CN113122576A

CN113122576A - Universal type TRUCK-T cell, preparation method and application thereof

Info

Publication number: CN113122576A
Application number: CN202011641112.7A
Authority: CN
Inventors: 袁鹏飞; 王飞; 牛立超; 付建强; 谷丰
Original assignee: Edigene Beijing Biotechnology Co ltd
Current assignee: Edigene Biotechnology Inc
Priority date: 2019-12-31
Filing date: 2020-12-31
Publication date: 2021-07-16

Abstract

The present invention relates to a method for preparing universal human TRUCK-T cells, comprising (i) disrupting, by gene editing techniques, in said human TRUCK-T cells: the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490; and the B2M genomic region from chromosome 15 from position 45003745 to position 45003788; and (ii) using the principle of homologous recombination to insert the CAR molecule and exogenous cytokine gene sequences into the TRAC and B2M cleavage sites, respectively, for expression using the T cell's own TRAC promoter and NFAT.

Description

Universal type TRUCK-T cell, preparation method and application thereof

Technical Field

The invention relates to the field of immunotherapy based on CAR-T technology, in particular to a universal type TRUCK-T cell (namely a U-TRUCK-T cell).

Background

CAR-T cell therapy, as one of the major means of tumor immunotherapy, exhibits a surprisingly bright therapeutic effect in the treatment of leukemia, lymphoma, multiple myeloma. Currently several large megahead companies with global CAR-T leaders: nowa, Kite, Juno and Cellectis have various characteristics in the aspects of target spots, co-stimulation areas, transfection modes and the like, and Nowa CTL019 is about to become the first CAR-T product on the market all over the world. The development of domestic CAR-T treatment is closely related to that of CAR-T clinical tests in the United states, wherein the CAR-T clinical tests are hundreds of tests.

With the introduction of two CAR-T products against CD19 into the market in 2017, the use of CAR-T in hematological tumors has also gained worldwide acceptance. However, blood tumor patients account for only 23% of tumor patients, and the rest are mostly solid tumors, but the current treatment means for solid tumors are limited, and even some solid tumors have no targeted drugs and treatment means. CAR-T is also widely applied to solid tumors, more than 200 cases of clinical trials are carried out on the solid tumors at home and abroad at present, and the CAR-T is mainly focused on high-grade cancers such as melanoma, liver cancer, lung cancer, breast cancer and the like. Although CAR-T has advanced in the use of solid tumors, the tumor microenvironment remains one of the major causes of many undesirable clinical effects. Therefore, how to make CAR-T cells better act on tumor foci to inhibit tumors, and even eliminate tumors is a problem to be solved.

The application is widely applied to the treatment of tumors by CAR-T, and the CAR-T is modified into a fourth generation CAR-T cell, namely universal TRUCK-T, by introducing exogenous cytokines on the basis of the third generation CAR-T, wherein the fourth generation CAR-T cell has the effects of recruiting other natural immune cells, increasing the release of killer factors, reducing the generation of tumor vessels, clearing extracellular matrix and the like and is used for treating refractory recurrent solid tumors.

CAR-T has wide application in solid tumors and achieves certain curative effect, but the curative effect is not satisfactory, so that CAR-T is limited in treating solid tumors. Based on the research on the factors that CAR-T can not achieve the treatment effect of hematological tumor in the treatment of solid tumor, the tumor microenvironment and CAR-T can not repeatedly enter the solid tumor are the main reasons for limiting the curative effect of CAR-T in the solid tumor. Aiming at the main factors, combining gene editing, introducing exogenous cell factors on the basis of the third generation CAR-T, transforming the CAR-T into a fourth generation CAR-T cell which can recruit other natural immune cells, increase the release of killer factors, reduce the generation of tumor vessels, clear extracellular matrix and the like, namely universal type TRUCK-T, and is used for treating refractory recurrent solid tumors.

According to the prior art, the CAR-T clinical research mostly adopts the second generation CAR-T technology, and the clinical research using the fourth generation CAR-T technology is not much reported. T cells prepared by the traditional TRUCK-T are mainly from patients, are subjected to in-vitro T cell separation, activation, CAR and cell factors are randomly inserted into a genome by using lentivirus, culture and amplification under a clean room (GMP) environment, and are finally returned to the patients through quality control. The problem of unsuitability for blood collection or difficulty in expansion after T cell isolation from blood collection due to the influence of the patient's own conditions may be accompanied. When the patient is critically ill, the waiting time of the whole process from the isolation of T cells to the reinfusion of CAR-T is also a significant problem to be faced with the reinfusion. These problems have limited the widespread use of CAR-T technology, and therefore an important current direction in CAR-T cell therapy is how to use T cells from a healthy donor to produce large numbers of CAR-T cells for clinical use in patients. The establishment of the technology can greatly reduce the cost of CAR-T therapy, better ensure the quality of uniformly prepared cells, and patients can be immediately treated by the CAR-T cells when needed.

The CAR gene segment targeting cancer cells and the gene segment expressing exogenous cytokines are respectively inserted behind TRAC and B2M promoters while TRAC and B2M are knocked out by using a gene editing technology, and the expression of CAR is regulated by using the original TRAC promoter of T cells, so that the T cells are more similar to natural T cells which are not subjected to gene modification. Meanwhile, the expression of the exogenous cell factor is regulated and controlled by using a nuclear factor of activated T cells (NFAT), so that the risk of factor syndrome caused by random insertion of lentivirus and random high expression of the exogenous cell factor is avoided. After the CAR recognizes the antigen, the T cell is activated, the exogenous cytokine is released at the moment, and the release of the exogenous cytokine such as IL12 can recruit other natural immune cells, increase the release of a killer factor, reduce the generation of tumor blood vessels, clear extracellular matrix, change cold tumor into hot tumor, release more tumor cells and antigens, and enable the tumor cells to be better recognized and eliminated under the action of the CAR-T and the natural cells, thereby greatly enhancing the curative effect.

