Method for efficiently screening ATP hydrolase antibody based on fluorescent probe

Abstract

The invention discloses an ATP hydrolase antibody efficient screening method based on a fluorescent probe, the method comprises two detection schemes of a biochemical system and a cell system, in the biochemical system, the antibody inhibition rate is directly calculated by quantifying the residual amount of ENTPD2 protein after ATP hydrolysis; in a cell system, cells over-expressing ENTPD2 are used for verifying the inhibition function of the antibody on transmembrane enzyme. According to the biochemical system method, antibody purification is not needed, primary screening can be completed only through 5-15 microliters of B cell supernate, the screening accuracy is high (the cell system can be used as an antibody obtained through verification and screening), the result is consistent with the in-vivo drug effect result, the method is suitable for development of anti-ENTPD2 antibody drugs, and the research and development cost is remarkably reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes, which is applicable to the initial screening stage in antibody drug development. Background Technology

[0002] Adenine triphosphate (ATP) is a core carrier of cellular energy metabolism, and its hydrolysis is precisely regulated by a series of ATP-hydrolyzing enzymes. Among them, ENTPD2, a member of the nucleoside triphosphate diphosphate hydrolase (NTPDase) family, is a transmembrane protein whose catalytic domain is directionally distributed on the outer side of the cell membrane, specializing in the hydrolysis of extracellular ATP to generate adenosine monophosphate (AMP). Recent extensive clinical and basic research has confirmed that dysregulation of ENTPD2 expression constitutes a key link in the progression of several major diseases.

[0003] In liver cancer, lung adenocarcinoma, and prostate cancer tissues, ENTPD2 overexpression accelerates tumor progression by remodeling the tumor microenvironment—particularly by promoting the immunosuppressive function of myeloid-derived suppressor cells (MDSCs). Gene knockdown experiments showed that inhibiting ENTPD2 significantly delayed tumor growth, confirming its feasibility as a therapeutic target. Animal model studies showed that ENTpd2-deficient mice exhibited a more severe inflammatory response in a dextran sulfate sodium-induced colitis model, with their colonic macrophages displaying a pro-inflammatory phenotype. In a carbon tetrachloride-induced liver fibrosis model, ENTpd2 deficiency exacerbated liver tissue damage, highlighting the anti-inflammatory protective effect of this gene. ENTPD2 mRNA expression was significantly increased in the entorhinal cortex of Alzheimer's disease patients, and peripheral blood ENTPD2 expression was abnormally high in schizophrenia patients without drug treatment, returning to normal levels after drug administration, suggesting its involvement in the pathological mechanisms of neurodegenerative and mental illnesses. The antipsychotic drug haloperidol reduced ENTpd2 transcription levels in the zebrafish brain, accompanied by a 35% decrease in ATP hydrolysis activity, revealing that ENTPD2 can serve as a molecular indicator for dynamic monitoring of drug effects.

[0004] The aforementioned chain of evidence collectively points to the central regulatory role of ENTPD2 in the disease network, making it an ideal candidate for developing targeted therapies (such as inhibitory antibodies).

[0005] Currently, the initial screening stage of antibody drug development faces two major technical bottlenecks: conventional methods such as enzyme-linked immunosorbent assay (ELISA) and Western blot can only detect the apparent binding affinity between antibodies and antigens, failing to directly reflect the spatial blocking effect of antibodies on enzyme active sites. The patent explicitly mentions that early data showed "inconsistency between antibody affinity ranking and inhibitory function ranking"—some high-affinity antibodies have low actual inhibitory efficiency because their binding sites deviate from the catalytic active domain. This lag in functional assessment leads to a large number of ineffective antibodies entering the subsequent development pipeline. Traditional processes require expression and purification of each candidate antibody (monoclonal antibody purification costs approximately $500-$1000), followed by in vivo efficacy validation using animal models. This process is time-consuming and resource-intensive, becoming a rate-limiting step in antibody drug development. Summary of the Invention

[0006] The purpose of this invention is to provide a highly efficient screening method for ATP hydrolase antibodies based on fluorescent probes, which is suitable for the initial screening stage in antibody drug development.

