Assays for quantification of drug and target concentrations

Abstract

The present invention generally relates to a method for determining the concentration of a drug and its target. In particular, the present invention relates, in part, to determining total drug and target concentrations using weakly acidic assay conditions to reduce either target or drug interference. The present invention also discloses a free target assay using a capture agent with a lower affinity that has much slower association and dissociation rates compared to the drug.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 249,417, filed September 28, 2021, and is incorporated herein by reference.

[0002] Field The present invention relates generally to providing total drug, free target and total target assays that are capable of accurately quantitating drug and target concentrations in clinical research samples. [Background technology]

[0003] background Measuring trace amounts of biotherapeutics in complex biological samples such as serum is of increasing clinical importance not only for basic science but also for patient management. For example, monoclonal antibodies (mAbs) are rapidly developing biotherapeutics aimed at treating various conditions such as diabetes, cancer, inflammation, and infections. Ligand binding assays (LBAs) are commonly used to quantify mAbs and their corresponding targets. Multiple forms of mAbs and targets exist in biological samples, including free mAb (i.e., not bound to target), free target (i.e., not bound to mAb or soluble physiological / endogenous binding partners), and monovalent and / or bivalent complexes of mAb and target. In nonclinical settings, total mAb concentrations in circulation are typically used to assess systemic exposure and evaluate potential drug toxicity, and to help determine safe starting and / or effective doses in first-in-human (FIH) studies.

[0004] In clinical evaluation, mAb concentration data are used to determine key pharmacokinetic (PK) parameters, characterize drug disposition, correlate exposure with safety and efficacy, and guide dosing regimen selection in later phase studies. Target concentrations can also be used in nonclinical development to determine effective mAb concentrations and enable model-based dosing. Target data in clinical practice can be used to characterize human PK profiles, define PK / pharmacodynamic (PD) relationships for safety and efficacy, and establish PK / PD models in patient populations. In particular, total target data provide information on the effect of the mAb on target accumulation and whether there is continued target engagement in circulation to achieve sustained complete target inhibition. Free target data helps determine effective doses and guide dose level / schedule selection. Free target can also be used in PK / PD modeling to help understand the outcomes of other PD / endpoints.

[0005] Although the measurement of drugs and targets provides important information, the accuracy of the drug and target measurements depends on the proper design of the bioanalytical method and the quality of the key reagents used. The capture and detection reagents are important components in determining the assay specificity of free and total target assays. Furthermore, if the anti-target antibodies used in the target assay have overlapping recognition regions with the biotherapeutic drug, the presence of the biotherapeutic drug may prevent accurate quantification of the total target. Therefore, specific strategies such as acid dissociation and the use of anti-drug antibodies must be used to mitigate drug interference. Furthermore, anti-target antibodies that do not compete with the drug for target binding can be used as capture and detection reagents. However, the drug in the target:drug complex may still affect the accurate detection of the total target due to steric hindrance. The pH of the assay can also be adjusted to selectively alter the binding of the target to the drug and / or capture and detection reagents to mitigate drug interference.

[0006] Developing a sensitive LBA method to measure free target in the presence of drug is also challenging because free target concentrations are often very low, have rapid turnover, and can vary with assay conditions. Because capture antibodies usually compete for the same binding epitope on the target as the drug, changes in the sample equilibrium between the drug-bound target and the capture antibody during the assay process can lead to an overestimation of the free target concentration. It is therefore important to fully understand the kinetics of biotherapeutic-target association. To minimize dissociation of existing drug-bound targets and accurately measure free targets, assay conditions need to be optimized. The affinity of the capture antibody for the target usually needs to be much weaker compared to that of the drug to minimize effects on sample equilibrium and artificial increases in free target concentrations. For similar reasons, the concentration of the capture antibody needs to be optimized. In addition, other factors such as sample collection, dilution, freeze / thaw cycles, and storage can also change the kinetics of association between biotherapeutic and target, for example, using buffers with high concentrations of salts or detergents and longer sample incubation times can affect dissociation between drug and target.

[0007] Free target assays can also be developed by removing bound targets using immunoprecipitation, solid phase extraction, or affinity separation. However, additional processes can introduce other changes by adsorption of targets to columns or filter / bead surfaces. Dissociation of target:drug complexes can still occur throughout the process. Furthermore, this procedure is labor intensive, has low throughput, and requires strict standardization of each step of the individual assay procedures. Other assay platforms such as Gyrolab technology can also be used, which usually allow quantification of free targets with minimal sample dilution and short sample incubation times. See Dysinger and Ma.AAPS J.,20(6),106(2018) (Non-Patent Document 1).

[0008] Finally, development of a whole drug assay can also be difficult, especially when the target is present at high concentrations, which can hinder the ability of anti-idiotypic capture or detection mAbs to bind to the drug. See Lee, JW, et al., AAPS J, 2011.13(1):p.99-110 (Non-Patent Document 2); Watanabe et al. AAPS J.23(1),21(2021) (Non-Patent Document 3).

[0009] LC-MS has emerged as a quantitative tool to measure the concentrations of biotherapeutics and their targets. See van de Merbel NC.Bioanalysis 11(7),629-644(2019) (Non-Patent Document 4). It offers a wide dynamic range with excellent precision and accuracy. It can also be used to quantify multiple analytes simultaneously, reducing the reliance on critical immunoreagents. This approach can also overcome assay interferences from drugs, targets, other endogenous binding proteins, etc., and can be easily used to measure the total amount of drugs and targets. However, LC-MS is typically a low-throughput method with limited sensitivity.

[0010] Accurate quantification of therapeutic proteins and their targets is critical for evaluating efficacy and safety, as well as for evaluating exposure-response relationships to support dose selection. Therefore, developing reliable and accurate bioanalytical methods is crucial to support drug development programs. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Dysinger and Ma.AAPS J.,20(6),106(2018) [Non-Patent Document 2] Lee, JW, et al., AAPS J, 2011.13(1):p.99-110 [Non-Patent Document 3] Watanabe et al.AAPS J.23(1),21(2021) [Non-Patent Document 4] van de Merbel NC.Bioanalysis 11(7),629-644(2019) Summary of the Invention

[0012] overview The exemplary embodiments disclosed herein fulfill the aforementioned needs by providing a method for determining drug concentration and target concentration.

[0013] The present disclosure provides a method for determining the concentration of free target in a sample. In an exemplary embodiment, the method includes the steps of adding the sample having bound target and the free target to a solid support coated with a capture agent, the capture agent having a lower affinity and a much slower association rate for the target compared to a drug, adding a detection agent having a detectable label, and measuring a signal from the detectable label of the detection agent to determine the concentration of the free target in the sample, the signal being proportional to the concentration of the free target in the sample.

