CN116478932A

CN116478932A - Genetically modified immune cells and uses thereof

Info

Publication number: CN116478932A
Application number: CN202310680241.4A
Authority: CN
Inventors: 马丽雅; 谢海涛; 张凯文; 刘�东
Original assignee: Shenzhen Xiankangda Life Science Co ltd
Current assignee: Shenzhen Xiankangda Life Science Co ltd
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2023-07-25

Abstract

The invention discloses a genetically modified immune cell and application thereof, wherein the immune cell adds a target gene with a nucleotide sequence shown as SEQ ID NO. 1 into an IFN-gamma gene of the immune cell in a gene editing mode of CRISPR-Cas9 and gRNA construction proteins so as to enhance the continuous amplification capability of the immune cell in vivo and the killing capability of the immune cell on tumors.

Description

Genetically modified immune cells and uses thereof

Technical Field

The invention relates to the field of biological cell gene modification, in particular to a genetically modified immune cell and application thereof.

Background

Tumors and/or cancers are important diseases that afflict human health and are life threatening to humans. In the long-term fight against tumor and/or cancer, various treatment means such as surgery, chemotherapy, targeted therapy, immunotherapy and the like have been developed, and certain progress has been made, so that the survival probability of tumor and/or cancer patients is remarkably improved. However, the current level of tumor and/or cancer treatment remains a significant distance from the health and life expectancy of a wide range of patients. Thus, the active development of new tumor and/or cancer treatment technologies remains the goal of continuing efforts in the medical field.

Cell therapy is the leading field and technical elevation of future medicine, and immune cell CAR therapy, such as CAR-T cell therapy technology, is more widely and more permanently applied to people because of more accurate targeting, and has a wider tumor killing range, although a certain clinical effect is achieved on hematological tumors. However, the technology has not yet achieved clinical breakthrough for solid tumor treatment. Thus, exploring the use of immune cell CAR therapies in solid tumor therapy is currently the main direction of research.

Chimeric Antigen Receptors (CARs) are typically scFv (single-chain variable fragment) comprising a transmembrane region and an intracellular co-stimulatory signaling region. T cells of a patient are transfected by means of gene transduction to target their Chimeric Antigen Receptor (CAR) to tumor cells. Extracellular scFv of CARs recognize a specific antigen, which is then transduced by intracellular domains, resulting in the corresponding expression of immune cells, such as T cell activation, proliferation, cytolytic toxicity and secretion of cytokines, which in turn eliminate target cells.

However, immune cell CAR treatment has some problems, the effect of immune cell CAR treatment on solid tumors is not as good as that of hematological tumors, and in immune cell CAR treatment, one of the important reasons is that immune cells have a short survival time in the microenvironment of solid tumors and cannot kill tumor cells continuously.

Disclosure of Invention

In order to solve the problem that the survival time of the immune cell CAR therapy in a solid tumor microenvironment is short, the gene editing technology of CRISPR-CAS9 and gRNA construction proteins is used, and the IFN-gamma genes of immune cells are subjected to gene editing in a mode of introducing exogenous genes, so that the activity and the continuous expansion of the immune cells are improved, and the continuous killing of cancer cells is realized.

The technical scheme of the invention is as follows:

a genetically modified immune cell has IFN-gamma gene added with target gene with nucleotide sequence as shown in SEQ ID No. 1.

Preferably, in one embodiment, the nucleotide sequence shown in SEQ ID NO. 1 is added to the IFN-gamma gene after the CRISPR-Cas9 and the gRNA together form a protein.

Preferably, in one embodiment, in the immune cell, the gRNA is selected from any one of the nucleotide sequences shown as SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30; preferably, the gRNA is selected from any of the nucleotide sequences set forth in SEQ ID NO. 21 or SEQ ID NO. 29.

Preferably, in one embodiment, the chimeric antigen receptor is targeted by one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, GUCY2C, nectin-4, and EBV by transfecting the immune cells with lentivirus into the above immune cells.

Preferably, in one embodiment, the immune cell is any one of a T cell, an NK cell, a CIK cell and a NKT cell.

