Method for detecting biological activity of human immunoglobulin ADCP

Abstract

The invention discloses a method for detecting the biological activity of human immunoglobulin ADCP, which is characterized in that CHO-K1 / SPIKESARS2 is taken as a target cell, THP-1 cells (human leukemia monocytes which naturally express CD32a (Fc gamma RIIa) and CD64a (Fc gamma RIIa) and stably transfect NF-kB-Luc luciferase reporter genes) modified by genetic engineering are taken as effector cells, and a fluorescence reporter gene method is adopted for detection. Based on specific target cells and effector cells, the ADCP biological activity of two different human immune globulin preparations, namely intravenous injection human immune globulin and subcutaneous injection human immune globulin, can be accurately and reliably reflected by a drawn dose-effect curve through a detection system matched with specific human immune globulin solution concentration, and the method is simple and easy to implement. Cost is low, and practical application and popularization value is achieved.

Description

Technical Field

[0001] The present invention specifically relates to a method for detecting the biological activity of human immunoglobulin ADCP. Background Art

[0002] Immunoglobulins are antibodies produced by plasma cells differentiated from B cells. Their molecules have a unique structure that allows them to specifically recognize antigenic determinants. Immunoglobulin preparations are prepared from the pooled plasma of numerous healthy blood donors (>1000 individuals per batch), separated and purified using low-temperature ethanol protein separation or other approved separation methods, and then subjected to viral removal and inactivation. my country only approves the use of intravenous immunoglobulin (IVIG), while subcutaneous immunoglobulin (SCIG) has not yet been approved for marketing in China. However, clinical use of SCIG products abroad has demonstrated that they have comparable or superior efficacy to intravenous immunoglobulin (IVIG). Human immunoglobulin preparations contain a vast array of diverse antibody repertoires, offering the potential for widespread use in a wide range of immune system diseases.

[0003] Cell-mediated phagocytosis (ADCP) occurs when the Fab segment of an antibody binds to an antigen on the surface of a target cell (a virus-infected cell or tumor cell). The Fc segment then interacts with immune cells (such as monocytes, macrophages, neutrophils, and dendritic cells) that have FcγRIIa (CD32a) and / or FcγRIa (CD64a) receptors, prompting these immune cells to phagocytose the target cell, thereby clearing it. The mechanism of action of this effect plays an important role in the body's immune defense and immune regulation. Therefore, detecting whether an antibody drug has ADCP biological activity and the extent of its biological activity has become a crucial step in the development and quality control of antibody drugs.

[0004] Currently, a variety of methods have been established to determine the biological activity of ADCP in antibody drugs, such as those based on primary peripheral blood mononuclear cells (hPBMCs) and fluorescent labeling (combined with flow cytometry). However, these methods are highly dependent on donor primary cells, are complex, time-consuming, labor-intensive, expensive, and have poor reproducibility. They also fail to more accurately reflect the complex immune environment in vivo. Therefore, there is an urgent need to develop a rapid, sensitive, and accurate method for detecting the biological activity of ADCP in human immunoglobulin drugs. Summary of the Invention

[0005] To solve the above problems, the present invention provides a method for detecting the biological activity of human immunoglobulin ADCP, which uses CHO-K1 / SPIKE_SARS2 as target cells, THP-1-NF-κB or GS-J2B-FCGR2A as effector cells, and adopts a fluorescent reporter gene method for detection.

[0006] Furthermore, the steps of the fluorescence reporter gene detection method are:

[0007] The target cell suspension was added to the well plate, and then a series of concentrations of human immunoglobulin solution were added for incubation. Finally, the effector cell suspension was added for incubation. After the incubation, the fluorescence was detected. The dose-effect curve was drawn with the relative luminescence unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa, and the half-maximal effect concentration was calculated.

[0008] Furthermore, the effector-target ratio of effector cells to target cells in each well of the well plate is 3:2.

[0009] Furthermore, the concentration range of the human immunoglobulin solution is 0.0012 to 5 mg / ml.

[0010] Furthermore, the human immunoglobulin solution was diluted to 6 concentrations using a 4-fold gradient dilution ratio with a starting working concentration of 5 mg / ml;

[0011] Or: Use 5 mg / ml as the starting working concentration and then dilute to 7 concentrations using a 3-fold serial dilution ratio.

