A method for evaluating the effect of antigen silencing on antibody drug efficacy and its application

Abstract

The present invention discloses a method for evaluating the effect of antigen silencing on the efficacy of antibodies, comprising the following steps: 1. subjecting the antibody to be tested to antigen silencing treatment; 2. incubating tumor cells expressing the TAA target after CFSE staining with the antibody solution after antigen silencing treatment, and analyzing the mean fluorescence intensity of the antibody bound to the cell surface. The method of the present invention improves the experimental signal window, significantly evaluates the effect of antigen silencing on the antibody action, and at the same time more realistically simulates the antigen silencing effect on solid tumor cells, and has a broader application prospect in the research and development of solid tumor therapeutic antibodies.

Description

Technical Field

[0001] The present invention relates to a method for evaluating the efficacy of an antibody, and more particularly to a method for evaluating the influence of antigen sink effect on the efficacy of an antibody. Background Art

[0002] Developing therapeutic antibodies against certain protein antigens highly expressed on the surface of tumor cells is a common form of drug research and development at present. However, a considerable number of druggable protein antigens are expressed not only in tumor cells but also in blood cells or normal tissue cells. Therefore, when the antibody drug enters the body, it will bind to non-tumor cells such as blood cells or normal tissue cells expressing the corresponding antigen, thus affecting the distribution and elimination of the antibody drug in the body and restricting the targeting of the antibody drug to the intended target tissue. This phenomenon is called antigen sink effect (Antigen Sink). Therefore, it is of great significance to effectively evaluate the influence of antigen sink effect on the efficacy of an antibody during the R & D stage.

[0003] Cluster of Differentiation 47 (CD47) was initially found to be expressed in red blood cells and later found to be expressed to varying degrees in almost all normal tissue cells. It acts as a natural immune checkpoint to regulate the phagocytosis of cells. CD47 is a 52kD glycoprotein with an immunoglobulin variable N-terminal domain, 5 transmembrane domains, and a short C-terminal intracellular domain. CD47 on the surface of normal tissue cells can interact with the receptor Signal Regulatory Protein alpha (SIRPα) on the surface of macrophages, initiating an inhibitory signaling pathway downstream of SIRPα, thereby blocking the clearance of normal cells by macrophages. However, this mechanism can also be exploited by tumor cells. Therefore, developing antibody drugs targeting CD47 to block the CD47-SIRPα signaling axis and restore the phagocytosis of tumor cells by macrophages has good clinical application prospects for anti-tumor treatment. However, CD47 is highly expressed on some normal tissue cells, especially red blood cells and lymphocytes, which will produce a relatively obvious antigen sink effect on the antibody drug, thus affecting the anti-tumor treatment effect. Summary of the Invention

[0004] The object of the present invention is to establish a method for evaluating the antigen sink effect of a test antibody. By pre-incubating the test antibody targeting CD47 with non-tumor cells expressing CD47 to simulate the antigen sink process, and further evaluating the influence of the antibody on the biological function of solid tumor cells, it can be used as an effective measure in the early R & D process of such drugs.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for evaluating the effect of antigen silencing on the efficacy of antibodies, comprising the following steps:

[0007] 1. The effect of antigen silencing on the binding of antibodies to cells

[0008] We use plasma-depleted human whole blood to simulate red blood cells and Jurkat cells to simulate T lymphocytes.

[0009] The antibody to be tested is diluted with 1% (v / v) BSA-PBS, starting from 300 nM and diluted 3-fold to 0.0051 nM, with a total of 11 points. Plasma-depleted human whole blood is diluted 10-fold with 1% BSA-PBS, and the Jurkat cell line is resuspended with 1% BSA-PBS to a density of 1.2×10 7 cells / mL. The 10-fold diluted blood cell solution or Jurkat cell suspension is inoculated into a 96-well round-bottom culture plate, 100 μL is added to each well, the supernatant is removed by centrifugation, and each well is resuspended with 100 μL of the diluted antibody solution and incubated at 4°C for 1 hour. After centrifugation, the supernatant is transferred to a new plate for standby, which is the antibody solution treated by antigen silencing.

