CN116218918A - Jurkat effector cell, construction method and application thereof - Google Patents

Abstract

The invention provides Jurkat effector cells, a construction method and application thereof. The construction of the effector cell is to knock two reporter genes GFP and luciferases into the coding frame of the Nur77 gene at fixed points in Jurkat cells while carrying out gene editing on the Nur77 gene termination codon region so as to ensure that the expression regulation of the reporter genes is consistent with the expression of endogenous Nur77 genes. The effector cells can react and evaluate specific functions of CAR and TCR molecules, including the strength of antigen-dependent and non-antigen-dependent Tonic signals, by Nur77 signal strength. The method is efficient, economical, convenient and fast, and the result is stable and accurate.

Description

A kind of Jurkat effector cell and its construction method and application

technical field

The present invention relates to an effector cell and its construction method and application, in particular to a Jurkat effector cell and its construction method and application.

Background technique

Tumor immunotherapy is to control and kill tumor cells by mobilizing the body's immune system and enhancing the anti-tumor immunity of the tumor microenvironment. In recent years, with the development of CAR-T and TCR-T technologies, tumor immunotherapy has been rejuvenated and has entered a stage of major outbreak. Among them, CAR-T has shown the potential to cure tumors in blood tumors. Chimeric antigen receptor (CAR) and T cell receptor (TCR) modified T cells are the latest and most fruitful technologies in the current adoptive cell therapy technology, realizing the research from basic immunological mechanism to clinical application of tumor immunotherapy change. Because of their ability to express artificially synthesized receptors and specifically recognize target cells, CAR-T and TCR-T are becoming exciting tumor treatments.

CAR and TCR-specific molecular sequence design and screening, as well as structure optimization and evaluation are the most critical issues faced by major companies and academic institutions in the development of CAR-T and TCR-T products. For example, the current traditional screening method uses high affinity as an indicator to select CAR molecules, which cannot distinguish tumor cells from normal cells. In the clinic, CAR-T cells with low tumor specificity will attack normal cells while killing cancer cells, thus causing side effects on the normal physiological functions of patients. In addition, in addition to specific recognition of tumor target antigens to activate T cells to play a killing function, CAR molecules themselves have structural characteristics such as expression density, folding conformation, and co-stimulatory factors on the surface of T cells, which may activate T cells in an antigen-independent manner. The signal is called Tonic signal. When this Tonic signal is too strong, without the stimulation of the target antigen, the signal can over-activate T cells, induce T cell differentiation, and accelerate T cell exhaustion, thereby reducing the persistence of CAR-T in the body and affecting its anti-tumor effect. Tumor function. Therefore, the development of an efficient and even high-throughput evaluation system that can simultaneously evaluate the affinity, specificity, and Tonic signal of CAR and TCR molecules is a key step to promote the further expansion of CAR-T and TCR-T therapies.

Jurkat cells are human leukemia T lymphocytes, which have the corresponding characteristics and functions of primary T lymphocytes, and express CD3 and other antigens on the surface. Through genetic engineering, they can become effector cells for CD3 double antibody and CAR or TCR molecular specificity assessment. Currently commonly used Jurkat-NFAT-LUC cells, the principle of effector cell construction is based on the phosphorylation and activation of intracellular PLC-γ after the TCR/CD3 complex or CAR molecule is targeted to activate the transcription of the NFAT pathway. , so that the fluorescence mediated by NFAT-RE is generated, and finally the intensity of the fluorescence signal is used to reflect the specificity of the antibody or CAR molecule. Similar to NFAT, there are Jurkat effector cells based on 41-BB or CD69 signaling pathways, but the expression regulation of these genes is not only activated by direct antigenic stimulation, but also by other inflammatory mediators (such as type I interferon and interleukins) are indirectly activated. Therefore, it is not very suitable for the evaluation of Tonic signal. Nur77 (NR4A1) is a nuclear receptor family transcription factor, and its expression can be used as a molecular marker of lymphocyte antigen-specific activation. It has been confirmed in human thymus tissue and gene reporter mice, and it has also been demonstrated in mouse models It does not respond to type I interferon or cytokine stimulation.