The invention also provides a microbial system, which contains the genetically modified immune cells.

The invention also provides a biological agent, which contains the genetically modified immune cells or microbial systems.

The invention also provides the application of the biological agent and immune cells in preparing medicaments for preventing and/or treating tumors and cancers.

The gene modified immune cell provided by the invention has the following advantages that the target gene is added into IFN-gamma gene of the immune cell by a gene editing technology:

1. under the stimulation of target cells, the immune cells can induce the expression of IL-15 after being activated by target antigens;

2. the immune cells can secrete IL-15 while secreting IFN-gamma, and the two are not mutually influenced, so that the continuous expansion capacity and the tumor killing capacity of the immune cells are improved;

3. through the gene editing technology of protein formed by CRISPR-CAS9 and gRNA, IFN-gamma gene can be knocked out accurately, and the off-target effect is low.

Drawings

FIG. 1 is a graph of IFN-gamma knock-out efficiency of the ith gRNA on T cells;

FIG. 2 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause ii;

FIG. 3 is a graph of IFN-. Gamma.knockout efficiency of the gRNA of clause iii for T cells;

FIG. 4 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause iv;

FIG. 5 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause v;

FIG. 6 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause vi;

FIG. 7 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause vii;

FIG. 8 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of clause viii;

FIG. 9 is a graph of IFN-gamma knock-out efficiency of the ix-th gRNA on T cells;

FIG. 10 is a graph of IFN-gamma knock-out efficiency of gRNA on T cells of the x th strip;

FIG. 11 is a graph showing the knock-in efficiency of the ith gRNA for a target gene;

FIG. 12 is a graph of knock-in efficiency of gRNA of item ii for a gene of interest;

FIG. 13 is a graph of the knock-in efficiency of the iii gRNA on the target gene;

FIG. 14 is a graph showing the knock-in efficiency of gRNA of item iv for a target gene;

FIG. 15 is a graph showing the knock-in efficiency of gRNA of clause v against a target gene;

FIG. 16 is a graph showing the knock-in efficiency of the gRNA of the vi th strip to the target gene;

FIG. 17 is a graph of knock-in efficiency of gRNA of clause vii against a gene of interest;

FIG. 18 is a graph of knock-in efficiency of the gRNA of clause viii against a gene of interest;

FIG. 19 is a graph of knock-in efficiency of the ix-th gRNA for a gene of interest;

FIG. 20 is a graph of knock-in efficiency of the x-th gRNA for a gene of interest;

FIG. 21 is a graph of expansion of NT cells, CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, and KI-GPC3-CAR-T cells;

FIG. 22 is a flow chart of the CAR positive expression of NT cells;

FIG. 23 is a flow chart of the CAR positive expression of CLDN18.2-CAR-T cells;

FIG. 24 is a flow chart of the CAR positive expression of GPC3-CAR-T cells;

FIG. 25 is a flow chart of the CAR positive expression of KI-CLDN18.2-CAR-T cells;

FIG. 26 is a graph of a CAR positive expression flow for KI-GPC3-CAR-T cells;

FIG. 27 is a flow chart of GFP positive expression of NT cells;

FIG. 28 is a flow chart of GFP positive expression of CLDN18.2-CAR-T cells;

FIG. 29 is a flow chart of GFP positive expression of GPC3-CAR-T cells;

FIG. 30 is a flow chart of GFP positive expression of KI-CLDN18.2-CAR-T cells;

FIG. 31 is a flow chart showing GFP-positive expression by KI-GPC3-CAR-T cells;

FIG. 32 is a histogram of INF-gamma secretion of NT cells, CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, and KI-GPC3-CAR-T cells;

FIG. 33 is a histogram of IL-15 secretion of NT cells, CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, and KI-GPC3-CAR-T cells;

FIG. 34 is a graph of specific killing rates of NT cells, CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, and KI-GPC3-CAR-T cells against tumors.