[0010] Tumor cells expressing the TAA target are stained with CFSE and resuspended with 1% BSA-PBS to a density of 0.8×10 6 cells / mL, inoculated into a 96-well round-bottom culture plate, 100 μL is added to each well, the supernatant is removed by centrifugation, and each well is resuspended with 100 μL of the antibody solution treated by antigen silencing and incubated at 4°C for 1 hour. After washing twice, each well is resuspended with 100 μL of the fluorescently labeled secondary antibody solution and incubated at 4°C in the dark for half an hour. After washing twice, it is resuspended with 1% BSA-PBS for flow cytometry detection. The CFSE-positive tumor cell population is circled, and the mean fluorescence intensity of the antibody binding on the cell surface is analyzed using FlowJo software.

[0011] 2. The effect of antigen silencing on the blocking of the binding of antibodies to SIRPα and cells

[0012] We use plasma-depleted human whole blood to simulate red blood cells and Jurkat cells to simulate T lymphocytes.

[0013] The antibody to be tested is diluted with 1% BSA-PBS, starting from 300 nM and diluted 3-fold to 0.0051 nM, with a total of 11 points. Plasma-depleted human whole blood is diluted 10-fold with 1% BSA-PBS, and the Jurkat cell line is resuspended with 1% BSA-PBS to a density of 0.6×10 7Dilute it to a density of 10-fold diluted blood cell solution or Jurkat cell suspension was inoculated into a 96-well round-bottom culture plate, 100 μl was added to each well. After centrifugation, the supernatant was discarded. Each well was resuspended with 100 μl of the diluted antibody solution and incubated at 4 °C for 1 hour. After centrifugation, the supernatant was transferred to a new plate for standby, which was the antibody solution treated by antigen silencing. The SIRPα protein with a mouse Fc fragment (mFc) tag was diluted with 1% BSA-PBS to a solution of 3 μg / ml.

[0014] The tumor cells expressing the TAA target were stained with CFSE and then resuspended with 1% BSA-PBS to a density of 2×10 6 cells / ml, inoculated into a 96-well round-bottom culture plate, 40 μl was added to each well. Then 40 μl of the SIRPα protein solution and 40 μl of the antibody solution treated by antigen silencing were added to each well. After mixing, it was incubated at 4 °C for 2 hours. After washing twice, each well was resuspended with 100 μl of the fluorescently labeled anti-mFc secondary antibody solution and incubated at 4 °C in the dark for half an hour. After washing twice, it was resuspended with 1% BSA-PBS for flow cytometry detection. The CFSE-positive tumor cell population was circled, and the mean fluorescence intensity of the SIRPα protein bound to the cell surface was analyzed using FlowJo software.

[0015] Furthermore, the antibody to be tested is: the bispecific antibody CD47 x TAA BsAb targeting CD47 and tumor-associated antigen (TAA) was constructed, expressed and purified by the Protein Science Department of the Biological New Drug R & D Service Business Unit of Shanghai WuXi Biologics Inc., and the variable region sequences of CD47 and TAA were both discovered by the Hybridoma Department of the Biological New Drug R & D Service Business Unit of Shanghai WuXi Biologics Inc. The monoclonal antibody CD47 mAb targeting CD47 was constructed, expressed and purified by the Protein Science Department of the Biological New Drug R & D Service Business Unit of Shanghai WuXi Biologics Inc., and the full-length sequence of this antibody was synthesized according to the sequence with the clone number 5F9 disclosed in the patent (US9017675).

