Method for quantifying antigen-binding molecules that bind to cells

Abstract

A method for quantifying an antigen-binding molecule that binds to a cell that expresses a target antigen that specifically binds to the antigen-binding molecule, the method comprising: a preparation step for preparing a biological sample that has been homogenized and is derived from the cell that expresses the target antigen that specifically binds to the antigen-binding molecule; the cell was exposed to the antigen-binding molecule; the method comprises an extraction step for obtaining an analysis sample by adding an extraction solution containing an organic acid and a nonionic surfactant and having a pH of 1-3 to the biological sample, and a quantification step for analyzing the analysis sample and quantifying the antigen-binding molecule.

Description

Technical Field

[0001] The present invention relates to a method for quantifying antigen-binding molecules conjugated to cells. Background Art

[0002] In recent years, the development of drugs using antibodies (hereinafter sometimes referred to as "antibody drugs") has been booming. Antibodies have the property of specifically binding to a specific antigen (antigen specificity). Utilizing this property, for example, by administering an antibody that binds only to a receptor (antigen) specifically expressed on cancer cells to a cancer tissue containing the cancer cells, an immune response is induced and cancer is treated. For example, cetuximab is known as an anticancer agent that specifically binds to the epidermal growth factor receptor (EGFR) and inhibits the proliferation of cancers such as colon cancer. Summary of the Invention

[0003] In general, in order for an antibody drug to exert its pharmacological effect, it first binds to the target antigen. Therefore, in order to evaluate an antibody drug under development, it is desired to develop a technique for accurately measuring the aggregation level of the antibody that has reached the target tissue or target cells. For example, the following technique is disclosed in Yakugaku Zasshi. 2017;137(5):535-544.: Based on the captured data obtained by microscopy, the XY-axis directions are shared with the mass spectrometry side, and thus the amount of ADC (antibody drug conjugate) distributed in the tissue is measured using a mass spectrometer to obtain its molecular distribution image. Clin Cancer Res. 2021 Jul 15;27(14):3970-3979. discloses a technique for analyzing the aggregation level of ADC using anti-human antibody and anti-payload antibody in formalin-fixed paraffin-embedded tissue sections (FFPE tissue sections) based on tissue staining images.

[0004] However, since the prior art indirectly detects the antibody bound to the target tissue, there is room for improvement in the quantification accuracy. The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for quantifying antigen-binding molecules conjugated to cells expressing a target antigen with good accuracy and sensitivity.

[0005] The inventors of the present invention conducted intensive studies and found that by adding a prescribed extraction solution to a biological sample derived from cells expressing a target antigen, an analysis sample can be obtained, and antigen-binding molecules conjugated to the cells can be quantified with good accuracy, thereby completing the present invention.

[0006] A first aspect of the present invention is a method for quantifying antigen-binding molecules conjugated to cells, which includes a preparation step, an extraction step, and a quantification step,

[0007] The preparation step prepares a biological sample derived from the cells, wherein,

[0008] The above biological sample has been homogenized,

[0009] The above cells express the target antigen specifically bound by the above antigen-binding molecule,

[0010] The above cells have been exposed to the above antigen-binding molecule;

[0011] In the above extraction step, an extraction solution is added to the above biological sample to obtain a test sample for analysis, wherein,

[0012] The above extraction solution contains an organic acid and a nonionic surfactant,

[0013] The pH of the above extract is 1 or more and 3 or less;

[0014] The above quantification step analyzes the above test sample for analysis and quantifies the above antigen-binding molecule.

[0015] From the following detailed description of the invention understood in conjunction with the accompanying drawings, the above and other objects, features, aspects and advantages of the invention will become apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 A schematic diagram showing one mode of the quantification method of the present embodiment.

[0017] Figure 2 A chart showing the results of the LCMS analysis in Experiment 1.

[0018] Figure 3 A chart showing the results of the LCMS analysis in Experiment 1.

[0019] Figure 4 A chart of the standard curve obtained from the analysis results of the standard curve samples in Experiment 1.

[0020] Figure 5 A chart showing the results of the LCMS analysis in Experiment 1.

[0021] Figure 6 A chart showing the quantification results of the antibody bound to the surface of the cell line in Experiment 2.

[0022] Figure 7 A schematic diagram (left) showing the steps for producing a model tumor using nude mice in Experiment 3 and an image of the tissue staining of the collected model tumor (right).

[0023] Figure 8 A chart (left) showing the image of the tissue staining of the collected model tumor (right) and the amount of antibody detected in the collected model tumor in Experiment 3.

[0024] Figure 9 A graph showing the amount of antibody detected in the collected model tumors ("antibody not bound to tissue", "amount of antibody in the whole tissue").

[0025] Figure 10 A graph showing the results of the LCMS analysis in Experiment 4.

[0026] Figure 11 A graph of the standard curve obtained from the analysis results of the standard curve samples in Experiment 4.

[0027] Figure 12 An image of the tissue staining of the collected model tumor (right side) and a graph showing the amount of antibody detected in the collected model tumor (left side) in Experiment 5.

[0028] Figure 13 A graph showing the amount of antibody detected in the collected model tumors ("antibody not bound to tissue", "amount of antibody in the whole tissue").

[0029] Figure 14 A graph showing the results of the LCMS analysis in Experiment 6.

[0030] Figure 15 A schematic diagram showing the steps for producing a model tumor using nude mice (left side) and an image of the tissue staining of the collected model tumor (right side) in Experiment 7.

[0031] Figure 16 An image of the tissue staining of the collected model tumor (right side) and a graph showing the amount of antibody detected in the collected model tumor (left side) in Experiment 7.

[0032] Figure 17 A graph showing the amount of antibody detected in the collected model tumors ("antibody not bound to tissue", "amount of antibody in the whole tissue"). Detailed Embodiment

[0033] One embodiment of the present invention (hereinafter referred to as "this embodiment") will be described below. However, this embodiment is not limited thereto. In this specification, a description in the form of "A to Z" represents the upper and lower limits of a range (i.e., A or more and Z or less). When there is no unit description in A and only a unit is described in Z, the unit of A is the same as the unit of Z.

[0034] "Quantification Method of Antigen-Binding Molecules Bound to Cells"

[0035] The first mode of this embodiment is a method for quantifying antigen-binding molecules bound to cells, which includes a preparation step, an extraction step, and a quantification step.

[0036] The above preparation step prepares a biological sample derived from the above cells, wherein,

[0037] the above biological sample has been homogenized,

[0038] the above cells express a target antigen specifically bound by the above antigen-binding molecule,

[0039] the above cells have been exposed to the above antigen-binding molecule,

[0040] The above extraction step adds an extraction solution to the above biological sample to obtain a sample for analysis, wherein,

[0041] the above extraction solution contains an organic acid and a nonionic surfactant,

[0042] the pH of the above extraction solution is 1 or more and 3 or less,

[0043] The above quantification step analyzes the above sample for analysis and quantifies the above antigen-binding molecule.

[0044] <Preparation step>

[0045] In this step, a biological sample derived from cells is prepared. In the present embodiment, the "biological sample derived from cells" refers to a biological sample containing the cells themselves or substances constituting the cells (cell membrane, cell nucleus, cytoplasm, etc.). The above biological sample has been homogenized. "Having been homogenized" means a state in which the components in the biological sample are uniform and there is no local deviation of the components. The above biological sample can be in a solid state or a liquid state, preferably in a liquid state.

