CN107254489B

CN107254489B - Method for detecting expression of chimeric antigen receptor or genetically modified T cell receptor and application

Info

Publication number: CN107254489B
Application number: CN201710464896.2A
Authority: CN
Inventors: 韦丹; 付苏雷; 连杰; 赵礼军; 李德志
Original assignee: Henan Hualong Biotechnology Co ltd
Current assignee: Henan Hualong Biotechnology Co ltd
Priority date: 2017-06-19
Filing date: 2017-06-19
Publication date: 2021-06-18
Anticipated expiration: 2037-06-19
Also published as: CN107254489A

Abstract

The invention relates to the field of immunology and molecular biology, in particular to a method for detecting expression of a chimeric antigen receptor or a genetically modified T cell receptor and application thereof, wherein the method mainly comprises the following steps: (1) infecting the packaged CAR or TCR lentivirus with adherent cells or suspension cells to obtain stable transfected adherent cells or suspension cells expressing the CAR or TCR; (2) paving adherent cells, and adding corresponding suspension cells when the fusion degree of the adherent cells reaches 70-90%; (3) and (4) continuing culturing and collecting the supernatant, counting the cells suspended in the supernatant and observing the phenomenon that the suspended cells are combined with adherent cells. The method for detecting the expression of the chimeric antigen receptor or the genetically modified T cell receptor can judge whether the CAR or the TCR is correctly expressed or not, and can prove whether the expressed CAR or the TCR can be specifically combined with the corresponding tumor antigen or not, so that the accuracy of CAR or TCR expression detection is further improved, and further guarantee is provided for tumor immunotherapy.

Description

Method for detecting expression of chimeric antigen receptor or genetically modified T cell receptor and application

Technical Field

The invention relates to the field of immunology and molecular biology, in particular to a method for detecting correct expression of a receptor, and particularly relates to a method for detecting expression of a chimeric antigen receptor or a genetically modified T cell receptor and application thereof.

Background

Immunotherapy of tumors falls into two broad categories: one is to relieve the tolerance or shielding effect of the tumor on immunity, allow the immune cells to "know" the tumor cells again, and then attack the tumor, such as PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, etc.; the other is to tell the characteristics of the tumor to immune cells, then the immune cells are searched and positioned to kill the tumor, such as T cells of chimeric antigen receptor (CAR-T), T cells of T cell receptor gene modification (TCR-T), Tumor Infiltrating Lymphocytes (TIL), specific cytotoxic T lymphocytes (DC-CTL) induced by dendritic cells loaded with tumor antigen, and the like.

Among them, chimeric antigen receptor T cells (CAR-T) and T cell receptor gene-modified T cells (TCR-T) are two of the latest technologies in current Adoptive Cell Therapy (ACT) therapy, develop very rapidly, and have achieved a shift from basic immunological mechanism research to clinical immunotherapy applications. The two technologies have the common feature that the recognition and attack ability of T cells to specific cancer cell antigens is improved by means of genetic modification, so the technologies are collectively called as "T cell receptor redirection" technologies.

T cells recognize killing of tumor cells by contact with tumor cells, and therefore correct expression of Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) genes on the T cell surface is undoubtedly an important place. Currently, there are two main approaches to whether CARs or TCRs are expressed: one is to detect specific genes in the CAR or TCR sequence using PCR, and the other is to detect specific fragments (e.g., Fab fragments, etc.) in the CAR or TCR structure using flow cytometry.

CN 106119411A discloses a method for detecting the infection efficiency of CAR-T cell virus, which comprises the following steps: infecting CAR-T cells with the virus; extracting the whole genome DNA of the CAR-T cell when the cell is stably expressed; detecting the copy number of the virus integrated into the whole genome DNA by using a fluorescent quantitative PCR technology; data processing: calculating the infection efficiency of the virus according to a Poisson distribution rule; wherein the formula for infection efficiency is: p (k) ═ 1-P (0); wherein P (0) ═ e-m; e is a natural constant; m is the MOI value, i.e., multiplicity of infection. Although this method can calculate the efficiency of viral infection, neither of the above methods can demonstrate whether an expressed CAR or TCR specifically binds to the corresponding tumor antigen, and the actual killing efficacy against tumors cannot be accurately predicted.

Therefore, it is important to accurately detect the expression of chimeric antigen receptors or genetically modified T cell receptors.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a method and application for detecting the expression of chimeric antigen receptor or genetically modified T cell receptor, which can not only determine whether CAR or TCR is correctly expressed, but also prove whether the expressed CAR or TCR can specifically bind to the corresponding tumor antigen.

