CN116482345A - Method for detecting occupancy of dual-target drug receptor - Google Patents

Method for detecting occupancy of dual-target drug receptor Download PDF

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CN116482345A
CN116482345A CN202310722864.3A CN202310722864A CN116482345A CN 116482345 A CN116482345 A CN 116482345A CN 202310722864 A CN202310722864 A CN 202310722864A CN 116482345 A CN116482345 A CN 116482345A
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target
binding moiety
detection reagent
drug
dual
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CN116482345B (en
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刘立月
刘运龙
欧伦
邹微微
武丽华
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Junke Zhengyuan Tianjin Biomedical Technology Co ltd
United Power Pharma Tech Co ltd
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United Power Pharma Tech Co ltd
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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Abstract

The present application provides methods for detecting first target receptor occupancy of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell.

Description

Method for detecting occupancy of dual-target drug receptor
Technical Field
The application belongs to the field of biological analysis and mainly relates to a method for detecting the occupancy of a first target receptor of a double-target drug.
Background
Receptor occupancy (Receptor Occupancy, RO) is the degree of drug binding to a cell surface target and is the most direct Pharmacodynamic (PD) index for evaluating the level of targeted drug target action. In preclinical studies, RO can reflect the relationship between drug concentration and drug efficacy, assessing the minimal biological effects of biological agents, and thus making dose and dosing regimen settings. In safety assessment studies, the binding of drug to target may be different from that in humans, RO may assess the binding level of drug to target in a xenogeneic animal and drug-resistant antibodies (ADA) are more readily produced in the xenogeneic animal, so detection of RO may assess the effect of ADA on drug efficacy and may also assess the relationship between high and sustained receptor occupancy and toxic side effects. In pharmacodynamic studies, RO is often combined with PK for model building to adjust experimental design, dose selection, and the like. Therefore, measurement of the receptor occupancy is receiving increasing attention and importance.
There are two basic detection modes of RO, namely binding mode (Bound) and Free mode (Free). Binding mode detection of receptor levels occupied by drug, i.e. fluorescence labeling or biotin labeling of non-competitive anti-drug antibodies as detection antibodies, for monoclonal antibodies, species antibodies may also be used, total receptor levels are detected by adding excess drug to the sample, receptor occupancy (%) = bound drug amount/total drug amount x 100%. The free mode detects the level of receptor unoccupied by the drug, the fluorescence-labeled drug is adopted, or an anti-target antibody with absolute competition relation with the drug is adopted, the free receptor level is detected, the total receptor level can be obtained through a sample before administration, and also can be obtained through an anti-target antibody with non-competition relation with the drug, and the receptor occupancy (%) = 1-free receptor/total receptor multiplied by 100%. The calculation of receptor occupancy is generally based on fluorescence Median (MFI) or positive rate. The mode is selected according to the specific circumstances, such as the nature of the receptor, the mechanism of action of the drug, the availability of the detection reagent, the affinity of the anti-drug antibody, the neutralizing activity of the neutralizing active antibody, etc. In addition, RO detection method development also needs to consider many other factors, such as the number of target cell subsets, which if small increases the difficulty of method development; and the abundance of expression of the target antigen, the robustness of the method can also be challenging if the abundance of expression is not high. Therefore, in the development of the RO method, various detection modes need to be tried, and various optimization and verification are performed to ensure the robustness and reliability of the method.
Development and application of double-target drugs (such as double-anti-drugs and antibody ligand protein drugs) are becoming more and more important, so that development of an RO detection method for the double-target drugs has important significance.
Disclosure of Invention
In a first aspect, the present application provides a method for detecting occupancy of a first target receptor of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to the first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell, the method comprising:
adding the dual-target drug to a whole blood sample at one or more concentrations for incubation;
adding a first detection reagent bearing a detectable label that binds to the second binding moiety, the second binding moiety having an affinity for the first detection reagent sufficient to block or abstract binding of the second binding moiety to the second target;
detecting the signal intensity of the detectable label of the immune cells expressing the first target point by taking single cells as detection units, and calculating one or more test medians corresponding to the one or more concentrations based on the signal intensity values of the respective cells;
determining the first target receptor occupancy of the dual-target agent by comparing the one or more test medians to a total signal intensity median, wherein the total signal intensity median is the median determined by the above steps under conditions in which the dual-target agent is added in an amount sufficient to saturation bind to the first target on the immune cell.
In some embodiments of the first aspect, the first target is CD40; and/or the second target is CD137.
In some embodiments of the first aspect, the immune cell expressing the first target is a B cell; the first binding moiety is a ligand for CD40 or a fragment thereof; and/or the second binding moiety is an anti-CD 137 antibody.
