Method for detecting neutralizing antibodies against interleukin-4 receptor antibody drugs

Abstract

The present application relates to methods of analyzing anti-drug neutralizing antibodies. In particular, the present application relates to methods of detecting neutralizing antibodies against an anti- interleukin-4 receptor alpha subunit (IL-4Ra) antibody drug in a biological sample from an individual administered the anti-interleukin-4 receptor alpha subunit (IL-4Ra) antibody drug.

Description

Technical Field

[0001] This application belongs to the field of biochemistry and mainly relates to the detection method of anti-drug neutralizing antibodies, and more specifically to the detection method of neutralizing antibodies against anti-interleukin-4 receptor antibody drugs. Background Technology

[0002] Interleukin-4 receptor (IL-4R) is a key target mediating the human Th2-type inflammatory response. IL-4 and IL-13 activate the downstream JAK-STAT6 signaling pathway by binding to the IL-4Rα subunit, thereby mediating the development of various allergic inflammatory diseases such as atopic dermatitis, bronchial asthma, and allergic rhinitis. Monoclonal antibody drugs targeting IL-4Rα can specifically block the binding of IL-4 and IL-13 to their receptors, inhibiting the abnormal activation of the Th2 inflammatory pathway, and have become important biological agents for the clinical treatment of moderate to severe allergic inflammatory diseases.

[0003] Therapeutic monoclonal antibodies (ADAs) can easily induce the production of anti-drug antibodies (ADAs) during in vivo application. As a type of ADA, neutralizing antibodies (NAb) can specifically bind to the active epitope of a drug, blocking the interaction between the drug and the IL-4R target. This directly leads to a decrease in the efficacy of drug therapy or even complete ineffectiveness, and in severe cases, can cause infusion reactions, hypersensitivity reactions, and other clinical safety risks. Therefore, establishing accurate, stable, and interference-resistant methods for detecting neutralizing antibodies against anti-IL-4R drugs is an essential core technical support for the non-clinical immunogenicity evaluation, clinical drug safety monitoring, and efficacy prediction of these drugs. Summary of the Invention

[0004] This application provides a method for detecting neutralizing antibodies against the anti-interleukin-4 receptor α subunit (IL-4Rα) antibody drug in biological samples from individuals who have been administered an anti-interleukin-4 receptor α subunit (IL-4Rα) antibody drug, the method comprising: (1) The biological sample is treated with a first acid hydrolysate, wherein the first acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.5, and the volume ratio of the first acid hydrolysate to the biological sample is 1:1; (2) Add a first neutralizing agent to a first solid-phase carrier coated with a deinterference agent that specifically binds IL-4Rα, then add the sample treated in step (1) and incubate to remove free IL-4Rα from the sample treated in step (1) to obtain a deinterference sample, wherein the deinterference agent that specifically binds IL-4Rα has different epitopes of IL-4Rα bound by the anti-IL-4Rα antibody drug; (3) The interference-removing sample in step (2) is treated with a second acid hydrolysate, wherein the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.0-2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:6-1:9. (4) Contact and incubate the sample treated in step (3) with the anti-IL-4Rα antibody drug immobilized on the second solid-phase support to capture the neutralizing antibody, wherein the second solid-phase support is pre-added with a second neutralizing reagent; (5) The neutralizing antibody is released by acid dissociation using a third acid hydrolysate to obtain a neutralizing antibody sample; (6) The neutralizing antibody sample, the third neutralizing reagent and the IL-4Rα antibody drug with a detectable label are mixed to obtain a sample for detection; (7) Contact the test sample and the control sample prepared with an equal amount of the anti-IL-4Rα antibody drug with a detectable label with IL-4Rα and incubate them respectively with IL-4Rα immobilized on the third solid phase support. (8) Determine the intensity of the first detectable marker signal of the test sample and the intensity of the second detectable marker signal of the control sample; (9) 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.

[0005] In some embodiments, in step (3), the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.2, and the volume ratio of the deinterference sample to the second acid hydrolysate is 1:9.

[0006] In some embodiments, the first neutralizing agent, the second neutralizing agent, and / or the third neutralizing agent are 1 M, pH 9.5 tris(hydroxymethyl)aminomethane solutions.

[0007] In some implementations, the volume ratio of the treated sample to the second neutralizing reagent in step (4) is 30:1.

