Methods for improving Anti-drug antibody assays

Abstract

Disclosed are methods of performing antibody-based / immunological assays having improved resolution; for example, to detect ADA antibodies of the IgM class. Also disclosed are methods for performing antibody-based / immunological assays having improved resolution / sensitivity to detect ADA antibodies of the IgG class. In particular, assays benefiting from the methods according to this disclosure may be performed on biological samples that contain, may contain, and / or are expected to contain rheumatoid factor (RF).

Description

[0001] METHODS FOR IMPROVING ANTI-DRUG ANTIBODY ASSAYS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel methods and uses of polypeptides which display protease activity against IgM immunoglobulins for the purpose of facilitating and improving the detection and / or characterization of anti-drug antibodies in anti-drug antibody assays.

[0004] BACKGROUND OF THE INVENTION

[0005] Anti-drug antibody (ADA) assays are used to detect the presence of ADAs generated by the immune system in response to therapeutic drugs, particularly biologies such as monoclonal antibodies or other protein-based treatments, and also drugs comprising recombinant AAV particles. ADAs may impact the safety and efficacy of a drug by neutralizing its therapeutic effects, altering its pharmacokinetics, facilitating cross-reactions with endogenous proteins, or triggering adverse immune reactions. As such, by identifying and characterizing ADAs, the immunogenicity of new and existing drugs can be interrogated and established, facilitating dosage optimisation and improvements in drug efficacy and overall drug safety.

[0006] The complexity of immune responses poses several challenges in assessing the impact of ADAs on the efficacy, pharmacokinetics, and safety profile of a drug, particularly given that ADAs raised in response to a drug of interest may fall under multiple different immunoglobulin (Ig) classes, such as IgG and IgM ADAs. Discriminating between IgG and IgM ADAs in an ADA assay is important as these two antibody classes play different roles in the immune response and can have varying impacts on drug efficacy and safety.

[0007] Typically, ADA assays are conducted using bridging ELISAs, which do not usually distinguish between antibody isotypes, or require the use of expensive and time-consuming assays such as the homogenous mobility shift assay (HMSA) which combines a ligand binding assay with size exclusion chromatography (SEC).

[0008] Thus, there is a recognized need for methods and systems to better characterize and identify ADAs, particularly the Ig class / isotype of ADAs, to enhance ADA detection techniques. These improved methods and systems can offer valuable insights into the immunogenicity effects relevant to pharmacokinetics, efficacy, and safety in clinical pharmacology, particularly in the administration of biologic drugs.

[0009] Furthermore, the presence of ‘heterophilic antibodies’, i.e. unwanted antibodies already present in a biological sample, which are capable of interfering in an antibody-based bridging assay, can significantly and adversely affect the ‘read-out’ from ADA assays and other immunoassays. As such, any ADA assays, (multiplex) immunoassays, or other diagnostic methods that rely on ELISA I antibody bridging can be affected adversely by heterophilic antibodies, i.e. heterophilic antibodies can interfere with the measurement of the target and result in false assay readouts. ‘Rheumatoid factor’ (RF), which is present in between about 65 to 80% of patients with rheumatoid arthritis (RA) (Bartels et al. (2011), Arthritis, 741071), encompasses a group of autoantibodies including IgM, IgG, IgA, IgD and / or IgE subclasses; of which IgM and IgG are the most abundant. For example, the accurate measurement of cytokine or therapeutic drug levels in a subject having RA can be problematic, potentially leading to misdiagnosis, affects on therapeutic drug monitoring and impaired immunogenicity evaluation. Since RA affects around 2% of the population, it would be desirable to have improved methods and systems for performing antibody-bridging assays, such as ELISA, ADA assays and (multiplex) immunoassays in most subjects; especially those also suffering from RA.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention relates to one or more methods of improving the sensitivity and resolution of an ADA assay and / or detecting an IgM ADA in an ADA assay or other similar assays that comprise IgM within a mix of components, such as in a sample to be analyzed. The methods comprise the use of an IgM specific protease to remove or inactivate IgM in a sample which enables the determination of the contribution of IgM to any observed assay activity.

[0012] In aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample is provided, the method comprising:

[0013] (a) contacting a first aliquot of the biological sample from a subject which comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,

[0014] (b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately

[0015] (b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;

[0016] (c) detecting the presence of an IgM complex in each of the first and second aliquot of the sample, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0017] (d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0018] In aspects of the invention, a method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample is provided, the method comprising:

[0019] (a) contacting a biological sample from a subject, wherein the biological sample comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0020] (b) contacting the treated sample with: a first drug moiety, and a second drug moiety;

[0021] (c) detecting the presence of an IgG complex in the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0022] In embodiments of the methods, the first drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

[0023] In embodiments of the methods, the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

[0024] In aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample from a subject is provided, the method comprising:

[0025] (a) contacting a first aliquot of the biological sample, wherein the sample comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,

[0026] (b)(i) contacting the first treated sample with an immobilised first drug moiety, (b)(ii) contacting a second aliquot of the sample which comprises or may comprise at least one anti-drug antibody of the IgM class with an immobilised first drug moiety,

[0027] (c) separately contacting the first and second aliquot of the sample with a second drug moiety,

[0028] (d) detecting the presence of an IgM complex in the first and second aliquot of the sample, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0029] (e) comparing the detection of the presence of the IgM complex in the first treated sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first treated sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0030] In aspects of the invention, a method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample is provided, the method comprising:

[0031] (a) contacting a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0032] (b) contacting the treated sample with an immobilised first drug moiety,

[0033] (c) contacting the treated sample of (b) with a second drug moiety,

[0034] (d) detecting the presence of an IgG complex in the treated sample of (c), wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0035] In embodiments of the methods, the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, such as between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. about 0.5 ng / pL.

[0036] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a detection moiety configured to specifically bind to the second drug moiety, optionally wherein the detection moiety is configured to specifically bind to a label of the second drug moiety.

[0037] In embodiments of the methods, the detection moiety is provided at a concentration in the sample of between about 5 to 50 mU / mL, such as between about 10 to 40 mU / mL, or between about 15 to 35 mU / mL; for example, between about 20 to 30 mU / mL; e.g. about 25 mU / mL.

[0038] In embodiments of the methods, the detection moiety is provided at a concentration in the sample of between about 100 to 1 ,000 ng / mL, between about 200 to 600 ng / mL, or between about 300 to 500 ng / mL; e.g. about 400 ng / mL.

[0039] In embodiments of the methods, contacting the first sample or portion of the sample with an IgM protease comprises contacting the first sample or portion of the sample with an IgM protease at a concentration between about 0.2 to 10 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL, e.g. about 0.91 U / pL.

[0040] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0041] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0042] (b) contacting the treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0043] (c) detecting the presence of an IgG complex in the treated sample of (b), wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0044] In aspects of the invention, a method for detecting at least one anti-AAV particle antibody of the IgM class in a biological sample from a subject is provided, the method comprising: (a) contacting a first aliquot of the biological sample, which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,

[0045] (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0046] (b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0047] (c) detecting the presence of an IgM complex in each aliquot of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle antibody of the IgM class, and the detection moiety; and

[0048] (d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0049] In aspects of the invention, a method for detecting at least one anti-AAV particle antibody of the IgM class in a biological sample from a subject is provided, the method comprising:

[0050] (a) contacting a first aliquot of the biological sample, which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,

[0051] (b)(i) contacting the first treated sample with: at least one AAV particle, and an AAV capture moiety;

[0052] (b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, and an AAV capture moiety;

[0053] (c) detecting the presence of an IgM complex in each aliquot of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle antibody of the IgM class; and

[0054] (d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0055] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0056] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0057] (b) contacting the treated sample with: at least one AAV particle, and an AAV capture moiety,

[0058] (c) detecting the presence of an IgG complex in the treated sample of (b), wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle antibody of the IgG class, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0059] In embodiments of the methods, contacting the first sample or portion of the first sample with an IgM protease comprises contacting the sample or portion of the sample with IgM protease; suitably wherein the IgM protease is at a concentration between about 0.4 and 1.4 U / pL, between about 0.6 and 1 .2 U / pL, or between about 0.8 and 1 .0 U / pL; for example, between about 0.8 and 1 .0 U / pL, e.g. about 0.9 U / pL.

[0060] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, comprises an AAV particle concentration of between about 1.0*109and 2.0*109vp / mL, between about 1.2*109and 1 .9*109vp / mL, between about 1.4*109and 1 .8*109vp / mL, or between about 1.6*109and 1.7*109vp / mL; for example, between about 1.6*109and 1.7*109vp / mL, e.g. about 1.65*109vp / mL. In embodiments of the methods, the AAV capture moiety is provided at a concentration of between about 0.5 and 3 U / pL, between about 0.7 and 1.5 U / pL, or between about 0.8 and 1.0 U / pL, e.g. about 0.91 U / pL.

[0061] In embodiments of the methods, the detection moiety is provided at a concentration of between about 100 and 1 ,000 ng / mL, between about 200 to 900 ng / mL, between about 300 to 700 ng / mL, between about 400 to 600 ng / mL, or between about 450 to 550 ng / mL; e.g. about 500 ng / mL.

[0062] In embodiments of the methods, contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety, comprises contacting the detection moiety with a recognition agent, suitably wherein the detection moiety comprises an anti-IgG or anti-IgM antibody configured to bind to the at least one anti-AAV particle antibody of the IgG or IgM class in each sample or portion or aliquot of the sample.

[0063] In embodiments of the methods, the IgM protease comprises SEQ ID NO: 2, suitably SEQ ID NO: 1.

[0064] In further aspects and embodiments, the methods provide for improvements in the accuracy, reliability, sensitivity and / or confidence in the results of antibody-based / immunological assays. For example, methods according to these aspects and embodiments may reduce non-specific signals and / or false positive signals in antibody-based / immunological assays.

[0065] Thus, in aspects of the invention, a method for improving the accuracy and / or sensitivity of an antibody-based or immunogenicity assay is provided, the method comprising: contacting a biological sample with an IgM protease to obtain a treated sample, thereby removing interfering compounds present in the sample and performing an antibody-based assay or immunogenicity assay on the treated sample.

[0066] In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis, wherein the method is according to any of aspects and / or embodiments disclosed therein.

[0067] In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having an autoimmune and / or infectious disease, wherein the method may be according to any of aspects and / or embodiments disclosed therein. In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis, wherein the method may be according to any of aspects and / or embodiments disclosed therein.

[0068] In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having elevated rheumatoid factor (RF), wherein the method may be according to any of aspects and / or embodiments disclosed therein.

[0069] In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising rheumatoid factor (RF), wherein the method may be according to any of aspects and / or embodiments disclosed therein.

[0070] In aspects of the invention, a method is provided for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL rheumatoid factor (RF), wherein the method may be according to any of aspects and / or embodiments disclosed therein.

[0071] In aspects of the invention, a method is provided for reducing non-specific signal and / or reducing false positive signals in an antibody-based or immunological assay of a biological sample, wherein the biological sample comprises or is expected to comprise rheumatoid factor (RF), wherein the method may be according to any of aspects and / or embodiments disclosed therein.

[0072] In embodiments, the biological sample was obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis. In embodiments, the biological sample was obtained from a subject having an autoimmune and / or infectious disease. In embodiments, the biological sample was obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[0073] In aspects of the invention, a use of an IgM protease for improving the accuracy, reliability, sensitivity and / or confidence of the results of antibody-based / immunological assays is provided.

[0074] In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis. In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having an autoimmune and / or infectious disease.

[0075] In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[0076] In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having elevated rheumatoid factor (RF).

[0077] In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising rheumatoid factor (RF).

[0078] In certain embodiments of the invention, the use is for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL rheumatoid factor (RF).

[0079] In certain embodiments of the invention, the use is for reducing non-specific signal and / or reducing false positive signals in an antibody-based or immunological assay of a biological sample, wherein the biological sample comprises or is expected to comprise rheumatoid factor (RF).

[0080] In certain embodiments, the biological sample was obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis. In embodiments, the biological sample was obtained from a subject having an autoimmune and / or infectious disease. In embodiments, the biological sample was obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[0081] BRIEF DESCRIPTION OF THE FIGURES

[0082] Figure 1 ADA bridging assay overview

[0083] Schematic overview of the ADA bridging assay: immune complexes, consisting of a first drug moiety comprising Drug-digoxigenin (Drug-Dig), an ADA (e.g. IgM-type) and second drug moiety comprising Drug-biotin (Drug-Bi), are able to bind to a binding agent conjugated to a solid support, e.g. a streptavidin (SA)-coated surface. The assay signal is generated by the addition of a recognition agent comprising anti-digoxigenin Fabs conjugated to horseradish peroxidase (Anti- Dig-HRP). Figure 2 Assay for the detection of pre-existing IgM-type anti-mAb1 antibodies

[0084] Schematic overview of the assay for the detection of pre-existing IgM-type anti-mAb1 antibodies: the first drug moiety comprising a Drug (mAb1) is non-specifically bound to a solid support comprising a MaxiSorp microtiter plate. Successfully bound IgM-type ADAs that are potentially contained in tested samples are specifically bound by a second drug moiety comprising a mouse- derived anti-IgM antibody. The assay signal is generated by the addition of a recognition agent, comprising an anti-mouse antibody conjugated to horseradish peroxidase (Anti-Mouse-HRP).

[0085] Figure 3 Assay for the detection of pre-existing IgG-type anti-mAb1 antibodies

[0086] Schematic overview of the assay for the detection of pre-existing IgG-type anti-mAb1 antibodies: the first drug moiety comprising a Drug (e.g. mAb1) is non-specifically bound to a solid support comprising a MaxiSorp microtiter plate. Successfully bound IgG-type ADAs that are potentially contained in tested samples are specifically bound by a second drug moiety comprising CD64- digoxigenin (FcgRI-Dig). The assay signal is generated by the addition of a recognition agent comprising anti-digoxigenin Fabs conjugated to horseradish peroxidase (Anti-Dig-HRP).

[0087] Figure 4 Results of using the ADA bridging assay, IgM-specific assay, and IgG- specific assay for the detection of pre-existing anti-mAb1 antibodies

[0088] Combined bar graph with the results of the analysis using the ADA bridging assay for the detection of pre-existing anti-mAb1 antibodies (A), the assay for the detection of pre-existing IgM-type anti- mAb1 antibodies (B) and the assay for the detection of pre-existing IgG-type anti-mAb1 antibodies (C). Samples from all donors were analyzed with and without IgMBRAZOR pre-treatment in each assay (bars denoted by treated / untreated).

[0089] Figure 5 AAV immune complex assay with IgG detection

[0090] Schematic overview of the AAV immune complex assay with IgG detection: formed immune complexes, consisting of AAV and IgG-type AAV-ADA, are captured via an AAV-capture moiety comprising a biotinylated anti-AAV antibody (anti-AAV-Bi) that is bound to a solid support comprising a streptavidin-coated microtiter plate (SA-MTP). Subsequently, immune complexes are bound by the detection moiety, comprising an anti-human IgG antibody conjugated to horseradish peroxidase (anti-human IgG-HRP). The assay signal is generated by the addition of a recognition agent comprising anti-human IgG antibody conjugated to horseradish peroxidase (anti-human IgG- HRP).

[0091] Figure 6 AAV immune complex assay with IgM detection

[0092] Schematic overview of the AAV immune complex assay with IgM detection: formed immune complexes, consisting of AAV and IgM-type AAV-ADA, are captured via an AAV-capture moiety comprising a biotinylated anti-AAV antibody (anti-AAV-Bi) that is bound to a solid support comprising a streptavidin-coated microtiter plate (SA-MTP). The assay signal is generated by the addition of the detection moiety comprising an anti-human IgM antibody conjugated to alkaline phosphatase (anti-human IgM-AP). Figure 7 Results of the analysis using the AAV immune complex assay with IgM detection and IgG detection

[0093] Combined graph with the results of the analysis using the AAV immune complex assay with IgM detection (A, C, E) and IgG detection (B, D, F). The analyzed samples are derived from animal 1 (A / B), animal 2 (C / D) and animal 3 (E / F). Samples from all time points and animals were analyzed with and without IgMBRAZOR pre-treatment in each assay (denoted by treated / untreated).

[0094] Figure 8 AAV de-masking approach using IgMBRAZOR

[0095] Schematic overview of the AAV de-masking approach using IgMBRAZOR: AAV that is masked / satu rated with anti-AAV antibodies of the IgM-type prevents co-existing anti-AAV antibodies of the IgG-type from binding due to competition for binding sites (inhibited IgG detection due to AAV masking). After treatment with IgMBRAZOR and subsequent digestion of anti-AAV antibodies of the IgM-type (de-masking procedure), it becomes possible for anti-AAV antibodies of the IgG-type to bind to the AAV. The AAV de-masking improves the binding of anti-AAV antibodies of the IgG-type and, thus, also detection of anti-AAV antibodies of the IgG-type.

[0096] Figure 9 Confirmation of IgM-type ADAs and improvement of the detection of IgG-type ADAs by specific digestion of IgM

[0097] Schematic overview of the specific digestion of IgM approach using IgMBRAZOR: Treatment of a sample comprising IgM ADAs with an IgM protease, e.g. IgMBRAZOR, and subsequent digestion of IgM-type ADAs results in reduced signal in a IgMBRAZOR treated sample compared to an untreated sample, indicating the presence of IgM ADAs in a sample. Additionally, target drugs that are masked / satu rated with antibodies of the IgM-type prevent co-existing ADAs antibodies of the IgG-type from binding due to competition for binding sites (inhibited IgG detection due to Drug masking). After treatment with IgMBRAZOR and subsequent digestion of antibodies of the IgM- type (de-masking procedure), it becomes possible for ADAs of the IgG-type to bind to the Drug. The Drug de-masking improves the binding of ADAs of the IgG-type and, thus, also detection of ADAs of the IgG-type.

[0098] Figure 10 IgMBRAZOR is an IgM specific protease

[0099] SDS-PAGE gel of the activity of IgMBRAZOR towards IgG from human and mouse (A). The activity of IgMBRAZOR was evaluated towards human IgG, IgA, and IgM. Lanes are labelled accordingly and molecular mass is indicated by reference to the molecular weight ladder.

[0100] Figure 11 Size exclusion analysis of IgM cleavage products

[0101] Size exclusion chromatography of the hydrolyzed IgM sample was performed alongside size standards consisting of Thyroglobulin (1), Apoferritin (2), and Human IgG (3). Intact IgM, as well as the hydrolysis products (pentameric CH3-CH4; CH1-CH1-CH2) are indicated on the chromatogram. Figure 12 AAV immune complex assay with IgG detection

[0102] Schematic overview of the AAV immune complex assay with IgG detection: formed immune complexes, consisting of AAV and IgG-type AAV-ADA, are captured via an AAV-capture moiety comprising a biotinylated anti-AAV antibody (anti-AAV-Bi) that is bound to a solid support comprising a streptavidin-coated microtiter plate (SA-MTP). Subsequently, immune complexes are bound by the detection moiety, comprising an anti-human IgG antibody-digoxigenin (anti-human IgG-Dig). The assay signal is generated by the addition of a recognition agent comprising anti- digoxigenin Fabs conjugated to horseradish peroxidase.

[0103] Figure 13 Mechanisms of rheumatoid factor (RF) interference

[0104] Schematic overview of the RF interference in immune complex assays. Unwanted or even false assay signals in the presence of RF (which are predominantly due to the presence of IgM), result from RF binding to Fc domains of drug molecules and, in an IgG assay, to endogenous drug- unspecific IgG. (a) interference in a bridging assay; (b) interference in an IgM assay; and (c) interference in an IgG assay.

[0105] Figure 14 Effects of rheumatoid factor (RF) on the ADA bridging assay, IgM-specific assay, and IgG-specific assay in the presence of absence of IgM-specific protease

[0106] Combined bar graph illustrating results of immunoassays based on samples taken from patients having rheumatoid arthritis (RA): (A) influence of RF on ADA bridging assay; (B) influence of RF on IgM assay; and (C) influence of RF on IgG assay. Samples from all donors were analyzed with and without IgM-specific protease (IgMBRAZOR) pre-treatment in each assay (bars denoted by treated / untreated).

[0107] DETAILED DESCRIPTION OF THE INVENTION

[0108] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0109] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein will be understood as having the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs, such as molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization. Standard techniques are used for nucleic acid synthesis.

[0110] The techniques and procedures are generally performed according to conventional methods in the art (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. 30 (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference). Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references. In case of conflict, the terms in this specification will control.

[0111] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps and features referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only.

[0112] As used herein, the term "comprising" means any of the recited elements are necessarily included and other elements may optionally be included as well. "Consisting essentially of’ means any recited elements are necessarily included, other elements may optionally be included, and that elements that would materially affect the basic and novel characteristics of the listed elements are excluded. “Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. As used herein, the term “contacting” means placement in direct physical association, for example both in solid form and / or in liquid form.

[0113] It should also be understood that the term "and / or" as used herein refers to and encompasses any or all possible combinations of one or more associated listed items.

[0114] As used herein, the term "unit", relates to the amount of an enzyme that catalyses the hydrolysis, suitably ‘full’ hydrolysis, e.g. at least about 90% hydrolysis, at least about 95% hydrolysis, at least about 98%, or at least about 99% hydrolysis of one microgram of a substrate per unit of time; e.g. per 30 minutes under specified conditions, such that at most, about 10% or less, about 5% or less, about 2% or less, or about 1 % or less of the substrate has not been hydrolysed after 30 minutes.

[0115] More specifically, the term “unit” may be used to define the amount of a protease, suitably an IgM protease, that catalyses the hydrolysis of one microgram of an immunoglobulin; such as one microgram of IgM per 30 minutes under specified conditions, e.g. 37°C. Accordingly, ’’units” of an enzyme as used herein may be considered to have the equivalent measurement, e.g. ‘pg / 30 mins’.

[0116] Those skilled in the art will appreciate that the quantity of protease corresponding to a unit of the protease may vary based on protease composition, protease production batch, and the target substrate. As such, a unit (U) of IgM protease of this disclosure may be provided at a wt:wt ratio of protease to target substrate of between about 1 :5 to 1 :200.

[0117] ADAs and ADA Assays

[0118] Anti-drug antibodies are antibodies, which are directed against any region of the drug antibody, including but not limited to, the variable region, the constant region or any linking region.

[0119] Such anti-drug antibodies may be generated during antibody therapy as an immunogenic reaction of a patient (see Pan, Y., et al., FASEB J. 9 (1995) 43-49), wherein the administration of biologies, such as monoclonal antibodies, may induce immune responses in (experimental) animal subjects and human patients, such as the development of ADAs towards a given therapeutic drug / antibody or to related proteins.

[0120] The generation of ADAs may lead to the induction of immunologically related adverse clinical events, including but not limited to the induction of binding antibodies, the induction of neutralizing antibodies, altered pharmacokinetics, reduced efficacy, increased toxicity, and safety concerns. The immunogenicity responses induced by therapeutic proteins can range from transient ADAs with no clinical significance to the generation of high titre, persistent ADAs which may lead to reduced drug exposure, lack or loss of efficacy and adverse events, such as hypersensitivity reaction, anaphylaxis and injection site reactions (see Koren et al., Recommendations on riskbased strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods, 2008. 333(1-2): p. 1-9).

[0121] The presence of ADAs is typically associated with a concordance between an increase in systemic clearance of pharmaceutical products and a reduction of efficacy. Some drug products have drugsustaining ADAs which resulted in a reduced clearance possibly due to the formation of ADA-drug complex, such as ADA binding of the drug (see Wang et al., Evaluating and Reporting the Immunogenicity Impacts for Biological Products - a Clinical Pharmacology Perspective. The AAPS Journal. 2016; 18(2): 395-403).

[0122] The neutralizing activity of ADAs and immunogenicity impacts in clinical pharmacology relevant to pharmacokinetics, efficacy, and safety, are well known in the art. The formation of ADAs during drug treatment may cause a decrease in drug concentration in patient's body, which may contribute to the reduced efficacy. Various ADAs which are capable of binding to different sites of the drugs can be present in patient's bodies, such as neutralizing or non-neutralizing ADAs. Neutralizing ADAs are capable of binding to the active site of the drug molecule, such as the binding site in drug molecule for binding to the drug target, or the (hyper)variable regions of an antibody drug, or may sterically interfere with the binding of the drug to its therapeutic target. When, e.g., the neutralizing ADA binds to the active site of a drug, it renders the drug inactive. The non-neutralizing ADA can be capable of binding to the non-active site of the drug molecule, such as the constant region or the scaffold of an antibody drug molecule. Even though the drug can be still active whilst bound to a non-neutralizing ADA, the presence of non-neutralizing ADAs may contribute to changes in clinical pharmacology.