In some embodiments of the first aspect, the first detection reagent is a neutralizing active antibody to an anti-CD 137 antibody; the concentration of the first detection reagent is 100 mug/mL; the detectable label is a biotin label or a fluorescent label; and/or the signal intensity is a fluorescent signal intensity.
In some embodiments of the first aspect, the method further comprises: after the incubation of the double-target drug and the whole blood sample is finished, adding erythrocyte lysate to carry out erythrocyte lysis; and/or after completion of erythrocyte lysis, the cell pellet is collected by centrifugation.
In some embodiments of the first aspect, the volume of the red blood cell lysate is 25-30 times the volume of the whole blood.
In some embodiments of the first aspect, the method further comprises adding a second detection reagent.
In some embodiments of the first aspect, the second detection reagent comprises PE-labeled streptavidin.
In some embodiments of the first aspect, the affinity of the second binding moiety for the first detection reagent is 5000-20000 times higher than the affinity of the second binding moiety for the second target.
In a second aspect, the present application provides a kit for detecting occupancy of a first target receptor of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to the first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell, the kit comprising: a first detection reagent with a detectable label bound to the second binding moiety;
wherein the affinity of the second binding moiety to the first detection reagent is sufficient to block or abstract binding of the second binding moiety to the second target.
In some embodiments of the second aspect, the kit further comprises instructions for use.
In some embodiments of the second aspect, the first target is CD40; and/or the second target is CD137.
In some embodiments of the second aspect, the immune cell expressing the first target is a B cell; the first binding moiety is a ligand for CD40 or a fragment thereof; and/or the second binding moiety is an anti-CD 137 antibody.
In some embodiments of the second aspect, the first detection reagent is a neutralizing active antibody to an anti-CD 137 antibody.
In some embodiments of the second aspect, the detectable label is a biotin label or a fluorescent label.
In some embodiments of the second aspect, the kit further comprises a red blood cell lysate, and/or a second detection reagent.
In some embodiments of the second aspect, the second detection reagent comprises PE-labeled streptavidin.
In some embodiments of the second aspect, the affinity of the second binding moiety for the first detection reagent is 5000-20000 times higher than the affinity of the second binding moiety for the second target.
Drawings
FIG. 1 is a schematic diagram of first target receptor occupancy detection of a dual-target drug. Wherein A is the binding receptor level of the first target of the dual-target drug and B is the total receptor level of the first target of the dual-target drug;
FIG. 2 is a schematic representation of a loop gate strategy for detecting first target receptor occupancy of a dual-target drug using a flow cytometer. Wherein A-F represent total cell population, single cell, lymphocyte population, CD3, respectively - CD19 + Cell population, CD3 - CD19 + Cell population PE-A positive signal peak and CD3 - CD19 + Cell population PE-A positive signal spots.
Detailed Description
For dual-target drugs (e.g., diabody drugs and antibody ligand fusion protein drugs), the development of RO detection methods faces greater challenges. Firstly, the action mechanism and the molecular structure of the double-target drug are more complex, and a proper mode is required to be selected by combining various factors; secondly, more than two target cell groups and target antigens exist in the system at the same time, interference exists between the two target cell groups and the target antigens, after medicines and detection antibodies are added, the binding reaction in the system is more complex, the binding balance is easier to break, and the robustness of the method faces challenges. Particularly when a labeled drug is used as a detection reagent, the affinity of the labeled drug has high requirements, and often it is difficult for a detection reagent with low affinity to achieve the required sensitivity and detection linearity range. The affinity of the ligand end in a dual-target drug, particularly an antibody ligand fusion protein drug, is often weak, so that the determination of the ligand end receptor occupancy of the drug is often difficult.
The inventors of the present application have developed a method for detecting the receptor occupancy of a dual-target drug (e.g., an antibody ligand fusion protein drug) through research and exploration.
The practice of the present application employs, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry and immunology techniques which are conventional in the art.
For ease of understanding the present application, certain terms used herein are first defined.
As used herein, an "antibody ligand fusion protein drug" comprises a first binding moiety that targets a first target and a second binding moiety that targets a second target, wherein the first binding moiety is a ligand of CD40 or a fragment thereof, and the second binding moiety is an anti-CD 137 antibody. In some embodiments, the first binding moiety is an extracellular domain sequence of a ligand of CD40 (CD 40L), and the second binding moiety is an Fab fragment of anti-CD 137.
