INKP cell for expressing CXCR4 as well as preparation method and application of iNKP cell

Abstract

The invention provides an iNKP cell (CXCR4-iNKP, R4-iNKP) for expressing CXCR4, and a preparation method and an application of the iNKP cell (CXCR4-iNKP, R4-iNKP). According to the invention, CXCR4 is expressed in stem cells, CXCR4-iNKP is induced, and the induced CXCR4-iNKP can efficiently differentiate NK cells in a mouse body. Specifically, the problems that in the NK cell therapy, large-dose infusion is needed, and the continuous existence time of the NK cells is short are innovatively solved. After the stem cell induced NK progenitor cells are infused into the body, the stem cell induced NK progenitor cells can be efficiently differentiated into the NK cells, and the NK cells can be output for a long time. Compared with the NK infusion dosage, the R4-iNKP infusion dosage is obviously reduced, the in-vivo output efficiency is high, the distribution is wide, and meanwhile, the NK cells generated after R4-iNKP infusion can exist in circulating blood for more than 80 days. In conclusion, the invention provides a new thought for the application of NK cell therapy.

Description

Technical Field

[0001] The present invention belongs to the field of stem cell biology, and in particular relates to an iNKP cell (induced NK progenitor cell) expressing CXCR4, and a preparation method and application thereof. Background Art

[0002] NK cells have a short persistence time in the body and require repeated large-dose infusions, which is costly. Under physiological conditions, the renewal time of NK cells in peripheral blood is about two weeks. Even after the autoimmune-compatible NK cells are reinfused, they can only exist in the peripheral blood for about two weeks. In clinical trials, the MD Anderson Cancer Center chose to use IL-15 to enhance the persistence of CD19 CAR-NK cells. Although the detection time of CD19 CAR at the molecular level can reach hundreds of days, the specific existence time of CAR-NK cells in the body at the cytological level is still unclear. The current infusion dose of NK cells has reached 10 7 / kg-10 9 / kg, among which NKaTa is using CD19 CAR-NK cells to treat B-cell tumors in a clinical trial (NCT05020678), with a single infusion dose of 1.5 billion and a total dose of 4.5 billion. Therefore, extending the in vivo duration of NK cells after infusion remains a core scientific issue that needs to be urgently addressed in the field of NK cell therapy. Currently, NK cells used for clinical treatment are mainly derived from peripheral blood or umbilical cord blood, etc., which have cumbersome processing procedures, large heterogeneity of preparations, low efficiency of multiple gene editing or genetic engineering modification, and high cost, making it difficult to achieve spot supply. Pluripotent stem cells (PSC) include embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC), which have the advantages of easy gene editing and unlimited sources of cloning, so NK cells derived from pluripotent stem cells are easier to achieve "spot" supply. Although NK cells derived from pluripotent stem cells have entered the clinical trial stage, the problem of short duration of NK cells still exists.

[0003] CXC chemokine receptor type 4 (CXCR-4) is a type of chemokine receptor that is widely expressed in blood cells, especially in T cells and NK cells and their corresponding progenitor cells. It can promote the migration, differentiation and activity of blood cells. However, existing data show that hematopoietic stem cells and NK cells derived from induced pluripotent stem cells do not express CXCR4. In summary, NK cell therapy has a short duration in vivo and requires multiple large-dose infusions, which is an important problem that needs to be solved in this field. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide an iNKP cell expressing CXCR4 and a preparation method and application thereof. Specifically, the present invention expresses CXCR4 in PSC, which is beneficial to the migration and chemotaxis of immune cells. The modified PSC is induced into a progenitor cell of NK cells, which can produce NK cells in vivo for a long time, thereby solving the problem of short duration after NK cell infusion.

[0005] In a first aspect of the present invention, a stem cell expressing CXCR4 is provided, characterized in that the stem cell comprises human embryonic stem cells (ESC), induced pluripotent stem cells (iPSC, CiPSC), mesoderm cells, induced hematopoietic stem progenitor cells or induced NK progenitor cells (iNKP).

[0006] Furthermore, the amino acid sequence of CXCR4 is shown in SEQ ID NO.1.

[0007] Furthermore, the stem cells are human embryonic stem cells (ESC) or induced NK progenitor cells (iNKP).