[0012] Furthermore, the target cell suspension, human immunoglobulin solutions of a series of concentrations and effector cell suspension are prepared using RPMI 1640 culture medium containing 10% FBS.

[0013] Furthermore, the concentration of the target cell suspension is 5×10 5 cells / ml, the volume is 40 μl / well; the volume of the human immunoglobulin solution of serial concentrations is 20 μl / well; the concentration of the effector cell suspension is 7.5×10 5 cells / ml, with a volume of 40 μl / well.

[0014] Furthermore, the target cell suspension was incubated with the human immunoglobulin solution at room temperature for 30 minutes; and the effector cell suspension was added and incubated at 37° C. and 5% CO 2 for 4 hours.

[0015] Furthermore, the fluorescence is detected using a luciferase detection kit.

[0016] Furthermore, the luciferase detection kit is Fire-Lumi TM Luciferase Assay Kit.

[0017] The present invention relates to a method for detecting the ADCP biological activity of human immunoglobulin. Specific target cells and effector cells are used in a reporter gene method to detect the ADCP biological activity of human immunoglobulin. By combining a specific concentration of a human immunoglobulin solution, a dose-effect curve can be drawn with a relative luminescence unit value as the ordinate and a logarithmic value of the human immunoglobulin solution concentration as the abscissa. The method can accurately and reliably reflect the ADCP biological activity of two different human immunoglobulin preparations, namely, intravenous human immunoglobulin and subcutaneous human immunoglobulin. The method is simple, low-cost, and has practical value for promotion and application.

[0018] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0019] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The experimental results of ADCP effector cell screening are shown in the figure. The data are expressed as relative luminescence units (RLU) mean ± SEM (n=3).

[0021] Figure 2 The experimental results of the optimization of the starting gradient of SCIG concentration are shown in the figure. The data are expressed as the mean ± SEM of relative luminescence units (RLU) (n=3).

[0022] Figure 3 The experimental results verify the target cell specificity.

[0023] Figure 4 The experimental results verify the effector cell specificity.

[0024] Figure 5 The experimental results are for sample specificity verification.

[0025] Figure 6 The data are expressed as relative luminescence units (RLU) mean ± SEM (n = 3).

[0026] Figure 7 The experimental results of the ADCP detection method applied to IVIG samples are shown in the figure. The data are expressed as the mean ± SEM of relative luminescence units (RLU) (n=3). DETAILED DESCRIPTION

[0027] In the following examples and experimental examples, reagents and raw materials not specifically described are commercially available. Example 1 Detection of ADCP Biological Activity of Intravenous Human Immunoglobulin IVIG of the Present Invention

[0028] 1) Solution Preparation

[0029] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 5×10 5 cells / mL of cell suspension;

[0030] Effector cell suspension: Take the revived THP-1-NF-kB and add RPMI 1640 containing 10% FBS to a concentration of 7.5×10 5 cells / mL of cell suspension;

[0031] IVIG solution series: Prepare a 5 mg / ml solution by adding IVIG to RPMI 1640 containing 10% FBS as the starting working concentration. Then, dilute the solution to 6 concentrations using RPMI 1640 containing 10% FBS in a 4-fold serial dilution ratio: 5 mg / ml, 1.25 mg / ml, 0.3125 mg / ml, 0.078125 mg / ml, 0.01953125 mg / ml, 0.004882813 mg / ml, and 0.0012207034 mg / ml, for a total of 7 IVIG solutions with a concentration range of 0.001 to 5 mg / ml.

[0032] 2) ADCP biological activity detection

[0033] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of IVIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 3:2. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-LumiNano was added at 80 μl / well. TM Incubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0034] 3) Calculation

[0035] The relative luminescence unit value was used as the ordinate and the logarithm of the IVIG solution concentration was used as the abscissa. The dose-effect curve of IVIG was fitted using a four-parameter curve equation regression model. The EC value of IVIG was obtained based on the dose-effect curve. 50 value.