[0016] The advantages of the present invention are as follows: The present invention can truly and effectively simulate the antigen silencing effect of a therapeutic antibody targeting a solid tumor during the blood circulation process after injection. The currently reported technical methods have a small signal window and are only applicable to blood tumor targets, and cannot truly simulate the antigen silencing effect against solid tumor targets. The present invention further improves the reported technology, increases the experimental signal window, significantly evaluates the influence of the antigen silencing effect on the antibody action, and at the same time more truly simulates the antigen silencing effect against solid tumor cells, and has a broader application prospect in the research and development of solid tumor therapeutic antibodies. Brief Description of the Drawings

[0017] Figure 1 It is the influence of the antigen silencing effect of red blood cells on the binding of antibodies to tumor cells.

[0018] Figure 2 It is the influence of the antigen silencing effect of lymphocytes on the binding of antibodies to tumor cells.

[0019] Figure 3 It is the influence of the antigen silencing effect of red blood cells on the blocking of the binding of SIRPα to tumor cells by antibodies.

[0020] Figure 4 It is the influence of the antigen silencing effect of lymphocytes on the blocking of the binding of SIRPα to tumor cells by antibodies.

[0021] Figure 5 It is the influence of the co-incubation of red blood cells and tumor cells on the blocking of the binding of SIRPα to tumor cells by antibodies. Detailed implementation mode

[0022] Example 1:

[0023] 1. Obtaining of antibodies

[0024] The bispecific antibody CD47 x TAA targeting CD47 and tumor-associated antigen (TAA) was constructed, expressed and purified by the Protein Science Department of the Biopharmaceutical R & D Services Business Unit of Shanghai WuXi Biologics Inc., and the variable region sequences of CD47 and TAA were discovered by the Hybridoma Department of the Biopharmaceutical R & D Services Business Unit of Shanghai WuXi Biologics Inc. The monoclonal antibody CD47 targeting CD47 was constructed, expressed and purified by the Protein Science Department of the Biopharmaceutical R & D Services Business Unit of Shanghai WuXi Biologics Inc., and the full-length sequence of this antibody was synthesized according to the sequence with the clone number 5F9 disclosed in the patent (US9017675).

[0025] 2. Influence of antigen silencing effect on the binding of antibodies to cells

[0026] We used human whole blood without plasma to simulate red blood cells and Jurkat cells to simulate T lymphocytes. The test antibodies (CD47 x TAA bispecific antibody and CD47 monoclonal antibody) were diluted with 1% (volume ratio) BSA-PBS, starting from 300 nM and diluted 3-fold to 0.0051 nM, with a total of 11 points. Human whole blood without plasma was diluted 10-fold with 1% BSA-PBS, and the Jurkat cell line was resuspended with 1% BSA-PBS to 1.2×10 7The density is 0 cells / mL. The 10-fold diluted blood cell solution or Jurkat cell suspension was inoculated into a 96-well round-bottom culture plate, 100 μL was added to each well. After centrifugation, the supernatant was discarded, and each well was resuspended with 100 μL of the diluted antibody solution to be tested (the concentration ranged from 300 nM to 0.0051 nM after 3-fold dilution, a total of 11 concentrations), incubated at 4 °C for 1 hour, after centrifugation, the supernatant was transferred to a new plate for standby, which was the antibody solution after antigen silencing treatment.

[0027] After the tumor cells expressing the TAA target were stained with CFSE, they were resuspended with 1% BSA-PBS to a density of 0.8×10 6 cells / mL, inoculated into a 96-well round-bottom culture plate, 100 μL was added to each well. After centrifugation, the supernatant was discarded, and each well was resuspended with 100 μL of the antibody solution after antigen silencing treatment, incubated at 4 °C for 1 hour, washed twice, then each well was resuspended with 100 μL of the fluorescently labeled secondary antibody solution, incubated at 4 °C in the dark for half an hour, washed twice, and resuspended with 1% BSA-PBS for flow cytometry detection. The CFSE-positive tumor cell population was circled, and the mean fluorescence intensity of the antibody binding on the cell surface was analyzed using FlowJo software.