[0008] In some embodiments, the third acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.5.

[0009] In some implementations, the detectable marker is a metallic marker.

[0010] In some implementations, the metal marker is a ruthenium marker.

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

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

[0013] To facilitate 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 neutralizing antibody binds to an antibody drug (equivalent to an antigen) coated on a microplate under slightly acidic conditions to achieve optimal capture of the neutralizing antibody. In some embodiments of this application, the neutralizing antibody is a neutralizing antibody against an anti-interleukin-4 receptor α subunit (IL-4Rα) antibody drug, and the antibody drug is an anti-IL-4Rα antibody drug.

[0015] As used herein, "acid dissociation" refers to the process of acidifying a sample to dissociate a drug-neutralizing antibody complex into antidrug-neutralizing antibodies. The primary purpose of acid dissociation is to make the antidrug-neutralizing antibodies that were previously bound to the drug detectable. In some embodiments of this application, the drug in the antidrug-neutralizing antibody is an anti-IL-4Rα antibody drug, and the antidrug-neutralizing antibody is a neutralizing antibody against the anti-IL-4Rα antibody drug.

[0016] As used herein, the term "drug resistance" refers to the level of neutralizing antibodies in a biological sample that tolerate high concentrations of free drug, which 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 some embodiments of this application, the antidrug-neutralizing antibody is a neutralizing antibody against an anti-IL-4Rα antibody drug.

[0017] Unless otherwise specified, “acid hydrolysate I” as used in this specification has the same meaning as “first acid hydrolysate”, and similarly, “acid hydrolysate II” and “acid hydrolysate III” have the same meaning as “second acid hydrolysate” and “third acid hydrolysate”, respectively.

[0018] Currently, methods for detecting neutralizing antibodies against anti-IL-4Rα drugs generally suffer from the following problems: The steady-state blood concentration of anti-IL-4Rα antibody drugs remains at a high level, and the serum samples being tested typically contain high concentrations of the free drug. This free drug competitively occupies IL-4Rα binding sites and antibody recognition epitopes within the detection system, masking the specific binding between the neutralizing antibody and the drug (e.g., anti-IL-4Rα antibody), leading to false negative results. Furthermore, existing detection systems also suffer from significant interference from the IL-4Rα target itself. IL-4Rα is widely expressed in human tissues and immune cells. Naturally occurring soluble IL-4Rα in the serum can non-specifically bind to anti-IL-4Rα drugs and detection probes in the detection system. This competitively occupies specific binding sites and causes increased background noise and signal drift, easily leading to false positive results and quantitative bias.

[0019] As understood after reviewing the working principle of the core invention of this application, it becomes clear that the anti-IL-4Rα antibody drug and the interference-removing reagent specifically binding to IL-4Rα are not limited to a single pair of antibodies with a specific sequence structure or binding property. In fact, suitable interference-removing reagents can be selected based on the different anti-IL-4Rα antibody drugs to be tested. The purpose of using interference-removing reagents is to remove free IL-4Rα from the sample, but simultaneously, the interference-removing reagents cannot bind to the neutralizing antibody of the anti-IL-4Rα antibody drug in the sample. The interference-removing reagent used in this application is also an anti-IL-4Rα antibody, but this antibody binds to different epitopes of IL-4Rα with the IL-4Rα antibody drug to be tested, and their variable regions are different. Because the neutralizing antibody specifically recognizes the variable region of the IL-4Rα antibody drug to be tested, the neutralizing antibody will not bind to the interference-removing reagent.

[0020] In some embodiments, the anti-IL-4Rα antibody drug used in this application is a biosimilar of dupilumab. The term "biosimilar" means that the drug is highly similar to the reference drug (dupilumab in this application) in terms of quality, safety, and efficacy, especially in terms of its binding epitopes to IL-4Rα. In some embodiments, the interference-removing agent used in this application is sprokitimab. Previous studies have shown that the epitopes of sprokitimab and dupilumab binding to IL-4Rα are completely separate and do not overlap (Liu, Wei, et al. "Stapokibart(CM310) targets IL-4Rα for the treatment of type 2 inflammation." iScience 27.9 (2024)). Dupilumab binds to the multi-segment loop region of the D2 domain of IL-4Rα, while sprokitimab binds to the key amino acids M39A, S95A, and L135A of the D1 domain of IL-4Rα. Therefore, when using a biosimilar of dupilumab as an anti-IL-4Rα antibody, septazibazide can be used as an interference-removing agent.

