CN112921004B - Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method - Google Patents
Cell strain for detecting CD3 receptor agonist, construction method thereof and CD3 receptor agonist detection method Download PDFInfo
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Abstract
The present application relates to the field of biological reagent detection, and more particularly, to a cell strain for detecting a CD3 receptor agonist, a construction method thereof and a detection method of the CD3 receptor agonist. Wherein the cell line for the detection of a CD3 receptor agonist is an immortalized T lymphocyte cell line naturally containing a TCR-CD3 complex, comprising in its cell an NFAT reporter comprising a binding sequence that binds NFAT linked to a downstream promoter linked to an open reading frame that expresses a reporter protein. The detection of the CD3 receptor agonist can be effectively realized by introducing the NFAT reporter gene into the immortalized T lymphocyte system which naturally contains the TCR-CD3 complex, the sensitivity is higher, the cell is not dependent on PBMC cells, and the method has good application prospect.
Description
Technical Field
The present application relates to the field of biological reagent detection, and more particularly, to a cell strain for detecting a CD3 receptor agonist, a construction method thereof and a CD3 receptor agonist.
Background
The TCR-CD3 complex on the surface of T lymphocytes plays a key role in the process of acquiring an immune response. When antigen presenting cells activate the CD3 receptor (TCR) on the surface of T cells, the TCR transmits the activated cells to the inside of T cells via the CD3 antigen, so that an immune response can be generated.
The CD3 targeted agonist drug can effectively activate T cells of human body, has the effect of killing tumors, and has wide application prospect. Currently, a large number of CD 3-targeted activator drugs have entered the clinical research and development stage, some of which have also been approved for the market, and thus, there is a great market need for a cap assay for CD3 receptor agonists and a neutralizing antibody assay for CD3 receptor agonists.
Existing activity assays for CD3 receptor agonists and neutralizing antibody assays for CD3 receptor agonists rely on human Peripheral Blood Mononuclear Cells (PBMCs) as a key reagent, stimulating PBMCs by CD3 receptor agonists to kill tumor cells and determining the survival of tumor cells to characterize relevant activity parameters of CD3 receptor agonists, or stimulating PBMCs by CD3 receptor agonists, determining IFNs of PBMCs by enzyme-linked immunosorbent assay (ELISA)-γSecretion of cytokines such as IL2 to determine relevant activity parameters of CD3 receptor agonists.
However, in the above prior art, PBMC cells need to be relied on, but the sources of PBMC cells are limited, and the differences of immunological characteristics of different batches are large, so that the requirements of drug development cannot be met.
Another approach is bioanalytical analysis by receptor binding assays (LBA), but the sensitivity of detection is lower because CD3 agonists, especially partial CD 3-targeted bispecific antibodies, have affinities between 10 and 100nM for CD3 antigen, which are weaker. However, in practical use, the dosage of CD3 agonist is generally low and the requirement for sensitivity is high, so that the sensitivity of the LBA experiment is generally difficult to meet the practical requirement.
Disclosure of Invention
In order to provide a method for detecting the CD3 receptor agonist with higher sensitivity and independent of PBMC cells, the application provides a cell strain for detecting the CD3 receptor agonist, a construction method thereof, and a method for detecting the CD3 receptor agonist and neutralizing antibodies.
In a first aspect, the present application provides a cell line for detecting a CD3 receptor agonist, which adopts the following technical scheme:
a cell line for use in the detection of a CD3 receptor agonist, the cell line being an immortalized T lymphocyte cell line naturally comprising a TCR-CD3 complex, comprising in its cell an NFAT reporter comprising a binding sequence that binds NFAT linked to a downstream promoter linked to an open reading frame that expresses a reporter protein.
In the above technical scheme, an immortalized T lymphocyte line naturally containing a TCR-CD3 complex is selected, and the TCR-CD3 structure can generate specific signals under the stimulation of a CD3 receptor agonist and form a signal path through an intracellular NFAT reporter gene. In the NFAT reporter gene, after receiving a stimulus signal through the NFAT, the stimulus signal is transmitted to a promoter, the promoter controls an open reading frame to express the reporter gene protein, and the content of the reporter gene protein is measured, so that the content and the activity of the CD3 receptor agonist can be used as a measurement standard.
