WO2025224166A1

WO2025224166A1 - Anti-drug antibody assays

Info

Publication number: WO2025224166A1
Application number: PCT/EP2025/061055
Authority: WO
Inventors: Gregor Bernd JORDAN; Thomas Bach; Roland Friedrich STAACK
Original assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Current assignee: F Hoffmann La Roche AG; Hoffmann La Roche Inc
Priority date: 2024-04-23
Filing date: 2025-04-23
Publication date: 2025-10-30
Anticipated expiration: 2026-10-23

Abstract

The present invention is in the field of immunoassays, particularly in the field of anti-drug antibody (ADA) assessment, and relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: (a) contacting the sample with a first and second labeled drug, and (b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti- drug antibody and the second labeled drug.

Description

ANTI-DRUG ANTIBODY ASSAYS ion is in the field of immunoassays, particularly in the field of anti-drug essment. ADA assays known in the art typically suffer from high rates of ts caused by residual drug targets present in the sample which can form ces with the assay reagents. In such cases, ADA assays with optimized are desired to remove these unwanted complexes formed by residual drug sample. The present invention is based, at least in part, on the a filtration step during sample processing to remove these unwanted a sample.

THE INVENTION

Immunogenicity assessment of therapeutic drug candidates is an important part of the drug development process. In cases of an immune response, appropriate interpretation of immunogenicity data is required to enable a correlation with clinical outcomes. Bioanalytical methods used for immunogenicity testing provide the required information by detecting and characterizing anti-drug antibodies (ADAs).

The "gold standard" assay format is the ADA bridging assay, where ADAs are complexed with labeled drug conjugates to form signal-giving complexes. However, this format is susceptible to interference (a) by residual drug in the sample that forms complexes with ADAs itself and therefore prevents complex formation of ADAs with assay reagents, and (b) by residual drug target in the sample that forms complexes with the assay reagents and therefore result in false positive readouts. The problem of residual drug in the sample is particularly relevant in studies involving the administration of high doses of biotherapeutics and/or biotherapeutics with long half-lives. In such cases, ADA assessment may be challenged by drug interference and ADA assays with optimized drug tolerance may be desired. This problem has been frequently addressed in the art.

To achieve high drug tolerance, many immunogenicity testing methods aim to break up the reversible, non-covalent binding interactions that hold together immune complexes formed by ADAs and drugs, in particular antibody drugs. Such interactions include electrostatic forces, hydrophobic interactions, Van der Waals forces and hydrogen bonds. A majority of such interactions can be weakened by high chaotropic salt concentrations, extremes of pH or detergents.

The use of low pH acid treatment has become a common method to dissociate immune complexes to achieve higher drug tolerance. For example, Butterfield, A.M., et al. (Butterfield, A.M., et al., Bioanal. 2 (2010) 1961-1969) compared three acid-based assay formats with respect to drug tolerance: Meso Scale Discovery® bridging assay format (Moxness, M., et al., Clin. Chem. 51 (2005) 1983-1985), solid-phase extraction with acid dissociation (SPEAD) (Smith, H.W., et al., Regul. Toxicol. Pharmacol. 49 (2007) 230-237) and affinity capture elution (ACE) (Bourdage, J.S., et al., J. Immunol. Meth. 327 (2007) 10-17). Another method that was developed to successfully eliminate drug interference uses a combination of precipitation and acid dissociation (PandA) (Zoghbi, J., et al., J. Immunol. Meth. 426 (2015) 62-69). Numerous other acid-based methods in many different variations have been successfully developed (Kelley, M., et al., AAPS. J. 15 (2013) 646-658; Sickert, D., et al., J. Immunol. Meth. 334 (2008) 29-36; Patton, A., et al., J. Immunol. Meth. 304 (2005) 189-195).

An alternative to pH mediated complex dissociation is the use of denaturing agents like guanidine hydrochloride. This chaotropic compound was successfully used to increase assay drug tolerance in a surface plasmon resonance-based method for the detection of ADAs (Barbosa, M.D., et al., Anal. Biochem. 441 (2013) 174-179). The use of such denaturing agents represents a powerful tool to dissociate immune complexes. However, they are generally considered as "harsh" conditions that can potentially damage protein structures, which again would be disadvantageous for ADA detection.

In contrast to that, non-denaturing ionic strength conditions are considered "gentle" on antibody function by causing only minimal or no changes in secondary and tertiary structures. This approach is commonly used in chromatographic methods, e.g. for gentle protein elution in the field of immunoaffinity purification. In chromatographic methods the salt magnesium chloride (MgCl2) is often used due to its relatively mild properties.

In WO 2021/224360, the use of a chaotropic salt, such as, e.g., MgCl2 or LiCI, was reported in high ionic strength dissociation assays (HISDA) to attain high drug tolerance while maintaining best possible structural integrity of ADAs.

While the problem of residual drug interference in ADA assays has been addressed in many studies, as evidenced above, the problem of interference by residual drug target in the sample remains unsolved. Hence, there is still a need in the art for improved ADA assays that address the problem of residual drug target interference.

The technical problem underlying the present invention can thus be formulated as the provisional of improved ADA assays.

SUMMARY OF THE INVENTION

The present invention is characterized in the herein provided embodiments and claims. In particular, the present invention relates, inter alia, to the following embodiments:

1. A method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, and b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, in the sample, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug. The method according to embodiment 1, wherein the sample is determined to comprise an anti-drug antibody, if the presence of the complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug can be detected in the sample. The method according to embodiment 1 or 2, wherein the sample is filtrated between steps (a) and (b). The method according to any one of embodiments 1 to 3, wherein the drug target is a virus. The method according to embodiment 4, wherein the virus is selected from the group consisting of: Hepatitis B virus (HBV), Hepatitis A virus (HAV), Hepatitis C virus (HCV), SARS-CoV-2, Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Ebola Virus, Influenza Virus, Human Cytomegalovirus (HCMV), Herpes Simplex Virus (HSV), Dengue Virus, Zika Virus, Papillomavirus, and Rabies Virus. The method according to any one of embodiments 1 to 5, wherein the filter has an average pore size of about 10 nm to about 60 nm, preferably of about 15 nm to about 55 nm, more preferably of about 20 nm to about 50 nm, even more preferably of about 25 nm to about 45 nm, even more preferably of about 30 to about 40 nm. The method according to any one of embodiments 1 to 6, wherein the drug comprised in the first and second labelled drug is a drug that has been administered to a patient from which the sample has been obtained. 8. The method according to any one of embodiments 1 to 7, wherein the drug comprised in the first and second labelled drug is an antibody, or an antigen-binding fragment thereof.

9. The method according to embodiment 8, wherein the first and/or second labelled drug antibody is added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 4 pg/mL or at least 10 pg/mL.

10. The method according to any one of embodiments 1 to 9, wherein the first labelled drug is labeled with a compound that allows immobilizing said drug on a solid surface or particle.

11. The method according to embodiment 10, wherein the first labelled drug is a biotinylated drug, in particular wherein the first labelled drug is a biotinylated antibody.

12. The method according to any one of embodiments 1 to 11, wherein the second labelled drug comprises a detectable marker or wherein the second labelled drug comprises a label that can be specifically bound by a binding molecule comprising a detectable marker, preferably wherein the binding molecule is an antibody or an antigen-binding fragment thereof.

13. The method according to embodiment 12, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug comprises a step of detecting the complex via the (i) detectable marker comprised in the second labelled drug or (ii) the binding molecule comprising the detectable marker.

14. The method according to embodiment 12 or 13, wherein the second labelled drug comprises a digoxigenin molecule, in particular wherein the second labelled drug is a dioxigenin-labelled antibody. 15. The method according to any one of embodiments 12 to 14, wherein the detectable marker is a marker that can generate a detectable signal, in particular wherein the detectable marker is selected from the group consisting of: a chromophore, an enzyme, an enzyme reactive compound whose cleavage product is detectable, a radioisotope, a fluorescent compound, a chemiluminescent compound, and derivatives and/or combinations of these markers.

16. The method according to embodiment 15, wherein the detectable marker is an enzyme, preferably wherein the enzyme is capable of converting a colorless form of a detection agent into a colored form of the detection agent, in particular wherein the enzyme is a peroxidase.

17. The method according to embodiment 15 or 16, wherein the sample is determined to comprise an anti-drug antibody, if the detectable signal generated by the detectable marker in the sample is higher than the detectable signal in a negative control that is free of anti-drug antibodies

18. The method according to any one of embodiments 1 to 17, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the antidrug antibody and the second labeled drug further comprises a step of immobilizing the complex on a solid surface or particle via the first labelled drug.

19. The method according to any one of embodiments 1 to 18, wherein the sample is a blood sample, in particular a whole blood sample, a plasma sample or a serum sample, or wherein the sample is an aqueous humour sample.

20. The method according to any one of embodiments 1 to 19, wherein the sample is pretreated prior to step (a) to dissociate existing complexes in the sample.

21. The method according to embodiment 20, wherein the sample is pretreated with a chaotropic agent prior to step (a). The method according to embodiment 21, wherein the chaotropic agent is magnesium chloride (MgCl2). The method according to any one of embodiments 1 to 22 comprising the steps of: i) providing a sample derived from a patient that has been administered with a drug, wherein the sample is suspected to comprise an anti-drug antibody against said drug, and wherein the sample may further comprise a residual drug target of said drug, ii) contacting the sample with a first and second labeled drug under suitable conditions to form a complex between the first labeled drug, the anti-drug antibody and the second labeled drug, iii) filtrating the sample to remove unwanted complexes formed between the first labeled drug, the drug target and the second labeled drug from the sample, iv) immobilizing the complexes formed between the first labeled drug, the anti-drug antibody and the second labeled drug comprised in the filtrate obtained in step (iii) to a solid surface or particle via the first labelled drug, v) removing non-complexed first and second labeled drug from the sample, and vi) detecting and/or quantifying the presence of an anti-drug antibody via a detectable marker comprised in the second labelled drug. A method for confirming the presence of an antibody drug antibody in a sample, the method comprising the steps of: a) providing a sample obtained from a patient; b) detecting and/or quantifying the presence of an anti-drug antibody in a first portion of the sample with the method according to any one of embodiments 1 to 23; c) repeating step (b) with a second portion of the sample, wherein the second portion of the sample is additionally contacted with excess unlabeled drug that competes with the first and second labeled drug for binding to the anti-drug antibody in the sample; and d) confirming the presence of an anti-drug antibody in the sample, if the presence of anti-drug antibodies can be detected in step (b) but not in step (c). 25. A kit for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the kit comprising at least: a first labeled drug, a second labeled drug and, optionally, a filter unit.

Accordingly, in a particular embodiment, the invention relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, and b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug and/or non-complexed anti-drug antibody.

That is, the present invention is directed to a bridging anti-drug antibody (ADA) assay comprising the use a first labeled drug and a second labeled drug that are capable of forming complexes with anti-drug antibodies in a sample. These complexes can be detected and/or quantified based on the label bound to the first and/or second drug.

A common problem with ADA assays is that the labelled drug cannot only form detectable complexes with ADAs, but also with other molecules that may be present in a sample, such as residual drug targets. Consequently, ADA assays inherently have high rates of false-positive results caused by unwanted interactions between the labelled drug reagents and the drug target.

The inventors have surprisingly found that adding a filtration step prior to detection/quantification of the desired complexes formed between the first and second labeled drug and the ADA can remove unwanted complexes formed between the first and/or second labeled drug and the drug target and/or non-complexed drug targets, thereby significantly reducing the frequency of false-positive results in ADA assays.

