Method for reducing target interference in Anti-drug antibody assay

Abstract

To provide a method and a system for detecting, quantifying, or characterizing an anti-drug antibody induced by administration of a pharmaceutical.SOLUTION: A method for identifying anti-drug antibodies in a sample includes: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a target binding partner; and detecting presence of a complex including the first labeled drug, an anti-drug antibody, and the second labeled drug, wherein the sample includes an anti-drug antibody and a target, and the target is a binding partner of the drug.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Field The present invention relates generally to methods and systems for characterizing, identifying, and / or measuring anti-drug antibodies induced by the administration of pharmaceutical agents, which methods and systems are based on competitive ligand binding. [Background technology]

[0002] background Anti-drug antibodies (ADAs) may contribute to several clinical outcomes, such as reduced drug efficacy, cross-reactivity to endogenous proteins, or altered pharmacokinetics of therapeutic proteins, raising concerns about drug efficacy and patient safety due to the presence of antibodies induced by pharmaceutical administration, for example, by the induction of ADAs. The FDA recommends adopting a risk-based approach to assess and mitigate immune responses related to adverse immunological responses associated with therapeutic protein products that affect safety and efficacy (Guidance for Industry: Immunogenicity Assessment for Therapeutic Protein Products, August 2014, USDapartment of Health and Human Services, Food and Drug Administration (Non-Patent Document 1)).

[0003] The prescribing information and FDA clinical pharmacology reviews of 121 FDA-approved biologics were reviewed to evaluate and report immunogenicity data, including monoclonal antibodies, enzyme products, cytokines, growth factors, and toxins. The incidence of immunogenicity was most frequently reported. The clinical significance of ADAs was unclear. Overall, there was a striking agreement between increased systemic clearance of the product and reduced efficacy associated with ADAs. (Wang et al., Evaluating and Reporting the Immunogenicity Impacts for Biological Products—a Clinical Pharmacology Perspective. The AAPS Journal. 2016;18(2):395-403)

[0004] The biological complexity of immune response presents a challenge in assessing the impact of ADA on pharmacokinetics, because pharmacokinetic exposure may be more sensitive than efficacy endpoints for assessing ADA effects.It will be recognized that there is a need for improved methods and systems for characterizing, identifying, and / or measuring ADA, for example, ADA detection methods and systems.These methods and systems can provide valuable information about immunogenicity effects in clinical pharmacology related to pharmacokinetics, efficacy, and safety for drug administration, for example, administration of biologics. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Guidance for Industry:Immunogenicity Assessment for Therapeutic Protein Products,August 2014,USDepartment of Health and Human Services,Food and Drug Administration [Non-patent document 2] Wang et al.,Evaluating and Reporting the Immunogenicity Impacts for Biological Products-a Clinical Pharmacology Perspective.The AAPS Journal.2016;18(2):395-403 Summary of the Invention

[0006] overview Biologics, such as monoclonal antibodies, are therapeutic proteins with clinical applications across a wide range of pathologies, including cancer, cardiovascular disease, infectious diseases, and autoimmune disorders. The emergence of immunogenicity in protein therapeutics has led to an increased demand for characterizing the presence of antibodies, such as anti-drug antibodies (ADAs), induced by the administration of protein therapeutics. Characterization and measurement data for ADAs can provide an understanding of the immunogenicity of pharmaceuticals to improve drug safety.

[0007] The exemplary embodiments disclosed herein fulfill the aforementioned needs by providing methods and systems for characterizing, identifying, and / or measuring ADA induced by administration of a pharmaceutical agent. The present disclosure provides a method for identifying anti-drug antibodies in a sample, comprising: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a binding partner of the target; detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; wherein the sample comprises an anti-drug antibody and a target, and the target is a binding partner of the drug.

[0008] In some exemplary embodiments, the method for identifying anti-drug antibodies in a sample further comprises contacting the sample with a cofactor to enhance binding between the target and the target's binding partner. In some embodiments, the method for identifying anti-drug antibodies in a sample is performed under a weakly acidic assay pH.

[0009] In some embodiments, the method of identifying anti-drug antibodies in a sample further comprises removing the target using an anti-target antibody, wherein the anti-target antibody is bound to a solid support.

[0010] In some embodiments, the first labeled drug or the second labeled drug of this method is a ruthenium-labeled drug or a biotinylated drug. In some embodiments, the binding partner of the target of this method is a natural binding partner or a receptor of the target, and the target is a soluble multimeric target.

[0011] In some embodiments, the weakly acidic assay pH of the present methods ranges from about pH 4.5 to about pH 6.5, is about pH 6.0, or is about pH 5.0.

[0012] In some embodiments, the drug of the method is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, an Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical. In some embodiments, the drug of the method is an antibody and the sample is a serum sample.

[0013] The present disclosure provides, at least in part, a system for identifying anti-drug antibodies in a sample, the system comprising: a first labeled drug; a second labeled drug; with the target's binding partner; an assay system for detecting the presence of a complex comprising a first labeled drug, an anti-drug antibody, and a second labeled drug; wherein the sample comprises an anti-drug antibody and a target, and the target is a binding partner of the drug.

[0014] In some exemplary embodiments, the system further comprises a cofactor capable of strengthening the binding between the target and the target's binding partner. In some embodiments, the sample of the system is treated with a solution having a weakly acidic assay pH. In some embodiments, the system further comprises an anti-target antibody, wherein the anti-target antibody is attached to a solid support.

[0015] In some embodiments, the first labeled drug or the second labeled drug of the system is a ruthenium-labeled drug or a biotinylated drug. In some other embodiments, the binding partner of the target of the system is a natural binding partner or a receptor of the target, and the target is a soluble multimeric target.

[0016] In some embodiments, the weakly acidic assay pH of the present system ranges from about pH 4.5 to about pH 6.5, is about pH 6.0, or is about pH 5.0.

[0017] In some embodiments, the drug of the system is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical. In other embodiments, the drug of the system is an antibody and the sample of the system is a serum sample.

[0018] [The present invention 1001] 1. A method for identifying anti-drug antibodies in a sample, comprising: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a binding partner of a target; detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; Including, the sample comprises the anti-drug antibody and the target; The method, wherein the target is a binding partner of the drug. [The present invention 1002] 1001. The method of claim 1001, further comprising contacting said sample with a cofactor to enhance binding between said target and said binding partner of said target. [The present invention 1003] 1001. The method of claim 1001, wherein the method is carried out under a weakly acidic assay pH. [The present invention 1004] 1001. The method of claim 1001, further comprising removing said target using an anti-target antibody. [The present invention 1005] 1005. The method of claim 1004, wherein said anti-target antibody is attached to a solid support. [The present invention 1006] 1001. The method of claim 1001, wherein said first labeled drug is a ruthenium labeled drug or a biotinylated drug. [The present invention 1007] 1001. The method of claim 1001, wherein said second labeled drug is a ruthenium labeled drug or a biotinylated drug. [The present invention 1008] 1002. The method of claim 1001, wherein said binding partner of said target is a natural binding partner. [The present invention 1009] 1001. The method of claim 10, wherein said binding partner of said target is a receptor for said target. [The present invention 1010] 1001. The method of claim 10, wherein said target is a soluble multimeric target. [The present invention 1011] 1003. The method of claim 1003, wherein said weakly acidic assay pH is in the range of about pH 4.5 to about pH 6.5. [The present invention 1012] 1003. The method of claim 1003, wherein said weakly acidic assay pH is about pH 6.0. [The present invention 1013] 1003. The method of claim 1003, wherein said weakly acidic assay pH is about pH 5.0. [The present invention 1014] 1001. The method of claim 1001, wherein said drug is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical. [The present invention 1015] 1001. The method of claim 10, wherein said drug is an antibody. [The present invention 1016] 1001. The method of claim 10, wherein said sample is a serum sample. [The present invention 1017] 1. A system for identifying anti-drug antibodies in a sample, comprising: a first labeled drug; a second labeled drug; with the target's binding partner; an assay system for detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; Including, the sample comprises the anti-drug antibody and the target; The system wherein the target is a binding partner of the drug. [The present invention 1018] The system of claim 1017, further comprising a cofactor capable of strengthening the binding between said target and said binding partner of said target. [The present invention 1019] The system of claim 1017, wherein the sample is treated with a solution having a weakly acidic assay pH. [The present invention 1020] The system of the present invention 1017 further comprising an anti-target antibody. [The present invention 1021] The system of claim 1020, wherein said anti-target antibody is attached to a solid support. [The present invention 1022] The system of the present invention 1017, wherein the first labeled drug is a ruthenium-labeled drug or a biotinylated drug. [The present invention 1023] The system of the present invention 1017, wherein the second labeled drug is a ruthenium-labeled drug or a biotinylated drug. [The present invention 1024] The system of claim 1017, wherein said binding partner of said target is a natural binding partner. [The present invention 1025] The system of claim 1017, wherein said binding partner of said target is a receptor of said target. [The present invention 1026] The system of the present invention 1017, wherein the target is a soluble multimeric target. [The present invention 1027] The system of the present invention, wherein the weakly acidic assay pH is in the range of about pH 4.5 to about pH 6.5. [The present invention 1028] The system of the present invention 1019, wherein the weakly acidic assay pH is about pH 6.0. [The present invention 1029] The system of the present invention 1019, wherein the weakly acidic assay pH is about pH 5.0. [The present invention 1030] The system of the present invention 1017, wherein the drug is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical. [The present invention 1031] The system of the present invention 1001, wherein the drug is an antibody. [The present invention 1032] The system of the present invention 1001, wherein the sample is a serum sample. These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, may be given by way of illustration and not limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the present invention. [Brief explanation of the drawings]

