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WO2025242112A1 - Cellule ingénierisée universelle, son procédé de production et son utilisation - Google Patents

Cellule ingénierisée universelle, son procédé de production et son utilisation

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Publication number
WO2025242112A1
WO2025242112A1 PCT/CN2025/096192 CN2025096192W WO2025242112A1 WO 2025242112 A1 WO2025242112 A1 WO 2025242112A1 CN 2025096192 W CN2025096192 W CN 2025096192W WO 2025242112 A1 WO2025242112 A1 WO 2025242112A1
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WO
WIPO (PCT)
Prior art keywords
cells
engineered
cell
hla
molecules
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Pending
Application number
PCT/CN2025/096192
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English (en)
Chinese (zh)
Inventor
梁德生
陈艳
唐齐玉
朱冠山
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Pinpoint Medical Technology Co Ltd
Original Assignee
Shanghai Pinpoint Medical Technology Co Ltd
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Publication of WO2025242112A1 publication Critical patent/WO2025242112A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This invention relates to the field of cell technology, and in particular to a universal engineered cell, its preparation method, and its application.
  • hPSCs Human pluripotent stem cells
  • MSCs mesenchymal stem cells
  • HLA genes as the major histocompatibility complex (MHC) provide T cells with exogenous and endogenous peptides.
  • MHC major histocompatibility complex
  • the MHC mainly consists of classical class I and class II molecules.
  • Classical class I genes HLA-A, HLA-B, and HLA-C
  • classical class II genes HLA-DP, HLA-DQ, and HLA-DR
  • HLA-DP human leukocyte antigens
  • Each polymorphic HLA gene contains numerous alleles, making it difficult to find cell, organ, or tissue donors that match a specific pair of HLA alleles.
  • existing technologies typically use immunosuppressants on patients to address immune rejection caused by HLA haplotype mismatch.
  • long-term use of immunosuppressive drugs often leads to serious side effects.
  • CD47 is a ubiquitous membrane protein that interacts with various cell surface receptors to suppress immune responses. It is involved in multiple functions, including neutrophil migration, axonal extension, and T-cell co-stimulation. Furthermore, CD47 can interact with the receptor SIRP ⁇ on immune cells to inhibit immune responses. Therefore, CD47 can provide a "don't eat me” signal to ensure that autologous cells are not recognized and mistakenly eliminated.
  • iPSCs low-immunogenic induced pluripotent stem cells
  • lentiviruses for transduction results in relatively low infection efficiency, especially in mesenchymal stem cells or iPSCs and their derivatives, where achieving high levels of CD47 expression may be difficult and may pose safety risks due to gene insertion mutations. This approach cannot achieve good and safe ultra-low immunogenicity.
  • this invention aims to construct a universal engineered super low immunogenic cells (SLIC) that can be used without any immunosuppressants, and to provide a universal cell source for allogeneic cell therapy.
  • SLIC super low immunogenic cells
  • this invention provides a universal engineered cell and its preparation method.
  • the present invention provides an engineered cell that, relative to wild-type cells, overexpresses CD47 molecules;
  • the CD47 molecule distribution density on the surface of the engineered cell is not less than 2700 molecules/ ⁇ m2 .
  • the density of CD47 molecules on the cell surface is not less than 2800, or not less than 2900, or not less than 3000 molecules/ ⁇ m2 .
  • the engineered cells have lower immunogenicity or higher immune tolerance compared to wild-type cells or cells with a CD47 molecule surface distribution density of less than 2700 molecules/ ⁇ m2 .
  • the engineered cells exhibit immune tolerance to a variety of immune cells.
  • the immune cells include, but are not limited to, one or more of the following: PBMC cells, T cells, NK cells, macrophages, neutrophils, eosinophils, mast cells, and dendritic cells.
  • the immune cells include one or more of PBMC cells, T cells, and NK cells. More preferably, the immune cells include all of T cells, PBMC cells, and NK cells.
