High Ionic Intensity Dissociation Assay for Advanced Drug Resistance Testing

Abstract

A method for determining the presence of anti-drug antibodies in a sample. [Solution] A method comprising the steps of: incubating a sample with MgCl2 at a final concentration in the range of 1N to 12N; adding a tracer antibody and then incubating the sample; incubating the isolated tracer antibody-anti-drug antibody conjugate with a detection antibody conjugated with a detectable label; and determining the presence of an anti-drug antibody if a signal exceeding a threshold level is obtained.

Description

Technical Field

[0001] The present invention is in the field of immunoassays, particularly in the field of anti-drug antibody (ADA) assessment. ADA assessment is often difficult, especially in studies involving administration of high-dose and / or long-half-life biologic therapeutic agents. In such cases, ADA assays with optimized drug tolerance are desired. The present invention is based, at least in part, on the use of chaotropic salts such as MgCl2 or LiCl in a High Ionic Strength Dissociation Assay (HISDA) to achieve high drug tolerance while maintaining the best possible structural integrity of ADA.

Background Art

[0002] Background of the Invention Immunogenicity assessment of therapeutic drug candidates is an important part of the drug development process. In the case of immune responses, appropriate interpretation of immunogenicity data is required to enable correlation with clinical outcomes. Bioanalytical methods used for immunogenicity testing provide the necessary information by detecting and characterizing anti-drug antibodies (ADA).

[0003] The "gold standard" assay format is the ADA bridging assay, in which ADA forms a complex with a labeled drug conjugate to generate a signal-conferring complex. However, this format is susceptible to interference from residual drug, which itself forms complexes with ADA and consequently prevents complex formation with assay reagents. For this reason, ADA assessment can be complicated by drug interference, especially in studies involving administration of high-dose and / or long-half-life biologic therapeutic agents[1]. In such cases, ADA assays with optimized drug tolerance are desired.

[0004] To achieve high drug resistance, many immunogenicity testing methods aim to break down the reversible non-covalent interactions that bind together immune complexes formed by ADA and drugs (e.g., antibodies). Such interactions include electrostatic, hydrophobic, van der Waals forces, and hydrogen bonds. Most of these interactions can be weakened by high chaotropic salt concentrations, extreme pH, or surfactants [2,3].

[0005] The use of low pH acid treatment has become a common method for dissociating immune complexes to achieve higher drug resistance. For example, Butterfield, AM et al. [4] compared three acid-based assay formats for drug resistance: Meso Scale Discovery® crosslinking assay format [5], solid-phase extraction by acid dissociation (SPEAD) [6], and affinity capture elution (ACE) [7]. Another novel method developed to successfully eliminate drug interference uses a combination of precipitation and acid dissociation (PandA) [8]. Numerous other acid-based methods of many different variations have been successfully developed [9, 10, 11].

[0006] However, the possibility of partial denaturation of the antibody / ADA and potential loss of binding function remains a limitation of such methods

[12] . With regard to immunogenicity testing, such loss of ADA's binding function to the assay reagent can potentially lead to erroneous assay results due to an underestimation of ADA.

[0007] An alternative to pH-mediated complex dissociation is the use of denaturants such as guanidine hydrochloride. This chaotropic compound has been successfully used to increase assay drug resistance in surface plasmon resonance-based methods for detecting ADA

[13] . The use of such denaturants represents a powerful tool for dissociating immune complexes. However, they are generally considered “harsh” conditions that can damage protein structure

[14] , which would also be detrimental to ADA detection.

[0008] In contrast, non-denaturing ionic strength conditions are considered “mild” to antibody function because they cause minimal or no changes in secondary and tertiary structure. This approach is commonly used in chromatography, for example, for mild protein elution in the field of immunoaffinity purification. In chromatography, magnesium chloride (MgCl2) salts are often used due to their relatively mild properties. For example, Durkee, KH et al. used a buffer containing 3.5 M MgCl2 and 0.05 M Tris-HCl at neutral pH for non-denaturing elution of the enzyme RVV-X from monoclonal antibodies, whereas low or high pH buffers resulted in enzyme inactivation

[15] .

[0009] International Publication No. 2019 / 105916 reports on targeted interference suppression anti-drug antibody assays. Zhong, ZD et al. outlined drug target interference—recommendation and mitigation strategies in immunogenicity assays (AAPS J., 19(2017) 1564-1575). Nath, N. et al. reported the development of a NanoLuc crosslinked immunoassay for detecting anti-drug antibodies (J.Immunol.Meth. 450(2017) 17-26). Brady, A. et al. described a novel multiplex binding activity assay for evaluating HPV antibodies (J.Immunol.Meth. 447(2017) 31-36). Xu, R. et al. reported the application of ELISA elution to dissociate digoxin-antibody complexes in immunoaffinity chromatography (Scan. J. Immunol. 71 (2010) 55-60). Hogben, DN et al. reported a method for separating components of HB5Ag: anti-HB immune complexes and evaluating antibody affinity (J. Immunol. Meth. 93 (1986) 29-36). Dandliker, WB et al. reported the effect of chaotropic ions on antigen-antibody complex dissociation (Biochem. 6 (1967) 1460-1467).

[0010] Tsang, VC et al. identified a composition of 3.0 M MgCl2*6H2O, 0.075 M HEPES / NaOH, and 25% ethylene glycol as suitable dissociation buffers for their immunoaffinity systems at pH 7.20, in terms of specific activity and total quantitative yield of eluted antibodies

[20] . Various other buffers, including guanidine hydrochloride, were tested in the study, but they did not yield high specific activity, possibly due to denaturing effects. [Overview of the project]

[0011] This invention is at least in part based on the finding that improved drug resistance can be achieved in anti-drug antibody (ADA) assays by adding chaotropic salts such as MgCl2 or LiCl. Unexpectedly, the structural integrity of the ADA present in the sample is maintained at the same time.

[0012] The present invention is at least in part based on the finding that, in anti-drug antibody (ADA) assays, by adding chaotropic salts such as MgCl2 or LiCl, higher signal-blank values ​​can be achieved with significantly shorter incubation times compared to, for example, overnight incubation or acid pretreatment.

[0013] Therefore, the addition and use of chaotropic salts, such as MgCl2 or LiCl, in ADA assays provides improvements in sensitivity, drug resistance, and handling time. Furthermore, the addition and use of chaotropic salts, such as MgCl2 or LiCl, provides an alternative method when acid pretreatment for antibody conjugate dissociation is impossible, undesirable, or results in sample distortion.

[0014] This specification reports the following:

[0015] Item 1. A method for detecting / determining the presence of a target antibody in a sample, comprising the following steps: a) Incubating the sample (or aliquots of the sample) with a chaotropic salt having a final cation charge normality in the range of 1N to 12N (including both values) / Adding a chaotropic salt to the sample to a final concentration in the range of 1N to 12N (including both values), and then incubating the sample; ab) Optionally, remove the precipitate formed in step a) from the sample, but do not remove the chaotropic salt; b) Adding a tracer antibody to the sample obtained in step a) (or step a), and then incubating the sample, which still contains the chaotropic salt, to form a tracer antibody-target antibody complex; bc) Optionally, a step of recovering the tracer antibody-target antibody complex from the sample obtained in step b); c) Incubating the tracer antibody-target antibody complex formed in step b) (or obtained in step bc) with a detection antibody conjugated with a detectable label to form a tracer antibody-target antibody-detection antibody complex; cd) Optionally, remove excess tracer antibody and detect the tracer antibody-target antibody-detection antibody complex. A method comprising the steps wherein the presence of the target antibody is determined if the tracer antibody-target antibody-detection antibody complex is detected in the sample obtained in step c) or optionally cd).

[0016] Item 2. The method according to Item 1, wherein the chaotropic salt is a moderate-strength chaotropic salt, preferably having a cation between a potassium cation and a calcium cation in the Hofmeister synthesizer series and an anion between a hydrogen phosphate anion and a nitrate anion in the Hofmeister synthesizer series.

[0017] Item 3. The method according to Item 1 or 2, wherein the chaotropic salt comprises a cation selected from the group of cations consisting of potassium cation, sodium cation, lithium cation, magnesium cation and calcium cation, and an anion selected from the group of anions consisting of (hydrogen) phosphate anion, acetate anion and chloride anion.

[0018] Item 4. The method according to any one of Items 1 to 3, wherein the chaotropic salt is MgCl2 or LiCl.

