Patent Application
20220380721
The present disclosure relates to bio-engineering, and in particular relates to a method for coupling an antibody to a surface of a cell and a method for applying a cell coupled with the antibody.
As tumor treatment enters the era of cellular immunotherapy, people have made major breakthroughs in the study of fighting cancer. The current mainstream tumor immunotherapy can be divided into adoptive cell therapy and immune checkpoint inhibitor therapy. The former can be divided into autologous cell therapy (cells from patients) and allogeneic cell therapy (cells from third-party donors) according to the cell source. Generally, immune cells are extracted from patients or other human beings, transformed and amplified, and then returned to patients to fight tumors. CAR-T (Chimeric Antigen Receptor T-cell) therapy is the leader of this type of therapy and has received extensive attention and research. Other immune checkpoint inhibitory therapies, such as PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, etc., are based on the principle of relieving the tumor's tolerance to immune cells and restoring the recognition and killing function of immune cells to tumors, thereby achieving the purpose of fighting cancer. Today, with the continuous investment of international pharmaceutical giants in cellular immunotherapy, related technologies will continue to make breakthroughs, and the cell therapy market has a bright future. But at the same time, it also faces many challenges, such as cell cytokine storm and off-target. At the same time, CAR-T is complicated and difficult to operate in the production process, and the resulting high treatment costs are also unbearable for ordinary families. PD 1 immune checkpoint blockade therapy has drug resistance and low efficacy, coupled with high treatment costs, and therefore continuous technological breakthroughs and innovations are still needed to overcome these difficulties.
An objective of the present disclosure is to solve the deficiencies of the existing techniques and provide a method for coupling an antibody to a surface of a cell and a method for applying the cell coupled with the antibody.
A technical solution of the present disclosure is as follows.
A method for coupling an antibody to a surface of a cell, comprising:
(1) chemically modifying sialic acid to obtain a sialic acid derivative containing an azide group;
(2) absorbing the sialic acid derivative by the cell, and expressing the azide group on a membrane surface of the cell to obtain a cell modified with the azide group through a sialic acid metabolic pathway of the cell;
(3) modifying the antibody with a conjunction compound to obtain a modified antibody, wherein a first end of the conjunction compound has a first active group configured to react with a sulfhydryl group or an amino group, and a second end of the conjunction compound has a second active group configured to bio-orthogonally react with the azide group, and the first active group is connected to the sulfhydryl group or the amino group of the antibody by a first reaction; and
(4) co-culturing the modified antibody with the cell modified with the azide group to enable the second active group of the conjunction compound of the modified antibody to be connected to the azide group of the cell modified with the azide group by a second reaction.
In a preferred embodiment of the present disclosure, the sialic acid derivative containing the azide group comprises a compound having any one of the following structural formulas:
In a preferred embodiment of the present disclosure, the first active group comprises a succinimide active ester group or a maleimide group.
In a preferred embodiment of the present disclosure, the conjunction compound comprises a compound having any one of the following structural formulas:
In a preferred embodiment of the present disclosure, the cell is an immune cell.
In a preferred embodiment of the present disclosure, the cell is an original human T cell or a natural killer cell.
A cell, a surface of the cell comprises an azide group expressed on the surface by absorbing a sialic acid derivative containing the azide group through a sialic acid metabolic pathway, the azide group is connected to an antibody through a conjunction compound, a first end of the conjunction compound has a first active group configured to react with a sulfhydryl group or an amino group, a second end of the conjunction compound has a second active group configured to react with the azide group by a bio-orthogonal reaction, the first active group is connected to the sulfhydryl group or the amino group of the antibody by a first reaction, and the second active group is connected to the azide group by a second reaction.
In a preferred embodiment of the present disclosure, the sialic acid derivative containing the azide group comprises a compound having any one of the following structural formulas:
In a preferred embodiment of the present disclosure, the first active group comprises a succinimide active ester group or a maleimide group.
In a preferred embodiment of the present disclosure, the conjunction compound comprises a compound having any one of the following structural formulas:
In a preferred embodiment of the present disclosure, the cell is an immune cell.
The present disclosure has the following advantages.
1. The present disclosure modifies natural sialic acid molecules in vitro through chemical synthesis methods, and utilizes modified natural sialic acid molecules to realize antibody modification on the surface of the cell.
