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WO2025156492A1 - Transposase et son utilisation - Google Patents

Transposase et son utilisation

Info

Publication number
WO2025156492A1
WO2025156492A1 PCT/CN2024/091741 CN2024091741W WO2025156492A1 WO 2025156492 A1 WO2025156492 A1 WO 2025156492A1 CN 2024091741 W CN2024091741 W CN 2024091741W WO 2025156492 A1 WO2025156492 A1 WO 2025156492A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
transposase
amino acid
cells
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/091741
Other languages
English (en)
Chinese (zh)
Inventor
黎凯明
陆金华
张�杰
翁靖杰
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.)
Triarm Therapeutics Shanghai Co Ltd
Triarm Inc
Original Assignee
Triarm Therapeutics Shanghai Co Ltd
Triarm Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Triarm Therapeutics Shanghai Co Ltd, Triarm Inc filed Critical Triarm Therapeutics Shanghai Co Ltd
Publication of WO2025156492A1 publication Critical patent/WO2025156492A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • the present application relates to the field of biomedicine, and specifically to a transposase polypeptide and its use.
  • Genetic engineering and gene therapy in most cases involve transferring nucleic acids into target cells or modifying the genes of target cells.
  • this process can be performed using viral vectors, non-viral vectors (such as naked DNA plasmids, RNA, or RNPs), and transposon systems.
  • the transposon system Compared with viral vector-mediated gene delivery, the transposon system has some advantages, such as low cost and safety.
  • the transposon system consists of a transposon element and a transposase, which can recognize specific sequences in the transposon and mediate the insertion of DNA into the cell genome.
  • the Sleeping Beauty transposon system has been widely used for various purposes in in vitro, ex vivo and in vivo genetic engineering.
  • polypeptides derived from the original SB transposase have been identified and determined to have different transposition activities. These transposase variants were obtained by random mutagenesis and then screening, so the mechanism of the different transposition activities is not well understood.
  • transposases obtained through random screening have high transposition efficiencies, their unclear mechanism may affect the integration site and the permissible transgene size.
  • This application screens transposases with different transposition activities based on the activity of the key step of transposase cleaving transposons.
  • the mechanism is clear, and the obtained enzymes can also be used to screen corresponding transposon sequences, thereby forming an efficient and safe transposition system.
  • Obtaining the target transposase polypeptide by improving a certain step has greater certainty and is also conducive to identifying the safety of the product obtained thereby.
  • This application uses an alanine scanning method to generate mutants of SB transposase, specifically in a region that has not been actively studied before. These mutants were evaluated for different excision activities, and the screening method was quick and simple. In addition, these mutants with single or combined mutations (Tri-FITase, a refined transposase for in vitro, in vivo, and in vivo) were determined to have different transposition activities and can therefore be used in different applications, such as T cell genetic engineering, which may require transposases with higher or lower activity, carry genetic material of different sizes, and/or have different insertion characteristics.
  • the present application provides a transposase polypeptide, the amino acid sequence of which contains at least one mutated amino acid residue compared to the amino acid sequence from positions 100 to 180 of the unmutated SB transposase as shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises 1 to 20 mutated amino acid residues.
  • the amino acid sequence of the transposase polypeptide comprises at least two mutated amino acid residues.
  • the mutation site of the at least one mutated amino acid residue is located at positions 110 to 120 and/or 170 to 180 of the amino acid sequence shown in SEQ ID NO:2.
  • the mutated amino acid residue is located at position 114, 116, 177 and/or 178 of the amino acid sequence shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at the G114 amino acid position compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at the S116 amino acid position compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at the K177 amino acid position compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at the P178 amino acid position compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the at least one mutated amino acid residue is mutated to an alanine residue.
  • amino acid residues that are mutated are all alanine residues.
  • the amino acid sequence of the transposase polypeptide comprises an amino acid mutation selected from the group consisting of G114A, S116A, K177A, and P178A, compared to the unmutated SB transposase as shown in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position G114 to alanine (G114A).
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position S116 to alanine (S116A) compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position K177 to alanine (K177A) compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the transposase polypeptide amino acid sequence comprises a mutation at the amino acid position P178 to alanine (P178A) compared to the unmutated SB transposase amino acid sequence as shown in SEQ ID NO:2.
  • the transposase polypeptide amino acid sequence comprises a combination of amino acid mutations selected from the group consisting of:
  • the present application provides a transposase polypeptide comprising a sequence that is at least 90% identical to SEQ ID NO:2, wherein the transposase polypeptide comprises alanine at a position corresponding to position 114 and/or alanine at a position corresponding to position 116.
  • the transposase polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO:1 and SEQ ID NO:3-11.
  • the present application provides a polynucleotide molecule comprising a nucleic acid sequence encoding the transposase polypeptide described herein.
  • it comprises RNA or DNA.
  • the DNA comprises a promoter for expressing the transposase polypeptide.
  • the polynucleotide molecule comprises a poly(A) sequence of 20 to 300 nucleotides.
  • the present application provides a construct comprising the polynucleotide molecule and regulatory sequence described in the present application, wherein The regulatory sequence is operably linked to the expressible polynucleotide to allow expression of the polynucleotide.
  • the present application provides a cell comprising the transposase polypeptide described herein, the polynucleotide molecule described herein, and/or the construct described herein.
  • the cells comprise mammalian cells.
  • the cells comprise human cells.
  • the cells are autologous.
  • the cells are allogeneic.
  • the cells comprise stem cells or induced pluripotent stem (iPS) cells.
  • iPS induced pluripotent stem
  • the cells comprise immune effector cells.
  • the cells comprise natural killer (NK) cells, T cells, or precursors of NK cells or T cells.