As used herein, "binding receptor level" refers to the addition of different concentrations of a dual-target drug (e.g., an antibody ligand fusion protein drug) comprising a first binding moiety (e.g., extracellular domain sequence of CD 40L) targeting a first target (e.g., CD 40L) and a second binding moiety (e.g., fab fragment of anti-CD 137) targeting a second target (e.g., CD 137) to a whole blood sample for incubation to mimic clinical administration, the addition of a drug with a detectable labelA first detection reagent (e.g., a neutralizing active antibody to a biotin-labeled anti-CD 137 antibody), followed by addition of a second detection reagent (e.g., a second detection reagent comprising PE-labeled streptavidin), detection of the target cell population (e.g., CD 3) by a flow cytometer via binding of the detectable label of the first detection reagent to the second detection reagent (e.g., binding of biotin to PE-labeled streptavidin) - CD19 + B cells) as binding receptor levels (e.g., expressed in fluorescence Median (MFI) (see fig. 1A).
As used herein, "total receptor level" refers to the addition of an excess of a dual-target drug (e.g., an antibody ligand fusion protein drug) comprising a first binding moiety (e.g., extracellular domain sequence of CD 40L) targeting a first target (e.g., CD 40) and a second binding moiety (e.g., fab fragment of anti-CD 137) targeting a second target (e.g., CD 137) to a whole blood sample such that all of the first target (e.g., CD 40) is occupied by the drug, as well as the sequential addition of a first detection reagent (e.g., a neutralizing active antibody of a biotin-labeled anti-CD 137 antibody) and a second detection reagent (e.g., a second detection reagent comprising PE-labeled streptavidin) with a detectable label for detection of total receptor level (e.g., expressed in MFI) (see fig. 1B).
In some embodiments, the calculation of receptor occupancy is performed as fluorescence Median (MFI), receptor occupancy (%) = bound receptor MFI value/total receptor MFI value x 100%.
In a first aspect, the present application provides a method for detecting occupancy of a first target receptor of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to the first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell, the method comprising:
adding the dual-target drug to a whole blood sample at one or more concentrations for incubation;
adding a first detection reagent bearing a detectable label that binds to the second binding moiety, the second binding moiety having an affinity for the first detection reagent sufficient to block or abstract binding of the second binding moiety to the second target;
detecting the signal intensity of the detectable label of the immune cells expressing the first target point by taking single cells as detection units, and calculating one or more test medians corresponding to the one or more concentrations based on the signal intensity values of the respective cells;
determining the first target receptor occupancy of the dual-target agent by comparing the one or more test medians to a total signal intensity median, wherein the total signal intensity median is the median determined by the above steps under conditions in which the dual-target agent is added in an amount sufficient to saturation bind to the first target on the immune cell.
In some embodiments of the first aspect, the first target is CD40.
In some embodiments of the first aspect, the second target is CD137.
In some embodiments of the first aspect, the immune cell expressing the first target is a B cell, e.g., CD3 - CD19 + B cells.
In some embodiments of the first aspect, the immune cell expressing the second target is a T cell, a natural killer cell, a dendritic cell, or the like.
In some embodiments of the first aspect, the first binding moiety is a ligand of CD40 or a fragment thereof, e.g., the first binding moiety is an extracellular domain sequence of a ligand of CD40 (CD 40L).
In some embodiments of the first aspect, the second binding moiety is an anti-CD 137 antibody, e.g., an Fab fragment of anti-CD 137.
In some embodiments of the first aspect, the first detection reagent is a neutralizing active antibody to an anti-CD 137 antibody (e.g., an Fab fragment of anti-CD 137).
In some embodiments of the first aspect, the concentration of the first detection reagent is 100 μg/mL.
In some embodiments of the first aspect, the detectable label is a biotin label.
In some embodiments of the first aspect, the detectable label is a fluorescent label, such as fluorescein isothiocyanate, tetrachlorofluorescein, hydroxyfluorescein, rhodamine, cyanine dyes, and the like.
In some embodiments of the first aspect, the signal intensity is a fluorescent signal intensity (e.g., a fluorescent signal of PE). In some embodiments, the signal intensity is detected by a flow cytometer.
In some embodiments of the first aspect, the method further comprises adding a red blood cell lysate to perform red blood cell lysis after the dual-target drug has been incubated with the whole blood sample.
In some embodiments of the first aspect, the method further comprises centrifuging to collect a cell pellet after completion of erythrocyte lysis.
In some embodiments of the first aspect, the volume of the red blood cell lysate is 25-30 times the volume of the whole blood, e.g., 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30 times, or a value or range between any two of the foregoing.
In some specific embodiments of the first aspect, the volume of the red blood cell lysate is 25 times or 30 times the volume of the whole blood.