Therefore, the present invention provides a method for constructing genetically engineered Jurkat effector cells based on the Nur77 signaling pathway and the specific use of the effector cells, which are of great significance to the functional evaluation of CAR or TCR molecules.

Contents of the invention

The purpose of the present invention is to solve the problems faced by traditional Jurkat effector cells, such as the inability to efficiently and accurately detect CAR and TCR molecular affinity, antigen-dependent specific signals and antigen-independent Tonic signals. Through a gene editing strategy, a Jurkat effector cell based on the Nur77 signaling pathway is constructed, which can efficiently, accurately, quickly and conveniently evaluate the antigen-dependent specific signals and antigen-independent Tonic signals of CAR or TCR molecules.

To achieve the above object, the present invention provides a method for constructing Jurkat effector cells, comprising the following specific steps:

By editing the stop codon region of the Nur77 gene in Jurkat cells, the reporter gene was targeted and knocked into the coding frame of the Nur77 gene in the same frame to ensure that the expression regulation of the reporter gene was consistent with the expression of the endogenous Nur77 gene.

Preferably, the gene editing method used to deliver the gene editing material to Jurkat cells can be ZFN, TALEN or/and CRISPR-Cas9, etc.; preferably CRISPR-Cas9, Cas9 can be SpCas9, SaCas9, SpCas9-HF, eSpCas9, xCas9 , cpf1 or other Cas9 of different bacterial genera; more preferably SpCas9.

Preferably, the gene editing substance can be plasmid, virus, DNA, mRNA or/and RNA protein complex; preferably RNA protein complex or/and template dsDNA for homologous repair.

Preferably, the delivery of gene editing substances can use liposome, calcium phosphate, DEAE-dextran, electroporation, microinjection or gene gun transfection methods; electroporation transfection is preferred.

In some embodiments, the CRISPR-Cas9 gene editing material includes Cas9 mRNA, Cas9 protein or/and sgRNA. Preferably, the RNP complex formed by Cas9 protein and sgRNA is used for electroporation. The RNP complex can be obtained by directly mixing and incubating Cas9 protein and sgRNA, or by mixing the two in a specific buffer (such as electroporation buffer).

In some embodiments, the sgRNA is designed by first identifying the genomic site to be knocked in, and designing it using the DNA sequence surrounding the site. The design principle is that the sequence of the PAM region is NGG, wherein N is any base in A, T, C and G. sgRNA includes targeted crRNA sequence and tracrRNA sequence, wherein crRNA can be 17, 18, 19, 20, 21 or 22 bases, preferably 20 bases.

In some embodiments, the sgRNA is unmodified or chemically modified. Chemical modifications include 2-O-methylation, 3-thioxo, 2-O-methylation combined with 3-thioxo, and the like.

In some embodiments, the 3 bases at the 5' and 3' ends are simultaneously 2-O-methylated and 3-thio-modified. Chemical modifications can occur between 1 and 10 bases at the 5' and 3' ends of the sgRNA. The designed sgRNA can be obtained by in vitro transcription of T7, or directly synthesized in vitro.

In some embodiments, the gene editing agent further includes template dsDNA for homologous repair at the edited site. The homology repair template dsDNA includes the left homology arm DNA, the knock-in exogenous reporter gene, and the right homology arm DNA. The left homology arm of the template DNA is homologous to the sequence at the 5' end of the DNA incision, and the right homology arm is homologous to the sequence at the 3' end of the DNA incision.

In some embodiments, the length of the left and right homology arm fragments can be selected from 50 to 1000 bases, preferably 150 bases. The knock-in reporter gene needs to be expressed in the same frame as the endogenous Nur77 gene, so that the expression level of the endogenous gene can be reflected by the expression of the reporter gene. Reporter gene can be at least one in fluorescent protein gene, luciferase gene, chloramphenicol acetyltransferase gene, dihydrofolate reductase gene, β-galactosidase gene and β glucuronidase gene; Preferably Dual reporter for the combination of the green fluorescent protein gene and the luciferase gene.