[0123] The increasing concerns of drug efficacy and patient safety due to immunogenicity incidences of protein pharmaceutical products have led to an increasing demand for characterizing ADAs. The demands of characterizing ADAs are driven by, for example, the needs of understanding the impacts of ADAs on reducing the drug efficacy, cross-reacting to endogenous proteins, or altering the pharmacokinetics of pharmaceutical products. The characterization data of ADAs can provide valuable information regarding immunogenicity of pharmaceutical products, and therefore to enhance the safety for drug administrations.

[0124] As such, immunogenicity assessment may be required by regulatory agencies as part of product safety, and the incidence of ADA and neutralizing antibody (NAb) are part of the prescribing information (US Department of Health and Human Services, U.F.C., CBER, Guidance for Industry — Assay Development for Immunogenicity Testing of Therapeutic Proteins. US Department of Health and Human Services, Washington, D.C., USA, 2009; European Medicines Agency, C.f.M.P.f.H.U., Guideline on Immunogenicity Assessment of Biotechnology-Drived Therapeutic Proteins. European Medicines Agency, London, U K, 2007).

[0125] Thus, the accurate detection of ADAs is an important aspect of any biological drug development programs (see Mire-Sluis, et al., Recommendations for the design and optimization of immunoassays used in the detection of host antibodies against biotechnology products. J Immunol Methods, 2004. 289(1-2): p. 1-16; and Shankar, G., et al., Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal, 2008. 48(5): p. 1267-81).

[0126] As used herein, the term “drug”, refers to a compound that has medicinal, performance-enhancing, and / or intoxicating effects when introduced into the body of a human or other animal. For example, the drug can be an organic or inorganic small molecule compound or a biologic therapeutic (e.g., an antibody (e.g., a drug antibody) or fragment thereof, multiple domain biotherapeutics, nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, or lipid).

[0127] In certain embodiments, the drug is immunogenic and capable of eliciting an immune response, and thus may be detected by an ADA assay. In alternate embodiments, the drug is not immunogenic and capable of eliciting an immune response. As such, an ADA assay may be negative for ADAs.

[0128] The term “drug antibody” denotes an antibody which can be administered to an individual for the treatment of a disease and as used herein distinguishes such antibodies from ADAs. As used herein, the term “pharmaceutical product” includes an active ingredient which can be fully or partially biological in nature or which has pharmaceutical activity. In some exemplary embodiments, the pharmaceutical product can comprise a drug, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, a peptide-drug conjugate, a Fc region of an antibody, an enzyme product, a cytokine, a growth factor, a pharmaceutical product, a toxin, a nucleic acid, DNA, RNA, a chemical compound, a cell, a tissue, an antigen, vaccine or any pharmaceutical ingredient which can be capable of inducing antibodies in a subject. In some other exemplary embodiments, the pharmaceutical product can comprise a recombinant, engineered, modified, mutated, or truncated version of a peptide, a protein, a fusion protein, an antibody, an antigen, vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate or combinations thereof.

[0129] Non-limiting examples of drug antibodies include, for example, an antibody selected from muromomab-CD3, abciximab, rituximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, etanercept, gemtuzumab, fresolimumab, alemtuzumab, ibritomomab, adalimumab, alefacept, omalizumab, tofacitinib, tositumomab, efalizumab, cetuximab, bevacizumab, natalizumab, ranibizumab, panitumumab, eculizumab mepolizumab, necitumumab, blinatumomab, nivolumab, dinutuximab, secukinumab, evolocumab, pembrolizumab, ramucirumab, vedoluzumab, siltuximab, opinutuzumab, adotrastuzumab emtansine, raxibacumab, pertuzumab, brentuximab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, catumaxomab, trontinemab and certolizumab.

[0130] The term “ADA”, as used herein, refers to antibodies to a particular drug, which are produced when any drug, suitably biological drugs, more suitably antibody drugs or drugs comprising anti-viral vectors, is administered to an organism, and which recognize the drug, suitably by binding to the drug. For example, in the case of the antibody drug ‘Infliximab’, examples of the corresponding ADAs include antibodies to Infliximab.

[0131] In some embodiments, the methods or uses of the invention may be used to identify anti-viral vector ADAs. For example, anti-AAV ADAs generated in response to administering a therapeutic viral vectors (e.g. adenovirus vectors or adeno-associated viral vectors) for gene therapy applications.

[0132] One of the primary challenges in gene therapy is the host immune response against the viral vectors used to deliver genes, including transgenes and edited genes. The immune system can recognize and target these vectors for inactivation, removal, and degradation, reducing their effectiveness and rendering repeated treatments less effective, i.e. the immune system can develop memory responses against viral vectors after the first administration, leading to rapid neutralization in subsequent doses. For example, IgG and IgM, e.g. IgG and IgM ADAs, have been demonstrated to significantly negatively affect gene transfer by adenovirus type 5, which is one of the most commonly used adenovirus vectors. For example, IgG and IgM reduces the transduction of the vector in the liver. IgG and IgM ADAs can bind to viral vectors and both sequester the vectors to non-target regions of the body, and also facilitate their clearance from the bloodstream. This sequestration and rapid clearance limit the distribution and time that therapeutic vectors have to reach their target cells.

[0133] Typically, IgG and IgM ADAs trigger inflammatory responses in response to the delivery of therapeutic vectors to a subject through the activation of immune cells such as macrophages and complement pathways. This inflammation can damage tissues and cause side-effects, which may reduce the overall effectiveness of gene therapy.

[0134] ADA assays may be performed on any suitable sample, e.g. a sample collected from a patient given a drug. Typically, the sample is a biological sample, suitably any fluid or tissue where the detection of ADAs may be of interest. In embodiments, the sample typically comprises or consists of a complex medium, such as a complex biological medium.

[0135] In embodiments, the sample may be a fluid sample, which includes but is not limited to bone marrow, tissue biopsies, whole blood, serum, plasma, blood cells, endothelial cells, circulating tumour cells, lymphatic fluid, ascites fluid, amniotic fluid, interstitial fluid (also known as "extracellular fluid" and encompasses the fluid found in spaces between cells, including, inter alia, gingival cervicular fluid), cerebrospinal fluid (CSF), saliva, tears, mucous, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluids, or any derivative thereof.

[0136] Alternatively, where the sample is solid or semi-solid in origin, it may subsequently be treated to provide a fluid sample. Examples of such samples are cell pellets, which may be resuspended in a liquid. Further examples of such samples are tissue biopsies, faecal matter, skin, tissue, hair, bone, muscle, and tumours.

[0137] In embodiments, the sample comprises or consists of whole blood, a blood derived fraction, serum, and / or plasma, and tumour tissues. In a preferred embodiment, the sample is blood serum. In embodiments, the sample is obtained during dialysis of a subject.

[0138] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has no recognised disease or disorder. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has no recognised disease or disorder associated with IgG ADAs or IgM ADAs.

[0139] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder associated with IgG ADAs or IgM ADAs.

[0140] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder and to which a drug has been administered at least one, twice or three times or more. In some embodiments the sample is a sample obtained from a human subject to which a drug has been administered at least one, twice or three times or more, wherein the subject has a recognised disease or disorder associated with IgG ADAs or IgM ADAs.

[0141] The sample may be appropriately diluted, solubilized, or concentrated after being collected from a patient, in such a way that any measurement generated according to the methods of the present invention is not, or not substantially, influenced thereby.

[0142] The disclosed methods may be carried out on any one or more samples that are known to contain or suspected to contain one or more ADAs. Alternatively, the invention may be carried out on one or more suitable samples to confirm the presence of one or more ADAs whose presence in the sample is unknown.

[0143] In embodiments, the first biological sample and second biological sample are from the same patient or animal.

[0144] In certain embodiments, a biological sample to be analysed by the methods of the invention may comprise more than one individual 'sample' collected from a patient or animal, for example, a biological sample may comprise a first biological sample taken at a first timepoint, and a second biological sample taken at a second timepoint, suitably wherein the timepoints are separated by less than about 1 minute, less than about 5 minutes, less than about 30 minutes, less than about an hour, less than about 8 hours, less than about 12 hours, less than about 24 hours, less than about 48 hours, less than about 1 week. Additionally, a biological sample may comprise a first biological sample comprising a first sample type, e.g. serum, and a second biological sample comprising a second sample type, e.g. plasma. Suitably, the first biological sample and second biological sample comprise the same sample type, e.g. serum.

[0145] Alternatively, in various aspects and embodiments, a first and second biological sample may be obtained by partitioning a single biological sample to obtain the first and second biological samples, or a first or second portions or aliquots of the biological sample.

[0146] Several ADA Assays for identifying or measuring ADAs are known in the art, including but not limited to pH-shift-anti-idiotype antigen binding test (PIA), Temperature-shift radioimmunoassay (TRIA), Acid-dissociation radioimmunoassay (ARIA), Homogeneous Mobility Shift Assays, Electrochemiluminescence Assays, direct ELISAs, indirect ELISAs, and bridging ELISAs. ADA Assays are usually bridging immunoassays, typically a bridging ELISA, a type of sandwich ELISA, typically incorporating a first drug moiety, which serves to ‘capture’ the ADA, and a second drug moiety or detection moiety, which serves, at least in part, to ‘detect’ the ADA, as the bridging components, resulting in the formation of an ADA-drug complex comprising the first drug moiety, the ADA, and the second drug moiety or detection moiety.

[0147] 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)). These are incorporated by reference herein.

[0148] In a bridging ELISA, a capture antibody, e.g. a first drug moiety, or an AAV capture moiety, is immobilized to a solid support, e.g. a microtiter plate well. Thereafter, a sample comprising an antigen or antibody to the immobilized capture antibody or capture moiety is added, e.g. a sample comprising an ADA, and if an antigen or antibody of interest, e.g. an ADA, is present in the sample, it is bound by, or binds to, the first drug moiety, which may be immobilised to a solid support. Alternatively, if an anti-AAV antibody is present in the sample, it may be bound or may bind to an AAV particle, which may be immobilised via an AAV capture moiety to a solid support. This is typically followed by washing, and then the subsequent addition of an antibody which can bind the ADA, e.g. a second drug moiety or detection moiety, and optionally the subsequent addition of a recognition agent which can bind the second drug moiety or detection moiety. This recognition agent can be a species-specific antibody (e.g., a mouse anti-human antibody). Alternatively, a recognition agent may bind the ADA directly.

[0149] Thus, a bridge is formed, comprising for example, solid support-first drug moiety-ADA-second drug moiety, or solid support-first drug moiety-ADA-second drug moiety-recognition agent, or solid support-AAV capture moiety-AAV particle-anti-AAV antibody-detection moiety- recognition agent, or solid support-AAV capture moiety-AAV particle-anti-AAV antibody-detection moiety.

[0150] Wherein the second drug moiety or recognition agent may comprise an enzyme, e.g. as a label, in a reaction catalysed by the enzyme, the activity of the enzyme is proportional to the ADA concentration in a sample.

[0151] The term “first drug moiety”, as used herein, relates to any chemical, suitably an antibody or antibody fragment or AAV particle, which comprises, consists, or consists essentially of a drug, suitably drug antibody or drug AAV particle, which binds to a corresponding ADA of interest, suitably wherein the ADA has been raised against said drug or drug antibody or drug AAV particle. In embodiments, the first drug moiety may comprise an antibody configured to bind to the at least one anti-drug antibody of the IgG class, or may comprise an antibody configured to bind to the at least one anti-drug antibody of the IgM class.

[0152] The term “second drug moiety”, as used herein, relates to any chemical, suitably an antibody or antibody fragment or AAV particle, which comprises, consists, or consists essentially of a drug, suitably drug antibody or a drug AAV particle, which binds to a corresponding ADA of interest, optionally wherein the ADA has been raised against said drug or drug antibody or drug AAV particle. In embodiments, the first drug moiety and the second drug moiety may comprise the "same" antibody molecule or antibody fragment or AAV particle, e.g. the same, or substantially the same amino acid sequence.

[0153] The term “detection moiety”, as used herein, relates to any chemical, suitably an antibody or antibody fragment, which binds to an ADA of interest. In embodiments, the detection moiety may comprise an anti-IgG antibody configured to bind to the at least one anti-AAV particle IgG antibody in each sample or portion of the sample, wherein the anti-IgG antibody may be derived from a species which is different to the species from which the sample to be analysed is derived.

[0154] The term “AAV capture moiety”, as used herein, relates to any chemical, suitably an antibody or antibody fragment, which may bind an AAV particle. In embodiments, the AAV capture moiety comprises an anti-AAV particle antibody, or antibody fragment, configured to bind to the at least one AAV particle. In embodiments wherein the AAV capture moiety is an antibody, said antibody may have been raised against a specific AAV or AAV fragment. In embodiments, the immobilised AAV particle is immobilised to a solid support via the immobilised AAV capture moiety.

[0155] An ADA assay according to the invention may comprise a solid-phase immunoassay.

[0156] Standard solid-phase immunoassays involve the formation of an ADA-first drug moiety complex between the first drug moiety, which may be adsorbed / immobilized on a solid support prior to contacting a first drug moiety, or after contacting a first drug moiety complex and formation of the ADA-first drug moiety complex.

[0157] The term “solid support”, as used herein, refers to a non-fluid substance, such as a stationary component, such as a tube, strip, cuvette or microtiter plate, e.g. a multi-well-plate, or may relate to non-stationary components, such as beads and microparticles. A variety of microparticles that allow either non-covalent or covalent attachment of proteins and other substances may be used. Such particles include polymer particles such as polystyrene and poly (methylmethacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See for example Martin, C. R., et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, J. E„ Methods 22 (2000) 4-23. Solid supports also encompass particles (including microparticles and beads) made from materials such as polymer, metal (paramagnetic, ferromagnetic particles), glass, and ceramic; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made of polymer, metal, glass, and / or ceramic; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers.

[0158] A solid support is distinguished from inert solid surfaces in that a “solid support” according to the invention comprises a binding agent at or on its surface, which interacts with a first drug moiety or AAV capture moiety, suitably binding to a label of a first drug moiety or AAV capture moiety.

[0159] The solid support may be coated or conjugated to a binding agent, suitably wherein the binding agent comprises, consists, or consists essentially of a first member of a binding pair, e.g. streptavidin, wherein the first drug moiety is conjugated, i.e. labelled, with a second member of a binding pair, e.g. biotin. As such, the term “binding agent”, as used herein, refers to one part of a specific binding pair. Any suitable binding pair can be used.

[0160] A polypeptide may be immobilised using methods known in the art, for example as described in Datta Set al., Enzyme immobilization: an overview on techniques and support materials, 3 Biotech, 3(1): 1-9 (2013). For example, the polypeptide may be immobilised by adsorption, covalent binding, affinity immobilization or entrapment. Materials that can be used as supports include but are not limited to for example, natural supports such as agarose, sepharose, collagen, gelatin, cellulose, pectin, sepharose, inorganic materials such as ceramics, silica, glass, activated carbon or charcoal, or synthetic polymers, such as Poly(styrene-divinylbenzene), or latex. Any of these may be provided as a resin or in any other suitable format. The polypeptide may be immobilised on magnetic beads.

[0161] The first drug moiety according to the invention may be conjugated to a solid support. The conjugation may be performed according to any suitable method in the art, e.g. by chemical binding via N-terminal and / or [epsilon]-amino groups (lysine), [epsilon]-amino groups of different lysines, carboxy-, sulfhydryl-, hydroxyl- and / or phenolic functional groups of the amino acid backbone of the drug antibody and / or sugar alcohol groups of the carbohydrate structure of the drug antibody.

[0162] In a preferred embodiment of the invention, the first drug moiety is immobilized to a solid support via a specific binding pair. Such a binding pair (first component / second component) is, for example, streptavidin or avidin / biotin, antibody / antigen (see, for example, Hermanson, G.T., et al., Bioconjugate Techniques, Academic Press, 1996), lectin / polysaccharide, steroid / steroid binding protein, hormone / hormone receptor, enzyme / substrate, IgG / Protein A and / or G, etc. Preferably, the first drug moiety is conjugated to biotin, e.g. the first drug moiety comprises a label, wherein the label is biotin, and immobilization is performed via immobilized avidin or streptavidin of a binding agent, which the solid support comprises. The term “label”, as used herein, refers to a ‘label’ moiety conjugated to a target moiety such as the first drug moiety, second drug moiety, or the recognition agent, wherein the conjugate may be a recombinant or chemical fusion between the label and the target moiety. The label may be covalently conjugated to the target moiety, and can be either directly conjugated or conjugated via a linker. Direct recombinant conjugation is by construction of a polypeptide fusion (i.e. by genetic fusion of the two genes, e.g., encoding an antibody and a detectable label or member of a binding pair and expressed as a single polypeptide. The label may comprise, but is not limited to, a first member of a binding pair, and / or a means for visualization or quantification, such as an enzyme.

[0163] The term “recognition agent”, as used herein, refers to an antibody or antibody fragment which is labelled, e.g. is conjugated to, a means for visualization or quantification. Such a means is typically an enzyme catalysing the formation of a coloured or fluorescent reaction product following the addition of a suitable substrate (detection reagent). These enzymes may include, but are not limited to, horseradish peroxidase, urease, alkaline phosphatase, glucoamylase or P- galactosidase. In certain embodiments, the recognition agent is a species-specific antiimmunoglobulin antibody. In certain embodiments, the recognition agent is conjugated to a label, such as, but not limited to: digoxygenin, biotin, fluorescein, theophylline, fluorescent markers, and radioisotopes. The recognition agent may be detected and / or quantified using this detection reagent. In certain embodiments, the recognition agent binds to the second drug moiety. In certain embodiments, the recognition agent binds to the detection moiety. In certain embodiments, the recognition agent binds to an ADA, e.g. an ADA of the IgM class or an ADA of the IgG class.

[0164] An “antibody fragment” refers to a molecule other than an intact or complete antibody that comprises a portion of an intact or complete antibody and that binds to the same antigen to which the intact or complete antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv, and scFab); single domain antibodies; and multi-specific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23: 1126-1136 (2005).

[0165] The term “detection reagent”, as used herein, refers to a reagent which permits the detection and / or quantification of an antibody or antibody fragment, e.g. the recognition agent or second drug moiety, bound to the ADA of interest. In embodiments, the detection reagent is a colorimetric substrate for an enzyme that has been conjugated to the recognition agent or second drug moiety, e.g. wherein the recognition agent or second drug moiety comprises a label, the label comprising an enzyme. Addition of a suitable substrate to the recognition agent or second drug moiety-enzyme conjugate results in the production of a colorimetric or fluorometric signal (e.g., following the binding of the conjugated antibody to the antigen of interest). Immunoglobulins (Ig) are heterodimeric protein molecules comprising two identical light chains and two identical heavy chains which are produced by plasma cells in response to an immunogen. Immunoglobulins can be separated into variable domains which bind antigens and constant domains that specify effector functions such as binding to Fc receptors. There are five main classes of heavy chain constant domains, the IgM, IgG, IgA, IgD, and IgE isotypes.

[0166] IgM is a class of immunoglobulin (Ig) that is typically characterised by its large size and pentameric structure. However, IgM may also exist in other structural forms such as a secreted hexamer or cell-surface displayed monomer that forms part of the B-cell receptor. The pentameric structure of IgM is such that it is made up essentially of five structures analogous to IgG. IgM comprises 10 potential antigen-binding sites (or paratopes), which facilitate high avidity target binding. This high avidity binding can allow target binding even when monovalent binding affinities are low. IgM is produced early during infection following antigen exposure and plays a key role in stimulating other effector functions of the immune system, such as activating complement and Fc receptor mediated activities.

[0167] In contrast to IgM, IgG is a class of immunoglobulin (Ig) that is typically characterised by its small size and monomeric structure. IgG polypeptides are typically composed of four polypeptide chains: two identical heavy (y) chains, comprising approximately 450 amino acids, and two identical light chains, comprising about 214 amino acids. A light chain and heavy chain are connected to one another by disulfide bonds. Similarly, the two heavy chains are connected in the hinge region by disulfide bonds to form a Y-shaped structure. In comparison with IgM, IgG is typically associated with the late-stage response to a disease or infection, whereas IgM production is observed immediately after the exposure to a particular antigen, representing the early-stage response to a disease or infection.

[0168] Discriminating between IgG and IgM in an ADA assay is important for gaining insights into the immune response to a therapeutic drug. IgM is typically the first antibody produced, signalling an early or acute phase of the immune response, while IgG appears later, indicating a more mature or prolonged response.

[0169] Examples where an understanding of isotyping and composition of an ADA response (including between IgG and IgM) can be highly important to predict future of disease progression or development of neutralizing ADAs can be found in Gorovits B, Current considerations for immunoglobulin isotype characterization of antibody response against biotherapeutics, AAPS J.22(6), 144 (2020).

[0170] Differentiating between IgG and IgM ADAs can facilitate assessment of the stage of the immune reaction where an ADA is raised against a target drug, which may facilitate an understanding of the potential impact on drug efficacy. For example, and without wishing to be bound by theory, IgG ADAs are typically higher-affinity and more specific, and therefore may be more likely to neutralize a target drug, potentially reducing its effectiveness.

[0171] In contrast, IgM ADAs, although present early, may not be as effective at neutralization, but detecting IgM ADAs can still signal that the immune system is reacting to the therapy, which may be important given that IgM can trigger complement activation and lead to hypersensitivity reactions. IgG can also cause hypersensitivity reactions through different mechanisms, such as immune complex formation (e.g., Type III hypersensitivity involving immune complex formation). As such, distinguishing the isotype can help predict or explain different types of adverse reactions.

[0172] Additionally, detecting IgM may indicate an early immune response, whilst IgG detection, particularly if neutralizing, could prompt adjustments in therapeutic strategy, such as dose adjustments or switching to a different drug to avoid loss of efficacy.

[0173] Discriminating between these two isotypes also provides mechanistic insights into the immune response and the immunogenicity of a target drug. For instance, if the ADA(s) is / are of the IgG class, or are predominately of the IgG class, this may be indicative of a T-cell dependent immune response, whereas if the ADA(s) is / are of the IgM class, or are predominately of the IgM class, this may be indicative of a T-cell independent pathway.

[0174] Finally, assay sensitivity and specificity may vary for different isotypes. Distinguishing between IgG and IgM ensures more accurate and clinically relevant results, aiding in better patient management and drug safety monitoring. Similarly, improved identification and discrimination between IgM and / or IgG ADAs be particularly useful in improving the efficiency, efficacy, and safety profile of gene therapy applications, in particular those utilising adenovirus type 5 vectors.

[0175] Whilst bridging ADA assays typically provide high throughput and sensitivity, as well as the ability to detect most ADA antibody isotypes, many ADA assays, including bridging ADA assays, do not typically distinguish between ADA classes or subclasses, e.g. between IgG and IgM.

[0176] Current methods to discriminate between Ig isotypes include anti-IgM isotype specific assays, such as Enzyme-Linked Immunosorbent Assays (ELISA), which are conventionally used for IgM isotyping (see for example, Kunzel, C. et al. 2021 , ‘Assay concept for detecting anti-drug IGM in human serum samples by using a novel recombinant human IGM positive control’, Bioanalysis, 13(4), pp. 253-263).

[0177] Additionally, Surface Plasmon Resonance (SPR) and Mass Spectrometry can also be used (see for example, Stubenrauch, K. et al. 2009, ‘Evaluation of a biosensor immunoassay for simultaneous characterization of Isotype and binding region of human anti-tocilizumab antibodies with control by surrogate standards’, Analytical Biochemistry, 390(2), pp. 189-196). In addition, multiplexing assays, for example Luminex-based multiplexing assays, can be used (see for example, Alleyn, M. et al. 2020, ‘Design and evaluation of a multiplexed assay to assess human immunogenicity against Humira®’, The AAPS Journal, 22(5)).

[0178] However, the current methods to discriminate between Ig isotypes are commonly limited by the requirement for additional individual assays to validate the results of the ADA screening assay, which typically entails the development of additional assays, such as an assay directed towards a specific ADA isotype to validate these results. For example, current methods typically require a two-step process. The initial ADA screening assay generates a total signal from mixed isotypes, followed by a second, IgM-specific assay to confirm the presence of IgM. Since these assays are independent, each requires separate development and validation.

[0179] In contrast, the methods of the invention do not require validation, or additional assay development, and can be easily integrated into existing assays via comparison of signals from treated versus untreated samples.

