Method for detecting neutralizing antibodies to biologic drugs

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biochemistry, and relates to a method for detecting neutralizing antibodies of a bispecific antibody drug, and more particularly to a method for detecting neutralizing antibodies against a vascular endothelial growth factor (VEGF) binding moiety in a bispecific antibody drug comprising a programmed cell death ligand-1 (PD-L1) binding moiety and a VEGF binding moiety. BACKGROUND

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

[0003] The present application provides a method for detecting neutralizing antibodies against a VEGF binding moiety in a biological sample from an individual administered with a bispecific antibody drug comprising a programmed cell death ligand-1 (PD-L1) binding moiety and a VEGF binding moiety (hereinafter referred to as a PD-L1 & VEGF bispecific antibody drug), the method comprising:

[0004] (1) treating the biological sample with a first acid solution having a concentration of 500-1000 mM and a pH of 2.0-3.5;

[0005] (2) The treated biological sample is contacted with an anti-VEGF monospecific antibody containing the VEGF binding portion immobilized on a first solid support to capture the neutralizing antibody in the biological sample;

[0006] (3) The neutralizing antibody is released by acid dissociation using a second acid hydrolysate to obtain a first neutralizing antibody sample;

[0007] (4) Contact the first neutralizing antibody sample with the vascular endothelial growth factor receptor (VEGFR) immobilized on the second solid-phase support to remove VEGF from the first neutralizing antibody sample and obtain the second neutralizing antibody sample;

[0008] (5) The second neutralizing antibody sample is mixed with the bispecific antibody drug with a detectable label to obtain a sample for detection;

[0009] (6) The test sample and the control sample prepared with an equal amount of the bispecific antibody drug with a detectable label are respectively contacted with VEGF immobilized on a third solid support and incubated.

[0010] (7) After incubation, wash the third solid support and measure the intensity of the first detectable label signal for the test sample and the intensity of the second detectable label signal for the control sample.

[0011] (8) The neutralizing antibody in the biological sample is detected by analyzing the intensity of the first detectable label signal and the intensity of the second detectable label signal.

[0012] In some implementations, the capture in step (2) includes adding a neutralizing agent to the biological sample.

[0013] In some embodiments, the neutralizing agent is a 1 M, pH 9.5 solution of tris(hydroxymethyl)aminomethane; and / or the volume ratio of the neutralizing agent to the biological sample used is approximately 3:10.

[0014] In some embodiments, the first acid hydrolysate is acetic acid with a concentration of 600 mM and a pH of 2.5.

[0015] In some embodiments, the first acid hydrolysate is acetic acid with a concentration of 800 mM and a pH of 3.0.

[0016] In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 3.0.

[0017] In some implementations, the detectable marker is a metallic marker.

[0018] In some implementations, the metal marker is a ruthenium marker.

[0019] In some implementations, the signal intensity is the electrochemiluminescence intensity.

[0020] In some embodiments, the biological sample is a plasma or serum sample. Detailed Implementation

[0021] To facilitate understanding of this application, some terms used herein are first defined.

[0022] 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 the antigen) coated on a microplate under neutral pH conditions, thus achieving antibody capture. In some embodiments of this application, the drug in the anti-drug neutralizing antibody is a PD-L1 & VEGF bispecific antibody. In some embodiments of this application, the anti-drug neutralizing antibody is a neutralizing antibody targeting the VEGF binding portion of the PD-L1 & VEGF bispecific antibody (hereinafter referred to as anti-VEGF neutralizing antibody).

[0023] As used herein, "acid dissociation" refers to the process of acidifying a sample to dissociate a drug-antibody neutralizing antibody complex into an antidrug-neutralizing antibody. The primary purpose of acid dissociation is to make the previously drug-bound antidrug-neutralizing antibody detectable. In some embodiments of this application, the drug in the antidrug-neutralizing antibody is a PD-L1 & VEGF bispecific antibody. In some embodiments of this application, the antidrug-neutralizing antibody is an anti-VEGF neutralizing antibody.

[0024] As used herein, the term "drug resistance" refers to the possibility that a biological sample may contain high concentrations of free drug that can compete with detection reagents for binding to antidrug-neutralizing antibodies, thereby interfering with the detection of antidrug-neutralizing antibodies and leading to false negative results. In the embodiments of this application, the antidrug-neutralizing antibody is an anti-VEGF neutralizing antibody.

