Method for detecting neutralizing antibody of double-antibody drug

Abstract

The invention relates to anti-drug neutralizing antibody analysis. In particular, the present application relates to methods for detecting neutralizing antibodies against a vascular endothelial growth factor (VEGF) binding moiety and a programmed cell death ligand-1 (PD-L1) binding moiety in a biological sample from an individual administered a bispecific antibody drug comprising the VEGF binding moiety.

Description

Technical Field

[0001] This application belongs to the field of biochemistry, mainly relates to a method for detecting neutralizing antibodies against bispecific antibody drugs, and more specifically relates to a method for detecting neutralizing antibodies against the vascular endothelial growth factor (VEGF) binding portion in a bispecific antibody drug comprising a programmed cell death ligand-1 (PD-L1) binding portion and a vascular endothelial growth factor (VEGF) binding portion. Background Art

[0002] Bispecific antibody drugs comprising a programmed cell death ligand-1 (PD-L1) binding portion and a vascular endothelial growth factor (VEGF) binding portion (hereinafter referred to as PD-L1 & VEGF bispecific antibody drugs) can, on the one hand, block the binding of PD-L1 to programmed cell death receptor-1 (PD-1), restore the ability of the immune system to recognize and kill tumor cells, and thus inhibit tumor cell immune escape; on the other hand, they can also inhibit tumor angiogenesis by neutralizing VEGF, improve the tumor microenvironment, and promote immune cell infiltration. PD-L1 & VEGF bispecific antibody drugs exhibit powerful anti-tumor effects by inhibiting these two pathways. However, the administration of antibody drugs for treatment may cause adverse immune reactions, resulting in the production of anti-drug antibodies (ADA). As a type of ADA, neutralizing antibodies (NAb) weaken the therapeutic effect of antibody drugs by preventing the drug from binding to the target or inhibiting downstream signal transduction after binding due to steric hindrance. Therefore, it is necessary to detect neutralizing antibodies during the treatment process. Summary of the Invention

[0003] This application provides a method for detecting neutralizing antibodies against the VEGF binding portion in a biological sample from an individual administered a bispecific antibody drug comprising a programmed cell death ligand-1 (PD-L1) binding portion and a vascular endothelial growth factor (VEGF) binding portion (abbreviated as PD-L1 & VEGF bispecific antibody drug), the method comprising: (1) treating the biological sample with a first acid digestion solution having a concentration of 500 - 1000 mM and a pH of 2.0 - 3.5; (2) contacting the treated biological sample with an anti-VEGF monospecific antibody comprising the VEGF binding portion immobilized on a first solid phase carrier to capture the neutralizing antibody in the biological sample; (3) Release the neutralizing antibody by acid dissociation using a second acid digestion solution to obtain a first neutralizing antibody sample; (4) Contact the first neutralizing antibody sample with vascular endothelial growth factor receptor (VEGFR) immobilized on a second solid-phase carrier to remove VEGF from the first neutralizing antibody sample and obtain a second neutralizing antibody sample; (5) Mix the second neutralizing antibody sample with the bispecific antibody drug with a detectable label to obtain a sample for detection; (6) Contact and incubate the sample for detection and a control sample prepared with an equal amount of the bispecific antibody drug with a detectable label with VEGF immobilized on a third solid-phase carrier; (7) Wash the third solid-phase carrier after incubation, and measure the intensity of the first detectable label signal for the sample for detection and the intensity of the second detectable label signal for the control sample; (8) Detect the neutralizing antibody in the biological sample by analyzing the intensity of the first detectable label signal and the intensity of the second detectable label signal.

[0004] In some embodiments, the capture in step (2) includes adding a neutralizing reagent to the biological sample.

[0005] In some embodiments, the neutralizing reagent is a tris(hydroxymethyl)aminomethane solution with a concentration of 1 M and a pH of 9.5; and / or the volume ratio of the neutralizing reagent to the volume of the biological sample used is about 3:10.

[0006] In some embodiments, the first acid digestion solution is acetic acid with a concentration of 600 mM and a pH of 2.5.

[0007] In some embodiments, the first acid digestion solution is acetic acid with a concentration of 800 mM and a pH of 3.0.

[0008] In some embodiments, the second acid digestion solution is acetic acid with a concentration of 300 mM and a pH of 3.0.