In the process, firstly, PBMC is avoided, the immortalized T lymphocyte line has a wider source, and a large number of passages can be carried out after immortalization, so that the method has better stability in the passage process and greatly saves the cost. Secondly, the activity of the CD3 receptor agonist is characterized by quantitatively measuring the reporter gene protein, so that the method is more visual and quantitative, has better precision compared with an LBA method, does not depend on the activity of exogenous cells, and has better stability between the same batches.
The cell strain for detecting the CD3 receptor agonist can be applied to biological activity detection and concentration detection of the CD3 receptor agonist, and can also be applied to detection of neutralizing antibodies of the CD3 receptor agonist and antagonist, thereby having wide application prospect.
Preferably, the blast cells of the immortalized T lymphocyte cell line naturally containing the TCR-CD3 complex are Jurkat cells.
Jurkat cells are immortalized T lymphocytes that naturally contain the TCR-CD3 complex and function in a manner similar to normal T cells in humans. The Jurkat cells are used as parent cells for expansion, so that the obtained cells have more stable properties, are wide in sources, have more stable properties and have smaller fluctuation in the measurement process.
Preferably, the open reading frame is a luciferase expression gene.
The promoter stimulates the luciferase expression gene to express the luciferase, the luciferase has stronger fluorescence reaction in the kit, the content of the luciferase can be accurately measured through fluorescence measurement, the measuring process is simpler, the accuracy is better, the detection limit is lower, and stronger fluorescence phenomenon can be generated under the condition of very low concentration, so that the cell has better sensitivity when being applied to the measurement of the CD3 receptor agonist.
Preferably, the cell strain is a cell strain with a preservation number of CCTCC NO: c2020264, the preservation institution is China center for type culture Collection.
In a second aspect, the present application provides a method for constructing the cell line for detecting a CD3 receptor agonist, which adopts the following technical scheme:
the construction method of the cell strain for detecting the CD3 receptor agonist comprises the following steps:
a1, transfecting an NFAT reporter gene containing a binding sequence, a promoter and an open reading frame of NFAT binding into an immortalized T lymphocyte line to obtain a transfected cell strain;
a2, carrying out transfection efficiency evaluation and function evaluation on the cell strain transfected in the step A1, and screening cell strains with better signal-to-noise;
a3, transfecting the cell strain with the better signal to noise ratio obtained by screening in the step A2 again, and screening to obtain a cell strain after secondary construction;
a4, carrying out TCR-CD3 abundance measurement and function evaluation on the cell strain obtained in the step A3 after the secondary construction, and screening the cell strain with higher TCR-CD3 to obtain the cell strain for detecting the CD3 receptor agonist.
In the technical scheme, through the steps of transfection-screening-secondary construction-secondary screening, on one hand, cell strains with more sufficient transfection can be selected, and the cell strains have better sensitivity and higher signal to noise ratio, on the other hand, after the primary screening, cells with a part of the cells having certain resistance to the transfection vector can be screened out, cell strains with easy transfection can be selected, other cells can be screened out, and the procedures in the processes of cell culture and cell line construction are reduced.
Meanwhile, in the process of twice screening, the NFAT reporter gene Jurkat cell strain with high kurtosis expression TCR-CD3 can be obtained, and cells can be amplified in a monoclonal mode later, so that the obtained cells have better uniformity and stability, and larger numerical fluctuation is not easy to generate in the measuring process.
Preferably, in step A2, cell lines are selected that have a signal to noise ratio greater than 5 and a better dose profile for the CD3 agonist.
The good dose curve is generally an S-shaped curve, has obvious upper and lower platforms and better linear parts, and a cell strain with good dose curve has better linearity when a standard curve is drawn, so that the concentration of the CD3 receptor agonist of a sample to be tested can be more accurately judged, and the sensitivity of the cell strain when the cell strain is applied to the detection of the CD3 receptor agonist is improved. The cell strain with the signal to noise ratio of more than 5 has better sensitivity, the measurement range of the cell strain is more fit with the actual use concentration of the CD3 receptor agonist, and the cell strain has better application prospect.
Preferably, in both step A1 and step A3, NFAT reporter genes comprising binding sequences, promoters and open reading frames for NFAT binding are transfected into the cell line to be transfected by means of lentiviral transfection.