[0036] Example 2 Detection of ADCP Biological Activity of Intravenous Human Immunoglobulin IVIG of the Present Invention

[0037] 1) Solution Preparation

[0038] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 5×10 5 cells / mL of cell suspension;

[0039] Effector cell suspension: Take the revived THP-1-NF-kB and add RPMI 1640 containing 10% FBS to a concentration of 7.5×10 5 cells / mL of cell suspension;

[0040] IVIG solution series: Prepare a 5 mg / ml solution by adding IVIG to RPMI 1640 containing 10% FBS as the starting working concentration. Then, dilute the solution to 7 concentrations using a 3-fold serial dilution in RPMI 1640 containing 10% FBS: 5 mg / ml, 1.67 mg / ml, 0.556 mg / ml, 0.185 mg / ml, 0.062 mg / ml, 0.021 mg / ml, 0.0069 mg / ml, and 0.0023 mg / ml, for a total of 8 IVIG solutions with a concentration range of 0.002 to 5 mg / ml.

[0041] 2) ADCP biological activity detection

[0042] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of IVIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 3:2. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-LumiNano was added at 80 μl / well. TM Incubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0043] 3) Calculation

[0044] The relative luminescence unit value was used as the ordinate and the logarithm of the IVIG solution concentration was used as the abscissa. The dose-effect curve of IVIG was fitted using a four-parameter curve equation regression model. The EC value of IVIG was obtained based on the dose-effect curve. 50 value.

[0045] Example 3 Detection of ADCP Biological Activity of Intravenous Human Immunoglobulin IVIG of the Present Invention

[0046] 1) Solution Preparation

[0047] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 5×10 5 cells / mL of cell suspension;

[0048] Effector cell suspension: Take the revived GS-J2B-FCGR2A and add RPMI 1640 containing 10% FBS to a concentration of 7.5×10 5 cells / mL of cell suspension;

[0049] IVIG solution series: Prepare a 5 mg / ml solution by adding IVIG to RPMI 1640 containing 10% FBS as the starting working concentration. Then, dilute the solution to 6 concentrations using RPMI 1640 containing 10% FBS in a 4-fold serial dilution ratio: 5 mg / ml, 1.25 mg / ml, 0.3125 mg / ml, 0.078125 mg / ml, 0.01953125 mg / ml, 0.004882813 mg / ml, and 0.0012207034 mg / ml, for a total of 7 IVIG solutions with a concentration range of 0.001 to 5 mg / ml.

[0050] 2) ADCP biological activity detection

[0051] The target cell suspension was added to a 96-well plate at 40 μl / well, and then a series of IVIG solutions were added at 20 μl / well. The cells were incubated at room temperature for 30 minutes. Finally, the effector cell suspension was added at 40 μl / well to make the effector-target ratio of each well 3:2. The cells were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was removed and Fire-LumiNano was added at 80 μl / well. TM Incubate with the luciferase assay kit working solution for 5-10 minutes and read the relative luminescence units using a PHERAstar FSX at room temperature.

[0052] 3) Calculation

[0053] The relative luminescence unit value was used as the ordinate and the logarithm of the IVIG solution concentration was used as the abscissa. The dose-effect curve of IVIG was fitted using a four-parameter curve equation regression model. The EC value of IVIG was obtained based on the dose-effect curve. 50 value.

[0054] Example 4 Detection of ADCP biological activity of subcutaneous immunoglobulin SCIG of the present invention

[0055] 1) Solution Preparation

[0056] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 5×10 5 cells / mL of cell suspension;

[0057] Effector cell suspension: Take the revived THP-1-NF-kB and add RPMI 1640 containing 10% FBS to a concentration of 7.5×10 5 cells / mL of cell suspension;

[0058] SCIG solution series: SCIG was prepared by adding RPMI 1640 containing 10% FBS to a concentration of 5 mg / ml as the starting working concentration. The solution was then diluted to six concentrations using RPMI 1640 containing 10% FBS in a 4-fold serial dilution ratio: 5 mg / ml, 1.25 mg / ml, 0.3125 mg / ml, 0.078125 mg / ml, 0.01953125 mg / ml, 0.004882813 mg / ml, and 0.0012207034 mg / ml, resulting in a total of seven SCIG solutions with a concentration range of 0.001 to 5 mg / ml.

[0059] 2) ADCP biological activity detection

[0060] Target cell suspension was added to a 96-well plate at 40 μl / well, followed by addition of a series of SCIG solutions at 20 μl / well. The plates were incubated at room temperature for 30 minutes. Finally, effector cell suspension was added at 40 μl / well to achieve an effector-target ratio of 3:2 in each well. The plates were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was then removed and 80 μl / well of Fire-LumiTM luciferase assay working solution was added. The plates were then incubated for 5-10 minutes. Relative luminescence units were read using a PHERAstar FSX at room temperature.

