EA045148B1 - METHODS FOR REDUCING THE INTERFERING INFLUENCE OF DRUG TARGET IN AN IMMUNOLOGICAL ASSAY FOR DRUG ANTIBODIES (ADA) - Google Patents

Description

Link to related applications

This invention claims benefit from U.S. Provisional Application No. 62/696,016, filed July 10, 2018. The entire contents of the foregoing application are incorporated herein by reference.

State of the art

Biotherapeutics (eg, biological agents such as proteins, peptides, nucleotides, etc.) are very well established in clinical practice. However, biotherapeutics, even fully human therapeutic monoclonal antibodies, can produce anti-drug antibodies (ADAs), which can cause undesirable effects such as loss of drug exposure, loss of efficacy, serious side effects, etc. (Koren, E. et al., Curr. Pharm. Biotechnol., 2002, 3(4): p. 349-60; Schellekens, H., Clin. Ther., 2002, 24(11): p. 1720- 40). Therefore, immunogenicity assessment is an important part of biotherapeutics safety testing; Several guidelines have been published on immunogenicity testing at various stages of drug development, including recommendations from regulatory authorities (Shankar, G. et al., J. Pharm. Biomed. Anal., 2008, 48(5): p. 1267-81; Shankar, G. et al., Nat. Biotechnol., 2007, 25(5): pp. 555-61; Mire-Sluis, A. R. et al., J. Immunol. Methods, 2004, 289(1-2): p. 1-16; Swanson, S. J., J. Bussiere, Curr. Opin. Microbiol., 2012, 15(3): p. 337-47; European Medicines Agency, C.f.M.P.f.H.U., Guideline on Immunogenicity Assessment of Biotechnology-Driven Therapeutic Proteins , European Medicines Agency, London, UK, 2007; and US Department of Health and Human Services, U.F.C., CBER, Guidance for Industry - Assay Development for Immunogenicity Testing of Therapeutic Proteins (Draft). US Department of Health and Human Services, Washington, DC, USA, 2009).

The essence of the invention

This invention relates to methods for reducing drug target interference in a bridging anti-drug antibody (ADA) immunoassay used to determine the presence of anti-drug ADA in a serum sample. These methods include contacting a serum sample with a capture drug labeled with a first label; a detection drug labeled with a second label; and a reagent that blocks the drug target. This contacting step is then followed by incubation of the capture drug, the detection drug, and the drug target blocking reagent, which allows the drug target blocking reagent to interact with the drug target present in the sample, thereby reducing interference. drug targets in ADA bridging immunoassay. A bridging immunoassay for anti-drug antibodies (ADA) may then be performed.

In one aspect of the present invention, the serum sample is a human serum sample, and in a particular aspect, the sample is collected from a subject being treated with the drug.

In another aspect of the invention, the drug target blocking reagent is a binding molecule, such as, for example, an antibody. In one embodiment of the invention, in which the binding molecule is an antibody, it may contain a human constant region. Alternatively, in another embodiment of the invention, the drug target blocking antibody comprises a mouse constant region.

In one aspect of the invention, the incubation step is carried out at room temperature. In another aspect of the invention, the incubation is carried out under moderately alkaline pH conditions.

In one aspect of the invention, the drug of this invention is a therapeutic protein used to treat humans, such as a therapeutic binding molecule, such as a monoclonal antibody (e.g., a fully human monoclonal antibody) or a therapeutic fusion protein, such as a receptor protein fused to an Fc domain immunoglobulin, for example the Fc domain of IgG1, intended for the treatment of humans. In a particular aspect of the invention, the drug is a therapeutic human monoclonal antibody.

In one aspect of the present invention, the drug target is a soluble protein, such as, for example, a receptor ligand. In a particular aspect of the invention, the drug target blocking reagent includes a receptor region fused to an IgG Fc domain. In a further aspect of the invention, the receptor site is an extracellular region of the receptor. The IgG Fc domain may be a mouse IgG Fc domain or a human IgG Fc domain.

In another aspect of the invention, the drug target of this invention is a soluble or excreted dimeric or multimeric drug target. In a particular aspect of the invention, the drug target is a homodimeric drug target.

In one aspect of the invention, the entrapment medicament of this invention is attached to a solid surface. In a particular aspect of the invention, the solid surface is a microtiter plate. In another aspect of the invention, the solid surface is coated with strepta-vidin.

In one aspect of the invention, the first tag is selected from the group consisting of a biotin tag, a protein A tag, a protein G tag, and a glutathionine S-transferase (GST) tag. In another aspect of the invention, the second label is selected from the group consisting of a ruthenium label, a radiological label, a photoluminescent label, a chemiluminescent label, a fluorescent label, an electrochemiluminescent label, and an enzyme label.

The method may further include contacting the serum sample with a second drug target blocking reagent. This second drug target blocking reagent is a drug target blocking binding molecule, such as, for example, an antibody. In one aspect of the invention, the second drug target blocking antibody comprises a human constant region. Alternatively, the second drug target blocking antibody contains a mouse constant region.

The present invention provides a method for reducing the interference of a drug target in an anti-drug antibody (ADA) bridging immunoassay for determining the presence of an anti-drug ADA in a serum sample, comprising contacting the serum sample with a capture drug labeled with a first label; a detection drug labeled with a second label; the first binding molecule that blocks the drug target; and a second drug target blocking binding molecule, incubating, under moderately alkaline pH conditions, the capture drug, the detection drug, the first drug target blocking binding molecule, and the second drug target blocking binding molecule, and causing the first binding molecule to interact a drug target blocking molecule and a second drug target blocking molecule binding to the drug target present in the sample, thereby reducing the interference of the drug target in the bridging ADA immunoassay. In a specific aspect of the invention, the first and second drug target blocking binding molecules are an antibody.

The present invention further provides a method for reducing the interference of a drug target in a bridging anti-drug antibody (ADA) immunoassay to determine the presence of an anti-drug ADA in a serum sample, wherein the drug target is a soluble protein such as, for example, a ligand receptor, and the method includes contacting a serum sample with a capture drug labeled with a first label; a detection drug labeled with a second label; one or more drug target blocking binding molecules (e.g., an antibody), incubation under moderately alkaline pH assay conditions, wherein the capture drug, detection drug, and one or more drug target blocking binding molecules react with the target drug present in the sample, thereby reducing interference with the drug target in the ADA bridging immunoassay.

This invention is illustrated by the following graphics and detailed description, which do not limit the scope of the invention as described in the claims.

Brief description of graphic materials

Fig. 1A-1C illustrate the interference of a soluble dimeric/multimeric target in bridging immunogenicity assays. (A) Positive controls or ADA in human serum samples bridge the biotinylated drug and the ruthenium-labeled drug and produce a true-positive ADA signal. (B) However, dimeric/multimeric targets can also bind to both biotinylated and ruthenium-labeled drugs and produce a target-mediated false positive signal. (C) Antibodies to the target or other target blocking reagents can prevent the binding of the dimeric target to the biotinylated drug and the ruthenium-labeled drug, eliminating the target-mediated false positive signal. The reagents designated in (C) represent an capture drug (1), a detection drug (2), and one or more drug target blocking molecules (3).

Fig. 2A-2C illustrate the effect of the target blocking antibody HuAb 1 and assay pH on the assay signal. (A) Without target-blocking antibodies, acid dissociation increased the background assay signal in an unexposed human serum sample, possibly due to cleavage of the target from its endogenous binding proteins. (B) Target tolerance increased with the addition of 100 μg/mL HuAb1 at neutral pH (from approximately 3 to 150 ng/mL) and increased further (to approximately 1.1 μg/mL) for the combination of HuAb1 and moderately alkaline pH. (C) Addition of 100 μg/mL HuAb1 under basal assay conditions (pH 8.3) significantly reduced mediation

- 2 045148 target signal in unexposed human serum samples.

Fig. 3A-3D illustrate that the ADA signal obtained from analyzing clinical study samples from various subjects using the target blocking antibody HuAb1 and an assay pH of 8.3 correlated with target levels but did not correspond to their pharmacokinetic profiles. (A) Target concentration for each sample from the clinical study. (B) ADA signal using 100 μg/ml target blocking antibody HuAb1 and pH 8.3. (C) Concentration profiles of drug X for the 3 test subjects. (D) Addition of a second target antibody (HuAb2 at 100 μg/mL) in combination with HuAb1 (100 μg/mL) and at moderately alkaline pH effectively reduced target-mediated background signal in clinical study samples.

Fig. 4A-4C illustrate the signal-to-noise ratio (assay sample signal compared to assay background signal) after further optimization of the assay to not only suppress target interference but also avoid possible false negative signals. (A) The combination of HuSR and HuAb2 was as effective as the combination of HuAb1 and HuAb2 in reducing target interference in clinical study samples. (B) and (C) Mouse Fc target blocking reagents (MsSR and MsAb2) were still effective in reducing target interference in clinical study samples.