[0008] In a second aspect of the present invention, a method for preparing stem cells expressing CXCR4 according to the first aspect of the present invention is provided, characterized in that the method comprises the following steps:

[0009] (1) constructing a recombinant vector containing the CXCR4 gene;

[0010] (2) transfecting the recombinant vector constructed in step (1) into stem cells;

[0011] (3) Isolate stem cells expressing CXCR4.

[0012] Furthermore, the nucleotide sequence of the CXCR4 gene is shown in SEQ ID NO.2.

[0013] Furthermore, the recombinant vector in step (1) is a piggyBac recombinant vector containing the CXCR4 gene.

[0014] Furthermore, the transfection method in step (2) includes pseudovirus infection, chemical transfection, and electroporation, preferably electroporation.

[0015] Furthermore, the sorting in step (3) is performed using a flow cytometer.

[0016] Furthermore, after the stem cells are introduced with CXCR4, the proportion of cells expressing CXCR4 is 10%-100%.

[0017] In the third aspect of the present invention, a method for preparing induced NK progenitor cells (iNKP) expressing CXCR4 is provided, characterized in that the induced NK progenitor cells (iNKP) expressing CXCR4 are obtained by induced culture of human embryonic stem cells (ESC), induced pluripotent stem cells or mesoderm cells expressing CXCR4 as described in the first aspect of the present invention.

[0018] Furthermore, the induced culture adopts an organoid culture method.

[0019] Furthermore, the induction culture specifically comprises the following steps:

[0020] (1) inducing and culturing the human embryonic stem cells (ESCs) expressing CXCR4 in culture medium I and culture medium II in sequence to obtain lateral plate mesoderm cells (CXCR4-LPM) expressing CXCR4;

[0021] (2) Inducing and culturing the lateral plate mesoderm cells expressing CXCR4 obtained in step (1) and trophoblast cells in culture medium III to obtain the induced NK progenitor cells (iNKP) expressing CXCR4.

[0022] Furthermore, the culture medium I is a basal culture medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, ACTIVIN, bFGF, a Wnt agonist and a PI3K inhibitor.

[0023] Furthermore, the culture medium I is an Essential 6 culture medium supplemented with the following substances: BMP4, ACTIVIN A, bFGF, CHIR-99021 and PIK-9; preferably, the Essential 6 culture medium contains 40 ng / mL BMP4, 30 ng / mL ACTIVIN A, 20 ng / mL bFGF, 6 μM CHIR-99021 and 100 nM PIK-90.

[0024] Furthermore, the culture medium II is a basal culture medium comprising the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and BMP4, a TGF-β inhibitor, and a Wnt inhibitor.

[0025] Furthermore, the culture medium II is Essential 6 culture medium supplemented with the following substances: BMP4, A-83-01 and C59; preferably, Essential 6 culture medium containing 30 ng / mL BMP4, 1 μM A-83-01 and 1 μM C59.

[0026] Furthermore, the culture medium III is a basic culture medium supplemented with one or more of the following substances: one or more serum replacements, one or more non-essential amino acids, stabilized dipeptide of glutamine or L-alanyl-L-glutamine, TGF-β inhibitor, hydrocortisone, ascorbic acid, and cell growth factors including IL-3 and IL-5.

[0027] Furthermore, the medium III is an Essential 6 medium supplemented with the following substances: one or more serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, SB431542, hydrocortisone, ascorbic acid, and cell growth factors including IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; preferably, the Essential 6 medium supplemented with the following substances: SB431542, hydrocortisone, ascorbic acid, IL-3, IL-5, Flt3L, TPO, SCF, EGF, VEGF, bFGF and IGF-1; preferably Essential 6 medium containing 10μM SB431542, 10μM hydrocortisone, 5ng / mL Flt3L, 5ng / mL TPO, 50ng / mL SCF, 50ng / mL EGF, 50ng / mL VEGF, 50ng / mL bFGF, 50ng / mL IGF-1, 5ng / mL IL-3, 10ng / mL IL-15, 50μg / mL ascorbic acid and 2% SUPERGROW Cell Culture Supplement.

[0028] Furthermore, the induction culture time in step (1) is 1-3 days, preferably 2 days.

[0029] Furthermore, in step (1), the lateral plate mesoderm cells are obtained by inducing culture in culture medium I and culture medium II for 1-2 days respectively, for example, inducing culture in culture medium I for 1 day and inducing culture in culture medium II for 1 day.

