Method for detecting anti-LAG-3 antibody drug receptor occupancy

Abstract

The present application relates to a method for detecting the receptor occupancy of an anti-LAG-3 antibody drug in a blood sample of a subject after administration of the anti-LAG-3 antibody drug.

Description

Technical Field

[0001] The present application relates to detecting the receptor occupancy of a drug. Specifically, the present application relates to detecting the receptor occupancy of an anti-LAG-3 antibody drug. Background Art

[0002] Receptor occupancy (RO) is an important research topic for evaluating the binding of drugs to receptors. Conducting receptor occupancy studies is an important part of clinical research. Receptor occupancy studies help verify the mechanism of action of drugs, select the best candidate drugs, and design the best dosage to ensure the safety and effectiveness of clinical research. However, this study also faces many technical and operational feasibility challenges. To establish an accurate and reproducible RO method, many factors need to be considered, such as: choosing the appropriate detection mode (direct or indirect method), detection reagents, sample type and target cell population, etc., and fully considering the impact of other factors such as receptor expression, receptor regulation and sample stability on the experimental results.

[0003] LAG-3 (lymphocyte activation gene-3) belongs to the immunoglobulin superfamily and is mainly expressed in activated T lymphocytes. By inhibiting the binding of LAG-3 and its ligand molecules, the activation and killing function of T cells can be promoted, and ultimately the immune system can effectively promote the killing of tumor cells.

[0004] Different from other antibody receptor occupancy studies, the receptor occupancy of anti-LAG-3 antibody drugs is very difficult. The challenges faced by this study mainly include that the target level expression in normal people is very low, the matrix interference is very large, and the variability of this type of cell-based biological analysis is large, resulting in very different results and inability to obtain effective data to establish a reliable method.

[0005] Currently, there is no report on a solution for LAG-3 target antibody drugs. Due to the difficulties of this type of method, many of these drugs cannot provide receptor occupancy data and cannot provide data support for early clinical safety and efficacy evaluation. Summary of the invention

[0006] In a first aspect, the present application provides a method for detecting the receptor occupancy of an anti-LAG-3 antibody drug in a blood sample of a subject after administration of the anti-LAG-3 antibody drug, the method comprising:

[0007] (1) obtaining a first leukocyte sample and a second leukocyte sample having substantially the same number of leukocytes from the blood sample, wherein the first leukocyte sample is used to obtain a test signal intensity, and the second leukocyte sample is used to obtain a total signal intensity; wherein:

[0008] Obtaining the test signal strength includes:

[0009] (a) adding a detectably labeled detection antibody for the anti-LAG-3 antibody drug to the first leukocyte sample, wherein the detection antibody is specific to the IgG subtype of the anti-LAG-3 antibody and binds to the constant region of the anti-LAG-3 antibody drug;

[0010] (b) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the first leukocyte sample to obtain the test signal intensity;

[0011] Obtaining the total signal strength includes:

[0012] (c) adding to the second leukocyte sample an amount of the anti-LAG-3 antibody drug sufficient to saturate binding to the LAG-3 target on the leukocytes in the second leukocyte sample;

[0013] (d) adding a detection antibody to the anti-LAG-3 antibody drug with a detectable label;

[0014] (e) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the second leukocyte sample to obtain the total signal intensity;

[0015] (2) comparing the test signal intensity with the total signal intensity to determine the receptor occupancy of the anti-LAG-3 antibody drug.

[0016] In some embodiments, the blood sample is a whole blood sample, and the method further comprises, before step (1), subjecting the whole blood sample to red blood cell lysis to obtain white blood cells.

[0017] In some embodiments, 1% ammonium chloride lysis solution is added at a volume ratio of 1:20 to 1:40 to perform red blood cell lysis treatment.

[0018] In some embodiments, 1% ammonium chloride lysis solution is added at a volume ratio of 1:30 to perform red blood cell lysis treatment.

[0019] In some embodiments, step (b) or step (e) is performed by flow cytometry.

[0020] In some embodiments, steps (a) to (b) are performed before steps (c) to (e), or steps (a) to (b) are performed after steps (c) to (e), or steps (a) to (b) are performed simultaneously with steps (c) to (e).

[0021] In some embodiments, the detectable label is a fluorescent labeling agent.