[0019] [Figure 1]According to exemplary embodiments, under the neutral assay pH for performing the crosslinked ADA assay, the multimeric target protein can simultaneously bind to the ruthenium-labeled drug and the biotinylated drug, thereby indicating the presence of a target-mediated signal due to the presence of soluble multimeric target in the serum sample. According to exemplary embodiments, the incorporation of the drug target's natural binding partner and cofactors into the crosslinked ADA assay can mitigate the target-mediated signal under the mildly acidic assay pH. [Figure 2] Figure 2A shows the screening of several anti-target antibodies, e.g., Ab1-Ab9, at 100 μg / mL compared to a control (Ctrl) for reducing target interference in naive monkey serum samples, according to an exemplary embodiment. Figure 2B shows the use of commercially available polyclonal anti-target antibodies for reducing target interference in naive monkey serum samples, according to an exemplary embodiment. [Figure 3] An exemplary embodiment demonstrates the incorporation of a target receptor into a cross-linked ADA assay to improve detection of ADA by mitigating target-mediated signal. [Figure 4] Figure 4A shows the incorporation of a target receptor and a cofactor into a crosslinking ADA assay to improve ADA detection by mitigating target-mediated signal, according to an exemplary embodiment. According to an exemplary embodiment, different concentrations of cofactor protein were added to a solution containing 50 μg / mL of soluble target receptor to perform a crosslinking ADA assay. Figure 4B shows that, according to an exemplary embodiment, a wide range of target-mediated assay signals was detected in the absence of any blocker protein in eight naive monkey serum samples (controls). The presence of target receptor (50 μg / mL) and cofactor (50 μg / mL) demonstrates effective mitigation of target-mediated assay signals in all monkey serum samples, according to an exemplary embodiment. [Figure 5]

[0033] In accordance with an exemplary embodiment, optimization of assay pH to mitigate target interference in a cross-linked ADA assay using four experimental designs is shown. The four experimental designs were: (1) four naive monkey serum samples at neutral pH (control), (2) four naive monkey serum samples at neutral pH with 50 μg / mL receptor and 50 μg / mL cofactor, (3) four naive monkey serum samples at slightly acidic pH (approximately pH 6.0), and (4) four naive monkey serum samples at approximately pH 6.0 with 50 μg / mL receptor and 50 μg / mL cofactor. [Figure 6] Figure 6A shows the determination of target tolerance levels using recombinant target protein under different assay pH conditions when an ADA assay is performed using 50 μg / mL of both receptor and cofactor proteins, according to an exemplary embodiment. Figure 6B shows the detection of ADA signal under different assay pH conditions using an early bleed from a MAB-Y Fab-immunized rabbit, according to an exemplary embodiment. [Figure 7] Figure 7A shows drug concentrations in serum samples from two monkeys administered a single dose of a drug, e.g., MAB-Y, according to an exemplary embodiment. LLOQ indicates lower limit of quantitation. Figure 7B shows target concentrations and ADA signals in samples from days 0, 28, and 52 with different assay conditions, including the incorporation of target receptors, cofactors, and a mildly acidic assay pH into the bridging ADA assay to improve ADA detection, according to an exemplary embodiment, using post-administration monkey samples. [Figure 8] Figure 1 shows drug concentrations in serum samples from three subjects given a single dose of MAB-Y according to an exemplary embodiment. LLOQ indicates lower limit of quantitation. [Figure 9] Exemplary embodiments show target concentrations and ADA signals in samples at day 0, day 29, and day 64 with different assay conditions, including the incorporation of target receptors, cofactors, and a mildly acidic assay pH into the bridging ADA assay to improve ADA detection. [Figure 10]Figure 10A shows immunodepletion of target proteins with MAB-A conjugated magnetic beads at a neutral assay pH to eliminate target-mediated signals in a drug-free naive human serum sample, according to an exemplary embodiment. Figure 10B shows immunodepletion of target proteins with MAB-A conjugated magnetic beads at a neutral assay pH to eliminate target-mediated signals in a baseline serum sample, according to an exemplary embodiment. [Figure 11] Exemplary embodiments show ADA assay signals in samples from four monkeys at days 1, 15, 29, and 57 measured under different assay conditions, e.g., no blocker at neutral assay pH, 100 μg / mL MAB-A at neutral assay pH, no blocker at slightly acidic pH (pH 6.0), and 100 μg / mL MAB-A at slightly acidic pH (pH 6.0). [Figure 12] An exemplary embodiment shows target concentrations and ADA assay signals in samples at days 1, 15, 29, and 57 before and after immunodepletion with MAB-A conjugated magnetic beads under a weakly acidic assay pH. [Figure 13] 1 shows immunodepletion using MAB-A conjugated magnetic beads in baseline and post-administration samples under different pH conditions according to an exemplary embodiment. [Figure 14] Exemplary embodiments demonstrate detection of true ADA signal in samples from ADA-positive subjects at day 1, day 15, day 29, and day 57 using a competitive blocker ADA method and a modified immunodepletion method containing soluble receptor (50 μg / mL) and cofactor (50 μg / mL) under a weakly acidic assay pH. DETAILED DESCRIPTION OF THE INVENTION

[0020] Detailed Description Increasing concerns about drug efficacy and patient safety due to the occurrence of immunogenicity of protein drugs have led to an increased demand for characterizing anti-drug antibodies (ADAs). The demand for characterizing ADAs is driven by the need to understand the impact of ADAs on, for example, reduced drug efficacy, cross-reactivity to endogenous proteins, or altered drug pharmacokinetics. ADA characterization data can provide valuable information about the immunogenicity of drugs and thus enhance the safety of drug administration.

[0021] Administration of biologics, such as monoclonal antibodies, can induce immune responses, such as the development of anti-drug antibodies (ADAs), in animal subjects and human patients. The immunogenic response induced by therapeutic proteins can range from transient ADAs with no clinical significance to the production of high-titer, persistent ADAs, which can lead to reduced drug exposure, lack of or loss of efficacy, and adverse events such as hypersensitivity reactions, anaphylaxis, and injection site reactions (Koren et al., Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. J Immunol Methods, 2008, 333(1-2):pp. 1-9). The occurrence of ADA neutralization activity has been reported, including the impact of immunogenicity on clinical pharmacology related to pharmacokinetics, efficacy, and safety. The formation of ADAs during drug treatment can cause a decrease in drug concentration in the patient's body, which can contribute to reduced efficacy. Various ADAs, such as neutralizing ADAs and non-neutralizing ADAs, that can bind to different sites on the drug may exist in the patient's body. Neutralizing ADA can bind to the active site of a drug molecule, such as the binding site in the drug molecule for binding to the drug target, or the variable region of an antibody drug. When neutralizing ADA binds to the active site of a drug, it inactivates the drug. Non-neutralizing ADA can bind to the non-active site of a drug molecule, such as the constant region or backbone of an antibody drug molecule. Although a drug can be active even when bound by non-neutralizing ADA, the presence of non-neutralizing ADA can contribute to certain changes in clinical pharmacology.