Through slow virus transfection, the transfection capacity is stronger, the cell strain can be more fully transfected, the signal generated by the obtained cell strain under the stimulation of the CD3 receptor agonist is further improved, and the sensitivity of the obtained cell strain in the process of measuring the CD3 receptor agonist is improved.
Preferably, in step A2, when the signal-to-noise ratio of the cell line is measured, the cell line to be measured is cultured, and then the cell line is measured with PMA and Ionomycin as stimulators, and the expression of the reporter protein is detected.
PMA and Ionomycin can stimulate NFAT in cells and further induce open reading frame expression of reporter proteins. The above procedure can reduce the exogenous influence, thus the transfection situation in the cell strain can be directly determined, so as to determine whether the NFAT reporter gene sequence to be transfected is actually transfected into the cell, and the cell can be more intuitively screened.
In a third aspect, the present application provides a method for detecting a CD3 receptor agonist, which adopts the following technical scheme:
the method for detecting the CD3 receptor agonist comprises a biological activity detection method, and comprises the following specific steps of:
b1-1, co-culturing the monoclonal of the cell strain for detecting the CD3 receptor agonist and the monoclonal of the stimulating cell for stimulating the monoclonal, carrying out gradient dilution on the CD3 receptor agonist, and respectively adding the diluted CD3 receptor agonist into a co-culture system to obtain a plurality of biological activity detection systems containing the CD3 receptor agonist with gradient concentration;
b1-2, culturing the biological activity detection system containing the CD3 receptor agonist with gradient concentration until the immune response is fully generated, measuring the generation amount of the reporter protein, and forming a dose curve according to the generation amount of the reporter protein and the concentration of the CD3 receptor agonist.
In the above technical scheme, by setting the gradient concentration, a dose curve of any cell strain for various CD3 receptor agonists can be formed, and the cell strain for detecting CD3 receptor agonists has good selectivity, so that the cell strain is not easy to be interfered by other exogenous substances, and accurate results can be obtained. The EC50 value and the signal to noise ratio can be obtained through a dose curve, the biological activity of the CD3 receptor agonist to be detected can be better judged, and the comparison and comparison can be carried out on different CD3 receptor agonists through the EC50 value, so that the method has better applicability.
Preferably, the method for detecting the concentration comprises the following steps:
b2-1, co-culturing the monoclonal cell strain for detecting the CD3 receptor agonist and the monoclonal cell for stimulating the monoclonal cell, selecting a part with better linearity according to the dose curve obtained in the step B1-2, carrying out gradient dilution on the CD3 receptor agonist according to the concentration of the part, and respectively adding the parts into the co-culture system to obtain a plurality of concentration detection systems containing the CD3 receptor agonist with gradient concentration;
b2-2, culturing the concentration detection system containing the CD3 receptor agonist with gradient concentration until immune response is fully generated, measuring the generation amount of the reporter gene protein, and forming a standard curve according to the generation amount of the reporter gene protein and the concentration of the CD3 receptor agonist;
b2-3, adding the random sample to be tested into a co-culture system of the monoclonal of the cell strain for detecting the CD3 receptor agonist and the monoclonal of the stimulated cells, which is the same as that in the step B1-1, culturing under the same condition in the step B1-2, measuring the generation amount of the reporter gene protein, and comparing a standard curve to judge the content of the CD3 receptor agonist in the sample to be tested.
The cell strain used for detecting the CD3 receptor agonist has good stability and sensitivity and has good dose curve, so that the situation of CD3 receptor excitation can be truly reflected by comparing the actual value of a sample to be detected with the actual value of the standard curve after the gradient concentration of the CD3 receptor agonist is set and the standard curve is drawn, and further the concentration difference of the same CD3 receptor agonist and the activity difference among different CD3 receptor agonists can be more accurately measured. In the experiment, all cells are taken from the same cell strain, and the mother cells of the cell strain are immortalized cells, so that the stability is better, the different batches have better identity, and more accurate results can be obtained in use. And the immortalized T lymphocytes are used as the parent cells of the cell lines, so that the source is wider, and the development cost of the CD3 receptor agonist is saved.
