Methods and kits for detecting the presence or amount of anti-drug antibodies to a nanobody in a biological sample

Abstract

This application relates to methods and kits for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples. The method includes steps of support adsorption, acid neutralization, specific binding, and electrochemiluminescence detection. The kit also provides a method for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples. The technical advantage of this application lies in reducing non-specific interference during nanobody detection.

Description

Technical Field

[0001] This application generally relates to the field of immunogenicity analysis of nanobodies, and more specifically, to methods and tools for detecting anti-drug antibodies against nanobodies. Background Technology

[0002] Nanobody drugs are a class of single-domain antibodies composed solely of the VHH variable region of the heavy chain, overcoming the limitations of traditional monoclonal antibody drugs in terms of tumor penetration and production costs. Due to their superior targeting ability and tissue penetration to specific tumor targets, nanobody drugs are considered a hot research area for next-generation antibody-based drugs and diagnostic reagents. Currently, several nanobodies have entered different stages of clinical research.

[0003] Nanobodies have small molecular weights, resulting in higher molar concentrations at the same dosage, which poses a challenge to drug resistance assays for immunogenicity. Furthermore, modified nanobodies readily bind to components in blank serum (e.g., targets), causing matrix interference and potentially leading to high false-positive rates. This phenomenon can mask the true immune response triggered by nanobodies after entry into the body, thus interfering with the interpretation of immunogenicity results and affecting the accurate assessment of drug safety.

[0004] Therefore, there is a need to develop methods that can effectively analyze the immunogenicity of nanobodies. Summary of the Invention

[0005] In a first aspect, this application provides a method for detecting the presence or amount of antidrug antibodies in a biological sample, comprising (a) contacting the biological sample with a support capable of binding the antidrug antibody but not binding the nanobody; (b) contacting the support with an acid-dissolving reagent to obtain an acid-dissolving sample containing dissociated antidrug antibodies; (c) mixing the acid-dissolving sample with a neutralizing reagent, biotin-labeled nanobody, and Ru derivative-labeled nanobody, thereby allowing the dissociated antidrug antibody to bind to the nanobody; and (d) contacting the mixture obtained in step (c) with a streptavidin-coated carrier for electrochemiluminescence detection to detect the presence or amount of the antidrug antibody based on the Ru derivative electrochemiluminescence.

[0006] Secondly, this application provides a kit for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples, comprising a support capable of binding the anti-drug antibody but not the nanobody, an acidification reagent, a neutralization reagent, biotin-labeled nanobody and Ru derivative-labeled nanobody, and a streptavidin-coated carrier for electrochemiluminescence detection.

[0007] The technical advantage of this application lies in its ability to reduce non-specific interference during nanobody detection. Detailed Implementation

[0008] Unless otherwise specified, this application is implemented using conventional molecular biology, microbiology, cell biology, biochemistry and immunology techniques in the art.

[0009] The terms "nanobody immunogenicity," "nanobody-resistant antibody," "anti-nanobody antibody," and similar terms used in this article refer to the biological process in which, when a nanobody is administered as a drug to an individual, the individual's immune system recognizes it as a foreign antigen, thereby producing immunoglobulins that specifically bind to the nanobody. That is, the aforementioned terms can refer to the inherent characteristic of nanobodies, as foreign substances, to induce an immune response in an individual's immune system (e.g., humoral immune response producing the aforementioned anti-nanobody antibodies, cellular immune response, etc.). The quantity or presence of "nanobody-resistant antibody" or "nanobody immunogenicity" is one of the core indicators for evaluating the feasibility and safety of nanobody-based drugs in clinical application.

[0010] The term "nanobody (Nb)" as used in this article refers to a class of antibodies composed solely of the variable region (VHH) of the heavy chain, also known as single-domain antibodies (sdAb) or VHH antibodies. Nanobodies can be classified into several different types, including monovalent nanobodies, bivalent nanobodies, bispecific nanobodies, multivalent nanobodies, and fusion nanobodies. Bivalent or multivalent nanobodies have two or more VHH structures and can recognize the same epitope of an antigen, exhibiting higher affinity than monovalent nanobodies. Bispecific nanobodies have two distinct VHHs and can bind to two different antigens, or different epitopes on the same antigen, demonstrating stronger antigen recognition capabilities than monovalent nanobodies.

[0011] As used in this article, the term "biological sample" refers to a sample taken from an individual (e.g., a human or other animal), such as a person receiving nanobody therapy. A biological sample can be any sample that may contain antibodies against nanobodies, such as bodily fluids, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites from the testes), vaginal douche fluid, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, fluid expelled from the nipple, fluid aspirated from different parts of the body (e.g., thyroid, breast), intraocular fluid (e.g., aqueous humor), etc.

