KR102626197B1 - How to accurately quantify the level of anti-drug antibodies - Google Patents

Abstract

The present invention relates to a method for accurately quantifying the level of anti-drug antibodies, comprising the steps of (a) adding an anti-drug antibody bound to a magnetic bead to a sample containing an anti-drug antibody and a drug; (b) removing magnetic beads from the sample that has undergone step (a); (c) dissociating the sample that has undergone step (b); (d) adding the sample that has gone through step (c) to a solution containing a drug bound to magnetic beads and a drug bound to a detector; (e) including adding a substrate after step (d).
According to the present invention as described above, the level of anti-drug antibodies present in the sample can be more accurately quantified by minimizing interference in the quantitative analysis of anti-drug antibodies by removing excess drug present in the sample in advance. there is.

Description

How to accurately quantify the level of anti-drug antibodies}

The present invention relates to a method for accurately quantifying the level of anti-drug antibodies, and more specifically, to a method for more accurately quantifying the level of anti-drug antibodies by minimizing interference by drugs present in a patient's sample.

The number of biotherapeutics in development has increased over the past decade. In the case of biotherapeutics, despite providing significant benefits to patients, almost all protein-based biotherapeutics induce unwanted immunogenicity, resulting in loss of efficacy, infusion reactions, anaphylaxis, and even life-threatening effects on endogenous proteins. There is a risk of side effects, such as reactions that threaten the body. Recent advances in immunogenicity analysis have revealed unexpectedly high levels of immunogenicity even in complete pharmaceutical products, creating new challenges in assessing immunogenicity and clinical relevance.

Therapeutic Drug Monitoring (TDM) refers to a series of activities that continuously observe drug response and provide feedback for appropriate drug treatment for individual patients. It is mainly used to maximize the treatment effect for each patient by monitoring drug concentration trends to maintain effective blood concentration and at the same time minimize the occurrence of drug toxicity. The main drugs covered by the service include several antibiotics, antiepileptics, and cardiotonic drugs with well-known therapeutic concentration ranges, and about 10,000 cases are performed annually. Monitoring of therapeutic drug concentration for optimal drug therapy circumvents individual differences in pharmacokinetics by adjusting drug dosage based on drug concentration data measured when administering a drug with a well-known therapeutic range of plasma concentration. It is possible. Recently, with the development of analytical chemistry to measure drugs and their metabolites in body fluids and the introduction of clinical pharmacokinetics, clinical monitoring of therapeutic drugs in individual patients has already become common, and this new medical field has become widespread. The need for research and clinical application is gradually increasing.

Anti-TNF agents are antibodies against Tumor necrosis factor α (TNF-α), a major inflammatory mediator that causes chronic inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. After being first used in Crohn's disease, it is now also used in ulcerative colitis. It is widely used. However, anti-TNF agents have the disadvantage that they are very expensive compared to other existing drugs and can cause opportunistic infections such as tuberculosis. Anti-TNF agents do not show a therapeutic effect in about 1/3 of patients, and the remaining patients It is known that if treatment is continued, its effectiveness decreases. According to a recently published meta-analysis, it is known that when antibodies against anti-TNF agents are formed and when the concentration of anti-TNF agents in the blood is low, there is a high possibility that the treatment response will be lost.

In the TAXIT (The Trough Concentration Adapted Infliximab Treatment) study published in 2015, the lowest drug level target (3-7 μg/mL) was set for inflammatory bowel disease patients treated with infliximab maintenance therapy. The dosing schedule was adjusted or the drug dose was adjusted. Looking at the results of the TAXIT study, there was no difference in clinical remission rates between the clinically based dosing group and the concentration-based dosing group in the maintenance phase, but the optimal phase ( In the optimization phase, the clinical remission rate increased from 65.1% to 88.4% for Crohn's disease patients. Additionally, in another study, when the lowest level of infliximab drug was maintained at 6 to 10 μg/mL in patients with inflammatory bowel disease, the mucosal healing rate evaluated using endoscopy was 80 to 90%. In this way, overseas, when infliximab is already administered to patients with inflammatory bowel disease, TDM is actively used and the test results are reflected in the treatment.

Meanwhile, in the case of a drug existing in the body, the lower the concentration of antibodies against the drug in the body, the less likely the immunogenicity of the drug will be, and in this case, the drug will be more effective. Therefore, it is necessary and very important to accurately measure the concentration of anti drug antibody (ADA), which is an antibody against drugs present in the body.