[0180] The methods of the invention enhance ADA screening assays by incorporating the ability to confirm IgM presence through signal reduction, suitably the reduction in signal originating from IgM. As a result, a second assay, e.g. a second assay type or second ADA assay, is not required, as the ADA assay according to the methods of the invention can be used for both initial screening of ADAs and confirmation of the presence of IgM ADAs. Since the ADA assays according to the methods of the invention (which comprise analysis of a sample post-IgM protease treatment) build upon the concept of a single / initial assay (which in the art typically comprises analysis of a sample pre-treatment), the ADA assays according to the invention do not require additional validation, and furthermore, the general principles of an ADA assay remain unchanged. As such, the skilled person would appreciate that, beneficially, the methods of the invention are suitable for use in enhancing ADA assays known in the art.

[0181] Furthermore, for methods such as SPR and Mass Spectrometry, the requirement for specialized equipment can be a barrier, which is associated with higher costs and potentially lower throughput compared to ELISA-based methods.

[0182] Additionally, isotype specific assays, e.g. anti-IgM isotype specific assays in particular, may suffer from low signal strength, leading to difficulties in distinguishing true positives from background noise.

[0183] In immunoassays, IgM has a propensity towards forming off-target interactions and aggregates, leading to potential nonspecific binding, which makes specific detection more challenging and complicates the differentiation between specific and nonspecific signals. To reduce this effect, assays can be modified through methods such as salt treatment or higher dilutions. However, these measures may (commonly) result in reduced signal strength. According to the methods of the invention, the sensitivity for IgM confirmation may be, or is, directly linked to the initial ADA screening assay. As such, the methods of the invention comprise a comparative analysis to identify IgM by comparing IgM protease treated samples versus untreated samples. Any signal reduction observed after treatment with a IgM protease is likely due to the presence of IgM. Consequently, the sensitivity is not dependent on a separate second assay but is directly coupled with the initial assay. This integration beneficially avoids the potential sensitivity issues associated with an additional assay.

[0184] Furthermore, the presence of IgM, e.g. IgM ADAs in a sample comprising IgG ADAs can interfere with the detection of IgG ADAs in an ADA assay. For example, IgM may bind to a target drug, or capture moiety, e.g. a first drug moiety, or AAV particle, and may physically block the sites where IgG may bind, e.g. by competing for the binding site or by steric hindrance, given that IgM antibodies are larger (pentameric) and as such whilst have lower affinity, have higher avidity (overall binding strength) than IgG, thus reducing the ability of the assay to accurately detect IgG ADAs, e.g. by IgM outcompeting IgG in binding to the drug of an ADA. Similarly, IgM may compete with IgG for binding to the second drug moiety.

[0185] A schematic representation of an example of the competition between IgM and IgG for the components of an ADA assay, including AAV masking in an assay to identify anti-AAV antibodies, is depicted in Figure 8 and Figure 9.

[0186] In addition, some assays may inadvertently result in the detection of IgM as if it were IgG due to cross-reactivity with detection reagents. This can generate false positives, complicating the interpretation of results and lowering the assay’s specificity for IgG ADAs.

[0187] IgM can also form larger immune complexes, in part due to its pentameric structure, which may aggregate non-specifically, leading to assay artifacts or signal interference that obscures the detection of true IgG ADAs.

[0188] A polypeptide which can degrade IgM may be used to remove or inactivate IgM in a sample and therefore facilitate increased resolution and / or sensitivity, particularly of IgG in ADA assays, or other similar assays that comprise IgM within a mix of components, by determining the contribution of IgM to any observed activity, and improving the signal of IgG ADA detection by removing / reducing IgM signal.

[0189] The term "polypeptide" as used herein relates to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term "polypeptide" thus includes short peptide sequences and also longer polypeptides and proteins. The terms "protein", "peptide" and "polypeptide" may be used interchangeably. The term "amino acid" may refer to either natural and / or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

[0190] A polypeptide suitable for use in any of the methods and uses of the invention has IgM protease activity.

[0191] It will be understood that, as used herein, a polypeptide having ‘IgM protease activity’ may refer to any protein, polypeptide, or fragment thereof, that is able to catalyze the cleavage of IgM.

[0192] The term ‘protease activity’, as used herein, which for the purposes of this application may be used interchangeably with simply ‘activity’, refers to the biochemical function of protease enzymes, which catalyse the hydrolysis of peptide bonds within proteins and peptides, leading to the breakdown of these molecules into smaller polypeptides or amino acids. This activity may be specific, wherein the protease recognises and hydrolyses / cleaves specific peptide bonds at particular amino acid sequences or structures.

[0193] Protease activity can be measured, and optionally quantified, using various assays and techniques that determine the rate and extent of peptide bond cleavage. Any suitable method known in the art may be used to analyse the activity of the protease, including spectrophotometric assays, Fluorescence Resonance Energy Transfer (FRET) assays, Kinetic Assays, Gel Electrophoresis, and HPLC or Mass Spectrometry. Gel Electrophoresis analysis of protease activity may comprise analysing the degradation pattern of protein substrates on SDS-PAGE, where the decrease in size or disappearance of specific protein bands indicates protease activity. HPLC or Mass Spectrometry analysis of protease activity may comprise quantifying the cleavage products of specific peptide or protein substrates to determine the extent and specificity of proteolysis, e.g. using liquid chromatography to separate and quantify peptide fragments followed by mass spectrometric identification, which may enable identification of cleavage sites present in the substrate.

[0194] In embodiments, polypeptides suitable for use in the methods have demonstrable protease activity in both simple and complex media, wherein the substrate may be considered substantially pure (e.g., a purified IgM sample) or substantially impure (e.g., a crude or complex sample comprising IgG and / or IgM).

[0195] In embodiments, polypeptides suitable for use in the methods and uses of the invention have IgM specific protease activity. In embodiments, the IgM specific protease activity may be endoprotease activity.

[0196] It will be understood that ‘IgM specific protease activity’ may refer to protease activity that is target (IgM) specific, and may not be considered to have a broad spectrum of target proteins, for example as trypsin does, and that the polypeptide may have residual or lower level activity for other targets, such as IgG, IgA, IgD, and IgE unless otherwise indicated. Furthermore, IgM specific activity may encompass activity against different IgM molecules, for example, IgM derived from different species or modified IgM molecules.

[0197] It will be understood that the term ‘human IgM specificity’ does not imply that the polypeptide is restricted to human IgM, rather, the protease is specific for IgM and human IgM is a recognised substrate, but not necessarily the only substrate. For example, IgM from other species, such as non-human primates, may also be a substrate.

[0198] The present invention provides methods of performing an ADA assay using polypeptides having IgM protease activity. That is, polypeptides are able to cleave an IgM molecule.

[0199] In embodiments, the IgM protease does not cleave, or exhibits lower cleavage activity against other classes of immunoglobulin.

[0200] In embodiments, the polypeptide may have low or negligible levels of detectable IgG protease activity. In another particularly suitable embodiment, the polypeptide has no detectable activity against IgG. This may be beneficial, as a protease, having IgG protease activity would degrade ADAs of the IgG class, first drug moieties of the IgG class, and second drug moieties of the IgG class, and therefore reduce the assay signal and effectiveness of an ADA assay, e.g. an IgG ADA assay, comprising these first drug moieties and second drug moieties.

[0201] In another particularly suitable embodiment, the polypeptide has no detectable activity against polypeptides other than IgM.

[0202] In embodiments, the polypeptide may have low levels of detectable IgG protease activity, wherein the level of IgG protease activity is less than the level of IgM protease activity; for example, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgM protease activity.

[0203] Determination of whether cleavage of IgM has occurred may be readily performed by the person skilled in the art, for example using common techniques in the art of protein biochemistry, such techniques including SDS-PAGE analysis, western blotting, chromatography (e.g., size-exclusion chromatography), and mass spectrometry.

[0204] In embodiments, the IgM protease may hydrolyse IgM below the CH2 region of human IgM (e.g. ...VPDQDT / AIRVFA...), into a mass-spectrometry detectable fragments corresponding to m / z 37499.6142 and a C-terminal fragment corresponding to m / z 25659.0200, the latter of which can be assigned to the theoretical mass value of amino acids 221 -453 (25657.6646 Da) of human IgM constant region (UniProt accession: P01871). In embodiments, the IgM protease may generate a F(ab’)2 (VH-CH1-CH2) and a pentameric Fc (CH3-CH4), due to the inter-monomer disulphide bonds between cysteine 413 residues in CH3 of IgM. Specific cleavage of IgM according to the methods can be verified, and the cleavage products isolated using any suitable method, such as in W02003051914 and W02009033670.

[0205] A polypeptide suitable for use in any of the methods or uses of the invention may comprise the sequence of any recently identified IgM specific proteases and modified IgM specific proteases derived from Lachnoanaerobaculum genus of commensal human bacteria, e.g.

[0206] Lachnoanaerobaculum umeaense and Lachnoanaerobaculum gingivalis, according to PCT / EP2024 / 065123 or GB2308197.9.

[0207] Exemplary polypeptide sequences are set out in Table 1 herein.

[0208] As such, in aspects and embodiments of the invention the sequence of a polypeptide suitable for use in any of the methods or uses of the invention may comprise SEQ ID NO: 1 , or a variant of the amino acid sequence of SEQ ID NO: 1 , in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of SEQ ID NO: 1 .

[0209] In aspects and embodiments of the invention the sequence of a polypeptide suitable for use in any of the methods or uses of the invention may comprise SEQ ID NO: 2.

[0210] Table 1 : Exemplary IgM protease sequences As such, in aspects and embodiments of the invention the sequence of a polypeptide suitable for use in any of the methods or uses of the invention comprising SEQ ID NO: 1 or a variant of SEQ ID NO: 1 , comprises the sequence of SEQ ID NO: 2.

[0211] Unless otherwise specified, the modifications are preferably conservative amino acid substitutions which do not ablate the IgM protease activity. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a preexisting aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains.

[0212] A polypeptide may be engineered or modified to assist with production, isolation or purification. For example, where a polypeptide of the invention is produced by recombinant expression in a bacterial host cell, the sequence of the polypeptide may include an additional methionine (M) residue at the N terminus to improve expression. As another example, the polypeptide of the invention may be engineered or modified by addition of protein purification tag at the N or C terminus, preferably at the C terminus.

[0213] The amino acid sequence of a polypeptide may be modified or engineered to include at least one non-naturally occurring amino acid, for example to increase stability. When the polypeptides are produced by synthetic means, such amino acids may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production.

[0214] Polypeptides may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such polypeptides. Preferred polypeptides of the invention may comprise at least one such unnatural or synthetic amino acid, or at least one non-L configuration amino acid.

[0215] A number of side chain modifications are known in the art and may be made to the side chains of the polypeptides, subject to the polypeptides retaining any further required activity or characteristic as may be specified herein. It will also be understood that polypeptides may be chemically modified, e.g. post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues. The polypeptide may be PEGylated. Accordingly, a polypeptide suitable for use in any of the methods and uses of the invention may be produced by any suitable method, including recombinant or synthetic methods. For example, the polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector which encodes said polypeptide. Expression of said polypeptides would be readily achievable by the person skilled in the art using standard techniques.

[0216] Those skilled in the art will appreciate that the amount of an IgM protease to be used to degrade IgM in a sample may be selected based on the Unit definition (e.g. as disclosed in this application) adjusted to anticipated total IgM serum level, see for example Ritchie, R.F. et al. (1998) ‘Reference distributions for Immunoglobulins A, G, and M: A comparison of a large cohort to the world’s literature’, Journal of Clinical Laboratory Analysis, 12(6), pp. 371-377.

[0217] Methods of the invention

[0218] Disclosed herein are methods to perform ADA assays, which are suitable for a broad range of ADAs. The disclosed methods of performing an ADA assay comprise cleaving an immunoglobulin, and may further comprise the detection or analysis of the cleavage products. For example, the methods may comprise a step of identifying and / or isolating the IgM specific cleavage products. The disclosed methods of performing an ADA assay comprise cleaving an immunoglobulin, and may further comprise the detection or analysis of the non-cleavage products. For example, the methods may comprise a step of identifying and / or isolating the IgG specific antibodies in a sample.

[0219] ADA assays performed according to the methods of the disclosure have improved diagnostic assay performance, such as higher IgG sensitivity, and beneficially facilitate / enable the identification of the contribution of IgM to any observed activity.

[0220] Furthermore, the methods disclosed herein have many advantages, e.g., simple to perform, short production cycle, low-cost, suitable for a wide range of ADAs, and can be readily and easily scaled up for large-scale production.

[0221] Any of the methods or uses herein may be performed in vitro or ex vivo.

[0222] Methods for performing anti-drug antibody (ADA) assays

[0223] Disclosed herein are methods to perform an anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti-mAb1 antibodies and supplementary assays for the determination of the corresponding ADA isotype (IgM or IgG). In aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample is provided, the method comprising:

[0224] (a) contacting a first biological sample from a subject which comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,

[0225] (b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately

[0226] (b)(ii) contacting a second biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;

[0227] (c) detecting the presence of an IgM complex in each of the first and second samples, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0228] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0229] In embodiments of the methods, the first and second biological samples are obtained by partitioning a biological sample into at least two portions.

[0230] In embodiments of the methods, the first and second biological samples are obtained separately from the same subject, wherein the samples may be obtained at different times or disease states, e.g. prior to a diagnosis of one or more diseases or during progression of one or more diseases.

[0231] In aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample is provided, the method comprising:

[0232] (a) partitioning a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class into at least a first portion and a second portion,

[0233] (b) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0234] (c) separately contacting each portion of the sample with: a first drug moiety, and a second drug moiety; (d) detecting the presence of an IgM complex in each portion of the sample, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0235] (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the biological sample.

[0236] In aspects of the invention, a method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample is provided, the method comprising:

[0237] (a) contacting a first biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a first treated sample,

[0238] (b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately

[0239] (b)(ii) contacting a second biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;

[0240] (c) detecting the presence of an IgG complex in the first treated and second samples, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0241] (d) comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample wherein the detection is improved compared to a method without step (a).

[0242] In aspects of the invention, a method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample is provided, the method comprising:

[0243] (a) contacting a biological sample from a subject, wherein the biological sample comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0244] (b) contacting the treated sample with: a first drug moiety, and a second drug moiety;

[0245] (c) detecting the presence of an IgG complex in the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0246] In aspects of the invention, a method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample is provided, the method comprising:

[0247] (a) providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class;

[0248] (b) partitioning the sample into at least a first portion and a second portion,

[0249] (c) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0250] (d) separately contacting the first portion and the second portion of the sample with: a first drug moiety, and a second drug moiety;

[0251] (e) detecting the presence of an IgG complex in each portion of the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0252] (f) comparing the detection of the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample.

[0253] In embodiments, the methods further comprise providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class. With respect to specific assay thresholds for indicating a higher level of IgM complex in a sample, those skilled in the art would appreciate that ADA assays may exhibit different variabilities. ADA assay variability is well known in the art. Indeed, according to the FDA guidelines (see: GUIDANCE DOCUMENT, Immunogenicity Testing of Therapeutic Protein Products — Developing and Validating Assays for Anti-Drug Antibody Detection, 2019, Docket number: FDA-2009-D-0539), assay variability is generally expected to be less than 20%.

[0254] The variability inherent to each assay is typically used as basis for determining appropriate thresholds. As such, the skilled person would further appreciate that the threshold for indicating a higher level of IgM complex in a sample may be adjusted or determined based on assay variability, or the variability of an assay run.

[0255] In view of the art, a reduction in assay signal (an inhibition), greater than 20% after IgM protease treatment may indicate the presence of IgM ADAs.

[0256] In embodiments, the higher level of IgM complex in the second sample relative to the first sample may be higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%; particularly by at least about 20%.

[0257] In embodiments, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class and / or IgG class.

[0258] In embodiments of the methods, contacting either the sample, each sample, or each portion of the sample, with a first drug moiety and a second drug moiety comprises contacting the sample, each sample, or each portion of the sample, with a first drug moiety and subsequently with a second drug moiety in a step-wise manner.

[0259] In embodiments of the methods, the sample comprises serum, suitably human or monkey serum; particularly human or cynomolgus serum.

[0260] The polypeptide may be administered to a sample containing IgM and incubated under conditions which permit immunoglobulin protease activity to occur.

[0261] The methods of the invention typically involve incubating a polypeptide having IgM protease activity with a sample under conditions which permit binding and cleavage of IgM. Suitable incubation conditions may take place at room temperature, preferably at approximately X, and more preferably at approximately 37°C.

[0262] The methods described may be carried out under any suitable pH. Suitable pH values include, for example, around pH 6.5 to around pH 8.5, preferably around pH 7.0 to around pH 8.0, most preferably at around pH 7.5.

[0263] In embodiments, contacting at least the first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0264] In embodiments, the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%; suitably by at least about 20%.

[0265] In embodiments of the methods, the IgM protease is an IgM specific protease, suitably having no detectable activity against polypeptides other than IgM.

[0266] In embodiments of the methods, the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0267] In embodiments of the methods, the IgM protease comprises SEQ ID NO: 2.

[0268] In embodiments of the methods, the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0269] In embodiments of the methods, contacting the first sample or the first portion of the sample with an IgM protease comprises contacting the first sample or the first portion of the sample with IgM protease at a concentration between about 0.2 to 10 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL, e.g. about 0.91 U / pL.

[0270] In embodiments of the methods, contacting each sample or portion of the sample with a first drug moiety and a second drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer. In embodiments of the methods, diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises diluting each sample or portion of the sample to a final serum concentration of between about 10% to 0.1 %, such as between about 7.5% to 0.5%, between about 5% to 1 %, or between about 2.5% to 1 .5%; e.g. about 2%.

[0271] In embodiments of the methods, the buffer, optionally a reaction buffer, comprises 1% (w / v) bovine serum albumin and / or 0.05% (v / v) Tween20 in PBS.

[0272] In embodiments of the methods, the at least one anti-drug antibody of the IgM class specifically binds to the first drug moiety anti-drug antibody of the IgM class.

[0273] In embodiments of the methods, the first drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgM class.

[0274] In embodiments of the methods, the at least one anti-drug antibody of the IgG class specifically binds to the first drug moiety anti-drug antibody of the IgG class.

[0275] In embodiments of the methods, the first drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgG class.

[0276] In embodiments of the methods, the antibody is derived from a species which is different to the species from which the biological sample is derived.

[0277] In embodiments of the methods, the first drug moiety comprises a detectable label.

[0278] In embodiments of the methods, the label comprises a biotin moiety.

[0279] In embodiments, the methods further comprise providing a solid support comprising a binding agent.

[0280] In embodiments of the methods, the binding agent of the solid support is configured to bind the biotin moiety of the label.

[0281] In embodiments of the methods, the binding agent of the solid support comprises streptavidin; for example, a streptavidin coated surface. In embodiments of the methods, the first drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

[0282] In embodiments of the methods, the second drug moiety specifically binds to the at least one antidrug antibody of the IgM class.

[0283] In embodiments of the methods, the second drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgM class.

[0284] In embodiments of the methods, the second drug moiety specifically binds to the at least one antidrug antibody of the IgG class.

[0285] In embodiments of the methods, the second drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgG class.

[0286] In embodiments of the methods, the antibody is derived from a species which is different to the species from which the biological sample is derived.

[0287] In embodiments of the methods, the second drug moiety comprises a detectable label.

[0288] In embodiments of the methods, the label comprises a digoxigenin moiety.

[0289] In embodiments of the methods, the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

[0290] In embodiments of the methods, detecting the presence of an IgM or IgG complex in each sample or portion of the sample comprises contacting the IgM or IgG complex with the solid support.

[0291] In embodiments of the methods, contacting the IgM or IgG complex with the solid support further comprises incubating the IgM or IgG complex with the solid support, suitably wherein the first drug moiety binds to the solid support.

[0292] In embodiments of the methods, contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises: (i) contacting the sample comprising the at least one anti-drug antibody of the IgM class with the first drug moiety, to generate an ‘anti-drug antibody of the IgM class- first drug moiety’ complex; and

[0293] (ii) contacting a portion of the ‘anti-drug antibody of the IgM class-first drug moiety’ complex from step (i) with the second drug moiety to generate the IgM complex.

[0294] In embodiments of the methods, contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0295] (i) contacting the sample comprising the at least one anti-drug antibody of the IgM class with the second drug moiety, to generate an ‘anti-drug antibody of the IgM class-second drug moiety’ complex; and

[0296] (ii) contacting a portion of the ‘anti-drug antibody ofthe IgM class-second drug moiety’ complex from step (i) with the first drug moiety to generate the IgM complex.

[0297] In embodiments of the methods, detecting the presence of an IgM complex in each sample further comprises:

[0298] (i) contacting a portion of the IgM complex corresponding to the first drug moiety with the binding agent of the solid support to immobilise the IgM complex, wherein the binding agent is configured to specifically bind the first drug moiety; and

[0299] (ii) contacting a portion of the immobilised IgM complex corresponding to the second drug moiety with the recognition moiety.

[0300] In embodiments of the methods, contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0301] (i) contacting the sample comprising the at least one anti-drug antibody of the IgG class with the first drug moiety, to generate an ‘anti-drug antibody ofthe IgG class- first drug moiety’ complex, and

[0302] (ii) contacting a portion of the ‘anti-drug antibody of the IgG class-first drug moiety’ complex from step (i) with the second drug moiety to generate the IgG complex.

[0303] In embodiments of the methods, contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0304] (i) contacting the sample comprising the at least one anti-drug antibody of the IgG class with the second drug moiety, to generate an ‘anti-drug antibody of the IgG class-second drug moiety’ complex, and

[0305] (ii) contacting a portion of the ‘anti-drug antibody of the IgG class-second drug moiety’ complex from step (i) with the first drug moiety to generate the IgG complex. In embodiments of the methods, detecting the presence of an IgG complex in each sample further comprises:

[0306] (i) contacting a portion of the IgG complex corresponding to the first drug moiety with the binding agent of the solid support to immobilise the IgG complex, wherein the binding agent is configured to specifically bind the first drug moiety; and

[0307] (ii) contacting a portion of the immobilised IgG complex corresponding to the second drug moiety with the recognition moiety.

[0308] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises one or more washing steps, comprising washing the samples with a wash buffer.

[0309] In embodiments of the methods, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0310] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a recognition agent configured to specifically bind to the second drug moiety, suitably wherein the recognition agent is configured to specifically bind to the label of the second drug moiety.

[0311] In embodiments of the methods, the recognition agent comprises a fragment antigen-binding region (Fab).

[0312] In embodiments of the methods, the fragment antigen-binding region (Fab) is configured to bind digoxigenin.

[0313] In embodiments of the methods, the recognition agent comprises a label.

[0314] In embodiments of the methods, the label comprises horseradish peroxidase.

[0315] In embodiments of the methods, the recognition agent is provided at a concentration in the sample or portion of the sample of between about 5 to 50 mU / mL, such as between about 10 to 40 mU / mL, or between about 15 to 35 mU / mL; for example, between about 20 to 30 mU / mL; e.g. about 25 mU / mL.

[0316] In embodiments of the methods, contacting the second drug moiety with the recognition agent comprises incubating the second drug moiety with the recognition agent; suitably wherein the incubation is up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour. In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0317] In embodiments of the methods, the detection reagent comprises 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid).

[0318] In embodiments of the methods, contacting the recognition agent with the detection reagent comprises incubating the second drug moiety with the recognition agent; suitably wherein the incubation is for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0319] In embodiments of the methods, detecting the presence of an IgM complex comprising: the first drug moiety, the anti-drug antibody of the IgM class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgM absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0320] In embodiments of the methods, detecting the presence of an IgG complex comprising: the first drug moiety, the anti-drug antibody of the IgG class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0321] In embodiments of the methods, the first specific wavelength is about 405 nm.

[0322] In embodiments of the methods, the second specific wavelength is about 490 nm.

[0323] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final absorbance. In embodiments of the methods, calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final absorbance.