[0046] The above cells express a target antigen specifically bound by the above antigen-binding molecule. In the present embodiment, the "target antigen" refers to a protein mainly expressed on the surface of the above cells. In one aspect of the present embodiment, the above target antigen can also be understood as a "target biomolecule" or a "target protein". As such a protein, for example, a receptor expressed on the surface of the above cells, an enzyme expressed inside the above cells, a tumor necrosis factor receptor, an insulin receptor, a vascular endothelial growth factor receptor, etc. can be cited. As the receptor expressed on the surface of the cells, for example, an epidermal growth factor receptor (EGFR), HER2, CD30, PD-L1, RANKL, a tumor necrosis factor receptor, an insulin receptor, a vascular endothelial growth factor receptor, etc. can be cited. In one aspect of the present embodiment, the above target antigen preferably contains at least 1 selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, a tumor necrosis factor receptor, an insulin receptor, and a vascular endothelial growth factor receptor.

[0047] The above-mentioned cells have been exposed to the above-mentioned antigen-binding molecule. In the present embodiment, the "antigen-binding molecule" refers to a protein or peptide that specifically binds to the above-mentioned target antigen. Examples of the antigen-binding molecule include, for example, an antibody, a peptide aptamer, etc. In one aspect of the present embodiment, the antigen-binding molecule preferably comprises an antibody or a peptide aptamer. In another aspect of the present embodiment, the antigen-binding molecule preferably comprises at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab vedotin, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab, and insulin analogs.

[0048] In the present embodiment, "having been exposed to an antigen-binding molecule" means that, before collecting the above-mentioned cells or the tissue containing the above-mentioned cells, the above-mentioned cells have been present in an environment where the above-mentioned antigen-binding molecule exists or have been artificially exposed to the antigen-binding molecule. For example, culturing the above-mentioned cells in a medium containing the above-mentioned antigen-binding molecule, administering the above-mentioned antigen-binding molecule to the tissue containing the above-mentioned cells belongs to "having been exposed to an antigen-binding molecule".

[0049] In the present embodiment, the above-mentioned cells are preferably cells derived from a cancer tissue or cells derived from a diseased tissue.

[0050] In the present embodiment, the method for homogenizing the above-mentioned biological sample is not particularly limited, and homogenization can be performed by a known method. Examples of the homogenization method include, for example, a method of homogenizing the above-mentioned cells or the tissue containing the above-mentioned cells (preferably a thin slice of the tissue) in a prescribed buffer (e.g., D-PBS) using beads for cell disruption (e.g., Figure 1 ), a method based on sonication, a method based on freeze-thawing, and a method using a French press, etc.

[0051] In one aspect of the present embodiment, the above-mentioned biological sample may further contain an enzyme inhibitor. When the biological sample contains an enzyme inhibitor, the decomposition of the above-mentioned antigen-binding molecule by an enzyme can be inhibited. Examples of the enzyme inhibitor include, for example, a phosphatase inhibitor, a protease inhibitor, etc.

[0052] <Extraction step>

[0053] In this step, an extraction solution is added to the above biological sample to obtain a sample for analysis. In the present embodiment, the "extraction solution" refers to a solution used to dissociate the above antigen-binding molecule bound to the above target antigen from the above target antigen. The above extraction solution contains an organic acid and a nonionic surfactant. In the present embodiment, the "sample for analysis" is a sample derived from the above biological sample and refers to a sample for analysis in the quantification step described later. It should be noted that in this specification, the pretreatment method performed in the above extraction step is sometimes referred to as the "ACES method" (Acidic Conditions Extraction supported with Surfactant method).

[0054] In the present embodiment, the "organic acid" refers to an organic compound that exhibits acidity. The above organic acid is preferably an organic compound having a carboxyl group. Examples of the above organic acid include arginine, citrulline, and glycine. In one aspect of the present embodiment, the above organic acid preferably contains at least one selected from the group consisting of arginine, citrulline, and glycine. When the above antigen-binding molecule is an antibody, from the viewpoint of maintaining the stability of the antibody, the above organic acid is preferably arginine.

[0055] Regarding the concentration of the above organic acid, it is preferably 20 mM or more and 1000 mM or less, more preferably 100 mM or more and 500 mM or less, relative to the above extraction solution.

[0056] In the present embodiment, the "nonionic surfactant" refers to a surfactant that does not ionize even when dissolved in water. Examples of the above nonionic surfactant include alkyl glycosides in which a sugar and a higher alcohol form a glycosidic bond. Examples of the above alkyl glycosides include n-octyl-β-D-thioglucopyranoside, n-octyl-β-D-glucoside, n-octyl-β-D-maltoside, n-decyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-glucoside, n-heptyl-β-D-thioglucopyranoside, and n-nonyl-β-D-thiomaltoside, trehalose C12, etc. In one aspect of the present embodiment, the above nonionic surfactant preferably contains an alkyl glycoside. The above alkyl glycoside preferably contains at least one selected from the group consisting of n-octyl-β-D-thioglucopyranoside, n-octyl-β-D-glucoside, n-octyl-β-D-maltoside, n-decyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-glucoside, n-heptyl-β-D-thioglucopyranoside, and n-nonyl-β-D-thiomaltoside. The above alkyl glycoside is more preferably n-octyl-β-D-thioglucopyranoside.

[0057] Regarding the concentration of the above nonionic surfactant, it is preferably 0.1% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, relative to the above extraction solution. In one aspect of the present embodiment, the concentration of the above nonionic surfactant is preferably a concentration higher than the critical micelle concentration of the nonionic surfactant.

[0058] When the above nonionic surfactant is n-octyl-β-D-thioglucopyranoside, the concentration of n-octyl-β-D-thioglucopyranoside is preferably 0.2% by mass or more and 10% by mass or less, more preferably 0.5% by mass or more and 5% by mass or less, relative to the above extraction solution.

[0059] The type and concentration of the above organic acid, and the type and concentration of the above nonionic surfactant can be determined by analyzing the above extraction solution using a liquid chromatography and mass spectrometry device.

[0060] The pH of the above extraction solution is 1 or more and 3 or less, preferably 1.5 or more and 2.5 or less. The pH of the above extraction solution can be measured using a commercially available pH meter.

[0061] In the present embodiment, the addition amount of the above extraction solution is not particularly limited as long as the effects of the present invention can be exhibited. For example, based on the mass of the above biological sample, it is preferably 10 times or more and 50 times or less, more preferably 20 times or more and 40 times or less. At this time, the addition amount of the above extraction solution is determined by regarding the density of the above extraction solution as 1 mg / 1 μL.

[0062] In the present embodiment, the above extraction solution can be directly added to the above biological sample to obtain an analysis sample (for example Figure 1 ). Alternatively, the above biological sample can be separated into a liquid component and a solid component and recovered separately, and then the above extraction solution can be added to the above solid component to obtain an analysis sample. As a method for separating the above biological sample into a liquid component and a solid component, there is no particular limitation, and examples thereof include a method of separating into a supernatant (liquid component) and a precipitate (solid component) by centrifugation.

[0063] In one aspect of the present embodiment, the above extraction step may include: separating the above biological sample into a liquid component and a solid component; adding the above extraction solution to the above solid component to obtain a first analysis sample; and obtaining the above liquid component as a second analysis sample.