  • the immune cells are autologous cells or allogeneic cells for the individual.
  • the engineered cells have a longer in vivo survival time compared to wild-type cells or cells with a CD47 molecule surface distribution density of less than 2700 molecules/ ⁇ m2 .
  • the in vivo survival time is at least 2 days.
  • the in vivo survival time is at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 2 weeks, 3 weeks, 4 weeks, or longer.
  • the engineered cells include engineered stem cells.
  • the engineered stem cells include engineered mesenchymal stem cells, engineered induced pluripotent stem cells and their derivative cells.
  • the engineered mesenchymal stem cells are derived from pluripotent stem cells, and more preferably, the pluripotent stem cells are selected from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the engineered mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood, or umbilical cord.
  • the derived cells include CAR-iNK, dopaminergic neural progenitor cells, CAR-iMac, cardiomyocytes, endothelial progenitor cells, iNK cells, retinal cells, nerve cells, osteoblasts, hematopoietic stem cells, blood cells, ⁇ cells, T cells, fibroblasts, hair cells, monocytes, macrophages, Treg cells, renal progenitor cells, lung epithelial cells, endothelial cells, megakaryocytes, smooth muscle cells, skeletal muscle cells, chondrocytes, osteocytes, adipocytes, hepatocytes, pancreatic islet cells, keratinocytes, melanocytes, and dendritic cells.
  • the engineered cells are engineered mesenchymal stem cells.
  • a single engineered mesenchymal stem cell expresses at least 1.9 million CD47 molecules.
  • a single engineered mesenchymal stem cell expresses at least 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.6 million, 2.7 million, 2.8 million, 2.9 million, or 3 million CD47 molecules.
  • the engineered mesenchymal stem cells have lower immunogenicity or higher immune tolerance compared to wild-type mesenchymal stem cells or mesenchymal stem cells with fewer than 1.9 million CD47 molecules distributed on the surface of a single cell.
  • the engineered mesenchymal stem cells have a longer in vivo survival time compared to wild-type mesenchymal stem cells or mesenchymal stem cells with fewer than 1.9 million CD47 molecules distributed on the surface of a single cell.
  • the CD47 molecule is selected from modified or unmodified natural CD47 molecules or variants thereof.
  • Natural CD47 molecules include, but are not limited to, naturally occurring different CD47 isotypes. Variants of CD47 molecules perform the same immune tolerance function as natural CD47 molecules, including, but not limited to, different forms of truncated CD47 molecules, fusion proteins that retain CD47 immune tolerance function, or CD47 mutants with insertions, substitutions, or deletions of one or more amino acids in the amino acid sequence of natural CD47 molecules.
  • Modifications to CD47 molecules include, but are not limited to, glycosylation, glutamine cyclization, phosphorylation, ubiquitination, and other post-translational modifications.
  • the expression of HLA-I and/or HLA-II molecules in the engineered cells is regulated.
  • the regulated expression is either non-expression or reduced expression.
  • the genes related to HLA-I and/or HLA-II molecules in the engineered cells include, but are not limited to, HLA-A, HLA-B, HLA-C, B2M, HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, CIITA, RFXANK, RFXAP, NLRC5, HDACs, PSMB, HLA-DM, HLA-DO, HLA-DRA, SUGT1, and FoxO1.
  • the genes related to HLA-I and/or HLA-II molecules in the engineered cells include B2M and CIITA.
  • the expression of CD54 and/or CD58 in the engineered cells is regulated.
  • the regulated expression is either non-expression or reduced expression.
  • nucleic acids encoding CD47 molecules are specifically integrated into one or more gene loci of the engineered cell genome.