[0019] Item 5. The method according to any one of Items 1 to 4, wherein the final normality of cation charge of the chaotropic salt is within the range of 5N to 10N.

[0020] Item 6. The method according to any one of Items 1 to 5, wherein the final normality of cation charge of the chaotropic salt is about 6.5N to 8.5N.

[0021] Item 7. The method according to any one of Items 1 to 6, wherein the incubation in steps a) and b) is 15 minutes to 180 minutes.

[0022] Item 8. The method according to any one of Items 1 to 7, wherein the incubation in steps a) and b) is 25 minutes to 75 minutes.

[0023] Item 9. The method according to any one of Items 1 to 8, wherein the incubation in steps a) and b) is about 30 minutes to 60 minutes, preferably about 30 minutes or about 60 minutes.

[0024] Item 10. The method according to any one of Items 1 to 9, wherein in step b), a capture antibody is further added together with the tracer antibody, before the tracer antibody, or after the tracer antibody but before incubating the sample.

[0025] Item 11. The method according to any one of Items 1 to 10, wherein in step b), the tracer antibody is conjugated to a label.

[0026] Item 12. The method according to Item 10 or 11, wherein the capture antibody, the tracer antibody and the detection antibody are conjugated to different labels, the label of the capture antibody does not interact with the label of the detection antibody, and vice versa.

[0027] Item 13. The method according to any one of Items 1 to 12, wherein the tracer antibody is conjugated to digoxigenin.

[0028] Item 14. The method according to any one of Items 10 to 13, wherein the capture antibody is conjugated to biotin or avidin / streptavidin.

[0029] Item 15. The method according to any one of Items 1 to 14, wherein the tracer antibody in step b) is added to a final concentration of 0.5 μg / mL to 5 μg / mL.

[0030] Item 16. The method according to any one of Items 1 to 15, wherein the tracer antibody in step b) is added to a final concentration of 0.9 μg / mL to 2.5 μg / mL.

[0031] Item 17. The method according to any one of Items 1 to 16, wherein the tracer antibody in step b) is added to a final concentration of about 1 μg / mL.

[0032] Item 18. The method according to any one of Items 10 to 17, wherein the capture antibody in step b) is added to a final concentration of 0.5 μg / mL to 5 μg / mL.

[0033] Item 19. The method according to any one of Items 10 to 18, wherein the capture antibody in step b) is added to a final concentration of 0.9 μg / mL to 2.5 μg / mL.

[0034] Item 20. The method according to any one of Items 10 to 19, wherein the capture antibody in step b) is added to a final concentration of about 1 μg / mL.

[0035] Item 21. The method according to any one of items 10 to 20, wherein the capture antibody and the tracer antibody are added to the same concentration.

[0036] Item 22. The method according to any one of items 1 to 21, wherein the detectable label of the detection antibody is an enzyme that can convert a colorless form of the detection agent into a colored form of the detection agent.

[0037] Item 23. The detection antibody is specifically bound to the label of the tracer antibody and is conjugated to horseradish peroxidase, and step c) of the method is c)b) The isolated tracer antibody-target antibody conjugate formed in c)b) is incubated with detection antibody conjugated with horseradish peroxidase and ABTS or HPPA. The method described in any one of items 1 to 22.

[0038] Item 24. The method according to any one of items 1 to 23, wherein the presence of a tracer antibody-target antibody-detection antibody complex in the sample obtained in step c) is detected when a signal exceeding the threshold cutoff level / cut point of the assay is obtained.

[0039] Item 25. The method according to any one of items 1 to 23, wherein the presence of a tracer antibody-target antibody-detection antibody complex in the sample obtained in step c) is detected when a signal exceeding a pre-set threshold level is obtained.

[0040] Item 26. The method according to any one of items 1 to 23, wherein the presence of a tracer antibody-target antibody-detection antibody complex in the sample obtained in step c) is detected when a signal is obtained that exceeds a predetermined signal level based on the nonspecific background level of the assay and the response of samples from the drug-untreated target population of subjects processed in steps a) to c).

[0041] The method according to any one of items 1 to 23, wherein the presence of a tracer antibody-target antibody-detection antibody complex in the sample obtained in step c) is detected when a statistically higher signal is obtained compared to a sample treated in steps a) to c) but without the target antibody.

[0042] The method according to any one of items 1 to 23, wherein the presence of a tracer antibody-target antibody-detection antibody complex in the sample obtained in step c) is detected when a signal at least twice as strong as the signal obtained in the sample treated in steps a) to c) but without the target antibody is obtained.

[0043] Item 29. Process c) is, c-1) Transferring the sample obtained in step b) to a solid surface containing an immobilized capture agent that can specifically bind to the capture antibody; c-2) Incubating the sample on the solid surface; c-3) Removing substances that are not bonded to the solid surface by washing; c-4) Incubating the immobilized tracer antibody-anti-drug antibody conjugate on the solid surface with a detection antibody conjugated to a detectable label; c-5) Removing substances not bound to the tracer antibody-antidrug antibody complex immobilized on the solid surface by washing; c-6) To detect a detectable label of the immobilized detection antibody. The method described in any one of items 10 to 28.

[0044] Item 30. The method according to any one of items 10 to 29, wherein the capture agent specifically binds to the label of the capture antibody.

[0045] Item 31. The method according to any one of items 10-30, wherein the capture agent is biotin and the capture antibody is conjugated with avidin / streptavidin, or vice versa.

[0046] Item 32. The method according to any of items 1 to 31, wherein the detection antibody is added so that the final enzyme activity is between 10 mU / mL and 100 mU / mL.

[0047] Item 33. Add the detection antibody so that the final enzyme activity is between 15 mU / mL and 50 mU / mL, according to any of the methods described in items 1 to 32.

[0048] Item 34. The method according to any of items 1 to 33, wherein the detection antibody is added so that the final enzyme activity is approximately 25 mU / mL.

[0049] Item 35. The method according to any one of items 1 to 34, wherein the tracer antibody-target antibody conjugate is isolated from 10 μL to 1000 μL of the solution obtained in step b).

[0050] Item 36. The method according to any one of items 1 to 35, wherein the tracer antibody-target antibody conjugate is isolated from 50 μL to 500 μL of the solution obtained in step b).

[0051] Item 37. The method according to any one of items 1 to 36, wherein the tracer antibody-target antibody conjugate is isolated from 75 μL to 125 μL of the solution obtained in step b).

[0052] Item 38. The method according to any one of items 1 to 37, wherein the tracer antibody-target antibody conjugate is isolated from 100 μL of the solution obtained in step b).

[0053] Item 39. The method according to any one of items 22 to 38, wherein the final concentration of the detection agent is approximately 20 mM.

[0054] Item 40. The method according to any one of items 1 to 39, wherein all steps are carried out at room temperature.

[0055] Item 41. The method according to any one of items 1 to 40, wherein the target antibody is an anti-drug antibody or a therapeutic antibody.

[0056] Item 42. The method described in any of Items 1 to 41, without performing an acid treatment / acid dissociation step.

[0057] Item 43. An immunoassay, as described in any of items 1-42.

