Macrophage derived from induced pluripotent stem cells as well as preparation method and application of macrophage

Abstract

The invention relates to macrophages derived from induced pluripotent stem cells as well as a preparation method and application of the macrophages. Specifically, the present invention relates to a macrophage (iPSC-CAR-M) derived from an induced pluripotent stem cell (iPSC), in which the macrophage expresses a chimeric antigen receptor (CAR) that specifically binds to a tumor-associated antigen; and a transcription factor combination comprising PU.1, IRF5 and BATF2 is overexpressed. The invention also relates to a pharmaceutical composition containing the macrophage derived from the induced pluripotent stem cells, and a preparation method and application of the pharmaceutical composition.

Description

Technical Field

[0001] This invention relates to the field of cell immunotherapy. Specifically, this invention relates to genetically engineered chimeric antigen receptor (CAR) macrophages capable of specifically recognizing and attacking tumor cells, particularly a type of macrophage derived from induced pluripotent stem cells (iPSCs) that stably maintains a highly effective anti-tumor M1 phenotype. Background Technology

[0002] In recent years, adoptive cell immunotherapy, represented by CAR-T cells, has achieved remarkable success in treating hematologic malignancies. However, for solid tumors, CAR-T therapy faces challenges such as low tumor infiltration efficiency and susceptibility to suppression in the tumor microenvironment.

[0003] As an important innate immune cell, macrophages possess a powerful phagocytic capacity and can penetrate deep into solid tumor tissues. Therefore, developing CAR-M-based macrophages has become a promising new direction for immunotherapy of solid tumors. Through the targeting action of CARs, CAR-Ms can specifically recognize and engulf tumor cells.

[0004] Currently, the preparation of CAR-M cells mainly relies on isolating monocytes from the peripheral blood of patients for in vitro induction and genetic modification. This autologous cell preparation method is costly, time-consuming, and the cell state is greatly affected by individual patient differences, making it difficult to achieve standardization and large-scale production. Induced pluripotent stem cell (iPSC) technology can provide a "off-the-shelf" cell source of uniform quality. This involves first genetically editing iPSCs and then directionally inducing their differentiation into CAR-macrophages (iPSC-CAR-M).

[0005] Macrophages possess high plasticity and can polarize into either a pro-inflammatory, anti-tumor M1 phenotype or an anti-inflammatory, tumor-promoting M2 phenotype based on microenvironmental signals. In the complex tumor microenvironment of solid tumors, macrophages are often induced into the M2 phenotype, thereby weakening their anti-tumor function. How to effectively and persistently induce and maintain iPSC-CAR-M in the M1 phenotype is a key bottleneck in improving its therapeutic efficacy for solid tumors. Current technologies typically induce M1 polarization by adding exogenous cytokines (such as IFN-γ and LPS), but the phenotype induced in this way is unstable. Once inside the tumor microenvironment, after the cytokines are metabolized or neutralized by inhibitory signals, iPSC-CAR-M still risks reverting to the M2 phenotype, leading to decreased anti-tumor ability and even promoting tumor progression.

[0006] Therefore, there is an urgent need in the field for a novel macrophage (iPSC-CAR-M) derived from induced pluripotent stem cells (iPSCs) that can endogenously and stably drive iPSC-CAR-Ms to M1 phenotype polarization and resist tumor microenvironment inhibitory signals. Summary of the Invention

[0007] In order to overcome the problems of unstable M1 phenotype of iPSC-CAR-M and its susceptibility to inhibition by the tumor microenvironment, which leads to weakened anti-tumor ability, the present invention aims to provide a technical solution that can stably drive and maintain iPSC-CAR-M in the M1 pro-inflammatory phenotype through endogenous mechanisms, thereby significantly enhancing its efficacy in the treatment of solid tumors.

[0008] In a first aspect, the present invention provides a macrophage derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-M), wherein the macrophages: a) Expressing chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Overexpression of a combination of transcription factors including PU.1, IRF5 and BATF2.

[0009] Compared to CAR macrophages that do not express the combination of the aforementioned transcription factors, the iPSC-CAR-M macrophages of the present invention exhibit enhanced M1 phenotype pro-inflammatory activity and reduced M2 phenotype immunosuppressive cytokine secretion.

[0010] In a second aspect, the present invention provides a modified induced pluripotent stem cell (iPSC) comprising: a) Polynucleotides encoding chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Polynucleotides encoding PU.1, IRF5 and BATF2.

[0011] In a third aspect, the present invention provides a method for preparing macrophages derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-M), the method comprising: Inducing the modified induced pluripotent stem cells (iPSCs) of the second aspect of the present invention to differentiate into macrophages.

[0012] In a fourth aspect, the present invention provides a pharmaceutical composition comprising macrophages derived from induced pluripotent stem cells (iPSC-CAR-M) according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0013] In a fifth aspect, the present invention provides the use of macrophages derived from induced pluripotent stem cells (iPSC-CAR-M) or pharmaceutical compositions comprising the same, according to the first aspect of the present invention, in the preparation of a medicament for treating cancer.

[0014] In a sixth aspect, the present invention provides a method for treating cancer, the method comprising administering to a subject in need a therapeutically effective amount of macrophages derived from induced pluripotent stem cells (iPSC-CAR-M) or a pharmaceutical composition comprising therethem.

[0015] Compared with existing technologies, the macrophages derived from induced pluripotent stem cells (iPSCs) of the present invention (iPSC-CAR-M) have the following significant advantages and positive effects: i) Powerful and stable M1 phenotypic polarization: By endogenously overexpressing the key transcription factor combination of PU.1, IRF5 and BATF2, the iPSC-CAR-M of the present invention can autonomously and continuously maintain pro-inflammatory M1 phenotypic characteristics (such as high expression of iNOS, IL-12, TNF-α, etc.) without relying on continuous stimulation by exogenous cytokines.

[0016] ii) Enhanced anti-tumor ability: The stable M1 phenotype enables the CAR-macrophage to have stronger phagocytic activity, antigen presentation ability and the ability to secrete pro-inflammatory cytokines, thereby more effectively clearing tumor cells and further activating adaptive immune responses (such as T cell responses) to form a synergistic anti-tumor effect.

[0017] iii) Resistance to tumor microenvironment inhibition: The endogenous M1 polarization driving force enables it to effectively resist the interference of immunosuppressive signals (such as IL-10, TGF-β, etc.) from the tumor microenvironment, and it is not easily "turned" into the tumor-promoting M2 phenotype, thus ensuring its long-term fighting ability in solid tumors.

[0018] iv) The potential of “off-the-shelf” treatment: Based on iPSC technology, genetically engineered iPSC cell lines with strict quality control can be prepared and stored in advance, and then thawed and differentiated when needed, thereby achieving standardized and large-scale production, reducing treatment costs, and benefiting more patients.

[0019] The embodiments of the present invention will be further described below with reference to the accompanying drawings and specific examples. However, it should not be construed as limiting the scope of the present invention to these specific examples. Attached Figure Description

[0020] Figure 1 This shows a schematic diagram of the control CAR vector and the multi-transcription factor (TF) vector.

[0021] Figure 2 The structure of the lentivirus vector is shown.

[0022] Figure 3 Indicates iPSC cell expansion. Magnification: 20×, Scale bar: 20 μm.

[0023] Figure 4 A shows the expression of surface markers on monocytes after iPSC virus transfection; B shows a bright-field plot of macrophage-induced macrophages. Magnification: 10×.

[0024] Figure 5 The analysis showed that macrophages from the control group (iPSC differentiation source without genetic modification), CAR-iPSC source, and CAR-TF-iPSC (co-expressing PU.1, IRF5, BATF2) source were induced with M1 and then analyzed. A. The percentage of cells positive for M1 markers (CD80, CD86) and M2 markers (CD163, CD206) on the cell surface of each group was quantitatively analyzed by flow cytometry. B. CAR molecules (targeting the G4S tag) on ​​the cell surface were detected by flow cytometry to verify CAR expression. C. The protein expression levels of transcription factors PU.1, BATF2, and IRF5 in each group of cells were detected by Western blotting. D. The relative mRNA expression levels of PU.1, IRF5, and BATF2 in each group of cells were detected by RT-qPCR.