[0030] Furthermore, in step (2), the trophoblast cells are selected from one or more of AFT024 cells, OP9 cells, MS5 cells, HS-5 cells or other fibroblasts, preferably OP9 cells.

[0031] Furthermore, in step (2), the ratio of the lateral plate mesoderm cells expressing CXCR4 to the trophoblast cells is 1:5-1:200, for example, 1:15, 1:10, 1:20, 1:30, 1:40, 1:60, 1:90, 1:100, 1:110, 1:120, 1:130, 1:140, 1:150, 1:160, 1:170, 1:180, 1:190 or 1:200, preferably 1:20.

[0032] Furthermore, the induction culture time in step (2) is 13-23 days, preferably 19 days.

[0033] Furthermore, the immunophenotype of the induced NK progenitor cells (iNKP) expressing CXCR4 is: CD45 + CD56 - CD34 + CXCR4 + (R4-iNKP).

[0034] In the fourth aspect of the present invention, an induced NK progenitor cell (iNKP) expressing CXCR4 is provided, characterized in that it is prepared by the preparation method of the induced NK progenitor cell (iNKP) expressing CXCR4 described in the third aspect of the present invention.

[0035] In the fifth aspect of the present invention, a method for preparing induced NK cells (iNK) expressing CXCR4 is provided, characterized in that the induced NK progenitor cells (iNKP) expressing CXCR4 described in the fourth aspect of the present invention are cultured in a bioreactor to separate the induced NK cells (iNK) expressing CXCR4.

[0036] Furthermore, the bioreactor is a mammal, such as a mouse, a rat, a pig, or a non-human primate.

[0037] In the sixth aspect of the present invention, an induced NK cell (iNK) expressing CXCR4 is provided, characterized in that it is prepared by the preparation method of the induced NK cell (iNK) expressing CXCR4 described in the fifth aspect of the present invention.

[0038] In the seventh aspect of the present invention, a pharmaceutical composition is provided, characterized in that it comprises the induced NK progenitor cells (iNKP) expressing CXCR4 described in the fourth aspect of the present invention, the induced NK cells (iNK) expressing CXCR4 described in the sixth aspect of the present invention, and a carrier or excipient.

[0039] Furthermore, the pharmaceutical composition is an injection, a microneedle, a mucosal patch, an enema, a suppository, a gel, an oral agent, an aerosol, a drop, an ointment, an implant, a capsule or an aerosol.

[0040] In the eighth aspect of the present invention, there is provided a use of the stem cell expressing CXCR4 according to the first aspect of the present invention, the induced NK progenitor cell (iNKP) expressing CXCR4 according to the fourth aspect of the present invention, the induced NK cell (iNK) expressing CXCR4 according to the sixth aspect of the present invention, or the pharmaceutical composition according to the seventh aspect of the present invention in the preparation of a drug, wherein the drug is used for:

[0041] (1) 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);

[0042] (2) 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;

[0043] (3) 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;

[0044] (4) anti-aging; or

[0045] (5) Treatment of NK cell-related immunodeficiency diseases, such as primary NK cell deficiency, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11 / CD18 deficiency) or severe combined immunodeficiency (SCID).

[0046] In the ninth aspect of the present invention, a method for anti-infection, anti-tumor, anti-viral infection, anti-aging or treatment of NK cell-related immunodeficiency diseases is provided, which comprises the step of administering or transplanting a therapeutically effective amount of the induced NK progenitor cells (iNKP) expressing CXCR4 described in the fourth aspect of the present invention, the induced NK cells (iNK) expressing CXCR4 described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention to a subject in need thereof.

[0047] Furthermore, the infection is caused by viruses, bacteria, fungi or parasites.

[0048] Furthermore, the infection is infection caused by various pathogens (such as viruses, bacteria, fungi or parasites) after radiotherapy and chemotherapy, and infection after bone marrow transplantation.

[0049] Furthermore, the bone marrow transplant is a bone marrow transplant after radiotherapy or chemotherapy.

[0050] 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.

[0051] 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.