For example, it has been shown in Example 1 that applying a filtration step during sample processing removes unwanted complexes formed between a labeled antibody drug and its drug target, the hepatitis B virus, from a sample with almost 100% efficiency. Accordingly, it has been demonstrated that the claimed method can be used to reduce the frequency of falsepositive results in bridging ADA assays caused by residual drug targets in the sample.

The term "anti-drug antibody" as used herein denotes an antibody produced by the innate immune system of the recipient of a therapeutic antibody against said therapeutic antibody after administration thereof.

To allow separation of the desired complexes formed between the first and second labeled drug and the ADA from the unwanted complexes formed between the first and second labeled drug and the drug target and/or the non-complexed drug target, the pore size of the filter has to be adjusted accordingly. That is, a filter pore size has to be selected that allows the desired complexes formed between the first and second labeled drug and the ADAs to pass through, but sufficiently retains any unwanted complexes formed between the first and second labeled drug and the drug target and/or the non-complexed drug target.

The pore size of the filter that is to be selected to allow sufficient retention of the unwanted complexes formed between the first and second labeled drug and the drug target and/or the non-complexed drug target depends mainly on the size and/or shape of the drug target, and to a lesser extent on the size and/or shape of the first and second labeled drug.

The skilled person is capable of selecting a filter pore size that allows for sufficient separation of the desired complexes formed between the first and second labeled drug and the ADA from the unwanted complexes formed between the first and/or second labeled drug and the drug target and/or from non-complexed drug target. Accordingly, the filter can be functionally defined as having "an average pore size that retains residual drug target, orcomplexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug and/or non-complexed anti-drug antibody".

The drug target is preferably a drug target that can form a complex with the first and second labeled drugs. That is, the drug target is preferably a drug target that can form covalent and/or non-covalent interactions with the first and second labeled drug. In certain embodiments, the drug target is a drug target that can form non-covalent interactions with the first and second labeled drug.

In certain embodiments, the drug target is significantly larger in molecular weight and/or diameter than an anti-drug antibody to allow sufficient retention of the drug target by the filter.

The anti-drug antibody that is to be detected with the method according to the invention is preferably an IgG antibody and typically has dimensions of approximately 14.5 nm x 8.5 nm x 4.0 nm. Accordingly, the filter used in the method according to the invention preferably has an average pore size of at least 10 nm to allow complexes comprising the anti-drug antibody and the first and second labeled drug to pass through the filter. In certain embodiments, the filter used in the method according to the invention has an average pore size of at least 10 nm, preferably at least 15 nm, more preferably at least 20 nm.

The term "average pore size" is widely used in the art and would be understood by a person skilled in the art. The pore size can be measured, without limitation, by electron microscopy and the average pore size may be calculated by known statistical methods.

The upper limit of the average pore size has to be selected based on the size of the drug target. That is, the average pore size of the filter is preferably smaller than the diameter or smallest dimension of the drug target.

Thus, in certain embodiments, the filter that is used to retain the drug target either alone or in complex with the first and/or second labeled drug is preferably a filter having an average pore size larger than 10 nm, 15 nm or 20 nm, to allow the anti-drug antibody in complex with the first and second labeled drug to pass through the filter, and smaller than the diameter of the drug target or a complex comprising the drug target and the first and/or second labeled drug, preferably at least 5 nm smaller than the diameter of the drug target or a complex comprising the drug target and the first and/or second labeled drug.

Alternatively, the average pore size of the filter may be defined as a molecular weight cut-off (MWCO). That is, in the method of the present invention, the MWCO of the filter may be selected such that it allows complexes of the first and second labeled drug with the ADA to pass through but retains unwanted complexes formed between the first and second drug and the drug target and/or the drug target alone. The term "molecular weight cut-off" means that dissolved substances having a molecular weight above the cut-off are retained at a level of at least 90%. IgGl antibodies, which are most common among anti-drug antibodies, have a molecular weight of approximately 150 kDa. Thus, in certain embodiments, the filter used in the method according to the invention has a molecular weight cut-off (MWCO) of at least 150 kDa, at least 200 kDa, at least 250 kDa, at least 300 kDa, at least 400 kDa or at least 500 kDa. In a preferred embodiment, the filter used in the method of the present invention has a MWCO of at least 300 kDa. In certain embodiments, the filter used in the method according to the invention has a molecular weight cut-off (MWCO) of about 150 to about 500 kDa, of about 200 to about 500 kDa, of about 250 to about 500 kDa, of about 250 to about 400 kDa, or of about 300 kDa.

The drug target may be any molecule or biological entity comprising several epitopes to which a drug can bind and that is present in the sample that is to be analyzed with the method according to the invention. Preferably, the drug target is significantly larger in size than the anti-drug antibody or the complex comprising the anti-drug antibody and the first and second labeled drug, to allow efficient retention of the drug target with the filter. The drug target may be a biological entity, such as, without limitation, a virus, a bacterial cell, or a eukaryotic cell, or fragments, vesicles, or particles thereof.

Alternatively, the drug target may be a molecule, for example a protein. In such embodiments, the drug target molecule preferably comprises one or more epitopes that can be specifically bound by a drug. When the drug target molecule is smaller or of similar size as the ADA, it is preferred that the drug target molecule is a molecule that aggregates under assay conditions. For example, the drug target molecule may be a molecule that would readily pass through the filterwhen in a monomeric state. However, if the molecule aggregates under assay conditions, the drug target molecule may be sufficiently retained by a filter that does not retain the wanted complexes formed between the ADA and the assay reagents. The skilled person is capable of identifying drug target molecules that aggregate under assay conditions or of adjusting the assay conditions such that a drug target molecule sufficiently aggregates.

The drug target, or a complex thereof comprising the first and second labeled drug, may be defined based on its hydrodynamic radius. The hydrodynamic radius is an ion's effective radius in a solution taking into account all H2O molecules it carries in its hydration shell.

In certain embodiments, the drug target may be a biological entity, a molecule or an aggregate comprising multiple molecules that, either alone or when in complex with the first and second labeled drug, has a hydrodynamic radius that is larger than the hydrodynamic radius of the complex formed by the ADA and the first and second labeled drug.

In certain embodiments, the drug target, i.e., the biological entity, the molecule or the aggregate comprising multiple molecules, either alone or when in complex with the first and second labeled drug, may have a hydrodynamic radius of at least 20 nm, at least 25 nm, at least 30 nm, at least 35 nm, at least 40 nm, at least 45 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm or at least 100 nm.

In certain embodiments, a filter having an average pore size of 35 nm is used in the method of the invention. In such embodiments, the drug target, i.e., the biological entity, the molecule or the aggregate comprising multiple molecules, either alone or when in complex with the first and second labeled drug, preferably has a hydrodynamic radius of at least 35 nm. However, the skilled person is aware that the filter pore size may be adjusted based on the size of the drug target.

Since the drug target of a drug that is tested in an ADA assay is known, the skilled person has no difficulties adjusting the average filter pore size according to the nature of the drug target. In a particular embodiment, the invention relates to the method according to the invention, wherein the drug target is a virus.

That is, in a preferred embodiment, the drug target is a virus. Most viruses vary in diameter from 20 nm to 250-400 nm; the largest, however, measure about 500 nm in diameter and are about 700-1,000 nm in length. Accordingly, an average filter pore size should be selected that sufficiently retains the virus and/or any unwanted complexes formed between the virus and the first and second labeled drug but allows the desired complex formed between the ADA and the first and second labeled drug to pass through.

Thus, in certain embodiments, the invention relates to the method according to the invention, wherein the filter has an average pore size of about 10 nm to about 500 nm, of about 10 nm to about 400 nm, of about 10 nm to about 250 nm, of about 10 nm to about 200 nm, of about 10 to about 150 nm, of about 10 to about 100 nm, of about 10 to about 75 nm, or of about 10 to about 50 nm.

In certain embodiments, the invention relates to the method according to the invention, wherein the filter has an average pore size of about 20 nm to about 500 nm, of about 20 nm to about 400 nm, of about 20 nm to about 250 nm, of about 20 nm to about 200 nm, of about 20 to about 150 nm, of about 20 to about 100 nm, of about 20 to about 75 nm, or of about 20 to about 50 nm.

In certain embodiments, the invention relates to the method according to the invention, wherein the filter has an average pore size of about 30 nm to about 500 nm, of about 30 nm to about 400 nm, of about 30 nm to about 250 nm, of about 30 nm to about 200 nm, of about 30 to about 150 nm, of about 30 to about 100 nm, of about 30 to about 75 nm, or of about 30 to about 50 nm.

In a particular embodiment, the invention relates to the method according to the invention, wherein the filter has an average pore size of about 10 nm to about 60 nm, preferably of about 15 nm to about 55 nm, more preferably of about 20 nm to about 50 nm, even more preferably of about 25 nm to about 45 nm, even more preferably of about 30 to about 40 nm. In a particular embodiment, the invention relates to the method according to the invention, wherein the virus is selected from the group consisting of: Hepatitis B virus (HBV), Hepatitis A virus (HAV), Hepatitis C virus (HCV), SARS-CoV-2, Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Ebola Virus, Influenza Virus, Human Cytomegalovirus (HCMV), Herpes Simplex Virus (HSV), Dengue Virus, Zika Virus, Papillomavirus, and Rabies Virus.

In a particularly preferred embodiment, the drug target is Hepatitis B virus (HBV). Hepatitis B is one of the world's most prevalent diseases. Although most individuals seem to resolve the infection following acute symptoms, approximately 30% of cases become chronic. According to current estimates, 350-400 million people worldwide have chronic hepatitis B, leading to 500,000-1,000,000 deaths per year due largely to the development of hepatocellular carcinoma, cirrhosis, and other complications. Despite the availability of an effective vaccine, immunoglobulin therapy, interferon, and antiviral drugs, hepatitis B remains a major global health problem.

The Hepatitis B virus is one of the smallest enveloped animal viruses with a virion diameter of 42 nm. Accordingly, to successfully retain complexes comprising Hepatitis B virions and the first and/or second labeled drug and/or non-complexed Hepatitis B virions with the filter, the filter preferably has an average pore size of 40 nm or less. In certain embodiments, the filter that is used to retain complexes comprising Hepatitis B virions has an average pore size of about 10 nm to about 40 nm, preferably of about 15 nm to about 40 nm, more preferably of about 20 nm to about 40 nm, even more preferably of about 25 nm to about 40 nm, even more preferably of about 35 nm.

The diameters of other viruses disclosed herein is provided in the table below:

These viruses, or complexes of these viruses with the first and second labeled drug, may be retained with a filter having an average pore size that is smaller than the diameter of the respective virus. More preferably, the average pore size of the filter is at least 5 nm smaller than the diameter of the respective virus.

For example, a Hepatitis C virus, or a complex thereof comprising the first and second labeled drug, may be retained with a filter having an average pore size smaller than 50 nm, preferably smaller than 45 nm.

Thus, in certain embodiments, the filter that is used to retain a viral drug target in complex with the first and/or second labeled drug is preferably a filter having an average pore size larger than 10 nm, 15 nm or 20 nm, to allow the anti-drug antibody in complex with the first and second labeled drug to pass through the filter, and smaller than the diameter of the viral drug target, or a complex comprising the viral drug target and the first and/or second labeled drug, preferably at least 5 nm smaller than the diameter of the viral drug target, or a complex comprising the viral drug target and the first and/or second labeled drug. Based on the teaching provided herein above, the skilled person has no difficulties selecting a suitable filter based on the nature of the viral drug target.

In certain embodiments, the drug target is a protein comprising at least one epitope that can be specifically bound by a drug. When the drug target is a protein, it is preferred that the protein forms aggregates under assay conditions that can be retained by the filter. Even if a protein only comprises a single epitope that can be bound by the drug, aggregation of the protein will result in a multiplication of epitopes that can form complexes with the assay reagents, which will increase the hydrodynamic radius of the drug target and thus facilitate retention of the drug target with the filter.

