CN115792242A - Method for determining capacity of anti-CD 70 monoclonal antibody for directly activating CD70 downstream pathway - Google Patents
Method for determining capacity of anti-CD 70 monoclonal antibody for directly activating CD70 downstream pathway Download PDFInfo
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Abstract
The invention discloses a method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate a CD70 downstream pathway, which comprises the following steps: the antibody solution was prepared by diluting the CD70 antibody with complete medium (RPMI-1640 +10% by weight of fetal bovine serum 90), and then diluting the antibody solution with 3-fold gradient of 8 gradients using complete medium, incubating the antibody solutions of 8 concentrations with Jurkat-CD70-NFkB-LUC cells, respectively, extracting cell lysate, adding luciferase substrate, and then measuring the chemiluminescence intensity. The invention has the beneficial effects that: the method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the CD70 downstream channel uses the Jurkat cells which are infected by lentivirus and stably express CD70, NFkB transcription factors and luciferase regulated by the NFkB transcription factors, extracts cell lysate and adds luciferase substrate after the Jurkat cells are incubated with the CD70 monoclonal antibody, determines the luminescence value of the Jurkat cells, determines the ability of CD70 monoclonal antibody molecules to activate the CD70 downstream channel, and provides a new direction for the screening and testing of the CD70 monoclonal antibody.
Description
Technical Field
The invention belongs to the technical field of biological activity determination, and particularly relates to a method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate a CD70 downstream pathway.
Background
CD70 and its receptor CD27 belong to the TNF ligand superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF), respectively. Under normal conditions, CD70 is typically only transiently expressed on the surface of antigen-activated B cells, T cells, natural killer cells (NK cells), and mature Dendritic cells (Dendritic cells), and its receptor CD27 is only expressed on some T cell populations: naive T cells (
T cell), regulatory T cell (regulatory T cell), and memory T cell (memory T cell), etc., memory B cell, and partial natural killer cell (NK cell), and is not co-expressed with CD 70. In cancer, however, CD70 is stably expressed on the surface of cancer cells. In solid tumors, CD70 is expressed on the surface of cancer cells alone, and in hematological tumors, CD70 is also co-expressed with CD 27.
Under normal conditions, CD70 and CD27 form trimers on the cell surface respectively, and realize the co-stimulation of immune response through the strictly regulated ligand receptor binding, help to regulate mature T cells, B cells and NK cells to realize various signal paths of immune response, and help to complete the immune response. The unregulated binding of CD70 to CD27 on the surface of tumor cells results in the proliferation of cancer cells, leading to the development of cancer. Meanwhile, CD70 on the surface of the cancer cell can be combined with CD27 on the surface of an immune cell in a tumor environment, and abnormal combination can lead to the suppression of the immune cell in the tumor environment, so that the immune escape of the tumor is realized.
The existing monoclonal antibody medicine aiming at the CD70 aims to combine the CD70 on the surface of a tumor cell and block the combination of the CD70 and the CD27 on the surface of an immune cell so as to achieve the aim of relieving immunosuppression. However, in mab therapy against CD70, some CD70 mabs, upon binding to cell surface CD70, may result in activation of the downstream pathway of CD 70.
Disclosure of Invention
The main purpose of the present application is to provide a method for determining different monoclonal antibodies against CD70, which can differentiate the ability of different monoclonal antibodies to activate the downstream pathway of CD 70.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway, comprising the steps of:
(1) Diluting CD70 antibody with complete culture medium to obtain three antibody solutions with concentration of 30 ug/ml; then, the three antibody solutions with the concentration of 30ug/ml are respectively subjected to 3-fold gradient dilution with 8 concentrations by using a complete culture medium to obtain 8 antibody solutions;
the 8-concentration 3-fold gradient dilution procedure was performed by diluting an antibody solution at a concentration of 30ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 10ug/ml, further diluting an antibody solution at a concentration of 10ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 3.3333ug/ml, further diluting an antibody solution at a concentration of 3.3333ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 1.1111ug/ml, further diluting an antibody solution at a concentration of 1.1111ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 0.3704ug/ml, further diluting an antibody solution at a concentration of 0.1111 ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 0.1235ug/ml, further diluting an antibody solution at a concentration of 0.5 ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 0.0412ug/ml, and further diluting an antibody solution at a concentration of 0.1235ug/ml 3-fold with complete medium to obtain an antibody solution at a concentration of 0.041/ml to obtain an antibody solution at a concentration of 0.0412 ug/ml.
