CN110982791A

CN110982791A - Preparation and screening method of hybridoma cells secreting AXL antibody

Info

Publication number: CN110982791A
Application number: CN201911371276.XA
Authority: CN
Inventors: 白先宏
Original assignee: Biotech Pharmaceuticals Co Ltd
Current assignee: Biotech Pharmaceuticals Co Ltd
Priority date: 2019-12-26
Filing date: 2019-12-26
Publication date: 2020-04-10

Abstract

The application provides a preparation and screening method of hybridoma cells secreting AXL antibody, belonging to the field of biology and antibody engineering. The method for preparing and screening hybridoma cells secreting AXL antibody according to the present application can produce spleen cells capable of producing high titer after primary immunization and several booster immunizations, and can produce hybridoma cells capable of continuously producing high titer and continuously dividing and proliferating by fusing spleen cells and myeloma cells.

Description

Preparation and screening method of hybridoma cells secreting AXL antibody

Technical Field

The application relates to the fields of biology and antibody engineering, in particular to a preparation and screening method of hybridoma cells secreting AXL antibody.

Background

AXL belongs to Tyro-3 family receptor tyrosine kinase, also called Ark, UFO or Tyro-7, the ligand is Gas6, Gas6 is a protein with the molecular weight of 70kDa, and is homologous with anticoagulant factor S, after the AXL is combined with the ligand and activated, a series of signal cascades are caused, and PI3K-Akt and Ras/Erk and β -catenin/TCF pathways are activated.

AXL is highly expressed abnormally in various tumor tissues, such as breast cancer, colon cancer, prostate cancer, gastric cancer, endometrial cancer, renal cancer, hepatocellular carcinoma, thymic tumor, esophageal sarcoma, chronic myelolymphoma, acute myelogenous lymphoma, melanoma, and head and neck tumors.

AXL has been shown to be abnormally high-surface in highly aggressive breast cancers and highly correlated with tumor migration. In vitro experiments have shown that tumor tissue migration and invasion is dependent on AXL activity, which is inhibited by treatment with antibodies (WO 04008147). Similarly, inhibition of AXL in vivo, such as expression of an inactive AXL or inhibition of AXL expression using siRNA, effectively blocks the growth of transplanted tumors in mice.

To date, several AXL antibodies have been developed for clinical treatment, such as US20190352407a1, and some AXL antibodies have been prepared as drug-conjugated antibodies, such as chinese patent CN110536703A, for direct killing of tumor tissues. AXL is considered to be a high-grade tumor tissue in various tumor tissues, and as a main driver in the process of generation and development of tumors, the drug development potential of AXL is not completely released. The main reasons are as follows: first, the blocking function of the existing antibodies is not high; secondly, the strategy of killing the tumor by relying on the European Union drug may limit the application range of the tumor due to potential toxicity. Based on the inference, the antibody with high blocking activity is obtained by screening, and therapeutic means except the ADC strategy is explored through a genetic engineering means, so that the potential druggy advantage is achieved, and an alternative solution is provided for the unmet clinical requirement.

Disclosure of Invention

The application discloses a method for preparing and screening hybridoma cells secreting AXL antibody, which comprises the following steps:

injecting 50 mu g of AXL immunogen mixture into an immunized mouse, detecting the antibody titer, stopping immunization when the antibody titer reaches 106;

taking a spleen of a mouse, and preparing splenocyte fluid; adding a PEG solution into the spleen cell fluid and the myeloma cell fluid under the condition of water bath at 40 ℃ for fusion, and centrifuging to obtain a hybridoma cell mixture;

resuspending the hybridoma cell mixture;

coating an AXL antigen, adding a hybridoma cell mixture, incubating for 1h, washing and patting dry, adding a secondary antibody, incubating and patting dry, and terminating reaction and screening to obtain a positive hybridoma cell secreting an AXL antibody; and the affinity of the hybridoma cells was detected by ELISA.