[0009] In some embodiments, the detectable label is a metal label.

[0010] In some embodiments, the metal label is a ruthenium label.

[0011] In some embodiments, the signal intensity is the electrochemiluminescence intensity.

[0012] In some embodiments, the biological sample is a plasma or serum sample. Detailed implementation manners

[0013] To facilitate the understanding of this application, some terms used herein are first defined.

[0014] As used herein, "affinity capture" refers to capture based on the principle of specific binding between antigen and antibody, such as using a specific antigen to capture an antibody. In some embodiments of this application, the anti-drug neutralizing antibody in the acid-treated sample binds to the drug (equivalent to an antigen) coated on the microplate under neutral pH conditions, thereby achieving the capture of the antibody. In some embodiments of this application, the drug in the anti-drug neutralizing antibody is a PD-L1 & VEGF bispecific antibody drug. In some embodiments of this application, the anti-drug neutralizing antibody is a neutralizing antibody against the VEGF-binding portion of the PD-L1 & VEGF bispecific antibody drug (hereinafter referred to as anti-VEGF neutralizing antibody).

[0015] As used herein, "acid dissociation" refers to dissociating the drug-antibody neutralizing antibody complex into the anti-drug neutralizing antibody by acidifying the sample. The maximum purpose of acid dissociation is to enable the anti-drug neutralizing antibody that was originally bound to the drug to be detected. In some embodiments of this application, the drug in the anti-drug neutralizing antibody is a PD-L1 & VEGF bispecific antibody drug. In some embodiments of this application, the anti-drug neutralizing antibody is an anti-VEGF neutralizing antibody.

[0016] As used herein, the term "drug resistance" means that the biological sample may contain a high concentration of free drug, which can compete with the detection reagent for binding to the anti-drug neutralizing antibody, thereby interfering with the detection of the anti-drug neutralizing antibody and resulting in false negative results. In the embodiments of this application, the anti-drug neutralizing antibody is an anti-VEGF neutralizing antibody.

[0017] Unless otherwise specified, "acid solution I" and "first acid solution" used in this specification have the same meaning, and "acid solution II" and "second acid solution" have the same meaning; "bispecific antibody drug comprising a PD-L1 binding portion and a VEGF binding portion" and "PD-L1 & VEGF bispecific antibody drug" have the same meaning, "anti-VEGF monospecific antibody comprising a VEGF binding portion" and "anti-VEGF monoclonal antibody" have the same meaning, and "anti-VEGF neutralizing antibody" refers to a neutralizing antibody against the VEGF-binding portion of the PD-L1 & VEGF bispecific antibody drug.

[0018] Currently, the detection of neutralizing antibodies targeting the VEGF-binding portion of PD-L1 & VEGF dual-antibody drugs faces the following challenges: Compared with single-target drugs, dual-antibody drugs have complex structures and target binding, which affects the detection sensitivity and drug tolerance of the method. Since the VEGF signaling pathway, when inhibited, upregulates VEGF expression through a negative feedback mechanism to maintain angiogenesis balance, some patients experience elevated VEGF concentrations during treatment with PD-L1 & VEGF dual-antibody drugs. In neutralizing antibody testing, acid hydrolysis can break open the drug-target complex in the sample, resulting in high levels of free VEGF. Free VEGF binds to the drug in the reaction system, making it prone to false-positive signals in the test. Furthermore, residual drug in the test sample may bind to the neutralizing antibody, leading to false-negative signals in the test.

[0019] Therefore, the method developed in the present invention for detecting neutralizing antibodies against the vascular endothelial growth factor (VEGF) binding portion of PD-L1 & VEGF dual-antibody drugs eliminates interference from targets and drugs while also meeting the sensitivity requirements of detection, which is of great significance.

[0020] The present application establishes a method for detecting neutralizing antibodies against programmed cell death ligand-1 (PD-L1) binding portion and vascular endothelial growth factor (VEGF) binding portion in biological samples from individuals who have been administered a bispecific antibody drug comprising the VEGF binding portion (hereinafter referred to as the PD-L1 & VEGF bispecific antibody drug). The advantages of the method include at least improvement in at least one of the detection sensitivity, drug tolerance, and target molecule interference issues.