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

in a first aspect, the invention provides a method of detecting CAR or TCR expression, comprising the steps of:

(1) infecting the packaged CAR or TCR lentivirus with adherent cells or suspension cells to obtain stable transfected adherent cells or suspension cells expressing the CAR or TCR;

(2) paving adherent cells, and adding suspended cells when the fusion degree of the adherent cells reaches 70-90%;

(3) continuously culturing and collecting supernatant, counting the cells suspended in the supernatant and observing the combination phenomenon of the suspended cells and adherent cells;

when the CAR or TCR-targeted tumor antigen is expressed in a suspension cell, the adherent cell of step (2) is the stably transfected adherent cell expressing the CAR or TCR of step (1), and the suspension cell is a CAR or TCR-targeted tumor antigen expressing suspension cell; when the CAR or TCR-targeted tumor antigen is expressed on an adherent cell, the adherent cell of step (2) is an adherent cell expressing the CAR or TCR-targeted tumor antigen, and the suspension cell is the stably transfected suspension cell expressing the CAR or TCR of step (1).

Specifically, the above method includes two cases:

(1) CAR or TCR-targeted tumor antigens are expressed in suspension cells: infecting the packaged CAR or TCR lentivirus with adherent cells to obtain stable transfected adherent cells expressing the CAR or TCR; then, plating the stable transformants, and adding CAR or TCR targeted tumor antigen expressed suspension cells when the fusion degree of adherent cells reaches 70-90%; and (4) continuing culturing and collecting the supernatant, counting the cells suspended in the supernatant and observing the phenomenon that the suspended cells are combined with adherent cells.

The adherent cells are 293T cells, stable transfer strains are easily obtained due to high infection efficiency of the 293T cells, and the adherent cell strains are 293T-CAR or 293T-TCR.

If the CAR or TCR is designed to be expressed correctly on the membrane of 293T cells, in the correct conformation, it will bind to the corresponding tumor antigen or MHC-polypeptide complex, fixing the two cells together; eventually, there was a reduction in suspended cells in the medium, and the adhesion of these cells to adherent cells was seen by microscopic observation.

(2) CAR or TCR-targeted tumor antigens are expressed on adherent cells: infecting the packaged CAR or TCR lentivirus into the suspension cells to obtain stable transfection suspension cells expressing CAR or TCR; then, plating adherent cells expressed by the CAR or TCR targeted tumor antigen, and adding suspension cells stably expressing the CAR or TCR when the fusion degree of the adherent cells reaches 70-90%; and (4) continuing culturing and collecting the supernatant, counting the cells suspended in the supernatant and observing the phenomenon that the suspended cells are combined with adherent cells.

The adherent cells are Sup-T1 cells, stable transformants are easily obtained due to high infection efficiency of the Sup-T1 cells, endogenous TCR is not expressed, and the suspension cell strain is Sup-T1-CAR or Sup-T1-TCR.

If the CAR or TCR is designed to be expressed correctly on the membrane of the suspended cell, in the correct conformation, it will bind to the corresponding tumor antigen or MHC-polypeptide complex, fixing the two cells together; eventually, there was a reduction in suspended cells in the medium, and the adhesion of these cells to adherent cells was seen by microscopic observation.

In the present invention, the CAR is a chimeric antigen receptor and the TCR is a genetically modified T cell receptor.

In the invention, when the fusion degree of adherent cells reaches 70-90%, the condition that the added suspension cells and the adherent cells have relatively large contact probability can be met, and if the fusion degree is less than 70%, the contact probability is too low; however, if the degree of fusion is greater than 90%, the adherent cells are detached due to excessive degree of fusion caused by the continued proliferation of the adherent cells during the co-culture after the addition of the suspension cells, and the cell count result in the supernatant is affected, and the degree of fusion of the stably transfected 293T cells may be, for example, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85%, preferably 80%.

Preferably, the plating in the step (2) is to spread adherent cells in an enzyme label plate, and culture medium is added for culture;

preferably, the elisa plate is any one or combination of at least two of a 6-hole elisa plate, a 12-hole elisa plate or a 24-hole elisa plate, for example, the elisa plate can be a 6-hole elisa plate, a 12-hole elisa plate, a combination of a 6-hole elisa plate and a 12-hole elisa plate or a combination of a 6-hole elisa plate, a 12-hole elisa plate and a 24-hole elisa plate, and is preferably a 6-hole elisa plate;

preferably, the number of adherent cells in the ELISA plate is (3-8) multiplied by 10⁵Number/hole, for example, may be 3X 10⁵4 x 10 pieces/hole⁵One/hole, 5X 10⁵6 x 10 pieces/hole⁵7 x 10 pieces/hole⁵One/hole or 8 x 10⁵One/hole, preferably 5X 10⁵Per well;

the culture medium is a mixed solution of a DMEM culture medium and fetal calf serum;

preferably, the fetal bovine serum is 5 to 15% by mass, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, preferably 8 to 12%, and more preferably 10%.