In some embodiments, the endogenous Nur77 gene and the reporter gene are separated by a cleavage peptide, which can be selected from at least one of T2A, P2A, E2A and F2A; preferably a combination of P2A and T2A.

In some embodiments, after the synthesis of the homologous repair template dsDNA is completed, it can be cloned and ligated into a plasmid vector. The plasmid vector can be any one, such as pUC57, pCDNA3.1, pCMV, etc., and the position of the ligation can also be flexible. It is also possible to proceed directly to the next steps without ligation to a plasmid vector.

In some embodiments, homologous repair dsDNA can be obtained by using unmodified or modified primers for PCR amplification, and the modified primers are modified with 5'-phosphorothioate combined with 5'-Biotin-TEG or 5'-locked nucleic acid. After the template dsDNA is amplified by PCR, the following methods can be used separately or in combination for purification and concentration: purification of DNA purification kits from various reagent companies, purification of various DNA binding magnetic beads, gel chromatography, ion chromatography, affinity chromatography, etc. And chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, etc.

In one embodiment, the present invention also discloses a method for establishing a Jurkat effector cell in which a double reporter gene of green fluorescent protein gene and luciferase gene is knocked in behind the Nur77 coding frame, and the synthesized gRNA is electrically charged with the repair template dsDNA. Jurkat cells were transfected, and the Jurkat effector cells were obtained through screening.

Preferably, it includes the following steps:

Step 1, design and evaluation of Nur77 gRNA sequence;

Step 2, molecular design and preparation of homologous repair template dsDNA;

Step 3, transfecting the cas9-sgRNA RNP complex and template dsDNA into Jurkat cells by electroporation;

Step 4, using the luciferase activity assay method to screen and verify the cell pool in which the reporter gene is knocked in;

Step 5. Screening and verifying the correct knock-in single cell clones from the reporter gene knock-in cell pool.

Wherein said sgRNA is as shown in at least one of SEQ ID NO:1～7, preferably as shown in SEQ ID NO:1, SEQ IDNO:3 or/and SEQ ID NO:6; Wherein said repair template dsDNA is as shown in SEQ ID NO:1 ID NO:8.

The present invention also provides an application of Jurkat effector cells. On the one hand, it can be used for CAR, TCR and BCR molecules and antibody-specific function screening and evaluation, especially for the precise precision of CAR molecule antigen-dependent signals and non-antigen-dependent Tonic signals. Detection; on the other hand, it can be used for high-throughput screening of CAR molecules of antibody display libraries.

The advantage of the present invention is that two reporter genes (GFP and luciferase gene) are simultaneously knocked into the coding frame of the Nur77 gene in Jurkat cells to construct a Jurkat effector cell based on the Nur77 signaling pathway. The effector cells can be used to measure the strength of Nur77 signal by flow cytometry and luciferase experiments to evaluate the specific function of CAR or TCR.

1. By selecting and targeting the Nur77 signaling pathway to construct effector cells, compared with existing technologies, it can more accurately measure the antigen-independent Tonic signal of CAR or TCR molecules, which is more conducive to rapid screening and evaluation of better CAR or TCR molecules for use In vivo or clinical verification will help accelerate the research and development process of CAR-T or TCR-T projects.

2. By choosing dual reporter genes, the choice of experimental methods will be more flexible and convenient. Since most CAR molecules will induce the endocytosis of CAR molecules on T cells under antigen stimulation, it is not suitable for accurate detection of antigen-specific signals of CAR-positive cell populations by flow cytometry. It is more conducive to the comprehensive evaluation of the optimal CAR or TCR molecule.

3. At present, CAR molecules are basically derived from scFv or VHH of antibody sequences that have been verified for affinity and specificity, and a large number of experimental results also show that excellent antibody molecules are not necessarily suitable for use in CAR-T. Therefore, a high-throughput screening technology platform for CARs molecules and structures suitable for CAR-T needs to be established and improved. The Jurkat effector cells constructed by the present invention are very suitable for high-throughput screening of CAR molecules initiated by antibody display libraries. Compared with multiple rounds of panning and verification of traditional antibody libraries, the screening process based on Jurkat effector cells will be more convenient , faster and more economical, the screened CAR molecules and structures are tailor-made for CAR-T.