  • NK natural killer
  • the cell comprises a transposase polypeptide as described herein.
  • the cells contain mRNA or DNA encoding the transposase polypeptide described herein.
  • transposon system comprising:
  • transposon unit containing inverted terminal repeats (ITRs) or direct terminal repeats (DTRs) flanking a target sequence to be inserted into the genome of a target cell;
  • the present application provides an in vitro use of the transposon system described herein for delivering genes to target cells.
  • the present application provides a method for genetically engineering cells, comprising the following steps:
  • the method further comprises: (c) isolating the engineered cell.
  • the target sequence encodes a screenable or selectable marker.
  • the target sequence encodes a therapeutic polypeptide or an inhibitory nucleic acid.
  • the target sequence encodes an antibody, a T cell receptor (TCR), or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the target sequence encodes a CAR.
  • the CAR targets a tumor antigen.
  • the tumor antigen is CD19, CD20, ROR1, CD22 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, prostate specific antigen, melanoma associated antigen, mutated p53, mutant ras, HER2/Neu, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD33, CD138, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R ⁇ , kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, VEGFR2, combined HER2-HER3, or combined HER1-HER2.
  • the target cell comprises a mammalian cell.
  • the target cell comprises a human cell.
  • the target cells are autologous.
  • the target cells are allogeneic.
  • the target cells comprise stem cells or induced pluripotent stem (iPS) cells.
  • iPS induced pluripotent stem
  • the target cells comprise immune effector cells or precursors thereof.
  • the target cells include natural killer (NK) cells, T cells, or precursors of NK cells or T cells.
  • NK natural killer
  • transfecting the target cell comprises using a polymer-, polypeptide-, or lipid-based transfection reagent.
  • transfecting the target cell comprises electroporating the cell.
  • the method further comprises:
  • the method further comprises:
  • the method further comprises: (c) culturing the engineered cells in a medium that selectively enhances the proliferation of CAR or TCR-expressing T cells.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the transposase polypeptide described herein, the polynucleotide molecule described herein, and/or the construct described herein, and a pharmaceutically acceptable carrier and/or excipient.
  • the present application provides a kit comprising
  • transposon unit containing inverted terminal repeats (ITRs) or DTRs flanking a target sequence to be inserted into the genome of a target cell;
  • the present application provides a use of the transposase polypeptide described herein, the polynucleotide molecule described herein, the construct described herein, the cell described herein, the transposase system described herein and/or the pharmaceutical composition described herein in the preparation of a medicament for treating a disease or condition.
  • the disease or condition is cancer.
  • the disease or condition is an infectious disease.
  • the present application provides a compound for treating a disease or condition, wherein the compound comprises the transposase polypeptide described herein, the polynucleotide molecule described herein, the construct described herein, the cell described herein, the transposase system described herein, the pharmaceutical composition described herein and/or the kit described herein.
  • FIG1A shows a schematic diagram of the excision assay used in the present application to distinguish SB transposases with known transposition activities.
  • Figure 1B shows the results of excision experiments using three commonly used SB transposases.
  • FIG2 shows that the transposase mutants prepared by the alanine scanning method have different transposase cleavage activities.
  • FIG3 shows the results of a transposase transposition assay using the Tri-FITase of the present application in Hela cells.
  • FIGS. 4A-4B show that the Tri-FITase of the present application achieves chimeric antigen receptor expression in T cells.
  • transposase generally refers to an enzyme that is a component of a functional nucleic acid-protein complex capable of transposition and mediates transposition.
  • the term “transposase” may also include integrases derived from retrotransposons or retroviral sources.
  • Transposition reaction as used herein generally refers to the reaction in which a transposon is inserted into a target nucleic acid.
  • the main components in the transposition reaction are transposons and transposases or integrases.
  • the transposase system according to the present invention is preferably derived from the so-called “Sleeping Beauty (SB)" transposase.
  • transposases are engineered enzymes with improved properties such as enhanced enzymatic function.
  • Some specific examples of engineered SB transposases include, but are not limited to, SB10, SB11 or SB100 ⁇ SB transposases (see, for example, Mates et al., Nat. Gen. 2009, incorporated herein by reference).
  • transposition systems such as Ty1 (Devine and Boeke, 1994 and WO 95/23875), Tn7 (Craig, 1996), Tn10 and IS10 (Kleckner et al., 1996), Mariner transposase (Lampe et al., 1996), Tc1 (Vos et al., 1996), Tn5 (Park et al., 1992), P elements (Kaufman and Rio, 1992), and Tn3 (Ichikawa and Ohtsubo, 1990), bacterial insertion sequences (Ohtsubo and Sekine, 1996), retroviruses (Varmus and Brown, 1989), and yeast retrotransposons (Boeke, 1989).
  • transposon unit refers to a nucleic acid construct comprising a transposon gene sequence and a target sequence to be introduced into the genome of a target cell.
  • a transposon unit is a nucleic acid and can be any form of vector suitable for transposition.
  • inverted terminal repeat refers to a sequence located at one end of a transposon unit that, when used in combination with a complementary sequence located at the opposite end of a vector or transposon unit, can be cleaved by a transposase polypeptide.
  • the pair of inverted terminal repeats is involved in the transposition activity of the transposon of the transposon unit of the present disclosure, particularly with respect to DNA addition or removal, as well as excision and integration of target DNA.
  • at least one pair of inverted terminal repeats appears to be the minimum sequence required for transposition activity in a plasmid.
  • the transposon unit of the present disclosure may include at least two, three, or four pairs of inverted terminal repeats.
  • the necessary terminal sequences may be as short as possible, thereby containing as few inverted repeats as possible.