In certain embodiments, the drug target is the protein Angiopoietin-2 (Ang2). Several antibody drugs directed against the target protein Ang2 have been proposed as angiogenesis inhibitors, for example in cancer therapy. The method of the present invention may thus be used to test whether a patient that has been treated with an anti-ANG2 antibody developed ADAs against said anti-Ang2 antibody. As it is known that Ang2 tends to form aggregates, Ang2 aggregates in a sample may form complexes with the assay reagents, i.e., the first and second labeled drug, and result in false-positive readouts. However, these complexes formed between the Ang2 aggregates, and the assay reagents may be removed from the sample with a suitable filter as described herein.

The filtration step may be performed at any step prior to the detection and/or quantification of complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug. In certain embodiments, the filtration step may be performed before the sample is contacted with the first and second labeled drug. That is, the sample may be filtrated to separate non-complexed ADAs or ADAs in complex with residual drug from residual drug target or residual drug target in complex with residual drug in the sample. Before the filtration step, the sample may be treated to dissociate existing complexes, for example with a chaotropic agent as described herein.

However, it is preferred that the filtration step is performed after the sample has been contacted with the first and second labeled drug and before detection and/or quantification of complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug.

Accordingly, in a particular embodiment, the invention relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, and b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample; characterized in that the method comprises a step of filtrating the sample between step (a) and step (b) with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug.

Adding the assay reagents, i.e., the first and second labeled drug, prior to the filtration step has the advantage that it will facilitate the separation of the wanted ADA complexes from the unwanted complexes comprising the drug target. That is, an ADA can bind one drug molecule with each of its two antigen-binding domains. Accordingly, contacting ADAs with an excess of the first and second labeled drug will result in the formation of trimers comprising the ADA and two drug molecules. That means that the first and second labeled drug can increase the size or hydrodynamic radius of the ADA only to a limited extent.

The drug target, on the other hand, typically comprises a large number of epitopes that can be bound by the drug. For example, when the drug target is a virus, or, more specifically, a protein present on the surface of the virus, multiple copies of the epitope exist which can be bound by the assay reagents. This significantly increases the hydrodynamic radius of the drug target and facilitates removal of complexes comprising the drug target.

Similarly, if the drug target is a protein that forms aggregates under assay conditions, a large number of epitopes will be present in the aggregate, even the protein only comprises a single epitope. Contacting these aggregates with the assay reagents prior to the centrifugation step will result in complex formation of the aggregate with multiple drug molecules, thereby increasing the size of the aggregate and facilitating the retention of the aggregates with the filter. As described above, adding the assay reagents prior to the filtration step has the advantage that the size (or hydrodynamic radius) of the drug target will increase, which will in turn facilitate separation of the drug target from the unwanted ADA complex. To further increase the size (or hydrodynamic radius) of the drug target, an additional reagent may be added that specifically binds to the drug target, but preferably does not compete with the assay reagents, i.e., the first and second labeled drug. This additional reagent may be any binding molecule that can specifically bind to the drug target.

Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the method comprises an additional step of adding a molecule to the sample that specifically binds to the drug target, but preferably does not compete for binding with the first and second labeled drug.

To guarantee efficient separation of the drug target from the ADAs, the filter used in the method according to the invention is preferably a centrifugal filter unit. That is, centrifugal forces may be used to used to facilitate the flow of the sample through the filter.

In certain embodiments, the filter unit comprising the sample is centrifuged for at least five minutes at about 500g, at about 1000g, at about 2000g, at about 3000g, 4000g, at about 5000g, at about 6000g, at about 7000g, at about 8000g, at about 9000g, at about 10000g, at about 11000g, at about 12000g, at about 13000 or at about 14000g to separate the drug target from the ADAs.

In certain embodiments, the filter unit comprising the sample is centrifuged for at least five minutes at about 500g to about 14000g, at about 500g to about 12000g, at about 500g to about 10000g, at about 500g to about 5000g, at about 500g to about 2500g, or at about 1000g to separate the drug target from the ADAs.

In certain embodiments, the filter unit comprising the sample is centrifuged for at least ten minutes at about 500g, at about 1000g, at about 2000g, at about 3000g, 4000g, at about 5000g, at about 6000g, at about 7000g, at about 8000g, at about 9000g, at about 10000g, at about 11000g, at about 12000g, at about 13000 or at about 14000g to separate the drug target from the ADAs.

In certain embodiments, the filter unit comprising the sample is centrifuged for at least fifteen minutes at about 500g, at about 1000g, at about 2000g, at about 3000g, 4000g, at about 5000g, at about 6000g, at about 7000g, at about 8000g, at about 9000g, at about 10000g, at about 11000g, at about 12000g, at about 13000 or at about 14000g to separate the drug target from the ADAs.

In certain embodiments, the filter unit comprising the sample is centrifuged for at least twenty minutes at about 500g, at about 1000g, at about 2000g, at about 3000g, 4000g, at about 5000g, at about 6000g, at about 7000g, at about 8000g, at about 9000g, at about 10000g, at about 11000g, at about 12000g, at about 13000 or at about 14000g to separate the drug target from the ADAs.

In a particular embodiment, the filter unit comprising the sample is centrifuged for at least five minutes at about 500g to about 5000g, at about 500g to about 4000g, at about 500g to about 3000g, at about 500g to about 2000g, or at about 1000g to separate the drug target from the ADAs.

In a particular embodiment, the filter unit comprising the sample is centrifuged for at least twenty minutes at about 500g to about 5000g, at about 500g to about 4000g, at about 500g to about 3000g, at about 500g to about 2000g, or at about 1000g to separate the drug target from the ADAs. The method according to the invention comprises a step of contacting a sample obtained from a patient with a first labeled drug and a second labeled drug. The first and second labeled drugs used in the method according to the invention are labeled variants of the drug that has been administered to the patient from which the sample has been obtained and against which the patient may have developed anti-drug antibodies.

Both the first and second labeled drugs comprise at least a drug portion and a label.

The drug portion comprised in the first and second labeled drug is preferably identical or at least highly similar to the drug that has been administered to the patient from which the sample has been obtained. In consequence, if a patient has developed anti-drug antibodies against a drug that has been administered to said patient, these anti-drug antibodies will also bind to the first and second labeled drugs used in the ADA assay of the present invention to form a detectable complex.

The drug portion comprised in the first and second labeled drug may be any drug that is capable of forming a complex with a drug target. That is, the drug portion comprised in the first and second labeled drug may be a drug that covalently or non-covalently interacts with the drug target.

In certain embodiments, the drug portion comprised in the first and second labeled drug is a binding molecule. The term "binding molecule", as used herein, refers to any molecule or part of a molecule that can specifically bind to an epitope of a drug target.

The term "binding" as used herein preferably relates to a specific binding. "Specific binding" means that a binding molecule (e.g., an antibody) binds stronger to a target, such as an epitope for which it is specific compared to the binding to another target. A binding molecule binds stronger to a first target compared to a second target if it binds to the first target with a dissociation constant (Kd) which is lower than the dissociation constant for the second target. Preferably the dissociation constant (Kd) for the target to which the binding molecule binds specifically is more than 10-fold, preferably more than 20-fold, more preferably more than 50- fold, even more preferably more than 100-fold, 200-fold, 500-fold or 1000-fold lower than the dissociation constant (Kd) for the target to which the binding molecule does not bind specifically.

As used herein, the term "Kd" (measured in "mol/L", sometimes abbreviated as "M") is intended to refer to the dissociation equilibrium constant of the particular interaction between a binding molecule (e.g., an antibody or fragment thereof) and a target molecule. Methods for determining binding affinities of compounds, i.e. for determining the dissociation constant KD, are known to a person of ordinary skill in the art and can be selected for instance from the following methods known in the art: Surface Plasmon Resonance (SPR) based technology, Bio-layer interferometry (BLI), quartz crystal microbalance (QCM), enzyme-linked immunosorbent assay (ELISA), flow cytometry, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA) and enhanced chemiluminescence (ECL).

The binding molecule comprised as a drug portion in the first and second labeled drug may be or may be derived, without limitation, from (a) an antibody or antigen-binding fragment thereof; (b) an aptamer; (c) an antibody-like protein; (d) a peptidomimetic, (e) a soluble T-cell receptor, or (f) a small molecule.

In a particular embodiment, the invention relates to the method according to the invention, wherein the drug comprised in the first and second labelled drug is an antibody or an antigenbinding fragment thereof.

The term "antibody or antigen-binding fragment thereof" as used herein refers to any type of antibody/antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity/binding activity. The antibody can be any type of immunoglobulin that is known in the art. For instance, the antibody can be of any isotype, i.e., IgA, IgD, IgE, IgG, IgM. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain FV, scFV), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T- cell epitope fusions (Troybodies) or Peptibodies.

In a preferred embodiment, the drug portion comprised in the first and second labeled drug is a monoclonal antibody. The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially similar and bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a variable region that binds a target, wherein the antibody was obtained by a process that includes the selection of the antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected antibody can be further altered, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered variable region sequence is also a monoclonal antibody of this disclosure. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 , (Elsevier, N. Y., 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1-2): 119-132 (2004) and technologies for producing human or human-like antibodies from animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., W098/24893, WO96/34096, W096/33735, and WO91/10741 , Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591 ,669 (all of GenPharm); 5,545,807; WO 97/17852, U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and Marks et al., Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995).

It is preferred herein that the drug portion comprised in the first and second labeled drug is an antibody, or an antigen-binding fragment thereof, that specifically binds to and neutralizes a viral drug target, i.e., an antibody that is used in the treatment of viral infections in a patient. Accordingly, when used in the method according to the present invention, such an antibody may undergo complex formation (a) with the viral drug target or (b) with an anti-drug antibody that may be present in a sample from a patient that has been treated with said anti-viral antibody. To reduce the risk of false positive results, unwanted complexes formed between the first and second labeled antibody and residual viral drug target can be removed by filtration with a suitable filter, as described herein above.

In a particularly preferred embodiment, the first and second labeled drug comprise an antibody portion that specifically binds to and neutralizes Hepatitis B virus. In certain embodiments, the first and second labeled drug are labeled variants of the anti-HBsAg antibody R07565020 (P1AG3782), as disclosed in WO 2021/249990, which is fully incorporated herein by reference. In certain embodiments, the anti-HBsAg antibody comprises a VH sequence having the amino acid sequence:

QVQLVESGGGVVQPGRSLRLSCEASGFTFSNYGMQWVRQAPGKGLEWVAIIWADGTKQYYGDSVKGR FTISRDNFKNTLYLQMNSLRGEDTAMYFCARDGLYASAPNDVWGQGTLVTVSS (SEQ ID NO:1); and a VL sequence having the amino acid sequence:

DIQMTQSPSSLSAYVGDRVTITCRASQRISTYLNWYHQRPGKSPSLLIYGASSLQSGVPSRFSASASGTDFT LTISSLRPEDLGTYYCQQTYTLPPNSGGGTKVEIK (SEQ ID NO:2).

In certain embodiments, the anti-HBsAg antibody comprises a heavy chain sequence having the amino acid sequence:

QVQLVESGGGVVQPGRSLRLSCEASGFTFSNYGMQWVRQAPGKGLEWVAIIWADGTKQYYGDSVKGR FTISRDNFKNTLYLQMNSLRGEDTAMYFCARDGLYASAPNDVWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO:3); and a VL sequence having the amino acid sequence:

DIQMTQSPSSLSAYVGDRVTITCRASQRISTYLNWYHQRPGKSPSLLIYGASSLQSGVPSRFSASASGTDFT LTISSLRPEDLGTYYCQQTYTLPPNSGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:4).