[0022] Immunogenicity refers to the ability of a therapeutic product to generate an immune response against itself and related proteins, such as inducing immunologically related adverse clinical events. Relevant immunogenicity information includes induction of binding antibodies, induction of neutralizing antibodies, altered pharmacokinetics, reduced efficacy, and safety concerns. However, the clinical significance of ADAs has been unclear. In addition, limited available data can hinder the determination of the impact of ADAs. ADAs may be associated with a concordance between increased systemic clearance of a drug and reduced efficacy. Some formulations have ADAs that retain the drug, resulting in reduced clearance, which may be due to the formation of ADA-drug complexes, such as ADA binding. (Wang et al., Evaluating and Reporting the Immunogenicity Impacts for Biological Products—a Clinical Pharmacology Perspective. The AAPS Journal. 2016;18(2):395-403).

[0023] Therefore, as part of product safety, immunogenicity assessment may be required by regulatory authorities, and the incidence of ADA and neutralizing antibodies (NAb) is part of the prescribing information (US Department of Health and Human Services, UFC, CBER, Guidance for Industry-Assay Development for Immunogenicity Testing of Therapeutic Proteins (Draft). US Department of Health and Human Services, Washington, DC, USA, 2009; European Medicines Agency, CfMPfHU, Guideline on Immunogenicity Assessment of Biotechnology-Driven Therapeutic Proteins. European Medicines Agency, London, UK, 2007). Accurate detection of ADA is therefore a critical aspect of any biological drug development program (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): pp. 1-16; 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): pp. 1267-81).

[0024] Assays for identifying or measuring ADA are typically bridging immunoassays that incorporate a biotinylated drug (Bio-drug) and a ruthenium-labeled drug (Ru-drug) as crosslinking components, for example, to form an ADA-drug complex containing the biotinylated drug, ADA, and the ruthenium-labeled drug. Bridging ADA assays offer high throughput and sensitivity, as well as the ability to detect most ADA antibody isotypes. However, bridging ADA assays can be susceptible to several interferences. The presence of certain molecules in the sample, such as free drug, soluble drug targets, and other matrix proteins in serum samples, can cause interference. Soluble drug targets can be dimeric or multimeric peptides or proteins. Due to the highly specific binding between the target and the drug, overcoming interference from soluble dimeric or multimeric targets is particularly challenging. This is even more challenging given the highly diverse biological properties of each target protein. (Zhong,et al.,Drug Target Interference in Immunogenicity Assays:Recommendations and Mitigation Strategies.AAPS J,2017.19(6):p.1564-1575.)

[0025] Incorporation of target-specific antibodies in bridging ADA assays has been used to reduce target interference by blocking the interfering signal (Liao, K., et al., Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody. J Immunol Methods, 2017. 441: pp. 15-23.; Zhong, et al., Identification and inhibition of drug target interference in immunogenicity assays. J Immunol Methods, et al., 2010. 355(1-2): pp. 21-8.). However, suitable blocking antibodies may not be readily available for many monoclonal antibody drugs. A suitable blocking antibody should be able to competitively bind to the target without affecting the bridging interaction between ADA and the labeled drug molecule. Other strategies to mitigate target interference include the use of target-binding proteins, target immunodepletion, and certain types of lectins to block interference from highly glycosylated target proteins (Carrasco-Triguero, et al., Overcoming soluble target interference in an anti-therapeutic antibody screening assay for an antibody-drug conjugate therapeutic. Bioanalysis, 2012.4(16):p.2013-26.).

[0026] Adjusting the pH conditions of a crosslinked ADA assay through acidic pretreatment or sample incubation under mildly acidic or basic pH conditions is an additional strategy for disrupting or enhancing various interactions contributed by the drug, ADA, drug target, or labeled drug molecule. Changes in pH can also have unintended consequences, such as the release of free drug from the target-drug complex or dimerization of the monomeric drug target, which can increase false-positive signals. Therefore, the specific pH conditions for each individual ADA assay must be carefully evaluated. (Dai, et al., "Development of a method that eliminates false-positive results due to nerve growth factor interference in the assessment of fluranumab immunogenicity." AAPS J, 2014, 16(3):464-77; Zoghbi, et al., "A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays." J Immunol Methods, 2015, 426:62-9)

[0027] The presence of drug targets in test samples can pose challenges in developing reliable cross-linking ADA assays because drug targets, especially dimeric or multimeric target proteins, can form cross-linked complexes between capture and detection molecules, resulting in false-positive ADA signals. The addition of either individual anti-target antibodies or a cocktail of such antibodies is a common approach to mitigating target interference signals. These antibodies typically offer high specificity and target affinity and are relatively easy to produce in large quantities. However, anti-target antibodies must meet certain criteria to be effective. To avoid cross-reactivity between these antibodies and ADA, anti-target antibodies should not share similar or overlapping CDR or framework sequences with the drug molecule. In addition, blocker antibodies should not contain human IgG constant region sequences, as these sequences may compete with ADA detection of similar sequences on the labeled drug molecule, resulting in false-negative ADA results.

[0028] The present disclosure provides methods and systems for characterizing, identifying, and measuring ADA induced by pharmaceutical administration, thereby fulfilling the aforementioned requirements. In particular, the methods and systems of the present application provide an improvement for reducing target interference by incorporating a natural binding partner of the drug target, such as a receptor for the drug target, into the crosslinked ADA assay. In some exemplary embodiments, a binding cofactor for the drug target is incorporated into the crosslinked ADA assay to reduce target interference, and the binding cofactor for the drug target can promote binding between the drug target and the natural binding partner. In some embodiments, the natural binding partner for the drug target is a target receptor that has high affinity for the drug target and can compete with the drug for target binding. In some embodiments, the target receptor and cofactor are incorporated into the crosslinked ADA assay to improve ADA detection. As a natural binding partner for the target, the receptor has high affinity for the target and can outcompete the drug for target binding. Intrinsically, cofactor molecules help maintain the structure of many receptor proteins and improve target-receptor binding.

[0029] The present application provides target-binding proteins, such as soluble target receptors, with or without their necessary cofactors, for inhibiting target interference. These proteins are natural binding partners of the target and usually exhibit high target affinity. Based on the glycosylation characteristics of soluble target proteins and the glycan-binding specificity of lectins, certain lectins can also be used to reduce target interference from highly glycosylated target proteins (Carrasco-Triguero, M., et al., Overcoming soluble target interference in an anti-therapeutic antibody screening assay for an antibody-drug conjugate therapeutic. Bioanalysis, 2012.4(16):p.2013-26).

[0030] The present application also provides strategies to modify the assay pH to reduce target interference, either by directly affecting dimeric or multimeric target protein formation or by altering drug binding affinity to the target. The present application provides that a weakly acidic assay pH alone can at least partially reduce target-mediated signaling, presumably by reducing target binding to the labeled drug.

[0031] In one embodiment, the present disclosure provides methods and systems for reducing target interference by incorporating the drug target's natural binding partner and cofactor into a crosslinked ADA assay under a weakly acidic assay pH. In some embodiments, in the presence of receptor and cofactor proteins under a weakly acidic assay pH for performing a crosslinked ADA assay, the drug target can no longer crosslink a labeled drug (e.g., a ruthenium-labeled drug and a biotinylated drug) because two different activities exist and these activities are synergistic. For example, as shown in Figure 1, because soluble multimeric targets exist in serum samples, this multimeric target protein can simultaneously bind to ruthenium-labeled drugs and biotinylated drugs (e.g., Bio-MAB-Y and Ru-MAB-Y) under a neutral assay pH for performing a crosslinked ADA assay, which can generate a target-mediated signal. In the presence of target receptor and cofactor proteins under a weakly acidic assay pH for performing a crosslinked ADA assay, the drug target can form a complex with the drug receptor and cofactor, as shown in Figure 1, thereby reducing the target-mediated signal. The use of a mildly acidic assay pH to perform a crosslinking ADA assay offers the advantage of reducing binding between the available drug target and the labeled drug. These competitive blockers, e.g., receptors and / or cofactors, synergistically inhibit target interference, increasing target tolerance levels, particularly when the assay is performed under mildly acidic conditions.

