CD19-targeting car-NK cell, preparation method therefor, and use thereof

Abstract

The present invention relates to a CD19-targeting CAR-NK cell, a preparation method therefor and the use thereof. The method comprises: introducing into CD34+ HSPC a nucleic acid encoding a CD19-targeting chimeric antigen receptor, mixing stromal cell with the cell after the introduction, and coculturing same by means of using an induction and amplification culture medium, so as to obtain CD19 CAR-NK precursor cells; and the CD19 CAR-NK precursor cells continuing undergoing induction and amplification culture to obtain mature CD19 CAR-NK cells. Provided is a novel method for inducing a CAR-NK cell on the basis of hematopoietic stem and progenitor cells, which greatly reduces the cost of genetic engineering or gene editing used in delivering a CAR element, and efficiently induces the production of CAR-NK cells, thus improving the efficiency of outputting CAR-NK cells. The CAR-NK cells obtained by the induction can efficiently target and kill tumor cells expressing a corresponding antigen and thus possess a broad application prospect.

Description

A CD19-targeting CAR-NK cell, its preparation method and application Technical Field

[0001] This application belongs to the field of stem cell biology and immune cell therapy technology, and relates to a CD19-targeting CAR-NK cell, its preparation method and application. Background Technology

[0002] Discovered in the early 1970s, NK (natural killer) cells were the first type of innate lymphoid cells (ILCs) to be identified. They can be found in various tissues and organs, such as bone marrow, lungs, thymus, lymph nodes, peripheral blood, spleen, liver, kidneys, and endometrium. NK cells account for 5%-20% of human peripheral blood lymphocytes. They can kill abnormal cells (such as tumor cells, pathogen-infected cells, and senescent cells) by releasing cytotoxic granules (granzyme B and perforin), activating apoptosis pathways, or through antibody-dependent cell-mediated cytotoxicity (ADCC). They can also produce various cytokines or chemokines to play an immunomodulatory role.

[0003] NK cell therapy can broadly kill a variety of tumor cells, has low toxicity and side effects, and does not require HLA matching. NK or chimeric antigen receptor (CAR) NK cell (CAR-NK) preparations have been widely used in clinical research and have achieved significant efficacy. For example, one study reported the use of retroviral vectors to introduce CD19 CAR into umbilical cord blood NK cells. Clinical results showed that among 11 patients, 8 achieved objective remission, of which 7 (3 patients with chronic B-lymphoblastic leukemia and 4 patients with B-lymphoma) achieved complete remission, and no toxic side effects caused by NK cell infusion were observed.

[0004] Because NK cells require high doses and multiple infusions to exhibit significant therapeutic effects, the cost of directly producing CAR-NK cells in large quantities from natural NK cells is extremely high. Currently, most clinically used natural NK cells are directly isolated from peripheral blood or umbilical cord blood, which carries the risks of high heterogeneity and T cell contamination. Inducing regenerative NK cells through in vitro induction of stem cells (hematopoietic stem cells (HPSCs) or pluripotent stem cells) holds promise for improving homogeneity and significantly reducing the production cost of CAR-NK cells. Some studies have utilized lentiviral transduction to deliver CAR elements to CD34. + After HSPC, cells can differentiate into targeted CD19 CAR-NK cells, but their induction efficiency has not been reported. Some studies have used CD19 CAR-NK lentivirus to transduce CD34 cells.+ After HSPC, transplantation into NSG severely immunodeficient mice yielded CD19 CAR-NK cells through in vivo differentiation. However, other CD19 CAR-positive blood lineage cells (such as T, B, and Myeloid) were also present, posing certain risks in real-world clinical applications. Furthermore, some studies have used CD19 CAR-NK retroviruses to transduce CD34 cells from the placenta. + HSPCs were subsequently differentiated in vitro for 35 days to obtain CD19 CAR-NK cells, which were then injected with single CD34 CAR-NK cells. + HSPC yielded only 6186±2847 cells, of which NK cells accounted for 93.8%±3.9%. To date, there have been no reports of more efficient artificial hematopoietic stem cell-induced CAR-NK cell technology.

[0005] In summary, the high cost and low induction efficiency severely limit the widespread clinical application of CAR-NK cells. Therefore, it is urgent to develop a technology that can efficiently induce stem cell differentiation to obtain high-purity, highly toxic CAR-NK cells, thereby further promoting the accessibility of CAR-NK cell drugs.

[0006] Summary of the Invention

[0007] This application provides a CD19-targeted CAR-NK cell (CD19 CAR-NK cell), its preparation method, and its application, enabling efficient induction of stem cell differentiation to obtain high-purity, highly toxic CD19 CAR-NK cells, and promoting the application of CAR-NK cells.

[0008] In a first aspect, this application provides a method for in vitro induction and preparation of CD19 CAR-NK cells, the method comprising:

[0009] To CD34 + The nucleic acid encoding a chimeric antigen receptor (CD19 CAR) targeting CD19 is introduced into HSPC, and the cells after introduction (CD19 CAR CD34) + HSPCs were mixed with stromal cells and co-cultured in an induction expansion medium to obtain CD19 CAR-NK precursor cells. The CD19 CAR-NK precursor cells were then further induced and expanded to obtain mature CD19 CAR-NK cells. The stromal cells included any one or a combination of at least two of AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, tissue-derived stromal cells, or primary cells.