[0025] Figure 6 The release of cytokines was visualized by ELISA. CAR-iPSC-derived macrophages, CAR-TF-iPSC-derived macrophages (co-expressing PU.1, IRF5, BATF2), and a control group (untransduced iPSC-derived macrophages) were co-cultured with HER2-negative (HER2-) or HER2-positive (HER2+) tumor cells, respectively. After 24 hours, the culture supernatant was collected, and the secretion levels (pg / ml) of various M1-phenotype pro-inflammatory cytokines (IL-1β, TNF-α, IL-12, IL-6) and M2-phenotype anti-inflammatory cytokines (IL-10, TGF-β) were detected by ELISA.

[0026] Figure 7 In vitro cytotoxicity was demonstrated. Control group (untransduced iPSC-derived macrophages), iPSC-CAR macrophages, and iPSC-TF-CAR macrophages were co-cultured with HER-2 negative or positive tumor cell lines at different effector-to-target ratios (E:T ratio, 1:1, 5:1, 10:1).

[0027] Figure 8The study demonstrated the in vivo tumor-killing effect in tumor-bearing mice. A HER-2 positive tumor model was established in immunodeficient (NSG) mice. Tumors were targeted until they reached approximately 100 mm². 3 Mice were divided into four groups and injected via tail vein with PBS (model control group), iPSC-M (cell control group), iPSC-CAR (standard treatment group), or iPSC-TF-CAR (intensified treatment group), respectively. A. Tumor growth curve. From the start of treatment (day 0), tumor volume (mm) was measured and calculated periodically. 3 B. Kaplan-Meier survival curves. The survival status of mice in each group was recorded until the end of the experiment. Detailed Implementation

[0028] To facilitate understanding of the macrophages, their preparation methods, and applications described in this invention, specific embodiments are further described below, and certain technical and scientific terms are defined. Unless otherwise defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0029] In one aspect, the present invention provides a macrophage derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-M), wherein the macrophages: a) Expressing chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Overexpression of a combination of transcription factors including PU.1, IRF5 and BATF2.

[0030] As used herein, the terms “induced pluripotent stem cells” and “iPSC” are used interchangeably and refer to a class of stem cells that are pluripotent and have unlimited proliferative capacity, obtained from somatic cells by introducing specific reprogramming factors (typically including Oct4, Sox2, Klf4, and c-Myc).

[0031] In some embodiments, the induced pluripotent stem cells used in this invention are human induced pluripotent stem cells (hiPSCs).

[0032] In some embodiments, the induced pluripotent stem cells used in this invention can be obtained from publicly available cell banks or commercial suppliers.

[0033] In some embodiments, the induced pluripotent stem cells (iPSCs) of the present invention are modified induced pluripotent stem cells (iPSCs) comprising: a) Polynucleotides encoding chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Polynucleotides encoding PU.1, IRF5 and BATF2.

[0034] In some embodiments, the modified induced pluripotent stem cells (iPSCs) of the present invention are exogenously introduced with polynucleotides encoding chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and polynucleotides encoding PU.1, IRF5 and BATF2.

[0035] In some implementations, gene delivery methods include, but are not limited to, the use of lentiviruses, adeno-associated viruses, transposon systems (such as PiggyBac or Sleeping Beauty), or gene editing technologies (such as CRISPR / Cas9).

[0036] As used herein, the terms “macrophages derived from induced pluripotent stem cells (iPSCs)” and “iPSC-CAR-M” are used interchangeably and refer to a population of cells with macrophage phenotypes and functions obtained by inducing differentiation, expansion and maturation of induced pluripotent stem cells (iPSCs) in vitro through a specific combination of cytokines and culture conditions.

[0037] In some embodiments, the iPSC-CAR-M of the present invention expresses a chimeric antigen receptor (CAR) that specifically binds to tumor-associated antigens on the cell surface; and expresses enhanced levels of PU.1, IRF5, and BATF2 transcription factors intracellularly.

[0038] In some embodiments, in the iPSC-CAR-M of the present invention, CD80 and / or CD86-positive M1 phenotype macrophages account for 80% or more of the total cell number, preferably 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, or 8% or more of the total cell number. 7% or higher, 88% or higher of the total number of cells, 89% or higher of the total number of cells, 90% or higher of the total number of cells, 91% or higher of the total number of cells, 92% or higher of the total number of cells, 93% or higher of the total number of cells, 94% or higher of the total number of cells, 95% or higher of the total number of cells, 96% or higher of the total number of cells, 97% or higher of the total number of cells, 98% or higher of the total number of cells, and 99% or higher of the total number of cells.

[0039] In some embodiments, in the iPSC-CAR-M of the present invention, CD163 and / or CD206 positive M2 phenotype macrophages account for 20% or less of the total number of cells, preferably 20% or less of the total number of cells, 19% or less of the total number of cells, 18% or less of the total number of cells, 17% or less of the total number of cells, 16% or less of the total number of cells, 15% or less of the total number of cells, 14% or less of the total number of cells, 13% or less of the total number of cells, 12% or less of the total number of cells, 11% or less of the total number of cells, 7% or less of the total number of cells, 6% or less of the total number of cells, 5% or less of the total number of cells, 4% or less of the total number of cells, 3% or less of the total number of cells, 2% or less of the total number of cells, or 1% or less of the total number of cells.

[0040] In some implementations, the iPSC-CAR-M of the present invention can maintain the M1 polarization state after exposure to the corresponding tumor-associated antigen.

[0041] In some embodiments, compared with the absence of exposure to tumor-associated antigens, the iPSC-CAR-M of the present invention significantly increases the levels of secreted cytokines IL-1β, TNF-α and IL-12 after exposure to the corresponding tumor-associated antigens.

[0042] Specifically, after exposure to the corresponding tumor-associated antigen, the level of the cytokine IL-1β secreted by the iPSC-CAR-M of the present invention is at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, or at least 100 times that when not exposed to the tumor-associated antigen.

[0043] Specifically, after exposure to the corresponding tumor-associated antigen, the level of the cytokine TNF-α secreted by the iPSC-CAR-M of the present invention is at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, or at least 100 times that when not exposed to the tumor-associated antigen.

[0044] Specifically, after exposure to the corresponding tumor-associated antigen, the level of the cytokine IL-12 secreted by the iPSC-CAR-M of the present invention is at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times, at least 55 times, at least 60 times, at least 65 times, at least 70 times, at least 75 times, at least 80 times, at least 85 times, at least 90 times, at least 95 times, or at least 100 times that when not exposed to the tumor-associated antigen.

[0045] In some embodiments, compared with the absence of exposure to tumor-associated antigens, the iPSC-CAR-M of the present invention does not show significant changes in the levels of secreted cytokines IL-10 and / or TGF-β after exposure to the corresponding tumor-associated antigens.

[0046] In some embodiments, after exposure to the corresponding tumor-associated antigen, the level of IL-10 secreted by the iPSC-CAR-M of the present invention is within the range of ±10, ±20, ±30, ±40, ±50, ±60, ±70, ±80, ±90, ±100, ±110, ±120, ±130, ±140, ±150, ±160, ±170, ±180, ±190, ±200 of the IL-10 level secreted when not exposed to the tumor-associated antigen.

[0047] In some embodiments, after exposure to the corresponding tumor-associated antigen, the level of the cytokine TGF-β secreted by the iPSC-CAR-M of the present invention is within the range of ±10, ±20, ±30, ±40, ±50, ±60, ±70, ±80, ±90, ±100, ±110, ±120, ±130, ±140, ±150, ±160, ±170, ±180, ±190, ±200 of the level of the cytokine TGF-β secreted when not exposed to the tumor-associated antigen.