[0025] Unless otherwise specified, the terms “acid hydrolysate I” and “first acid hydrolysate” are equivalent in this specification, as are “acid hydrolysate II” and “second acid hydrolysate”; “bispecific antibody drug containing PD-L1 binding and VEGF binding moieties” is equivalent to “PD-L1 & VEGF bispecific antibody drug”; “anti-VEGF monospecific antibody containing VEGF binding moieties” is equivalent to “anti-VEGF monoclonal antibody”; and “anti-VEGF neutralizing antibody” refers to a neutralizing antibody against the VEGF binding moieties of the PD-L1 & VEGF bispecific antibody drug.

[0026] Currently, the detection of neutralizing antibodies targeting the VEGF binding site in PD-L1 & VEGF bispecific anti-inflammatory drugs faces the following challenges: Bispecific anti-inflammatory drugs have more complex structures and binding mechanisms with the target compared to single-target drugs, affecting the detection sensitivity and drug tolerability of the method; because 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 PD-L1 & VEGF bispecific anti-inflammatory drug treatment. In neutralizing antibody detection, acid hydrolysis opens the drug-target complex in the sample, resulting in higher levels of free VEGF. Free VEGF binds to the drug in the reaction system, easily leading to false positive signals; simultaneously, residual drug in the test sample may bind to the neutralizing antibody, resulting in false negative signals.

[0027] Therefore, the method developed in this invention for detecting neutralizing antibodies against the vascular endothelial growth factor (VEGF) binding site in PD-L1 & VEGF bispecific anti-drugs removes interference from the target and the drug, while also meeting the sensitivity requirements for detection, and is of great significance.

[0028] This application establishes a method for detecting neutralizing antibodies against the VEGF binding moiety in biological samples from individuals who have been administered a bispecific antibody drug containing a programmed death ligand-1 (PD-L1) binding moiety and a vascular endothelial growth factor (VEGF) binding moiety (hereinafter referred to as PD-L1 & VEGF bispecific antibody drug). The advantages of the method include at least improvements in at least one of the following: detection sensitivity, drug tolerance, and target molecule interference.

[0029] As a specific example, the detection method of this application can be based on the angiotensin-converting enzyme (ACE) method using the competitive ligand binding assay (CLBA) technology on the MSD (Meso Scale Discovery) platform:

[0030] (1) First, a 96-well ELISA plate was coated with an anti-VEGF monospecific antibody (hereinafter referred to as anti-VEGF monoclonal antibody) targeting the VEGF binding portion of the PD-L1 & VEGF bispecific antibody drug to ensure that the anti-VEGF monoclonal antibody was firmly captured on the ELISA plate. The sample was acidified with acid hydrolysis solution I (e.g., 600 mM, pH 2.5 acetic acid). Then, the neutralizing reagent and the pretreated sample were added to the pre-captured and blocked ELISA plate and incubated overnight at room temperature with shaking to ensure that the neutralizing antibody (NAb) in the sample formed an NAb-anti-VEGF monoclonal antibody complex with the anti-VEGF monoclonal antibody in the ELISA plate, and that the target protein VEGF in the sample formed a VEGF-anti-VEGF monoclonal antibody with the drug in the ELISA plate.

[0031] (2) After washing the plate, add acid hydrolysis solution II (e.g., 300 mM, pH 3.0 acetic acid) to dissociate NAb-antiVEGF monoclonal antibody and VEGF-antiVEGF monoclonal antibody complex. Add the neutralizing reagent and the sample after dissociation in the previous step to a 96-well microplate that has been pre-captured for VEGFR and sealed, and incubate at room temperature with shaking to remove interference and eliminate VEGF interference in the sample.

[0032] (3) Take the supernatant and the detection reagent (e.g., a bispecific antibody drug with detectable label PD-L1 & VEGF) and mix them in an incubation plate. After shaking the reaction, add the mixture to a blocked MSD microplate that has been pre-captured with VEGF. Incubate at room temperature with shaking. After washing the plate, 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, the VEGF in the system can fully bind to the detection reagent. The instrument response value (ECLU) read on the electrochemiluminescence detection instrument is high. The higher the signal-to-noise ratio of the sample compared to the negative control sample, the lower the inhibition rate. If the sample contains neutralizing active antibody, the ECLU value is low. The lower the signal-to-noise ratio, the higher the inhibition rate.