[0324] In embodiments of the methods, comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample comprises comparing the IgM absorbance value in the first sample to the IgM absorbance value in the second sample, wherein a lower IgM absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0325] In embodiments of the methods, comparing the detection of the presence of the IgM complex in the first portion of the sample with the detection of the presence of the IgM complex in the second portion of the sample comprises comparing the IgM absorbance value in the first portion of the sample to the IgM absorbance value in the second portion of the sample, wherein a lower IgM absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0326] In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG absorbance value in the first sample to the IgG absorbance value in the second sample, wherein a lower IgG absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0327] In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG absorbance value in the first portion of the sample to the IgG absorbance value in the second portion of the sample, wherein a lower IgG absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0328] In alternative aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample is provided, the method comprising:

[0329] (a) contacting a first biological sample from a subject, wherein the sample comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample, (b)(i) contacting the first treated sample with an immobilised first drug moiety,

[0330] (b)(ii) contacting a second sample which comprises or may comprise at least one anti-drug antibody of the IgM class with an immobilised first drug moiety,

[0331] (c) separately contacting the first and second samples with a second drug moiety,

[0332] (d) detecting the presence of an IgM complex in the first and second samples, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0333] (e) comparing the detection of the presence of the IgM complex in the first treated sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0334] In alternative embodiments, the first and second biological samples are obtained by partitioning a biological sample into at least two portions.

[0335] In alternative embodiments, the first and second biological samples are obtained separately from the same subject.

[0336] In alternative aspects of the invention, a method for detecting at least one anti-drug antibody of the IgM class in a biological sample is provided, the method comprising:

[0337] (a) partitioning a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class into at least a first portion and a second portion,

[0338] (b) contacting the first portion of the sample with an IgM protease, to obtain a first treated portion of the sample,

[0339] (c) separately contacting each portion of the sample with an immobilised first drug moiety,

[0340] (d) separately contacting each portion of the sample with a second drug moiety,

[0341] (e) detecting the presence of an IgM complex in each portion of the sample, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0342] (f) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second sample portion relative to the first portion is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample In alternative aspects of the invention, a method for improving the detection of at least one antidrug antibody of the IgG class in a biological sample is provided, the method comprising:

[0343] (a) contacting a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0344] (b) contacting the treated sample with an immobilised first drug moiety,

[0345] (c) contacting the treated sample with a second drug moiety,

[0346] (d) detecting the presence of an IgG complex in the treated sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0347] In alternative aspects of the invention, a method for improving the detection of at least one antidrug antibody of the IgG class in a biological sample is provided, the method comprising:

[0348] (a) contacting a first biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a first treated sample,

[0349] (b)(i) contacting the first treated sample with an immobilised first drug moiety; and separately

[0350] (b)(ii) contacting a second biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an immobilised first drug moiety; and

[0351] (c) separately, contacting the first treated and second samples with a second drug moiety,

[0352] (d) detecting the presence of an IgG complex in each sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0353] (e) comparing the detection of the presence of the IgG complex in the first treated sample with the detection of the presence of the IgG complex in the second sample, wherein the detection is improved compared to a method without step (a). In alternative aspects of the invention, a method for improving the detection of at least one antidrug antibody of the IgG class in a biological sample, the method comprising:

[0354] (a) partitioning a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; into at least a first portion and a second portion,

[0355] (b) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0356] (c) separately contacting the first treated and second portions of the sample with an immobilised first drug moiety,

[0357] (d) separately contacting the first treated and second portions of the sample with a second drug moiety,

[0358] (e) detecting the presence of an IgG complex in each portion of the sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0359] (f) comparing the detection of the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample, wherein the detection is improved compared to a method without step (b).

[0360] In alternative embodiments, the methods further comprise providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class.

[0361] In alternative embodiments, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class.

[0362] In alternative embodiments, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgG class.

[0363] In alternative embodiments, the methods further comprise providing a solid support configured to bind a first drug moiety.

[0364] In alternative embodiments, the methods further comprise contacting a solid support configured to bind a first drug moiety with a first drug moiety to generate a solid support comprising the immobilised first drug moiety. In alternative embodiments, the methods further comprise contacting the solid support configured to bind the first drug moiety with the first drug moiety is performed before the step of contacting the first drug moiety with the sample, each sample, or portion of the sample.

[0365] In alternative embodiments, the immobilised first drug moiety comprises a first drug moiety immobilised on a solid support.

[0366] In alternative embodiments, the solid support comprises the immobilised the first drug moiety.

[0367] In alternative embodiments, the methods further comprise providing the solid support comprising the immobilised first drug moiety.

[0368] In alternative embodiments, separately contacting each sample with the immobilised first drug moiety comprises contacting the sample comprising the at least one anti-drug antibody of the IgM class with the immobilised first drug moiety, to generate an ‘anti-drug antibody of the IgM class- immobilised first drug moiety’ complex.

[0369] In alternative embodiments, separately contacting each sample with a second drug moiety further comprises contacting a portion of the ‘anti-drug antibody of the IgM class-immobilised first drug moiety’ complex corresponding to the anti-drug antibody of the IgM class with the second drug moiety to generate the IgM complex.

[0370] In alternative embodiments, detecting the presence of an IgM complex in each sample or each portion of the sample further comprises contacting a portion of the IgM complex corresponding to the second drug moiety with the recognition moiety.

[0371] In alternative embodiments, separately contacting each sample with the immobilised first drug moiety comprises contacting the sample comprising the at least one anti-drug antibody of the IgG class with the immobilised first drug moiety, to generate a ‘anti-drug antibody of the IgG class- immobilised first drug moiety’ complex.

[0372] In alternative embodiments, separately contacting each sample with a second drug moiety further comprises contacting a portion of the ‘anti-drug antibody of the IgG class-immobilised first drug moiety’ complex corresponding to the anti-drug antibody of the IgG class with the second drug moiety to generate the IgG complex. In alternative embodiments, detecting the presence of an IgG complex in each sample further comprises contacting a portion of the IgG complex corresponding to the second drug moiety with the recognition moiety.

[0373] In alternative embodiments, the sample comprises serum, suitably human serum or monkey serum; particularly human or cynomolgus serum.

[0374] In alternative embodiments, contacting at least the first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0375] In embodiments, the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%; suitably by at least about 20%.

[0376] In alternative embodiments, contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class is performed at a temperature of between about 4 and 25°C, between about 4 and 20°C, between about 4 and 15°C, or between about 4 and 10°C; suitably at a temperature of about 4 °C.

[0377] In alternative embodiments, the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0378] In alternative embodiments, contacting the first sample or portion of the sample with an IgM protease comprises contacting the first sample or portion of the sample with IgM protease at a concentration between about 0.25 to 103 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL, e.g. about 0.91 U / pL.

[0379] In alternative embodiments, separately contacting each sample or portion of the sample with the immobilised first drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer. In alternative embodiments, separately contacting each sample or portion of the sample with the second drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0380] In alternative embodiments, diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises a dilution of between about 50-fold and 150-fold, between about 70-fold and 140-fold, between about 8-fold and 130-fold, between about 90-fold and 120-fold, or between about 100-fold and 110-fold; e.g. about 105-fold.

[0381] In alternative embodiments, the solid support comprises a charged polystyrene surface, suitably having high affinity to the first drug moiety.

[0382] In alternative embodiments, contacting the first drug moiety with the solid support configured to bind a first drug moiety comprises incubating the first drug moiety with the with the solid support configured to bind a first drug moiety.

[0383] In alternative embodiments, the methods further comprise blocking the immobilised first drug moiety after contacting the first drug moiety with the solid support configured to bind a first drug moiety.

[0384] In alternative embodiments, blocking the immobilised first drug moiety comprises contacting the immobilised first drug moiety with a blocking buffer.

[0385] In alternative embodiments, contacting the immobilised first drug moiety with the blocking buffer comprises incubating the immobilised first drug moiety with the blocking buffer.

[0386] In alternative embodiments, the blocking buffer comprises LowCross buffer from CANDOR Bioscience GmbH.

[0387] In alternative embodiments, the methods further comprise one or more washing steps after blocking the immobilised first drug moiety prior to contacting each sample or portion of the sample with a second drug moiety, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0388] In alternative embodiments, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0389] In alternative embodiments, the second drug moiety comprises an anti-IgM antibody. In alternative embodiments, the anti-IgM antibody is derived from a species which is different to the species from which the biological sample is derived.

[0390] In alternative embodiments, the anti-IgM antibody is derived from mouse and the biological sample is derived from human.

[0391] In alternative embodiments, the second drug moiety comprises an anti-IgG antibody.

[0392] In alternative embodiments, the anti-IgG antibody is derived from a species which is different to the species from which the biological sample is derived.

[0393] In alternative embodiments, the anti-IgG antibody is derived from mouse and the biological sample is derived from human.

[0394] In alternative embodiments, the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, such as between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. about 0.5 ng / pL.

[0395] In alternative embodiments, contacting each sample or portion of the sample with the second drug moiety comprises incubating each sample or portion of the sample with the second drug moiety; suitably, wherein the incubation is up to about 6 hours, up to about 4 hours, for between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0396] In alternative embodiments, the methods further comprise one or more washing steps after contacting each sample or portion of the sample with a second drug moiety prior to detecting the presence of a complex in each sample or portion of the sample, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0397] In alternative embodiments, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0398] In alternative embodiments, the second drug moiety comprises a label.

[0399] In alternative embodiments, detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a recognition agent configured to specifically bind the second drug moiety; suitably the detectable label of the second drug moiety. In alternative embodiments, contacting the second drug moiety with the recognition agent comprises incubating the second drug moiety with the recognition agent; suitably, wherein the incubation is up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0400] In alternative embodiments, the recognition agent comprises an antibody configured to bind to the second antibody.

[0401] In alternative embodiments, the recognition agent antibody is derived from a species which is different to the species from which the second drug moiety is derived.

[0402] In alternative embodiments, the second drug moiety is derived from mouse and the recognition agent comprises an anti-mouse antibody.

[0403] In alternative embodiments, the recognition agent comprises a label.

[0404] In alternative embodiments, the label comprises horseradish peroxidase.

[0405] In alternative embodiments, the recognition agent is provided at a concentration of between about 100 to 1 ,000 ng / mL, between about 200 to 600 ng / mL, or between about 300 to 500 ng / mL; e.g. about 400 ng / mL.

[0406] In alternative embodiments, detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0407] In alternative embodiments, the detection reagent comprises 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid).

[0408] In alternative embodiments, contacting the recognition agent with the detection reagent comprises incubating the second drug moiety with the recognition agent, suitably wherein the incubation is performed for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0409] In alternative embodiments, detecting the presence of an IgM complex comprising: the first drug moiety, the anti-drug antibody of the IgM class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgM absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0410] In alternative embodiments, detecting the presence of an IgG complex comprising: the first drug moiety, the anti-drug antibody of the IgG class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0411] In alternative embodiments, the first specific wavelength is about 405 nm.

[0412] In alternative embodiments, the second specific wavelength is about 490 nm.

[0413] In alternative embodiments, detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final absorbance.

[0414] In alternative embodiments, calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final IgM absorbance.

[0415] In alternative embodiments, calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final IgG absorbance.

[0416] In alternative embodiments, comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample comprises comparing the IgM absorbance value in the first sample to the IgM absorbance value in the second sample, wherein a lower IgM absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample. In alternative embodiments, comparing the detection of the presence of the IgM complex in the first portion of the sample with the detection of the presence of the IgM complex in the second portion of the sample comprises comparing the IgM absorbance value in the first portion of the sample to the IgM absorbance value in the second portion of the sample, wherein a lower IgM absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0417] In alternative embodiments, comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG absorbance value in the first sample to the IgG absorbance value in the second sample, wherein a lower IgG absorbance in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0418] In alternative embodiments, comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG absorbance value in the first portion of the sample to the IgG absorbance value in the second portion of the sample, wherein a lower IgG absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0419] The invention also provides methods and uses comprising alternative second drug moieties and recognition agents.

[0420] In alternative embodiments, the second drug moiety comprises an Fc receptor, which may comprise, consist, or consist essentially of CD64.

[0421] In alternative embodiments, the second drug moiety comprises a label, which may comprise, consist, or consist essentially of digoxigenin.

[0422] In alternative embodiments, the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. 0.5 ng / pL.

[0423] In alternative embodiments, the recognition agent comprises a fragment antigen-binding region (Fab) may be configured to bind digoxigenin, and which may comprise, consist, or consist essentially of an anti-digoxigenin Fab fragment. In alternative embodiments, the recognition agent comprises a label, which may comprise, consist, or consist essentially of horseradish peroxidase.

[0424] In alternative embodiments, the recognition agent is provided at a concentration of between about 20 to 120 mU / mL, between about 20 to 100 mU / mL, or between about 40 to 80 mU / mL, or between about 40 to 60 mU / mL; e.g. about 50 mU / mL.

[0425] Methods for performing Anti-AAV antibody immune complex assays

[0426] Also disclosed herein are methods to perform Anti-AAV antibody immune complex assays for the detection of anti-AAV antibodies of the IgG class and supplementary assay for the determination of the corresponding anti-AAV antibodies of the IgM class which comprise the use of a detection moiety.

[0427] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample, the method comprising:

[0428] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0429] (b) contacting the treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0430] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0431] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0432] (a) contacting a first biological sample from a subject which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample, (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; and separately

[0433] (b)(ii) contacting a second biological sample from the subject, which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample;

[0434] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without step (a).

[0435] In embodiments of the methods, the first and second biological samples are obtained a partitioning a biological sample into at least two portions.

[0436] In embodiments of the methods, the first and second biological samples are obtained separately from the same subject.

[0437] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle IgG antibody in a biological sample is provided, the method comprising:

[0438] (a) partitioning a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody, into at least a first portion and a second portion,

[0439] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0440] (c) separately contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0441] (d) detecting the presence of an IgG complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without step (b).

[0442] In aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample is provided, the method comprising:

[0443] (a) contacting a first biological sample from a subject, which comprises or may comprise at least one anti-AAV particle IgM antibody, with an IgM protease to obtain at least a first treated sample,

[0444] (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0445] (b)(ii) contacting a second biological sample from the subject which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0446] (c) detecting the presence of an IgM complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody, and the detection moiety; and

[0447] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0448] In aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample is provided, the method comprising:

[0449] (a) partitioning a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody into at least a first portion and a second portion,

[0450] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0451] (c) separately contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[0452] (d) detecting the presence of an IgM complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody, and the detection moiety; and

[0453] (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second portion relative to the first portion is indicative for the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0454] In embodiments, the methods further comprise providing a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody.

[0455] In embodiments, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody.

[0456] In embodiments, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-AAV particle IgG antibody.

[0457] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; is performed separately and in a step-wise manner.

[0458] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0459] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle’ complex, and

[0460] (ii) contacting a portion of the ‘anti-AAV particle antibody ofthe IgG class-AAV particle’ complex of step (i) with an AAV capture moiety to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex, and

[0461] (iii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgG class-AAV particle- AAV capture moiety’ complex of step (ii) with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgG class-AAV particle- AAV capture moiety’, wherein the binding agent is configured to specifically bind the AAV capture moiety,

[0462] (iv) contacting a portion of the immobilised ‘anti-AAV particle antibody ofthe IgG class- AAV particle-AAV capture moiety’ of step (iii) with the detection moiety to generate the IgG complex.

[0463] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0464] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle’ complex, and

[0465] (ii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgG class-AAV particle’ complex of step (i) with an immobilised AAV capture moiety, wherein the AAV capture moiety is immobilised to a solid support, and

[0466] (iii) contacting a portion of the immobilised ‘anti-AAV particle antibody ofthe IgG class- AAV particle’ of step (ii) with the detection moiety to generate the IgG complex.

[0467] In embodiments of the methods, the immobilised AAV capture moiety is immobilised on a solid support via a binding agent.

[0468] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0469] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one immobilised AAV particle, and

[0470] (ii) contacting the anti-AAV particle antibody of the IgG class with the detection moiety to generate the IgG complex.

[0471] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0472] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex,

[0473] (ii) contacting a portion of the ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex of step (i) with an AAV capture moiety to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle-AAV capture moiety’ complex,

[0474] (iii) contacting a portion of the ‘anti-AAV particle antibody of the IgG class-AAV particle- AAV capture moiety’ complex of step (ii) with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgM class-AAV particle-AAV capture moiety’, wherein the binding agent is configured to specifically bind the AAV capture moiety, and

[0475] (iv) contacting a portion of the immobilised ‘anti-AAV particle antibody of the IgM class- AAV particle-AAV capture moiety’ of step (iii) with the detection moiety to generate the IgM complex.

[0476] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0477] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex,

[0478] (ii) contacting a portion of the ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex of step (i) with an immobilised AAV capture moiety, wherein the AAV capture moiety is immobilised to a solid support, and

[0479] (iii) contacting a portion of the immobilised ‘anti-AAV particle antibody of the IgM class- AAV particle’ of step (ii) with the detection moiety to generate the IgM complex.

[0480] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[0481] (viii) contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with an immobilised AAV particle, and

[0482] (ix) contacting the anti-AAV particle antibody of the IgM class with the detection moiety to generate the IgM complex.

[0483] In embodiments of the methods, the immobilised AAV particle is immobilised to a solid support via the immobilised AAV capture moiety.

[0484] In embodiments of the methods, the immobilised AAV capture moiety is immobilised on a solid support via a binding agent.

[0485] In embodiments of the methods, contacting the portion of the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ comprises: incubating the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex with the binding agent conjugated to a solid support; suitably wherein the incubation is for up to about 2 hours, up to about 1.5 hours; up to about 1 hour, up to about 30 minutes; between about 5 and 30 minutes, or between about 10 and 20 minutes; for example about 15 minutes; preferably at room temperature. In embodiments, the methods, further comprise one or more washing steps, after contacting the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex with the binding agent conjugated to a solid support prior to contacting the portion of the immobilised ‘anti- AAV particle antibody of the IgG class-AAV particle’ with the detection moiety to generate the IgG complex, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0486] In embodiments of the methods, the immobilised AAV particle is immobilised to a solid support via the immobilised AAV capture moiety.

[0487] In embodiments of the methods, the sample comprises serum, suitably human serum or monkey serum; particularly human or cynomolgus serum.

[0488] In embodiments of the methods, contacting the first sample or portion of the first sample with an IgM protease comprises contacting the sample or portion of the sample with IgM protease; suitably wherein the IgM protease is at a concentration between about 0.4 and 1.4 U / pL, between about 0.6 and 1 .2 U / pL, or between about 0.8 and 1 .0 U / pL; for example, between about 0.8 and 1 .0 U / pL, e.g. about 0.9 U / pL.

[0489] In embodiments of the methods, contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0490] In embodiments, the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%; suitably by at least about 20%.

[0491] In embodiments of the methods, contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class is performed at a temperature of between about 4 and 25°C, between about 4 and 20°C, between about 4 and 15°C, or between about 4 and 10°C; suitably at a temperature of about 4 °C.

[0492] In embodiments of the methods, the IgM protease is an IgM specific protease, suitably having no detectable activity against polypeptides other than IgM.

[0493] In embodiments of the methods, the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0494] In embodiments of the methods, the IgM protease comprises SEQ ID NO: 2.

[0495] In embodiments of the methods, the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0496] In embodiments of the methods, contacting each sample or portion of the sample with a AAV capture moiety and a detection moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0497] In embodiments of the methods, diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises a dilution to between about 0.1 % and 2.0% (v / v) final serum concentration, such as between about 0.2 and 1.8% (v / v), between about 0.4 and 1.6% (v / v), between about 0.6 and 1 .4% (v / v), or between about 0.8 and 1 .2 % (v / v) final serum concentration; for example, between about 0.8 and 1 .2 % (v / v), e.g. about a 1 % (v / v) final serum concentration.

[0498] In embodiments of the methods, the buffer, optionally a reaction buffer, comprises Low Cross Buffer® from Candor Bioscience.

[0499] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, a AAV capture moiety, and a detection moiety; comprises an AAV particle concentration of between about 1 .0x109and 2.0x109vp / mL, between about 1 .2x109and 1 .9x109vp / mL, between about 1 .4x109and 1.8x109vp / mL, or between about 1 .6x109and 1 .7x109vp / mL; for example, between about 1.6x109and 1 .7x109vp / mL, e.g. about 1 .65x109vp / mL.

[0500] In embodiments of the methods, the AAV particle comprises rAAV2.

[0501] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, a AAV capture moiety, and a detection moiety; further comprises incubating each sample or portion of the sample with the at least one AAV particle; wherein the incubation is for up to about 3 hours, up to about 2 hours; e.g. from about 5 minutes to 1 .5 hours, about 10 minutes to 1 hour, about 15 minutes to 45 minutes; e.g. about 30 minutes; preferably wherein the incubation is performed at about room temperature.

[0502] In embodiments of the methods, the AAV capture moiety comprises a label.

[0503] In embodiments of the methods, the label comprises a biotin moiety.

[0504] In embodiments of the methods, the biotin moiety is configured to bind to the binding agent conjugated to a solid support.

[0505] In embodiments of the methods, the binding agent conjugated to a solid support comprises a streptavidin coated surface.

[0506] In embodiments of the methods, the AAV capture moiety comprises an anti-AAV particle antibody configured to bind to the at least one AAV particle.

[0507] In embodiments of the methods, the anti-AAV particle antibody is derived from a species which is different to the species from which the sample is derived.

[0508] In embodiments of the methods, the anti-AAV particle antibody is derived from mouse and the sample is derived from primate, suitably human.

[0509] In embodiments of the methods, the anti-AAV particle antibody comprises biotinylated murine monoclonal anti-AAV2 antibody A20R (e.g. from Progen).

[0510] In embodiments of the methods, the AAV capture moiety is provided at a concentration of between about 0.5 and 3 U / pL, between about 0.7 and 1.5 U / pL, or between about 0.8 and 1.0 U / pL, e.g. about 0.91 U / pL.

[0511] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, a AAV capture moiety, and a detection moiety; comprises incubating the at least one AAV particle with the AAV capture moiety; wherein the incubation is for up to about 3 hours, up to about 2 hours; e.g. from about 5 minutes to 1 .5 hours, about 10 minutes to 1 hour, about 15 minutes to 45 minutes; e.g. about 30 minutes; preferably wherein the incubation is performed at about room temperature.

[0512] In embodiments of the methods, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS. In embodiments of the methods, contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety, further comprises contacting the detection moiety with a recognition agent.

[0513] In embodiments of the methods, the detection moiety comprises an anti-IgG antibody configured to bind to the at least one anti-AAV particle IgG antibody in each sample or portion of the sample.

[0514] In embodiments of the methods, the detection moiety comprises an anti-IgM antibody configured to bind to the at least one anti-AAV particle IgM antibody in each sample or portion of the sample.

[0515] In embodiments of the methods, the anti-IgG antibody is derived from a species which is different to the species from which the sample is derived.

[0516] In embodiments of the methods, the anti-IgG antibody is derived from goat and the sample is derived from primate, suitably human.

[0517] In embodiments of the methods, the detection moiety comprises a label.

[0518] In embodiments of the methods, the label comprises digoxigenin.

[0519] In embodiments of the methods, the detection moiety is provided at a concentration of between about 100 and 1 ,000 ng / mL, between about 200 to 900 ng / mL, between about 300 to 700 ng / mL, between about 400 to 600 ng / mL, or between about 450 to 550 ng / mL; e.g. about 500 ng / mL.

[0520] In embodiments of the methods, the recognition agent comprises a fragment antigen-binding region (Fab) configured to bind to the recognition agent, suitably the label of the recognition agent, suitably digoxigenin.

[0521] In embodiments of the methods, the recognition agent comprises a label.

[0522] In embodiments of the methods, the label comprises horseradish peroxidase.

[0523] In embodiments of the methods, the recognition agent is provided at a concentration of between about 100 and 1 ,000 ng / mL, between about 200 and 900 ng / mL, between about 300 and 700 ng / mL, between about 400 and 600 ng / mL, or between about 450 and 550 ng / mL; for example, about 500 ng / mL. In embodiments of the methods, the recognition agent is provided at a concentration of between about 20 to 120 mU / mL, between about 20 to 100 mU / mL, or between about 40 to 80 mU / mL, or between about 40 to 60 mU / mL; e.g. about 50 mU / mL.

[0524] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises contacting each sample or portion of the sample with at least one AAV particle, an AAV capture moiety, and subsequently incubating the detection moiety with each contacted sample, suitably wherein the incubation is performed for up to about 6 hours, up to about 4 hours, between about 0.5 and 3 hours, between about 0.5 and 2 hours, or between about 0.5 and 1.5 hours; e.g. about 1 hour; suitably at about room temperature.