[0012] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. The terms "individual," "subject," "patient," and "subject" are used interchangeably herein.

[0013] The term “a” or “an” entity refers to one or more of the entity; for example, “a pharmaceutical composition” is understood to mean one or more pharmaceutical compositions.

[0014] As used herein, the term "and / or" is considered a specific disclosure of each of the two specified features or components. Therefore, when used, for example, in the phrase "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when used, for example, in the phrase "A, B, and / or C," the term "and / or" is intended to include each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0015] As used herein, the term "about" is used to indicate the inherent variation of a numerical value, including errors in the apparatus or methods used to determine that value, or differences between the subjects under study. The term "about" includes the exact figures listed. In some embodiments, "about" means plus or minus 10% of a given value or range. In some embodiments, "about" means a difference of ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about". In some embodiments, "about" means a difference of ±1%, ±0.5%, ±0.2%, or ±0.1% of the value indicated by "about".

[0016] Throughout this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is for convenience and brevity only and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the scope description should be considered to have specifically disclosed all possible sub-ranges within that range and each numerical value. For example, a description of a range such as 1 to 6 should be considered to include sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and each number within said range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. The above rules apply regardless of how wide the range.

[0017] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0018] This application aims to provide an analytical method for the immunogenicity of nanobodies. For ease of understanding, without constituting a limitation of this application, its basic concept is briefly described as follows: Antibodies against nanobodies (i.e., anti-drug antibodies) in biological samples such as serum are captured by a support (e.g., Protein A) that can bind to anti-drug antibodies but not to nanobodies; after the anti-drug antibodies are captured by Protein A, other components that do not bind to Protein A are removed by washing, thereby eliminating non-specific interference and improving the specificity and drug resistance of the method; then, acid hydrolysis solution is added for dissociation, and the dissociated and neutralized sample is mixed with biotin and ruthenium-labeled nanobodies and incubated to form an immune complex of "biotinylated nanobodies - anti-nanobody antibodies - ruthenium derivative-labeled nanobodies". Then, the immune complex is transferred to streptavidin-coated microplates, the plates are washed to remove unbound components, substrates are added, and electrochemical signal values ​​are read on the MSD. The strength of the signal value is proportional to the content of anti-drug antibodies.

[0019] The embodiments of this application described below are intended to be exemplary only; many variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to fall within the scope of the invention as defined in any of the appended claims.

[0020] In a first aspect, this application provides a method for detecting the presence or amount of antidrug antibodies in a biological sample, comprising (a) contacting the biological sample with a support capable of binding the antidrug antibody but not binding the nanobody; (b) contacting the support with an acid-dissolving reagent to obtain an acid-dissolving sample containing dissociated antidrug antibodies; (c) mixing the acid-dissolving sample with a neutralizing reagent, biotin-labeled nanobody, and Ru derivative-labeled nanobody to allow the dissociated antidrug antibody to bind to the nanobody; and (d) contacting the mixture obtained in step (c) with a streptavidin-coated carrier for electrochemiluminescence detection to detect the presence or amount of the antidrug antibody based on Ru derivative electrochemiluminescence.

[0021] In some embodiments, the support may be a resin or magnetic beads containing Protein A, Protein G, or Protein A / G. In a preferred embodiment, the support may be a resin containing Protein A.

[0022] In some embodiments, the acid hydrolysis reagent may contain 100-500 mM of HAc or glycine. For example, the acid hydrolysis reagent may contain about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM of HAc.

[0023] In some embodiments, the neutralizing agent may contain 0.5–1.5 M of Trizma or Tris-HCl. For example, the neutralizing agent may contain about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, or about 1.5 M of Trizma. For example, the pH of the neutralizing agent may be about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, or about 9.5.

[0024] In some implementations, the carrier may be a streptavidin-coated MSD plate.

[0025] In some implementations, the biological sample can be serum, plasma, or whole blood.

[0026] Secondly, this application provides a kit for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples, comprising a support capable of binding the anti-drug antibody but not the nanobody, an acidification reagent, a neutralization reagent, biotin-labeled nanobody and Ru derivative-labeled nanobody, and a streptavidin-coated carrier for electrochemiluminescence detection.

[0027] In some embodiments, the support may be a resin or magnetic beads containing Protein A, Protein G, or Protein A / G. In a preferred embodiment, the support may be a resin containing Protein A.

