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WO2024239143A1 - Cellule modifiée et son utilisation - Google Patents

Cellule modifiée et son utilisation Download PDF

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Publication number
WO2024239143A1
WO2024239143A1 PCT/CN2023/095270 CN2023095270W WO2024239143A1 WO 2024239143 A1 WO2024239143 A1 WO 2024239143A1 CN 2023095270 W CN2023095270 W CN 2023095270W WO 2024239143 A1 WO2024239143 A1 WO 2024239143A1
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Prior art keywords
cells
cell
xcl1
receptor
antigen
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PCT/CN2023/095270
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English (en)
Chinese (zh)
Inventor
邢芸
杜靖雯
任江涛
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香港北恒生物科技有限公司
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Priority to PCT/CN2023/095270 priority Critical patent/WO2024239143A1/fr
Publication of WO2024239143A1 publication Critical patent/WO2024239143A1/fr

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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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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

  • the present invention belongs to the field of immunotherapy. More specifically, the present invention relates to an engineered cell expressing a cell surface molecule that specifically recognizes an antigen and exogenous XCL1 and Fc, and also relates to the use of the engineered cell in treating diseases such as cancer, infection or autoimmunity.
  • the present invention provides a novel engineered cell that expresses cell surface molecules that specifically recognize antigens as well as exogenous XCL1 and Fc.
  • cell surface molecule that specifically recognizes an antigen refers to a molecule that is expressed on the cell surface and can specifically bind to a target molecule (eg, an antigen).
  • target molecule eg, an antigen
  • Such surface molecules generally include an antigen binding domain that can specifically bind to an antigen, a transmembrane domain that anchors the surface molecule to the cell surface, and an intracellular domain responsible for signal transduction.
  • CARs chimeric antigen receptors
  • TCRs T cell receptors
  • T cell receptor fusion proteins T cell antigen couplers
  • ImmTACs immune mobilization monoclonal T cell receptors
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide, which generally includes an antigen (e.g., tumor antigen) binding domain (e.g., a ligand of an antibody or antigen), a transmembrane domain, a primary signaling domain, and an optional co-stimulatory domain, each domain being connected by a linker.
  • an antigen e.g., tumor antigen
  • binding domain e.g., a ligand of an antibody or antigen
  • transmembrane domain e.g., a ligand of an antibody or antigen
  • transmembrane domain e.g., a ligand of an antibody or antigen
  • primary signaling domain e.g., a primary signaling domain
  • co-stimulatory domain e.g., a co-stimulatory domain
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises a tumor antigen binding domain, a transmembrane domain, a primary signaling domain, and optionally one or more co-stimulatory domains.
  • the chimeric antigen receptor also includes one or more of the following structures: a signal peptide, a hinge region, a suicide gene, a switch structure, and the like.
  • T cell receptor refers to a membrane protein complex that responds to antigen presentation and participates in T cell activation.
  • MHC major histocompatibility complex molecule
  • TCR consists of six peptide chains that form heterodimers, which are generally divided into ⁇ type and ⁇ type. Each peptide chain includes a constant region and a variable region, wherein the variable region is responsible for binding to a specific antigen and MHC molecule of specificity.
  • the variable region of TCR may include an antigen binding domain or be operably connected to an antigen binding domain, wherein the definition of the antigen binding domain is as described below.
  • T cell antigen coupler includes three functional domains: (1) a tumor targeting domain, which may include a single-chain antibody, a designed ankyrin repeat protein (DARPin), or other targeting moieties; (2) an extracellular domain, which is a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with the TCR receptor; and (3) a transmembrane domain and an intracellular domain of the CD4 co-receptor, wherein the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) of the TCR complex as the initial step in T cell activation.
  • TAC immunoreceptor tyrosine-based activation motif
  • T cell receptor fusion protein refers to a recombinant polypeptide derived from various components of TCR, which is usually composed of a TCR subunit and an antigen binding domain connected thereto and expressed on the cell surface.
  • the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular signaling domain.
  • ImmTAC immune mobilizing monoclonal T cell receptor
  • TCR T cell receptor
  • anti-CD3 scFv an engineered T cell receptor (TCR) and an anti-CD3 scFv, wherein: the engineered TCR can specifically recognize and bind to the HLA-peptide complex on the surface of tumor cells with significantly improved affinity, and promote T cell-mediated effector function through the interaction of the scFv antibody fragment with CD3.
  • the cell surface molecule comprises an extracellular domain that specifically recognizes an antigen (e.g., a tumor antigen).
  • the extracellular domain comprises an antibody that specifically binds to an antigen or a ligand of the antigen.
  • the antigen is selected from: ALK, ADRB3, AKAP-4, APRIL, ASGPR1, BCMA, B7H3, B7H4, B7H6, bcr-abl, BORIS, BST2, BAFF-R, BTLA, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD24, CD25, CD28, CD30, CD33, CD38, CD40, CD44, CD44v6 , CD44v7/8, CD47, CD52, CD56, CD57, CD58, CD70, CD72, CD79a, CD79b, CD80, CD81, CD86, CD97, CD123, CD133, CD 137.