(2) Respectively adding 8 kinds of antibody solutions obtained in the step (1) into corresponding holes of a 96-well plate;
(3) Making Jurkat-CD70-NFkB-LUC cells into single cell suspension by using complete culture medium; adding the single cell suspension into the corresponding hole of the 96-hole plate in the step (2);
(4) Supplementing a complete culture medium into the corresponding hole of the 96-hole plate in the step (3) for incubation;
(5) And (5) adding luciferase substrates into corresponding wells of the 96-well plate in the step (4), incubating, and detecting chemiluminescence intensity.
Method for determining the ability of anti-CD 70 monoclonal antibody to directly activate CD70 downstream pathway as a preferred embodiment, in the above method for determining the ability of anti-CD 70 monoclonal antibody to directly activate CD70 downstream pathway, in step (1), the complete medium comprises the following raw materials in parts by weight: RPMI-1640 90 parts, and fetal calf serum 10 parts.
RPMI is an abbreviation of Roswell Park Memorial Institute, designated Rosevir Pake Institute, RPMI is a type of cell culture medium developed by the Institute, 1640 is the medium's code; fetal bovine serum is serum taken from blood of a fetal bovine born by caesarean section.
As a preferred embodiment of the above method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the downstream CD70 pathway, in step (2), the antibody solution is added to each well in an amount of 30ul.
As a preferred embodiment, in the above method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the downstream pathway of CD70, in step (3), the concentration of the Jurkat-CD70-NFkB-LUC single cell suspension is 3X 10^6cells/ml; the amount of single cell suspension added per well was 30ul.
The preparation method of the single cell suspension comprises the following steps: jurkat-CD70-NFkB-LUC cells were placed in a centrifuge tube, centrifuged at 300Xg (300 times gravity) for 5 minutes, the liquid in the centrifuge tube was discarded, the complete medium was aspirated using a pipette gun, and the cell pellet in the centrifuge tube was gently blown to suspend each cell in the complete medium individually without adhesion to other cells.
In the above method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway, as a preferred embodiment, in step (4), each well is supplemented with complete medium in an amount of 30ul; the incubation was performed by placing the 96-well plate at 37 ℃ for 5 hours.
In the above method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway, as a preferred embodiment, in step (5), the amount of luciferase substrate added per well is 90ul; the incubation is as follows: the 96-well plate was incubated at 37 ℃ for 5 minutes.
The luciferase substrate adopted by the application is a luciferase substrate mixed liquor with the product number of E2520 produced by Promega, wherein a cell lysis substance and a luciferase substrate are added, so that two operations of cell lysis solution extraction and luciferase substrate reaction luminescence can be completed in one step.
The beneficial effects of the invention are as follows: the method for determining the capacity of the anti-CD 70 monoclonal antibody for directly activating the CD70 downstream channel uses the Jurkat cells which are infected by lentivirus and stably express CD70, NFkB transcription factors and luciferase (luciferase) regulated and controlled by the NFkB transcription factors, extracts cell lysate and adds luciferase substrate after the Jurkat cells are incubated with the CD70 monoclonal antibody, determines the luminous value of the cell lysate and the activation capacity of CD70 monoclonal antibody molecules on the CD70 downstream channel, and provides a new direction for the screening and testing of the CD70 monoclonal antibody.
The method for determining the capability of the anti-CD 70 monoclonal antibody for directly activating the CD70 downstream channel has short experimental period, and can quickly, effectively and accurately determine the biological activity of the CD70 monoclonal antibody on the activation of the CD70 downstream channel.
The method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the downstream pathway of CD70 uses a stable transfer cell strain infected by slow virus, can stably express the required gene, and can be stably cultured for a long time.