Fig. 5 illustrates the elimination of target interference in samples from a clinical study with 100 μg/mL MsSR, 100 μg/mL MsAb2, and assay pH 8.3. Only background assay signal was detected in clinical study samples from 7 subjects at 4 different time points (day 1, 29, 57, and 113).

Fig. 6A, 6B illustrate the minimal effect of mildly alkaline pH assays and target blocking reagents on the detection of polyclonal ADA in sera from immunized rabbits and in rat toxicology samples. (A) A moderately alkaline pH of 8.3 had no negative effect on the detection of ADA in the serum of a rabbit immunized with drug Fab. (B) An improved assay format attenuates target-mediated signals and allows detection of ADA signals in real toxicology study samples in rats.

Fig. 7A, 7B illustrate target-drug binding in the absence and presence of target blocking reagents and at various assay pH values. (A) Binding association and dissociation curves under different assay conditions. The wavelength shift (Δ nm) is directly proportional to the change in the thickness of the biosensor tip resulting from target-drug binding. Target-drug association and dissociation are shown at pH 7.3 and 8.2, in the presence of MsAb2 and MsSR at pH 7.3, and in the presence of MsAb2 and MsSR at pH 8.2. Control buffer solutions at pH 7.3 and 8.2 are also shown. Target-drug binding is partially inhibited by pH 8.3 alone. Although the combination of MsAb2 and MsSR at a neutral pH assay can significantly reduce drug target binding, complete inhibition of binding is achieved with the combination of MsAb2, MsSR and a moderately alkaline pH. (B) Target-mediated signals in clinical study samples were partially abolished by moderately alkaline pH alone or by the presence of MsAb2 and MsSR at neutral pH. However, complete elimination of the interfering influence is achieved by a combination of MsAb2, MsSR and pH 8.3.

Detailed description of the invention

The immunogenicity of drugs, especially therapeutic proteins, is a major concern in clinical and preclinical research because it can lead to potentially serious side effects, loss of efficacy, and changes in drug exposure, complicating the interpretation of toxicity, pharmacokinetics (PK), and pharmacodynamics (PD) data. . Anti-drug antibody (ADA) immunoassays to detect and quantify ADA are important for determining the immunogenicity of biotherapeutics. However, drug targets can interfere with ADA immunoassays and lead to target-mediated false-positive results (see Fig. 1B). Target levels can be high based on up-regulation of the target and release of the target from target drug and/or target: binding protein complexes during the acid dissociation step commonly used in ADA assays to increase the tolerable level of drug in the assay.

ADA is typically tested using a tiered approach to detect, confirm, and titrate ADA. A screening assay typically uses a floating cutoff point to identify samples that are potentially ADA positive, while a confirmatory assay uses a confirmatory cutoff point to determine whether the observed positive response in the screening assay may be suppressed by the presence of excess drug (as confirmed by the sample as ADA positive). The titration cutoff point is used in titration assays to estimate ADA levels in positive samples. ADA assays typically use a bridge format using drugs as capture and detection reagents. These tests are relatively simple to set up and perform,

- 3 045148 detects responses from most isotypes, with the exception of most IgG4, and provides excellent sensitivity. They are independent of species and have high throughput. However, soluble or released dimeric or multimeric drug targets can interfere with the assay and lead to target-mediated false-positive results. For example, elevated levels of IL-5 homodimer in post-dose samples from patients treated with mepolizumab contributed to the observed increase in positive cases in the ADA assay due to target-mediated false-positive signals in the ADA bridge assay (Liao, K. et al., J. Immunol. Methods, 2017, 441: pp. 15-23). The presence of NGF, a homodimer, also produced false positive assay signals in samples from patients treated with fulranumab due to bridging of fulranumab labeled with biotin and ruthenium (Dai, S. et al., AAPS J., 2014, 16(3): pp. 464-77). CD20 present on cell membrane fragments has also been reported to result in matrix interference in the ADA assay for ofatumumab (Chen, K. et al., J. Immunol. Methods, 2013, 394(1-2): p. 22 -31). In addition, acid dissociation has recently been reported to dimerize the monomeric target in serum samples, which also leads to false-positive signals (Zoghbi, J. et al., J. Immunol. Methods, 2015, 426: p. 62-9).

Some attempts have been reported to limit the interfering influence of the target. For example, pretreatment with target-blocking antibodies or blocking with target-binding proteins, as well as immunodepletion of the target, have been used to reduce the interference of a soluble target (Liao, K. et al., J. Immunol. Methods, 2017, 441: p. 15 -23; Dai, S. et al., AAPS J., 2014, 16(3): p. 464-77; Zhong, Z. D. et al., J. Immunol. Methods, 2010, 355(1-2): pp. 21-8; Weeraratne, D. K. et al., J. Immunol. Methods, 2013, 396(1-2): pp. 44-55; and Maria M. L. J., Wakshull E., Quarmby V., AAPS National Biotechnology Conference , Seattle, WA, USA, 2009). Other strategies have been reported, including the use of wheat germ agglutinin (WGA) lectin to block the interference of a highly glycosylated target protein without affecting ADA detection (Carrasco-Triguero, M. et al., Bioanalysis, 2012, 4(16): p. 2013-26). Another alternative assay format that uses the human soluble Fcy I receptor (hsFcyRI) to detect the ADA Fc region has also been reported to reduce interference from soluble targets, however this format does not detect all possible ADA isotypes (Wessels, U. et. al., Bioanalysis, 2016, 8(20): p. 2135-45). A white paper was recently published describing a number of strategies to reduce interference with drug targets in ADA and neutralizing antibody (NAb) assays (Zhong, Z.D. et al., AAPS J., 2017, 19(6): p. 1564 -1575). Therefore, it is important to develop reliable testing methods that can overcome target interference and provide reliable immunogenicity assessments in both preclinical and clinical studies.

The present invention provides methods for reducing target interference in ADA immunoassays to reduce the number of false positives in ADA immunoassays, such as bridging immunogenicity assays. In particular, this disclosure is based, at least in part, on the discovery that the combination of one or more target blocking reagents, such as antibodies or target receptor-Fc IgG fusion proteins, under mildly alkaline pH assay conditions results in high acceptable levels of recombinant target protein and a reduction in false-positive results in study samples with PK profiles that did not indicate a significant ADA response. Accordingly, the methods described herein provide a reduction in target interference when standard acid dissociation procedures and target blocking antibodies alone are ineffective.

I. Definitions.

To make the present invention easier to understand, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter is given, it is understood that values and ranges intermediate to the listed values are also part of this invention.

In the following description, specific numbers, materials, and configurations are set forth for purposes of explanation in order to provide a thorough understanding of the invention. However, it will be apparent to one skilled in the art that the invention can be practiced without these specific details. In some cases, well-known features may be omitted or simplified so as not to obscure the present invention.

The singular is used herein to denote one or more than one (ie at least one) singular grammatical entity. By way of example, an element means one element or more than one element.

The term antibody includes an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains: CH1, CH2 and CH3. Each light chain consists of a variable

- 4 045148 light chain region (abbreviated herein as LCVR or VL) and light chain constant region. The light chain constant region consists of a single CL domain. 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 consists of three CDRs and four FRs, located in the amino-terminal to carboxy-terminal direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The term antigen, as used herein, means any substance that causes the immune system to produce antibodies or specific cell-mediated immune responses against it. A disease-associated antigen is any substance associated with a disease that causes the immune system to produce antibodies or a specific cell-mediated response against it.

A binding domain (also referred to as a binding region or binding fragment), as used herein, refers to a molecule or part thereof (eg, peptide, oligopeptide, polypeptide, protein) that has the ability to specifically and non-covalently associate, bind, or join with a target. A binding domain includes any naturally occurring, synthetic, semi-synthetic or recombinant binding partner for a biological molecule, molecular complex (ie, a complex containing two or more biological molecules) or other target of interest. Examples of binding domains include single chain immunoglobulin variable regions (eg, scTCR, scFv), receptor ectodomains, ligands (eg, cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind a biological molecule, molecular complex, or other target of interest.

A binding molecule, as contemplated herein, is a molecule that specifically interacts with a particular target. Examples of such binding molecules include, but are not limited to, antibodies (including monoclonal antibodies) and fragments thereof, engineered antibodies, fusion proteins and other similar antigen binding molecules well known to those skilled in the art. Additionally, the term binding molecule, as used herein, includes a receptor or receptor-like molecule that can interact with a target.

