Method for detecting neutralizing antibodies against tgf-beta antibody drugs

Abstract

The present application is in the field of biochemistry and relates primarily to methods for detecting anti-drug neutralizing antibodies, and more particularly to methods for detecting neutralizing antibodies against anti-TGF-beta antibody drugs.

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-TGF-β antibody drugs. Background Technology

[0002] Transforming growth factor-β (TGF-β) is a multifunctional cytokine that plays a crucial role in physiological and pathological processes such as cell proliferation, differentiation, immune regulation, wound repair, and extracellular matrix formation. TGF-β has a dual role in tumor development: in the early stages of tumorigenesis, it inhibits tumor growth by suppressing cell cycle progression and inducing apoptosis; while in the later stages, TGF-β acts as a major inducing factor of epithelial-mesenchymal transition (EMT), enhancing tumor migration and invasion, and also helps tumor cells escape immune responses by influencing immune cells.

[0003] Currently, various targeted drugs that inhibit TGF-β have been developed, including anti-TGF-β monoclonal antibodies and bispecific antibodies that target TGF-β, such as PD-L1 / TGF-β bispecific antibodies and VEGF / TGF-β bispecific antibodies. However, treatment with antibody drugs may cause adverse immune responses, leading to the production of anti-drug antibodies (ADAs). As a type of ADA, neutralizing antibodies (NAb) weaken the therapeutic effect of antibody drugs by preventing the binding of antibody drugs to their targets or by inhibiting downstream signaling after binding due to steric hindrance. Therefore, it is necessary to detect neutralizing antibodies during treatment. To date, there are no studies on neutralizing antibodies against anti-TGF-β antibody drugs. Summary of the Invention

[0004] This invention develops a method for detecting neutralizing antibodies against anti-TGF-β antibody drugs, the method comprising: (1) The biological sample was treated with acetic acid. (2) The biotin-labeled anti-transforming growth factor-β antibody drug is contacted with and incubated with the biological sample to allow the anti-transforming growth factor-β antibody drug to bind to the neutralizing antibody against the anti-transforming growth factor-β antibody drug in the biological sample to form an anti-transforming growth factor-β antibody drug-neutralizing antibody complex; (3) Add magnetic beads that can specifically bind the biotin to the reactants obtained in step (2); (4) The magnetic beads obtained in step (3) are subjected to acid hydrolysis to release the neutralizing antibody; (5) In a reaction vessel with a bottom coated with transforming growth factor-β receptor (TGFβR), a neutralizing agent and the acid hydrolysis product obtained in step (4) are added and incubated to remove transforming growth factor-β (TGF-β) from the acid hydrolysis product, and the reaction solution is taken out from the reaction vessel as a test sample; (6) Contact and incubate the test sample with the anti-transforming growth factor-β antibody drug to obtain the first reaction sample; (7) The first reaction sample is contacted with transforming growth factor-β (TGF-β) and incubated to obtain the second reaction sample; (8) Contact and incubate the second reaction sample with cells carrying the luciferase reporter gene; (9) Add a detection reagent that can quantify the expression level of the luciferase reporter gene to the cells in step (8), and determine the neutralizing antibody in the biological sample based on the comparison with the quantification result of the expression level of the luciferase reporter gene when no test sample is added in step (6).

[0005] In some embodiments, the volume ratio of the biological sample to acetic acid is 1:1.

[0006] In some embodiments, the magnetic beads are magnetic beads coupled with streptavidin (SA).

[0007] In some implementations, the acid hydrolysis treatment in step (4) uses a cell culture medium containing 2% fetal bovine serum at pH 2.0.

[0008] In some embodiments, the neutralizing agent is a solution of tris(hydroxymethyl)aminomethane.

[0009] In some implementations, the incubation time for step (3) is 2 hours.

[0010] In some implementations, the incubation time for step (6) is more than 8 hours.

[0011] In some implementations, the incubation in step (8) is carried out at 37°C and 5% CO2 for 6 to 7 hours.

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

[0013] In some embodiments, the concentration of the acetic acid is 300 mM. Attached Figure Description

[0014] Figure 1The sensitivity curves for detecting neutralizing antibodies against anti-TGF-β antibody drugs using the method of the present invention are shown. Detailed Implementation

[0015] To facilitate understanding of this application, some terms used herein are first defined.