Because biotherapeutics, such as infliximab, generally have a long half-life and are almost always present in the test sample at most sampling points, sensitivity analysis of the drug becomes almost meaningless without knowing the resistance limits for the drug in the ADA assay. If the level of resistance to the drug in the ADA assay is lower than the drug concentration in the test sample, the ADA assay will not be able to detect the ADA present in the sample due to the interference of the drug, and the incidence of ADA will be underestimated. Therefore, there is a need to develop a method that has a high level of resistance to the target drug and can accurately detect the amount of ADA present in the body.

The purpose of the present invention is to more accurately quantify the level of anti-drug antibodies present in a sample by minimizing interference in the quantitative analysis of anti-drug antibodies by removing excess drugs present in the sample in advance.

To achieve the above object, the present invention includes the steps of (a) adding an anti-drug antibody bound to a magnetic bead to a sample containing an anti-drug antibody and a drug; (b) removing magnetic beads from the sample that has undergone step (a); (c) dissociating the sample that has undergone step (b); (d) adding the sample that has gone through step (c) to a solution containing a drug bound to magnetic beads and a drug bound to a detector; (e) adding a substrate after step (d); One aspect of the present invention is to provide a method for accurately quantifying the level of anti-drug antibodies including.

Preferably, the drug may be an antibody drug.

Preferably, in step (b), a complex in which a drug is bound to an anti-drug antibody to which a magnetic bead is bound can be removed.

According to the present invention as described above, the level of anti-drug antibodies present in the sample can be quantified more accurately by removing excess drug present in the sample in advance and minimizing interference in the quantitative analysis of anti-drug antibodies.

Figure 1 shows the result error due to the interference effect caused by excess free drug (infliximab) when the present invention is not applied.
Figure 2 illustrates a method for detecting drug (infliximab) antibodies by removing excess free drug (infliximab) using anti-drug antibodies coupled to magnetic beads according to an embodiment of the present invention. will be.
Figure 3 is a graph showing the affinity of anti-infliximab monoclonal antibodies and anti-infliximab monoclonal antibodies for infliximab according to an embodiment of the present invention.
Figure 4 is a table and graph showing the drug resistance of the quantitative measurement method of the anti-infliximab antibody of the present invention when the removal step of excess free drug (infliximab) is not performed.
Figure 5 is a table and graph showing the drug resistance of the quantitative measurement method of the anti-infliximab antibody of the present invention when the excess free drug (infliximab) is removed according to an embodiment of the present invention. .

Hereinafter, the present invention will be described in detail with reference to the attached drawings.

Referring to Figures 1 and 2, one embodiment of the present invention includes the steps of (a) adding an anti-drug antibody (magnetic bead-antibody) to which a magnetic bead is bound to a sample containing an anti-drug antibody and a drug; (b) removing magnetic beads from the sample that has undergone step (a); (c) dissociating the sample that has undergone step (b); (d) adding the sample that has gone through step (c) to a solution containing a drug bound to magnetic beads and a drug bound to a detector; (e) adding a substrate after step (d); Provides a method for accurately quantifying the level of anti-drug antibodies containing.

The drug may be an antibody drug. Biological products (biopharmaceuticals) are largely divided into protein drugs, antibody drugs, vaccines, and cell therapy products. Among these, antibody drugs refer to biopharmaceuticals that use monoclonal antibodies that target and bind to protein antigens involved in the immune cell signaling system or markers expressed on the surface of cancer cells. Antibody drugs can be classified into monoclonal antibodies (mab), antibody-drug conjugates (ADC), and immunoconjugates. Among these, monoclonal antibodies include mouse monoclonal antibodies, chimeric monoclonal antibodies, and humanized antibodies. They are divided into monoclonal antibodies and human monoclonal antibodies.

In addition, the monoclonal antibodies include infliximab, trastuzumab, pertuzumab, cetuximab, bevacizumab, ramucirumab, and rituximab, depending on the target. Among them, infliximab is a chimeric monoclonal antibody among monoclonal antibodies. It exhibits anti-inflammatory and immunosuppressive effects by inhibiting tumor necrosis factor, and is used to treat excessive and persistent inflammatory diseases caused by abnormalities in the immune system. It's a drug. It is mainly used in the form of injections for rheumatoid arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease in adults, Crohn's disease in children, psoriasis, and psoriatic arthritis.

In addition to the above-mentioned drugs such as infliximab, trastuzumab, pertuzumab, cetuximab, bevacizumab, ramucirumab, and rituximab, the drug may be any drug that is an antibody drug.