[0032] In some exemplary embodiments, the drug target is a multimeric protein present in serum, and the drug target may generate a target-mediated false-positive signal that may interfere with the quantification of ADA. For example, the ADA of a MAB-Y (e.g., a drug) in a serum sample can be detected using Ru-MAB-Y (ruthenium-labeled MAB-Y) and Bio-MAB-Y (biotinylated MAB-Y), for example, by using an ADA to crosslink Ru-MAB-Y and Bio-MAB-Y to form a complex containing Ru-MAB-Y, ADA, and Bio-MAB-Y. However, because the target of MAB-Y is a multimeric protein that is expressed at different levels in monkey and human naive serum samples, for example, using a target to crosslink Ru-MAB-Y and Bio-MAB-Y can cause the target in serum to form a complex with Ru-MAB-Y and Bio-MAB-Y, contributing to a target-mediated false-positive signal. In some exemplary embodiments, anti-target antibodies are used to remove drug targets through immunodepletion, thereby reducing the interference of false positive signals caused by the cross-linking effect of drug targets.Immunodepletion of target proteins can be performed using magnetic beads conjugated with anti-target antibodies, which, combined with a weakly acidic assay pH, is effective in reducing target-mediated signals.These methods allow the detection of true ADA signals in post-administration serum samples from monkeys and humans.

[0033] This application provides two different approaches for reducing multimeric target interference in monkey and human serum samples, including competition for target binding by soluble target receptor and cofactor proteins, and immunodepletion using anti-target antibody-conjugated magnetic beads. For both approaches, mildly acidic assay conditions (such as a pH of approximately 6.0) can selectively inhibit target binding to labeled drug molecules or enhance target binding to anti-target antibodies. The combination of target receptor and cofactor proteins under mildly acidic assay pH can significantly reduce target-mediated signals to background levels in post-administration monkey serum samples and human clinical trial samples. Immunodepletion under mildly acidic assay conditions results in a 50-fold or greater reduction in target levels, eliminating target interference in clinical research samples while maintaining true-positive ADA detection.

[0034] In one embodiment, methods and systems are provided for characterizing, identifying, and / or measuring anti-drug antibodies in samples. These fulfill a long-felt need for characterizing the antibodies induced by the administration of drugs or pharmaceuticals, which can be used to study preclinical or clinical toxicology and pharmacokinetics. These methods and systems can be applied to preclinical toxicology or pharmacokinetic studies to monitor ADA over time after administration of pharmaceuticals.

[0035] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although any method and material similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials will now be described.All publications mentioned are incorporated herein by reference.

[0036] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation as understood by one of ordinary skill in the art, and when ranges are provided, endpoints are included.

[0037] As used herein, the terms "include," "includes," and "including" are intended to be open-ended and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0038] In some exemplary embodiments, the present disclosure provides a method for identifying anti-drug antibodies in a sample, the method comprising: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a binding partner of the target; detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; wherein the sample comprises an anti-drug antibody and a target, and the target is a binding partner of the drug. In some exemplary embodiments, the drug of the method is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, an Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical.

[0039] As used herein, the term "peptide" or "protein" includes any amino acid polymer having covalently linked amide bonds. A protein comprises one or more amino acid polymer chains, commonly known in the art as "peptides" or "polypeptides." A protein may comprise one or more polypeptides to form a single functional biomolecule. In some exemplary embodiments, the protein may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a host cell-derived protein, or a combination thereof.

[0040] As used herein, the term "pharmaceutical" includes an active ingredient that may be fully or partially biological in nature or have pharmaceutical activity. In some exemplary embodiments, a pharmaceutical may include a drug, peptide, protein, fusion protein, antibody, antibody fragment, Fab region of an antibody, antibody-drug conjugate, peptide-drug conjugate, Fc region of an antibody, enzyme product, cytokine, growth factor, medicine, toxin, nucleic acid, DNA, RNA, chemical compound, cell, tissue, antigen, vaccine, or any pharmaceutical component that may be capable of inducing antibodies in a subject. In some other exemplary embodiments, a pharmaceutical may include a recombinant, engineered, modified, mutated, or truncated version of a peptide, protein, fusion protein, antibody, antigen, vaccine, peptide-drug conjugate, antibody-drug conjugate, protein-drug conjugate, or combinations thereof.

[0041] As used herein, "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fc fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, while an ScFv protein is a recombinant single-chain polypeptide molecule in which the variable regions of an immunoglobulin light and heavy chains are connected by a peptide linker. Antibody fragments can be produced by various means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or they can be recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments may be wholly or partially synthetically produced. Antibody fragments may optionally comprise single-chain antibody fragments. Alternatively or additionally, antibody fragments may comprise multiple chains linked together, for example, by disulfide bonds. Antibody fragments may optionally comprise multimolecular complexes.

[0042] As used herein, the term "antibody-drug conjugate" or "ADC" may refer to an antibody linked to a biologically active drug via a linker having a labile bond. An ADC may contain several molecules of a biologically active drug (or payload) that can be covalently attached to the side chains of amino acid residues of the antibody (Siler Panowski et al., Site-specific antibody drug conjugates for cancer therapy, 6 mAbs 34-45 (2013)). The antibody used in the ADC may be able to bind with sufficient affinity for selective accumulation and sustained retention at the target site. Most ADCs can have Kd values ​​in the nanomolar range. The payload may have potency in the nanomolar / picomolar range and be able to reach achievable intracellular concentrations following distribution of the ADC to the target tissue. Finally, the linker forming the connection between the payload and the antibody can take advantage of the pharmacokinetic properties (e.g., long half-life) of the antibody moiety, allowing the payload to remain attached to the antibody as it distributes within tissues, yet be sufficiently stable in the circulation to allow efficient release of the biologically active drug once the ADC is internalized within target cells. The linker can be non-cleavable during cellular processing and cleavable once the ADC reaches the target site. In a non-cleavable linker, the biologically active drug released on demand includes the payload and all elements of the linker that remain attached to amino acid residues, typically lysine or cysteine ​​residues, of the antibody after complete proteolysis of the ADC in the lysosome. A cleavable linker is one whose structure includes a cleavage site between the payload and the amino acid binding site on the antibody. Cleavage mechanisms can include hydrolysis of acid-labile bonds in acidic intracellular compartments, enzymatic cleavage of amide or ester bonds by intracellular proteases or esterases, and reductive cleavage of disulfide bonds in the reducing environment within the cell.

[0043] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL can consist of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes, but is not limited to, those prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. IgG includes a subset of antibodies.

[0044] Illustrative Embodiments Embodiments disclosed herein provide compositions, methods, and systems for identifying anti-drug antibodies in a sample.

[0045] In some exemplary embodiments, the present disclosure provides a method for identifying anti-drug antibodies in a sample, the method comprising: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a binding partner of the target; detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; In some embodiments, the method for identifying an anti-drug antibody in a sample is performed under a weakly acidic assay pH.

[0046] In some embodiments, the weakly acidic assay pH of the present methods is in the range of about pH 4.5-6.5, about pH 3-6.9, about pH 4-6.5, about pH 4.5-6.5, about pH 5-6.5, about pH 5.5-6.5, about pH 5.9-6.2, about pH 5.0, or preferably about pH 6.0.

[0047] In some embodiments, the method of identifying anti-drug antibodies in a sample further comprises removing the target using an anti-target antibody, wherein the anti-target antibody is bound to a solid support.

[0048] In some embodiments, the solid support in the methods or systems of the present application can be a bead, a magnetic bead, a chromatography resin, a polymer, or a chromatography matrix.

[0049] It is understood that the present system is not limited to any of the aforementioned drugs, peptides, proteins, antibodies, anti-drug antibodies, protein conjugates, or pharmaceuticals.