In some of the foregoing embodiments, the AXL immunogen mixture is obtained by mixing AXL-mFc fusion protein and freund's complete adjuvant in a mass ratio of 1: 1;

the method also comprises 3 times of boosting immunity before the detection of the antibody titer; the immunogen for strengthening immunity is obtained by mixing AXL-mFc fusion protein and Freund's incomplete adjuvant according to the mass ratio of 1: 1; the booster immunization was performed once every two weeks.

In the embodiment, after the immunogen and Freund's complete adjuvant are used for immunizing the mouse, the boosting immunization is carried out for multiple times, so that the binding effect of the antibody can be improved, and the affinity of the generated antibody and the antigen can be improved.

In some of the foregoing embodiments, the myeloma cells are SP20 cells, and the concentration of SP20 cells is 1 × 10⁷one/mL.

In some of the foregoing embodiments, the ratio of spleen cells to myeloma cells is 3: 1.

In the examples, it is advantageous that the spleen cells are fully homozygously fused with myeloma cells by mixing an excess of spleen cells with myeloma cells.

In some of the foregoing embodiments, the PEG solution is preheated to 40 ℃.

In the examples, preheating the PEG solution to 40 deg.C also facilitates cell fusion, and since the heating temperature becomes high, the fluidity of cell membrane is enhanced, and the fusion of spleen cells and myeloma cells can be accelerated.

In some of the foregoing embodiments, resuspending the hybridoma cell mixture comprises diluting the cells to 5 × 10 with 20% FBS medium⁴Concentration per well, 5% CO at 37 ℃₂Culturing for 7-10 days under the condition.

In some of the foregoing embodiments, the amount of coated AXL antigen is 2 μ g/well.

In some of the foregoing embodiments, the conditions for screening for positive secreted AXL antibodies are OD₄₅₀The value is greater than 1.

In some of the foregoing embodiments, the antigen to detect the affinity of the hybridoma cells is Gas 6-His.

In some of the foregoing embodiments, 0.5. mu.g/mL AXL-mFc is mixed with the hybridoma cells at a ratio of 1:1 as the primary antibody.

Compared with the prior art, the beneficial effect of this application is: the method for preparing and screening hybridoma cells secreting AXL antibody according to the present application can produce spleen cells capable of producing high titer after primary immunization and several booster immunizations, and can produce hybridoma cells capable of continuously producing high titer and continuously dividing and proliferating by fusing spleen cells and myeloma cells.

Drawings

FIG. 1 is a graph of an antibody binding experiment of Experimental example 3;

FIG. 2 is a graph showing the results of an antibody blocking test in Experimental example 4;

FIG. 3 is a graph showing the results of the flow cytometric screening experiment in Experimental example 5.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present application are described in further detail below with reference to examples.

Example 1

This example provides a method for preparing and screening hybridoma cells that secrete AXL antibody, comprising the steps of:

the selected mice are BALA/C, healthy female mice of 6-8 weeks of age. The specific immunization operation steps are as follows:

1.1 Freund's complete adjuvant is adopted for the first immunization, the ratio of AXL-mFc immune antigen (fusion protein) to the immune adjuvant (Freund's complete adjuvant) is 1:1, the antigen and the adjuvant are only required to be mixed softly before the immunization (note: the antigen and the adjuvant are placed on ice before the immunization), the abdominal subcutaneous multi-point injection mode is adopted, and 50 mu g of each point is injected;

1.2 consecutive 3 boosts two weeks apart: injection of 30. mu.g per spot with Freund's incomplete adjuvant;

1.3, detecting the antibody titer, diluting the antigen AXL-hFc with a coating solution, and coating 1 mu g of antigen in each hole;

1.4 adding 50 mu L/hole into the plate hole of the enzyme label, and placing the plate hole at 4 ℃ overnight or 37 ℃ for coating for 2 hours; discarding the liquid in the hole, washing with 200 μ L of washing solution for 3 times, and drying; adding 200 μ L of blocking solution per well, and blocking at 4 deg.C overnight or 37 deg.C for 2 hr