[0014] In one aspect of this embodiment, the method further comprises determining the amount of free target from the signal by comparing the signal to a standard calibration curve, the standard calibration curve being generated by performing the method by substituting at least three standard solutions having three different concentrations of the free target in place of the sample.

[0015] In one aspect of this embodiment, the sample is incubated with the capture agent for about 15 minutes. In another aspect of this embodiment, the sample is incubated with the capture agent for about 30 minutes. In yet another aspect of this embodiment, the sample is incubated with the capture agent for about 45 minutes. In yet another aspect of this embodiment, the sample is incubated with the capture agent for about 60 minutes.

[0016] In one aspect of this embodiment, the sample is incubated with the detection agent for about 15 minutes. In another aspect of this embodiment, the sample is incubated with the detection agent for about 30 minutes. In yet another aspect of this embodiment, the sample is incubated with the detection agent for about 45 minutes. In yet another aspect of this embodiment, the sample is incubated with the detection agent for about 60 minutes.

[0017] In one aspect of this embodiment, the affinity of the capture agent is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold lower than the drug. In another aspect of this embodiment, the affinity of the capture agent is more than about 50-fold lower than the drug.

[0018] In one aspect of this embodiment, the half-life of the capture agent is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times longer than the drug. In another aspect of this embodiment, the half-life of the capture agent is more than about 10 times longer than the drug.

[0019] In one aspect of this embodiment, the solid support is coated with streptavidin.

[0020] In one aspect of this embodiment, the capture agent is biotinylated.

[0021] In one aspect of this embodiment, the detectable label is ruthenium.In another aspect of this embodiment, the detectable label is an electrochemiluminescent substrate.

[0022] In one aspect of this embodiment, the signal is obtained by applying a voltage.

[0023] In one aspect of this embodiment, the detection agent is different from the capture agent. In another aspect of this embodiment, the detection agent is the same as the capture agent.

[0024] The present disclosure provides a method for determining the concentration of a total target in a sample. In an exemplary embodiment, the method includes contacting the sample with an acidic solution, adding the sample to the solid support coated with a capture agent, adding a detection agent having a detectable label, and measuring a signal from the detection agent to determine the concentration of the total target in the sample, the total target including a target complexed with a drug and a free target, and the signal is proportional to the concentration of the total target in the sample.

[0025] In one aspect of this embodiment, the method further comprises determining the amount of total target from the signal by comparing the signal to a standard calibration curve, the standard calibration curve being generated by performing the method by substituting at least three standard solutions having three different concentrations of free target in place of the sample.

[0026] In one aspect of this embodiment, the signal is obtained by applying a voltage.

[0027] In one aspect of this embodiment, the acidic solution has a pH of about 5.0 to about 7.0. In a preferred embodiment, the acidic solution has a pH of about 6.0.

[0028] In one aspect of this embodiment, the acidic solution contains about 50 to 500 mM acetic acid. In a preferred embodiment, the acidic solution contains about 300 mM acetic acid. In another preferred embodiment, the acidic solution contains about 30 mM acetic acid.

[0029] In one aspect of this embodiment, the capture agent has a lower dissociation rate and a larger t 1 / 2 has.

[0030] In one aspect of this embodiment, the off-rate of the capture agent is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold slower than the drug. In another aspect of this embodiment, the off-rate of the capture agent is more than about 50-fold slower than the drug.

[0031] In one aspect of this embodiment, the t 1 / 2 is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold longer than the drug. 1 / 2 is approximately 10 times longer than that of the drug.

[0032] In one aspect of this embodiment, the detection agent has a lower dissociation rate and a larger t 1 / 2 has.

[0033] In one aspect of this embodiment, the off-rate of the detection agent is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 15 times, about 20 times, about 25 times, about 30 times, about 35 times, about 40 times, about 45 times, or about 50 times slower than the drug. In another aspect of this embodiment, the off-rate of the detection agent is more than about 50 times slower than the drug.

[0034] In one aspect of this embodiment, the detection material t 1 / 2 is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, or about 10 times longer than the drug. 1 / 2 is approximately 10 times longer than that of the drug.

[0035] In one aspect of this embodiment, the sample is incubated with the capture agent for about 15 minutes. In another aspect of this embodiment, the sample is incubated with the capture agent for about 30 minutes. In yet another aspect of this embodiment, the sample is incubated with the capture agent for about 45 minutes. In yet another aspect of this embodiment, the sample is incubated with the capture agent for about 60 minutes.

[0036] In one aspect of this embodiment, the sample is incubated with the detection agent for about 15 minutes. In another aspect of this embodiment, the sample is incubated with the detection agent for about 30 minutes. In yet another aspect of this embodiment, the sample is incubated with the detection agent for about 45 minutes. In yet another aspect of this embodiment, the sample is incubated with the detection agent for about 60 minutes.

[0037] In one aspect of this embodiment, the detection agent is different from the capture agent. In another aspect of this embodiment, the detection agent is the same as the capture agent.

[0038] In one aspect of this embodiment, the solid support is coated with streptavidin.

[0039] In one aspect of this embodiment, the capture agent is biotinylated.

[0040] In one aspect of this embodiment, the detectable label is ruthenium.In another aspect of this embodiment, the detectable label is an electrochemiluminescent substrate.

[0041] The present disclosure provides a method for determining the concentration of a total drug in a sample. In an exemplary embodiment, the method includes adding the sample to the solid support coated with a capture agent, adding a detection agent having a detectable label, the capture agent being different from the capture agent, and measuring a signal from the detection agent to determine the concentration of the total target in the sample, the total drug including the drug complexed with the target and the free drug, and the signal is proportional to the concentration of the free target in the sample.

[0042] In one aspect of this embodiment, the method further comprises the step of adding a substrate specific for binding to the detection substance, said substrate bound to the detection substance providing said signal.

[0043] In one aspect of this embodiment, the capture agent is a monoclonal antibody that binds to the drug.

[0044] In one aspect of this embodiment, the detection agent is different from the capture agent.

[0045] In one aspect of this embodiment, the detection agent is biotinylated.

[0046] In one aspect of this embodiment, the acidic solution has a pH of about 5.0 to about 7.0. In a preferred embodiment, the acidic solution has a pH of about 6.0.