In some embodiments of the first aspect, centrifugation (e.g., 400×g,5 min) is performed after incubation with the addition of the red blood cell lysate, and if red blood cells remain at the bottom, the lysis step is repeated once. The erythrocyte lysis method improves the quality of cell suspension, greatly reduces the background and improves the signal to noise ratio.
In some embodiments of the first aspect, the red blood cell lysate comprises the following components: NH (NH) 4 Cl、KHCO 3 EDTA and ultrapure water. In some embodiments, the 10 x red blood cell lysate is formulated as follows: 8.99g NH 4 Cl；1.00g KHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the 37.0mg EDTA;100mL of ultrapure water. In some embodiments, 10-fold dilution prior to use is 1 x red blood cell lysate.
In some embodiments of the first aspect, the method further comprises adding a second detection reagent to detect the detectably labeled first detection reagent.
In some embodiments of the first aspect, the second detection reagent comprises PE-labeled streptavidin. In some embodiments, the fluorescence intensity of the PE linked to streptavidin is detected by flow cytometry by pairing biotin with streptavidin and the median fluorescence intensity is calculated.
In some embodiments of the first aspect, the median is the median of fluorescence intensities.
In some embodiments of the first aspect, the affinity of the second binding moiety for the first detection reagent is 5000-20000 times higher than the affinity of the second binding moiety for the second target, e.g., 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 times, or a value or range between any two of the foregoing.
In some embodiments of the first aspect, the affinity of the second binding moiety for the first detection reagent is 10000-fold higher than the affinity of the second binding moiety for the second target.
In some embodiments of the first aspect, the detection concentration of the dual-target agent is 0.4-30 μg/mL, such as 0.4, 0.625, 0.8, 1.0, 1.25, 2.5, 3.5, 4.5, 5, 10, 15, 20, 25, 30 μg/mL, or a value or range between any two of the foregoing.
In some specific embodiments of the first aspect, the method for detecting first target receptor occupancy of a dual-target drug comprises:
adding the dual-target drug to a whole blood sample at one or more concentrations for incubation;
after the incubation is completed, adding erythrocyte lysate (for example, erythrocyte lysate which is 25-30 times of the whole blood volume) for lysis;
after the erythrocyte lysis is finished, centrifugally collecting cell sediment;
adding to the cell pellet a detectably labeled first detection reagent (e.g., a biotin-labeled neutralizing active antibody to an anti-CD 137 antibody) that binds to the second binding moiety with an affinity sufficient to block or abstract binding incubation of the second binding moiety to the second target;
adding a second detection reagent (e.g., a second detection reagent comprising PE streptavidin) for incubation;
detecting immune cells (e.g., CD 3) expressing the first target (e.g., CD 40) using the single cells as detection units - CD19 + B cells) and calculating one or more test medians (e.g., fluorescence medians) corresponding to the one or more concentrations based on the signal intensity values of the respective cells;
determining the first target (e.g., CD 40) receptor occupancy of the dual-target agent by comparing the one or more test medians to a total signal intensity median, wherein the total signal intensity median is the median (e.g., fluorescence median) determined by the above steps under conditions in which the amount of dual-target agent added is sufficient to saturation bind to the first target on an immune cell.
In a second aspect, the present application provides a kit for detecting occupancy of a first target receptor of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to the first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell, the kit comprising: a first detection reagent with a detectable label bound to the second binding moiety;
wherein the affinity of the second binding moiety to the first detection reagent is sufficient to block or abstract binding of the second binding moiety to the second target.
In some embodiments of the second aspect, the kit further comprises instructions for use.
In some embodiments of the second aspect, the kit further comprises a red blood cell lysate.
In some embodiments of the second aspect, the detectable label is a biotin label.
In some embodiments of the first aspect, the detectable label is a fluorescent label, such as fluorescein isothiocyanate, tetrachlorofluorescein, hydroxyfluorescein, rhodamine, cyanine dyes, and the like.
In some embodiments of the second aspect, the kit further comprises a second detection reagent.
In some embodiments of the second aspect, the second detection reagent comprises PE-labeled streptavidin.
In some embodiments of the second aspect, the first target is CD40.
In some embodiments of the second aspect, the second target is CD137.
In some embodiments of the second aspect, the first binding moiety is a ligand of CD40 or a fragment thereof, e.g., the first binding moiety is an extracellular domain sequence of a ligand of CD40 (CD 40L).
In some embodiments of the second aspect, the second binding moiety is an anti-CD 137 antibody, e.g., an Fab fragment of anti-CD 137.
In some embodiments of the second aspect, the first detection reagent is a neutralizing active antibody to an anti-CD 137 antibody.
In some embodiments of the second aspect, the concentration of the first detection reagent may be 100 μg/mL.