[0021] As a specific example, the detection method of the present application can be based on the angiotensin-converting enzyme (ACE) method of the competitive ligand binding assay (CLBA) technology of the MSD (Meso Scale Discovery) platform: (1) First, coat a 96-well ELISA plate with an anti-VEGF monospecific antibody (hereinafter referred to as anti-VEGF mAb) against the VEGF-binding portion of the PD-L1 & VEGF bispecific antibody drug, so that the anti-VEGF mAb is firmly captured on the ELISA plate. Treat the sample with acid digestion solution I (e.g., acetic acid at 600 mM, pH 2.5), and then add the neutralization reagent and the pretreated sample to the ELISA plate that has pre-captured the test drug and has been blocked, and incubate overnight with shaking at room temperature to ensure that the neutralizing antibody (abbreviated as NAb) in the sample forms an NAb-anti-VEGF mAb complex with the anti-VEGF mAb on the ELISA plate. At the same time, the target protein VEGF in the sample forms a VEGF-anti-VEGF mAb complex with the drug on the ELISA plate.

[0022] (2) After washing the plate, add acid digestion solution II (e.g., acetic acid at 300 mM, pH 3.0) to dissociate the NAb-anti-VEGF mAb and VEGF-anti-VEGF mAb complexes. Add the neutralization reagent and the sample dissociated in the previous step to a 96-well ELISA plate that has pre-captured VEGFR and has been blocked, and incubate with shaking at room temperature to remove interference, so as to remove the interference of VEGF in the sample.

[0023] (3) Mix the supernatant with a detection reagent (e.g., a PD-L1 & VEGF bispecific antibody drug with a detectable label) in an incubation plate for an oscillating reaction, and then add it to an MSD microplate that has been blocked and has pre-captured VEGF. Incubate with shaking at room temperature, wash the plate, and then add MSD Read Buffer T (2×), and read the instrument signal on a MESO QUICKPLEX SQ120; if there is no neutralizing antibody in the sample, VEGF in the system can fully bind to the detection reagent, and the instrument response value (ECLU) read on the electrochemiluminescence detection instrument is high, and the signal-to-noise ratio for the negative control sample is higher and the inhibition rate is lower; if the sample contains a neutralizing active antibody, its ECLU value is low, then its signal-to-noise ratio is lower and the inhibition rate is higher.

[0024] Unless otherwise specified, the implementation of this application uses conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques in the art.

[0025] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0026] This application provides a method for detecting neutralizing antibodies against the VEGF-binding portion in a biological sample from an individual administered a bispecific antibody drug comprising a PD-L1-binding portion and a VEGF-binding portion, the method comprising: (1) Treating the biological sample with a first acid digestion solution at a concentration of 500-1000 mM and a pH of 2.0-3.5; (2) Contact the processed biological sample with an anti-VEGF monospecific antibody comprising the VEGF-binding moiety immobilized on a first solid-phase carrier to capture the neutralizing antibody in the biological sample; (3) Release the neutralizing antibody by acid dissociation using a second acidolysis solution to obtain a first neutralizing antibody sample; (4) Contact the first neutralizing antibody sample with a vascular endothelial growth factor receptor (VEGFR) immobilized on a second solid-phase carrier to remove VEGF from the first neutralizing antibody sample and obtain a second neutralizing antibody sample; (5) Mix the second neutralizing antibody sample with the bispecific antibody drug with a detectable label to obtain a sample for detection; (6) Contact and incubate the sample for detection and a control sample prepared with an equal amount of the bispecific antibody drug with a detectable label with VEGF immobilized on a third solid-phase carrier respectively; (7) Wash the third solid-phase carrier after incubation and measure the intensity of the first detectable label signal for the sample for detection and the intensity of the second detectable label signal for the control sample; (8) Detect the neutralizing antibody in the biological sample by analyzing the intensity of the first detectable label signal and the intensity of the second detectable label signal.

[0027] In some embodiments, the first acidolysis solution is acetic acid at a concentration of 300 mM, 600 mM or 800 mM. In some embodiments, the pH of the first acidolysis solution is 1.5, 2.0, 2.5, 3.0, 3.5 or 4.0. Preferably, in some embodiments, the first acidolysis solution is acetic acid at a concentration of 600 mM and pH 2.5 and / or the first acidolysis solution is acetic acid at a concentration of 800 mM and pH 3.0.