Detailed Description

The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

The invention provides a gene editing technology of proteins formed by gRNA and CRISPR-Cas9 of various different nucleotide sequences, which is used for knocking in target genes of the nucleotide sequences shown as SEQ ID NO. 1 into IFN-gamma genes of immune cells, carrying out gene editing on the IFN-gamma genes of the immune cells, leading the immune cells to be induced to express IL-15 after being activated by target antigens and improving the continuous expansion capacity and tumor killing effect of the immune cells, and the target genes can be knocked in any position of the IFN-gamma genes according to the needs.

Wherein, the nucleotide sequence shown in SEQ ID NO. 1 is as follows:

TTTATAATTCCTATATCCTGTGACTGTCTCACTTAATCCTTTGTTTTCTGACTAATTAGGCAAGGCTATGTGATTACAAGGCTTTATCTCAGGGGCCAACTAGGCAGCCAACCTAAGCAAGATCCCATGGGTTGTGTGTTTATTTCACTTGATGATACAATGAACACTTATAAGTGAAGTGATACTATCCAGTTACTGCCGGTTTGAAAATATGCCTGCAATCTGAGCCAGTGCTTTAATGGCATGTCAGACAGAACTTGAATGTGTCAGGTGACCCTGATGAAAACATAGCATCTCAGGAGATTTCATGCCTGGTGCTTCCAAATATTGTTGACAACTGTGACTGTACCcccctctccctcccccccccctaacgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattgtatgggatctgatctggggcctcggtacacatgctttacatgtgtttagtcgaggttaaaaaaacgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaaccatgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagtaactgggtgaatgtaataagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcaccccagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggagatgcaagtattcatgatacagtagaaaatctgatcatcctagcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttctgactacaaggacgacgatgacaaaggcagcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggccgcttcgagcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttCAAATGGAAAGTAACTCATTTGTTAAAATTATCAATATCTAATATATATGAATAAAGTGTAAGTTCACAACTACTTATGCTGTGTTGGACTTTTTCTAAGTGAGACCTGGAGTGAAAGAACTACCTATTAATGAATTAGTAGGGAGGGGAGTCTTCTTAGCTGTGGAAATTTTAGAGTTGCATTTGGTTCCATTAAATGTGGTATTTCTTTCCACTAGCATTTTGTTGGCTTTCGCTTTTCCAGTTAGCAGCTCTTTGAATTATCTTTCTAAGATACAGATTTAATTATGTCACTATTCAATTCAGAGGTTCTGCTATGGAATGTAGTTTAAACTGCTTAGCTTGGCACAC。

in the present invention, the gRNA may be selected from any one of the nucleotide sequences shown below:

i. the nucleotide sequence shown in SEQ ID NO. 21 is:

5'-CTTCTTTTACATATGGGTCC-3';

the nucleotide sequence shown in SEQ ID NO. 22 is as follows:

5'-GTTCATGTATTGCTTTGCGT-3'；

the nucleotide sequence shown in SEQ ID NO. 23 is:

5'-CAACTGTGACTGTACCCAAA-3';

iv the nucleotide sequence shown as SEQ ID NO. 24 is:

5'-TGTCGCCAGCAGCTAAAACA-3';

v, the nucleotide sequence shown as SEQ ID NO:25 is:

5'-AGGAGTCAGATGCTGTTTCG-3';

vi, the nucleotide sequence shown as SEQ ID NO. 26 is:

5'-CTTTATCTCAGGGGCCAACT-3';

the nucleotide sequence shown in SEQ ID NO. 27 is as follows:

5'-CCAACCTAAGCAAGATCCCA-3';

viii the nucleotide sequence shown as SEQ ID NO. 28 is:

5'-CAACCTAAGCAAGATCCCAT-3';

the nucleotide sequence shown in SEQ ID NO. 29 is shown as follows:

5'-GCGACAGTTCAGCCATCACT-3';

the nucleotide sequence shown in the SEQ ID NO. 30 is:

5'-CTGACATGCCATTAAAGCAC-3'；

wherein 5 'and 3' represent the two asymmetric ends of each of the above nucleotide sequence fragments, respectively.