In certain embodiments, the drug portion comprised in the first and second labeled drug is an aptamer. The term "aptamer" as used herein refers to single-stranded nucleic acid molecules with secondary structures that facilitate high-affinity binding to a target molecule. In certain embodiments, the single-stranded nucleic acid is ssDNA, RNA or derivatives thereof to improve bioavailability.

In certain embodiments, the drug portion comprised in the first and second labeled drug is an antibody-like protein. As used herein, the term "antibody-like protein" refers to a protein that has been engineered (e.g. by mutagenesis of loops) to specifically bind to a target molecule, such as a drug target. Typically, such an antibody-like protein comprises at least one variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the antibody-like protein to levels comparable to that of an antibody. The length of the variable peptide loop typically consists of 10 to 20 amino acids. The scaffold protein may be any protein having good solubility properties. Preferably, the scaffold protein is a small globular protein. Antibody-like proteins include without limitation affibodies, anticalins, and designed ankyrin repeat proteins (for review see: Binz H.K. et al. (2005) Engineering novel binding proteins from nonimmunoglobulin domains. Nat. Biotechnol. 23(10): 1257-1268). Antibody-like proteins can be derived from large libraries of mutants, e.g. be panned from large phage display libraries and can be isolated in analogy to regular antibodies. Also, antibody-like binding proteins can be obtained by combinatorial mutagenesis of surface-exposed residues in globular proteins. Antibody-like proteins are sometimes referred to as "peptide aptamers".

In certain embodiments, the drug portion comprised in the first and second labeled drug is a peptidomimetic. As used herein, a "peptidomimetic" is a small protein-like chain designed to mimic a peptide. Peptidomimetics typically arise from modification of an existing peptide in order to alter the molecule's properties. For example, they may arise from modifications to change the molecule's stability or biological activity. This can have a role in the development of drug-like compounds from existing peptides. These modifications involve changes to the peptide that will not occur naturally (such as altered backbones and the incorporation of nonnatural amino acids).

In certain embodiments, the drug portion comprised in the first and second labeled drug is a soluble T cell receptor. As used herein, the term "soluble T cell receptor" refers to heterodimeric truncated variants of native TCRs, which comprise extracellular portions of the TCR a-chain and -chain linked by a disulfide bond, but which lack the transmembrane and cytosolic domains of the native protein. The terms "soluble T cell receptor a-chain sequence and soluble T cell receptor -chain sequence" referto TCR a-chain and |3- chain sequences that lack the transmembrane and cytosolic domains. The sequence (amino acid or nucleic acid) of the soluble TCR a-chain and -chains may be identical to the corresponding sequences in a native TCR or may comprise variant soluble TCR a-chain and -chain sequences, as compared to the corresponding native TCR sequences. The term "soluble T cell receptor" as used herein encompasses soluble TCRs with variant or non-variant soluble TCR a-chain and p-chain sequences. The variations may be in the variable or constant regions of the soluble TCR a- chain and p-chain sequences and can include, but are not limited to, amino acid deletion, insertion, substitution mutations as well as changes to the nucleic acid sequence, which do not alter the amino acid sequence.

In certain embodiments, the drug portion comprised in the first and second labeled drug is a small molecule. The term "small molecule", as used herein, generally refers to an organic molecule that is less than about 2000 g/mol in molecular weight, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. The small molecule is preferably a small molecule drug that bind specifically to a drug target, such as any of the drug targets disclosed herein.

It is to be understood that the type of the drug portion affects the size of the complexes that are formed. While the cutoff values provided herein are mostly designed for antibody drugs, the skilled person would have no difficulties adjusting the filter pore size so that complexes formed between the first and second labeled drug and the ADA can pass through the filter and complexes formed between the first and second labeled drug and the drug target and/or the non-complexed drug target are retained.

The method of the present invention is a bridging assay in which an anti-drug antibody is complexed with a first and a second labeled drug to form a signal-giving complex. To form this signal-giving complex, one of the first or second labeled drugs comprises a capturing moiety with which the complex can be immobilized to a surface and the other of the first or second labeled drugs comprises a detection moiety that enables detection and/or quantification of the immobilized complex.

Preferably, the drug portions of the first and second labeled drugs are identical, whereas the labels that are conjugated to the drug portions are different and do not interact with each other. One of the first or second labeled drug comprises a capturing moiety.

Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the first labelled drug is labeled with a compound that allows immobilizing said drug on a solid surface or particle.

That is, the first labeled drug comprises a label that allows immobilizing or "capturing" the drug on a solid surface or a particle. The label that allows immobilizing the drug on a solid surface or particle may therefore also be referred to as "capturing moiety". Capturing or immobilizing the first labeled drug on a solid surface or particle has the advantage that excess reagents that do not bind to the solid surface or particle, either directly or indirectly via the first labeled drug, can be easily removed in a washing step.

Thus, in a particular embodiment, the invention relates to the method according to the invention, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug further comprises a step of immobilizing the complex on a solid surface or particle via the first labelled drug.

In certain embodiments, the capturing moiety is a chemical or biological moiety that can undergo covalent or non-covalent bond formation with another chemical or biological moiety present on or comprised in a solid surface or particle.

Preferably, the first labeled drug comprises a biotin moiety which can form a strong non- covalent bond with a streptavidin (or avidin or neutravidin) molecule linked to a solid surface or particle. Alternatively, the first labeled drug may comprise a chemical moiety that can undergo a click reaction with a compatible chemical moiety present on a solid surface or particle.

The solid surface or particle preferably has been functionalized with a chemical or biological moiety that can undergo covalent or non-covalent bond formation with a compatible label of the first labeled drug. In certain embodiments, the solid surface or particle may be a bead of any suitable material and/or size that has optionally been functionalized with a chemical or biological moiety that can undergo covalent or non-covalent bond formation with the label of the first labeled drug.

In a preferred embodiment, the solid surface or particle on which the first labeled drug can be immobilized is the bottom of a well of a microtiter plate that has optionally been functionalized with a chemical or biological moiety that can undergo covalent or non-covalent bond formation with the label of the first labeled drug. In certain embodiments, the bottom of the well of the microtiter plate has been functionalized with (strept)avidin molecules. Streptavidin-coated microtiter plates are well-known in the art and commercially available.

In a particular embodiment, the invention relates to the method according to the invention, wherein the first labelled drug is a biotinylated drug, in particular wherein the first labelled drug is a biotinylated antibody.

That is, the first labeled drug preferably comprises a biotin moiety with which the drug portion, preferably in complex with the ADA, can be immobilized to a solid surface or particle that has been functionalized with avidin, or a variant thereof, such as streptavidin.

In a particularly preferred embodiment, the first labeled drug is a biotinylated antibody, also referred to as a capture antibody, which can be immobilized to a solid surface or particle that has been functionalized with avidin, or a variant thereof, such as streptavidin, via its biotin moiety.

The first labeled drug comprising the biotin moiety can be immobilized on solid surfaces or particles that have been functionalized with a streptavidin (or avidin or neutravidin) molecule. In a particular embodiment, the first labeled drug comprising the biotin moiety, for example a biotinylated antibody, can be immobilized on a streptavidin-coated microtiter plate.

Alternatively, the first labeled drug may be labeled with a streptavidin (or avidin or neutravidin) molecule and may be immobilized on a solid surface or particle that has been functionalized with a biotin molecule. Alternative capturing moieties that allow immobilizing drugs to a solid surface or particle are known in the art and may be used within the present invention to capture ADA-drug complexes on said solid surface or particle.

The skilled person is aware of methods to label a drug with a capturing moiety, in particular any of the capturing moieties disclosed herein. Moreover, the skilled person is aware of suitable linkers that may be used to connect the drug portion to the capturing moiety. In particular, the skilled person is well aware of methods to conjugate antibody drugs to a capturing moiety, such as a biotin moiety.

The other of the first or second labeled drug comprises a detection moiety.

In a particular embodiment, the invention relates to the method according to the invention, wherein the second labelled drug comprises a detectable marker or wherein the second labelled drug comprises a label that can be specifically bound by binding molecule comprising a detectable marker.

That is, in certain embodiments, the second labeled drug may comprise a detectable marker with which the second labeled drug can be directly detected and/or quantified, preferably when in complex with the anti-drug antibody and the first-labeled drug, more preferably when the complex is immobilized on a solid surface or particle. Various labels that can be detected and/or quantified are known in the art and comprise, without limitation, dyes, fluorophores, and so forth.

Alternatively, the second labeled drug may comprise a label that can be specifically bound by a binding molecule, such as an antibody or antibody fragment, comprising a detectable marker. Preferably, the label that can be specifically bound by the binding molecule, e.g., an antibody or antibody fragment, comprising a detectable marker has been linked or conjugated to the drug portion comprised in the second labeled drug.

Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug comprises a step of detecting the complex via the (i) detectable marker comprised in the second labelled drug or (ii) the binding molecule comprising the detectable marker.

The label that can be specifically bound by a binding molecule, e.g., an antibody or antibody fragment, comprising a detectable marker may be any molecule comprising an epitope that can be specifically bound by the binding molecule comprising the detectable marker.

In a particular embodiment, the invention relates to the method according to the invention, wherein the second labeled drug comprises a digoxigenin molecule.

That is, in certain embodiments, the second labeled drug preferably comprises a digoxigenin molecule that can be specifically bound by an anti-digoxigenin (anti-DIG) antibody or antibody fragment comprising a detectable moiety.

As mentioned elsewhere herein, the drug used in the method according to the invention is preferably an antibody drug. Accordingly, in embodiments where the drug is an antibody drug, the second labeled drug is preferably a digoxigenin-labeled antibody. Hence, in a particular embodiment, the invention relates to the method according to the invention, wherein the second labelled drug is a digoxigenin-labeled antibody. Methods of labeling antibodies with a digoxigenin molecule are well known in the art.

To allow detection and/or quantification of the complex comprising the anti-drug antibody and the first and second labeled drug, either the second labeled drug or the binding molecule, e.g., the antibody or antibody fragment, that specifically binds to the second labeled drug has to comprise a detectable marker.

The term "detectable marker", as used herein, refers to any agent that can produce a diagnostic signal detectable by any suitable means. A "detectable marker" may be, without limitation, a chromophore, an enzyme, an enzyme reactive compound whose cleavage product is detectable, a radioisotope, a fluorescent compound, a chemiluminescent compound, and derivatives and/or combinations of these markers. In a particular embodiment, the invention relates to the method according to the invention, wherein the detectable marker is an enzyme.

The enzyme that is used as detectable marker is preferably an enzyme that catalyzes the conversion of a substrate into a colored or fluorescent reaction product. Suitable enzymes are known in the art and include, without limitation: peroxidases, such as horseradish peroxidase, urease, alkaline phosphatase, glucoamylase and p-galactosidase.

In a particular embodiment, the invention relates to the method according to the invention, wherein the detectable marker is a peroxidase.

Peroxidases are a large family of enzymes which catalyze the transfer of either one or two electrons via a single electron transfer from an organic substrate, using hydrogen peroxide as electron acceptor. The substrate may be any substrate that produces a detectable signal when converted by a peroxidase, such as a colorimetric or fluorescent signal.

Various substrates for peroxidases are known in the art. A particularly preferred substrate is 3-(4-dihydroxy phenyl) propionic acid (HPPA), which produces a high fluorescence signal when converted by a peroxidase. Another suitable substrate that may be used with a peroxidase is ABTS (2,2'-azino-bis[3-ethylbenziazoline-6-sulfonic acid]). However, other suitable substrates of peroxidases are known in the art.