[0033] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0034] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0035] This application provides a method for detecting neutralizing antibodies against the VEGF binding moiety in biological samples from individuals who have been administered a bispecific antibody drug comprising a PD-L1 binding moiety and a VEGF binding moiety, the method comprising:

[0036] (1) Treat the biological samples with a first acid hydrolysate with a concentration of 500-1000 mM and a pH of 2.0-3.5;

[0037] (2) The treated biological sample is contacted with an anti-VEGF monospecific antibody containing the VEGF binding portion immobilized on a first solid support to capture the neutralizing antibody in the biological sample;

[0038] (3) The neutralizing antibody is released by acid dissociation using a second acid hydrolysate to obtain a first neutralizing antibody sample;

[0039] (4) Contact the first neutralizing antibody sample with the vascular endothelial growth factor receptor (VEGFR) immobilized on the second solid-phase support to remove VEGF from the first neutralizing antibody sample and obtain the second neutralizing antibody sample;

[0040] (5) The second neutralizing antibody sample is mixed with the bispecific antibody drug with a detectable label to obtain a sample for detection;

[0041] (6) The test sample and the control sample prepared with an equal amount of the bispecific antibody drug with a detectable label are respectively contacted with VEGF immobilized on a third solid support and incubated.

[0042] (7) After incubation, wash the third solid support and measure the intensity of the first detectable label signal for the test sample and the intensity of the second detectable label signal for the control sample.

[0043] (8) The neutralizing antibody in the biological sample is detected by analyzing the intensity of the first detectable label signal and the intensity of the second detectable label signal.

[0044] In some embodiments, the first acid hydrolysate is acetic acid with a concentration of 300 mM, 600 mM, or 800 mM. In some embodiments, the pH of the first acid hydrolysate is 1.5, 2.0, 2.5, 3.0, 3.5, or 4.0. Preferably, in some embodiments, the first acid hydrolysate is acetic acid with a concentration of 600 mM and a pH of 2.5 and / or the first acid hydrolysate is acetic acid with a concentration of 800 mM and a pH of 3.0.

[0045] In some embodiments, the volume ratio of the biological sample to the first acid hydrolysate is 1:1 to 1:100, for example, 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 a range between any two of the above ratios. In some embodiments, the volume ratio of the biological sample to the first acid hydrolysate is 1:1. The ratio is 1:75, for example, 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 ratios. In some specific embodiments, the volume ratio of the biological sample to the first acid hydrolysate with a concentration of 600 mM and a pH of 2.5 is 1:5.

[0046] In some embodiments, acid treatment of biological samples with the first acid hydrolysate can improve detection sensitivity. In some embodiments, acid treatment of biological samples with the first acid hydrolysate can improve drug resistance.

[0047] In some implementations, a neutralizing agent needs to be added to the first solid-phase support before adding the acid-treated biological sample to the first solid-phase support for the capture of anti-VEGF neutralizing antibodies.

[0048] In some embodiments, the capture includes adding a neutralizing agent and an acid-treated biological sample to a first solid-phase carrier coated with an anti-VEGF monoclonal antibody, wherein the neutralizing agent is a 1 M, pH 9.5 solution of tris(hydroxymethyl)aminomethane (Trizma), and the volume ratio of the neutralizing agent to the acid-treated sample is 3:10. In some embodiments, the capture includes adding 30 μL of neutralizing agent and 100 μL of acid-treated biological sample to a first solid-phase carrier coated with an anti-VEGF monoclonal antibody.

[0049] In some embodiments, the neutralizing agent may be a pH reagent capable of neutralizing the binding process between the anti-VEGF neutralizing antibody and the anti-VEGF monoclonal antibody in the PD-L1 & VEGF bispecific antibody drug. In some embodiments, the neutralizing agent is a 1 M, pH 9.5 solution of tris(hydroxymethyl)aminomethane.