[0064] In another aspect of the present embodiment, the above preparation step and the above extraction step can be performed simultaneously. For example, a method of adding the above extraction solution to the above biological sample and then homogenizing can be cited.

[0065] In one aspect of the present embodiment, the extraction solution may further contain an enzyme inhibitor, an internal standard substance, or an adsorption inhibitor. Here, the "adsorption inhibitor" refers to a reagent that inhibits the physical adsorption of the above antigen-binding molecule to the wall surface of the microtube or the like. Examples of the enzyme inhibitor include a phosphatase inhibitor, a protease inhibitor, and the like. Examples of the internal standard substance include an isotope-labeled antigen-binding molecule and the like. Examples of the adsorption inhibitor include BSA, mouse IgG, and the like.

[0066] <Quantification step>

[0067] In this step, the above analysis sample is analyzed to quantify the above antigen-binding molecule. In one aspect of the present embodiment, "analyzing the above analysis sample" includes directly subjecting the above analysis sample to analysis, or removing the solid components (such as the remaining above precipitate, etc.) in the above analysis sample and then subjecting the analysis sample to analysis.

[0068] In the present embodiment, the method for quantifying the above antigen-binding molecule may be to directly measure the above antigen-binding molecule for quantification, or to detect a peptide derived from the above antigen-binding molecule for quantification.

[0069] In the present embodiment, the method for quantifying the above antigen-binding molecule is not particularly limited, and examples thereof include a quantification method using ELISA, a quantification method using a liquid chromatography tandem mass spectrometry device (LC-MS device or LC-MS / MS device), and the like.

[0070] The column of LC in the LC-MS device is not particularly limited, and hydrophobic columns such as C30, C18, C8, C4, etc. commonly used in the analysis of proteins or peptides, columns containing carriers for hydrophilic affinity chromatography, etc. can be appropriately selected and used. If necessary, the sample can be used for mass spectrometry after undergoing treatments such as desalting, solubilization, extraction, concentration, drying, etc.

[0071] The ionization method in mass spectrometry is not particularly limited, and methods such as electron ionization (EI) method, chemical ionization (CI) method, field desorption (FD) method, fast atom bombardment (FAB) method, matrix-assisted laser desorption ionization (MALDI) method, electrospray ionization (ESI) method, etc. can be adopted. The analysis method of the ionized sample is also not particularly limited, and it can be appropriately determined according to the ionization method as magnetic field deflection type, quadrupole (Q) type, ion trap (IT) type, time of flight (TOF) type, Fourier transform ion cyclotron resonance (FT-ICR) type, etc. A triple quadrupole type mass spectrometry device or the like can also be used to perform MS / MS analysis, multi-stage mass spectrometry of MS3 or higher, or multiple reaction monitoring (MRM).

[0072] The device particularly suitable for the quantification method of the present embodiment is not particularly limited, and examples thereof include LCMS-8030, LCMS-8040, LCMS-8050, LCMS-8060, LCMS-9030, and LCMS-IT-TOF (all manufactured by Shimadzu Corporation).

[0073] In one aspect of the present embodiment, when the antigen-binding molecule is an antibody, the quantification step preferably includes determining a peptide derived from the variable region of the antibody using the nSMOL method and analyzing the peptide using LC-MS / MS analysis. The nSMOL method can be implemented, for example, using the nSMOL Antibody BA Kit (product name, manufactured by Shimadzu Corporation).

[0074] In the prior art, the antigen-binding molecule (such as an antibody) bound to the cell expressing the target antigen is indirectly detected, so there is room for improvement in the quantification accuracy. In the present invention, by recovering the antigen-binding molecule bound to the cell from the cell using an extraction solution containing an organic acid and a non-surfactant, the antigen-binding molecule can be directly detected, and the quantification accuracy can be improved. According to the quantification method of the present invention, not only the aggregation state of the antigen-binding molecule (such as an antibody drug) in the target tissue (such as a cancer tissue) can be analyzed, but also whether the antigen-binding molecule has aggregated in an undesired tissue (such as a normal tissue) can be analyzed. Therefore, the quantification method of the present invention is also useful for elucidating the mechanism of side effects in normal tissues.

[0075]

Examples

[0076] The following examples are given to illustrate the present invention in more detail, but the present invention is not limited thereto.

[0077] (Experiment 1: Quantification of antibodies present in cancer tissues)

[0078] The quantification of the antibody present in the cancer tissue was carried out through the following steps. Here, the cancer tissue corresponds to the tissue containing the cell expressing the target antigen, and the antibody corresponds to the antigen-binding molecule.

[0079] (Example: Extraction using the ACES method)

[0080] (Preparation step, extraction step)

[0081] First, the following cancer tissue and preparation solution were prepared.

[0082] Cancer tissue:

[0083] Cancer tissue (esophageal cancer) collected from a patient administered cetuximab and cancer tissue of an experimental model mouse administered cetuximab

[0084] (1) Tissue washing solution:

[0085] D-PBS

[0086] Phosphatase inhibitor (1:50 (volume ratio), PhosSTOP)

[0087] Protease inhibitor mixture (1:50 (volume ratio), cOmplete ULTRA)

[0088] (2) Inhibitory solution:

[0089] 200 mM L-arginine hydrochloride aqueous solution (pH 2.1)

[0090] Phosphatase inhibitor (1:50 (volume ratio), PhosSTOP)

[0091] Protease inhibitor mixture (1:50 (volume ratio), cOmplete ULTRA)

[0092] (3) Internal standard IS solution:

[0093] Inhibitory solution

[0094] SILuMAb cetuximab (final concentration 0.25 μg / ml)

[0095] BSA (final concentration 0.1%)

[0096] Mouse IgG1 (clone 2545) (final concentration 50 μg / ml)

[0097] (4) Extraction solution:

[0098] 200 mM L-arginine hydrochloride aqueous solution (pH 2.1)

[0099] n-Octyl β-D-thioglucopyranoside (final concentration 2%)

[0100] First, the above cancer tissue is sectioned with a microtome to prepare thin sections (thickness 5 μm). The above thin sections (10 mg) are transferred to a low-attachment microtube, and cooled tissue washing solution (200 μl) is added thereto. Further, beads for tissue disruption (zirconia beads, diameter 2 mm, 8 pieces) are added to the above microtube.

[0101] Under cooling (below 2°C), the cancer tissue in the above microtube was disrupted (homogenized) using a microbead homogenizer (MicroSmash MS-100R) at 5,000 rpm for 1 minute. After that, it was left standing in the device for 1 minute to cool the above microtube. This operation was performed a total of 4 times. Through the above steps, a sample containing disrupted cancer tissue (homogenized biological sample) was prepared (preparation step). The above biological sample is a sample derived from cancer cells.

[0102] All of the sample containing disrupted cancer tissue (homogenized biological sample) was transferred to another low-adsorption microtube. To the above microtube, tissue washing solution (100 μl) was further added to wash the disrupted cancer tissue, and it was recovered (2 times). For the recovered sample, ultrasonic treatment was performed for 5 minutes using an ultrasonic cleaner under ice cooling (0°C) to defoam. For the microtube containing this sample, centrifugation was performed (16,000 g × 5 minutes, 4°C) to separate it into a supernatant and a precipitate. All of the supernatant was recovered into another low-adsorption microtube as the "free antibody sample" (sample for the second analysis). The "free antibody sample" was stored at 4°C.