[0010] This application designs novel artificial hematopoietic stem and progenitor cells (HSPC, CD34). + The method of inducing CD19 CAR-NK cells by HSPC, and then inducing CD34 + The encoding nucleic acid of CD19 CAR is introduced into HSPC, and CD34 is delivered via stromal cells. + HSPCs were efficiently induced into CD19 CAR-NK precursor cells, and further induction of precursor cell maturation led to the expansion of a large number of CD19 CAR-NK cells, significantly improving the induction of CD34. + The efficiency of HSPC in producing CD19 CAR-NK cells, 10 6 CD34 + HSPC can get 10 13 -10 14 This method yields CD19 CAR-NK cells, reducing the cost of introducing CARs through gene editing or genetic engineering. The resulting CD19 CAR-NK cells are highly mature and possess good natural killer activity and tumor killing ability.

[0011] The CD34 described in this application + HSPCs are hematopoietic stem and progenitor cells, derived from sources including but not limited to umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, and CD34 cells obtained through pluripotent stem cell induction. + HSPC or blood cells undergoing dedifferentiation or transdifferentiation to obtain CD34 + HSPC, etc.

[0012] Preferably, the tissue includes bone marrow and / or liver tissue.

[0013] This application focuses on the introduction of CD19 CAR expression elements into CD34. + After HSPC, the cells are rapidly induced into NK cells, thus obtaining CD19 CAR-NK cells. The CD19 CAR expression element does not participate in the induction process. Therefore, the CD19 CAR expression elements commonly used in this field are applicable to this application, including all CD19 CAR types that can recognize human CD19 protein, such as FMC63, Hu1E7, and SJ25C1 (different antibody sources that can recognize CD19 protein).

[0014] Preferably, the vector encoding the chimeric antigen receptor nucleic acid includes a lentiviral vector, a retroviral vector, an adenoviral vector, a transposon expression vector, a homologous recombination expression vector, or a non-integrative expression vector.

[0015] Preferably, the method of introduction includes viral infection, chemical transfection, or electroporation transfection. More specific introduction methods include, but are not limited to: lentiviral infection, retroviral infection, adenovirus infection, Sendai virus infection, liposome transfection, transposon integration, and genome-directed integration.

[0016] Preferably, the induction amplification medium includes any one or a combination of at least two of the following: KBM581 medium, Opti-MEM medium, APEL2 medium, Essential 6 medium, DMEM-high glucose medium, DMEM / F12 medium, α-MEM medium, F-12 medium, EBM2 medium, MEM medium, BME medium, RPMI 1640 medium, G-MEM medium, or a basal medium containing active substances.

[0017] Preferably, the active substance comprises any one or a combination of at least two of the following: serum substitute, insulin-like growth factor 1 (IGF-1), non-essential amino acids, glutamine, a stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonist, ascorbic acid, or cell growth factors.

[0018] Preferably, the cell growth factor includes any one or a combination of at least two of IL-3, IL-7, IL-2, SCF, IL-12, IL-15, IL-21, hTPO, IL-12, IL-18 or Flt3L.

[0019] Preferably, the co-culture method includes 3D culture.

[0020] It is understood that the 3D culture refers to the three-dimensional cell culture method commonly used in this field.

[0021] Preferably, the stromal cells also overexpress factors that promote lymphocyte development, maturation, proliferation, or survival.

[0022] Preferably, the factor includes human Notch ligand and / or human cytokines.

[0023] Preferably, the human Notch ligand comprises any one or a combination of at least two of DLL1, DLL1 active fragment, DLL4, or DLL4 active fragment.

[0024] Preferably, the human cytokines include any one or a combination of at least two of IL-15, IL-12, IL-2, or IL-21.

[0025] Preferably, the induced amplification culture further includes the step of adding feeder cells.

[0026] Preferably, the feeder cells include any one or a combination of at least two of the following: K562-mbIL-21 amplified cell line, K562-mbIL-15-mbIL-21 amplified cell line, K562-mbIL-15-4-1BBL amplified cell line, or K562-mbIL-21-4-1BBL amplified cell line.

[0027] Preferably, the CD34 + The number of HSPCs is 5×10 2 -1×10 6 10×10 2 10×10 3 Or 10×10 4 wait.

[0028] Preferably, the CD34 + The ratio of HSPCs to stromal cells is 1:(10-500), including but not limited to 1:12, 1:15, 1:20, 1:30, 1:50, 1:80, 1:100, 1:200, 1:300, 1:350, 1:400 or 1:450, etc.

[0029] Preferably, the co-culture and induced amplification culture times are each independently 5-30 days, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 25, 26, 27, 28 or 29 days.

[0030] Preferably, the method for in vitro induction of CAR-NK cells may further include CD34... + The steps of HSPC / or cell expansion after introduction.

[0031] Preferably, the amplification includes:

[0032] CD34 + HSPC and / or introduced cells are seeded into cell expansion medium for culture, or,

[0033] CD34 + HSPC and / or introduced cells are co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells.

[0034] Preferably, the cell expansion culture medium includes StemMACS medium and StemSpan medium. TM SFEM II medium, IMDM medium, Opti-MEM medium, Stemline medium, StemPro medium, QBSF-60 medium, XVIVO-15 medium, HPGM medium, RPMI medium, Hematopoietic Progenitor Expansion Medium XF medium (PromoCell) Any one or a combination of at least two of the following: hPSC XF medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion Media (CELPROGEN) medium, and basal media containing active substances.

[0035] Preferably, the active substance comprises any one or a combination of at least two of the following: serum substitute, insulin-like growth factor 1, non-essential amino acids, glutamine, a stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, aromatic hydrocarbon receptor antagonists, ascorbic acid, or cell growth factors.

[0036] Preferably, the cell growth factor includes any one or a combination of at least two of IL-2, IL-12, IL-3, IL-7, SCF, IL-6, IL-10, IL-11, hTPO, or Flt3L.