Drawings
FIG. 1 is a diagram: three CD70 antibodies were tested using the assay: CD70mAb1, CD70mAb2, and CD70mAb3 activated the ability of Jurkat-CD70-NFkB-LUC cells to activate the CD70 downstream pathway.
Detailed Description
In order to make the technical solutions in the embodiments of the present application better understood, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to examples, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. 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.
Example 1
Example 1 provides a method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway, comprising the steps of:
(1) Three CD70 antibodies were diluted using complete medium: CD70mAb1, CD70mAb2, and CD70mAb3, each made a 30ug/ml antibody solution. Then, the three antibody solutions with the concentration of 30ug/ml are respectively diluted by 3 times of 8 gradients in complete culture medium to obtain 24 antibody solutions.
The complete culture medium comprises the following raw materials in parts by weight: 90% RPMI-1640 medium (RPMI is an abbreviation of Roswell Park Memori Institute, and refers to Rosevir Park Pake Memorial institute.RPMI is a type of cell culture medium developed by the Institute, and 1640 is the code of the medium), 10% fetal bovine serum (serum taken from blood of a fetal calf born by Caesarean section).
The CD70mAb1 is an anti-human CD70 monoclonal antibody developed by argenx under the name cusatuzumab (ARGX-110).
CD70mAb2 was a humanized anti-human CD70 antibody designated SGN-70 developed by Seattle Genetics Inc.
The CD70mAb3 is preferably a humanized IgG1 CD70 monoclonal antibody named IMM40H developed by Oncon biomedical technology (Shanghai) Inc.
The procedure of the 8-step 3-fold gradient dilution was to dilute 30ug/ml antibody solution 3-fold with complete medium to obtain 10ug/ml antibody solution, dilute 10ug/ml antibody solution 3-fold with complete medium to obtain 3.3333ug/ml antibody solution, dilute 3.3333ug/ml antibody solution 3-fold with complete medium to obtain 1.1111ug/ml antibody solution, dilute 1.1111ug/ml antibody solution 3-fold with complete medium to obtain 0.3704ug/ml antibody solution, dilute 0.4 ug/ml antibody solution 3-fold with complete medium to obtain 0.1235ug/ml antibody solution, dilute 0.5 ug/ml antibody solution 3-fold with complete medium to obtain 0.0412ug/ml antibody solution, and dilute 0.1235ug/ml antibody solution 3-fold with complete medium to obtain 0.0412ug/ml antibody solution 0.04137-fold with complete medium to obtain 0.04137 ug/ml antibody solution.
(2) The 24 antibody solutions obtained in step (1) were added to the corresponding wells of a 96-well plate, 30ul was added to each well, and 3 wells were added to each antibody solution (average at the later stage).
(3) Jurkat-CD70-NFkB-LUC cells were made into single cell suspensions of 3X 10^6cells/ml (i.e., 3000000 cells per ml) using complete culture.
The preparation method of the single cell suspension comprises the following steps: placing Jurkat-CD70-NFkB-LUC cells in a centrifuge tube, centrifuging for 5 minutes under the condition of 300Xg (300 times gravity) centrifugation, discarding the liquid in the centrifuge tube, sucking the complete culture medium by using a pipette gun, slightly blowing and beating the cell sediment in the centrifuge tube to suspend each cell in the complete culture medium independently and without adhesion with other cells
(4) And (3) adding the single cell suspension obtained in the step (3) into corresponding holes of the 96-hole plate in the step (2), and adding 30ul of cell suspension into each hole.
(5) And (5) adding 30ul of complete culture medium into each corresponding well of the 96-well plate obtained in the step (4). The 96-well plate was incubated at 37 ℃ for 5 hours.
(6) And (4) respectively adding 90ul of luciferase substrate into corresponding wells of the 96-well plate obtained in the step (5), incubating for 5 minutes at 37 ℃, and detecting chemiluminescence intensity by using a microplate reader.
The luciferase substrate is a luciferase substrate mixed liquor with the product number of E2520 produced by Promega, and a cell lysis substance and the luciferase substrate are added, so that two operations of cell lysis solution extraction and luciferase substrate reaction luminescence can be completed in one step.
