CN114966061B

CN114966061B - Method for detecting biological activity of anti-OX 40 antibody

Info

Publication number: CN114966061B
Application number: CN202210895161.6A
Authority: CN
Inventors: 李萌; 杨雅岚; 刘春雨; 付志浩; 于传飞; 赵雪羽; 王兰; 王军志
Original assignee: National Institutes for Food and Drug Control
Current assignee: National Institutes for Food and Drug Control
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2022-10-21
Anticipated expiration: 2042-07-28
Also published as: CN114966061A

Abstract

The invention discloses a method for detecting the biological activity of an anti-OX 40 antibody, which comprises the steps of incubating HEK293-Fc gamma RIIB cells and cells over expressing OX40 with the anti-OX 40 antibody, and detecting the biological activity of the anti-OX 40 antibody by using a luciferase reporter gene method, wherein the method has higher specificity, sensitivity, accuracy and precision and is suitable for detecting different anti-OX 40 antibodies.

Description

Method for detecting biological activity of anti-OX 40 antibody

Technical Field

The invention belongs to the field of biological medicine, and relates to a method for detecting the biological activity of an anti-OX 40 antibody.

Background

OX40 (also called CD 134) and its interacting ligand OX40L (also called CD 252) are members of TNF (R) SF. OX40 is a type I transmembrane glycoprotein comprising 275 amino acids and consisting of 3 complete cysteine-rich domains (CRDs) and 1 partial shuttle-terminated CRDs with a molecular weight of about 50 kD. OX40, whether human or mouse, is predominantly expressed on activated T cells, including CD4 ⁺ /CD8 ⁺ T cells, neutrophils, NK cells, and NKT cells play an important role in the activation and proliferation of T cells.

As we know, immune checkpoints play an important role both in tumors and in infectious diseases. It has recently been reported that most of these cell surface molecules exist in soluble form, can be detected in the serum of patients and may be related to the severity of the disease and inflammatory activity, etc. Although OX40 has become a hotspot in research as a representative target of the second generation of immune checkpoints, no drugs against OX40 are on the market, and anti-OX 40 mabs currently used in tumor immunotherapy are in clinical stage I or II, such as MEDI-6469, PF-04518600, GSK-3174998, INCAGNO-1949, BMS-986178, tavolixizumab, etc.

The biological activity is the key quality attribute of antibody drugs, and the accurate determination of the biological activity is the basic prerequisite for ensuring the safety and the effectiveness of the drugs. At present, the biological activity of antibody drugs is determined mainly by establishing a corresponding cell evaluation model in vitro according to an in vivo action mechanism of the antibody drugs, performing linear or curvilinear fitting on an experimental result, and comparing the experimental result with a reference substance to evaluate the relative biological activity of the antibody drugs. In vitro, the activity determination method aiming at OX40 target spot mainly comprises enzyme-linked immunosorbent assay, surface plasmon resonance titer determination, human umbilical vein endothelial cell proliferation inhibition method and Transwell cell migration experiment. Among them, the Elisa and SPR titer assay can only reflect the binding capacity of antibody and antigen, and the Transwell cell migration assay is complicated to operate and difficult to quantify. The HUVEC proliferation inhibition method which is most widely applied at present has the defects of difficult culture of primary cells, long experimental period and high mutation degree. In the early days, a method for detecting the biological activity of OX40 (patent application No. 2019103249216) was developed, but the method has a good detection effect on an early process product (a sample with the purity of 93%), but has a poor detection effect on a final process product (a sample with the purity of 98.3%), and a high-point platform cannot be formed. In order to solve the problems, a lot of research works such as a search for sample concentration, a search for cell plate density, a search for CD3/CD28 concentration, etc. were conducted, but the results were still not ideal, and a stable high plateau and a high recovery rate could not be obtained. Based on the above, the detection method of the application is developed.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a rapid, stable, simple, accurate, specific and widely applicable method for detecting anti-OX 40 antibody activity.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides a method of detecting a biological activity of an anti-OX 40 antibody, comprising:

HEK293-Fc gamma RIIB cells and OX40 overexpressing cells were incubated with anti-OX 40 antibody and the biological activity of the anti-OX 40 antibody was detected by luciferase reporter gene method.

Further, the OX40 overexpressing cell is selected from a naturally overexpressing OX40 cell or an engineered OX40 overexpressing cell.

Further, the OX40 overexpressing cells are selected from engineered OX40 overexpressing cells.

Further, the engineered OX40 overexpressing cells also comprise a luciferase reporter gene.

Further, the engineered OX40 overexpressing cell further comprises a response element.