[0007] This invention proposes a highly efficient screening method for ATP hydrolase antibodies based on fluorescent probes, comprising: directly detecting the antibody's inhibitory ability on ATP hydrolysis by mixing ENTPD2 protein, the antibody to be tested, and ATP, overcoming the limitations of traditional binding force detection; precisely quantifying antibody function using inhibition rate = (RLUx - RLU1) / (RLU2 - RLU1) (RLUx: antibody group; RLU1: enzyme reaction control group; RLU2: ATP control group); supporting direct detection in B cell culture supernatant, significantly reducing initial screening costs. It directly screens highly active antibodies at the functional level, avoiding invalid candidates with "high affinity, low inhibition" in traditional methods, improving initial screening efficiency by 10 times.

[0008] Furthermore, the ENTPD2 protein concentration was fixed at 1.667 ng / μL (final concentration 0.357 ng / μL), and the ATP concentration was 333 nM (final concentration 190–222 nM) to ensure the detection window was within the linear range (inhibition rate 50%–100%). 5–15 μL of unpurified B cell supernatant was used, and the reaction conditions were 37°C with shaking for 40 minutes; treatment at 60°C for 45 minutes eliminated complement interference. The parameter combination was rigorously validated, accurately distinguishing between potent and weak antibodies (e.g., mAb1 inhibition rate 84.7% vs. mAb13 inhibition rate 0%), reducing the false negative rate to below 5%.

[0009] Furthermore, ENTPD2 overexpressing cells (B16F10 hEN OE or HEK293 hEN OE) were used, with gene knockout cells (B16F10 mEN KO) or wild-type cells as negative controls. The amount of ATP remaining on the cell membrane surface was measured by centrifugation and supernatant collection. The inhibition rate was calculated as (RLUx - RLU1) / (RLU2 - RLU1) (RLU1: overexpressing cell group; RLU2: negative cell group). This simulated the in vivo enzyme spatial conformation to verify the inhibitory effect of the antibody on transmembrane proteins, providing dual protection in conjunction with the biochemical system.

[0010] Furthermore, a stepwise incubation process was employed: antibody binding was performed at 4°C for 15 minutes (to inhibit non-target reactions); the reaction was repeated twice at 37°C (15 minutes each time) to activate ENTPD2 hydrolytic activity; cell density: 1×10⁻⁶. 5 cells / mL (system contains 1×10⁶ cells) 4 (Number of cells); ATP concentration: 4440 nM (final concentration 190–222 nM). The short-term temperature control strategy effectively avoided interference from spontaneous ATP release by cells, reducing background signal by 70%.

[0011] Furthermore, B cell culture medium or DMEM is heat-inactivated at 60°C for 45 minutes to destroy complement activity; sample addition and incubation are carried out on ice throughout to inhibit the hydrolysis of ATP by miscellaneous enzymes (or inhibit the generation of ATP by cells); before enzyme labeling, air bubbles are punctured with a heated needle tip to systematically eliminate environmental interferences (such as complement fixation and miscellaneous enzyme activity) and ensure data reliability.

[0012] Furthermore, the selected antibodies were used to treat diseases associated with abnormal ENTPD2 expression, including: tumors (liver cancer, lung adenocarcinoma, etc.); inflammatory diseases (colitis, liver fibrosis); and neurodegenerative diseases (Alzheimer's disease). Antibodies selected based on functional screening were validated in vivo, showing a tumor inhibition rate exceeding 56% (7B12 antibody), demonstrating clear potential for clinical translation.

[0013] Furthermore, in a tumor-bearing mouse model (B16F10 hEN OE), the tumor volume in the antibody treatment group was reduced by ≥30% compared to the PBS group; using the patented antibody mAb1 as a benchmark (tumor inhibition rate of 43.6%). This provides a quantitative standard for efficacy, accelerating the entry of antibodies into preclinical studies.

[0014] Further, the following are required: ENTPD2 protein (1.667 ng / μL, final concentration 0.357 ng / μL) or overexpressing cell lines; ATP working solution (333 nM / 4440 nM, final concentration 190–222 nM); CCL2.0 fluorescent detection reagent; control antibodies (mAb1 and mAb13); 96-well microplate (flat-bottomed, opaque black). Ready to use out of the box, supporting high-throughput detection in 96-well plates, allowing for initial screening of hundreds of antibody strains in a single run.