Alternatively, the second labeled drug or the binding molecule comprising the detectable marker may be labeled with a fluorescent compound or fluorophore to allow detection of complexes comprising ADAs. The term "fluorophore" as used herein refers to a fluorescent molecule or a portion of a molecule which gives rise to fluorescent properties. There are a number of parameters which together describe the fluorescence characteristics of a fluorophore. These include, for example, characteristic wavelengths, such as the wavelengths of excitation and emission maxima, the breadth of the peaks for excitation and emission, the difference between the excitation and emission maxima (the "Stokes shift"), fluorescence intensity, quantum yield, and extinction coefficient. The skilled person is aware of methods for conjugating a label to a drug, in particular to an antibody drug. Covalent conjugation can be either direct or via a variety of linkers, including chemical linkers and peptide linkers.

The first and second labeled drug may be added to the sample at any suitable concentration that allows formation of complexes with antibody-drug antibodies comprised in said sample.

However, the inventors have surprisingly found that adding high concentrations of the first and second labeled drug to the sample reduces incubation times without significantly affecting sensitivity of the assay. Furthermore, addition of high concentrations of the first and second labeled drug increases the chances that ADAs form complexes with the first and second labeled drugs but not with residual drug present in the sample.

In certain embodiments, the first and second labeled drug are added to the sample such that complexation of the ADAs in the sample can be achieved in less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 2 minutes or less than 1 minute, when incubated at room temperature in a suitable buffer.

In certain embodiments, the first and second labeled drug may be added to the sample at a concentration of at least 0.1 nM, at least 0.5 nM, at least 1 nM, at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, or at least 100 nM.

Preferably, the first and second labeled drugs comprise a monoclonal antibody having a molecular weight of approximately 150 g/mol. In such embodiments, the first and/or second labeled drug may each be added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 3 pg/mL, at least 4 pg/mL, at least 5 pg/mL, at least 6 pg/mL, at least 7 pg/mL, at least 8 pg/mL, at least 9 pg/mL or at least 10 pg/mL. In a preferred embodiment, the first and/or second labeled drug may each be added to the sample at a final concentration of about 10 pg/mL. In certain embodiments, the first labeled drug is a capture antibody that is added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 3 pg/mL, at least 4 pg/mL, at least 5 pg/mL, at least 6 pg/mL, at least 7 pg/mL, at least 8 pg/mL, at least 9 pg/mL or at least 10 pg/mL, preferably at a concentration of about 10 pg/mL.

In certain embodiments, the second labeled drug is a detection antibody that is added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 3 pg/mL, at least

4 pg/mL, at least 5 pg/mL, at least 6 pg/mL, at least 7 pg/mL, at least 8 pg/mL, at least 9 pg/mL or at least 10 pg/mL, preferably at a concentration of about 10 pg/mL.

In a particular embodiment, the invention relates to the method according to the invention, wherein the first and/or second labeled drug antibody is added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 4 pg/mL, at least 10 pg/mL or at least 50pg/mL.

It is preferred herein that the first and second labelled drug are added to the sample in equal concentrations. That is, the molar ratio between the first labeled drug and the second labeled drug after mixing with the sample preferably ranges from about 1:10 to about 10:1, preferably from about 1:5 to about 5:1, more preferably from about 1:2 to about 2:1. In a particularly preferred embodiment, the molar ratio between the first labeled drug and the second labeled drug after mixing with the sample is about 1:1.

It is preferred that the sample is incubated with the first and second labeled drug for at least

5 minutes, for at least 10 minutes, for at least 15 minutes, for at least 20 minutes, for at least 25 minutes or for at least 30 minutes. In certain embodiments, the sample is incubated with the first and second labeled drug between 10 min. +/- 10 % to 30 min. +/- 10 %.

Within the present invention, complexes comprising an ADA which are immobilized on a solid surface or particle via the capturing moiety of the first labeled drug may be detected and/or quantified via the detectable moiety of the second labeled drug. The term "detect" as used herein means the detection of whether or not a complex comprising an ADA is present in a sample. That is, detecting the presence of a complex comprising an ADA in a sample provides information as to whether a patient sample contains ADAs and thus whether a patient has developed ADAs to a drug administered to said patient.

The method according to the invention may be a qualitative detection assay in which the presence or absence of ADAs in a sample is determined. Forthis, a sample may be determined to comprise ADAs if immobilized complexes comprising the first and second labeled drugs and the ADA can be detected via the detectable marker that produces a detectable signal. More specifically, a sample may be determined to comprise ADAs if the detectable signal generated by the detectable marker comprised in the immobilized complex can be detected above a defined threshold. This threshold may vary depending on the type of detectable marker comprised in the labeled drug. Preferably, a negative control that is known to be free of ADAs is used define the threshold. That is, the signal that is detected with a negative control without ADAs may be used as a threshold to determine whether or not a patient sample comprises ADAs. More specifically, a patient sample may be determined to comprise ADAs if the detectable signal obtained with the patient sample is higher than the detectable signal obtained with the negative control.

Accordingly, in certain embodiments, the invention relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, and b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample, and c) determining the sample to comprise an anti-drug antibody, if the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug can be detected in the sample; characterized in that the method comprises a step of filtrating the sample with a filter, wherein the filter has an average pore size that retains residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug and/or non-complexed anti-drug antibody.

In certain embodiments, the invention relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, wherein at least one of the first or second labeled drug comprises a detectable marker that can generate a detectable signal, b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample via the detectable signal generated by the detectable marker; and c) determining the sample to comprise an anti-drug antibody, if the detectable signal in the sample is higher than the detectable signal obtained with a negative control that is free of anti-drug antibodies; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug and/or non-complexed anti-drug antibody.

In certain embodiments, a patient sample may be determined to comprise ADAs if a statistically elevated signal is obtained with respect to a negative control that is free of ADAs.

In certain embodiments, a patient sample may be determined to comprise ADAs if the detectable signal obtained with the patient sample is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900% or at least 1000% higher than the detectable signal obtained with the negative control that is free of ADAs. Preferably, the negative control and the patient sample are analyzed in parallel under identical conditions, preferably in the same microtiter plate. However, the threshold may also be calculated based on the signal(s) obtained with one or more previously analyzed negative controls.

In certain embodiments, the method according to the invention may be used to quantify the concentration of ADAs in a sample. The term "quantifying", as used herein, refers to any method for obtaining a quantitative measure.

To allow quantification of ADAs in a sample, a calibration curve may be used. The calibration curve may be generated by analyzing control samples comprising known amounts of ADAs with the method according to the invention. Based on the slope of the generated calibration curve, the concentration of ADAs in a patient sample can be calculated. Preferably, the samples comprising known amounts of ADAs are analyzed in parallel under identical conditions as the patient samples, preferably in the same microtiter plate.

Complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug may be detected and/or quantified with any suitable method. Typically, the method with which the complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug are detected and/or quantified depends on the type of detectable marker comprised in the first and/or second labeled drug and thus on the type of detectable signal that is generated by the detectable marker. The skilled person is aware of suitable read-out methods to detect and/or quantify a detectable signal.

In certain embodiments, the detectable signal generated by the detectable marker is a colorimetric, a fluorescent or a luminescent signal. In such embodiments, the samples (including negative and positive control samples) may be analyzed in a microtiter plate and the detectable signal may be detected and/or quantified with a suitable microtiter plate reader.

The method according to the invention comprises at least one washing step to remove labeled drugs that have not formed complexes with ADAs before the detection/quantification step. The washing step typically takes place when the complex comprising the ADA and the first and second labeled drug is immobilized on a solid surface or particle.

Accordingly, in certain embodiments, the invention relates to a method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug to form a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug, b) removing non-complexed first and second labeled drug from the sample, c) detecting and/or quantifying the presence of the complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample, and d) determining the sample to comprise an anti-drug antibody, if the presence of said complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample can be detected; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug and/or non-complexed anti-drug antibody.

Removing first and second labeled drugs from the sample that did not form a complex with the ADA may be achieved with any suitable method known in the art. Preferably, the complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug is immobilized to a solid surface or particle, such as a bottom of a well of a microtiter plate, via a capturing moiety comprised in the first or second labeled drug. Excess first and second labeled drug may then be removed by replacing the liquid fraction of the sample with a suitable buffer solution not comprising the first and second labeled drug. For example, complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug may be immobilized at the bottom of a well of a microtiter plate. The liquid fraction of the sample may then be aspirated and replaced with a suitable buffer not comprising the first and second labeled drug.

The washing step to remove excess non-complexed first and second labeled drug may be repeated several times, such as 2 times, 3 times, 4 times or 5 times to guarantee efficient removal of non-complexed first and second labeled drug.

A suitable washing buffer with which the liquid fraction of the sample is replaced may be lxPBS with 0.05% Tween 20. However, the skilled person is able to identify other suitable buffers.

In certain embodiments, the method according to the invention is an immunoassay, more specifically an in vitro immune assay. The term "in vitro" denotes either an artificial environment as such or that a process or reaction is performed within such an artificial environment.

The term "immunoassay" denotes any technique that utilizes specifically binding molecules, such as antibodies, to capture and/or detect a specific target for qualitative or quantitative analysis. In general, an immunoassay is characterized by the following steps: 1) immobilization or capture of the analyte and 2) detection and measuring the analyte. The analyte can be captured, i.e. bound, on any solid surface, such as e.g. a membrane, plastic plate, or some other solid surface.

In certain embodiments, the method according to the invention is an enzyme-linked immunosorbent assay (ELISA).

The term "ELISA" denotes an enzyme-linked immunosorbent assay. Different ELISA formats and applications are known in the art (see, e.g., Crowther, "Enzyme- Linked Immunosorbent Assay (ELISA)," in Molecular Biomethods Handbook, Rapley et al. [eds.], pp. 595-617, Humana Press, Inc., Totowa, NJ (1998); Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988); Ausubel et al. (eds.), Current Protocols in Molecular Biology, Ch. 11, John Wiley & Sons, Inc., New York (1994)).

One specific ELISA format is a so-called "direct ELISA". In this ELISA format a target, e.g. a polypeptide, present in a sample is detected. In a direct ELISA the sample containing the target is brought in contact with a solid phase, such as e.g., stationary or immobilized support (e.g., a microtiter plate well). The target, if present in the sample, becomes immobilized to the solid phase, and is thereafter detected directly using an enzyme-conjugated detection molecule. If the target is an antigen the detection molecule is an antibody specific for the antigen, or if the target is an antibody specific for an antigen the detection molecule is an enzyme- conjugated antibody specific for the antigen.

Another specific ELISA format is a so-called "indirect ELISA". In this ELISA format an antigen (or an antibody) is immobilized to a solid phase (e.g., a microtiter plate well). Thereafter an antigen-specific antibody (or antigen) is added followed by the addition of a detection antibody specific for the antibody that specifically binds the antigen. This detection antibody can be a „species-specific" antibody (e.g., a goat anti-rabbit antibody).

Another specific ELISA format is a so-called "sandwich ELISA". In this format the antigen is immobilized on a solid phase (e.g., a microtiter plate well) via capture by an antibody specifically binding to the antigen (i.e., a capture antibody), which is (covalently or via a specific binding pair) immobilized on the solid phase. Generally, a sample comprising the antigen is added to the solid phase, followed by washing. If the antigen of interest is present in the sample, it is bound by the capture antibody to the solid phase. When the antigen of interest is an antibody, such as an ADA, the "sandwich ELISA" may also be referred to as a "bridging ELISA".

The above-specified ELISA formats can be combined. A sandwich ELISA can be a "direct sandwich ELISA", wherein the captured antigen is detected directly by using an enzyme- conjugated antibody directed against the antigen. A sandwich ELISA can be an "indirect sandwich ELISA", wherein the captured antigen is detected indirectly by using an antibody directed against the antigen, which is then detected by another enzyme-conjugated antibody which binds the antigen-specific antibody either directly or via an attached label. With a reporter reagent, the third antibody is detected.

