SCOTR-iNK cell as well as preparation method and application thereof

Abstract

The invention provides an sCOTR-iNK cell as well as a preparation method and application thereof. Specific TCR molecules and co-receptor molecules CD8 and co-stimulatory molecules CD28 thereof are simultaneously introduced into the pluripotent stem cells, and sCOTR-iNK capable of simultaneously expressing TCR, CD8 and CD28 can be output at high yield through induced differentiation. The sCOTR-iNK cell obtained by the preparation method disclosed by the invention has more remarkable tumor poisoning activity. According to the method, the NK cells are obtained through in-vitro induction after gene engineering / gene editing is carried out in the pluripotent stem cell stage, materials do not need to be taken from human tissues, multiple rounds of gene transfection do not need to be carried out, the gene editing efficiency can be greatly improved, and the TCR-NK preparation cost is reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biological technology, and in particular relates to a sCOTR-iNK cell and a preparation method and application thereof. Background Art

[0002] At present, CAR-T, TCR-T and CAR-NK immune cell therapies have shown effective anti-tumor ability in the treatment of hematological malignancies and solid tumors (PMID: 36423279, PMID: 35165422, PMID: 32822573). TCR-T cells specifically recognize antigen peptide-major histocompatibility complex (pMHC) on the surface of antigen presenting cells or target cells to target and kill abnormal cells (PMID: 357255704). However, T cell therapy has certain disadvantages in its application, such as easy to induce cytokine release syndrome (CRS), graft-versus-host disease (GVHD), immune effector cell-associated neurotoxicity syndrome (ICANS), and inability to effectively kill tumor cells with downregulated HLA-I molecules (PMID: 37454745).

[0003] In contrast, NK cells have many advantages in immune cell therapy, including low toxicity, targeted killing of tumor or virus-infected cells without pre-sensitization, and no strict requirements for HLA matching (PMID: 30361801). In addition, NK cells can also be genetically modified to introduce CAR or TCR elements to enhance their anti-tumor effects. TCR and CAR molecules mainly transduce signals through CD3ζ molecules in NK cells (PMID: 35879429). Studies have shown that TCR cannot be expressed on the plasma membrane of NK cells alone (PMID: 28878363), but when combined with the transmembrane and signaling domains of CD28 and CD3ζ, TCR can effectively trigger target cell killing in an MHC-restricted and antigen-specific manner. Studies overexpressing CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) and TCR molecules in NK-92 cell lines have shown that NK cells can express and assemble TCRs (PMID: 30665853, 31054264). In addition, there are also studies reporting that TCR, CD8 co-receptor and CD3 molecules can be introduced into primary NK cells and TCR assembled through two-step retroviral transfection (PMID: 35288464). However, NK cell lines (such as NK-92) have low expression of activating receptors and must be irradiated before infusion to prevent tumorigenesis, affecting efficacy, and TCR-NK-92 cells have low cytotoxic activity and are not the most ideal source of TCR-NK cells. NK cells derived from human tissues face high heterogeneity and need to be obtained from human tissues. The single batch preparation quantity is limited, and the cells need to undergo additional transfection processes such as viruses. The preparation cost of TCR-NK cells is high. The efficiency of multiple gene editing / genetic engineering of primary NK cells (from umbilical cord blood or peripheral blood) is low, and multiple rounds of gene transfection are required. The reported transfection efficiency of introducing TCR into natural NK cells is only 42%. Summary of the invention

[0004] In view of this, the present invention aims to propose a sCOTR-iNK cell and a preparation method and application thereof, so as to solve the problems of low gene editing efficiency, high TCR-NK cell preparation cost and low cytotoxic activity.

[0005] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0006] In a first aspect, the present invention provides a stem cell expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, wherein the stem cell includes human embryonic stem cell (hESC), human induced pluripotent stem cell (hiPSC) or chemically reprogrammed induced pluripotent stem cell (CiPSC).

[0007] Furthermore, the TCR includes EBV TCR, (NY-ESO-1)-TCR or WT1 TCR.