  • the transposon unit of the present disclosure may contain no more than one, no more than two, no more than three, or no more than four pairs of inverted terminal repeats.
  • the transposon unit of the present disclosure may include only one inverted terminal repeat.
  • having more than one inverted terminal repeat may be disadvantageous because it may result in non-specific transposases binding to multiple inverted terminal repeats and resulting in removal of the desired sequence or insertion of an undesired sequence.
  • the inverted terminal repeats of the present disclosure can form perfect inverted terminal repeats (or interchangeably referred to as “perfect inverted repeats") or imperfect inverted terminal repeats (or interchangeably referred to as “imperfect inverted repeats").
  • perfect inverted repeat refers to two identical DNA sequences placed in opposite orientations.
  • transposon units having ITRs also applies to transposon units having DTRs.
  • Transposon systems that can be used with the transposon polypeptides/nucleic acids of the invention are disclosed, for example, in WO 2017/050448 A1, which is incorporated into the present disclosure by reference.
  • CAR chimeric antigen receptor
  • CAR generally refers to a group of polypeptides, which are generally two in the simplest embodiment, and when in immune effector cells, provide cell specificity to target cells (usually cancer cells) and generate intracellular signals.
  • CAR includes at least one extracellular antigen binding domain (such as VHH, scFv or part thereof), a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain”), which includes a functional signaling domain derived from a stimulatory molecule and/or a costimulatory molecule as defined below.
  • the group of polypeptides is in the same polypeptide chain (for example, comprising a chimeric fusion protein). In some embodiments, the group of polypeptides is discontinuous with each other, for example, in different polypeptide chains. In some aspects, the group of polypeptides includes a dimerization switch, which can couple polypeptides to each other in the presence of a dimerization molecule, for example, the antigen binding domain can be coupled to the intracellular signaling domain.
  • the stimulatory molecule of CAR is a ⁇ chain associated with a T cell receptor complex.
  • the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3- ⁇ ).
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule defined below.
  • the costimulatory molecule can be selected from 4-1BB (i.e., CD137), CD27, ICOS and/or CD28.
  • CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • CAR comprises a chimeric fusion protein that may include an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain
  • the intracellular signaling domain comprises a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule
  • CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain
  • the intracellular signal transduction domain includes a functional signal transduction domain derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule.
  • CAR includes a chimeric fusion protein, which may include an extracellular antigen recognition domain, a transmembrane domain and an intracellular signal transduction domain, and the intracellular signal transduction domain includes at least two functional signal transduction domains derived from one or more costimulatory molecules and a functional signal transduction domain derived from a stimulatory molecule.
  • CAR includes an optional leader sequence on the amino terminus (N-ter) of the CAR fusion protein.
  • CAR is further included in a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally removed from the antigen recognition domain (eg, VHH) during cell processing, and CAR is positioned at the cell membrane.
  • antibody is generally used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170: 4854-4861).
  • Antibodies can be mouse, human, humanized, chimeric, or derived from other species.
  • homologous sequences may include amino acid sequences that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the subject sequence.
  • a homologue will contain the same active sites etc. as the subject amino acid sequence.
  • Homology may be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions) or may be expressed in terms of sequence identity.
  • a sequence having a percentage identity to any of the SEQ ID NOs of an amino acid sequence or nucleotide sequence refers to a sequence having the stated percentage identity over the entire length of the SEQ ID NO mentioned.
  • sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment over the full-length sequences being compared.
  • upstream and downstream are functional definitions and generally refer to the direction or polarity of a chain of coding nucleotide sequences.
  • the "upstream” direction refers to nucleotides located in the 5' direction of a given polynucleotide sequence, i.e., toward the starting nucleotide.
  • upstream is interpreted as/refers to amino acids located in the N-terminal direction, i.e., toward the start of the polypeptide chain.
  • isolated nucleic acid molecule generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length or their analogs, separated from their natural environment or artificially synthesized.
  • the term "construct” generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and/or between host cells.
  • the construct may include a vector that is primarily used for inserting DNA or RNA into a cell, a vector that is primarily used for replicating DNA or RNA, and a vector that is primarily used for expression of the transcription and/or translation of DNA or RNA.
  • the vector also includes a vector with multiple of the above functions.
  • the construct may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product by cultivating a suitable host cell that contains the vector.
  • the term "host cell” or “cell” generally refers to an individual cell, cell line or cell culture that may or already contain a vector comprising the isolated nucleic acid molecule described in the present application, or that can express the isolated antigen-binding fragment described in the present application.
  • the host cell may include the offspring of a single host cell. Due to natural, accidental or intentional mutations, the offspring cells may not necessarily be completely identical in morphology or genome to the original parent cell, but they can express the isolated antigen-binding fragment described in the present application.
  • the host cell can be obtained by transfecting cells in vitro using the vector described in the present application.
  • the host cell may be a prokaryotic cell (such as Escherichia coli) or a eukaryotic cell (such as a yeast cell, such as COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell or myeloma cell).
  • the host cell may be an Escherichia coli cell.
  • the The host cell can be a yeast cell.
  • the host cell can be a mammalian cell.
  • the mammalian cell can be a CHO-K1 cell.
  • T cell can be any T cell, such as a cultured T cell, for example a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTI, etc., or a T cell obtained from a mammal (preferably a primate, species including monkeys, dogs, or humans). If obtained from a mammal, the T cell can be obtained from many sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell can also be enriched or transfected. The T cell can be obtained by maturing hematopoietic stem cells into T cells in vitro or in vivo.
  • a cultured T cell for example a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupTI, etc.
  • a T cell obtained from a mammal preferably a primate, species including monkeys, dogs, or humans.
  • the T cell can be obtained from many sources, including
  • the T cell is a human T cell.