Step (a) is a step of adding an anti-drug antibody (magnetic bead-antibody) bound to a magnetic bead to a sample containing an anti-drug antibody and a drug. More specifically, the sample contains the free anti-drug antibody and the drug. It may include bound anti-drug antibodies and free drug. The sample may be not only serum, but also plasma and whole blood. Then, a complex (magnetic bead-antibody) in which a magnetic bead is bound to an anti-drug antibody with high affinity for the drug is added to the sample. As a result, most of the free drug present in the sample binds to the anti-drug antibody (magnetic bead-antibody) to which the magnetic beads are bound.

In step (b), the complex (magnetic bead-antibody-drug) containing the anti-drug antibody bound to the magnetic bead and the drug may be removed. More specifically, the magnetic bead-bound anti-drug antibody and the drug-bound complex (magnetic bead-antibody-drug) can be removed using a magnetic rod. A commonly used analysis method for anti-drug antibodies is bridge analysis, which forms a multivalent bridge between a capture drug (unlabeled or biotin-labeled form) and a labeled detection drug. These assays are characterized as susceptible to endogenous drug interference (false negative ADA) and/or drug target interference (false positive ADA). Therefore, in the field of analytical method development, many approaches have been used to mitigate the above vulnerabilities, such as acid or base dissociation, inhibition of third-party binding partner competition and/or elimination of interfering factors, and solid phase extraction. In the case of the present invention, free drugs that may cause interference in the measurement of anti-drug antibodies are removed in advance, so a specific immune complex consisting of a drug bound to an antibody and a magnetic bead can be easily formed, resulting in interference from the free drug. It is possible to accurately quantify the level of anti-drug antibodies (both free anti-drug antibodies and drug-bound anti-drug antibodies) present in a sample with greater specificity and sensitivity.

In step (c), the sample from which the magnetic beads were removed in step (b) can be dissociated. In order to accurately measure the level of anti-drug antibodies in a sample, drug-bound anti-drug antibodies (drug-antibodies) must be dissociated into free anti-drug antibodies. After removing the magnetic bead-bound anti-drug antibody and drug-bound complex (magnetic bead-antibody-drug) from the sample, a dissociation buffer containing a sufficient amount of acid to dissociate the drug-bound anti-drug antibody Bring the solution into contact with the sample. Here, the acid may include an organic acid or an inorganic acid. The dissociation step may take 10 minutes or less, preferably 5 minutes or less, and more preferably 2 minutes or less.

In step (d), the sample that has undergone the dissociation process can be added to a solution containing a drug bound to a magnetic bead and a drug bound to a detector. More specifically, the sample that has undergone the dissociation process is brought into contact with a neutralization buffer containing a drug bound to magnetic beads and a drug bound to a detector.

The detector is a means that can generate a signal by binding to an antigen, antibody, or protein, such as an enzyme or enzyme protein such as HRP (Horseradish peroxidase), ALP (Alkaline phosphatase), GO (Glucose oxidase); Fluorescent dyes of organic derivatives such as xanthene derivatives, cyanine derivatives, squaraine derivatives, naphthalene derivatives, coumarin derivatives, oxidazole derivatives, anthracene derivatives, arylmethine derivatives, and dipyrromethene derivatives; Inorganics such as CdS/ZnS Quantum dot, CdSe Quantum dot, CdSe/ZnS Quantum dot, CdSSe/ZnS Quantum dot, CdTe/CdSe/ZnS Quantum dot, CdS Quantum dot, ZnCdSe/ZnS Quantum dot, CdTe Quantum dot, Eu, Tb, etc. derivative fluorescent dye; Microparticles such as latex and polystyrene colored with fluorescent dyes, etc.; and nanoparticles composed of Au, Ag, Fe, Pt, Co, or a combination thereof, but are not limited thereto.

In the case of drugs and anti-drug antibodies, their three-dimensional structures are slightly modified through a dissociation process using acid, so the original structures of the drugs and antibodies can be restored through the neutralization process.

Meanwhile, during the neutralization reaction, it may take a considerable amount of time for the original drug and antibody structures to recover and for the drug and anti-drug antibody to bind again. At this time, the addition of the capture drug (magnetic bead-drug) and the detection drug (detector-drug) can enable the formation of a complex with the anti-drug antibody faster than the recovery of the structure of the existing drug present in the sample. In addition, unlike conventional ELISA products, by using magnetic beads with large particles with a diameter of about 200 nm to 5 μm, it is possible to manufacture a complex in which a large amount of drug is bound to magnetic beads with a large surface area. Therefore, as the molecular size expands and mobility increases, the specific reaction rate with anti-drug antibodies can be increased, thereby reducing the interference regression phenomenon of the dissociated free (unlabeled) drug.