[0061] 3) Calculation

[0062] The relative luminescence unit value was used as the ordinate and the logarithm of the SCIG solution concentration was used as the abscissa. The dose-effect curve of SCIG was fitted using a four-parameter curve equation regression model. The EC value of SCIG was obtained based on the dose-effect curve. 50 value.

[0063] Example 5 Detection of ADCP biological activity of subcutaneous immunoglobulin SCIG of the present invention

[0064] 1) Solution Preparation

[0065] Target cell suspension: Take the revived CHO-K1 / SPIKE_SARS2 and add RPMI 1640 containing 10% FBS to a concentration of 5×10 5 cells / mL of cell suspension;

[0066] Effector cell suspension: Take the revived THP-1-NF-kB and add RPMI 1640 containing 10% FBS to a concentration of 7.5×10 5 cells / mL of cell suspension;

[0067] SCIG solution series: SCIG was prepared by adding RPMI 1640 containing 10% FBS to a 5 mg / ml solution as the starting working concentration. The solution was then diluted to seven concentrations using RPMI 1640 containing 10% FBS in a 3-fold serial dilution ratio: 5 mg / ml, 1.67 mg / ml, 0.556 mg / ml, 0.185 mg / ml, 0.062 mg / ml, 0.021 mg / ml, 0.0069 mg / ml, and 0.0023 mg / ml, for a total of eight SCIG solutions with a concentration range of 0.002 to 5 mg / ml.

[0068] 2) ADCP biological activity detection

[0069] Target cell suspension was added to a 96-well plate at 40 μl / well, followed by addition of a series of SCIG solutions at 20 μl / well. The plates were incubated at room temperature for 30 minutes. Finally, effector cell suspension was added at 40 μl / well to achieve an effector-target ratio of 3:2 in each well. The plates were then incubated at 37°C, 5% CO2 for 4 hours. The 96-well plate was then removed and 80 μl / well of Fire-LumiTM luciferase assay working solution was added. The plates were then incubated for 5-10 minutes. Relative luminescence units were read using a PHERAstar FSX at room temperature.

[0070] 3) Calculation

[0071] The relative luminescence unit value was used as the ordinate and the logarithm of the SCIG solution concentration was used as the abscissa. The dose-effect curve of SCIG was fitted using a four-parameter curve equation regression model. The EC value of SCIG was obtained based on the dose-effect curve. 50 value.

[0072] The technical solution of the present invention is further illustrated below through experimental examples. The information of human immunoglobulin samples and reference substances involved in the experimental examples is shown in the following table:

[0073] Table 1 Sample and reference substance information

[0074]

[0075] Experimental Example 1 Study on the activity detection method of human immunoglobulin against SARS-CoV-2 ADCP

[0076] 1. Selection of effector cells in ADCP activity assays of human immunoglobulins

[0077] 1. Experimental methods

[0078] 1.1 Cell culture and passaging

[0079] Cell Thawing: Add 4 ml of prewarmed culture medium (Probio, RD00941, RPMI 1640 + 10% FBS) to a 15 ml centrifuge tube. Thaw the cryovial rapidly in a 37°C water bath. Transfer the thawed cell suspension to the aforementioned centrifuge tube. Centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium (Probio, RD00941, RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B). Count the cells, seed the cells at an appropriate density in a cell culture dish, gently shake to mix, and incubate in a 5% CO2, 37°C incubator.

[0080] Cell passaging: (1) Passaging of effector cells THP-1-NF-κB, GS-J2B-FCGR2A, and GS-J8: Pipette the cells into a 15 ml centrifuge tube and centrifuge at 800 rpm for 5 minutes. Resuspend the cell pellet in complete culture medium (90% RPMI 1640 + 10% FBS + 400 μg / ml Hygromycin B) and count the cells. Then, seed the cells at an appropriate density into a cell culture dish, shake gently to mix, and culture in a 5% CO2, 37°C incubator.

