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

Jurkat effector cell, construction method and application thereof

Technical Field

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

Background

Tumor immunotherapy is to control and kill tumor cells by mobilizing the immune system of the organism 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 is rejuvenated and enters a large burst phase. Among them, CAR-T has shown potential for curing tumors on hematological tumors. Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR) modified T cells are the latest and most productive techniques in current adoptive cell therapy technologies, enabling the transition from basic immunological mechanism studies to clinical tumor immunotherapy applications. Because of their ability to express synthetic receptors and to specifically recognize target cells, CAR-T and TCR-T are becoming methods of tumor treatment that are exciting.

CAR and TCR specific molecular sequence design and screening, and structural optimization and evaluation are currently the most critical issues faced by large companies and academic structures in developing CAR-T and TCR-T products. For example, currently traditional screening methods select CAR molecules with high affinity as an indicator, and cannot distinguish between tumor cells and normal cells. In clinic, CAR-T cells with low tumor specificity attack normal cells while killing cancer cells, thereby producing side effects on normal physiological functions of patients. In addition, the CAR can specifically recognize tumor target antigen to activate T cells to perform a killing function, and the structural characteristics of the CAR molecule, such as the expression density, folding conformation and costimulatory factors on the surface of T cells, can activate non-antigen dependent signals of T cells, which are called as Tonic signals. When the Tonic signal is too strong, the signal can excessively activate T cells without target antigen stimulation, induce T cell differentiation and accelerate T cell exhaustion, thereby reducing the persistence of CAR-T in the body and further affecting the anti-tumor function of the CAR-T. Therefore, developing a high-efficiency and even high-throughput evaluation system that can evaluate both CAR and TCR molecule affinity, specificity, and Tonic signals is a key step in driving the further expansion of CAR-T and TCR-T therapies.

Jurkat cells are human leukemia T lymphocytes, have the corresponding characteristics and functions of primary T lymphocytes, express antigens such as CD3 and the like on the surface, and can be used for the specific evaluation of CD3 double antibodies and CAR or TCR molecules through genetic engineering. The principle of effector cell construction such as Jurkat-NFAT-LUC cells commonly used at present is based on that the TCR/CD3 complex or CAR molecule is targeted to stimulate and cause phosphorylation and activation of intracellular PLC-gamma, thereby activating transcription of the NFAT pathway, thereby leading to NFAT-RE mediated fluorescence generation, and finally utilizing the intensity of fluorescence signals to react the specificity of antibodies or CAR molecules. Similar to NFAT are also Jurkat effector cells as constructed based on 41-BB or CD69 signaling pathways, but the expression regulation of these genes is not only activated by direct antigen stimulation, but also indirectly by other inflammatory mediators (e.g. type I interferons and interleukins). Therefore, it is not very suitable for the evaluation of the tronic signal. Nur77 (NR 4A 1) as a nuclear receptor family transcription factor, whose expression can be used as a molecular marker for lymphocyte antigen specific activation, has been demonstrated in human thymus tissue and in gene reporter mice, and also demonstrated in a mouse model to be unresponsive to type I interferon or cytokine stimulation.

Therefore, the invention provides a construction method of a Nur77 signal pathway-based genetically engineered Jurkat effector cell and a specific application of the effector cell, which have great significance for functional assessment of CAR or TCR molecules.

Disclosure of Invention

The invention aims to solve the problems that the traditional Jurkat effector cells cannot detect CAR and TCR molecule affinity, antigen-dependent specific signals, non-antigen-dependent Tonic signals and the like at the same time with high efficiency and accuracy. Through a gene editing strategy, a Jurkat effector cell based on a Nur77 signal path is constructed, and the antigen-dependent specific signal and the non-antigen-dependent Tonic signal of the CAR or TCR molecule can be evaluated efficiently, accurately, quickly and conveniently.

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

the gene editing is carried out on the Nur77 gene termination codon region in Jurkat cells, and meanwhile, a reporter gene is fixed point and knocked into the coding frame of the Nur77 gene in a same frame, so that the expression regulation of the reporter gene is consistent with the expression of the endogenous Nur77 gene.

Preferably, the gene editing method used to deliver the gene editing substance to the Jurkat cells can be ZFN, TALEN, or/and CRISPR-Cas9, etc.; preferably CRISPR-Cas9, cas9 may be SpCas9, saCas9, spCas9-HF, eSpCas9, xCas9, cpf1 or Cas9 of other different bacteria; more preferably SpCas9.