[0037] As a preferred technical solution, the method for in vitro induction of NK cells includes the following steps:

[0038] (1) CD34 + HSPCs are inoculated into amplification medium for culture, or...

[0039] CD34 + HSPCs were co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells.

[0040] (2) Obtain CD34 from step (1) + HSPCs were used to introduce the coding nucleic acid of a chimeric antigen receptor targeting CD19. The introduced cells were then mixed with stromal cells and co-cultured to obtain CD19 CAR-NK precursor cells.

[0041] The stromal cells include any one or a combination of at least two of the following: AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, stromal cells or primary cells derived from bone marrow or liver tissue; and

[0042] (3) Take the CD19 CAR-NK precursor cells obtained in step (2) and induce expansion culture to obtain CD19 CAR-NK cells.

[0043] Secondly, this application provides an induced CD19 CAR-NK cell, which is prepared by the in vitro induction method for preparing CD19 CAR-NK cells described in the first aspect.

[0044] Thirdly, this application provides the method for in vitro induction of CD19 CAR-NK cells as described in the first aspect or the use of the induced CD19 CAR-NK cells as described in the second aspect in the preparation of drugs for treating tumors.

[0045] Preferably, the tumor includes, but is not limited to, B-cell lymphomas (including Hodgkin's and non-Hodgkin's lymphomas), chronic or acute leukemias (including acute lymphoblastic leukemia and chronic lymphoblastic leukemia), and B-cell-related autoimmune diseases (including lupus erythematosus and type II diabetes).

[0046] Compared with the prior art, this application has at least the following beneficial effects:

[0047] This application designs a novel artificial hematopoietic stem cell (CD34). + The method of inducing CAR-NK cells by HSPC, targeting CD34 + The encoding nucleic acid of CD19 CAR is introduced into HSPC, and CD34 is delivered via stromal cells. + HSPCs were efficiently induced into CD19 CAR-NK precursor cells, and further induction of precursor cell maturation led to the expansion of a large number of CD19 CAR-NK cells, significantly improving the induction of CD34. + The efficiency of HSPC in producing CD19 CAR-NK cells, 10 6 CD34 + HSPC can get 10 13 -10 14 This method yields CD19 CAR-NK cells, reducing the cost of introducing CARs through gene editing or genetic engineering. The resulting CD19 CAR-NK cells are highly mature and possess good natural killer activity and tumor killing ability. Attached Figure Description

[0048] Figure 1 shows CD34 + Flowchart of HPSC differentiation into CD19 CAR-HiNK cells.

[0049] Figure 2 shows the separated CD34. + After HSPCs are infected with CD19 CAR retroviruses, CD19 CARs are located on CD34. + HSPC (CD19 CAR-CD34) + The representation diagram in HSPC).

[0050] Figure 3 shows the amplification of CD19 CAR-CD34 using a co-culture method with stromal cells (AFT024 cells). + HSPC, statistical analysis of CD19 CAR-CD34 on days 7 and 14 during the amplification process. + Amplification fold results of HSPC.

[0051] Figure 4 shows the overall morphological changes of cells during the 3D system induction phase (day 0, day 7, and day 8).

[0052] Figure 5 shows the CD19 CAR-CD34 levels on day 14 after 3D system induction. + CD19CAR-HiNK precursor cells differentiated from HSPCs (CD45) + CD56 - CD34 + CD7 + CD45 + CD34 - CD7 + CD56 + Immunophenotypic results of CD19 CAR expression.

[0053] Figure 6 shows microscopic images of CD19 CAR-HiNK precursor cells on days 7, 14, and 21 during the maturation and expansion phase.

[0054] Figure 7 shows the immunophenotypes of CD19 CAR-HiNK precursor cells (CD56) on days 7, 14, and 21 of the maturation and expansion phase. + ) and expression of CD19 CAR (CD19 CAR) + The result image.

[0055] Figure 8 shows 10. 6 CD19 CAR-CD34 + A statistical chart showing the number of CD19 CAR-HiNK cells obtained from HSPC.

[0056] Figure 9 shows the results of CD19 CAR-HiNK cells killing the K562 hematologic malignancy cell line (myeloid leukemia) in vitro.

[0057] Figure 10 shows the results of CD19 CAR-HiNK cells killing the CD19-expressing B-cell hematologic malignancy cell line Nalm-6 (acute B-lymphoblastic leukemia) in vitro.

[0058] Figure 11 shows the results of in vitro killing of tumor tissue extracted from patients with B-lymphoma by CD19 CAR-HiNK cells.

[0059] Figure 12 shows the results of CD19 CAR-HiNK cells killing bone marrow cells from a patient with B-lymphoblastic leukemia in vitro.

[0060] Figure 13 shows the results of CD19 CAR-HiNK cells killing the CD19-expressing B-cell hematologic malignancy cell line Nalm-6 (acute B-lymphoblastic leukemia) in vivo. Detailed Implementation

[0061] The technical solution of this application will be further described below with reference to the accompanying drawings and specific embodiments. However, the examples below are merely simplified examples of this application and do not represent or limit the scope of protection of this application. The scope of protection of this application shall be determined by the claims.

[0062] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0063] This application presents a novel method for preparing CAR-NK cells. It is understood that the focus of this application is on the introduction of the CD19 CAR expression element into CD34. + Following HSPC, the cells are rapidly induced into NK cells, thus obtaining CAR-NK cells. The CAR expression element does not participate in the induction process; therefore, commonly used CAR expression elements in this field are applicable to this application. Taking CD19 CAR as an example, the flowchart is shown in Figure 1, which can be performed in four steps (the CD19 CAR introduction step can be combined with the CD34 step). + HSPC amplification steps reversed), five-step or three-step process (removal of amplified CD34) + HSPC or CD19 CAR-CD34 + The HSPC amplification step is included in this application. In this application, the NK cells obtained after induction are further named HiNK cells, and this name will be used to refer to the obtained NK cells thereafter.

