WO2022012531A1 - Procédé de préparation d'une cellule immunitaire modifiée - Google Patents

Procédé de préparation d'une cellule immunitaire modifiée Download PDF

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Publication number
WO2022012531A1
WO2022012531A1 PCT/CN2021/106007 CN2021106007W WO2022012531A1 WO 2022012531 A1 WO2022012531 A1 WO 2022012531A1 CN 2021106007 W CN2021106007 W CN 2021106007W WO 2022012531 A1 WO2022012531 A1 WO 2022012531A1
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Prior art keywords
protein
cells
nucleic acid
cell
acid molecule
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PCT/CN2021/106007
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English (en)
Chinese (zh)
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贾璐盈
林彦妮
袁慧
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苏州克睿基因生物科技有限公司
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Publication of WO2022012531A1 publication Critical patent/WO2022012531A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/26—Universal/off- the- shelf cellular immunotherapy; Allogenic cells or means to avoid rejection

Definitions

  • the present application relates to the field of biomedicine, in particular to a method for preparing a universal chimeric antigen T cell, including knocking out Fas protein and transferring FasL protein.
  • the method comprises administering a Cas protein to the immune cell.
  • the CD24 protein comprises the amino acid sequence set forth in SEQ ID NO:59.
  • the vector comprises a viral vector.
  • the present application provides a population of cells comprising said immune cells, wherein at least 20% of the immune cells in said cell population do not substantially express Fas and at least 5% of the immune cells overexpress FasL.
  • Figure 5 shows the fold expansion of the modified immune cells described in the present application and allogeneic T cells co-cultured on day 6.
  • Figure 7 shows the expansion fold of the modified immune cells described in the present application after co-culture with allogeneic T cells.
  • gene silencing generally refers to the inhibition of the expression of a gene, which can be inhibited by blocking the transcription or translation of the gene.
  • chimeric antigen receptor generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain.
  • CAR is the core component of chimeric antigen receptor T cells (CAR-T), which can include targeting moieties (eg, moieties that bind tumor-associated antigens (TAAs)), hinge regions, transmembrane regions, and cells internal domain.
  • targeting moieties eg, moieties that bind tumor-associated antigens (TAAs)
  • TAAs tumor-associated antigens
  • CRISPR/Cas system or “CRISPR-Cas system” generally refers to a nuclease system consisting of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related proteins (i.e. Cas proteins), It can cut almost all genomic sequences adjacent to the protospacer-adjacent motif (PAM) in eukaryotic cells.
  • CRISPR/Cas system may be used to collectively refer to transcripts involving CRISPR-associated (“Cas”) genes, as well as other elements involved in their expression or directing their activity, and may include sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g.
  • the present application provides a method for preparing a modified immune cell, the modification comprising the steps of: a) down-regulating the expression and/or activity of Fas protein in the immune cell, b) up-regulating the expression and/or activity of the Fas protein in the immune cell FasL protein expression and/or activity such that Fas protein expression and/or activity in the modified immune cells is reduced or eliminated compared to immune cells without the modification, and the modified Elevated expression and/or activity of FasL protein in immune cells.
  • the method can include administering to the immune cell one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas systems, RNA editing systems such as RNA adenosine deaminase (ADAR), RNA-Guided Endonuclease, Zinc Finger Nuclease (ZFN), Mega-TAL Nuclease, Transcription Activator-Like Effector Nuclease (TALEN), Meganuclease, Base Editing, CRISPR interference (or CRISPRi), and, Zinc finger gene repressor and/or transcription activator-like effector (TALE) gene repressor-mediated transcriptional repression.
  • the method may include administering to immune cells using inhibitory proteins, which may include substances capable of inhibiting the activity or function of the Fas protein, eg, inhibitory ligands, receptors, antibodies of the Fas protein and/or enzymes.
  • the target polynucleotide of the CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide that resides in the nucleus of a eukaryotic cell.
  • the backbone sequence of the present application can be derived from the backbone sequence described in PCT application publication WO2019011118A1, for example, can be the nucleotide sequence shown in any one of SEQ ID NOs: 34-45 (listed as 5' to 3') , where the first region in lowercase font represents the tracr mate sequence, and the second region in lowercase font represents the tracr sequence, and the last poly-U sequence represents the transcription terminator.
  • the number of U in the poly-U is not limited to what is shown in this example, and can be increased or decreased. In some cases, poly-U can be removed without affecting activity.
  • a crRNA targeting a nucleic acid molecule encoding a Fas protein described herein may comprise at least 80% (eg, at least 85%, at least 90%) of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotides of sequence identity sequence.
  • proteins that can be fused to Cas proteins include, but are not limited to, epitope tags, reporter genes, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • the FasL protein may also comprise a hinge region.
  • the hinge region may contain fewer protease cleavage sites than the hinge region of the native FasL protein.
  • the hinge region may be a hinge region derived from a transmembrane protein other than FasL.
  • the hinge region may be a hinge region derived from the tumor necrosis factor superfamily.
  • the hinge region can be a hinge region derived from TNFSF10 or OX40L.
  • nucleic acid molecules described herein can also be linked to many types of vectors.
  • the nucleic acid can be ligated to, including but not limited to, plasmids, phagemids, phages, viruses and/or cosmids.
  • Particular vectors of interest can include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the methods of the present application may comprise introducing the vectors described herein into immune effector cells.
  • the vectors described herein can be introduced into the immune effector cells, such as T lymphocytes, B cells, macrophages, or natural killer (NK) cells.
  • each or each cell may comprise one or one of the vectors described herein.
  • each or each cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the vectors described herein.
  • the vectors described herein can be introduced into the cells.
  • immune effector cells can be transfected with a retroviral vector, and the viral genome with the nucleic acid encoding the fusion protein can be integrated into the host genome to ensure long-term and stable expression of the target gene.
  • a transposon using a transposon, a plasmid carrying a nucleic acid encoding the fusion protein and a plasmid carrying a transposase are introduced into target cells.
  • Gene or protein expression levels can be detected by a variety of methods, including methods at the nucleic acid level (including by reverse transcriptase polymerase chain reaction (RT-PCR) or by Southern blotting, in situ hybridization for mRNA quantification, next-generation sequencing ) and methods at the protein level (including immunofluorescence labeling and analysis by flow cytometry, histochemistry, immunoblot analysis, and in vitro binding studies).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • protein expression levels can be quantified by ELISA techniques well known to those skilled in the art. Quantitative measurements can be done using a number of standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis. Immunohistochemical staining and flow cytometry, Western blot analysis can also be used to assess whether and how much Fas protein and/or Fas gene is present.
  • FasL and its variants FasL-M1 and FasL-M2 can protect allogeneic T cells from killing, so that T cells expressing FasL, FasL-M1 and FasLF-M2 can survive more under the killing effect of allogeneic T cells. .