[0022] In some embodiments, the detectable label is AF647, FITC, BV421, or BV785.

[0023] In some embodiments, the anti-LAG-3 antibody drug is a non-IgG1 antibody drug.

[0024] In some embodiments, the anti-LAG-3 antibody drug is an IgG4 antibody drug, and the detection antibody of the anti-LAG-3 antibody drug with a detectable label is an anti-antibody of the IgG4 antibody conjugated to AF647.

[0025] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of CD3-positive T cells, CD8-negative T cells, or CD8-positive T cells is detected.

[0026] In some embodiments, the detection antibodies comprise 5 μg / mL of FITC-CD45, 0.5 ug / mL of BV421-CD3, 1 ug / mL of BV785-CD8, and 1.65 ug / mL of AF647-IgG4. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the detection principle diagram of this application;

[0028] Figure 2 The flow cytometry images after PBMC stimulation and culture. A is the flow cytometry image after 5 days of PBMC stimulation and culture; B is the flow cytometry image after 14 days of PBMC stimulation and culture;

[0029] Figure 3 Schematic diagram of sample preparation for verification. DETAILED DESCRIPTION

[0030] In response to the problem of receptor occupancy, after repeated exploration, the applicant established a new method for detecting receptor occupancy of anti-LAG-3 target monoclonal antibodies, completed methodological validation, and applied it to the evaluation of this type of drugs.

[0031] The present application utilizes flow cytometry to determine the occupancy rate of anti-LAG-3 antibody drugs binding to LAG-3 targets on the surface of human peripheral lymphocytes.

[0032] The principle of the receptor occupancy detection technology for anti-LAG-3 antibody drugs is as follows:

[0033] See also Figure 1, select whole blood samples at different time points before and after drug administration, and make two tubes (tube A and tube B) for each sample. Tube A is incubated with an excess of antibody drugs to ensure that all LAG-3 sites are occupied. It is used as a total tube to reflect the total receptor level (total signal); tube B is used as a binding tube to reflect the actual situation of receptor occupancy at different time points before and after drug administration (test signal). Tubes A and B are incubated with detection antibodies with detectable markers (e.g., AF647-IgG4; optionally, FITC-CD45, BV421-CD3 and / or BV786-CD8) to detect LAG-3 epitopes (binding epitopes) bound to antibody drugs. The average fluorescence intensity and percentage of detection antibodies with detectable markers (e.g., AF647-IgG4) in leukocytes (e.g., T cells) are detected by flow cytometry. The LAG-3 receptor occupancy of samples at different time points on T cells is calculated by a formula.

[0034] This study used a drug binding receptor assay format, and antibodies against IgG (eg, IgG4 type) were selected as detection antibodies.

[0035] The method of the present application optimizes the analytical conditions, selects a lower concentration of LAG-3 antibody, and shortens the incubation time of the detection antibody with a detectable label.

[0036] The method of the present application can not only evaluate the receptor occupancy of the anti-LAG-3 antibody drug in the blood sample of the subject after administration of the anti-LAG-3 antibody drug, but can also be used to evaluate the receptor occupancy of the anti-LAG-3 antibody drug during the research and development process of the anti-LAG-3 antibody drug.

[0037] In a first aspect, the present application provides a method for detecting the receptor occupancy of an anti-LAG-3 antibody drug in a blood sample of a subject after administration of the anti-LAG-3 antibody drug, the method comprising:

[0038] (1) obtaining a first leukocyte sample and a second leukocyte sample having substantially the same number of leukocytes from the blood sample, wherein the first leukocyte sample is used to obtain a test signal intensity, and the second leukocyte sample is used to obtain a total signal intensity; wherein:

[0039] Obtaining the test signal strength includes:

[0040] (a) adding a detectably labeled detection antibody for the anti-LAG-3 antibody drug to the first leukocyte sample, wherein the detection antibody is specific to the IgG subtype of the anti-LAG-3 antibody and binds to the constant region of the anti-LAG-3 antibody drug;

[0041] (b) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the first leukocyte sample to obtain the test signal intensity;

[0042] Obtaining the total signal strength includes:

[0043] (c) adding to the second leukocyte sample an amount of the anti-LAG-3 antibody drug sufficient to saturate binding to the LAG-3 target on the leukocytes in the second leukocyte sample;

[0044] (d) adding a detection antibody to the anti-LAG-3 antibody drug with a detectable label;

[0045] (e) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the second leukocyte sample to obtain the total signal intensity;

[0046] (2) comparing the test signal intensity with the total signal intensity to determine the receptor occupancy of the anti-LAG-3 antibody drug.