[0048] As used herein, in a numerical context, the terms “significant increase,” “significant decrease,” or “no significant change” are not limited to specific statistical methods and, unless otherwise stated, can be reasonably judged by those skilled in the art based on conventional experimental repeatability, error range, and actual observations.

[0049] As used herein, the terms "chimeric antigen receptor" and "CAR" are used interchangeably, referring to a genetically engineered artificial receptor that can be expressed on the cell surface. It typically possesses the following functional domains: a target antigen-binding domain for specific recognition of the target antigen; a transmembrane domain for anchoring the receptor to the cell membrane; and an intracellular signaling domain containing T cell activation signaling domains (such as CD3ζ) and intracellular domains of one or more co-stimulatory molecules (such as CD28, 4-1BB, OX40, etc.), enabling the receptor to trigger cell activation, proliferation, and effector functions upon binding to the antigen.

[0050] In some implementations, the CAR used herein has a target antigen binding region that specifically binds to tumor-associated antigens.

[0051] The term "tumor-associated antigen" as used in this article refers to antigens that are expressed at levels significantly higher in tumor tissues than in normal tissues, or that are expressed only in tumor tissues, including but not limited to: HER-2, CD33, CD30, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, etc. SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, phosphatidylinositol polysaccharide 3 (Glypican-3), or combinations thereof.

[0052] In some embodiments, the target antigen binding region comprises a heavy chain variable region and a light chain variable region of a monoclonal antibody. For example, the target antigen binding region is an scFv that specifically binds to tumor-associated antigens, and more specifically, the target antigen binding region is an scFv that specifically binds to HER-2.

[0053] In some implementations, the CAR described herein also includes a CD8α hinge and transmembrane region, a CD3ζ intracellular domain, and a MyD88 intracellular domain.

[0054] In some embodiments, the iPSC-CAR-M of the present invention overexpresses transcription factors PU.1, IRF5, and BATF2.

[0055] In the context of gene expression, the term "overexpression" refers to a significant increase in the expression level of a target gene relative to its endogenous expression level in control or untreated cells. This increase can be achieved by increasing gene copy number, enhancing transcriptional activity, or using strong promoters to drive the expression of exogenous genes.

[0056] Overexpression of a target gene can be achieved through a variety of means, including but not limited to: introducing exogenous expression vectors, expression cassettes, or promoters to drive stronger transcription; using viral vectors, plasmids, or other genetic engineering techniques to increase gene copy number; altering the cellular environment or using inducers to enhance gene expression; and deleting or inhibiting repressive regulatory elements to upregulate the expression of the target gene.

[0057] In some embodiments, compared with control cells that have not transduced the gene encoding the transcription factor PU.1, the iPSC-CAR-M of the present invention exhibits 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, and 32-fold increases in efficacy. Overexpression of transcription factor PU.1 at 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 times.

[0058] In some embodiments, compared with control cells that have not been transduced to encode the transcription factor IRF5, the iPSC-CAR-M of the present invention exhibits 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, and 32-fold increases in efficacy. Overexpression of transcription factor IRF5 at 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 times.

[0059] In some embodiments, compared with control cells that have not transduced the gene encoding the transcription factor BATF2, the iPSC-CAR-M of the present invention exhibits 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, and 32-fold improvements. BATF2 transcription factor overexpressed at 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, and 70 times.

[0060] As used in this article, the term "PU.1", short for Purine-rich box 1, also known as SPI1, is a transcription factor belonging to the ETS family. It regulates the differentiation of hematopoietic stem cells into specific blood cell lines, such as monocytes / macrophages and B cells, and is closely related to the regulation of immune cell development and function.

[0061] As used in this article, the term "IRF5," short for Interferon Regulatory Factor 5, is also a transcription factor that regulates the expression of genes involved in antiviral and inflammatory responses. In macrophages, IRF5 can promote the pro-inflammatory phenotype of the M1 phenotype, producing inflammatory factors such as TNF-α and IL-6.

[0062] As used in this article, the term "BATF2," short for Basic Leucine Zipper ATF-Like Transcription Factor 2, is a transcription factor belonging to the AP-1 / ATF family. In the immune system, BATF2 participates in regulating antiviral and antitumor responses. It can regulate the expression of inflammation-related genes and enhance the effector function of immune cells.

[0063] The iPSC-CAR-M provided by this invention can continuously maintain the pro-inflammatory M1 phenotype.

[0064] In another aspect, the present invention provides modified induced pluripotent stem cells (iPSCs) comprising: a) Polynucleotides encoding chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Polynucleotides encoding PU.1, IRF5 and BATF2.

[0065] One or more exogenous gene expression cassettes can be introduced into the genome of iPSCs using techniques well known in the art, thereby obtaining modified induced pluripotent stem cells (iPSCs). Such gene introduction methods include, but are not limited to, using lentiviruses, adeno-associated viruses, transposon systems (such as PiggyBac or Sleeping Beauty), or gene editing technologies (such as CRISPR / Cas9) to introduce exogenous sequences into the host cell chromosome.

[0066] In a specific implementation, the present invention provides a method for preparing modified induced pluripotent stem cells (iPSCs), which includes the following steps: i) Obtain induced pluripotent stem cells, ii) Transducing a lentiviral expression vector containing the nucleotide sequence encoding CAR into iPSCs yields CAR-iPSCs; and iii) A lentiviral expression vector containing nucleotide sequences encoding PU.1, IRF5 and BATF2 is transduced into the CAR-iPSC obtained in step ii) to obtain modified induced pluripotent stem cells CARTF-iPSC.

[0067] In some implementations, the order of steps ii) and iii) above can be interchanged.

[0068] In another aspect, the present invention provides a method for preparing iPSC-CAR-M, the method comprising the following steps: i) Obtain induced pluripotent stem cells, ii) Transducing a lentiviral expression vector containing a nucleotide sequence encoding CAR into iPSCs yields CAR-iPSCs; iii) A lentiviral expression vector containing nucleotide sequences encoding PU.1, IRF5, and BATF2 was transduced into the CAR-iPSCs obtained in step ii) to obtain modified induced pluripotent stem cells CARTF-iPSCs; and iv) Induce the differentiation of CARTF-iPSCs from step iii) into macrophages.

[0069] In a specific implementation scheme, step iv) includes: iv-1) The step of culturing modified induced pluripotent stem cells in vitro to induce them into monocytes; iv-2) The monocytes from step iv-1) were cultured in vitro to induce differentiation into M0 macrophages; and iv-3) In vitro exposure of M0 macrophages to appropriate polarizing factors under in vitro conditions to induce differentiation into a specific subtype M1.

[0070] Furthermore, step iv) also includes: iv-4) Perform phenotypic or functional verification on the macrophages.

[0071] In some embodiments, the iPSC-CAR-M provided by the present invention exhibits an enhanced M1 polarization state after recognizing a target antigen: it has an enhanced ability to secrete key anti-tumor cytokines IL-1β, TNF-α and IL-12; and an blocked ability to secrete key immunosuppressive cytokines IL-10 and TGF-β.

[0072] In another aspect, the present invention provides the use of macrophages derived from induced pluripotent stem cells (iPSC-CAR-M) or pharmaceutical compositions comprising them, according to the present invention, in the preparation of a medicament for treating cancer.

[0073] In some implementations, the cancer is selected from solid tumors or hematologic malignancies, such as breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, bladder cancer, kidney cancer, endometrial cancer, cervical cancer, glioma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms.

[0074] As used herein, the terms “subject” and “patient” are used interchangeably and include any living organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, and rabbit), and most preferably a human.

[0075] As used herein, the term "antibody" refers to any form of antibody that exhibits the desired biological or binding activity. Therefore, it is used in the broadest sense and specifically encompasses, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelified single-domain antibodies. "Parental antibody" is an antibody obtained by exposing the immune system to an antigen before modifying the antibody for its intended use, such as the humanization of antibodies used as human therapeutic agents.