Further, the reactive element is selected from NF- κ B.

Further, the engineered OX40 overexpressing cells include Jurkat cells, HUVEC cells, 293 cells, COS7 cells, L929 cells, hepG2 cells, CHO cells, 3T3 cells.

Further, the engineered OX40 overexpressing cells are selected from Jurkat cells.

Further, the engineered OX40 overexpressing cells are selected from

A cell.

Further, the method may further comprise diluting the OX 40-overexpressing cells with a buffer to which a stimulating factor is added.

Further, the stimulating factor is selected from an anti-CD 3 antibody and/or an anti-CD 28 antibody.

Further, the stimulating factor is selected from the group consisting of antibodies against CD 3.

Further, the concentration of the anti-CD 3 antibody is selected from 2 to 4 mu g/mL.

Further, the concentration of the anti-CD 3 antibody is selected from 4 μ g/mL.

Further, the buffer is RPMI-1640 medium.

Further, the RPMI-1640 medium was supplemented with 10% FBS.

Further, the density of OX 40-overexpressing cells after dilution was 8X 10 ⁵ Each/ml.

Further, OX40 overexpressing cells were seeded at a number of 8X 10 ⁵ Per well.

Furthermore, HEK293-Fc gamma RIIB cells were cultured for 5 to 6h, and then incubated with OX 40-overexpressing cells and anti-OX 40 antibody.

Further, the method comprises:

1) Planting HEK293-Fc gamma RIIB cells into a detection plate for culturing for 5 to 6 hours;

2) Adding the diluted OX 40-overexpressing cells to a test panel in which HEK293-Fc γ RIIB cells are cultured;

3) Adding serially diluted anti-OX 40 antibody for culture;

4) A detection reagent is added to each well, and enzyme-labeled detection is performed.

Further, the detection reagent is a luciferase detection reagent.

Furthermore, HEK293-Fc γ RIIB cells were diluted and added to the assay plate.

Furthermore, the dilution density of HEK293-Fc gamma RIIB cells was 5.0X 10 ⁵ One per ml.

Furthermore, the number of HEK293-Fc γ RIIB cells inoculated was 5.0X 10 ⁴ Per well.

Further, the buffer for diluting HEK293-Fc γ RIIB was RPMI-1640 medium.

Further, 10% FBS was added to the RPMI-1640 medium.

Further, the detection plate is a 96-hole enzyme label plate.

Further, the anti-OX 40 antibody was diluted by a factor of 3.

Further, the buffer for diluting the anti-OX 40 antibody was RPMI-1640 medium.

Further, 10% FBS was added to the RPMI-1640 medium.

In a specific embodiment of the invention, the method comprises:

HEK 293-Fc. Gamma. RIII B cells were diluted to a density of 5.0X 10 using 10% FBS-added RPMI-1640 medium ⁵ Inoculating the diluted HEK293-Fc gamma RIIB cells into a 96-well enzyme label plate for culturing for 5-6h;

using RPMI-1640 medium supplemented with anti-CD 3 antibody and 10% FBS

Diluting the cells to a density of 8.0X 10 ⁵ Per ml, diluted

Adding the cells into an ELISA plate cultured with HEK293-Fc gamma RIIB cells;

adding 3-fold serial dilutions of anti-OX 40 antibody at 37 ℃ and 5% CO ₂ The culture box is cultured for 16-18h, and luciferase detection reagent is added for enzyme labeling detection.

A second aspect of the invention provides the use of any one of:

1) Application of HEK293-Fc gamma RIIB cells in detecting the biological activity of anti-OX 40 antibodies;

2) Use of HEK293-Fc γ RIIB cells in combination with anti-CD 3 antibodies for the detection of the biological activity of anti-OX 40 antibodies.

In a third aspect, the invention provides a product for detecting anti-OX 40 antibodies, the product comprising HEK293-Fc γ RIIB cells, OX40 overexpressing cells, and anti-CD 3 antibodies.

Further, the product also comprises a detection reagent.

Further, the detection reagent is a luciferase detection reagent.

Further, the OX40 overexpressing cells are selected from

。

In a fourth aspect, the invention provides an apparatus for detecting the biological activity of an anti-OX 40 antibody, the apparatus comprising means for performing the method according to the first aspect of the invention.

In an alternative embodiment of the invention, the apparatus comprises a cell culture device, a sample application device, and a detection device for performing the method according to the first aspect of the invention.

In an alternative embodiment of the invention, the apparatus comprises a cell culture device, a sample application device, a detection device and an output device for performing the method according to the first aspect of the invention.