Anti-drug antibodies, or ADAs, are antibodies that can be directed against any region of the drug, such as, for example, the variable domain, constant domains, or glycostructure of the drug. Such drug antibodies may arise during antibody therapy as an immunogenic response in the patient (see Pan, Y. et al., FASEB J., 9 (1995) 43-49). Most anti-drug antibodies bind to one or more complementarity-determining regions of the drug. The affinity of drug antibodies for their drug antigen is generally lower compared to the affinity of a drug for its target antigen.

The term bridging immunoassay or bridging ADA immunoassay, as used herein, refers to a sandwich-type immunoassay in which divalent ADA is coupled to two different binding molecules (i.e., a capture drug and a detection drug), each of which binds to different non-overlapping or non-interfering ADA epitopes. In this assay, a sandwich is formed containing a capture antibody, an ADA, and a detection antibody, and thus the ADA bridges the two antibodies that bind to it (see Fig. 1A). The capture antibody can be attached to a solid surface, such as a microtiter plate or other solid surface. The bridging immunoassay may be a high throughput assay. In one aspect of the invention, a bridging immunoassay for ADA, as described herein, includes two drug antibodies, a capture drug and a detection drug. In one embodiment of the invention, the detection drug and the capture drug comprise the same antibody molecule, for example, recombinantly produced using the same expression vector and containing the same amino acid sequence.

pH is a logarithmic scale used to determine the acidity or basicity of an aqueous solution. It approximately represents the negative value of the decimal logarithm of the molar concentration of hydrogen ions, measured in moles per liter. More precisely, it represents the negative value of the decimal logarithm of the activity of hydrogen ions. Solutions with a pH less than 7 are acidic, and solutions with a pH greater than 7 are basic. The term moderately alkaline pH, as used herein in relation to assay conditions, refers to a pH from about pH 7.5 to about pH 9.5. In one aspect of the invention, a moderately alkaline pH value includes a pH from about pH 8.0 to about pH 9.0. In another aspect of the invention, a moderately alkaline pH value includes a pH from about pH 8.5 to about pH 9.5. In another aspect of the invention, a moderately alkaline pH value includes a pH of about pH 7.5 to about pH 8.5. In another aspect of the invention, a moderately alkaline pH includes a pH of about 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, or 8.4.

The term drug, as used herein, refers to a therapeutic protein or a therapeutically effective portion thereof that can be administered to a subject to treat a disease. In one aspect of the invention, the drug is a human therapeutic protein, such as a therapeutic human monoclonal antibody or a human therapeutic fusion protein, such as a receptor protein fused to an immunoglobulin Fc domain, such as an IgG1 Fc domain. In another aspect of the invention, the drug is a humanized therapeutic monoclonal antibody. In another aspect of the invention, the drug is a chimeric antibody. In yet another aspect of the invention, the drug is a mouse antibody. In one aspect of the invention, a therapeutic drug is evaluated in a clinical study.

Therapeutics such as antibody drugs are widely used to treat various diseases such as cancer, immunological diseases, central nervous system diseases, vascular diseases and infectious diseases. Antibody drugs that are included in the methods of the invention include any therapeutic antibodies approved by a regulatory agency or in clinical or preclinical trials. Antibody drugs approved in the US and EU as of 2018 include, but are not limited to, bezlotoxumab, avelumab, dupilumab, durvalumab, ocrelizumab, brodalumab, reslizumab, olaratumab, daratumumab, elotuzumab, necitumumab, infliximab, obiltoxaximab , atezolizumab, secukinumab, mepolizumab, nivolumab, alirocumab, idarucizumab, evolocumab, dinutuximab, bevacizumab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, alemtuzumab, pertuzumab, infliximab, obinutuzumab, brentuximab, raxiba cumab, belimumab, ipilimumab, denosumab, ofatumumab, besilesomab, tocilizumab a lemtuzumab, trastuzumab, gemtuzumab ozogamicin, infliximab, palivizumab, necitumumab, basiliximab, rituximab.

The drug used in the methods of the invention also includes a therapeutic drug that is in development or undergoing preclinical or clinical trials, i.e. evaluated in clinical trials.

Antibody drugs may include antibodies targeting any antigen, including, for example, IL-4R, IL-6R, IL-33, PD-1, CD20 X CD3, LAG-3, IL-33, Fel d 1, C5, ANGPTL-3, activin A, GDF8, PCSK9, VEGF, NGF, or a viral antigen such as Ebola virus or MERS-CoV.

Additional therapeutic agents that can be used in the methods of the invention include, for example, evinacumab, alarmrumab, cemiplimab, alirocumab, aflibercept, fasinumab, rilonacept and sarilumab.

Therapeutics also include biosimilar versions of approved drugs, such as antibodies or therapeutic fusion proteins. For example, biosimilars to aflibercept are being developed, including ALT-L9 (Alteogen), M710 (Momenta/Mylan), FYB203 (Formycon (DE)/Santo Holding GmbH), and CHS-2020 (Coherus).

Drug target, as used herein, refers to the target of a drug. For example, in one embodiment of the invention, the drug target is a dimeric target. In one embodiment of the invention, the drug target is a homodimeric drug target. In another embodiment of the invention, the drug target is a multimeric drug target. In another embodiment of the invention, the drug target is a soluble or excreted drug target. The drug target may interfere with the ADA immunoassay and result in false-positive assay results.

A drug target blocking reagent, as used herein, refers to any reagent that is capable of binding to and/or blocking a drug target in an immunoassay, thereby preventing the drug target from binding to the capture drug or drug target. detection. In one embodiment of the invention, the drug target blocking reagent is a target blocking antibody. The target blocking antibody may comprise a human constant region or a mouse constant region. In another embodiment of the invention, the drug target blocking reagent is a target receptor-IgG Fc fusion protein, wherein the fusion protein includes a portion of the target receptor linked or fused to an IgG Fc domain. In one aspect of the invention, the target receptor site is an extracellular receptor site. In a specific aspect of the invention, the IgG Fc domain is a human IgG Fc domain. In another aspect of the invention, the Fc domain of an IgG is a mouse IgG Fc domain. How

- 6 045148 described in detail herein, an ADA immunoassay, such as a bridging ADA immunoassay, may include one or more drug target blocking reagents that reduce interference with the drug target in the immunoassay. For example, in one embodiment of the invention, the immunoassay may include a single drug target blocking reagent. In another embodiment, the immunoassay may include two drug target blocking reagents. In one embodiment of the invention, both drug target blocking reagents may contain drug target blocking antibodies. In another embodiment, the assay may include one or more drug target blocking antibodies and one or more target receptor-IgG Fc fusion proteins.

As used herein, an agent or reagent (e.g., binding molecule, drug capture, drug detection, anti-drug antibody (ADA), drug, protein, enzyme, antibody, antibody fragment or related molecules) that modified by the term labeled, includes any agent conjugated to another molecule or chemical agent that is experimentally detectable (eg, a detectable label). Chemical agents suitable as labels for the labeling agents include, but are not limited to, ruthenium, radiological label, photoluminescent label, chemiluminescent label, fluorescent label, electrochemiluminescent label, enzyme label, quantum dots or optical dye label. Other tags include, for example, biotin, protein A, protein G, glutathionine S-transferase (GST). These tags can be used to label drug antibodies for capture, which can then be attached to a solid surface.

As used herein, the terms fluorescent tag and fluorophore may be used interchangeably and refer to any substance that emits electromagnetic energy, such as light at a certain wavelength (emission wavelength), when the substance is illuminated by radiation at a different wavelength (excitation wavelength). , and are intended to cover a chemical or biochemical molecule or fragments thereof that are capable of interacting or reacting specifically with the analyte of interest in a sample to provide one or more optical signals.

As used herein, target tolerance is defined as the amount of target required to generate a target-mediated false positive signal in the assay (with assay signal above the plate cutoff point).

The term sample includes, but is not limited to, any quantity of a substance from a living or previously living organism. Such living organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits and other animals. In one embodiment of the invention, such samples include, but are not limited to, whole blood, serum, or plasma from a subject. In one embodiment of the invention, the sample, such as a serum sample, is a sample obtained from a subject during clinical or preclinical testing of a drug. For example, a sample may be obtained from a subject after administration of a drug during a clinical trial.

As used herein, the term subject refers to a human or non-human organism. Thus, the methods and fusion complexes described herein are applicable to diseases and pathological conditions of both humans and animals. Subjects can be patients, i.e. living persons or non-human organisms receiving medical care for a disease or condition, or persons or non-human organisms without a specified disease that are being examined for signs of pathology or the presence/absence of a specified disease condition. Subjects also include participants in clinical trials of a drug where the subject is administered the drug for the purpose of the trial.