  • the gene locus is selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, genes related to HLA-I molecules, genes related to HLA-II molecules, or rDNA regions; more preferably, the gene locus is selected from one or more of genes related to HLA-I molecules, AAVS1, genes related to HLA-II molecules, or rDNA regions; even more preferably, the gene locus is multiple genes related to HLA-I molecules, AAVS1, genes related to HLA-II molecules, or rDNA regions; even more preferably, genes related to HLA-I molecules and/or genes related to HLA-II molecules are used; most preferably, the gene locus is B2M and/or CIITA.
  • the engineered cells are autologous or allogeneic to the individual.
  • the present invention provides a method for preparing the above-mentioned engineered cells, comprising introducing a nucleic acid encoding the CD47 molecule into cells to obtain the engineered cells.
  • the nucleic acid encoding the CD47 molecule is site-specifically integrated into one or more gene loci in the cell genome, and the engineered cells are obtained by screening the expression level of CD47 on the surface of monoclonal cells.
  • the gene locus is selected from one or more of AAVS1, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, genes related to HLA-I molecules, genes related to HLA-II molecules, or rDNA regions; more preferably, the gene locus is selected from one or more of genes related to HLA-I molecules, AAVS1, genes related to HLA-II molecules, or rDNA regions; even more preferably, the gene locus is multiple genes related to HLA-I molecules and/or genes related to HLA-II molecules; most preferably, the gene locus is B2M and/or CIITA.
  • the invention further includes knocking out one or more of the following genes in the cell: genes related to HLA class I molecules, genes related to HLA class II molecules, CD54 gene, and CD58 gene.
  • the genes related to HLA-I and/or HLA-II molecules include, but are not limited to, HLA-A, HLA-B, HLA-C, B2M, HLA-DP, HLA-DQ, HLA-DR, TAP1, TAP2, LMP2, LMP7, RFX5, CIITA, RFXANK, RFXAP, NLRC5, HDACs, PSMB, HLA-DM, HLA-DO, HLA-DRA, SUGT1, FoxO1, etc.
  • the introduction of the nucleic acid encoding the CD47 molecule and the knockout of the gene are carried out simultaneously or in steps in the cell.
  • the nucleic acid encoding the CD47 molecule is integrated into gene loci associated with HLA-I and/or HLA-II molecules.
  • the nucleic acid encoding the CD47 molecule is integrated into the B2M site and/or the CIITA site.
  • gene editing tools are used to introduce nucleic acids or knock out genes.
  • the gene editing tool includes the Cre-lox system, Zinc Finger Nucleases (ZFNs), CRISPR-Cas or Transcription Activator-Like Effector Nucleases (TALENs), preferably CRISPR-Cas or TALENs.
  • ZFNs Zinc Finger Nucleases
  • TALENs Transcription Activator-Like Effector Nucleases
  • a non-viral method is used to introduce nucleic acids or knock out genes.
  • the present invention provides a composition comprising the engineered cells described above or cells obtained by the preparation method described above.
  • the present invention provides the use of the above-described engineered cells and compositions in the preparation of medicaments for treating diseases.
  • the disease includes, but is not limited to, tumors, infectious diseases, immune rejection, autoimmune diseases, genetic diseases, neurological diseases, metabolic diseases, fibrotic diseases, or tissue regeneration.
  • the tumors include, but are not limited to, skin cancer, lung cancer, breast cancer, prostate cancer, stomach cancer, colon cancer, rectal cancer, malignant mesenchymal tumors originating from muscle, bone, cartilage, fat, or connective tissue, leukemia, lymphoma, myeloma, leiomyosarcoma, or osteosarcoma, as well as brain cancer, thyroid cancer, ovarian cancer, pancreatic cancer, and kidney cancer.
  • infectious diseases include, but are not limited to, hepatitis B, tuberculosis, HIV/AIDS, influenza, tuberculosis, rabies, hepatitis B, human papillomavirus infection (associated with cervical cancer), malaria, syphilis, mycoplasma infection, diphtheria, tetanus, scarlet fever, hand-foot-and-mouth disease, acute rheumatic fever, gas gangrene, sepsis, cholecystitis, appendicitis, boils, gingivitis, infective endocarditis, and chronic rhinitis.