[0058] Item 44. Enzyme-linked immunosorbent assay (ELISA), as described in any one of items 1 to 43. [Invention 1001] A method for detecting a target antibody in a sample, comprising the following steps: a) Incubating the sample with a chaotropic salt having a final cation charge normality in the range of 1N to 12N (including the values ​​at both ends); b) Adding a tracer antibody to the sample obtained in step a), and then incubating the sample to form a tracer antibody-target antibody complex in the presence of the chaotropic salt; c)b) The tracer antibody-target antibody complex formed in c)b) is incubated with a detection antibody conjugated with a detectable label to form a tracer antibody-target antibody-detection antibody complex. A method comprising detecting the target antibody when the tracer antibody-target antibody-detection antibody complex is detected in the sample obtained in step c). [Invention 1002] The method of the present invention 1001, wherein the chaotropic salt is a moderate-strength chaotropic salt, preferably having a cation between a potassium cation and a calcium cation in the Hofmeister synthesizer series and an anion between a hydrogen phosphate anion and a nitrate anion in the Hofmeister synthesizer series. [Invention 1003] The method of the present invention 1001 or 1002, wherein the chaotropic salt comprises a cation selected from the group consisting of potassium cations, sodium cations, lithium cations, magnesium cations, and calcium cations, and an anion selected from the group consisting of phosphate (hydrogen) anions, acetate anions, and chloride anions. [Invention 1004] The method according to any one of the present invention 1001 to 1003, wherein the chaotropic salt is MgCl2 or LiCl. [Invention 1005] The method according to any one of the present invention 1001 to 1004, wherein the final normality of the cation charge of the chaotropic salt is in the range of 6.5N to 8.5N (including the values ​​at both ends). [Invention 1006] The method of the present invention 1004 or 1005, wherein the final cation charge normality of MgCl2 is 7.2N±10%, corresponding to a final concentration of MgCl2 of 3.6M±10%, or the final cation charge normality of LiCl is 8N±10%, corresponding to a concentration of 8M±10%. [Invention 1007] The method according to any one of the present invention 1001 to 1006, wherein the incubation in steps a) and b) is 30 minutes ± 10% to 60 minutes ± 10%. [Invention 1008] The method according to any one of the 1001 to 1007 of the present invention, wherein in step b), a capture antibody is further added together with the tracer antibody, either before the tracer antibody or after the tracer antibody, but before the sample is incubated. [Invention 1009] The method according to any one of the 1001 to 1008 of the present invention, wherein in step b), the tracer antibody is conjugated to a label. [Invention 1010] The method of the present invention 1008 or 1009, wherein the capture antibody, the tracer antibody, and the detection antibody are conjugated to different labels, and the label of the capture antibody does not interact with the label of the detection antibody, and vice versa. [Invention 1011] The method according to any one of the 1001 to 1010 of the present invention, wherein the tracer antibody in step b) is added up to a final concentration of 0.9 μg / mL to 2.5 μg / mL. [Invention 1012] The method according to any one of the 1008 to 1011 of the present invention, wherein the capture antibody in step b) is added up to a final concentration of 0.9 μg / mL to 2.5 μg / mL. [Invention 1013] The detection antibody is specifically bound to the label of the tracer antibody and is conjugated to horseradish peroxidase, and step c) of the method is c)b) The isolated tracer antibody-target antibody complex formed in c)b) is incubated with a detection antibody conjugated with horseradish peroxidase and ABTS or HPPA. The present invention relates to any of the methods described in 1001 to 1012. [Invention 1014] Step c) is, c-1) Transferring the sample obtained in step b) to a solid surface containing an immobilized capture agent that can specifically bind to the capture antibody; c-2) Incubating the sample on the solid surface; c-3) Removing substances that are not bonded to the solid surface by washing; c-4) Incubating the immobilized tracer antibody-target antibody complex on the solid surface with a detection antibody conjugated to a detectable label; c-5) Removing substances not bound to the tracer antibody-target antibody complex immobilized on the solid surface by washing; c-6) To detect a detectable label of the immobilized detection antibody. The present invention relates to any of the methods described in 1008 to 1013. [Invention 1015] The method according to any one of items 1001 to 1014 of the present invention, wherein the target antibody is an anti-drug antibody or a therapeutic antibody. [Modes for carrying out the invention]

[0059] Specific embodiments of the present invention In anti-drug antibody (ADA) assays, many different assay formats and methods have been used to address interference by residual therapeutic antibodies (i.e., drugs). In particular, methods that use an acid step to dissociate ADA-drug immune complexes are commonly used to improve assay drug resistance [4, 8, 10, 11]. A common drawback of these methods is that assay conditions, such as pH and acid treatment exposure time, must be carefully optimized to achieve a balance between eliminating drug interference and potential antibody denaturation.

[0060] Here, it was found that improved drug resistance could be achieved in anti-drug antibody (ADA) assays by adding chaotropic salts such as MgCl2 or LiCl. Unexpectedly, the structural integrity of the ADA present in the sample was maintained at the same time.

[0061] For example, it was further found that by adding chaotropic salts such as MgCl2 or LiCl, higher signal-blank values ​​can be achieved with significantly shorter incubation times compared to, for example, overnight incubation or acid pretreatment.

[0062] Therefore, the addition and use of chaotropic salts, such as MgCl2 or LiCl, in ADA assays provides improvements in sensitivity, drug resistance, and handling time. Furthermore, the addition and use of chaotropic salts, such as MgCl2 or LiCl, provides an alternative method when acid pretreatment for drug-ADA complex dissociation is impossible, undesirable, or results in sample distortion.

[0063] I. Definition Unless otherwise specifically defined herein, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specifically required by context, singular terms shall include plural forms and plural terms shall include singular forms. The methods and techniques of this disclosure are generally carried out in accordance with conventional methods well known in the art. Generally, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular biology, and cell biology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, described herein are well known and commonly used in the art.

[0064] Unless otherwise defined herein, the term “comprising of” includes the term “consisting of.”

[0065] As used herein in relation to a specific value (e.g., temperature, concentration, time, etc.), the term "approximately" refers to a variation of + / - 1% of the specific value to which the term "approximately" refers.

[0066] In this specification, the term "antibody" is used in its broadest sense and includes, but is not limited to, a variety of antibody structures, including monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0067] Antibodies generally consist of two so-called light chain polypeptides (light chains) and two so-called heavy chain polypeptides (heavy chains). Each heavy and light chain polypeptide contains a variable domain (variable region) (generally the amino-terminal portion of the polypeptide chain) that contains a binding region that can interact with an antigen. Each heavy and light chain polypeptide also contains a constant region (generally the carboxyl-terminal portion). The constant region of the heavy chain mediates the binding of the antibody to i) cells possessing the Fc gamma receptor (FcγR), such as phagocytes, or ii) cells possessing the neonatal Fc receptor (FcRn), also known as the Brambell receptor. The constant region of the heavy chain also mediates binding to several factors, including classical complement factors such as component (C1q). The constant domain of the antibody heavy chain contains the CH1, CH2, and CH3 domains, while the light chain contains only one constant domain CL, which can be a kappa isotype or a lambda isotype.

[0068] The variable domains of the light or heavy chains of immunoglobulins contain different segments, namely four framework regions (FRs) and three hypervariable regions (HVRs).

[0069] An "antibody fragment" refers to a molecule other than an intact antibody, including a portion of the intact antibody that binds to the antigen to which the intact 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 (dAb); and multispecific antibodies formed from antibody fragments. For a review of specific antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0070] The term "capture antibody" refers to an antibody used in a sandwich ELISA format to bind to (i.e., capture) a target substance present in the sample being detected. A secondary antibody (i.e., detection antibody) then binds to the captured target, enabling the detection of an antibody-target-antibody complex (forming an antibody-target-antibody "sandwich").

[0071] The "class" of an antibody refers to the type of constant domain or constant region it possesses in its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In certain contexts, the antibody is the IgG1 isotype. In certain contexts, the antibody is the IgG1 isotype with P329G, L234A, and L235A mutations to reduce the effector function of the Fc region. In other contexts, the antibody is the IgG2 isotype. In certain contexts, the antibody is the IgG4 isotype with the S228P mutation in the hinge region to improve the stability of the IgG4 antibody. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The light chain of an antibody can be assigned to one of two types, called kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain.

[0072] A "conjugate" is a fusion protein of the present invention conjugated to one or more heterologous molecules, including but not limited to labels, neurotoxic agents, or cytotoxic agents.

[0073] The term "detection antibody" refers to an antibody for which there are means for visualization or quantification. Such means are typically enzymes (catalyzing a colored or fluorescent reaction product after the addition of a suitable substrate), such as horseradish peroxidase, urease, alkaline phosphatase, glucoamylase, and β-galactosidase. In some embodiments, the detection antibody is against the antigen of interest. In some embodiments, the detection antibody is an anti-species antibody. In some embodiments, the detection antibody is conjugated to a detectable label such as biotin, a fluorescent marker, or a radioisotope, and is used for detection and / or quantification.

[0074] The term “detection reagent” refers to a reagent that enables the detection and / or quantification of an antibody bound to an antigen. In some embodiments, the detection reagent is a colorimetric substrate for an enzyme conjugated to an antibody. The addition of a suitable substrate to the antibody-enzyme conjugate results in the generation of a colorimetric or fluorescence measurement signal (e.g., after the conjugated antibody has bound to the antigen of interest). This definition also includes the use of biotin and avidin compounds (e.g., neutraavidin and streptavidin) as part of a detection system.