Summary of The Invention

1. A method of making universal human TRUCK-T cells, comprising:

(i) obtaining T cells from a subject with healthy lymphatic system, wherein the disruption of the T cells in the human by gene editing techniques:

the TRAC gene region of chromosome 14; and

the B2M gene region of chromosome 15; and is

(ii) The CAR molecule and the exogenous cytokine gene sequence are inserted into the TRAC and B2M cleavage sites respectively by using the principle of homologous recombination to express by using the T cell's own TRAC promoter and NFAT.

2. The method of item 1, wherein the CAR molecule uses the T cell's own TRAC promoter to initiate expression, and the foreign cytokine gene sequence further comprises an NFAT sequence.

3. The method of item 1, wherein said TRAC gene region is chromosome 14 from position 23016448 to position 23016490; the B2M gene region is from position 45003745 to position 45003788 of chromosome 15.

4. The method of item 1, wherein the CAR molecule is inserted into the TRAC cleavage site using the principle of homologous recombination; the foreign cytokine gene sequence was inserted into the B2M cleavage site.

5. The method of item 1, wherein the gene editing technology comprises a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRRISPR/Cas gene editing technology.

6. The method of item 5, wherein the CRRISPR/Cas gene editing technology comprises CRRISPR/Cas9 technology.

7. The method of item 6, wherein, when using the CRRISPR/Cas9 technique, the sgRNA used for the target TRAC gene region to be edited comprises the sequence of SEQ ID No.:2-5, and the sgRNA used for the target B2M gene region to be edited comprises the sgRNAs of SEQ ID Nos. 15-22.

8. The method of item 6, wherein, when using the CRRISPR/Cas9 technique, the sgRNA used for the target TRAC gene region to be edited comprises the sequence of SEQ ID No.:2, and the sgRNA used for the target B2M gene region to be edited includes the sgRNA of SEQ ID No. 20.

9. The method of item 1, wherein the target targeted by the CAR comprises mesothelin, CD19, CD123, CD20, GPC3, Her2, EGFR, NY-ESO-1, MUC1, EBV, PMSA, GD2, TAA.

10. The method of item 1 or 9, wherein the exogenous cytokine comprises: IL-12, IL17, CCL 19.

11. A universal type TRUCK-T cell made using the method according to any one of claims 1 to 10.

12. Use of the universal TRUCK-T cell according to item 11 in the manufacture of a medicament for treating cancer in a patient.

13. The use of item 12, wherein the cancer is a solid tumor.

14. The use according to item 13, wherein the solid tumor is a mesothelin-highly expressed solid tumor, including ovarian, gastric and lung cancers.

Drawings

FIG. 1 shows the measurement of the expression level of IL12 in U-TRUCK-T.

FIG. 2 shows the detection of the CAR expression level of universal type TRUCK-T.

FIG. 3 shows the knockout efficiency of universal type TRUCK-T.

FIG. 4 shows the measurement of the ratio of CD4/CD 8.

FIG. 5 shows the expression assay of memory cells.

FIG. 6 is a cell expansion experiment.

FIG. 7 shows the Knock-in (KI) assay of IL12 in the genome.

FIG. 8 is a Knock-in (KI) test of the CAR in the genome.

FIG. 9 shows cell killing experiments.

Detailed Description

In one aspect, the invention provides methods for preparing universal TRUCK-T cells. In some embodiments, the method comprises introducing a CAR molecule encoding nucleotide or vector into any of the genetically engineered T cells described herein (e.g., universal T cells).

In some embodiments, there is provided a method of making universal TRUCK-T cells, comprising:

(i) introducing into a T cell a sgRNA comprising a TRAC gene targeting chromosome 14 from position 23016448 to position 23016490 to disrupt the TRAC gene region; and/or

(ii) Introducing a sgRNA comprising a B2M gene targeting chromosome 15 from position 45003745 to position 45003788 into the T cell to disrupt the B2M gene region; and/or

(iii) Introducing a sgRNA comprising a PD-1 gene region targeting chromosome 2 from position 242800936 to position 242800978, or a sgRNA targeting a PD-1 gene region from position 242795009 to position 242795051 of chromosome 2, into the T cell to disrupt said PD-1 gene region; and

(iv) introducing into the T cell a nucleic acid encoding a Chimeric Antigen Receptor (CAR).

The CAR molecule or nucleic acid encoding it and the sgRNA targeting TRAC, the sgRNA targeting B2M and the CAR molecule or nucleotides encoding it may be introduced into the T cell in any suitable order. In some embodiments, the sgRNA targeting TRAC, the sgRNA targeting B2M, and the CAR molecule, or its encoding nucleotides, are introduced into the T cell simultaneously. In some embodiments, the CAR molecule or its encoding nucleotide precedes the sgRNA targeted to TRAC, and the sgRNA targeted to B2M is introduced into the T cell. In some embodiments, the coding nucleotides of the CAR molecule are introduced into a T cell in which gene editing has been achieved, the TRAC, B2M gene region of the T cell having been disrupted by editing. In some embodiments, the method further comprises introducing Cas9 or its encoding nucleotide into the T cell with the sgRNA.

In some embodiments, the CAR molecule refers to a gene that expresses a chimeric antigen receptor, which may be used interchangeably with "CAR gene fragment".

TRAC gene refers to a gene encoding the constant region of the α chain of the T cell receptor, whose disruption prevents α β type TCR expression. The CAR transgene is expressed at the site of the TRAC gene while disrupting native TCR expression of the T cells, under the control of the native TCR gene promoter.

The B2M gene refers to a gene encoding beta 2 microglobulin (B2M), a beta light chain of human leukocyte antigen class I (HLA-I). Its main function is to participate in recognition of lymphocytes with target cell surface antigens, and thus B2M is closely related to histocompatibility. Almost all nucleated cells in the body synthesize β 2 microglobulin, which attaches to the cell surface. The loss of beta 2 microglobulin can cause the abnormal polymerization of HLA-I molecules, so that complete functional molecules cannot be formed. Knocking out a B2M gene in the CAR-T cell by a gene editing technology, so that the CAR-T cell cannot express a normal HLA-I molecule, and the transplantation rejection effect of the CAR-T cell is further reduced.