In some embodiments of the second aspect, the immune cell expressing the first target is a B cell, e.g., CD3 - CD19 + B cells.
In some embodiments of the second aspect, the immune cells expressing the second target are T cells, natural killer cells, dendritic cells, and the like.
In some embodiments of the second aspect, the affinity of the second binding moiety for the first detection reagent is 5000-20000 times higher than the affinity of the second binding moiety for the second target, e.g., 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000 times, or a value or range between any two of the foregoing.
In some embodiments of the second aspect, the affinity of the second binding moiety for the first detection reagent is 10000 times higher than the affinity of the second binding moiety for the second target.
The application establishes a robust detection method for the receptor occupancy of CD40L, and the detection mode adopted by the method is a combination mode, and experiments prove that the combination mode is superior to a free mode. The principle is explained as follows: the whole blood sample is incubated with different concentrations of drug to simulate clinical administration, a biotin-labeled neutralizing antibody against a second binding moiety (e.g., anti-CD 137 antibody) is added, followed by PE-labeled streptavidin, which binds to biotin, and the target cell population (e.g., CD 3) is detected by flow cytometry - CD19 + B cells) as binding receptor levels (e.g., expressed as fluorescence Median (MFI) (see fig. 1A); adding an excess of drug to the whole blood sample such that all of the first target (e.g., CD 40) is occupied by the drug, and likewise sequentially adding a biotin-labeled neutralizing antibody against the second binding moiety (e.g., an antibody against CD 137) and PE-labeled streptavidin for detection of total receptor levels (e.g., expressed as MFI) (see fig. 1B); the calculation of receptor occupancy was performed as fluorescence Median (MFI), receptor occupancy (%) = bound receptor MFI value/total receptor MFI value x 100%.
The present application provides methods for detecting receptor occupancy for dual-target drugs, particularly antibody ligand fusion protein drugs.
The application adopts a combination mode to detect, and has at least one of the following advantages: good linearity, high sensitivity (drug concentration of 0.4. Mu.g/mL) and wide detection range (drug concentration of 0.4-30. Mu.g/mL).
The application uses the biotin-labeled neutralizing active antibody of the second binding site (such as an anti-CD 137 antibody) as a first detection reagent, wherein the affinity of the neutralizing active antibody with the second binding site (such as an anti-CD 137 antibody) is enough to block or abstract the binding (10000 times higher affinity) of the second binding site (such as an anti-CD 137 antibody) with the second target (such as CD 137), so that interference caused by another target is avoided in the detection process, and the accuracy and stability of the method are improved.
The application adopts the same detection antibody in the detection of the combined receptor and the total receptor, so that the data has direct comparability, and the data variation caused by factors such as affinity difference and the like by adopting different detection reagents is avoided.
The method uses the biotin-labeled neutralizing active antibody as a first detection reagent and then uses PE-labeled streptavidin as a second detection reagent, so that the detection signal is amplified for the second time, and higher sensitivity is achieved.
It should be understood that the foregoing detailed description is only for the purpose of making the contents of the present application more clearly apparent to those skilled in the art, and is not intended to be limiting in any way. Various modifications and changes to the described embodiments will occur to those skilled in the art.
The following examples are for the purpose of illustration only and are not intended to limit the scope of the present application.
Examples
An antibody ligand fusion protein drug comprising the extracellular domain of CD40L and a CD 137-targeting antibody Fab fragment and a neutralizing active antibody as a first detection reagent was prepared in the laboratory of the present inventors. Wherein the binding of the neutralizing active antibody prepared as the first detection reagent to the CD 137-targeting antibody Fab fragment is sufficient to block or abstract the binding of the CD 137-targeting antibody Fab fragment to the CD137 expressed by the cell.