4. The method of the present invention can efficiently establish a stable cell line in which LUC and GFP dual reporter genes are knocked in at the Nur77 site in-frame.

Description of drawings

Figure 1 is a schematic diagram of the construction of Jurkat effector cells based on the Nur77 signaling pathway,

Figure 2 is a schematic diagram of the working principle of Jurkat effector cells based on the Nur77 signaling pathway,

Figure 3 is the expression detection of gene edited cell pool luciferase,

Fig. 4 is the expression detection of single cell clone luciferase,

Figure 5 is the genotype identification of single cell clones of Jurkat effector cells,

Figure 6 shows MSLN CAR expression and antigen-independent Tonic signal detection,

Figure 7 is the detection of MSLN CAR antigen-dependent signal,

Figure 8 is a comparative analysis of MSLN CAR antigen-dependent and antigen-independent signals.

Detailed ways

In order to understand the technical content of the present invention more clearly, the following embodiments are given in detail in conjunction with the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. Various commonly used biological or chemical reagents used in the examples are all commercially available products.

Example 1: Design, synthesis and screening of sgRNA targeting Nur77 site

In order to achieve efficient insertion of the reporter gene into the coding frame of the Nur77 gene, about 50 bp before and after the stop codon of Nur77 will be selected, with a total of about 100 bp sequence, and the sgRNA design will be carried out through the online sgRNA design website (http://crispor.tefor.net/) , and screened 7 sequences with higher scores for in vitro synthesis (sgRNA synthesis service provided by Nanjing GenScript). Preferably, in this embodiment, all sgRNAs are modified with 3'-sulfo and 2'-O-methylation at the 3' end and 3' end respectively, so as to make the sgRNA more stable and less prone to After transfection into cells, it is rapidly degraded by nucleases. The chemically synthesized sgRNA sequence is shown in Table 1 (Nur77 sgRNA list and cutting efficiency)

Table 1

serial number serial number PAM Indel% sgRNA-01 SEQ ID NO:1 GGG 81 sgRNA-02 SEQ ID NO:2 TGG 13 sgRNA-03 SEQ ID NO:3 TGG 67 sgRNA-04 SEQ ID NO:4 TGG twenty two sgRNA-05 SEQ ID NO:5 GGG 15 sgRNA-06 SEQ ID NO:6 AGG 63 sgRNA-07 SEQ ID NO:7 AGG 34

In order to screen the best sgRNA, the synthetic 200pmol sgRNA was mixed with 100pmol TrueCut ^TM Cas9 protein (purchased from Thermo Fisher) and incubated at room temperature for 20min; using LONZA Nucleofector System, according to the electroporation recommended procedure CL-120, RNP was electroporated into 5E5 Jurkat cells In , the cells after electroporation were immediately transferred to a 12-well plate containing pre-warmed medium, and the names were marked. After 3 to 5 days, the cells after electroporation were taken to extract genomic DNA according to the instructions of the genomic DNA extraction kit (TIANGEN), and the unedited wild-type Jurkat genome was used as a control. Using specific primers Nur77-SF (SEQ ID NO: 9) and Nur77-SR (SEQ ID NO: 10) designed before and after the cutting site, the gene editing region was amplified by PCR for Sanger sequencing. The sequencing results were compared with the control sequence to detect the specific editing efficiency of the corresponding sgRNA. The specific editing efficiency was analyzed through the online analysis tool ICE Analysis (https://ice.synthego.com), and the analysis results are listed in Table 1. The results showed that SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6 had higher editing efficiency.