In summary, the present application has the following beneficial effects:
1. in the application, a cell strain for detecting a CD3 receptor agonist is provided, and by introducing an NFAT reporter gene sequence containing a binding sequence, a promoter and an open reading frame of NFAT binding into an immortalized T lymphocyte cell line naturally containing a TCR-CD3 complex, the detection of the CD3 receptor agonist can be realized, so that the cell strain has better stability and higher sensitivity, meanwhile, PBMC cells are avoided, the research and development cost is reduced, meanwhile, the fluctuation of detection results caused by the difference between cell batches is reduced, and the stability of the detection results is improved.
2. In the application, the cell strain for detecting the CD3 receptor agonist is provided, and is subjected to transfection-screening-secondary construction-secondary screening, so that the cell strain with better dose response curve and higher sensitivity is obtained, and the cell strain has higher sensitivity and better stability.
Drawings
FIG. 1 is a graph showing the results of evaluating transfection efficiency of cell lines measured in step A2 of example 1 of the present application.
FIG. 2 shows the results of evaluating the functions of the cell lines measured in step A2 of example 1 of the present application.
FIG. 3 shows the results of functional evaluation of the cell lines secondarily constructed in step A4 of example 2 of the present application.
FIG. 4 shows the result of CD3 receptor abundance determination of the cell line secondarily constructed in step A4 of example 2 of the present application.
FIG. 5 is a dose curve of a portion of a monoclonal cell line versus OKT3 in examples 1 and 2 of the present application.
FIG. 6 is a dose curve of OKT3 antibodies, foraluab antibodies and Anti-CD3CD19 diabodies obtained from the J3-5 cell assay of example 3 of the present application.
FIG. 7 is a standard curve of Anti-CD3CD19 diabodies measured by DB1-8 in example 3 of the present application.
Detailed Description
The present application is described in further detail below with reference to figures 1-7 and examples.
For the following examples, the sources of some of the materials are shown in Table 1.
TABLE 1 Material Source List
| Material | Source |
| NFAT reporter lentivirus | Jin Weizhi customization |
| Jurkat cells | ATCC，TIB-152 |
| Ionomycin | Cayman 11932 |
| PMA | sigma P1-585 |
| Bright-Glo tm Luciferase Assay reagent | Promega E2620 |
| Raji cells | ATCC, CCL-86 |
| OKT3 | Thermo Fisher，14-0037-82 |
| PE anti-human CD3 (UCHT1) | Biolegend，300408 |
Example 1
Cell lines for the detection of CD3 receptor agonists were obtained by lentiviral transfection using an immortalized T lymphocyte line naturally containing the TCR-CD3 complex as the master cell, in this example Jurkat cells were selected.
The cell line contains an NFAT reporter gene comprising an NFAT binding sequence linked to a downstream promoter linked to an open reading frame that expresses a reporter protein. In this example, the open reading frame is a luciferase expression gene, the reporter protein expressed is luciferase, the sequence of the promoter connected downstream by the NFAT binding sequence is SEQ ID NO. 1, the specific sequence is:
GGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTGGAGGAAAAACTGTTTCATACAGAAGGCGTAGATCTAGACTCTAGAGGGTATATAATGGAAGCTCGAATTCCAG。
the cell lines described above for CD3 receptor agonist detection were incubated by the following steps:
a1, transferring the NFAT reporter sequence into the immortalized T lymphocyte cell line naturally containing the TCR-CD3 complex by slow virus transfection.
The specific operation mode of the step A1 is as follows: lentiviruses containing the above nucleotide sequences were infected with 3E 4/well Jurkat cells in 24 well plates at a multiplicity of infection of 50 in a total volume of 400ul; performing cell centrifugation and liquid exchange on the next day to remove slow viruses; the culture was continued until the fourth day, transferred into T25 cell flasks and pressure culture was performed by adding 1640 medium containing 0.5. Mu.g/ml puromycin. After maintaining 0.5 μg/ml of puromycin1640 medium for growth passage pressurization, cells were plated at 1 per well in 96 well plates on day 10; the observed monoclone in the 96-well plate is transferred to a 24-well plate, and then is transferred to a 6-well plate after evaluation and screening, a T25 cell culture bottle, a T75 cell culture bottle and a T175 cell culture bottle are sequentially cultured to obtain monoclone, so that a plurality of cell strains are obtained, and the number of the monoclone is sequentially J1-1, J1-2, J2-3, J2-4, J2-5, J3-1, J3-4, J3-6, J4-1, J4-2, J4-3, J4-5, J4-6, J5-1, J5-3, J5-4, J5-6, J6-1, J6-3, J6-4, J6-5, J6-6, J7-1, J7-2, J7-3, J7-4, J7-6, J8-1, J8-2, J8-3, J8-5.