  • the autoimmune diseases mentioned include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), multiple sclerosis, type 1 diabetes, thyroid autoimmune diseases, and psoriasis.
  • the genetic diseases mentioned include, but are not limited to, Down syndrome, cystic fibrosis, hereditary muscular dystrophy, phenylketonuria, sickle cell anemia, Huntington's disease, and congenital deafness.
  • the neurological diseases mentioned include, but are not limited to, Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, and amyotrophic lateral sclerosis (ALS).
  • the metabolic diseases mentioned include, but are not limited to, diabetes, gout, Wilson's disease (copper metabolism disorder), phenylketonuria, and congenital hypothyroidism.
  • the fibrotic diseases mentioned include cirrhosis, idiopathic pulmonary fibrosis, and systemic sclerosis (scleroderma).
  • the tissue regeneration mentioned includes, but is not limited to, chronic wound healing, bone regeneration after fractures, and stem cell therapy, such as using stem cells to repair myocardial tissue.
  • the present invention provides the use of CD47 molecules in the preparation of ultra-low immunogenic cells, wherein CD47 molecules are overexpressed on the cell surface; and the CD47 molecules are distributed at a density of not less than 2700 molecules/ ⁇ m2 on the cell surface.
  • the present invention has the following beneficial effects:
  • This invention provides an engineered cell with ultra-low immunogenicity. Based on the density of CD47 molecules on the cell surface, the density of CD47 molecules on the engineered cell surface is no less than 2700, 2800, 2900, and 3000 molecules/ ⁇ m2 .
  • This engineered cell exhibits good immune tolerance against multiple allogeneic immune cells (including but not limited to PBMCs, T cells, and NK cells), overcoming the problem in existing technologies where engineered cells expressing low levels of CD47 molecules only exhibit limited immunosuppressive capabilities.
  • the engineered cells in this invention are mesenchymal stem cells, an important cell pool participating in tissue regeneration during normal tissue damage repair.
  • Mesenchymal stem cells have a much stronger in vitro proliferation capacity than cardiomyocytes and endothelial cells, and they retain multi-lineage differentiation capacity, making them more advantageous in damage repair and transdifferentiation colonization processes.
  • cells with a CD47 molecule distribution density of no less than 2700, 2800, 2900, and 3000 molecules/ ⁇ m2 on the surface of mesenchymal stem cells were selected, or mesenchymal stem cells expressing at least 2 million, 2.1 million, 2.2 million, 2.3 million, 2.4 million, 2.5 million, 2.6 million, 2.7 million, 2.8 million, 2.9 million, and 3 million CD47 molecules were selected. This achieved good immune tolerance against various allogeneic immune cells, better meeting the practical application needs of allogeneic cell therapy, providing a new universal cell source, and is expected to significantly reduce the cost of allogeneic cell therapy.
  • This invention simultaneously knocks out HLA-I/II genes and overexpresses CD47 in mesenchymal stem cells. Only when the CD47 expression level exceeds a certain threshold can the mesenchymal stem cells exhibit good ultra-low immunogenicity, which can simultaneously avoid the killing by T cells, PBMC cells, and NK cells, and significantly prolong the survival time of transplanted cells in vivo.
  • the ultra-low immunogenicity mesenchymal stem cells prepared in the embodiments of this invention are expected to generate universal cell products and solve the problem of immune rejection.
  • Figure 1 illustrates a technical flowchart of an embodiment of the present invention.
  • Figure 2 shows the Sanger sequencing diagram of the CIITA knockout clone in Example 1.
  • Figure 3 shows the expression of HLA-II by the CIITA knockout clone in Example 1, detected by flow cytometry.
  • Figure 4 is a schematic diagram of the fixed-point integration of the CD47 expression frame in Example 2.
  • Figure 5 shows the CD47 site-specific integration diagram identified by PCR in Example 2.