[0028] 3. Influence of antigen silencing effect on the antibody blocking the binding of SIRPα to cells

[0029] We used plasma-depleted human whole blood to simulate red blood cells and Jurkat cells to simulate T lymphocytes. The antibodies to be tested (CD47 x TAA bispecific antibody and CD47 monoclonal antibody) were diluted with 1% BSA-PBS, starting from 300 nM, 3-fold diluted to 0.0051 nM, a total of 11 points. Plasma-depleted human whole blood was 10-fold diluted with 1% BSA-PBS, and the Jurkat cell line was resuspended with 1% BSA-PBS to a density of 0.6×10 7 cells / mL. The 10-fold diluted blood cell solution or Jurkat cell suspension was inoculated into a 96-well round-bottom culture plate, 100 μL was added to each well. After centrifugation, the supernatant was discarded, and each well was resuspended with 100 μL of the diluted antibody solution to be tested (CD47 x TAA bispecific antibody and CD47 monoclonal antibody) (the concentration ranged from 300 nM to 0.0051 nM after 3-fold dilution, a total of 11 concentrations), incubated at 4 °C for 1 hour, after centrifugation, the supernatant was transferred to a new plate for standby, which was the antibody solution after antigen silencing treatment. The SIRPα protein with a mouse Fc fragment (mFc) tag was diluted with 1% BSA-PBS to a solution with a concentration of 3 μg / ml.

[0030] After the tumor cells expressing the TAA target were stained with CFSE, they were resuspended with 1% BSA-PBS to a density of 2×10 6Cells were inoculated into a 96-well round-bottom culture plate at a density of 6×10 cells / mL, with 40 μL added to each well. Then, 40 μL of SIRPα protein solution with a mouse Fc fragment (mFc) tag and 40 μL of antigen-silenced antibody solution were added to each well. After mixing, the plate was incubated at 4°C for 2 hours. After washing twice, each well was resuspended with 100 μL of fluorescently labeled anti-mFc secondary antibody solution and incubated at 4°C in the dark for half an hour. After washing twice, the cells were resuspended with 1% BSA-PBS for flow cytometry analysis. The CFSE-positive tumor cell population was circled, and the mean fluorescence intensity of SIRPα protein binding on the cell surface was analyzed using FlowJo software.

[0031] 4. Effect of red blood cells on antibody blocking of SIRPα binding to tumor cells

[0032] We used plasma-depleted human whole blood to simulate red blood cells. The antibody to be tested (CD47 monoclonal antibody) was diluted with 1% BSA-PBS starting from 40 nM and serially diluted 3-fold to 0.018 nM, with a total of 8 points. Plasma-depleted human whole blood was diluted with 1% BSA-PBS. SIRPα protein with a mouse Fc fragment (mFc) tag was diluted with 1% BSA-PBS to a solution of 6 μg / mL. Tumor cells expressing the TAA target were stained with CFSE and then resuspended with 1% BSA-PBS at a density of 6×10 6 cells / mL. In a 96-well round-bottom plate, 20 μL of CFSE-stained tumor cells expressing the TAA target, 20 μL of SIRPα protein solution with a mouse Fc fragment (mFc) tag, 40 μL of the antibody to be tested (CD47 monoclonal antibody) solution (diluted from 40 nM starting and serially diluted 3-fold to 0.018 nM, with a total of 8 points), and 40 μL of blood cells (20 times the number of tumor cells) or 40 μL of 1% BSA-PBS were added to each well. After mixing, the plate was incubated at 4°C for 2 hours. After washing twice, each well was resuspended with 100 μL of fluorescently labeled anti-mFc secondary antibody solution and incubated at 4°C in the dark for half an hour. After washing twice, the cells were resuspended with 1% BSA-PBS for flow cytometry analysis. The CFSE-positive tumor cell population was circled, and the mean fluorescence intensity of SIRPα protein binding on the cell surface was analyzed using FlowJo software.