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

Preferably, the delivery of the gene editing substance may be by using a transfection method of liposome, calcium phosphate, DEAE-dextran, electroporation, microinjection or gene gun; electroporation transfection is preferred.

In some embodiments, the CRISPR-Cas9 gene editing substance comprises Cas9 mRNA, cas9 protein, or/and sgRNA. Preferably, the electroporation is performed using an RNP complex formed by Cas9 protein and sgRNA. The RNP complex can be obtained by directly mixing incubation of Cas9 protein and sgRNA, or by mixing both in a specific buffer (such as an electrotransport buffer).

In some embodiments, the sgrnas are designed by first specifying the genomic site to knock in, using the DNA sequence surrounding that site. The design principle is that the PAM region sequence is NGG, wherein N is any base of A, T, C and G. sgrnas include targeted crRNA sequences and tracrRNA sequences, where crrnas can be 17, 18, 19, 20, 21, or 22 bases, preferably 20 bases.

In some embodiments, the sgrnas are unmodified or chemically modified. Chemical modifications include 2-O-methylation, 3-thio, 2-O-methylation in combination with 3-thio, 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 modification can occur at 1 to 10 bases at the 5 'and 3' ends of the sgrnas. The designed sgRNA can be obtained through T7 in vitro transcription, and can also be directly synthesized in vitro.

In some embodiments, the gene editing substance further comprises template dsDNA for homologous repair at the editing site. The homologous repair template dsDNA comprises left homology arm DNA, a knocked-in exogenous reporter gene and right homology arm DNA. The left homology arm of the template DNA is homologous to the 5 'end sequence of the DNA cut, and the right homology arm is homologous to the 3' end sequence of the DNA cut.

In some embodiments, the length of the left and right homology arm fragments may be selected from 50 to 1000 bases, preferably 150 bases. The knocked-in reporter gene needs to be expressed in frame with the endogenous Nur77 gene to reflect the expression level of the endogenous gene by expression of the reporter gene. The reporter gene may be at least one of a fluorescent protein gene, a luciferase gene, a chloramphenicol acetyl transferase gene, a dihydrofolate reductase gene, a beta-galactosidase gene, and a beta-glucuronidase gene; preferably a dual reporter gene of 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 may be selected from at least one of T2A, P2A, E a and F2A; combinations of P2A and T2A are preferred.

In some embodiments, the homologous repair templates dsDNA can be clonally ligated into a plasmid vector after synthesis is complete, which can be any of, for example pUC57, pcdna3.1, pCMV, etc., and the position of the ligation can also be flexible. It is also possible to proceed directly to the subsequent step without ligation to the plasmid vector.

In some embodiments, the homologous repair dsDNA can be obtained by PCR amplification using unmodified or modified primers modified with 5' -phosphorothioate in combination with 5' -Biotin-TEG or 5' -locked nucleic acid. After PCR amplification, the template dsDNA can be purified and concentrated by the following methods respectively or jointly: purification of DNA purification kit of each reagent company, purification of various DNA combined magnetic beads, gel chromatography, ion chromatography, affinity chromatography, ultrafiltration tube ultrafiltration, dialysis membrane dialysis, etc.

In one embodiment, the invention also discloses a method for establishing the Jurkat effector cells by knocking in the green fluorescent protein gene and the luciferase gene dual reporter gene at fixed points after Nur77 coding frame, the synthesized gRNA and repair template dsDNA are electrically transfected into the Jurkat effector cells, and the Jurkat effector cells are obtained through screening.

Preferably, it comprises the steps of:

step 1, designing and evaluating Nur77 gRNA sequences;

step 2, designing and preparing molecules of a homologous repair template dsDNA;

step 3, transfecting cas9-sgRNA RNP complex and template dsDNA into Jurkat cells by using an electrotransfection method;

step 4, screening and verifying a cell pool in which a reporter gene is knocked in by adopting a luciferase activity measurement method;

and 5, screening and verifying correctly knocked-in single cell clones from a cell pool knocked-in by the reporter gene.

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

The invention also provides application of the Jurkat effector cell, which can be used for screening and evaluating the specific functions of CAR, TCR and BCR molecules and antibodies, in particular for accurately detecting the antigen-dependent signals and the non-antigen-dependent Tonic signals of the CAR molecules; on the other hand, it can be used for high throughput screening of CAR molecules of antibody display libraries.