  • IGF1R IGF1R, KIT, Kappa Light Chain, KDR, LewisY, LMP2, LY6K, LAGE-1a, legumain, LCK, LAIR1, LILRA2, LY75, M SLN, MUC1, MUC16, MAGE-A1, MAGE3, MAD-CT-1, MelanA/MART1, ML-IAP, MYCN, mut hsp70-2, NCAM, NY-BR-1, NY- ESO-1, NA17, Notch-1-4, nAchR, NKG2D, NKG2D ligand, OY-TES1, OR51E2, OX40, PRSS21, PSCA, PD1, PD-L1, PD-L2, PSMA, Prostase, PAP, PDGFR- ⁇ , PCTA-1/galectin 8, p53, p53 mutant, prostein, PLAC1, PANX3, PAX3, PAX5, PTCH1, RANK, RAGE-1, ROR1, Ras mutant, RhoC,
  • the antigen is selected from CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA and GPRC5D, more preferably selected from CD19, Claudin 18.2, MSLN, GPRC5D, ROR1, CD7 and BCMA.
  • the functional exogenous receptor comprises an extracellular domain that specifically recognizes CD19, such as an antibody targeting CD19.
  • Antibodies targeting CD19 known in the art can be used in the present invention.
  • the antibody comprises a light chain variable region and a heavy chain variable region, wherein the CDR1-H, CDR2-H and CDR3-H contained in the heavy chain variable region are the same as the CDR1-H, CDR2-H and CDR3-H contained in SEQ ID NO: 10 or 19; wherein the CDR1-L, CDR2-L and CDR3-L contained in the light chain variable region are the same as the CDR1-L, CDR2-L and CDR3-L contained in SEQ ID NO: 11 or 20.
  • the CDR1-H contained in the heavy chain variable region is as shown in SEQ ID NO: 4
  • the CDR2-H is as shown in SEQ ID NO: 5
  • the CDR3-H is as shown in SEQ ID NO: 6
  • the CDR1-L contained in the light chain variable region is as shown in SEQ ID NO: 7
  • the CDR2-L is as shown in SEQ ID NO: 8
  • the CDR3-L is as shown in SEQ ID NO: 9.
  • the heavy chain variable region comprises CDR1-H as shown in SEQ ID NO:13, CDR2-H as shown in SEQ ID NO:14, and CDR3-H as shown in SEQ ID NO:15
  • the light chain variable region comprises CDR1-L as shown in SEQ ID NO:16, CDR2-L as shown in SEQ ID NO:17, and CDR3-L as shown in SEQ ID NO:18.
  • the antibody targeting CD19 comprises a light chain variable region and a heavy chain variable region
  • the heavy chain variable region is at least 90%, 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 100% identical to SEQ ID NO: 10 or 19
  • the light chain variable region is at least 90%, 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 100% identical to SEQ ID NO: 11 or 20.
  • the antibody targeting CD19 in the present invention comprises a heavy chain variable region as shown in SEQ ID NO:10 and a light chain variable region as shown in SEQ ID NO:11, or comprises a heavy chain variable region as shown in SEQ ID NO:19 and a light chain variable region as shown in SEQ ID NO:20.
  • the antibody targeting CD19 is at least 90%, 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 100% identical to SEQ ID NO: 12 or 21.
  • the antibody targeting CD19 is as shown in SEQ ID NO: 12 or 21.
  • the functional exogenous receptor comprises an extracellular domain that specifically recognizes Claudin 18.2, such as an antibody targeting Claudin 18.2.
  • Antibodies targeting Claudin 18.2 known in the art can be used in the present invention.
  • the antibody targeting Claudin 18.2 comprises VHH, wherein the CDR1, CDR2 and CDR3 contained in the VHH are the same as the CDR1, CDR2 and CDR3 contained in SEQ ID NO:25.
  • the CDR1 contained in the VHH is as shown in SEQ ID NO:22
  • the CDR2 is as shown in SEQ ID NO:23
  • the CDR3 is as shown in SEQ ID NO:24.
  • the antibody targeting Claudin 18.2 comprises an amino acid sequence that is at least 90%, 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 100% identical to SEQ ID NO: 25.
  • the antibody targeting Claudin 18.2 in the present invention comprises an amino acid sequence as shown in SEQ ID NO: 25.
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which comprises an antigen binding domain, a transmembrane domain, a primary signaling domain, and optionally one or more co-stimulatory domains.
  • the functional exogenous receptor of the present invention is a chimeric antigen receptor, which targets CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, MSLN, AFP, folate receptor ⁇ , CEA, PSCA, PSMA, Her2, EGFR, IL-13Ra, GD2, NKG2D, Claudin 18.2, ROR1, EGFRvIII, CS1, BCMA, GPRC5D, or any combination thereof, more preferably targets CD19, Claudin 18.2, MSLN, ROR1, GPRC5D, CD7, BCMA, or any combination thereof.
  • the chimeric antigen receptor further comprises a signal peptide and/or
  • antigen-binding domain refers to any structure (such as an antibody, etc.) or a functional variant thereof that can bind to an antigen.
  • the antigen binding domain in the present invention is selected from an antibody.
  • antibody has the broadest meaning understood by those skilled in the art, and includes complete antibodies such as monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that can exhibit the desired biological activity, which may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).
  • antibody fragment refers to at least a portion of a complete antibody or a variant thereof, and refers to a binding domain (e.g., the antigen binding domain of a complete antibody) sufficient to confer recognition and specific binding to a target (e.g., an antigen) to the antibody fragment.