[0050] The sequential labeling of the method steps provided herein with numbers and / or letters is not intended to limit the method or any embodiment thereof to the particular indicated order. Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers, and academic papers, are cited throughout this specification. Each of these cited documents is incorporated herein by reference in its entirety for all purposes. The present disclosure will be more fully understood by reference to the following examples, which are provided to more fully illustrate the present disclosure. They are intended to be illustrative and should not be construed as limiting the scope of the present disclosure. [Example]

[0051] Reagent preparation Solutions used for all drug and target assays were prepared in assay dilution buffer (ADB: 0.5% BSA, 0.05% Tween-20, 1x PBS). Solutions used for the ADA assay were prepared in 1% BSA, 1x PBS. (ADB is assay dilution buffer, BSA is bovine serum albumin, and PBS is phosphate-buffered saline.) PBS was purchased from Gibco (Grand Island, NY). 1.5 M Trizma base was purchased from Sigma (St Louis, MO). Glacial acetic acid was purchased from Thermo Fisher Scientific (Waltham, MA). Monkey and human sera were purchased from Bioreclamation (Westbury, NY). Streptavidin-coated microplates were purchased from Meso Scale Discovery (Rockville, MD). Dynabeads Antibody Coupling Kit was purchased from Thermo Fisher Scientific (Vilnius, Lithuania). Recombinant human target protein, rat anti-target monoclonal antibody, biotinylated sheep anti-target polyclonal antibody, and horseradish peroxidase-conjugated streptavidin were purchased from R&D Systems (Minneapolis, MN). Soluble target receptor and cofactor proteins were purchased from Sigma (St Louis, MO). Black microwell plates, horseradish peroxidase-conjugated NeutrAvidin, and SuperSignal ELISA Pico chemiluminescent substrate were purchased from Thermo Fisher Scientific (Rockford, IL). MAB-Y is a fully human monoclonal antibody drug.

[0052] method 1. pH measurement pH measurements were performed using a calibrated Mettler Toledo meter (Columbus, Ohio) equipped with an InLab Expert Pro-ISM electrode. Pooled human serum was diluted 10-fold with 300 mM acetic acid. The acidified samples were then diluted 5-fold with different concentrations of Tris base solution, using 50 μg / mL of receptor and 50 μg / mL of cofactor. pH measurements were performed on the final assay solutions shown in Table 1.

[0053] Table 1. Evaluation of pH conditions for detecting anti-MAB-Y ADA TIFF2025176059000002.tif61128

[0054] 2. Coupling of magnetic beads with anti-target antibodies To deplete target proteins, such as those that can be recognized by antibody drugs, anti-target antibodies were coupled to magnetic beads to perform immunodepletion. The anti-target antibody MAB-A was coupled to Dynabeads according to the manufacturer's instructions. An appropriate amount of Dynabeads was washed with 1 mL of C1 solution from the kit and then resuspended in an appropriate volume of anti-target antibody MAB-A diluted in C1 solution. An equal volume of C2 solution was then added to this mixture, followed by incubation at 37°C for 16–24 hours. The coupled beads were washed sequentially with HB, LB, and SB buffers from the kit. The coupled beads were then resuspended in SB buffer and incubated at room temperature for approximately 15 minutes. The supernatant was removed. The MAB-A-conjugated Dynabeads were resuspended in SB buffer at a concentration of 10 mg / mL and stored at 4°C until use.

[0055] 3. Cross-linking ADA Assay An immunoassay was developed to detect the presence of ADA in serum samples. The presence of ADA, such as anti-MAB-Y antibodies, in monkey and human serum samples was detected using a bridging immunoassay, e.g., a bridging ADA assay. A mouse anti-drug monoclonal antibody, such as a mouse anti-MAB-Y antibody, was used as a positive control. Biotinylated drugs (Bio-drugs) and ruthenium-labeled drugs (Ru-drugs), e.g., biotinylated MAB-Y (Bio-MAB-Y) and ruthenium-labeled MAB-Y (Ru-MAB-Y), were used as components to establish bridge complexes, e.g., bridge complexes containing ADA, Bio-drugs, and Ru-drugs (e.g., bridge complexes containing anti-MAB-Y antibodies, Bio-MAB-Y, and Ru-MAB-Y).

[0056] Serum samples containing anti-MAB-Y antibodies were acidified with acetic acid before performing the cross-linking ADA assay, such as by performing a 10-fold dilution with 300 mM acetic acid followed by incubation at room temperature for at least 10 minutes. To achieve a cross-linking ADA assay with a neutral pH, Bio-MAB-Y (1.0 μg / mL) and Ru-MAB-Y (1.0 μg / mL) were prepared in an assay buffer containing 75 mM Tris base before incorporating them into the serum samples.

[0057] The acid-treated serum samples were diluted 5-fold with a solution containing a labeled drug, e.g., Bio-MAB-Y and / or Ru-MAB-Y, and then the samples were incubated at room temperature for approximately 60 minutes. After incubation, the samples were transferred to a (5% BSA) blocked streptavidin Multi-Array® 96-well plate (MSD, i.e., Meso Scale Discovery, LLC) and incubated at room temperature for approximately 60 minutes. The plate was washed. To read the plate using an MSD plate reader, Read Buffer was added to the plate.

[0058] 4. Total Drug Assay An assay method was developed to detect the presence of drugs in serum samples. To analyze the presence of drugs in monkey serum samples, e.g., the total amount of drug in a serum sample, a microtiter plate was coated with a mouse anti-human IgG4 antibody (2 μg / mL). MAB-Y was used as a standard for the total drug assay. Acidification was used, using acetic acid to dissociate soluble target-drug complexes. Monkey serum samples, standards, and controls were treated with 30 mM acetic acid to dissociate soluble target-drug complexes present in the serum samples. In addition, acidification treatment was used to improve drug detection in the presence of soluble targets in serum. After capturing MAB-Y on the microtiter plate, it was detected using biotinylated mouse anti-human Ig, a kappa light chain-specific monoclonal antibody (100 ng / mL) combined with NeutrAvidin-conjugated horseradish peroxidase (NeutrAvidin-HRP, 50 ng / mL). All incubations were performed at room temperature for approximately 60 minutes. A luminol-based substrate, a peroxidase-specific substrate, was then used to generate a detection signal. The signal intensity was proportional to the total MAB-Y concentration. The plate was read using a microplate luminometer.

[0059] To analyze the presence of drugs in human serum samples, e.g., the total amount of drugs in serum samples, microtiter plates were coated with mouse anti-MAB-Y monoclonal antibody (2 μg / mL). MAB-Y was used as a standard for the total drug assay. Acidification was performed using acetic acid to dissociate soluble target-drug complexes. Human serum samples were treated with 30 mM acetic acid to dissociate soluble target-drug complexes present in the serum samples. After capturing MAB-Y on the microtiter plate, different biotinylated mouse anti-MAB-Y-specific monoclonal antibodies (100 ng / mL) were used in conjunction with NeutrAvidin-conjugated horseradish peroxidase (NeutrAvidin-HRP, 50 ng / mL) to detect MAB-Y. All incubations were performed at room temperature for approximately 60 minutes. A luminol-based substrate, a peroxidase-specific substrate, was then used to generate the detection signal. The signal intensity was proportional to the total MAB-Y concentration. The plates were read in a microplate luminometer.

[0060] 5. Total Target Assay An assay method was developed to detect the presence of target proteins in serum samples. To analyze the presence of target proteins in serum samples, e.g., the total amount of target protein in serum samples, microtiter plates were coated with rat anti-target monoclonal antibody (4 μg / mL). Recombinant target protein was used as a standard. Acidification was performed using acetic acid to dissociate soluble target-drug complexes. Serum samples, standards, and controls were diluted 1:10 with 300 mM acetic acid to dissociate soluble target-drug complexes that may be present in the serum samples, followed by neutralization with a 1:5 dilution in 75 mM Tris solution. The neutralized standards, controls, and samples were then added to the microtiter plate. The target proteins captured on the plate were detected with a combination of biotinylated sheep anti-target polyclonal antibody (100 ng / mL) and streptavidin-conjugated horseradish peroxidase (streptavidin-HRP, diluted 1:200 in ADB). All incubations were carried out at room temperature for approximately 60 minutes. A luminol-based substrate, a specific substrate for peroxidase, was then added to generate a detection signal. The signal intensity was proportional to the total target concentration. The plate was read using a microplate luminometer.