Prior to the filtration step, the first and second labeled drugs are to be mixed with a sample. The sample is preferably a sample derived from a patient who has been administered a drug and may be suspected of having developed anti-drug antibodies against said drug. Accordingly, the sample may be any sample that may comprise anti-drug antibodies.

The term "patient", as used herein, refers to animals, including mammals, preferably humans.

The term "sample" as used herein, refers to a volume or mass obtained, provided, and/or subjected to analysis. In certain embodiments, the sample is a liquid sample that has been obtained from a patient, e.g., with a needle.

In certain embodiments, the sample is a blood sample or a processed blood sample. That is, the sample may be a whole blood sample. It is, however, preferred that the sample is a processed blood sample, such as a plasma or serum sample.

The term "whole blood sample", as used herein, refers to blood material which is not part of the body of an individual anymore. A whole blood sample may be provided by removing blood from an individual, but may also be provided by using previously isolated blood material. The whole blood sample may be removed from an individual using conventional blood collection techniques. For example, the whole blood may be extracted from a vein in the arm of an individual using a needle, or via finger prick.

The term "plasma", as used herein, refers to the pale yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. This makes plasma the extracellular matrix of blood cells. It is a fluid which is composed of about 92% water, 7% vital proteins such as albumin, gamma globulin, anti-hemophilic factor, and other clotting factors, and 1% mineral salts, sugars, fats, hormones and vitamins. The term "serum", as used herein, refers to the blood component that is neither a blood cell (serum does not contain white or red blood cells) nor a clotting factor. It is the blood plasma not including the fibrinogens. Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances (e.g., drugs and microorganisms).

Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the sample is a blood sample, preferably a whole blood sample, a plasma sample or a serum sample. In a particularly preferred embodiment, the sample is a serum sample.

The skilled person is aware of techniques to obtain a blood sample from a subject and process said blood sample to obtain a plasma or serum sample.

Alternatively, the sample may be another liquid sample derived from a patient. For example, the sample may be an aqueous humour sample. The term aqueous humour refers to a transparent water-like fluid similar to blood plasma, but containing low protein concentrations. It is secreted from the ciliary body, a structure supporting the lens of the eyeball. It fills both the anterior and the posterior chambers of the eye, and is not to be confused with the vitreous humour, which is located in the space between the lens and the retina, also known as the posterior cavity or vitreous chamber. Accordingly, in some embodiments, the invention relates to the method according to the invention, wherein the sample is an aqueous humour sample. The skilled person is aware of techniques to collect an aqueous humour sample from a patient.

In certain embodiments, the sample is directly contacted with the first and second labeled drug to allow formation of ADA complexes.

However, it is preferred herein that the sample is pre-treated before contacting with the first and second labeled drug to dissociate existing complexes between residual drug and ADAs and/or residual drug target in the sample. That is, the method according to the invention may comprise a step of reducing residual drug interference. Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the sample is pretreated prior to step (a) to dissociate existing complexes in the sample.

Existing complexes in the sample, such as complexes comprising residual drug and ADAs or residual drug target, may be dissociated with any suitable method known in the art. For example, complexes in the sample may be dissociated by low pH acid treatment, as described by Butterfield, A.M., et al. (Bioanal. 2 (2010) 1961-1969), Moxness, M., et al. (Clin. Chem. 51 (2005) 1983-1985), Smith, H.W., et al. (Regul. Toxicol. Pharmacol. 49 (2007) 230-237), Bourdage, J.S., et al. (J. Immunol. Meth. 327 (2007) 10-17), Zoghbi, J., et al. (J. Immunol. Meth. 426 (2015) 62-69), Kelley, M., et al. (AAPS. J. 15 (2013) 646-658), Sickert, D., et al. (J. Immunol. Meth. 334 (2008) 29-36) and Patton, A., et al. (J. Immunol. Meth. 304 (2005) 189-195), which are all incorporated herein by reference in their entirety.

Alternatively, dissociation of existing complexes may be achieved by treating the sample with denaturing agents, such as guanidine hydrochloride, before contacting the sample with the first and second labeled drug. Respective methods have been described by Barbosa, M.D., et al. (Anal. Biochem. 441 (2013) 174-179), which is incorporated herein by reference in its entirety.

Preferably, existing complexes in the sample are dissociated with chaotropic salts, such a MgCl2 or LiCI. Using chaotropic salts has the advantage that the structural integrity of the ADAs is maintained, resulting in more robust assay results. The dissociation of complexes comprising ADAs and residual drug with chaotropic salts is disclosed in WO 2021/224360, which is fully incorporated herein by reference.

Accordingly, in a particular embodiment, the invention relates to the method according to the invention, wherein the sample is pretreated with a chaotropic agent prior to step (a).

The term "chaotropic agent" means, in accordance with the present invention, any substance that disturbs the three-dimensional structure of the hydrogen bonds in water. Thus, intramolecular binding forces, which are involved in the formation of spatial structures of biological molecules, are also weakened, causing the solubilization or even denaturation of biological molecules.

Preferably, the chaotropic agent is a weak or medium strength chaotropic salt that is capable of disrupting protein complexes but does not result in complete denaturation of the ADAs. That is, in certain embodiments, the chaotropic agent is a medium strength chaotropic salt, preferably with a cation between potassium and calcium in the lyotrophic series according to Hofmeister and an anion between hydrogen phosphate and nitrate in the lyotrophic series according to Hofmeister.

The term "lyotrophic series according to Hofmeister" denotes the ranking of anions and cations based on their chaotropic properties as first described by Hofmeister (Arch. Path. Anatom. Pathobiol. 24 (1888) 247-260). This lyotrophic series is for anions as follows:

F < SO₄ ²’ < HPO₄ ²’ < CH₃COO’ < Ch < NO₃’ < Br < CIO₃’ < I’ < CIO₄’ < SCN’, <CI₃CCOO’ and for cations as follows:

NH4 < K⁺ < Na⁺ < Li⁺ < Mg²⁺ < Ca²⁺ < guanidinium.

Accordingly, in some embodiments, the chaotropic salt has a cation selected from the group of cations consisting of potassium, sodium, lithium, magnesium and calcium, and an anion selected from the group of anions consisting of (hydrogen)phosphate, acetate and chloride.

Preferred chaotropic salts that may be used to disrupt complexes in the sample may be, without limitation, magnesium chloride (MgCl2) or lithium chloride (LiCI).

In a particular embodiment, the invention relates to the method according to the invention, wherein the chaotropic agent is magnesium chloride (MgCh). It is preferred herein that the sample is incubated with a chaotropic salt at a final cation charge normality in the range of and including 1 N to 12 N, preferably in the range of and including 6.5 N to 8.5 N.

In some embodiments, the final MgCl2 cation charge normality is 7.2 N +/- 10 % corresponding to a final MgCl2 concentration of 3.6 M +/- 10 % or the final LiCI cation charge normality is 8 N +/- 10 % corresponding to a concentration of 8 M +/- 10 %.

The term "normality" denotes the measure of concentration equal to the gram equivalent weight of solute per liter of solution. The normality formula of interest is N = M*n, with n = the number of equivalents/the number of single-charge ions the species can react with. To convert normality to molarity or vice-versa in the case of MgCl2, it has to be taken into account a 1 M solution will generate a 2 M solution of chloride ions and a 1 M solution of Mg²⁺ ions, which because of their charge also have a value of 2 for n. Thus, in this case, N = (1 M)(2) = 2 N, i.e. MgCb has a cation charge normality of 2 N.

In certain embodiments, the sample is incubated in a solution comprising about 1 - 10 M MgCb, preferably about 2 - 5 M MgCb, more preferably about 3 - 4 M MgCb.

Most preferably, the sample is diluted ten-fold in a 4 M MgCb solution to achieve dissociation of complexes in the sample, resulting in a final MgCb concentration of approximately 3.6 M. Thus, in a particularly preferred embodiments, the sample is incubated in about 3.6 M M MgCb to achieve dissociation of complexes comprising ADAs and residual drug.

The sample may be incubated with the chaotropic agent for any amount of time that allows for sufficient dissociation of existing complexes comprising ADAs and residual drug. In certain embodiments, the sample may be incubated with the chaotropic agent between 20 min. +/- 10 % to 60 min. +/- 10 %. In certain embodiments, the sample may be incubated with the chaotropic agent for about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes or about 60 minutes. Subsequent to the incubation step, the sample comprising the chaotropic agent may be directly contacted with the first and second labeled drug without additional treatment of the sample. However, it is preferred herein that mixing the sample comprising the chaotropic agent with the first and second labeled drug reduces the concentration of the chaotropic agent in the sample to allow complex formation between the ADA in the sample and the first and second labeled drug.

In certain embodiments, mixing the sample comprising the chaotropic agent with the first and second drug reduces the concentration of the chaotropic agent in the sample at least by a factor of 2, more preferably by a factor of 5, most preferably by a factor of 10.

In certain embodiments, the chaotropic agent is MgCb. It is preferred that after mixing the sample comprising the chaotropic agent MgCb with the first and second labeled drug, the concentration of MgC decreases to below IM, preferably below 0.5 M.

In certain embodiments, the MgCb concentration in the sample after addition of the chaotropic agent is about 3.6 M. Aten-fold dilution of the sample with first and second labeled drug will then result in an MgCb concentration in the sample of about 0.36 M.

In certain embodiments, pre-treatment of the sample for complex dissociation and contacting of the sample with the first and second drugs results in dilution of the sample by a factor of about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400 or about 500. In a preferred embodiment, pretreatment of the sample for complex dissociation and contacting of the sample with the first and second drugs results in dilution of the sample by a factor of about 100.

In a particular embodiment, the invention relates to the method comprising the steps of: i) providing a sample derived from a patient that has been administered with a drug, wherein the sample is suspected to comprise an anti-drug antibody against said drug, and wherein the sample may further comprise a residual drug target of said drug, ii) contacting the sample with a first and second labeled drug under suitable conditions to form a complex between the first labeled drug, the anti-drug antibody and the second labeled drug, iii) filtrating the sample to remove unwanted complexes formed between the first labeled drug, the drug target and the second labeled drug from the sample, iv) immobilizing the complexes formed between the first labeled drug, the antidrug antibody and the second labeled drug comprised in the filtrate obtained in step (iii) to a solid surface via the first labelled drug, v) removing non-complexed first and second labeled drug from the sample, and vi) detecting and/or quantifying the presence of an anti-drug antibody via a detectable marker comprised in the second labelled drug.

In another particular embodiment, the invention relates to the method comprising the steps of: i) providing a sample derived from a patient that has been administered with a drug, wherein the sample is suspected to comprise an anti-drug antibody against said drug, and wherein the sample may further comprise a residual drug target of said drug, ii) contacting the sample with a first and second labeled drug under suitable conditions to form a complex between the first labeled drug, the anti-drug antibody and the second labeled drug, iii) filtrating the sample with a filter to remove unwanted complexes formed between the first labeled drug, the drug target and the second labeled drug from the sample, wherein the filter has an average pore size that retains unwanted complexes formed between the first labeled drug, the drug target and the second labeled drug but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug, iv) immobilizing the complexes formed between the first labeled drug, the antidrug antibody and the second labeled drug comprised in the filtrate obtained in step (iii) to a solid surface via the first labelled drug, v) removing non-complexed first and second labeled drug from the sample, and vi) detecting and/or quantifying the presence of an anti-drug antibody via a detectable marker comprised in the second labelled drug. It is preferred herein that the steps of the method according to the invention are performed at a temperature ranging from 15 to 25°C.