[0076] As used herein, the term “treatment” includes treating a disease or condition described herein in a subject, such as a person, and includes: (i) suppressing the disease or condition, i.e. preventing its occurrence; (ii) alleviating the disease or condition, i.e. causing the condition to subside; (iii) slowing the progression of the disease; and / or (iv) suppressing, alleviating or slowing the progression of one or more symptoms of the disease or condition.

[0077] The terms “patient,” “subject,” “individual,” etc., are used interchangeably herein and, whether in vitro or in situ, refer to any animal or its cells that are suitable for the methods described herein. In some non-limiting embodiments, the patient, subject, or individual is a human being.

[0078] In the context of pharmaceutical compositions described herein, the term "simultaneous administration" means that the drugs may be administered at the same time, with a difference of no more than 30 minutes, through the same formulation or different formulations; The term "separate administration" means that each drug is administered separately, but designed as a combination therapy; the administration time may exceed 30 minutes.

[0079] The term "sequential administration" refers to the administration of drugs in a predetermined order; the second drug is administered within a certain time period after the first drug.

[0080] The routes of drug administration may be the same or different, including but not limited to: oral administration, intravenous administration, local administration, intratumoral administration, intramuscular administration, subcutaneous injection, intradermal administration, and intraperitoneal administration.

[0081] The present invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention. Experimental methods in the embodiments of the present invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material or product manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0082] Example 1: Construction of a lentiviral vector expressing a combination of CAR and transcription factors In this embodiment, the inventors constructed the following core expression carrier: i) Chimeric antigen receptor (CAR) sequence vectors: Their structure includes an extracellular antigen-binding domain (e.g., scFv), a transmembrane domain, and one or more intracellular signaling domains (e.g., CD3ζ chain and co-stimulatory molecule signaling domains such as 4-1BB or CD28); and ii) M1 polarization transcription factor combined sequence vector: This vector contains the coding sequences of three genes: PU.1, IRF5, and BATF2. These three genes can be co-expressed under a single promoter or driven by independent promoters by linking them using elements such as IRES (internal ribosome entry site) or 2A self-cleaving peptides. 。

[0083] CAR / Lentiviral vector CAR sequence design: Taking the scFv targeting human HER-2 as an example, a CAR structure was constructed containing the hinge region and transmembrane region of human CD8α, as well as the human TIR co-signaling domain. See the CAR structure section. Figure 1 See the lentiviral vector structure containing CAR for details. Figure 2 A.

[0084] Vector construction: The above-mentioned CAR sequence, P2A self-cleaved peptide sequence and ID3 sequence were synthesized using synthetic technology and delivered to Yunzhou Biotechnology Co., Ltd. (Guangzhou) for the construction of a third-generation lentiviral expression vector.

[0085] Lentiviral vectors with a combination of transcription factors The CDS sequences of PU.1, IRF5, and BATF2 were linked via a 2A peptide and synthesized, then delivered to Yunzhou Biotechnology Co., Ltd. (Guangzhou) for the construction of a third-generation lentiviral expression vector. For the structures of the multiple transcription factors, please refer to [link to relevant documentation]. Figure 1 For the structure of lentiviral vectors containing multiple transcription factors, see [link to relevant documentation]. Figure 2 B.

[0086] Each protein moiety has the following amino acid sequence: HER-2 scFv protein sequence: VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID NO: 1 G4S GGGGSGGGGSGGGGS SEQ ID NO: 2 VL DIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK SEQ ID NO: 3 CD8α hinge and transmembrane protein sequences (UniProt: P01732, aa 138-206): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDTYIWAPLAGTCGVLLLSLVITLYC SEQ ID NO: 4 CD3ζ intracellular domain protein sequence (UniProt: P20963, aa 31-163): LCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 5 MyD88 intracellular domain protein sequence (UniProt: Q99836, aa 148-296): FLSLPALEWTRLAQRAKRLLAQRAQRAWRRLAQRAQRVLLGAPGGAGGAAPAPGAASQAPAWQLLSRWPAPSRPAQPAPGPAPASSFSQLPLLSLVLPLAPAPAARPALSLAPASSLSPGSPASPAPQEPLSLKSNGIVTLVGFLRDPWCKYQMIWPRQQEACVWRSLEPGRPLGSLSL SEQ ID NO: 6 PU.1 protein sequence (UniProt: P17947): MLQACKMEGFPLVPPPSEDLVPYDTDLYQRQTHEYYPYLSSDGESHSDHYWDFHPHHVHSEFESFAENNFTELQSVQPPQLQQLYRHMELEQMHVLDTPMVPPHPSLGHQVSYLPRMCLQYPSLSPAQPSSDEEEGERQSPPLEVSDGEADGLEPGPGLLPGETGSKKKIRLYQFLLDLLRSGDMKDSIWWVDKDKGTFQFSSKHKEALAHRWGIQKGNRKKMTYQKMARALRNYGKTGEVKKVKKKLTYQFSGEVLGRGGLAERRHPPH SEQ ID NO: 7 IRF5 protein sequence (UniProt: Q13568): MNQSIPVAPTPPRRVRLKPWLVAQVNSCQYPGLQWVNGEKKLFCIPWRHATRHGPSQDGDNTIFKAWAKETGKYTEGVDEADPAKWKANLRCALNKSRDFRLIYDGPRDMPPQPYKIYEVCSNGPAPTDSQPPEDYSFGAGEEEEEEEELQRMLPSLSLTEDVKWPPTLQPPTLRPPTLQPPTLQPPVVLGPPAPDPSPLAPPPGNPAGFRELLSEVLEPGPLPASLPPAGEQLLPDLLISPHMLPLTDLEIKFQYRGRPPRALTISNPHGCRLFYSQLEATQEQVELFGPISLEQVRFPSPEDIPSDKQRFYTNQLLDVLDRGLILQLQGQDLYAIRLCQCKVFWSGPCASAHDSCPNPIQREVKTKLFSLEHFLNELILFQKGQTNTPPPFEIFFCFGEEWPDRKPREKKLITVQVVPVAARLLLEMFSGELSWSADSIRLQISNPDLKDRMVEQFKELHHIWQSQQRLQPVAQAPPGAGLGVGQGPWPMHPAGMQ SEQ ID NO: 8<s BATF2 protein sequence (UniProt: Q8N1L9): MHLCGGNGLLTQTDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQSLQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSCYPAQPLS PGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLTAQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLSSAQVHF SEQ ID NO: 9 T2A protein sequence: EGRGSLLTCGDVEENPG SEQ ID NO: 10 P2A protein sequence: GSGATNFSLLKQAGDVEENPGP SEQ ID NO: 11 Example 2: Preparation of a stably expressing genetically engineered iPSC mononuclear cell line In this embodiment, the inventors integrated the CAR vector targeting a specific tumor antigen constructed in Example 1 with a combination of key transcription factors that can synergistically promote macrophage polarization toward the M1 phenotype: PU.1, IRF5, and BATF2, into the same expression framework and introduced it into iPSCs.

[0087] 2.1 The resurgence of human-derived iPSCs Matrigel / VTN-coated six-well plates were brought to room temperature in a biosafety cabinet beforehand. 5 mL of hiPSC complete medium was added, along with 0.5 μl of Rho kinase inhibitor at a 1:10000 ratio, and brought to room temperature. One vial of frozen cells was removed and gently thawed in a 37°C water bath using forceps. The cells were removed once the ice crystals in the cell suspension were almost completely dissolved. The vial was wiped with 75% alcohol-based lint-free paper and transferred to the biosafety cabinet. The cell suspension was transferred to a pre-prepared 15 mL centrifuge tube. 5 mL of DMEM / F12 was pipette-loaded and added dropwise to the frozen cell suspension, gently agitating the cells during mixing. The tube was centrifuged at 200 g for 3 min. The supernatant was discarded, and 1 mL of pre-warmed Rho kinase inhibitor + hiPSC complete medium was added and mixed thoroughly, avoiding pipetting as much as possible. Cell counting was then performed. Discard the Matrigel / VTN coating medium from the six-well plate. First, seed 2 mL of Rho kinase inhibitor + hiPSC complete medium into each well. Seed the cells at a rate of 15-20 weeks per well. Mix three times horizontally using a cross-hatching method. Incubate at 37°C, 5% CO2 concentration, and saturated humidity. Mix three more times horizontally using a cross-hatching method. Incubate for 18-24 hours, then replace with fresh hiPSC complete medium. Change the medium every other day thereafter.