  • a binding domain e.g., the antigen binding domain of a complete antibody
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide bond-linked Fv (sdFv), linear antibodies, "diabodies” with two antigen binding sites, single domain antibodies (sdAb) (e.g., heavy chain variable region VH, light chain variable region VL, nanobody VHH, etc. of antibodies).
  • sdAb single domain antibodies
  • the antigen binding domain of the present invention is selected from ligands, receptors and functional fragments thereof (i.e., functional fragments with antigen binding ability, such as extracellular domains).
  • ligand or receptor refers to any molecule or atom that can interact with an antigen after binding.
  • a ligand or receptor can be a naturally occurring molecule, such as an organic or inorganic molecule, or a synthetic molecule.
  • the "antigen binding domain" of the present invention encompasses antibodies, ligands, receptors and functional fragments thereof as described above. Therefore, the antigen binding domain described in the present invention is selected from a complete antibody, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, sdFv, linear antibody, diabody, sdAb, a functional fragment of a ligand or a receptor.
  • the term "functional variant” or “functional fragment” refers to a variant or fragment that contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the parent amino acid sequence but retains the biological activity of the parent amino acid.
  • the functional fragment of a ligand or receptor in the present invention generally refers to a fragment of a ligand or receptor that is capable of binding to a corresponding immunosuppressive protein, such as an extracellular region.
  • heavy chain refers to the larger of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules and generally determines the class to which the antibody belongs.
  • light chain refers to the smaller of the two types of polypeptide chains that occur in naturally occurring conformations in antibody molecules. Kappa ( ⁇ ) and lambda ( ⁇ ) light chains refer to the two major antibody light chain isotypes.
  • CDR complementarity determining region
  • CDR or FR The precise amino acid sequence of a given CDR or FR may be different due to the different numbering schemes selected, and it should be understood that a "CDR" or "FR" of a given antibody or its region (such as its variable region) covers a CDR or FR defined by any of the above schemes or other known schemes, and in the case where a specified CDR or FR contains a given amino acid sequence, it should be understood that such CDR or FR can also have a sequence of a corresponding CDR or FR defined by any of the above schemes or other known schemes.
  • the numbering scheme used herein to define the boundaries of CDRs and FRs is the Chothia scheme.
  • single-chain antibody refers to a fusion protein comprising at least one light chain variable region (VL) and at least one heavy chain variable region (VH), wherein the light chain variable region and the heavy chain variable region are adjacent (e.g., connected via a linker) and can be expressed in the form of a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived.
  • the scFv herein may have the VL and VH in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or VH-linker-VL from the N-terminus to the C-terminus.
  • the term "linker” refers to a molecular sequence connecting two molecules or two sequences on the same molecule.
  • the linker is a peptide linker.
  • the linker does not adversely affect the expression, secretion or biological activity of the polypeptide.
  • the linker is preferably not antigenic and does not induce an immune response.
  • the linker may be an endogenous amino acid sequence, an exogenous amino acid sequence (e.g., a sequence rich in GS) or a non-peptide chemical linker.
  • the linker described in the present invention has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:41 or 42.
  • single domain antibody refers to a single antigen-binding polypeptide having three complementary determining regions (CDRs), including full-length antibodies (e.g., HCAbs) and antigen-binding fragments thereof (e.g., VH, VL, VHH).
  • CDRs complementary determining regions
  • the single domain antibody is selected from or engineered from HCAbs of camel or shark origin, and its heavy chain variable domain is referred to herein as "VHH”.
  • VHH has the following basic structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementary determining regions 1 to 3.
  • identity refers to the extent to which two (nucleotide or amino acid) sequences have the same residue at the same position in an alignment, and is usually expressed as a percentage. Preferably, the identity is determined over the entire length of the compared sequences. Thus, sequences with complete identity are considered to be identical. Two copies of the same sequence have 100% identity.
  • sequence identity such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.
  • transmembrane domain refers to a polypeptide structure that enables cell surface molecules to be expressed on the cell surface and anchors the antigen binding domain to the cell membrane.
  • the transmembrane domain can be natural or synthetic, and can also be derived from any membrane-bound protein or transmembrane protein. When the antigen binding domain binds to the target, the transmembrane domain can carry out signal transduction.
  • transmembrane domains in the present invention can be derived from, for example, TCR alpha chains, TCR beta chains, TCR gamma chains, TCR delta chains, CD3 zeta subunits, CD3 epsilon subunits, CD3 gamma subunits, CD3 delta subunits, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
  • the transmembrane domain is derived from the following molecules: CD8 ⁇ , CD4, CD28 or 4-1BB, or, the transmembrane domain may be synthetic and may mainly contain hydrophobic residues such as leucine and valine.
  • the transmembrane domain is derived from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 26, or the transmembrane domain is derived from CD8 ⁇ , which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 27 or 28.
  • the chimeric antigen receptor further comprises a hinge region between the antigen binding domain and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the antigen binding domain. Specifically, the hinge region is used to provide greater flexibility and accessibility for the antigen binding domain.
  • the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region may be derived in whole or in part from natural molecules, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.
  • the hinge region is derived from the hinge region of CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably from the hinge region of CD8 ⁇ , CD28 or IgG4.
  • the hinge region is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 29.
  • the hinge region is from CD8 ⁇ , which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 30 or 31.
  • the hinge region is from IgG4, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 32.
  • costimulatory domain refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response such as an effector function, which is an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular region of the costimulatory molecule, or a functional fragment thereof.