[0028] In some embodiments, the volume ratio of the biological sample to the first acidolysis solution is 1:1 - 1:100, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, or the range between any two of the above ratio values. In some embodiments, the volume ratio of the biological sample to the first acidolysis solution is 1:1 1:75, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or a range between any two of the above ratio values. In some specific embodiments, the volume ratio of the biological sample to the first acid digestion solution with a concentration of 600 mM and pH 2.5 is 1:5.

[0029] In some embodiments, acid-treating the biological sample with the first acid digestion solution can improve the detection sensitivity. In some embodiments, acid-treating the biological sample with the first acid digestion solution can improve the drug resistance.

[0030] In some embodiments, before adding the acid-treated biological sample to the first solid phase carrier for capturing the anti-VEGF neutralizing antibody, a neutralizing reagent needs to be added to the first solid phase carrier.

[0031] In some embodiments, the capture includes adding a neutralizing reagent and the biological sample acid-treated with the first acid digestion solution to the first solid phase carrier coated with anti-VEGF monoclonal antibody, wherein the neutralizing reagent is a tris(hydroxymethyl)aminomethane (Trizma) solution with a concentration of 1 M and pH 9.5, and the volume ratio of the neutralizing reagent to the acid-treated sample is 3:10. In some embodiments, the capture includes adding 30 μL of the neutralizing reagent and 100 μL of the acid-treated biological sample to the first solid phase carrier coated with anti-VEGF monoclonal antibody.

[0032] In some embodiments, the neutralizing reagent can be a pH reagent that can make the binding process between the anti-VEGF neutralizing antibody and the anti-VEGF monoclonal antibody in the PD-L1 & VEGF bispecific drug neutral. In some embodiments, the neutralizing reagent is a tris(hydroxymethyl)aminomethane solution with a concentration of 1 M and pH 9.5.

[0033] In some embodiments, the biological sample in the captured first solid phase carrier is dissociated using a second acid digestion solution. The purpose is to release (1) the anti-VEGF neutralizing antibody captured by the anti-VEGF monoclonal antibody in the first solid phase carrier, and (2) the anti-VEGF neutralizing antibody originally bound to the PD-L1 & VEGF bispecific drug, thereby obtaining a first neutralizing antibody sample.

[0034] In some embodiments, the second acid digestion solution is acetic acid with a concentration of 300 mM and pH 3.0.

[0035] In some embodiments, the interference removal process needs to be carried out under neutral pH conditions. In some embodiments, before adding the first neutralized antibody sample after acid dissociation to the second solid-phase carrier for VEGF removal treatment, a neutralizing reagent needs to be added to the second solid-phase carrier. In some embodiments, the neutralizing reagent can be a reagent that can make the binding process between VEGFR that specifically binds VEGF and VEGF neutral at pH. In some embodiments, the neutralizing reagent is a tris(hydroxymethyl)aminomethane solution with a concentration of 1 M and a pH of 9.5. The sample after VEGF removal is called the second neutralized antibody sample.

[0036] In some embodiments, the detectable label is a metal label, such as a ruthenium (Ru) label. In some embodiments, the bispecific antibody drug is labeled with ruthenium to make the reaction system more stable.

[0037] In some embodiments, the signal intensity is the electrochemiluminescence intensity. In some embodiments, an exemplary example of the electrochemiluminescence method based on the ruthenium label is the MSD method, which uses an MSD plate coated with streptavidin and an MSD Read Buffer T working solution for electrochemiluminescence detection. In some embodiments, an electrochemiluminescence detection is performed using a plate reader, program, and kit commercially available from Meso ScaleDiscovery Inc.

[0038] In some embodiments, the signal intensity of the first detectable label is the signal intensity of the test sample containing the anti-VEGF neutralizing antibody to be detected; the signal intensity of the second detectable label is the signal intensity of the serum (such as human serum) sample that does not contain the anti-VEGF neutralizing antibody to be detected.

[0039] In some embodiments, the solid-phase carrier can be a microplate, where the first solid-phase carrier and the second solid-phase carrier are ELISA plates, and the third solid-phase carrier is an MSD plate.