[0008] Furthermore, the amino acid sequence of the EBV TCR is:

[0009]

[0010]

[0011] The amino acid sequence of the (NY-ESO-1)-TCR is:

[0012]

[0013] The amino acid sequence of the WT1 TCR is:

[0014]

[0015] Further, the CD8 includes CD8A or CD8B;

[0016] The amino acid sequence of the CD8A is:

[0017]

[0018] The amino acid sequence of the CD8B is:

[0019]

[0020] The amino acid sequence of the CD28 is:

[0021]

[0022] The second aspect of the present invention provides a method for preparing stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to the first aspect of the present invention, comprising the following steps:

[0023] (1) constructing a recombinant vector containing a specific TCR molecule and its co-receptor molecule CD8 and co-stimulatory molecule CD28;

[0024] (2) introducing the recombinant vector constructed in step (1) into stem cells (hPSCs);

[0025] Furthermore, the specific TCR molecule includes EBV TCR-GFP, (NY-ESO-1)-TCR-GFP or WT1TCR-GFP (with GFP as a reporter gene).

[0026] Furthermore, the nucleotide sequence of the EBV TCR is shown in SEQ ID NO.8;

[0027] The nucleotide sequence of the GFP is shown in SEQ ID NO.9;

[0028] The nucleotide sequence of (NY-ESO-1)-TCR-GFP is shown in SEQ ID NO.15;

[0029] The nucleotide sequence of the WT1 TCR-GFP is shown in SEQ ID NO.17.

[0030] Furthermore, the CD8 includes CD8A or CD8B.

[0031] Furthermore, the nucleotide sequence of the CD8A is shown in SEQ ID NO.10;

[0032] The nucleotide sequence of CD8B is shown in SEQ ID NO.11.

[0033] Furthermore, the nucleotide sequence of CD28 is shown in SEQ ID NO.12.

[0034] Furthermore, the recombinant vector in step (1) is a piggyBac recombinant vector containing a specific TCR molecule and its co-receptor molecule CD8 and co-stimulatory molecule CD28.

[0035] Furthermore, the introduction method in step (2) includes lentiviral infection, retroviral infection, adenoviral infection, Sendai virus infection, electrofection or chemical transfection, preferably electrofection.

[0036] The third aspect of the present invention provides a method for preparing NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, wherein the NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 are obtained by induced differentiation of the stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in the first aspect of the present invention.

[0037] Furthermore, the expression mode includes constitutive expression, endogenous gene-driven expression, and small molecule compound or drug-induced expression.

[0038] Furthermore, the differentiation induction method includes a monolayer induction method, an embryoid body induction method or an organoid induction method.

[0039] The fourth aspect of the present invention provides a NK cell (sCOTR-iNK) expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, which is prepared by the preparation method of NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 described in the third aspect of the present invention.

[0040] The fifth aspect of the present invention provides a pharmaceutical composition, comprising the NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in the fourth aspect of the present invention, and a carrier or excipient.

[0041] Furthermore, the pharmaceutical composition is an injection, microneedle, mucosal patch, enema, suppository, gel, oral agent, aerosol, drop, ointment, implant, capsule or aerosol.

[0042] The sixth aspect of the present invention provides the use of the stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 described in the first aspect of the present invention, the NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 described in the fourth aspect of the present invention, or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of a drug, wherein the drug is used for:

[0043] (1) Anti-tumor, preferably, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer;

[0044] (2) Antiviral (e.g., coronaviruses such as SARS-CoV-2, human HIV, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV, herpes zoster virus) infections, for example, novel coronavirus infection (COVID-19), viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital and urinary tract infections, central nervous system infections or liver diseases caused by CMV, hepatitis B, blistering dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infections or neonatal infections caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus;

[0045] (3) Anti-infection (including infections caused by viruses, bacteria, fungi and parasites). Preferably, the infections include infections caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and early infections after bone marrow transplantation (such as bone marrow transplantation after radiotherapy or chemotherapy).

[0046] The seventh aspect of the present invention provides an anti-tumor, anti-viral infection, and anti-infection method, comprising the step of administering or transplanting a therapeutically effective amount of NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in the fourth aspect of the present invention or the pharmaceutical composition as described in the fifth aspect of the present invention to a subject in need thereof.

[0047] Furthermore, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer.

[0048] Furthermore, the virus is a coronavirus such as SARS-CoV-2, human HIV virus, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV or herpes zoster virus.

[0049] Furthermore, the viral infection is novel coronavirus infection (COVID-19), viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital and urinary tract infection, central nervous system infection or liver disease caused by CMV, hepatitis B, blistering dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infection or neonatal infection caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus.