Example 2: Molecular Design and Preparation of Homologous Repair Template dsDNA

The genetic modification scheme of the Jurkat effector cells constructed in the present invention is to first use CRISPR-Cas9 to generate a DNA nick in the stop codon region of the Nur77 genomic site, and then use the homologous repair template dsDNA (including the reporter gene) to perform homologous recombination on the DNA nick Repair, and finally knock the reporter genes LUC and GFP into the coding frame of the Nur77 gene. This genetic modification can make the LUC and GFP genes into the site, and use the endogenous gene Nur77 promoter to transcribe and express. The specific construction diagram is shown in Figure 1. Homologous repair template dsDNA consists of two parts:

a. Left and right homology arms: The left and right homology arms are mainly used to identify the target DNA and carry out recombination and exchange. In order to ensure the correct expression of the knock-in gene, the knock-in gene is targeted to the 3' end region of the last exon of the Nur77 gene . At the same time, in order to ensure that the selected sgRNA will not edit the repair template, a synonymous mutation of individual bases was carried out in the complementary region of the left homology arm. In addition, the length of the homology arm is more important to the efficiency of the entire gene knock-in. Generally, the longer the homology arm is, the higher the knock-in efficiency will be. However, the length of the entire gene knock-in sequence is limited. The length of the reporter gene is relatively long, which is preferred, so in this embodiment, a sequence about 150 bp upstream and downstream of the cleavage site is selected as the homology arm.

b. Reporter gene: In the present invention, two reporter genes, luciferase gene LUC and green fluorescent protein gene GFP, are selected to be knocked in simultaneously. In order to ensure that the two inserted genes can be expressed consistently with the endogenous Nur77 gene, the present invention added a cleavage peptide between these three genes, and the combination of P2A and T2A was selected in this embodiment.

After all the components of the homologous repair template dsDNA are sequentially designed, its nucleotide sequence is shown in SEQ ID NO:8. After gene synthesis of this sequence (gene synthesis service provided by Shanghai Sangong), it was directly cloned into the cloning plasmid pUC57. Using the plasmid as a template and the designed unmodified homologous template primers Nur77-LG-F (SEQ ID NO: 11) and Nur77-LG-R (SEQ ID NO: 12), a large amount of PCR amplification for homologous repair dsDNA molecules. The PCR product was purified and recovered by the MN kit to obtain high-concentration and high-purity homologously repaired dsDNA (SEQ ID NO: 8).

Embodiment 3: Construction of Jurkat effector cells

1) Electroporation of Jurkat cells

For each sgRNA, 1E6 Jurkat cells were electroporated to deliver spCas9 protein (purchased from ThermoFisher), sgRNA and homologous repair dsDNA (PCR purification product obtained in Example 2), so as to cut the Nur77 genomic site at one time And knocking in the reporter gene, the electroporation process uses the Lonza electroporation kit (product number: V4XP-3024) and the Lonza electroporation equipment (4D-Nucleofector). The specific process is as follows: after preparing Lonza electroporation buffer, mix 100 pmol spCas9 protein with 200 pmol sgRNA-01 and sgRNA-03 designed and synthesized, and incubate at room temperature for 20 minutes, then add 6 μg homologous repair dsDNA. In order to further enhance the editing efficiency, you can choose to add 200 pmol Alt-RElectroporation Enhancer (purchased from IDT), mix it at room temperature, mix it with the 1E6 Jurkat cell pellet, and add it to the electroporation cup. Electric shock according to the recommended procedure CL-120, add after electroporation Transfer to a 6-well plate containing 2ml of pre-warmed medium and mark the name. You can choose to repeat the above electroporation process 2 to 3 times in a row.