[0075] The term "ELISA" stands for enzyme-linked immunosorbent assay. Various ELISA formats and applications are known in the art (see, for example, Crowther, "Enzyme-Linked Immunosorbent Assay (ELISA)," 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)).

[0076] One specific ELISA format is the so-called "direct ELISA." In this ELISA format, a target present in the sample, such as a polypeptide, is detected. In direct ELISA, the sample containing the target is brought into contact with a solid phase, such as an immobilized or immobilized support (e.g., a microtiter plate well). If the target is present in the sample, it is immobilized on the solid phase and then directly detected using an enzyme-conjugated detection molecule. If the target is an antigen, the detection molecule is an antibody specific to that antigen, or if the target is an antibody specific to the antigen, the detection molecule is an enzyme-conjugated antibody specific to that antigen.

[0077] Another specific ELISA format is the so-called "indirect ELISA." In this ELISA format, the antigen (or antibody) is immobilized on a solid phase (e.g., a microtiter plate well). Then, an antigen-specific antibody (or antigen) is added, followed by a detection antibody specific to the antibody that specifically binds to the antigen. This detection antibody may be a "species-specific" antibody (e.g., a goat anti-rabbit antibody).

[0078] Another specific ELISA format is the so-called "sandwich ELISA." In this format, the antigen is immobilized on a solid phase (e.g., a microtiter plate well) via capture by an antibody that specifically binds to the antigen (i.e., a capture antibody), which is then immobilized on the solid phase (either covalently or via specific binding pairs). Generally, after adding the sample containing the antigen to the solid phase, it is washed. If the target antigen is present in the sample, it is bound to the solid phase by the capture antibody.

[0079] The above ELISA formats can be combined. A sandwich ELISA can be a "direct sandwich ELISA," where the captured antigen is detected directly using an enzyme-conjugated antibody against the antigen. A sandwich ELISA can be an "indirect sandwich ELISA," where the captured antigen is detected indirectly using an antibody against the antigen, and then detected by another enzyme-conjugated antibody that is either directly bound to the antigen-specific antibody or bound via a label. A third antibody is detected using a reporter reagent.

[0080] The "framework" or "FR" refers to variable domain residues other than the complementarity-determining region (CDR). The variable domain FR generally consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0081] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein synonymously to refer to antibodies having a structure substantially similar to that of a natural antibody or having a heavy chain containing an Fc region as defined herein.

[0082] A "human antibody" is an antibody produced by a human or human cell, or an antibody that has an amino acid sequence corresponding to a non-human antibody that utilizes a sequence encoding a human antibody repertoire or other human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0083] The term "in vitro" refers to either such an artificial environment, or the process or reaction taking place within such an artificial environment.

[0084] The term "in vivo" refers to a compound taking place in its natural environment (e.g., an animal or cell), or a process or reaction taking place within that natural environment.

[0085] The term "immunoassay" refers to any technique that uses specifically binding molecules, such as antibodies, to capture and / or detect specific targets for qualitative or quantitative analysis. Generally, an immunoassay is characterized by the following steps: 1) immobilization or capture of the analyte, and 2) detection and measurement of the analyte. The analyte can be captured, or bound, to any solid surface, such as a membrane, plastic plate, or any other solid surface.

[0086] The term "linker" refers to a chemical linker or single-chain peptide linker that covalently links various entities of the blood-brain barrier shuttle module and / or fusion polypeptide and / or conjugate described herein. A linker, for example, links a brain effector entity to a monovalent binding entity. For example, if the monovalent binding entity includes a CH2-CH3 Ig entity and a scFab directed to a blood-brain barrier receptor, the linker causes the scFab to conjugate to the C-terminus of the CH3-CH2 Ig entity. The linker that conjugates the brain effector entity to the monovalent binding entity (first linker) and the linker that conjugates the scFab to the C-terminus of the CH2-CH3 Ig domain (second linker) may be the same or different.

[0087] A single-chain peptide linker containing 1 to 20 amino acid residues linked by peptide bonds may be used. In certain embodiments, the amino acids are selected from 20 native amino acids. In certain other embodiments, one or more amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In some embodiments, the linker is a chemical linker. In certain embodiments, the linker is a single-chain peptide linker having an amino acid sequence having a length of at least 25 amino acid residues, preferably 32 to 50 amino acid residues in one embodiment. In one embodiment, the peptide linker is a (GxS)n linker where G=glycine, S=serine, (x=3, n=8, 9, or 10) or (x=4 and n=6, 7, or 8), in one embodiment x=4, n=6, or 7, and in a preferred embodiment x=4, n=7.

[0088] Conjugation can be carried out using various chemical linkers. For example, monovalent binding entities or fusion polypeptides and brain effector entities can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidylsberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). The linker may be a “cleavable linker” that facilitates the release of effector entities upon delivery to the brain. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al, Cancer Res. 52 (1992) 127-131; U.S. Patent No. 5,208,020) may be used.

[0089] Covalent conjugation can be either direct or via a linker. In certain embodiments, direct conjugation is by constructing a polypeptide fusion (i.e., by gene fusion of two genes that encode a monovalent binding entity to BBBR and an effector entity and are expressed as a single polypeptide (chain)). In certain embodiments, direct conjugation is by the formation of a covalent bond between a reactive group in one of the two portions of the monovalent binding entity to BBBR and a corresponding group or acceptor on the brain effector entity. In certain embodiments, direct conjugation is by modifying (i.e., genetically modifying) one of the two molecules to be conjugated to contain a reactive group (as a non-limiting example, a sulfhydryl group or a carboxyl group) that forms a covalent bond to the other molecule to be conjugated under appropriate conditions. As one non-limiting example, a molecule (i.e., an amino acid) having the desired reactive group (i.e., a cysteine ​​residue) can be introduced, for example, into the disulfide bond formed by the monovalent binding entity to the BBBR antibody and the neurotherapeutic antibody. Methods for covalent conjugation of nucleic acids and proteins are also known in the art (i.e., photocrosslinking, see, e.g., Zatsepin et al. Russ. Chem. Rev. 74:77-95 (2005)). Conjugation can also be carried out using various linkers.For example, monovalent entities and effector entities can be conjugated using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imide esters (e.g., dimethyl HCl adipiimidoate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). Peptide linkers consisting of 1 to 20 amino acid residues linked by peptide bonds may also be used. In certain such embodiments, the amino acid residues are selected from 20 naturally occurring amino acids. In certain other such embodiments, one or more amino acid residues are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. The linker may be a “cleavable linker” that facilitates the release of effector entities upon delivery to the brain. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al, Cancer Res. 52 (1992) 127-131; U.S. Patent No. 5,208,020) may be used.

[0090] The term "Hofmeister's synergy series" refers to the ranking of anions and cations based on their chaotropic properties, first described by Hofmeister (Arch. Path. Anatom. Pathobiol. 24(1888) 247-260). For anions, this synergy series is as follows: The following applies to the TIFF2026143806000002.tif11142 cation. TIFF2026143806000003.tif5128

[0091] The chaotropic properties of each ion and the salts containing them increase from left to right. Ions presented further to the left are shown as antichaotropic or comotropic ions. These have precipitation properties, i.e., they result in the precipitation of proteins from solution. Ions presented further to the right are shown as chaotropic ions. These have denaturing properties, i.e., they result in the denaturation of proteins in solution.

[0092] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies in the population are identical, except for, for example, naturally occurring mutations or mutant antibodies that may arise during the production of a monoclonal antibody preparation, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed toward different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed toward a single determinant on one antigen. Thus, the modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous collection of antibodies and is not constructed to require antibody production by any particular method. For example, monoclonal antibodies according to the present invention can be produced by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, including but not limited to such methods and other exemplary methods for producing monoclonal antibodies described herein.

[0093] The term "normality" represents a measure of concentration equal to the gram equivalent weight of solute per liter of solution. The formula for normality is N = M * n, where n = number of equivalents / number of single-charged ions that can react. In the case of MgCl2, to convert normality to molar concentration, or vice versa, a 1M solution equals a 2M chloride ion solution and a 1M Mg 2+ We must take into account the formation of an ionic solution, which also has a value of n of 2 due to its charge. Therefore, in this case, N = (1M)(2) = 2N, i.e., MgCl2 has a final normality of 2N of cation charge.

[0094] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to the antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen may be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain of the antibody that binds to the antigen.