[0103] On the other hand, for the above precipitate, an inhibition solution (160 μl), an internal standard IS solution (40 μl), and an extraction solution (200 μl) were added to obtain a suspension of the precipitate. The obtained suspension was left standing under ice cooling for 1 hour. After that, the above suspension was resuspended by pipetting and then left standing overnight at 4°C (extraction step, ACES method). For the tube containing the above suspension, centrifugation was performed (16,000 g × 30 minutes, 4°C) to separate it into a supernatant and a precipitate. The supernatant was recovered and centrifugal filtration was performed using Ultrafree MC (0.22 μm) (10,000 g × 2 minutes, 4°C) to obtain a filtrate. The obtained filtrate was recovered into another low-adsorption microtube as the "bound antibody sample" (sample for analysis or the first sample for analysis). The bound antibody sample was stored at 4°C.

[0104] (Quantification step)

[0105] Prepare the following preparation solutions.

[0106] (1) Standard dilution solution:

[0107] Inhibition solution

[0108] BSA (final concentration 0.1%)

[0109] Mouse IgG1 (clone 2454) (final concentration 50 μg / ml)

[0110] (2) Washing solution:

[0111] D-PBS

[0112] n-Octyl β-D-thioglucopyranoside (final concentration 0.1%)

[0113] (3) Neutralize the solution:

[0114] 250 mM Na 2 HPO 4 aqueous solution

[0115] n-Octyl β-D-thioglucopyranoside (final concentration 1%)

[0116] (4) Reaction solution:

[0117] 25 mM Tris-HCl aqueous solution (pH 8.0)

[0118] (5) Initial mobile phase:

[0119] MilliQ water (0.1% formic acid) + acetonitrile (volume ratio 99:1)

[0120] Prepare standard curve samples and QC samples with the following compositions.

[0121] Standard curve samples: All are prepared with the standard dilution solution.

[0122] Blank sample 0 ng / ml cetuximab

[0123] Standard curve 1 313 ng / ml cetuximab

[0124] Standard curve 2 625 ng / ml cetuximab

[0125] Standard curve 3 1.25 μg / ml cetuximab

[0126] Standard curve 4 2.50 μg / ml cetuximab

[0127] Standard curve 5 5.00 μg / ml cetuximab

[0128] Standard curve 6 10.0 μg / ml cetuximab

[0129] Standard curve 7 20.0 μg / ml cetuximab

[0130] Standard curve 8 40.0 μg / ml cetuximab

[0131] QC samples: All are prepared with the standard dilution solution.

[0132] LLOQ 313 ng / ml cetuximab

[0133] LQC 938 ng / ml cetuximab

[0134] Cetuximab at 3.75 μg / ml of MQC

[0135] Cetuximab at 12.5 μg / ml of HQC

[0136] Cetuximab at 40.0 μg / ml of HLOQ

[0137] Add 20 μL each of the above standard curve samples and QC samples to 160 μL of a matrix solution (a solution obtained by subjecting interfering components derived from the measurement tissue to an extraction treatment using an inhibition solution and an extraction solution in the same manner as for the measurement sample), and then add 20 μL of an internal standard IS solution. These correspond to the "standard curve samples (with IS)" and "QC samples (with IS)" described below. In addition, in the above preparation, samples were also prepared by adding 20 μL of the matrix solution in place of 20 μL of the internal standard IS solution. These correspond to the "standard curve samples (without IS)" and "QC samples (without IS)" described below.

[0138] Quantitative analysis of cetuximab (antigen-binding molecule) in the analytical sample is performed by generating a cetuximab-specific peptide (peptide derived from the variable region) using the nSMOL method and detecting the peptide using a mass spectrometry device. The specific steps are as described below.

[0139] (Generating the peptide using the nSMOL method)

[0140] Dispense 200 μl each of the bound antibody sample, standard curve samples (with or without IS), and QC samples (with or without IS) into low-binding microtubes. Then, add a washing solution (600 μl) and a neutralization solution (200 μl) to each microtube.

[0141] In addition, transfer 200 μl of the free antibody sample to a low-binding microtube, and then add 20 μl of the internal standard IS solution. Then, add a washing solution (760 μl) and a neutralization solution (20 μl) to the above microtube.

[0142] After thoroughly suspending the immunoglobulin collection resin in the nSMOL Antibody BA Kit (manufactured by Shimadzu Corporation), take 12.5 μl and add it to each tube. Stir each tube with a self-supporting tube mixer for 30 minutes. Then, centrifuge each tube (15,000 g × 5 minutes, 4 °C). Remove 700 μl of the supernatant smoothly from each tube without sucking up the resin. Transfer the remaining suspension (resin and remaining liquid) in each tube to Ultrafree MC (0.22 μm) respectively. For each tube after transferring the above suspension, add the washing solution (300 μl), recover the remaining resin in each tube, and transfer it to the above Ultrafree MC (0.22 μm) respectively.

[0143] For each Ultrafree MC, perform centrifugal filtration (10,000 g × 1 minute, 25 °C) to remove the filtrate. Add the washing solution (300 μl) to each Ultrafree MC, perform centrifugal filtration (10,000 g × 1 minute, 25 °C), and remove the filtrate (a total of 2 times). Add D-PBS (300 μl) to each Ultrafree MC, perform centrifugal filtration (10,000 g × 1 minute, 25 °C), and remove the filtrate (a total of 3 times).

[0144] Add the reaction solution (85 μl) to each Ultrafree MC. After thoroughly suspending the FGbeads Trypsin DART in the nSMOL Antibody BA Kit, take 5 μl and add it to each Ultrafree MC. Then, incubate at 52 °C under saturated vapor pressure for 5 hours to carry out the nSMOL reaction.

[0145] Add the termination solution (10 μl) in the nSMOL Antibody BA Kit to each Ultrafree MC to stop the nSMOL reaction. Then, perform centrifugal filtration on each Ultrafree MC (10,000 g × 1 minute, 25 °C), and collect all the filtrates into other microtubes respectively. Stand each microtube on a magnetic stand and let it stand for 2 minutes to remove the remaining magnetic beads. Then, collect the supernatant (90 μl) in each microtube into a low-binding microtube respectively. Add the initial mobile phase (90 μl) to the above low-binding microtube. Place it in an HPLC polypropylene vial and remove the bubbles. Through the above steps, cetuximab-specific peptides (peptides derived from the variable region) are generated.

[0146] (Detect the peptide using a mass spectrometry device)

[0147] The above-mentioned cetuximab-specific peptide (Sequence No. 1: ASQSIGTNIHWYQQR (single-letter symbols)) was quantified by LCMS analysis under the conditions shown below.