The drug bound to magnetic beads (magnetic bead-drug) and the drug bound to the detector (detector-drug) can each bind to both arms (variable regions) of the free anti-drug antibody present in the sample, In this case, the complex in the form of magnetic bead-drug and detector-drug bound to both arms of the anti-drug antibody, respectively, can be collected using a magnetic rod. And, thanks to the magnetic beads, the collection and washing of the complex can be easily controlled and the sensitivity and specificity of the analysis can be promoted.

In step (e), a substrate that reacts with the detector may be added to the sample that has passed step (d). In this case, the level of the anti-drug antibody can be quantified through the reaction between the detector and the substrate. As a technology for quantifying the level of the anti-drug antibody, an immunoassay technology, also called EIA (Enzyme ImmunoAssay), based on specific binding between antigens and antibodies can be used. The EIA immunoassay technology includes a color change measurement method (chromogenic, or colorimetric), a chemiluminescence method, and a fluorescence method that measures the color reaction by absorbance, depending on the type of substrate used to detect the analyte.

In this way, by going through steps (a), (b), (c), (d), and (e), it is possible to accurately quantify the level of anti-drug antibodies present in the sample.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Selection of anti-infliximab antibody

Reactivity with the drug is confirmed for the 16 clones obtained by immunizing mice with infliximab, and the 1 clone with the highest reactivity is selected. To check reactivity with the drug, first, the infliximab drug is mixed with 0.1M NaHCO ₃ containing biotin-NHS (N-hydroxysuccinimide) ester and stirred at room temperature for 2 hours. After the reaction is completed, the biotin-labeled drug is separated from the solution using a size exclusion dialysis membrane. 50μL (100%) magnetic beads (Thermofisher) with a functional group of streptavidin and 5μg/mL biotin-conjugated infliximab were mixed and reacted at 37 degrees for 2 hours to form infliximab bound to the magnetic beads ( manufacture magnetic beads-infliximab). Meanwhile, in order to manufacture an alkalinephosphatase (ALP)-labeled drug, an ALP-labeled drug (ALP-drug) is prepared using an ALP labeling kit (Abcam Alkaline phosphatase conjugation kit).

Infliximab (0.02%) coupled with the prepared magnetic beads, ALP-labeled infliximab (1 μg/mL), and each clone (50 μL) were mixed and reacted for 7 minutes. Afterwards, the magnetic beads are recovered, washed, 600uM 4-MUP (4-Methylumbelliferyl - phosphate) is reacted with the magnetic beads, and the fluorescence intensity is measured after 7 minutes.

Each clone was used at two concentrations, 10ng/mL and 250ng/mL, and the fluorescence intensity and ratio of low and high concentrations are shown in Figure 3. 2F5 of clone sr No.11 was selected as it has relatively high reactivity compared to the commercial antibody (sr No.1).

Example 2

1. Buffer

The dissociation buffer is prepared with 0.1M Glycine-HCl at pH 1.92. The neutralization buffer was prepared by adding 0.05% preservative to 1M Tris-HCl, pH 8.5 solution. Each buffer was dispensed into each cartridge well prior to use.

2. Preparation (manufacturing) of conjugate

The conjugate of infliximab and magnetic beads and the conjugate of infliximab and enzyme (ALP) were prepared in the same manner as in Example 1, and were used by dispensing into each well of the cartridge. Sr No. selected in Example 1 to remove excess free drug present in the sample. A conjugate combining magnetic beads with the 2F5 clone antibody in 11 was used. The bonding method is the same as the magnetic bead bonding method in Example 1 above. The 2F5 clone antibody was mixed with 0.1M NaHCO ₃ containing biotin-NHS (N-hydroxysuccinimide) ester and stirred at room temperature for 2 hours. After the reaction is completed, biotin-labeled clones are separated from the solution using a size exclusion dialysis membrane. 2F5 clone antibody bound to magnetic beads was prepared by mixing magnetic beads (Thermofisher) with a streptavidin functional group and 5 μg/mL biotin-conjugated infliximab and reacting at 37 degrees for 2 hours.

3. Preparation of samples

(1) Reference sample (free)

Anti-infliximab (Biorad) at a concentration of 1 mg/ml was serially diluted into human plasma (healthy human) to produce anti-infliximab (Biorad) at concentrations of 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL, respectively. A map solution was prepared.