2) Single cell clone screening verification

a. Cell pool verification: Jurkat cells were cultured for 7 days after electroporation, and luciferase assay was used to detect the knock-in effect of the reporter gene in the cell pool. Under the stimulation of two agonists, PMA and Ionomycin, T cells will induce and activate the expression of Nur77 gene. Therefore, when the reporter gene is successfully knocked into the Nur77 locus, the reporter gene will also be expressed simultaneously with the activation of Nur77, so 1E5 all edited cells, including the unedited control Jurkat cells, will be taken in 96 wells plate. For each type of cell, two groups and two duplicate wells were set up, one group was added with 2.5 μM Ionomycin and 20ng/mL PMA (purchased from Beyond) as the experimental group, and the other group was not added as the control group. After being placed in a 37-degree incubator for 2 hours, 50 uL of fluorescent substrate One-Glo (purchased from Promega) was added to each well, and the fluorescence value was measured with a fluorophotometer (Envision), and the results were analyzed. The result analysis is shown in Figure 3. The results show that in the cell pools of the sgRNA-01 and sgRNA-03 groups, there are cell clones with correct knock-in compared to the control, and in the two repetitions, the editing efficiency of the sgRNA-01 group is higher than that of the sgRNA-03 Group.

b. Screening of single-cell clones: Inoculate the cells of the sgRNA-01 group into 96-well plates at 0.5 and 1 per well by limiting dilution method, culture them in a 37-degree CO ₂ incubator for 10-14 days, and select the cells with obvious monoclonal Cells were transferred to 48-well plates to continue to scale up. After culturing for 4 days, half of the cells were taken out from each well for primary screening by the luciferase assay method, the specific method being the same as above. The general process is as follows: from the 58 clones picked, take 100uL (about half of the cells) into a black opaque 96-well plate, add 2.5μM Ionomycin and 20ng/mL PMA to each well, and react for 2 hours. 50uL of fluorescent substrate One-Glo (purchased from Promega) was added, and the fluorescence value was measured with a fluorophotometer (Envision). The analysis results are shown in FIG. 4 . Finally, single-cell clones NLG-1, NLG-8, NLG-31, NLG-46, and NLG-57 with better cell proliferation and higher fluorescence signals were selected for further amplification and cryopreservation, and the genome was extracted at the same time. DNA for further genotyping and sequencing analysis. The genotyping primers are: Nur77-SF, Nur77-SR and LUC-GFP-R (SEQ ID NO: 13). The PCR electrophoresis graph is shown in Figure 5. The results showed that NLG-8 was homozygous, that is, both alleles were inserted into the reporter gene, while the other four clones were heterozygous, that is, only one allele was successfully inserted into the reporter gene, and The other allele is wild type. Further sequencing results confirmed that all five clones were correctly inserted with the designed reporter gene at the 3' end of the last exon of Nur77.

Example 4: Jurkat effector cells are used for functional screening and evaluation of CAR molecules

In this example, the functional screening process of the MSLN-BBz CAR molecule is selected as an example, and the functional screening and evaluation processes of other CAR or TCR molecules or structures are basically similar.

Preparation of CAR-Jurkat cells

The constructed and prepared different MSLN-BBz CAR molecules (6 in total, including a BMK molecule M5 clinically verified by Novartis) were infected with the Jurkat effector cell line NLG-8# clone verified in Example 3 with an MOI equal to 1. The infected cells were transferred to a 37°C, 5% CO ₂ cell incubator for 3 to 7 days, and the medium was changed or passaged every 2 days.

MSLN CAR expression and detection of non-antigen-dependent Tonic signal

The MSLN CAR-Jurkat cells cultured for 3 to 7 days were taken, and the expression of MSLN CAR and the non-antigen-dependent Tonic signal were detected by FACS. The specific detection process was as follows:

a. Antibody staining: Take 1E5 cells from each group in a 96-well plate, centrifuge at 350g for 5 minutes, remove the supernatant, add PBS to wash once, add Fc-labeled Mesothelin antigen (purchased from Acro) and incubate at 4 degrees for 30 minutes. Centrifuge, wash once with PBS, add AF647-labeled anti-human Fc secondary antibody (purchased from Jackson) and incubate at 4 degrees. Centrifuge after incubation for 30min, and resuspend the cells with 100uL PBS, and perform flow cytometric detection. Two groups of BMK were set up in parallel, one group was the positive control group, and 2.5 μM Ionomycin and 20 ng/mL PMA were added to the 96-well plate for pretreatment for 1 h before antibody incubation, while the other group was not treated.