[0047] In one aspect of this embodiment, the acidic solution contains about 50 to 500 mM acetic acid. In a preferred embodiment, the acidic solution contains about 300 mM acetic acid. In another preferred embodiment, the acidic solution contains about 30 mM acetic acid. [Brief description of the drawings]

[0048] [Figure 1A]Shown are standard curve signals using neutral buffer (assay dilution buffer, no acid), acid treatment with neutralization (300 mM, pH approx. 3.2), and weak acid (30 mM) according to an exemplary embodiment. [Figure 1B] 1 shows the assay signal of LLOQ spiked samples with acid treatment (300 mM) and weak acid (30 mM) according to an exemplary embodiment. [Figure 2A] 1 shows total target levels in three human plasma samples in the absence and presence of 1 mg / mL drug at neutral assay pH, according to an exemplary embodiment. [Figure 2B] 1 shows total target assay signal with neutral assay pH, acid treatment (300 mM acetic acid, pH approx. 3.2) and neutralization, and 30 mM acetic acid in 5% BSA (pH approx. 6.0) according to an exemplary embodiment. [Figure 2C] 1 shows total target levels in four human plasma samples in the absence and presence of 1 mg / mL drug under a weakly acidic assay pH (pH approx. 6.0), according to an exemplary embodiment. [Figure 2D] 1 shows the concentration of total target in four human plasma samples with or without 1 mg / mL drug, in the presence of 200 μg / mL or 1 mg / mL anti-drug antibody blocking agent under a weakly acidic assay pH (pH approx. 6.0), according to an exemplary embodiment. [Figure 2E] 1 shows total target levels in three human plasma samples with and without 1 mg / mL drug using a new capture and detection antibody pair and a mild assay pH (pH approx. 6.0) according to an exemplary embodiment. [Figure 3A] FIG. 1 shows the LLOQ signal from a free target assay and assay signals from target:drug complexes at molar ratios of 1:5, 1:2, and 1:1, using samples diluted 1:2 in 5% BSA and Mab-2 or Mab-3 as capture antibodies, according to an exemplary embodiment. [Figure 3B]1 shows the LLOQ signal from a free target assay and assay signals from target:drug complexes at molar ratios of 1:5, 1:2, and 1:1, using samples diluted 1:50 in 5% BSA and Mab-2 or Mab-3 as capture antibodies, according to an exemplary embodiment. [Figure 3C] 1 shows the predicted (green bars) and measured (blue bars) concentrations of free target from target:drug complexes when Mab-1, Mab-2, or Mab-3 are used as capture antibodies according to an exemplary embodiment. [Figure 3D] FIG. 1 shows the recovery of free target (%AR) from a 1:0.25 molar ratio target:drug complex when 1:50 diluted samples were incubated with Mab-1, Mab-2 or Mab-3, respectively, for approximately 15 or 45 minutes, according to an exemplary embodiment. [Figure 3E] 1 shows the recovery of free target by target:drug complex (1:50 sample dilution) for samples subjected to 1 or 7 freeze / thaw cycles using Mab-3 as the capture antibody, according to an exemplary embodiment. [Figure 4] FIG. 1 shows a schematic diagram of a free target assay using different capture antibodies, according to an exemplary embodiment. [Figure 5A] 1 shows concentrations of drug, total target, and free target in human serum and plasma samples from individuals participating in a single-dose clinical study with 1 mg / kg intravenous drug, according to an exemplary embodiment. [Figure 5B] 1 shows concentrations of drug, total target, and free target in human serum and plasma samples from individuals participating in a single-dose clinical study with 30 mg / kg intravenous drug, according to an exemplary embodiment. [Figure 5C] 1 shows concentrations of drug, total target, and free target in human serum and plasma samples from individuals participating in a single-dose clinical study with 300 mg / kg subcutaneous drug, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description Reliable bioanalytical methods to measure circulating mAbs and their targets are important for evaluating efficacy and safety, as well as for assessing exposure-response relationships to support dose selection. Lee, JW, et al.,AAPS J,2011.13(1):p.99-110;Lee,JWand H.Salimi-Moosavi.Bioanalysis,2012.4(20):p.2513-23;Zheng,S.,T.McIntosh,and W.Wang.J Clin Pharmacol,2015.55 Suppl 3:p.S75-84;Gupta,S.,et al.,2017(Part 3-LBA:immunogenicity,biomarkers and PK assays).Bioanalysis,2017.9(24):p.1967-1996;Yang,J.and V.Quarmby.Bioanalysis,2011.3(11): p.1163-5.

[0050] Free mAb levels provide information about the active drug available to bind to the target, while total mAb levels help characterize the dynamic interaction between the mAb and the target. Target concentrations are also used during preclinical development to determine effective mAb concentrations and allow model-based dosing. See Betts, AM, et al. J Pharmacol Exp Ther, 2010. 333(1):p.2-13.

[0051] Clinical-phase target data are used to characterize the human PK profile, define PK / pharmacodynamic (PD) relationships for safety and efficacy, and establish PK / PD models in the target population. In particular, total target data provide information on the effect of the mAb on target accumulation and whether there is continued target engagement during circulation to achieve sustained complete target inhibition. Monitoring free target during administration is beneficial to determine effective doses and guide dose level / schedule selection. Free target can also be used for PK / PD modeling to help understand the outcomes of other PD / endpoints.

[0052] The present invention provides a whole drug assay using a mildly acidic assay pH to reduce target interference. The present invention also provides a drug that exhibits a low dissociation rate to the target compared to the drug, and a very large t under acidic assay conditions. 1 / 2 The present invention provides similar mildly acidic assay conditions for the whole target assay, along with a paired anti-target antibody having a similar affinity for the target and a much slower association rate compared to the drug. Thus, these assays can accurately quantitate drug and target concentrations in clinical research samples, supporting PK / PD modeling of biotherapeutics.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are now described. All publications mentioned are incorporated herein by reference.

[0054] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations as understood by one of ordinary skill in the art, and endpoints are included when ranges are provided.

[0055] As used herein, the term "protein" includes any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues linked through peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. A synthetic peptide or polypeptide can be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known. A protein can contain one or more polypeptides to form a single functional biomolecule. Proteins can include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In an exemplary alternative embodiment, the protein can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a review discussing biotherapeutic proteins and their production, see Ghaderi et al. "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," (BIOTECHNOL. GENET. ENG. REV. 147-175 (2012)). In some exemplary embodiments, the proteins include modifications, adducts, and other covalently attached moieties.These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, other dyes, etc. Proteins can be classified based on their composition and solubility, and thus include simple proteins, such as globular proteins, fibrous proteins, complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, lipoproteins, and derived proteins, such as primary derived proteins, secondary derived proteins, etc.