  • the T cell is a T cell isolated from humans.
  • T cells can be any type of T cell, including NKT cells, and can be of any developmental stage, including but not limited to CD4+/CD8+ double positive T cells; CDA+ helper T cells; such as Th1 and Th2 cells, CD8+ T cells (such as cytotoxic T cells); peripheral blood mononuclear cells (PBMC); peripheral blood leukocytes (PBL); tumor infiltrating cells (TIL); memory T cells; untreated T cells and the like.
  • T cells are CD8+ T cells or CD4+ T cells.
  • the T cells are allogeneic (different donors from the same species) to the recipient of the cells or the cells to be received (such as the cells are in the form of a therapeutic composition); in some alternatives, the T cells are autologous (the donor and the recipient are the same); in some alternatives, the T cells are syngeneic (the donor and the recipient are different, but are identical twins).
  • immune effector cell generally refers to an immune cell that participates in an immune response and performs an effector function.
  • the effector function may include clearing foreign antigens or promoting immune effector responses.
  • Immune effector cells may include plasma cells, T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.
  • the immune effector cells of the present application can be autologous/self (autologous/autogeneic) ("one's own”) or non-autologous ("non-one's own", such as allogeneic, isogenic or allogeneic).
  • autologous generally refers to cells from the same subject.
  • Allogenic generally refers to cells that are of the same species but genetically different from the compared cells.
  • Isogenic generally refers to cells of a different subject that are genetically identical to the compared cells.
  • Allogenic generally refers to cells of a species different from the compared cells.
  • the cells of the present application are autologous or allogenic.
  • the term "modification” generally refers to changing the state or structure of a cell and/or a change in the state or structure of a cell.
  • the change is usually compared to the state or structure of a corresponding cell without the modification.
  • the change may include a change in the expression level or function of an endogenous gene, such as downregulating, upregulating or not expressing the endogenous gene expression level of the cell through genetic engineering means, such as homologous recombination, CRISPR/Cas9 system gene editing, etc.
  • the change may also include a change in the expression, structure or function of a cell protein, such as by changing the expression level or function of the endogenous gene.
  • the changes in protein expression, structure or function achieved by the corresponding protein can be, for example, changes in protein expression, structure or function achieved by regulating protein translation or post-translational modification; the changes can also include the introduction of exogenous genes, the expression of exogenous proteins, etc.
  • nucleic acid or “polynucleotide” or “nucleic acid molecule” generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form.
  • the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to a reference nucleic acid (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides.
  • sequence of a nucleic acid may include variants thereof modified in a conservative manner, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.
  • operably linked generally refers to placing regulatory sequences necessary for expression of a coding sequence at an appropriate position relative to the coding sequence so as to achieve expression of the coding sequence.
  • operably linked may also refer to the arrangement of coding sequences and regulatory sequences (e.g., promoters, enhancers, and termination elements) in an expression vector. This definition sometimes also applies to the arrangement of nucleic acid sequences of a first and a second nucleic acid molecule in which a hybrid nucleic acid molecule is generated.
  • regulatory sequence generally includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that regulate the transcription or translation of the antibody chain genes.
  • promoters e.g., promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that regulate the transcription or translation of the antibody chain genes.
  • expression control elements e.g., polyadenylation signals
  • Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be understood by those skilled in the art that the design of expression vectors, including the selection of regulatory sequences, depends on factors such as the choice of the host cell to be transformed and the desired level of protein expression.
  • Regulatory sequences for mammalian host cell expression can include viral elements that direct high-level protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., adenovirus major late promoter (AdMLP)), and polyoma virus.
  • CMV cytomegalovirus
  • SV40 simian virus 40
  • AdMLP adenovirus major late promoter
  • regulatory elements may contain sequences from different sources, such as the SR ⁇ promoter system, which includes sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).
  • expression generally refers to the transcription and/or translation of a specific nucleotide sequence.
  • tumor and cancer are used interchangeably and generally refer to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.
  • cancer or “tumor” include pre-malignant and malignant cancers and tumors, and also encompass solid tumors and non-solid tumors.
  • tumor antigen includes the meaning known in the art, including any molecule expressed on tumor cells (or associated with tumor cell development), known or believed to have an effect on the tumorigenic properties of tumor cells. Many tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays familiar to those skilled in the art, such as clonogenic assays, transformation assays, in vitro or in vivo tumor formation assays, gel migration assays, gene knockout analysis, etc.
  • tumor antigen when used herein refers to human transmembrane proteins, that is, cell membrane proteins anchored in the lipid bilayer of the cell.
  • the human transmembrane proteins used herein will typically include an "extracellular domain” that can bind to a ligand, a lipophilic transmembrane domain, a conserved intracellular domain, such as a tyrosine kinase domain, and a carboxyl-terminal signaling domain having several tyrosine residues that can be phosphorylated.
  • Tumor antigens include molecules such as EGFR, HER2/neu, HER3, HER4, EpCAM, CEA, TRAIL, TRAIL receptor 1, TRAIL receptor 2, lymphotoxin beta receptor, CCR4, CD19, CD20, CD22, CD28, CD33, CD40, CD80, CSF-1R, CTLA-4, fibroblast activation protein (FAP), hepsin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), prostate-specific membrane antigen (PSMA), VEGF receptor 1, VEGF receptor 2, IGF1-R, TSLP-R, TIE-1, TIE-2, TNF- ⁇ , TNF-like weak inducer of apoptosis (TWEAK), or IL-1R.
  • FAP fibroblast activation protein
  • MCSP melanoma-associated chondroitin sulfate proteoglycan
  • PSMA prostate-specific membrane antigen
  • VEGF receptor 1 VEGF receptor 2
  • the term "pharmaceutically acceptable” generally refers to those compounds, materials, compositions and/or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • the term "pharmaceutically acceptable carrier” generally refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration.