[0052] Furthermore, the viral infection is novel coronavirus infection (COVID-19), viral pneumonia, AIDS, acute respiratory disease caused by rhinovirus, genital and urinary system infection, central nervous system infection or liver disease caused by CMV, hepatitis B, vesicular dermatitis, gingivostomatitis, keratoconjunctivitis, encephalitis, reproductive system infection or neonatal infection caused by HSV, or chickenpox, encephalomyelitis or conjunctivitis caused by herpes zoster virus.

[0053] Furthermore, the NK cell-related immunodeficiency disease is a primary NK cell deficiency disease, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11 / CD18 deficiency) or severe combined immunodeficiency (SCID).

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0055] Compared with the prior art, the present invention has the following advantages and progress:

[0056] The present invention expresses CXCR4 in stem cells and induces CXCR4-iNKP, and the induced CXCR4-iNKP can efficiently differentiate into NK cells in mice and can persist in peripheral blood for more than 80 days. Specifically, the present invention innovatively solves the problem of large-dose infusion required in NK cell therapy. After infusion into the body, NK progenitor cells derived from stem cell induction can efficiently differentiate into NK cells. The infusion dose of R4-iNKP (1-5×10 5 / ) is lower than the NK infusion dose (100 times lower), has a high in vivo output efficiency (quoted from the contribution rate of NK infusion one week / two weeks later), and is widely distributed (various tissues and organs, including but not limited to bone marrow, lymph nodes, spleen, thymus, liver and lungs). At the same time, the present invention also solves the problem of the short duration of NK cells in NK cell therapy. After NK progenitor cells derived from stem cell induction are infused into the body, NK cells can be output for a long time. R4-iNKP infusion solves the problem of short duration after NK cell infusion. The NK cells produced after R4-iNKP infusion exist in the circulating blood for more than 80 days. In summary, the iNKP cells expressing CXCR4 provided by the present invention, and their preparation method and application, provide a new idea for the application of NK cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The accompanying drawings, which constitute 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 improper limitations on the present invention.

[0058] Figure 1 The overall process of preparing NK cell progenitors (iNKP) using PSCs and using iNKPs is shown.

[0059] Figure 2 The expression of introduced CXCR4 in ESCs is shown (detected by flow cytometry).

[0060] Figure 3 The flow cytometric phenotype of CXCR4-expressing ESCs (R4-ESCs) induced into CXCR4-iNKP cells (R4-iNKPs) is shown.

[0061] Figure 4 The phenotype of NK cells differentiated from R4-iNKP was shown after R4-iNKP was infused into mice and detected in the peripheral blood in large quantities.

[0062] Figure 5 It was shown that after R4-iNKP expression was infused into mice, differentiated cells could be distributed to the bone marrow, lung, spleen, liver and other parts.

[0063] Figure 6The phenotype of NK cells differentiated from R4-iNKP detected in the bone marrow, lung, spleen and liver after R4-iNKP was infused into mice is shown.

[0064] Figure 7 The figure shows the statistical graph of the proportion of NK cells differentiated by R4-iNKP detected in the peripheral blood at different time points after R4-iNKP was infused into mice.

[0065] Figure 8 The figure shows the statistical graph of the proportion of NK cells differentiated by R4-iNKP detected in the peripheral blood at different time points after R4-iNKP was infused into mice.

[0066] Fig. 9 It was shown that NK cells differentiated by R4-iNKP in mice expressed genes related to natural NK cells, such as those encoding classical activating receptors, inhibitory receptors, and cytotoxic effector molecules.

[0067] Fig.10 It was shown that NK cells differentiated by R4-iNKP in mice were able to kill K562 cells (chronic myeloid leukemia cell line) in vitro. DETAILED DESCRIPTION

[0068] The present invention provides an iNKP cell expressing CXCR4 (CXCR4-iNKP, R4-iNKP) and a preparation method and application thereof. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in this field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0069] Example 1 Construction of ESC expressing CXCR4 (R4-ESC)

[0070] TM Medium, A1517001, Gibco brand) to obtain ESCs expressing CXCR4 (CXCR4-ESC, R4-ESC). Figure 2 , the proportion of R4-ESC expressing CXCR4 was 99.4% as detected by flow cytometry.