[0056] In some exemplary embodiments, the protein of interest may be an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, or an Fc fusion protein.

[0057] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (herein HCVR or V H The heavy chain constant region is C H 1. C H2 , and C H Each light chain comprises three domains: a light chain variable region (herein LCVR or V L The light chain constant region comprises one domain (C L1 ) is included. V H and V L The regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different exemplary embodiments, the FRs of the anti-big-ET-1 antibody (or antigen-binding portion thereof) may be identical to the human germline sequence or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of an intact antibody molecule. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, include any naturally occurring, enzymatically accessible, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies may be derived from intact antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated using chemical or molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate orientation, introduce codons, create cysteine ​​residues, modify, add or delete amino acids.

[0058] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as, for example, an antigen-binding or variable region of an 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, and 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 recombinantly produced from a gene encoding a partial antibody sequence. Optionally, or in addition, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Optionally, or in addition, an antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds. An antibody fragment may optionally comprise a multimolecular complex.

[0059] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0060] The term "Fc fusion protein" as used herein includes parts or all of two or more proteins that are not naturally fused, one of which is the Fc portion of an immunoglobulin molecule. The preparation of fusion proteins including certain heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, by Ashkenazi et al., Proc. Natl. Acad. Sci USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins," in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11, 1992. A "receptor-Fc fusion protein" includes one or more of one or more extracellular domain(s) of a receptor linked to an Fc portion, which in some embodiments includes the hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein comprises two or more different receptor chains that bind to a single or multiple ligands. For example, the Fc fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which comprises an IL-1 RAcP ligand binding domain fused to an IL-1R1 extracellular domain fused to an Fc of hIgG1, see US Pat. No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept, which comprises an Ig domain 2 of the VEGF receptor Flt1 fused to an Ig domain 3 of the VEGF receptor Flk1 fused to an Fc of hIgG1, see SEQ ID NO: 1, US Pat. Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

[0061] As used herein, the term "affinity" refers to the strength of interaction between a protein of interest or a protein and a target. Affinity is K D It is measured in.

[0062] The term "sample" as used herein includes any biological specimen obtained from a patient. Samples include, but are not limited to, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, stool (i.e., feces), sputum, bronchoalveolar lavage fluid, tears, nipple aspirates, lymph (such as disseminated tumor cells in lymph nodes), fine needle aspirates, other bodily fluids, tissue samples (e.g., tumor tissue), such as tumor biopsies (e.g., needle biopsies), and cellular extracts thereof. In some embodiments, the sample is whole blood or a portion thereof, such as plasma, serum, cell pellets, etc. In a preferred embodiment, the sample is obtained by isolating circulating cells of a solid tumor from whole blood or a cellular fraction thereof using any technique known in the art and preparing a cellular extract of the circulating cells. In another embodiment, the sample is a formalin-fixed paraffin-embedded (FFPE) tumor tissue sample, for example, from a solid tumor of the lung, colon, or rectum.

[0063] In another embodiment, the sample may include whole blood, serum, plasma, urine, sputum, bronchoalveolar lavage fluid, tears, nipple aspirate, lymph, saliva, and / or fine needle aspirate samples. In a particular example, a whole blood sample is separated into a plasma or serum fraction and a cellular fraction (i.e., a cell pellet). The cellular fraction typically contains red blood cells, white blood cells, and / or circulating cells of solid tumors, such as circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPCs), cancer stem cells (CSCs), and combinations thereof. The plasma or serum fraction typically contains, among other things, nucleic acids (e.g., DNA, RNA) and proteins released by circulating cells of solid tumors.

[0064] As used herein, the term "capture reagent" is used herein to refer to a binding reagent that is immobilized on a surface to form a binding surface for use in an assay. The assay modules and methods may also use or include another binding reagent, a "detection reagent," whose participation in a binding reaction on the binding surface can be measured. The detection reagent may be measured by measuring an intrinsic property of the reagent, such as color, luminescence, radioactivity, magnetic field, charge, refractive index, mass, chemical activity, etc. Alternatively, the detection reagent may be labeled with a detectable label and measured by measuring a property of the label. Suitable labels include, but are not limited to, labels selected from the group consisting of electrochemiluminescent labels, luminescent labels, fluorescent labels, phosphorescent labels, radioactive labels, enzymatic labels, electroactive labels, magnetic labels, and light scattering labels.

[0065] The capture or detection reagents can either directly bind (or compete) with the analyte of interest (drug or target) or indirectly interact through one or more bridging ligands. Thus, the dry assay reagents may include such bridging ligands. As an example, streptavidin or avidin may be used as a capture or detection reagent by using a biotin-labeled bridging reagent that binds or competes with the analyte of interest. Similarly, an anti-hapten antibody may be used as a capture or detection reagent by using a hapten-labeled binding reagent that binds or competes with the analyte of interest. In another example, an anti-species antibody or an Fc receptor (such as protein A, G, or L) is used as a capture or detection reagent by its ability to bind to an analyte-specific antibody. Such techniques are well established in the field of binding assays, and one of skill in the art can easily identify a bridging ligand suitable for a particular application.

[0066] Certain embodiments of the assay module / plate include a capture reagent immobilized on the surface of the module / plate to form a binding surface. The immobilization can be performed using immobilization techniques well established in the field of solid-phase binding assays, such as techniques established for performing ELISA assays or array-based binding assays. In one example, the binding reagent can be non-specifically adsorbed to the surface of a well of a multi-well plate. The surface can be untreated or can be treated (e.g., with plasma or a charged polymer) to enhance the surface's adsorption properties. In another example, the surface can have active chemical functional groups that allow the covalent attachment of the binding reagent. After the reagent is immobilized, the surface can optionally be contacted with a reagent that includes a blocking agent to block uncoated sites on the surface. To perform multiplexed measurements, a binding surface with an array of different capture reagents can be used. Various techniques for forming arrays of capture reagents are now well established in the field of array-based assays.