[0061] 6. Immunodepletion of Target Proteins Two methods, Methods A and B, were developed for immunodepletion of target proteins in serum samples. In Method A, serum samples were diluted 10-fold with 300 mM acetic acid and incubated at room temperature for at least 10 minutes. The acidified samples were then neutralized with 150 mM Tris solution at a 1:3 dilution. Magnetic beads conjugated with anti-target antibody MAB-A were washed once with 1x PBS and resuspended in the neutralized serum samples. After incubation at room temperature for approximately 60 minutes, the supernatant was collected and then mixed with a solution containing 2 μg / mL Bio-MAB-Y and 2 μg / mL Ru-MAB-Y. After incubation at room temperature for approximately 60 minutes, the samples were transferred to a streptavidin Multi-Array® 96-well plate (MSD) blocked with 5% BSA and further incubated at room temperature for approximately 60 minutes. The plate was washed, and Read Buffer was added. Plates were read using an MSD plate reader.

[0062] In Method B for immunodepletion of target proteins, serum samples were diluted 10-fold with 30 mM acetic acid and then incubated with magnetic beads conjugated with anti-target antibody MAB-A at room temperature for approximately 30 minutes. The supernatant was collected and then diluted 3-fold with 10 mM Tris solution containing 1 μg / mL Bio-MAB-Y and 1 μg / mL Ru-MAB-Y. After approximately 60 minutes of incubation at room temperature, the samples were transferred to a (5% BSA) blocked streptavidin Multi-Array® 96-well plate (MSD) and further incubated for approximately 60 minutes at room temperature. The plate was washed, and Read Buffer was added. The plate was read using an MSD plate reader.

[0063] Example 1. Use of anti-target antibodies to improve ADA detection If the drug target is a multimeric protein present in serum, the drug target may generate a target-mediated false-positive signal that can interfere with ADA detection. The ADA of MAB-Y in serum samples can be detected using Ru-MAB-Y and Bio-MAB-Y, for example, by using an ADA to crosslink Ru-MAB-Y and Bio-MAB-Y to form a complex containing Ru-MAB-Y and Bio-MAB-Y. However, because the target of MAB-Y (e.g., a drug) is a multimeric protein expressed at different levels in monkey and human naive serum samples, for example, using a target to crosslink Ru-MAB-Y and Bio-MAB-Y, the target in serum may form a complex with Ru-MAB-Y and Bio-MAB-Y, which contributes to a target-mediated false-positive signal. Anti-target antibodies were used to reduce the interference of false-positive signals caused by the crosslinking effect of the drug target.

[0064] Anti-target antibodies are frequently used to mitigate target interference in crosslinking ADA assays (Liao, et al., Inhibition of interleukin-5 induced false positive anti-drug antibody responses against mepolizumab through the use of a competitive blocking antibody. J Immunol Methods, 2017. 441: pp. 15-23; Zhong, et al., Identification and inhibition of drug target interference in immunogenicity assays. J Immunol Methods, 2010. 355(1-2): pp. 21-8; Dai, et al.; Weeraratne, et al., Development of a biosensor-based immunogenicity assay capable of blocking soluble drug target interference. J Immunol Methods, 2013. 396(1-2): pp. 44-55; Maria, et al., A novel strategy for elimination of soluble-ligand interference in immunogenicity assays. AAPS National Biotechnology Conference. Seattle, WA, USA, 2009). As shown in Figure 2A, to mitigate target interference in monkey naive serum samples, several anti-target antibodies, e.g., Ab1-Ab9, were screened at 100 μg / mL compared to a control (Ctrl). As shown in Figure 2A, among the screened anti-target antibodies, only one, e.g., Ab4, was able to partially inhibit target-mediated false-positive signals. Two of the tested antibodies, e.g., Ab8 and Ab9, actually enhanced (increased) target-mediated false-positive signals.Various combinations of these antibodies were also evaluated but were unable to sufficiently inhibit target interference in monkey serum samples (data not shown).

[0065] Several rounds of immunization were further performed to generate more anti-target antibodies. However, all of the screened anti-target antibodies could compete well with MAB-Y. One commercially available polyclonal anti-target antibody was used to reduce target interference. As shown in Figure 2B, this polyclonal anti-target antibody showed a specific effect of reducing target interference in a dose-dependent manner. However, polyclonal antibodies may exhibit batch-to-batch variation in target interference reduction, which may negatively impact assay development.

[0066] Example 2. Use of Targeted Receptors to Improve ADA Detection The incorporation of target receptors into the crosslinked ADA assay improved ADA quantification by reducing target-mediated signal. Soluble target receptors (e.g., 50 μg / mL) were included in the labeled drug solution. The labeled drug solution was prepared in 50 mM Tris solution to adjust the assay pH to a slightly acidic state of approximately pH 6.0. The slightly acidic state also minimizes target binding to both Bio-MAB-Y and Ru-MAB-Y. The results showed that the addition of soluble target receptors significantly reduced target-mediated signal in naive monkey serum samples. As shown in Figure 3, the soluble target receptors were able to reduce target-mediated signal in a dose-dependent manner. In Figure 3, the Y-axis represents the average ADA counts, and the X-axis represents the concentration of target receptor (μg / mL). The soluble target receptors effectively blocked target interference in naive monkey serum samples at 100 μg / mL.

[0067] Example 3. Use of target receptors and cofactors to improve ADA detection Target receptors and cofactors were incorporated into the crosslinking ADA assay to improve ADA detection by reducing target-mediated signals. Soluble target receptors (e.g., 50 μg / mL) and cofactor proteins (e.g., 50 μg / mL) were included in the labeled drug solution. The labeled drug solution was prepared in 50 mM Tris solution to adjust the assay pH to a slightly acidic condition of approximately pH 6.0. The slightly acidic condition also minimizes target binding to both Bio-MAB-Y and Ru-MAB-Y. As shown in Figure 4A, different concentrations of cofactor proteins were added to a solution containing 50 μg / mL of soluble target receptor to perform the crosslinking ADA assay. In Figure 4A, the Y axis indicates the average ADA counts, and the X axis indicates the concentration of target receptor and / or cofactor in μg / mL. The results showed that the combination of soluble target receptors and cofactor proteins can significantly reduce target-mediated signals in naive monkey serum samples. The two proteins, e.g., the target receptor and the cofactor, worked synergistically together to effectively reduce background signals in monkey naive samples. As shown in Figure 4A, the results indicated that the combination of 50 μg / mL of receptor and 50 μg / mL of cofactor was the most effective.

[0068] As shown in Figure 4B (control), in the eight naive monkey serum samples, a wide range of target-mediated assay signals was detected in the absence of any blocker protein, which may reflect natural variations in endogenous target levels. As shown in Figure 4B, the presence of target receptor and cofactor, e.g., a combination of 50 μg / mL receptor and 50 μg / mL cofactor, effectively reduced target-mediated assay signals in all monkey serum samples. However, one serum sample still had a signal with an average count of approximately 400.

[0069] Example 4. Optimization of assay pH to reduce target interference The binding affinity of MAB-Y to its target was significantly reduced under acidic conditions (approximately pH 6.0) compared with neutral pH. To test whether a weakly acidic assay pH could inhibit target-mediated signals in the cross-linked ADA assay, four experimental designs were performed to optimize the assay pH. The four experimental designs were: (1) four naive monkey serum samples without any blocker at neutral pH (control), (2) four naive monkey serum samples with 50 μg / mL receptor and 50 μg / mL cofactor at neutral pH, (3) four naive monkey serum samples without any blocker at weakly acidic pH (approximately pH 6.0), and (4) four naive monkey serum samples with 50 μg / mL receptor and 50 μg / mL cofactor at weakly acidic pH (approximately pH 6.0).

[0070] As shown in Figure 5, the results indicated that the high background signal from naive monkey serum samples was partially inhibited by only a weakly acidic pH (pH approximately 6.0). The pH optimization results indicated that a weakly acidic assay pH could inhibit target interference in monkey serum samples. The combination of receptor and cofactor proteins was able to significantly reduce target-mediated signals under both pH conditions, e.g., neutral and weakly acidic pH. In particular, the combination of receptor, cofactor, and weakly acidic assay conditions (pH approximately 6.0) provided a synergistic effect that completely inhibited target-mediated signals, as shown in Figure 5.