[0064] Four-step process: Step 1: In CD34+ HSPC introduces CAR elements to prepare CD19 CAR-CD34 + HSPC; Step 2: Amplification of CD19 CAR-CD34 + HSPC; Step 3: 3D induction of CD19 CAR-CD34 + HSPCs differentiate into CD19 CAR-HiNK precursor cells; Step 4: Induce CD19 CAR-HiNK precursor cell maturation and expand CD19 CAR-HiNK cells.

[0065] Four-step process optional: Step 1: Amplify CD34 + HSPC; Step 2: In the amplified CD34 + HSPC introduces CAR elements to prepare CD19 CAR-CD34 + HSPC; Step 3: 3D induction of CD19CAR-CD34 + HSPCs differentiate into CD19 CAR-HiNK precursor cells; Step 4: Induce CD19 CAR-HiNK precursor cell maturation and expand CD19 CAR-HiNK cells.

[0066] Five-step process: Step 1: Amplify CD34 + HSPC; Step 2: In the amplified CD34 + HSPC introduces CAR elements to prepare CD19 CAR-CD34 + HSPC; Step 3: Amplification of CD19 CAR-CD34 + HSPC; Step 4: 3D Induction of CD19 CAR-CD34 + HSPCs differentiate into CD19 CAR-HiNK precursor cells; Step 5: Induce CD19 CAR-HiNK precursor cell maturation and expand CD19 CAR-HiNK cells.

[0067] Three-step process: Step 1: In CD34 + HSPC introduces CAR elements to prepare CD19 CAR-CD34 + HSPC; Step 2: 3D induction of CD19 CAR-CD34 + HSPCs differentiate into CD19 CAR-HiNK precursor cells; Step 3: Induce CD19 CAR-HiNK precursor cell maturation and expand CD19 CAR-HiNK cells.

[0068] In CD34 + Steps for importing CD19 CAR into HSPC: Introduce CD19 CAR using viral infection, chemical transfection, or electroporation transfection methods into CD34. +Importing the CD19 CAR component into HSPC yields the CD19 CAR-CD34. + HSPC, this process takes 1-5 days to complete.

[0069] 1. The antigen-binding domain in the CD19 CAR structure can be a single-chain antibody sequence (scFv) of any origin (such as mouse, human, synthetic, etc.) that can target human CD19 protein, including but not limited to FMC63 and Hu1E7.

[0070] 2. Plasmid vectors for delivering CD19 CAR elements include, but are not limited to: lentiviral vectors, retroviral vectors, transposon expression vectors, homologous recombination expression vectors, and non-integrative expression vectors.

[0071] 3. To CD34 + The methods for introducing CD19 CAR elements in HSPC include, but are not limited to: viral infection, chemical transfection, and electroporation transfection.

[0072] 4. More specific import methods include, but are not limited to: lentiviral infection, retroviral infection, adenovirus infection, Sendai virus infection, liposome transfection, transposon integration, and genome-directed integration.

[0073] 5.CD34 + After CD19 CAR elements are introduced into HSPC, purification can be performed before proceeding to the next step, or purification can be performed first. Purification methods include, but are not limited to, magnetic column enrichment, flow cytometry sorting, and drug screening.

[0074] 6.CD34 + HSPC sources include, but are not limited to, umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, and CD34 obtained from pluripotent stem cell induction. + HSPC, blood cell dedifferentiation, transdifferentiation to obtain CD34 + HSPC.

[0075] Amplification of CD34 + HSPC or CD19 CAR-CD34 + HSPC steps: CD34 + HSPC or CD19CAR-CD34 + HSPCs are expanded using amplification medium and / or feeder cells for 1-30 days.

[0076] CD34 + HSPC or CD19 CAR-CD34 +HSPC amplification medium is any artificial blood stem cell / progenitor cell amplification medium, including but not limited to: StemMACS, StemSpan. TM SFEM II, IMDM, Opti-MEM, Stemline, StemPro, QBSF-60, XVIVO-15, HPGM, RPMI, Hematopoietic Progenitor Expansion Medium XF, hPSC XF Medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion Media, and any combination thereof. The basal culture medium may contain one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, UM171, and one, two, or more of the following cell growth factors: TPO, IL-7, IL-10, IL-2, IL-3, SCF, Flt3L, IL-11, and IL-6.

[0077] Amplification of CD34 + HSPC or CD19 CAR-CD34 + HSPC methods include, but are not limited to, the following:

[0078] 1. Using amplification medium, CD19 CAR-CD34 + HSPCs are cultured and amplified using culture medium, with an amplification time of 1-30 days.

[0079] 2. Transfer CD19 CAR-CD34 + HSPCs were co-cultured with irradiated AFT024 cells, or AFT024-DLL1 (AFT024-Delta Like Canonical Notch Ligand 1) cells, or AFT024-DLL4 (AFT024-Delta Like Canonical Notch Ligand 4) cells, or AFT024-DLL1-DL4 cells to amplify CD34. + Cells, expansion time is 1-30 days.

[0080] 3D-induced CD19 CAR-CD34 + Steps for differentiating HSPCs into CD19 CAR-HiNK precursor cells and CD19 CAR-HiNK cells: CD19 CAR-CD34 +A 3D induction system was prepared by mixing HSPC cells and stromal cells in a certain ratio, and then cultured on an exogenous matrix or scaffold. The culture time was 5-30 days.