The invention has the advantages that: the simultaneous site-directed knock-in of two reporter genes (GFP and luciferase genes) into the coding box of the Nur77 gene in Jurkat cells was selected to construct a Jurkat effector cell based on the Nur77 signaling pathway. The effector cells can be assayed for Nur77 signal strength by flow and luciferase assays to assess specific function of the CAR or TCR.

1. Compared with the prior art, the method can more accurately measure the non-antigen-dependent Tonic signal of the CAR or TCR molecules by selecting the targeting Nur77 signal path to construct effector cells, is more favorable for rapidly screening and evaluating the better CAR or TCR molecules for in vivo or clinical verification, and is favorable for accelerating the research and development process of the CAR-T or TCR-T project.

2. By selecting dual reporter genes, the experimental method is more flexible and convenient to select. Because most of CAR molecules induce endocytosis of CAR molecules on T cells under antigen stimulation, the method is not suitable for accurately detecting antigen specific signals of CAR positive cell groups through flow, and therefore luciferase can be selectively measured to replace GFP signals through flow detection, and comprehensive evaluation of optimal CAR or TCR molecules is facilitated.

3. Currently CAR molecules are essentially derived from scFv or VHH of antibody sequences that have been validated for affinity and specificity, while a number of experimental results also indicate that superior antibody molecules are not necessarily suitable for use on CAR-T. Thus, a platform of high throughput screening technology for CARs-T compatible CARs molecules and structures needs to be established and perfected. Compared with the traditional antibody library, the Jurkat effector cell constructed by the invention is very suitable for the high-throughput screening of the CAR molecules initiated by the antibody display library, and compared with the traditional antibody library, the screening process based on Jurkat effector cells is more convenient, quicker and more economical, and the screened CAR molecules and structures are customized for the CAR-T.

4. The method can efficiently establish a stable cell line of the Nur77 site fixed point in-frame knock-in LUC and GFP dual reporter genes.

Drawings

FIG. 1 is a schematic diagram of Jurkat effector cell construction based on Nur77 signaling pathway,

figure 2 is a schematic diagram of Jurkat effector cell operation based on the Nur77 signaling pathway,

FIG. 3 shows the detection of the expression of Chi Yingguang luciferase by gene editing cells,

FIG. 4 shows the detection of expression of a monoclonal luciferase,

FIG. 5 is a Jurkat effect cell single cell clone genotyping,

figure 6 is MSLN CAR expression and antigen-independent detection of the tronic signal,

figure 7 is the detection of MSLN CAR antigen dependent signals,

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

Detailed Description

In order that the technical contents of the present invention may be more clearly understood, the following embodiments are specifically described with reference to the accompanying drawings. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.

The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The various commonly used biological or chemical reagents used in the examples are commercially available products.

Example 1: sgRNA design synthesis and screening of Nur77 locus

In order to realize efficient fixed-point insertion of the reporter gene into the coding frame of the Nur77 gene, about 50bp before and after the Nur77 stop codon is selected, the total sequence is about 100bp, the sgRNA design is carried out through an online sgRNA design website (http:// crispor. Tefor. Net /), and 7 sequences with higher scores are screened for in vitro synthesis (sgRNA synthesis service is provided by Nanjing Jinsri). Preferably, in this example all sgrnas are 3 '-thio and 2' -O-methylation modified at each of the 3 bases at the 5 'and 3' ends to make the sgrnas more stable and less susceptible to rapid degradation by nucleases after transduction into cells. The chemically synthesized sgRNA sequences are shown in Table 1 (Nur 77 sgRNA list and cleavage efficiency)

TABLE 1

Numbering device	Sequence numbering	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	22
				sgRNA-05	SEQ ID NO:5	GGG	15
sgRNA-06	SEQ ID NO:6	AGG	63
				sgRNA-07	SEQ ID NO:7	AGG	34