[0071] When the ADA assay was performed using 50 μg / mL of both receptor and cofactor proteins, the target tolerance level was determined using recombinant target protein under different assay pH conditions. The target tolerance level was defined as the amount of target required to obtain an assay signal above the plate cut point. As shown in Figure 6A, when the ADA assay was performed using 50 μg / mL of both receptor and cofactor proteins under neutral assay pH conditions, the target tolerance level was determined to be approximately 94 ng / mL. When the assay pH was approximately 6.5, the target tolerance level increased to approximately 380 ng / mL using the same concentrations of receptor and cofactor. When the assay pH was approximately 6.0, the target tolerance level was even higher, approximately 5.0 μg / mL. These results indicated that a weakly acidic assay pH can significantly improve target-mediated signal reduction.

[0072] To ensure that a weakly acidic pH had minimal impact on the stability and / or detection of true ADA signals, early bleeds from MAB-Y Fab-immunized rabbits were analyzed under different assay pH conditions. Bleed 1 was collected approximately 30 days after immunization, and the induced antibody response in this bleed typically contained low-affinity polyclonal ADAs, the detection of which may be more affected by harsh assay conditions. As shown in Figure 6B, ADA mean count values ​​were similar across assay conditions, regardless of assay pH. The results indicated that a weakly acidic pH had minimal or no impact on the stability and / or detection of true ADAs in these samples. The results indicated that a weakly acidic assay pH improved target acceptance levels and had minimal impact on true ADA detection.

[0073] Example 5. Combination of target receptor, cofactor, and weakly acidic assay pH The combination of target receptor, cofactor, and mildly acidic assay pH was incorporated into the crosslinked ADA assay to improve ADA detection by mitigating target-mediated signaling. Post-administration monkey samples, which typically contain higher levels of target protein, were used to perform the crosslinked ADA assay. Serum samples from two monkeys 0, 28, and 52 days after a single dose of drug (e.g., MAB-Y) were tested using the crosslinked ADA assay. Drug concentrations in serum samples representing the pharmacokinetic (PK) profiles of these two monkeys are shown in Figure 7A (LLOQ indicates the lower limit of quantification). Monkey 1 exhibited a linear PK profile. Monkey 2 showed a significant decrease in drug levels, e.g., accelerated drug clearance, beginning on day 21, which may indicate a significant ADA response in monkey 2.

[0074] The concentrations of target and ADA in monkey serum samples were measured. Figure 7B shows the target concentrations and ADA signals on days 0, 28, and 52 using post-dose monkey samples under different assay conditions according to an exemplary embodiment. Compared to baseline, target levels increased approximately 10-15-fold in the day 28 samples from both monkeys, and target concentrations remained high in the day 52 sample from monkey 1. When the bridging ADA assay was performed using post-dose samples under control conditions at a neutral assay pH and without the presence of any competitive blocker, strong assay signals were obtained from these serum samples, as shown in Figure 7B. The addition of receptor and cofactor molecules in the bridging ADA assay at a neutral assay pH partially inhibited the assay signal in these samples. The baseline sample showed a more significant decrease in signal. However, because the ADA mean counts remained well above the plate cutpoint for all samples under these conditions, it was difficult to distinguish true ADA signals from target-mediated false-positive signals.

[0075] When monkey serum samples were tested in a bridged ADA assay in the presence of 50 μg / mL receptor and 50 μg / mL cofactor at a mildly acidic assay pH (e.g., approximately pH 6.0), low background signals were detected for all samples from monkey 1, as shown in Figure 7B. For monkey 2, approximately 2-fold and 200-fold increases in ADA signal were observed for the 28-day and 52-day samples, respectively, compared to baseline, as shown in Figure 7B. The results indicated that the combination of soluble receptor, cofactor, and mildly acidic assay pH can reduce target interference and detect true ADA in monkey samples after administration. The results also indicated that the serum sample from monkey 1 had no ADA, and the serum sample from monkey 2 had an ADA response, supporting the drug concentration profiles of these two monkeys, as shown in Figure 7A.

[0076] Clinical study samples (days 0, 29, and 64) from three subjects in a Phase I clinical study were also tested using cross-linked ADA by incorporating a combination of target receptors, cofactors, and a mildly acidic assay pH to improve ADA detection by mitigating target-mediated signals. Drug concentrations in these clinical study samples were measured. As shown in Figure 8, drug concentrations were measured in serum samples from three subjects administered a single dose of MAB-Y. LLOQ indicates the lower limit of quantitation. The PK profiles of these samples did not suggest a significant ADA response. However, as shown in Figure 9, when these samples were tested in the cross-linked ADA assay under a neutral assay pH and without the presence of a blocker molecule, high assay signals were observed for all samples. Figure 9 shows the target concentrations and ADA signals in samples on days 0, 29, and 64 with different assay conditions, including the incorporation of target receptors, cofactors, and a mildly acidic assay pH into the cross-linked ADA assay to improve ADA detection, according to an exemplary embodiment.

[0077] These assay signals appeared to correlate with target levels in these samples. Target concentrations increased in post-dose samples from all three subjects. Under neutral assay pH, high target-mediated signals were detected in all samples without the presence of blockers. When these clinical trial samples were retested under a mildly acidic assay pH (approximately pH 6.0) in the presence of soluble receptor (50 μg / mL) and cofactor (50 μg / mL), only background signals were detected, as shown in Figure 9. A larger set of clinical trial samples (day 0, day 29, and day 64 samples from 11 subjects) was subsequently tested, and all samples showed only background signals (data not shown). These results indicated that an assay format incorporating two competing blocker proteins (e.g., target receptor and cofactor) under mildly acidic assay conditions can effectively mitigate target interference in post-dose human serum samples. This result also supported a PK profile that did not suggest a positive ADA response.

[0078] Example 6. Immunodepletion of target proteins at neutral assay pH Immunodepletion has been used to remove various target proteins from serum samples to inhibit target-mediated assay signals (Dai, et al.). A similar approach was explored to remove multimeric target proteins from human serum samples. An anti-target antibody, MAB-A, was used to perform immunodepletion, which was unable to compete with the drug MAB-Y for target binding at neutral assay pH. Instead, MAB-A could enhance target-mediated signals in human serum samples (data not shown).

[0079] A different approach was implemented using MAB-A-conjugated magnetic beads for immunodepletion, which successfully removed target proteins from five human naive serum samples, thereby inhibiting target-mediated signals, as shown in Figure 10A. Target protein immunodepletion with MAB-A-conjugated magnetic beads at a neutral assay pH eliminated target-mediated signals in drug-free naive human serum samples. However, one sample still showed a signal with an average count of approximately 450 even after target removal. MAB-A-conjugated magnetic beads were also used to remove target proteins from clinical study samples, for example, from samples on days 1, 15, 29, and 57 from two subjects who received a single dose of MAB-Y. Results showed that only the baseline sample showed a reduction to background signals. Significant levels of target-mediated signals were still detected in the post-administration samples, as shown in Figure 10B. These post-administration samples contained high concentrations of the drug MAB-Y. Because MAB-A does not compete with MAB-Y for target binding at neutral assay pH, anti-target antibody-coupled beads may not be able to completely deplete target proteins from serum samples in the presence of high concentrations of MAB-Y. In post-administration samples, when the drug was still present at high concentrations, the target-mediated assay signal was not completely inhibited. These results indicate that immunodepletion by MAB-A-conjugated magnetic beads under neutral assay pH conditions is not sufficient to alleviate target interference in post-administration human samples.