Abstract

L'invention concerne un procédé de préparation d'une cellule immunitaire modifiée. La modification comprend les étapes suivantes : a) la réduction de l'expression et/ou de l'activité d'une protéine Fas dans la cellule immunitaire, et b) l'augmentation de l'expression et/ou de l'activité d'une protéine FasL dans la cellule immunitaire. Ainsi, par rapport à une cellule immunitaire qui n'est pas soumise à la modification, l'expression et/ou l'activité de la protéine Fas dans la cellule immunitaire modifiée sont réduites ou supprimées, et l'expression et/ou l'activité de la protéine FasL dans la cellule immunitaire modifiée sont augmentées.
PCT/CN2021/106007 2020-07-14 2021-07-13 Procédé de préparation d'une cellule immunitaire modifiée WO2022012531A1 (fr)

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CN202010676239 2020-07-14
CN202010676239.6 2020-07-14

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022187663A1 (fr) * 2021-03-04 2022-09-09 Allogene Therapeutics, Inc. Expression de fasl et inactivation du gène fasr pour protéger les cellules thérapeutiques contre le rejet allogénique et la mort cellulaire induite par activation

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CN104046593A (zh) * 2013-03-14 2014-09-17 浙江大学 一种低免疫原性的人细胞及其制备方法
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CN107206024A (zh) * 2014-10-31 2017-09-26 宾夕法尼亚大学董事会 改变cart细胞中的基因表达及其用途
CN109722437A (zh) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 一种通用型car-t细胞及其制备方法和用途
US20190345446A1 (en) * 2018-04-25 2019-11-14 The Broad Institute, Inc. Compositions and methods for modulating th-17 and th-1 cell balance
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WO1999020308A1 (fr) * 1997-10-23 1999-04-29 Biocrystal Ltd. Procede pour inhiber les metastases et la croissance des cellules tumorales par modulation clinique de l'expression tumorale du ligand fas
CN104822384A (zh) * 2012-03-30 2015-08-05 南加州大学 用于间充质干细胞诱导的免疫调节的组合物和治疗方法
CN104046593A (zh) * 2013-03-14 2014-09-17 浙江大学 一种低免疫原性的人细胞及其制备方法
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022187663A1 (fr) * 2021-03-04 2022-09-09 Allogene Therapeutics, Inc. Expression de fasl et inactivation du gène fasr pour protéger les cellules thérapeutiques contre le rejet allogénique et la mort cellulaire induite par activation

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