[0525] In embodiments, the methods further comprise one or more washing steps, after contacting each sample or portion of the sample with at least one AAV particle, a AAV capture moiety, and a detection moiety; prior to detecting the presence of a complex in each sample or portion of the sample; wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0526] In embodiments of the methods, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0527] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0528] In embodiments of the methods, the detection reagent comprises ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid); suitably wherein the concentration of ABTS is between about 10 and 150 pM, between about 20 and 100 pM, or between about 30 and 70 pM; for example, about 50 pM.

[0529] In embodiments of the methods, contacting the recognition agent with the detection reagent comprises incubating the IgM complex or IgG complex with the detection reagent; suitably wherein the incubation is performed for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1.5 hours; e.g. about 1 hour.

[0530] In embodiments of the methods, detecting the presence of a complex comprising: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG complex absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0531] In embodiments of the methods, the first specific wavelength is about 405 nm.

[0532] In embodiments of the methods, the second specific wavelength is about 490 nm.

[0533] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final IgG complex absorbance value.

[0534] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises using an ELISA reader to detect one or more specific wavelengths of light to obtain an IgG complex absorbance value.

[0535] In embodiments of the methods, calculating an IgG complex absorbance value comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength, suitably wherein calculating an IgG complex absorbance value comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain a final IgG absorbance.

[0536] In embodiments, the methods further comprise comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample.

[0537] In embodiments, the methods further comprise comparing the detection of the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample.

[0538] In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG complex absorbance value of the first sample to the IgG complex absorbance value of the second sample, wherein a lower IgG complex absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample. In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG complex absorbance value in the first portion of the sample to the IgG complex absorbance value in the second portion of the sample, wherein a lower IgG complex absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0539] Also disclosed herein are methods to perform Anti-AAV antibody immune complex assays for the detection of anti-AAV antibodies of the IgG class and supplementary assay for the determination of the corresponding anti-AAV antibodies of the IgM class which do not comprise the use of a detection moiety.

[0540] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0541] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0542] (b) contacting the treated sample with: at least one AAV particle, and an AAV capture moiety,

[0543] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0544] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0545] (a) contacting a first biological sample from a subject which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,

[0546] (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety; and separately

[0547] (b)(ii) contacting a second biological sample from the subject, which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample;

[0548] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without step (a).

[0549] In embodiments of the methods, the first and second biological samples are obtained a partitioning a biological sample into at least two portions.

[0550] In embodiments of the methods, the first and second biological samples are obtained separately from the same subject.

[0551] In aspects of the invention, a method for improving the detection of at least one anti-AAV particle IgG antibody in a biological sample is provided, the method comprising:

[0552] (a) partitioning a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody, into at least a first portion and a second portion,

[0553] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0554] (c) separately contacting each portion of the sample with: at least one AAV particle, and an AAV capture moiety;

[0555] (d) detecting the presence of an IgG complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without step (b). In aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample is provided, the method comprising:

[0556] (a) contacting a first biological sample from a subject, which comprises or may comprise at least one anti-AAV particle IgM antibody, with an IgM protease to obtain at least a first treated sample,

[0557] (b)(i) contacting the first treated sample with: at least one AAV particle, and an AAV capture moiety;

[0558] (b)(ii) contacting a second biological sample from the subject which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, and an AAV capture moiety;

[0559] (c) detecting the presence of an IgM complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgM antibody; and

[0560] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0561] In aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample is provided, the method comprising:

[0562] (a) partitioning a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody into at least a first portion and a second portion,

[0563] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0564] (c) separately contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety;

[0565] (d) detecting the presence of an IgM complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody; and

[0566] (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second portion relative to the first portion is indicative for the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0567] In embodiments of the methods, the methods further comprise providing a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody.

[0568] In embodiments of the methods, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody.

[0569] In embodiments of the methods, the methods further comprise providing a biological sample which comprises or may comprise at least one anti-AAV particle IgG antibody.

[0570] In embodiments of the methods, contacting each sample with: at least one AAV particle, and an AAV capture moiety; is performed separately and in a step-wise manner.

[0571] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety; comprises:

[0572] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle’ complex, and

[0573] (ii) contacting a portion of the ‘anti-AAV particle antibody ofthe IgG class-AAV particle’ complex of step (i) with an AAV capture moiety to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex, and

[0574] (i) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgG class-AAV particle- AAV capture moiety’ complex of step (ii) with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgG class-AAV particle- AAV capture moiety’, to generate the IgG complex, wherein the binding agent is configured to specifically bind the AAV capture moiety.

[0575] In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety; comprises:

[0576] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle’ complex, and

[0577] (ii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgG class-AAV particle’ complex of step (i) with an immobilised AAV capture moiety, to generate the IgG complex, wherein the AAV capture moiety is immobilised to a solid support. In embodiments of the methods, contacting each sample with: at least one AAV particle, an AAV capture moiety; comprises: contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one immobilised AAV particle, and contacting the at least one immobilised AAV particle with the anti-AAV particle antibody of the IgG class to generate the IgG complex.

[0578] In embodiments of the methods, contacting each sample with: at least one AAV particle, and an AAV capture moiety; comprises:

[0579] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex, and

[0580] (ii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgM class-AAV particle’ complex of step (i) with an AAV capture moiety to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle-AAV capture moiety’ complex, and

[0581] (iii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgG class-AAV particle- AAV capture moiety’ complex of step (ii) with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgM class-AAV particle- AAV capture moiety’, to generate the IgM complex, wherein the binding agent is configured to specifically bind the AAV capture moiety.

[0582] In embodiments of the methods, contacting each sample with: at least one AAV particle, and an AAV capture moiety, comprises:

[0583] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgM class-AAV particle’ complex, and

[0584] (ii) contacting a portion ofthe ‘anti-AAV particle antibody ofthe IgM class-AAV particle’ complex of step (i) with an immobilised AAV capture moiety, to generate the IgM complex, wherein the AAV capture moiety is immobilised to a solid support.

[0585] In embodiments of the methods, contacting each sample with: at least one AAV particle, and an AAV capture moiety; comprises: contacting the sample comprising the at least one anti-AAV particle antibody of the IgM class with an immobilised AAV particle to generate the IgM complex.

[0586] In embodiments of the methods, the immobilised AAV particle is immobilised to a solid support via the immobilised AAV capture moiety. In embodiments of the methods, the immobilised AAV capture moiety is immobilised on a solid support via a binding agent.

[0587] In embodiments of the methods, contacting the portion of the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex or ‘anti-AAV particle antibody of the IgM class- AAV particle-AAV capture moiety’ with a binding agent conjugated to a solid support to immobilise the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ or ‘anti-AAV particle antibody of the IgM class-AAV particle-AAV capture moiety’ comprises: incubating the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ complex with the binding agent conjugated to a solid support; suitably wherein the incubation is for up to about 2 hours, up to about 1 .5 hours; up to about 1 hour, up to about 30 minutes; between about 5 and 30 minutes, or between about 10 and 20 minutes; for example about 15 minutes; preferably at room temperature.

[0588] In embodiments of the methods, the methods further comprise one or more washing steps, after contacting the ‘anti-AAV particle antibody of the IgG class-AAV particle-AAV capture moiety’ or ‘anti-AAV particle antibody of the IgM class-AAV particle-AAV capture moiety’ complex with the binding agent conjugated to a solid support, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0589] In embodiments of the methods, the sample comprises serum, suitably human serum or monkey serum, suitably human or cynomolgus serum.

[0590] In embodiments of the methods, contacting the first sample or portion of the first sample with an IgM protease comprises contacting the sample or portion of the sample with IgM protease; suitably wherein the IgM protease is at a concentration between about 0.4 and 1.4 U / pL, between about 0.6 and 1 .2 U / pL, or between about 0.8 and 1 .0 U / pL; for example, between about 0.8 and 1 .0 U / pL, e.g. about 0.9 U / pL.

[0591] In embodiments of the methods, contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0592] In embodiments of the methods, the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, e.g. by at least about 20%.

[0593] In embodiments of the methods, contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the sample, first sample, or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class is performed at a temperature of between about 4 and 25°C, between about 4 and 20°C, between about 4 and 15°C, or between about 4 and 10°C; suitably at a temperature of about 4 °C.

[0594] In embodiments of the methods, the IgM protease is an IgM specific protease, suitably having no detectable activity against polypeptides other than IgM.

[0595] In embodiments of the methods, the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0596] In embodiments of the methods, the IgM protease comprises SEQ ID NO: 2.

[0597] In embodiments of the methods, the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0598] In embodiments of the methods, contacting each sample or portion of the sample with a AAV capture moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0599] In embodiments of the methods, diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises a dilution to between about 0.1 % and 2.0% (v / v) final serum concentration, such as between about 0.2 and 1.8% (v / v), between about 0.4 and 1.6% (v / v), between about 0.6 and 1 .4% (v / v), or between about 0.8 and 1 .2 % (v / v) final serum concentration; for example, between about 0.8 and 1 .2 % (v / v), e.g. about a 1 % (v / v) final serum concentration.

[0600] In embodiments of the methods, the buffer, optionally a reaction buffer, comprises Low Cross Buffer® from Candor Bioscience.

[0601] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, and a AAV capture moiety, comprises an AAV particle concentration of between about 1.0x109and 2.0*109vp / mL, between about 1.2*109and 1.9*109vp / mL, between about 1.4x109and 1 .8x109vp / mL, or between about 1.6x109and 1 .7x109vp / mL; for example, between about 1 .6x109and 1 .7x109vp / mL, e.g. about 1 .65x109vp / mL.

[0602] In embodiments of the methods, the AAV particle comprises rAAV2.

[0603] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, and a AAV capture moiety, further comprises incubating each sample or portion of the sample with the at least one AAV particle; wherein the incubation is for up to about 3 hours, up to about 2 hours; e.g. from about 5 minutes to 1.5 hours, about 10 minutes to 1 hour, about 15 minutes to 45 minutes; e.g. about 30 minutes; preferably wherein the incubation is performed at about room temperature.

[0604] In embodiments of the methods, the AAV capture moiety comprises a label.

[0605] In embodiments of the methods, the label comprises a biotin moiety.

[0606] In embodiments of the methods, the biotin moiety is configured to bind to the binding agent conjugated to a solid support.

[0607] In embodiments of the methods, the binding agent conjugated to a solid support comprises a streptavidin coated surface.

[0608] In embodiments of the methods, the AAV capture moiety comprises an anti-AAV particle antibody configured to bind to the at least one AAV particle.

[0609] In embodiments of the methods, the anti-AAV particle antibody is derived from a species which is different to the species from which the sample is derived.

[0610] In embodiments of the methods, the anti-AAV particle antibody is derived from mouse and the sample is derived from primate, suitably human.

[0611] In embodiments of the methods, the anti-AAV particle antibody comprises biotinylated murine monoclonal anti-AAV2 antibody A20R (e.g. from Progen).

[0612] In embodiments of the methods, the AAV capture moiety is provided at a concentration of between about 0.5 and 3 U / pL, between about 0.7 and 1.5 U / pL, or between about 0.8 and 1.0 U / pL, e.g. about 0.91 U / pL. In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, and a AAV capture moiety; comprises incubating the at least one AAV particle with the AAV capture moiety; wherein the incubation is for up to about 3 hours, up to about 2 hours; e.g. from about 5 minutes to 1 .5 hours, about 10 minutes to 1 hour, about 15 minutes to 45 minutes; e.g. about 30 minutes; preferably wherein the incubation is performed at about room temperature.

[0613] In embodiments of the methods, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0614] In embodiments of the methods, contacting each sample, or portion of the sample with: at least one AAV particle, and an AAV capture moiety, further comprises contacting the sample or portion of the sample with a recognition agent.

[0615] In embodiments of the methods, the recognition agent comprises an anti-IgG antibody configured to bind to the at least one anti-AAV particle IgG antibody in each sample or portion of the sample.

[0616] In embodiments of the methods, the recognition agent comprises an anti-IgM antibody configured to bind to the at least one anti-AAV particle IgM antibody in each sample or portion of the sample.

[0617] In embodiments of the methods, the anti-IgG or anti-IgM antibody is derived from a species which is different to the species from which the sample is derived.

[0618] In embodiments of the methods, the anti-IgG or anti-IgM antibody is derived from goat and the sample is derived from primate, suitably human.

[0619] In embodiments of the methods, the recognition agent comprises a label.

[0620] In embodiments of the methods, the label comprises digoxigenin.

[0621] In embodiments of the methods, the recognition agent is provided at a concentration of between about 100 and 10000 ng / mL, between about 200 to 5000 ng / mL, between about 300 to 1000 ng / mL, between about 400 to 750 ng / mL, or between about 450 to 550 ng / mL; e.g. about 500 ng / mL.

[0622] In embodiments of the methods, the recognition agent comprises a fragment antigen-binding region (Fab) configured to bind to the recognition agent, suitably the label of the recognition agent.

[0623] In embodiments of the methods, the recognition agent comprises a label. In embodiments of the methods, the label comprises horseradish peroxidase.

[0624] In embodiments of the methods, the recognition agent is provided at a concentration of between about 100 and 1 ,000 ng / mL, between about 200 and 900 ng / mL, between about 300 and 700 ng / mL, between about 400 and 600 ng / mL, or between about 450 and 550 ng / mL; for example, about 500 ng / mL.

[0625] In embodiments of the methods, the recognition agent is provided at a concentration of between about 20 to 120 mU / mL, between about 20 to 100 mU / mL, or between about 40 to 80 mU / mL, or between about 40 to 60 mU / mL; e.g. about 50 mU / mL.

[0626] In embodiments of the methods, contacting each sample or portion of the sample with at least one AAV particle, an AAV capture moiety, and a recognition agent; comprises contacting each sample or portion of the sample with at least one AAV particle, an AAV capture moiety, and subsequently incubating the recognition agent with each contacted sample.

[0627] In embodiments of the methods, the incubation of incubating the recognition agent with each contacted sample is performed for up to about 6 hours, up to about 4 hours, between about 0.5 and 3 hours, between about 0.5 and 2 hours, or between about 0.5 and 1.5 hours; e.g. about 1 hour; optionally at about room temperature.

[0628] In embodiments of the methods, the methods further comprise one or more washing steps, after contacting each sample or portion of the sample with at least one AAV particle, a AAV capture moiety, and a recognition agent; prior to detecting the presence of a complex in each sample or portion of the sample; wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0629] In embodiments of the methods, the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0630] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0631] In embodiments of the methods, the detection reagent comprises ABTS (2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid); suitably wherein the concentration of ABTS is between about 10 and 150 pM, between about 20 and 100 pM, or between about 30 and 70 pM; for example, about 50 pM. In embodiments of the methods, contacting the recognition agent with the detection reagent comprises incubating the IgM complex or IgG complex with the detection reagent; suitably wherein the incubation is performed for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1.5 hours; e.g. about 1 hour.

[0632] In embodiments of the methods, detecting the presence of a complex comprising: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG complex absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0633] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises using an ELISA reader to detect one or more specific wavelengths of light to obtain an IgG complex absorbance value.

[0634] In embodiments of the methods, the first specific wavelength is about 405 nm.

[0635] In embodiments of the methods, the second specific wavelength is about 490 nm.

[0636] In embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final IgG complex absorbance value.

[0637] In embodiments of the methods, calculating a final IgG complex absorbance value comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength, suitably wherein calculating an IgG complex absorbance value comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain a final IgG absorbance value.

[0638] In embodiments of the methods, the methods further comprise comparing the detection of the presence of the IgG complex in each sample, suitably the detection of the presence of the IgG complex in the first treated sample with the detection of the presence of the IgG complex in the second sample, or the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample. In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first sample or portion with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG complex absorbance value of the first sample to the IgG complex absorbance value of the second sample, wherein a lower IgG complex absorbance value in the first sample relative to the second sample is indicative of the presence of at least one antidrug antibody of the IgG class in the sample.

[0639] In embodiments of the methods, comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG complex absorbance value in the first portion of the sample to the IgG complex absorbance value in the second portion of the sample, wherein a lower IgG complex absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0640] In alternative aspects of the invention, a method for improving the detection of at least one anti- AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0641] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0642] (b) contacting the treated sample with: at least one AAV particle, and an AAV capture moiety,

[0643] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0644] In alternative aspects of the invention, a method for improving the detection of at least one anti- AAV particle antibody of the IgG class in a biological sample is provided, the method comprising:

[0645] (a) contacting a first biological sample from a subject which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample, (b)(i) contacting the first treated sample with: at least one AAV particle, and an AAV capture moiety; and separately

[0646] (b)(ii) contacting a second biological sample from the subject, which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample;

[0647] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without step (a).

[0648] In alternative embodiments of the methods, the first and second biological samples are obtained a partitioning a biological sample into at least two portions.

[0649] In alternative embodiments of the methods, the first and second biological samples are obtained separately from the same subject.

[0650] In alternative aspects of the invention, a method for improving the detection of at least one anti- AAV particle IgG antibody in a biological sample is provided, the method comprising:

[0651] (a) partitioning a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody, into at least a first portion and a second portion,

[0652] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0653] (c) separately contacting each portion of the sample with: at least one AAV particle, and an AAV capture moiety;

[0654] (d) detecting the presence of an IgG complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgG antibody, wherein the detection is improved compared to a method without step (b).

[0655] In alternative aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample, the method comprising:

[0656] (a) contacting a first biological sample from a subject, which comprises or may comprise at least one anti-AAV particle IgM antibody, with an IgM protease to obtain at least a first treated sample,

[0657] (b)(i) contacting the first treated sample with: at least one AAV particle, and an AAV capture moiety;

[0658] (b)(ii) contacting a second biological sample from the subject which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, and an AAV capture moiety;

[0659] (c) detecting the presence of an IgM complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgM antibody; and

[0660] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0661] In alternative aspects of the invention, a method for detecting at least one anti-AAV particle IgM antibody in a biological sample, the method comprising:

[0662] (a) partitioning a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody into at least a first portion and a second portion,

[0663] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[0664] (c) separately contacting each portion of the sample with: at least one AAV particle, and an AAV capture moiety;

[0665] (d) detecting the presence of an IgM complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle IgM antibody; and (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second portion relative to the first portion is indicative for the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0666] In alternative embodiments of the methods, the sample comprises serum, suitably monkey serum; particularly cynomolgus serum.

[0667] In alternative embodiments of the methods, contacting each portion of the sample with: at least one AAV particle, and an AAV capture moiety, further comprises contacting the sample with a recognition agent.

[0668] In alternative embodiments of the methods, the recognition agent comprises an anti-IgM antibody configured to bind to the at least one anti-AAV particle IgM antibody in each sample or portion of the sample.

[0669] In alternative embodiments of the methods, the anti-IgM antibody is derived from a species which is different to the species from which the sample is derived.

[0670] In alternative embodiments of the methods, the anti-IgM antibody is derived from goat and the sample is derived from primate, suitably human.

[0671] In alternative embodiments of the methods, the recognition agent comprises a label.

[0672] In alternative embodiments of the methods, the label comprises alkaline phosphatase.

[0673] In alternative embodiments of the methods, the recognition agent is provided at a concentration of between about 100 and 1 ,000 ng / mL, between about 200 and 900 ng / mL, between about 300 and 700 ng / mL, between about 400 and 600 ng / mL, or between about 450 and 550 ng / mL; for example, about 500 ng / mL.

[0674] In alternative embodiments of the methods, the label comprises horseradish peroxidase.

[0675] In alternative embodiments of the methods, the detection reagent comprises ABTS (2,2'-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid); suitably wherein the concentration of ABTS is between about 10 and 150 pM, between about 20 and 100 pM, or between about 30 and 70 pM; for example, about 50 pM. In alternative embodiments of the methods, the detection reagent comprises Alkaline Phosphatase Yellow (pNPP), suitably wherein the concentration of pNPP is between about 10 and 150 pM, between about 20 and 100 pM, or between about 30 and 70 pM; for example, about 50 pM.

[0676] In alternative embodiments of the methods, contacting the recognition agent with the detection reagent comprises incubating the detection moiety with the detection reagent, suitably wherein the incubation is up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0677] In alternative embodiments of the methods, detecting the presence of an IgM complex comprising: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody, and the detection moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgM complex absorbance value for each sample or portion of the sample; optionally wherein measuring an absorbance of the detection reagent to obtain an IgM complex absorbance value for each sample or portion of the sample comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0678] In alternative embodiments of the methods, the first specific wavelength is about 405 nm.

[0679] In alternative embodiments of the methods, the second specific wavelength is about 490 nm.

[0680] In alternative embodiments of the methods, detecting the presence of a complex in each sample or portion of the sample further comprises using an ELISA reader to detect one or more specific wavelengths of light.

[0681] In alternative embodiments of the methods, calculating an IgM complex absorbance value comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength, suitably wherein calculating an IgM complex absorbance value comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain a final IgM absorbance.

[0682] In alternative embodiments of the methods, comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample comprises comparing the IgM complex absorbance value of the first sample to the IgM complex absorbance value of the second sample, wherein a lower IgM complex absorbance value in the first sample relative to the second sample is indicative of the presence of at least one antidrug antibody of the IgM class in the sample.

[0683] In alternative embodiments of the methods, comparing the detection of the presence of the IgM complex in the first portion of the sample with the detection of the presence of the IgM complex in the second portion of the sample comprises comparing the IgM complex absorbance value of the first portion of the sample to the IgM complex absorbance value of the second portion of the sample, wherein a lower IgM complex absorbance value of the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0684] Further embodiments of this alternative aspect of the invention comprise the features of any of the embodiments of the previous (non-alternative) aspects of the invention.

[0685] Rheumatoid Arthritis (RA) and Rheumatoid Factor (RF)

[0686] Rheumatoid arthritis (RA) is a well-known and relatively common (affecting around 2% of the population) autoimmune condition in which self-antibodies attack the lining of joints, and surrounding tissues. This causes the thin layer of cells (synovium) covering the joints to become sore and inflamed; and the subsequent release of inflammatory cytokines and chemokines can then cause further damage to nearby tissues. Despite its frequency, the causes / triggers of RA are not fully understood; but are thought to reside in a combination of genetics, hormones (RA is more common in women), and environmental / chemical factors (e.g. smoking).

[0687] RA is a long-term condition that causes pain, swelling and stiffness in the joints. The condition usually affects the hands, feet and wrists. However, RA may also cause more general symptoms, and inflammation in other parts of the body, such as: tiredness and a lack of energy; high temperature and / or sweating; poor appetite and / or weight loss; dry eyes; and chest pain, when the heart and / or lungs are affected.

[0688] There is no cure for RA, but many different types of treatment are available to help reduce inflammation in the joints, relieve pain, prevent or slow down joint damage, and aim to reduce disability. T reatments for RA include: disease-modifying anti-rheumatic drugs (DMARDs); steroids; and biological / antibody treatments. DMARDs include, for example, methotrexate, leflunomide, hydroxychloroquine and sulfasalazine. Current biological treatments include, for example, adalimumab, etanercept and infliximab, which may be taken in combination with a DMARD. Painkillers, such as paracetamol, or a non-steroidal anti-inflammatory drug (NSAID; e.g. ibuprofen, naproxen or diclofenac) may also be prescribed. Rheumatoid factors (RFs) are autoantibodies which can be of various immunoglobulin classes, including IgM, IgG, IgA, IgD and IgE, which recognise antigenic determinants on the Fc region of IgG. Different RFs will likely recognise different parts of the IgG-Fc. The predominant form of RF is IgM. In a patient, RF and the targeted IgG join to form immune complexes that can contribute to the disease process, for example, causing chronic inflammation and joint destruction at the synovium and cartilage.

[0689] Despite its name, the presence of RF, however, does not necessarily mean that a subject has RA. Whereas RFs are found in upwards of 70% of patients with RA, RFs are also found in around 15 to 30% of patients with systemic lupus erythematosus (SLE); almost 100% of patients with Sjogrens syndrome; around 50 to 60% of patients with mixed connective tissue disease (MCTD); and about 20 to 30% of patients with systemic sclerosis, amongst others. Indeed, low levels of RF are common in patients with a wide range of autoimmune or infectious diseases especially those associated with hypergammaglobulinaemia including viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy, and pulmonary fibrosis. Furthermore, around 1 in 20 people without diagnosed RA also test positive for RF.

[0690] Having regard to the prevalence of RF in the population, therefore, adverse affects on immune complex assays performed on patient samples may be widespread and unexpected, potentially leading to a range of undesirable consequences, including misdiagnosis, inaccurate therapeutic drug monitoring and impaired immunogenicity evaluation.