[0047] In some embodiments, the blood sample is a whole blood sample, and the method further comprises, before step (1), subjecting the whole blood sample to red blood cell lysis to obtain white blood cells.

[0048] In some embodiments, the blood sample is a whole blood sample.

[0049] In some embodiments, the method further comprises, before step (1), subjecting the whole blood sample to red blood cell lysis to obtain white blood cells.

[0050] In some embodiments, 1% ammonium chloride lysis solution is added at a volume ratio of 1:20 to 1:40 to perform red blood cell lysis treatment.

[0051] In some embodiments, 1% ammonium chloride lysis solution is added to perform red blood cell lysis treatment at a volume ratio of 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40 or any range therebetween.

[0052] In some embodiments, 1% ammonium chloride lysis solution is added at a volume ratio of 1:30 to perform red blood cell lysis treatment.

[0053] In some embodiments, step (b) or step (e) is performed by flow cytometry.

[0054] In some embodiments, step (b) is performed by flow cytometry.

[0055] In some embodiments, step (e) is performed by flow cytometry.

[0056] In some embodiments, step (b) and step (e) are performed by flow cytometry.

[0057] In some embodiments, flow cytometry may include multiple fluorescent labeling reagents for distinguishing different cells, such as FITC-CD45, BV421-CD3, BV650-CD19, BV785-CD8, AF647-CD14, and / or 7-AAD.

[0058] In some embodiments, steps (a) to (b) are performed prior to steps (c) to (e).

[0059] In some embodiments, steps (a) to (b) are performed after steps (c) to (e).

[0060] In some embodiments, steps (a) to (b) are performed simultaneously with steps (c) to (e).

[0061] In some embodiments, the detectable label is a fluorescent labeling agent.

[0062] In some embodiments, the detectable label is AF647, FITC, BV421, or BV785.

[0063] In some embodiments, the detection antibody of the anti-LAG-3 antibody drug with a detectable label is an anti-antibody of the IgG4 type antibody conjugated to AF647.

[0064] In some embodiments, the anti-LAG-3 antibody drug is a non-IgG1 antibody drug.

[0065] In some embodiments, the anti-LAG-3 antibody drug is an IgG4 antibody drug.

[0066] In some embodiments, the anti-LAG-3 antibody drug is an IgG4 antibody drug, and the detection antibody of the anti-LAG-3 antibody drug with a detectable label is an anti-antibody of the IgG4 antibody conjugated to AF647.

[0067] In some embodiments, the anti-LAG-3 antibody drug is ZK001. ZK001 is an anti-LAG-3 IgG4 antibody drug.

[0068] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of CD3-positive T cells, CD8-negative T cells, or CD8-positive T cells is detected.

[0069] T cells are detected using CD3-positive, helper T cells are detected using CD8-negative, and cytotoxic T cells are detected using CD8-positive.

[0070] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of at least one of CD3-positive T cells, CD8-negative T cells and CD8-positive T cells is detected.

[0071] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of at least two T cells among CD3-positive T cells, CD8-negative T cells and CD8-positive T cells is detected.

[0072] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as a detection unit, the signal intensity of the detectable marker of CD3-positive T cells, CD8-negative T cells, and CD8-positive T cells is detected.

[0073] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of the CD3-positive T cell is detected.

[0074] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of the CD8-negative T cell is detected.

[0075] In some embodiments, when the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of the CD8-positive T cell is detected.

[0076] In some embodiments, the test signal intensity is divided by the total signal intensity to determine receptor occupancy of the anti-LAG-3 antibody drug.

[0077] In some embodiments, the receptor occupancy of the anti-LAG-3 antibody drug is calculated as follows:

[0078]

[0079] In some embodiments, the detection antibodies comprise 5 μg / mL of FITC-CD45, 0.5 ug / mL of BV421-CD3, 1 ug / mL of BV785-CD8, and 1.65 ug / mL of AF647-IgG4. Example

[0080] The application will be described in more detail by way of specific examples. The following examples are provided for illustrative purposes only, and are not intended to limit the application in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially the same result.