In a particular embodiment, the invention relates to a method for confirming the presence of an antibody drug antibody in a sample, the method comprising the steps of: a) providing a sample obtained from a patient; b) detecting and/or quantifying the presence of an anti-drug antibody in a first portion of the sample with the method according to the invention; c) repeating step (b) with a second portion of the sample, wherein the second portion of the sample is additionally contacted with excess unlabeled drug that competes with the first and second labeled drug for binding to the anti-drug antibody in the sample; and d) confirming the presence of an anti-drug antibody in the sample, if the presence of anti-drug antibodies can be detected in step (b) but not in step (c).

That is, in another aspect, the present invention relates to an assay in which the presence of ADAs in a sample can be confirmed. For that, a first portion of a sample derived from a patient is tested for the presence of ADAs as disclosed herein above. If this sample was tested positive for the presence of ADAs, the assay is repeated with another portion of the patient sample, but this time the sample is spiked with excess unlabeled drug, i.e., the drug that was administered to the patient from which the sample has been obtained. Spiking the sample with excess unlabeled drug will increase the likelihood of complex formation between ADAs in the sample and the unlabeled drug and will decrease the likelihood of formation of signalgiving complexes comprising ADAs and the first and second labeled drug. That is, spiking the sample with excess unlabeled drug will reduce the sensitivity of the method according to the invention. In consequence, spiking the sample with excess unlabeled drug will prevent or at least significantly reduce the formation of signal-giving complexes comprising ADAs and the first and second labeled drug.

The presence of ADAs in a sample can be confirmed by comparing the outcome of the assay that has been performed in the absence of excess unlabeled drug with the outcome of the assay that has been performed in the presence of excess unlabeled drug. That is, if the presence of anti-drug antibodies has been detected in the absence of excess unlabeled drug, these anti-drug antibodies should not be detectable in the presence of excess unlabeled drug. If, however, anti-drug antibodies are still detectable in the presence of excess unlabeled drug, it is likely that the results have been caused by unspecific binding of the second labeled drug and thus can be considered false positive.

The presence of anti-drug antibodies is preferably detected as disclosed herein above. That is, the presence of anti-drug antibodies is preferably detected through the formation of signalgiving complexes comprising ADAs and the first and second labeled drug. Detecting a signal in the absence of excess unlabeled drug is a strong indication that a sample comprises ADAs directed against said drug. To confirm this finding, a comparable sample from the same patient can be re-analyzed in the presence of excess unlabeled drug. In the presence of excess unlabeled drug, formation of the signal-giving complex comprising the ADA and the first and second labeled drug would be expected to be significantly impeded. Thus, in certain embodiments, the presence of ADAs in a sample can be confirmed, if the detectable signal generated by the signal giving complex is reduced by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 95% or by at least 99% in the presence of excess unlabeled drug.

The unlabeled drug is preferably the drug that has been administered to the patient from which the sample has been obtained. Moreover, the unlabeled drug is preferably identical to the drug portion of the first and/or second labeled drug used in the method according to the invention.

To confirm the presence of ADAs in a sample, the unlabeled drug is to be added to the sample in excess. Preferably, the unlabeled drug is to be added to the sample in molar excess compared to the first and second labeled drug. In certain embodiments, the unlabeled drug is added to the sample in about 2-fold molar excess, in about 3-fold molar excess, in about 4- fold molar excess, in about 5-fold molar excess, in about 6-fold molar excess, in about 7-fold molar excess, in about 8-fold molar excess, in about 9-fold molar excess, in about 10-fold molar excess, in about 15-fold molar excess, in about 20-fold molar excess or in about 100- fold excess compared to the first and second labeled drug. In certain embodiments the drug is an antibody drug, in particular a monoclonal antibody drug. In such embodiments, the first and second labeled drug may be added to the sample at a concentration of 2 pg/mL each, or 4 pg/mL in total. In such embodiments, the unlabeled drug may be added at a concentration of 50 pg/mL to prevent formation of signal-giving complexes comprising ADAs and the first and second labeled drug.

The sample may be any sample defined herein above. Preferably, the sample is a sample obtained from a patient who has been administered a drug and is suspected of developing anti-drug antibodies to that drug. To confirm the presence of ADAs in said sample, the sample may be analyzed at least twice, once in the absence of excess unlabeled drug and once in the presence of excess unlabeled drug. For that, a first portion of a sample and a second portion of a sample may be analyzed.

For that, a sample may have been derived from a patient and then divided in two or more portions that are to be analyzed in separate assays. Alternatively, the first portion of a sample may be a first sample that has been obtained from a patient and the second portion of a sample may be a second sample that has been obtained from the same patient, preferably in the same manner and at a comparable time point, e.g., within one week.

In a particular embodiment, the invention relates to a kit for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the kit comprising at least: a first labeled drug, a second labeled drug and, optionally, a filter unit.

The present invention further relates to a kit for detecting and/or quantifying the presence of anti-drug antibodies in a sample. Preferably, the kit is drug specific and thus can be used to detect whether a patient has developed ADAs against a specific drug that has been administered to said patient. Accordingly, the kit according to the invention comprises at least a first and a second labeled variant of said drug, as defined herein above. That is, the first labeled drug is preferably a variant of the drug that is labeled with a capturing moiety that allows immobilizing complexes comprising the first labeled drug and the ADA on a solid surface or particle. The second labeled drug is preferably a variant of the drug that is labeled with a detectable marker that allows detecting and/or quantifying immobilized complexes comprising the second labeled drug and the ADA. Suitable labels of the first and second labeled drug are disclosed herein.

In a particular embodiment, the first labeled drug comprised in the kit according to the present invention is a drug that is labeled with a capturing moiety, preferably wherein the capturing moiety is biotin.

In another particular embodiment, the second labeled drug comprised in the kit according to the present invention is a drug that is labeled with a detectable marker or is a drug that is conjugated to a label that can be specifically bound by a binding molecule comprising a detectable marker, preferably wherein the label that can be specifically bound by a binding molecule comprising a detectable marker is a digoxigenin molecule.

In a further particular embodiment, the drug is an antibody drug, preferably an antiviral antibody drug, more preferably an anti-HBsAg antibody.

The kit may comprise further components, such as a filter unit that allows separation of wanted complexes comprising the first and second labeled drug and ADAs from unwanted complexes comprising the first and second labeled drug and residual drug target. Preferably, the filter unit is a centrifugal filter unit. It is disclosed herein above how the filter can be selected based on the nature of the drug target.

The kit may further comprise other components, such as microtiter plates, buffers and solutions.

All numeric ranges provided herein are inclusive of narrower ranges; delineated upper and lower range limits are interchangeable to create further ranges not explicitly delineated. The number of significant digits conveys neither limitation on the indicated amounts nor on the accuracy of the measurements.

In this document, the terms "a" or "an" are used to include one or more than one and the term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The term "about," as used herein, means approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 10%. Therefore, about 50% means in the range of 45%-55%. Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." BRIEF DESCRITPION OF THE DRAWINGS

FIG.l: Overview of the steps of the method according to the invention.

FIG.2: Signal obtained with ADA positive controls before and after filtration.

FIG.3: Signal to blank ratio (S/B) at different capture/detection reagent concentrations is shown in relation to the ADA positive control concentration in the serum sample.

EXAMPLES

The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Test procedure for the detection of anti-drug antibodies

This Example describes an ELISA-based test procedure for the determination of anti-drug antibodies against R07565020 in human serum samples. R07565020 is an antibody that specifically binds to hepatitis B surface antigen (HBsAg).

To detect anti-drug antibodies against R07565020 in human serum, an enzyme linked immunosorbent assay (ELISA) has been established. FIG.l schematically shows the different steps of the assay procedure.

In short, negative controls (NC, human pool serum), positive controls/quality controls (QCs) and study samples were diluted by a factor of 10 with 4M MgCl2 and incubated for 30 min. The MgCl2 treated samples were diluted by a factor of 10 with capture antibody mAb<HBsAg>rH-lgG-Bi and detection antibody mAb<HBsAg>rH-lgG-Dig, mixed and incubated for 20 min at RT and 500 rpm on a microtiter plate (MTP)-shaker, followed by a filtration with 300 K Pall filters for 5 min at 1000 g (to remove HBV). Formed immune complexes, present in the filtrate, were then transferred to a streptavidin (SA)-coated MTP and incubated for additional 30 min to immobilize the immune complexes via the biotin-labeled capture antibody. Following aspiration of the supernatant, unbound substances were removed by repeated washing. Immobilized immune complexes were incubated with an anti-digoxigenin Fab fragment conjugated to horseradish peroxidase (Anti-Dig-POD). After that, formed immobilized complexes were visualized by addition of HPPA solution, a POD substrate, which is converted to a fluorescent reaction product. Finally, the fluorescent intensity was measured after approx. 20min.

A detailed protocol of the ELISA procedure is provided below (also see FIG. 1):

Step 1: Incubation of samples with 4M MgCI2 solution (complex dissociation)

A 96-well pre-incubation plate was filled with 10 pL of QCs, NCs (pooled blank human serum) or study samples and mixed with 90 pL 4M MgCl2 . Subsequently, the plate was covered with adhesive cover foil (without glue) and centrifuged at 370 ref for approximately ten seconds. The plate was then mixed two times for roughly 30 seconds at ~1300 rpm and incubated at RT and 500 rpm on an MTP-shaker for 30 minutes.

Step 2: Preparation of capture and detection antibodies

The capture antibody "mAb<HBsAg>rH-lgG-Bi" is a monoclonal IgG antibody that specifically binds to hepatitis B surface antigen (HBsAg) and is labelled with a biotin (Bi) molecule. The detection antibody "mAb<HBsAg>rH-lgG-Dig" is a monoclonal IgG antibody that specifically binds to hepatitis B surface antigen (HBsAg) and is labelled with a digoxigenin (Dig) molecule.

To obtain the capture/detection solution, a lyophilisate of each antibody was dissolved in 1.1 mL H20dest. 792 pL of each antibody solution was mixed with 7.42 mL assay buffer (0.5% BSA in lxPBS). Final concentration of each antibody in the capture/detection solution was 20000 ng/mL.

Step 3: Incubation of MgCl2-sample mixture with capture/detection solution

After 30 minutes incubation, the adhesive cover foil was removed from the plate, 30 pL of the salty sample was transferred to a new plate and 120pL assay buffer (0.5% BSA in lxPBS) and 150pL of the capture/detection solution (20000 ng/mL of capture antibody and 20000 ng/mL of detection antibody) were added on top into the salty-sample mixture. The plate was again covered with an adhesive cover foil, mixed two times for roughly two seconds at ~1300 rpm and incubated at RT and 500 rpm on an MTP-shaker for 20 minutes.

Step 4: Filtration of samples and binding of analyte complex to SA-MTP

After 20 minutes of incubation, the samples were filtrated for 5 minutes at 1000 g with Pall 300 kDa Nanosep Centrifugal Devices (Pall Cat No.: OD300C33). After the filtration step, the filtered samples were pipetted into a Streptavidin-coated microtiter plate (SA-MTP; Microcoat Cat No.: 11643673001). For that, 100 pL of the pre-incubated QCs, NCs and samples retained by the filter were pipetted in duplicates to the designated wells of a 96-well SA-MTP plate. The SA-MTP was covered with adhesive foil and incubated for 30 minutes on an MTP-shaker (500 rpm). The MTP was washed three times with 300 pL washing buffer (lxPBS and 0.05% Tween 20) per well and residual washing buffer was carefully removed by tapping the MTP on a paper towel.