[0691] Immune complex assays in samples comprising rheumatoid factor (RF)

[0692] Also disclosed herein are methods to perform antibody-based or immunological assays with improved accuracy or sensitivity; or which reduce non-specific assay signals and / or false positive results by use of an IgM specific proteases according to this disclosure. Such aspects and embodiments are particularly beneficial for providing enhanced reliability and / or confidence in data and analytics when the sample on which the assay is to be performed comprises, is suspected of comprising or may comprise rheumatoid factor (RF) or any other agent that may interfere with the antibody-based or immunological assay.

[0693] Thus, in accordance with aspects of the invention, a method or / and use is provided for improving the accuracy and / or sensitivity of an antibody-based or immunogenicity assay. The assay method may comprise: contacting a biological sample with an IgM protease to obtain a treated sample, and performing an antibody-based assay or immunogenicity assay on the treated sample.

[0694] In aspects and embodiments, the biological sample may be obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis; a subject having an autoimmune and / or infectious disease; a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[0695] Methods and uses according to these aspects and embodiments may be particularly beneficial where the biological sample has been obtained from a subject having elevated rheumatoid factor (RF), or where the biological sample may comprise or is expected to comprise elevated RF. For example, where the concentration of RF in the sample is at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL. Suitably, the sample may be blood or serum. For example, human or monkey blood or serum; particularly human or cynomolgus blood or serum; more particularly serum.

[0696] According to these aspects and embodiments, the benefits of the invention may be achieved in any method / assay / use that requires specific binding of an antibody to a target antigen, in which RF or another binding agent comprising an IgM may be present in the sample to be assayed. In embodiments, for example, the immunogenicity assay is an anti-drug antibody (ADA) assay, for example, an ADA bridging assay. In particular aspects and embodiments, the benefits of the invention may be realised, for example, when the subject from which the biological sample to be assayed has not previously been exposed to the drug that is the subject of the anti-drug antibody assay. In embodiments, the drug is an antibody. In various aspects and embodiments, the immunogenicity assay may be a multiplex immunoassay. In aspects and embodiments, the immunogenicity assay may be an ELISA assay. Indeed, any of the assays described throughout this disclosure may be performed in accordance with these aspects and embodiments.

[0697] Aspects and embodiments of these methods may beneficially be used for therapeutic drug monitoring, immunogenicity evaluation or disease diagnosis.

[0698] The IgM protease may be according to any of the aspects and embodiments described elsewhere herein; and may be used as described elsewhere herein.

[0699] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis, the method according to any of aspects and embodiments described throughout this disclosure.

[0700] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having an autoimmune and / or infectious disease, the method according to any of aspects and embodiments described throughout this disclosure.

[0701] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis, the method according to any of aspects and embodiments described throughout this disclosure.

[0702] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample obtained from a subject having elevated rheumatoid factor (RF), the method according to any of aspects and embodiments described throughout this disclosure.

[0703] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising rheumatoid factor (RF), the method according to any of aspects and embodiments described throughout this disclosure.

[0704] In further aspects and embodiments, there is provided a method for improving the accuracy or sensitivity of an antibody-based or immunogenicity assay in a biological sample comprising at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL rheumatoid factor (RF), the method according to any of aspects and embodiments described throughout this disclosure.

[0705] In further aspects and embodiments, there is provided a method for reducing non-specific signal and / or reducing false positive signals in an antibody-based or immunological assay of a biological sample, wherein the biological sample comprises or is expected to comprise rheumatoid factor (RF), the method according to any of aspects and embodiments described throughout this disclosure. In aspects and embodiments, the biological sample may have been obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis. In aspects and embodiments, the biological sample may have been obtained from a subject having an autoimmune and / or infectious disease. In aspects and embodiments, the biological sample may have been obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis. EXAMPLES

[0706] Materials and Methods

[0707] Assay for the detection of pre-existing anti-mAb1 antibodies

[0708] The following method describes an ADA assay for the detection of pre-existing anti-mAb1 antibodies in serum samples of human individual donors.

[0709] All incubation steps were performed for 1 hour at room temperature with shaking at 450 rpm except where indicated otherwise.

[0710] 70 pL of individual sera of healthy human donors were mixed together with 7 pL of 10 U / pL IgMBRAZOR (“treated”) or 7 pL of 1x phosphate-buffered saline (PBS; Roche Diagnostics GmbH) (“untreated”) and incubated overnight (16 hours). Subsequently, all samples were diluted 50-fold using 1x PBS containing 1 % (w / v) bovine serum albumin (BSA, Merck Chemicals GmbH) and 0.05% (v / v) Tween20 (Roche Diagnostics GmbH) (assay buffer) together with 1000 ng / mL mAb1- Biotin and 1000 ng / mL mAb1-digoxigenin and incubated for 2 hours.

[0711] Formed immune complexes were transferred to a streptavidin-coated microtiter plate (MTP) and incubated to immobilize immune complexes via the Biotin-labelled capture antibody. After three washing step each using 300 pL 1x PBS containing 0.05% (v / v) Tween20, 100 pL / well assay buffer containing 25 mU / mL horseradish peroxidase-labelled anti-digoxigenin Fab (Roche Diagnostics GmbH) were added to the MTP followed by incubation.

[0712] After three washing steps, the substrate reaction was executed by adding 100 pL / well 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) solution (ABTS; Roche Diagnostics GmbH) and the optical density was measured at a wavelength of 405 nm (reference wavelength at 490 nm) on a microplate reader (Sunrise; Tecan) after 10 minutes incubation time. The final absorbance of each analyzed sample was calculated by subtraction of the absorbance at 490 nm from the absorbance at 405 nm.

[0713] Assay for the determination of the corresponding anti-mAb1 isotype (IgM)

[0714] The following method describes an assay for the detection of pre-existing IgM-type anti-mAb1 antibodies.

[0715] To the wells of a MaxiSorp MTP (Fisher Scientific GmbH) 100 pL / well of 2000 ng / mL mAb1 diluted in 1x PBS was added and incubated. After three washing steps, the MTP was blocked by adding 250 pL / well LowCross buffer (CANDOR Bioscience GmbH) followed by incubation. All previously prepared treated and untreated samples (Example 1.A above) were diluted 100-fold using LowCross buffer and added to the MTP using 100 pL / well followed by incubation. After three washing steps, 100 pL / well of 500 ng / mL anti-IgM, mouse origin (first detection reagent) diluted in LowCross buffer was added to the MTP followed by incubation. After three washing steps, 100 pL / well of 2000-fold diluted horseradish peroxidase-labeled anti-mouse antibody (second detection reagent) diluted in LowCross buffer was added to the MTP followed by incubation and three washing steps. For the substrate reaction, the same procedure was followed as for the anti-drug antibody bridging assay for detection of pre-existing anti-mAb1 antibodies outlined in Example 1 .A. As a negative control, the assay is performed with 1x PBS (without mAb1 addition) at the initial MTP coating step.

[0716] Assay for the determination of the corresponding anti-mAb1 isotype (IgG)

[0717] The following method describes an assay for the detection of pre-existing IgG-type anti-mAb1 antibodies.

[0718] The same procedure was followed as for the assay for detection of pre-existing IgM-type anti- mAb1 antibodies described before, except using 100 pL / well of 500 ng / mL CD64-digoxigenin as first detection reagent and 100 pL / well of 50 mU / mL horseradish peroxidase-labelled anti- digoxigenin Fab fragments as second detection reagent.

[0719] Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgG class

[0720] The following method describes an assay for resolving the IgM interference in an anti-AAV antibody immune complex assay for the detection of anti-AAV antibodies of the IgG class by demasking of AAV particles covered by (co-existing) anti-AAV antibodies of the IgM class. The surface of AAV particles becomes accessible for binding of anti-AAV antibodies of the IgG class by IgM-specific digestion / cleavage.

[0721] Cynomolgus serum samples to be analysed for anti-AAV antibodies of the IgG class were either treated or not treated prior to analysis in the assay.

[0722] For treatment, 10 pl serum samples were mixed with 1 pl of a 10 U / pL IgMBRAZOR dilution and incubated over night at 4 °C. Forthe control group (untreated), 10 pl of serum samples were mixed with 1 pl Low Cross buffer and incubated at 4 °C.

[0723] After pretreatment, all samples (treated and untreated) were diluted with Low Cross Buffer® to a final serum concentration of 1 % (v / v), spiked with capsid particles (rAAV2 or dosing material for the analysis of the cynomolgus study samples) at a concentration of 1.65*10E+09 vp / mL and incubated for 30 min at room temperature. Thereafter, a capture antibody (biotinylated murine monoclonal anti-AAV2 antibody A20R, 0.1 pg) was added to the samples, the samples were incubated for 30 min at room temperature and then transferred onto a SA-MTP. The microtiter plate was then incubated for 15 min at room temperature and washed with PBS / 0.05% Tween. A detection antibody (HRP-labelled goat anti-human IgG antibody, 0.5 pg / mL) was added to the plate and allowed to bind to the captured immune complexes of the rAAV2p and the anti-AAV2 antibody of the IgG class for 1 h at room temperature. The plate was washed with PBS / 0.05% Tween, and the bound immune complexes were detected by colour reaction with ABTS (50 pM) using an ELISA reader.

[0724] Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgM class

[0725] The following method describes an assay for resolving the IgM interference in an anti-AAV antibody immune complex assay for the detection of anti-AAV antibodies of the IgG class by demasking of AAV particles covered by (co-existing) anti-AAV antibodies of the IgM class. The surface of AAV particles becomes accessible for binding of anti-AAV antibodies of the IgG class by IgM-specific digestion / cleavage.

[0726] Cynomolgus serum samples to be analyzed for anti-AAV antibodies of the IgM class were either treated or not treated prior to analysis in the assay.

[0727] For treatment, 10 pl serum samples were mixed with 1 pl of a 10 U / pL IgMBRAZOR dilution and incubated over night at 4 °C. Forthe control group (untreated), 10 pl of serum samples were mixed with 1 pl LowCross buffer and incubated at 4°C.

[0728] After pretreatment, all samples (treated and untreated) were diluted with Low Cross Buffer® to a final serum concentration of 1 % (v / v), spiked with capsid particles (rAAV2 or dosing material for the analysis of the cynomolgus study samples) at a concentration of 1.65*10E+09 vp / mL and incubated for 30 min at room temperature. Thereafter, a capture antibody (biotinylated murine monoclonal anti-AAV2 antibody A20R, 0.1 pg) was added to the samples, the samples were incubated for 30 min at room temperature and then transferred onto a SA-MTP. The microtiter plate was then incubated for 15 min at room temperature and washed with PBS / 0.05% Tween. A detection antibody (alkaline phosphatase-labelled goat anti-human IgM, 0.5 pg / mL) was added to the plate and allowed to bind to the captured immune complex of the rAAV2p and the anti-AAV2 antibody of the IgM class for 1 h at room temperature. The plate was washed with PBS / 0.05% Tween, and the bound immune complexes were detected by colour reaction with Alkaline Phosphatase Yellow (pNPP) (Sigma Aldrich) using an ELISA reader.

[0729] ADA bridging assay for anti-drug X antibodies - samples from patients with rheumatoid arthritis

[0730] Human serum samples (70 pL) were mixed with either 7 pL of the IgM cleaving protease (10 U / pL) or 7 pL PBS and incubated overnight at room temperature for 16 h (the amount of protease was based on manufacturer’s instructions and anticipated total IgM levels as per Ritchie et al., (1998), J. Clin. Lab. Anal. 12, 371-377. Thereafter, the samples were diluted 10-fold with assay buffer (Roche universal buffer), biotinylated Drug X and digoxigenin-labeled Drug X were added (1 pg / mL each) and the samples were incubated at room temperature for 2 h. The samples were transferred onto a SA-MTP (100 pL / well) and the plate was incubated and then washed. HRP-conjugated anti- digoxigenin Fab fragments were added to each well (25 mU / mL in assay buffer; 100 pL / well) and the plate was incubated and then washed again. ABTS was added and colorimetric signals at 405 nm (reference 490 nm) were measured using a microplate reader (Sunrise, Tecan, Mannedorf, Switzerland).

[0731] In both steps, the plate was incubated at room temperature with shaking (450 rpm) for 1 h and washed 3 times with PBS containing 0.05% v / v Tween 20.

[0732] Human IgG against Drug X (10 pg / mL in PBS) was used as a positive control. Pooled human serum from healthy donors was used as a negative control.

[0733] The assay format is schematically shown in Figure 1.

[0734] IgG- and IgM-specific assays for anti-drug X antibodies - samples from patients with rheumatoid arthritis)

[0735] Human serum samples were pretreated as described for the bridging anti-Drug X antibody assay from patients with rheumatoid arthritis and then diluted 100-fold with LowCross-Buffer®. A MaxiSorp plate was coated with unlabeled Drug X (2 pg / mL Drug X in PBS, 100 pL / well, incubated at room temperature with shaking [450 rpm] for 1 h). After washing, the plate was blocked with LowCross-Buffer® (incubated with 250 pL / well) and the diluted samples were added to the plate (100 pL / well). The plate was incubated and washed, and then the first detection reagent (‘second drug moiety’) was added. After incubation and washing, the second detection reagent ("recognition agent’) was added, and the plate was incubated and then washed again. Signal detection was performed using ABTS as described for the bridging anti-Drug X antibody assay.

[0736] For the IgG-specific assay, FcyRI-digoxigenin was used as the first detection reagent (0.5 pg / mL in LowCross-Buffer®, 100 pL / well) and HRP-conjugated anti-digoxigenin Fab fragments were used as the second detection reagent (50 mU / mL, 100 pL / well).

[0737] For the IgM-specific assay, mouse anti-human IgM antibody was used as the first detection reagent (0.5 pg / mL in LowCross-Buffer®, 100 pL / well) and HRP-labeled anti-mouse antibody was used as the second detection reagent (400 ng / mL, 100 pL / well).

[0738] In all steps, plate incubation and washing were performed as described for the bridging anti-Drug X antibody assay. Human IgG against Drug X (10 pg / mL in PBS) was used as a positive control. Pooled human serum from healthy donors was used as a negative control.

[0739] The assay formats are schematically shown in Figure 2.

[0740] ADA bridging assay for anti-drug Y antibodies - samples from healthy donors

[0741] Sample pretreatment and analysis were performed as described for the bridging anti-Drug X antibody assay with samples from rheumatoid arthritis patients, with the following modifications: PBS containing 1 % w / v BSA and 0.05% v / v Tween 20 was used as assay buffer and the samples were diluted 50 fold; biotinylated Drug Y and digoxigenin-labeled Drug Y (1 pg / mL each) were used as capture and detection antibodies.

[0742] IgG- and IgM-specific assays for anti-drug Y antibodies - samples from healthy donors

[0743] Sample pretreatment and analysis were performed as described for anti-Drug X antibodies with unlabeled Drug Y from patients with rheumatoid arthritis (2 pg / mL) used for coating.

[0744] Example 1 : Anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti- mAb1 antibodies - Anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti-mAb1 antibodies

[0745] The following described immunological assays were used to assess the impact of IgM-specific enzymatic cleavage of ADAs in human serum samples using IgMBRAZOR. Due to the specificity of IgMBRAZOR towards IgM with no impact on other Ig isotypes such as IgG, a signal reduction after enzymatic treatment could be used for confirmation of presence of IgM in the sample (e.g. proof of IgM response in immunogenicity testing).

[0746] The assay was performed according to the method ’’Assay for the detection of pre-existing anti- mAb1 antibodies” described above.

[0747] The results of this assay and analysis are depicted in Table 2 and Figure 4A, wherein the signal is substantially reduced from the IgM protease ‘untreated’ to the ‘treated’ condition in Donor nos. 1 to 6, indicative of the presence of IgM antibodies in these sample, and successful degradation of these antibodies by IgM protease treatment. Notably, Donor 8 did not exhibit a high signal in the ‘untreated’ condition, indicative of low IgM present in the sample, supported by little to no observable change in signal in response to IgM protease treatment. Table 2: Absorbance data from ADA assay for the detection of pre-existing anti-mAb1 antibodies

[0748] A schematic overview of the described assay is shown in Figure 1. Example 2: Anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti- mAb1 antibodies - Supplementary assays for the determination of the corresponding anti- mAb1 isotype (IgM)

[0749] The assay was performed according to the method “Assays for the determination of the corresponding anti-mAb1 isotype (IgM)” as described above.

[0750] A schematic overview of the described assay is shown in Figure 2.

[0751] The results of the analysis using the assays described in Examples 2 (IgM) and 3 (IgG) are depicted in Table 3 and summarised in Figure 4B and 4C, wherein the signal is substantially reduced from the IgM protease ‘untreated’ to the ‘treated’ condition in Donor nos. 1 to 6, indicative of the presence of IgM antibodies in these sample, and successful degradation of these antibodies by IgM protease treatment. Notably, Donors 7 and 8 did not exhibit a high signal in the IgM ‘untreated’ condition, indicative of low IgM present in the sample, supported by little to no observable change in response to IgM protease treatment. Little to no observable change in signal was observed in response to IgG treatment, supporting the IgM target specificity of the IgM protease IgMBRAZOR. Table 3: Results of ADA assay for the detection of pre-existing anti-mAb1 antibodies and determination of ADA isotype Example 3: Anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti- mAb1 antibodies - Supplementary assays for the determination of the corresponding anti- mAb1 isotype (IgG)

[0752] The assay was performed according to the method “Assays for the determination of the corresponding anti-mAb1 isotype (IgG)” as described above.

[0753] A schematic overview of the described assay is shown in Figure 3. The results of the analysis using the assays described in Examples 2 and 3 (detection of preexisting IgM-type or IgG-type anti-mAb1 antibodies) are depicted in Table 2 above.

[0754] Example 4: Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgG class

[0755] The assay was performed according to the method “Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgG class” as described above.

[0756] A schematic overview of the described assay is shown in Figure 5.

[0757] The results of the analysis using the assays described in Examples 4 (anti-AAV antibodies of the IgG class) and Example 5 (anti-AAV antibodies of the IgM class) are depicted in Table 4. As can be seen in Table 4, the signal in the IgM untreated condition for all animals generally increases from day 1 to day 12, plateauing around 12 to 28, before decreasing from around day 28 to day 56, and returning to or towards signal levels observed on day 1 around day 84. In contrast, the signal in the IgM treated condition does not exhibit a comparatively substantial increase from day 1 at any of the measured timepoints, indicative that a substantial portion / the majority of the signal in the IgM untreated condition relates to an increase in IgM, e.g. IgM ADAs.

[0758] With respect to the results for IgG untreated condition, all animals exhibit a substantial increase of signal, increasing dramatically at day 56 reaching a maximum around day 84. The IgG treated condition exhibits similar results, however, a lower baseline reading is observed, which may indicate the reduction of false positive signal; the increased signal is observed earlier compared to the untreated condition, e.g. by 8-12 hours, (particular examples are shown by means of shaded cells) and generally the signal is greater across the majority of the timepoints as in the treated condition vs the untreated condition, wherein the maximum signal is also improved in the treated condition. The results of table 4 clearly demonstrate improved sensitivity of the assay, resulting in earlier detection of IgG and greater detectable signal in the assay, indicating that the presence of IgM antibodies was reducing assay sensitivity, and that IgM protease treatment removes IgM and increases sensitivity. Furthermore, the assay comprising IgM protease treatment clearly evidences an IgM response which can be discriminated from the IgG response.

[0759] Table 4: Anti-AAV IgG immune complex assay results for animals 1 , 2, and 3

[0760] Example 5: Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgM class

[0761] The assay was performed according to the method “Anti-AAV immune complex assay for the specific detection of anti-AAV antibodies of the IgM class” as described above.

[0762] A schematic overview of the described assay is shown in Figure 6.

[0763] Example 6: Activity of IgM protease IgMBRAZOR

[0764] Methods of assessing IgM protease activity were performed as described in PCT / EP2024 / 065123 and GB2308197.9.

[0765] Briefly, IgMBRAZOR was separately incubated with human serum IgG, IgA, and IgM. As can be seen in Figure 10, IgMBRAZOR is active against IgM, and does not exhibit IgA or IgG-specific activity.

[0766] Additionally, to investigate the site of hydrolysis Myeloma IgM was deglycosylated with PNGaseF and incubated with IgMBRAZOR. As can be seen from figure 11 , only the heavy chain was hydrolyzed, while the light chain and the J-chain remained intact. The intact mass sum of the deglycosylated heavy chain fragment (63140.6167 Da) aligned well with the measured mass of the reduced IgM heavy chain (63140.9971 Da). Upon addition of IgMBRAZOR, the heavy chain was fragmented into m / z 37499.6142 and m / z 25659.0200. The C-terminal fragment (25659.0200 Da) could be assigned to the theoretical mass value of amino acids 221-453 (25657.6646 Da) of human IgM constant region (UniProt accession: P01871). The results shown in Figure 11 indicate that digestion takes place below the CH2 region of human IgM (...VPDQDT / AIRVFA...), similar to the porcine IgM protease IdeSSuis. The native reaction leaves F(ab’)2 (VH-CH1-CH2) and a pentameric Fc (CH3-CH4), due to the inter-monomer disulphide bonds between cysteine 413 residues in CH3.

[0767] Example 7: Anti-drug antibody (ADA) bridging assay for the detection of and compensation for rheumatoid factor (RF)

[0768] In this Example, the utility of IgM-specific proteolytic cleavage for the improvement of routine immunogenicity assays was tested. Rheumatoid factor, mostly IgM, can interfere with conventional immunogenicity assays for monoclonal antibodies (mAbs) and Fc-fusion proteins, leading to increased assay signals and false positive results (Figure 13). Assays were therefore performed to determine whether the IgM-specific protease could mitigate rheumatoid factor (RF) interference, which has been found to provide a challenge for various immunoassays, both bioanalytical and clinical, and is a significant regulatory concern.

[0769] The assay was performed according to the method “ADA bridging assay for anti-drug X antibodies - samples from patients with rheumatoid arthritis” described above.

[0770] In view of the specificity of the IgM protease (IgMBRAZOR) towards IgM with no impact on other Ig isotypes such as IgG, a signal reduction after enzymatic treatment could be used for confirmation of the presence of IgM in the sample (e.g. proof of IgM response in immunogenicity testing).

[0771] The results of this assay and analysis are depicted in Table 5 and Figure 14A, wherein the signal is substantially reduced from the IgM protease ‘untreated’ to the ‘treated’ condition in Donor nos. 1 to 5 (including the ‘pool’), indicative of the presence of IgM antibodies in these sample, and successful degradation of these antibodies by IgM protease treatment. Notably, the sample from a ‘healthy’ donor did not exhibit a high signal in the ‘untreated’ condition, indicative of low IgM present in the sample, supported by little to no observable change in signal in response to IgM protease treatment. Similarly, the positive control sample containing IgG did not show any change in signal from IgM protease treatment.

[0772] In particular, this assay demonstrates that significant IgM binding can be observed for samples taken from patients with rheumatoid arthritis in a bridging assay for ADAs against mAb Drug X (Figure 14A; Table 5). Since these patients were not exposed to Drug X and no signals were seen in a serum pool from healthy donors, this finding indicates signal generation caused by RF interference and not by ADAs. Protease treatment abolished assay signals in all patient samples but not in the positive control (human anti-Drug X IgG), demonstrating that the signals in patient samples originated from IgM antibodies. Example 8: Anti-drug antibody (ADA) bridging assay for the detection of and compensation for rheumatoid factor (RF) - Supplementary assays for the determination of the corresponding anti-mAb1 isotype (IgM)

[0773] The assay was performed according to the method “IgG- and IgM-specific assays for anti-drug X antibodies - samples from patients with rheumatoid arthritis” as described above.

[0774] The results of the assays are depicted in Table 5 below and summarised in Figure 14B.

[0775] The data show that the signal is substantially reduced in the IgM protease ‘treated’ samples (compared to ‘untreated’) in all of Donor nos. 1 to 5 (including the ‘pool’), indicative of the presence of IgM antibodies in these sample, and successful degradation of these antibodies by IgM protease treatment. Notably, the sample from a ‘healthy’ donor did not exhibit a high signal in the ‘untreated’ condition, indicative of low IgM present in the sample, and the signal was insignificant in the IgM ‘treated’ assay.

[0776] As expected, essentially no signal was measured in the IgG control assay specific for detection of IgM.

[0777] This IgM ADA assay data further confirms the presence of IgM / RF-based interference, with only patient samples showing strong and protease-sensitive signals (Figure 14B). As in the bridging assay, IgM digestion suppressed the elevated signal with no effect on the positive control.