[0050] In some embodiments, a second acid hydrolysate is used to dissociate the biological sample in the first solid-phase carrier after capture. 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 that was originally bound to the PD-L1 & VEGF bispecific antibody, thereby obtaining a first neutralizing antibody sample.

[0051] In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 3.0.

[0052] In some embodiments, the interference removal process needs to be performed under neutral pH conditions. In some embodiments, a neutralizing agent needs to be added to the second solid-phase support before adding the acid-dissociated first neutralizing antibody sample to the second solid-phase support for VEGF removal. In some embodiments, the neutralizing agent can be a reagent capable of neutralizing the binding process of VEGFR, which specifically binds to VEGF, to VEGF. In some embodiments, the neutralizing agent is a 1 M solution of tris(hydroxymethyl)aminomethane at pH 9.5. The sample after VEGF removal is referred to as the second neutralizing antibody sample.

[0053] In some embodiments, the detectable label is a metallic label, such as a ruthenium (Ru) label. In some embodiments, using ruthenium to label the bispecific antibody drug makes the reaction system more stable.

[0054] In some embodiments, the signal intensity is the electrochemiluminescence intensity. In some embodiments, an exemplary example of a ruthenium-labeled electrochemiluminescence method is the MSD method, which uses an MSD plate coated with streptavidin and MSD Read Buffer T working solution for electrochemiluminescence detection. In some embodiments, electrochemiluminescence detection is performed using plate readers, programs, and kits commercially available from Meso ScaleDiscovery Inc.

[0055] In some embodiments, the first detectable label signal intensity is the signal intensity of the test sample containing anti-VEGF neutralizing antibodies; the second detectable label signal intensity is the signal intensity of the serum (e.g., human serum) sample that does not contain anti-VEGF neutralizing antibodies.

[0056] In some embodiments, the solid support may be a microplate, wherein the first and second solid supports are ELISA plates, and the third solid support is an MSD plate.

[0057] In some embodiments, a PD-L1 & VEGF bispecific antibody (hereinafter referred to as PD-L1 & VEGF bispecific antibody) is used to capture anti-VEGF neutralizing antibodies in the biological sample. In some embodiments, an anti-VEGF monoclonal antibody has a higher coating efficiency than a PD-L1 & VEGF bispecific antibody. In some embodiments, an anti-VEGF monoclonal antibody has a stronger ability to capture anti-VEGF neutralizing antibodies than a PD-L1 & VEGF bispecific antibody.

[0058] In some implementations, the anti-VEGF neutralizing antibody is diluted with serum (e.g., human serum) to the following concentrations in the test sample: 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. 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, anti-VEGF neutralizing antibodies are diluted with serum (e.g., human serum) to 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 of the biological sample to be tested. In some specific embodiments, anti-VEGF neutralizing antibodies are diluted with serum (e.g., human serum) to 100, 200, 400, 800, 1600, 3200, or 6400 ng / mL of the biological sample to be tested.

[0059] In some implementations, the method is based on the ACE method of CLBA technology on the MSD platform, which has at least one of the following advantages:

[0060] The interference of endogenous VEGF was resolved;

[0061] The impact of high-dose drug tolerance on the detection of anti-VEGF neutralizing antibody activity was resolved; and

[0062] This improved the method's sensitivity.

[0063] In some implementations, the sensitivity of the method is 100 ng / mL.

[0064] In some implementations, the tolerable concentration of the PD-L1 & VEGF bispecific antibody can be increased to 200 μg / mL when the concentration of the anti-VEGF neutralizing antibody is 500 ng / mL.

[0065] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0066] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0067] Example

[0068] This application will be described in more detail by way of specific examples. The following embodiments are provided for illustrative purposes only and are not intended to limit this application in any way. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same results.

[0069] Unless otherwise specified, the reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0070] I. Materials and Methods

[0071] The drugs and reagents used in this application are as follows:

[0072] ELISA plate 1 capture reagent working solution: anti-VEGF monoclonal antibody (commercially purchased, lot number: 20240831 CSX, concentration: 2.53 mg / mL), diluted to 10 μg / mL with 1× carbonate buffer (CBS).

[0073] Neutralizing reagent: Trizma neutralization buffer.