[0021] Therefore, the detection method for neutralizing antibodies in anti-IL-4Rα antibody drugs proposed in this application removes the interference of free drug and its own target, while also meeting the sensitivity requirements of detection, which is of great significance.

[0022] This application establishes a method for detecting neutralizing antibodies against the anti-IL-4Rα antibody drug in biological samples from individuals who have been administered the anti-IL-4Rα 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.

[0023] 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: (1) First, the sample was acid-treated with acid hydrolysate I (300 mM, pH 2.5 acetic acid). The acid-treated sample was then added to a 96-well microplate containing neutralizing reagent coated with anti-IL-4Rα monoclonal antibody (septinomycin in this paper) and incubated for a period of time to remove IL-4Rα interference in the sample.

[0024] (2) Take the supernatant and dilute the sample with acid hydrolysis solution II (e.g., 300 mM, pH 2.2 acetic acid) at a certain ratio (e.g., 1:9) to perform a second acid treatment on the sample so that the pH of the reaction system is slightly acidic, which is more conducive to affinity capture.

[0025] (3) The sample after the second acid treatment was aspirated into a 96-well microplate coated with anti-IL-4Rα antibody drug and pre-added with neutralizing reagent. The plate was shaken and incubated overnight to capture the neutralizing antibody against anti-IL-4Rα antibody drug (hereinafter referred to as neutralizing antibody, NAb) in the sample, forming a neutralizing antibody-anti-IL-4Rα antibody drug complex.

[0026] (4) After washing the plate, acid hydrolysate III was added to the above 96-well microplate to acid dissociate the neutralizing antibody-anti-IL-4Rα antibody-drug complex.

[0027] (5) Mix the above acid-dissociated sample, neutralizing reagent and detection reagent (e.g., anti-IL-4Rα antibody drug with detectable label) and perform a shaking reaction. Add the reaction mixture to an MSD microplate pre-captured IL-4Rα, incubate with shaking at room temperature, wash the plate and 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, IL-4Rα in the system can fully bind to the detection reagent, and the instrument response value (ECLU) read on the electrochemiluminescence detection instrument is high. The higher the signal-to-noise ratio for the negative control sample, the lower the inhibition rate. If the sample contains neutralizing active antibody, its ECLU value is low, and the lower the signal-to-noise ratio, the higher the inhibition rate.

[0028] Unless otherwise specified, this application employs conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques in the art.

[0029] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0030] This application provides a method for detecting neutralizing antibodies against the anti-IL-4Rα antibody drug in biological samples from individuals who have been administered the anti-IL-4Rα antibody drug, the method comprising: (1) The biological sample is treated with a first acid hydrolysate, wherein the first acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.5, and the volume ratio of the first acid hydrolysate to the biological sample is 1:1; (2) Add a first neutralizing agent to a first solid-phase carrier coated with a deinterference agent that specifically binds IL-4Rα, then add the sample treated in step (1) and incubate to remove free IL-4Rα from the sample treated in step (1) to obtain a deinterference sample, wherein the deinterference agent that specifically binds IL-4Rα has different epitopes of IL-4Rα bound by the anti-IL-4Rα antibody drug; (3) The interference-removing sample in step (2) is treated with a second acid hydrolysate, wherein the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.0-2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:6-1:9. (4) Contact and incubate the sample treated in step (3) with the anti-IL-4Rα antibody drug immobilized on the second solid-phase support to capture the neutralizing antibody, wherein the second solid-phase support is pre-added with a second neutralizing reagent; (5) The neutralizing antibody is released by acid dissociation using a third acid hydrolysate to obtain a neutralizing antibody sample; (6) The neutralizing antibody sample, the third neutralizing reagent and the IL-4Rα antibody drug with a detectable label are mixed to obtain a sample for detection; (7) Contact the test sample and the control sample prepared with an equal amount of the anti-IL-4Rα antibody drug with a detectable label with IL-4Rα and incubate them respectively with IL-4Rα immobilized on the third solid phase support. (8) Determine the intensity of the first detectable marker signal of the test sample and the intensity of the second detectable marker signal of the control sample; (9) 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.