[0778] Example 9: Anti-drug antibody (ADA) bridging assay for the detection of and compensation for rheumatoid factor (RF) - Supplementary assays for the determination of the corresponding anti-mAb1 isotype (IgG)

[0779] The assay was performed according to the method “IgG- and IgM-specific assays for anti-drug X antibodies - samples from patients with rheumatoid arthritis” as described above.

[0780] The results of the assays are depicted in in Table 5 below and summarised in Figure 14C.

[0781] The data demonstrate, as expected that treatment with the IgM protease does not have any impact on the expected low level signal in the samples from Donor nos. 1 , 2, 3 and 5; and in the ‘pool’ sample, indicative of the lack of IgG antibodies in these sample. The signals from the ‘healthy’ subject were similarly negligible; and the positive IgG control gave a high signal which was not affected by the presence of IgM protease. The sample from Donor 4 gave a relatively high signal in the ‘untreated’ assay condition, which was substantially reduced with addition of IgM protease, suggestive of cross-reactivity of Igs in this sample.

[0782] Thus, interference was observed in one patient sample in the IgG-specific assay (Donor 4). Lack of an effect on anti-Drug X IgG in both assays confirms IgM-specificity of the protease and demonstrates that IgM digestion has no impact on assay performance. IgM digestion can thus eliminate RF interference in bridging and IgG assays with no effect on IgG ADA determination (Figure 14C).

[0783] Table 5: Absorbance data from ADA bridging in samples from patients with rheumatoid arthritis

[0784] Sample Signal, AU

[0785] Bridging assay IgM assay IgG assay

[0786] Untreated Treated Untreated Treated Untreated Treated

[0787] Donor 1 0.740 0.079 1.804 0.036 0.033 0.026

[0788] Donor 2 0.839 0.079 1.875 0.077 0.061 0.027

[0789] Donor 3 0.779 0.077 1.650 0.036 0.056 0.036

[0790] Donor 4 0.189 0.078 1.904 0.274 0.865 0.105

[0791] Donor 5 0.272 0.095 1.819 0.035 0.067 0.034

[0792] RA pool 0.676 0.079 2.003 0.346 0.147 0.037

[0793] Healthy pool 0.084 0.081 0.111 0.020 0.022 0.023

[0794] IgG 1.063 1.059 0.018 0.018 1.278 1.284

[0795] Schematic overviews of the described assays are shown in Figures 1 and 2.

[0796] CLAUSES

[0797] Clause Group A: First embodiments of Example 1:

[0798] Anti-drug antibody (ADA) bridging assay for the detection of pre-existing anti-mAb 1 antibodies and supplementary assays for the determination of the corresponding ADA isotype (IgM or IgG).

[0799] A1 . A method for detecting at least one anti-drug antibody of the IgM class in a biological sample, the method comprising:

[0800] (a) contacting a first biological sample from a subject which comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,

[0801] (b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately

[0802] (b)(ii) contacting a second biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;

[0803] (c) detecting the presence of an IgM complex in each of the first and second samples, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0804] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0805] A2. The method according to clause A1 , wherein the first and second biological samples are obtained by partitioning a biological sample into at least two portions.

[0806] A3. The method according to clause A1 , wherein the first and second biological samples are obtained separately from the same subject.

[0807] A4. A method for detecting at least one anti-drug antibody of the IgM class in a biological sample, the method comprising:

[0808] (a) partitioning a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class into at least a first portion and a second portion,

[0809] (b) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0810] (c) separately contacting each portion of the sample with: a first drug moiety, and a second drug moiety;

[0811] (d) detecting the presence of an IgM complex in each portion of the sample, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0812] (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the biological sample. A5. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0813] (a) contacting a first biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a first treated sample,

[0814] (b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately

[0815] (b)(ii) contacting a second biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;

[0816] (c) detecting the presence of an IgG complex in the first treated and second samples, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0817] (d) comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample wherein the detection is improved compared to a method without step (a).

[0818] A6. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0819] (a) contacting a biological sample from a subject, wherein the biological sample comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0820] (b) contacting the treated sample with: a first drug moiety, and a second drug moiety;

[0821] (c) detecting the presence of an IgG complex in the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0822] A7. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class.

[0823] A8. The method according to any one of clauses A1 to A6, further comprising providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class.

[0824] A9. The method according to any one of clauses A1 to A6, further comprising providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgG class.

[0825] A10. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0826] (a) providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class;

[0827] (b) partitioning the sample into at least a first portion and a second portion,

[0828] (c) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0829] (d) separately contacting the first portion and the second portion of the sample with: a first drug moiety, and a second drug moiety;

[0830] (e) detecting the presence of an IgG complex in each portion of the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0831] (f) comparing the detection of the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample.

[0832] A11 . The method according to any of the preceding clauses, wherein contacting either the sample, each sample, or each portion of the sample, with a first drug moiety and a second drug moiety comprises contacting the sample, each sample, or each portion of the sample, with a first drug moiety and subsequently with a second drug moiety in a step-wise manner.

[0833] A12. The method according to any of the preceding clauses, wherein the sample comprises serum; suitably human or monkey serum; suitably human or cynomolgus serum.

[0834] A13. The method according to any of the preceding clauses, wherein contacting at least the first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0835] A14. The method according to any one of clauses A1 to A4, A7 to A9, A11 , or A12 when dependent on clause A1 or clause A4, wherein the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%; suitably by at least about 20%.

[0836] A15. The method according to any of the preceding clauses, wherein the IgM protease is an IgM specific protease, suitably having no detectable activity against polypeptides other than IgM.

[0837] A16. The method according to any of the preceding clauses, wherein the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0838] A17. The method according to any of the preceding clauses, wherein the IgM protease comprises SEQ ID NO: 2.

[0839] A18. The method according to any of the preceding clauses, wherein the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0840] A19. The method according to any of the preceding clauses, wherein contacting the sample, first sample, or the first portion of the sample with an IgM protease comprises contacting the sample, first sample, orthe first portion of the sample with IgM protease at a concentration between about 0.2 to 10 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1.0 U / pL; e.g. about 0.91 U / pL. A20. The method according to any of the preceding clauses, wherein contacting each sample or portion of the sample with a first drug moiety and a second drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0841] A21. The method according to any of clauses A13 to A20 when dependent on clause A12, wherein diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises diluting each sample or portion of the sample to a final serum concentration of between about 10% to 0.1 %, such as between about 7.5% to 0.5%, between about 5% to 1 %, or between about 2.5% to 1 .5%; e.g. about 2%.

[0842] A22. The method according to clause A21 , wherein the buffer, optionally a reaction buffer, comprises 1% (w / v) bovine serum albumin and / or 0.05% (v / v) Tween20 in PBS.

[0843] A23. The method according to any of clauses A1 to A4, A7 to A9, or A11 to A22 when dependent on clause A1 or clause A4, wherein the at least one anti-drug antibody of the IgM class specifically binds to the first drug moiety.

[0844] A24. The method according to any of clauses A1 to A4, A7 to A9, or A11 to A23 when dependent on clause A1 or clause A4, wherein the first drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgM class.

[0845] A25. The method according to clause A5, or any of clauses A6 to A22, when dependent on any of clauses A5, A6, or A10, wherein the at least one anti-drug antibody of the IgG class specifically binds to the first drug moiety.

[0846] A26. The method according to clause A5, or any of clauses A6 to A22 or A25 when dependent on any of clauses A5, A6, or A10, wherein the first drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgG class.

[0847] A27. The method according to any of the preceding clauses, wherein the antibody is derived from a species which is different to the species from which the biological sample is derived.

[0848] A28. The method according to any of the preceding clauses, wherein the first drug moiety comprises a label.

[0849] A29. The method according to clause A28, wherein the label comprises a biotin moiety. A30. The method according to any of the preceding clauses, further comprising providing a solid support comprising a binding agent.

[0850] A31 . The method according to clause A30 when dependent on clause A29, wherein the binding agent of the solid support is configured to bind the biotin moiety of the label.

[0851] A32. The method according to clause A30 or A31 , wherein the binding agent of the solid support comprises streptavidin; for example, a streptavidin coated surface.

[0852] A33. The method according to any of the preceding clauses, wherein the first drug moiety is provided at a concentration of between about 100 to 10,000 ng / mL, such as between about 250 to 7,500 ng / mL, between about 500 to 5,000 ng / mL, between about 750 to 2,500 ng / mL, or between about 800 to 1 ,250 ng / mL; e.g. between about 900 to 1 ,100 ng / mL, e.g. 1 ,000 ng / mL.

[0853] A34. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, or A27 to A33 when dependent on clause A1 or clause A4, wherein the second drug moiety specifically binds to the at least one anti-drug antibody of the IgM class.

[0854] A35. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, or A27 to A34 when dependent on clause A1 or clause A4, wherein the second drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgM class.

[0855] A36. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33 when dependent on any of clauses A5, A6, or A10, wherein the second drug moiety specifically binds to the at least one anti-drug antibody of the IgG class.

[0856] A37. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33 when dependent on any of clauses A5, A6, or A10, wherein the second drug moiety comprises an antibody configured to bind to the at least one anti-drug antibody of the IgG class.

[0857] A38. The method according to any of the preceding clauses, wherein the antibody is derived from a species which is different to the species from which the biological sample is derived.

[0858] A39. The method according to any of the preceding clauses, wherein the second drug moiety comprises a label.

[0859] A40. The method according to clause A39, wherein the label comprises a digoxigenin moiety. A41 . The method according to any of the preceding clauses, wherein the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

[0860] A42. The method according to any of the preceding clauses, wherein detecting the presence of an IgM or IgG complex in each sample or portion of the sample comprises contacting the IgM or IgG complex with the solid support.

[0861] A43. The method according to clause A42, wherein contacting the IgM or IgG complex with the solid support further comprises incubating the IgM or IgG complex with the solid support, suitably wherein the first drug moiety binds to the solid support.

[0862] A44. The method according to any of the preceding clauses, wherein detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a recognition agent configured to specifically bind to the second drug moiety, suitably wherein the recognition agent is configured to specifically bind to the label of the second drug moiety.

[0863] A45. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, A27 to A35, or A38 to A44 when dependent on clause A1 or clause A4, wherein contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0864] (i) contacting the sample comprising the at least one anti-drug antibody of the IgM class with the first drug moiety, to generate an ‘anti-drug antibody of the IgM class- first drug moiety’ complex; and

[0865] (ii) contacting a portion of the ‘anti-drug antibody of the IgM class-first drug moiety’ complex from step b(i) with the second drug moiety to generate the IgM complex.

[0866] A46. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, A27 to A35, or A38 to A44 when dependent on clause A1 or clause A4, wherein contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0867] (i) contacting the sample comprising the at least one anti-drug antibody of the IgM class with the second drug moiety, to generate an ‘anti-drug antibody of the IgM class-second drug moiety’ complex; and

[0868] (ii) contacting a portion of the ‘anti-drug antibody of the IgM class-second drug moiety’ complex from step (i) with the first drug moiety to generate the IgM complex. A47. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, A27 to A35, or A38 to A44 when dependent on clause A1 or clause A4, wherein detecting the presence of an IgM complex in each sample further comprises:

[0869] (i) contacting a portion of the IgM complex corresponding to the first drug moiety with the binding agent of the solid support to immobilise the IgM complex, wherein the binding agent is configured to specifically bind the first drug moiety; and

[0870] (ii) contacting a portion of the immobilised IgM complex corresponding to the second drug moiety with the recognition moiety.

[0871] A48. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33, or A36 to A44 when dependent on any of clauses A5, A6, or A10, wherein contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0872] (i) contacting the sample comprising the at least one anti-drug antibody of the IgG class with the first drug moiety, to generate an ‘anti-drug antibody of the IgG class- first drug moiety’ complex, and

[0873] (ii) contacting a portion of the ‘anti-drug antibody of the IgG class-first drug moiety’ complex from step (i) with the second drug moiety to generate the IgG complex.

[0874] A49. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33, A36 to A44, or A48 when dependent on any of clauses A5, A6, or A10, wherein contacting each portion of the sample with: a first drug moiety, and a second drug moiety; further comprises:

[0875] (i) contacting the sample comprising the at least one anti-drug antibody of the IgG class with the second drug moiety, to generate an ‘anti-drug antibody of the IgG class-second drug moiety’ complex, and

[0876] (ii) contacting a portion of the ‘anti-drug antibody of the IgG class-second drug moiety’ complex from step (i) with the first drug moiety to generate the IgG complex.

[0877] A50. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33, A36 to A44, A48, or A49, when dependent on any of clauses A5, A6, or A10, wherein detecting the presence of an IgG complex in each sample further comprises:

[0878] (i) contacting a portion of the IgG complex corresponding to the first drug moiety with the binding agent of the solid support to immobilise the IgG complex, wherein the binding agent is configured to specifically bind the first drug moiety; and

[0879] (ii) contacting a portion of the immobilised IgG complex corresponding to the second drug moiety with the recognition moiety. A51 . The method according to any of the preceding clauses, wherein detecting the presence of a complex in each sample or portion of the sample further comprises one or more washing steps, comprising washing the samples with a wash buffer.

[0880] A52. The method according to clause A51 , wherein the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0881] A53. The method according to clause A44, wherein the recognition agent comprises a fragment antigen-binding region (Fab).

[0882] A54. The method according to clause A53, wherein the fragment antigen-binding region (Fab) is configured to bind digoxigenin.

[0883] A55. The method according to any of clauses A44, A53, or A54, wherein the recognition agent comprises a label.

[0884] A56. The method according to clause A55, wherein the label comprises horseradish peroxidase.

[0885] A57. The method according to any of clauses A44, A53 to A56, wherein the recognition agent is provided at a concentration in the sample or portion of the sample of between about 5 to 50 mU / mL, such as between about 10 to 40 mU / mL, or between about 15 to 35 mU / mL; for example, between about 20 to 30 mU / mL; e.g. about 25 mU / mL.

[0886] A58. The method according to any of clauses A44, A53 to A57, wherein contacting the second drug moiety with the recognition agent comprises incubating the second drug moiety with the recognition agent; suitably wherein the incubation is up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0887] A59. The method according to any of clauses A44, A53 to A58, wherein detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0888] A60. The method according to clause A59, wherein the detection reagent comprises 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid).

[0889] A61. The method according to clause A59 or clause A60, wherein contacting the recognition agent with the detection reagent comprises incubating the second drug moiety with the recognition agent; suitably wherein the incubation is for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0890] A62. The method according to any of clauses A1 to A4, A7 to A9, A11 to A24, A27 to A35, A38 to A47, or A51 to A61 when dependent on clause A1 or clause A4, wherein detecting the presence of an IgM complex comprising: the first drug moiety, the anti-drug antibody of the IgM class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgM absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0891] A63. The method according to clause A5, or any of clauses A6 to A22 or A25 to A33, A36 to A44, or A48 to A61 when dependent on any of clauses A5, A6, or A10, wherein detecting the presence of an IgG complex comprising: the first drug moiety, the anti-drug antibody of the IgG class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0892] A64. The method according to clause A62 or clause A63, wherein the first specific wavelength is about 405 nm.

[0893] A65. The method according to any of clauses A62 to A64, wherein the second specific wavelength is about 490 nm.

[0894] A66. The method according to any of clauses A62 to A65, wherein detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final absorbance.

[0895] A67. The method according to any of clauses A62 to A66, wherein calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final absorbance.

[0896] A68. The method according to clause A62, or any of clauses A64 to A67 when dependent on clause A62, wherein comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample comprises comparing the IgM absorbance value in the first sample to the IgM absorbance value in the second sample, wherein a lower IgM absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0897] A69. The method according to clause A62, or any of clauses A64 to A67 when dependent on clause A62, wherein comparing the detection of the presence of the IgM complex in the first portion of the sample with the detection of the presence of the IgM complex in the second portion of the sample comprises comparing the IgM absorbance value in the first portion of the sample to the IgM absorbance value in the second portion of the sample, wherein a lower IgM absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[0898] A70. The method according to clause A63, or any of clauses A64 to A67 when dependent on clause A63, wherein comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG absorbance value in the first sample to the IgG absorbance value in the second sample, wherein a lower IgG absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0899] A71 . The method according to clause A63, or any of clauses A64 to A67 when dependent on clause A63, wherein comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG absorbance value in the first portion of the sample to the IgG absorbance value in the second portion of the sample, wherein a lower IgG absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[0900] A72. The method according to any of clauses A1 to A71 , wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis. A73. The method according to any of clauses A1 to A72, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having an autoimmune and / or infectious disease.

[0901] A74. The method according to any of clauses A1 to A73, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[0902] A75. The method according to any of clauses A1 to A74, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having elevated rheumatoid factor (RF).

[0903] A76. The method according to clause A75, wherein the level of rheumatoid factor (RF) in the blood of the subject is at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL.

[0904] A77. Use of an IgM protease in a method according to any of clauses A1 to A76.

[0905] A78. Use of an IgM protease for improving the accuracy, reliability, sensitivity and / or confidence of the results of an antibody-based and / or immunological assay; optionally wherein the method is according to any of clauses A1 to A76.

[0906] A79. The use of an IgM protease according to clause A77 or clause A78, wherein the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0907] A80. The use of an IgM protease according to any of clauses A77 to A79, wherein the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0908] A81 . The use of an IgM protease according to any of clauses A77 to A80, wherein the IgM protease is used at a concentration between about 0.2 to 10 U / pL, between about 0.5 to 3 U / pL, between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL; e.g. about 0.91 U / pL.

[0909] Clause Group B: Alternative embodiments of Example 1:

[0910] B1 . A method for detecting at least one anti-drug antibody of the IgM class in a biological sample, the method comprising: (a) contacting a first biological sample from a subject, wherein the sample comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,

[0911] (b)(i) contacting the first treated sample with an immobilised first drug moiety,

[0912] (b)(ii) contacting a second sample which comprises or may comprise at least one anti-drug antibody of the IgM class with an immobilised first drug moiety,

[0913] (c) separately contacting the first and second samples with a second drug moiety,

[0914] (d) detecting the presence of an IgM complex in the first and second samples, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0915] (e) comparing the detection of the presence of the IgM complex in the first treated sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[0916] B2. The method according to clause B1 , wherein the first and second biological samples are obtained by partitioning a biological sample into at least two portions.

[0917] B3. The method according to clause B1 , wherein the first and second biological samples are obtained separately from the same subject.

[0918] B4. A method for detecting at least one anti-drug antibody of the IgM class in a biological sample, the method comprising:

[0919] (a) partitioning a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class into at least a first portion and a second portion,

[0920] (b) contacting the first portion of the sample with an IgM protease, to obtain a first treated portion of the sample,

[0921] (c) separately contacting each portion of the sample with an immobilised first drug moiety,

[0922] (d) separately contacting each portion of the sample with a second drug moiety,

[0923] (e) detecting the presence of an IgM complex in each portion of the sample, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and

[0924] (f) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second sample portion relative to the first portion is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample

[0925] B5. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0926] (a) contacting a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,

[0927] (b) contacting the treated sample with an immobilised first drug moiety,

[0928] (c) contacting the treated sample with a second drug moiety,

[0929] (d) detecting the presence of an IgG complex in the treated sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[0930] B6. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0931] (a) contacting a first biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a first treated sample,

[0932] (b)(i) contacting the first treated sample with an immobilised first drug moiety; and separately

[0933] (b)(ii) contacting a second biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an immobilised first drug moiety; and

[0934] (c) separately, contacting the first treated and second samples with a second drug moiety,

[0935] (d) detecting the presence of an IgG complex in each sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and (e) comparing the detection of the presence of the IgG complex in the first treated sample with the detection of the presence of the IgG complex in the second sample, wherein the detection is improved compared to a method without step (a).

[0936] B7. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:

[0937] (a) partitioning a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; into at least a first portion and a second portion,

[0938] (b) contacting the first portion of the sample with an IgM protease to obtain a first treated portion of the sample,

[0939] (c) separately contacting the first treated and second portions of the sample with an immobilised first drug moiety,

[0940] (d) separately contacting the first treated and second portions of the sample with a second drug moiety,

[0941] (e) detecting the presence of an IgG complex in each portion of the sample, wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; and

[0942] (f) comparing the detection of the presence of the IgG complex in the first treated portion of the sample to the second portion of the sample, wherein the detection is improved compared to a method without step (b).

[0943] B8. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class.

[0944] B9. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class.

[0945] B10. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise at least one anti-drug antibody of the IgG class. B11 . The method according to any of the preceding clauses, further comprising providing a solid support configured to bind a first drug moiety.

[0946] B12. The method according to any of the preceding clauses, further comprising contacting a solid support configured to bind a first drug moiety with a first drug moiety to generate a solid support comprising the immobilised first drug moiety.

[0947] B13. The method according to clause B12, wherein contacting the solid support configured to bind the first drug moiety with the first drug moiety is performed before the step of contacting the first drug moiety with the sample, each sample, or portion of the sample.

[0948] B14. The method according to any of the preceding clauses, wherein the immobilised first drug moiety comprises a first drug moiety immobilised on a solid support.

[0949] B15. The method according to any of clauses B11 to B14, wherein the solid support comprises the immobilised the first drug moiety.

[0950] B16. The method according to clause B15, further comprising providing the solid support comprising the immobilised first drug moiety.

[0951] B17. The method according to any of clauses B1 to B4, or B8 to B16 when dependent on clause B1 or clause B4, wherein separately contacting each sample with the immobilised first drug moiety comprises contacting the sample comprising the at least one anti-drug antibody of the IgM class with the immobilised first drug moiety, to generate an ‘anti-drug antibody of the IgM class- immobilised first drug moiety’ complex.

[0952] B18. The method according to clause B17, wherein separately contacting each sample with a second drug moiety further comprises contacting a portion of the ‘anti-drug antibody of the IgM class-immobilised first drug moiety’ complex corresponding to the anti-drug antibody of the IgM class with the second drug moiety to generate the IgM complex.

[0953] B19. The method according to any of clauses B1 to B4, or B8 to B18 when dependent on clause B1 or clause B4, wherein detecting the presence of an IgM complex in each sample or each portion of the sample further comprises contacting a portion of the IgM complex corresponding to the second drug moiety with a recognition moiety.

[0954] B20. The method according to any of clauses B5 to B7, or any of clauses B8 to B16 when dependent on any of clauses B5 to B7, wherein separately contacting each sample with the immobilised first drug moiety comprises contacting the sample comprising the at least one antidrug antibody of the IgG class with the immobilised first drug moiety, to generate a ‘anti-drug antibody of the IgG class-immobilised first drug moiety’ complex.

[0955] B21 . The method according to clause B20, wherein separately contacting each sample with a second drug moiety further comprises contacting a portion of the ‘anti-drug antibody of the IgG class-immobilised first drug moiety’ complex corresponding to the anti-drug antibody of the IgG class with the second drug moiety to generate the IgG complex.

[0956] B22. The method according to any of clauses B5 to B7, or any of clauses B8 to B16, B20, or B21 when dependent on any of clauses B5 to B7, wherein detecting the presence of an IgG complex in each sample further comprises contacting a portion of the IgG complex corresponding to the second drug moiety with the recognition moiety.

[0957] B23. The method according to any of the preceding clauses, wherein the sample comprises serum; suitably human serum or monkey serum; suitably human or cynomolgus serum.

[0958] B24. The method according to any of the preceding clauses, wherein contacting at least the first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class; suitably wherein the incubation is for up to about 36 hours, up to about 30 hours; e.g. from about 1 to 24 hours, about 2 to 24 hours, about 4 to 20 hours, about 6 to 20 hours, about 8 to 16 hours, or about 10 to 16 hours; such as about 12 hours or about 16 hours.

[0959] B25. The method according to any of clauses B1 to B4, B8 to B19, B23, or B24, when dependent on clause B1 or clause B4, wherein the higher level of IgM complex in the second sample relative to the first sample is higher by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%, e.g. by at least about 20%.

[0960] B26. The method according to any of the preceding clauses, wherein contacting at least a first sample or portion of the sample with an IgM protease comprises incubating the IgM protease with the first sample or portion of the sample to hydrolyse the at least one anti-drug antibody of the IgM class is performed at a temperature of between about 4 and 25°C, between about 4 and 20°C, between about 4 and 15°C, or between about 4 and 10°C; suitably at a temperature of about 4 °C. B27. The method according to any of the preceding clauses, wherein the IgM protease is an IgM specific protease, suitably having no detectable activity against polypeptides other than IgM.