[0067] The binding surface is optionally coated with a reconstitutable dry protective layer. The protective layer stabilizes the binding surface, protects it from contact with detection reagents during manufacturing and storage, or simply serves as a place to store assay reagents such as cross-linking reagents, blocking reagents, pH buffers, salts, surfactants, electrochemiluminescence coreactants, etc. Stabilizers that may be found in the protective layer include, but are not limited to, sugars (sucrose, trehalose, mannitol, sorbitol, etc.), polysaccharides and sugar polymers (dextran, ficoll, etc.), polymers (polyethylene glycol, polyvinylpyrrolidone, etc.), zwitterionic osmolytes (glycine, betaine, etc.) and other stabilizing osmolytes (trimethylamine-N-oxide). Blocking agents are substances that prevent non-specific binding of assay components, particularly detection reagents, to the binding surface and include proteins (such as serum albumin, gamma globulins, immunoglobulins, milk powder or purified casein, gelatin, etc.), polymers (such as polyethylene oxide and polypropylene oxide), surfactants (e.g., classes of non-ionic detergents or surfactants known under the trade names BRIJ™, TRITON™, TWEEN®, THESIT®, LUBROL, GENAPOL®, PLURONIC®, TETRONIC®®, and SPAN), etc. Certain embodiments include a protective layer that includes ammonium phosphate as a buffer component, includes other ammonium salts, and / or includes less than 1% or 0.1% (w / w) sodium or potassium ions.

[0068] The solid support may comprise any suitable substrate for immobilizing proteins. Examples of solid supports include, but are not limited to, glass (e.g., glass slides), plastic, chips, pins, filters, beads (e.g., magnetic beads, polystyrene beads, etc.), paper, membranes, fiber bundles, gels, metals, ceramics, etc. Membranes such as nylon (Biotrans™, ICN Biomedicals, Inc. (Costa Mesa, Calif.), Zeta-Probe™, Bio-Rad Laboratories (Hercules, Calif.)), nitrocellulose (Protran™, Whatman Inc. (Florham Park, NJ)), PVDF (Immobilon™, Millipore Corp. (Billerica, Mass.)), etc. are suitable for use as solid supports in the arrays of the present invention. Preferably, the capture antibody is bound onto a glass slide coated with a nitrocellulose polymer, e.g., FAST™ Slides available from Whatman Inc. (Florham Park, NJ).

[0069] The term "incubate" is used synonymously with "contact" and "exposure" and does not imply specific time or temperature requirements unless specifically indicated.

[0070] Various publications, including patents, patent applications, published patent applications, accession numbers, technical papers and journal articles, are cited throughout the specification. Each of these references is incorporated by reference in its entirety.

[0071] The present invention may be more fully understood by reference to the following examples, which should not be construed as limiting the scope of the invention. EXAMPLES

[0072] Materials and Reagents. For the total drug, total target, and free target assays, all solutions were prepared in assay dilution buffer (0.5% bovine serum albumin [BSA], 0.05% Tween-20, 1× phosphate-buffered saline [PBS]) PBS from Gibco (Grand Island, NY) unless otherwise specified. Glacial acetic acid was obtained from Thermo Fisher Scientific (Waltham, MA). Human serum and plasma were obtained from BioIVT (Westbury, NY). Streptavidin-coated microplates were obtained from Meso Scale Discovery (MSD, Rockville, MD). Black microwell plates, NeutrAvidin conjugated with horseradish peroxidase (NeutrAvidin-HRP), and SuperSignal ELISA Pico chemiluminescent substrate were obtained from Thermo Fisher Scientific (Rockford, IL). Purified native human target was obtained from EMD Millipore (Burlington, MA). Drug (fully human mAb), mouse anti-drug mAb, biotinylated mouse anti-drug mAb, and all human anti-target mAb (used in target assays) were manufactured by Regeneron Pharmaceuticals (Tarrytown, NY).

[0073] Total drug assay. This assay involves mild acid treatment of serum samples to dissociate soluble target:drug complexes and improve detection of drugs while soluble targets are present in serum. The procedure uses microtiter plates coated with mouse anti-drug mAb (2 μg / mL) and drugs as standards. Standards, controls, and samples were diluted 1:50 in 30 mM acetic acid in ADB (pH ≈5.0) and added to the plate. Drug captured on the plate was detected using another non-competitive biotinylated mouse anti-drug mAb (200 ng / mL) followed by NeutrAvidin HRP (200 ng / mL). All incubations were performed at room temperature for approximately 60 min. Finally, a luminol-based substrate specific for peroxidase was added to achieve a signal intensity proportional to the concentration of total drug.

[0074] All-target assay. This assay involves a mild acid pretreatment of plasma samples to dissociate soluble target:drug complexes present in the plasma sample and improve detection of target in the presence of drug. The procedure uses streptavidin-coated MSD plates, biotinylated human anti-target mAb (5 μg / mL) as the capture reagent, and purified target as the standard. Standards, controls, and samples (samples prediluted 1:5 in 5% BSA) were diluted 1:10 with 30 mM acetic acid in 5% BSA and further diluted 1:5 with 30 mM acetic acid in 5% BSA (pH approx. 6.0, 5% BSA was used to lower the assay background) containing 6.25 μg / mL of capture mAb before addition to the plate. After the 1:5 dilution, the final concentration of capture mAb in the sample / capture antibody mixture was 5 μg / mL. Target bound to the capture reagent and captured on the plate was detected using another ruthenium-labeled human anti-target mAb (2 μg / mL). All incubations were performed at room temperature for approximately 60 min. Finally, the target concentration was measured by the electrochemiluminescence signal generated by the ruthenium label when a voltage was applied to the plate by the MSD plate reader. The resulting electrochemiluminescence signal (e.g., counts) is proportional to the concentration of the total target in the plasma sample.

[0075] Free target assay. This procedure uses streptavidin-coated MSD plates, biotinylated human anti-target mAb (5 μg / mL) as the capture reagent, and purified target as the standard. Standards, quality controls (QCs), and samples were diluted 1:2 or 1:50 in 5% BSA and added to the plate. The plate was incubated at room temperature for approximately 15 or 45 minutes. Target bound to the capture mAb and captured on the plate was detected using another ruthenium-labeled human anti-target mAb (2 μg / mL), which does not compete with the drug or a different capture antibody for target binding. The plate was incubated at room temperature for approximately 15 minutes. Finally, the target concentration was measured by the electrochemiluminescence signal generated by the ruthenium label when a voltage was applied to the plate by the MSD plate reader. The resulting electrochemiluminescence signal (e.g., counts) obtained is proportional to the concentration of free target in the plasma sample.

[0076] Preparation of target:drug conjugates. Target:drug conjugates with molar ratios of 1:5, 1:2, 1:1, 1:0.5, 1:0.25 and 1:0.125 were prepared in naive human plasma samples with known target concentrations based on analysis using the whole-target assay. Target:drug conjugate samples were prepared by spiking specific amounts of drug into plasma samples based on the molecular weights of the target and drug. The obtained target:drug mixtures were incubated at room temperature for approximately 1-3 hours before being used in the target assay.