A2, carrying out transfection efficiency evaluation and function evaluation on the cell strain obtained in the step A1, and selecting a cell strain with better signal to noise ratio.
Transfection efficiency was assessed as follows: after the transfected cell line obtained in the step A1 is subjected to pressure culture for a period of time by using 1640 culture medium of 0.5 mug/ml puromycin, 80uL of cell suspension is taken into each hole of a white impermeable 96-well plate, the cell adding amount is 10000 cells/hole, and then 20uL of culture medium or 20uL of PMA with the working concentration of 300nM and 10uM of Ionomycin are added into each hole, wherein the hole added with the culture medium is used as a control group, and after uniform mixing, the cells are put into a culture box with 5% CO2 at 37 ℃ for culture and incubation for 5 hours. After the completion of the incubation, 100ul bright-Glo ™ Luciferase Assay reagent was added, mixed at 1000rpm at room temperature for 5 minutes, and the mixture was subjected to detection by a chemiluminescent detector, and the detection result was shown in FIG. 1. Cell lines with a signal to noise ratio greater than 5 were selected, and the cell lines remaining after the selection were numbered J1-1, J3-5, J4-6.
The specific operation of the function evaluation is as follows: for cell lines J1-1, J3-5 and J4-6, seeding a white, non-bottom well plate at 40 ul/well, and seeding Raji cells 1E5 per well, 40 ul/well, into a white, non-bottom well plate; raji cells 1E 5/well, 40 ul/well seeded into wells of a white, impermeable 96-well plate containing Jurkat cells; the CD3 agonist OKT3 is prepared into 10000ng/ml culture medium solution, 20 ul/hole is added into the cell suspension after being subjected to 3-time gradient dilution for 7 points, and after being uniformly mixed, the cell suspension is placed into a culture box with 5% CO2 at 37 ℃ for culture and incubation for 5 hours. 100ul of Bright-Glo ™ Luciferase Assay reagent was added, mixed at 1000rpm at room temperature for 5 minutes, and detected by a chemiluminescent detector, the results of which are shown in FIG. 2. The three monoclonal cell lines all had a distinct dose-signal response curve upon stimulation with the CD3 agonist OKT 3.
Example 2
Cell lines for the detection of CD3 receptor agonists were obtained by further processing J1-1, J3-5 and J4-6 obtained in example 1 by the following procedure.
A3, performing secondary construction on the cell strains J1-1, J3-5 and J4-6 to obtain the cell strain after secondary construction.
The specific operation of the secondary construction is as follows: the cell line with better signal is taken as a mother cell, 3E 4/hole J3-5 cells are infected in a 24-well plate through NFAT reporter gene slow virus with the infection complex number of 50 and 4 mug/ml Polybrene, and the total volume is 400ul; culturing for the fourth day, and performing cell centrifugation and liquid exchange to remove slow viruses; cells were plated at 1/well in 96-well plates until day six; on day 20, the observed monoclone in the 96-well plate is transferred to a 24-well plate, and then sequentially transferred to a 6-well plate, a T25 culture flask, a T75 culture flask and a T175 culture flask to obtain the monoclone, wherein the monoclone is used as a cell strain after secondary construction, and is numbered as DA1-1, DA1-3, DA1-4, DA1-6, DA1-8, DA1-10, DA1-14, DA1-15, DA1-16, DA1-18, DA1-20, DA2-1, DA2-2, DA2-3, DA2-10, DA2-13, DA2-15, DA2-20, DA2-21, DA2-22, DA2-23, DB1-1, DB1-2, DB1-3, DB1-4, DB1-5, DB1-6, DB1-8, DB1-10, DB1-11, DB1-13, DB1-14, DB1-15, DB1-16, DB2-2, DB2-3, DB1-2, DB2-3, DB 1-9, DB2-2, DB2-3, DB 1-9, DB2-2, DB2-9, DB2-2, DB 2-9.