[0961] B28. The method according to any of the preceding clauses, wherein the IgM protease hydrolyses IgM below the CH2 region of IgM.

[0962] B29. The method according to any of the preceding clauses, wherein the IgM protease comprises SEQ ID NO: 2.

[0963] B30. The method according to any of the preceding clauses, wherein the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[0964] B31 . The method according to any of the preceding clauses, wherein contacting the first sample or portion of the sample with an IgM protease comprises contacting the first sample or portion of the sample with IgM protease at a concentration between about 0.2 to 10 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL, e.g. about 0.91 U / pL.

[0965] B32. The method according to any of the preceding clauses, wherein contacting each sample or portion of the sample with the immobilised first drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0966] B33. The method according to any of the preceding clauses, wherein contacting each sample or portion of the sample with the second drug moiety further comprises diluting each sample or portion of the sample in a buffer, for example, wherein the buffer is a reaction buffer.

[0967] B34. The method according to clause B32 or clause B33, wherein diluting each sample or portion of the sample in a buffer, optionally a reaction buffer, comprises a dilution of between about 10-fold and 150-fold, between about 50-fold and 140-fold, between about 80-fold and 130-fold, between about 90-fold and 120-fold, or between about 95-fold and 110-fold; e.g. about 100-fold or about 105-fold.

[0968] B35. The method according to clause B11 , or any of clauses B12 to B34 when dependent on clause B11 , wherein the solid support comprises a charged polystyrene surface, suitably having high affinity to the first drug moiety.

[0969] B36. The method according to clause B12, or any of clauses B13 to B35 when dependent on clause B12, wherein contacting the first drug moiety with the solid support configured to bind a first drug moiety comprises incubating the first drug moiety with the with the solid support configured to bind a first drug moiety.

[0970] B37. The method according to clause B12, or any of clauses B13 to B36 when dependent on clause B12, further comprising blocking the immobilised first drug moiety after contacting the first drug moiety with the solid support configured to bind a first drug moiety.

[0971] B38. The method according to clause B37, wherein blocking the immobilised first drug moiety comprises contacting the immobilised first drug moiety with a blocking buffer.

[0972] B39. The method according to clause B38, wherein contacting the immobilised first drug moiety with the blocking buffer comprises incubating the immobilised first drug moiety with the blocking buffer.

[0973] B40. The method according to clause B38 or clause B39, wherein the blocking buffer comprises LowCross buffer from CANDOR Bioscience GmbH.

[0974] B41 . The method according to any of clauses B37 to B40, further comprising one or more washing steps after blocking the immobilised first drug moiety prior to contacting each sample or portion of the sample with a second drug moiety, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0975] B42. The method according to clause B41 , wherein the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0976] B43. The method according to any of clauses B1 to B4, B8 to B19, or B23 to B42, when dependent on clause B1 or clause B4, wherein the second drug moiety comprises an anti-IgM antibody.

[0977] B44. The method according to clause B43, wherein the anti-IgM antibody is derived from a species which is different to the species from which the biological sample is derived.

[0978] B45. The method according to clause B44, wherein the anti-IgM antibody is derived from mouse and the biological sample is derived from human.

[0979] B46. The method according to any of clauses B5 to B16, B20 to B23, or B26 to B42 when dependent on any of clauses B5 to B7, wherein the second drug moiety comprises an anti-IgG antibody. B47. The method according to clause B46, wherein the anti-IgG antibody is derived from a species which is different to the species from which the biological sample is derived.

[0980] B48. The method according to clause B46 or clause B47, wherein the anti-IgG antibody is derived from mouse and the biological sample is derived from human.

[0981] B49. The method according to any of clauses B46 to B48, wherein the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, such as between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. about 0.5 ng / pL.

[0982] B50. The method according to any of the preceding clauses, wherein contacting each sample or portion of the sample with the second drug moiety comprises incubating each sample or portion of the sample with the second drug moiety; suitably, wherein the incubation is up to about 6 hours, up to about 4 hours, for between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0983] B51. The method according to any of the preceding clauses, further comprising one or more washing steps after contacting each sample or portion of the sample with a second drug moiety prior to detecting the presence of a complex in each sample or portion of the sample, wherein the one or more washing steps comprise washing the samples with a wash buffer.

[0984] B52. The method according to clause B51 , wherein the wash buffer comprises 0.05% (v / v) Tween20 in 1x PBS.

[0985] B53. The method according to any of the preceding clauses, wherein the second drug moiety comprises a label.

[0986] B54. The method according to any of the preceding clauses, wherein detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a recognition agent configured to specifically bind the second drug moiety; suitably the label of the second drug moiety.

[0987] B55. The method according to clause B54, wherein contacting the second drug moiety with the recognition agent comprises incubating the second drug moiety with the recognition agent; suitably, wherein the incubation is up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour. B56. The method according to clause B54 or clause B55, wherein the recognition agent comprises an antibody configured to bind to the second antibody.

[0988] B57. The method according to any of clauses B54 to B56, wherein the recognition agent antibody is derived from a species which is different to the species from which the second drug moiety is derived.

[0989] B58. The method according to any of clauses B54 to B57, wherein the second drug moiety is derived from mouse and the recognition agent comprises an anti-mouse antibody.

[0990] B59. The method according to any of clauses B54 to B58, wherein the recognition agent comprises a label.

[0991] B60. The method according to clause B59, wherein the label comprises horseradish peroxidase.

[0992] B61 . The method according to any of clauses B54 to B60, wherein the recognition agent is provided at a concentration of between about 100 to 1 ,000 ng / mL, between about 200 to 600 ng / mL, or between about 300 to 500 ng / mL; e.g. about 400 ng / mL.

[0993] B62. The method according to any of clauses B54 to B61 , wherein detecting the presence of a complex in each sample or portion of the sample further comprises contacting the recognition agent with a detection reagent.

[0994] B63. The method according to clause B62, wherein the detection reagent comprises 2,2'-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid).

[0995] B64. The method according to clause B62 or clause B63, wherein contacting the recognition agent with the detection reagent comprises incubating the second drug moiety with the recognition agent, suitably wherein the incubation is performed for up to about 6 hours, up to about 4 hours, between about 0.5 to 3 hours, between about 0.5 to 2 hours, or between about 0.5 to 1 .5 hours; e.g. about 1 hour.

[0996] B65. The method according to any of clauses B1 to B4, B8 to B19, B23 to B45, or B49 to B64 when dependent on clause B1 or clause B4, wherein detecting the presence of an IgM complex comprising: the first drug moiety, the anti-drug antibody of the IgM class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgM absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0997] B66. The method according to any of clauses B5 to B16, B20 to B23, B26 to B42, or B46 to B64 when dependent on any of clauses B5 to B7, wherein detecting the presence of an IgG complex comprising: the first drug moiety, the anti-drug antibody of the IgG class, and the second drug moiety; in each sample or portion of the sample further comprises measuring an absorbance of the detection reagent at one or more specific wavelengths of light to obtain an IgG absorbance value; optionally wherein measuring an absorbance of the detection reagent comprises measuring an absorbance at a first specific wavelength and at a second specific wavelength.

[0998] B67. The method according to clause B65 or clause B66, wherein the first specific wavelength is about 405 nm.

[0999] B68. The method according to any of clauses B65 to B67, wherein the second specific wavelength is about 490 nm.

[1000] B69. The method according to any of clauses B65 to B68, wherein detecting the presence of a complex in each sample or portion of the sample further comprises calculating a final absorbance.

[1001] B70. The method according to clause B69, wherein calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final IgM absorbance.

[1002] B71. The method according to clause B69, wherein calculating a final absorbance comprises subtracting the absorbance at the second specific wavelength from the absorbance at the first specific wavelength; suitably wherein calculating a final absorbance comprises subtracting the absorbance at about 490 nm from the absorbance at about 405 nm, to obtain the final IgG absorbance. B72. The method according to any of clauses B65 to B71 , wherein comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample comprises comparing the IgM absorbance value in the first sample to the IgM absorbance value in the second sample, wherein a lower IgM absorbance value in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[1003] B73. The method according to any of clauses B65 to B71 , wherein comparing the detection of the presence of the IgM complex in the first portion of the sample with the detection of the presence of the IgM complex in the second portion of the sample comprises comparing the IgM absorbance value in the first portion of the sample to the IgM absorbance value in the second portion of the sample, wherein a lower IgM absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgM class in the sample.

[1004] B74. The method according to any of clauses B65 to B71 , wherein comparing the detection of the presence of the IgG complex in the first sample with the detection of the presence of the IgG complex in the second sample comprises comparing the IgG absorbance value in the first sample to the IgG absorbance value in the second sample, wherein a lower IgG absorbance in the first sample relative to the second sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[1005] B75. The method according to any of clauses B65 to B71 , wherein comparing the detection of the presence of the IgG complex in the first portion of the sample with the detection of the presence of the IgG complex in the second portion of the sample comprises comparing the IgG absorbance value in the first portion of the sample to the IgG absorbance value in the second portion of the sample, wherein a lower IgG absorbance value in the first portion of the sample relative to the second portion of the sample is indicative of the presence of at least one anti-drug antibody of the IgG class in the sample.

[1006] Alternate second drug moiety and recognition agent:

[1007] B76. The method according to any of the preceding clauses, wherein the second drug moiety comprises an Fc receptor.

[1008] B77. The method according to clause B76, wherein the Fc receptor comprises CD64. B78. The method according to clause B76 or clause B77, wherein the second drug moiety comprises a label.

[1009] B79. The method according to clause B78, wherein the label comprises digoxigenin.

[1010] B80. The method according to any of clauses B76 to B79, wherein the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. 0.5 ng / pL.

[1011] B81. The method according to any of clauses B76 to B80, wherein the recognition agent comprises a fragment antigen-binding region (Fab).

[1012] B82. The method according to clause B81 , wherein the fragment antigen-binding region (Fab) is configured to bind digoxigenin.

[1013] B83. The method according to any of clauses B76 to B82, wherein the recognition agent comprises a label.

[1014] B84. The method according to clause B83, wherein the label comprises horseradish peroxidase.

[1015] B85. The method according to any of clauses B76 to B84, wherein the recognition agent comprises an anti-digoxigenin Fab fragment.

[1016] B86. The method according to any of clauses B76 to B85, wherein the recognition agent is provided at a concentration of between about 20 to 120 mU / mL, between about 20 to 100 mU / mL, or between about 40 to 80 mU / mL, or between about 40 to 60 mU / mL; e.g. about 50 mU / mL.

[1017] B87. The method according to any of clauses B1 to B86, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), and / or systemic sclerosis.

[1018] B88. The method according to any of clauses B1 to B87, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having an autoimmune and / or infectious disease. B89. The method according to any of clauses B1 to B88, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

[1019] B90. The method according to any of clauses B1 to B89, wherein the biological sample, first biological sample and / or second biological sample has been obtained from a subject having elevated rheumatoid factor (RF).

[1020] B91. The method according to clause B90, wherein the level of rheumatoid factor (RF) in the blood of the subject is at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL.

[1021] B92. Use of an IgM protease in a method according to any of clauses B1 to B91 .

[1022] B93. Use of an IgM protease for improving the accuracy, reliability, sensitivity and / or confidence of the results of an antibody-based and / or immunological assay; optionally wherein the method is according to any of clauses B1 to B91 .

[1023] B94. The use of an IgM protease according to clause B92 or clause B93, wherein the IgM protease hydrolyses IgM below the CH2 region of IgM.

[1024] B95. The use of an IgM protease according to any of clauses B92 to B94, wherein the IgM protease is IgMBRAZOR™ (Genovis) comprising SEQ ID NO: 1.

[1025] B96. The use of an IgM protease according to any of clauses B92 to B95, wherein the IgM protease is used at a concentration between about 0.2 to 10 U / pL, between about 0.5 to 3 U / pL, between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL; e.g. about 0.91 U / pL.

[1026] Clause Group C: Predominantly first (alternate) embodiment (A) of Example 2:

[1027] Anti-AAV immune complex assay for the detection of anti-AAV IgG antibodies and supplementary assay for the determination of the corresponding anti-AAV IgM antibodies

[1028] C1 . A method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample, the method comprising:

[1029] (a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample, (b) contacting the treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[1030] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

[1031] C2. A method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample, the method comprising:

[1032] (a) contacting a first biological sample from a subject which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,

[1033] (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; and separately

[1034] (b)(ii) contacting a second biological sample from the subject, which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample;

[1035] (c) detecting the presence of an IgG complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without step (a). C3. The method according to clause C2, wherein the first and second biological samples are obtained a partitioning a biological sample into at least two portions.

[1036] C4. The method according to clause C2, wherein the first and second biological samples are obtained separately from the same subject.

[1037] C5. A method for improving the detection of at least one anti-AAV particle IgG antibody in a biological sample, the method comprising:

[1038] (a) partitioning a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody, into at least a first portion and a second portion,

[1039] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[1040] (c) separately contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[1041] (d) detecting the presence of an IgG complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without step (b).

[1042] C6. A method for detecting at least one anti-AAV particle IgM antibody in a biological sample, the method comprising:

[1043] (a) contacting a first biological sample from a subject, which comprises or may comprise at least one anti-AAV particle IgM antibody, with an IgM protease to obtain at least a first treated sample,

[1044] (b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[1045] (b)(ii) contacting a second biological sample from the subject which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[1046] (c) detecting the presence of an IgM complex in each sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody, and the detection moiety; and

[1047] (d) comparing the detection of the presence of the IgM complex in the first sample with the detection of the presence of the IgM complex in the second sample, wherein a higher level of IgM complex in the second sample relative to the first sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

[1048] C7. A method for detecting at least one anti-AAV particle IgM antibody in a biological sample, the method comprising:

[1049] (a) partitioning a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody into at least a first portion and a second portion,

[1050] (b) contacting at least the first portion of the sample with an IgM protease to obtain at least a first treated portion of the sample,

[1051] (c) separately contacting each portion of the sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;

[1052] (d) detecting the presence of an IgM complex in each portion of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgM antibody, and the detection moiety; and

[1053] (e) comparing the detection of the presence of the IgM complex in the first treated portion of the sample to the second portion of the sample, wherein a higher level of IgM complex in the second portion relative to the first portion is indicative for the presence of the at least one anti-drug antibody of the IgM class in the sample.

[1054] C8. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise: at least one anti-AAV particle IgG antibody, and at least one anti-AAV particle IgM antibody. C9. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody.

[1055] C10. The method according to any of the preceding clauses, further comprising providing a biological sample which comprises or may comprise at least one anti-AAV particle IgG antibody.

[1056] C11. The method according to any of the preceding clauses, wherein contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; is performed separately and in a step-wise manner.

[1057] C12. The method according to any of clauses C1 to C5, or C8 to C11 when dependent on any of clauses C1 , C2, or C5, wherein contacting each sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety; comprises:

[1058] (i) contacting the sample comprising the at least one anti-AAV particle antibody of the IgG class with the at least one AAV particle, to generate an ‘anti-AAV particle antibody of the IgG class-AAV particle’ complex, and

[1059] (ii) contacting a port...

Claims

CLAIMS1. A method for detecting at least one anti-drug antibody of the IgM class in a biological sample, the method comprising:(a) contacting a first aliquot of the biological sample from a subject which comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,(b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately(b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;(c) detecting the presence of an IgM complex in each of the first and second aliquot of the sample, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and(d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

2. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:(a) contacting a biological sample from a subject, wherein the biological sample comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with: a first drug moiety, and a second drug moiety;(c) detecting the presence of an IgG complex in the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, andthe second drug moiety; wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

3. The method according to Claim 1 or Claim 2, wherein the first drug moiety is provided at a concentration of between about 0.1 to 1 ng / mL, such as between about 0.125 to 0.75 ng / mL, between about 0.15 to 0.5 ng / mL, between about 0.2 to 0.4 ng / mL, or between about 0.25 to 0.3 ng / mL; e.g. between about 0.25 to 0.26 ng / mL.

4. A method for detecting at least one anti-drug antibody of the IgM class in a biological sample from a subject, the method comprising:(a) contacting a first aliquot of the biological sample, wherein the sample comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,(b)(i) contacting the first treated sample with an immobilised first drug moiety,(b)(ii) contacting a second aliquot of the sample which comprises or may comprise at least one anti-drug antibody of the IgM class with an immobilised first drug moiety,(c) separately contacting the first and second aliquot of the sample with a second drug moiety,(d) detecting the presence of an IgM complex in the first and second aliquot of the sample, wherein the IgM complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and(e) comparing the detection of the presence of the IgM complex in the first treated sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first treated sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

5. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample, the method comprising:(a) contacting a biological sample which comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with an immobilised first drug moiety,(c) contacting the treated sample of (b) with a second drug moiety,(d) detecting the presence of an IgG complex in the treated sample of (c), wherein the IgG complex comprises: the immobilised first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

6. The method according to Claim 4 or Claim 5, wherein the second drug moiety is provided at a concentration of between about 0.1 to 1 ng / pL, such as between about 0.1 to 0.8 ng / pL, between about 0.2 to 0.8 ng / pL, between about 0.3 to 0.7 ng / pL, or between about 0.4 to 0.6 ng / pL; for example, between about 0.4 to 0.6 ng / pL, e.g. about 0.5 ng / pL.

7. The method according to any one of Claims 1 to 6, wherein detecting the presence of a complex in each sample or portion of the sample comprises contacting the second drug moiety with a detection moiety configured to specifically bind to the second drug moiety, optionally wherein the detection moiety is configured to specifically bind to a label of the second drug moiety.

8. The method according to any one of Claims 1 to 7, wherein contacting the first sample or portion of the sample with an IgM protease comprises contacting the first sample or portion of the sample with an IgM protease at a concentration between about 0.2 to 10 U / pL, such as between about 0.5 to 3 U / pL, or between about 0.7 and 1 .5 U / pL, or between about 0.8 and 1 .0 U / pL, e.g. about 0.91 U / pL.

9. A method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample, the method comprising:(a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;(c) detecting the presence of an IgG complex in the treated sample of (b), wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, andthe detection moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

10. A method for detecting at least one anti-AAV particle antibody of the IgM class in a biological sample from a subject, the method comprising:(a) contacting a first aliquot of the biological sample, which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,(b)(i) contacting the first treated sample with: at least one AAV particle, an AAV capture moiety, and a detection moiety;(b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with: at least one AAV particle, an AAV capture moiety, and a detection moiety;(c) detecting the presence of an IgM complex in each aliquot of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle antibody of the IgM class, and the detection moiety; and(d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

11. A method for detecting at least one anti-AAV particle antibody of the IgM class in a biological sample from a subject, the method comprising:(a) contacting a first aliquot of the biological sample, which comprises or may comprise at least one anti-AAV particle antibody of the IgM class, with an IgM protease to obtain at least a first treated sample,(b)(i) contacting the first treated sample with: at least one AAV particle, and an AAV capture moiety;(b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-AAV particle IgM antibody, with: at least one AAV particle, and an AAV capture moiety;(c) detecting the presence of an IgM complex in each aliquot of the sample, wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle antibody of the IgM class; and(d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relative to the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

12. A method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample, the method comprising:(a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with: at least one AAV particle, and an AAV capture moiety,(c) detecting the presence of an IgG complex in the treated sample of (b), wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, and the at least one anti-AAV particle antibody of the IgG class, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

13. The method according to any one of Claims 9 to 12, wherein contacting the first sample or portion of the first sample with an IgM protease comprises contacting the sample or portion of the sample with IgM protease; suitably wherein the IgM protease is at a concentration between about 0.4 and 1.4 U / pL, between about 0.6 and 1.2 U / pL, or between about 0.8 and 1.0 U / pL; for example, between about 0.8 and 1 .0 U / pL, e.g. about 0.9 U / pL.

14. The method according to any one of Claims 11 to 13 when dependent on either Claim 11 or 12, wherein contacting each sample, or aliquot of the sample with: at least one AAV particle, and an AAV capture moiety, further comprises contacting the sample or portion of the sample with a recognition agent, suitably wherein the recognition agent comprises: an anti-IgG antibody configured to bind to the at least one anti-AAV particle IgG antibody in each sample or aliquot of the sample; and / or an anti-IgM antibody configured to bind to the at least one anti-AAV particle IgM antibody in each sample or aliquot of the sample.

15. A method for improving the accuracy and / or sensitivity of an antibody-based or immunogenicity assay, the method comprising: contacting a biological sample with an IgM protease to obtain a treated sample, and performing an antibody-based assay or immunogenicity assay on the treated sample.

16. The method according to Claim 15, wherein the biological sample has been obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), systemic sclerosis, an autoimmune, an infectious disease, hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

17. The method according to Claim 15 or Claim 16, wherein the biological sample has been obtained from a subject having elevated rheumatoid factor (RF).

18. The method according to Claim 17, wherein the level of rheumatoid factor (RF) in the biological sample is at least 20 U / mL; at least 40 U / mL; at least 60 U / mL, at least 80 U / mL or at least 100 U / mL.

19. The method according to any one of Claims 15 to 18, wherein the immunogenicity assay is an anti-drug antibody (ADA) assay, for example, an ADA bridging assay.

20. The method according to Claim 19, wherein the drug is an antibody.21 . The method according to any one of Claims 15 to 20, wherein the immunogenicity assay is a multiplex immunoassay or ELISA assay.

22. The method according to any one of Claims 15 to 21 , which is for therapeutic drug monitoring, immunogenicity evaluation or disease diagnosis.

23. The method according to any one of Claims 1 to 22, wherein the IgM protease comprises SEQ ID NO: 2, or SEQ ID NO: 1.

24. A method for reducing non-specific signal and / or reducing false positive signals in an antibody-based or immunological assay of a biological sample, wherein the biological sample comprises or is expected to comprise rheumatoid factor (RF), the method according to any of claims 15 to 23.

25. The method according to Claim 24, wherein the biological sample was obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), systemic sclerosis, an autoimmune disease, an infectious disease, hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis.

26. A method for detecting at least one anti-drug antibody of the IgM class in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), systemic sclerosis, an autoimmune disease, an infectious disease, hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis, the method comprising:(a) contacting a first aliquot of the biological sample from a subject which comprises or may comprise at least one anti-drug antibody of the IgM class, with an IgM protease to obtain a first treated sample,(b)(i) contacting the first treated sample with: a first drug moiety, and a second drug moiety; and separately(b)(ii) contacting a second aliquot of the biological sample which comprises or may comprise at least one anti-drug antibody of the IgM class, with: a first drug moiety, and a second drug moiety;(c) detecting the presence of an IgM complex in each of the first and second aliquot of the sample, wherein the IgM complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgM class, and the second drug moiety; and(d) comparing the detection of the presence of the IgM complex in the first aliquot of the sample with the detection of the presence of the IgM complex in the second aliquot of the sample, wherein a higher level of IgM complex in the second aliquot of the sample relativeto the first aliquot of the sample is indicative of the presence of the at least one anti-drug antibody of the IgM class in the sample.

27. A method for improving the detection of at least one anti-drug antibody of the IgG class in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), systemic sclerosis, an autoimmune disease, an infectious disease, hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis, the method comprising:(a) contacting a biological sample from a subject, wherein the biological sample comprises or may comprise: at least one anti-drug antibody of the IgG class, and at least one anti-drug antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with: a first drug moiety, and a second drug moiety;(c) detecting the presence of an IgG complex in the sample, wherein the IgG complex comprises: the first drug moiety, the at least one anti-drug antibody of the IgG class, and the second drug moiety; wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

28. A method for improving the detection of at least one anti-AAV particle antibody of the IgG class in a biological sample obtained from a subject having rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sjogrens syndrome, mixed connective tissue disease (MCTD), systemic sclerosis, an autoimmune disease, an infectious disease, hypergammaglobulinaemia, viral hepatitis, chronic liver disease, syphilis, sarcoidosis, leprosy and / or pulmonary fibrosis, the method comprising:(a) contacting a biological sample which comprises or may comprise: at least one anti-AAV particle antibody of the IgG class, and at least one anti-AAV particle antibody of the IgM class; with an IgM protease to obtain a treated sample,(b) contacting the treated sample with: at least one AAV particle, an AAV capture moiety, anda detection moiety;(c) detecting the presence of an IgG complex in the treated sample of (b), wherein the complex comprises: the AAV capture moiety, the at least one AAV particle, the at least one anti-AAV particle IgG antibody, and the detection moiety, wherein the detection is improved compared to a method without contacting a biological sample with an IgM protease.

29. Use of an IgM protease in a method or assay according to any one of claims 1 to 28.

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