[0095] As used herein, the terms “hypervariable region” or “HVR” mean each of the regions of an antibody variable domain, such as “complementarity-determining regions” (CDRs), that are hypervariable within a sequence and determine antigen-binding specificity. These regions form paratopes or binding sites.

[0096] Generally, antibodies contain six antigen-binding specificity-determining regions: three in the VH region (H1, H2, H3) and three in the VL region (L1, L2, L3). Exemplary antigen-binding specificity-determining regions as used herein include: (a) Hypervariable loops (HVRs) present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)); (b) Complementarity-determining regions (CDRs) located at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); (a+b) HVR combined with CDR present at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 26-35 (H1), 50-65 (H2), and 95-102 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987) + Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); Furthermore (c) Antigen contact present at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J.Mol.Biol.262:732-745 (1996)).

[0097] Unless otherwise specified, HVR is determined according to Kabat et al. above. Those skilled in the art will understand that the notation of the antigen-binding specificity region may also be determined according to Chothia above, McCallum above, or any other scientifically acceptable nomenclature system.

[0098] As used herein, the term “signal” encompasses any detectable physical change that can be used to indicate that a reaction has occurred, for example, the binding of an antibody to its antigen. Signals in the form of fluorescent or colorimetric products / reagents are specific forms of signals intended to be used in the methods of the present invention. In some embodiments of the present invention, the signal is evaluated quantitatively.

[0099] "Solid phase" refers to non-fluid matter and includes particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic and ferromagnetic particles), glass, and ceramics; gel materials such as silica, alumina, and polymer gels; capillaries which may be made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. "Solid support" distinguishes the solid phase components of an assay from inert solid surfaces in that it contains at least one portion on its surface that is intended to chemically interact with molecules. The solid phase may be stationary components such as tips, tubes, strips, cuvettes, or microtiter plates, or non-stationary components such as beads and microparticles. Various microparticles that enable either non-covalent or covalent bonding of proteins and other substances may be used. Such particles include polymer particles such as polystyrene and poly(methyl methacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. For example, see Martin, CR, et al., Analytical Chemistry - News & Features, 70(1998) 322A-327A, or Butler, JE, Methods 22(2000) 4-23.

[0100] The terms “therapeutic (monoclonal) antibody” and “drug” are used interchangeably herein. The term “antibody” as used herein is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0101] As used herein, the term “sample” refers to any biological matrix on which the ADA response can be determined. Exemplary samples, though not limited to them, include serum, plasma, aqueous humor, vitreous fluid, retinal tissue lysate, and tumor tissue. In one preferred embodiment, the sample is plasma.

[0102] As used herein, the term “anti-drug antibody” refers to an antibody against a therapeutic antibody that is produced by the innate immune system of a recipient of a therapeutic antibody after administration of the therapeutic antibody.

[0103] As used herein, the term "immunogenicity" refers to the ability of a therapeutic antibody to induce an immune response in humans or animals. During drug development, immunogenicity is primarily assessed by measuring the binding and neutralizing anti-drug antibodies.

[0104] II. Specific compositions and methods according to the present invention This specification provides a method for detecting antibodies, particularly anti-drug antibodies (ADAs), in serum or plasma samples, with a focus on high sensitivity and drug resistance (drug resistance refers to the ability of the assay to analyze ADAs in the presence of residual drugs) (ADA screening assays).

[0105] The assay according to the present invention differs from those known in the art in its assay setup; that is, a complex containing a target antibody and a tracer antibody is formed in the presence of the same chaotropic salt used to dissociate immune complexes in the sample being analyzed. This increases the reactivity of the assay according to the present invention, thereby increasing the sensitivity of the assay.

[0106] The method of the present invention involves the formation and detection of a complex of ADA and a tracer antibody in solution. By using a chaotropic salt, the complex of ADA with residual drugs is predissociated (in the case of samples from patients still undergoing drug treatment), and the tracer antibody is incubated simultaneously with ADA.

[0107] The methods described in this art have significant drawbacks when applied to clinical samples containing circulating residual therapeutic agents. For example, when chronic diseases are treated and therapeutic drug levels are established, high concentrations of residual drugs are very often present in the sample. In these cases, anti-drug antibodies bind to circulating therapeutic agents. As a result, in the case of circulating drugs, the ADA epitope is masked by the circulating drug (e.g., by the formation of a dimeric complex), and the tracer drug cannot form a complex with ADA. Therefore, these complexed ADAs in the sample cannot be used for detection, and only free ADA can be detected.

[0108] The ability of an assay to detect ADA in the presence of residual drugs is known as drug resistance.

[0109] One aspect of the present invention is a robust, high-throughput compatible high ionic strength dissociation assay (HISDA) / method in which MgCl2 is used as a non-denaturing ionic strength modifier to achieve high drug resistance in the assay. In two case studies, the method of the present invention has been shown to be effective in improving drug resistance without adversely affecting assay reagents. Therefore, the HISDA method according to the present invention provides a highly sensitive, drug-resistant, and easy-to-use method that can be used to improve drug resistance in any assay.

[0110] One aspect of the present invention is a method for determining the presence of an anti-drug antibody in a sample, comprising the following steps: a) Incubating the sample (or aliquots of the sample) with MgCl2 or LiCl at a final concentration in the range of 1M to 6M / Adding MgCl2 to the sample to a final concentration in the range of 1M to 6M, and then incubating the sample; b) Adding tracer antibody to the sample obtained in step a), and then incubating the sample; c)b) The isolated tracer antibody-anti-drug antibody conjugate formed in c)b) is incubated with a detection antibody conjugated with a detectable label. This method includes step c), and the presence of an anti-drug antibody is determined if a tracer antibody-anti-drug antibody-detection antibody complex is detected in the sample obtained in step c).

[0111] In the first step of sample analysis, all samples are analyzed to determine whether they are positive or negative for anti-drug antibodies (final dilution of 1:100). This screening assay yields a yes / no answer based on a predetermined cutoff point. The cutoff point should be defined to allow for a maximum of 5% false positives.

[0112] In the second step, all positive samples are analyzed for specificity using an additional confirmatory assay to filter out false positives from the initial screening assay.

[0113] The following is presented as an example of a general method according to the present invention using MgCl2 as the chaotropic salt. This is merely illustrative and should not be treated as an limitation. The true scope is described in the appended claims.

[0114] The high ionic strength dissociation assay (HISDA) according to the present invention was evaluated in two studies to achieve high drug resistance while maintaining the best possible structural integrity of ADA.

[0115] The improvement in drug resistance by MgCl2 treatment was demonstrated using two investigational antibodies: mAb-1, an anti-latent myostatin antibody, and mAb-2, an anti-human Aβ antibody (see mAb-1-based ADA assay and mAb-2-based ADA assay – Examples 1 and 2, respectively). Each assay was first compared using three different variations: overnight incubation (Example 4), acid treatment (Example 3), and MgCl2 treatment (Examples 1 or 2, respectively). Overnight incubation was used as a “benchmark” in terms of PC stability and drug resistance to the other two treatments, as it is a commonly used method with no risk of antibody damage.

[0116] First, we evaluated the negative effects of acid vs. MgCl2 treatment on the quantification of PC compared to overnight incubation. The evaluation was based on formal acceptance criteria for bioanalytical methods [1,18].

[0117] To enable direct comparison of different treatments, signal-blank (SB) values ​​were plotted against PC1 serum concentration (Figure 2, left). Furthermore, all values ​​were normalized against the benchmark "overnight incubation" and plotted against PC1 serum concentration to evaluate potential signal inhibition exceeding 20% ​​(Figure 2, right).

[0118] As shown in Figure 2 (right), samples treated with acid produced significantly lower SB values ​​compared to samples incubated overnight or treated with MgCl2. This observation demonstrated a negative effect of low pH treatment on PC1 quantification. While not bound by this theory, this is likely due to the denaturation of PC1 and / or the assay reagent (mAb-1-biotin / mAb-1-digoxigenin). Compared to acid treatment, MgCl2 addition by the present invention showed a higher dynamic range while having the same SB as the overnight approach at low PC1 concentrations.

[0119] Therefore, using the method of the present invention offers several advantages by eliminating the need for acid treatment. First, time-consuming evaluation work, such as scouting pH and exposure time to find conditions that are not too harsh on key assay components, is no longer necessary. Second, the risk that such optimized acid treatment conditions using artificial PCs may not be transferable to human ADA

[10] , which has been considered a major limitation of this approach, is no longer present. Third, the risk of targeted dimerization that can lead to false-positive results in cross-linking assays[8] is no longer present. Fourth, the risk of antibody deamidation that can negatively affect binding efficacy

[19] is no longer present.