To screen for optimal sgRNA, 200pmol of the synthesized sgRNA was combined with 100pmol TrueCut, respectively ^TM Cas9 protein (purchased from Thermo Fisher) was mixed well and incubated for 20min at room temperature; using LONZA Nucleofector System, cells were electrotransferred to RNP-5E 5 Jurkat cells according to electrotransfer recommendation program CL-120, and immediately transferred to 12-well plates containing pre-warmed medium, labeled with the name. After 3-5 days, the cells after the electrotransformation were taken to extract genomic DNA according to the genomic DNA extraction kit instructions (TIANGEN), and the unedited wild-type Jurkat genome was used as a control. The gene editing region was PCR amplified for Sanger sequencing by specific primers Nur77-SF (SEQ ID NO: 9) and Nur77-SR (SEQ ID NO: 10) designed before and after the cleavage site. The sequencing results were compared with control sequences to detect the specific editing efficiency of the corresponding sgrnas. The specific editing efficiency was analyzed by the on-line Analysis tool ICE Analysis (https:// ICE. Synthon. Com) and the Analysis results are shown in Table 1. The results show that SEQ ID NO. 1, SEQ ID NO. 3 and SEQ ID NO. 6 have higher editing efficiency.

Example 2: molecular design and preparation of homologous repair template dsDNA

The gene modification scheme of the Jurkat effector cell constructed by the invention is that firstly, a CRISPR-Cas9 is utilized to generate a DNA incision on a termination codon region of a Nur77 genome locus, then, a homologous repair template dsDNA (comprising a reporter gene) is utilized to carry out homologous recombination repair on the DNA incision, finally, the reporter gene LUC and GFP are knocked into a Nur77 gene coding frame at fixed points, and the gene modification can lead the LUC and GFP genes to be led in at fixed points and be transcribed and expressed by utilizing an endogenous gene Nur77 promoter. A specific construction schematic is shown in FIG. 1. Homologous repair template dsDNA comprises two major parts:

a. left and right homology arms: the left and right homology arms are mainly used for identifying target DNA and carrying out recombination exchange, so that the knock-in gene can be correctly expressed, and the knock-in gene is fixed to the 3' end region of the last exon of the Nur77 gene. Meanwhile, to ensure that the selected sgrnas will not edit the repair template, synonymous mutations of individual bases were performed in the complementary region of the left homology arm. In addition, the length of the homology arm is important for the efficiency of the whole gene knock-in, and generally, the longer the homology arm is, the higher the knock-in efficiency is, but the length of the whole gene knock-in sequence is limited, and in view of the fact that the length of the reporter gene to be knocked in is longer, it is preferable that the sequence about 150bp upstream and downstream of the cleavage site is selected as the homology arm in this example.

b. Reporter gene: the invention selects two reporter genes, namely luciferase gene LUC and green fluorescent protein gene GFP, which are knocked in simultaneously. In order to ensure that the two inserted genes can be consistent with the endogenous Nur77 gene expression, a cleavage peptide is added between the three genes, and the combination of P2A and T2A is selected in the embodiment.

All components of the homologous repair template dsDNA are designed in sequence, and the nucleotide sequence of the homologous repair template dsDNA is shown as SEQ ID NO. 8. After gene synthesis (served by Shanghai, ind.) the sequence was cloned directly into cloning plasmid pUC 57. Using this plasmid as a template, the designed unmodified homologous template primers Nur77-LG-F (SEQ ID NO: 11) and Nur77-LG-R (SEQ ID NO: 12) were used to PCR heavily amplify dsDNA molecules for homologous repair. The PCR product was recovered by purification using MN kit to obtain high concentration and high purity homologous repair dsDNA (SEQ ID NO: 8).

Example 3: construction of Jurkat effector cells

1) Electrotransformation of Jurkat cells

For each sgRNA, 1E6 Jurkat cells were electrotransferred to deliver spCas9 protein (purchased from ThermoFisher), sgRNA and homologous repair dsDNA (PCR purified product obtained in example 2), thereby achieving cleavage of the Nur77 genomic locus and knock-in reporter gene at one time, using the Lonza electrotransfer kit (cat No. V4 XP-3024) and Lonza electrotransfer device (4D-nucleoactor). The specific flow is as follows: after preparing the Lonza electrotransfer buffer, 100pmol of spCas9 protein was mixed with 200pmol of sgRNA-01 and sgRNA-03 designed for synthesis, respectively, and incubated at room temperature for 20min, and 6. Mu.g of homologous repair dsDNA was added. To further enhance the editing efficiency, 200pmol Alt-R Electroporation Enhancer (purchased from IDT) was optionally added, mixed with 1E6 Jurkat cell pellet at room temperature, added to the electric rotating cup, electric shocked according to recommended procedure CL-120, added to a 6-well plate containing 2ml pre-heated medium after electric shocking, and labeled with the name. The above-mentioned electric conversion process can be continuously repeated 2-3 times.