In some embodiments, the CAR expressed by the universal type TRUCK-T cell of the present invention can target any one target, thereby exerting a killing effect.

In some embodiments, the CAR expressed in the universal type TRUCK-T cell of the present invention comprises a signal peptide, an extracellular binding region, a hinge region, a transmembrane region, and an intracellular signal region, connected in sequence. The term "signal peptide" as used herein refers to a short (e.g., 5-30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. In the present invention, signal peptides of various proteins in the human body, for example, signal peptides of cytokine proteins secreted in the body, leukocyte differentiation antigen (CD molecule) can be used.

In some embodiments, the signal peptide is a CD8 signal peptide, for example the amino acid sequence of which is as shown in the patent application US20140271635a 1.

In some embodiments, the hinge region may employ the hinge region of a variety of different antibodies or antigen receptors, particularly the hinge region of a CD molecule. In a particular embodiment, the hinge region may be selected from the hinge region of proteins such as CD8 or CD 28. The CD8 or CD28 is a natural marker of the T cell surface.

In the present invention, transmembrane domains of various human proteins, particularly those of various antigen receptors, can be used. The preferred transmembrane region used is that of the CD molecule. In one embodiment, the transmembrane region is selected from the transmembrane region of the CD8 protein.

In some embodiments, the hinge region is a CD8 a hinge region (CD8-hinge), the amino acid sequence of which is shown in the inventive patent application US20140271635a 1.

The "extracellular binding domain" refers to a domain that comprises a region that specifically recognizes a target antigen. In some embodiments, the extracellular binding region comprises a region that specifically recognizes a target tumor cell surface antigen. For example, this region may be an antigen-binding fragment of an scFv or other antibody. The term "scFv" as used herein refers to a recombinant protein of variable heavy chain regions (VH and light chain variable regions (VL)) linked by a linker region (linker) that allows the two domains to associate and ultimately form an antigen binding site.

The CARs and domains thereof used in the invention can be further modified by using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations and/or other modifications, alone or in combination. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, e.g., Sambrook molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.). The modification is preferably carried out at the nucleic acid level.

The term "specifically recognizes" as used herein means that the antigen recognition region of the present invention does not cross-react or does not substantially cross-react with any polypeptide other than the antigen of interest. The degree of specificity can be determined by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.

In some embodiments, the extracellular binding region is an antigen binding region that specifically recognizes mesothelin.

In the present invention, the intracellular signaling region is that of the CD137(4-1BB) protein. The CD3 molecule consists of five subunits, among which the CD3 zeta subunit (also called CD3 zeta, abbreviated zeta) contains 3 Immunoreceptor tyrosine-based activation motifs (ITAMs), which are important signaling regions in the tcr (t cell receptor) -CD3 complex. Fcsri γ is distributed primarily on the surface of mast cells and basophils, contains an ITAM motif and is similar in structure, distribution and function to CD3 ζ. In addition, as mentioned above, CD137 is a costimulatory signaling molecule whose intracellular signaling segment produces costimulation upon binding to its respective ligand, resulting in sustained T cell proliferation and can increase the levels of cytokines such as IL-2 and IFN- γ secreted by T cells, as well as increase the survival cycle and antitumor effects of CAR-T cells in vivo. In certain embodiments, the signal generated by the CAR alone is insufficient to fully activate native T cells, requiring initiation of antigen-dependent primary activation sequences (primary intracellular signaling domains) by the TCR and sequences that act in an antigen-independent manner to provide a costimulatory signal (costimulatory domains). The primary signaling domain regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. The primary intracellular signaling domain, which acts in a stimulatory manner, may contain signaling motifs known as Immunoreceptor Tyrosine Activation Motifs (ITAMs). The primary cytoplasmic signaling sequence of the invention containing ITAMs is CD3 ζ. In one embodiment, the primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain with altered (e.g., increased or decreased) activity compared to the native ITAM domain, or a primary intracellular signaling domain of a truncated ITAM. In one embodiment, the primary signaling domain comprises one or more ITAM motifs.

The costimulatory signaling domain refers to the portion of the TCR that comprises the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for efficient response of lymphocytes to an antigen. The costimulatory molecule region of the present invention is 4-1BB (CD 137).

In some embodiments, the exogenous cytokine of the present invention may be any cytokine. In an embodiment, the inserted exogenous cytokine of the present invention may be interleukin 12(IL12), and IL12 uses NFAT as a transcription factor for transcription and expression of IL12 protein. Here, IL12 serves an exemplary, but not limiting, role, and the cytokine herein may also be any other cytokine that is beneficial for tumor therapy, such as IL17, CCL 19.

In some embodiments, the present invention provides a method of making a TRUCK-T cell, e.g., a universal-type TRUCK-T cell, comprising the steps of:

1) introduction of sgRNA molecule and Cas9 molecule in T cells:

in some embodiments, the sgRNA molecule comprises a promoter sequence that hybridizes to an alpha chain constant coding region (i.e., TRAC) gene from a TCR, an HLA constant coding region B2M gene, and optionally: a targeting domain complementary to a target region of a gene encoding a constant region of PD-1.

2) Introducing a CAR molecule and an exogenous cytokine gene sequence in the T cell;

the CAR molecule may be inserted into the TRAC cleavage site;

the foreign cytokine gene sequence may be inserted into the B2M cleavage site, optionally into the PD-1 cleavage site.

TRUCK-T cells refer to CAR-T cells that are accompanied by a transgenic "payload", a fourth generation CAR-T cell technology that shapes the tumor microenvironment through the induced release of transgenic immunomodulators. This TRUCK-T cell technology is applicable in cancer therapy, including the treatment of viral infections, autoimmune diseases or metabolic diseases.

In some embodiments, the CAR molecule and the exogenous cytokine gene sequence are inserted into the TRAC and B2M cleavage sites, respectively, using the principle of homologous recombination, and expressed using the T cell's own TRAC promoter and NFAT, respectively.