  • Cosmetic molecule refers to a cognate binding partner that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response (e.g., proliferation and survival).
  • the costimulatory signaling domain of any costimulatory molecule is suitable for use in the immunosuppressive molecules described herein.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.
  • the costimulatory domain of the present invention includes but is not limited to the intracellular region derived from the following proteins:CD94 ⁇ LTB ⁇ TLR1 ⁇ TLR2 ⁇ TLR3 ⁇ TLR4 ⁇ TLR5 ⁇ TLR6 ⁇ TLR7 ⁇ TLR8 ⁇ TLR9 ⁇ TLR10 ⁇ CARD11 ⁇ CD2 ⁇ CD7 ⁇ CD8 ⁇ CD18 ⁇ CD27 ⁇ CD28 ⁇ CD30 ⁇ CD40 ⁇ CD54 ⁇ CD83 ⁇ CD134(OX40) ⁇ CD137(4-1BB) ⁇ CD270(HVEM) ⁇ CD272(BTLA) ⁇ CD276(B7-H3) ⁇ CD278(ICOS) ⁇ CD357(GITR) ⁇ DAP10 ⁇ DAP12 ⁇ LAT ⁇ NKG2C ⁇ SLP76 ⁇ PD-1 ⁇ LIGHT ⁇ TRIM ⁇ ZAP70 ⁇ Preferably, the costimulatory domain of CAR of the present invention is 4-1BB and
  • the costimulatory domain is from CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 33.
  • the costimulatory domain is from 4-1BB, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 34 or 35.
  • the chimeric antigen receptor of the present invention also comprises a primary signaling domain.
  • the term "primary signaling domain” refers to a protein structure that works together to initiate primary signaling after antigen-receptor binding, which is generally an intracellular sequence of a T cell receptor and a co-receptor.
  • the primary signaling domain generally comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the primary signaling domain in the present invention includes, but is not limited to, intracellular regions derived from the following proteins: FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the chimeric antigen receptor of the present invention comprises a CD3 ⁇ primary signaling domain, for example, a CD3 ⁇ primary signaling domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 36 or 37.
  • the chimeric antigen receptor of the present invention further comprises a signal peptide so that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain a long hydrophobic amino acid segment that has a tendency to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually an amino acid segment that is recognized and cleaved by a signal peptidase.
  • the signal peptidase can be cleaved during or after the translocation to produce a free signal peptide and a mature protein.
  • the free signal peptide is then digested by a specific protease.
  • Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8 ⁇ , IgG1, GM-CSFR ⁇ , etc.
  • the signal peptide that can be used in the present invention is from B2M, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:38.
  • the signal peptide useful in the present invention is derived from CD8 ⁇ , which has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:39 or 40.
  • XCL1 is also known as lymphoid chemokine 1, which is a member of the C-type chemokine family and is mainly produced by CD8+T cells and natural killer cells.
  • XCL1 has unique sequence characteristics and two mutually convertible protein spatial conformations, which makes XCL1 different from other chemokines and plays a unique function.
  • the XCL1-specific receptor XCR1 is a member of the G protein-coupled receptor family. The interaction between the two not only plays an important role in the negative selection of the thymus and the establishment of autoimmune tolerance, but also can initiate cross-antigen presentation and mediate cytotoxic immune responses.
  • XCL1 can not only regulate the balance of the immune system and maintain intestinal immune homeostasis, but also is associated with a variety of diseases, such as autoimmune diseases, nephritis, tuberculosis and human immunodeficiency virus infection.
  • the XCL1 used in the present invention may be wild-type XCL1, a variant thereof, or a functional fragment thereof, wherein the variant or functional fragment has the same or similar biological function as the wild-type XCL1.
  • the XCL1 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2.
  • Fc refers to the C-terminal region of the immunoglobulin heavy chain, which contains at least part of the constant region. Fc has no antigen binding activity and is the site where the immunoglobulin interacts with effector molecules or cells.
  • the term includes native Fc and variant Fc.
  • Native Fc refers to a molecule or sequence containing a non-antigen binding fragment produced by digesting a complete antibody, whether in monomeric or multimeric form.
  • the immunoglobulin source that produces native Fc is preferably derived from humans.
  • Native Fc is composed of monomeric polypeptides that can be connected to dimers or multimers by covalent linkage (e.g., disulfide bonds) and non-covalent linkages.
  • a native Fc is a disulfide-linked dimer produced by digesting IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9).
  • the term "native Fc" as used herein generally refers to monomeric, dimer and multimeric forms.
  • variant Fc also known as “Fc variant” refers to a variant or fragment that contains at least one amino acid modification (i.e., substitution, deletion or insertion) compared to the amino acid sequence of a native Fc but retains the biological activity (e.g., receptor binding activity) of the native Fc.
  • the Fc of the present invention is derived from a natural Fc or variant Fc of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD, or a hybrid Fc of any two of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, IgD.
  • it is derived from a natural Fc or variant Fc of IgG1, IgG2, IgG3, IgG4, or a hybrid Fc of IgG4 and IgD.
  • the Fc of the present invention has a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% with SEQ ID NO: 3.
  • the expression of the exogenous gene XCL1 and/or Fc in the present invention can be constitutive expression or conditional expression.
  • the expression of the exogenous gene XCL1 and/or Fc is conditional expression.