[0081] (2) Passaging of target cells CHO-K1 / SPIKE_SARS2 (purchased from ATCC): Collect the supernatant in a 15 ml centrifuge tube, wash once with preheated DPBS, add an appropriate amount of preheated Accutase digestion solution and digest in a 37°C incubator for a certain time (the specific time depends on the cell state), add an appropriate amount of complete culture medium (Probio, RD00819, F-12 + 10% FBS + 8 μg / ml Puromycin) to terminate the digestion, centrifuge at 800 rpm for 5 minutes, add an appropriate amount of complete culture medium, count, and inoculate at an appropriate density in a cell culture dish. After gently shaking to mix, place in a 5% CO2, 37°C incubator for culture.

[0082] 1.2 ADCP reporter gene experimental steps

[0083] (1) Target cells CHO-K1 / SPIKE_SARS2 were collected by centrifugation and resuspended in 1 ml of ADCP assay buffer (RPMI 1640 + 10% FBS).

[0084] (2) Based on the corresponding concentration of human immunoglobulin as the sample starting concentration, prepare the corresponding concentration sample solution using ADCP experimental buffer.

[0085] (3) Adjust the target cell density (5E5 cells / ml) with ADCP assay buffer and transfer the target cell suspension into a 96-well assay plate (40 μl / well).

[0086] (4) Transfer the prepared sample solution (see Table 2 for the sample solution concentration in this experiment) at 20 μl / well to the corresponding wells of a 96-well plate.

[0087] (5) Incubate the test plate at room temperature for 30 minutes.

[0088] (6) Collect the effector cells (THP-1-NF-κB, GS-J2B-FCGR2A, GS-J8) and resuspend them in ADCP assay buffer.

[0089] (7) Based on an E / T ratio of 3:2 (effector-target ratio, the ratio of effector cells to target cells), the effector cell density (7.5E5 cells / ml) was adjusted with ADCP assay buffer and the effector cell suspension was transferred to the corresponding wells of a 96-well assay plate (40 μl / well).

[0090] (8) Incubate the experimental plate in a cell culture incubator (37°C / 5% CO2) for 4 hours.

[0091] (9) After the incubation, take out the 96-well test plate and add Fire-Lumi TM Luciferase assay kit working solution (80 μl / well) was added to the corresponding wells and incubated for 5-10 minutes.

[0092] (10) Chemiluminescence values were read using PHERAstar FSX at room temperature.

[0093] Table 2 Experimental design for exploring effector cells

[0094]

[0095] Note: Sources and genetic modification information of effector cells: (1) THP-1-NF-κB, purchased from Probio, this cell naturally expresses CD32a (FcγRIIa) and CD64a (FcγRIa), and is stably transfected with the NF-κB-Luc luciferase reporter gene; (2) GS-J2B-FCGR2A (Jurkat / FCGR2A / NFAT-Luc), purchased from Probio, this cell is a genetically engineered Jurkat cell, and is stably transfected with the NF-κB-Luc luciferase reporter gene; (3) GS-J8 (Jurkat / NF-κB-Luc), purchased from Probio, this cell is a genetically engineered Jurkat cell, and is stably transfected with the NF-κB-Luc luciferase reporter gene.

[0096] 1.3 ADCP data analysis

[0097] The raw data of ADCP experiments were exported and analyzed using Microsoft Office Excel.

[0098] 2. Results

[0099] CHO-K1 / SPIKE_SARS2 was used as the target cell and SCIG was used as the sample. Under the condition of E / T = 3:2, SCIG was prepared according to the scheme in Table 2. 1.333 mg / ml was used as the starting working concentration, and SCIG was serially diluted. A 6-fold serial dilution ratio was used to dilute 6 concentration points. The luciferase reporter gene method was used to carry out ADCP effect detection experiments of SCIG on the target cell CHO-K1 / SPIKE_SARS2 on three different effector cells (THP-1-NF-κB, GS-J8, and GS-J2B-FCGR2A).

[0100] The experimental results are as follows Figure 1 As shown, at a starting working concentration of 1.333 mg / ml, SCIG induced ADCP in both effector cell types (THP-1-NF-κB and GS-J2B-FCGR2A) against target CHO-K1 / SPIKE_SARS2 cells, resulting in positive cytotoxicity. However, the response curves for GS-J8 effector cells showed no clear dose-response relationship, indicating that SCIG was ineffective in inducing ADCP against target cells. THP-1-NF-κB and GS-J2B-FCGR2A effector cells exhibited distinct S-shaped dose-response curves, with the ADCP effect becoming increasingly pronounced and increasing exponentially with increasing SCIG dose. The presence or absence of a dose-response curve is closely related to the reliability and validity of the research method, and therefore GS-J8 was not selected as an effector cell in this method.