The term Fc domain, or immunoglobulin Fc, or Ig Fc, is intended to designate the crystallizable fragment of the heavy chain region of an immunoglobulin. Typically, the Fc domain is able to interact with a second Fc domain to form a dimeric complex. The Fc domain may be capable of binding cell surface receptors called Fc receptors and/or complement proteins or may be modified to decrease or increase such binding ability. The Fc domain may be derived from IgG, IgA, IgD, IgM, or IgE antibody isotypes (referred to herein as the IgG Fc domain, IgA Fc domain, IgD Fc domain, IgM Fc domain, and IgE Fc domain, respectively). The Fc domain can influence immune activity, including opsonization, cell lysis, degranulation of mast cells, basophils and eosinophils, and other Fc receptor-dependent processes; activation of the complement pathway; and protein stability in vivo.

The term polypeptide is intended to refer to any polymer containing any of the 20 naturally occurring amino acids, regardless of size. Although the term protein is often used to refer to relatively large proteins, peptide is often used to refer to small polypeptides

- 7 045148 tides, the use of these terms in the field often overlaps. The term polypeptide generally refers to proteins, polypeptides, and peptides unless otherwise noted. Peptides useful in accordance with this disclosure typically range from about 0.1 to 100 kDa or more to about 1000 kDa, preferably about 0.1, 0.2, 0.5, 1.2, 5, 10, 20, 30 and 50 kDa, as assessed using standard molecular sizing methods such as centrifugation or SDS-polyacrylamide gel electrophoresis.

As used herein, the term soluble means that the fusion molecule is soluble if it remains in aqueous solution at a temperature above about 5-37°C and at or near neutral pH in the presence of low or no concentration of anionic or nonionic surfactant substances. Under these conditions, the soluble protein will often have a low sedimentation number, for example, less than about 10 to 50 Svedberg units. The aqueous solutions mentioned herein typically contain a buffer compound to adjust the pH, typically in the pH range of about 5-9 and in the ionic strength range of about 2 to 500 mM. Sometimes a protease inhibitor or a mild nonionic surfactant is added. Additionally, a carrier protein such as bovine serum albumin (BSA) may be added, if desired, up to several mg/ml. Examples of aqueous buffers include standard phosphate-buffered saline, Tris-buffered saline, or other well-known buffers and cell media formulations.

The term solid surface means a non-flowing substance and includes particles (including microparticles and beads) made from materials such as polymer, metal (paramagnetic, ferromagnetic particles), glass and ceramic; gelling agents such as silica, alumina and polymer gels; capillaries, which can be made of polymer, metal, glass and/or ceramic; zeolites and other porous substances; electrodes; microtiter plates; solid strips; cuvettes, test tubes, or other spectrometer sample containers. The term isolated refers to a composition, compound, substance or molecule modified by man from its natural state. For example, a naturally occurring composition or substance is isolated if it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide naturally present in a living animal is not isolated, but the same polynucleotide or polypeptide separated from coexisting substances in its natural state is isolated, as that term is used herein.

II. Reducing the interfering influence of the target.

The ADA bridging immunoassay for detecting ADA in a sample, as detailed below, is sensitive to interference from the drug target (false positive ADA signal). In a bridging ADA immunoassay, a sample is incubated with a capture drug (labeled or unlabeled) and a detection drug containing a detectable label. After incubation of the sample, a sandwich is formed containing the capture drug, ADA and detection drug, and thus the ADA bridges the two binding drugs and the bound ADA can be detected (see Fig. 1A). The true positive signal in the ADA bridging assay results from the divalent binding of ADA to the capture drug and detection drug to form a bridge. However, a false positive occurs when a dimeric or multimeric target bridges the capture drug and the detection drug, thereby bridging the target (see FIG. 1B and, for example, Liao, K. et al., J. Immunol. Methods, 2017, 441: pp. 15-23).

As shown in the example provided herein, the present invention provides methods for reducing the interference of a drug target in a bridging immunoassay for ADA, thereby reducing the number of false positives. The methods include incubating the sample with at least one drug target blocking reagent under moderately alkaline pH assay conditions.

In one embodiment, the invention provides a method for reducing the interference of a drug target in an ADA bridging immunoassay to determine the presence of drug ADA in a serum sample. This method involves contacting a serum sample with a capture drug, a detection drug, and one or more drug target blocking reagents. These components are then incubated under the mildly alkaline pH conditions of the assay, allowing the drug target blocking reagent to interact with the drug target present in the sample, thereby reducing the interference of the drug target in the ADA bridging immunoassay.

In one aspect of the invention, the method further includes performing a bridging immunoassay for ADA, as described below.

In another aspect of the invention, one or more drug target blocking reagents are capable of binding to and/or blocking the drug target in an immunoassay, thereby preventing the drug target from binding to the capture drug and/or the drug target. detection. In a particular aspect of the invention, the one or more target blocking reagents are a target blocking binding molecule, such as an antibody. In a further aspect of the invention, the target blocking antibody comprises a human constant region.

If the target-blocking binding molecule (eg, antibody) and the drug (eg, drug antibody) have the same human constant regions, ADAs in serum samples that are specific to the Fc region of the drug can bind to the human target-blocking antibody and potentially difficult to detect in analysis. Replacing the human Ig regions of a target-blocking antibody with mouse Ig regions may result in a reduction in the interfering effect of the drug target-blocking antibody on ADA detection. Thus, in one aspect of the invention, the target blocking antibody comprises a mouse constant region.

The ADA bridging immunoassay may include one or more drug target blocking reagents. In one aspect of the invention, the immunoassay includes two drug target blocking reagents. In a specific aspect of the invention, the two drug target blocking reagents are drug target blocking antibodies, eg, a first drug target blocking antibody and a second drug target blocking antibody. In a more specific aspect of the invention, the first and/or second target blocking antibodies contain human constant regions. In another specific aspect of the invention, the first and/or second target blocking antibodies contain mouse constant regions. In yet another aspect of the invention, both the first and second target blocking antibodies contain human constant regions. In yet another aspect of the invention, both the first and second target blocking antibodies contain mouse constant regions. In another aspect of the invention, the first target blocking antibody contains a human constant region and the second target blocking antibody contains a mouse constant region. In yet another aspect of the invention, the first target blocking antibody contains a mouse constant region and the second target blocking antibody contains a human constant region.

In one embodiment, the invention provides methods for reducing drug target interference in an ADA bridging immunoassay to determine the presence of drug ADA in a serum sample, comprising contacting the serum sample with a capture drug, a detection drug, a first antibody , blocking the drug target, and a second antibody, blocking the drug target. These components are incubated under moderately alkaline pH assay conditions, which allows the first drug target blocking antibody and the second drug target blocking antibody to interact with the drug target present in the sample, thereby reducing interference from the drug target in bridging immunoassay. analysis for ADA.

As described herein, we have discovered that target blocking antibodies used in ADA bridging immunoassays may share some CDR VH rearrangements with the drug of interest. Accordingly, ADAs specific to these VH rearrangement regions may bind to the target blocking antibody in the sample, potentially making them difficult to detect in the assay. To optimize detection, a target receptor fused to a human IgG Fc was constructed and used in the methods of the present invention in place of one of the target blocking antibodies.

In one embodiment, one or more drug target blocking reagents used in the methods of the invention comprise a target receptor region fused to an IgG Fc domain (a target receptor-IgG Fc fusion protein). In one aspect of the invention, the target receptor site is an extracellular receptor site. In another aspect of the invention, the target receptor-IgG Fc fusion protein is soluble. In a specific aspect of the invention, the IgG Fc domain of the target receptor-IgG Fc fusion protein is a human IgG Fc domain.

If the target receptor-IgG Fc fusion protein and drug have human IgG Fc regions, ADAs in serum samples specific to the drug Fc region may bind to the target receptor-IgG Fc fusion protein and potentially hamper detection in the assay. Replacing the human Ig Fc regions of the target receptor fusion protein and IgG Fc with mouse Ig regions may result in a reduction in the interference of the target receptor fusion protein and IgG Fc on ADA detection. Therefore, in another aspect of the invention, the IgG Fc domain of the target receptor-IgG Fc fusion protein is the Fc domain of a mouse IgG.

The assay may include one or more drug target blocking antibodies and one or more target receptor-IgG Fc fusion proteins. In addition, the assay may include a target blocking antibody and a target receptor-IgG Fc fusion protein.

Accordingly, in one embodiment, the invention provides a method for reducing the interference of a drug target in a bridging immunoassay for ADA

- 9 045148 for determining the presence of a drug ADA in a serum sample, wherein the drug target is a soluble protein, such as, for example, a receptor ligand, and the method includes contacting the serum sample with an capture drug, a detection drug, a drug target blocking reagent containing an extracellular portion of the receptor fused to an IgG Fc domain, and a drug target blocking antibody. These components are incubated under moderately alkaline pH conditions of the assay, allowing the drug target blocking reagent and drug target blocking antibody to interact with the drug target present in the sample, thereby reducing interference from the drug target in the bridging ADA immunoassay .