b. Flow cytometry detection and analysis: The stained cells were detected by FACS with BD flow cytometer to detect APC and FITC channels, and the flow cytometry results were analyzed by FlowJo, and the results are shown in Figure 6. FACS results showed that except for the poor expression of CAR-4# molecule, other CAR molecules were well expressed. Since the expression of CAR is detected by antigen, the affinity of its antibody sequence can be reflected by the intensity of CAR expression, and this result shows that CAR-1# has a higher expression intensity than BMK, while CAR-5# has a similar and CAR-2# have lower expression intensity. Therefore, the use of Jurkat effector cells, compared with primary T cells and traditional antibody affinity testing, has a high infection efficiency, is simple, convenient and more economical. At the same time, by detecting the expression level of background GFP in CAR-Jurkat cells, it can reflect the self-activation degree of CAR without antigen stimulation, that is, Tonic signal. The results of this experiment show that BMK CAR has a very low expression of GFP, indicating that its Tonic signal is weak. After being stimulated by the agonist Ionomycin and PMA, the CAR-positive cells will turn from GFP-negative to positive, indicating that the Jurkat effector cells constructed in this patent invention Specific is good. Further GFP expression of CAR molecules in MSLN found that CAR-3# molecules had high GFP expression in the CAR-positive group, indicating that their Tonic signal was high and not suitable as CAR molecules, while CAR-1# and CAR-5# It has the same low GFP expression level as BMK, indicating that its Tonic signal is weak, and can be used as a candidate CAR molecule for further development.

Detection of MSLN CAR antigen-dependent signaling

Since the CAR molecules of most targets will mediate the endocytosis of CAR molecules on the cell membrane after being stimulated by antigens, the FACS method cannot be directly used for the detection of CAR molecule antigen-dependent signals. Therefore, the present invention uses CAR-Jurkat effector cells The intensity of luciferase expression under antigen and non-antigen stimulation reflects the antigen-dependent signal of CAR molecule, and this signal can further reflect the targeting specificity and killing function of CAR. The specific experimental procedure is as follows: MSLNCAR-Jurkat cells cultured for 3 to 7 days were taken, and the CAR expression levels of all groups were adjusted to a consistent level according to the CAR expression levels. According to the effect-to-target ratio of 1:1 or 2:1, CAR-Jurkat cells were co-incubated with target cells with different MSLN expression levels for 3-5 hours. In this example, the effect-to-target ratio of 1:1 was selected, and the CAR-Jurkat cells in each group after the CAR expression levels were adjusted to be consistent were compared with target cells A2780 with no MSLN expression, target cells with weak MSLN expression SKOV3, and target cells with high MSLN expression. Target cells OVCAR3 were co-incubated for 4 hours in a black opaque 96-well plate, and Jurkat cells not infected with CAR were used as a control group. The number of effector cells or target cells in each well is 4E4, and 2 duplicate wells are set. After co-incubating for 4 hours, 50 uL of fluorescent substrate One-Glo (purchased from Promega) was added to each well, and the fluorescence value was measured with a fluorophotometer (Envision). The analysis results are shown in FIG. 7 . The results of luciferase expression detection showed that CAR-1# and CAR-5# had target-specific activation similar to BMK, showing extremely low luciferase activity under the stimulation of MSLN-negative cell A2780, and in MSLN high-expressing cells showed higher luciferase activity under stimulation of OVCAR3, while weakly expressing cells of SKOV3 showed relatively weaker luciferase activity. Comprehensive analysis of the experimental data of non-antigen-dependent signals in Figure 6 and antigen-dependent signals in Figure 7 (as shown in Figure 8), it is not difficult to evaluate that CAR-1# and CAR-5# have functions similar to BMK CAR molecules, showing The lower non-antigen-dependent Tonic signal and the higher and more specific antigen-dependent signal can be used as candidate molecules for further in vivo verification or clinical development, while other CAR molecules will not be worthy of further development.

Therefore, it can be seen from the above example data that the Jurkat effector cells developed based on the present invention can be very economical, fast and efficient to screen out more suitable CAR molecules for further CAR-T project development, and to accelerate the development of CAR-T projects is of great significance.