In some embodiments, the sgRNA molecule refers to a nucleic acid sequence comprising a targeting domain complementary to a target region of a gene to be knocked out, which recognizes the target DNA sequence and directs the Cas9 molecule to cleave the target site, which can achieve a one-step efficient (knock out efficiency of 84% or more) knock out of the corresponding site.

In some embodiments, the Cas9 molecule refers to Cas9 mRNA that is capable of cleaving a target site under the guidance of the sgRNA.

In some specific embodiments, the sgRNA molecule comprises a targeting domain having a sequence as set forth in one of table 1. In some preferred embodiments, the targeting domain has the sequence shown as T2, B6 (table 1).

TABLE 1

In some preferred embodiments, the sgRNA molecule and mRNA encoding Cas9 are introduced into the T cell by electrotransfer techniques.

In some embodiments, the CAR molecule and exogenous cytokine gene sequence are introduced into the T cell, directionally inserted into the TRAC and B2M knockout sites, respectively, by, for example, line-related viral transfection techniques. In some specific embodiments, comprising the step of isolating and/or activating T cells from peripheral blood or cord blood of a healthy person; preferably, the method further comprises a step of sorting universal type TRUCK-T cells after the step 2) above; more preferably, the sorting is followed by functional validation of the resulting TRUCK-T cells, e.g., universal type TRUCK-T cells.

In some embodiments, the invention provides the use of the above-described TRUCK-T cells for the preparation of a medicament for treating a disease (e.g., a tumor).

In one aspect, the invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of a TRUCK-T cell according to the invention. The subject may include cancer and/or HIV/AIDS. In some embodiments, the disease is a tumor. In some embodiments, the CAR targets mesothelin to treat some mesothelin-highly expressing solid tumors, such as ovarian, gastric, and lung cancers. In some embodiments, the T cell is not obtained from a subject. For example, the T cells may be derived from a healthy donor.

The universal type TRUCK-T cells of the present invention may be administered to a subject in need thereof by a route conventionally used for administering pharmaceutical preparations containing cellular components, such as intravenous infusion route. The dosage administered may be specifically determined based on the condition and general health of the subject.

T cell source

Prior to amplification and genetic modification, a source of T cells is obtained from the subject. The term "subject" is intended to include living organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors (with the exception of T-lineage lymphomas). The T cells of the invention may also be derived from hematopoietic stem cells at various stages of differentiation. Hematopoietic stem cells are differentiated into T cells under committed differentiation culture conditions. In certain aspects of the invention, a variety of T cell lines available in the art may be used.

In certain aspects of the invention, T cells can be obtained using a variety of techniques known to the skilled artisan, such as Ficoll^TMSeparating blood collected from the subject. Cells may also be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.

Can be prepared by lysing erythrocytes and, for example, by PERCOLL^TMDepletion of monocytes by gradient centrifugation or countercurrent centrifugation elutriation separates T cells from peripheral blood lymphocytes. Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA +, CCR7+, CD62L +, and CD45RO + T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are obtained by coupling to anti-CD 3/anti-CD 28 (e.g., 3 x 28) beads, such as DYNABEADS^TMM-450CD3/CD28T were isolated by incubating together for a period of time sufficient to positively select for the desired T cells. Tumor Infiltrating Lymphocytes (TILs) can be isolated from tumor tissue.

sgRNA

In one aspect of the invention, there is provided a sgRNA targeted to TRAC, a sgRNA targeted to B2M. The sgRNA contains any one nucleotide sequence selected from SEQ ID NO. 2-5 or 15-22. In some embodiments, the sgRNA is chemically modified.

Also encompassed by the invention are sgRNA compositions, kits or articles of manufacture comprising the sgrnas or vectors thereof to which the invention relates. In some embodiments, the kit comprises: (i) comprises a sequence selected from SEQ ID No.: 2-5; (ii) comprises a sequence selected from SEQ ID No.: 6-14; and/or (iii) comprises a sequence selected from SEQ ID No.: 15-22. In some embodiments, the kit comprises: (i) comprises SEQ ID No.: 3 sequence sgRNA; (ii) comprises a sequence selected from SEQ ID No.: 16 sequence of sgRNA; and (iii) comprises SEQ ID No.: 7 sequence sgRNA. In some embodiments, the kit further comprises a Cas 6-encoding nucleic acid or a vector thereof. In some embodiments, the sgRNA is chemically modified.

SEQ ID No.: 6-14 are targeting domains complementary to the target region of the constant coding region gene of PD-1.

SEQ ID No.:2-5 are targeting domains complementary to the target region of the constant coding region gene for TRAC.

SEQ ID No.: 15-22 are targeting domains complementary to the target region of the constant coding region gene of B2M.

In some embodiments, the T cell genetic engineering methods of the invention use chemically modified sgrnas. The chemically modified sgrnas employed herein are believed to have the following two advantages. First, because sgrnas are single-stranded RNA, their half-lives are very short and degrade rapidly after entering cells (up to 12 hours), whereas Cas9 protein requires at least 48 hours for sgRNA to bind to and perform gene editing. Therefore, the chemically modified sgRNA is stably expressed after entering cells, and can efficiently edit genomes to generate Indels (Indels) after being combined with the Cas9 protein. Second, unmodified sgrnas have poor ability to penetrate cell membranes and cannot effectively enter cells or tissues to perform their functions. Whereas the ability of chemically modified sgrnas to penetrate cell membranes is generally enhanced. In the present invention, chemical modification methods commonly used in the art may be employed, as long as the stability (half-life extension) of sgRNA and the ability to enter cell membranes can be improved. In addition to the specific chemical modifications used in the examples, other modification methods are also contemplated, for example, Deleavey GF1, Damha mj. design chemical modified oligonucleotides for targeted gene immunization.chem biol.2012, 24 months; 937-54, and Hendel et al chemical ly modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells. nat Biotechnol.2015, 9 months; 33(9) 985-989.