Step 5: Second Detection Antibody

A second detection solution comprising 5 mll/mL of polyclonal peroxidase-labelled anti- digoxigenin Fab fragment (pAb<Dig>S-Fab-POD(p); Sigma-Aldrich Catalog No.: 11633716001) was prepared and 100 pL of said solution were pipetted into each MTP well. The MTP was covered with adhesive cover foil and incubate for 30 minutes on an MTP-shaker (500 rpm). The MTP was washed three times with 300 pL washing buffer (lxPBS and 0.05% Tween 20) per well and residual washing buffer was carefully removed by tapping the MTP on paper towel.

Step 6: Substrate Reaction and Detection

2pL of 30% hydrogen peroxi de was added to freshly prepared 3-(4-Hydroxyphenyl)propionic acid (HPPA, Aldrich Catalog No.: H52406-25G) solution (43 mg of HPPA in 13 mL 0.1 M Tris buffer) and mixed well. 100 pL of HPPA solution comprising hydrogenperoxide was pipetted into each MTP well. The plate was incubated for 20min on an MTP-shaker (500rpm). Fluorescent intensity was read out with an ELISA Reader for 96 well MTPs (Tecan Infinite) using the following parameters:

• Excitation Wavelength: 320 nm

• Emission Wavelength: 400 nm

• Gain: Optimal/Calculated at HPC

• Number of Flashes: 10

• Integration Time: 20 ps

• Lag Time: O ns

• Settle Time: 0 ms

Step 7: Confirmation assay (optional)

Samples that were identified as ADA positive in the screening assay were tested in a subsequent confirmation assay to discard the theoretical 5% false-positive results generated by design of the screening assay.

To confirm positive screening assay results, samples were spiked and pre-incubated with excess drug and compared to the corresponding un-spiked sample on the same plate. Using a 50 pg/mL assay-concentration of R07565020 is recommended.

The experimental procedure for sample pre-incubation was identical to that described above for the ADA screening assay. Only the 120 pL assay buffer described in Step 3 was modified by the addition of 125 pg/mL of R07565020 (providing a final concentration after addition of 30pL sample and 150pL reagent of 50 pg/mL).

Step 8: Data Analysis

Result interpretation of samples was based on qualitative interpretation of the fluorescent units of the samples and corresponding positive controls.

Example 2: Analysis of HBV-positive samples with the method of the invention

Three HBV-positive serum samples (SP 5, SP 13 and SP 15) and one negative control (pooled blank human serum) were analyzed with the method described in Example 1. It could be demonstrated that HBV particles in the serum samples resulted in a strong signal when no filtration step was applied, indicating that residual virus particles can cause falsepositive signals in ADA assays. In contrast, filtration of the HBV-positive serum samples with a 300 kDa (35 nm) filter resulted in a drastic decrease of the signal, indicating that the HBV particles were efficiently removed from the sample in the filtration step (see Table 1 below).

Table 1: Analysis of HBV-positive samples in ADA assay with and without filtration

Example 3: Confirmation that ADA complexes are not retained by the filter

A calibration curve of the drug specific ADA positive control was prepared by addition of ADA positive control at 10 ng/mL, 25 ng/mL, 60 ng/mL, 145 ng/mL, 347 ng/mL, 833 ng/mL and 2000 ng/mL (serum concentration) followed by mixing. The serum samples were diluted by a factor of 100 with capture/detection solution. The final capture and detection concentration was 2 pg/mL each. Samples were separated into two separate aliquots. One aliquot was filtrated (Pall Cat No.: OD300C33) and the filtrate was added onto a streptavidin coated microtiter plate (SA-MTP; Microcoat Cat No.: 11643673001). The second, unfiltrated, sample was added to a separate well on the same plate. Visualization of the formed immune complexes was performed as described in step 5,6 and 8 in Example 1. The comparison of each calibrator with and without filtration shows only minor signal differences and is in the range of 2-16% lower signal intensity for the filtrated sample (see FIG.2). Example 4: Optimization of capture and detection antibody concentrations

To evaluate the optimal concentration of the detection and capture solution in combination with the filtration procedure, results of two approaches were considered. Firstly, the impact of a sensitivity loss on the detectability of the ADA positive control in case of rising concentrations of detection and capture solution was assessed. Secondly, the impact of reducing the false positivity of remaining HBsAg in the filtered sample was assessed.

4.1 Impact of rising capture and detection concentration on detectability of the ADA positive control:

A calibration curve of the drug specific ADA positive control was prepared by addition of ADA positive control at Ong/mL, 10 ng/mL, 25 ng/mL, 60 ng/mL, 145 ng/mL, 347 ng/mL, 833 ng/mL and 2000 ng/mL (serum concentration) followed by mixing. The serum samples were diluted by a factor of 100 with capture/detection solution at varying concentrations. The final capture and detection antibody concentration was either 2, 5 or 10 pg/mL each. Visualization of the formed immune complexes was performed as described in step 5, 6 and 8 in Example 1. The comparison of each calibrator is shown if FIG.3 where signal to blank (S/B) is shown in relation to the ADA positive control concentration in the serum sample. As can be seen, the impact of rising concentration of the reagent has only a minor impact on the detectability of the ADA positive control.

4.2 Impact of rising capture and detection concentration on the false positive signal of HBV positive serum samples:

Five individual HBV-positive samples and one negative control sample were processed as described in Example 1, except for the final detection/capture concentration (step 3). Final concentrations of 2 and 10 pg/mL were applied. S/B was calculated based on each applied reagent concentration by dividing the individual signal by the negative control. Table 2 summarizes the results with an improved S/B value at 10 pg/mL condition. The average S/B for the five individual HBV-positive samples could be reduced from 2.27 to 1.23 by applying lOpg/mL conditions. Table 2: Analysis of HBV-positive samples in ADA assay at 2pg/mL and 10 pg/mL capture/detection reagent concentration The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

Claims

1. A method for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the method comprising the steps of: a) contacting the sample with a first and second labeled drug, and b) detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug in the sample; characterized in that the method comprises a step of filtrating the sample with a filter to remove residual drug target from the sample, wherein the filter has an average pore size that retains the residual drug target, or complexes thereof, in the sample, but does not retain complexes comprising the first labeled drug, the anti-drug antibody and the second labeled drug.

2. The method according to claim 1, wherein the sample is determined to comprise an antidrug antibody, if the presence of the complex comprising the first labeled drug, the antidrug antibody and the second labeled drug can be detected in the sample.

3. The method according to claim 1 or 2, wherein the sample is filtrated between steps (a) and (b).

4. The method according to any one of claims 1 to 3, wherein the drug target is a virus.

5. The method according to claim 4, wherein the virus is selected from the group consisting of: Hepatitis B virus (HBV), Hepatitis A virus (HAV), Hepatitis C virus (HCV), SARS-CoV-2, Respiratory Syncytial Virus (RSV), Human Immunodeficiency Virus (HIV), Ebola Virus, Influenza Virus, Human Cytomegalovirus (HCMV), Herpes Simplex Virus (HSV), Dengue Virus, Zika Virus, Papillomavirus, and Rabies Virus.

6. The method according to any one of claims 1 to 5, wherein the filter has an average pore size of about 10 nm to about 60 nm, preferably of about 15 nm to about 55 nm, more preferably of about 20 nm to about 50 nm, even more preferably of about 25 nm to about 45 nm, even more preferably of about 30 to about 40 nm.

7. The method according to any one of claims 1 to 6, wherein the drug comprised in the first and second labelled drug is a drug that has been administered to a patient from which the sample has been obtained.

8. The method according to any one of claims 1 to 7, wherein the drug comprised in the first and second labelled drug is an antibody, or an antigen-binding fragment thereof.

9. The method according to claim 8, wherein the first and/or second labelled drug antibody is added to the sample at a final concentration of at least 1 pg/mL, at least 2 pg/mL, at least 4 pg/mL or at least 10 pg/mL.

10. The method according to any one of claims 1 to 9, wherein the first labelled drug is labeled with a compound that allows immobilizing said drug on a solid surface or particle.

11. The method according to claim 10, wherein the first labelled drug is a biotinylated drug, in particular wherein the first labelled drug is a biotinylated antibody.

12. The method according to any one of claims 1 to 11, wherein the second labelled drug comprises a detectable marker or wherein the second labelled drug comprises a label that can be specifically bound by a binding molecule comprising a detectable marker, preferably wherein the binding molecule is an antibody or an antigen-binding fragment thereof.

13. The method according to claim 12, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug comprises a step of detecting the complex via the (i) detectable marker comprised in the second labelled drug or (ii) the binding molecule comprising the detectable marker.

14. The method according to claim 12 or 13, wherein the second labelled drug comprises a digoxigenin molecule, in particular wherein the second labelled drug is a dioxigenin- labelled antibody.

15. The method according to any one of claims 12 to 14, wherein the detectable marker is a marker that can generate a detectable signal, in particular wherein the detectable marker is selected from the group consisting of: a chromophore, an enzyme, an enzyme reactive compound whose cleavage product is detectable, a radioisotope, a fluorescent compound, a chemiluminescent compound, and derivatives and/or combinations of these markers.

16. The method according to claim 15, wherein the detectable marker is an enzyme, preferably wherein the enzyme is capable of converting a colorless form of a detection agent into a colored form of the detection agent, in particular wherein the enzyme is a peroxidase.

17. The method according to claim 15 or 16, wherein the sample is determined to comprise an anti-drug antibody, if the detectable signal generated by the detectable marker in the sample is higher than the detectable signal in a negative control that is free of antidrug antibodies

18. The method according to any one of claims 1 to 17, wherein the step of detecting and/or quantifying the presence of a complex comprising the first labeled drug, the anti-drug antibody and the second labeled drug further comprises a step of immobilizing the complex on a solid surface or particle via the first labelled drug.

19. The method according to any one of claims 1 to 18, wherein the sample is a blood sample, in particular a whole blood sample, a plasma sample or a serum sample, or wherein the sample is an aqueous humour sample.

20. The method according to any one of claims 1 to 19, wherein the sample is pretreated prior to step (a) to dissociate existing complexes in the sample.

21. The method according to claim 20, wherein the sample is pretreated with a chaotropic agent prior to step (a).

22. The method according to claim 21, wherein the chaotropic agent is magnesium chloride (MgCb).

23. The method according to any one of claims 1 to 22 comprising the steps of: i) providing a sample derived from a patient that has been administered with a drug, wherein the sample is suspected to comprise an anti-drug antibody against said drug, and wherein the sample may further comprise a residual drug target of said drug, ii) contacting the sample with a first and second labeled drug under suitable conditions to form a complex between the first labeled drug, the anti-drug antibody and the second labeled drug, iii) filtrating the sample to remove unwanted complexes formed between the first labeled drug, the drug target and the second labeled drug from the sample, iv) immobilizing the complexes formed between the first labeled drug, the anti-drug antibody and the second labeled drug comprised in the filtrate obtained in step (iii) to a solid surface or particle via the first labelled drug, v) removing non-complexed first and second labeled drug from the sample, and vi) detecting and/or quantifying the presence of an anti-drug antibody via a detectable marker comprised in the second labelled drug.

24. A method for confirming the presence of an antibody drug antibody in a sample, the method comprising the steps of: a) providing a sample obtained from a patient; b) detecting and/or quantifying the presence of an anti-drug antibody in a first portion of the sample with the method according to any one of claims 1 to 23; c) repeating step (b) with a second portion of the sample, wherein the second portion of the sample is additionally contacted with excess unlabeled drug that competes with the first and second labeled drug for binding to the anti-drug antibody in the sample; and d) confirming the presence of an anti-drug antibody in the sample, if the presence of anti-drug antibodies can be detected in step (b) but not in step (c).

25. A kit for detecting and/or quantifying the presence of an anti-drug antibody in a sample, the kit comprising at least: a first labeled drug, a second labeled drug and, optionally, a filter unit.