2) Monoclonal screening verification

a. Cell pool verification: jurkat cells were electroporated and cultured for 7 days, and luciferase assay was used to examine the knock-in effect of the reporter gene in the cell pool. Since T cells are stimulated by both PMA and Ionomycin agonists, the Nur77 gene expression is induced and activated. Thus, when the reporter gene is successfully knocked into the Nur77 locus, the reporter gene is also simultaneously expressed along with the activation of Nur77, so 1E5 all edited cells will be taken, as well as the control Jurkat cells including the unedited cells in 96 well plates. For each cell, two groups and two duplicate wells were set, one with 2.5 μm Ionomycin and 20ng/mL PMA (purchased from bi-cloudy days) added as experimental groups and the other with no control group. After a 2-hour incubation period in a 37-degree incubator, 50uL of the fluorogenic substrate One-Glo (purchased from Promega) was added to each well, and the fluorescence value was measured using a fluorescence photometer (Envision) and the results were analyzed. The analysis of the results is shown in FIG. 3. The results show that in both the pools of sgRNA-01 and the group of sgRNA-03, cell clones with correct knockins compared to the control exist, wherein the editing efficiency of the group of sgRNA-01 was higher in both replicates than in the group of sgRNA-03.

b. Single cell clone selection: inoculating cells of sgRNA-01 group into 96-well plate at 0.5 and 1 per well by limiting dilution method, and inoculating 37 degree CO ₂ Culturing in incubator for 10-14 days, selecting obvious monoclonal cells to 48-well plate for further amplifying culture. After 4 days of culture, half of the cells were removed from each well and subjected to primary screening by the luciferase assay method, as described above. The general flow is as follows: from the 58 clones picked, 100uL (approximately 1 half of the cells) were removed from the black opaque 96-well plate, 2.5. Mu.M Ionomycin and 20ng/mL PMA were added to each well, and after 2 hours of reaction, 50uL of the fluorogenic substrate One-Glo (purchased from Promega) was added to each well, and the fluorescence value was measured using a fluorescence photometer (Envision), and the analysis results are shown in FIG. 4. Final pickingThe single cell clone with better cell proliferation and higher fluorescent signals, NLG-1, NLG-8, NLG-31, NLG-46 and NLG-57, is selected for further amplification and cryopreservation, and meanwhile, genome DNA is extracted for further genotyping and sequencing analysis. The genotyping primers are: nur77-SF, nur77-SR and LUC-GFP-R (SEQ ID NO: 13). The PCR electrophoretogram is shown in FIG. 5, and shows that NLG-8 is homozygous, i.e., both alleles are inserted into the reporter gene, while the other 4 clones are heterozygotes, i.e., only one allele is successfully inserted into the reporter gene, and the other allele is wild-type. Further sequencing results confirmed that 5 clones were correctly inserted into the designed reporter gene at the 3' end of the last exon of Nur 77.

Example 4: functional screening and assessment of Jurkat effector cells for CAR molecules

This example describes, as an example, a functional screening procedure for MSLN-BBz CAR molecules, and a functional screening and evaluation procedure for other CAR or TCR molecules or structures, which are substantially similar.

Preparation of CAR-Jurkat cells

Lentiviral infection of the different MSLN-BBz CAR molecules constructed and prepared (6 total, including one BMK molecule M5 that was clinically validated by Novartis) was screened for clones of the validated Jurkat effector cell line NLG-8# in example 3 with a MOI equal to 1. Infected cells were transferred to 37 ℃,5% co ₂ The cell incubator is cultured for 3 to 7 days, and the liquid is changed or passaged every 2 days.