[0088] 2.2. Generation of Human-Derived iPSCs Prepare Matrigel / VTN-coated six-well plates by bringing them to room temperature in a biosafety cabinet beforehand. Prepare 2 mL / well of hiPSC complete medium according to the number of wells for subculturing, and add Rho kinase inhibitor at a 1:10000 ratio, then bring to room temperature. Aspirate the medium from the iPSC wells, add 2 mL / well of DPBS (calcium- and magnesium-free), gently shake, and aspirate. Add 1 mL / well of TrypLE express preheated to 37°C to completely cover the bottom of the well. Incubate at 37°C for 2 min. After digestion, gently return the cell culture plate to the biosafety cabinet, avoiding shaking the cells, and aspirate the TrypLE express solution by tilting. Immediately add 1 mL / well of preheated hiPSC complete medium, and gently shake the six-well plate horizontally in a cross shape to detach the cells from the substrate. Centrifuge, transferring the cell suspension to a 15 mL centrifuge tube at 300 g for 3 min. After counting and seeding, aspirate the Matrigel / VTN solution from the six-well plate, add 2 ml / well of pre-warmed Rho kinase inhibitor + hiPSC complete medium, and seed the desired cell volume into the six-well plate. Mix the six-well plate horizontally three times, incubate at 37°C, 5% CO2, and saturated humidity, and then mix again horizontally three times. Incubate overnight. Replace with fresh hiPSC complete medium after 18-24 hours, and then change the medium every other day. After 4-5 days, passage or cryopreserve.

[0089] Microscopic morphology of expanded iPSC cells is shown in Figure 1. Figure 3 .

[0090] 2.3 Lentiviral transduction The lentiviral vector constructed in Example 1 was co-transfected with the packaging plasmid into HEK293T cells, and the viral supernatant was collected and concentrated. This viral supernatant was then used to infect iPSC-mono cells in the logarithmic growth phase.

[0091] 2.3.1 Virus Packaging A four-plasmid coating system (pMD21, REV, vsvg, and the CAR lentiviral vector constructed in Example 1) was used. On the first day, cell passage was performed, with 5-6 × 10⁶ cells seeded in a 10 cm cell culture dish. 6293T cells were added to 9 ml of complete culture medium. The next day, 10 μg of the target plasmid was taken from a 1.5 mL EP tube. Volume x = 10 μg / plasmid concentration. In the same EP tube, 5 μg MD21, 2.5 μg REV, and 3.5 μg VSVG were added. In the same EP tube, 42 μl PEI (concentration 1 mg / ml) was added (42 μl PEI is used for co-transfection of 21 μg of plasmid, plasmid:PEI = 1:2). In the same EP tube, y volumes of transfection medium (opti-MEM or serum-free DMEM) were added, for a total transfection volume of 1 mL. After mixing thoroughly, the mixture was incubated for 10 min. The cells were tilted, and the entire contents were added to the cell supernatant along the cell wall.

[0092] On the morning of the third day, cell morphology and status were observed. In the afternoon, the medium was changed, all supernatant was discarded, and 5 mL of complete culture medium was added. After 36 hours, the virus was collected, all supernatant was collected, filtered through a 0.45 μm filter membrane, flash-frozen in liquid nitrogen, and then transferred to -80°C.

[0093] 2.3.2 Virus purification The virus was centrifuged at 120,000g for 2.5 hours using an ultra-high-speed centrifuge, and then resuspended in PBS.

[0094] 2.3.4 Lentiviral transfection Select iPSCs with good growth status and a confluence of approximately 70-80%. Plant the cells at a density of 2-5 x 10⁻⁶ cells / cm². 5 Inoculate pre-coated 6-well plates at a density of 10 cells / well. Add ROCK inhibitor to the inoculated culture medium to a final concentration of 10 μM. Calculate the viral load: Calculate the required viral volume based on the multiple of infection (MOI).

[0095] Calculation formula: Required viral volume (μL) = (MOI × total cell count) / viral titer (TU / mL) × 1000 If 2.5×10 5 One cell, with a viral titer of 1×10⁻⁶. 8 TU / mL, desired MOI of 20, then required virus volume = (20 × 2.5 × 10⁻⁶) / mL 5 ) / (1×10 8 ) × 1000 = 50 μL.

[0096] In a sterile centrifuge tube, dilute the calculated virus solution with fresh iPSC complete medium (without ROCK inhibitor). Add Polybrene to a final concentration of 5 μg / mL. Mix gently.

[0097] Next, aspirate the old culture medium from the iPSC wells. Slowly add the prepared virus and polybrene medium (approximately 1-1.5 mL per well) to the wells. Gently shake the culture plate to distribute the virus evenly. Place the culture plate back in a 37°C incubator and incubate for 18-24 hours. Forty-eight hours after infection, 1 ug / ml of puromycin was added for selection. Selection was continued for 7-10 days until all uninfected control cells died. Surviving single clones were picked, expanded, and cultured to establish a stable CAR-iPSC cell line.

[0098] A stable CAR-iPSC cell line was established by transfecting the CAR-iPSC cell line with TF virus according to the lentiviral transfection conditions described above. Forty-eight hours after infection, 400 ug / ml G418 was added for selection, and selection was continued for 7-10 days until all uninfected control cells died. Surviving single clones were selected, expanded, and cultured to establish a stable CAR-TF-iPSC cell line expressing PU.1, IRF5, and BATF2.

[0099] Example 3: Inducing stable CAR-iPSC cell lines to differentiate into CAR-macrophages In this embodiment, the inventors directed the differentiation of genetically modified iPSCs to obtain macrophages capable of co-expressing CAR and the above-mentioned transcription factor combination.

[0100] Monocyte induction On days -9 to -5, the cell line of Example 2 is cultured to 80-90% confluence. Under a microscope, the cell line of Example 2 should have obvious thickness and show no differentiation or aging.

[0101] On day -4, iPSCs in 1.2 × 6-well plates, 90% confluence. Wash once with 1 ml PBS / well, add 1 ml RelesR / well, let stand for 30 seconds, then discard the RelesR and incubate at 37°C for 4 minutes. Add 1 ml mTesR (with 5 μM Rocki) / well, gently agitate and pipette to detach the cells. Collect the cells using a 5 ml pipette and evenly distribute them into two 15 ml centrifuge tubes, avoiding blowing the cells apart. Centrifuge for 5 minutes to allow the cells to pellet. If cell pelleting is too slow, centrifuge at 50g for 30-60 seconds. Remove the supernatant using a pipette, leaving approximately 1 ml of culture medium + cells at the bottom of each tube. Take two 10 cm hypoadsorbent culture dishes and add 17 ml mTesR (with 5 μM Rocki) to each. Slowly add 2 ml of mTesR (with 5 μM Rocki) to two centrifuge tubes, and transfer the cell pellet to a 10 cm low-adsorption culture dish using a 5 ml centrifuge tube (final volume approximately 20 ml). Label the culture dish 1 / 2.

[0102] On day -3, mix 15 ml of mTesR medium with 5 ml of EBM. Shake the low-adsorption culture dish to concentrate the EB to the center, and aspirate the old medium using a 10 ml centrifuge tube. (If EB was aspirated, transfer the aspirated old medium to a 15 ml centrifuge tube, allow it to settle, discard the supernatant, add the solution, and then transfer the EB back to the culture dish). Add 20 ml of mTesR to culture dish 1. Add 20 ml of a 3:1 mTesR / EBM solution to culture dish 2. (The ratio after adding the solution is 3:1) On day -2, aspirate 10 ml of old culture medium from culture dish 1 and add 10 ml of mTesR. Aspirate 10 ml of old culture medium from culture dish 2 and add 10 ml of 1:3 mTesR / EBM solution. (The ratio after adding the solution is 1:1).