Example 5: Screening process of Jurkat effector cells for displaying library CAR molecules

The Jurkat effector cells of the invention can be used for the screening and evaluation of scFv or VHH derived from antibody sequences, and are also very suitable for high-throughput screening of CAR molecules of antibody display libraries. Since the screened CAR molecules and structures are tailor-made for CAR-T, it will be more convenient, economical and efficient. The recommended efficient screening process is:

文库)以富集特定靶点的抗体序列。Panning and enrichment: 1-2 rounds of preliminary panning of antibody (scFv) display libraries (including but not limited to immune libraries or

library) to enrich for antibody sequences for specific targets.

Construction of the CAR virus library: Design universal primers to amplify the scFv sequence of the antibody using the genomic DNA of the antibody display library as a template, and assemble it into a conventional second-generation CAR structure, such as CD8sp-scFv-CD8Hinge-CD8TM-41BB-CD3z. Finally, transform Escherichia coli, extract the plasmids of mixed clones, and package lentiviruses or retroviruses to construct CAR virus libraries.

Construction of CAR-Jurkat cell bank: Infect Jurkat cells with packaged CAR virus to construct CAR-Jurkat cell bank.

Screening of CAR molecules: Take an appropriate CAR-Jurkat cell bank and incubate with specific target cells, sort out GFP-positive cells by flow cytometry, further culture or extract DNA, and finally use CAR primers to amplify the CAR sequence, TA clone, and sequence To obtain the CAR sequence for primary screening. If too many sequences are obtained, two or more rounds of repeated screening can be selected, and the obtained sequences can be further screened and verified according to Example 4.

Using the Jurkat effector cells invented by this patent, according to the recommended screening process, better CAR molecules can be screened within one month at the fastest for further development of CAR-T projects, which is very economical and efficient.

Claims (10)

1. A method for constructing Jurkat effector cells, characterized in that it comprises the following steps: while carrying out gene editing in the Nur77 gene stop codon region in Jurkat cells, the reporter gene is targeted and knocked into the coding of the Nur77 gene in the same frame box to ensure that the expression regulation of the reporter gene is consistent with the expression of the endogenous gene Nur77. 2. A method for constructing Jurkat effector cells according to claim 1, characterized in that, the gene editing method selected from ZFN, TALEN or/and CRISPR-Cas9 is used to deliver gene editing substances. 3. A method for constructing Jurkat effector cells according to claim 1 or 2, wherein the gene editing substance is homologous repair template dsDNA, and its nucleotide sequence is shown in SEQ ID NO.8. 4. A method for constructing Jurkat effector cells according to claim 1 or 2, wherein the RNP complex formed by Cas9 protein and sgRNA is used for electroporation. 5. A method for constructing Jurkat effector cells according to claim 4, wherein the sgRNA targeting nucleotide sequence is shown in at least one of SEQ ID NO:1-7. 6. A kind of method of constructing Jurkat effector cell according to claim 1 or 2, is characterized in that, described reporter gene is the double reporter gene of green fluorescent protein and luciferase gene combination. 7. a kind of method of constructing Jurkat effector cell according to claim 1 or 2, is characterized in that, described method comprises the following specific steps: 1) Design and evaluation of Nur77 gRNA sequence; 2) Molecular design and preparation of homologous repair template dsDNA; 3) Transfecting the cas9-sgRNA RNP complex and template dsDNA into Jurkat cells by electroporation; 4) Using the luciferase activity assay method to screen and verify the cell pool in which the reporter gene is knocked in; 5) Screen and verify correct knock-in single cell clones from the reporter gene knock-in cell pool. 8. A Jurkat effector cell based on Nur77 signaling pathway, characterized in that it is prepared according to the method of claim 1 or 2. 9. the application of the Jurkat effector cell described in claim 8 in CAR and TCR molecule-specific functional screening and assessment. 10. the application of the Jurkat effector cell described in claim 8 in antibody display library high-throughput screening CAR molecule.

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