[0040] In some embodiments, a PD-L1 & VEGF bispecific antibody (hereinafter referred to as PD-L1 & VEGF bisAb) is used to capture the anti-VEGF neutralizing antibody in the biological sample. In some embodiments, the anti-VEGF monoclonal antibody has a higher coating efficiency than the PD-L1 & VEGF bispecific antibody. In some embodiments, compared with using the PD-L1 & VEGF bispecific antibody, the ability to capture the anti-VEGF neutralizing antibody using the anti-VEGF monoclonal antibody is stronger.

[0041] In some embodiments, serum (such as human serum) is used to dilute the anti-VEGF neutralizing antibody into test samples at the following concentrations, such as 1 - 10 ng / mL, 1 - 20 ng / mL, 1 - 30 ng / mL, 1 - 40 ng / mL, 1 - 50 ng / mL, 1 - 60 ng / mL, 1 - 70 ng / mL, 1 - 80 ng / mL, 1 - 90 ng / mL, 1 - 100 ng / mL, 1 - 150 ng / mL, 1 - 200 ng / mL, 1 - 250 ng / mL, 1 - 300 ng / mL, 1 - 350 ng / mL, 1 - 400 ng / mL, 1 - 450 ng / mL, 1 - 500 ng / mL, 1 - 550 ng / mL, 1 - 600 ng / mL, 1 - 650 ng / mL, 1 - 700 ng / mL, 1 - 750 ng / mL, 1 - 800 ng / mL, 1 - 850 ng / mL, 1 - 900 ng / mL, 1 - 950 ng / mL, 1 - 1000 ng / mL, 1 - 2000 ng / mL, 1 - 3000 ng / mL, 1 - 4000 ng / mL, 1 - 5000 ng / mL, 1 - 6000 ng / mL or 1 - 7000 ng / mL. In some embodiments, serum (such as human serum) is used to dilute the anti-VEGF neutralizing antibody into test biological samples at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2000, 3000, 4000, 5000, 6000 or 7000 ng / mL. In some specific embodiments, serum (such as human serum) is used to dilute the anti-VEGF neutralizing antibody into test biological samples at 100, 200, 400, 800, 1600, 3200, 6400 ng / mL.

[0042] In some embodiments, the method is based on the ACE method of the MSD platform CLBA technology and has at least one of the following advantages: It solves the interference of endogenous VEGF; It solves the influence of high-dose drug tolerance on the detection of anti-VEGF neutralizing antibody activity; and It improves the sensitivity of the method.

[0043] In some embodiments, the sensitivity of the method is 100 ng / mL.

[0044] In some embodiments, when the concentration of the anti-VEGF neutralizing antibody is 500 ng / mL, the tolerable concentration of the PD-L1 & VEGF bispecific antibody drug can be increased to 200 μg / mL.

[0045] It should be understood that the above detailed description is only to make those skilled in the art more clearly understand the content of the present application, and is not intended to limit in any way. Those skilled in the art can make various changes and modifications to the described embodiments.

[0046] The following examples are only for the purpose of illustration and not for limiting the scope of the present application.

[0047] Examples

[0048] The present application will be described in more detail by way of specific examples. The following examples are provided only for illustrative purposes and are not intended to limit the present application in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially the same results.

[0049] Unless otherwise specified, the reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0050] I. Materials and Methods

[0051] The drugs and reagents used in the present application are as shown below: ELISA Plate 1 Capture Reagent Working Solution: Anti-VEGF monoclonal antibody (commercially purchased, batch number: 20240831 CSX, concentration: 2.53 mg / mL), diluted to 10 μg / mL using 1× carbonate buffer solution (CBS).

[0052] Neutralizing Reagent: Tris (hydroxymethyl) aminomethane (Trizma) neutralizing buffer solution.

[0053] ELISA Plate 2 Capture Reagent (Interference Removal Reagent) Working Solution: Vascular endothelial growth factor receptor (VEGFR) (manufacturer: R&D Systems, product number: 321-FL-050 / CF, 5 mg / vial), diluted to 10 μg / mL using 1× carbonate buffer solution (CBS).

[0054] MSD Plate Capture Reagent Working Solution: Vascular endothelial growth factor (VEGF) (manufacturer: Acro BiosystemsAcro Biosystems, product number: VE5-H5248, batch number: 2410-234HF1-1CB

[0055] Specification: 50 μg / vial, dissolve to 1 mg / mL according to the instruction manual, and dilute to 10 μg / mL with 1% BSA.