1.5 washing solution 200. mu.L for 3 times; at this time, the coating plate can be stored at-20 ℃ or 4 ℃ for later use; adding 50 μ L primary antibody (mouse serum to be detected) into each well, and simultaneously setting positive and negative control wells; incubating at 37 ℃ for 1 h; washing with 200 μ L of washing solution for 5 times, and drying;

1.6 adding enzyme-labeled secondary antibody, incubating at 37 ℃ for 1h with 50 mu L of each well, washing with 200 mu L of washing solution for 5 times, and patting dry; adding 50 mu L of substrate solution TMB, and developing for 2-15min at 37 ℃; at 2mol/L H₂SO₄Stopping the reaction, and reading an OD value by an enzyme-labeling instrument at 450 nm; as a result, cell fusion was carried out so that the antibody titer reached more than the power of 6 of 10.

1.7 the puncture immunization is carried out at two weeks intervals, at the moment, the adjuvant is not added, only the antigen is used for carrying out the abdominal cavity immunization, each point is injected with 50 mu g, and the cell fusion can be carried out after 3 days of the puncture immunization

Example 2

In this example, spleen cells and myeloma cells were fused, and the specific procedures were as follows:

1.1 removing one side of eyeball to bleed, matching with vertebra dislocation, and putting the killed mouse in 70% alcohol; the skin inside the left hind thigh groove of the mouse was lifted with forceps, and the ligature was exposed by lateral and vertical scissors or by hand tearing. For lymph nodes in the groin, they were removed and immersed in 10ml of MPI 1640 medium (in a 9cm dish). Carefully taking off the spleen, immersing in the culture medium, carefully cutting off excessive white fat or connective tissue, clamping and immersing in fresh culture medium, and shaking to wash the spleen for 1-2 minutes;

1.2 the washed spleen and lymph nodes were transferred into a cell sieve, which was previously immersed in 20mL fresh medium; grinding spleen in cell sieve with syringe push head, transferring culture medium (containing cells) to clean centrifuge tube; slowly adding 20mL of culture medium into the cell sieve for washing; transferring the filtered cells into a centrifuge tube, gently blowing and beating liquid in the centrifuge tube, and centrifuging; 1000rpm, 5min, abandoning the supernatant; adding 40mL of culture medium, re-suspending the cells, centrifuging again, and discarding the supernatant;

1.3 add 1-5mL erythrocyte lysate (Sigma, R7757-100ML) into the tube, resuspend, carefully blow for 1-2 min; adding 40mL of culture medium to stop the cracking reaction; centrifuging: 1000rpm, 5min, abandoning the supernatant; the cells were resuspended by adding 10mL of medium;

1.4 Take 10. mu.L of cell suspension to transfer to a clean EP tube and add 90. mu.L of medium to dilute, take 10. mu.L of this dilution to a new EP tube and add 10. mu.L of 4% tryptan blue. Counting the cells under an inverted microscope and calculating the cell viability;

1.5 the majority of the remaining cells were brought to 30mL volume in culture medium, centrifuged, and the supernatant discarded (at this point water bath 40C was set up and PEG solution (Sigma 1450) was preheated);

1.6 resuspend splenocytes in 10mL of medium, as 1:3 was added SP20 (previously harvested, washed, resuspended in media,. about.10 ^ 7/mL). Wherein SP 20: splenocytes as 1: 3(SP20, preferably 10-20%);

1.7 the volume is adjusted to 20mL with medium and centrifuged. Discarding the supernatant; injecting 40C water into the beaker, and placing the tube filled with the cells in the beaker in water bath for about 2 min; slowly dripping 1mL of preheated PEG into the tube within 60s, adding and uniformly mixing, and keeping in a water bath;

1.8 sucking the cell suspension, slowly dripping the cell suspension back into the tube, and keeping a water bath; slowly adding 1mL of culture medium within 60s, uniformly mixing while adding, and keeping in a water bath; slowly adding 10mL of culture medium, and continuously mixing and bathing; slowly adding a culture medium, and fixing the volume to 20 mL; mixing, and water bathing for 10 min. (the water temperature is slowly reduced to the room temperature), centrifuging and discarding the supernatant; obtaining a hybridoma cell mixture.