[0016] As used herein, the term "reporter gene" refers to a class of marker genes used for real-time monitoring of gene expression or transformation efficiency. Common reporter genes include, for example, luciferase (LUC), green fluorescent protein (GFP), red fluorescent protein (RFP), β-glucuronidase, and antibiotic resistance genes. Antibody drugs act on cells, driving reporter gene expression. Antibody activity can be indirectly reflected by detecting the reporter gene product (e.g., fluorescence, chemiluminescence, etc.). The reporter gene used in the method of this application is the Luc luciferase reporter gene.

[0017] As used herein, "acid dissociation" refers to the process of dissociating a drug-antidrug neutralizing antibody complex into an antidrug neutralizing antibody by acidifying a sample. In some embodiments of this application, the drug in the antidrug neutralizing antibody is an anti-TGF-β antibody drug. In some embodiments of this application, the antidrug neutralizing antibody is a neutralizing antibody against an anti-TGF-β antibody drug.

[0018] 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 a neutralizing antibody against an anti-TGF-β antibody drug.

[0019] In some cases, the terms "antibody" and "antibody drug" used in this article may be used interchangeably, for example, anti-TGF-β antibody and anti-TGF-β antibody drug may be used interchangeably.

[0020] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0021] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0022] This application proposes a method for detecting neutralizing antibodies against anti-TGF-β antibody drugs based on reporter gene assay, the principle of which is as follows: First, the neutralizing antibody against the anti-TGF-β antibody drug (hereinafter referred to as neutralizing antibody) is captured and released using the Bead Extraction with Acid Dissociation (BEAD) method. Specifically, magnetic beads that specifically bind to biotin, such as streptavidin (SA) beads, are used to capture the neutralizing antibody and TGF-β bound to the biotin-labeled anti-TGF-β antibody drug. After capture, the magnetic beads are repeatedly washed and eluted with acid to release the neutralizing antibody and TGF-β bound to the anti-TGF-β antibody drug. The magnetic beads are then magnetically adsorbed, and the supernatant after elution is added to an ELISA plate coated with transforming growth factor-β receptor (TGFβR) for incubation to remove endogenous TGF-β interference from the sample. The de-interference sample is then mixed with tris(hydroxymethyl)aminomethane solution (Tris alkaline solution) to restore the pH of the solution to neutral. The above mixture was then combined with the anti-TGF-β antibody working solution and the TGF-β working solution. After the reaction was complete, it was used to incubate HEK293 cells carrying the SBE-Luc reporter gene. After incubation, a luciferase reporter gene assay reagent (e.g., One-Lite) was added. TM The Luciferase Assay System reagent detects the signal value of the reporter gene. By comparing this signal value with that of the reporter gene in a sample without neutralizing antibodies, the neutralizing antibodies against anti-TGF-β drugs in the biological sample can be analyzed. If the sample contains neutralizing antibodies, TGF-β in the working solution can bind to the TGF-β receptor on the cell surface and activate the downstream Smad signaling pathway, driving luciferase expression through the Smad binding element (SBE), thereby increasing the signal value of the reporter gene. Conversely, if the sample does not contain neutralizing antibodies, TGF-β in the working solution binds to the anti-TGF-β antibody drug, and the signal value of the reporter gene decreases.

[0023] Specifically, this application provides a method for detecting neutralizing antibodies against anti-TGF-β antibody drugs, the method comprising: (1) The biological sample was treated with acetic acid. (2) The biotin-labeled anti-transforming growth factor-β antibody drug is contacted with and incubated with the biological sample to allow the anti-transforming growth factor-β antibody drug to bind to the neutralizing antibody against the anti-transforming growth factor-β antibody drug in the biological sample to form an anti-transforming growth factor-β antibody drug-neutralizing antibody complex; (3) Add magnetic beads that can specifically bind the biotin to the reactants obtained in step (2); (4) The magnetic beads obtained in step (3) are subjected to acid hydrolysis to release the neutralizing antibody; (5) In a reaction vessel with a bottom coated with transforming growth factor-β receptor (TGFβR), a neutralizing agent and the acid hydrolysis product obtained in step (4) are added and incubated to remove transforming growth factor-β (TGF-β) from the acid hydrolysis product, and the reaction solution is taken out from the reaction vessel as a test sample; (6) Contact and incubate the test sample with the anti-transforming growth factor-β antibody drug to obtain the first reaction sample; (7) The first reaction sample is contacted with transforming growth factor-β (TGF-β) and incubated to obtain the second reaction sample; (8) Contact and incubate the second reaction sample with cells carrying the luciferase reporter gene; (9) Add a detection reagent that can quantify the expression level of the luciferase reporter gene to the cells in step (8), and determine the neutralizing antibody in the biological sample based on the comparison with the quantification result of the expression level of the luciferase reporter gene when no test sample is added in step (6).