[0074] ELISA Plate 2 Capture Reagent (Interference Removal Reagent) Working Solution: Vascular Endothelial Growth Factor Receptor (VEGFR) (Manufacturer: R&D Systems, Catalog No.: 321-FL-050 / CF, 5 mg / vial), diluted to 10 μg / mL with 1× Carbonate Buffered (CBS).

[0075] MSD plate capture reagent working solution: Vascular endothelial growth factor (VEGF) (Manufacturer: Acro Biosystems, Catalog No.: VE5-H5248, Lot No.: 2410-234HF1-1CB)

[0076] Specification: 50 μg / bottle (dissolve to 1 mg / mL according to instructions), or dilute to 10 μg / mL with 1% BSA.

[0077] The working solution for the test reagent is a Ru-labeled PD-L1 & VEGF bispecific antibody, abbreviated as Ru-drug (manufacturer: Andu Biotechnology, batch number: 20220711, concentration: 351 μg / mL), diluted to 100 ng / mL with PBS containing 1% BSA.

[0078] Neutralizing antibody: Recombinant anti-VEGFR1 monoclonal antibody (manufacturer: Yiming Onco, batch number: 20230913, concentration: 6.15 mg / mL).

[0079] The specific experimental steps for detecting neutralizing antibodies against the VEGF binding site in PD-L1 & VEGF bispecific anti-antibody drugs in biological samples using anti-VEGF monoclonal antibodies are shown in the table below:

[0080]

[0081] II. Experimental Results

[0082] The formula for calculating "% inhibition" in sensitivity and drug resistance is as follows:

[0083]

[0084] The concentration of anti-VEGF neutralizing antibody in the control sample was 0 ng / mL.

[0085] 1. The effect of treating samples with acetic acid solutions of different concentrations or pH on sensitivity and drug resistance.

[0086] Table 1. Sensitivity data at different pH values ​​when samples are acid-treated with 300 mM acetic acid.

[0087]

[0088] Table 2. Sensitivity data at different pH values ​​when samples are acid-treated with 600 mM acetic acid.

[0089]

[0090] Table 3. Sensitivity data at different pH values ​​when samples are acid-treated with 800 mM acetic acid.

[0091]

[0092] Table 4. Drug resistance data at different pH levels when samples are acid-treated with 300 mM acetic acid (neutralizing antibody concentration is 500 ng / mL).

[0093]

[0094] Table 5. Drug resistance data at different pH levels when samples are acid-treated with 600 mM acetic acid (neutralizing antibody concentration is 500 ng / mL).

[0095]

[0096] Table 6. Drug resistance data at different pH levels when samples are acid-treated with 800 mM acetic acid (neutralizing antibody concentration is 500 ng / mL).

[0097]

[0098] The data in Table 1-6 show that, through comparison, it was found that in all systems at pH 1.5, the signal value did not show a gradient, meaning there was no sensitivity or drug resistance data. When acid treatment with 300 mM acetic acid was used, the sensitivity of all systems except pH 1.5 met the requirements, reaching 100 ng / mL, but drug resistance was poor, and resistance showed a reverse inhibition phenomenon with increasing drug concentration. When acid treatment with 600 mM acetic acid was used, the sensitivity of all systems except pH 1.5 met the requirements, reaching 100 ng / mL. The best drug resistance was observed at pH 2.5, reaching 200 μg / mL, while resistance was poor at other pH values. Specifically, at pH 3.0, 3.5, or 4.0, a reverse inhibition phenomenon of drug resistance appeared with increasing drug concentration. When treated with 800 mM acetic acid, the sensitivity of all addition systems except pH 1.5 met the requirements, reaching 100 ng / mL. The best resistance was observed at pH 3.0, reaching 200 μg / mL, while resistance was poor at other pH values. Specifically, at pH 2.5, 3.5, or 4.0, resistance was reversed with increasing drug concentration. The % inhibition threshold ranged from 15% to 20%.

[0099] 2. The effect of using PD-L1 & VEGF bispecific antibodies and anti-VEGF monoclonal antibodies to capture neutralizing antibodies.

[0100] Table 7. Comparison of sensitivity data for ELISA plates coated with PD-L1 & VEGF bispecific antibodies and anti-VEGF monoclonal antibodies.