[0028] In some embodiments, the acid hydrolysis reagent may contain 100-500 mM of HAc or glycine. For example, the acid hydrolysis reagent may contain about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM of HAc.

[0029] In some embodiments, the neutralizing agent may contain 0.5–1.5 M of Trizma or Tris-HCl. For example, the neutralizing agent may contain about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, or about 1.5 M of Trizma. For example, the pH of the neutralizing agent may be about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, or about 9.5.

[0030] In some implementations, the carrier may be a streptavidin-coated MSD plate.

[0031] In some implementations, the biological sample can be serum, plasma, or whole blood.

[0032] Unless otherwise stated, the embodiments provided below are for illustrative purposes only and are not intended to be limiting. Therefore, the invention should not be construed as being limited to the following embodiments, but should be construed as including any and all variations that become apparent from the teachings provided herein.

[0033] Where specific techniques or conditions are not specified in the experimental examples, the techniques or conditions described in the literature in this field or in accordance with the product instructions shall be followed. Reagents or instruments whose manufacturers are not specified are all commercially available standard products.

[0034] Example

[0035] Example 1. Study on the effect of Protein A treatment on the immunogenicity detection of nanobodies

[0036] Step 1: The Protein A Resin FF (GenScript, catalog number: L00464-50) was washed 6 times with 1×PBS, and then an equal volume of 1×PBS and Protein A Resin FF were mixed. The mixed Protein A Resin FF was added to a DVfree 96-well filter plate (catalog number 009602-20, manufacturer: Biocomma).

[0037] Step 2: All samples were pretreated with 1×PBS at a volume ratio of 1:20. The preparation and composition of the samples are shown in Tables 1 to 3 below.

[0038] Step 3: Add approximately 100 μL / well of the sample from step 2 to the DV-free 96-well filter plate from step 1, seal the plate, and incubate overnight (at least 16 hours) at room temperature with shaking at approximately 350 rpm.

[0039] Step 4: After incubation, the sample in the DV-free 96-well filter plate from step 3 was removed by centrifugation at 2000 rpm. Protein A Resin FF was then washed three times with 1×PBS. After the final wash, 1×PBS was removed by vacuum filtration.

[0040] Step 5: Add approximately 300 mM HAc to the washed DV-free 96-well filter plate from step 4 and dissociate it by oscillation at approximately 600 rpm for 20-30 min at room temperature.

[0041] Step 6: Biotin-labeled nanobody (Caplacizumab (His Tag), purchased from MCE, catalog number HY-P99227) and Ru-labeled nanobody were mixed in PBS containing 1% BSA to prepare a final concentration of 250 ng / mL (Master mix, MM solution, 1:1).

[0042] The biotin labeling method is as follows: The nanobody is replaced in PBS buffer and incubated with Biotin tag (Thermofisher, 21338) at a molar ratio of 1:20 for 1 hour. Then, Biotin-Nb is obtained by purification using a purification column.

[0043] The Ru tag (Sulfo tag) method is as follows: The nanobody is replaced in PBS buffer and incubated with Sulfo tag (Mesoscale, R32AD-1) at a molar ratio of 1:12 for 1 hour. Then, it is purified with a purification column to obtain RU-Nb.

[0044] The labeled nanobodies can be aliquoted and frozen at -80 degrees Celsius. When needed, they can be taken out and stored at 2-8 degrees Celsius for up to one month.

[0045] Step 7: Add 20 μL / well of 1 M Trizma, 90 μL / well of MM solution, and 50 μL / well of the dissociated sample from step 5 to a round-well polypropylene plate and incubate with shaking at room temperature for 2–2.5 hours. For confirmatory experiments, in addition to the biotin-labeled and Ru-labeled nanobodies described in step 6, the MM solution also contains 10 μg / mL of the unlabeled nanobodies to test whether the antidrug antibody is a drug-specific antidrug antibody (data expressed as inhibition rate).

[0046] Step 8:Block the MSD SA plate (Mmesoscale, catalog number L15SA) at room temperature with PBS containing 3% BSA at a rate of 150 μL / well for at least 30 min by shaking.

[0047] Step 9: Wash the streptavidin (SA) coated MSD plate (MSD SA plate), and then transfer the sample from the round-well polypropylene plate of step 7 into the MSD SA plate at 50 μL / well, and incubate at room temperature with shaking for 1-1.5 hr.

[0048] Step 10: Wash the MSD SA plate, then add 150 μL of MSD Read Buffer (2×) (Mesoscale, catalog number R92TD-1) to each well.