[0050] Furthermore, the infection is caused by viruses, bacteria, fungi or parasites.

[0051] Furthermore, the infection includes infections caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and early infections after bone marrow transplantation.

[0052] Furthermore, the bone marrow transplant is a bone marrow transplant after radiotherapy or chemotherapy.

[0053] Compared with the prior art, the sCOTR-iNK cells and the preparation method and application thereof described in the present invention have the following advantages:

[0054] (1) The present invention simultaneously introduces specific TCR molecules and their co-receptor molecules CD8 and co-stimulatory molecules CD28 into pluripotent stem cells, and through organoid induced differentiation, sCOTR-iNK cells expressing TCR, CD8 and CD28 can be output in high yield. Pluripotent stem cells can be used to regenerate TCR-NK cells with high uniformity and unlimited sources in batches;

[0055] (2) The present invention performs genetic engineering / gene editing at the pluripotent stem cell stage and then induces NK cells in vitro, without the need to obtain materials from human tissues or undergo multiple rounds of gene transfection, which can greatly improve gene editing efficiency and reduce TCR-NK preparation costs;

[0056] (3) The present invention introduces the co-stimulatory molecule CD28 on the basis of the traditional molecular combination (CD3+TCR+CD8). Compared with NK and TCR-NK, sCOTR-iNK has stronger targeted killing ability and persistence;

[0057] (4) The TCR-NK cells obtained by the present invention can be used to treat and prevent tumors, resist viral infections, and resist infections;

[0058] (5) The pluripotent stem cell-derived NK cells of the present invention do not have endogenous TCR, and the introduction of exogenous TCR will not cause mismatching. They can be used to accurately evaluate the affinity of specific TCRs and their cell activation / toxic response capabilities, and to screen specific target antigens for new TCRs. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0060] Figure 1 Schematic diagram of the preparation strategy and activity evaluation of sCOTR-iNK;

[0061] Figure 2 The expression levels of CD3, CD8, and CD28 in primary NK cells and iNK cells were analyzed for transcriptome analysis;

[0062] Figure 3 Design diagram for piggyBac recombinant vectors of EBV TCR-GFP, CD8 co-receptor and CD28 co-stimulatory molecules;

[0063] Figure 4 The expression level diagram of EBV TCR-GFP, CD8 and CD28 molecules in EBV-sCOTR-hESC was detected by flow cytometry;

[0064] Figure 5 This is the immunophenotype diagram of EBV-sCOTR-iNK cells detected by flow cytometry;

[0065] Figure 6 This is a graph showing the expression levels of CD8α, CD8β, CD28 and CD3ε in EBV-sCOTR-iNK cells detected by flow cytometry;

[0066] Figure 7 This is the result of the activity test of EBV-sCOTR-iNK cells killing Raji-1101-EBV peptide cells in vitro;

[0067] Figure 8This is the result of the activity detection of EBV-sCOTR-iNK cells continuously killing Raji-1101-EBV peptide cells in vitro;

[0068] Fig. 9 This is a graph showing the expression levels of EBV TCR-GFP, CD8 and CD28 molecules in EBV-sCOTR-iPSC detected by flow cytometry;

[0069] Fig.10 This is the result of the activity test of iPSC-derived EBV-sCOTR-iNK cells killing Raji-1101-EBV peptide cells in vitro;

[0070] Fig.11 This is a graph showing the expression levels of (NY-ESO-1)-TCR-GFP, CD8 and CD28 molecules in (NY-ESO-1)-sCOTR-hPSC detected by flow cytometry;

[0071] Fig.12 This is a graph showing the results of the activity test of (NY-ESO-1)-sCOTR-iNK cells killing T2 cells loaded with (NY-ESO-1) antigen peptide in vitro;

[0072] Fig.13 This is a graph showing the expression levels of WT1 TCR-GFP, CD8 and CD28 molecules in WT1-sCOTR-hPSC detected by flow cytometry;

[0073] Fig.14 This is a graph showing the results of the activity test of WT1-sCOTR-iNK cells killing BV173 cells in vitro;

[0074] Fig.15 Diagram of the strategy for evaluating the anti-tumor activity of EBV-sCOTR-iNK cells in vivo;

[0075] Fig.16 This is the dynamic in vivo imaging of EBV-sCOTR-iNK cells' anti-tumor activity;