Preferably, the culture conditions are CO₂The concentration is 3-10%, for example 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%, preferably 5-7%.

Preferably, the temperature of the culture is 30-42 ℃, for example, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, 41 ℃ or 42 ℃, preferably 35-40 ℃, more preferably 37 ℃.

Preferably, the addition amount of the suspension cells in the step (2) is (0.5-3). times.10⁶Number/hole, for example, may be 0.5X 10⁶1 x 10 per hole⁶1.5X 10 pieces/hole⁶2 x 10 pieces/hole⁶2.5X 10 pieces/hole⁶One/hole or 3X 10⁶One/hole, preferably 1X 10⁶Per well.

Preferably, the culture medium used in the step (3) is a mixed solution of 1640 culture medium and fetal bovine serum;

preferably, the fetal bovine serum is 5 to 15% by mass, for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, preferably 8 to 12%, and more preferably 10%;

preferably, the period of time for continuing the culture in step (3) is 6-7h, for example, 6.1h, 6.2h, 6.3h, 6.4h, 6.5h, 6.6h, 6.7h, 6.8h, 6.9h or 7h, preferably 6 h.

Preferably, the counting in step (3) is performed by using a blood counting chamber and/or an automatic counter.

As a preferred technical scheme, the method comprises the following steps:

(1) infecting adherent cells or suspension cells with packaged lentiviruses of the CAR or the TCR to obtain stably transfected adherent cells or suspension cells expressing the CAR or the TCR;

(2) plating adherent cells on a six-hole enzyme label plate, wherein the number of the adherent cells is (3-8) multiplied by 10⁵Adding DMEM culture medium and fetal calf serum with mass fraction of 5-15% into each well, adding into the mixture in CO₂Culturing at 30-42 deg.C and 3-10% concentration;

when the fusion degree of the adherent cells reaches 70-90%, removing the culture medium, and adding (0.5-3). times.10⁶1640 culture medium of suspension cells of each hole and fetal calf serum with the mass fraction of 5-15%;

(4) continuously culturing for 6-7h, collecting supernatant, and counting cells in the supernatant by adopting a blood counting chamber and/or an automatic counter;

In a second aspect, the present invention provides a method for detecting expression of a chimeric antigen receptor or a genetically modified T cell receptor according to the first aspect for use in the preparation of a tumor immunizing drug.

Compared with the prior art, the invention has at least the following beneficial effects:

the method for detecting the expression of the chimeric antigen receptor or the genetically modified T cell receptor can judge whether the CAR or the TCR is correctly expressed or not, and can prove whether the expressed CAR or the TCR can be specifically combined with the corresponding tumor antigen or not, so that the accuracy of CAR or TCR expression detection is further improved, and further guarantee is provided for tumor immunotherapy.

Drawings

FIG. 1 is a histogram of the number of supernatant cells obtained in example 1 and comparative example 1 in the case of culturing the cell mixture for 1 hour, 2 hours, and 4 hours;

FIG. 2 is a graph showing the results of microscopic observation of suspension cells adhering to adherent cells at different time points.

Detailed Description

For the purpose of facilitating an understanding of the present invention, the present invention will now be described by way of examples. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

Verification of a lentivirus (pCDH-CAR19) carrying the CAR gene of CD19 molecule was selected, and Raji cells, B lymphoma cells expressing CD19, were selected as corresponding tumor cells.

(1) Infecting 293T cells with pCDH-CAR19 to obtain stably transfected 293T cells expressing chimeric antigen receptor;

(2) stably transfected 293T cells were plated and the first medium (DMEM medium mixed with 10% by mass fetal bovine serum) was added at 5% CO₂Culturing at 37 deg.C under the condition;

(3) when the fusion degree of the stably transfected 293T cells reaches 80%, removing the first culture medium, and adding a second culture medium (mixed solution of 1640 culture medium and fetal bovine serum) containing Raji cells to obtain a cell mixed solution; initial concentration of Raji cells was 1.0X 10⁶；

(4) Culturing the cell mixed solution for 1h, 2h, 4h and 6h, and collecting supernatant;

(5) counting the suspension cells in the supernatant, and observing the condition that the suspension cells adhere to adherent cells after the supernatant is discarded.