[0120] Because elevated drug levels are increasingly common in the clinical population, assay drug resistance was tested at antibody concentrations up to 500 μg / mL serum concentration. To address this, different amounts of PC1 were tested for positivity in the presence of scalding concentrations of mAb-1 based on a screening cutpoint (see Example 6).

[0121] Both methods showed good sensitivity to PC1 serum concentrations of at least 16 ng / mL in the absence of mAb-1. At high PC1 concentrations in the absence of mAb-1, the present invention's method using MgCl2 addition showed improved SB values ​​(41 vs. 88 for 4000 ng / mL PC1 for overnight incubation and MgCl2 treatment, respectively).

[0122] Low mAb-1 serum concentrations of 1 μg / mL were generally well tolerated across the entire PC1 concentration range in the present invention's method using MgCl2 supplementation. However, in the case of the overnight approach, the lowest PC1 concentration (16 ng / mL) was determined to be negative, which is a false negative result.

[0123] In the presence of increasing mAb-1 levels, the present invention's method using MgCl2 addition showed improved sensitivity compared to overnight incubation. With 63 ng / mL PC1 and 500 μg / mL mAb-1, analysis yielded SB values ​​of 1.03 (overnight incubation) and 1.29 (MgCl2 addition).

[0124] The drug resistance achieved (detection of 63 ng / mL PC in the presence of 500 μg / mL drug) was within a similar range to that of novel acid-based methods such as PandA (detection of 14 ng / mL PC in the presence of 100 μg / mL drug) [8], but the method of the present invention does not carry the same risks as those associated with acid-based methods.

[0125] The method of the present invention, using the addition of MgCl2, achieved both the sensitivity and the required drug resistance necessary for assay certification under FDA guidelines. Therefore, the method of the present invention was successfully validated.

[0126] In a second case study using an mAb-2-based ADA assay, different assay variations (overnight incubation, acid treatment, and MgCl2 treatment) were compared in a similar manner based on the PC2 calibration curve in the absence of mAb-2 (Figure 4).

[0127] For mAb-2, either PC2 and / or the assay reagent (mAb-2-biotin / mAb-2-digoxigenin) was clearly more resistant to acid treatment, similar to mAb-1 (Figure 4, left vs. Figure 2, left). This is not an unusual phenomenon. Different antibodies have been reported to react differently to acid treatment. Kavita, U. et al. showed that some low-affinity and medium-affinity monoclonal antibodies were sensitive to glycine pH 2.2 treatment by losing activity in the ADA assay, while others were unaffected

[12] . Nevertheless, the method of the present invention still yielded higher SB values ​​compared to both overnight incubation and acid treatment.

[0128] Compared to overnight incubation, both MgCl2 addition and acid treatment by the method of the present invention consistently produced higher SB values ​​across the entire PC2 concentration range. This observation can be explained by the decrease in the blank value: absorbance units decreased from 0.064 (overnight incubation) to 0.028 (MgCl2 addition) and 0.040 (acid treatment).

[0129] Because elevated drug levels are increasingly common in the clinical population, assay drug resistance was tested using antibody concentrations up to 100 μg / mL serum using all three variations of the mAb-2-based ADA assay.

[0130] High sensitivity for PC2 plasma concentrations of at least 25 ng / mL was achieved in the absence of mAb-2 using all three methods.

[0131] Analysis of PC2 at 25 ng / mL and mAb-2 at 100 μg / mL yielded SB values ​​of 0.94 (overnight incubation), 0.94 (acid treatment), and 2.22 (MgCl2 addition). Based on a screening cutpoint of 1.08, the improvement with MgCl2 addition was significant and even more pronounced than in the initial case study.

[0132] In overnight incubation or acid treatment, all relevant samples were screened as false negatives, making it impossible to achieve the desired sensitivity for a PC2 plasma concentration of 100 ng / mL, even in the presence of a low mAb-2 concentration of 1 μg / mL. In contrast, with the method of the present invention using MgCl2 addition, the same mAb-2 concentration was very well tolerated across the entire PC2 concentration range.

[0133] Both sensitivity and the required drug tolerance threshold level were achieved in this second case study using the method of the present invention with MgCl2 addition. The method was subsequently deemed successful.

[0134] The method of the present invention was used for the analysis of clinical trial samples. In a study using mAb-1-like antibodies, a false positive error rate (FPER) of 2.6% was determined (39 samples). In two further studies using mAb-2-like antibodies, FPERs of 4.9% (366 samples) and 3.4% (354 samples) were determined, respectively.

[0135] The above demonstrates further advantages of the method of the present invention. Generally, methods for determining target antibodies in a sample are validated using a predetermined number of samples obtained from untreated healthy humans (a drug-free target population). This determines the cutpoint, i.e., the threshold for distinguishing negative and positive samples. The same applies to clinical samples. However, if the observed FPER of clinical baseline samples under study is within the range of 2-11% after excluding samples with pre-existing ADA, the cutpoint of the same screening assay and the confirmation cutpoint values ​​determined from pre-study validation can be applied to the evaluation of clinical study samples. However, if the FPER is less than 2% or greater than 11%, new test-specific SCPs and CCPs should be determined using clinical trial baseline samples (see, e.g., Devanarayan, V., et al., AAPS J.19(2017)1487-1498). Thus, the method of the present invention has been shown to be close to or even below the target of 5%. This eliminates the need to redo assay development.

[0136] Therefore, in two different case studies and clinical trials, the addition of MgCl2 was successfully used to dissociate immune complexes formed by ADA and therapeutic agents, resulting in improved assay drug resistance.

[0137] Maintaining ADA binding activity is equally important regarding complex dissociation in immunogenicity studies, otherwise screening results may be falsified. In both case studies, there was no negative effect of adding MgCl2 by the method of the present invention on PC and / or assay reagents.

[0138] The simplicity of the HISDA protocol according to the present invention makes this method less prone to errors compared to methods with more steps and / or those requiring precise setting of pH values ​​and exposure times. The combination of a relatively small number of adjustable parameters and a short incubation time also makes this method suitable for high-throughput applications.

[0139] As the number of therapeutic drug candidates increases, the demand for immunogenicity testing will continue to grow. The biological analytical methods used in immunogenicity testing are particularly important because they are used to generate important clinical data. Such methods must be developed to be free from technical biases such as interference from the drug. The use of the method of the present invention enables the generation of meaningful and clear data, thus enabling improved and robust immunogenicity testing. The principle of the HISDA method according to the present invention can be applied to any type of immunoassay whose performance is influenced by its binding partner. This is of particular interest for bioanalytical support of the pharmacokinetic evaluation of novel drug candidates when clear-defined total drug information

[21] is required in the presence of relevant concentrations of soluble ligands or anti-drug antibodies [9,22].

[0140] The following embodiments and drawings are provided to aid in understanding the present invention, and the true scope of the invention is described in the appended claims. It is understood that modifications to the described procedures can be made without departing from the spirit of the invention.