[0031] In some embodiments, a neutralizing agent is provided to adjust the pH of the capture system to improve affinity capture efficiency. The neutralizing agent used in the affinity capture step is a 1 M, pH 9.5 tris(hydroxymethyl)aminomethane solution. In some embodiments, a neutralizing agent is provided to ensure that the deinterference step is performed under neutral conditions. In some embodiments, a neutralizing agent is added to provide a neutral binding environment for IL-4Rα and the anti-IL-4Rα antibody drug. The neutralizing agent used to provide the neutral environment can be the same tris(hydroxymethyl)aminomethane solution as described above, or any suitable buffer, salt solution, etc., such as 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), phosphate buffered saline (PBS), glycine-sodium hydroxide buffer, etc.

[0032] In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.5-2.5, for example, pH values ​​of 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, or any range between two of the above values. In some embodiments, the volume ratio of the deinterference sample to the second acid hydrolysate is 1:1 to 1:15, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, or any range between two of the above ratios. In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.5-1.7, and the volume ratio of the deinterference sample to the second acid hydrolysate is 1:4. In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.7-2.0, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:6. In some embodiments, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.0-2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:9. In a preferred embodiment, the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:9.

[0033] In some embodiments, the third acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.5.

[0034] In some embodiments, the detectable label is a metallic label, such as a ruthenium (Ru) label. In some embodiments, using ruthenium to label the anti-IL-4Rα antibody drug makes the reaction system more stable.

[0035] 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.

[0036] In some embodiments, the first detectable label signal intensity is the signal intensity of the test sample containing a neutralizing antibody against the anti-IL-4Rα antibody drug; the second detectable label signal intensity is the signal intensity of the serum (e.g., human serum) sample that does not contain a neutralizing antibody against the anti-IL-4Rα antibody drug.

[0037] 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.

[0038] In some embodiments, the biological sample is a blood, plasma, or serum sample.

[0039] In some implementations, the neutralizing antibody against the IL-4Rα antibody drug 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. 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, 1-1500 ng / mL, 1-2000 ng / mL, 1-2500 ng / mL, 1-3000 ng / mL, 1-3500 ng / mL, 1-4000 ng / mL. In some embodiments, the neutralizing antibody is 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, 1500, 2000, 2500, 3000, 3500, or 4000 ng / mL of the biological sample to be tested. In some specific embodiments, the neutralizing antibody is diluted with serum (e.g., human serum) to 50, 100, 200, 400, 800, 1600, or 3200 ng / mL of the biological sample to be tested.

[0040] 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: The interference of endogenous IL-4Rα was resolved; The impact of high-dose drug tolerance on the detection of neutralizing antibody activity against anti-IL-4Rα antibody drugs was resolved; and This improved the method's sensitivity.

[0041] In some implementations, the sensitivity of the method is 50 ng / mL.

[0042] In some embodiments, a tolerable anti-IL-4Rα antibody concentration of 200 μg / mL is achieved when the neutralizing antibody concentration is 500 ng / mL. In some preferred embodiments, a tolerable anti-IL-4Rα antibody concentration of 200 μg / mL is achieved when the neutralizing antibody concentration is 200 ng / mL.

[0043] 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.

[0044] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0045] Example

[0046] This application will be described in more detail through specific embodiments. 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.

[0047] 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.

[0048] I. Materials and Methods

[0049] The drugs and reagents used in this application are as follows: ELISA Plate 1 Capture Reagent Working Solution: Anti-IL-4Rα antibody drug (biosimilar of dupilumab), diluted to 10 μg / mL with 1× carbonate buffer (CBS).

[0050] ELISA Plate 2 Capture Reagent (Interference Removal Working Solution): Sipazimab Injection, diluted to 10 μg / mL with 1 × CBS.

[0051] Neutralization reagent: Trizma neutralization buffer.

[0052] MSD plate capture reagent working solution: Human IL-4Rα / CD124 protein, His tag (MALS verified) (manufacturer: ACROBio sysetms, lot number: 381-251AF1-1ZZ, concentration 1 µg / mL).

[0053] The working solution of the test reagent is a Ru-labeled anti-IL-4Rα antibody drug (Ru-drug for short) with a labeling ratio of 20:1.

[0054] Neutralizing antibody: Antiduple neutralizing antibody (manufacturer: Antibody System, batch number: 90986, concentration: 2.4 mg / mL).

