly associated with poor patient prognosis. Therefore, the specific antibody provided by this invention is not only a research tool but also directly translates into a potential prognostic assessment reagent. Detecting the level of FBW7 pT680 in cancer patient tissue samples using IHC is expected to be used to assist in tumor subtyping, prognostic risk stratification, and provide a reference for the development of personalized treatment strategies, demonstrating clear clinical translational potential. Attached Figure Description

[0035] Figure 1 Western blot validation of the anti-FBW7 pT680 antibody of this invention in different cell lines;

[0036] Figure 2 Immunofluorescence localization of the anti-FBW7 pT680 antibody of the present invention in HCT116 cells;

[0037] Figure 3 Immunohistochemical staining of human colon cancer tissue with the anti-FBW7 pT680 antibody of this invention;

[0038] Figure 4 This study provides an analysis of the SPR binding kinetics between the anti-FBW7 pT680 antibody and the phosphorylated peptide of FBW7 in this invention.

[0039] Figure 5 This invention relates to the IHC staining and prognostic analysis of the anti-FBW7 pT680 antibody in colorectal cancer tissue microarray. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] The following are examples of the FBW7 phosphorylation-specific antibody and its application in assessing the prognosis of colorectal cancer:

[0042] Please see Figures 1-5An FBW7 phosphorylation-specific antibody, wherein the FBW7 phosphorylation-specific antibody is an antibody or its antigen-binding fragment that specifically recognizes the phosphorylation site of threonine at position 680 of the FBW7 protein, and the antibody or its antigen-binding fragment satisfies at least one of the following characteristics:

[0043] It can specifically bind to human FBW7 protein or its peptide containing phosphorylated threonine 680; it has no significant cross-reactivity with the unphosphorylated FBW7 T680 peptide; it recognizes endogenous FBW7 protein bands with a molecular weight of approximately 70 kDa in Western blotting; it localizes to the cell nucleus in immunofluorescence assays; it specifically labels the cell nuclei in formalin-fixed, paraffin-embedded tissue sections in immunohistochemical staining; and its equilibrium dissociation constant with the FBW7 pT680 peptide, Kd, ​​is ≤ 1 nM.

[0044] The antibody is a monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, or fully human antibody; the antigen-binding fragment is selected from Fab, F(ab')2, scFv, dsFv, nanobody, or single-domain antibody.

[0045] A nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof.

[0046] A recombinant expression vector containing the nucleic acid molecule.

[0047] A host cell containing the recombinant expression vector.

[0048] A method for preparing the antibody or its antigen-binding fragment thereof includes the following steps: synthesizing an immunogenic peptide containing phosphorylated threonine at position 680 of the FBW7 protein; immunizing a mammal with the immunogenic peptide; isolating B cells or serum from the immunized animal; and screening for antibodies or their antigen-binding fragments that specifically recognize FBW7 pT680.

[0049] A detection kit comprising the antibody or its antigen-binding fragment, and auxiliary reagents for immunoassay. The immunoassay is selected from Western blotting, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or flow cytometry.

[0050] Use of the antibody or its antigen-binding fragment, and the detection kit, in the preparation of a reagent for detecting the phosphorylation level of threonine at position 680 of the FBW7 protein.

[0051] The application of an FBW7 phosphorylation-specific antibody in assessing the prognosis of colorectal cancer includes the use of the FBW7 phosphorylation-specific antibody and the detection kit in the preparation of a product for assessing the prognosis of colorectal cancer. The assessment of colorectal cancer prognosis involves detecting the expression level of FBW7 pT680 in colorectal cancer tissue samples using immunohistochemistry and correlating it with the patient's disease-free survival or overall survival.

[0052] One of the methods is a way to assess the prognosis of colorectal cancer, comprising the following steps: obtaining a colorectal cancer tissue sample from a subject; performing immunohistochemical staining on the sample using the antibody or its antigen-binding fragment; evaluating the staining results to determine the expression level of FBW7 pT680; and associating high expression levels with poor prognostic risk.

[0053] Immunogen design and antibody preparation

[0054] This invention utilizes a short peptide containing phosphorylated threonine (pT680) at position 680 of the FBW7 protein as an immunogen, the amino acid sequence of which is shown in SEQ ID NO:1. The immunogen is conjugated to a carrier protein (such as KLH or BSA) and then used to immunize Balb / c mice or New Zealand rabbits. Using conventional hybridoma techniques or phage display library screening techniques, monoclonal antibodies or their antigen-binding fragments that specifically recognize FBW7 pT680 are obtained.