Example 1 determination of the concentration of the first detection reagent
The first detection reagent neutralization activity antibody concentration of the detection method of CD40 receptor occupancy of the antibody ligand fusion protein drug comprising the extracellular domain of CD40L and the antibody Fab fragment targeting CD137 is determined as follows:
1. adding excessive medicine for incubation: preparing 2 centrifuge tubes of 15 mL for each blood sample for detecting the concentration of the antibody, respectively marking as a tube A and a tube B, respectively and correspondingly adding 200 mu L of whole blood sample, then adding 20 mu L of medicine storage liquid (the concentration is 5.0 mg/mL) into the tube A, aiming at adding excessive medicine to enable all receptors to be occupied by the medicine, adding 20 mu L of PBS into the tube B, uniformly mixing, and incubating for 30+/-5 min at room temperature in a dark place;
2. erythrocyte lysis: 15 volumes of 1 Xerythrocyte lysate (10 Xerythrocyte lysate fraction: 8.99g NH) were added to each tube of step 1 4 Cl；1.00g KHCO 3 The method comprises the steps of carrying out a first treatment on the surface of the 37.0mg EDTA;100mL of ultrapure water; diluting 10 times before using to obtain 1 Xerythrocyte lysate), mixing, standing at room temperature in dark for 15+ -5 min, centrifuging at room temperature 400 Xg for 5 min, discarding supernatant, and repeating the steps once if erythrocyte is present at the bottom of the centrifuge tube;
3. adding 1 mL flow buffer to each tube, re-suspending cells, centrifuging at 400 Xg for 5 min at room temperature, and discarding the supernatant;
4. incubation of detection antibody: respectively adding 100 mu L of first detection reagent-biotin-labeled anti-CD 137 antibody neutralizing active antibodies (the concentrations are 200, 100, 80, 50 and 40 mu g/mL respectively) into the A pipe and the B pipe, replacing the 0 mu g/mL group with PBS, and incubating for 30+/-5 min at 2-8 ℃ in a dark place;
5. adding 1 mL flow buffer to each tube, re-suspending cells, centrifuging at 400 Xg at room temperature for 5 min, and discarding the supernatant to perform twice;
6. 100 mu L of flow buffer solution is added into each tube, 8 mu L of second detection reagent-detection antibody mixed solution (1 mu L of FITC-CD45, 1 mu L of BV421-CD3, 1 mu L of BV650-CD19 and 5 mu L of PE-streptavidin (1:20 configuration and on-the-fly) is respectively added into each sample tube, and the sample tubes are incubated for 30+/-5 min at 2-8 ℃ in a dark place;
7. adding 1 mL flow buffer to re-suspend the cells, and centrifuging at 400 Xg at room temperature for 5 min;
8. after the supernatant is discarded, 200 mu L of flow buffer solution is added to resuspend the cells, and the cells are detected by an upper machine;
9. flow cytometer detection: open flow cytometer, device calibration: according to the self-carried program of the instrument, adopting CST Setup loads to correct the fluorescence channel;
10. in the first experiment, voltage and fluorescence compensation are determined according to a blank control tube and a fluorescence channel adjusting tube and are set up as templates; the number of collections per sample was CD3 - CD19 + 10000 cells; collecting the whole volume (200 [ mu ] L) if the cell number is insufficient;
11. circled CD3 - CD19 + Target cell populations, recording MFI values;
wherein, the A tube is the total receptor level, the B tube is the nonspecific background signal, the B tube signal is lower, the A tube signal is higher, namely the signal to noise ratio is as large as possible, wherein, the experimental result corresponding to the 100 mug/mL neutralizing activity antibody is optimal, the difference value between the total receptor level signal and the background signal is ideal, and the result is shown in the table 1.
TABLE 1
Example 2 optimization of the volume of erythrocyte lysate used
2.1 15 times volume of 1 x erythrocyte lysate
1. Configuration of series gradient concentration drugs: 10 centrifuge tubes, 1.5 mL, were prepared and the drug was formulated with PBS in the following concentration gradients: 100. 50, 25, 12.5, 10, 8, 6.25, 4, 2 and 0 μg/mL numbered STD1 to STD10, respectively;
2. configuration of samples with different drug concentrations: preparing 10 5 mL centrifuge tubes, respectively adding 100 mu L of the drug solution in the step 1 into 900 mu L of whole blood sample, so that the final concentration of the drug in the sample is respectively 10, 5, 2.5, 1.25, 1, 0.8, 0.625, 0.4, 0.2 and 0 mu g/mL, uniformly mixing, and incubating at room temperature for 1 h;
3. adding excessive medicine for incubation: preparing 2 15 mL centrifuge tubes for each gradient blood sample, respectively marking as a tube A and a tube B, respectively and correspondingly adding 200 mu L of whole blood sample in the step 2, then adding 20 mu L of drug storage solution (with the concentration of 5.0 mg/mL) into the tube A, aiming at adding excessive drug to enable all receptors to be occupied by the drug, adding 20 mu L of PBS into the tube B, uniformly mixing, and incubating for 30+/-5 min at room temperature in a dark place;
4. erythrocyte lysis: respectively adding 15 times of 1 Xerythrocyte lysate to each tube in the step 3 for cracking, uniformly mixing, standing at room temperature in a dark place for 15+/-5 min, centrifuging at room temperature of 400 Xg for 5 min, discarding supernatant, and repeating the step once if erythrocytes are arranged at the bottom of the centrifuge tube;
5. adding 1 mL flow buffer to each tube, re-suspending cells, centrifuging at 400 Xg for 5 min at room temperature, and discarding the supernatant;
6. incubation of detection antibody: respectively adding 100 mu L of a first detection reagent, namely a neutralizing active antibody (100 mu g/mL) of a biotin-labeled anti-CD 137 antibody into the A pipe and the B pipe, and incubating for 30+/-5 minutes at 2-8 ℃ in a dark place;
7. subsequent experimental steps while steps 5-11 of example 1;
the binding mode receptor occupancy (RO%) is calculated as follows:
receptor occupancy (%) = bound receptor MFI value/total receptor MFI value x 100%.