Detection of MSLN CAR expression and antigen-independent Tonic Signal

MSLN CAR-Jurkat cells cultured for 3-7 days are taken, and the expression of the MSLN CAR and the non-antigen-dependent Tonic signal are detected by a FACS method, wherein the specific detection flow is as follows:

a. staining of antibodies: 1E5 cells from each group were centrifuged at 350g for 5min in 96-well plates, the supernatant removed, washed 1-fold with PBS and incubated at 4℃for 30min with Fc-labeled Mesothelin antigen (purchased from Acro). After centrifugation and 1 pass of PBS wash, AF 647-labeled anti-human Fc secondary antibody (purchased from Jackson) was added for 4-degree incubation. After 30min incubation, the cells were centrifuged and resuspended in 100uL PBS for flow detection. Wherein BMK was placed in parallel in two groups, one positive control group, pretreated with 2.5. Mu.M Ionomycin and 20ng/mL PMA in 96-well plates for 1h prior to antibody incubation, and the other group was untreated.

b. Flow detection and analysis: the stained cells were subjected to FACS detection of APC and FITC channels by BD flow, and the flow results were analyzed by FlowJo, if shown at 6. FACS results showed that other CAR molecules were better expressed except for CAR-4# molecule which was poorly expressed. Because the antigen is adopted to detect the expression of the CAR, the affinity of the antibody sequence can be reflected by the strength of the expression of the CAR, and the result shows that CAR-1# has higher expression intensity compared with BMK, and CAR-5# has similar expression intensity as CAR-2# has lower expression intensity. Therefore, the Jurkat effector cells are adopted, and compared with primary T cells and traditional antibody affinity detection, the method has the advantages of high infection efficiency, simplicity, convenience and economy. Meanwhile, by detecting the expression level of background GFP in the CAR-Jurkat cells, the self-activation degree of the CAR under the condition of not receiving antigen stimulation, namely the Tonic signal, can be reflected. The experimental result shows that the BMK CAR has very low GFP expression, which indicates that the Tonic signal is weak, and the CAR positive group cells are converted from GFP negative to positive after being stimulated by adding the agonists Ionomycin and PMA, thus indicating that the Jurkat effector cells constructed by the invention have good specificity. Further GFP expression of MSLN CAR molecules, CAR-3# molecules were found to have very high GFP expression in the CAR positive population, indicating that their Tonic signals were very high and unsuitable as CAR molecules, whereas CAR-1# and CAR-5# had GFP expression levels as low as BMK, indicating that their Tonic signals were both weak and could be used as candidate CAR molecules for further development.

Detection of MSLN CAR antigen dependent Signal

Since most target CAR molecules mediate endocytosis of CAR molecules on cell membranes after being stimulated by antigens, the detection of CAR molecule antigen-dependent signals cannot be directly performed by adopting a FACS method, and therefore, the invention adopts the strength of expression of luciferase of CAR-Jurkat effector cells under antigen and non-antigen stimulation to reflect CAR molecule antigen-dependent signals, and the signals can further reflect the targeting specificity and killing function of CARs. The specific experimental procedure is as follows: MSLN CAR-Jurkat cells cultured for 3-7 days were taken and CAR expression levels of all groups were adjusted to consistent levels depending on the level of CAR expression. CAR-Jurkat cells were incubated with target cells of different MSLN expression levels for 3-5 hours at 1:1 or 2:1 effective target ratio, respectively. In this example, CAR-Jurkat cells obtained by adjusting the CAR expression level of each group to be consistent were co-incubated with target cells a2780 not expressing MSLN, target cells SKOV3 weakly expressing MSLN, and target cells OVCAR3 highly expressing MSLN in a black opaque 96-well plate for 4 hours, respectively, in which the Jurkat cells not infected with CAR were used as control groups, at an effective target ratio of 1:1. The number of effector cells or target cells per well was 4E4, 2 multiplex wells were set. After 4h of incubation, 50uL of fluorogenic substrate One-Glo (purchased from Promega) was added to each well and the fluorescence value was determined using a fluorescence photometer (Envision) and the analysis results are shown in FIG. 7. The results of luciferase expression assays showed that CAR-1# and CAR-5# had a targeted specific activation similar to BMK, and exhibited very low luciferase activity upon stimulation by MSLN negative cells A2780, higher luciferase activity upon stimulation by MSLN high expressing cells OVCAR3, and relatively weaker luciferase activity upon stimulation by weakly expressing cells SKOV 3. By comprehensively analyzing the experimental data of the non-antigen dependent signals in fig. 6 and the antigen dependent signals in fig. 7 (as shown in fig. 8), it is easy to evaluate that CAR-1# and CAR-5# have functions similar to those of BMK CAR molecules, and are expressed as a lower non-antigen dependent Tonic signal and a higher and more specific antigen dependent signal, so that the CAR-1# and the CAR-5# can be used as candidate molecules for further in vivo verification or clinical development, while other CAR molecules will not be worth further development.