Example 2

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells in step (3) was 60%, as compared with example 1.

Example 3

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells in step (3) was 70% as compared with example 1.

Example 4

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells in step (3) was 90% as compared with example 1.

Example 5

And (3) selecting slow virus (pCDH-CAR-CEA) carrying the CAR gene of the CEA (carcinoembryonic antigen) antibody for verification, and selecting a human gastric cancer cell-MKN-45 cell expressing the CEA from corresponding tumor cells.

(1) Infecting Sup-T1 cells with pCDH-CAR-CEA to obtain a stably transfected cell strain Sup-T1-CAR expressing a chimeric antigen receptor;

(2) plating MKN-45 adherent cells, adding first culture medium (mixed solution of RPMI-1640 culture medium and 10% fetal calf serum), and adding CO at concentration of 5%₂Culturing at 37 deg.C under the condition;

(3) removing the first culture medium when the fusion degree of the MKN-45 adherent cells reaches 80%, and adding a second culture medium (mixed solution of 1640 culture medium and fetal calf serum) containing Sup-T1-CAR to obtain a cell mixed solution; Sup-T1-CAR cells at an initial concentration of 1.0X 10⁶；

Example 6

Compared with example 5, the fusion degree of MKN-45 adherent cells in the step (3) is 60%, and the method is the same as that in example 1.

Example 7

Compared with example 5, the fusion degree of MKN-45 adherent cells in the step (3) is 70%, and the method is the same as that in example 5.

Example 8

Compared with example 5, the fusion degree of MKN-45 adherent cells in the step (3) is 90%, and the method is the same as that in example 5.

Comparative example 1

Compared with example 1, the procedure was the same as example 1 except that PCDH-CAR19 was replaced with PCDH-GFP.

Comparative example 2

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells in step (3) was 30% as compared with example 1.

Comparative example 3

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells in step (3) was 40% as compared with example 1.

Comparative example 4

The same procedure as in example 1 was repeated except that the degree of fusion of 293T cells obtained in step (3) was 50% as compared with example 1.

Comparative example 5

The same procedure as in example 1 was repeated, except that the degree of fusion of 293T cells obtained in step (3) was 98%.

The suspension cells in the supernatants of examples 1-8 and comparative examples 1-5 were counted and the results are shown in Table 1.

TABLE 1 number of suspension cells in supernatant of examples 1 to 8 and comparative examples 1 to 5 (unit:. times.10)⁵)

	1h	2h	4h	6h
					Example 1	8.0	6.4	4.7	3.0
Example 2	8.1	6.2	4.4	3.3
					Example 3	7.9	6.3	4.7	3.1
Example 4	8.2	.6.4	4.6	3.1
					Example 5	7.9	6.7	4.7	3.1
Example 6	7.8	6.2	4.4	3.4
					Example 7	8.1	6.7	4.4	3.3
Example 8	8.0	6.6	4.5	3.3
					Comparative example 1	10.8	11.7	12.2	16.0
Comparative example 2	10.2	8.9	7.8	6.0
					Comparative example 3	10.0	8.7	7.4	5.3
Comparative example 4	9.3	8.5	7.0	4.7
					Comparative example 5	10.3	8.6	8.1	9.1

As can be seen from Table 1, the number of suspension cells in the supernatant of examples 1-8 was less than the original number at 1h, whereas comparative example 1 was greater than the original number and comparative examples 2-5 were slightly higher than the original number. The number of suspended cells in the supernatant of examples 1 to 8 showed a marked decrease in time, a marked increase in comparative example 1 and a slight decrease in comparative examples 2 to 5.

Compared with comparative examples 2-5, examples 1-4 further enhance the specificity of T cell receptors by controlling the fusion degree of adherent cells in step (3) to 70-90%, therefore, the method of examples 1-4 can not only determine whether CAR19 is correctly expressed, but also prove whether the expressed CAR19 can be specifically bound with corresponding tumor antigens, thereby further improving the accuracy of CAR19 expression detection and providing further guarantee for tumor immunotherapy. When the fusion degree of the adherent cells in step (3) is not within the range of the present invention, the number of suspended cells in the supernatant does not correctly reflect the specific binding of the two cells, which may lead to misjudgment of the result and is not suitable for detecting whether the CAR or TCR is correctly expressed.

FIG. 1 is a histogram of the cell numbers of the supernatants of example 1 (i.e., pCDH-CAR19 group) and comparative example 1 (i.e., pCDH-GFP group) cultured in the cell mixture for 1h, 2h, 4h, and 6 h; it can be obviously seen that the T cell receptor screened by the method has obvious adhesion effect on Raji cells and extremely strong specificity; while Raji cells in the supernatant of comparative example 1 were not decreased but increased, it was found that they did not bind specifically.