[0071] Example 2 Inducing R4-expressing ESCs into NKP cells

[0072] R4-ESC and ESC (as a control that does not express CXCR4) were first induced into NKP cells using the organoid culture method. The specific operation was to first use culture medium I and II for two days to induce (specifically, first use culture medium I for one day and then use culture medium II for one day) into lateral plate mesoderm cells (CXCR4-LPM). In this embodiment, the composition of culture medium I is: Essential 6 culture medium containing 40ng / mL BMP4, 30ng / mL ACTIVIN A, 20ng / mL bFGF, 6μM CHIR-99021 and 100nM PIK-90. The composition of culture medium II is: Essential 6 culture medium containing 30ng / mL BMP4, 1μM A-83-01 and 1μM C59. CXCR4-LPM was mixed with OP9 cells (CRL-2749, ATCC) to prepare organoids, and the mixing ratio of the two cells was 1:20. After the organoids were inoculated into the transwell culture plate, they were induced using medium III. In this example, the composition of medium III was: Essential 6 medium containing 10μM SB431542, 10μM hydrocortisone, 5ng / mL Flt3L, 5ng / mL LTPO, 50ng / mL SCF, 50ng / mL EGF, 50ng / mL VEGF, 50ng / mL bFGF, 50ng / mL IGF-1, 5ng / mL IL-3, 10ng / mL IL-15, 50μg / mL ascorbic acid and 2% SUPERGROW cell culture supplement. On the 19th day of culture, R4-iNKP cells and control group iNKPs were collected and the flow cytometry phenotype was detected. See Figure 3 Flow cytometry was used to detect iNKP cells and R4-iNKP cells. The immunophenotype of iNKP cells was CD45 + CD34 + CD56 - The immunophenotype of R4-iNKP cells is CD45 + CD34 + CD56 - CXCR4 + .

[0073] Example 3 After the CXCR4-iNKP expressing cells were infused into mice, NK cells were detected in the peripheral blood

[0074] The immunophenotype of R4-iNKP cells (CD45 + CD34 + CD56 - CXCR4 + ) and iNKP cells (CD45 + CD34 + CD56 - ) using flow cytometry (BD FACSAria TM Fusion (BD Biosciences)) and the two types of cells were infused into irradiated (1.5 Gy) immunodeficient mice (B-NDGhIL15) expressing human IL-15 (hIL-15) through the tail vein at a volume of 2 × 10 per mouse. 5 cells. 14 days after the infusion, peripheral blood of mice was collected through the ophthalmic vein. After the red blood cells were lysed, the cells were stained with human CD45 (clone number: HI30, Biolegend brand), CD3 (clone number: UCHT1, Biolegend brand), CD56 (clone number: HCD56, Biolegend brand), CD16 (clone number: 3G8, Biolegend brand) and CXCR4 antibodies (clone number: 12G5, Biolegend brand). The specific operation is to first use ACK red blood cell lysis buffer (Cat. No.: A1049201, Gibcop brand) to lyse the red blood cells in the peripheral blood, centrifuge to remove the red blood cell fragments after lysis, resuspend the cell pellet with PBS, and add the above-mentioned flow cytometry antibody (the amount of antibody is used according to the instructions), incubate on ice for 15 minutes, centrifuge to remove excess antibodies, resuspend the cells with DAPI solution, and use a flow cytometer (BDLSRFortessa TM (BD Biosciences)) conducted an analysis and found that R4-iNKP-derived NK cells were detected in the peripheral blood of recipient mice infused with R4-iNKP cells. However, NK cells were almost undetectable in the peripheral blood of recipient mice infused with iNKP. Figure 4, flow cytometry was used to detect the immune phenotype and proportion of peripheral blood NK cells in mice infused with R4-iNKP cells and mice infused with iNKP cells. A large number of NK cells were present in mice infused with R4-iNKP cells, and they expressed CD45, CD56 and / or CD16, some of them expressed CXCR4, and did not express CD3 (T cell-associated protein). A very small number of NK cells were present in mice infused with iNKP cells, and they expressed CD45, CD56 and / or CD16, and did not express CD3 (T cell-associated protein).

[0075] Example 4: After infusing mice expressing R4-iNKP, the distribution of CXCR4-iNKP and differentiated cells in tissues and organs was tracked using in vivo imaging technology

[0076] 5 Cells were detected by in vivo imaging technology to detect the distribution of CXCR4-iNKP and differentiated cells in tissues and organs. Figure 5 In vivo imaging of these recipient mice in different body positions (supine, prone, left lateral decubitus) was performed on the 5th, 8th, 11th, 14th, 21st, and 28th days after infusion of iNKP-luci and R4-iNKP-luci. The results showed that R4-iNKP-luci and its differentiated cells were distributed in the mouse bone marrow, liver, spleen, lung and other organs. However, iNKP-luci could hardly colonize and differentiate in mice.