2. The modification method of the present disclosure is simple, low-cost, safe, and efficient, does not need complex gene editing or enzyme catalytic operation, and has universality. In theory, the modification method can realize the coupling of any antibody or macro-molecular substance on the surface of the cell.
The present disclosure will be further described below in combination with the accompanying drawings and embodiments.
1. A Mechanism of a Coupling Reaction
Referring to
A preferred structural formula of the sialic acid derivatives modified with the azide group is as follows:
A preferred structural formula of the conjunction compound is as follows:
2. Steps for Synthesizing Related Molecules
9N3-SA is synthesized according to the literature (Cheng B, et al. ACS chemical biology, 2019, 14, 2141, which is incorporated herein by reference). Synthesis methods of 5N3-SA and diN3-SA are as follows:
A Synthesis of 5N3-SA
1) 500 mg of a compound 1 (the compound 1 is synthesized step according to the literature: Abdu-Allah, et al. Journal of Medicinal Chemistry, 2008, 51, 6665, which is incorporated herein by reference) is dissolved in 10 mL of N,N-Dimethylformamide (DMF), then 736 mg of (2-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) (HATU), 500 mg of N,N-Diisopropylethylamine (DIEA), and 156 mg of 2-azidoacetic acid are added to obtain a first reaction solution. The first reaction solution is stirred overnight at room temperature (e.g., 20-25° C.), and the first reaction solution is then concentrated and is purified with a silica gel column to obtain 300 mg of an intermediate 2.
2) 300 mg of the intermediate 2 is added in 30 mL of deionized water, 323 mg of I2 is then added to obtain a second reaction solution, and the second reaction solution is stirred for 24 hours at room temperature. The second reaction solution is then extracted, concentrated, and purified using a high pressure liquid chromatography to obtain 100 mg of the 5N3-SA.
A Synthesis of diN3-SA
1) 500 mg of a compound 3 (the compound 3 is synthesized according to the literature: Peng W, Paulson J C. Journal of the American Chemical Society, 2017, 139, 12450, which is incorporated herein by reference) is dissolved in 10 mL of N,N-Dimethylformamide (DMF), then 691 mg of the HATU, 500 mg of DIEA, and 156 mg of 2-azidoacetic acid are added to obtain a third reaction solution. The third reaction solution is stirred overnight at room temperature, and the third reaction solution is then concentrated and is purified with a silica gel column to obtain 280 mg of an intermediate 4.
2) 280 mg of the intermediate 4 is dissolved in 30 mL of deionized water, and 323 mg I2 is added to obtain a fourth reaction solution. The fourth reaction solution is stirred for 24 hours at room temperature. The fourth reaction solution is extracted, concentrated, and is purified using a high pressure liquid chromatography to obtain 80 mg of the diN3-SA.
The conjunction compound can be purchased from some reagent websites (such as Chengdu Boerkang Biotechnology Co., LTD., Shanghai Bide Pharmaceutical Technology Co., LTD., etc.) or prepared by conventional organic synthesis methods.
For example, a route for synthesizing the linker-4 is as follows:
A preferred synthesis method is as follows: 500 mg of Stau is dissolved in 10 mL of dichloromethane, then 317 mg of DIEA, and 123 mg of succinic anhydride are added to obtain a fifth reaction solution, and the fifth reaction solution is stirred at room temperature for 1 hour. After the fifth reaction solution is concentrated, Stau-1 is obtained. The Stau-1 is directly ready for the next step without purification. 212 mg of N-hydroxysuccinimide (NHS), 354 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbondiimide hydrochloride (EDC), and the Stau-1 are dissolved in 10 mL of dichloromethane, reacted at room temperature for 3 hours, and then extracted with ethyl acetate and water to obtain an organic phase. The organic phase is concentrated and purified by a silica gel column to obtain 120 mg of the Linker-4.
3. General Coupling Steps
1. Metabolic Incorporation of an Azido Analogues Sialic Acid in Cells
An appropriate amount of the azido analogues sialic acid is added into a normal cell culture medium with a final concentration of 100 μM (μMol/L). Cells are cultured in the normal cell culture medium under normal conditions for 24-48 hours to obtain azide modified cells (cell-N3), and the azide modified cells are then washed with phosphate-buffered saline (PBS) for 2-3 times and replaced with another normal cell culture medium for later use.