[0015] Furthermore, the antibody names are: 7B12, 11A2, 12G4, 13A4, and 16F8; functional data: inhibition rate >70% in cellular systems (see), and tumor inhibition rate >40% in vivo. Top-tier antibodies validated in both systems (such as 7B12 with a tumor inhibition rate of 56.4%) can be directly used for drug development.

[0016] Further indications: melanoma, cholangiocarcinoma, colorectal cancer, gastric cancer, breast cancer, esophageal cancer, leukemia, pancreatic cancer, lymphoma, liver cancer, lung cancer, bladder / urethral tumors, ovarian / fallopian tube tumors, prostate cancer, and head and neck tumors; Dosage regimen: anti-ENTPD2 antibody 10 mg / kg intravenously, once; combined with anti-mouse PD1 antibody 10 mg / kg intraperitoneally, twice, with a 3-day interval between doses. Targeting ENTPD2 reshapes the tumor immune microenvironment, overcoming the bottleneck of drug resistance in existing immunotherapies.

[0017] 1. Biochemical system testing (cell-free system)

[0018] Material:

[0019] Human ENTPD2 protein (concentration 1.667 ng / μL)

[0020] ATP working solution (333 nM)

[0021] Fluorescent probe CCL2.0 (Novizan, catalog number DD1101)

[0022] Heat-inactivated B cell culture medium (treated at 60°C for 45 minutes)

[0023] Operating procedures:

[0024] 1. Grouped sample addition (operation on ice):

[0025]

[0026] 2. Incubate at room temperature (25℃) for 30 minutes.

[0027] 3. Add 40 μL of ATP working solution to ice.

[0028] 4.37℃, oscillation reaction for 40 minutes

[0029] 5. Add 20 μL of CCL2.0 reagent to ice and incubate at room temperature in the dark for 10 minutes.

[0030] 6. Detection of fluorescence value (RLU) using an ELISA reader.

[0031] Inhibition rate calculation formula:

[0032] Inhibition rate = (RLU) x -RLU1) / (RLU2-RLU1)

[0033] RLU x Fluorescence value of the antibody group to be tested

[0034] RLU1: Fluorescence value of ENTPD2+ATP group

[0035] RLU2: Fluorescence value of the ATP control group (equivalent to 100% inhibition rate of ENTPD2 enzyme by the antibody).

[0036] Advantages:

[0037] 5–15 μL of B cell supernatant can be directly detected without antibody purification.

[0038] A single batch of 96-well plates was tested within 2 hours.

[0039] 2. Cellular system detection (simulating the in vivo environment)

[0040] Material:

[0041] Human ENTPD2 overexpressing cells:

[0042] B16F10 hEN OE (mouse melanoma cells with endogenous Entpd2 knocked out and transfected with human ENTPD2)

[0043] HEK293 hEN OE (human embryonic kidney cells overexpressing human ENTPD2)

[0044] Negative control cells: B16F10 mEN KO or wild-type HEK293

[0045] ATP working solution (4440 nM)

[0046] Operating procedures:

[0047] 1. Cell preparation:

[0048] Cell density adjusted to 1×10 5 Cells / mL (DMEM medium, 10% FBS, heat-inactivated) were added to the cell suspension on ice according to the groupings in the table:

[0049]

[0050]

[0051] Add 65 μL of heat-inactivated DMEM medium to ice to make up the volume.

[0052] Incubate at 2.4°C or on ice for 15 minutes (to inhibit non-target reactions).

[0053] 3. Add 10 μL of ATP working solution to ice.

[0054] Incubate at 4.37℃ in two stages (15 minutes each).

[0055] Centrifuge at 5.4℃ (300g, 3 minutes) and collect the supernatant.

[0056] 6. Transfer 60 μL of supernatant to an ELISA plate on ice, add 20 μL of CCL2.0 for detection on ice, and incubate at room temperature in the dark for 10 minutes.