And A4, performing functional evaluation and CD3 receptor abundance determination on the cell strain obtained in the step A3 after the secondary construction, and screening the cell strain with higher CD3 receptor abundance, thereby obtaining the cell strain for detecting the CD3 receptor agonist.
The specific steps of the function evaluation are as follows: transferring the cell strain obtained in the step A3 after secondary construction into a 24-well plate, culturing until the density is high, inoculating 1E5 cells/well, 40ul cells/well into a white opaque 96-well plate, inoculating Raji cells at 1E5 cells/well, 40ul cells/well into the white opaque 96-well plate, adding 20ul of CD3 agonist OKT3 with the working concentration of 1000ng/ml or 20ul of culture medium (control group) into each well, uniformly mixing, and culturing in a culture box with 5% CO2 at 37 ℃ for 5 hours. 100ul of Bright-Glo ™ Luciferase Assay reagent is added, the mixture is uniformly mixed at room temperature and 1000rpm for 5 minutes, a chemiluminescent detector is used for detection, the measurement result of the cell strain is shown in figure 3, clones DA1-15, DB1-8, DB1-13, DB1-15, DB2-23, DC1-19, DC1-20, DC2-22 and DB2-3 with better OKT 3/culture medium signal to noise ratio are selected for further amplification culture and evaluation.
The specific steps for determining CD3 receptor abundance are as follows: the monoclonal cell lines obtained by screening in the above steps were further amplified and prepared into 100. Mu.L of a cell suspension of 1000000 cells/tube, while 5. Mu.L of PE anti-human CD3 with a concentration of 10. Mu.g/mL was added as a control group to the cell suspension of Jurkat cells of 100. Mu.L of 1000000 cells/tube, and after half an hour incubation at 4℃the cell lines were washed with PBS for the third time, and the signal values obtained for each monoclonal cell line were determined by using a flow cytometer, the measurement results are shown in FIG. 4, and the signal values obtained for each monoclonal cell line are shown in Table 2.
TABLE 2 CD3 receptor abundance determination results Table
| Monoclonal cell line numbering | Signal value (Mean PE-A) |
| DA1-15 | 13860 |
| DB1-8 | 31031 |
| DB1-13 | 35987 |
| DB1-15 | 9262 |
| DB2-23 | 11097 |
| DC1-19 | 7204 |
| DC1-20 | 11021 |
| DC2-22 | 12259 |
| DB2-3 | 31207 |
| J3-5 | 13578 |
| Jurkat blast cells | 40485 |
| J3-5 (undyed) | 229 |
Cell lines were selected and maintained in which the signal values were higher than 30000, and as can be seen from Table 2 and FIG. 4, the monoclonal cell lines numbered DB1-8, DB1-13, DB2-3 displayed, were maintained and used as cell lines for CD3 receptor agonist detection. Wherein, the cells with the numbers of DB1-8 are preserved, and the preservation number is No: c2020264, the preservation unit is China center for type culture collection, the preservation address is Chinese Wuhan, the classification name is NFAT reporter gene Jurkat stable transgenic cell strain DB8-1, and the preservation date is 2020, 12 months and 22 days.
In examples 1 and 2, jurkat cells were selected as the initial parent cells. Jurkat cells naturally contain a CD3 receptor, and an NFAT reporter gene containing an NFAT binding sequence, a promoter and an open reading frame is transfected into Jurkat cells by a slow virus transfection mode, and the slow virus has stronger transfection capability, so that the Jurkat cells can be more effectively transfected, fully receive the NFAT reporter gene and can respond to a CD3 receptor agonist.
In the step A2, through NFAT reporter gene transfection abundance evaluation and transfected cell function evaluation, selecting the cell receiving Jurkat with larger NFAT reporter gene quantity, further screening Jurkat cells which can fully express reporter gene protein after receiving NFAT reporter gene in the function evaluation, and obtaining the cell screened by the process with good dose-signal curve. Cell lines with better dose-signal curves can more accurately determine the amount of CD3 receptor agonist in the sample to be tested in an actual assay.