[0081] 1. CD19 CAR-CD34 + HSPC refers to freshly prepared unamplified, amplified fresh, or frozen unamplified, or amplified frozen.

[0082] 2. The types of stromal cells include, but are not limited to: AFTO24, MS5, OP9, HS-5, MSC, MUTZ-3, bone marrow-derived stromal cell lines or primary cells; the above stromal cells can be modified in any way to express factors that are beneficial to the development, maturation, expansion and survival of lymphocytes, including, but not limited to, human Notch ligands: DLL1 (Delta Like Canonical Notch Ligand 1) or / and DLL4 (Delta Like Canonical Notch Ligand 4), or their active fragments, human cytokines: IL-15, IL-2, IL-21, the cytokines are expressed in secretory form or anchored to the cell membrane (membrane-bound).

[0083] 3. CD19 CAR-CD34 + The mixing ratio of HSPCs to stromal cells is 1:10-1:500, CD19 CAR-CD34 + The number of HSPC cells can reach 5×10 2 -1×10 6 Within a certain range.

[0084] 4. The exogenous matrix or scaffold is a filter membrane made of any material with a pore size not exceeding 3 μm. Materials include, but are not limited to, polycarbonate or polyethylene terephthalate.

[0085] 5.3D induction time: 5-30 days; the induced cell phenotype is CD34. + or / and CD34 + CD7 + or / and CD34 - CD7 + or / and CD7 + CD56 + or / and CD7 - CD56 + .

[0086] 6. CD19 CAR-HiNK Induction Medium: The basal medium can be selected from KBM581, Opti-MEM, APEL2, Essential 6, DMEM-high glucose, DMEM / F12, α-MEM, F-12, EBM2, MEM, BME, RPMI 1640, G-MEM, and any combination thereof. It can also be a basal medium supplemented with the following substances: one or more serum substitutes, one or more non-essential amino acids, a stabilized dipeptide of glutamine or L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, ascorbic acid, and cell growth factors such as IL-3, IL-7, IL-2, SCF, IL-15, IL-21, IL-12, IL-18, and Flt3L, one, two, or more.

[0087] Steps for inducing CD19 CAR-HiNK precursor cell maturation and expanding CD19 CAR-HiNK cells: Prepare single-cell suspensions of cells induced by the 3D system, collect them in centrifuge tubes, discard the cell supernatant after centrifugation, resuspend the cell pellet in induction maturation and expansion medium, transfer it into a culture vessel, and induce maturation and expansion of CD19 CAR-HiNK cells using CD19 CAR-HiNK induction medium for 5-30 days.

[0088] 1. The culture medium used in this step is CD19 CAR-HiNK induction medium.

[0089] 2. The methods of inducing maturation and expansion include, but are not limited to: directly using culture medium for induction of maturation and expansion; and using feeder cells for induction of maturation and expansion, including, but not limited to: K562-mIL-21 expanded cell line and K562-mIL-15-mIL-21 expanded cell line; the feeder cells can be added at any time point during the induction of maturation and expansion phase.

[0090] 3. The culture containers used for inducing maturation and amplification include, but are not limited to: culture dishes of any size / model, culture plates of any size / model, culture flasks of any size / model, culture bags of any specification, fermenters or automated culture devices of any specification.

[0091] Example 1

[0092] In this embodiment, a retroviral infection method is used to deliver the CD19 CAR element to the CD34. + HSPC. First, CD34 enriched with magnetic beads (CD34 MicroBead Kit, human, Miltenyi Biotec, 130-046-702) +Cells were cultured in hematopoietic stem and progenitor cell expansion medium for 24 h. Then, CD19 CAR retroviruses were used to stimulate the cultured CD34 cells. + Cells were infected, and CD19-CAR was detected on CD34 on day 3 post-infection. + The positivity rate in HSPC is shown in Figure 2, indicating that CD19 CAR is effective against CD45. + The positive rate in CD34 cells was 65.2%.

[0093] Example 2

[0094] This embodiment amplifies CD19 CAR CD34 + HSPC. First, irradiated AFT024 cells were seeded at a density of 100,000 cells / well in 24-well plates and cultured. Then, CD19 CAR CD34... + HSPCs (10,000) were seeded onto the above-mentioned stromal cells for co-culture. CD34 levels were counted on day 7 and day 14 of expansion. + The number of HSPCs and the conversion to amplification fold are shown in Figure 3. The results indicate that after 7 days of amplification, CD19 CAR CD34 + HSPC amplified 59-67 times. After 14 days of amplification, CD19 CAR CD34... + HSPC expanded 931-1126 times in a stromal cell co-culture environment.

[0095] Example 3

[0096] This embodiment uses a 3D culture system to generate CD19 CAR HiNK precursor cells. The CD19 CAR CD34 cells obtained in Example 2 were used... + HSPC cells were mixed with OP9 (ATCC, catalog number CRL-2749) feeder cells at a ratio of 1:20, resulting in a total cell count of 210,000, to prepare a 3D induction system. Each 3D system occupied a culture surface area of ​​0.5 cm². 2 The prepared 3D system was laid on a filter membrane made of polycarbonate material, and the filter membrane was supported in a culture plate. CD19 CAR-HiNK induction medium (KBM581 basal medium with 15% SUPERGROW cell culture additive and 2mM GlutaMAX) was added to the culture plate. TMThe 3D system was induced for 14 days with the following media: 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 1% Penicillin-Streptomycin Solution, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15, and 10 ng / mL Flt3L. The medium was changed every 2 days. The results are shown in Figure 4. The overall cell morphology of the 3D system changed at different time points (day 0, day 7, and day 14). Flow cytometry was performed on day 14 of induction to detect CD19 CAR HiNK cell precursor cells (CD45). + CD56-CD34 + CD7 + CD45 + CD56-CD34 + CD7-, CD45 + CD34-CD7 + CD56 + The results of the CD19 CAR positivity rate are shown in Figure 5. The results indicate that after 14 days of 3D culture, the CD19 CAR CD34 positivity rate... + HSPC can effectively induce CD19-CAR HiNK precursor cells (CD45). + CD56-CD34 + / -CD7 + ) and HiNK cells (CD45) + CD56 + The positive rates of CD19CAR in CD19-CAR HiNK precursor cells and HiNK cells were 75.8% and 77.4%, respectively.