[0055] Antibody performance verification

[0056] This invention systematically validates the prepared antibodies through the following five aspects:

[0057] (1) Western Blot detection of endogenous FBW7 pT680 protein in various cell lines

[0058] Six cell lines were selected: human colorectal cancer cells HCT116, human breast cancer cells MCF-7, human non-small cell lung cancer cells A549, human normal breast epithelial cells MCF10A, human normal colonic epithelial cells NCM460, and human embryonic kidney cells HEK293T. Cell lysates were subjected to SDS-PAGE electrophoresis, transferred to a membrane, blocked, and then immunohybridized using the antibody of this invention (1:1000 dilution) as the primary antibody. Results showed:

[0059] In cancer cells such as HCT116, MCF-7, and A549, the antibody specifically recognizes a single, clear band with a molecular weight of approximately 70 kDa.

[0060] In normal cells such as MCF10A and NCM460, the signal intensity was significantly weaker;

[0061] Total FBW7 antibody detection showed that the pT680 signal intensity did not completely match the trend of total protein abundance, suggesting that the antibody recognizes the phosphorylation modification state.

[0062] (2) Immunofluorescence (IF) detection of FBW7 pT680 subcellular localization

[0063] HCT116 cells were seeded in confocal dishes, fixed, permeabilized, and blocked. Cells were stained with the antibody of this invention (1:200 dilution) as the primary antibody, Alexa Fluor 488-labeled secondary antibody, and DAPI-counted nuclei. Laser confocal microscopy revealed the following:

[0064] The green fluorescence signal is mainly concentrated in the cell nucleus, and appears as a punctate or diffuse distribution;

[0065] The cytoplasmic region shows almost no specific signals;

[0066] It is highly consistent with the nuclear localization characteristics of the FBW7 protein.

[0067] (3) Immunohistochemical (IHC) detection of paraffin-embedded tissue sections

[0068] After antigen retrieval, endogenous peroxidase blockade, and serum blocking of paraffin sections of human colon cancer and adjacent normal tissue, the antibody of this invention (1:50 dilution) was used as the primary antibody, followed by DAB staining and hematoxylin counterstaining. Results showed:

[0069] The colon cancer cells showed a clear brownish-red positive signal within their nuclei.

[0070] The nuclear signal of normal epithelial cells adjacent to cancer is extremely weak or negative;

[0071] The background is clean and there is no nonspecific staining.

[0072] (4) Surface plasmon resonance (SPR) determination of affinity

[0073] Biotinylated FBW7 pT680 peptide was immobilized on a streptavidin chip, and antibody binding kinetics were determined using a Biacore T200 system. Antibody concentration gradients were 0.078, 0.156, 0.312, 0.624, and 1.248 nM. Using a 1:1 Langmuir model, the equilibrium dissociation constant Kd = 0.43 nM was calculated, indicating that the antibody of this invention possesses picomolar-level ultra-high affinity for the target epitope.

[0074] (5) Tissue microarray (TMA) and prognostic correlation analysis

[0075] Standardized IHC staining was performed on TMA composed of 80 cases of colorectal cancer and paired adjacent normal tissue. IHC scoring (staining intensity × percentage of positive cells) was performed by a double-blind pathologist. Statistical analysis showed:

[0076] The FBW7 pT680 IHC score in colorectal cancer tissue was significantly higher than that in adjacent normal tissue (p < 0.001).

[0077] Patients were divided into a high expression group (n=40) and a low expression group (n=40) based on the median score.

[0078] Kaplan-Meier survival analysis showed that the disease-free survival (DFS) and overall survival (OS) of patients in the high expression group were significantly lower than those in the low expression group (Log-rank p < 0.01).

[0079] This study is the first to confirm that FBW7 pT680 is an independent poor prognostic factor for colorectal cancer.

[0080] Antibody sequence information (optional)

[0081] The present invention provides a preferred antibody with a heavy chain variable region (VH) amino acid sequence as shown in SEQ ID NO:2 and a light chain variable region (VL) amino acid sequence as shown in SEQ ID NO:3. The nucleotide sequences encoding the VH and VL are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0082] Example 1: Validation of the broad reactivity and specificity of the FBW7 pT680 antibody