[0077] Biacore surface plasmon resonance analysis. The binding kinetics and binding affinity of anti-target antibodies were assessed using surface plasmon resonance technology on a Biacore T200 (Cytiva, MA, USA) instrument using a Series S CM5 sensor chip in filtered and degassed PBS-T running buffer (0.01 M Na2HPO4 / NaH2PO4, 0.15 M NaCl, 0.05% v / v Tween-20, pH 7.4 or pH 6.0). The capture sensor surface was prepared by covalently immobilizing mouse anti-human Fc mAb using standard amine coupling chemistry as previously reported. Different concentrations of target (PBS-P, pH 7.4 running buffer, prepared in the range of 100-11.11 nM, 3-fold diluted) were injected over the anti-target antibody capture surface for 3 min at a flow rate of 50 pl / min and their dissociation in two running buffers, PBS-T, pH 7.4 and PBS-T, pH 6.0, was monitored for 6 min. At the end of each cycle, the anti-human Fc surface was regenerated by a 12 s injection of 20 mM phosphate. All of the specific surface plasmon resonance binding sensorgrams were double reference subtracted as previously reported. Kinetic parameter constants (kd and ka) were determined by fitting the real-time sensorgrams to a 1:1 binding model using Scrubber 2.0c (BioLogic Software, Campbell, Australia) curve fitting software. The dissociation rate constant (kd) was determined by fitting the change in binding response during the dissociation phase, and the association rate constant (ka) was determined by globally fitting the association of analyte at various concentrations. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. The dissociation half-life (t1 / 2) in minutes was calculated as ln2 / (kd*60).

[0078] Example 1. Reducing target interference in whole drug assays by using a weakly acidic assay pH To quantify total drug concentrations, a sandwich ELISA assay format was studied using two non-competing anti-drug antibodies as capture and detection reagents. Initial experiments with standards prepared in human serum produced unexpectedly poor signals (Figure 1A), much lower than standards prepared in monkey serum (data not shown). This result suggested that the drug target, present at high concentrations (80-100 μg / mL) in serum bound to the drug, may interfere with the ability of anti-idiotypic capture or detection mAbs to recognize the drug.

[0079] To minimize potential target interference, serum samples were acidified (300 mM acetic acid) to dissociate target:drug complexes and neutralized prior to analysis. The acid pretreatment step followed by neutralization significantly improved the assay signal, although it was not uniform across the standard curve range. At the ULOQ, acid pretreatment increased the signal approximately 10-fold, whereas at the LLOQ, the signal increased approximately 4-fold (Figure 1A). Furthermore, the assay produced variable signal responses with individual serum samples spiked at the LLOQ (Figure 1B), suggesting that endogenous target levels may interfere at the lower end of the calibration range.

[0080] Published data indicate that in weakly acidic assay conditions (pH ≈5), some target may dissociate from the drug while anti-idiotypic mAbs retain binding capacity (Partridge, MA, et al., Minimizing target interference in PK immunoassays: new approaches for low-pH-sample treatment. Bioanalysis, 2013.5(15):p.1897-910.). To test this approach in a whole-drug assay, standards in human serum were diluted in weak acetic acid (30 mM) and analyzed in a sandwich ELISA without a neutralization step. As with the standard acidification / neutralization pretreatment, the assay signal of samples diluted in weak acid was significantly increased compared to samples analyzed without acid pretreatment. However, with the weak acid approach, the improvement in the assay signal was uniform across the range of the standard curve (Figure 1A). Furthermore, the assay produced more consistent signal responses with individual serum samples spiked at the LLOQ level ( Fig. 1B ), and the %AR was within ±20% of the nominal spiked drug concentration for all spiked samples (data not shown).

[0081] Example 2. Selection of capture and detection antibodies and use of a mildly acidic assay pH to reduce drug interference in a whole target assay The target concentration in naive human plasma samples is about 80-100 μg / mL, but may be even higher in post-dose samples from clinical studies. Thus, a significant amount of drug is usually administered to achieve sustained target inhibition. Therefore, a significant level of target:drug complex is usually formed in the circulation, which may affect the accurate detection of total targets. Two anti-target antibodies that do not compete with the drug for target binding were initially selected as capture and detection reagents for measuring total targets (Table 1).

[0082] Table 1. Characterization of original and new capture and detection antibodies for the whole target assay. TIFF2024534638000001.tif42155 *Under the current experimental conditions, no dissociation of the target from the captured monoclonal antibody was observed, and the kd value was fixed at 1.0E-05.

[0083] Total target levels in three naïve human plasma samples ranged from 80 to 150 μg / mL (Figure 2). However, the target concentration was reduced by approximately 20% in the presence of 1 mg / mL of drug (Figure 2A), suggesting that even if the two anti-target antibodies do not compete with the drug for target binding, steric hindrance may affect the binding of the capture and / or detection antibodies to the target when the drug is present at high concentrations. Next, acid dissociation was used to dissociate the target:drug complex. However, the assay signal was significantly reduced after acid treatment (300 mM acetic acid) and neutralization compared to the signal obtained under neutral conditions (Figure 2B), suggesting that the target protein may not be stable under this harsh acidic condition. Similar to the total drug assay, weakly acidic assay conditions (pH approximately 6.0) were used to dissociate the target:drug complex. The standard curve signal at weakly acidic pH was comparable to the signal without acid treatment (Figure 2B). However, in the presence of 1 mg / mL of drug, a 15%–25% decrease in target concentration was still observed (Figure 2C).

[0084] Anti-drug antibodies have been successfully used to reduce drug interference in the determination of total target concentrations (unpublished data). Anti-drug antibodies that can effectively block target-drug binding were tested in a total target assay using a weakly acidic assay pH (pH 6.0). Neither 200 μg / mL nor 1 mg / mL of anti-drug antibodies could effectively inhibit drug interference under the weakly acidic assay conditions (Figure 2D), possibly because the weakly acidic assay conditions could not completely dissociate the target:drug complex and / or the antibodies may not be able to effectively bind to the drug under these acidic assay conditions.

[0085] The whole-target assay was redeveloped using two new anti-target antibodies as capture and detection reagents (second generation assay). The two anti-target antibodies selected have a slow dissociation rate from the target and a t at pH ≈6.0 when compared to the drug.1 / 2 is much larger (Table 1). Indeed, at weakly acidic pH, the dissociation rate of the drug increases by about 5-fold compared to neutral pH, and t 1 / 2 is significantly shorter. These results indicate that under acidic conditions, even though the binding of the drug to the target is greatly reduced, both the new capture and detection antibodies can still effectively bind to the target. Finally, similar target concentrations were obtained in 10 human plasma samples (Figure 2E) using the second generation assay format in the absence and presence of 1 mg / mL of drug, indicating that drug interference was successfully mitigated by the new capture and detection antibody pair and the weakly acidic assay pH (pH ∼6.0).