[0056] Detection reagent working solution: Ru-labeled PD-L1 & VEGF bispecific antibody drug, abbreviated as Ru-drug, (manufacturer: Antengene, batch number: 20220711, concentration: 351 μg / mL) dilute to 100 ng / mL with PBS containing 1% BSA.

[0057] Neutralizing antibody: Recombinant anti-VEGFR1 monoclonal antibody (manufacturer: Immunooncology Biopharma, batch number: 20230913, concentration: 6.15 mg / mL).

[0058] The specific experimental steps for detecting the neutralizing antibody (abbreviated as anti-VEGF neutralizing antibody) against the VEGF-binding part in the PD-L1 & VEGF bispecific antibody drug in biological samples using anti-VEGF monoclonal antibody are shown in the following table:

[0059] II. Experimental Results

[0060] The calculation formula for "% inhibition" in sensitivity and drug resistance is as follows:

[0061] Among them, the concentration of anti-VEGF neutralizing antibody in the control sample is 0 ng / mL.

[0062] 1. Effects of treating samples with acetic acid solutions of different concentrations or different pH values on sensitivity and drug resistance.

[0063] Table 1. Sensitivity data at different pH values when the sample is acid-treated with acetic acid at a concentration of 300 mM.

[0064]

[0065] Table 2. Sensitivity data at different pH values when the sample is acid-treated with acetic acid at a concentration of 600 mM.

[0066]

[0067] Table 3. Sensitivity data at different pH values when the sample is acid-treated with acetic acid at a concentration of 800 mM.

[0068]

[0069] Table 4. Drug resistance data at different pH values when the sample is acid-treated with acetic acid at a concentration of 300 mM (neutralizing concentration antibody is 500 ng / mL).

[0070]

[0071] Table 5. Drug resistance data at different pH values when the sample is acid-treated with acetic acid at a concentration of 600 mM (neutralizing concentration of antibody is 500 ng / mL).

[0072]

[0073] Table 6. Drug resistance data at different pH values when the sample is acid-treated with acetic acid at a concentration of 800 mM (neutralizing concentration of antibody is 500 ng / mL).

[0074]

[0075] The data in Tables 1-6 show that: By comparison, it is found that in all systems with a pH of 1.5, the signal values do not show a gradient, that is, there are no sensitivity and drug resistance data. When the acid treatment is carried out with 300 mM acetic acid in the other sample addition systems except pH 1.5, the sensitivity meets the requirements and can reach 100 ng / mL, but the drug resistance is poor, and as the drug concentration increases, there is a reverse inhibition phenomenon of drug resistance. When the acid treatment is carried out with 600 mM acetic acid, the sensitivity of the other sample addition systems except pH 1.5 meets the requirements and can reach 100 ng / mL. Among them, when the pH is 2.5, the drug resistance is the best and can reach 200 μg / mL, while the drug resistance is poor at other pH values. Among them, when the pH is 3.0, 3.5 or 4.0, there is a reverse inhibition phenomenon of drug resistance as the drug concentration increases. When the acid treatment is carried out with 800 mM acetic acid, the sensitivity of the other sample addition systems except pH 1.5 meets the requirements and can reach 100 ng / mL. Among them, when the pH is 3.0, the drug resistance is the best and can reach 200 μg / mL, while the drug resistance is poor at other pH values. Among them, when the pH is 2.5, 3.5 or 4.0, there is a reverse inhibition phenomenon of drug resistance as the drug concentration increases. The threshold range of % inhibition is 15%-20%.

[0076] 2. Effect of using PD-L1 & VEGF bispecific antibody and anti-VEGF monoclonal antibody to capture neutralizing antibody.

[0077] Table 7. Comparison results of sensitivity data of ELISA plates coated with PD-L1 & VEGF bispecific antibody and anti-VEGF monoclonal antibody.

[0078]

[0079] Table 8. Comparison results of drug resistance data when the concentration of neutralizing antibody is 500 ng / mL, using the bispecific antibody of PD-L1 & VEGF and anti-VEGF monoclonal antibody to coat the ELISA plate.