The invention has the advantages and beneficial effects that:

the invention provides a method for detecting the biological activity of an anti-OX 40 antibody, which has the advantages of low cost, simple and convenient operation, short period, no need of animal experiments, stable and reliable result, high accuracy and good specificity, is suitable for detecting OX40 antibodies with different purities, is favorable for promoting the research and development of medicines, controlling the quality and clinically applying, and has higher application value.

Drawings

FIG. 1 is a diagram showing the specificity of the bioactivity detection method.

FIG. 2 is a linear verification diagram of the bioactivity detection method.

Detailed Description

The present invention will be described in further detail with reference to examples.

The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 establishment of a method for detecting the biological Activity of an antibody Using HEK293-Fc γ RIIB cells

1. Optimization of cell culture density

To determine the biological activity assay, cell culture density was optimized.

The specific method comprises the following steps:

(1) Preparation of assay buffer: RPMI 1640 basal medium was added with 10% volume of FBS.

(2) Preparation of an analysis medium:

addition of CD3 and CD28: the RPMI 1640 basic medium was added with 10% FBS by volume, 4. Mu.g/mL anti-human CD3 antibody at the final concentration, and 4. Mu.g/mL anti-human CD28 antibody at the final concentration.

(3) Cell plating:

HEK293-Fc γ RIIB cells (donated to the Central Hospital) were digested, centrifuged and resuspended in DMEM complete medium at a density of 5X 10 ⁵ one/mL or 3X 10 ⁵ 100 mul/well of each cell were added to a white opaque cell culture plate at 37 ℃ in a carbon dioxide incubator with 5% CO ₂ Culturing for 5-6h under the condition, discarding the supernatant,

resuspending the above assay medium after centrifugation of the cells (laboratory preparation, see patent 2019103249216) by pipetting and adjusting the density to 1X 10 ⁶ 8X 10 pieces/mL ⁵ 4X 10 units/mL ⁵ piece/mL, 50 μ l/well was added to the above white opaque cell culture plate.

(4) Sample dilution: the sample (pre-process sample, purity 93%) was diluted 3-fold with 1.5 μ g/ml as starting concentration using assay buffer for a total of 10 concentration points.

(5) Sample adding: correspondingly adding a gradient dilution sample, 50 ul per hole. The cell plates were placed in a carbon dioxide incubator at 37 ℃ with 5% CO ₂ Culturing under the condition for 16-18h.

(6) And (3) detection: the Bright-Glo Luciferase Assay System, which had been returned to room temperature, was added at 100. Mu.l/well to a 96-well leukocyte culture plate. Incubating at room temperature in dark for 3-5min, measuring the fluorescence intensity of each hole with a multifunctional microplate reader, and adopting a Luminescence mode with integration time of 500-1000ms; and fitting a four-parameter S-shaped curve by taking the concentration of the antibody as an abscissa and the chemiluminescence value as an ordinate.

Detection of different titers 50. Mu.g/mL anti-OX 40 antibody was used as a control, and cell activity assays were repeated for samples with 64%,80%,100%,125%,156% active titer levels, each titer level was independently determined 3 times.

(7) The cell activity assay was repeated for each of the test samples at 100% titer, 64% titer, and 156% titer activity titer levels, each titer level was independently measured 3 times.

(8) Results

The results showed that when Jurkat-OX40 cell density was 8X 10 ⁵ Number/ml, larger curve window, jurkat-OX40 cell density set at 8X 10 ⁵ HEK293-Fc gamma RIIB cell density of 5X 10/ml ⁵ At the time of individual/ml, no cross phenomenon appears at the high-point plateau phase, the recovery rate is good, the RSD is less than 10, and the density of HEK293-Fc gamma RIIB cells is determined to be 5 multiplied by 10 ⁵ Each/ml.

2. Optimization of assay media

To determine the bioactivity assay, the assay medium was optimized using HEK293-Fc γ RIIB cells (5X 10) at an optimized cell density ⁵ Individuals/ml) (donation to the midrange),

cells (8X 10) ⁵ Individual/ml) (see patent 2019103249216) was optimized for analysis of the medium.

The specific method comprises the following steps:

(1) Preparation of assay buffer: RPMI 1640 basal medium was taken and 10% FBS by volume was added.

(2) Preparation of an analysis medium:

a. addition of CD3 alone: adding 10% FBS (FBS) by volume and an anti-human CD3 antibody with the final concentration of 4 mu g/mL into an RPMI 1640 basic culture medium;

b. addition of CD3 and CD28: taking an RPMI 1640 basic culture medium, and adding 10% FBS, an anti-human CD3 antibody with a final concentration of 4 mug/mL and an anti-human CD28 antibody with a final concentration of 4 mug/mL;

c. without addition of CD3 and CD28: RPMI 1640 basal medium was taken and 10% FBS by volume was added.