[0033] Example 2:

[0034] 1. Effect of antigen silencing on antibody binding to tumor cells

[0035] As Figure 1 shown in and Table 1, under the condition of not pre-incubating with whole blood solution, the affinity (EC 50 ) of the CD47xTAA bispecific antibody binding to tumor cells was 4.80 nM, and the affinity (EC 50) is 0.36 nM. Under the condition of pre-incubation with whole blood solution, the affinity (EC 50 ) of the CD47xTAA bispecific antibody for binding to tumor cells is 4.73 nM, showing basically no change, indicating that it is not affected by the antigen silencing effect. However, the affinity (EC 50 ) of the CD47 monoclonal antibody for binding to tumor cells is 6.41 nM, showing a change of about 18-fold, indicating that it is affected by a relatively severe antigen silencing effect. Table 1 shows the influence of the antigen silencing effect of red blood cells on the binding of antibodies to tumor cells.

[0036]

[0037] Table 1

[0038] In addition, as Figure 2 and Table 2 show, under the condition of not pre-incubating with Jurkat cells, the affinity (EC 50 ) of the CD47xTAA bispecific antibody for binding to tumor cells is 2.65 nM, and the affinity (EC 50 ) of the CD47 monoclonal antibody for binding to tumor cells is 0.19 nM. Under the condition of pre-incubating with Jurkat cells, the affinity (EC 50 ) of the CD47xTAA bispecific antibody for binding to tumor cells is 2.82 nM, showing basically no change, indicating that it is not affected by the antigen silencing effect. However, the affinity (EC 50 ) of the CD47 monoclonal antibody for binding to tumor cells is 1.53 nM, showing a change of about 8-fold, indicating that it is affected by a certain degree of antigen silencing effect. Table 2 shows the influence of the antigen silencing effect of lymphocytes on the binding of antibodies to tumor cells.

[0039]

[0040] Table 2

[0041] 2. Influence of antigen silencing effect on the ability of antibodies to block the binding of SIRPα to tumor cells

[0042] As Figure 3 and Table 3 show, under the condition of not pre-incubating with whole blood solution, the ability (IC 50 ) of the CD47xTAA bispecific antibody to block the binding of SIRPα to tumor cells is 0.09 nM, and the ability (IC 50 ) of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells is 0.06 nM. Under the condition of pre-incubating with whole blood solution, the ability (IC 50) was 0.09 nM, with little change, indicating that it was not affected by the antigen silencing effect. However, the ability of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.93 nM, with a change of about 15-fold, indicating that it was affected by a relatively severe antigen silencing effect. Table 3 shows the effect of the antigen silencing effect of red blood cells on the ability of antibodies to block the binding of SIRPα to tumor cells.

[0043]

[0044] Table 3

[0045] In addition, as Figure 4 and Table 4 show, under the condition of not pre-incubating with Jurkat cells, the ability of the CD47xTAA bispecific antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.19 nM, and the ability of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.17 nM. Under the condition of pre-incubating with Jurkat cells, the ability of the CD47xTAA bispecific antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.26 nM, with little change, indicating that it was not affected by the antigen silencing effect. However, the ability of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.44 nM, with a change of about 2.5-fold, indicating that it was affected by a certain degree of antigen silencing effect.

[0046] Table 4 shows the effect of the antigen silencing effect of lymphocytes on the ability of antibodies to block the binding of SIRPα to tumor cells.

[0047]

[0048] Table 4

[0049] 3. Effect of red blood cells on the ability of antibodies to block the binding of SIRPα to tumor cells

[0050] As Figure 5 and Table 5 show, under the condition of not co-incubating with whole blood cells, the ability of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.07 nM. Under the condition of co-incubating with whole blood cells, the ability of the CD47 monoclonal antibody to block the binding of SIRPα to tumor cells (IC 50 ) was 0.16 nM, with a change of about 2-fold. Compared with the CD47 monoclonal antibody in Figure 3 and Table 3, it indicates that it was less affected by blood cells. Table 5 shows the effect of whole blood cells on the ability of antibodies to block the binding of SIRPα to tumor cells.