1.8 at 5X 10 per well⁴Per cell, 100. mu.L culture solution calculation required 96-well plate(Corning COSTAR 3599) and medium (complete medium, P/S, 20% FBS, HAT), suspending the cells at full medium weight and dispensing into culture plates; culturing at 37 deg.C with 5% CO2 for 7-10 days; taking 50ul of supernatant, and performing ELISA detection; the positive well broth was transferred to about 1ml of HT medium and cultured in 24-well plates and assayed 2-3 days later

Example 3

This example opens and verifies the antibody binding method. In order to detect the affinity of the antibody AXL-his and the antibody Anti-AXL-10G5(hIgG1/mIgG2a), the ELISA experimental steps are optimized as follows:

1.1 AXL-his (0.7322mg/ml LOT: 20191112 frozen at-80 ℃) (96-well Fbottom plate manufacturer: Constar); coating concentration: 0.5 mu g/mL; 1 mu g/mL; 2 mu g/mL; 5 μ g/mL, 50 μ L/well, 16 wells each (PBS dilution), incubation: at 37 ℃ for 2 h; washing: PBST plate washing 3 times;

1.25% skimmed milk powder, 200 μ L/well, sealed overnight at 4 deg.C; washing: PBST plate washing 3 times;

1.3 immersing the polished electrode in ethanol for 5s of short-time ultrasonic treatment, then immersing the electrode in deionized water, cleaning the electrode with the deionized water for 5s of short-time ultrasonic treatment, and airing the electrode in air for later use;

1.4 Primary antibody: Anti-AXL-10G5(hIgG1/mIgG2a) is diluted in a gradient way, the sample is added, the reaction is carried out for 1h at 37 ℃, and a chessboard titration method is adopted;

TABLE 1 chessboard titration method

The plates were washed 5 times with PBST at 50. mu.L/well, 37 ℃ for 1h

1.5 Goat-Anti-mouse Fc-HRP 1:50000, 50 μ L/well, 37 deg.C, 1 h; wash plate 5 times with PBST solution

Goat-Anti-human Fc-HRP 1:50000, 50 μ L/well, 37 deg.C, 1 h; washing: PBST washing plate 5 times;

1.6 TMB substrate color development solution (manufacturer: Sigma, cat # T4444), 50 muL/hole, developing for 2-10 min at 37 ℃ or room temperature;

1.7 with 2M H₂SO₄At 50. mu.L/wellDosing into 96-well plates; reading an OD value by an enzyme-labeling instrument at 450 nm; and carrying out experimental results and data analysis.

The result analysis of the actual data is shown in fig. 1, and it can be seen that the experimental method obtained by the experiment is stable and reliable.

Example 4

In this example, the optimization of ELISA assay for detecting the blocking effect of Anti-AXL-mIgG2a on the binding of receptor AXL-mFc and ligand Gas6-his is as follows:

1.1 Using the antigen Gas6-his (LOT: 20191015 at 1mg/mL frozen at-20 ℃) (96-well Fbottom plate manufacturer: Constar) at a coating concentration of 4. mu.g/mL and adding the solution to each well at 50. mu.L/well for 8 wells (and diluting with PBS solution); and (3) incubation: at 37 ℃ for 2 h; washing: PBST plate washing 3 times;

1.25% of skimmed milk powder, 200 mu L/hole, sealing at 37 ℃ for 2 h; washing: PBST plate washing 3 times;