In one aspect of the invention, the Fc domain of an IgG is a mouse IgG Fc domain. In another aspect of the invention, the Fc domain of an IgG is a human IgG Fc domain. In yet another aspect of the invention, the drug target blocking antibody includes a human constant region. In yet another aspect of the invention, the drug target blocking antibody comprises a mouse constant region. In one aspect of the invention, the target receptor-IgG Fc fusion protein comprises a mouse IgG Fc and the target blocking antibody comprises a mouse constant region.

In one embodiment, each of the target blocking reagents has a concentration in the ADA bridging immunoassay from about 10 μg/ml to about 200 μg/ml. In one aspect of the invention, each of the target blocking reagents has a concentration of from about 20 μg/ml to about 175 μg/ml. In another aspect of the invention, each of the target blocking reagents has a concentration of from about 30 μg/ml to about 150 μg/ml. In yet another aspect of the invention, each of the target blocking reagents has a concentration of from about 40 μg/ml to about 125 μg/ml. In yet another aspect of the invention, each of the target blocking reagents has a concentration of from about 50 μg/ml to about 100 μg/ml. In another aspect, each of the target blocking reagents has a concentration of about 50, 60, 70, 75, 80, 85, 90, 95, or 100 μg/ml. In another aspect of the invention, each of the target blocking reagents has a concentration of about 100 μg/ml.

Methods described herein to reduce target interference include performing an ADA immunoassay under moderately alkaline pH assay conditions. Moderately alkaline pH includes a range of pH values slightly above neutral pH. In one embodiment of the invention, a moderately alkaline pH value refers to a pH of from about 7.5 to about 9.5. In one aspect of the invention, a moderately alkaline pH value includes a pH from about pH 7.5 to about pH 8.5. In one aspect of the invention, a moderately alkaline pH value includes a pH from about pH 8.5 to about pH 9.0. In one aspect of the invention, a moderately alkaline pH value includes a pH from about pH 8.0 to about pH 9.0. In another aspect of the invention, a moderately alkaline pH value includes a pH from about pH 8.5 to about pH 9.5.

III. Immunological tests for ADA.

Immunological assays are well known to one skilled in the art. Methods for performing such assays, as well as practical applications and techniques, are well known in the art and are described, for example, in Colowick, S.P., Caplan, N.O. (eds.), Methods in Enzymology, Academic Press, covering immunological detection methods, especially volumes 70, 73, 74, 84, 92 and 121. The principles of various immunoassays are described, for example, Hage, D. S. (Anal. Chem. 71 (1999) ) 294R-304R); Lu, V. et al. (Analyst, 121 (1996) 29R-32R), which describes oriented immobilization of antibodies for use in immunoassays. Avidin-biotin-mediated immunoassays are described, for example, in Wilchek, M., Bayer, E.A., Methods Enzymol., 184 (1990) 467-469.

A commonly used ADA assay is the bridging immunoassay (see, for example, Liao, K. et al., J. Immunol. Methods, 2017, 441: p. 15-23; Dai, S. et al., AAPS J., 2014, 16(3): pp. 464-77; and Zhong, Z.D. et al., AAPS J., 2017. 19(6): pp. 1564-1575, the contents of which are incorporated herein by reference) . The ADA bridging immunoassay is a sandwich-type immunoassay in which a multivalent ADA binds to two different drug antibodies (a capture drug and a detection drug), each of which binds to different non-overlapping or non-interfering ADA epitopes. Specifically, in this assay, a sample is incubated with a capture drug (labeled or unlabeled) and a detection drug containing a detectable label. After incubation of the sample, a sandwich is formed containing the capture drug, ADA and detection drug, and thus the ADA bridges the two binding drugs and the bound ADA can be detected (see Fig. 1A). In one aspect of the invention, the immunoassay is a high throughput assay.

The ADA bridging immunoassay further includes determining the presence or amount of ADA. Therefore, this disclosure provides a detection drug conjugated to a detectable label. Non-limiting Examples of Detectable Markers

- 10 045148 for any of the methods of the invention include ruthenium, a radiological label, a photoluminescent label, a chemiluminescent label, a fluorescent label, a fluorophore, a hapten, an electrochemiluminescent label or an enzyme label. The detectable mark can be measured using equipment and devices known to those skilled in the art.

Exemplary fluorophores for use in the methods presented herein include, for example, green fluorescent protein, blue fluorescent protein, red fluorescent protein, fluorescein, fluorescein-5-isothiocyanate (FITC), cyanine dyes (Cy3, Cy3.5, Cy5, Cy5.5, Cy7), Bodipy dyes (Invitrogen) and/or Alexa Fluor dyes (Invitrogen), dansyl, dansyl chloride (DNS-Cl), 5-(iodoacetamide)fluorescein (5-IAF), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-1,3-diazol-4-yl chloride (NBD-Cl), ethidium bromide, Lucifer yellow, rhodamine dyes (5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamine-B-sulfonyl chloride, rhodamine B -isothiocyanate (RITC (rhodamine-B-isothiocyanate), rhodamine 800); tetramethylrhodamine 5- (and 6-) isothiocyanate (TRITC)), Texas Red, sulfonyl chloride, naphthalamine sulfonic acids including, but not limited to, 1-anilinonaphthalene-8 -sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene2-sulfonic acid (TNS), anthroyl fatty acid, DPH, parinaric acid, TMA-DPH, fluorenyl fatty acid, fluorescein-phosphatidylethanolamine, Texas red phosphatidylethanolamine, pyrenyl-phosphatidylcholine, fluorenyl- phosphotidylcholine, merocyanine 540, naphthylstyryl, 3,3'-dipropylthiadicarbocyanine (diS-C3-(5)), 4-(p-dipentylaminostyryl)-1methylpyridinium (di-5-ASP), Cy-3-iodoacetamide, Cy-5- N-hydroxysuccinimide, Cy-7-isothiocyanate, IR-125, Thiazole orange, Azur-B, Nile blue, Al phthalocyanine, Oxaxine 1,4',6-diamidino-2phenylindole (DAPI), Hoechst 33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2, calcium green, carboxy-SNARF-6, BAPTA, coumarin, phytofluors, coronene and metal-ligand complexes.

Haptens for use in the methods presented herein include, for example, digoxigenin and biotin.

Enzymes for use in the methods presented herein include, for example, alkaline phosphatase (AP), β-galactosidase, horseradish peroxidase (HRP), soybean peroxidase (SBP), urease, β-lactamase and glucose oxidase.

In one embodiment of the invention, the entrapment drug is conjugated to a solid surface. In one aspect of the invention, conjugation of the capture drug to a solid surface is accomplished through a specific binding pair, wherein the capture drug is labeled or conjugated. In one aspect of the invention, the specific binding pair (first component/second component) is selected from streptavidin or avidin/biotin, biotin/neutravidin, biotin/captavidin, antibody/antigen (see, for example, Hermanson, G.T. et al., Bioconjugate Techniques, Academic Press, 1996), epitope/antibody, protein A/immunoglobulin, protein G/immunoglobulin, protein L/immunoglobulin, GST/glutathione, His tag/nickel, FLAG/M1 antibody, maltose binding protein/maltose, calmodulin binding protein/calmodulin , enzyme/enzyme substrate, lectin/polysaccharide, steroid/steroid binding protein, hormone/hormone receptor, and receptor-ligand binding pairs. In one aspect of the invention, the capture drug is conjugated to biotin (as the first component of a specific binding pair). In this case, conjugation with the solid phase occurs through immobilized avidin or streptavidin (see Fig. 1A).

In some embodiments, the GlcNac binding protein is conjugated to the first member of the binding pair (e.g., biotin, avidin, neutravidin, captavid, antibody, antigen, protein A, protein G, protein L, GST, His tag, FLAG, MBP, calmodulin binding protein, enzyme, receptor or ligand).

In one embodiment of the invention, the sample tested in the ADA immunoassay is a serum sample. In one aspect of the invention, the serum sample contains from 1 to 20% serum. In one aspect of the invention, the serum sample contains from about 1% to about 10% serum. In another aspect of the invention, the serum sample contains from about 10% to about 15% serum. In yet another embodiment of the invention, the serum sample contains from about 10% to about 20% serum. In yet another embodiment of the invention, the serum sample contains from about 15% to about 20% serum. In a particular aspect of the invention, the serum sample contains about 1% serum. In one aspect of the invention, the serum is human serum.

In one embodiment of the invention, the ADA bridging immunoassay includes an acid dissociation step. In one aspect of the invention, the serum sample is diluted 10-fold with an acid, such as acetic acid, and incubated at room temperature before incubation with the capture drug and detection drug.