[0076] Fig.17 This is a survival curve analysis of tumor-bearing mice after EBV-sCOTR-iNK cell treatment. DETAILED DESCRIPTION

[0077] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0078] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0079] Example 1 Construction of hESCs containing specific EBV TCR, co-receptor molecule CD8 and co-stimulatory molecule CD28 expression elements (EBV-sCOTR-hESC)

[0080] TCR and CD3 molecules are expressed on the cell surface in the form of a complex. To successfully assemble TCR in NK cells, both CD3 and TCR elements need to be expressed. Unlike primary NK cells, iNK cells obtained from hPSCs through the organoid differentiation system (PMID: 36344493) express a complete set of four CD3 subunits (CD3δ, CD3γ, CD3ε, CD3ζ) (see Figure 2 ), it is speculated that the assembly and successful expression of TCR in iNK does not require the additional introduction of CD3. In order to explore whether the co-receptor molecule CD8 and the co-stimulatory molecule CD28 can enhance the function of TCR-NK, this example first designed a piggyBac vector expressing EBV TCR-GFP (TCR cannot be assembled in the hPSC stage, and GFP is used as a reporter gene; EBV TCR recognizes EBV-LMP2peptide presented by HLA-A*11:01, and the amino acid sequence is shown in SEQ ID NO.7, specifically SSCSSCPLSK), CD8, and CD28 (see Figure 3 and Table 1). The above vector elements were then introduced into hESCs by electroporation, and the genetically engineered hESCs were defined as EBV-sCOTR-hESCs. In addition, EBV TCR-hESCs obtained by transfection with EBV TCR-GFP alone were used as controls for subsequent evaluation of the enhanced functions of CD8 and CD28 molecules. Figure 4 Flow cytometry was used to detect the expression of the above exogenous molecules in EBV TCR-hESC and EBV-sCOTR-hESC. More than 99% of EBV TCR-hESC expressed EBV TCR-GFP, and more than 98% of EBV-sCOTR-hESC expressed EBV TCR-GFP, CD8 and CD28 molecules.

[0081] Table 1. Sequence information of each element of piggyBac recombination vector

[0082]

[0083]

[0084]

[0085] Example 2 Inducing EBV-sCOTR-hESC differentiation in vitro to obtain EBV-sCOTR-iNK cells

[0086] The hESCs in Example 1 were gradually induced into EBVTCR-iNK cells and EBV-sCOTR-iNK cells using the organoid differentiation system (PMID: 36344493). The generated EBV-sCOTR-iNK cells expressed EBV-TCR molecules at a rate of more than 85% (see Figure 5 ). In addition, EBV-sCOTR-iNK cells highly expressed CD3, CD8, and CD28 molecules. At the same time, EBVTCR-iNK cells also successfully assembled TCR, but lacked CD8 and CD28 expression (see Figure 6 ).

[0087] Example 3 hESC-derived EBV-sCOTR-iNK cells can enhance the activity of targeting and killing EBV-positive tumors in vitro

[0088] In order to simulate the antigen presentation process in tumor cells, we introduced HLA-A*11:01 and EBV-LMP2 peptide expression elements (amino acid sequence as shown in SEQ ID NO.7, specifically SSCSSCPLSK) into Raji cells (Raji-1101-EBV peptide cells), and then co-incubated with three different types of hESC-induced iNK cells (iNK, EBV TCR-iNK and EBV-sCOTR-iNK) for 6 hours. The results showed that EBV-sCOTR-iNK cells showed the best tumor cell cytotoxic activity (see Figure 7 ). In addition, we performed serial killing experiments using Raji-1101-EBVpeptide cells. EBV-sCOTR-iNK cells consistently showed higher cytotoxic activity than iNK and EBV TCR-iNK cells (see Figure 8 ).

[0089] Example 4 Inducing EBV-sCOTR-iPSC differentiation in vitro to obtain EBV-sCOTR-iNK cells

[0090] Using a method similar to Example 1, we introduced the piggyBac vector elements expressing EBV TCR-GFP, CD8, and CD28 into iPSC cells to construct EBV-sCOTR-iPSC, and the EBV TCR-iPSC obtained by transfecting EBV TCR-GFP alone was used as a control. Fig. 9Flow cytometry was used to detect the expression of the above exogenous molecules in EBV TCR-iPSC and EBV-sCOTR-iPSC. More than 99% of EBV TCR-iPSC expressed EBV TCR-GFP, and more than 98% of EBV-sCOTR-iPSC expressed EBV TCR-GFP, CD8 and CD28 molecules.