[0101] Within the linear concentration range of the sigmoidal dose-response curve, changes in drug dose and effect exhibit a relatively stable proportional relationship. When GS-J2B-FCGR2A cells were used as effector cells to detect the SCIGADCP effect, the linear range for the SCIG sample was limited to 0.00617284–0.2222222 mg / ml (determined by the concentrations at which the upper and lower plateaus of the curve began to appear), encompassing only three effective concentration points. In contrast, when THP-1-NF-κB cells were used as effector cells to detect the SCIGADCP effect, the linear range for the SCIG sample extended to at least 0.001028807–1.333333 mg / ml (because the curve still did not reach an upper plateau), encompassing at least five effective concentration points. This wider effective concentration range reflects the advantages of the method, including lower sample requirements, the ability to accommodate a wider range of samples, and superior sensitivity. Therefore, THP-1-NF-κB cells were selected as effector cells for subsequent system development.

[0102] 2. Optimization of sample concentration in ADCP activity assay of human immunoglobulin

[0103] 1. Experimental methods

[0104] In the experiment of effector cells, the initial concentration and gradient of SCIG were not well designed, which resulted in the failure of the ADCP dose-effect curve to have an upper plateau (see Figure 1 ), therefore, CHO-K1 / SPIKE_SARS2 was used as the target cell and THP-1-NF-κB was used as the effector cell. The starting concentration and dilution gradient of SCIG were redesigned, and the ADCP activity of SCIG was further detected according to the ADCP reporter gene experimental steps in the effector cell exploration experiment. The experimental design is shown in Table 3.

[0105] Table 3 Experimental design for confirmation of optimal sample starting concentration

[0106]

[0107] 2. Experimental results

[0108] Under the condition of E / T=3:2, SCIG was used as the starting working concentration of 5 mg / ml, and SCIG was serially diluted. When 8 concentration points were diluted with a 3-fold serial dilution ratio, the ADCP dose-response effect of human immunoglobulin on the effector cells THP-1-NF-κB against the target cells CHO-K1 / SPIKE_SARS2 was detected.

[0109] like Figure 2As shown in the results, SCIG can induce the ADCP effect of effector cells THP-1-NF-κB on target cells CHO-K1 / SP IKE_SARS2, and the killing result is positive. The dose-effect curve has a good fit and reaches the expected saturation state (i.e., it has upper and lower platforms). Therefore, the starting working concentration of subcutaneous injection of human immunoglobulin is 5 mg / ml, which is the optimal starting working concentration for ADCP activity detection. Further gradient dilution can obtain a dose-effect curve with a good fit.

[0110] 3. Methodological Validation Experiment of the ADCP Activity Detection Method for Human Immunoglobulin

[0111] When the ADCP activity was tested at a starting working concentration of 5 mg / ml for subcutaneous injection of human immunoglobulin and 8 concentration points were diluted in a 3-fold gradient, SCIG could induce the ADCP effect of effector cells THP-1-NF-κB on target cells CHO-K1 / SPIKE_SARS2. The dose-effect curve had a good fit, but the platform under the dose-effect curve was too long (see Figure 2 ), the lower plateau is too long, which may overestimate the stability of the effect of human immunoglobulin at higher doses. Therefore, we retained the upper plateau concentration at 5 mg / ml, reduced one dilution point, and adjusted the dilution factor to 4 for methodological validation experiments. The experimental design is shown in Table 4.

[0112] Table 4 Method validation experimental design

[0113]

[0114] According to the experimental design in Table 4, the sample concentrations (mg / mL) of human immunoglobulin (SCIG) were set at: 5 mg / ml, 1.25 mg / ml, 0.3125 mg / ml, 0.078125 mg / ml, 0.01953125 mg / ml, 0.004882813 mg / ml, and 0.0012207034 mg / ml. Methodological validation of ADCP activity assays for SCIG was performed using the ADCP reporter gene protocol used in the effector cell exploration experiment, with three replicates performed for each sample.

[0115] 1. Specificity verification experiment

[0116] 1.1 Experimental Methods

[0117] 1.1.2 Target cell specificity

[0118] 293T cells were used as target cells instead of CHO-K1 / SPIKE_SARS2 cells, and the CHO-K1 / SPIKE_SARS2 cell line was set as a control. ADCP activity was detected using the ADCP reporter gene experimental steps in the effector cell exploration experiment.