[0083] To evaluate the ability of the antibody of this invention to recognize target phosphorylated proteins under different cellular backgrounds, six representative human cell lines were selected and detected by Western blotting. HCT116 (human colorectal cancer cells), MCF-7 (human breast cancer cells), A549 (human non-small cell lung cancer cells), MCF10A (human normal breast epithelial cells), NCM460 (human normal colon epithelial cells), and HEK293T (human embryonic kidney cells) were cultured. When the cells reached 80-90% confluence, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. 200 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (such as PhosSTOP) was added to every 10⁷ cells, and the cells were lysed on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected as total cellular protein. Protein concentration was determined using the BCA method. 30 μg of total protein was subjected to SDS-PAGE electrophoresis (8-10% separating gel), followed by wet transfer (300 mA constant current, 90 min) to a PVDF membrane. The membrane was blocked with TBST containing 5% skim milk at room temperature for 1 hour. The anti-FBW7 pT680 antibody (1:1000 dilution) prepared in this invention was added as the primary antibody, and the membrane was incubated overnight at 4°C. After washing three times with TBST (10 min each time), the corresponding species secondary antibody labeled with HRP (1:5000 dilution) was added, and the membrane was incubated at room temperature for 1 hour. After thorough washing with TBST, the membrane was developed using ECL chemiluminescent substrate, and images were acquired using a chemiluminescent imaging system.

[0084] The results showed that in lysates from various cancer cells, including HCT116, MCF-7, and A549, the antibody specifically recognized a single, clear band with a molecular weight of approximately 70 kDa, consistent with the expected molecular weight of FBW7 protein. The signal intensity varied among different cell lines, reflecting differences in the phosphorylation level of FBW7 at the T680 site in different cells. Corresponding bands were also detected in normal epithelial cells such as MCF10A and NCM460, but the signal intensity was usually weaker. Re-incubation of the same membrane with the anti-total FBW7 antibody as a control showed that the trend of FBW7 pT680 signal change was not entirely the same as that of total FBW7 protein level, suggesting that this antibody detects a specific phosphorylation modification state rather than total protein abundance.

[0085] like Figure 1 As shown, total protein was extracted from six cell lines: HCT116, MCF-7, A549, MCF10A, NCM460, and HEK293T, and Western blotting was performed using the anti-FBW7 pT680 antibody of this invention.

[0086] Example 2: Subcellular localization verification of FBW7 pT680 antibody

[0087] As a component of the nuclear-localized E3 ubiquitin ligase, FBW7's phosphorylation modification may affect its nucleocytoplasmic shuttle. Immunofluorescence (IF) experiments were performed using human colorectal cancer cells HCT116 to clarify the subcellular localization of phosphorylated FBW7 recognized by the antibody of this invention. HCT116 cells were seeded in confocal microscopy dishes and allowed to adhere and grow to an appropriate density. After gentle washing with PBS, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes. Permeabilization was performed with 0.2% Triton X-100 for 10 minutes, followed by blocking with 3% BSA at room temperature for 30 minutes. Anti-FBW7 pT680 antibody (1:200 dilution) was added as the primary antibody, and the cells were incubated overnight at 4°C. After washing three times with PBS, Alexa Fluor 488-labeled secondary antibody (1:500 dilution) was added, and the cells were incubated at room temperature in the dark for 1 hour. After washing with PBS, the nuclei were stained with DAPI for 5 minutes. Finally, the cells were mounted with anti-fluorescence quenching mounting medium, and images were observed and acquired under a laser confocal microscope.

[0088] like Figure 2 As shown, HCT116 cells were immunofluorescence stained using the anti-FBW7 pT680 antibody of this invention. Green fluorescence (Alexa Fluor 488) represents the antibody binding signal, and blue fluorescence (DAPI) labels the cell nucleus. The results showed that the green fluorescence signal was mainly concentrated in the cell nucleus, exhibiting a clear punctate or diffuse distribution pattern, highly consistent with the nuclear localization characteristics of FBW7. Only a weak background signal was observed in the cytoplasm. This result confirms that this antibody can specifically recognize and bind to phosphorylated FBW7 protein in cells, and that it is mainly localized in the cell nucleus.

[0089] Example 3: Validation of the recognition ability of FBW7 pT680 antibody in clinical pathological samples

[0090] To evaluate the application value of the antibody of this invention in complex pathological tissue environments, immunohistochemical (IHC) staining was performed on paraffin sections of human colon cancer tissue. Surgically resected human colon cancer and paired adjacent normal tissue samples were collected, fixed with 4% paraformaldehyde, dehydrated, cleared, and embedded in paraffin using standard procedures, and cut into 4μm thick sections. After dewaxing with xylene and hydration with graded ethanol, antigen retrieval was performed using a high-pressure heat retrieval method (sodium citrate retrieval solution, pH 6.0). Incubation with 3% H2O2 at room temperature for 10 minutes was used to block endogenous peroxidase activity. Blocking with 10% normal goat serum at room temperature for 30 minutes was performed. Anti-FBW7pT680 antibody (1:50 dilution) was added, and incubation was carried out overnight at 4°C. After washing with PBS, HRP-labeled polymeric secondary antibody was added, and incubation was carried out at room temperature for 30 minutes. DAB staining was performed, followed by hematoxylin counterstaining, hydrochloric acid ethanol differentiation, tap water blueing, graded ethanol dehydration, xylene clearing, and mounting with neutral resin. Observe and photograph under an optical microscope.