In one embodiment of the invention, the capture drug and the detection drug have a concentration in the immunoassay of about 0.5 μg/ml to

- 11 045148 about 10 µg/ml. In one aspect of the invention, the capture drug and the detection drug have a concentration of greater than 0.5 μg/ml to less than 10 μg/ml. In another aspect of the invention, the capture drug and the detection drug have a concentration of from about 0.5 μg/ml to about 5 μg/ml. In yet another aspect of the invention, the capture drug and detection drug have a concentration of from about 0.5 μg/ml to about 2.0 μg/ml. In yet another aspect of the invention, the capture drug and the detection drug have a concentration of from about 0.5 μg/ml to about 1 μg/ml. In a preferred aspect of the invention, the capture drug and detection drug have a concentration of about 0.5 μg/ml.

In one embodiment of the invention, incubation of the sample, capture drug, and detection drug is carried out at room temperature. In one aspect of the invention, the incubation time for the sample, capture drug, and detection drug is at least 0.5 hours. In another aspect of the invention, the incubation time is at least 1 hour. In one aspect of the invention, the incubation time is at least 1 hour .5 hours. In one aspect of the invention, the incubation time is up to 2 hours. In yet another aspect of the invention, the incubation time is from 0.5 to 12 hours. In one aspect of the invention, the incubation time is from 0.5 to 5 hours. In another aspect of the invention the incubation time is from 1 to 12 hours. In one aspect of the invention, the incubation time is from 1 to 5 hours. In another aspect of the invention, the incubation time is from 5 to 12 hours.

In one embodiment of the invention, after incubation of the sample, the capture drug, and the detection drug, the sample is transferred to a labeled hard surface, such as a streptavidin-labeled hard surface, and incubated further to allow the capture drug to attach to the hard surface. In one aspect of the invention, the incubation is carried out at room temperature. In another aspect of the invention, after incubation, samples are assayed for ADA binding using any method known in the art for detecting labeled antibodies, wherein the detection drug is detected based on detection of the drug tag, eg, ruthenium. The true positive signal in the ADA bridging assay results from the divalent binding of ADA to the capture drug and detection drug to form a bridge.

Solid surfaces for immunoassays described herein are widely described in the art (see, for example, Butler, J.E., Methods, 22 (2000) 4-23, which is incorporated herein by reference). The solid surface component of the assay differs from inert solid surfaces with which the assay mixture may come into contact in that the solid surface contains at least one moiety on its surface that is designed to interact with the drug for entrapment. The solid surface may be a stationary component, such as a tube, strip, cuvette, or microtiter plate, or may be a non-stationary component, such as beads and microparticles. Microparticles can also be used as a solid phase for uniform surface formats. Various microparticles can be used that allow the non-covalent or covalent attachment of proteins and other substances. 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. See, for example, Martin, C.R. et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, which is incorporated herein by reference.

The present invention is further illustrated by the following examples, which are not intended to be limiting in any way. The entire contents of all references, patents and published patent applications cited in this invention, as well as drawings, are hereby incorporated herein by reference.

Examples

Example. Reduce target interference in bridging immunogenicity assays with target blocking reagents and at moderately alkaline pH.

Materials and methods

Materials and reagents.

For the drug functional assay and target assay described herein, all solutions, unless otherwise noted, were prepared in assay buffer (0.5% BSA, 0.05% Tween-20, 1X PBS). For the ADA assay described herein, all solutions, unless otherwise noted, were prepared in 1% BSA, 1X PBS. FSB was from Life Technologies (Grand Island, NY). Trizma base, 1.5 M was from Sigma (St. Louis, MO). Glacial acetic acid was from Thermo Fisher Scientific (Waltham, MA). HBS-EP+(10X) buffer was from GE Life Science (Marlborough, MA). Human serum was purchased from Bioreclamation (Hicksville, NY). Streptavidin-coated microplates were from Meso Scale Discovery

- 12 045148 (Rockville, MD). Recombinant human protein target was from R&D System (Minneapolis, MN). Black microwell plates, horseradish peroxidase (HRP)-conjugated NeutrAvidin™, and picochemiluminescent ELISA substrate SuperSignal™ were from Thermo Fisher Scientific (Rockford, IL). HRP-conjugated goat anti-mouse IgG Fc fragment-specific antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). ANS (human IgG Fc capture) biosensors were from Pall ForteBio (Fremont, CA). Fully human monoclonal drug antibody, mouse monoclonal drug antibody, biotinylated drug, ruthenium-labeled drug, and all human and mouse monoclonal anti-drug antibodies (referred to herein as HuAb1, HuAb2 (human), and MsAb2 (mouse)), soluble receptor human-mouse fusion proteins (herein referred to as HuSR and MsSR, respectively) and biotinylated human monoclonal anti-target antibody (used in the ADA and target assays) were obtained from Regeneron Pharmaceuticals (Tarrytown, NY).

Determination of pH.

pH measurements were performed using a calibrated Metier Toledo meter (Columbus, OH) with an InLab Expert Pro-ISM™ electrode. Mixed human serum was diluted 10 times with 300 mM acetic acid. The acidified samples were then buffered 10-fold with various concentrations of Tris base solutions. pH measurements were performed on the final assay solutions as shown in the table below.

pH Conditions Evaluated for Drug ADA Detection

Solution pH Mix human serum 1:10 in 300 mM acetic acid, then 1:10 in master mix 70 mM Tris 8.43 60 mM Tris 8.30 50 mM Tris 8.13 40 mM Tris 7.83

Analysis for ADA.

Anti-drug antibodies (ADA) in human serum samples were detected using a non-quantitative ADA bridging immunoassay (Figures 1A-1C). This ADA bridging assay uses a mouse anti-drug monoclonal antibody as a positive control and a biotinylated drug and a ruthenium-labeled drug as bridging components (Figure 1A). Serum samples were acidified by diluting them 10-fold in 300 mM acetic acid and incubating at room temperature (RT) for at least 10 min. Biotinylated drug and ruthenium-labeled drug (0.5 μg/mL) were prepared in assay buffer containing 60 mM Tris base before addition to serum samples. The acid-treated serum samples were then diluted 10-fold with the labeled drug solution. After incubation for approximately 60 min at room temperature, samples were transferred to blocked (5% BSA) 96-well Streptavidin (MSD) Multi-Array™ plates and incubated for approximately 60 min at room temperature. The plate was washed and reading buffer was added, and the plates were read using an MSD plate reader, such as the SECTOR Imager 2400. To block target-mediated interference (Figure 1B), monoclonal anti-antibodies were also included in the labeled drug solution. target (100 μg/ml) and/or soluble receptor protein (100 μg/ml) (Fig. 1C). In addition, a solution of the labeled drug was prepared in 60 mM Tris to adjust the pH to a moderate alkaline value, which also minimizes target binding to both biotinylated and ruthenium-labeled drug.

Target identification.

This technique uses a microtiter plate coated with a mouse monoclonal antibody to the target (1 μg/ml), and a recombinant target protein is used as a standard. Standards and controls were prepared in a medium well known to those skilled in the art to eliminate interference from endogenous target protein from human serum. Standards, controls, and samples were diluted 10-fold with 300 mM acetic acid and incubated at room temperature for about 30 min. Acid-treated samples were neutralized (1:2 dilution) with a 300 mM Tris base solution spiked with anti-drug monoclonal antibody (100 μg/ml) to minimize drug interference before adding to the plate. The target protein captured on the plate was detected using another biotinylated human monoclonal anti-target antibody (200 ng/ml) followed by NeutrAvidin™ conjugated to horseradish peroxidase (NeutrAvidin-HRP™) (100 ng/ml). A peroxidase-specific luminol-based substrate was added to achieve a signal intensity that was proportional to the total target concentration.

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Functional analysis of the drug.

In a drug functional assay, the level of a drug antibody that is either not bound to a target or has only one arm bound to a target is quantified. This way it can still bind to the target molecule. The procedure used a microtiter plate coated with target (0.5 μg/ml) and used the drug antibody as a standard. Drug captured on the plate was detected using mouse monoclonal anti-human IgG4 antibody (250 ng/ml) followed by horseradish peroxidase-conjugated goat anti-mouse IgG Fc-specific antibody (anti-mouse IgG-HRP) (100 ng/ml ). A peroxidase-specific luminol-based substrate was then added to achieve a signal intensity that is proportional to the concentration of the functional drug.

Biolayer interferometry.

Target-drug antibody binding in the absence and presence of the mouse target-blocking antibody MsAb2 and soluble mouse receptor fusion protein MsSR at pH 7.3 or 8.3 was studied on an Octet RED96 system (Pall Forte Bio) at 30°C with shaking speed 1000 rpm ANS biosensors were pre-equilibrated with a dilution buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.02% (v/v) sodium azide, 1 mg/mL BSA, pH 7.3, for 30 min. .