[0081]

[0082]

[0083] Example 1: PBMC preparation

[0084] After obtaining healthy human PBMC cells, CD3 / CD28 monoclonal antibody-coupled magnetic beads were used to activate T cells in PBMC to obtain T cells with high expression of LAG-3, which facilitated the subsequent verification of the preparation of samples containing validation samples with high expression of LAG-3.

[0085] PBMC resuscitation

[0086] Preheat a water bath to 37°C, prepare a 15 mL sterile centrifuge tube, and add 5 mL PBS;

[0087] Take out the frozen PBMC from the liquid nitrogen tank, immediately put the frozen PBMC into a 37°C water bath, and gently shake the cryotube to completely thaw it within 60 to 120 seconds;

[0088] Transfer the thawed PBMCs to the prepared 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 minutes at room temperature;

[0089] Discard the supernatant, add 5 mL of PBS, and gently pipette to resuspend the cells to avoid bubbles; take 20 µL of the cell suspension in a centrifuge tube and count the cells using a cell analyzer (for example: take 20 µL of the cell suspension, add 20 µL of AOPI dye, pipette to mix, and take 20 µL of the mixture and add it to the well of the cell counting plate); record the cell viability, total cell count and number of live cells, and calculate the total number of live cells in the sample for later use.

[0090] CD3 / CD28 monoclonal antibody coupled to magnetic beads to activate T cells

[0091] Washing AMMS® CD3 / CD28 mAb-coupled magnetic beads:

[0092] Resuspend the magnetic beads (vortex for more than 30 seconds) and take out the required volume of magnetic beads into a 1.5 mL EP tube according to the ratio of 1:1 between magnetic beads and cells;

[0093] Add an equal volume of PBS buffer (containing 10% FBS) and resuspend;

[0094] Place the EP tube on the magnetic stand for 1 minute and discard the supernatant;

[0095] Transfer the EP tube from the magnetic stand and resuspend the magnetic beads with the same volume of PBS buffer (containing 10% FBS) (the initial volume of magnetic beads in the second step above);

[0096] The counted PBMC cells were centrifuged at room temperature, 1000 rpm, for 5 minutes, and the cell density was adjusted to 2 to 5×10 with PBS buffer (containing 10% FBS). 7 Pieces / mL;

[0097] Mix the magnetic beads and PBMC cells in a 1:1 ratio, place the sample on a shaker at 100-200 rpm / min, and incubate at room temperature for 30 minutes;

[0098] Dilute the mixture of magnetic beads and cells with PBS buffer (containing 10% FBS) to ensure that the sorting volume of the magnetic rack is 1mL (for example: if the volume of the suspension of cells and magnetic beads is 100μL, 900μL PBS buffer (containing 10% FBS) should be added). At the same time, place the centrifuge tube on the magnetic rack for 1 minute.

[0099] Discard the supernatant and resuspend the mixture of magnetic beads and cells in 1640 complete medium containing 1000 IU / mL IL-2, and adjust the cell density to 0.5×10 6 / mL to 1×10 6 Pieces / mL.

[0100] The cell suspension was placed in a 6-well cell culture plate, cultured in an incubator at 37°C and 5% CO2, and the cell status was observed.

[0101] See the culture results for Figure 2 The culture results showed that when PBMC was stimulated and cultured for 14 days, the expression level of LAG-3 was lower. When PBMC was stimulated and cultured for 5 days, the expression level of LAG-3 was better. In this embodiment, the final culture time was 4 to 5 days.

[0102] Take about 2×10 5 PBMCs were washed twice with 1 mL of flow cytometry buffer. After discarding the supernatant, 200 μL of flow cytometry buffer was added to each tube to resuspend the cells. The positive rate of LAG-3 positive / CD3 positive T lymphocytes in PBMCs was detected.

[0103] The positive rate of LAG-3 positive / CD3 positive T lymphocytes in PBMC was 14.2%.

[0104] Example 2: Whole blood sample preparation

[0105] Use BC-5100CRP fully automatic blood cell analyzer to count the number of cells in healthy human whole blood samples. Take at least 100 µL of whole blood into an EP tube, mix well and load the EP tube. Record the number of white blood cells and count the total number of white blood cells in the sample.