For the purposes of the present invention, when the immune cell expresses a chimeric antigen receptor CAR, the immune cell is selected from the group consisting of NK cells, NKT cells, TIL, gamma-delta T cells, and is equivalent to a T cell (or a T cell may replace an NK cell). In the present invention, T cells are preferred.

The preparation and application of the immune cells of the present invention will be described in detail typically using CAR-T cells as an example, namely, genetically edited immune cells will be described in detail using CAR-T as an example. Immune cells are not limited to CAR-T cells described in the context, and the cells of the invention have the same or similar technical features and benefits as CAR-T cells described in the context.

The immune cells after gene editing provided by the invention comprise the steps of carrying out gene editing on the immune cells by a gene editing technology of CRISPR-CAS9 and gRNA constituent proteins, such as: the target gene with the nucleotide sequence shown as SEQ ID NO. 1 is knocked into IFN-gamma gene, and the IFN-gamma gene of immune T cell is edited, so that the immune cell can induce and express IL-15 after being activated by target antigen, and the continuous expansion and tumor killing effects of the immune cell are improved.

A chimeric antigen receptor CAR expressed by the immune cell (e.g., T cell) comprising an antigen binding region, a transmembrane domain, a co-stimulatory domain, and a stimulatory domain cd3ζ; and the chimeric antigen receptor is expressed as a chimeric antigen receptor that targets one or more targets; or the chimeric antigen receptor is expressed as a first generation CAR cell, a second generation CAR cell, a third generation CAR cell, a fourth generation CAR cell that targets any target.

The CAR structure adopted by the invention is a second generation CAR, and consists of a single chain variable fragment (scFv), a transmembrane domain, a co-stimulatory domain 4-1BB and a signal domain CD3 zeta; the scFv fragment targets any target, e.g., the target may be one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, GUCY2C, nectin-4, and EBV.

The immune cells of the invention can improve the continuous expansion capability in the tumor microenvironment and the capability of killing tumor cells

The immune cells using the invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and (3) a preservative. The compositions of the present invention are preferably formulated for intravenous infusion and intratumoral injection.

Pharmaceutical compositions made using the immune cells of the invention may be administered in a manner appropriate for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, and by the clinical regimen. When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be 1 x 10 ⁴ ~1×10 ⁹ A dose of individual per kg body weight, preferably 1X 10 ⁵ ~1×10 ⁶ A dose of individual per kg body weight. T cell compositions may also be administered multiple times at these doses. Optimal dosages and treatment regimens for a particular patient can be determined by one of skill in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordinglyAnd (5) setting.

Administration of the biological agents of the present invention may be carried out in any convenient manner, including by injection, infusion, and the like. The compositions described herein can be injected into a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous infusion, and intratumorally.

The following examples illustrate the genetic editing of IFN-gamma genes in immune cells and their technical effects, using immune cell CAR-T as an example.

Example 1 sorting and culture of T cells

Sorting T cells from normal donor peripheral blood, activating and expanding T cells using anti-CD3/anti-CD28 coupled magnetic beads; culturing in a constant temperature incubator to obtain primary T cells for later use. This part is prior art and will not be described in detail here.

Example 2 IFN-y Gene knockout and knock-in from T cells

1.1 knockout efficiency

In this example, the T cells prepared in example 1 were subjected to knockdown experiments using grnas corresponding to the above-described i to x different nucleotide sequences.

Sequencing was performed by knocking out IFN-gamma gene on T cells, and the sequencing results are shown in FIGS. 1 to 10.

From the gene sequencing results shown in FIGS. 1 to 10, the IFN-. Gamma.gene knockout efficiencies of the T cells by the above-mentioned ith to x. Sup. Th gRNAs were: 73.3%, 71.5%, 75.2%, 89.3%, 84.7%, 84.3%, 81.9%, 91.4%, 67.3%; the gene knockout efficiency is higher, especially the IFN-gamma gene knockout efficiency of the ix-th gRNA on T cells is highest and reaches 91.4%, which indicates that the IFN-gamma gene knockout of the gRNA corresponding to the ix-th group on the T cells is more thorough.