[0103] On day -1, aspirate 10 ml of old culture medium from culture dish 1 and add 10 ml of mTesR. Aspirate 10 ml of old culture medium from culture dish 2 and add 10 ml of EBM. (The ratio after replenishment is 1:3). Take two six-well plates, coat 18 wells with 1 ml / well of 0.1% Gelatin, and incubate at 37°C for 12-18 hours. Prepare 200 ml of monocyte basal medium (MBM): 200 ml DMEM; 22.4 ml FBS (heat-inactivated at 56°C for 30 minutes); 2.24 ml MEM-NEAA; 2.24 ml L-glutamine; 224 μl 2-mercaptoethanol, 55 mM. Filter through a 0.22 μM filter and store at 4°C for 4 weeks.

[0104] On day 0, prepare 40 ml of Monocyte Differentiation Medium 1 (MDM1): 40 ml of basal monocyte culture medium; 400 μl of M-CSF, 10 μg / ml; 100 μl of IL-3, 10 μg / ml. Collect all liquid from a 10 cm hypoadsorbent culture dish into a 50 ml centrifuge tube and centrifuge for 5 minutes to allow cell pelleting. If cell pelleting is too slow, centrifuge at 50 g for 30-60 seconds. Remove the supernatant with a pipette, leaving approximately 2 ml of EB suspension at the bottom. Add fresh EBM to approximately 5 ml. Remove the gelatin-coated six-well plate, discard the gelatin, and add 3 ml of MDM1 to each well. Seed a certain number of hematopoietic stem / progenitor cells (EB) into each well, and shake the plate to distribute them evenly. The diameter of the EB can be observed using a scale bar in photography software; 250-500 μm is ideal. Take a 24-well plate and add 0.5 ml of PBS to 3-6 wells. After disinfecting the scissors with 75% alcohol, cut off 2-3 mm from the tip of the 200 μl pipette. Shake the EB suspension to mix thoroughly, then take the EB. Inoculate one six-well plate into each of culture dishes 1 and 2.

[0105] On days 3, 6, and 8, without tilting the plate, aspirate half of the culture medium (1.5 ml / well) with a pipette and prepare and replenish with fresh MDM1 (20 ml).

[0106] On days 10 and 12, prepare 40 ml of monocyte differentiation medium 2 (MDM2): 40 ml monocyte basal medium; 200 μl M-CSF, 10 μg / ml; 100 μl IL-3, 10 μg / ml. Without tilting the plate, aspirate half of the medium (1.5 ml / well) with a pipette and add fresh MDM2.

[0107] On days 16, 19, and 23, all culture medium was collected and fresh MDM2 was added. After cell counting, the cells were centrifuged at 100g for 5 minutes, and the supernatant was discarded to obtain CAR-expressing mononuclear cells (CAR-iPSC-mono), which were then cryopreserved.

[0108] Macrophage induction: CAR-expressing monocytes derived from iPSCs differentiate into M0 macrophages; Prepare the macrophage differentiation medium, which contains... RPMI-1640; 10% FBS; 1% penicillin-streptomycin, and 50 ng / mL M-CSF.

[0109] Harvesting and counting monocytes: Gently pipette the culture dish to collect suspended and loosely adherent iPSC-derived monocytes. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 300 x g for 5 minutes. Discard the supernatant, resuspend the cells in pre-chilled PBS, and centrifuge again to wash the cells. Resuspend the cells in a small amount of differentiation medium and perform viable cell counting using trypan blue staining.

[0110] Planking: Based on the cell count results, seed monocytes at a density of 0.5 - 1.0 x 10⁶ cells / mL in tissue culture plates. Gently shake the culture plate to distribute the cells evenly.

[0111] Induced differentiation: Place the culture plate in a 37°C, 5% CO2 incubator. Day 3: Half medium change. Carefully aspirate half of the old culture medium and add an equal volume of fresh, preheated differentiation medium (containing 50 ng / mL M-CSF). Days 5-7: Observe cell morphology. At this time, the cells should be fully adhered to the plate, have increased in size, and exhibit typical round "fried egg" or irregular amoeboid morphology as M0 macrophages.

[0112] Polarization of M0 macrophages towards the M1 subtype This stage occurs after M0 macrophage differentiation is complete and takes approximately 24-48 hours.

[0113] Prepare polarization medium: M1 polarization medium: Add 100 ng / mL LPS and 20 ng / mL IFN-γ to fresh M0 differentiation medium. Polarization: Carefully aspirate all the old culture medium from the 6-well plate. Gently rinse the cells once with sterile PBS to remove residual cytokines and metabolic waste. Add M1 polarization medium to the corresponding wells according to the experimental group. Return the culture plate to the incubator and continue culturing for 24-48 hours.

[0114] Amplification culture was carried out for subsequent experiments.

[0115] The expression of monocyte surface markers after iPSC virus transfection and the bright-field plot of macrophage induction are shown in the figure. Figure 4In samples A and B, the results showed that CAR-iPSC and CAR-TF-iPSC cells could still maintain a monocyte state. Through macrophage induction culture, CAR-iPSC and CAR-TF-iPSC cells could be successfully induced to differentiate into macrophages.

[0116] Example 4: Characterization of iPSC-derived macrophages (iPSC-CAR-M) Phenotypic identification: The expression rate of CARs on the surface of differentiated mature macrophages, as well as the expression levels of M1 phenotypic markers (CD80, CD86) and M2 phenotypic markers (CD163, CD206), were detected by flow cytometry. The expression of intracellular transcription factors (PU.1, IRF5, BATF2) proteins or mRNAs was verified by Western blotting or RT-qPCR.

[0117] 4.1 Macrophage phenotype and CAR protein identification Collect cells from the experimental and control groups in the logarithmic growth phase. Gently resuspend / scrape adherent cells using a pipette or a gentle cell scraper. Wash the cell suspension twice with pre-chilled PBS (centrifuge at 300g for 5 minutes at 4°C) to remove residual culture medium. Resuspend the cells in 100 μL of pre-chilled FACS Buffer and perform cell counting, adjusting the cell concentration to approximately 5 × 10⁶ cells per tube. 5 - 1×10 6 Each cell.

[0118] The following tubes are prepared for antibody staining: Unstained tubes: Contain only cells.

[0119] Single-positive tubes (for compensation regulation): single-staining tubes containing CD45, CD80, CD186, CD163, CD206, and CAR antibodies, respectively.

[0120] Isotype Control: All Isotype antibodies that match the full-stain tubes are added.

[0121] Full-stain tubes: After adding CD45, antibodies against CD80, CD186, CD163, CD206, and CAR were added separately. Full-stain tubes were prepared for both experimental and control groups.

[0122] Add the corresponding fluorescent antibody or isotype control antibody to each tube at the concentration recommended in the antibody's instructions. Incubate at 4°C in the dark for 30 minutes. Add 1 mL of FACS buffer and centrifuge at 300g for 5 minutes at 4°C. Discard the supernatant. Repeat the washing process once. Resuspend the cells in 300-500 μL of FACS buffer.

[0123] Flow cytometry was used for detection. Cell voltage was first adjusted using blank tubes, followed by fluorescence compensation using single-positive tubes (or compensating microspheres). Data were collected for each sample, with at least 10,000 cellular events recorded per sample.

[0124] The positivity rate of CAR protein was analyzed in a population of CD45+ macrophages.