[0049] Test results

[0050] Table 1. Detection results of samples doped with different concentrations of rabbit polyclonal antibodies

[0051] Note: PC01-07: Positive samples prepared by artificially doping different concentrations of rabbit polyclonal antibody (GenScript, # C231UGC310-1, as an anti-nanobody antibody) into human mixed serum; only screening experiments were conducted.

[0052] NC: Negative human mixed serum samples without rabbit polyclonal antibodies; used for screening and confirmatory experiments.

[0053] Both screening and confirmatory experiments were conducted according to steps 1-10 above.

[0054] NC inhibition rate = (1 - ECLU confirmation / ECLU screening) 100

[0055] As can be seen from the data in the table above, the sensitivity of the method increases after pretreatment with Protein A. The sensitivity is approximately 7.81 ng / mL without Protein A treatment, while it increases to 1.95 ng / mL after Protein A treatment.

[0056] A qualified immunogenicity assay is generally considered to require an inhibition rate close to 0 after drug administration (i.e., the MM solution also contains unlabeled nanobodies), meaning that individuals not treated with the drug do not exhibit immunogenicity. The data in the table above show that the blank sample treated with Protein A had a low response value and an inhibition rate of 0%, indicating that the method can eliminate non-specific binding and meets the requirements for immunogenicity assays. However, without Protein A treatment, the background ECLU value was high, and the inhibition rate was 30.7%, failing to meet the requirements for immunogenicity assays.

[0057] Table 2. Detection results of serum samples from healthy individuals

[0058] Note: NC01-08 are serum samples from different healthy individuals who did not receive the drug.

[0059] Both screening and confirmatory experiments were conducted according to steps 1-10 above.

[0060] Inhibition rate = (ECLU screening - ECLU confirmation) / ECLU screening A positive inhibition rate of 100% indicates organic interference or the presence of pre-existing antibodies. The inhibition rate decreased after method optimization, which is presumably due to a mechanism effect. Negative values ​​within 10% can be considered as analytical errors. A higher inhibition rate indicates stronger matrix interference.

[0061] The data in the table above show that Protein A can reduce non-specific interference, as evidenced by a lower inhibition rate compared to untreated Protein A, indicating that Protein A treatment can effectively reduce the influence of matrix serum.

[0062] Table 3. Results of drug resistance test (simultaneous doping with nanobodies and rabbit polyclonal antibodies)

[0063] Note: DT01-08 is a sample prepared by doping different concentrations of nanobodies (Caplacizumab (His Tag), MCE) into human mixed serum containing 100 ng / mL of the above rabbit polyclonal antibody.

[0064] The drug resistance results in Table 3 show that 100 ng / mL of rabbit polyclonal antibody (i.e., the positive control in this example) can tolerate 50 μg / mL of the drug.

Claims

1. A method for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples, including (a) Contact the biological sample with a support that can bind the antidrug antibody but cannot bind the nanobody, the support being a resin containing Protein A; (b) Contact the support with an acid hydrolysis reagent to obtain an acid hydrolyzed sample containing dissociated antidrug antibodies; (c) The acid-hydrolyzed sample is mixed with a neutralizing agent, the biotin-labeled nanobody, and the Ru derivative-labeled nanobody, thereby allowing the dissociated antidrug antibody to bind to the nanobody; as well as (d) Contact the mixture obtained in step (c) with a streptavidin-coated carrier for electrochemiluminescence detection to detect the presence or amount of the antidrug antibody based on the Ru derivative.

2. The method of claim 1, wherein: The acid hydrolysis reagent contains 100-500 mM HAc or glycine; and / or The neutralizing agent contains 0.5-1.5 M Trizma or Tris-HCl.

3. The method of claim 1, wherein the carrier is a streptavidin-coated MSD plate.

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

5. A kit for detecting the presence or amount of anti-drug antibodies against nanobodies in biological samples, comprising a support capable of binding the anti-drug antibody but not the nanobody, an acidification reagent, a neutralization reagent, biotin-labeled nanobodies and Ru derivative-labeled nanobodies, and a streptavidin-coated carrier for electrochemiluminescence detection, wherein the support is a resin containing Protein A.

6. The kit of claim 5, wherein the acid hydrolysis reagent comprises 100-500 mM of HAc or glycine; and / or The neutralizing agent contains 0.5-1.5 M Trizma or Tris-HCl.

7. The kit of claim 5, wherein the carrier is a streptavidin-coated MSD plate.

8. The kit of claim 5, wherein the biological sample is serum, plasma or whole blood.

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