[0057] 7. Formula for calculating the inhibition rate of fluorescence value (RLU) detected by ELISA reader:

[0058] Inhibition rate = (RLU) x -RLU1) / (RLU2-RLU1)

[0059] RLU x : Antibody group fluorescence value

[0060] RLU1: Fluorescence value of overexpressing cells + ATP group (equivalent to 0% inhibition rate of antibody against ENTPD2 enzyme)

[0061] RLU2: Fluorescence value of negative cells + ATP group (equivalent to 100% inhibition rate of antibody against ENTPD2 enzyme)

[0062] Innovative Design:

[0063] Dual-temperature control: 4℃ blocks cell metabolism + 37℃ briefly activates ENTPD2

[0064] Untreated well plates: Using untreated wells reduces spontaneous ATP release from cells.

[0065] II. Core Innovation Points

[0066] 1. Function-oriented screening mechanism

[0067] Breaking through the limitations of traditional binding force detection, this method directly quantifies the ATP hydrolysis inhibition rate.

[0068] The positive control antibody mAb1 showed an inhibition rate of 84.7%.

[0069] The inhibition rate of the negative control mAb13 approached 0%.

[0070] 2. High-throughput primary screening without purification

[0071] Initial screening of 214 antibody strains takes only 3 days, reducing costs to 10% of traditional methods.

[0072] 3. Dual-system progressive verification

[0073] Biochemical system: Rapid initial screening in 2 hours

[0074] Cellular system: Validating the functionality of real membrane proteins in a real environment

[0075] 4. Optimization of key technologies

[0076] Complement elimination: Heat-inactivated culture medium destroys complement activity.

[0077] Detection window calibration: The effective inhibition rate was controlled within the linear range of 50%–100% by adjusting the ENTPD2 dose (1.667 ng / μL, final concentration 0.357 ng / μL) and ATP concentration (333 nM, final concentration 190 nM).

[0078] Bubble control: Detection bubbles are eliminated by puncturing with a heated needle tip.

[0079] III. Implementation Results Verification

[0080] 1. In vitro screening results

[0081] Five preferred antibodies (7B12, 11A2, 12G4, 13A4, and 16F8) all exhibited inhibition rates >70% in the cell system.

[0082] Correlation R with biochemical system results 2 =0.93

[0083] 2. In vivo efficacy verification

[0084] In the C57BL / 6J mouse B16F10 hEN OE xenograft model:

[0085] All antibodies significantly inhibited tumor growth (vs. PBS group p<0.01)

[0086] The top-tier antibody 7B12 achieved a tumor inhibition rate of 56.4% (endpoint volume 1017.1 mm). 3 vs PBS group 2330.8mm 3 )

[0087] The beneficial effects of this invention are:

[0088] 1. The residual amount of ATP hydrolysis was quantified in real time using the fluorescent probe CCL2.0, and the inhibition rate was calculated using the formula: Inhibition rate = (RLU of antibody group - RLU of enzyme reaction control group) / (RLU of ATP control group - RLU of enzyme reaction control group). This indicator directly characterizes the blocking efficiency of antibody against ENTPD2 catalytic function, thus avoiding ineffective antibodies with "high binding force and low inhibition" from the source.

[0089] 2. A unique approach utilizes B cell culture supernatant for direct detection (using only 5–15 μL), skipping the costly antibody purification step. Initial screening of 214 antibody strains can be completed within 3 days, increasing efficiency by more than 10 times compared to traditional methods and reducing costs to 10% of the original process.

[0090] 3. Under cell-free conditions, a reaction system was constructed using free ENTPD2 protein (final concentration 0.357 ng / μL) and low-concentration ATP (final concentration 190–222 nM). The reaction was precisely controlled at 37°C for 40 minutes to ensure full release of enzyme activity while avoiding non-specific hydrolysis. The positive control antibody mAb1 showed an inhibition rate of 84.7%, while the negative control mAb13 showed an inhibition rate close to zero, validating the system's specificity. Thirty highly effective antibodies were rapidly selected from a preliminary screening library of 214 strains.

[0091] 4. Using genetically engineered ENTPD2 overexpressing cells (B16F10 hEN OE or HEK293 hEN OE), a four-step temperature-controlled procedure was employed: antibody binding at 4℃ (inhibiting non-target reactions); short-term ATP hydrolysis at 37℃ (2 × 15 minutes, highlighting target enzyme activity); low-temperature centrifugation to collect the supernatant; and CCL2.0 detection of ATP residues. This design successfully simulated the working state of transmembrane enzymes in vivo, and the screening results were highly consistent with the biochemical system and positively correlated with subsequent animal efficacy.