2. Modification of the Antibody
The antibody is prepared into an antibody solution with a concentration of 6 mg/mL by PBS for use. 50 μL of a solution of the linker (the conjunction compound) prepared by dimethyl sulphoxide (DMSO) (a concentration of an original solution of the linker is 10 mM (mMol/L)) is added into 1 mL of the antibody solution and incubated at room temperature for 30 minutes. Then, 50 μL of a Tris buffer (pH 8.0, 1 M (Mol/L)) is added for quenching and quenched at room temperature for 5 minutes to obtain a solution of the IgG B antibody modified with the linker. The solution is ready for next cell-coupling steps.
3. Procedure for Coupling Cells with Antibody
100 μL of the solution of the IgG B antibody modified with the linker obtained in Step 2 is added into 1 mL of the cell-N3 (a density of the cells is 1×106 cells/mL) obtained in Step 1 and incubated at 37° C. for 2 hours. During this process, a bio-orthogonal reaction between an active group at a terminal of the linker of the IgG B antibody and the azide group on the cell surface is performed. The bio-orthogonal reaction can be complete under physiological conditions, and the IgG B antibody modified with the linker and the azide group on the cell surface does not react with the medium or other molecules in organisms; a principle is shown in
4. Tumor Cells are Killed Using the Immune Cells with a Surface of the Immune Cells Coupled with the Antibodies
The present disclosure realizes the surface of the immune cells coupled with specific antibodies using the general coupling steps. In this embodiment, for example, a surface of NK92 cells coupled with antibodies of receptors (Her2) of breast cancer surface cells (anti-Her2 Ab) is used. The conjunction compound is Linker1, and the sialic acid derivative containing the azide group is 9N3-SA.
1. Modification of the Antibodies
The anti-Her2 Ab is prepared into 6 mg/mL of an antibody solution by PBS, and 50 μL of linker1 original solution (10 mM) is added into 1 mL of the antibody solution and coupled at room temperature for 30 minutes. 50 μL of Tris buffer (pH 8.0, 1M) is then added and quenched at room temperature for 5 minutes to obtain a solution of the anti-Her2 Ab coupled with the linker1(anti-Her2-linker1), and the solution is stored at 4° C. for later use.
2. Metabolic Incorporation of the Azido Sialic Acid in NK92 Cells
NK92 cells normally cultured are distributed into a 24-well plate and then cultured in a NK92 cell medium containing 9N3-SA with a final concentration of 100 μM for 24-48 hours. The NK92 cells are washed by PBS for 3 times, and the NK92 cell medium containing 9N3-SA is then replaced by a normal NK92 medium to obtain NK92 cells modified with the azide groups. In order to verify that the NK92 cells have been modified with the azide groups, in this embodiment, Fluorescein-Stau (FITC-Stau) (as shown in
A route for synthesizing FITC-Stau is as follows:
The FITC is a commercial product, the Stau is synthesized according to the literature (Chang, Pamela V., et al. Journal of the American Chemical Society. 2007, 129, 8400, which is incorporated herein by reference). A synthesis method is as follows: 200 mg of FITC is dissolved in 5 mL of DMF, 209 mg of the Stau and 200 mg of the DIEA are then added to obtain a sixth reaction solution, and the sixth reaction solution is stirred at room temperature for 5 hours. The sixth reaction solution is concentrated and then purified by a high pressure liquid chromatography to obtain 100 mg of FITC-Stau.