(2) Reference sample (composite)

Anti-infliximab (Biorad) at a concentration of 1 mg/ml was serially diluted with human plasma (from a healthy person) to give anti-infliximab (Biorad) at two-fold concentrations of 0 ng/mL, 10 ng/mL, 50 ng/mL and 250 ng/mL. After preparing the simab solution, it was mixed with 100μg/ml of infliximab in a 1:1 ratio (v/v) to obtain a final concentration of 0ng/mL, 10ng/mL, and 50μg/mL of infliximab. Prepare 50ng/mL and 250ng/mL anti-infliximab solutions.

Example 3. Immunoassay

The following immunoassay was performed using the buffer, conjugate, and reference sample prepared in Example 2.

The reference sample (complex) prepared in Example 2 was dispensed in an amount of 50 μl into the sample well contained in the cartridge. After adding the reference sample to the sample well, 50 μl of the conjugate of the magnetic beads and anti-infliximab antibody prepared in Example 2 above was added to the sample and reacted for 5 minutes, and then the magnetic beads were removed and discarded. . Then, 50 μl of the sample in the sample well was transferred to the reaction well containing the dissociation buffer mix of Example 2 and waited for 2 minutes. After the dissociation reaction, 150 μl of the dissociated sample was transferred to the neutralization buffer well of Example 2 containing 25 μl of the infliximab drug bound to magnetic beads and the drug infliximab bound to the enzyme (ALP). Then mixed and waited for 5 minutes. Afterwards, the immune complexes were collected using a magnetic rod, washed twice, waited for 7 minutes for final detection, and then the washed immune complexes were transferred to the final well containing the substrate 4-MUP (4-Methylumbelliferyl phosphate).

Measurement example 1 . Drug Tolerance

When the concentration of the infliximab drug present in the sample is 0, 10, 50, and 100 μg/ml, the magnetic beads prepared by Example 2 and anti-infliximab (selected in Example 1) Antibodies present in the reference sample (complex) in the same manner as in Example 3, except that the step of removing excess infliximab drug was not performed using conjugation of the 2F5 antibody clone of Sr No. 11). The concentration of Inflixmap antibody was measured. As a result, as shown in Figure 4, for each antibody concentration present in the sample, as the concentration of the drug present in the sample increases, the ratio of recovered antibodies gradually decreases, and the signal value indicating the presence of the antibody present in the sample also decreases. You can see that it is getting smaller. This means that if the excess infliximab drug is not removed, the immune complex (a combination of the drug and the anti-drug antibody) goes through the dissociation and neutralization step, and then the excess drug present in the sample binds to the anti-drug antibody. This is because an interference reaction occurs.

However, when the concentration of the infliximab drug present in the sample is 0, 10, 50, and 100 μg/ml, the magnetic beads prepared in Example 2 and anti-infliximab ( When a step was taken to remove excess infliximab drug using conjugation of the 2F5 antibody clone of Sr No. 11 selected in Example 1, looking at FIG. 5, each measured antibody concentration present in the sample As the concentration of the drug present in the sample increases, the decrease in recovery rate is much less compared to the case where the excess infliximab drug removal step is not performed, and the signal value indicating the presence of antibodies present in the sample also shows a significant change. You can see that it is not.

Therefore, by going through the step of removing excess infliximab drug as described above, the immune complex (a form in which the drug and anti-drug antibody are combined) goes through a dissociation and neutralization step and then interferes with the drug's binding to the anti-drug antibody again. It can be seen that almost no reaction occurred.

Claims (3)

(a) adding an anti-drug antibody bound to a magnetic bead to a sample containing an anti-drug antibody and a drug;
(b) removing the complex of the anti-drug antibody bound to the magnetic bead and the drug from the sample after step (a) using a magnetic rod;
(c) dissociating the drug-bound anti-drug antibody present in the sample that has undergone step (b) into a free anti-drug antibody by contacting it with a dissociation buffer solution containing acid;
(d) adding the sample after step (c) to a neutralization buffer containing a drug bound to a magnetic bead with a diameter of 200 nm to 5 μm and a drug bound to a detector, and reacting within 5 minutes;
(e) collecting the immune complex formed after step (d) using a magnetic rod and adding it to a substrate that allows quantification of the level of the anti-drug antibody through reaction with a detector; A method for accurately quantifying the level of anti-drug antibodies containing.
According to paragraph 1,
A method for accurately quantifying the level of anti-drug antibodies, wherein the drug is an antibody drug.
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