The chemically modified sgRNA and the Cas9 encoding gene are jointly electrically transferred into the T cell to generate high-efficiency gene editing efficiency (for example, the high-efficiency gene editing efficiency is expressed by TCR alpha/beta-/B2M-%).

The present invention will be described with reference to specific examples. It should be understood that the specific embodiments are for illustrative purposes only and are not meant to limit the disclosure to only specific embodiments.

Throughout this specification, several documents are cited. Each document herein (including any journal articles or abstracts, published or unpublished patent applications, issued patents, manufacturer's specifications, instructions for use, etc.) is incorporated by reference. However, there is no admission that the documents cited herein are in fact prior art to the present invention.

Examples

Example 1 preparation of Universal type TRUCK-T cells

1. Isolation and activation of healthy donor T cells

Collecting cord blood of healthy donors, namely taking the cord blood from a blood bank, temporarily storing the cord blood in a refrigerator at 4 ℃, and transporting the cord blood to a GMP laboratory for separation of T cells in 24 hours by a transport vehicle equipped with constant temperature equipment.

1.1 cord blood mononuclear cell preparation, which is to use a 10ml pipette (manufacturer: Corning, goods number: 4488, batch number: 32317601) to absorb physiological saline (for medical use) and add the physiological saline into the cord blood transported in the step (1), dilute the cord blood and the physiological saline according to the ratio of 1:1(V/V), slowly add the blood cell diluent into a lymphocyte separation tube (manufacturer: Dake, goods number: DKW-LST-25050SK), centrifuge for 20 minutes at 800g, absorb white membrane layer cells above the lymphocyte separation liquid, transfer the white membrane layer cells into a new 50 ml centrifuge tube (manufacturer: Corning, goods number: 430828), add Lonza X-vivo15 culture medium (manufacturer: Lonza, goods number: 04-418Q), centrifuge for 5 minutes, discard supernatant, reserve the cell sediment at the bottom of the centrifuge tube, and obtain the peripheral blood mononuclear cell.

1.2 isolation and activation of T cells the cord blood mononuclear cells obtained were counted in a cytotechnologist (Nexcelom, model: Cellomer Auto T4) and T Cell sorting was carried out using the StemShell easy Sep Human T Cell Iso Kit (manufacturer: StemShell, cat # 17951) as follows:

1.2.1. the cell pellet was adjusted to a density of 5X 10 with Easy buffer (manufacturer: StemCell, cat # 16F72331)⁷Per ml, cells were transferred to a 5ml flow tube with a 5ml pipette;

1.2.2. adding the isolation Cocktail (manufacturer: StemCell, cat # 17951) at a concentration of 50. mu.l/ml, and incubating at room temperature for 5 minutes after the addition;

1.2.3. adding sorting magnetic beads (manufacturer: StemCell, cat # 17951), mixing magnetic beads in 30S, adding 40 μ l/ml;

1.2.4 using Easy buffer solution to supplement the cell sap to 2.5 ml, directly placing on a magnetic column for 3 minutes after the cell sap is added, and then pouring the cells into a 15 ml centrifuge tube to obtain the T cells;

1.2.5. after sorting, the cells were counted by mixing with a 1000. mu.l pipette (manufacturer: Thermo, model: 4642090), centrifuged (400G, 5min) and the supernatant removed to obtain a T cell pellet.

The T cell pellet was resuspended in complete medium (Lonza X-vivo15 + IL-2) (Lonza X-vivo15 medium (manufacturer: Lonza, cat # 04-418Q); IL-2 (manufacturer: Beijing tetracyclic organism, batch: 20170745a)), Dynabeads Human T-Activator CD3/CD28 (manufacturer: Gibco, cat # 602101) was added at a ratio of 1:1, and the T cells were placed in an incubator to continue the expansion culture.

Knock-out of TCR and B2M genes

Knocking out TCR and B2M genes in the T cells obtained in the step 1.2 by using a CRISPR/Cas9 gene editing technology, and specifically operating steps are as follows:

2.1 sgRNA design and plasmid construction for the α chain constant coding region (i.e., TRAC) gene of TCR and the constant coding region gene of HLA constant coding region (B2M) gene.

Sgrnas designed for TRAC and B2M were both designed by CRISPR RGEN Tools, and the sgRNA sequences selected according to the highest scores are shown in table 1.

2.2 the sgRNA is modified by 2 '-O-methyl analogues and/or 3' thio between nucleotides by a chemical method, so that the sgRNA with high knockout efficiency and stability is prepared.

2.3 using electrotransfer technology, sgRNA and Cas9 mRNA were introduced into T cells, using a line-associated virus containing CAR molecule at MOI of 3E5, 6E5, 9E5 and 1.2E6 and a line-associated virus containing IL12 sequence at MOI of 3E5, 8E5 and 1E6, respectively, the target fragments were introduced into the edited T cells, site-specific insertion was performed, CAR molecule was inserted into the TRAC cleavage site, IL12 was inserted into the B2M cleavage site, and U-TRUCK-T cells were obtained.

2.4 taking out the knockout T cell, using antibody to detect the expression level of TCR, B2M, CAR and IL12, it can be seen from the results of FIG. 3 that the DKO efficiency is stably maintained at about 84%, and the expression level of IL12 is about 50% as shown in FIG. 1. Under the system, the CRAC gene of TCR and the B2M gene of HLA can be successfully edited by using the CRISPR/Cas9 technology, wherein the CRAC gene and the B2M gene of HLA comprise insertion mutation and deletion mutation, and both of the insertion mutation and the deletion mutation cause frame shift mutation, thereby inhibiting the expression of TCR and HLA from the gene level. Meanwhile, by utilizing a homologous recombination mechanism, a homology arm is added to the gene sequence for expressing the CAR and the gene sequence for expressing the IL12 at the same time, and the sequences can be inserted into a fixed site in a fixed point manner.

2.5 predicting the potential off-target sites on the whole human genome of TRAC-sg3(T3) and B2M-sg2(B6) at the same time, and performing amplification analysis on the predicted off-target site regions which may influence the expression of other genes, aiming at confirming that the knockout of TCR and HLA does not introduce off-target (off-target) non-specific gene from the molecular level, and the results are shown in Table 2, which shows that the TRAC and B2M genes do not have any mutation, thereby indicating that the specificity of the system meets the requirements.