[0055] The specific experimental steps for detecting neutralizing antibodies (hereinafter referred to as neutralizing antibodies) against anti-IL-4Rα antibody drugs in biological samples are shown in the table below:

[0056] II. Experimental Results

[0057] The formula for calculating "% inhibition" in sensitivity and drug resistance is as follows:

[0058] The neutralizing antibody concentration in the control sample was 0 ng / mL.

[0059] 1. Effect of different pH levels on affinity capture efficiency

[0060] In the preliminary experiments, different volumes of Trizma neutralization buffer (Tris) were added to biological samples treated with acid hydrolysate I (300 mM, pH 2.5 HAc) to explore the optimal pH for affinity capture. The capture efficiency was expressed by sensitivity and drug resistance. The experimental results are shown in Table 1 and Table 2, respectively.

[0061] Table 1. Sensitivity data under different pH conditions (acid-hydrolyzed sample 150 μL)

[0062] Table 2. Drug resistance data under different pH conditions (acid-hydrolyzed sample 150 μL, neutralizing antibody concentration 500 ng / mL)

[0063] The results in Tables 1 and 2 show that when the incubation system contains 5 μL of Tris solution, the sensitivity and drug resistance of the detection method meet the requirements, with a sensitivity reaching 50 ng / mL. In the presence of 500 ng / mL neutralizing antibody, the method can tolerate at least 200 μg / mL of anti-IL-4Rα antibody. When the incubation system contains only 150 μL of acid-digested sample and no Tris solution, the sensitivity reaches 100 ng / mL, but the drug resistance is poor. The sensitivity and drug resistance of the other incubation systems do not meet the detection requirements. In summary, the affinity capture incubation system is most effective when it is slightly acidic. Furthermore, since interference removal needs to be performed under neutral conditions, this application selected to perform the interference removal step first, followed by capture of the neutralizing antibody. The % inhibition threshold range is 15%-20%.

[0064] 2. The removal effect of the interference-eliminating reagent (spritzibamab) on IL-4Rα.

[0065] The removal effect of the interference-removing reagent on IL-4Rα and its influence on sensitivity are shown in Tables 3 and 4.

[0066] Table 3. Sensitivity of the method when using different concentrations of spritzibimab

[0067] Table 4. Interference-reducing effects of different concentrations of spritzyl

[0068] Table 3 shows that the sensitivity of the method was essentially unaffected before and after the interference removal operation, indicating that the interference removal reagent (sipazimab) itself does not affect the detection results. Table 4 shows that without interference removal, the method can only tolerate 10 ng / mL IL-4Rα. When the interference removal reagent concentration reaches 15 μg / mL or 20 μg / mL, the method can tolerate up to 2000 ng / mL IL-4Rα, indicating that the interference removal reagent at these two concentrations can remove most of the influence of IL-4Rα in the sample. The % inhibition threshold range is 15%-20%.

[0069] 3. The impact of different acid treatment systems on detection methods

[0070] In the above experiments, it was found that the affinity capture effect was best under slightly acidic conditions. Therefore, after the interference removal operation steps, the sample needs to be treated with acid again. This experiment explored the effect of different pH values ​​of acid hydrolysate II (300 mM HAc, pH 1.5-2.2) and dilution ratios on the detection method. The experimental results are shown in Tables 5-7.

[0071] Table 5. Sensitivity and resistance data at different pH values ​​when the volume ratio of the interference-removing sample to acid hydrolysate II is 1:4.

[0072] Note: In the drug resistance analysis, all samples contained 500 ng / mL of neutralizing antibody.

[0073] Table 6. Sensitivity and resistance data at different pH values ​​when the volume ratio of the interference-removing sample to acid hydrolysate II is 1:6.

[0074] Note: In the drug resistance analysis, all samples contained 500 ng / mL of neutralizing antibody.

[0075] Table 7. Sensitivity and resistance data at different pH values ​​when the volume ratio of the interference-removing sample to acid hydrolysate II is 1:9.

[0076] Note: In the drug resistance analysis, all samples contained 500 ng / mL of neutralizing antibody.