[0077] Example 5 R4-iNKP can detect NK cells in the mouse bone marrow, liver, spleen and lung using flow cytometry

[0078] After the R4-iNKP cells were sorted, they were infused into irradiated (1.5 Gy) B-NDG hIL15 cells at a volume of 2 × 10 per mouse. 5 After 21 days of infusion, the mice were euthanized by cervical dislocation, and the bone marrow, lungs, spleen and liver of the mice were separated and ground into single cell suspensions. The cells were stained with human CD45, CD56 and CD16 antibodies, and analyzed by flow cytometry. NK cells derived from R4-iNKP were detected in the above tissues and organs. Figure 6 , flow cytometry was used to detect the expression of CD45, CD56 and / or CD16 in R4-iNKP differentiated NK cells in the bone marrow, liver, spleen and lung of mice.

[0079] Example 6 After the expression of R4-iNKP was infused into mice, the proportion of NK cells differentiated by R4-iNKP in peripheral blood was detected at different time points

[0080] After the R4-iNKP cells were sorted, they were infused into irradiated (1.5 Gy) IL-15 humanized immunodeficient mice (B-NDG hIL15) at a volume of 2 × 10 per mouse. 5 On the 14th, 21st and 28th day after infusion, peripheral blood of mice was collected through the ophthalmic vein, and the red blood cells were lysed and stained with human CD45, CD56 and CD16 antibodies. After staining, the cells were analyzed by flow cytometry and the proportion of NK cells derived from R4-iNKP detected in the peripheral blood was counted. Figure 7The proportion of NK cells in the peripheral blood of R4-iNKP-infused mice (3 mice) at each time point was statistically analyzed. The average proportion on the 14th day was 44.7%, the average proportion on the 21st day was 40.3%, and the average proportion on the 28th day was 25.2%.

[0081] After the R4-iNKP cells were sorted, they were infused into irradiated (1.5 Gy) IL-15 humanized immunodeficient mice (B-NDG hIL15) at a volume of 2 × 10 per mouse. 5 On the 38th, 45th, 52nd and 80th day after infusion, peripheral blood of mice was collected through the ophthalmic vein, and after lysing the red blood cells, the cells were stained with human CD45, CD56 and CD16 antibodies. After staining, the cells were analyzed by flow cytometry, and the proportion of NK cells derived from R4-iNKP detected in the peripheral blood was counted. Figure 8 The proportion of NK cells in the peripheral blood of R4-iNKP-infused mice (3 mice) at each time point was statistically analyzed. The average proportion on the 38th day was 7.4%, the average proportion on the 45th day was 5.8%, the average proportion on the 52nd day was 7.4%, and the average proportion on the 80th day was 0.9%.

[0082] Example 7 Single-cell sequencing of NK cells differentiated from R4-iNKP

[0083] Single-cell sequencing technology (10x genomics) was used to sequence NK cells differentiated from R4-iNKP in peripheral blood (PB), bone marrow (BM), liver (Liver), lung (Lung) and spleen (Spleen) (the data has been uploaded to the China Omics Raw Data Archive, data number: HRA008662). After sequencing, the expression of NK cell classic activating receptor encoding genes, inhibitory receptor encoding genes, and killing effector molecule-related genes was analyzed. Fig. 9, Violin plots were used to show the expression of genes, and the vertical axis showed the expression value. PB-NK, BM-NK, Liver-NK, Lung-NK and Spleen-NK cells all expressed genes encoding activating receptors: NCAM1 (encoding CD56), FCGR3A (encoding CD16), NCR1 (encoding NKp46), NCR3 (encoding NKp30), KLRK1 (encoding NKG2D), CD69, KLRC2 (encoding NKG2C), KLRC3 (encoding NKG2E), SLAMF7 (encoding CD319), CD226, CD244; genes encoding inhibitory receptors: KLRB1 (encoding CD161), KLRC1 (encoding NKG2A), KLRD1 (encoding CD94); NK cell function regulatory molecules: CD96, cytotoxic effector molecules: GZMB, PRF1 (encoding Perforin), GNLY, FASLG (encoding CD178).