[0086] Example 3. Selection of capture antibodies and optimization of assay format in the development of free target assays A free target assay was also developed to support this program, as free target data can help determine effective doses and guide dose selection. To accurately measure free target concentrations, special considerations must be taken, including choice of capture antibody, sample dilution, sample incubation time, and stability of the target:drug complex. (See Hansen, RJ, et al. MAbs, 2013.5(2):p.288-96; Liu, Y., et al., Bioanalysis, 2021.13(7):p.575-585; Colbert, A., et al. MAbs, 2014.6(4):p.1103-13; Peng, K., et al. AAPS J, 2018.21(1):p.9.) Three anti-target antibodies, Mab-1, Mab-2, and Mab-3, that compete with the drug for target binding, were tested as capture antibodies in the free target assay. Mab-2 has a similar K D However, Mab-1 and Mab-3 have approximately 5- to 7-fold lower affinity for the target compared to the drug (Table 2).

[0087] Table 2. Characterization of antibodies Mab-1, Mab-2 and Mab-3 for free target assays TIFF2024534638000002.tif29128

[0088] To prevent possible dissociation of the target:drug complex, target:drug complexes with molar ratios of 1:1, 1:2, and 1:5 were diluted 1:2 prior to analysis. In complexes with excess drug (e.g., 1:5 and 1:2 molar ratios), little or no detectable levels of free target are expected, and therefore the assay signal from these complexes should be at or below the lower assay limit of quantitation (LLOQ, 1.56 μg / mL).

[0089] When Mab-1 or Mab-2 were used as capture antibodies, the assay signals from these complexes were greater than or close to the assay LLOQ (Figure 3A), indicating that higher concentrations of free target were measured. However, when Mab-3 was used as the capture reagent, the assay signals from these complexes were below the assay LLOQ signal (Figure 3A). When the target and drug are present at equimolar concentrations (e.g., 1:1 complex), small amounts of free target can be detected, but much higher signals were observed with Mab-1 and Mab-2 when compared to Mab-3. These results suggest that as the target dissociates from the drug in solution, Mab-1 and Mab-2 (which have a higher rate of association with the target than Mab-3) may compete with the drug more effectively, capturing the free target on the plate surface and removing it from solution, preventing reassociation with the drug and further disrupting the equilibrium.

[0090] To further evaluate these capture antibodies and ensure that samples could be further diluted so that the signal fell within the range of the standard curve, larger sample dilutions (1:50) were performed to quantify free target in the presence of various concentrations of drug. Similar to previous findings, when Mab-1 was used as the capture antibody, the signal from these target:drug complexes was above the assay LLOQ signal (Figure 3B), indicating that there was still complex dissociation even at this high sample dilution. When Mab-2 was used as the capture reagent, the assay signal from the 1:1 complexes was above the assay LLOQ. However, when Mab-3 was used as the capture antibody, the assay signals from the 1:5, 1:2, and 1:1 complexes were below the assay LLOQ signal (Figure 3B), suggesting that this assay format can more accurately measure the free target concentration present in the sample.

[0091] When measurable levels of free target were expected, the free target concentrations were also measured by target:drug complexes at molar ratios of 1:0.5, 1:0.25, and 1:0.125. The amount of free target detected using Mab-3 as the capture antibody was very similar to the free target concentration predicted based on the target-mediated pharmacokinetic PK model developed using total drug and target concentrations in the clinical setting (Figure 3C). However, the concentrations measured using Mab-1 or Mab-2 as the capture antibodies were greater than the model predictions (Figure 3C), further suggesting that these antibodies have a greater effect on the equilibrium, favoring dissociation of the target:drug complex.

[0092] Finally, the free target concentration in these complexes was also measured when the sample incubation time was varied to approximately 15 or 45 min. When Mab-3 was used as the capture antibody, the free target concentration was similar regardless of the sample incubation time (Figure 3D), indicating that longer sample incubation times had minimal effect on the complexes in solution. However, when Mab-1 or Mab-2 were used as the capture antibody, the free target concentration increased with longer sample incubation times (Figure 3D), suggesting further dissociation of the complexes. Finally, similar free target concentrations were obtained when these samples were subjected to one or seven freeze / thaw cycles using Mab-3 as the capture antibody (Figure 3E), indicating that the target:drug complexes and free target are stable in these samples even when the samples are exposed to stress.

[0093] The choice of capture antibody is important to accurately measure free target concentration. See Liu, Y., et al., Development of a Meso Scale Discovery ligand-binding assay for measurement of free (drug-unbound) target in nonhuman primate serum. Bioanalysis, 2021.13(7):p.575-585. In this study, Mab-3 showed a higher affinity (K D values) and a much slower association rate, making it less likely to disrupt the equilibrium of the target:drug complex in solution. Thus, at equilibrium, there is target:drug dissociation for the 1:5, 1:2, and 1:1 complexes, but most of the target can quickly reassociate with the drug and therefore is not detected in the free target assay. Only the free target is captured by Mab-3 and detected in the assay (Figure 4). However, Mab-1 also has a similar association rate (k a). Some of the target dissociated from the target:drug complex may bind to Mab-1 on the plate rather than reassociating with the drug, and thus be detected in the free target assay (Figure 4), resulting in an overestimation of the free target concentration. The top panel of Figure 4 shows a schematic of the free target assay when Mab-1 or Mab-2 are used as capture antibodies, since they have similar ka values ​​as the drug (despite the lower affinity of Mab-1), some of the target newly dissociated from the target:drug complex may bind to Mab-1 or Mab-2 on the plate surface rather than reassociating with the drug in solution, resulting in an overestimation of the free target concentration. The bottom panel shows a schematic when Mab-3, which has a lower affinity and slower rate of association with the target compared to the drug, is used as the capture reagent, and the newly dissociated target may quickly reassociate with the drug and is not detected in the free target assay. Only the free target is captured by Mab-3 and detected in the assay.

[0094] Example 4. Measurement of total drug, total target and free target concentrations in clinical study samples Samples from individuals receiving 1 mg / kg drug intravenously (i.v.), 30 mg / kg drug (i.v.), or 300 mg drug subcutaneously (sc.) in the Phase I single-dose clinical trial were tested for total drug concentrations in serum, and total and free target concentrations in plasma (Figure 5).