  • Figure 6 is an analysis of the surface markers of iMSCs and the HLA-ABC flow cytometry detection results in Example 2.
  • Figure 7 shows the T-cell killing of iMSCs in Example 4.
  • Figure 8 shows the PBMC killing iMSCs in Example 4.
  • Figure 9 shows the NK cell killing of iMSCs in Example 4.
  • Figure 10 shows the retention of iMSCs in live imaging monitoring in Example 5.
  • CD47 molecules are expressed at a high level on the membrane surface of engineered cells.
  • the density of CD47 molecules on the cell surface is no less than 2700 molecules/ ⁇ m2 .
  • This high-level expression of CD47 molecules achieves ultra-low immunogenicity in the engineered cells, resulting in good immune tolerance when these engineered cells are co-cultured with allogeneic PBMC cells, T cells, NK cells, and other cells.
  • HLA-I and HLA-II molecules are further reduced by knocking out the B2M and CIITA genes.
  • HLA-I molecules are composed of highly polymorphic heavy chain ⁇ and ⁇ 2-microglobulin (B2M). ⁇ 2-microglobulin forms a heterodimer with HLA-I proteins and is essential for the expression of HLA-I molecules on the cell surface.
  • Knockout of the B2M gene can further limit the immune response of cytotoxic CD8+ T cells by consuming HLA-I molecules.
  • the loss of expression of the HLA-II transactivator CIITA gene leads to low expression of HLA-II molecules, thus inhibiting the function of CD4+ T cells.
  • the simultaneous high expression of CD47 molecules further suppresses the immune rejection response, establishing an ultra-low immunogenicity strategy to overcome the immune rejection problem in allogeneic cell therapy.
  • iPSCs also known as induced pluripotent stem cells, refer to the same thing in this document. They are stem cells reprogrammed from somatic cells, such as differentiated somatic cells, and possessing higher potential compared to said somatic cells. iPS cells are capable of self-renewal and differentiation into mature cells, such as smooth muscle cells.
  • the induced pluripotent stem cells used in the specific embodiments of this invention are not strictly limited; they can be commercially available products or reprogrammed using known methods disclosed in the prior art.
  • iMSCs also known as iMSCs, are mesenchymal stem cells derived from induced pluripotent stem cells.
  • CD47 protein also known as CD47 molecule
  • CD47 molecule is selected from modified or unmodified natural CD47 molecules or their variants.
  • Natural CD47 molecules include, but are not limited to, naturally occurring different CD47 isotypes.
  • Variants of CD47 molecules perform the same immune tolerance function as natural CD47 molecules, including but not limited to different forms of truncated CD47 molecules, fusion proteins that retain CD47 immune tolerance function, or CD47 mutants with one or more amino acids inserted, replaced, or deleted in the amino acid sequence of natural CD47 molecules.
  • the ultra-low immunogenicity of cells refers to the ability of cells to tolerate multiple allogeneic immune cells simultaneously, including but not limited to allogeneic PBMC cells, T cells, and NK cells, rather than tolerance to a single type of allogeneic immune cell.
  • CD47 is preferably expressed by site-directed integration into the genome.
  • the integration mentioned in this embodiment can be performed at any safe site within the genome, as long as it enables the safe and efficient expression of the CD47 protein.
  • the safe integration site can be one or more of AAVS1, B2M, CCR5, HTRP, H11, GAPDH, TCR, ROSA26, RUNX1, CIITA, or rDNA regions. Integration minimizes the impact on normal host cell function and maintains stable transgene expression.
  • CD47-expressing gene into the B2M site of the genome to simultaneously achieve B2M knockout and high CD47 expression, reducing gene editing operations, lowering off-target risks, and further improving the safety of engineered cells.
  • expression vector refers to a vector containing recombinant polynucleotides, which includes an expression control sequence that effectively links the nucleotide sequence to be expressed.
  • Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
  • gene editing refers to the editing (directed modification) of a target gene and its transcription products to achieve the addition, deletion, deletion, substitution, or insertion of specific DNA fragments, thereby altering the sequence, expression level, or function of the target gene or regulatory element.
  • stem cell refers to undifferentiated cells that can be induced to proliferate. Stem cells are self-sustaining, meaning that with each cell division, a daughter cell will also be a stem cell. Stem cells can be obtained from embryonic, fetal, neonatal, adolescent, or adult tissues.
  • overexpression refers to the expression of a target gene or protein at a higher level than normal compared to parental or “wild-type” cells that do not contain the specified genetic modifications.
  • Gene editing methods including but not limited to Cre-lox system, Zinc Finger Nucleases (ZFN), CRISPR-Cas or TALEN, preferably CRISPR-Cas or TALEN-mediated gene editing.
  • ZFN Zinc Finger Nucleases
  • CRISPR-Cas or TALEN preferably CRISPR-Cas or TALEN-mediated gene editing.
  • This invention first synthesized a CD47 expression vector driven by the broad-spectrum promoter EF1 ⁇ (its nucleotide sequence is shown in SEQ ID NO: 1) by Sangon Biotech (Shanghai) Co., Ltd., which completely matched the target sequence after Sanger sequencing. Then, CIITA-sgRNA (its nucleotide sequence is shown in SEQ ID NO: 2) and B2M-sgRNA (its nucleotide sequence is shown in SEQ ID NO: 3) were synthesized by Genscript Biotech Co., Ltd.
  • Figure 1 illustrates the technical flow diagram of a specific embodiment of this invention.
  • Example 1 Preparation of normal human iPSCs with CIITA gene knocked out
  • iPSCs Normal human iPSCs, i.e., hiPSCs (Chinese Academy of Sciences Stem Cell Bank, DYR0100), were selected for nuclear transfection targeting.
  • the CRISPR/Cas9 gene editing tool (with the nucleotide sequence of CIITA-sgRNA shown in SEQ ID NO: 2) was used to disrupt the CIITA gene to achieve HLA-II knockout, thereby inhibiting T helper cell activation and antibody production.
  • Single-cell nuclear transfection of iPSCs was performed using the Neon transfection kit (Invitrogen, MPK10096). The kit was preheated to room temperature for 30 min before nuclear transfection. The specific method is as follows:
  • the cells were blown up and resuspended into a single-cell suspension. After counting the cells, they were centrifuged at 300g for 5 minutes at room temperature.
  • iPSCs were inoculated into six-well plates pre-coated with Matrigel (Corning, 354277), and 10 ⁇ M Y27632 was added. After mixing in a cross shape, the plates were placed in a 37°C, 5% CO2 saturated humidity incubator and cultured in mTeSR Plus medium.
  • iPSCs After expanding the cells, use the Nanocellect WOLF cell sorter to seed single cells. In about 10-12 days, iPSCs can grow from a single cell into a large clone and can be passaged and expanded.
  • iPSCs were directed to differentiate into iMSCs according to the method in its instruction manual.
  • iMSCs were cultured with 100 ng/ml IFN- ⁇ for 48 h to stimulate HLA-II molecule expression.
  • the culture medium of iMSCs was discarded, washed twice with DPBS, digested with TrypLE Express at room temperature for 1 min, resuspended with an appropriate amount of complete culture medium, and transferred to a 15 mL centrifuge tube and centrifuged at 300 g for 5 min.
  • HLA-II knockout was achieved by disrupting the CIITA gene.
  • Example 2 uses CRISPR/Cas9 technology to site-specifically integrate an expression cassette of exogenous CD47 molecules at the B2M site.
  • CRISPR/Cas9 technology was used to disrupt the B2M gene to achieve HLA-I knockout, aiming to inhibit T cell proliferation and activation.
  • an expression cassette for exogenous CD47 molecules was site-specifically integrated into the B2M site using a CD47 expression vector, enabling the edited cells to overexpress CD47.