[0106] According to the cell counting results, add 1× red blood cell lysis buffer to lyse the corresponding amount of whole blood (100 μL whole blood + 3 mL 1× red blood cell lysis buffer) to obtain the required white blood cells, so that the number of white blood cells in each tube is about 1×10 6 Incubate at room temperature in the dark for about 10 minutes (12 ± 3 minutes). Centrifuge at 400g for 5 minutes at room temperature. Discard the supernatant, add 2 mL PBS to each tube to resuspend the cells, centrifuge at 400g for 5 minutes at room temperature, and discard the supernatant.

[0107] Example 3: Preparation and testing of verification samples

[0108] Material

[0109] Detection antibody preparation (per 100 μL system): 1 μL FITC-CD45, 1 μL BV421-CD3, 1 μL BV785-CD8, 3.3 μL AF647-IgG4 (1:10) and 93.7 μL flow buffer, of which 3.3 μL AF647-IgG4 (1:10) is the optimized antibody concentration.

[0110] The final concentrations of the detection antibodies were 5 μg / mL FITC-CD45, 0.5 ug / mL BV421-CD3, 1 ug / mL BV785-CD8, and 1.65 ug / mL AF647-IgG4.

[0111] During the sample processing process, the antibody incubation was performed at a low temperature of 2-8°C, and dead cells were removed by adding live and dead dyes. The optimized conditions greatly reduced nonspecific interference and background signals. The specific signal was achieved by combining the optimized AF647-IgG4 specific concentration with the LAG-3 drug.

[0112] Preparation of validation samples

[0113] Compared with healthy people, the expression of LAG-3 on the surface of T cells in diseased people (e.g., long-term exposure to pathological antigens) is enhanced. Therefore, in order to simulate the actual state of LAG-3 expression in the blood under diseased conditions, according to literature reports and the inventors' research results, the PBMCs stimulated with LAG-3-positive / CD3-positive T lymphocytes in Example 1 were added to the leukocytes obtained after lysis in Example 2 to obtain a mixed matrix of leukocytes and PBMCs, which was used as a verification sample (see Figure 3 ), and PBS was used to make up the volume and mixed evenly. In the final mixed matrix, the positive rate of LAG-3 positive / CD3 positive T lymphocytes was about 8.6% (the positive rate of LAG-3 positive / CD3 positive T lymphocytes in healthy people is about 1% or less than 1%).

[0114] Verification of sample testing

[0115] Prepare two 1.5 mL EP tubes, labeled as A and B, and add 100 µL of cell suspension (verification sample) to each EP tube. Tube A is used to obtain the total signal intensity; tube B is used to obtain the test signal intensity.

[0116] For the first experiment, prepare 6 more single-label tubes, labeled as "Blank", FITC-CD45, BV421-CD3, BV785-CD8, AF647-CD14, and 7-AAD. Add 100 µL of lysed white blood cells from whole blood to each tube; AF647-CD14 represents the single-stained tube of AF647, which is used to set voltage, compensation and other conditions when the machine is on; 7-AAD is used to distinguish dead cells from live cells.

[0117] After adding the verification sample, add 100 µL of ZK001 (100 μg / mL solution, anti-LAG-3 antibody drug, IgG4 antibody drug, the amount added is sufficient to saturate the binding of LAG-3 targets on leukocytes in the verification sample) to tube A, and add 100 µL of PBS to tube B; add 100 µL of PBS to each single-label tube, mix well, and incubate at 2 to 8°C for 30 minutes ± 5 minutes;

[0118] Add 1 mL of flow cytometry buffer to each tube, resuspend the cells, and centrifuge at 400 g for 5 minutes at 2 to 8°C;

[0119] After discarding the supernatant, add 1 mL of flow cytometry buffer to each tube to resuspend the cells and centrifuge at 400 g at 2 to 8°C for 5 minutes;

[0120] After discarding the supernatant, add 100 µL of detection antibody (i.e., detection antibody of anti-LAG-3 antibody drug with detectable label) to each tube of tube A and tube B respectively; do not add any reagent to the single-label tubes "Blank" and 7-AAD, add 1 µL FITC-CD45 to the "FITC-CD45" tube, add 1 µL BV421-CD3 to the "BV421-CD3" tube, add 1 µL BV785-CD8 to the "BV785-CD8" tube, and add 1 µL AF647-CD14 to the "AF647-CD14" tube, and incubate at 2-8°C in the dark for 30 ± 5 minutes;