The chemiluminescence intensity detection method is characterized in that a microplate reader which is manufactured by BMG LABTECH and has the model of ClaRIOstar Plus is used, and the chemiluminescence intensity of a 96-well plate is measured according to the instrument use instruction of the microplate reader.
The detection results are shown in fig. 1:
as can be seen from fig. 1:
Jurkat-CD70-NFkB-LUC cells emit fluorescence after being incubated with CD70 antibody, cell lysate is extracted, and the fluorescence intensity is higher when the antibody concentration is higher after the cell lysate reacts with luciferase substrate. It was demonstrated that the NFkB pathway of Jurkat-CD70-NFkB-LUC cells is activated by the CD70 antibody, the higher the antibody concentration is, the more strongly the NFkB pathway is activated, and 3 different antibodies have different activation abilities to the pathway.
With increasing antibody concentration, CD70mAb3 produced activation of the NFkB pathway of Jurkat-CD70-NFkB-LUC cells earliest and reached the plateau of activation earliest (chemiluminescence intensity no longer increased with increasing concentration, i.e. the maximum activation capacity of the antibody for the NFkB pathway of Jurkat-CD70-NFkB-LUC cells), CD70mAb2 produced activation of the NFkB pathway of Jurkat-CD70-NFkB-LUC cells latest, and CD70mAb1 produced activation of the NFkB pathway of Jurkat-CD70-NFkB-LUC cells latest and reached the plateau at concentrations between those of the other two antibodies. After the antibody concentration reached the plateau concentration, the highest chemiluminescence intensity was produced by Jurkat-CD70-NFkB-LUC cells co-incubated with CD70mAb2, meaning that CD70mAb2 was most active on the NFkB pathway of Jurkat-CD70-NFkB-LUC cells, the second to activation of CD70mAb3, and the weakest activation of CD70mAb1 after reaching the plateau concentration.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for a person skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be considered as the protection scope of the present invention.
Claims (7)
1. A method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway, comprising the steps of:
(1) Diluting the CD70 antibody by using a complete culture medium to prepare an antibody solution with the concentration of 30 ug/ml; then using complete culture medium to carry out 3-fold gradient dilution of 8 concentrations on the antibody solution with the concentration of 30ug/ml to obtain 8 antibody solutions;
(2) Respectively adding 8 antibody solutions obtained in the step (1) into corresponding holes of a 96-well plate;
(3) Making Jurkat-CD70-NFkB-LUC cells into single cell suspension by using complete culture medium; adding the single cell suspension into the corresponding hole of the 96-hole plate in the step (2);
(4) Supplementing a complete culture medium into the corresponding hole of the 96-well plate in the step (3), and incubating;
(5) And (4) adding luciferase substrates into the corresponding wells of the 96-well plate in the step (4), incubating, and detecting the chemiluminescence intensity.
2. The method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the downstream pathway of CD70 according to claim 1, wherein in step (1), the complete culture medium comprises the following raw materials in parts by weight: RPMI-1640 parts, 10 parts of fetal bovine serum.
3. The method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate the downstream CD70 pathway of claim 1, wherein in step (2), the antibody solution is added to each well in an amount of 30ul.
4. The method for determining the ability of the anti-CD 70 monoclonal antibody to directly activate CD70 downstream pathway according to claim 1, wherein in step (3), the concentration of Jurkat-CD70-NFkB-LUC single cell suspension is 3 x 10^6cells/ml; the amount of single cell suspension added per well was 30ul.
5. The method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate a CD70 downstream pathway of claim 1, wherein in step (4), each well is supplemented with complete medium in an amount of 30ul; the incubation was performed by placing the 96-well plate at 37 ℃ for 5 hours.
6. The method for determining the ability of an anti-CD 70 monoclonal antibody to directly activate the CD70 downstream pathway as claimed in claim 1, wherein the amount of luciferase substrate added per well in step (5) is 90ul.
7. The method of claim 1, wherein in step (5), the incubation is as follows: the 96-well plate was incubated at 37 ℃ for 5 minutes.
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