[0148] List of prepared solutions:

[0149] Mobile phase A: MilliQ (LCMS grade)

[0150] Mobile phase B: 0.1% formic acid + acetonitrile (LCMS grade)

[0151] LCMS analysis conditions:

[0152] Column used: Shimpack GISS C18, 2.1 mm ID × 50 mm

[0153] Column temperature: 50 °C

[0154] Sample injection volume: 10 μl

[0155] Nebulizing gas flow rate: 3 l / min

[0156] Drying gas flow rate: 10 l / min

[0157] Heating gas flow rate: 10 l / min

[0158] ESI interface temperature: 300 °C

[0159] Heating block temperature: 400 °C

[0160] DL temperature: 250 °C

[0161] CID gas pressure: 270 kPa

[0162] HPLC conditions:

[0163] HPLC: Shimadzu Nexera X2

[0164] Flow rate: 0.4 ml / min

[0165] 0 - 1.5 minutes: 1% B

[0166] 1.5 - 4.9 minutes: 1 - 38% B gradient

[0167] 4.9 - 6.5 minutes: 38% B

[0168] 6.5 - 7.0 minutes: 38 - 95% B gradient

[0169] 7.0 - 9.0 minutes: 95% B

[0170] 9.0 - 9.5 minutes: 95 - 1% B

[0171] 9.5 - 13.5 minutes: 1%B

[0172] LCMS analysis time: 3.0 - 6.0 minutes

[0173] LCMS MRM analysis transition conditions:

[0174] LCMS: Shimadzu LCMS - 8060

[0175] Cetuximab: ASQSIGTNIHWYQQR

[0176] 596.95(+++) > 651.80(y10++) for quantification

[0177] 596.95(+++) > 751.85(y12++) confirmation ion

[0178] 596.95(+++) > 708.35(y11++) confirmation ion

[0179] 13C6,15N4 - Cetuximab, 13C6,15N4 - ASQSIGTNIHWYQQR

[0180] 600.45(+++) > 657.10(y10++) for quantification (Ab_IS)

[0181] 600.45(+++) > 757.30(y12++) confirmation ion

[0182] 600.45(+++) > 713.15(y11++) confirmation ion

[0183] (Comparative example)

[0184] The thin - section slices (about 10 mg) of the above - mentioned cancer tissue were enzymatically treated using ProteinWorks Auto - eXpress Digest Kits (manufactured by Waters). The obtained enzymatically treated product was centrifuged (800 g × 15 minutes, 10°C), and the supernatant (about 160 μl) was recovered. The recovered supernatant was analyzed by LCMS analysis under the same conditions as above.

[0185] (Results)

[0186] The results of LCMS analysis of the binding antibody samples in the examples and the samples in the comparative example are shown in Figure 2 . Figure 2 in which the horizontal axis represents the retention time in LC, and the vertical axis represents the detection intensity of the peptide corresponding to its retention time during mass spectrometry analysis. From Figure 2The results show that the methods using the ACES method and the sMOL method only detect the cetuximab-specific peptide ( Figure 2 the left chart). That is, the methods of the examples can detect cetuximab present in cancer cells. On the other hand, multiple peaks were detected in the sample obtained by enzymatically treating the whole cancer tissue ( Figure 2 the right chart).

[0187] Figure 3 The LCMS analysis results of the standard curve samples (blank) (with IS (the second chart from the top) or without IS (the first chart from the top)) and QC samples (LLOQ) (with IS (the fourth chart from the top) or without IS (the third chart from the top)) in the examples are shown. From Figure 3 the results, it can be seen that cetuximab can also be detected at a concentration of 313 ng / ml under the analysis conditions of the examples. It should be noted that the same results were also confirmed when the ions (596.95(+++)>751.85(y12++)) were confirmed ( Figure 5 ).

[0188] The graph of the standard curve obtained from the analysis results of the standard curve samples (blank, standard curves 1-8) (with IS) in the examples is shown in Figure 4 . Figure 4 In it, the horizontal axis represents the concentration ratio of cetuximab in each standard curve sample (based on the concentration of IS), and the vertical axis represents the ratio of the peak area of the analyte to the peak area of IS in mass spectrometry analysis. Figure 4 The results show that cetuximab can be quantified under the analysis conditions of the examples.

[0189] (Experiment 2: Quantification of antibodies bound to the surface of cell lines)

[0190] Four cell lines (A-431, FaDu, TE-4, and SW620) were used to quantify the antibodies bound to the surface of each cell line by the following steps. First, each cell was seeded at 4×10 5 cells / well in a 12-well plate and allowed to adhere in an incubator (37 °C, 5% CO 2 ). 1 ml of 5 mg / ml (excess) cetuximab was added to each well, and the mixture was left standing at ice-cooling for 30 minutes. After the reaction, the cetuximab was aspirated, and each well was washed and aspirated twice with D-PBS.

[0191] For each well, add the inhibition solution (160 μl), the internal standard IS solution (40 μl), and the extraction solution (200 μl). After sufficient pitting, leave it to stand for 1 hour under ice cooling (extraction step, ACES method). Recover the above suspension into a tube and perform centrifugation (16,000 g × 30 minutes, 4 °C) to separate it into a supernatant and a precipitate. Recover the supernatant and perform centrifugal filtration using Ultrafree MC (0.22 μm) (10,000 g × 2 minutes, 4 °C) to obtain a filtrate. Thereafter, perform treatment and quantification in the same manner as the quantification step in "Experiment 1: Quantification of antibodies present in tissue samples". The results are shown in Figure 6 (the lower left chart and the lower right chart). Figure 6 The results of Figure 6 show that it is related to the expression level of EGFR in

[0192] (Experiment 3: Quantification of antibodies bound to tissues in model tumors)

[0193] Subcutaneously transplant the SW620 strain or the A431 strain into nude mice (BALB / c, female, 5 - 8 weeks old). The number of cells transplanted at this time is 1 × 10 7 cells per mouse. Conduct conventional breeding for 14 - 21 days until the transplanted cells survive. Thereafter, intravenously inject cetuximab (0 - 1.0 mg / body) into the above mice. 24 hours after the intravenous injection, collect the transplanted cells (model tumors) from the mice ( Figure 7 , left side).

[0194] Stain the collected model tumors with HE and anti - EGFR antibodies ( Figure 7 , right side). Confirm that the model tumors collected from the mice transplanted with the A431 strain express EGFR.

[0195] In addition, stain the collected model tumors with DAPI and anti - human IgG antibodies ( Figure 8 , right side). Confirm that in the model tumors collected from the mice transplanted with the A431 strain, the amount stained by the anti - human IgG antibody increases depending on the amount of cetuximab administered.

[0196] Treat the collected model tumors in the same manner as in Experiment 1 and quantify the cetuximab bound to the model tumors. The sample used in the quantification is the sample corresponding to the "bound antibody sample" in Experiment 1. The results are shown in Figure 8(Left). It was confirmed that the quantitative value of cetuximab using the ACES method in the model tumors collected from mice transplanted with A431 strain increased depending on the amount of cetuximab administered. This result was also correlated with the result of the existing method - tissue staining( Figure 8 ). On the other hand, cetuximab was hardly detected in the model tumors collected from mice transplanted with SW620 strain.

[0197] Using the specimen corresponding to the "free antibody sample" in Experiment 1, cetuximab that was present in the model tumor but not bound to EGFR was quantified( Figure 9 , left). In the model tumors collected from mice transplanted with SW620 strain, more cetuximab was detected compared to the model tumors collected from mice transplanted with A431 strain.

[0198] (Experiment 4: Extraction by the ACES method from cancer tissues administered with Trastuzumab-Deruxtecan (T-DXd))

[0199] (Preparation process, extraction process)

[0200] First, the following cancer tissues and preparation solutions were prepared.

[0201] Cancer tissues:

[0202] Cancer tissues of experimental model mice administered with T-DXd

[0203] (1) Tissue washing solution, (2) Inhibitory solution, and (4) Extraction solution used solutions with the same composition as in "Experiment 1: Quantification of antibodies present in tissue specimens".