[0125] 4.2 Confirmation of PU.1, IRF5, and BATF2 expression by protein imprinting Collect approximately 2-5 × 10 6 Cells were divided into experimental and control groups. The cells were washed twice with PBS. 100-200 μL of RIPA lysis buffer containing the inhibitor was added to each tube, and the tubes were incubated on ice for 15-30 minutes, vortexing several times during lysis. The tubes were centrifuged at 12,000g for 15 minutes at 4°C, and the supernatant was collected as the total protein solution. Protein concentration was determined using a BCA assay kit, and the protein concentration of all samples was adjusted to be consistent based on the results.

[0126] Mix an equal amount of protein (20-30 μg / well) with the loading buffer and heat at 95-100°C for 5-10 minutes to denature it. Load the sample and protein marker onto an SDS-PAGE gel and perform SDS-PAGE electrophoresis.

[0127] The proteins in the gel were electrotransferred to the PVDF membrane. After the transfer was completed, the PVDF membrane was placed in 5% skim milk or BSA (diluted with TBST) and blocked at room temperature for 1-2 hours.

[0128] Wash the membrane three times with TBST, 5-10 minutes each time. Incubate the membrane overnight at 4°C with diluted primary antibodies (anti-PU.1, IRF5, BATF2 antibodies, and anti-internal control antibody). The next day, wash the membrane three times with TBST, 10 minutes each time. Add HRP-labeled secondary antibody and incubate at room temperature for 1-2 hours. Wash the membrane thoroughly three to four times with TBST, 10 minutes each time. Add ECL chemiluminescence buffer, expose the membrane on a chemiluminescence imaging system, and acquire images.

[0129] 4.3 Confirmation of PU.1, IRF5, and BATF2 expression by qPCR Collect approximately 1-2 × 10 6 There were 10 experimental and control groups of cells. Total RNA was extracted using the Trizol method or centrifugation column method. The concentration and purity of RNA were determined using an ultra-micro spectrophotometer (such as NanoDrop) (A260 / A280 should be between 1.8 and 2.0).

[0130] Take 1 μg of total RNA and, according to the reverse transcription kit instructions, remove genomic DNA (gDNA) and synthesize first-strand cDNA.

[0131] Real-time quantitative PCR (qPCR) was performed as follows: In a qPCR reaction tube, SYBR GreenMaster Mix, forward primer, reverse primer, cDNA template, and nuclease-free water were added sequentially. Groups were set up: experimental group and control group, with three technical replicates for each sample. A template-free control (NTC) was also included to monitor for contamination.

[0132] The qPCR reaction conditions and primers used are shown in Tables 1 and 2 below.

[0133] Table 1 Table 2 Raw Ct values ​​were exported for data analysis. The relative expression levels of PU.1, IRF5, and BATF2 mRNA were calculated using the 2-ΔΔCt method.

[0134] ΔCt = Ct(PU.1 or IRF5 or BATF2) - Ct(internal reference gene) ΔCt = ΔCt(experimental group) - ΔCt(control group) Relative expression factor = 2^(-ΔΔCt).

[0135] The results show Figure 5 middle.

[0136] Example 4: Detection of cytokine secretion from iPSC-derived macrophages (iPSC-CAR-M) The levels of pro-inflammatory cytokines (IL-1β, TNF-α, IL-12, IL-6) and anti-inflammatory cytokines (IL-10, TGF-β) secreted by macrophages after stimulation by tumor cells were detected using ELISA.

[0137] Experimental Groups: Background control group 1: CAR-iPSC cultured alone.

[0138] Background control group 2: CAR-TF-iPSC cultured alone.

[0139] Background control group 3: HER-2 positive tumor cells (MDA-MB-231) cultured alone.

[0140] Background control group 4: HER-2 negative tumor cells (MDA-MB-468) cultured alone.

[0141] Experimental group A: CAR-iPSC + MDA-MB-231.

[0142] Experimental group B: CAR-TF-iPSC + MDA-MB-231.

[0143] Negative control group A: CAR-iPSC + HER-2 negative tumor cells (MDA-MB-468).

[0144] Negative control group B: CAR-TF-iPSC + HER-2 negative tumor cells (MDA-MB-468).

[0145] Non-specific activation control group: Untransduced iPSC-macrophages (control) + MDA-MB-231.

[0146] Specifically, prepare each group of effector cells (CAR-M) and target cells (tumor cells). In a 96-well plate, mix effector cells and target cells at an effector-to-target ratio (E:T ratio) of 5:1. The total volume per well is 200 μL. Set up all control groups simultaneously. Incubate the culture plate at 37°C in a 5% CO2 incubator for 24 hours. After incubation, centrifuge the culture plate at 400g for 5 minutes, carefully aspirate 150 μL of the supernatant into a new EP tube, and store at -80°C for later use. Perform ELISA assays according to the kit instructions to detect the concentrations of IL-1β, TNF-α, IL-12, IL-60, IL-10, and TGF-β in the supernatant.

[0147] The results show Figure 6 middle.

[0148] In the detection of all six cytokines, the secretion levels of both the negative control group (co-cultured with HER2- cells) and the non-specific control group (Control + HER2+) were at very low baseline levels. This further confirms that cytokine secretion is strictly dependent on the specific binding of CAR molecules to the HER2 antigen, ruling out the possibility of non-specific activation. The results reveal a key shortcoming of conventional CAR-M therapy: its activation is an incomplete, M1 / M2 mixed functional state. While attacking the tumor, it also secretes immunosuppressive factors, which may "self-limit" and affect long-term efficacy. Transcription factor engineering successfully interrupted the M2-related pathway, solving this problem. Compared with conventional CAR-iPSC macrophages, CAR-TF-iPSC macrophages exhibit a more functionally pure and ideal M1 polarization state after recognizing the target antigen: pro-inflammatory function is greatly enhanced: its ability to secrete key anti-tumor cytokines (IL-1β, TNF-α, IL-12) is amplified several times. The anti-inflammatory function was almost completely shut down: its ability to secrete key immunosuppressive cytokines (IL-10, TGF-β) was effectively blocked.

[0149] Example 5: In vitro killing effect of iPSC-derived macrophages (iPSC-CAR-M) iPSC-CAR and iPSC-TF-CAR were co-cultured with HER-2 positive or negative tumor cells at different effector-target ratios, and their specific killing ability against tumor cells was detected by the CCK-8 killing assay.

[0150] Experimental Groups: Experimental groups: iPSC-CAR + HER-2+ group, iPSC-TF-CAR + HER-2+ group.

[0151] Specific control groups: iPSC-CAR + HER-2- group, iPSC-TF-CAR + HER-2- group.

[0152] Control group: Untransduced iPSC-M + HER-2+, Untransduced iPSC-M + HER-2-.

[0153] Background group: Only HER-2+ tumor cells (added to culture medium).

[0154] Natural release group: only HER-2+ tumor cells (with added lysis buffer).

[0155] Specifically, target cells were seeded in 96-well white-walled plates, with 1 x 10 cells per well. 4Target cells expressing luciferase were selected. Serially diluted CAR-M cell suspensions were added to wells containing target cells at effector-target ratios of 10:1, 5:1, and 1:1. All control groups were set up. The cells were incubated at 37°C in a 5% CO2 incubator for 24–48 hours. After incubation, CCK-8 was added to each well, and the cells were incubated at room temperature in the dark for 10 minutes. The chemiluminescence signal was detected using a multi-mode microplate reader.

[0156] The kill rate is calculated using the following formula: Specific kill rate (%) = [1 - (experimental group - background) / (natural release group - background)] x 100% The results show Figure 7 middle.