[0121] Add 1 mL of flow cytometry buffer to each tube to resuspend the cells and centrifuge at 400 g for 5 minutes at 2 to 8°C;

[0122] After discarding the supernatant, add 1 mL of flow cytometry buffer to each tube to resuspend the cells and centrifuge at 400 g at 2 to 8°C for 5 minutes;

[0123] After discarding the supernatant, add 200 μL of flow cytometry buffer to each tube to resuspend the cells and store at 2 to 8°C until testing. Add 2.5 μL of 7-AAD to each sample tube and "7-AAD" single-label tube, incubate at room temperature in the dark for at least 5 minutes before flow cytometry detection.

[0124] For the first experiment, determine the voltage and light compensation based on the "blank" tube and the fluorescent channel adjustment tube, and set up the acquisition analysis template. Collect 100,000 CD3-positive T cells for each sample; if the number of cells is insufficient, collect the entire volume (200 µL). Complete the flow cytometer test and record the completion time.

[0125] RO calculation

[0126] After the flow cytometer detection was completed, the analysis template obtained by setting the gate was obtained using BD FACS Diva software, single cells were selected using FSC-A / FSC-H, singlets were excluded, live cell populations (Live) were gated from Singlets using 7-AAD-A / SSC-A, lymphocyte populations (LYM) were gated from Live using CD45-A / SSC-A, CD3-positive T cell populations were gated from LYM, CD8-negative T and CD8-positive T cell populations were gated from CD3-positive, AF647-IgG-positive cell populations were circled under CD3-positive T cell populations, CD8-negative T cell populations, and CD8-positive T cell populations; the average fluorescence intensity of AF647-IgG of CD3-positive cell populations, CD8-negative cell populations, and CD8-positive cell populations and the percentages of AF647-IgG-positive cell populations under CD3-positive, CD8-negative, and CD8-positive were obtained, and the data were processed by Microsoft Excel 2013.

[0127] Receptor occupancy calculation

[0128] The receptor occupancy calculation formula for anti-LAG-3 antibody drugs (taking ZK001 as an example) is as follows:

[0129] MFI average calculation formula:

[0130]

[0131] Percentage calculation formula:

[0132]

[0133] Drug titer linearity

[0134] Samples 1 to 5 are samples of whole blood added to stimulated PBMC (i.e., validation samples) with different gradients of drug ZK001 added (i.e., blood samples of subjects simulating administration of different concentrations of anti-LAG-3 antibody drugs), and the preparations simulate the binding of different concentrations of ZK001 to cells. By detecting the RO value of MFI (mean fluorescence intensity) and the percentage of RO, it can be seen that the RO value increases in a concentration-dependent manner with the increase of drug concentration. The results are shown in Table 3.

[0135]

[0136] Table 3 shows the results of drug titer linearity test. T cells were tested using CD3 positive; helper T cells were tested using CD8 antibody negative selection, i.e. CD8 negative; and cytotoxic T cells were tested using CD8 positive. As shown in Table 3, as the drug concentration continued to decrease, the RO% of CD3 positive cells continued to decrease, and CD8 positive cells and CD8 negative cells had similar trends.

[0137] Example 4: Run-to-run precision testing

[0138] Samples 6 to 8 are samples of stimulated PBMCs prepared with a certain amount of ZK001 in whole blood (i.e., validation samples, different batches from Example 3), and blood samples of subjects after administration of different concentrations of anti-LAG-3 antibody drugs were simulated by adding different gradients of drug ZK001. Each precision analysis includes 3 concentration gradients: sample 6 (containing about 5.0 μg / mL of ZK001), sample 7 (containing about 0.5 μg / mL of ZK001) and sample 8 (containing about 0.0 μg / mL of ZK001), and each validation sample is repeated 3 times. It is concluded that the inter-run precision results meet the validation acceptance criteria, and the precision (CV%, coefficient of variation) should not be greater than 30%.