[0204] (3) Internal standard IS solution:

[0205] Inhibitory solution

[0206] SILuMAb Trastuzumab (final concentration 0.25 μg / ml)

[0207] BSA (final concentration 0.1%)

[0208] Mouse IgG1 (clone 2545) (final concentration 50 μg / ml)

[0209] First, slice the above-mentioned cancer tissue using a microtome to prepare thin sections (5 μm thick). Transfer the above thin sections (10 mg) to a low-adsorption microtube and process them in the same manner as in "Experiment 1: Quantification of Antibodies Present in Tissue Specimens". Through the above steps, a specimen containing disrupted cancer tissue (homogenized biological specimen) is prepared (preparation step). The above biological specimen is a specimen derived from cancer cells. Subsequently, process it in the same manner as in "Experiment 1: Quantification of Antibodies Present in Tissue Specimens" to obtain a "bound antibody sample" (analytical specimen or first analytical specimen) and a "free antibody sample" (second analytical specimen) from the specimen containing the above disrupted cancer tissue.

[0210] (Quantification step)

[0211] Prepare the following preparation solutions.

[0212] (1) Standard dilution solution, (2) washing solution, (3) neutralization solution, and (4) reaction solution use the same solutions as in "Experiment 1: Quantification of Antibodies Present in Tissue Specimens".

[0213] (5) Initial mobile phase:

[0214] MilliQ water (0.1% acetic acid) + acetonitrile (volume ratio 99:1)

[0215] Prepare standard curve samples and QC samples with the following compositions.

[0216] Standard curve samples: All are prepared using the standard dilution solution.

[0217] Blank sample 0 ng / ml trastuzumab

[0218] Standard curve 1 250 ng / ml trastuzumab

[0219] Standard curve 2 500 ng / ml trastuzumab

[0220] Standard curve 3 1.00 μg / ml trastuzumab

[0221] Standard curve 4 2.00 μg / ml trastuzumab

[0222] Standard curve 5 4.00 μg / ml trastuzumab

[0223] Standard curve 6 8.0 μg / ml trastuzumab

[0224] Standard curve 7 16.0 μg / ml trastuzumab

[0225] Standard curve 8 32.0 μg / ml trastuzumab

[0226] QC samples: All were prepared by diluting with the standard solution.

[0227] LLOQ 250 ng / ml trastuzumab

[0228] LQC 750 ng / ml trastuzumab

[0229] MQC 2.50 μg / ml trastuzumab

[0230] HQC 12.0 μg / ml trastuzumab

[0231] HLOQ 32.0 μg / ml trastuzumab

[0232] Add 20 μL each of the above standard curve samples and QC samples to 160 μL of the matrix solution (a solution obtained by extracting interfering components from the measurement tissue using the suppression solution and extraction solution in the same manner as for the measurement sample), and then add 20 μL of the internal standard IS solution. These correspond to the "standard curve samples (with IS)" and "QC samples (with IS)" described below. In addition, in the preparation of the above standard curve samples, samples were also prepared by adding 20 μL of the matrix solution instead of 20 μL of the internal standard IS solution. This corresponds to the "standard curve samples (without IS)" described below.

[0233] The quantitative analysis of trastuzumab (antigen-binding molecule) in the analytical sample is performed by generating a trastuzumab-specific peptide (peptide derived from the variable region) using the nSMOL method and detecting the peptide using a mass spectrometry device. The specific steps are as follows.

[0234] (Generating the peptide using the nSMOL method)

[0235] Dispense 200 μl each of the binding antibody sample, standard curve samples (with or without IS), and QC samples (with IS) into low-binding microtubes, and then perform the same treatment as in "Experiment 1: Quantification of antibodies present in tissue samples". The trastuzumab-specific peptide (peptide derived from the variable region) is generated through the above steps.

[0236] (Detecting the peptide using a mass spectrometry device)

[0237] Quantify the above trastuzumab-specific peptide (sequence number 2: IYPTNGYTR (single-letter symbol)) using LCMS analysis under the conditions shown below.

[0238] List of prepared solutions:

[0239] Mobile phase A: MilliQ water (0.1% acetic acid) (LCMS grade)

[0240] Mobile phase B: Acetonitrile (LCMS grade)

[0241] LCMS analysis conditions:

[0242] Perform in the same manner as "Experiment 1: Quantification of Antibodies Present in Tissue Specimens".

[0243] HPLC conditions:

[0244] HPLC: Shimadzu Nexera X2

[0245] Flow rate: 0.4 ml / min

[0246] 0 - 1.5 minutes: 1% B

[0247] 1.5 - 5.5 minutes: 1 - 42% B gradient

[0248] 5.5 - 6.5 minutes: 95% B

[0249] 6.5 - 8.5 minutes: 1% B

[0250] LCMS analysis time: 2.5 - 6.5 minutes

[0251] LCMS MRM analysis transition conditions:

[0252] LCMS: Shimadzu LCMS - 8060

[0253] Trastuzumab, IYPTNGYTR

[0254] 542.90(++) > 404.80(y7++) for quantification

[0255] 542.90(+++) > 808.50(y7+) confirmation ion

[0256] 13C6,15N4 - Trastuzumab, 13C6,15N4 - IYPTNGYTR

[0257] 547.90(++) > 409.70(y7++) for quantification (Ab_IS)

[0258] 547.90(++) > 818.50(y7+) confirmation ion

[0259] (Results)

[0260] Figure 10 Show the results of LCMS analysis of the standard curve samples (blank) (with IS (second chart from the top) or without IS (first chart from the top)) and QC samples (LLOQ) (with IS (third chart from the top)) in the examples. By Figure 10From the results, it can be known that under the analysis conditions of the examples, trastuzumab can also be detected at a concentration of 250 ng / ml, similar to the examples of cetuximab.

[0261] The graph of the standard curve obtained from the analysis results of the standard curve samples (blank, standard curves 1 - 8) (with IS) in the examples is shown in Figure 11 . Figure 11 In it, the horizontal axis represents the concentration ratio of trastuzumab in each standard curve sample (based on the concentration of IS), and the vertical axis represents the ratio of the peak area of the analyte to the peak area of IS in mass spectrometry. Figure 11 The results show that trastuzumab can be quantified under the analysis conditions of the examples.

[0262] (Experiment 5: Quantification of the antibody part (trastuzumab) of ADC (T-DXd) bound to tissues in model tumors)

[0263] SW620 strain or OE-19 strain was subcutaneously transplanted into nude mice (BALB / c, female, 5 - 8 weeks old). Here, the SW620 strain is a cell line that does not express HER2 (the antigen of trastuzumab), and the OE-19 strain is a cell line that expresses HER2. The number of cells transplanted at this time was 1×10 7 cells per mouse. Normal feeding was carried out for 14 - 21 days until the transplanted cells survived. After that, T-Dxd (0 or 10 mg / Kg) was intravenously injected into the above mice. 24 hours after the intravenous injection, the transplanted cells (model tumors) were collected from the mice in the same way as in "Experiment 3: Quantification of antibodies bound to tissues in model tumors".

[0264] In addition, the collected model tumors were subjected to tissue staining with DAPI and anti-human IgG antibody ( Figure 12 , right side). In the model tumors collected from the mice transplanted with the OE-19 strain, the antibody part of T-DXd in the tissues administered with T-DXd was stained.