1.3 AXL-mFc (0.5 mu g/ml) and Anti-AXL-mIgG2a are diluted in a gradient way for 1:1 mixing and preincubation at 37 ℃ for 60min, sample adding and reaction at 37 ℃ for 1 h; PBST washing plate 5 times at 37 deg.C and 1h in 50 μ L/well;

table 2 method of dilution titration is as follows:

1.4 washes with Goat-Anti-mouse Fc-HRP 1:50000, 50. mu.L/well, 37 ℃, 1 h: PBST washing plate 5 times; developing color of TMB substrate (manufacturer: Sigma, goods number: T4444) at 50 μ L/hole at 37 deg.C or room temperature for 2-10 min; the reaction was stopped with 2M H2SO4, 50. mu.L/well; and reading OD value by a microplate reader at 450nm and analyzing the experimental result and data.

The experimental results are shown in table 3 and fig. 2; the results of the experiment were analyzed using Graphpad Prism software.

It can be seen that the inhibition rate of the antibody becomes higher as the concentration increases.

TABLE 3 inhibition experiments

Example 5

In this example, the method of screening cells by flow cytometry was investigated and improved to detect the affinity of HeLa cells to the antibody Anti-AXL-10G5(mIgG2a), and the detection method included:

1.1 Hela cells were digested with pancreatin, counted, and the cell density was adjusted to 3X 10⁶Per mL transfer 100. mu.L of cell suspension to a round bottom 96-well plate (manufacturer: Constar) for a total of 17 wells; placing on an ice box for later use;

1.2 centrifuge at 1000rpm for 2min, Buffer resuspend wash once, centrifuge off supernatant (ice box operation) Buffer: DMEM + 1% BSA;

1.3 Anti-AXL-10G5(mIgG2a) (1.9303mg/mL) antibody gradient dilution, 8-point calibration of the curve, and setting multiple holes at the same time, and performing incubation on an ice box; adding 50 μ L of the suspension into the cell precipitate, re-suspending the cells, mixing, and incubating on ice for 30 min; centrifuging at 1000rpm for 2min, performing once buffer heavy suspension washing, centrifuging to remove supernatant, and centrifuging to obtain 100 μ L; buffer resuspension (on ice box operation);

table 4 methods of antibody dilution are as follows:

1.4 adding 50 mu L of the mixture into the cell suspension by a PE-Goat-Anti-mouse IgG1:300 suction method, incubating the mixture on ice for 30min, centrifuging to remove the supernatant, performing buffer resuspension washing once, centrifuging to remove the supernatant, and performing buffer resuspension of about 200 mu L (ice box operation);

1.5 transfer the cell suspension to a 1.5mL EP tube; detecting a PE fluorescence signal value by adopting a flow cytometer; and carrying out experimental results and data analysis.

The experimental results are shown in fig. 3, and it can be seen that the screening method obtained by the present embodiment is more effective.

Example 6

In this example, hybridoma cells were selected, and the method for selecting hybridoma cells includes the following steps:

1.1 AXL-his antigen was diluted with coating solution, and 2. mu.g of antigen was coated in each well; adding 50 mu L/hole into the hole of an enzyme label plate, and coating for 2 hours at 4 ℃ overnight or 37 ℃; discarding the liquid in the hole, washing with washing solution for 3 times, patting to dry, adding 200 μ L of sealing solution into each hole, sealing at 4 deg.C overnight or 37 deg.C for 2 hr;

1.2 washing with a washing solution for 3 times; at this time, the coating plate can be stored at-20 ℃ or 4 ℃ for later use; adding 50 mu L of hybridoma cell culture supernatant to be detected in each hole, and simultaneously setting positive and negative control holes; incubation at 37 ℃ for 1 hour; washing and drying;

1.3 adding enzyme-labeled secondary antibody, incubating at 37 ℃ for 1 hour with 50 mu L of each well, washing, and patting dry; adding 50 μ L of substrate solution TMB, and developing at 37 deg.C for 2-15 min; at 1mol/L H₂SO₄Stopping the reaction, and reading an OD value by an enzyme-labeling instrument at 450 nm;

1.4 judging the result: the result shows that 80 hybridoma cell high positive antibody strains are obtained by screening, and the 450nm positive OD values are all more than 1.