[0101]

[0102] Table 8. Comparison of drug resistance data for ELISA plates coated with PD-L1 & VEGF bispecific antibody and anti-VEGF monoclonal antibody when the neutralizing antibody concentration is 500 ng / mL.

[0103]

[0104] The data in Tables 7 and 8 show that, compared to systems coated with PD-L1 & VEGF bispecific antibodies, the inhibition rate was higher in all acid-hydrolyzed systems. This indicates that the monospecific antibody can capture more neutralizing antibodies, resulting in better sensitivity and resistance. Specifically, the best resistance was observed with 600 mM acetic acid at pH 2.5, while treatment with other concentrations of acetic acid resulted in resistance-induced inhibition. The % inhibition threshold ranged from 15% to 20%.

[0105] 3. The effect of interference removal reagent on the removal of free VEGF.

[0106] Table 9. Comparison of data with and without the use of interference removal reagent (VEGFR).

[0107]

[0108] Table 10. Comparison of sensitivity data with and without the application of the interference removal reagent (VEGFR).

[0109]

[0110] Table 11. Comparison of drug resistance data with and without the use of the VEGFR (Video Anti-interference Reagent) when the neutralizing antibody concentration is 500 ng / mL.

[0111]

[0112] Table 9-11 shows that the VEGFR (vegetative interference remover) can specifically bind to free VEGF, effectively removing VEGF interference in the reaction system. Furthermore, comparisons revealed that neither sensitivity nor drug resistance was affected after using the VEGFR; sensitivity reached 100 ng / mL, while resistance reached 200 μg / mL at a drug concentration of 500 ng / mL. The % inhibition threshold ranged from 15% to 20%.

[0113] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

Claims

1. A method for detecting neutralizing antibodies against the vascular endothelial growth factor (VEGF) binding moiety in a biological sample from an individual who has been administered a bispecific antibody drug comprising a programmed death ligand-1 (PD-L1) binding moiety and a VEGF binding moiety, the method comprising: (1) Treat the biological samples with a first acid hydrolysate with a concentration of 500-1000 mM and a pH of 2.0-3.5; (2) Add a neutralizing agent and the treated biological sample to the first solid-phase carrier, so that the treated biological sample is in contact with the anti-vascular endothelial growth factor monospecific antibody containing the vascular endothelial growth factor binding portion fixed on the first solid-phase carrier, so as to capture the neutralizing antibody in the biological sample, wherein the neutralizing agent is a 1 M, pH 9.5 tris(hydroxymethyl)aminomethane solution, and wherein the volume ratio of the neutralizing agent to the treated biological sample is 3:10; (3) The neutralizing antibody is released by acid dissociation using a second acid hydrolysate to obtain a first neutralizing antibody sample; (4) Contact the first neutralizing antibody sample with the vascular endothelial growth factor receptor (VEGFR) immobilized on the second solid-phase support to remove the vascular endothelial growth factor in the first neutralizing antibody sample and obtain the second neutralizing antibody sample. (5) The second neutralizing antibody sample is mixed with a bispecific antibody drug with a detectable label to obtain a sample for detection; (6) The test sample and the control sample prepared with an equal amount of the bispecific antibody drug with a detectable label are respectively contacted with vascular endothelial growth factor immobilized on a third solid-phase carrier and incubated. (7) After incubation, wash the third solid support and measure the intensity of the first detectable label signal for the test sample and the intensity of the second detectable label signal for the control sample. (8) The neutralizing antibody in the biological sample is detected by analyzing the intensity of the first detectable label signal and the intensity of the second detectable label signal.

2. The method of claim 1, wherein the first acid hydrolysate is acetic acid with a concentration of 600 mM and a pH of 2.

5.

3. The method of claim 1, wherein the first acid hydrolysate is acetic acid with a concentration of 800 mM and a pH of 3.

0.

4. The method of claim 1, wherein the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 3.

0.

5. The method of claim 1, wherein the detectable marker is a metallic marker.

6. The method of claim 5, wherein the metal marker is a ruthenium marker.

7. The method of claim 1, wherein the signal intensity is electrochemiluminescence intensity.

8. The method of any one of claims 1-7, wherein the biological sample is a plasma or serum sample.

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