[0265] The collected model tumors were processed by the same method as in Experiment 1 to quantify the antibody part of T-DXd bound to the model tumors. The sample used for quantification was the sample corresponding to the "bound antibody sample" in Experiment 1. The results are shown in Figure 12 (left side). In the model tumors collected from the mice transplanted with the OE-19 strain, trastuzumab was detected only in the tissues administered with T-DXd. On the other hand, almost no trastuzumab was detected in the model tumors collected from the mice transplanted with the SW620 strain.

[0266] Using the sample corresponding to the "free antibody sample" in Experiment 4, the antibody part of T-DXd that exists in the model tumor but is not bound to HER2 was quantified (Figure 13 ., left). In the model tumors collected from the mice transplanted with the SW620 strain, the antibody portion of T-DXd equivalent to that in the model tumors transplanted with the OE-19 strain was also detected (where, for the value at the lower limit of quantification, calculation was performed by substituting half of the LLOQ (12.5 ng / ml)).

[0267] (Experiment 6: Extraction from cancer tissues administered with trastuzumab using the ACES method)

[0268] (Preparation process, extraction process)

[0269] First, prepare the following cancer tissues and preparation solutions.

[0270] Cancer tissue:

[0271] Cancer tissue of experimental model mice administered with trastuzumab

[0272] (1) Tissue washing solution, (2) Inhibitory solution, and (4) Extraction solution use solutions with the same composition as in "Experiment 1: Quantification of antibodies present in tissue specimens".

[0273] (3) Internal standard IS solution:

[0274] Inhibitory solution

[0275] SILuMAb trastuzumab (trastuzumab) (final concentration 0.25 μg / ml)

[0276] BSA (final concentration 0.1%)

[0277] Mouse IgG1 (clone 2545) (final concentration 50 μg / ml)

[0278] First, slice the above cancer tissue with a microtome to prepare thin sections (thickness 5 μm). Transfer the above thin sections (10 mg) to a low-adsorption microtube and process them in the same manner as in "Experiment 1: Quantification of antibodies present in tissue specimens". Through the above steps, prepare a specimen containing disrupted cancer tissue (homogenized biological specimen) (preparation process). The above biological specimen is a specimen derived from cancer cells. Thereafter, process it in the same manner as in "Experiment 1: Quantification of antibodies present in tissue specimens" to obtain a "bound antibody sample" (analytical specimen or first analytical specimen) and a "free antibody sample" (second analytical specimen) from the specimen containing disrupted cancer tissue.

[0279] (Quantification process)

[0280] Prepare the following preparation solutions.

[0281] (1) Standard dilution solution, (2) cleaning solution, (3) neutralization solution, and (4) reaction solution use the same solutions as in "Experiment 1: Quantification of Antibodies Present in Tissue Specimens".

[0282] (5) Initial mobile phase:

[0283] MilliQ water (0.1% acetic acid) + acetonitrile (volume ratio 99:1)

[0284] Prepare standard curve samples and QC samples with the following compositions.

[0285] Standard curve samples: All are prepared using the standard dilution solution.

[0286] Blank sample: 0 ng / ml trastuzumab

[0287] Standard curve 1: 250 ng / ml trastuzumab

[0288] Standard curve 2: 500 ng / ml trastuzumab

[0289] Standard curve 3: 1.00 μg / ml trastuzumab

[0290] Standard curve 4: 2.00 μg / ml trastuzumab

[0291] Standard curve 5: 4.00 μg / ml trastuzumab

[0292] Standard curve 6: 8.0 μg / ml trastuzumab

[0293] Standard curve 7: 16.0 μg / ml trastuzumab

[0294] Standard curve 8: 32.0 μg / ml trastuzumab

[0295] QC samples: All are prepared using the standard dilution solution.

[0296] LLOQ: 250 ng / ml trastuzumab

[0297] LQC: 750 ng / ml trastuzumab

[0298] MQC: 2.50 μg / ml trastuzumab

[0299] HQC: 12.0 μg / ml trastuzumab

[0300] HLOQ: 32.0 μg / ml trastuzumab

[0301] Add 20 μL each of the above standard curve samples and QC samples to 160 μL of a matrix solution (a solution obtained by subjecting interfering components derived from the measurement tissue to an extraction treatment using an inhibition solution and an extraction solution in the same manner as for the measurement sample), and then add 20 μL of an internal standard IS solution. These correspond to the "standard curve sample (with IS)" and "QC sample (with IS)" described later. In addition, in the preparation of the above standard curve samples, a sample was also prepared by adding 20 μL of the matrix solution in place of 20 μL of the internal standard IS solution. This corresponds to the "standard curve sample (without IS)" described later.

[0302] Quantitative analysis of trastuzumab (antigen-binding molecule) in the test sample for analysis is performed by generating a trastuzumab-specific peptide (peptide derived from the variable region) using the nSMOL method and detecting the peptide using a mass spectrometry device. The specific steps are as follows.

[0303] (Generating peptide using the nSMOL method)

[0304] Dispense 200 μl each of the binding antibody sample, standard curve sample (with or without IS), and QC sample (with or without IS) into low-adsorption microtubes, and then perform the same treatment as in "Experiment 1: Quantification of antibodies present in tissue samples". A trastuzumab-specific peptide (peptide derived from the variable region) is generated through the above steps.

[0305] (Detecting peptide using a mass spectrometry device)

[0306] For the above trastuzumab-specific peptide (SEQ ID NO: 2: IYPTNGYTR (single-letter code)), quantification is performed by LCMS analysis under the conditions shown below.

[0307] List of prepared solutions:

[0308] Mobile phase A: MilliQ water (0.1% acetic acid) (LCMS grade)

[0309] Mobile phase B: acetonitrile (LCMS grade)

[0310] LCMS analysis conditions:

[0311] Perform in the same manner as in "Experiment 1: Quantification of antibodies present in tissue samples".

[0312] HPLC conditions:

[0313] HPLC: Shimadzu Nexera X2

[0314] Flow rate: 0.4 ml / min

[0315] 0 - 1.5 minutes: 1% B

[0316] 1.5 - 5.5 minutes: 1 - 42% B gradient

[0317] 5.5 - 6.5 minutes: 95% B

[0318] 6.5 - 8.5 minutes: 1% B

[0319] LCMS analysis time: 2.5 - 6.5 minutes

[0320] LCMS MRM analysis transition conditions:

[0321] LCMS: Shimadzu LCMS - 8060

[0322] Trastuzumab, IYPTNGYTR

[0323] 542.90(++) > 404.80(y7++) for quantification

[0324] 542.90(+++) > 808.50(y7+) confirmation ion

[0325] 13C6,15N4 - Trastuzumab, 13C6,15N4 - IYPTNGYTR

[0326] 547.90(++) > 409.70(y7++) for quantification (Ab_IS)

[0327] 547.90(++) > 818.50(y7+) confirmation ion

[0328] (Results)

[0329] Figure 14 Shows the results of LCMS analysis of the standard curve samples (blank) (with IS (the second chart from the top) or without IS (the first chart from the top)) and QC samples (LLOQ) (with IS (the fourth chart from the top) or without IS (the third chart from the top)) in the examples. From Figure 14 the results, it can be seen that under the analysis conditions of the examples, similar to the examples of cetuximab, trastuzumab can also be detected at a concentration of 250 ng / ml.