[0024] In some embodiments, the volume ratio of the biological sample to the acetic acid is 1:2 to 1:100, for example, 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 specific embodiments, the acetic acid concentration is 300 mM, and its volume ratio to the biological sample is 1:1.

[0025] In some embodiments, the magnetic beads are streptavidin (SA) magnetic beads.

[0026] In some implementations, the acid hydrolysis in step (4) uses a medium containing 2% fetal bovine serum at pH 2.0 (detection medium).

[0027] In some embodiments, the neutralizing agent is a tris(hydroxymethyl)aminomethane solution (Tris alkali). The volume ratio of the acid-hydrolyzed sample obtained in step (4) to the Tris alkali is 1:1 to 10:1, for example, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any range between two of the above ratios. In some specific embodiments, the volume ratio of the acid-hydrolyzed sample to the Tris alkali is 3:1.

[0028] In some implementations, the incubation time for step (3) is 2 hours.

[0029] In some implementations, the incubation time for step (6) is more than 8 hours.

[0030] In some implementations, the incubation in step (8) is carried out at 37°C and 5% CO2 for 6 to 7 hours.

[0031] In some implementations, the neutralizing antibody against the anti-TGF-β antibody drug is diluted with serum (e.g., human mixed serum) to the following concentrations in the test sample, such as 1-100 ng / mL, 1-200 ng / mL, 1-300 ng / mL, 1-400 ng / mL, 1-500 ng / mL, 1-600 ng / mL, 1-700 ng / mL, 1-800 ng / mL, 1-900 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, 1-7000 ng / mL, and 1-8000 ng / mL. In some embodiments, neutralizing antibodies against anti-TGF-β antibody drugs are diluted with serum (e.g., human mixed serum) to 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 1000, 8000 ng / mL, or any two of the above values, in the biological sample to be tested. In some specific embodiments, neutralizing antibodies against anti-TGF-β antibody drugs are diluted with serum (e.g., human mixed serum) to 537, 671, 839, 1049, 1311, 1638, 2048, 2560, 3200, 4000, 5000 ng / mL, in the biological sample to be tested.

[0032] In some implementations, the sensitivity of the method is 377 ng / mL.

[0033] In some implementations, the method is resistant to at least 200 μg / mL of anti-TGF-β antibody drugs.

[0034] In some embodiments, the biological sample is a blood, serum, or plasma sample.

[0035] This application proposes for the first time a method for detecting neutralizing antibodies against anti-TGF-β antibody drugs, which has the advantages of high sensitivity and strong specificity. At the same time, the use of the BEAD method improves the drug resistance of the method and can effectively remove endogenous TGF-β interference in the sample.

[0036] 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.

[0037] The following examples are for illustrative purposes only and are not intended to limit the scope of this application.

[0038] Example

[0039] 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.

[0040] 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.

[0041] I. Experimental Methods

[0042] The specific experimental steps for detecting neutralizing antibodies against anti-TGF-β antibody drugs are as follows: (a) Reagent preparation The preparation methods for some of the reagents used in this article are shown below: Acid hydrolysate (test medium): Add 2% FBS and 50 μg / mL hygromycin to MEM Alpha (Gibco, catalog number: 12561072) medium, with a pH of 2.0.

[0043] Washing solution: 1 × PBST, prepared by adding 0.05% Tween 20 to 1 × PBS.

[0044] Blocking solution: I-Block blocking buffer, prepared by dissolving I-Block blocking reagent (ThermoFisher, catalog number: T2015) in 1 × PBS, followed by the addition of 0.05% Tween 20.