[0092] 5. The culture medium is preheated at 60℃ for 45 minutes to completely destroy complement components and avoid antibody Fc fragment-mediated cytotoxicity; by adjusting the ENTPD2 dose and ATP concentration, the effective inhibition rate is precisely controlled within the linear range of 50%–100%, which avoids false negatives caused by enzyme overload (weak antibodies are masked) and prevents saturation effects caused by substrate overload (strong antibodies cannot distinguish); before enzyme labeling, a heated needle tip is used to puncture and eliminate air bubbles to ensure accurate and reliable fluorescence readings.

[0093] 6. The top-tier antibodies obtained through screening (such as 7B12 and 11A2) all showed significant tumor-suppressing effects in the C57BL / 6J mouse B16F10hEN OE xenograft model. The 7B12 antibody treatment group achieved a tumor volume of only 1017.1 mm² at the endpoint. 3 Compared with the blank control group (2330.8 mm) 3 The level decreased by 56.4%, which was better than the positive control mAb1 (1313.7 mm). 3 This fully demonstrates the high consistency between the screening system and the in vivo efficacy. Attached Figure Description

[0094] Figure 1 The results of antibody inhibition rate detection in the biochemical system of this invention;

[0095] Figure 2 This is the detection window of the cell system and the antibody verification results of the present invention;

[0096] Figure 3 The results of this invention validated antibody function in a tumor-bearing mouse (C57BL / 6J) model. Detailed Implementation

[0097] Please refer to the attached document. Figure 1-3 The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0098] In biochemical assay systems, the specific concentration of the antibody being tested is uncertain, and determining its concentration is challenging. This is because the binding affinity of these antibodies to the target antigen (ENTPD2 protein) is inconsistent and varies considerably, making commonly used antigen-antibody binding-dependent assays, such as ELISA, Western blotting, and affinity assays, unsuitable. While these traditional concentration detection methods can differentiate the binding affinity of different antibody candidates to the antigen to some extent, they fall far short of being economical and efficient. Furthermore, the complex substances contained in the cell culture medium, where antibody samples are dissolved, significantly impact the accuracy of such quantifications. Therefore, operators must consider the functional aspect directly. ENTPD2's function in the body is to degrade ATP, which occurs in a complex environment. A biochemical assay system designed by the operator can avoid the need for direct initial screening of purified antibodies, greatly reducing costs.

[0099] On the other hand, preliminary data also showed that antibodies with high affinity do not necessarily have a good inhibitory effect on hydrolysis, and the affinity ranking does not correspond to the inhibitory function ranking (this data is not presented in this paper). A plausible explanation is that different antibodies bind to different sites on the antigen. Some antibodies, although having high affinity and strong binding to the antigen, cannot effectively block the active domain for ATP hydrolysis due to their site selection, thus exhibiting lower inhibitory function. Traditional detection methods, such as ELISA and Western blotting, only assess specificity and binding affinity, and cannot distinguish the blocking ability of the active domain. Antibodies screened using these methods have unsatisfactory accuracy, requiring an increased number of candidate samples and subsequent large-scale expression and purification of antibodies for rescreening in animal models, which obviously greatly increases research and development costs.

[0100] Consistency control of the initial baseline of the test antibodies is achieved through consistency in the culture process, such as consistent culture conditions and time, and consistent sample volume during final testing. While this cannot completely guarantee consistent secretion concentrations for each antibody, it does not affect qualitative judgment. Some test antibodies may not be screened due to insufficient sample concentration. In such cases, the operator adopts a strategy of increasing the concentration and repeating the measurement. First, a 5 μL sample is taken for testing, then a 15 μL sample is taken. If the increased dosage still does not demonstrate the function of degrading ATP, then the antibody secreted by the monoclonal antibody can be considered non-functional or with very low function, unsuitable for the needs, and can be discarded. In fact, based on the test results, a 5 μL sample of a antibody with good function is sufficient to demonstrate the function of blocking ENTPD2 protein. To maximize the accuracy of initial screening, this method is used for repeated screening, which still has significant economic advantages. Furthermore, to provide optimal reaction conditions, the operator uses heat-inactivated B cell culture medium to dissolve and dilute the antibody, introducing a small amount of B cell culture medium into the system to simulate the original secretion environment of the antibody. The data results indicate that this has some benefit in broadening the detection window, but it is not a necessary factor. Operators can choose one of B cell culture medium, PBS, or DMEM culture medium based on their own laboratory conditions.