  • the exogenous gene of the present invention can be operably linked to an inducible, repressible or tissue-specific promoter to regulate the expression level of the introduced exogenous gene at a specific time or in a specific tissue or cell type.
  • the promoter is an inducible promoter, that is, a promoter that initiates transcription only under specific environmental conditions, developmental conditions or the presence of an inducer.
  • the promoter is a repressible promoter, that is, in the presence of a repressor specific to the repressible promoter, the expression of the exogenous gene in the cell is inhibited or not expressed.
  • the exogenous gene XCL1 and/or Fc can be operably linked to a localization domain, which can localize the exogenous gene of the present invention to a specific cell location for expression, such as a cell membrane, etc.
  • the localization domain includes, but is not limited to, a nuclear localization signal, a guide peptide, a transmembrane domain, etc.
  • the exogenous gene XCL1 and/or Fc of the present invention is operably linked to a transmembrane domain, thereby anchoring the expression on the surface of the engineered cell.
  • the present invention also provides an engineered cell, which expresses cell surface molecules that specifically recognize antigens and exogenous XCL1 and Fc.
  • the engineered cells of the invention are engineered immune cells.
  • immune cell refers to any cell with one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC) of the immune system.
  • effector functions e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC
  • immune cells can be B cells, T cells, macrophages, neutrophils, dendritic cells, monocytes, NK cells or NKT cells.
  • Immune cells can be obtained from a variety of sources, such as from a subject (e.g., from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor, etc.), or from a cell line (e.g., Jurkat, SupT1, NK92, etc.) cultured in vitro, or from stem cell differentiation (e.g., derived from cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells, adult stem cells, embryonic stem cells, pluripotent stem cells, iPSC, etc.).
  • a subject e.g., from a subject's peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, tumor, etc
  • immune cells are T cells or NK cells, more preferably T cells.
  • T cells can also be concentrated or purified.
  • T cells may be at any developmental stage, including, but not limited to, CD4+CD8+T cells, CD4+T cells (e.g., Th1 and Th2 cells), CD8+T cells (e.g., cytotoxic T cells), CD4-CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells, and T cells may be obtained using a variety of techniques known to those skilled in the art, such as separating T cells from the blood of a subject using Ficoll.
  • the engineered cells of the invention are engineered stem cells.
  • stem cell is a primitive cell with self-replication, multidirectional differentiation and homing potential. It is the origin cell of the body and the ancestral cell that forms various tissues and organs of the human body.
  • a daughter cell irreversibly moves to the terminal of differentiation and becomes a differentiated cell with a single function, until it completely loses the ability to divide again and eventually aging and dies.
  • the body retains a part of the undifferentiated primitive cells, which retain the characteristics of the parent. This part of the undifferentiated primitive cells that are retained is called stem cells. Once needed, these stem cells can produce differentiated cells by division according to the developmental pathway.
  • the stem cells described in the present invention can be embryonic stem cells, adult stem cells (such as umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells, etc.) and pluripotent stem cells (such as induced pluripotent stem cells iPSC, etc.).
  • adult stem cells such as umbilical cord blood stem cells, bone marrow stem cells, hematopoietic stem cells, mesenchymal stem cells, etc.
  • pluripotent stem cells such as induced pluripotent stem cells iPSC, etc.
  • the immune cells of the present invention further comprise at least one endogenous gene whose expression is inhibited or silenced, and the endogenous gene is selected from the following: CD52, GR, TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , HLA-I, HLA-II, B2M, immune checkpoint genes such as PD1, CTLA-4, LAG3 and TIM3. More particularly, the expression of at least TCR components (including TCR ⁇ , TCR ⁇ genes) or CD3 components (including CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ) in immune cells is inhibited or silenced. Silencing. This strategy is particularly useful for avoiding graft-versus-host disease (GvHD).
  • GvHD graft-versus-host disease
  • Methods for inhibiting or silencing genes are known in the art, such as by large-range nucleases, zinc finger nucleases, TALEN nucleases, or Cas enzymes in CRISPR systems to mediate DNA fragmentation, thereby knocking out the gene; or inhibiting gene expression by shRNA, RNAi, etc.
  • the present invention also provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding a cell surface molecule that specifically recognizes an antigen, a nucleic acid sequence encoding Fc, and a nucleic acid sequence encoding XCL1.
  • nucleic acid molecule includes sequences of ribonucleotides and deoxyribonucleotides, such as modified or unmodified RNA or DNA, each in single-stranded and/or double-stranded form, linear or circular, or mixtures thereof (including hybrid molecules). Therefore, nucleic acids according to the present invention include DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA).
  • the nucleic acid is DNA or RNA, more preferably mRNA.
  • the present invention also provides a vector comprising the nucleic acid described in the present invention.
  • the nucleic acid sequence encoding the cell surface molecule that specifically recognizes the antigen, the nucleic acid sequence encoding Fc and the nucleic acid sequence encoding XCL1 can be located in one or more vectors. When located in one vector, each nucleic acid sequence can be operably linked via a 2A peptide.
  • vector is a nucleic acid molecule used as a medium for transferring (foreign) genetic material into a host cell where it can, for example, be replicated and/or expressed.
  • Targeting vector is a medium that delivers separated nucleic acid to the interior of a cell by, for example, homologous recombination or using a hybrid recombinase of a sequence at a specific targeting site.