When the lysis was performed by adding 15 volumes of 1 x red blood cell lysate, the effect was not ideal, the final sample showed poor cell dispersion, cell mass formation, and the flow detection background signal was high, and the results are shown in table 2.
TABLE 2
2.2 20 times volume of 1 x erythrocyte lysate
The experimental procedure is the same as section 2.1 except that the final drug concentrations are 20, 15, 10, 5, 2.5, 1.25, 1.0, 0.8, 0.625, 0.4 and 0 μg/mL, respectively.
When 20 volumes of 1 x red blood cell lysate were added for lysis, the effect was not ideal, the final sample showed poor cell dispersion, cell mass formation, and the flow detection background signal was high, and the results are shown in table 3.
TABLE 3 Table 3
2.3 25 times volume of 1 x erythrocyte lysate
The experimental procedure is the same as section 2.1 except that the final drug concentrations are 30, 20, 10, 5, 3.5, 2.5, 1.25, 0.625, 0.4 and 0 μg/mL, respectively.
When the lysis was performed by adding 25 volumes of 1 x red blood cell lysate, the effect was ideal, which was shown in that the final sample cells were well dispersed, no cell clusters were formed, and the flow detection background signal was low, and the results are shown in table 4. Fig. 2 is a schematic view of a round door strategy.
TABLE 4 Table 4
The same effect is ideal when lysis is performed by adding 30 volumes of 1 x red blood cell lysate, which is manifested in good cell dispersion of the final sample, no cell mass formation, and low background signal for flow detection.
In addition, the stability of the receptor occupancy assay was verified using the whole blood sample using the optimal method of section 2.3, with 25 volumes of 1 x red blood cell lysate added. The results are shown in tables 5-7, and the samples are stored for 5 days at the temperature of 2-8 ℃, and compared with RO% on the 0 th day, the coefficient of variation CV% is less than or equal to 20, and more accurate results are obtained, so that the stability of the method is good.
Table 5 day 1
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TABLE 6 day 2
TABLE 7 day 5
Comparative example receptor occupancy detection Using free mode
The CD40 receptor occupancy of an antibody ligand fusion protein drug comprising the extracellular domain of CD40L and an antibody Fab fragment targeting CD137 was detected using the free mode, as follows:
1. configuration of series gradient concentration drugs: 9 centrifuge tubes, 1.5 mL, were prepared and the drug was formulated with PBS in the following concentration gradients: 100. 50, 12.5, 10, 8, 6.25, 1.56, 0.78 and 0 μg/mL numbered STD1-STD9, respectively;
2. configuration of samples with different drug concentrations: preparing 9 5 mL centrifuge tubes, respectively adding 100 mu L of the drug solution in the step 1 into 900 mu L of whole blood sample, so that the final concentration of the drug in the sample is respectively 10, 5, 1.25, 1.0, 0.8, 0.625, 0.156, 0.078 and 0 mu g/mL, uniformly mixing, and incubating at room temperature for 1 h;
3. adding excessive medicine for incubation: preparing 2 15 mL centrifuge tubes for each gradient blood sample, respectively marking the centrifuge tubes as a tube A and a tube B, respectively and correspondingly adding 200 mu L of whole blood sample in the step 2, then adding 20 mu L of drug storage solution (the concentration is 5.0 mg/mL) into the tube A, aiming at adding excessive drug to enable all receptors to be occupied by the drug, finally measuring the drug to be a background value, adding 20 mu L of PBS into the tube B, uniformly mixing, and incubating for 30+/-5 min at room temperature in a dark place;
4. erythrocyte lysis: respectively adding 25 times of 1 Xerythrocyte lysate to each tube in the step 3 for cracking, uniformly mixing, standing at room temperature in a dark place for 15+/-5 min, centrifuging at room temperature of 400 Xg for 5 min, discarding supernatant, and repeating the step once if erythrocytes are arranged at the bottom of the centrifuge tube;
5. adding 1 mL flow buffer to each tube, re-suspending cells, centrifuging at 400 Xg for 5 min at room temperature, and discarding the supernatant;
6. incubation of detection antibody: adding 100 mu L of a first detection reagent, namely a biotin-labeled drug (100 mu g/mL), into the tube A and the tube B respectively, and incubating for 30+/-5 minutes at 2-8 ℃ in a dark place;
7. adding 1 mL flow buffer to each tube, re-suspending cells, centrifuging at 400 Xg at room temperature for 5 min, and discarding the supernatant to perform twice;
8. 100 mu L of flow buffer solution is added into each tube, 8 mu L of second detection reagent-detection antibody mixed solution (1 mu L of FITC-CD45, 1 mu L of BV421-CD3, 1 mu L of BV650-CD19 and 5 mu L of PE-streptavidin (1:20 configuration and on-the-fly) is respectively added into each sample tube, and the sample tubes are incubated for 30+/-5 min at 2-8 ℃ in a dark place;