[0097] Example 4

[0098] This embodiment describes the induction, maturation, and expansion of CD19 CAR-HiNK cell precursor cells. Cells cultured in the 3D system to day 14 in Example 3 were prepared into single-cell suspensions and transferred to culture bags. The cells were cultured in CD19 CAR-HiNK induction medium (KBM581 basal medium supplemented with 15% SUPERGROW cell culture additive and 2 mM GlutaMAX). TMThe cells were cultured using 1 μM β-mercaptoethanol, 5 ng / mL sodium selenite, 50 μM ethanolamine, 20 μg / mL ascorbic acid, 1% Penicillin-Streptomycin Solution, 5 ng / mL IL-3, 20 ng / mL SCF, 20 ng / mL IL-7, 10 ng / mL IL-15, and 10 ng / mL Flt3L. The cells were replenished with fluid every 3 days to maintain a cell density of 1.5 × 10⁻³ L / mL. 6 This step can effectively induce CD19 CAR-HiNK cells (CD45) to be produced. + CD56 + CD19 CAR + The results, shown in Figure 6, illustrate the cell morphology of CD19 CAR-HiNK precursor cells on days 7, 14, and 21 of maturation and expansion. Flow cytometry results, shown in Figure 7, analyzed the immunophenotype (CD56) of CD19 CAR-HiNK cells on days 7, 14, and 21 of maturation and expansion. + CD19CAR + The results showed that CD56 + The proportion gradually increased, eventually reaching 100%. The proportion of CD19 CAR remained above 70% and eventually stabilized at 86.6%. + CD56 + The number gradually increased, eventually reaching 10 6 CD34 + Cells can obtain 10 13 -10 14 10 CD19 CAR-HiNK cells. Specifically, as shown in Figure 8, the number of CD19 CAR-HiNK cells induced increases over time, reaching 10... 6 CD19 CAR CD34 + HSPC can get 10 13 -10 14 One CD19 CAR HiNK cell.

[0099] Example 5

[0100] This embodiment statistically analyzed the viral load used in three independent inductions of CD19 CAR-HiNK cells. Specifically, CD19 CAR retrovirus (1×10⁻⁶) was used. 8 TU / mL, MOI=10) infected CD34 + HSPC (1×10 6 After preparing CD19 CAR-CD34, +HSPC, CD19 CAR-CD34 + HSPC amplification was followed by induction of CD19 CAR-HiNK cells. The viral load (μL) used in three independent inductions and the number of induced CD19 CAR-HiNK cells were recorded. Simultaneously, CD19 CAR-iNK cells were prepared using mature NK cells derived from umbilical cord blood, and CD19 CAR retrovirus (1×10⁻⁶) was used. 8 TU / mL, MOI=5) infected NK cells (1×10⁻⁶) 6 CD19 CAR-NK cells were induced after three independent inductions, and the amount of virus used (μL) and the number of CD19 CAR-NK cells prepared were recorded. The results are shown in Table 1. The three inductions involved CD34... + Results of HSPC-induced CD19 CAR-HiNK cell production showed that CD19 CAR-CD34 was prepared... + A viral load of 100 μL for HSPC can induce 3.6 × 10⁻⁶ cells / mL. 13 (CAR positivity rate: 68.1%), 3.8 × 10 13 (CAR positivity rate: 78.7%) and 4.4 × 10 13 (CAR positivity rate: 86.1%) CD19 CAR-HiNK cells, averaging 10 after conversion. 6 The viral load corresponding to one CD19 CAR-HiNK cell is 1.0 × 10⁻⁶. -1 TU. Results of CD19 CAR-NK cell preparation after three-stage infection of NK cells showed that the viral load used for CD19 CAR-NK cell preparation was 50 μL, and the resulting cells could be expanded to 2.2 × 10⁻⁶. 7 (CAR positivity rate: 52.0%), 2.7 × 10 7 (CAR positivity rate: 72.0%) and 3.3 × 10 7 (CAR positivity rate: 60.0%) CD19 CAR-NK cells, averaging 10 after conversion. 6 The viral load corresponding to each CD19 CAR-NK cell is 1.9 × 10⁻⁶. 5 TU. It can be obtained by utilizing CD34. + The viral load used for HSPC-induced CD19 CAR-HiNK cells was 7.2 × 10⁻⁶ less than that used to prepare CD19 CAR-NK cells from umbilical cord blood mature NK cells. 5 times.