[0080] Example 7. Immunodepletion of target proteins under weakly acidic assay pH The anti-target antibody (MAB-A) and drug MAB-Y have similar K DAlthough the t values ​​are shown, the t of MAB-A is slightly longer than that of MAB-Y, as shown in Table 2. However, at pH ≈ 6.0, this anti-target antibody exhibits much better binding to the target, with a much longer t (Table 2). To test whether MAB-A can compete with MAB-Y for target binding at a slightly acidic pH (pH ≈ 6.0), clinical trial samples from days 1, 15, 29, and 57 were tested with or without MAB-A at either a neutral or slightly acidic (pH ≈ 6.0) assay pH. At the neutral assay pH, the addition of MAB-A failed to inhibit the target-mediated signal. Instead, this antibody slightly enhanced the observed target interference in the test samples, as shown in Figure 11. At a slightly acidic pH alone, the assay signal decreased, particularly in the baseline sample. When MAB-A was added to a weakly acidic assay solution, inhibition of target-mediated signaling was observed in post-administration samples, even in samples in which MAB-Y was still present at high concentrations, as shown in Figure 11. Figure 11 shows the ADA assay signal in samples on days 1, 15, 29, and 57 without blocker at neutral assay pH, with 100 μg / mL MAB-A at neutral assay pH, without blocker at weakly acidic pH (pH 6.0), and with 100 μg / mL MAB-A at weakly acidic pH (pH 6.0). The results indicated that MAB-A can compete with MAB-Y when the assay pH is weakly acidic.

[0081] (Table 2) K of MAB-Y and MAB-A D value and t1 / 2 value TIFF2025176059000003.tif23128

[0082] Compared to the baseline sample, target concentrations in samples taken on days 15, 29, and 57 increased approximately 3-5 fold before returning to baseline levels on day 183, as shown in Figure 12. Figure 12 shows target concentrations and ADA assay signals in samples taken on days 1, 15, 29, and 57 before and after immunodepletion with MAB-A conjugated magnetic beads under a weakly acidic assay pH, according to an exemplary embodiment. To ensure that the modified immunodepletion method was capable of depleting the target protein in these samples, target concentrations were measured before and after immunodepletion. Before immunodepletion, target levels ranged from 150 ng / mL to 750 ng / mL, whereas target concentrations were only approximately 2-5 ng / mL after immunodepletion. As shown in Figure 12, the results demonstrated that the target protein was efficiently removed. When clinical trial samples were tested in the modified immunodepletion ADA assay, only background signal (average counts approximately 200) was detected, compared to the average counts of 1500 to 2300 observed without immunodepletion, as shown in Figure 12. These results indicated that when the assay pH was approximately 5.0, MAB-A was able to effectively compete with MAB-Y for target binding and thus completely deplete the target protein from the analyzed clinical trial samples, as shown in Figure 13. Immunodepletion of multimeric target proteins from clinical trial samples using MAB-A-conjugated magnetic beads was performed under a mildly acidic assay pH.

[0083] The results demonstrated that immunodepletion with MAB-A-conjugated magnetic beads under mildly acidic assay conditions can reduce target interference in post-administration human samples. As shown in Figure 13, for the baseline sample, MAB-A-conjugated magnetic beads were able to effectively remove the target protein. For the post-administration sample, even when MAB-Y was present at high concentrations, acid treatment with 300 mM acetic acid was able to dissociate the target-drug complex. After neutralization and addition of MAB-A-conjugated magnetic beads, MAB-A was unable to compete with MAB-Y at neutral assay pH, so some of the target protein was able to bind to MAB-A on the beads, and some of them were able to reassociate with MAB-Y. Therefore, there was still a sufficient amount of reformed target-drug complex in the supernatant that could generate target-mediated signals. For the same post-administration sample, acid treatment with 30 mM acetic acid was able to dissociate the target-drug complex. Under this acidic condition, when the acidified sample was incubated with MAB-A-conjugated magnetic beads, the free target protein could preferably bind to MAB-A on the beads, rather than to MAB-Y in the supernatant, because MAB-A has a much better affinity for target binding compared to MAB-Y when the assay pH was 5-6 and the t was much larger. Therefore, only unbound REGN-Y was present in the supernatant.

[0084] Using the competitive blocker ADA method under mildly acidic pH, samples from ADA-positive subjects identified using competitive blockade with, for example, soluble receptor (50 μg / mL) and cofactor (50 μg / mL) were tested on days 1, 15, 29, and 57. With the competitive blocker ADA method, the ADA signal was found to increase approximately 8-fold in the day 15 sample and approximately 3-fold in the day 29 sample compared to the day 1 sample, as shown in Figure 14. With the immunodepletion method, a similar increase in ADA signal was observed for the day 15 and day 29 samples. As shown in Figure 14, the results indicated that this method also allows for the detection of true ADA signals. For the day 57 sample, the assay signal remained at background levels, despite the serum target concentration being approximately 450 ng / mL. The results also indicated that both methods can successfully inhibit target interference signals. These results indicated that the modified immunodepletion method can detect true ADA responses.

Claims

1. 1. A method for identifying anti-drug antibodies in a sample, comprising: contacting the sample with a first labeled drug; contacting the sample with a second labeled drug; contacting the sample with a binding partner of a target; detecting the presence of a complex comprising the first labeled drug, the anti-drug antibody, and the second labeled drug; Including, the sample comprises the anti-drug antibody and the target; The method, wherein the target is a binding partner of the drug.

2. 10. The method of claim 1, further comprising contacting the sample with a cofactor to enhance binding between the target and the binding partner of the target.

3. 10. The method of claim 1, wherein the method is carried out under a weakly acidic assay pH.

4. 10. The method of claim 1, further comprising removing the target using an anti-target antibody.

5. The method of claim 4 , wherein the anti-target antibody is attached to a solid support.

6. 10. The method of claim 1, wherein the first labeled drug is a ruthenium-labeled drug or a biotinylated drug.

7. The method of claim 1 , wherein the second labeled drug is a ruthenium-labeled drug or a biotinylated drug.

8. The method of claim 1 , wherein the binding partner of the target is a natural binding partner.

9. The method of claim 1 , wherein the binding partner of the target is a receptor of the target.

10. The method of claim 1 , wherein the target is a soluble multimeric target.

11. 4. The method of claim 3, wherein the weakly acidic assay pH is in the range of about pH 4.5 to about pH 6.

5.

12. 4. The method of claim 3, wherein the weakly acidic assay pH is about pH 6.

0.

13. 4. The method of claim 3, wherein the weakly acidic assay pH is about pH 5.

0.

14. 10. The method of claim 1, wherein the drug is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical.

15. The method of claim 1 , wherein the drug is an antibody.

16. The method of claim 1 , wherein the sample is a serum sample.

17. 1. A system for identifying anti-drug antibodies in a sample, comprising: a first labeled drug; a second labeled drug; with a binding partner of the target; an assay system for detecting the presence of a complex comprising said first labeled drug, said anti-drug antibody, and said second labeled drug; Including, the sample comprises the anti-drug antibody and the target; The system wherein the target is a binding partner of the drug.

18. 20. The system of claim 17, further comprising a cofactor capable of strengthening the binding between the target and the binding partner of the target.

19. 20. The system of claim 17, wherein the sample is treated with a solution having a weakly acidic assay pH.

20. The system of claim 17 further comprising an anti-target antibody.

21. 21. The system of claim 20, wherein the anti-target antibody is attached to a solid support.

22. 18. The system of claim 17, wherein the first labeled drug is a ruthenium-labeled drug or a biotinylated drug.

23. 18. The system of claim 17, wherein the second labeled drug is a ruthenium-labeled drug or a biotinylated drug.

24. The system of claim 17 , wherein the binding partner of the target is a natural binding partner.

25. The system of claim 17 , wherein the binding partner of the target is a receptor of the target.

26. The system of claim 17 , wherein the target is a soluble multimeric target.

27. 20. The system of claim 19, wherein the weakly acidic assay pH is in the range of about pH 4.5 to about pH 6.

5.

28. 20. The system of claim 19, wherein the weakly acidic assay pH is about pH 6.

0.

29. 20. The system of claim 19, wherein the weakly acidic assay pH is about pH 5.

0.

30. 18. The system of claim 17, wherein the drug is a chemical compound, a nucleic acid, a toxin, a peptide, a protein, a fusion protein, an antibody, an antibody fragment, a Fab region of an antibody, an antibody-drug conjugate, or a pharmaceutical.

31. The system of claim 1 , wherein the drug is an antibody.

32. The system of claim 1 , wherein the sample is a serum sample.

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