1.2, knock-in efficiency

The target gene of the nucleotide sequence shown as SEQ ID NO. 1 is knocked in after IFN-gamma gene in human immune T cells is knocked out by using the gRNA from the ith to the x of the invention, and the knockin efficiency results are shown in figures 11 to 20.

As can be seen from fig. 11 to 20, when target genes are knocked in to T cells in IFN- γ gene by the ith to x pieces of gRNA, the knocking-in efficiencies are respectively: 24.01%, 9.20%, 18.20%, 10.32%, 11.80%, 8.82%, 7.17%, 6.58%, 42.37%, 10.41%. Wherein, the ix-th nucleotide sequence shown as SEQ ID NO. 29 has the highest knocking-in efficiency of 42% corresponding to the gRNA. Therefore, the gene editing after constructing the protein by using gRNA and CRISPR-CAS9 corresponding to the ith to the x nucleotide sequences has better gene knock-in performance for knocking out IFN-gamma genes and target gene knock-in.

Example 3 preparation, detection and analysis of immune CAR-T cells

3.1 CAR-T cell preparation

3.1.1 lentiviral transfection

After the primary T cells obtained in example 1 were activated by magnetic beads, lentiviral vectors of target CLDN18.2 and GPC3 sequences were transferred to two cell culture flasks, respectively, and placed at 37℃in 5% CO ₂ Respectively culturing in a constant temperature incubator to obtain two lentivirus infected T cells with different targets, which are respectively called CLDN18.2-CAR-T cells and GPC3-CAR-T cells for later use. This part is the prior art and will not be described in detail here.

3.1.2 Gene editing

CLDN18.2-CAR-T cells and GPC3-CAR-T cells were cultured respectively to day 3, and then electrotransformation was performed in CLDN18.2-CAR-T cells and GPC3-CAR-T cells respectively, using grnas corresponding to any one of the above-described nucleotide sequences of groups i to x, e.g., grnas corresponding to the nucleotide sequence shown in SEQ ID No. 29 of ix and CRISPR Cas9 to form a protein compound.

After electrotransformation is completed, two target gene fragments with nucleotide sequences shown as SEQ ID NO. 1 are respectively transferred into the electrotransformed CLDN18.2-CAR-T cells and GPC3-CAR-T cells through AAV (adeno-associated viral vector) to obtain two CAR-T cells, namely KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells.

KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells were transferred to 24-well plates, respectively, and placed at 37℃in 5% CO ₂ And (5) continuously culturing in a constant-temperature incubator for later use.

3.2 culture of T cells and CAR-T cells

NT cells (blank control), CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, KI-GPC3-CAR-T cells and 5T cells were cultured, and the number of cells was measured by sampling on days 5, 7, 9, 11 and 13, respectively, and the CAR positive rate, GFP expression, IFN- γ secretion amount and IL-15 secretion amount were measured by sampling T cells, respectively. Wherein, in the process of T cell culture, culture medium is supplemented for passage every 1-2 days. This part is the prior art and will not be described in detail here.

The results of the amplification assay for 5T cell growth are shown in figure 21. As can be seen from fig. 21, the expansion of NT cells (blank control), CLDN18.2-CAR-T cells, GPC3-CAR-T cells were not greatly different, indicating that lentiviral transfection had less effect on T cell expansion; and KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells are subjected to electrotransformation, so that the cell expansion is affected to a certain extent, but the total expansion of the cells can also meet the requirement of subsequent experiments.

The results of CAR positive rate detection for 5T cells are shown in figures 22 to 26. As can be seen from fig. 22 to 26, the results of CAR positive rate detection for NT cells (blank control), CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells were 0.93%, 99.76%, 99.77%, 98.23%, 99.03%, respectively; thus, electrotransport has less effect on CAR expression, and CAR-T cells do not lose expression of their function due to electrotransport.