[0139] The analysis of inter-run precision is shown in Tables 4 and 5. T cells were detected using CD3 positive selection; helper T cells were negatively selected using CD8 antibody, i.e., CD8 negative; and cytotoxic T cells were detected using CD8 positive selection.

[0140] Table 4 shows the statistical summary of the RO values ​​calculated using the mean fluorescence intensity. The precision experiment was performed by three analysts who completed three independent analysis runs in one day with different time periods: long, medium and short. Each analyst completed one analysis run.

[0141] Table 5 shows a statistical summary of RO values ​​calculated using percentage results, the operation is equivalent to Table 4.

[0142]

[0143]

[0144] The LAG-3 target is expressed at very low levels on healthy human lymphocytes, making detection very difficult. This application uses stimulated PBMCs to highly express LAG-3 and then added to whole blood to simulate samples of high-expression LAG-3 in patient whole blood. At the same time, to avoid nonspecific interference, the sample processing steps are all completed at low temperatures, and antibodies specific to the LAG-3 IgG4 subtype are used to establish a linear range of 0.01~5 μg / mL, with a sensitivity of 0.01 μg / mL drug concentration level.

[0145] The method of the present application avoids data variation caused by inconsistent total receptor expression such as up-regulation or down-regulation of expression in actual patient sample detection. The present application establishes an accurate and reliable method for detecting receptor occupancy of anti-LAG-3 antibody drugs. The method can well solve the influence of factors such as low LAG-3 target level and large endogenous interference on receptor occupancy detection, thereby accurately obtaining receptor occupancy data.

Claims

1. A method for detecting the receptor occupancy of an anti-LAG-3 antibody drug in a blood sample of a subject after administration of the anti-LAG-3 antibody drug, the method comprising: (1) obtaining a first leukocyte sample and a second leukocyte sample having substantially the same number of leukocytes from the blood sample, wherein the first leukocyte sample is used to obtain a test signal intensity, and the second leukocyte sample is used to obtain a total signal intensity; wherein: Obtaining the test signal strength includes: (a) adding a detectably labeled detection antibody for the anti-LAG-3 antibody drug to the first leukocyte sample, wherein the detection antibody is specific to the IgG subtype of the anti-LAG-3 antibody and binds to the constant region of the anti-LAG-3 antibody drug; (b) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the first leukocyte sample to obtain the test signal intensity; Obtaining the total signal strength includes: (c) adding to the second leukocyte sample an amount of the anti-LAG-3 antibody drug sufficient to saturate binding to the LAG-3 target on the leukocytes in the second leukocyte sample; (d) adding a detection antibody to the anti-LAG-3 antibody drug with a detectable label; (e) using a single cell as a detection unit, detecting the signal intensity of the detectable marker of the second leukocyte sample to obtain the total signal intensity; (2) comparing the test signal intensity with the total signal intensity to determine the receptor occupancy of the anti-LAG-3 antibody drug; wherein the anti-LAG-3 antibody drug is an IgG4 antibody drug; and When the signal intensity of the detectable marker of the leukocyte sample is detected using a single cell as the detection unit, the signal intensity of the detectable marker of CD3-positive T cells, CD8-negative T cells or CD8-positive T cells is detected.

2. The method of claim 1, wherein the blood sample is a whole blood sample, and the method further comprises, before step (1), subjecting the whole blood sample to a red blood cell lysis treatment to obtain white blood cells.

3. The method of claim 2, wherein 1% ammonium chloride lysis solution is added at a volume ratio of 1:20 to 1:40 to perform red blood cell lysis treatment.

4. The method of claim 2, wherein 1% ammonium chloride lysing solution is added at a volume ratio of 1:30 to perform red blood cell lysis treatment.

5. The method of claim 1, wherein step (b) or step (e) is performed by flow cytometry. The method of claim 1 , wherein the detectable label is a fluorescent labeling agent.

7. The method of claim 1, wherein the detectable label is AF647, FITC, BV421 or BV785.

8. The method of claim 1, wherein: The detection antibody of the anti-LAG-3 antibody drug with a detectable label is an anti-antibody of the IgG4 antibody conjugated to AF647.

9. The method of claim 1, wherein the detection antibodies comprise 5 μg / mL of FITC-CD45, 0.5 ug / mL of BV421-CD3, 1 ug / mL of BV785-CD8, and 1.65 ug / mL of AF647-IgG4.

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