[0091] like Figure 3 As shown, immunohistochemical staining results revealed that the antibody of this invention clearly displayed a brownish-red positive signal within the nuclei of tumor cells in colon cancer tissue sections, with positive cells appearing scattered or focally distributed. A weaker positive signal was also observed in the nuclei of some adjacent normal mucosal epithelial cells. The location of the positive signal (nucleus) was consistent with the aforementioned immunofluorescence results, and the background staining was clean, demonstrating high specificity. These results prove that this antibody can be effectively applied to formalin-fixed, paraffin-embedded clinicopathological samples, and can be used to detect the phosphorylation status of the FBW7 T680 site in tissues, possessing potential diagnostic or prognostic value.

[0092] Example 4: Verification of high affinity binding of FBW7 pT680 antibody to target antigen

[0093] We employed surface plasmon resonance (SPR) technology to precisely determine the interaction kinetics between the antibody of this invention and synthetic phosphorylated peptides (corresponding to the pT680 sequence in the FBW7 protein) or recombinant phosphorylated FBW7 protein (if available) in a cell-free system. Using the Biacore T200 system as an example, the biotinylated FBW7 pT680 phosphorylated peptide was captured and immobilized using a streptavidin (SA) chip. The running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4). The purified anti-FBW7 pT680 antibody was sequentially injected at concentrations ranging from low to high (0.078 nM, 0.156 nM, 0.312 nM, 0.624 nM, 1.248 nM) at a flow rate of 30 μL / min, with a binding time of 180 seconds and a dissociation time of 300 seconds. After each analysis, the chip was regenerated under mild regeneration conditions (e.g., 10 mM glycine-HCl, pH 2.0). The sensor spectrum was fitted using a 1:1 Langmuir binding model, and the equilibrium dissociation constant (Kd) was calculated. Steady-state fitting analysis showed that the antibody has a high affinity for the target phosphorylated peptide, with Kd values ​​ranging from picomolar to low nanomolar levels (Kd = 0.43 nM). This data directly confirms from a biophysical perspective that the antibody of this invention has extremely high specificity and binding strength to its target epitope (FBW7 pT680), which is the molecular basis for its ability to achieve high sensitivity and high specificity detection in complex biological samples.

[0094] like Figure 4 As shown, the sensor graph displays the real-time response curves of antibodies of different concentrations flowing through the surface of a chip immobilized with FBW7 pT680 peptides. With increasing antibody concentration, the binding response value (RU) increases in a dose-dependent manner and decreases slowly during the dissociation phase, indicating strong binding.

[0095] Example 5: Validation of the recognition ability and clinical relevance of FBW7 pT680 antibody in clinical pathological samples.

[0096] To comprehensively evaluate the clinical translational potential of the antibody of this invention, two levels of validation were conducted: First, the antibody was used to perform standardized immunohistochemical (IHC) staining on a high-throughput tissue microarray (TMA) containing 80 pairs of colorectal cancer (CRC) tissues and their paired adjacent normal (NT) tissues. The aforementioned standard IHC procedure was adopted: dewaxing of sections, antigen heat retrieval, endogenous peroxidase blockade, serum blocking, incubation with primary antibody (anti-FBW7 pT680 antibody, 1:50 dilution) at 4°C overnight, incubation with secondary antibody, DAB staining, counterstaining, dehydration, and mounting. After staining, two independent pathologists, under double-blind conditions, scored the staining based on staining intensity (0: negative; 1: weakly positive; 2: moderately positive; 3: strongly positive) and the percentage of positive cells, calculating the IHC score (staining intensity × percentage of positive cells).