  • CIITA-KO-iPSCs underwent further B2M knockout and CD47 targeting.
  • the experimental method was the same as above.
  • the nuclear transfer buffer was prepared as follows: 12 ⁇ g Cas9 protein, 120 pmol B2M-sgRNA, and 6 ⁇ g CD47 expression vector, and the volume was brought up to 20 ⁇ L using Neon Resuspension Buffer R. After the Cas9 protein cleaved the B2M gene, the CD47 expression cassette could be site-directedly integrated into the B2M site via homologous recombination through the FHA and BHA homologous arms on the CD47 expression vector (as shown in Figure 4).
  • Primers F1/R1 (PCR product size 1.1kb) were designed to cross the upstream homologous arm FHA, and primers F2/R2 (PCR product size 1.2kb) were designed to cross the downstream homologous arm BHA.
  • positive monoclonal antibodies were identified by PCR.
  • a total of five monoclonal antibodies with CD47 site-directed integration were obtained (as shown in Figure 5), and named M2, M3, M4, M5, and M6, respectively.
  • iPSCs cells with the CIITA gene and B2M gene knocked out and not targeting CD47 were screened as a control group for subsequent differentiation detection and named M1.
  • the above monoclonal cells were differentiated into iMSCs, and the resulting iMSCs were still named and numbered as described above.
  • Monoclonal cells of site-integrated iPSCs were directed to differentiate into iMSCs, and the surface markers of each iMSC were identified by flow cytometry, using the same experimental method as above.
  • the iMSC surface marker antibodies used were PE-CD105 (Biolegend, 800504), FITC-CD73 (Biolegend, 400114), FITA-CD90 (BD Biosciences, 555595), BV421-CD11b (BD Biosciences, 562632), BV421-CD19 (BD Biosciences, 562440), BV421-CD34 (BD Biosciences, 562577), BV421-CD45 (BD Biosciences, 563879), and BV421-HLA-DR (BD Biosciences, 562804), and PE-HLA-ABC (BD Biosciences, 555553) was used to detect HLA-I molecule expression.
  • CD47 was quantitatively detected using PE quantitative microspheres.
  • the CD47 expression level of iMSCs was detected by flow cytometry according to the instructions for use of PE quantitative microspheres (BD Biosciences, 340495).
  • the flow cytometry operation steps were the same as above, except that the antibody used was PE-CD47 (BD Biosciences, 556046).
  • the quantitative data results are shown in Table 1.
  • the CD47 expression level per unit area i.e., CD47 density, was obtained by dividing the flow cytometry data of CD47 by the corresponding cell surface area (as shown in Table 1).
  • M0 WT-iMSC
  • M1 HLA-I/II double knockout iMSC.
  • M2-M6 are HLA-I/II double knockout iMSCs with site-specific integration of CD47 overexpression.
  • M2 and M3 are high-expression strains with CD47 expression levels exceeding 2 million molecules per cell, while M4, M5, and M6 are low-expression strains with CD47 expression levels below 2 million molecules per cell.
  • M0 and M1 have very low levels of endogenous CD47 expression.
  • Example 4 Detection of low immunogenicity of cells using a cell co-culture killing experiment.
  • multiple immune cells are used for co-culture killing experiments, such as T cells, PBMC cells, and NK cells, to make it closer to the actual application environment in allogeneic cell therapy.
  • the co-culture medium used in the cell co-culture killing assay was RPMI 1640 supplemented with 10% FBS and 100 IU/mL IL-2.
  • the killing assay was performed on an xCELLigence RTCA DP cell analyzer.
  • iMSCs were treated with 100 ng/mL IFN- ⁇ for 48 h, and 100 ng/mL IFN- ⁇ was also added to the co-culture medium.
  • the specific method is as follows:
  • the RTCA system automatically performs a scan (“Scan Plate”) to check for good contact (the “Message” page will display Connection OK).