  • the use of such media and agents for pharmaceutical active substances is well known in the art.
  • the composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions.
  • the term “effective amount” or “effective dose” generally refers to an amount sufficient to achieve, or at least partially achieve, a desired effect.
  • a “therapeutically effective amount” or “therapeutically effective dose” of a drug or therapeutic agent is generally any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes regression of a disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from the disease.
  • the term "about” generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.
  • subject generally refers to humans or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.
  • the term "marker gene” refers to a nucleic acid sequence that is capable of expressing an activity that causes a positive or negative selection to occur in a host cell, such as to the presence or absence of a sequence.
  • the marker sequence or gene may optionally display positive and negative selectivity.
  • Important positive selection markers are, for example, antibiotic resistance genes.
  • Selective markers may be positive, negative, or bifunctional. Positive selection markers allow the selection of cells that carry the marker, while negative selection markers allow selective elimination of cells that carry the marker. Typically, selective markers will confer resistance to drugs or confer metabolic or catabolism defects in the compensatory cell.
  • Selectable markers that can be used in eukaryotic cells include, for example, genes for aminoglycoside phosphotransferases (APHs), such as hygromycin phosphotransferase (hyg), neomycin and G418 APHs, dihydrofolate reductase (DHFR), thymidine kinase (tk), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (selection agent indole), histidinol dehydrogenase (selection agent histidinol D), and genes that confer resistance to puromycin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid.
  • APHs aminoglycoside phosphotransferases
  • hygromycin phosphotransferase hygromycin phosphotransferase
  • DHFR dihydrofo
  • the present application provides a transposase polypeptide, wherein the transposase polypeptide is derived from Sleeping Beauty (SB) transposase, and the amino acid sequence of the transposase polypeptide contains at least one mutated amino acid residue compared to the amino acid sequence from positions 100 to 180 of the unmutated SB transposase as shown in SEQ ID NO:2.
  • SB Sleeping Beauty
  • the number of amino acid residues mutated is 1 to 20.
  • the number of amino acid residues mutated is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
  • the at least one mutated amino acid residue is at least two mutated amino acid residues.
  • the mutated amino acid residues are 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3.
  • the mutated amino acid residues are 2.
  • the mutation site of the at least one mutated amino acid residue is located at positions 110 to 120 and 170 to 180 of the amino acid sequence as shown in SEQ ID NO: 2.
  • the mutation site is located at position 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 and/or 180 of the amino acid sequence as shown in SEQ ID NO: 2.
  • the mutated amino acid residue is located at position 114, 116, 177 and/or 178 of the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position G114, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position S116, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position K177, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2.
  • the amino acid sequence of the transposase polypeptide comprises a mutation at amino acid position P178, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO: 2.
  • the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and S116, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and K177, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions G114 and P178, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions S116 and K177, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide comprises mutations at amino acid positions S116 and P178, compared to the unmutated SB transposase amino acid sequence as set forth in SEQ ID NO:2.
  • the amino acid sequence of the transposase polypeptide contains mutations at the K177 and P178 amino acid positions.
  • the at least one mutated amino acid residue is mutated to an alanine residue.
  • amino acid residues that are mutated are all mutated to alanine residues.
  • the amino acid sequence of the transposase polypeptide comprises an amino acid mutation selected from the group consisting of G114A, S116A, K177A, and P178A, compared to the unmutated SB transposase as shown in SEQ ID NO:2.
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position G114 to alanine (G114A).
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position S116 to alanine (S116A).
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position K177 to alanine (K177A).
  • the transposase polypeptide amino acid sequence comprises a mutation at amino acid position P178 to alanine (P178A).
  • the transposase polypeptide amino acid sequence comprises a combination of amino acid mutations selected from the group consisting of:
  • the present application provides a transposase polypeptide comprising an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2, wherein the transposase polypeptide comprises alanine at a position corresponding to position 114 and/or alanine at a position corresponding to position 116.
  • the transposase polypeptide comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:3-11, or a sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:3-11.
  • the present application provides a polynucleotide molecule comprising a nucleic acid sequence encoding the aforementioned transposase polypeptide.
  • the molecule can be a DNA expression vector.
  • the DNA expression vector can include a transposase encoding sequence operably connected to a promoter (such as T7 or SP6 promoter) for expressing polypeptides in vitro or a promoter for expressing polypeptides in mammalian cells.
  • the polynucleotide molecule can be RNA or mRNA.
  • RNA can include a 5'-cap, an IRES motif, (heterologous) 5'UTR, (heterologous) 3'UTR and/or a poly (A) sequence.
  • RNA can additionally include a poly (A) sequence of 20 to 300 nucleotides.
  • the application provides a method for preparing a transposase polypeptide as described above, comprising transfecting cells with a polynucleotide encoding a transposase polypeptide and expressing the polypeptide from the polynucleotide.
  • the invention provides a host cell comprising the polypeptide or polynucleotide molecule of the embodiment.
  • the cell is a mammalian cell, such as a human cell.
  • the cell is a stem cell or an induced pluripotent stem (iPS) cell.
  • the cell is a natural killer (NK) cell, a precursor of a NK cell, a T cell, a precursor of a T cell, or an immune cell.
  • the cell may comprise an RNA encoding the transposase polypeptide of the embodiment.
  • a cell colony is provided, the cell comprising the polypeptide or polynucleotide molecule of the embodiment.