[0119] 1.1.2 Effector cell specificity

[0120] THP-1 original cell line (THP-1) was used instead of THP-1-NF-κB cells as effector cells, and THP-1-NF-κB cells were set as controls. ADCP activity was detected using the ADCP reporter gene experimental steps in the effector cell exploration experiment.

[0121] 1.1.3 Specificity of test samples

[0122] Excipients (polysorbate 80 + glycine, negative control) were used instead of SCIG as the sample, and SCIG was set as the control. ADCP activity was detected using the ADCP reporter gene experimental steps in the effector cell exploration experiment.

[0123] 1.2 Experimental Results

[0124] The results are as follows Figure 3 As shown, CHO-K1 / SPIKE_SARS2 cells were used as target cells to detect the ADCP activity of human immunoglobulin, and had significantly better fluorescence response values than 293T cells, indicating that 293T cells were not suitable for detecting the ADCP effect of human immunoglobulin as target cells.

[0125] The results are as follows Figure 4 As shown, THP-1-NF-κB cells used as effector cells to detect the ADCP activity of human immunoglobulin have significantly better fluorescence response values than THP-1 cells, indicating that THP-1 cells are not suitable for detecting the ADCP effect of human immunoglobulin as effector cells.

[0126] The results are as follows Figure 5 As shown, only SCIG showed ADCP activity, and the negative control group did not show an upward trend in the detection signal, and had no ADCP activity under this experimental system, indicating that the method of the present invention can specifically and effectively detect the ADCP activity of SCIG and has good specificity.

[0127] The above results show that CHO-K1 / SPIKE_SARS2 has the best positive detection results as a target cell, reflecting the specificity of this experimental system for target cells; only THP-1-NF-kB cells have a positive detection result as an effector cell, reflecting the specificity of this experimental system for effector cells THP-1-NF-kB; only human immunoglobulin samples have a positive detection result, reflecting the specificity of this experimental system for the detection sample. Therefore, the method of the present invention can effectively detect the ADCP activity of human immunoglobulin and has good specificity.

[0128] 2. Accuracy verification test

[0129] 2.1 Experimental Methods

[0130] According to the experimental design in Table 4, the concentration of human immunoglobulin (SCIG) samples was within the sensitivity range of this method. Within this concentration range, ADCP accuracy was tested on SCIG using the ADCP reporter gene protocol used in the effector cell exploration experiment, with three replicates performed for each sample.

[0131] The data analysis method is as follows: select the detection signal value corresponding to the concentration point of the curve for linear analysis, calculate the theoretical signal value according to the curve fitting formula, perform linear fitting on the calculated detection value and the theoretical value, and obtain R 2 The values were then compared for the three replicates R 2 The RSD of the values.

[0132] 2.2 Experimental Results

[0133] The results are as follows Figure 6 As shown in Table 5, the relative standard deviation of the signal values of three repeated detections of this method is within 5%, which has good accuracy.

[0134] Table 5 Verification of the accuracy of the ADCP reporter gene assay

[0135]

[0136] 3. Precision verification test

[0137] 3.1 Experimental Methods

[0138] SCIG with a starting working concentration of 5 mg / ml was used for intra-day and inter-day precision testing of ADCP detection. SCIG was serially diluted, and when a 4-fold serial dilution ratio was used to dilute 7 concentration points, the ADCP reporter gene experimental steps in the effector cell exploration experiment were used. Three independent tests were set up within one day, and one 96-well cell culture plate was used for each test. The intra-day precision was tested, and the RLU of the starting working concentration was counted to verify the precision of the method.

[0139] 3.2 Experimental Results

[0140] The results are as follows Figure 6 As shown in Table 6, the relative standard deviation of the detection signal values of the three plates of this method is about 15% (precision acceptance standard: the deviation must be within the range of ±30%), indicating that this method has good precision and can be used for ADCP detection of SCIG.

[0141] The above results show that the detection method of the present invention has good accuracy and precision.