  • Expression vector is a vector for transcription of heterologous nucleic acid sequences (such as those encoding chimeric antigen receptor polypeptides of the present invention) in suitable host cells and translation of their mRNA. Suitable vectors that can be used for the present invention are known in the art, and many are commercially available.
  • the vectors of the present invention include but are not limited to plasmids, viruses (such as retroviruses, oncolytic viruses, slow viruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses, polyoma viruses and adeno-associated viruses, etc.), phages, phagemids, cosmids and artificial chromosomes (including BAC and YAC).
  • viruses such as retroviruses, oncolytic viruses, slow viruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses, polyoma viruses and adeno-associated viruses, etc.
  • phages phagemids
  • cosmids and artificial chromosomes including BAC and YAC.
  • the vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgenic) and a larger sequence as a vector "skeleton".
  • Engineered vectors are also generally included in the starting point of autonomous replication in host cells (if stable expression of polynucleotides is required), selection markers and restriction enzyme cleavage sites (such as multiple cloning sites, MCS).
  • the vector may further comprise elements such as a promoter, a polyadenylic acid tail (polyA), a 3'UTR, an enhancer, a terminator, an insulator, an operator, a selection marker, a reporter gene, a targeting sequence and/or a protein purification tag.
  • the vector is an in vitro transcription vector.
  • the present invention also provides a pharmaceutical composition, which comprises the engineered cells, nucleic acid molecules or vectors of the present invention as active agents, and one or more pharmaceutically acceptable excipients. Therefore, the present invention also covers the use of the nucleic acid molecules, vectors or engineered cells in the preparation of pharmaceutical compositions.
  • pharmaceutically acceptable excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of inducing the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, antiadherents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers, and tension modifiers. It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention.
  • Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose and water.
  • the selection of a suitable excipient depends, inter alia, on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention can be applied to a variety of routes of administration. Typically, administration is accomplished parenterally.
  • Parenteral delivery Methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.
  • composition according to the present invention may also be administered in combination with one or more other pharmaceutical agents suitable for the treatment and/or prevention of the disease to be treated.
  • the present invention also provides a method for treating a subject suffering from cancer, infection or autoimmune disease, comprising administering to the subject an effective amount of the nucleic acid molecule, vector, engineered cell or pharmaceutical composition according to the present invention. Therefore, the present invention also covers the use of the nucleic acid molecule, vector, engineered cell in the preparation of a drug for treating cancer, infection or autoimmune disease.
  • the method of treatment comprises administering to the subject an effective amount of the engineered cells and/or pharmaceutical compositions of the invention.
  • the term "subject" is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer.
  • the subject is a human.
  • the cancer is a cancer associated with expression of a target to which a functional exogenous receptor binds.
  • the cancer includes, but is not limited to, brain gliomas, blastomas, sarcomas, leukemias, basal cell carcinomas, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancers, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, liver tumors, lung cancers (e.g., small cell lung cancer, non-small cell lung cancer, adenocarcinoma and squamous cell lung cancer), lymphoma (including Hod
  • the infection includes, but is not limited to, infections caused by viruses, bacteria, fungi, and parasites.
  • the autoimmune diseases include but are not limited to type I diabetes, celiac disease, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia and systemic lupus erythematosus.
  • the method further comprises administering one or more additional chemotherapeutic agents, biologics, drugs or treatments to the subject.
  • the chemotherapeutic agent, biologic, drug or treatment is selected from radiation therapy, surgery, antibody reagents and/or small molecules and any combination thereof.
  • Figure 1 CAR expression levels of each CAR-T cell targeting CD19 determined by flow cytometry.
  • Figure 2 Mouse XCL1 levels in the supernatant of each CD19-targeting CAR-T cell culture until D5.
  • Figure 3 Target cell killing levels after co-incubation of various CAR-T cells targeting CD19 with target cells at effector-target ratios of 5:1, 1:1, 1:5, and 1:10.
  • Figure 4 IFN- ⁇ release levels of various CAR-T cells targeting CD19 after co-culture with target cells.
  • FIG. 5 Tumor growth in mice in each CAR-T cell treatment group in the B6 mouse Panc02-mCD19 subcutaneous tumor model.
  • FIG. 6 Body weight of mice in each CAR-T cell treatment group in the Panc02-mCD19 subcutaneous tumor model of B6 mice.
  • Figure 7 CAR expression levels of each CAR-T cell targeting Claudin 18.2 were measured by flow cytometry.
  • Figure 8 Mouse XCL1 levels in the supernatant of various CAR-T cells targeting Claudin 18.2 cultured until D5.
  • Figure 9 Target cell killing levels after co-incubation of various CAR-T cells targeting Claudin 18.2 with target cells at effector-target ratios of 5:1, 1:1, 1:5, and 1:10.
  • Figure 10 IFN- ⁇ release levels of various CAR-T cells targeting Claudin 18.2 after co-culture with target cells.
  • FIG. 11 Tumor growth of mice in each CAR-T cell treatment group in the B6 mouse Panc02-18.2 subcutaneous tumor model.
  • Figure 12 Body weight of mice in each CAR-T cell treatment group in the Panc02-18.2 subcutaneous tumor model of B6 mice.