[0095] In individuals administered 1 mg / kg of drug intravenously (Figure 5A), circulating drug concentrations decreased over time, reaching a peak around day 57, with total target levels ranging from 80 to 100 μg / mL at all time points tested, without significant target accumulation (Figure 5A). However, free target levels were significantly reduced following drug administration, remaining at low levels for approximately 1 week after administration, and then gradually returning to baseline levels.

[0096] In individuals receiving 30 mg / kg of drug intravenously, drug concentrations reached fairly high levels within the first 4 weeks, began to decrease around day 29, and were very low by day 99 (Figure 5B). Total target concentrations appeared to increase around days 2 to 4, reached highest levels from days 22 to 29, and then returned to baseline levels (Figure 5B). Once drug was administered, free target was undetectable until day 57, when drug concentrations declined markedly (Figure 5B). There was a strong correlation between the decrease in free target levels and the increase in drug concentrations at both the 1 mg / kg and 30 mg / kg doses. A similar correlation was observed in individuals receiving 300 mg of drug subcutaneously (Figure 5C). These data indicate that the total target and free target assays can accurately quantify target concentrations in clinical study samples, and the results correlate well with total drug concentrations.

[0097] In this study, a whole drug assay, a whole target assay and a free target assay were developed to support the development program of a therapeutic mAb drug. Due to high endogenous target concentrations, drugs are administered at very high concentrations. To reduce target interference in the whole drug assay, weakly acidic assay conditions were used. The original whole target assay had problems with drug interference that could not be reduced by weakly acidic conditions or the use of anti-drug antibodies. Therefore, the whole target assay was redeveloped to reduce drug interference by using (1) a much higher affinity for the target compared to the drug, and (2) a longer half-life (t ) under weakly acidic conditions. 1 / 2A new pair of anti-target antibodies with much longer q-values ​​are used as capture and detection reagents. The new assay format can accurately quantify total target concentration in human plasma samples in the presence of drug. In addition, a free target assay was also developed using a capture antibody that has a much slower association rate to the target compared to the drug. Measuring free target concentration can be even more challenging. As shown above, multiple anti-target antibodies that compete with the drug for target binding were evaluated in the development of the free target assay. This assay format was not affected by sample dilution, incubation time of the sample with the capture antibody, or freeze / thaw cycles of the sample. Finally, these assays showed precision when used to determine the concentrations of total drug, total target, and free target in a subset of Phase I clinical trial samples to demonstrate precision.

Claims

1. A method for determining the concentration of free target in a sample, comprising: a. adding the sample having a drug-bound target and the free target to a solid support coated with a capture agent, wherein the capture agent has a lower affinity and a slower association rate for the target compared to the drug; b. adding a detection substance having a detectable label; c) measuring a signal from the detectable label of the detection agent to determine the concentration of the free target in the sample, wherein the signal is proportional to the concentration of the free target in the sample.

2. The method of claim 1 , wherein the solid support is coated with streptavidin.

3. The method of claim 1 , wherein the capture agent is biotinylated.

4. 10. The method of claim 1, wherein the sample of (a) is incubated for about 15 minutes or about 45 minutes.

5. The method of claim 1 , wherein the detectable label is ruthenium.

6. The method of claim 1 , wherein the detectable label is an electrochemiluminescent substrate.

7. The method of claim 1 , wherein the signal in (c) is obtained by applying a voltage.

8. 2. The method of claim 1, further comprising determining the amount of free target from the signal by comparing the signal with a standard calibration curve, wherein the standard calibration curve is prepared by using at least three standard solutions having three different concentrations of the free target in place of the sample.

9. A method for determining the concentration of a total target in a sample, comprising: a. contacting the sample with an acidic solution; b. adding the sample to a solid support coated with a capture agent; c. adding a detection substance having a detectable label; d. Measuring a signal from the detection substance to determine the concentration of the total target in the sample, the total targets include targets complexed with a drug and free targets; the capture agent has a slower dissociation rate and a greater t 1 / 2 than the drug with respect to the target; and The signal is proportional to the concentration of the total target in the sample.

10. The method of claim 9 , wherein the capture agent is biotinylated.

11. 10. The method of claim 9, wherein the detectable label is ruthenium.

12. 10. The method of claim 9, wherein the detectable label is an electrochemiluminescent substrate.

13. 10. The method of claim 9, wherein the signal in (c) is obtained by applying a voltage.

14. 10. The method of claim 9, wherein step (b) is incubated for about 60 minutes.

15. 10. The method of claim 9, wherein step (c) is incubated for about 60 minutes.

16. 10. The method of claim 9, wherein the acidic solution has a pH of about 5.0 to about 7.

0.

17. 10. The method of claim 9, wherein the acidic solution has a pH of about 6.

0.

18. 10. The method of claim 9, wherein the acidic solution comprises 300 mM acetic acid.

19. The method of claim 9, wherein the acidic solution contains 30 mM acetic acid.

20. The detection substance has a lower dissociation rate and a larger t １／２ 10. The method of claim 9, comprising:

21. 10. The method of claim 9, further comprising determining the amount of total target from the signal by comparing the signal with a standard calibration curve, wherein the standard calibration curve is generated by performing the method of claim 9 by using at least three standard solutions having three different concentrations of free target in place of the sample.

22. 1. A method for determining the concentration of a total drug in a sample, comprising: a. contacting the sample with an acidic solution; b. adding the sample to a solid support coated with a capture agent; c. adding a detection agent having a detectable label, wherein the detection agent is different from the capture agent; d. Measuring a signal from the detection substance to determine the concentration of the total target in the sample, the total drug includes the drug complexed with the target and the free drug; The signal is proportional to the concentration of the free target in the sample.

23. 2. The method of claim 1, further comprising determining the amount of free drug from the signal by comparing the signal with a standard calibration curve, wherein the standard calibration curve is created by using at least three standard solutions having three different concentrations of the free drug in place of the sample.

24. 23. The method of claim 22, wherein the capture agent is a monoclonal antibody that binds to the drug.

25. 23. The method of claim 22, wherein the detection agent is different from the capture agent.

26. 23. The method of claim 22, wherein the detection agent is biotinylated.

27. 23. The method of claim 22, further comprising the step of adding a substrate specific for binding to the detection substance, wherein the substrate bound to the detection substance provides the signal of (d).

28. 23. The method of claim 22, wherein the acidic solution has a pH of about 5.0 to about 7.

0.

29. 23. The method of claim 22, wherein the acidic solution has a pH of about 6.

0.

30. 23. The method of claim 22, wherein the acidic solution comprises 30 mM acetic acid.

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