[0101] Table 1

[0102] Example 6

[0103] This embodiment verifies that the induced CD19 CAR-HiNK cells can maintain the natural killing activity of NK cells in vitro and kill non-CD19 target hematologic malignancies. The CD19CAR-HiNK cells obtained in Example 4 were co-incubated with K562 (human chronic myeloid leukemia cell line) to evaluate the natural killing ability (cytotoxicity) of CD19CAR-HiNK cells against tumor cells. The specific results are shown in Figure 9, where the X-axis represents different E:T ratios (CD19 CAR-HiNK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells. After 4 hours of co-incubation, the percentage of dead tumor cells in each sample was detected by flow cytometry; this percentage represents the cytotoxicity (%) of CD19 CAR-HiNK cells. (Each E:T sample was repeated three times; the cytotoxicity of CD19 CAR-HiNK cells was: K562: 48.4% (1:1), 67.0% (5:1), 87.6% (10:1), indicating that CD19 CAR-HiNK cells...) CAR-HiNK cells have a natural killer ability that is no weaker than HiNK and umbilical cord blood NK cells (UCB-NK).

[0104] Example 7

[0105] This embodiment verifies that the induced CD19 CAR-HiNK cells can specifically kill hematological malignancies expressing CD19 antigen in vitro. The CD19 CAR-HiNK cells obtained in Example 4 were co-incubated with Nalm-6 (human B-lymphoblastic leukemia cell line) to evaluate the ability of CD19 CAR-HiNK cells to specifically kill tumor cells (cytotoxicity). The specific results are shown in Figure 10, where the X-axis represents different E:T ratios (CD19 CAR-HiNK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after 4 hours of co-incubation, the proportion of dead tumor cells in each sample was detected by flow cytometry. This proportion is the killing ability (Cytotoxicity%) of CD19 CAR-HiNK cells (each E:T sample was repeated 3 times, CD19...). The killing capacity of CAR-HiNK cells was as follows: Nalm-6: 51.0% (0.2:1), 76.7% (0.4:1), 89.4% (0.8:1), 93.3% (1.6:1), 96.7% (5:1), indicating that CD19 CAR-HiNK cells have a stronger killing capacity than HiNK and umbilical cord blood NK cells (UCB-NK).

[0106] Example 8

[0107] This embodiment verifies that the induced CD19 CAR-HiNK cells can specifically kill primary patient B lymphoma cells and B lymphoblastic leukemia cells expressing CD19 antigen in vitro. The CD19 CAR-HiNK cells obtained in Example 4 were co-incubated with primary patient B lymphoma cells and B lymphoblastic leukemia cells to evaluate the specific cytotoxicity of CD19 CAR-HiNK cells. Specific results are shown in Figures 11 and 12, where the X-axis represents different E:T ratios (CD19 CAR-HiNK cells (Effector cells, E): Tumor cells (Target, T)); the Y-axis shows the percentage of dead tumor cells out of the total number of tumor cells; after 12 hours of co-incubation, flow cytometry was used to detect the percentage of dead tumor cells in each sample. This percentage represents the killing ability (cytotoxicity%) of CD19 CAR-HiNK cells (each E:T sample was repeated 3 times, CD19...). The killing capacity of CAR-HiNK cells was as follows: Killing human B lymphoma cells (Figure 11): 22.7% (0.2:1), 29.8% (0.4:1), 32.7% (0.8:1), 37.4% (1.6:1), 45.0% (5:1); Killing human B lymphoblastic leukemia cells-1 (Figure 12): 53.7% (0.2:1), 65%. 3% (0.4:1), 70.3% (0.8:1), 70.9% (1.6:1), 73.0% (5:1); Human B-lymphoblastic leukemia cells-2: 9.6% (0.2:1), 13.0% (0.4:1), 20.8% (0.8:1), 29.0% (1.6:1), 43.5% (5:1); indicating that CD19-CAR HiNK cells have a stronger ability to kill CD19-positive primary B-lymphoma cells than HiNK cells.

[0108] Example 9

[0109] This embodiment verifies the in vivo tumor-killing effect of CD19 CAR-HiNK cells. To evaluate the in vivo anti-tumor function of CD19 CAR-HiNK cells, a tumor model was constructed in 8-week-old female B-NDG mice (severely immunodeficient mice, Biocytogen) using Nalm-6 cells (human B-lymphoblastic leukemia cell line, 100,000 cells injected into each mouse via the tail vein). After observing the tumor burden of Nalm-6 in mice using a small animal imaging system, the mice were randomly divided into 3 groups of 5 mice each. CD19 CAR-HiNK cells were injected into the tumor model via the tail vein, with each mouse receiving 10 million CD19 CAR-HiNK cells via the tail vein. An equivalent amount of HiNK cells was injected as a control. The tumor burden of the treated mice was then detected weekly using a small animal imaging system. As shown in Figure 13, CD19 CAR-HiNK cells effectively alleviated the tumor burden in mice, while the tumor burden in mice that were not injected and those injected with HiNK cells continued to increase. This indicates that CD19 CAR-HiNK cells can kill tumor cells in vivo and reduce the tumor burden in mice.

[0110] In summary, this application presents a novel method for preparing CD19-targeted CAR-NK cells. Utilizing artificial hematopoietic stem cells, this method significantly reduces the cost of genetic engineering or gene editing required for CD19 CAR element delivery, efficiently induces the generation of CD19 CAR-NK cells, and increases the efficiency of single CD34 cells. + The efficiency of HSPC in outputting CD19 CAR-NK cells, and the induced CD19 CAR-NK cells can more efficiently target and kill tumor cells expressing CD19 antigen, which has broad application prospects.

[0111] The applicant declares that the above description is only a specific implementation of this application, but the protection scope of this application is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application fall within the protection and disclosure scope of this application.

Claims

1. A method for in vitro induction of CD19 CAR-NK cells, comprising: To CD34 + The HSPC introduces the coding nucleic acid of the chimeric antigen receptor targeting CD19, mixes the introduced cells with stromal cells, and co-cultures them in an induction expansion medium to obtain CD19 CAR-NK precursor cells. The CD19 CAR-NK precursor cells are then further induced and expanded to obtain mature CD19 CAR-NK cells. The stromal cells include any one or a combination of at least two of the following: AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, tissue-derived stromal cells, or primary cells.