[0045] (II) Pretreatment for magnetic bead extraction and acid dissociation (BEAD)

[0046] 1. Sample pretreatment: Human mixed serum was pretreated with 300 mM acetic acid, with a volume ratio of acetic acid to human mixed serum of 1:1, and incubated at room temperature with shaking for 40-45 min.

[0047] 2. Reaction of capture reagent with NAb: Mix 10 μL / well of biotin-labeled anti-TGF-β antibody (candidate drug, working solution concentration: 100 μg / mL) with 100 μL / well of pretreated human mixed serum and incubate at room temperature with shaking for 2 hours.

[0048] 3. Magnetic bead capture: Add 25 μL / well of SA magnetic beads (Promega, catalog number: V7820) to the above reaction system and incubate with shaking at room temperature for 2 hours.

[0049] 4. Washing and elution: Place the plate that has been captured as described above on a magnetic rack, wash three times with PBS, and use the magnetic beads to completely remove the buffer solution from the wells. Then, add acid hydrolysis buffer for elution, 80 μL / well, and incubate at room temperature with shaking for 30-40 min.

[0050] (III) Removal of endogenous TGF-β interference

[0051] 1. TGF-β receptor (TGFβR) coating: Add 50 μL of TGFβR working solution (manufacturer: Sino Biological, catalog number: 10358-H08B, working solution concentration: 5 ng / mL) to the ELISA plate and incubate at 37°C for 2.5 hours for coating.

[0052] 2. Washing the plate: Remove the ELISA plate and wash it 3 times with washing solution.

[0053] 3. Blocking: Add blocking solution to the ELISA plate, 300 μL / well, and incubate at room temperature with shaking for at least 2 hours.

[0054] 4. Wash the plate: Remove the ELISA plate and wash it 3 times with washing solution.

[0055] 5. Removal of endogenous TGF-β interference: Add Tris alkali solution, 20 μL / well, to the ELISA plate; place the eluted plate on a magnetic rack, add the sample from the supernatant to the wells of the ELISA plate containing Tris alkali solution at 60 μL / well, and incubate at room temperature with shaking for 2 hours.

[0056] (iv) Detection of neutralizing antibodies against anti-TGF-β antibodies based on reporter gene assays

[0057] 1. Neutralizing antibody reaction with anti-TGF-β antibody: In a new polypropylene plate, the sample after removing endogenous TGF-β interference was mixed with an equal volume of anti-TGF-β antibody working solution (working solution concentration: 500 ng / mL) and incubated overnight at room temperature with shaking.

[0058] 2. Reaction of neutralizing antibody / anti-TGF-β antibody mixture with TGF-β working solution: In a new polypropylene plate, mix the neutralizing antibody / anti-TGF-β antibody mixture that has been incubated above with TGF-β (Genscript, catalog number: Z03411) working solution (working solution concentration: 16 ng / mL) at a volume ratio of 1:1 and incubate at room temperature for 2 hours.

[0059] 3. Cell seeding: In 96-well cell culture plates, seed 1×10⁶ cells per well. 7 Cells / mL containing the reporter gene (SBE-Luc / HEK293) were mixed with the above-mentioned neutralizing antibody / anti-TGF-β antibody / TGF-β reaction mixture at a volume ratio of 1:1.

[0060] 4. Incubation: Place the 96-well culture plate with the added samples in a 37℃, 5% CO2 incubator for 6-7 hours.

[0061] 5. Detection: Remove the 96-well cell culture plate from the plate, allow it to equilibrate to room temperature, and add 80 μL / well of the detection reagent (One-Lite). TM The Luciferase Assay System was incubated at room temperature in the dark for 15 minutes, and the readings were taken on a multi-functional microplate reader.

[0062] II. Experimental Results

[0063] This article uses the signal-to-noise ratio (S / N) to reflect the content of neutralizing antibodies against anti-TGF-β (hereinafter referred to as neutralizing antibodies) in biological samples (human mixed serum). The formula for calculating S / N is:

[0064] The negative control (NC) sample was a mixed serum of humans that did not contain neutralizing antibodies.