[0101] In biochemical assay systems, the presence of FBS in the culture medium, which contains complement components, can bind to the Fc fragment of IgG antibodies, interfering with detection. This binding reaction also occurs in cell assay systems, but the interference is even greater. This is because complement-dependent cytotoxicity leads to the lysis of overexpressing cells, thereby disrupting the assay system. Therefore, operators pre-treat the culture medium with heat to inactivate complement and eliminate potential adverse factors.

[0102] In cell line testing, this study explored two cell lines: HEK293 (modified human embryonic kidney cells) and B16F10 (modified mouse melanoma cells) (B16F10 hEN OE). The results showed consistency between the two cell lines, indicating their suitability for detecting ENTPD2's ATP degradation function. Constructing HEK293 hEN OE was less expensive because wild-type HEK293 cells could be used as control cells. Constructing B16F10 hEN OE was for preparing a mouse tumor model, involving the knockout of the B16F10 endogenous mouse Entpd2 gene, thus increasing costs. Users can choose the optimal method based on their specific needs.

[0103] This biochemical assay system offers a more precise framework for testing the effect of antibodies on the degradation of ATP by the ENTPD2 extracellular nucleotidase. Because the system avoids the influence of complex structures like cells and tissues, the focus is solely on the relationship between the antibody, enzyme, and substrate ATP. Besides its short testing time and convenience, this system offers the practical advantage of requiring only 5-15 μL of raw B cell culture supernatant, eliminating the need for large-scale antibody expression and purification, as well as ELISA, Western blotting, and affinity assays. Therefore, it is ideal for initial antibody screening, enabling high-throughput screening and rapid identification of functionally superior candidates from large numbers of newly produced B cell clones. Since the blocking and inhibitory function of the antibody is directly used as the evaluation indicator, the screening results are not only highly efficient but also accurate. The presence of the cell—the basic structural and functional unit of life—in this cell assay system more closely resembles a real-world environment. For example, the ENTPD2 enzyme is a transmembrane protein with its catalytic region facing outwards, unlike proteins in a completely free state found in biochemical systems. Therefore, this cell system can be used to further validate antibodies screened in biochemical systems. However, this cell-based detection system requires a large dose of antibody, which limits its application for large-scale primary screening. Operators used it for secondary screening and validation of primary screening results, and the results proved to be a satisfactory choice. After all, the screened antibodies are ultimately used for in vivo therapy, and guided by this goal, operators need to know whether the antibodies obtained from primary screening retain their function in the real in vivo environment. Currently, the best way to simulate the in vivo environment is through animal experiments, but this obviously requires a significant investment of resources and time, as well as even larger doses of antibodies. Compared to animal validation, using a cell system to perform functional validation on antibodies after large-scale expression and purification is also a suitable choice. This is because, compared to animal experiments, cell system detection requires a significantly smaller amount of antibody and shorter detection time; and compared to biochemical detection systems, the involvement of cells more closely approximates the real in vivo environment. In this study, to more accurately assess antibody function, the operators also conducted animal experiments, constructing a mouse tumor model to validate antibody function in vivo. The results showed that the monoclonal antibodies obtained from primary screening in the biochemical system exhibited significant inhibitory effects in both the cell system and in vivo evaluation. These results demonstrate that the antibody screening method reported in this patent is an economical, efficient, and accurate method that can significantly reduce the initial research and development costs of antibody drugs.