In the evaluation of the transfection abundance of the NFAT reporter gene, two agents, namely PMA and Ionomycin, are adopted for co-stimulation, and the PMA and the Ionomycin can enter cells and activate the NFAT in the cells, so that the abundance evaluation can directly determine the degree of the transfection of the NFAT reporter gene sequence in the cells, and the fully transfected cells can be selected. In the functional evaluation, the actual situation is simulated by using OKT3 as an agonist through Raji auxiliary stimulation, and when the NFAT reporter gene sequence is transfected in the cell, the cell with TRC-CD3 on the surface can respond to the OKT3 and express the luciferase through external stimulation. In the process of testing the abundance of the CD3 receptor, PE anti-human CD3 is added to stimulate the CD3 receptor on the cell surface, so that cell strains with more surface CD3 receptors are selected.
For example 1 and example 2, cell lines numbered J3-5, DB1-8, DB1-13, DB2-3 were selected and the following comparison experiments were performed:
1E5 cells/well, 40ul cells/well are planted into a white non-bottom permeable 96-well plate, 1E5 cells/well, 40ul cells/well are planted into a white non-bottom permeable 96-well plate hole containing Jurkat cells; gradient diluting the OKT3 antibody, wherein the initial working concentration of the OKT3 antibody is 5000ng/ml, 12 points are added in 2 times of gradient dilution, 20 ul/hole is added into a hole with stable transgenic cell strain clone, and the mixture is uniformly mixed; the 96-well plate is placed in an incubator with 5% CO2 at 37 ℃ for incubation for 5 hours, 100ul of Bright-Glo ™ Luciferase Assay reagent is added, the mixture is uniformly mixed for 5 minutes at room temperature of 1000rpm, and the mixture is placed in a chemiluminescent detector for detection, and the obtained luminescence intensity-dose curve is shown in FIG. 5.
As can be seen from the results in FIG. 5, the four monoclonal cell lines J3-5, DB1-8, DB1-13 and DB2-3 have better dose curves for OKT3, and the monoclonal cell lines DB1-8, DB1-13 and DB2-3 after secondary construction have obvious advantages in signal intensity and signal to noise ratio for J3-5.
Example 3
Methods for detecting CD3 receptor agonists, including biological activity assays and concentration assays. Wherein, the biological activity determination method comprises the following steps:
b1-1, implanting a monoclonal cell strain with the number of J3-5 into a white impermeable 96-well plate at the amount of 1E 5/well and 40 mu/L/well, adding 1E 5/well of Raji cells, co-culturing 40 ul/well as a stimulating cell for stimulating the monoclonal, dividing a culture system into three groups, respectively carrying out gradient dilution on an OKT3 antibody, a foraluab antibody and an Anti-CD3CD19 double antibody, and respectively adding the obtained products into the three groups at the adding amount of 20 ul/well, wherein the initial working concentration of the OKT3 antibody and the foraluab antibody is 5000ng/ml, the initial working concentration of the Anti-CD3CD19 antibody is 1500ng/ml, and carrying out gradient dilution on the three at 2 times for 8 points, thereby obtaining a plurality of biological activity detection systems containing the CD3 receptor agonist with gradient concentration after the drug addition is completed.
B1-2, placing the biological activity detection system containing the CD3 receptor agonist with gradient concentration into an incubator with 5% CO2 at 37 ℃ for culture and incubation for 5 hours, adding 100ul Bright-Glo ™ Luciferase Assay reagent, mixing for 5 minutes at room temperature of 1000rpm, placing into a chemiluminescent detector for detection, taking the concentration of the added reagent as an abscissa and the luminous intensity as an ordinate, and obtaining the double antibody curves of OKT3 antibody, foraluab antibody and Anti-CD3CD19, as shown in figure 6.
FIG. 6 shows that the monoclonal cell line numbered J3-5, when used for the detection of CD3 receptor agonists, has a better dose curve for both OKT3 and Anti-CD3CD19 bispecific antibodies, and can be calculated from the dose curve, whereas the non-agonist CD3 binding antibody Foralumab has no dose curve, demonstrating that the above protocol can be better used for the activity assay of CD3 agonists and can distinguish between agonistic CD3 antibodies and non-agonistic CD3 antibodies.