  • T cells prepare an appropriate concentration of effector cells (T cells).
  • the effector-to-target ratio of T cells is 5:1.
  • T cells effector cells
  • iMSCs target cells
  • PBMCs are mononuclear cells found in peripheral blood, containing various immune cells and reflecting the overall killing effect of peripheral immune cells.
  • the results showed that PBMCs exhibited strong killing activity against M0 and M1 cells, while CD47-high expression lines (M2 and M3) showed the best survival (Figure 8).
  • M2 and M3 CD47-high expression lines
  • Figure 8 This indicates that iMSCs can only sustainably resist PBMC killing and possess low immunogenicity when their cell surface expression exceeds 2 million CD47 molecules.
  • CD47 expression is below 2 million, iMSCs are gradually killed by PBMCs and do not possess low immunogenicity.
  • This also demonstrates that iMSCs with low CD47 expression levels (insufficient CD47 distribution density on the iMSC cell membrane surface) cannot provide sufficient immune tolerance when facing PBMC killing.
  • NK cells exhibited the strongest killing effect against M1 (HLA-I/II double knockout); they also showed relatively strong killing effect against CD47-low expression iMSCs (M4, M5, and M6); while CD47-high expression lines (M2 and M3) showed similar survival to M0 (WT-iMSCs).
  • HLA knockout can lead to NK cell activation and killing.
  • Low-level CD47 overexpression cannot effectively salvage this killing effect
  • high-level CD47 overexpression (more than 2 million molecules per cell) can effectively avoid NK cell activation caused by HLA knockout, thus enabling iMSCs to acquire low immunogenicity.
  • iMSCs with different genotypes and CD47 expression levels were used to detect their cytotoxicity against T cells, PBMCs, and NK cells.
  • the results showed that iMSCs with high CD47 expression levels (over 2 million CD47 molecules per cell) effectively avoided NK cell activation induced by HLA knockout, and could simultaneously evade the killing effects of T cells, PBMCs, and NK cells.
  • Low CD47 expression levels were insufficient to salvage NK cell-induced cytotoxicity.
  • HLA-I/II double knockout iMSCs with high CD47 expression levels represent ultra-low immunogenicity cells capable of evading immune killing.
  • Example 5 Monitoring cell survival after iMSCs were transplanted into humanized mice.
  • iMSC strains M0, M2, and M4, were transduced with firefly luciferase using lentivirus and then transplanted into HSC-NCG-hIL15 humanized mouse models (provided by Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). Each mouse received 1.5 ⁇ 106 iMSCs cells. The cells were resuspended in 140 ⁇ L of DPBS, mixed with 70 ⁇ L of Matrigel gel, and injected subcutaneously into the hind limbs of the mice.

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Abstract

La présente invention concerne une cellule ingénierisée, son procédé de préparation et son utilisation. Par comparaison avec une cellule de type sauvage, la cellule ingénierisée surexprime une molécule CD47 ; de plus, en matière de densité de la molécule CD47 à la surface cellulaire, la densité de distribution de la molécule CD47 à la surface de la cellule ingénierisée est supérieure ou égale à 2700 molécules/μm2. La cellule ingénierisée selon la présente invention présente une excellente immunogénicité ultra-faible, peut simultanément éviter d'être détruite par un lymphocyte T, une cellule PBMC et une cellule NK, et prolonge considérablement la durée de survie d'une cellule transplantée in vivo. Elle devrait permettre de créer un produit cellulaire universel pour résoudre le problème du rejet immunitaire.
PCT/CN2025/096192 2024-05-22 2025-05-21 Cellule ingénierisée universelle, son procédé de production et son utilisation Pending WO2025242112A1 (fr)

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CN202410641320.9A CN121006322A (zh) 2024-05-22 2024-05-22 一种通用型工程化细胞及其制备方法与应用
CN202410641320.9 2024-05-22

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