[0077] As shown in Tables 5-7, when the volume ratio of the deinterference sample to acid hydrolysate II is 1:4, secondary acid treatment with 300 mM HAc at pH 1.5 or 1.7 is more effective, with pH 1.5 showing the best sensitivity and drug resistance. When the volume ratio of the deinterference sample to acid hydrolysate II is 1:6, secondary acid treatment with 300 mM HAc at pH 1.7 or 2.0 is more effective, with pH 1.7 showing the best sensitivity and drug resistance. When the volume ratio of the deinterference sample to acid hydrolysate II is 1:9, secondary acid treatment with 300 mM HAc at pH 2.0 or 2.2 is more effective, with pH 2.2 showing the best sensitivity and drug resistance. The % inhibition threshold range is 15%-20%.

[0078] To select the optimal acid treatment conditions from the six effective acid treatment systems mentioned above, the drug resistance of the method was investigated when the neutralizing antibody concentration in the sample was 200 ng / mL. Furthermore, in another set of experiments, different concentrations of IL-4Rα were added to the samples to investigate the specificity (i.e., anti-interference ability) of the method under the six acid treatment systems. The drug resistance and specificity data are shown in Tables 8-9.

[0079] Table 8. Drug resistance data under different acid treatment systems with a neutralizing antibody concentration of 200 ng / mL.

[0080] Table 9. Specific data under different acid treatment systems

[0081] The results in Tables 8 and 9 show that the method met the requirements for drug resistance and specificity only when the volume ratio of acid hydrolysate II was 1:9 and the pH was 2.2. The method was resistant to 200 μg / mL anti-IL-4Rα antibody and 200 μg / mL IL-4Rα. The % inhibition threshold ranged from 15% to 20%.

[0082] In summary, this application provides a highly sensitive, drug-resistant, and highly specific method for detecting neutralizing antibodies against anti-IL-4Rα drugs by combining an anti-IL-4Rα monoclonal antibody (septazibazide) with an applied anti-IL-4Rα drug (a biosimilar of dupilumab) with an anti-IL-4Rα monoclonal antibody (septazibazide) with a different epitope for interference removal, and by subjecting the interference-removed sample to secondary acid treatment.

[0083] 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 said anti-IL-4Rα antibody drug in a biological sample from an individual who has been administered an anti-IL-4Rα antibody drug, the method comprising: (1) The biological sample is treated with a first acid hydrolysate, wherein the first acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.5, and the volume ratio of the first acid hydrolysate to the biological sample is 1:

1. (2) Add a first neutralizing agent to a first solid-phase carrier coated with a deinterference agent that specifically binds IL-4Rα, then add the sample treated in step (1) and incubate to remove free IL-4Rα from the sample treated in step (1) to obtain a deinterference sample, wherein the deinterference agent that specifically binds IL-4Rα has different epitopes of IL-4Rα bound by the anti-IL-4Rα antibody drug; (3) The interference-removing sample in step (2) is treated with a second acid hydrolysate, wherein the second acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 2.0-2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:6-1:

9. (4) Contact and incubate the sample treated in step (3) with the anti-IL-4Rα antibody drug immobilized on the second solid-phase support to capture the neutralizing antibody, wherein the second solid-phase support is pre-added with a second neutralizing reagent; (5) The neutralizing antibody is released by acid dissociation using a third acid hydrolysate to obtain a neutralizing antibody sample; (6) The neutralizing antibody sample, the third neutralizing reagent and the IL-4Rα antibody drug with a detectable label are mixed to obtain a sample for detection; (7) Contact the test sample and the control sample prepared with an equal amount of the anti-IL-4Rα antibody drug with a detectable label with IL-4Rα and incubate them respectively with IL-4Rα immobilized on the third solid phase support. (8) Determine the intensity of the first detectable marker signal of the test sample and the intensity of the second detectable marker signal of the control sample; (9) 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 according to claim 1, wherein the second acid hydrolysate in step (3) is acetic acid with a concentration of 300 mM and pH 2.2, and the volume ratio of the interference-removing sample to the second acid hydrolysate is 1:

9.

3. The method according to claim 1, wherein the first neutralizing agent, the second neutralizing agent and / or the third neutralizing agent is a 1 M, pH 9.5 solution of tris(hydroxymethyl)aminomethane.

4. The method according to claim 3, wherein the volume ratio of the treated sample and the second neutralizing reagent in step (4) is 30:

1.

5. The method according to claim 1, wherein the third acid hydrolysate is acetic acid with a concentration of 300 mM and a pH of 1.

5.

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

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

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

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

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