The concentration detection method specifically comprises the following steps:
b2-1, accession number No: c2020264, cell lines of China center for type collection are planted in a white impermeable bottom 96-well plate at the amount of 1E5 cells/well and 40 mu/L cells/well, raji cells 1E5 cells/well and 40 mu/well are added as stimulating cells for stimulating the monoclonal, the monoclonal cells are co-cultured, anti-CD3CD19 bispecific antibodies are diluted by 8 points in a 2-fold gradient manner at the initial working concentration of 1000pg/ml, and 20 mu L/well are added into the system, and the concentration detection system containing the CD3 receptor agonist with the gradient concentration is obtained after uniform mixing.
B2-2, the above cell culture system containing CD3 receptor agonist at gradient concentration was cultured in an incubator containing 5% CO2 at 37℃for 5 hours until the immune reaction was sufficiently developed, then 100 ul/well Bright-Glo ™ Luciferase Assay reagent was added, and mixed at room temperature 1000rpm for 5 minutes, and the 96-well plate was put into a chemiluminescent detector to detect its luminescence intensity, and a standard curve was drawn with the luminescence intensity and OKT3 concentration, and the standard curve was drawn as shown in FIG. 7.
B2-3, the cell lines with the numbers DB1-8 are planted into a white impermeable 96-well plate at the volume of 1E5 cells/well and 40 mu/L per well, raji cells are added for 1E5 cells/well and 40 mu/well as the stimulated cells for stimulating the monoclonal, the cells are cultured together, the anti-CD3CD19 bispecific antibody is diluted by 8 points at the initial working concentration of 1000pg/ml in a 2-fold gradient manner, 20 mu L/well are added into the system, the mixture is uniformly mixed, the 96-well plate is placed into a culture box with 5% CO2 at 37 ℃ for culture and incubation for 5 hours, 100 mu L of Bright-Glo ™ Luciferase Assay reagent is added, the mixture is evenly mixed at room temperature of 1000rpm for 5 minutes, a chemiluminescent detector is added for detection, and the measurement results are compared with a standard curve, and are shown in Table 3.
TABLE 3 comparison deviation table of return test result and standard curve
| anti-CD3CD19 bispecific antibody concentration (pg/ml) | RUL signal value | Returning the result | Deviation% |
| 1000.000 | 7487 | 1014.425 | 1.44 |
| 500.000 | 6351 | 488.097 | -2.38 |
| 250.000 | 5092 | 254.179 | 1.67 |
| 125.000 | 3689 | 123.539 | -1.17 |
| 62.500 | 2645 | 64.315 | 2.90 |
| 31.250 | 1837 | 30.887 | -1.16 |
| 15.625 | 1343 | 14.279 | -8.50 |
| 7.813 | 1112 | 7.417 | -5.07 |
Wherein, deviation= (back measurement result-anti-CD 3CD19 bispecific antibody theoretical concentration)/anti-CD 3CD19 bispecific antibody theoretical concentration is 100%
From the data, it is clear that the concentration of the CD3 receptor agonist is measured by using DB1-8 monoclonal cells, the result is accurate, the deviation is smaller, the sensitivity is excellent, 7.813pg/mL can be achieved, and the sensitivity and accuracy advantages are obvious compared with the sensitivity and accuracy advantages of the sensitivity and accuracy measured by using PBMC for cytological activity detection and using LBA method.
In addition, the cell lines obtained in example 1 and example 2 can be used to detect neutralizing antibodies to CD3 receptor agonists or antagonists, and have a wide application prospect.
The present embodiment is merely illustrative of the present application and is not intended to be limiting, and those skilled in the art, after having read the present specification, may make modifications to the present embodiment without creative contribution as required, but is protected by patent laws within the scope of the claims of the present application.
Sequence listing
<110> Ningbo Xining detection technology Co., ltd
<120> cell line for detecting CD3 receptor agonist, construction method thereof and method for detecting CD3 receptor agonist
<130> WZF006PTA01F1906704
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<170> SIPOSequenceListing 1.0
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Claims (1)
1. A cell line for the detection of a CD3 receptor agonist, characterized in that the cell line is an immortalized T lymphocyte cell line naturally containing a TCR-CD3 complex, comprising in its cell an NFAT reporter comprising a binding sequence that binds NFAT linked to a downstream promoter linked to an open reading frame that expresses a reporter protein; the cell strain is a cell strain with a preservation number of CCTCC NO: c2020264, the preservation institution is China center for type culture Collection.
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