[0051]

[0052] Table 5

[0053] 3. Experimental Summary and Discussion

[0054] The present invention provides a method. By pre-incubating the antibody to be detected targeting CD47 with non-tumor cells such as red blood cells or lymphocytes that highly express CD47 to simulate the antigen silencing process, and further detecting the biological function of the antibody in solid tumor cells, thereby evaluating the influence of antigen silencing effect on the antibody function, which has important significance for the early research and development of antibody drugs involving targeting CD47.

Claims

1. A method for evaluating the effect of antigen silencing on the efficacy of antibodies, characterized in that, Comprising the following steps: 1). Dilute the antibody to be tested with BSA-PBS, starting from 300 nM, and perform 3-fold dilution to 0.0051 nM, with a total of 11 points; dilute human whole blood without plasma 10-fold with BSA-PBS, and resuspend the Jurkat cell line with BSA-PBS to a density of 1.2×10 7 cells / mL; inoculate the 10-fold diluted blood cell solution or Jurkat cell suspension into a 96-well round-bottom culture plate, add 100 μL to each well, centrifuge and discard the supernatant, add 100 μL of the diluted antibody solution to each well to resuspend, incubate at 4°C for 1 hour, centrifuge and then transfer the supernatant to a new plate for standby, which is the antibody solution treated by antigen silencing; 2). After tumor cells expressing the TAA target were stained with CFSE, they were resuspended in BSA-PBS at a density of 0.8×10 6 cells / mL, seeded into a 96-well round-bottom culture plate, 100 μL was added to each well. After centrifugation, the supernatant was discarded. Each well was resuspended with 100 μL of the antibody solution treated with antigen silencing and incubated at 4°C for 1 hour. After washing twice, each well was resuspended with 100 μL of the fluorescently labeled secondary antibody solution and incubated in the dark at 4°C for half an hour. After washing twice, it was resuspended with BSA-PBS for flow cytometry detection. The CFSE-positive tumor cell population was circled, and the mean fluorescence intensity of the antibody binding on the cell surface was analyzed using FlowJo software; 3) Comparing the mean fluorescence intensity of the antibody bound to the cell surface under the condition of not pre-incubating with whole blood solution or Jurkat cells with the mean fluorescence intensity of the antibody bound to the cell surface under the condition of pre-incubating with whole blood solution or Jurkat cells to evaluate the effect of antigen silencing on the binding of the antibody to tumor cells.

2. The method for evaluating the effect of antigen silencing on the efficacy of an antibody according to claim 1, characterized in that, The volume concentration of the BSA-PBS is 1%.

3. The method for evaluating the effect of antigen silencing on the efficacy of an antibody according to claim 1 or 2, characterized in that, The antibody to be tested is a bispecific antibody CD47 x TAA targeting CD47 and a tumor-associated antigen and / or a monoclonal antibody targeting CD47.

4. Use of a method for evaluating the effect of antigen silencing on antibody efficacy as described in claim 1 in detecting the effect of antigen silencing on the ability of an antibody to block the binding of SIRPα to cells, characterized in that, Comprising the following steps: Dilute the SIRPα protein with an mFc tag into a 3 μg / ml solution using BSA-PBS; after tumor cells expressing the TAA target are stained with CFSE, resuspend them with BSA-PBS at a density of 2×10 6 cells / mL, seed them into a 96-well round-bottom culture plate, add 40 μl to each well, then add 40 μl of the SIRPα protein solution and 40 μl of the antibody solution treated by antigen silencing to each well. After mixing, incubate at 4°C for 2 hours. After washing, resuspend each well with 100 μl of a fluorescently labeled anti-mFc secondary antibody solution, incubate at 4°C in the dark for half an hour. After washing, resuspend with BSA-PBS for flow cytometry detection. Gate the CFSE-positive tumor cell population, and analyze the mean fluorescence intensity of the SIRPα protein bound to the cell surface using FlowJo software.

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