An application step was performed at a drug concentration of 5 μg/mL in the same dilution buffer for 20 seconds (to achieve ~0.5 nm thickness) followed by a 60 second background step in the same dilution buffer before the biosensors were immersed in wells containing 200 μl of target at a concentration of 60 nM with or without 60 nM MsAb2 and MsSR at pH 7.3. The same procedure was also carried out in a pH 8.3 environment. Association curves were obtained for a total of ∼200 s followed by a dissociation phase of ∼300 s.

Results and discussion.

Moderately alkaline pH assay conditions with the target blocking antibody HuAb1 increase target tolerance levels and reduce background signals in unexposed human serum samples

Reported levels of the target in normal human serum are approximately 10 ng/mL. The target forms complexes with the drug antibody and several inhibitory binding proteins in the bloodstream. Anti-drug antibodies were measured by an ADA immunogenicity bridge assay using an acid dissociation step (Figures 1A-1C). Although acid treatment generally increases tolerable drug levels, it releases the target from its complex with the drug and any target-binding proteins, resulting in high background signals in an unexposed human serum sample (Figure 2A). Similar results have been reported previously when acid pretreatment was used to dissociate ADA and fulranumab. NGF-fulranumab complexes also dissociated, releasing free NGF, which interfered with the ADA assay and produced false-positive results (Dai, S. et al., AAPS J., 2014, 16(3): p. 464-77).

In the ADA assay without target blocking antibody and at a neutral assay pH, the target tolerance level (defined as the amount of target required to produce an assay signal above the plate cutoff point) determined using recombinant target protein is about 3 ng/mL (Fig. 2B). The target tolerance level increases to about 150 ng/ml in the presence of 100 μg/ml target blocking antibody HuAb1 at a neutral pH assay. Interestingly, at an assay pH of 8.3 and the same concentration of the human target blocking antibody HuAb1, the target tolerance level increased to about 1.1 μg/ml (Fig. 2B). The acceptable target level is even higher, about 7.5 μg/mL, when the assay pH is 8.9. Sensitivity and drug tolerance limit (DTL) values in the assay were similar at both neutral pH and pH 8.3. The assay signal in unexposed human serum samples was also reduced to background levels in the presence of 100 μg/ml HuAb1 and at moderately alkaline pH (Fig. 2C).

A moderately alkaline pH in combination with two target blocking reagents suppresses target-mediated background signals in clinical study samples.

To test whether the combination of human target blocking antibody HuAb1 and mild alkaline pH could also inhibit target interference in clinical trial samples, phase I clinical trial samples from three subjects (from the single-dose study) were analyzed. ) at 4 different time points (days 1, 29, 57 and 113). A 10- to 12-fold increase in target levels was observed in the day 29 and day 57 samples (Fig. 3A), with target levels similar to baseline levels in the day 113 samples as measured by an in-house target assay. The highest target concentration observed in these samples was approximately 60 ng/mL, which is much lower than the acceptable target level in the ADA assay. However, when these samples were tested in an ADA assay using 100 μg/ml human target blocking antibody HuAb1 at pH 8.3, positive signals were detected in samples at days 29 and 57, with assay signals close to background levels in the samples at day 113 (Fig. 3B).

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These subjects received a single dose of drug antibody, and their PK profiles did not suggest a significant ADA response (Fig. 3C). In addition, ADA signals in these samples appear to correlate with the levels of the target within them. Subsequently, a larger group of post-dose samples was also tested, most of which demonstrated positive ADA signals. Therefore, it is likely that the presence of elevated levels of the target is responsible for the positive signal in the assay, creating a target-mediated bridge with the labeled drugs.

To further reduce the apparent target interference observed in these clinical samples, a second human target blocking antibody, HuAb2, was added to HuAbl in combination with either a neutral pH or a moderately alkaline pH. Interestingly, the target-mediated background signal was significantly reduced by using 100 μg/ml target blocking antibodies, HuAb1 and HuAb2, in a neutral pH assay, although not to baseline levels. However, a subset of post-dose samples still produced signals in the assay above the plate cutoff. When these samples were tested again with the same concentration of target blocking antibodies, HuAb1 and HuAb2, but at an assay pH of 8.3, they all exhibited assay signals below the plate cutoff point (Fig. 3D).

In the original assay format with alkaline pH and 100 μg/ml target blocking antibody, HuAb1 appears to be unaffected by approximately 1.1 μg/ml recombinant target protein (Fig. 2A). However, in clinical study samples that contained relatively low levels of target protein (<60 ng/mL), target interference was observed. This indicates that the recombinant target protein may behave differently in the assay than the native protein and/or that patients may express different forms of the target protein with different binding properties compared to the recombinant protein, making the target-blocking antibody HuAb1 was less effective at inhibiting target interference in clinical study samples. This discrepancy in ADA assay results between recombinant and endogenous protein targets highlights the importance of using actual test samples to characterize assay performance.

Another interesting finding was the effect of moderately alkaline pH on target tolerance levels. The combination of HuAb1 and HuAb2 did not completely suppress target interference in these clinical study samples under neutral pH conditions. To completely eliminate target interference, slightly alkaline conditions were required along with two target blocking antibodies.

The combination of the target receptor MsSR and the target blocking antibody MsAb2 also reduces target interference.

The target blocking antibody HuAb1 shares some rearrangements in the VH CDRs with the therapeutic drug antibody, so any ADAs specific to these VH rearrangement regions may bind to HuAb1 in the assay buffer rather than to the labeled drugs, potentially making them difficult to detect in analysis. To overcome this possible problem, HuSR, which is a target receptor fused to human IgG Fc, was initially used to replace the target blocking antibody HuAb1. As shown in FIG. 4A, the combination of 100 μg/mL HuAb2 and 100 μg/mL HuSR can effectively inhibit target-mediated background signals in samples from a phase I clinical study. In fact, similar target tolerance levels were obtained with either the HuAb2/HuSR combination or the HuAb1/HuAb2 combination.

Additionally, the target blocking antibody HuAb2 has the same human IgG4 constant region as the drug antibody, and the target receptor HuSR contains the human IgG Fc. Any anti-drug antibodies in patient serum samples that are specific to the Fc region of the drug antibody may bind to HuAb2 and HuSR, which could also potentially make them difficult to detect in the assay. To overcome this potential problem, the Fc domain of HuSR and the entire constant region of HuAb2 were converted from human to murine to further reduce their possible interference with ADA detection. The combination of MsAb2 (HuAb2 with mouse constant region) and MsSR (HuSR with mouse Fc) was still effective in reducing target interference in clinical study samples (Figures 4B and 4C). Samples from the clinical study on days 1, 29, 57, and 113 showed only background signals with this new assay format, even though they contained high serum levels of target (Figure 5).

Moderately alkaline pH and target blocking reagents have minimal impact on the detection of ADA in real rabbit blood samples and in samples from rat toxicology studies.

To ensure that mildly alkaline pH and target blocking reagents had minimal impact on the stability and/or detection of ADA, rabbit blood samples obtained shortly after immunization with drug Fab were analyzed using this assay format.

Blood sample 1 and blood sample 2 from two rabbits, collected approximately 30-40 days after immunization, were analyzed using two target blocking reagents, either at neutral pH or at pH 8.3. Such rabbit blood collected at short notice typically contains low-affinity polyclonal drug antibodies, the detection of which may be more affected by more stringent assay conditions. As shown in FIG. 6A, the mean ADA amounts were similar for each assay condition regardless of assay pH, indicating that mildly alkaline pH and addition of target blocking reagents had little or no effect on the stability or detection of ADA in these samples.

To further confirm that the dual target blocking reagent format at pH 8.3 was capable of detecting ADA in post-dose samples, rat serum samples from 2 rats in a toxicology study were analyzed for both target and ADA levels. Target levels increased at least 10-fold in the day 28 and day 85 samples from rat 1 and about 10-fold in the day 28 sample from rat 2 compared to baseline samples, however these samples did not show an unusual increase in ADA signal in the assay , indicating that the interfering influence of the target has been reduced. At the same time, high levels of ADA were detected in both animals starting at day 85, indicating that the addition of two target blocking reagents and the mildly alkaline pH of the assay did not interfere with the detection of ADA (Fig. 6B).

Moderately alkaline pH, together with target blocking reagents, inhibit target-drug binding.