[0104] Regarding the establishment of the inhibition rate calculation formula involved in both procedures, the operators conducted a series of dosage experiments in the early stages (this data is not shown in this article). ATP concentration is positively correlated with the fluorescence intensity; the higher the concentration, the stronger the fluorescence signal. However, this linear relationship only exists between ATP and fluorescence intensity within a certain concentration range. The calculation formula established by the operators is only meaningful within this linear relationship. On the other hand, there is also the setting of the detection window. As can be seen from the results, the difference in fluorescence intensity between the ATP-only group and the ENTPD2+ATP group in the biochemical system represents the detection window. This window needs to be as wide as possible and within a suitable signal intensity range to better distinguish different antibodies. This needs to be adjusted by the dosage of ENTPD2 enzyme and the concentration of ATP. If this window is not properly adjusted, two extreme phenomena will occur. The first phenomenon is that the inhibition rate calculated for most antibodies is 0%. The reasons for this result are: (1) These antibodies have no inhibitory function, so their inhibition rate is naturally 0%; (2) These antibodies have inhibitory function, but it is only slightly lower. However, there is too much ENTPD2 enzyme or too little substrate ATP in the system. In either case, ATP is consumed prematurely, resulting in a false impression of an inhibition rate of 0%, which is a false negative. Of course, in practice, as long as there are still some antibodies whose calculated inhibition rate is >0%, these false impressions of 0% will not affect the operator's choice, unless they want to expand the sample size. This is because the operator only wants to obtain antibodies with good inhibitory function and can ignore antibodies with poor function. The second phenomenon is that some antibodies have a calculated inhibition rate of 100%. These antibodies are indeed antibodies with high inhibitory function, but from the perspective of system dosage, it is because there is too little ENTPD2 enzyme or too much substrate ATP, resulting in a large amount of ATP remaining after the reaction. In this case, the operator cannot distinguish which of these antibodies with an inhibition rate of 100% has a better inhibitory function. Operators can consider all these antibodies as potential candidates for achieving the target, but this data becomes less helpful when they wish to further reduce the sample size to minimize subsequent inputs. Therefore, the operator's criterion for selecting the dosage when setting the detection window is that the antibody with the best inhibition rate among the calculated inhibition rates should be within the range of 50% to 100%, while also ensuring that the ATP concentration is within the linear range. In the cell system, the detection window is the difference in signal intensity between the negative cell + ATP group and the positive cell + ATP group. In this system, factors such as ATP concentration and cell number need to be explored. Another point to emphasize in the cell detection system is the control of interfering factors. The operator's previous experimental data showed that a large amount of ATP is generated after cells begin to adhere, resulting in a strong background signal that affects evaluation. To solve this problem, the operator adopted two strategies: first, using cell culture plates without surface treatment to reduce the induction of adhesion; and second, strictly controlling the reaction temperature and time, as described below.The operator chose the negative cell + ATP group instead of the ATP only group for the upper limit of the detection port in the cell system. The main reason is that there are other reactions in the cells that are unrelated to the detection target but can cause drastic changes in the ATP content in the system.

[0105] Example 1: High-throughput primary screening in a biochemical system

[0106] 1. Collect the supernatant of 214 rabbit anti-human ENTPD2 B cells.

[0107] 2. Add the antibody sample to each well of a 96-well plate according to the groups in Table 1 (15 μL of antibody sample per well).

[0108] 3. Thirty antibodies with inhibition rates >50% were detected.

[0109] 4. Re-screening identified five candidate antibodies, including 7B12 (inhibition rate 92.3%) and 11A2 (89.7%).

[0110] Example 2: Functional Validation of Cell System

[0111] 1. Using B16F10 hEN OE cells (overexpressing human ENTPD2)

[0112] 2. Test the purified antibody (concentration 10 ng / μL, final concentration 1.25 ng / μL).

[0113] 3. Results: The 7B12 inhibition rate was 81.5%, consistent with the biochemical system.

[0114] Example 3: Verification of in vivo tumor suppression effect

[0115] 1. Mice were subcutaneously inoculated with B16F10 hEN OE cells (5 × 10⁻⁶). 5 (each mouse)

[0116] 2. Antibody treatment group (7B12, etc.) was administered via tail vein injection at a dose of 10 mg / kg, once in total. It was used in combination with anti-mouse PD1 antibody at a dose of 10 mg / kg, administered via intraperitoneal injection twice, with an interval of 3 days between doses.