  • the present application provides a method for genetically engineering a cell, comprising: transfecting the cell with a transposase polypeptide as described above or a nucleic acid encoding a transposase polypeptide and a DNA vector comprising a sequence encoding a selected genetic element flanked by transposon repeat sequences, and then incubating the cell under conditions suitable for (transient or stable) transposase activity, thereby integrating the selected genetic element into the genome of the cell and producing an engineered cell.
  • the sequence encoding the transposase polypeptide is flanked by transposon repeat sequences.
  • the DNA vector of the selected genetic element also includes the sequence of the transposase polypeptide of the coding embodiment. Therefore, in some aspects, the method of the embodiment includes transfecting cells with a DNA vector, wherein the DNA vector includes the sequence of the selected genetic element that encodes transposons and the sequence of the transposase of the coding embodiment (which is under the control of a promoter sequence), and then incubating cells under conditions suitable for transposase expression and activity, thereby integrating the genetic element selected in the genome of the cell and producing engineered cells. In some aspects, the method includes the third step of separating or cultivating engineered cells. In some aspects, the genetic element selected is a screenable or selectable marker.
  • the genetic element selected can encode an antibody, inhibitory nucleic acid (for example, small interfering RNA (siRNA)), therapeutic polypeptide, T cell receptor (TCR), chimeric antigen receptor (CAR) or an enhancer for immune cell function.
  • the genetic element encoding CAR or TCR selected can be a gene or part thereof for replacing or modifying the corresponding gene from the cell (for example, changing the sequence or expression of the gene or "knockout" gene expression in the cell).
  • the transfected cells are mammalian cells, such as human cells.
  • the cells can be stem cells or iPS cells.
  • the cells can be immune system cells or precursors thereof, such as NK cells, T cells, precursors of NK cells, or precursors of T cells.
  • transfection cells can include the use of chemical-based transfection reagents, electroporation of cells or other technologies for providing nucleic acid and/or protein to the cytoplasm and nucleus of cells.
  • salt precipitates such as CaPO4 precipitates
  • lipids such as charged or non-polar lipids
  • cationic polymers such as PEG-complexes and/or protein complexes (such as cationic polypeptides)
  • transfection can involve the use of liposomes, such as phospholipid liposomes (for example, liposomes incorporating glycerophospholipids or sphingolipids).
  • cells can be transduced with viral vectors (for example, adenovirus, adeno-associated virus, retrovirus (such as slow virus) or vaccinia virus vectors).
  • viral vectors for example, adenovirus, adeno-associated virus, retrovirus (such as slow virus) or vaccinia virus vectors.
  • the viral vector used according to the embodiment is a non-integrating viral vector.
  • the transposase of the embodiment can be delivered to cells together with or separately from the nucleic acid encoding the transposon repetition.
  • protein transfection reagents can be used to deliver transposase to cells as recombinant polypeptides, and nucleic acid transfection systems or viral vectors can be used to deliver the nucleic acid molecules comprising the transposon repetitions.
  • RNA encoding the transposase is co-transfected with DNA comprising the transposon repeats and the selected genetic element.
  • the method further comprises transfecting a cell population with a transposase polypeptide of the embodiment or a nucleic acid encoding a transposase polypeptide and a DNA vector comprising a sequence encoding the selected genetic element flanked by transposon repeats, and incubating the population under conditions suitable for transposase activity, thereby integrating the selected genetic element in the genome of the cell and producing an engineered cell population.
  • the method comprises transfecting a population of T cells or T cell precursors with a transposase polypeptide or a nucleic acid encoding a transposase polypeptide and a DNA vector comprising a sequence encoding a CAR flanked by transposon repeats, and incubating the population under conditions suitable for transposase activity, thereby integrating the CAR in the genome of the cell and producing an engineered T cell or T cell precursor population.
  • the method comprises transfecting a population of T cells or T cell precursors with a transposase polypeptide or a nucleic acid encoding a transposase polypeptide and a DNA vector comprising a sequence encoding a CAR flanked by transposon repeats, and incubating the population under conditions suitable for transposase activity, thereby integrating the CAR in the genome of the cell and producing an engineered T cell or T cell precursor population.
  • a method of providing a T cell response in a human subject having a disease comprising first obtaining a population of engineered T cells or T cell precursors according to the embodiments, optionally culturing the cells in a medium that selectively enhances the proliferation of CAR-expressing T cells, and then administering an effective amount of CAR-expressing T cells to the subject to provide a T cell response.
  • the method of the embodiment includes:(a) obtaining a cell sample from a subject, the sample comprising T cells or T cell progenitors;(b) DNA transfection cells are transfected with a chimeric antigen receptor (CAR) having a transposon flanking and a transposase encoding the embodiment of the invention integrated into the cell genome to provide a transgenic CAR expressing cell colony;(c) optionally, ex vivo culture transgenic CAR cell colonies in a culture medium that selectively enhances CAR expression T cell proliferation;And (d) applying an effective amount of transgenic CAR cells to the subject to provide T cell responses.
  • CAR chimeric antigen receptor
  • transgenic CAR cells are cultured in vitro for less than 21 days, for example, less than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or less.
  • CAR cells are cultured in vitro for no more than 3 to 5 days.
  • steps (a)-(d) of the method i.e., obtaining the cell sample to administering the CAR T cells
  • the cell sample from the subject can be a sample of peripheral blood or umbilical cord blood that is less than about 200 ml.
  • the sample can be collected by apheresis.
  • the sample is collected by a method that does not involve apheresis (e.g., by venipuncture).
  • the initial volume of the cell sample is less than 175 ml, less than about 175 ml, less than 150 ml, less than about 150 ml, less than 125 ml, less than about 125 ml, less than 100 ml, less than about 100 ml, less than 75 ml, less than about 75 ml, less than 50 ml, less than about 50 ml, less than 25 ml, or less than about 25 ml (e.g., when obtained from a subject, the cell sample has an initial volume of between about 50 to about 200 ml, between about 50 to about 100 ml, or between about 100 to about 200 ml).