[0141] References List of References TIFF2026143806000004.tif182142 [Brief explanation of the drawing]

[0142] [Figure 1]A 4M MgCl2*6H2O solution is added to all samples and incubated at room temperature for 30 minutes to allow dissociation of potentially present immune complexes formed by anti-drug antibodies (ADA) and monoclonal antibodies (mAb), indicated by the dashed lines. After the initial incubation, the released ADA is complexed by adding labeled assay reagents (mAb-biotin and mAb-digoxigenin) and incubated at room temperature for 30 minutes. In the subsequent step, the formed immune complexes are captured using a streptavidin-coated microtiter plate and detected using a horseradish peroxidase-labeled secondary antibody. [Figure 2] Comparison of assay variations: Left: Different amounts of pAb tested in three variations of the mAb-1-based ADA assay (overnight incubation, MgCl2 addition and acid treatment according to the present invention) plotted against PC1 serum concentration. <mab-1>Signal versus blank (SB) values ​​for samples treated with Rb(PC1). Right: All SB values ​​were normalized to the SB values ​​of samples incubated overnight and plotted against PC1 serum concentration to assess potential signal inhibition exceeding 20%. [Figure 3] Drug resistance assessment: Different doses of pAb <mab-1>Samples treated with Rb(PC1) and mAb-1 were analyzed using two variations of the mAb-1-based ADA assay: overnight incubation (left) and MgCl2 addition according to the present invention (right). Corresponding signal-blank (SB) values ​​were plotted against PC1 serum concentration and compared to the cutoff point to assess assay drug resistance. [Figure 4] Comparison of assay variations: Left: Different amounts of mAb tested in three variations of the mAb-2-based ADA assay (overnight incubation, MgCl2 addition and acid treatment according to the present invention) plotted against PC2 plasma concentration. <mab-2>Signal-blank (SB) values ​​of samples treated with M(PC2). Right: All SB values ​​were normalized to the SB values ​​of samples incubated overnight and plotted against PC2 serum concentration to evaluate potential signal inhibition exceeding 20%. [Figure 5] Drug tolerance assessment: Different amounts of mAb <mab-2>Samples treated with M(PC2) and mAb-2 were analyzed using three variations of the mAb-2-based ADA assay: overnight incubation (left), acid treatment (middle), and MgCl2 addition according to the present invention (right). Corresponding signal-blank (SB) values ​​were plotted against PC2 plasma concentration and compared to the cutoff point to assess assay drug resistance. [Figure 6] A typical calibration curve for the method of the present invention. [Examples]

[0143] material Positive control Against therapeutic monoclonal mAb-1, rabbit-derived polyclonal antibody (pAb <mab-1>Rb (Roche Diagnostics GmbH, Germany) was used as a positive control (PC1) in an mAb-1 based ADA assay. It was dissolved at 2.0 mg / mL in 1× phosphate-buffered saline (PBS; Roche Diagnostics GmbH, Germany).

[0144] Against therapeutic monoclonal mAb-2, mouse-derived monoclonal antibody (mAb <mab-2>M (Roche Diagnostics GmbH, Germany) was used in an mAb-2 based ADA assay (PC2). It was dissolved at a concentration of 5.4 mg / mL in aqueous solutions of 50 mM potassium phosphate (Merck Chemicals GmbH, Germany) and 150 mM potassium chloride (Merck Chemicals GmbH, Germany), pH 7.5.

[0145] Human Matrix Human pooled serum and human pooled K3 EDTA plasma (both mixed from males and females) were obtained from TRINA Bioreactives AG in Switzerland.

[0146] Example 1 mAb-1-based ADA assay according to the present invention (with MgCl2 treatment) For the qualitative detection of antibodies against therapeutic monoclonal mAb-1, ELISA (enzyme-linked immunosorbent assay) was used.

[0147] Quality control samples were prepared from pooled human serum using PC1. The quality control, negative control, and test samples were diluted 1:10 (5 μL + 45 μL) with 4 M MgCl2*6H2O solution (VWR International bvba, Belgium) and incubated at room temperature for 30 minutes with shaking at 450 rpm. Then, all samples were diluted 1:10 (30 μL + 270 μL) with 1x PBS containing 1x Western blocking reagent (Merck Chemicals GmbH, Germany) along with 900 ng / mL mAb-1-biotin and 900 ng / mL mAb-1-digoxigenin, and incubated at room temperature for 30 minutes with shaking at 450 rpm. The formed immunocomplexes (100 μL) were transferred to streptavidin (SA) coated microtiter plates (MTP) and incubated at room temperature for 1 hour with shaking at 450 rpm to immobilize the immunocomplexes via biotin-labeled capture antibody.

[0148] After three washing steps using 300 μL of 1×PBS (phosphate-buffered saline) containing 0.05% (v / v) Tween 20, 100 μL of 25 mU / mL horseradish peroxidase (HRP)-labeled anti-digoxigenin Fab fragment (<digoxigenin>HRP; Roche Diagnostics GmbH, Germany), diluted in 1×PBS containing 0.5% (w / v) BSA (Merck Chemicals GmbH, Germany), was added to MTP and incubated at room temperature for 1 hour with shaking at 450 rpm.

[0149] After three washing steps, the substrate reaction was carried out by adding 100 μL / well of 20 mM 3-p-hydroxyphenylpropionic acid (HPPA; Merck Chemicals GmbH, Germany) supplemented with a 0.1 M tris(hydroxymethyl)aminomethane (TRIS; Merck Chemicals GmbH, Germany) solution and a 0.02% (v / v) hydrogen peroxide solution dissolved at pH 8.5 with 30% (w / w) (H2O2; Merck Chemicals GmbH, Germany), and incubated at room temperature for 10 minutes with shaking at 450 rpm

[16] . Fluorescence intensity was determined at optimal gain using an excitation wavelength of 320 nm and an emission wavelength of 400 nm on a microplate reader (Infinite F200; Tecan, Switzerland).

[0150] A typical calibration curve is shown in Figure 6. The values ​​are shown in the table below.

[0151] TIFF2026143806000005.tif30143

[0152] A sample was defined as "potentially ADA positive" if the associated signal was above a screening cut-point value calculated to produce a 5% false-positive rate, based on the assumption of a 5% coefficient of variation for screening individual donors. The assay had sensitivity for at least 16 ng / mL of PC1 in 100% human serum. Potentially ADA positive results were confirmed in a second confirmatory assay identical to the screening assay, except that the test samples were incubated with excess therapeutic monoclonal mAb-1 (100 μg / mL final assay concentration) in mAb-1-Bi-containing buffer and mAb-1-Dig-containing buffer.

[0153] This method was developed and certified in accordance with recommendations [1, 17] and has been successfully validated.

[0154] Example 2 mAb-2-based ADA assay according to the present invention (with MgCl2 treatment) Cross-linked ELISA was used for the qualitative detection of antibodies against therapeutic monoclonal mAb-2.

[0155] Quality control samples were prepared from human pooled K3EDTA plasma using PC2. 5 μL of quality control, negative control, and test samples were diluted in 45 μL of 4M MgCl2*6H2O solution to obtain a 1:10 dilution and incubated at room temperature for 30 minutes with shaking at 450 rpm. Subsequently, 30 μL of the total sample was diluted 1:10 with 270 μL of Roche Universal Buffer (Roche Diagnostics GmbH, Germany) along with 2000 ng / mL mAb-2-biotin and 2000 ng / mL mAb-2-digoxigenin and incubated at room temperature for 30 minutes with shaking at 450 rpm. The formed immunocomplexes were transferred to SA-coated MTP (100 μL / well) and incubated at room temperature for 1 hour with shaking at 450 rpm.

[0156] After three washing steps using 300 μL of 1×PBS containing 0.05% (v / v) Tween 20, a 25 mU / mL anti-digoxigenin antibody-HRP conjugate diluted in Roche Universal Buffer (100 μL / well) was added to the MTP and incubated at room temperature for 1 hour with shaking at 450 rpm.

[0157] After three washing steps, the substrate reaction was carried out by adding 100 μL / well of 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid solution (ABTS; Roche Diagnostics GmbH, Germany). The absorbance of the quality control sample containing 1600 ng / mL of PC2 was measured at a wavelength of 405 nm with 490 nm as the reference wavelength using a microplate reader (Sunrise; Tecan, Switzerland) until the absorbance unit reached 2.0 ± 0.1. The final absorbance was calculated as follows: absorbance(405 nm) - absorbance(490 nm).

[0158] A sample was defined as "potentially ADA positive" if the associated signal was above a screening cut-point value calculated to produce a 5% false-positive rate, based on the assumption of a 5% coefficient of variation for screening individual donors. The assay had sensitivity for at least 25 ng / mL of PC2 in 100% human plasma. Potentially ADA positive results were confirmed in a second confirmatory assay identical to the screening assay, except that the test samples were incubated with excess therapeutic monoclonal mAb-2 (100 μg / mL final assay concentration) in mAb-2-Bi-containing buffer and mAb-2-Dig-containing buffer.

[0159] This method was developed and certified in accordance with the recommendations [1, 17].