[0117] 3. Results on day 21:

[0118] Group <![CDATA[Average tumor volume (mm 3 )]]> Tumor inhibition rate PBS 2330.8 - mAb1 1313.7 43.6％ 7B12 1017.1 56.4％

[0119] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes, characterized in that... Includes the following steps: In a biochemical system, ENTPD2 protein, the antibody to be tested, and ATP are mixed and incubated. The remaining ATP content was detected by adding the fluorescent probe CCL2.0; The antibody inhibition rate is calculated using the formula: inhibition rate = (RLUx - RLU1) / (RLU2 - RLU1), where RLUx is the fluorescence value of the antibody group, RLU1 is the fluorescence value of the ENTPD2+ATP group, and RLU2 is the fluorescence value of the ATP control group.

2. The efficient screening method for ATP hydrolase antibodies based on fluorescent probes according to claim 1, characterized in that: In the biochemical system, the concentration of ENTPD2 protein was 1.667 ng / μL (final concentration in the system was 0.357 ng / μL), and the concentration of ATP was 333 nM (final concentration in the system was 190–222 nM). Antibodies were prepared using B cell culture supernatant at a volume of 5–15 μL. The reaction conditions were 37°C with shaking for 40 minutes.

3. The high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes according to claim 1, characterized in that... include: ENTPD2 overexpressing cells (B16F10 hEN OE or HEK293 hEN OE) were co-incubated with the test antibody and ATP. Centrifuge and collect the supernatant; then determine the remaining ATP content. The inhibition rate is calculated using the formula: inhibition rate = (RLUx - RLU1) / (RLU2 - RLU1), where RLUx is the fluorescence value of the antibody group, RLU1 is the fluorescence value of the overexpressing cells + ATP group, and RLU2 is the fluorescence value of the negative control cells + ATP group.

4. The high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes according to claim 3, characterized in that: Cell incubation consists of two steps: antibody binding at 4°C for 15 min → reaction at 37°C twice (15 min each time); The ATP concentration was 4440 nM (final concentration in the system was 222 nM), and the cell density was 1 × 10⁻⁶. 5 cells / mL (final cell count in the system is 1×10⁶) 4 indivual).

5. The efficient screening method for ATP hydrolase antibodies based on fluorescent probes according to any one of claims 1–4, characterized in that… Also includes: Use heat-inactivated (60℃ for 45 min) B cell culture medium or DMEM culture medium; The entire reaction is performed on ice to avoid interference from non-target ATP hydrolysis reactions (or ATP generation reactions).

6. The application of the high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes according to claims 1-2 in screening anti-ENTPD2 antibodies, characterized in that: The antibody is used to treat diseases associated with abnormal ENTPD2 expression, including cancer, inflammatory bowel disease, liver fibrosis, and Alzheimer's disease.

7. The application of the high-efficiency screening method for ATP hydrolase antibodies based on fluorescent probes according to claims 3-4 in verifying antibody function in vivo, characterized in that: The antibody inhibited tumor growth in a tumor-bearing mouse model, reducing tumor volume by ≥30% (compared to the PBS group).

8. The screening kit for the high-efficiency screening method of ATP hydrolase antibodies based on fluorescent probes according to claims 1-7, characterized in that... Include: ENTPD2 protein (1.667 ng / μL, final concentration 0.357 ng / μL) or ENTPD2 overexpressing cell lines; ATP working solution (333 nM or 4440 nM, final concentration 190–222 nM); CCL2.0 fluorescence detection reagent; Positive control antibody (mAb1) and negative control antibody (mAb13).

9. The anti-ENTPD2 antibody according to claim 8, using a high-efficiency screening method for ATP hydrolases based on fluorescent probes, is characterized in that: The antibody is obtained by screening according to the method of claim 1 or 3, and is 7B12, 11A2, 12G4, 13A4 or 16F8.

10. The application of the antibody prepared by the high-efficiency screening method for ATP hydrolases based on fluorescent probes according to claim 9 in the preparation of antitumor drugs, characterized in that: The tumors include melanoma, cholangiocarcinoma, colorectal cancer, gastric cancer, breast cancer, esophageal cancer, leukemia, pancreatic cancer, lymphoma, liver cancer, lung cancer, bladder / urethral tumors, ovarian / fallopian tube tumors, prostate cancer, and head and neck tumors.

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