  • the method of the embodiment relates to transfecting cells with DNA encoding a chimeric antigen receptor (CAR) and a transposase.
  • CAR chimeric antigen receptor
  • a transposase a chimeric antigen receptor
  • Cell transfection methods are well known in the art, but in some aspects, efficient transfection methods such as electroporation are used.
  • a nuclear transfection device can be used to introduce nucleic acid into cells.
  • the transfection step does not involve infecting or transducing cells with viruses, which can cause genotoxicity and/or lead to an immune response for cells containing viral sequences in treated subjects.
  • the CAR expression vector is a DNA expression vector, such as a plasmid, a linear expression vector, or a vector encoding a CAR.
  • the vector comprises additional sequences, such as sequences that promote CAR expression, such as promoters, enhancers, poly A signals and/or one or more introns.
  • the CAR coding sequence is flanked by transposon sequences such that the presence of a transposase allows the coding sequence to be integrated into the genome of the transfected cell.
  • transposase of the embodiment of the transfected cell genome is further transfected with promoting CAR encoding sequence to be integrated.
  • transposase is provided as DNA expression vector.
  • transposase is provided as expressible RNA or protein so that the long-term expression of transposase does not occur in transgenic cells.
  • mRNA for example, mRNA comprising cap and poly-A tail.
  • the method of the embodiment can be used to prepare (for example, for clinical trials) CAR+T cells with binding specificity to various tumor antigens (such as CD19, ROR1, CD56, EGFR, CD33, CD123, c-met, GD2).
  • the CAR+T cells produced using this technology can be used to treat patients with leukemia (such as AML, ALL, CML), infection and/or solid tumors.
  • the method of the embodiment can be used to treat cell proliferative diseases, fungi, viruses, bacteria or parasitic infections.
  • Pathogens that can be targeted include but are not limited to Plasmodium, Trypanosoma, Aspergillus (Aspergillus), Candida (Candida), HSV, RSV, EBV, CMV, JC virus, BK virus or Ebola pathogens.
  • antigens that can be targeted by the CAR cells of the embodiments include, but are not limited to, CD19, CD20, carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant ras, HER2/Neu, ERBB2, folate binding protein, HIV-1 envelope glycoprotein gp120, HIV-1 envelope glycoprotein gp41, GD2, CD123, CD23, CD30, CD56, c-Met, mesothelin, GD3, HERV-K, IL-11R ⁇ , kappa chain, lambda chain, CSPG4, ERBB2, EGFRvIII, or VEGFR2.
  • CD19, CD20 carcinoembryonic antigen, alpha-fetoprotein, CA-125, 5T4, MUC-1, epithelial tumor antigen, melanoma-associated antigen, mutant p53, mutant r
  • CAR cells are delivered to an individual in need thereof, such as an individual with cancer or infection.
  • the cells then enhance the individual's immune system to attack the corresponding cancer or pathogenic cells.
  • one or more doses of antigen-specific CAR T cells are provided to the individual.
  • the duration between administrations should be sufficient to allow for proliferation in the individual, and in specific embodiments, the duration between doses is 1, 2, 3, 4, 5, 6, 7 or more days.
  • the source of allogeneic or autologous T cells modified to contain a chimeric antigen receptor can be of any kind, but in a specific embodiment, for example, the cells are obtained from a reservoir of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells.
  • a suitable dose for therapeutic effect will be at least 10 5 or about 10 5 to about 10 10 cells per dose, for example, preferably in a series of dosing cycles.
  • An exemplary dosing regimen consists of four weekly dosing cycles of increasing doses, starting with at least about 10 5 cells on day 0, for example, gradually increasing to a target dose of about 10 10 cells within a few weeks of starting a dose escalation regimen within the patient.
  • Suitable modes of administration include intravenous, subcutaneous, intracavitary (e.g., through a reservoir access device), intraperitoneal, and direct injection into a tumor mass.
  • compositions of the present invention can be used alone or in combination with other established agents useful in treating cancer. Whether delivered alone or in combination with other agents, the pharmaceutical compositions of the present invention can be delivered to various sites in mammals, particularly humans, by various routes to achieve a specific effect. Those skilled in the art will recognize that, although more than one route can be used for administration, a particular route can provide a more direct and more effective response than another route. For example, intradermal delivery can be more advantageously used to treat melanoma relative to inhalation.
  • Local or systemic delivery can be achieved by administration, including application or instillation of the formulation into a body cavity, inhalation or insufflation of an aerosol, or by parenteral introduction, including intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, or intradermal administration.
  • each dosage unit e.g., injection
  • each dosage unit contains a predetermined amount of the composition alone or in appropriate combination with other active agents.
  • unit dosage form refers to a physically discrete unit suitable as a unit dose for human and animal subjects, each unit containing a predetermined amount of the composition of the present invention alone or in combination with other active agents, which is calculated in an amount sufficient to produce a desired effect in combination with a pharmaceutically acceptable diluent, carrier, or vehicle where appropriate.
  • the specification of the unit dosage form of the present invention depends on the specific pharmacodynamics associated with the pharmaceutical composition in a particular subject.
  • An effective amount or sufficient number of isolated transduced T cells are present in a composition and introduced into a subject such that a long-term, specific anti-tumor response is established to reduce the size of the tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment.
  • the amount of transduced T cells reintroduced into the subject results in a reduction in tumor size of about or at least about 10%, about or at least about 20%, about or at least about 30%, about or at least about 40%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 95%, about or at least about 98%, or about 100% or 100% when compared to the original or initial (e.g., "treatment day 0") size of the tumor.