2. The method for in vitro induction of CD19 CAR-NK cells according to claim 1, wherein, The CD34 + HSPCs can be derived from umbilical cord blood, placenta, mobilized peripheral blood, bone marrow, and CD34 obtained through pluripotent stem cell induction. + HSPC or blood cells undergoing dedifferentiation or transdifferentiation to obtain CD34 + Any one or at least two of the HSPCs.

3. The method for in vitro induction and preparation of CD19 CAR-NK cells according to claim 1, wherein, The tissues include bone marrow and / or liver tissue; Preferably, the vector encoding the chimeric antigen receptor nucleic acid includes a lentiviral vector, a retroviral vector, an adenoviral vector, a transposon expression vector, a homologous recombination expression vector, or a non-integrative expression vector. Preferably, the method of introduction includes viral infection, chemical transfection, or electroporation transfection; Preferably, the induction amplification medium includes any one or a combination of at least two of the following: KBM581 medium, Opti-MEM medium, APEL2 medium, Essential 6 medium, DMEM-high glucose medium, DMEM / F12 medium, α-MEM medium, F-12 medium, EBM2 medium, MEM medium, BME medium, RPMI 1640 medium, G-MEM medium, or a basal medium containing active substances. Preferably, the active substance comprises any one or a combination of at least two of the following: serum substitute, insulin-like growth factor 1, non-essential amino acids, glutamine, a stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonists, ascorbic acid, or cell growth factors. Preferably, the cell growth factor includes any one or a combination of at least two of IL-3, IL-7, IL-2, SCF, IL-12, IL-15, IL-21, hTPO, IL-12, IL-18 or Flt3L.

4. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-3, wherein, The co-cultivation method includes 3D cultivation.

5. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-4, wherein, The stromal cells also overexpress factors that promote lymphocyte development, maturation, proliferation, or survival. Preferably, the factor includes human Notch ligand and / or human cytokines; Preferably, the human Notch ligand comprises any one or a combination of at least two of DLL1, DLL1 active fragment, DLL4, or DLL4 active fragment; Preferably, the human cytokines include any one or a combination of at least two of IL-15, IL-12, IL-2, or IL-21.

6. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-5, wherein, The induced amplification culture also includes the step of adding feeder cells; Preferably, the feeder cells include any one or a combination of at least two of the following: K562-mbIL-21 amplified cell line, K562-mbIL-15-mbIL-21 amplified cell line, K562-mbIL-15-4-1BBL amplified cell line, or K562-mbIL-21-4-1BBL amplified cell line.

7. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-6, wherein, The CD34 + The number of HSPCs is 5×10 2 -1×10 6 indivual; Preferably, the CD34 + The ratio of HSPCs to stromal cells is 1:(10-500); Preferably, the co-culture and induced amplification cultures each last independently for 5-30 days.

8. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-7, wherein, The method also includes CD34 + Steps for HSPC and / or cell expansion after introduction; Preferably, the amplification includes: CD34 + HSPC and / or introduced cells are seeded into cell expansion medium for culture, or, CD34 + HSPCs and / or introduced cells are co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells. Preferably, the cell expansion culture medium includes StemMACS medium and StemSpan medium. TM SFEM II medium, IMDM medium, Opti-MEM medium, Stemline medium, StemPro medium, QBSF-60 medium, XVIVO-15 medium, HPGM medium, RPMI medium, Hematopoietic Progenitor Expansion Medium XF medium. Any one or a combination of at least two of the following: hPSC XF medium, Human Embryonic Hematopoietic Stem Cell Serum Free Expansion Media medium, and basic culture media containing active substances; Preferably, the active substance comprises any one or a combination of at least two of the following: serum substitute, insulin-like growth factor 1, non-essential amino acids, glutamine, a stabilized dipeptide of L-alanyl-L-glutamine, β-mercaptoethanol, sodium selenite, ethanolamine, aryl hydrocarbon receptor antagonists, ascorbic acid, or cell growth factors. Preferably, the cell growth factor includes any one or a combination of at least two of IL-2, IL-12, IL-3, IL-7, SCF, IL-6, IL-10, IL-11, hTPO, or Flt3L.

9. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-8, wherein, The method includes the following steps: (1) CD34 + HSPCs are inoculated into amplification medium for culture, or... CD34 + HSPCs were co-cultured with AFT024 cells, AFT024-DLL1 cells, AFT024-DLL4 cells, or AFT024-DLL1-DLL4 cells. (2) Obtain CD34 from step (1) + HSPCs were used to introduce the coding nucleic acid of a chimeric antigen receptor targeting CD19. The introduced cells were then mixed with stromal cells and co-cultured to obtain CD19 CAR-NK precursor cells. The stromal cells include any one or a combination of at least two of the following: AFT024 cells, MS5 cells, OP9 cells, HS-5 cells, MSC cells, MUTZ-3 cells, stromal cells or primary cells derived from bone marrow or liver tissue; and (3) Take the CD19 CAR-NK precursor cells obtained in step (2) and induce expansion culture to obtain CD19 CAR-NK cells.

10. An induced CD19 CAR-NK cell, wherein, The induced CD19 CAR-NK cells are prepared by the method for in vitro induction of CD19 CAR-NK cells as described in any one of claims 1-9.

11. The method for in vitro induction of CD19 CAR-NK cells according to any one of claims 1-9, or the use of the induced CD19 CAR-NK cells according to claim 10 in the preparation of a medicament for treating tumors.

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