To understand why moderately alkaline pH and target-blocking reagents may help reduce target interference in clinical study samples, experiments were conducted using the Octet system to test target-drug binding under different pH conditions, with reagents blocking the target, or without them. The results are shown in Fig. 7A. As can be seen from the binding association curves, in the absence of MsAb2 and MsSR, the response was slightly lower at pH 8.2 compared to pH 7.3, indicating that the target-drug association is not significantly affected by the pH of the assay. However, a higher dissociation rate was observed at pH 8.2 compared to pH 7.3, indicating that the binding affinity between the target and drug is weaker at pH 8.2. At pH 7.3, in the presence of MsAb2 and MsSR, a higher dissociation rate was also observed, similar to alkaline pH only. However, the presence of target-blocking reagents also significantly influenced target-drug association. Finally, complete inhibition of binding was achieved by the combination of MsAb2 and MsSR at pH 8.2. pH-dependent differences in binding potentially relate to either a conformational change in the target at moderately alkaline pH or better binding of target blocking reagents to the target at pH 8.2, thereby inhibiting target-drug association.

These binding data further support the ADA assay data, which show that alkaline pH alone can partially inhibit target-mediated signaling in clinical study samples. While the combination of MsAb2 and MsSR at neutral pH significantly inhibited the target-mediated signal, complete inhibition of target interfering was achieved only for the combination of MsAb2, MsSR and pH 8.3 (Fig. 7B).

Ligand binding assays (LBAs) are very sensitive to interfering molecules that can distort assay results, either by producing artificial signals or blocking desired interactions in the assay. Target interference is a common problem and difficult to overcome due to its drug specificity and the highly variable biological properties of each target protein. The target typically forms strong complexes with the drug and its binding proteins in the bloodstream, which may be difficult to disrupt. In addition, target levels may increase to relatively high levels in the bloodstream due to the formation of drug target and target:binding protein complexes or through feedback mechanisms inherent in the biological pathway. In ADA bridging assays, the presence of dimeric or multimeric targets can lead to false-positive results and complicate the assessment of immunogenicity. This effect may be enhanced by acid dissociation, a commonly used strategy to increase drug tolerance in ADA assays, which can also potentially disrupt target-containing complexes, releasing the target and leading to target-mediated false-positive signals. Therefore, careful consideration must be given when developing an analysis, weighing the benefits of each strategy against the risk of introducing possible bias into the analysis. Based on biological properties, target-specific approaches need to be evaluated to reduce their interfering effects on the assay.

Pretreatment with target-blocking antibodies (as individual antibodies or in combination), receptors, cofactors, and target-binding proteins can be used to reduce target interference. In addition, the pH of the assay can also be altered to reduce target interference, either by directly affecting the dimeric or multimeric target or by altering the binding affinity of the drug to the target. In addition, it is important to evaluate the effectiveness of the chosen assay strategy/format in reducing target interference by testing real samples after dosing, since the native target protein in serum samples may behave differently than its recombinant variant. Use

Claims (8)

Knowledge of PK data and target levels may also help distinguish actual ADA responses from target-mediated signals. To reduce the interfering influence of the target, the addition of target blocking reagents can be used. However, in the case of therapeutic antibodies, it is necessary to ensure that these reagents do not share CDR sequences with the drug (in the case of target antibodies) or do not contain human IgG constant sequences (in the case of antibodies, receptors and binding proteins), since these common sequences may impair detection of ADAs specific to these sequences. Finally, the use of low-affinity ADA-positive samples obtained from animals with a polyclonal drug response may represent a tool to confirm that truly low-affinity, low-titer ADA responses, the detection of which are more likely to be affected by assay modifications, however less may be detected under final assay conditions. In this disclosure, the drug target is a homodimer that forms multiple inactive complexes with its inhibitory binding proteins in the bloodstream. While acid dissociation increases the drug tolerance limit (DTL) in the ADA assay, it releases the drug-bound target and the target bound to its inhibitory binding proteins. The released target will interfere with the ADA assay. Additionally, treating mice with the drug increases expression of the target. Western blot analysis using antibodies specific for the precursor and mature form of the target showed that different forms of the target were activated in mice 28 days after drug injection. Therefore, a reliable ADA assay with the highest target tolerance possible is required. By incorporating a target blocking antibody (HuAb1) and under moderately alkaline pH assay conditions (pH 8.3), we were able to obtain high tolerable levels of recombinant target protein. The assay was optimized for the addition of a second target blocking antibody, HuAb2. This significantly increased the target tolerance level, and the positivity rate was significantly reduced in clinical trial samples. The assay was further optimized by replacing the target antibody with HuAb1. Because HuAb1 shares some complementarity determining region (CDR) sequences with the drug, it could potentially bind ADA to the drug, thereby impairing ADA detection. Therefore, instead of HuAb1, the external region of the target receptor fused to the Fc of human IgG (HuSR) was used. When combined with HuAb2, HuSR was able to block target interference, similar to the HuAb1/HuAb2 combination. The Fc domains of HuSR and the entire constant region of HuAb2 were then converted from human to mouse (MsAb2 and MsSR) to further reduce potential interference with ADA detection. Characterization experiments using the Octet system showed that target-drug binding is inhibited by only moderately alkaline pH or only by the presence of target blocking reagents. However, complete inhibition of binding is achieved using a combination of MsAb2, MsSR and a moderately alkaline pH. Analysis of low-titer ADA blood from immunized rabbits and known ADA-positive samples from preclinical studies in rats confirmed the ability to detect ADA-positive samples in the assay and the minimal influence of alkaline pH and target blocking reagents on ADA detection. These results provide alternative strategies to overcome target interference in bridging immunogenicity assays when standard target blocking antibodies are ineffective. Equivalents. Those skilled in the art will understand or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims. The contents of all references, patents and published patent applications cited in this invention are incorporated herein by reference. CLAIM 1. A method for reducing the interference of a drug target in an anti-drug antibody (ADA) bridging immunoassay for determining the presence of an anti-drug ADA in a serum sample, comprising contacting the serum sample with a capture drug labeled with a first drug label. for detection, labeled with a second label, a first drug target blocking reagent containing an extracellular portion of the receptor fused to an IgG Fc domain, and a second drug target blocking reagent containing an extracellular - 17 045148 a receptor region fused to an IgG Fc domain, wherein the first drug target blocking reagent and the second drug target blocking reagent are different; incubating the serum sample under moderately alkaline pH assay conditions of pH 7.5-9.5 with an capture drug, a detection drug, a first drug target blocking reagent, and a second drug target blocking reagent; causing the capture drug and detection drug to bind to the ADA; and causing the first drug target blocking reagent and the second drug target blocking reagent to bind to the drug target present in the sample, thereby reducing interference with the drug target agents in the bridging immunoassay for ADA. 2. A method for reducing the interference of a drug target in an anti-drug antibody (ADA) bridging immunoassay for determining the presence of an anti-drug ADA in a serum sample, comprising contacting the serum sample with a capture drug labeled with a first drug label. for detecting, labeled with a second label, a first drug target blocking antibody and a second drug target blocking antibody, wherein the first drug target blocking antibody and the second drug target blocking antibody are different; incubating the serum sample with a capture drug, a detection drug, a first drug target blocking antibody, and a second drug target blocking antibody under moderately alkaline pH assay conditions of pH 7.5-9.5; causing the capture drug and detection drug to bind to the ADA; and causing the first drug target blocking antibody and the second drug target blocking antibody to bind to the drug target present in the sample, thereby reducing interference with the drug target agents in the bridging immunoassay for ADA. 3. A method of reducing the interference of a drug target in an anti-drug antibody (ADA) bridging immunoassay to determine the presence of an anti-drug ADA in a serum sample, wherein the drug target is a soluble protein, the method comprising contacting the serum sample with an capture drug labeled with a first label, a detection drug labeled with a second label, a drug target blocking reagent comprising an extracellular portion of the receptor fused to an IgG Fc domain, and a drug target blocking antibody; incubating the serum sample with a capture drug, a detection drug, a drug target blocking reagent, and a drug target blocking antibody under moderately alkaline pH assay conditions of pH 7.5-9.5; causing the capture drug and the detection drug to bind to the ADA; and causing the drug target blocking reagent and the drug target blocking antibody to bind to the drug target present in the sample, thereby reducing the interference of the drug target in bridging immunoassay for ADA. 4. The method according to any one of claims 1 to 3, further comprising performing a bridging immunoassay for anti-drug antibodies (ADA). 5. Method according to claim 1 or 2, characterized in that the drug target is a soluble protein, such as, for example, a ligand. 6. Method according to any one of claims 1 to 3, characterized in that the medicinal product is a human therapeutic monoclonal antibody or a humanized therapeutic monoclonal antibody, preferably wherein the human or humanized therapeutic monoclonal antibody is evaluated in a clinical trial. 7. The method according to claim 1 or 2, characterized in that the drug target is a soluble or excreted multimeric drug target or a homodimeric drug target. 8. A method according to any one of claims 1 to 3, wherein the moderately alkaline assay pH value includes a pH value of about 8.3 to about 8.9, preferably wherein the moderately alkaline assay pH value includes a pH value of about 8.3 or 8.9. -