[0142] Table 6 Verification of the precision of ADCP reporter gene assay

[0143]

[0144] IV. Application of ADCP Detection Method on IVIG Samples

[0145] 1. Experimental methods

[0146] To explore the applicability of the ADCP assay to IVIG samples, we conducted a dose-response experiment to investigate the ADCP effect of human immunoglobulin (IVIG) on THP-1-NF-κB effector cells against target CHO-K1 / SPIKE_SARS2 cells. Using an E / T ratio of 3:2, IVIG was serially diluted to seven concentrations using a 4-fold dilution ratio. The ADCP assay was performed using the same ADCP reporter gene protocol as used in the effector cell assay, with three replicates per sample. The experimental design is shown in Table 7.

[0147] Table 7 Experimental design for confirmation of optimal starting concentration of IVIG samples

[0148]

[0149] 2. Experimental results

[0150] like Figure 7 As shown in the figure, IVIG can also induce the ADCP effect of effector cells THP-1-NF-κB on target cells CHO-K1 / SPIKE_SARS2. The killing result is positive, the dose-effect curve has a good fit, and reaches the expected saturation state (i.e., with upper and lower platforms).

[0151] The above results show that when testing the antiviral ADCP bioactivity of human immunoglobulin products, the detection system uses THP-1-NF-κB (human leukemic mononuclear cells that naturally express CD32a (FcγRIIa) and CD64a (FcγRIa) and are stably transfected with the NF-κB-Luc luciferase reporter gene) as effector cells; the starting concentration of the human immunoglobulin sample is set at 5 mg / ml, with 4-fold dilution and 7 concentration points; the effector-target ratio is 3:2, which can produce an ADCP bioeffect on the virus-infected target cells CHO-K1 / SPIKE_SARS2. The dose standard curve drawn based on this includes a sufficient dose range that can cause the human immunoglobulin to have a range from no effect to a maximum effect, achieving the expected saturation state. The dose-response curve has a good fit, realizing accurate detection of the ADCP bioactivity of human immunoglobulin products.

[0152] In summary, the detection method of the present invention is simple to operate, has good specificity, and high precision and accuracy. It fills the current gap in the field of ADCP detection of human immunoglobulin products and is of great significance for improving the quality control and clinical application of human immunoglobulin products.

Claims

1. A method for detecting the biological activity of human immunoglobulin ADCP, characterized in that: It uses CHO-K1 / SPIKE_SARS2 as target cells, THP-1-NF-κB or GS-J2B-FCGR2A as effector cells, and adopts fluorescence reporter gene method for detection.

2. The detection method according to claim 1, wherein: The steps of the fluorescent reporter gene detection method are as follows: The target cell suspension was added to the well plate, and then a series of concentrations of human immunoglobulin solution were added for incubation. Finally, the effector cell suspension was added for incubation. After the incubation, the fluorescence was detected. The dose-effect curve was drawn with the relative luminescence unit value as the ordinate and the logarithm of the human immunoglobulin solution concentration as the abscissa, and the half-maximal effect concentration was calculated.

3. The detection method according to claim 2, wherein: The effector-target ratio of effector cells to target cells in each well of the well plate is 3:

2.

4. The detection method according to claim 2, wherein: The concentration of the human immunoglobulin solution ranges from 0.0012 to 5 mg / ml.

5. The detection method according to claim 4, wherein: The human immunoglobulin solution was diluted to 6 concentrations using a 4-fold gradient dilution ratio with 5 mg / ml as the initial working concentration; Or: Use 5 mg / ml as the starting working concentration and then dilute to 7 concentrations using a 3-fold serial dilution ratio.

6. The detection method according to claim 2, wherein: The target cell suspension, human immunoglobulin solutions with a series of concentrations and effector cell suspension are prepared using RPMI 1640 culture medium containing 10% FBS.

7. The detection method according to claim 6, wherein: The concentration of the target cell suspension was 5×10 5 cells / ml, the volume is 40 μl / well; the volume of the human immunoglobulin solution of serial concentrations is 20 μl / well; the concentration of the effector cell suspension is 7.5×10 5 cells / ml, with a volume of 40 μl / well.

8. The detection method according to claim 2, wherein: The target cell suspension was incubated with the human immunoglobulin solution at room temperature for 30 minutes; the effector cell suspension was added and incubated at 37° C. and 5% CO 2 for 4 hours.

9. The detection method according to claim 2, wherein: The fluorescence was detected using a luciferase detection kit.

10. The detection method according to claim 9, characterized in that: The luciferase detection kit is Fire-Lumi TM Luciferase Assay Kit.

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