[0091] Example 5 iPSC-derived EBV-sCOTR-iNK cells can enhance the activity of targeting and killing EBV-positive tumors in vitro

[0092] Using a method similar to Example 2, EBV TCR-iPSC and EBV-sCOTR-iPSC were successfully induced to obtain EBVTCR-iNK and EBV-sCOTR-iNK. The three iPSC-derived iNKs (iNK, EBV TCR-iNK and EBV-sCOTR-iNK) were co-incubated with Raji-1101-EBV peptide for 6 hours. The results showed that the iPSC-derived EBV-sCOTR-iNK cells exhibited the best tumor cell cytotoxic activity (see Fig.10 ).

[0093] Example 6 Construction of hPSCs containing specific (NY-ESO-1)-TCR, co-receptor molecule CD8 and co-stimulatory molecule CD28 expression elements ((NY-ESO-1)-sCOTR-hPSCs)

[0094] Using a method similar to Example 1, the piggyBac vector elements containing (NY-ESO-1)-TCR-GFP, CD8, and CD28 were introduced into hPSC, and the nucleotide sequence of (NY-ESO-1)-sCOTR-hPSC ((NY-ESO-1)-TCR-GFP) was successfully constructed as shown in SEQ ID NO. 15, specifically

[0095]

[0096] (NY-ESO-1)-TCR recognizes (NY-ESO-1) peptide presented by HLA-A*02:01, the amino acid sequence of which is shown in SEQ ID NO.16, specifically SLLMWITQC). Fig.11Flow cytometry was used to detect the expression of the above-mentioned exogenous molecules in (NY-ESO-1)-TCR-hPSC and (NY-ESO-1)-sCOTR-hPSC. More than 99% of (NY-ESO-1)-TCR-hPSC expressed (NY-ESO-1)-TCR-GFP, and more than 99% of (NY-ESO-1)-sCOTR-hPSC expressed (NY-ESO-1)-TCR-GFP, CD8 and CD28 molecules.

[0097] Example 7 (NY-ESO-1)-sCOTR-iNK cells have a stronger ability to target and kill T2 cells loaded with (NY-ESO-1) antigen peptide in vitro

[0098] Using a method similar to Example 2, (NY-ESO-1)-TCR-hPSC and (NY-ESO-1)-sCOTR-hPSC were successfully induced to obtain (NY-ESO-1)-TCR-iNK and (NY-ESO-1)-sCOTR-iNK. T2 cells (HLA-A*02 restricted) were co-incubated with synthetic (NY-ESO-1) peptide (amino acid sequence as shown in SEQ ID NO.16, specifically SLLMWITQC) for 20 hours, and then the T2 cells loaded with (NY-ESO-1) peptide were harvested. Three different types of iNK cells (iNK, (NY-ESO-1)-TCR-iNK and (NY-ESO-1)-sCOTR-iNK) were co-incubated with T2 cells loaded with (NY-ESO-1) peptide for 4 hours. The results showed that (NY-ESO-1)-sCOTR-iNK cells showed the strongest tumor cell cytotoxicity compared with iNK and (NY-ESO-1)-TCR-iNK (see Fig.12 ).

[0099] Example 8 Construction of hPSCs containing specific WT1-TCR, co-receptor molecule CD8 and co-stimulatory molecule CD28 expression elements (WT1-sCOTR-hPSCs)

[0100] Using a method similar to Example 1, the piggyBac vector elements containing WT1 TCR-GFP, CD8, and CD28 were introduced into hPSCs, and WT1-sCOTR-hPSCs were successfully constructed (the nucleotide sequence of WT1TCR-GFP is shown in SEQ ID NO. 17, specifically

[0101]

[0102] WT1TCR recognizes the WT1 peptide presented by HLA-A*02, the amino acid sequence of which is shown in SEQ ID NO.18, specifically RMFPNAPYL). Fig.13 Flow cytometry was used to detect the expression of the above exogenous molecules in WT1 TCR-hPSC and WT1-sCOTR-hPSC. More than 98% of WT1 TCR-hPSC expressed WT1 TCR-GFP, and more than 98% of WT1-sCOTR-hPSC expressed WT1 TCR-GFP, CD8 and CD28 molecules.