[0065] (a) Method sensitivity

[0066] A series of neutralizing antibody-positive samples (537, 671, 839, 1049, 1311, 1638, 2048, 2560, 3200, 4000, 5000 ng / mL) were prepared using human mixed serum and commercially available neutralizing antibodies (GenScript, catalog number: A01860). The neutralizing antibodies in these positive samples were detected using the method of this invention, thereby verifying the sensitivity. The sensitivity curve is shown below. Figure 1 As shown. The experiment was repeated twice. The sensitivity of the method was 377 ng / mL.

[0067] (ii) Interference removal effect

[0068] Different concentrations of TGF-β (GenScript, catalog number: Z03411) (1000, 100, 10, 1.0, 0.1 ng / mL) were added to human mixed serum samples lacking neutralizing antibodies. Using the method of this invention, neutralizing antibody detection was performed on the above samples with and without an interference removal step. The results are shown in Table 1. It can be seen that without interference removal, the detection result was positive when TGF-β reached 100 ng / mL. However, after interference removal using TGFβR, the detection results in all five concentrations of TGF-β serum samples were negative. This sufficiently demonstrates that the method of this invention can effectively remove endogenous TGF-β interference in biological samples.

[0069] Table 1. Comparison of detection signal values ​​before and after interference removal

[0070] (III) Method resistance

[0071] Table 2 shows a comparison of drug resistance at different concentrations of neutralizing antibodies. The data shows that when the neutralizing antibody concentration is 1000 ng / mL, it can tolerate at least 50 μg / mL of anti-TGF-β antibody; when the anti-TGF-β antibody concentration reaches 100 μg / mL, the test results become abnormal. When the neutralizing antibody concentration is 4000 ng / mL, it can tolerate at least 200 μg / mL of anti-TGF-β antibody.

[0072] Table 2. Resistance of different concentrations of neutralizing antibodies to TGF-β antibodies

[0073] 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 anti-transforming growth factor-β (TGF-β) antibody drugs in biological samples, the method comprising: (1) The biological sample was treated with acetic acid. (2) The biotin-labeled anti-transforming growth factor-β antibody drug is contacted with and incubated with the biological sample to allow the anti-transforming growth factor-β antibody drug to bind to the neutralizing antibody against the anti-transforming growth factor-β antibody drug in the biological sample to form an anti-transforming growth factor-β antibody drug-neutralizing antibody complex; (3) Add magnetic beads that can specifically bind the biotin to the reactants obtained in step (2); (4) The magnetic beads obtained in step (3) are subjected to acid hydrolysis to release the neutralizing antibody; (5) In a reaction vessel with a bottom coated with transforming growth factor-β receptor (TGFβR), a neutralizing agent and the acid hydrolysis product obtained in step (4) are added and incubated to remove transforming growth factor-β (TGF-β) from the acid hydrolysis product, and the reaction solution is taken out from the reaction vessel as a test sample; (6) Contact and incubate the test sample with the anti-transforming growth factor-β antibody drug to obtain the first reaction sample; (7) The first reaction sample is contacted with transforming growth factor-β (TGF-β) and incubated to obtain the second reaction sample; (8) Contact and incubate the second reaction sample with cells carrying the luciferase reporter gene; as well as (9) Add a detection reagent that can quantify the expression level of the luciferase reporter gene to the cells in step (8), and determine the neutralizing antibody in the biological sample based on the comparison with the quantification result of the expression level of the luciferase reporter gene when no test sample is added in step (6).

2. The method of claim 1, wherein the volume ratio of the biological sample to the acetic acid is 1:

1.

3. The method of claim 1, wherein the magnetic beads are magnetic beads coupled with streptavidin (SA).

4. The method of claim 1, wherein the acid hydrolysis in step (4) is performed using a cell culture medium containing 2% fetal bovine serum at pH 2.

0.

5. The method of claim 1, wherein the neutralizing agent is a tris(hydroxymethyl)aminomethane solution.

6. The method of claim 1, wherein the incubation time in step (3) is 2 hours.

7. The method of claim 1, wherein the incubation time in step (6) is 8 hours or more.

8. The method of claim 1, wherein the incubation in step (8) is carried out at 37°C and 5% CO2 for 6 to 7 hours.

9. The method of claim 1, wherein the biological sample is a blood, serum, or plasma sample.

10. The method of claim 1, wherein the concentration of acetic acid is 300 mM.

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