[0097] Tissue microarray analysis revealed the following differences: ① Expression profile differences: In the vast majority of CRC tissue samples, FBW7 pT680 showed significant nuclear positivity (high IHC score), while in the corresponding NT tissues, the positive signal was extremely weak or almost undetectable (low IHC score). This difference was statistically significant (p < 0.001), indicating that FBW7 pT680 is specifically highly expressed in colorectal cancer. ② Prognostic value assessment: Based on the median IHC score, 80 CRC patients were divided into a low-expression group (Low, n=40) and a high-expression group (High, n=40). Kaplan-Meier survival curves were plotted by querying and correlating the patients' clinical follow-up data. Statistical analysis showed that the disease-free survival (DFS) and / or overall survival (OS) of patients in the high-expression group were significantly shorter than those in the low-expression group (Log-rank test, p < 0.01). This key result indicates that the FBW7pT680 level detected by the antibody of this invention is not only a tumor-specific marker, but also a potential independent prognostic factor.

[0098] like Figure 5As shown, A: Representative IHC staining images, demonstrating the expression of FBW7 pT680 in adjacent normal tissue (NT, top row) and colon cancer tissue (CRC, bottom row). Almost no positive signal was observed in NT tissue, while significant nuclear positive staining (brownish) was visible in CRC tissue. B: Statistical analysis of FBW7 T680 phosphorylation levels in NT and CRC tissues. C: Kaplan-Meier survival curves. Eighty CRC patients were divided into a low-expression group (Low, blue curve) and a high-expression group (High, red curve) based on their FBW7 pT680 IHC scores. The vertical axis represents survival probability, and the horizontal axis represents survival time (months).

[0099] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An FBW7 phosphorylation-specific antibody, wherein the FBW7 phosphorylation-specific antibody is an antibody or its antigen-binding fragment that specifically recognizes the phosphorylation site of threonine at position 680 of the FBW7 protein, characterized in that, The antibody or its antigen-binding fragment satisfies at least one of the following characteristics: (1) It can specifically bind to human FBW7 protein or its peptide containing phosphorylated threonine 680; (2) No significant cross-reactivity with the unphosphorylated FBW7 T680 peptide; (3) Identify an endogenous FBW7 protein band with a molecular weight of approximately 70 kDa in Western Blot; (4) In immunofluorescence experiments, it is located in the cell nucleus; (5) Specific labeling of cell nuclei in formalin-fixed and paraffin-embedded tissue sections during immunohistochemical staining; (6) Its equilibrium dissociation constant Kd with the FBW7 pT680 peptide is ≤ 1 nM.

2. The FBW7 phosphorylation-specific antibody according to claim 1, characterized in that, The antibody is a monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, humanized antibody, or fully human antibody; the antigen-binding fragment is selected from Fab, F(ab')2, scFv, dsFv, nanobody, or single-domain antibody.

3. A nucleic acid molecule encoding the antibody or an antigen-binding fragment thereof as described in claim 1 or 2.

4. A method for preparing the antibody or antigen-binding fragment thereof as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Synthesize an immunogenic peptide containing phosphorylated threonine at position 680 of the FBW7 protein; (2) Immunize mammals with the immunogenic peptides described above; (3) Isolate B cells or serum from immunized animals; (4) Screen for antibodies or antigen-binding fragments that can specifically recognize FBW7 pT680.

5. A test kit, characterized in that, It comprises the antibody or antigen-binding fragment thereof as described in claim 1, and auxiliary reagents for immunoassay.

6. The detection kit according to claim 5, characterized in that, The immunoassay is selected from Western blotting, immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, immunoprecipitation, or flow cytometry.

7. The application of an FBW7 phosphorylation-specific antibody in assessing the prognosis of colorectal cancer, characterized in that: This includes the use of the FBW7 phosphorylation-specific antibody as described in claim 1 or 2, and the detection kit as described in claim 5 or 6, in the preparation of products for assessing the prognosis of colorectal cancer.

8. The application of the FBW7 phosphorylation-specific antibody according to claim 7 in assessing the prognosis of colorectal cancer, characterized in that, The assessment of colorectal cancer prognosis involves detecting the expression level of FBW7 pT680 in colorectal cancer tissue samples using immunohistochemistry and then performing a correlation analysis between this level and the patient's disease-free survival or overall survival.

9. The application of the FBW7 phosphorylation-specific antibody according to claim 7 in assessing the prognosis of colorectal cancer, comprising a method for assessing the prognosis of colorectal cancer, characterized in that, Includes the following steps: (1) Obtain colon cancer tissue samples from the subjects; (2) The sample is subjected to immunohistochemical staining using the antibody or its antigen-binding fragment; (3) Evaluate the staining results and determine the expression level of FBW7 pT680; (4) High expression levels are associated with poor prognostic risk.

Images