3. NK92 Cells with the Cell Surface Coupled with Her2 Antibody
100 μL of the solution of the anti-Her2-linker1 obtained in step 1 is added into 1 mL of the cells modified with the azide groups obtained in step 2 (a cell density is about 1×106 cells/mL), incubated at 37° C. for 2-8 hours, and washed with PBS for 2-3 times to obtain the NK92 cells coupled with the Her2 antibody: anti-Her2-NK92 cells. In order to verify that the surface of the NK92 cells have been connected to the Her2 antibody, in this embodiment, the NK92 cells coupled with the Her2 antibody and the NK92 cells not coupled with antibodies are further treated by Allophycocyanin (APC)-anti-Fc, and a fluorescence signal of the APC is detected by a flow cytometry. Referring to
4. Killing Activity of the Anti-Her2-NK92 Cells Against SK-BR-3 Cells In Vitro
In this experiment, SK-BR-3 cells (Her2 protein is overexpressed, human breast cancer cells) are used as target cells to verify the killing activity of the anti-Her2-NK92 cells in vitro. The SK-BR-3 cells are mixed with the NK92 cells or the anti-Her2-NK92 cells in a ratio of 1:5 and then incubated in a 37° C. incubator for 4 hours. A release of lactate dehydrogenase in a supernatant is detected by a lactate dehydrogenase detection kit, and a cytotoxicity is calculated. The results are shown in
5. Antitumor Activity of the Anti-Her2-NK92 Cells in Mice In Vivo
SK-BR-3 human breast cancer cells overexpressing Her2 are used to establish a subcutaneous tumor model of breast cancer in Balb/c nude mice in vivo, and the anti-Her2-NK92 cells (in an experimental group) are injected via a tail vein once a week from a seventh day after tumor inoculation (a tumor volume in the mice is about 200 mm3), and the number of cells is 107 each time. The mice in a control group are injected with original NK92 cells, and the mice in a blank group are injected with a PBS solution at the same volume for three continuous weeks, and changes of the tumor volume are recorded. Referring to
5. Coupling Different Antibodies on a Same Cell Surface
The technology of the present disclosure differs from chimeric antibodies on the cell surface using gene editing. Different species of antibodies are easily coupled to the same cell surface using this technology.
5.1 Coupling Different Species of Antibodies on the Surface of the NK92 Cells.
Three species of antibodies (hIgG, mIgG, rIgG) derived from human, mouse, and rabbit are respectively selected, and the antibodies are coupled to the surface of the NK92 cells according to the aforementioned coupling method. A corresponding secondary antibody having a fluorescent label is then used for fluorescent staining, and a fluorescent signal on the cell surface is detected by a flow cytometer. The experimental results are shown in
5.2 Coupling Multiple Species of the Antibodies Simultaneously on the Same Cell Surface
Another advantage of the technology of the cell surface coupled with the antibody of the present disclosure is that the multiple species of the antibodies can be simultaneously coupled to the same cell surface. In order to verify this conclusion, three species of the antibodies (hIgG, mIgG, rIgG) derived from human, mouse, and rabbit are respectively selected. In antibody-cell coupling steps, two (or three) species of the antibody prepared in advance are added to the NK92 cells with the surface modified with the azide group according to the aforementioned coupling method. After the coupling is complete, corresponding secondary antibodies with different fluorescent molecules are then used for fluorescent labeling. Finally, fluorescence signals on the cell surface are observed by a laser confocal microscopy. The experimental results are shown in
5.3 Retention Time of the Antibodies on the Cell Surface
In order to detect a retention time of the antibodies coupled to the cell surface, cetuximab, which can inhibit a proliferation of human tumor cells expressing EGFR and induce apoptosis of human tumor cells, is coupled to the NK92 cells using the method of the present disclosure. Anti-Fc-APC is used to label the cetuximab on the cell surface, and fluorescence signals are detected by a flow cytometry. The fluorescence signals on the cell surface are then detected at different time points. The experimental results are shown in
5.4 Killing Effects of the NK92 Cells Coupled with Cetuximab Against SW480 Cells In Vitro
As the cetuximab can inhibit the proliferation of the tumor cells expressing EGFR and induce apoptosis of the tumor cells, the SW480 cells are used to verify a killing ability of the NK92 cells coupled with the cetuximab (NK92-CET cells). The experimental results are shown in
5.5 Antitumor Activities of the NK92-CET Cells in Mice In Vivo
The SW480 cells are subcutaneously injected into nude mice to establish a model of a subcutaneous tumor. The NK92-CET cells are then respectively injected via a tail vein on day 5, 8 and 11, and growth states of the subcutaneous tumor are recorded (
The aforementioned embodiments are merely some embodiments of the present disclosure, and the scope of the disclosure is not limited thereto. Thus, it is intended that the present disclosure cover any modifications and variations of the presently presented embodiments provided they are made without departing from the appended claims and the specification of the present disclosure by those skilled in the art.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201911196567.X | Nov 2019 | CN | national |
This application is a continuation of and claims priority to International patent application number PCT/CN2020/132028, filed Nov. 27, 2020, which claims priority to Chinese patent application number 201911196567.X, filed on Nov. 28, 2019. International patent application number PCT/CN2020/132028 and Chinese patent application number 201911196567.X are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/132028 | Nov 2020 | US |
| Child | 17828811 | US |