For subsequent control experiments, a portion of healthy donor T cells were retained; knocking out TCR and B2M genes in the T cell obtained in the step 1.2 by using a CRISPR/Cas9 gene editing technology, and preparing a U-T cell; and (3) introducing the CAR molecules into the T cells and the U-T cells respectively by using an adenovirus transfection technology to obtain the CAR-T cells and the U-CAR-T cells. Similarly, the IL12 sequence was introduced into U-T cells to obtain U-IL12-T cells.

TABLE 2

The above results show that TCR and HLA genes in universal T cells obtained by gene editing and screening T cells using TRAC-sg2(T2) and B2M-sg6(B6) as sgrnas were completely knocked out, and no gene mutation at a potential off-target site was found.

3. Expansion of universal CAR-T cells

Sorted T cells were activated using Dynabeads Human T-Activator CD3/CD28 (manufacturer: Gibco, cat # 602101) coupled with CD3/CD28 antibody, and then cell density was adjusted to 1X 10 using Lonza X-vivo15 (manufacturer: Lonza, cat # 04-418Q) medium⁶Individual cells/mL. The state of the cells was observed after 72 hours, and the cell suspension was collected, Dynabeads Human T-Activator CD3/CD28 was removed, centrifuged at 300g for 7min, the supernatant was discarded, washed 2 times with DPBS (manufacturer: Gibco; cat # 1924294), and the cell density was adjusted to 2.5X 10 using Opti-MEM medium⁷Individual cells/mL. Cas9 mRNA prepared using HiScribe TM T7 ARCA mRNA Kit (tailed) (manufacturer: NEB, cat # E2060S) and synthetic sgRNA were mixed to a final concentration of 2.5X 10 RNA per 100. mu.L of T cells and RNA⁶Individual cells and 8. mu.g of RNA (Cas9 mRNA and sgRNA each 4. mu.g) were introduced into the cells using a transter BTX Agile pulse MAXThen, the culture is carried out. Line-related viruses (CAR structure composed of scFv: mesothelin, Linker region (Linker), CD8 alpha hinge region (CD8 alpha hinge), CD8 transmembrane region (CD8 transmberane domain), 4-11BB signal region (4-11BB signaling domain) and CD3 zeta) packaged with IL12 sequence (IL12 sequence is preceded by NFAT) were added at MOI of 8 within 2h after electrotransformation, and the sequence is shown in Table 3.

TABLE 3

Delivering the gene of interest into the electroporated T cell. The growth of the cells was observed every day, cell counting and fluid replacement were performed every other day, and phenotyping of the gene-edited T cells was performed, as shown in fig. 3, with an efficiency of knocking out TCR and B2M of about 84%, and the results of fig. 1 and 2 indicate that the CAR molecule knock-in efficiency was about 16%, the IL12 sequence knock-in efficiency was about 50%, and the resultant T cells were monitored for quality control (i.e., QC) after 14 days of cell culture. The growth status of the cells can be monitored by techniques, as shown in FIG. 6, after 2 weeks of culture, T cells and CAR-T cells did not undergo gene editing and grew the best, while the gene-edited cell samples expanded slightly less than the cells that did not undergo gene editing.

FIG. 1 shows the measurement of the expression level of U-TRUCK-T IL 12. As shown in the figure, when T cells were cultured to day 3, TCR and HLA were knocked out by electrotransfer, a line-related virus of different MOIs (MOI ═ 3E5, 8E5, 10E5) with a foreign gene to be inserted was added to the gene editing cells 2 hours after electrotransfer, the CAR sequence was inserted behind the TRAC promoter, and the IL12 sequence was inserted behind the B2M knock-out site, to obtain universal TRUCK-T. After adding the stimulator, the universal type TRUCK-T highly expresses IL12 to achieve the effects of recruiting macrophages, natural killer cells and other effects of preventing angiogenesis and the like.

FIG. 2 shows the expression amount of CAR of universal type TRUCK-T. As shown in the figure, when T cells were cultured to day 3, TCR and HLA were knocked out by electrotransfer, a line-associated virus with a different MOI (MOI ═ 3E5, 6E5, 9E5, 12E5) was added to the gene editing cells 2 hours after electrotransfer, the virus contained a foreign gene to be inserted, the CAR sequence was inserted behind the TRAC promoter, and the IL12 sequence was inserted behind the knock-out site of B2M, to obtain universal TRUCK-T. CAR expression was highest at an MOI of 9E5, reaching about 16%.

FIG. 3 shows the knockout efficiency of universal type TRUCK-T. As shown in the figure, T cells are cultured to 3 days, TCR and HLA knockout is carried out in an electrotransfer mode, phenotype detection is carried out on 4 days after editing, the proportion of CD 3-/TCR-accounts for 84.4%, and knockout efficiency is stable and efficient.

4. Screening of Universal type TRUCK-T cells

T cells that were TCR and/or HLA-I negative, CD4 and CD8 positive were screened as follows.

T cells which are negative in TCR and/or HLA-I and positive in CD4 and CD8 are screened by an immunomagnetic bead technology, and the state of the edited T cells is monitored by the survival rate of the T cells, and the method comprises the following specific steps:

in the first step, the T cells after electroporation were collected at day 12-14, 400G, centrifuged for 5min, the supernatant was discarded, and the cells were lysed with Easy buffer to 1X 10⁸And then transferring the cells to a 5ml flow tube, removing cells which still express CD3(TCR) and HLA-I from the T cells by using an Easy Sep Human PE positive Selection Kit (manufacturer: Easy Sep, cat # 17G81322), and screening to obtain a final product, namely universal type TRUCK-T cells.