[0080]

[0081] The data in Table 7 - Table 8 show that: By comparison, in all acid hydrolysis systems, compared with coating with the bispecific antibody of PD-L1&VEGF, the inhibition rate in the system coated with anti-VEGF monoclonal antibody is higher, indicating that monospecific antibodies can capture more neutralizing antibodies and have better sensitivity and drug resistance. Among them, the drug resistance is the best when treated with acetic acid at 600 mM, pH 2.5, while the phenomenon of reverse inhibition of drug resistance occurs when treated with acetic acid at other concentrations. The threshold range of % inhibition is 15% - 20%.

[0082] 3. Removal effect of the interference removal reagent on free VEGF.

[0083] Table 9. Data comparison of applying the interference removal reagent (VEGFR) and not applying the interference removal reagent (NA).

[0084]

[0085] Table 10. Data comparison of sensitivity of applying the interference removal reagent (VEGFR) and not applying the interference removal reagent (NA).

[0086]

[0087] Table 11. Data comparison of drug resistance when the concentration of neutralizing antibody is 500 ng / mL, applying the interference removal reagent (VEGFR) and not applying the interference removal reagent (NA).

[0088]

[0089] The data in Table 9 - Table 11 show that: The interference removal reagent (VEGFR) can specifically bind to free VEGF and effectively remove the interference of VEGF in the reaction system. At the same time, through comparison, it is found that the sensitivity and drug resistance are not affected after using the interference removal reagent. The sensitivity can reach 100 ng / mL, and when the drug concentration is 500 ng / mL, the drug resistance can reach 200 μg / mL. The threshold range of % inhibition is 15% - 20%.

[0090] All patents, patent application publications, and non-patent literature mentioned and / or listed in this application are hereby incorporated by reference in their entirety. The exemplary embodiments of the inventions of this application have been described above. However, without departing from the essence and scope of this application, those skilled in the art can modify or improve the exemplary embodiments described in this application, and the resulting variant embodiments or equivalent embodiments also fall within the scope of this application.

Claims

1. A method for detecting neutralizing antibodies against a vascular endothelial growth factor (VEGF) binding portion in a biological sample from an individual administered with a bispecific antibody drug comprising a programmed cell death ligand-1 (PD-L1) binding portion and a VEGF binding portion, the method comprising: (1) treating the biological sample with a first acid digestion solution having a concentration of 500 - 1000 mM and a pH of 2.0 - 3.5; (2) contacting the treated biological sample with a VEGF-specific antibody comprising the VEGF binding portion immobilized on a first solid phase carrier to capture the neutralizing antibodies in the biological sample; (3) releasing the neutralizing antibodies by acid dissociation using a second acid digestion solution to obtain a first neutralizing antibody sample; (4) contacting the first neutralizing antibody sample with a vascular endothelial growth factor receptor (VEGFR) immobilized on a second solid phase carrier to remove VEGF from the first neutralizing antibody sample and obtain a second neutralizing antibody sample; (5) mixing the second neutralizing antibody sample with the bispecific antibody drug with a detectable label to obtain a test sample; (6) separately contacting and incubating the test sample and a control sample prepared with an equal amount of the bispecific antibody drug with the detectable label with VEGF immobilized on a third solid phase carrier; (7) washing the third solid phase carrier after incubation, and measuring a first detectable label signal intensity for the test sample and a second detectable label signal intensity for the control sample; (8) detecting the neutralizing antibodies in the biological sample by analyzing the first detectable label signal intensity and the second detectable label signal intensity.

2. The method according to claim 1, wherein the capture in step (2) comprises adding a neutralizing reagent to the biological sample.

3. The method according to claim 2, wherein the neutralizing reagent is a tris(hydroxymethyl)aminomethane solution having a concentration of 1 M and a pH of 9.5; and / or the volume ratio of the neutralizing reagent to the volume of the biological sample used is 3:

10.

4. The method according to claim 1, wherein the first acid digestion solution is acetic acid having a concentration of 600 mM and a pH of 2.

5.

5. The method according to claim 1, wherein the first acid digestion solution is acetic acid having a concentration of 800 mM and a pH of 3.

0.

6. The method according to claim 1, wherein the second acid digestion solution is acetic acid having a concentration of 300 mM and a pH of 3.

0.

7. The method according to claim 1, wherein the detectable label is a metal label.

8. The method according to claim 7, wherein the metal label is a ruthenium label.

9. The method according to claim 1, wherein the signal intensity is an electrochemiluminescence intensity.

10. The method according to any one of claims 1 - 9, wherein the biological sample is a plasma or serum sample.

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