  • kits can include a variety of additional elements, such as a DNA vector encoding transposon repeats, a transfection reagent, cells, a CAR expression construct, culture medium, aAPCs, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells), and/or a plasmid encoding a CAR or a transposase.
  • additional elements such as a DNA vector encoding transposon repeats, a transfection reagent, cells, a CAR expression construct, culture medium, aAPCs, growth factors, antibodies (e.g., for sorting or characterizing CAR T cells), and/or a plasmid encoding a CAR or a transposase.
  • the kit comprises a transposase polypeptide of the embodiment or a nucleic acid encoding a transposase polypeptide, one or more reagents for producing a CAR-expressing construct (with flanking transposon repeats), cells for transfecting the expression construct, and/or one or more instruments for obtaining cells for transfection expressing the construct (such instruments may be syringes, pipettes, tweezers, and/or any such medically approved device).
  • a transfection device such as an electroporation device is included.
  • the kit may include one or more suitable aliquots of the compositions of the present invention or reagents for producing the compositions of the present invention.
  • the components of the kit may be packaged in aqueous media or lyophilized form.
  • the container means of the kit may include at least one vial, test tube, flask, bottle, syringe or other container means into which the compositions of the present invention may be placed and preferably into which suitable aliquots of the compositions may be taken.
  • the kit will typically also include a second, third, or other additional container in which the additional components can be separately placed.
  • various combinations of components can be included in vials.
  • the kit of the present invention will typically also include a device for containing the chimeric receptor construct and any other reagent containers in a sealed confinement for commercial sale.
  • such containers can include injection or blow-molded plastic containers that retain the desired vials.
  • FIG. 1A shows a schematic diagram of a transposase enzyme activity assay.
  • the streptomycin resistance gene in the plasmid is split by a transposon encoding a zeocin resistance gene.
  • This plasmid and the transposase plasmid are co-transfected into HEK293T cells. Plasmid DNA is extracted from the HEK293T cells and used to transform E. coli. Plasmids cleaved by the transposase lose zeocin resistance but gain streptomycin resistance.
  • Figure 1B shows the results of testing the enzymatic activity of different transposases.
  • HEK293T cells were seeded on a 6-well plate and cultured overnight. The cells were then transfected with equal amounts of transposon plasmids, plasmids containing no transposase, an inactivated transposase mutant (E279D), SB11 transposase, and SB100X transposase. After 72 hours, the cells were harvested, plasmid DNA was extracted using the Plasmid Mini Kit, and Escherichia coli was transformed. The transformed bacteria were plated on streptomycin-containing plates, cultured overnight, and monoclonal colonies were counted. The number of colonies reflects the activity of the transposase.
  • Example 2 Transposase mutants prepared by alanine scanning method have different enzymatic activities
  • Transposase mutants were obtained by site-directed mutagenesis chain amplification. After sequencing and identification, plasmids encoding different transposase mutants were co-transfected with transposon plasmids into HEK293T cells, and then the transposase cleavage activity was tested using the method described in Example 1. The E279D transposase mutant was used as a negative control. The results were normalized with SB32X transposase (SEQ ID NO: 2). All error bars represent S.E.M. of three independent replicates. The results showed that the enzymatic cleavage activity of the multiple transposase mutants provided in this application was higher than that of SB32X.
  • HeLa cells were cultured in DMEM (Gibco, NY, USA) containing fetal bovine serum. 250 ng of plasmid DNA encoding each transposase mutant and 750 ng of transposon plasmid DNA encoding the Zeocin resistance gene were co-transfected into the cells. After 24 hours, Cells were transferred to 10-cm culture dishes. Resistant cell clones were selected using Zeocin-containing culture medium. Two weeks later, cells were fixed with 4% paraformaldehyde, stained, and the number of cell clones was counted. Results were normalized to those of SB32X transposase (SEQ ID NO: 2). All error bars represent the mean average (SEM) of three independent replicates. The results showed that the multiple transposase mutants provided herein exhibited higher enzymatic activity than SB32X.
  • T cells were sorted from PBMC using magnetic beads, and minicircle DNA expressing CD19-CAR was electroporated into T cells simultaneously with three transposase plasmids, SB11, SB100X, or Tri-FITase, using an electroporator (Lonza). After culturing overnight in RPMI-1640 medium containing 10% FBS and 0.1% DMSO, live cells were enriched by OptiPrep (Axis-Shield) gradient density centrifugation.
  • CAR-T cells were then expanded and cultured in X-VIVO15 medium containing 10% FBS and cytokines (50 IU/mL IL-2, 50 IU/mL IL-7, and 100 IU/mL IL-15), and the culture medium was refreshed every 2 days. The number of live cells was counted at different times after electroporation, and the CAR-T positive rate and MFI were detected by flow cytometry. Dead cells were excluded with 7-AAD, T cells were labeled with PE-Cy7-Anti-CD3, and CAR-T cells were labeled with FITC-CD19-Fc. T cells transfected with different transposases maintained high viability. The CAR-positive rate in T cells transfected with Tri-FITase transposase was higher than that in T cells transfected with SB11 transposase, and similar to that in T cells transfected with SB100X.

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Abstract

L'invention concerne un polypeptide de transposase. Le polypeptide de transposase est dérivé d'une transposase Sleeping Beauty (SB), et par comparaison avec la séquence d'acides aminés aux positions 100-180 de la transposase SB non mutée représentée dans SEQ ID NO : 2, la séquence d'acides aminés du polypeptide de transposase comprend au moins un résidu d'acide aminé muté.
PCT/CN2024/091741 2024-01-23 2024-05-08 Transposase et son utilisation Pending WO2025156492A1 (fr)

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