In the second step, a small amount of T cells are taken and subjected to flow detection to detect the sub-population of the T cells, such as FIG. 4, and simultaneously stained with CD4 and CD8 cell surface biomarkers, and the experimental result shows that the gene editing in vitro culture has no significant difference on the proportion change of CD4 and CD 8. It was reported that memory T cells play an important role in the therapeutic effect, and therefore in this example, as shown in figure 5, CD62L and CCR7 were stained simultaneously, and as shown in the figure, there was a higher proportion of memory cells compared to T cells that were not genetically edited and CAR-T cells, that were genetically edited universal T cells, universal T cells that were knocked in CAR molecules, universal T cells that were knocked in IL12 sequences, and universal TRUCK-T cells, indicating that gene editing had no significant effect on a subset of memory cells.

FIG. 4 is a graph of the CD4/CD8 ratio. By monitoring CD4 and CD8 on different days, the ratio of CD4/CD8 was almost unchanged, both at about 1.6:1, by day 5 and by day 10 of the culture. The results indicate that gene editing in vitro culture has no significant difference to the change in CD4 and CD8 ratios.

FIG. 5 is an expression assay of memory cells. As shown, the ratio of universal T cells with gene editing, universal T cells with knockin CAR molecules, universal T cells with knockin IL12 sequences, and universal T cells with knockin TRUCK-T cells was higher compared to T cells without gene editing and CAR-T cells, indicating that gene editing had no significant effect on a subset of memory cells.

And thirdly, after the final harvested finished cells are extracted from the genome, the knock-in fragments are subjected to gene level detection, and the result is shown in fig. 7 and 8, and the gene fragments of the knock-in IL12 and CAR are specifically amplified, and a specific band is shown on a gel electrophoresis picture. Further described is the targeted insertion of a knocked-in gene into a knock-out double strand break site. The method effectively avoids potential safety problems caused by random insertion of viruses into genomes, and simultaneously avoids introduction of foreign promoter genes by using the promoter of the T cell, so that the prepared product is closer to the T cell in a natural state.

FIG. 6 is a cell expansion experiment. As shown, after 2 weeks of culture, T cells and CAR-T cells did not undergo gene editing and grew the best, while gene-edited cell samples expanded slightly less than non-gene-edited cells.

FIG. 7 shows KI detection of IL12 in the genome. Sample No. 1 is a T cell sample, i.e., a sample that has not been subjected to any treatment; no. 2 and No. 3 are universal type TRUCK-T samples, i.e. the CAR and IL12 gene fragments are knocked in simultaneously. MOI of No. 2 is 3E5, MOI of No. 3 is 8E 5; samples No. 4 and No. 5 were knock-in CAR gene fragment only, sample No. 4 had an MOI of 12E5, and sample No. 5 had an MOI of 6E 5; m is a DNA marker. According to gel electrophoresis, two groups of cell samples of knock-in IL12 sequences have expression of IL12 sequence bands, which indicates that the IL12 sequences stably knock-in into cell genomes.

FIG. 8 is KI detection of CAR in the genome. Number 1 is universal T cell sample; no. 2 is a sample in which only the IL12 gene fragment was knocked in; no. 3 is a sample in which only CAR gene fragments were knocked in; no. 4 is a lentivirus-infected sample containing CAR molecules; no. 5 is a universal type TRUCK-T sample, namely, a CAR and IL12 gene fragment are knocked in simultaneously; sample No. 6, T cell sample, i.e. sample without any treatment; m is a DNA marker. As can be seen by gel electrophoresis, both sets of cell samples knocked-in CAR molecules had expression of CAR molecule bands, indicating that the CAR molecules were stably knocked-in into the cell genome.

Example 2 general type TRUCK-T functional verification

The killing effect of the universal type TRUCK-T cells (i.e., effector cells) obtained in example 1 on cancer cells highly expressing mesothelin was observed.

In vitro killing effect on specific tumor cells

The experimental steps of the invention are as follows:

first step, target cell inoculation

1. Target cells (A549 high-expression mesothelin stable Cell line, all cells from ATCC) were inoculated into a 96-well plate containing a Real-Time Cell Analyzers (Real-Time Cell Analyzers, RTCA (manufacturer: Ason)) killing detection instrument containing R1640+ 10% FBS culture solution according to 10000 cells, the volume was 100. mu.l, and the target cells were just spread over the bottom of the plate.

2. And (3) observing a cell proliferation curve in real time, and when the cell proliferation reaches a logarithmic phase, putting the effector T cells, the universal T cells, the CAR-mesothelin T cells, the universal IL 12T cells and the universal TRUCK-T cells obtained in the steps into a pore plate added with the target cells according to an effective target ratio (E: T is 10: 1; 5: 1; 2.5: 1; 1.25: 1; 0.625:1), wherein the volume is 100 mu l, the rest three pores are used as negative controls, the effector cells are not added, only the culture medium containing R1640+ 10% FBS is supplemented, and the final culture volume is 200 mu l. And then put into RTCA for observation. The process should be accurate and rapid, and the temperature of the culture medium should be kept at 37 ℃.

3. By monitoring CO-cultured cells in real time, 5% CO at 37 ℃₂The result of the culture for 100 hours is shown in FIG. 9, in which the universal type TRUCK-T cells had the best killing effect on specific target cells and almost no killing function on non-specific target cells compared to other T cells. In addition, the killing effect and the action starting time of effector cells on target cells can be well tracked through the real-time monitoring of RTCA.

FIG. 9 is a cell killing experiment. As shown in the figure, the killing effect of the effector cells was monitored in real time by co-culturing for 100 hours according to different effector-target ratios (effector T cell sample: target cell Huh-7: 10:1, 5:1, 2.5:1, 1.25:1, 0.625:1), and 10: the sample with a high target ratio of 1 killed the target cells best. From the cell killing curve, the killing effect can be seen: TRUCK-MSLN > DKO-TRAC-MSLN > CAR-T-MSLN > DKO-B2M-IL 12T cells are more than or equal to DKO cells, and the killing effect is reduced along with the reduction of the effective target ratio. Huh-7 grew normally compared to the blank control NC group without any T cells.