[0103] Example 9 WT1-sCOTR-iNK cells have a stronger ability to target and kill WT1-positive tumors in vitro

[0104] Using a method similar to Example 2, WT1 TCR-hPSC and WT1-sCOTR-hPSC were successfully induced to obtain WT1TCR-iNK and WT1-sCOTR-iNK. BV173 cells (WT1 positive, HLA-A*02 restricted) were co-incubated with three different types of iNK cells (iNK, WT1 TCR-iNK and WT1-sCOTR-iNK) for 6 hours. The results showed that compared with iNK and WT1 TCR-iNK, WT1-sCOTR-iNK cells showed stronger tumor cell cytotoxic activity (see Fig.14 ).

[0105] Example 10 EBV-sCOTR-iNK cells can effectively kill EBV-positive tumor cells in vivo and prolong the life span of tumor-bearing mice

[0106] On Day-1, Raji-1101-EBV peptide cells expressing luciferase (Raji-1101-EBV peptide-LUC, 1×10 5 The human tumor mouse model was constructed by injecting iNK or EBV sCOTR iNK cells (5×10 cells / mouse) into the tail vein of the tumor model mice on Day 0, Day 3, and Day 6. 6 cells / mouse) and weekly intravital imaging was performed to assess tumor burden (see Fig.15 In vivo imaging results showed that EBV-sCOTR-iNK cells exhibited significantly stronger tumor-killing ability in vivo than iNK cells (see Fig.16 Mice treated with EBV-sCOTR-iNK cells had a longer median survival time than mice treated with iNK cells or PBS (PBS: 24 days; iNK: 25 days; EBV-sCOTR-iNK: 45 days; p<0.001) (see Fig.17 ).

[0107] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A stem cell expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, characterized in that: The stem cells include human embryonic stem cells, human induced pluripotent stem cells or chemically reprogrammed induced pluripotent stem cells.

2. The method for preparing stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to claim 1, characterized in that: The following steps are involved: (1) constructing a recombinant vector containing a specific TCR molecule and its co-receptor molecule CD8 and co-stimulatory molecule CD28; (2) Introducing the recombinant vector constructed in step (1) into stem cells.

3. The method for preparing stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to claim 2, characterized in that: The introduction method in step (2) includes lentiviral infection, retroviral infection, adenoviral infection, Sendai virus infection, electrofection or chemical transfection, preferably electrofection.

4. A method for preparing NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, characterized in that: The NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 are obtained by inducing differentiation of the stem cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in claim 1.

5. The method for preparing NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to claim 4, characterized in that: The differentiation induction method includes a monolayer induction method, an embryoid body induction method or an organoid induction method.

6. A NK cell expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28, characterized in that: The NK cells are prepared by the method for preparing NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in claim 4 or 5.

7. A pharmaceutical composition, characterized in that: It comprises the NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in claim 6, and a carrier or excipient.

8. Use of the stem cell expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to claim 1, the NK cell expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 according to claim 6, or the pharmaceutical composition according to claim 7 in the preparation of a drug, wherein the drug is used for: (1) Anti-tumor, preferably, the tumor is a blood tumor or a solid tumor, for example, leukemia, lymphoma, breast cancer, ovarian cancer, liver cancer, glioma or pancreatic cancer; (2) Antiviral (e.g., coronaviruses such as SARS-CoV-2, human HIV, rhinovirus, cytomegalovirus CMV, hepatitis B virus HBV, herpes simplex virus HSV, herpes zoster virus) infections, for example, novel coronavirus infection (COVID-19), viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital and urinary tract infections, central nervous system infections or liver diseases caused by CMV, hepatitis B, blistering dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infections or neonatal infections caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus; (3) Anti-infection (including infections caused by viruses, bacteria, fungi and parasites). Preferably, the infections include infections caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and early infections after bone marrow transplantation (such as bone marrow transplantation after radiotherapy or chemotherapy).

9. An anti-tumor, anti-viral infection, and anti-infection method, characterized in that: The method comprises the steps of administering or transplanting a therapeutically effective amount of the NK cells expressing TCR and its co-receptor molecule CD8 and co-stimulatory molecule CD28 as described in claim 6 or the pharmaceutical composition as described in claim 7 to a subject in need thereof.

Images