[0330] A chart (not shown) of the standard curve obtained from the analysis results of the standard curve samples (blank, standard curves 1 - 8) (with IS) in the examples was made. The results of this chart indicate that trastuzumab can be quantified under the analysis conditions of the examples.

[0331] (Experiment 7: Quantification of trastuzumab bound to tissues in model tumors

[0332] Subcutaneously transplant the SW620 strain or the OE-19 strain into nude mice (BALB / c, female, 5 - 8 weeks old). Here, the SW620 strain is a cell line that does not express HER2 (the antigen of trastuzumab), and the OE-19 strain is a cell line that expresses HER2. At this time, the number of transplanted cells is 1×10 7 cells per mouse. Conduct routine feeding for 14 - 21 days until the transplanted cells survive. After that, intravenously inject trastuzumab (0, 10, or 20 mg / Kg) into the above-mentioned mice. 24 hours after the intravenous injection, collect the transplanted cells (model tumors) from the mice in the same manner as in "Experiment 3: Quantification of Antibodies Bound to Tissues in Model Tumors" ( Figure 15 , left side).

[0333] In addition, stain the collected model tumors with DAPI and anti-human IgG antibody ( Figure 16 , right side). In the model tumors collected from the mice transplanted with the OE-19 strain, trastuzumab is stained in the tissues administered with trastuzumab.

[0334] Process the collected model tumors in the same manner as in Experiment 1, and quantify the antibody portion of trastuzumab bound to the model tumor. The specimen used for quantification is the specimen corresponding to the "bound antibody sample" in Experiment 1. Show the results in Figure 16 (left side). In the model tumors collected from the mice transplanted with the OE-19 strain, trastuzumab is detected only in the tissues administered with trastuzumab. On the other hand, in the model tumors collected from the mice transplanted with the SW620 strain, almost no trastuzumab is detected.

[0335] Use the specimen corresponding to the "free antibody sample" in Experiment 6 to quantify the trastuzumab present in the model tumor but not bound to HER2 ( Figure 17 , left side). In the model tumors collected from the mice transplanted with the SW620 strain, trastuzumab equivalent to that in the model tumors transplanted with the OE-19 strain is also detected (wherein, for the value of the lower limit of quantification, calculate by substituting half of the LLOQ value (12.5 ng / ml)).

[0336] [Mode]

[0337] Those skilled in the art can understand that the above-mentioned multiple exemplary embodiments and examples are specific examples of the following modes.

[0338] (Item 1)

[0339] A method for quantifying an antigen-binding molecule of one type is a method for quantifying an antigen-binding molecule bound to cells, which includes a preparation step, an extraction step, and a quantification step. The preparation step prepares a biological sample derived from the cells, where the biological sample has been homogenized, the cells express a target antigen to which the antigen-binding molecule specifically binds, and the cells have been exposed to the antigen-binding molecule. The extraction step adds an extraction solution to the biological sample to obtain an analysis sample, where the extraction solution contains an organic acid and a non-ionic surfactant, and the pH of the extraction solution is 1 or more and 3 or less. The quantification step analyzes the analysis sample and quantifies the antigen-binding molecule. According to the quantification method described in item 1, a method for accurately quantifying an antigen-binding molecule bound to cells expressing a target antigen can be provided.

[0340] (Item 2)

[0341] In the quantification method described in item 1, the organic acid includes at least one selected from the group consisting of arginine, citrulline, and glycine. According to the quantification method described in item 2, the antigen-binding molecule can be extracted in a stable state.

[0342] (Item 3)

[0343] In the quantification method described in item 1 or item 2, the non-ionic surfactant includes alkyl glycoside. According to the quantification method described in item 3, the antigen-binding molecule can be extracted in a stable state.

[0344] (Item 4)

[0345] In the quantification method described in any one of items 1 to 3, the target antigen includes at least one selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, tumor necrosis factor receptor, insulin receptor, and vascular endothelial growth factor receptor. According to the quantification method described in item 4, an antigen-binding molecule bound to a specific target antigen can be accurately quantified.

[0346] (Item 5)

[0347] In the quantification method described in any one of items 1 to 4, the antigen-binding molecule includes an antibody or a peptide aptamer. According to the quantification method described in item 5, an antibody or a peptide aptamer can be accurately quantified.

[0348] (Item 6)

[0349] In the quantitative determination method according to item 5, the antigen-binding molecule comprises at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab and insulin analogs. According to the quantitative determination method according to item 6, the predetermined antibody can be quantified with good accuracy.

[0350] (Item 7)

[0351] In the quantification method according to any one of items 1 to 6, the cells are cells derived from cancer tissue or cells derived from lesion tissue. According to the quantification method according to item 7, the amount of antigen-binding molecules accumulated in cancer tissue or lesion tissue can be quantified with high accuracy.

[0352] (Item 8)

[0353] In the quantitative method according to any one of items 1 to 7, the antigen-binding molecule is an antibody, and the quantitative step comprises: generating a peptide derived from the variable region of the antibody by the nSMOL method; and analyzing the peptide by an LC-MS analyzer. According to the quantitative method according to item 8, the antibody can be quantified with higher accuracy.

[0354] Although the embodiments and examples of the present invention have been described above, it is intended from the outset that the configurations of the above-described embodiments and examples may be appropriately combined.

[0355] The embodiments and examples disclosed this time should be considered as illustrative in all aspects and not restrictive. The scope of the present invention is indicated by the claims rather than the embodiments and examples, and is intended to include all modifications within the meaning and scope equivalent to the claims.

Claims

1. A method for quantifying an antigen-binding molecule bound to a cell, comprising a preparation step, an extraction step, and a quantification step. The preparing step comprises preparing a biological sample derived from the cell, wherein: The biological sample is homogenized. The cell expresses the target antigen to which the antigen-binding molecule specifically binds, The cell has been exposed to the antigen-binding molecule; The extraction step adds an extraction solution to the biological sample to obtain an analytical sample, wherein: The extraction solution comprises an organic acid and a nonionic surfactant, The pH of the extract is 1 or more and 3 or less, The quantification step analyzes the analytical sample to quantify the antigen-binding molecule.

2. The quantitative method according to claim 1, wherein: The organic acid includes at least one selected from the group consisting of arginine, citrulline, and glycine.

3. The quantitative method according to claim 1, wherein The nonionic surfactant comprises an alkyl polyglycoside.

4. The quantitative method according to claim 1, wherein: The target antigen comprises at least one selected from the group consisting of EGFR, HER2, CD30, PD-L1, RANKL, tumor necrosis factor receptor, insulin receptor and vascular endothelial growth factor receptor.

5. The quantitative method according to claim 1, wherein The antigen-binding molecule comprises an antibody or a peptide aptamer.

6. The quantitative method according to claim 5, wherein: The antigen-binding molecule comprises at least one selected from the group consisting of cetuximab, trastuzumab, brentuximab, denosumab, infliximab, adalimumab, etanercept, aflibercept, ramucirumab, atezolizumab, avelumab, and insulin analogs.

7. The quantitative method according to claim 1, wherein: The cells are cells derived from cancer tissue or cells derived from diseased tissue.

8. The quantitative method according to claim 1, wherein: The antigen binding molecule is an antibody, The quantification step includes the steps of generating peptides derived from the variable region of the antibody by the nSMOL method and analyzing the peptides by an LC-MS analyzer.

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