Second, the in vivo killing effect of universal type TRUCK-T to specific tumor cells

1. Cell line Huh 7-mesothelin (luciferase-GFP)

The Huh7 human hepatoma cell line is established by Naka bayashi et al, the cell is derived from a Japanese male well-differentiated liver cell, mesothelin and luciferase-GFP expression genes are introduced through lentivirus, a stable transfer cell line is established, and the mesothelin expression is 99% positive and can be used as a target cell of CAR-T cells. Huh 7-mesothelin (luciferase-GFP) was modified to express both GFP and luciferase. A mouse human high-expression mesothelin solid tumor canceration model can be constructed by an abdominal subcutaneous injection mode, and the tumor formation area is counted by the fluorescence displayed by XenoLight D-fluorescein sylvite (manufacturer: PerkinElmer; product number: 122799) in cooperation with a small animal living body imager.

Culture of Huh 7-mesothelin (luciferase-GFP) cells

The Huh 7-mesothelin (luciferase-GFP) cell line is an adherent cell line that can grow rapidly in RPMI 1640 medium (manufacturer: Hyclone, cat # SH30809.01) containing 10% FBS. Passages were required when the cells were 90% confluent at the bottom of the plate. At passage, the cells were digested with trypsin for 1 minute, neutralized with RPMI 1640 medium containing 10% FBS, the cell suspension was centrifuged at 300g for 6 minutes in a centrifuge tube, and the supernatant was discarded. Adjusting cell density to 1X 10⁵The cells/mL were added to a 10 mL-10 cm petri dish and the culture was continued.

3. Mouse model

7-10 weeks old NPG female mice 25, single subcutaneous injection of tumor cells (Huh 7-mesothelin (luciferase-GFP)) 5X 10⁵The cells were weighed every other day, observed once a day, and 3-5 days after inoculation of tumor cells, randomly divided into 6 groups by using the area of tumor formation and the tumor enrichment degree as indexes, which were presented by XenoLight D-fluorescein potassium salt (manufacturer: Perkinelmer; Cat. No.: 122799) in combination with a small animal in-vivo imager: saline group, T cell group, CAR-T cell group, U-CAR-T group, U-IL12-T group, U-TRUCK-T group.

4. Mouse lymphoma model dosing

Record the day of molding as D0. The cells were transfused with 200. mu.L of physiological saline and 200. mu.L of human T cells (total 2X 10) by tail vein injection⁶Individual cell/individual), CAR-T cells 200. mu.L (2X 10 in total)⁶/), U-CAR-T cells 200. mu.L (2X 10 in total)⁶/), U-IL12-T cells 200. mu.L (total 2X 10)⁶Per), U-TRUCK-T cells 200. mu.L (2X 10 in total)⁶/only) all mice were dosed once.

5. Post-dose monitoring in mice

The mice after administration are monitored every day, and the weight of the mice is recorded every 2 days, including the weight, the skin integrity, the hair, the mental state, the activity frequency, the activity coordination and the like of the mice, the mice are continuously observed for 37 days, the elimination of the tumor area and the reduction of the tumor enrichment are taken as the judgment indexes of the effector cell function, and the safety of the universal TRUCK-T is judged according to the skin integrity, the hair, the mental state, the activity frequency and the activity coordination. As shown in FIG. 8, the mice did not lose weight, had intact skin and hair, were mentally active, and were coordinated, and showed no GVHD response.

Sequence listing

<110> Boya Yingyin (Beijing) Biotechnology Ltd

<120> a universal type TRUCK-T cell, its preparation method and use

<130> PD01299

<150> 2019114099706

<151> 2019-12-31

<160> 28

<170> PatentIn version 3.5

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<213> Artificial sequence

<220>

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agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900

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Claims

1. A method of making universal human TRUCK-T cells, comprising:

the TRAC gene region of chromosome 14; and

the B2M gene region of chromosome 15; and is

(ii) The CAR molecule and/or exogenous cytokine gene sequence is inserted into the TRAC and/or B2M cleavage site using the principle of homologous recombination.

2. The method of claim 1, wherein the CAR molecule uses the T cell's own TRAC promoter to initiate expression, and the foreign cytokine gene sequence further comprises an NFAT sequence.

3. The method of claim 1 wherein said TRAC gene region is chromosome 14 from position 23016448 to position 23016490; the B2M gene region is from position 45003745 to position 45003788 of chromosome 15.

4. The method of claim 1, wherein the CAR molecule is inserted into the TRAC cleavage site using the principle of homologous recombination; the foreign cytokine gene sequence was inserted into the B2M cleavage site.

5. Method according to claim 1, wherein said gene editing technology comprises a zinc finger nuclease based gene editing technology, a TALEN gene editing technology or a CRRISPR/Cas gene editing technology, preferably said CRRISPR/Cas gene editing technology comprises a CRRISPR/Cas9 technology.

6. The method of claim 5, wherein, when using the CRRISPR/Cas9 technique, the sgRNA used for the target TRAC gene region to be edited comprises the sequence of SEQ ID No.:2-5, and the sgRNA used for the target B2M gene region to be edited comprises the sgRNAs of SEQ ID Nos. 15-22

Preferably, when using the CRRISPR/Cas9 technique, the sgRNA used for the target TRAC gene region to be edited includes SEQ ID No.:2, and the sgRNA used for the target B2M gene region to be edited includes the sgRNA of SEQ ID No. 20.

7. The method of claim 1, wherein the CAR-targeted target comprises mesothelin, CD19, CD123, CD20, GPC3, Her2, EGFR, NY-ESO-1, MUC1, EBV, PMSA, GD2, TAA.

8. The method of claim 1 or 7, wherein the exogenous cytokine comprises: IL-12, IL17, CCL 19.

9. Universal type TRUCK-T cells made using the method of any one of claims 1 to 8.

10. Use of the universal TRUCK-T cell according to claim 9 in the manufacture of a medicament for treating cancer in a patient.

11. The use according to claim 10, wherein the cancer is a solid tumor, preferably the solid tumor is a mesothelin-highly expressed solid tumor, including ovarian, gastric and lung cancer.