[0157] In the control group, unmodified macrophages showed very low tumor cell killing rates (<10%) at all effector-to-target ratios, indicating that they lacked effective anti-tumor capabilities. The iPSC-CAR group exhibited clear effector-to-target ratio-dependent killing. As the effector cell ratio increased, the killing rate steadily increased from approximately 25% at a 1:1 ratio to approximately 70% at a 10:1 ratio. This demonstrates that conventional CAR-iPSC macrophages can effectively and specifically kill target cells. The iPSC-TF-CAR group exhibited the strongest killing activity. At every effector-to-target ratio, its killing rate was significantly higher than that of the conventional iPSC-CAR group. Particularly at the highly efficient 10:1 effector-to-target ratio, its killing rate approached 100%, almost completely eliminating tumor cells. Compared to the killing effect shown in Figure A, in Figure B, all three cell groups (including the most functional iPSC-TF-CAR) showed very low killing rates against HER-2 negative tumor cells (generally below 20%), with no significant difference from the control group. Compared with conventional CAR-iPSC macrophages, iPSC-TF-CAR macrophages showed significantly enhanced in vitro antitumor activity, exhibiting an overwhelming advantage in both killing efficiency (low E:T ratio) and killing potential (high E:T ratio).

[0158] Example 6: In vivo efficacy of iPSC-derived macrophages (iPSC-CAR-M) in animals A human tumor xenograft model (HER-2 positive tumor cells, MDA-MB-231) was established in immunodeficient mice. Once the tumor reached a certain size, the iPSC-CAR-M of this invention was injected via the tail vein or intratumorally, and multiple control groups were set up (e.g., injection of unmodified macrophages, injection of iPSC-M expressing only CAR, etc.). Tumor volume was measured periodically, and Kaplan-Meier survival curves were plotted.

[0159] Experimental groups (n=8 mice per group): Immunodeficient mice (NSG mice, 6-8 weeks old): Model control group (Vehicle): Tumor-bearing mice injected with PBS.

[0160] Cell control group: Tumor-bearing mice injected with untransduced iPSC-M.

[0161] Standard treatment group: tumor-bearing mice injected with iPSC-CAR.

[0162] Intensive treatment group: Tumor-bearing mice injected with iPSC-TF-CAR.

[0163] Specifically, 5x10 mg was injected subcutaneously into the right side of the mouse. 6 A number of tumor cells were used to establish a tumor model. The tumor was further developed when its average volume reached approximately 100 mm². 3 Mice were randomly divided into 4 groups. Each group was injected with 1x10g of the drug via the tail vein. 7 Macrophages (or PBS) of the corresponding group were collected. The long diameter (L) and short diameter (W) of the tumor were measured using calipers 2-3 times per week, and the tumor volume (V = L x W) was calculated. 2 / 2). Simultaneously monitor mouse weight and health status. When the tumor volume in the model control group mice reaches the preset endpoint (e.g., 2000 mmHg). 3 All mice were euthanized when the experiment reached the predetermined number of days (e.g., 4-6 weeks).

[0164] The results show Figure 8 middle.

[0165] Compared to conventional iPSC-CAR therapy, transcription factor-engineered iPSC-TF-CAR can not only control tumors more effectively, but can even completely eliminate established solid tumors, thus bringing more significant and longer-term survival benefits.

Claims

1. An induced pluripotent stem cell (iPSC)-derived macrophage (iPSC-CAR-M), wherein, The macrophages: a) express a chimeric antigen receptor (CAR) that specifically binds to a tumor- associated antigen; and b) overexpress a combination of transcription factors comprising PU.1, IRF5 and BATF2; Preferably, The macrophages derived from induced pluripotent stem cells (iPSCs) are capable of maintaining the M1 phenotype before or after contacting the corresponding tumor-associated antigen.

2. The macrophages derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-Ms) according to claim 1, wherein, 80% or more, preferably 85% or more, more preferably 90% or more, further more preferably 95% or more of the macrophages in the macrophages derived from induced pluripotent stem cells (iPSCs) exhibit the CD80 and / or CD86 positive M1 phenotype, and 20% or less, preferably 15% or less, more preferably 10% or less, further more preferably 5% or less of the macrophages exhibit the CD163 and / or CD206 positive M2 phenotype; Preferably, the levels of the cytokines IL-1 β, TNF-α and TNF-α secreted by the macrophages derived from induced pluripotent stem cells (iPSCs) after contacting the corresponding tumor-associated antigen are at least 40-fold, at least 50-fold and at least 30-fold, respectively, of the levels when not contacting the tumor-associated antigen; and, the levels of the cytokines IL-10 and / or TGF-β secreted by the macrophages derived from induced pluripotent stem cells (iPSCs) after contacting the corresponding tumor-associated antigen are unchanged.

3. The macrophages derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-Ms) according to claim 1, wherein, the induced pluripotent stem cells (iPSCs) are human induced pluripotent stem cells (iPSCs); Preferably, the induced pluripotent stem cells (iPSCs) are modified induced pluripotent stem cells (iPSCs); More preferably, the modified induced pluripotent stem cells (iPSCs) comprise: a) a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds to a tumor-associated antigen; and b) a polynucleotide encoding PU.1, IRF5 and BATF2.

4. A modified induced pluripotent stem cell (iPSC) comprising: a) a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically binds to a tumor-associated antigen; and b) a polynucleotide encoding PU.1, IRF5 and BATF2.

5. The macrophages derived from induced pluripotent stem cells (iPSCs) (iPSC-CAR-Ms) according to any one of claims 1-3, or the modified induced pluripotent stem cells (iPSCs) according to claim 4, wherein: the chimeric antigen receptor (CAR) comprises: a target antigen binding region that specifically binds to a tumor-associated antigen, a CD8a hinge and transmembrane region, a CD3 zeta endodomain and a MyD88 endodomain; Preferably, the target antigen binding region is a scFv that specifically binds to a tumor-associated antigen; More preferably, the tumor associated antigen is selected from the group consisting of: HER-2, CD33, CD30, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor alpha), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-alpha, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN9, CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, phosphatidylinositol-glycan 3 (Glypican-3), or a combination thereof.

6. A method of preparing the induced pluripotent stem cell (iPSC)-derived macrophage (iPSC-CAR-M) of any one of claims 1-3, the method comprising: inducing the modified induced pluripotent stem cell (iPSC) of claim 4 to differentiate into a macrophage; optionally, the method comprises the steps of: i) obtaining an induced pluripotent stem cell, ii) transducing a lentiviral expression vector comprising a nucleotide sequence encoding a CAR into the iPSC, resulting in a CAR-iPSC; iii) differentiating the CAR-iPSC into a macrophage. iii) A lentiviral expression vector containing nucleotide sequences encoding PU.1, IRF5, and BATF2 was transduced into the CAR-iPSCs obtained in step ii) to obtain modified induced pluripotent stem cells CARTF-iPSCs; and iv) Induce the differentiation of CARTF-iPSCs from step iii) into macrophages; The order of steps ii) and iii) can be interchanged.

7. The method of claim 6, wherein Step iv) includes: iv-1) The step of culturing modified induced pluripotent stem cells in vitro to induce them into monocytes; iv-2) The monocytes from step iv-1) were cultured in vitro to induce differentiation into M0 macrophages; and iv-3) In vitro exposure of M0 macrophages to appropriate polarizing factors under in vitro conditions to induce differentiation into a specific subtype M1; Optionally, step iv) further includes: iv-4) Perform phenotypic or functional verification on the macrophages.

8. A pharmaceutical composition comprising macrophages derived from induced pluripotent stem cells (iPSC-CAR-M) as described in any one of claims 1-3 and a pharmaceutically acceptable carrier.

9. Use of the macrophage derived from induced pluripotent stem cells (iPSC-CAR-M) according to any one of claims 1-3 or the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating cancer; Preferably, the cancer is selected from: Solid tumors or hematologic malignancies; More preferably, the cancer is selected from: Breast cancer, colorectal cancer, gastric cancer, non-small cell lung cancer, small cell lung cancer, melanoma, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, bladder cancer, kidney cancer, endometrial cancer, cervical cancer, glioma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, and myeloproliferative neoplasms.

10. A macrophage (CAR-M), wherein, The macrophages: a) Expressing chimeric antigen receptors (CARs) that specifically bind to tumor-associated antigens; and b) Overexpression of a combination of transcription factors including PU.1, IRF5, and BATF2; The macrophages are able to maintain the M1 phenotype before or after contact with the corresponding tumor-associated antigens.

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