The GFP-positive rate test results for 5T cells are shown in FIGS. 27 to 31. Since GFP positive rate indicates the knock-in efficiency of the target gene, it was found from FIGS. 27 to 31 that NT cells (blank control), CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells were expressed, and that only KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells were expressed when gene editing was performed using a protein compound composed of a gRNA having the nucleotide sequence shown in SEQ ID NO:29 and CRISPCas 9, and that expression did not significantly affect the CLDN18.2 and GPC3 targets.

The results of IFN-gamma secretion assays for 5T cells are shown in FIG. 32. As can be seen from fig. 32, after the target gene fragment was knocked into the IFN- γ gene, the amounts of IFN- γ secretion by CLDN18.2-CAR-T cells, GPC3-CAR-T cells, KI-CLDN18.2-CAR-T cells, and KI-GPC3-CAR-T cells were similar, indicating whether the target gene fragment was knocked into the IFN- γ gene of CAR-T cells, and the amounts of IFN- γ secretion by CAR-T cells were not affected, and the effects of target CLDN18.2 and GPC3 on the amounts of IFN- γ secretion were also not great.

The results of measuring the content of secreted IL-15 in 5T cells are shown in FIG. 33. As can be seen from fig. 33, only KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells after gene editing will secrete IL-15 factor, while NT cells, CLDN18.2-CAR-T cells and GPC3-CAR-T cells without gene editing will not secrete IL-15 factor, and different targets will have less effect on IL-15 secretion. Therefore, the target gene knocked in the IFN-gamma gene of the immune cell can be expressed in the CAR-T cell, and the continuous expansion capacity and the tumor killing capacity of the CAR-T cell are further improved.

The specific tumor killing rates of 5T cells in the effective target ratios (E: t=0.3:1, 1:1, 3:1) are shown in figure 34. As can be seen from fig. 34, KI-CLDN18.2-CAR-T cells and KI-GPC3-CAR-T cells after gene editing were significantly better in killing efficiency or tumor killing rate than NT cells, CLDN18.2-CAR-T cells and GPC3-CAR-T cells without the edited genes. The reason is that the target gene knocked in IFN-gamma gene of immune cells is induced to express after being stimulated by target antigen and has positive effect on CAR-T cells, so that the killing efficiency is superior to that of T cells without gene editing.

In summary, in the invention, the target gene can be inserted into IFN-gamma gene of immune cells by using gene editing technology of protein formed by CRISPR-Cas9 and gRNA together, and the target gene can be induced to be expressed after target antigen activation, so that the continuous expansion capacity of immune cells and the effect of killing tumors are improved, and the invention has wide application prospect in the fields of cell therapy and gene editing.

It is to be understood that the foregoing description of the preferred embodiments of the invention is not to be considered as limiting the scope of the invention, which is defined by the appended claims.

Claims

1. A genetically modified immune cell is characterized in that a target gene with a nucleotide sequence shown as SEQ ID NO. 1 is added into IFN-gamma genes of the immune cell.

2. The immune cell of claim 1, wherein the nucleotide sequence shown in SEQ ID NO. 1 is added to the IFN-. Gamma.gene after the protein is formed by CRISPR-Cas9 and gRNA together.

3. The immune cell of claim 2, wherein the gRNA is selected from any one of the nucleotide sequences set forth in SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29 and SEQ ID NO. 30, respectively.

4. The immune cell of claim 2, wherein the gRNA is selected from the nucleotide sequence set forth in SEQ ID No. 21 or SEQ ID No. 29.

5. The immune cell of claim 1, wherein the chimeric antigen receptor-targeted target in the immune cell is one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, GUCY2C, nectin-4, and EBV.

6. The immune cell of claim 1, wherein the immune cell is any one of a T cell, NK cell, CIK cell, and NKT cell.

7. A microbial system comprising an immune cell according to any one of claims 1 to 6.

8. A biological agent comprising an immune cell according to any one of claims 1 to 6 or a microbial system according to claim 7.

9. The use of the biological agent of claim 8 for the preparation of a medicament for the prevention and/or treatment of tumors and cancers.

10. Use of an immune cell according to any one of claims 1 to 6 for the preparation of a medicament for the prophylaxis and/or treatment of tumors and cancers.