[0084] Example 8 NK cells differentiated from R4-iNKP in vivo have the ability to kill tumor cells (K562) in vitro

[0085] B-NDG hIL15 mice were taken 21 days after infusion of R4-iNKP, and NK cells were taken out after being killed by cervical dislocation. NK cells (Effector, E) and tumor cells (Target, T) were co-incubated at different ratios (E:T=0.2:1, 0.4:1, 0.8:1, 1.6:1), and 4 technical replicates were set for each E / T. After 4 hours of co-incubation, the killing efficiency (Cytotoxicity%) of NK cells at different ratios against tumor cells was detected. Fig.10 The results show that when E:T=0.5:1, the average killing efficiency is 14.9%, when E:T=1:1, the average killing efficiency is 25.4%, when E:T=2.5:1, the average killing efficiency is 31.1%, and when E:T=5:1, the average killing efficiency is 34.3%.

[0086] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A stem cell expressing CXCR4, characterized in that: The stem cells include human embryonic stem cells (ESCs), induced pluripotent stem cells, mesodermal cells or induced hematopoietic stem progenitor cells.

2. The method for preparing stem cells expressing CXCR4 according to claim 1, characterized in that: The steps include: (1) constructing a recombinant vector containing the CXCR4 gene; (2) transfecting the recombinant vector constructed in step (1) into stem cells; (3) Isolate stem cells expressing CXCR4.

3. A method for preparing induced NK progenitor cells (iNKP) expressing CXCR4, characterized in that: The induced NK progenitor cells (iNKP) expressing CXCR4 are obtained by inducing and culturing the human embryonic stem cells (ESC), induced pluripotent stem cells or mesodermal cells expressing CXCR4 as claimed in claim 1.

4. The preparation method according to claim 3, characterized in that: The induction culture adopts an organoid culture method, which specifically includes the following steps: (1) inducing and culturing the human embryonic stem cells (ESCs) expressing CXCR4 in culture medium I and culture medium II in sequence to obtain lateral plate mesoderm cells (CXCR4-LPM) expressing CXCR4; (2) Inducing and culturing the lateral plate mesoderm cells expressing CXCR4 obtained in step (1) and trophoblast cells in culture medium III to obtain the induced NK progenitor cells (iNKP) expressing CXCR4.

5. An inducible NK progenitor cell (iNKP) expressing CXCR4, characterized in that: The induced NK progenitor cells (iNKP) expressing CXCR4 are prepared by the preparation method of claim 3 or 4.

6. A method for preparing induced NK cells (iNK) expressing CXCR4, characterized in that: The induced NK progenitor cells (iNKP) expressing CXCR4 according to claim 5 are cultured in a bioreactor to isolate the induced NK cells (iNK) expressing CXCR4.

7. An inducible NK cell (iNK) expressing CXCR4, characterized in that The induced NK cells (iNK) expressing CXCR4 are prepared by the preparation method of claim 6.

8. A pharmaceutical composition, characterized in that The method comprises the induced NK progenitor cell (iNKP) expressing CXCR4 according to claim 5 and / or the induced NK cell (iNK) expressing CXCR4 according to claim 7, and a carrier or an excipient.

9. Use of the stem cell expressing CXCR4 according to claim 1, the induced NK progenitor cell (iNKP) expressing CXCR4 according to claim 5, the induced NK cell (iNK) expressing CXCR4 according to claim 7 or the pharmaceutical composition according to claim 8 in the preparation of a drug, wherein the drug is used for: (1) 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); (2) 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; (3) 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; (4) anti-aging; or (5) Treatment of NK cell-related immunodeficiency diseases, such as primary NK cell deficiency, Chédiak-Higashi syndrome, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency (CD11 / CD18 deficiency) or severe combined immunodeficiency (SCID).

10. A method for anti-infection, anti-tumor, anti-viral infection, anti-aging or treatment of NK cell-related immunodeficiency diseases, comprising the step of administering or transplanting a therapeutically effective amount of the induced NK progenitor cells (iNKP) expressing CXCR4 according to claim 5, the induced NK cells (iNK) expressing CXCR4 according to claim 7, or the pharmaceutical composition according to claim 8 to a subject in need thereof.

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