Therefore, as can be seen from the above example data, the Jurkat effector cells developed based on the invention can screen more suitable CAR molecules for further CAR-T project development very economically, rapidly and efficiently, and have great significance in accelerating the development of the CAR-T project.

Example 5: screening procedure of Jurkat effector cells for displaying library CAR molecules

The Jurkat effector cells disclosed by the invention can be used for screening and evaluating scFv or VHH derived from antibody sequences, and are also very suitable for screening CAR molecules of an antibody display library in high throughput. Since the CAR molecules and structures selected are inherently tailored for CAR-T, it would be more convenient, economical and efficient. The recommended efficient screening process is as follows:

panning enrichment: display libraries (including but not limited to immune libraries or target cells) by 1-2 rounds of preliminary panning of antibodies (scFv) using specific antigens or target cells

Library) to enrich for antibody sequences for specific targets.

Construction of CAR virus library: universal primers were designed to amplify the scFv sequences of the antibodies using the antibody display library genomic DNA as template and assembled into conventional second generation CAR structures, such as CD8sp-scFv-CD8 finger-CD 8TM-41BB-CD3z. Finally, E.coli is transformed, and the plasmids of the mixed clones are extracted, and lentiviruses or retroviruses are packaged to construct a CAR virus library.

Construction of CAR-Jurkat cell banks: the packaged CAR virus was infected with Jurkat cells to construct a CAR-Jurkat cell bank.

Screening of CAR molecules: incubating a proper CAR-Jurkat cell bank with specific target cells, separating GFP positive group cells by flow, further culturing or extracting DNA, amplifying CAR sequence by using CAR primer, TA cloning, and sequencing to obtain the initially screened CAR sequence. If the acquired sequences are more than one, two or more rounds of repeated screening can be selected, and further screening verification can be performed on the acquired sequences according to example 4.

By adopting the Jurkat effector cells disclosed by the invention, according to the recommended screening flow, the better CAR molecules can be screened out in one month at maximum for further development of CAR-T projects, so that the method is quite economical and efficient.

Claims (10)

1. A method of constructing Jurkat effector cells comprising the steps of: the gene editing is carried out on the Nur77 gene termination codon region in Jurkat cells, and the report gene is fixed point and knocked into the coding frame of the Nur77 gene in a same frame, so that the expression regulation of the report gene is consistent with the expression of the endogenous gene Nur 77.

2. The method of claim 1, wherein the gene editing substance is delivered using a gene editing method selected from ZFNs, TALENs, or/and CRISPR-Cas 9.

3. The method of constructing Jurkat effector cells according to claim 1 or 2, wherein the gene editing substance is a homologous repair template dsDNA having a nucleotide sequence shown in SEQ ID No. 8.

4. A method of constructing Jurkat effector cells according to claim 1 or 2, characterized in that the electroporation is performed using RNP complexes formed by Cas9 protein and sgRNA.

5. The method for constructing Jurkat effector cells according to claim 4, wherein the targeting nucleotide sequence of the sgRNA is shown in at least one of SEQ ID NOs 1 to 7.

6. The method of constructing Jurkat effector cells according to claim 1 or 2, wherein said reporter gene is a dual reporter gene of a combination of green fluorescent protein and luciferase gene.

7. A method of constructing Jurkat effector cells according to claim 1 or 2, comprising the specific steps of:

1) Designing and evaluating Nur77 gRNA sequence;

2) Molecular design and preparation of homologous repair template dsDNA;

3) Transfecting cas9-sgRNA RNP complex and template dsDNA into Jurkat cells by electrotransfection;

4) Screening and verifying the cell pool knocked in by the reporter gene by adopting a luciferase activity measurement method;

5) The correctly knocked-in single cell clones were selected and validated from the pool of reporter knockins.

8. Jurkat effector cells based on the Nur77 signaling pathway characterized by being prepared according to the method of claim 1 or 2.

9. Use of the Jurkat effector cells of claim 8 in CAR and TCR molecule specific functional screening and assessment.

10. Use of Jurkat effector cells according to claim 8 for high throughput screening of CAR molecules from antibody display libraries.

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