  • the MSCV-mCD19 scFv plasmid was constructed, which contained the following structures connected in sequence: anti-mouse CD19 scFv (SEQ ID NO: 12), CD8 ⁇ hinge region (SEQ ID NO: 31), CD8 ⁇ transmembrane region (SEQ ID NO: 28), and CD3 ⁇ intracellular region (SEQ ID NO: 37).
  • T cells were activated with anti-CD3 and anti-CD28 antibodies (Thermofisher) and cultured for 2 days at 37°C and 5% CO 2. Then, retroviral supernatant containing MSCV-mCD19 scFv was added, and after continuous culture for 3 days, CAR-T cells (CAR19-T) containing anti-CD19 scFv were obtained.
  • anti-CD3 and anti-CD28 antibodies Thermofisher
  • MSCV-XCL1 plasmid containing the following structure: mouse XCL1 (SEQ ID NO: 1).
  • Retrovirus was prepared according to the method of Example 1.1, and activated T cells were infected with a combination of retrovirus containing MSCV-mCD19 scFv prepared in Example 1.1 and retrovirus containing MSCV-XCL1 to obtain CAR19+XCL1-T cells (expressing anti-CD19 CAR and mouse secretory XCL1).
  • MSCV-XCL1 Fc plasmid which contains the following structures connected in sequence: mouse XCL1 (SEQ ID NO: 1), IgD+IgG4 Fc (SEQ ID NO: 3).
  • the retrovirus was prepared according to the method of Example 1.1, and the activated T cells were infected with a combination of a retrovirus containing MSCV-mCD19 scFv prepared in Example 1.1 and a retrovirus containing MSCV-XCL1 Fc to obtain CAR19+XCL1 Fc-T cells (expressing anti-CD19 CAR and XCL1 Fc fusion protein).
  • the target cells Panc02-mCD19-luci cells were plated into a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR19-T cells, CAR19+XCL1-T cells, and CAR19+XCL1 Fc-T cells were plated into a 96-well plate for co-culture at different effector-target ratios of 5:1, 1:1, 1:5, and 1:10.
  • the fluorescence value was measured using an ELISA reader after 18 hours.
  • the killing efficiency was calculated according to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean ⁇ 100%, and the results are shown in Figure 3.
  • CAR19+XCL1 Fc-T cells expressing XCL1 Fc fusion protein showed higher target cell killing activity compared with CAR19+XCL1 T cells and CAR19-T cells expressing conventional XCL1. This indicates that CAR19+XCL1 Fc-T cells expressing XCL1 Fc fusion protein can enhance the killing activity of CART cells against target cells.
  • the target cells Panc02-mCD19-luci cells and non-target cells Pan02-luci were plated into 96-well plates at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR19-T cells, CAR19+XCL1-T cells, and CAR19+XCL1 Fc-T cells were plated into 96-well plates for co-culture at an effector-target ratio of 20:1. After culturing at 37°C for 18 hours, the cell supernatant was collected and the IFN- ⁇ content was detected using the mouse IFN-gamma DuoSet ELISA Kit (R&D systems). The results are shown in Figure 4.
  • Example 5 Detection of the therapeutic ability of various CAR-T cells targeting CD19 expressing XCL1 Fc fusion protein in the Panc02-mCD19 cell tumor-bearing model injected subcutaneously in B6 mice
  • mice 24 5-week-old B6 mice were selected and 100 ⁇ L of PBS suspension of Panc02-mCD19 tumor cells at a concentration of 10e6/mL was subcutaneously injected into the right upper back. The tumor growth was observed and recorded every day.
  • NT cells, CAR19-T cells, CAR19+XCL1-T cells, and CAR19+XCL1 Fc-T cells were prepared according to Example 1.
  • the mice were randomly divided into 4 groups according to the tumor volume, with 6 mice in each group, namely NT group, CAR19 group, CAR19+XCL1 group, and CAR19+XCL1 Fc group.
  • each mouse was injected with 5 mg CPA to remove its own immune cells.
  • each group of CART cells was prepared into a PBS suspension with a concentration of 5e6/mL, and 200 ⁇ l CART cell suspensions were injected into the corresponding groups of mice through the tail vein. The state of the mice was observed every day, and the weight and tumor growth were recorded.
  • the tumor growth is shown in Figure 5. It can be seen that the tumor growth rate of the CAR19+XCL1 Fc group is significantly reduced compared with the CAR19+XCL1 group and the CAR19 group. This indicates that in the B6 mouse Panc02-mCD19 tumor-bearing model, CAR19+XCL1 Fc-T cells expressing XCL1 Fc fusion protein can significantly improve the killing activity of CART cells against target tumors.
  • MSCV-Claudin 18.2 VHH plasmid which contains the following structures connected in sequence: anti-mouse Claudin 18.2 VHH (SEQ ID NO: 25), CD8 ⁇ hinge region (SEQ ID NO: 31), CD8 ⁇ transmembrane region (SEQ ID NO: 28), 4-1BB Costimulatory domain (SEQ ID NO: 35), CD3 ⁇ intracellular region (SEQ ID NO: 37).
  • Retrovirus containing MSCV-Claudin 18.2 VHH was prepared according to the method of Example 1.1.
  • T cells were activated with anti-CD3 and anti-CD28 antibodies (Thermofisher) and cultured for 2 days at 37°C and 5% CO 2. Then, retroviral supernatant containing MSCV-Claudin 18.2 VHH was added, and after continuous culture for 3 days, CAR-T cells (CAR18.2) containing Claudin 18.2 VHH were obtained.