9. adding 1 mL flow buffer to re-suspend the cells, and centrifuging at 400 Xg at room temperature for 5 min;
10. after the supernatant is discarded, 200 mu L of flow buffer solution is added to resuspend the cells, and the cells are detected by an upper machine;
11. flow cytometer detection: open flow cytometer, device calibration: according to the self-carried program of the instrument, adopting CST Setup loads to correct the fluorescence channel;
12. in the first experiment, voltage and fluorescence compensation are determined according to a blank control tube and a fluorescence channel regulating tube and are set up as templates, and the collection number of each sample is CD3 - CD19 + 10000 cells; collecting the whole volume (200 [ mu ] L) if the cell number is insufficient;
13. circled CD3 - CD19 + Target cell population, recording MFI value, and calculating receptor occupancy;
the formula for the free mode RO% is as follows:
RO% = 100% -free receptor MFI value/total receptor MFI value x 100%
Wherein, free receptor MFI value = post-dosing B-tube MFI value-post-dosing a-tube MFI value;
total receptor MFI value = pre-dose B-tube MFI value-pre-dose a-tube MFI value.
The results are shown in Table 8, where the RO% results are not linear.
TABLE 8
All patents, patent application publications, and non-patent documents mentioned and/or listed in this application are incorporated herein by reference in their entirety. While exemplary embodiments of the inventions of the present application have been described above, those skilled in the art will be able to make modifications or improvements to the exemplary embodiments described herein, and variations or equivalents thereto, without departing from the spirit and scope of the application.

Claims (10)

1. A method for detecting occupancy of a first target receptor of a dual-target drug, wherein the dual-target drug comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target, the first target and the second target being proteins expressed on the surface of an immune cell, the method comprising:
adding the dual-target drug to a whole blood sample at one or more concentrations for incubation;
adding a first detection reagent bearing a detectable label that binds to the second binding moiety, the second binding moiety having an affinity for the first detection reagent sufficient to block or abstract binding of the second binding moiety to the second target;
detecting the signal intensity of the detectable label of the immune cells expressing the first target point by taking single cells as detection units, and calculating one or more test medians corresponding to the one or more concentrations based on the signal intensity values of the respective cells;
determining the first target receptor occupancy of the dual-target agent by comparing the one or more test medians to a total signal intensity median, wherein the total signal intensity median is the median determined by the above steps under conditions in which the dual-target agent is added in an amount sufficient to saturation bind to the first target on the immune cell.
2. The method of claim 1, wherein
The first target is CD40; and/or
The second target is CD137.
3. The method of claim 1, wherein
The immune cells expressing the first target are B cells;
the first binding moiety is a ligand for CD40 or a fragment thereof; and/or
The second binding moiety is an anti-CD 137 antibody.
4. A method as claimed in claim 3, wherein
The first detection reagent is a neutralizing activity antibody of an anti-CD 137 antibody;
the concentration of the first detection reagent is 100 mug/mL;
the detectable label is a biotin label or a fluorescent label; and/or
The signal intensity is a fluorescent signal intensity.
5. The method of claim 1, wherein the method further comprises:
after the incubation of the double-target drug and the whole blood sample is finished, adding erythrocyte lysate to carry out erythrocyte lysis; and/or
After completion of erythrocyte lysis, cell pellet was collected by centrifugation.
6. The method of claim 5, wherein
The volume of the erythrocyte lysate is 25-30 times of the whole blood volume.
7. The method of claim 4, wherein the method further comprises adding a second detection reagent.
8. The method of claim 7, wherein the second detection reagent comprises PE-labeled streptavidin.
9. The method of claim 1, wherein the affinity of the second binding moiety for the first detection reagent is 5000-20000 times higher than the affinity of the second binding moiety for the second target.
10. The method of claim 1, wherein the affinity of the second binding moiety for the first detection reagent is 10000-fold higher than the affinity of the second binding moiety for the second target.
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