(3) Cell plating: HEK293-Fc gamma RIIB cells were digested, centrifuged and resuspended in DMEM complete medium at a density of 5X 10 ⁵ Adding 100 mul/well of each cell into a white opaque cell culture plate, and placing the white opaque cell culture plate in a carbon dioxide incubator at 37 ℃ and 5% CO ₂ Culturing for 5-6h under the condition, discarding the supernatant,

after cell centrifugation, the cells were respectively blown out and resuspended in the three analytical media mentioned above, the density was adjusted to 8X 10 ⁵ pieces/mL, 50 μ l/well were added to the above white opaque cell culture plates.

(4) Sample dilution: the sample (pre-process sample, purity 93%) was diluted 3-fold with 9 μ g/ml as starting concentration using analysis buffer for a total of 10 concentration points.

(5) Sample adding: and correspondingly adding a gradient dilution sample, wherein each well is 50 mu l. The cell plates were placed in a carbon dioxide incubator at 37 ℃ with 5% CO ₂ Culturing under the condition for 16-18h.

(6) And (3) detection: the Bright-Glo Luciferase Assay System, which had been returned to room temperature, was added at 100. Mu.l/well to a 96-well leukocyte culture plate. Incubating at room temperature in dark place for 3-5min, measuring the fluorescence intensity of each well with a multifunctional microplate reader, and adopting a Luminescence mode to integrate the time of 500-1000ms generally; and fitting a four-parameter S-shaped curve by taking the concentration of the antibody as an abscissa and the chemiluminescence value as an ordinate.

(7) As a result, the

As shown in Table 1, the signal-to-noise ratio was highest in the CD3 group, and the test for OX40 antibody was performed by selecting the assay medium to which only anti-human CD3 antibody was added.

Based on the results of the above parameter optimization experiments, the experimental parameters collated to obtain a reporter gene based approach to the biological activity of anti-OX 40 antibodies are shown in Table 2:

example 2 detection of antibody Activity in Raji cells

And detecting the influence of different analysis culture media on the detection of the activity of the antibody by using Raji cells and HEK293-Fc gamma RIIB cells of different cell lines respectively by using optimized parameters. The method comprises the following steps:

raji cells + Jurkat-OX40-NF κ B-Luc cells:

(2) Preparation of an analysis medium:

a. addition of CD3 alone: adding 10% FBS (FBS) by volume and an anti-human CD3 antibody with the final concentration of 8 mug/mL into an RPMI 1640 basic culture medium;

b. addition of CD3 and CD28: taking an RPMI 1640 basic culture medium, and adding 10% FBS, an anti-human CD3 antibody with a final concentration of 8 mug/mL and an anti-human CD28 antibody with a final concentration of 8 mug/mL;

c. without CD3 and CD28: RPMI 1640 basal medium was added with 10% volume of FBS.

(3) Cell plating: raji cell centrifugation is followed by pipetting with assay buffer and resuspension adjusted to a density of 2X 10 ⁶ Per mL;

after centrifugation, the cells were resuspended by pipetting with the three analytical media mentioned above, adjusted to a density of 4X 10 ⁵ One per mL. Two cells were added to a white opaque cell culture plate at 25 μ l/well, respectively.

(4) Sample dilution: the sample (earlier process sample, purity 93%) was diluted with an assay buffer at 200 μ g/ml as the starting concentration, 2-fold gradient at the second concentration point, and 4-fold dilutions were made at the following 8 concentration points, for a total of 10 concentration points. .

The detection method of HEK293-Fc gamma RIIB cells + Jurkat-OX40-NF kappa B-Luc cells is the same as that in example 1, and the specific steps are as follows:

(2) Preparation of an analysis medium:

after the cells are centrifuged, the cells are respectively blown by the three analysis culture media for heavy suspension, and the density is adjusted to be 8 multiplied by 10 ⁵ pieces/mL, 50 μ l/well were added to the above white opaque cell culture plates.

(4) Sample dilution: the sample (earlier process sample, purity 93%) was diluted 3-fold with 9 μ g/ml as starting concentration using assay buffer for a total of 10 concentration points.

(6) And (3) detection: the Bright-Glo Luciferase Assay System, which had been returned to room temperature, was added to a 96-well leukocyte culture plate at 100. Mu.l/well. Incubating at room temperature in dark for 3-5min, measuring the fluorescence intensity of each hole with a multifunctional microplate reader, and adopting a Luminescence mode with integration time of 500-1000ms; and fitting a four-parameter S-shaped curve by taking the concentration of the antibody as an abscissa and the chemiluminescence value as an ordinate.