[0160] Example 3 - Comparative Example Acid treatment Acid dissociation was performed using mAb-1-based and mAb-2-based ADA assays according to the corresponding assay protocols, with the following differences compared to the MgCl2 treatment in Examples 1 and 2: 3 μl each of the quality control, negative control, and test samples were diluted in 17 μl of the corresponding assay buffer, followed by the addition of 100 μl of 0.1 M glycine-HCl pH 2.0 (Merck Chemicals GmbH, Germany) to obtain a final dilution of 1:40, and incubated at room temperature for 30 minutes with shaking at 450 rpm. Subsequently, all samples were adjusted to neutral pH by 2.5-fold dilution with 0.5 M Tris-HCl pH 8.5 (Merck Chemicals GmbH, Germany) along with the corresponding concentrations of biotin-labeled and digoxigenin-labeled assay reagents, and incubated at room temperature for 30 minutes with shaking at 450 rpm (120 μl acidified sample; 30 μl labeling reagent; 150 μl 0.5 M Tris buffer). Next, the formed immune complexes (100 μL / well) were transferred to SA-coated MTP as described in the corresponding MgCl2 assay protocols of Examples 1 and 2.

[0161] Example 4 - Comparative Example Incubate overnight Assay modification "overnight incubation" was performed using mAb-1-based and mAb-2-based ADA assays according to the corresponding assay protocols, but differed from the MgCl2 treatment in Examples 1 and 2 in the following respects: 3 μL of quality control sample, negative control sample, and test sample were diluted 1:50 with 147 μL of the corresponding assay buffer. Then, all 50-fold diluted samples were further diluted 1:2 by adding 150 μL of assay buffer containing the corresponding concentrations of biotin-labeled assay reagent and digoxigenin-labeled assay reagent, and incubated overnight at room temperature with shaking at 450 rpm. The following day, the formed immunocomplexes were transferred to SA-coated MTP (100 μL / well) as described in the corresponding MgCl2 assay protocols of Examples 1 and 2.

[0162] Example 5 Drug resistance The ability to detect ADA in the presence of therapeutic agents (assay drug resistance) was determined in each ADA assay using the respective PC and drug. The combinations tested were PC1 / mAb-1 and PC2 / mAb-2. PC concentrations were selected to meet the FDA's recommended sensitivity of at least 100 ng / mL[1] for ADA assays, and drug concentrations were selected based on the expected level of circulating drug in the test sample.

[0163] Different amounts of each PC were added to ADA-negative human serum or plasma samples in the presence or absence of different drug concentrations, and incubated at room temperature for 3 hours with shaking at 450 rpm to form immune complexes. The samples were then frozen and stored overnight at -80°C, and analyzed the following day according to the corresponding assay protocol. The highest drug concentration that yielded an average signal above the screening cut-off point was considered assay drug resistance to a given PC concentration.

[0164] Example 6: Determination of screening cut points The sensitivity of the assay was evaluated by screening 64 healthy volunteer serum samples, according to the description provided for mAb1 (Example 1). The signals were normalized to the corresponding serum pool values. The normalized values ​​are shown in the table below.

[0165] TIFF2026143806000006.tif91132

[0166] The normalized values ​​were checked for normality using R (version 3.5.1 (2018-07-02) "Shapiro-Wilk normality test") to determine if they were non-normal. Next, 64 values ​​were analyzed using an outlier test based on 1.5 IQR, and three values ​​were excluded (ID: 23, 25, 49). The test was then retested for a normal distribution with p-value = 0.01, but the distribution remained non-normal. Because of the non-normal distribution, the sensitivity was calculated based on 95 outlier values ​​(quantil function, Microsoft Office Standard 2016), resulting in 1.115. By inverse calculation (extrapolation), a sensitivity of 4.96 ng / mL PC1 in 100% serum was obtained. Calibration data based on normalized signal versus PC1 concentration are shown in the table below.

[0167] TIFF2026143806000007.tif16130

[0168] Example 7: Bond disruption and maintenance of analyte activity by using LiCl An antibody with a molecular weight of 145.8 kDa was used as the analyte. The antibody could bind to recombinant human (rh) mesothelin in either a "1:1" or "1:2" complex. To evaluate the complex disruption properties of LiCl, the antibody was incubated at different concentrations, with different ratios of rh-mesothelin forming a total of 50% of the total free and partially free antibodies, based on the total concentration of antibody used in the sample. Horse serum was used as the sample matrix. These samples were analyzed by a homogeneous ligand-binding assay using biotinylated rh-mesothelin as a capture reagent and a human IgG-specific detection antibody conjugated to ruthenium for signal generation. QC samples were analyzed in two ways: one by incubation with 8M LiCl to disrupt the complex and analyze the total antibody concentration, and the other without LiCl to analyze the free antibody concentration by confirming the stability of the complex during the assay procedure.

[0169] TIFF2026143806000008.tif89143

[0170] The results demonstrate sufficient complex disruption by maintaining antibody binding properties within the investigated serum range of 100–200,000 ng / mL.

Claims

1. A method for detecting a target antibody in a sample, comprising the following steps: a) Incubating the sample with a chaotropic salt having a final cation charge normality in the range of 1 N to 12 N (including the values ​​at both ends); b) Adding a tracer antibody to the sample obtained in step a), and then incubating the sample to form a tracer antibody-target antibody complex in the presence of the chaotropic salt; c) The tracer antibody-target antibody complex formed in b) is incubated with a detection antibody conjugated with a detectable label to form a tracer antibody-target antibody-detection antibody complex. A method comprising step c) wherein the target antibody is detected when the tracer antibody-target antibody-detection antibody complex is detected in the sample obtained in step c).

2. The method according to claim 1, wherein the chaotropic salt is a moderate-strength chaotropic salt, preferably having a cation between a potassium cation and a calcium cation in the Hofmeister synthesizer series and an anion between a hydrogen phosphate anion and a nitrate anion in the Hofmeister synthesizer series.

3. The method according to claim 1 or 2, wherein the chaotropic salt comprises a cation selected from the group consisting of potassium cations, sodium cations, lithium cations, magnesium cations, and calcium cations, and an anion selected from the group consisting of phosphate (hydrogen) anions, acetate anions, and chloride anions.

4. The aforementioned chaotropic salt is MgCl ２ The method according to any one of claims 1 to 3, wherein the material is LiCl.

5. The method according to any one of claims 1 to 4, wherein the final normality of the cation charge of the chaotropic salt is in the range of 6.5 N to 8.5 N (including the values ​​at both ends).

6. MgCl ２ The final normality of the cation charge of MgCl is ２ The method according to claim 4 or 5, wherein the final concentration of is 7.2 N ± 10%, corresponding to a final concentration of 3.6 M ± 10%, or the final cation charge normality of LiCl is 8 N ± 10%, corresponding to a concentration of 8 M ± 10%.

7. The method according to any one of claims 1 to 6, wherein the incubation in steps a) and b) is 30 minutes ± 10% to 60 minutes ± 10%.

8. The method according to any one of claims 1 to 7, wherein in step b), a capture antibody is further added together with the tracer antibody, either before the tracer antibody or after the tracer antibody, but before the sample is incubated.

9. The method according to any one of claims 1 to 8, wherein in step b), the tracer antibody is conjugated with a label.

10. The method according to claim 8 or 9, wherein the capture antibody, the tracer antibody, and the detection antibody are conjugated to different labels, and the label of the capture antibody does not interact with the label of the detection antibody, and vice versa.

11. The method according to any one of claims 1 to 10, wherein the tracer antibody in step b) is added to a final concentration of 0.9 μg / mL to 2.5 μg / mL.

12. The method according to any one of claims 8 to 11, wherein the capture antibody in step b) is added to a final concentration of 0.9 μg / mL to 2.5 μg / mL.

13. The detection antibody is specifically bound to the label of the tracer antibody and is conjugated to horseradish peroxidase, and step c) of the method is c) The isolated tracer antibody-target antibody conjugate formed in b) is incubated with a detection antibody conjugated with horseradish peroxidase and ABTS or HPPA. The method according to any one of claims 1 to 12.

14. Step c) is, c-1) Transferring the sample obtained in step b) to a solid surface containing an immobilized capture agent that can specifically bind to the capture antibody; c-2) Incubating the sample on the solid surface; c-3) Removing substances that are not bonded to the solid surface by washing; c-4) Incubating the immobilized tracer antibody-target antibody complex on the solid surface with a detection antibody conjugated to a detectable label; c-5) Removing substances that are not bound to the tracer antibody-target antibody complex immobilized on the solid surface by washing; c-6) Detecting a detectable label of the immobilized detection antibody. The method according to any one of claims 8 to 13.

15. The method according to any one of claims 1 to 14, wherein the target antibody is an anti-drug antibody or a therapeutic antibody.

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