Second round of screening was performed

2.1 Gas6-his (LOT: 20191015 at 1mg/mL frozen at-20 ℃ C.) (96-well F bottomplate manufacturer: Constar); coating concentration: 2 μ g/mL, 50 μ L/well, 8 wells (PBS dilution): at 37 ℃ for 2 h; washing: PBST plate washing 3 times;

2.2 sealing with 5% skimmed milk powder at 200 μ L/well at 37 deg.C for 2 h; washing the plate with PBST solution for 3 times;

2.3 mixing AXL-mFc (0.5 mu g/mL) with hybridoma cell supernatant at a ratio of 1:1, simultaneously setting up positive and negative control holes, pre-incubating at 37 ℃ for 30min, transferring the plate, 50 mu L/hole, 37 ℃, 1h, and washing the plate with PBST for 5 times;

2.4 using Goat-Anti-mouse Fc-HRP 1:50000, 50 μ L/hole, 37 deg.C, 1 h; washing the plate with PBST solution for 5 times;

2.5 developing with TMB substrate developing solution (manufacturer: Sigma, cat # T4444) at 50 μ l/well at 37 deg.C or room temperature for 2-15 min; 2M of H₂SO₄，50μ L/well; reading an OD value by an enzyme-labeling instrument at 450 nm; and analyzing the experimental results and data.

According to the experimental results, two hybridoma cell strains with higher diagnosis efficiency are obtained by screening, and the blocking efficiency is respectively as follows: 41.93% and 40.08%.

The embodiments described above are some, but not all embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Claims

1. A method for preparing and screening hybridoma cells that secrete an AXL antibody, comprising the steps of:

injecting 50 mu g of AXL immunogen mixture into an immunized mouse, and detecting the antibody titer, wherein the antibody titer reaches 10⁶Stopping immunization;

resuspending the hybridoma cell mixture;

coating an AXL antigen, adding the hybridoma cell mixture, incubating for 1h, washing and patting to be dry, adding a secondary antibody, incubating and patting to be dry, and terminating reaction and screening to obtain a positive hybridoma cell secreting an AXL antibody; and detecting the affinity of the hybridoma cells by ELISA.

2. The method for preparing and screening hybridoma cells secreting AXL antibody according to claim 1, wherein the AXL immunogen mixture is obtained by mixing AXL-mFc fusion protein and freund's complete adjuvant in a mass ratio of 1: 1;

3. The method for screening hybridoma secreting AXL antibody according to claim 1, wherein said myeloma cells are SP20 cells, and the concentration of SP20 cells is 1 x 10⁷one/mL.

4. The method of claim 1, wherein the ratio of said spleen cells to said myeloma cells is 3: 1.

5. The method of claim 1, wherein the PEG solution is pre-heated to 40 ℃.

6. The screening method of claim 5, wherein resuspending the hybridoma cell mixture comprises diluting the cells to 5 x 10 with 20% FBS medium⁴Concentration per well, 5% CO at 37 ℃₂Culturing for 7-10 days under the condition.

7. The method for screening hybridoma cells secreting AXL antibody according to claim 1, wherein the amount of AXL antigen coated is 2 μ g/well.

8. The method of claim 7, wherein the conditions for screening positive secreted AXL antibody are OD₄₅₀The value is greater than 1.

9. The method for screening hybridoma secreting AXL antibody according to claim 1, wherein said antigen for detecting the affinity of said hybridoma is Gas 6-His.

10. The method for screening hybridoma secreting AXL antibody according to claim 9, wherein 0.5 μ g/mL AXL-mFc is mixed with said hybridoma at a ratio of 1:1 as primary antibody.