The above tables and figures illustrate that the method of examples 1-4 has a significant effect on detecting the correct expression of CAR19, whereas the T cells prepared in comparative example 1 are not specific for Raji cells and the fusion degree of the T cells prepared in comparative examples 2-5 is less adherent to Raji cells.

FIG. 2 is a view showing the microscopic observation results of the state in which suspended cells adhered to adherent cells after the supernatant of example 1 and comparative example 1 was discarded, and it can be seen that, after 6 hours of culture, significantly more cells were attached to the adherent cells at the bottom of the culture plate in example 1, whereas substantially no cells were attached to the adherent cells in comparative example 1.

The applicant states that the present invention is illustrated by the above examples to show the detailed process equipment and process flow of the present invention, but the present invention is not limited to the above detailed process equipment and process flow, i.e. it does not mean that the present invention must rely on the above detailed process equipment and process flow to be implemented. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims

1. A method of detecting expression of a chimeric antigen receptor or a genetically modified T cell receptor comprising the steps of:

wherein, when the CAR or TCR-targeted tumor antigen is expressed in a suspension cell, the adherent cell of step (2) is the stably transfected adherent cell expressing the CAR or TCR of step (1), and the suspension cell is a CAR or TCR-targeted tumor antigen expressing suspension cell;

when the CAR or TCR-targeted tumor antigen is expressed in an adherent cell, the adherent cell of step (2) is an adherent cell expressing the CAR or TCR-targeted tumor antigen, and the suspension cell is the stably transfected suspension cell expressing the CAR or TCR of step (1);

the plating in the step (2) is to spread adherent cells in an enzyme label plate and add a culture medium for culture;

the number of adherent cells in the ELISA plate is (3-8) multiplied by 10⁵Per well;

the mass fraction of the fetal calf serum is 5-15%.

2. The method as claimed in claim 1, wherein the ELISA plate is any one of 6-well ELISA plate, 12-well ELISA plate or 24-well ELISA plate or the combination of at least two of them.

3. The method of claim 2, wherein the microplate is a 6-well microplate;

the number of adherent cells in the ELISA plate is 5 multiplied by 10⁵Per well;

the mass fraction of the fetal calf serum is 8-12%.

4. The method of claim 3, wherein the fetal bovine serum is present in an amount of 10% by weight.

5. The method of claim 2, wherein the culturing conditions are CO₂The concentration is 3-10%;

the temperature of the culture is 30-42 ℃.

6. The method of claim 5, wherein the culturing conditions are CO₂The concentration is 5-7%;

the temperature of the culture is 35-40 ℃.

7. The method of claim 6, wherein the temperature of the culturing is 37 ℃.

8. The method according to any one of claims 1 to 7, wherein the suspension cells are added in step (2) in an amount of (0.5 to 3) x 10⁶Per well;

the fusion degree of the adherent cells is 75-85%.

9. The method of claim 8, wherein the suspension cells of step (2) are added in an amount of 1 x 10⁶Per well;

the degree of fusion of the adherent cells was 80%.

10. The method according to any one of claims 1 to 7, wherein the culture medium used in the step (3) is a mixed solution of 1640 medium and fetal bovine serum;

the mass fraction of the fetal calf serum is 5-15%.

11. The method of claim 10, wherein the fetal bovine serum is present in an amount of 8-12% by weight.

12. The method of claim 11, wherein the fetal bovine serum is present in an amount of 10% by weight.

13. The method according to any one of claims 1 to 7, wherein the incubation in step (3) is continued for a period of 6 to 7 hours.

14. The method of claim 13, wherein the culturing in step (3) is continued for 6 hours.

15. The method according to any one of claims 1 to 7, wherein the counting in step (3) is performed by a hemocytometer and/or an automatic counter.

16. The method according to one of claims 1 to 7, comprising the steps of:

when the CAR or TCR-targeted tumor antigen is expressed in a suspension cell, the adherent cell of step (2) is the stably transfected adherent cell expressing the CAR or TCR of step (1), and the suspension cell is a CAR or TCR-targeted tumor antigen expressing suspension cell; when the CAR or TCR-targeted tumor antigen is expressed on an adherent cell, the adherent cell of step (2) is an adherent cell expressing the CAR or TCR-targeted tumor antigen, and the suspension cell is a stably transfected suspension cell of the CAR or TCR of step (1).