As shown in Table 3, the signal-to-noise ratio was highest with only anti-human CD3 antibody using HEK293-Fc γ RIIB cells, and was significantly higher with HEK293-Fc γ RIIB cells than with Raji cells. Finally, HEK293-Fc gamma RIIB and anti-human CD3 antibody are added as detection reagents.

Example 3 validation of the method of measuring biological Activity

1. Attribute validation

The PDL1/VEGF double-antibody sample, PDL1/OX40 blank Buffer are used as control samples, and the PDL1/OX40 double-antibody sample (later process sample, purity is 98.3%) is used for biological activity detection, and the specificity of the optimized method is examined.

Acceptance criteria: PDL1/VEGF diabody samples and PDL1/OX40 blank Buffer should have no specific sigmoid curve and no dose-effect relationship, while PDL1/OX40 diabody samples have specific sigmoid curves.

As shown in FIG. 1, the PDL1/VEGF double antibody sample (group 2) and the PDL1/OX40 blank Buffer (group 3) have no specific sigmoid curve, while the PDL1/OX40 double antibody sample (group 1) has a specific sigmoid curve and the specificity meets the acceptance criteria.

2. Accuracy verification

The cell activity test was repeated for the samples (later process samples, purity 98.3%) with 64%,80%,100%,125%,156% activity titer levels, respectively, and each titer level was independently measured 3 times.

Acceptance criteria: the relative bias of each titer level to titer measurement should be within ± 20%; the mass standard range for the relative titer (recovery) per titer level was between 70% and 130%. Relative activity (%) = reference EC ₅₀ Sample EC ₅₀ X100%. Recovery (%) = measured relative activity/theoretical relative activity × 100%.

As a result: the results are shown in tables 4 and 5, with recovery rates in the range of 80% -120%, relative bias in the range of ± 20%, and relative accuracy meeting the acceptable standards.

3. Precision verification

Different testers repeatedly perform biological activity detection on the test samples with 64%,80%,100%,125% and 156% activity titer levels on different dates, and each titer level is independently measured for 3 times.

Acceptance criteria: intermediate precision was assessed as Geometric Standard Deviation (GSD) or geometric coefficient of variation (% GCV) of the relative titers measured at each titer level. The geometric coefficient of variation (% GCV) of each titer level relative to the titer measurement is not greater than 20.0.

As shown in Table 6, the result was that the geometric coefficient of variation (% GCV) was not more than 10.0, and that the precision was high.

4. Linear verification

The logarithm of the theoretical titer value (abscissa) is plotted against the logarithm of the corresponding titer measurement value (ordinate), and linear regression is performed by using the least square method.

Acceptance criteria: the slope of the regression equation should be in the range of 0.70-1.40; the correlation coefficient of the linear regression equation should not be less than 0.95.

As a result: the linear regression equation of the titer theoretical value pairs and the logarithm of the measured value is shown in fig. 2, the fitted linear regression equation is y =1.0145x-0.0079, the gradient of the regression equation is 1.0145, and the correlation coefficient is 0.95.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims

1. A method of detecting a biological activity of an anti-OX 40 antibody comprising:

2. The method of claim 1, wherein the OX40 overexpressing cells are selected from natural OX40 overexpressing cells or engineered OX40 overexpressing cells.

3. The method of claim 2, wherein the engineered OX40 overexpressing cell further comprises a luciferase reporter gene.

4. The method of claim 3, wherein the engineered OX 40-overexpressing cells are selected from

A cell.

5. The method of any one of claims 1-4, further comprising diluting the OX 40-overexpressing cells with a stimulus-added buffer.

6. The method of claim 5, wherein the stimulating factor is selected from the group consisting of anti-CD 3 antibodies and/or anti-CD 28 antibodies.

7. The method of claim 6, wherein the concentration of the anti-CD 3 antibody is selected from 2 to 4 μ g/mL.

8. The method of claim 5, wherein the density of OX 40-overexpressing cells after dilution is 8 x 10 ⁵ Each/ml.

9. The method of claim 1, wherein HEK293-Fc γ RIIB cells are cultured for 5 to 6h and then incubated with OX 40-overexpressing cells and anti-OX 40 antibody.

Use of HEK293-Fc γ RIIB cells for the detection of biological activity of anti-OX 40 antibodies.

Use of HEK293-Fc γ RIIB cells in combination with anti-CD 3 antibodies for the detection of biological activity of anti-OX 40 antibodies.