  • anti-CD3 and anti-CD28 antibodies Thermofisher
  • MSCV-XCL1 plasmid containing the following structure: mouse XCL1 (SEQ ID NO: 1).
  • Retrovirus was prepared according to the method of Example 1.1, and activated T cells were infected with a combination of retrovirus containing MSCV-Claudin 18.2 VHH and retrovirus containing MSCV-XCL1 to obtain CAR18.2+XCL1-T cells (expressing anti-Claudin 18.2 CAR and mouse secretory XCL1).
  • MSCV-XCL1 Fc plasmid which contains the following structures connected in sequence: mouse XCL1 (SEQ ID NO: 1), IgD+IgG4 Fc (SEQ ID NO: 3).
  • Retrovirus was prepared according to the method of Example 1.1, and activated T cells were infected with a combination of retrovirus containing MSCV-Claudin 18.2 VHH and retrovirus containing MSCV-XCL1 Fc to obtain CAR18.2+XCL1 Fc-T cells (expressing anti-Claudin 18.2 CAR and XCL1 Fc fusion protein).
  • Example 7 Detection of XCL1 levels in each CAR-T cell targeting Claudin 18.2 by ELISA
  • CAR18.2+XCL1 Fc-T cells is significantly higher than that of other groups. This indicates that CAR18.2+XCL1 Fc-T cells expressing XCL1 Fc fusion protein can promote the secretion of XCL1.
  • Example 8 Detection of the killing level of target cells by each CAR-T cell targeting Claudin 18.2
  • the target cells Panc02-Claudin 18.2-luci cells were plated into a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR18.2-T cells, CAR18.2+XCL1-T cells, and CAR18.2+XCL1Fc-T cells were plated into a 96-well plate for co-culture at different effector-target ratios of 5:1, 1:1, 1:5, and 1:10. After 18 hours, the fluorescence value was measured using an ELISA reader. According to the calculation formula: (target cell fluorescence mean - sample fluorescence mean) / target cell fluorescence mean ⁇ 100%, the killing efficiency was calculated, and the results are shown in Figure 9.
  • CAR18.2+XCL1 Fc-T cells expressing XCL1 Fc fusion protein have higher killing activity against target cells than CAR18.2+XCL1 T cells and CAR18.2-T cells expressing conventional XCL1. This indicates that CAR18.2+XCL1 Fc-T cells expressing XCL1 Fc fusion protein can enhance the killing activity of CART cells against target cells.
  • the target cells Panc02-Claudin 18.2-luci cells and non-target cells Pan02-luci were plated into 96-well plates at a concentration of 1 ⁇ 10 4 cells/well, and then NT cells, CAR18.2-T cells, CAR18.2+XCL1-T cells, and CAR18.2+XCL1 Fc-T cells were plated into 96-well plates for co-culture at an effector-target ratio of 20:1. After culturing at 37°C for 18 hours, the cell supernatant was collected and the IFN- ⁇ content was detected using the mouse IFN-gamma DuoSet ELISA Kit (R&D systems). The results are shown in Figure 10.
  • Example 10 Detection of the therapeutic ability of CAR-T cells targeting Claudin 18.2 expressing XCL1 Fc fusion protein in a subcutaneous injection of Panc02-Claudin 18.2 cell tumor-bearing model in B6 mice
  • NT cells 24 5-week-old B6 mice were selected and 100 ⁇ L of PBS suspension of Panc02-Claudin 18.2 tumor cells at a concentration of 10e6/mL was subcutaneously injected into the right upper back. The tumor growth was observed and recorded every day. On the 8th day, NT cells, CAR18.2-T cells, CAR18.2+XCL1-T cells, and CAR18.2+XCL1 Fc-T cells were prepared according to Example 1.
  • mice were randomly divided into 4 groups according to the tumor volume, with 6 mice in each group, namely NT cells, CAR18.2 group, CAR18.2+XCL1 group, and CAR18.2+XCL1 Fc group. Each mouse was injected intraperitoneally with 5 mg CPA to remove its own immune cells.
  • each group of CART cells was prepared into a PBS suspension with a concentration of 5e6/mL, and 200ul CART cell suspensions were injected into the corresponding groups of mice through the tail vein. The status of mice was observed every day, and the body weight and tumor growth were recorded.
  • the tumor growth is shown in Figure 11. It can be seen that the tumor growth rate of the CAR18.2+XCL1 Fc group was significantly reduced compared with the CAR18.2 group and the CAR18.2+XCL1 group. This indicates that in the Panc02-Claudin 18.2 tumor-bearing model of B6 mice, CAR18.2+XCL1 Fc-T cells expressing XCL1 Fc fusion protein can significantly improve the killing activity of CART cells against target tumors.

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Abstract

La présente invention concerne une cellule modifiée, qui exprime une molécule de surface cellulaire reconnaissant spécifiquement un antigène et XCL1 et Fc exogènes. La présente invention concerne également l'utilisation de la cellule modifiée dans le traitement de maladies telles que des cancers, des infections ou des maladies auto-immunes. Par comparaison avec les cellules modifiées traditionnelles, la cellule modifiée de la présente invention présente une activité d'élimination tumorale considérablement améliorée.
PCT/CN2023/095270 2023-05-19 2023-05-19 Cellule modifiée et son utilisation WO2024239143A1 (fr)

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