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WO2025148750A1 - Il-10 exprimé par membrane et son utilisation - Google Patents

Il-10 exprimé par membrane et son utilisation

Info

Publication number
WO2025148750A1
WO2025148750A1 PCT/CN2024/144113 CN2024144113W WO2025148750A1 WO 2025148750 A1 WO2025148750 A1 WO 2025148750A1 CN 2024144113 W CN2024144113 W CN 2024144113W WO 2025148750 A1 WO2025148750 A1 WO 2025148750A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
cancer
domain
cell
car
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/CN2024/144113
Other languages
English (en)
Chinese (zh)
Inventor
张骅
华坚
徐峰
余婧
崔雨竹
宛玉花
邓林燕
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.)
Sph Biotherapeutics Hk Ltd
Sph Biotherapeutics Shanghai Ltd
Original Assignee
Sph Biotherapeutics Hk Ltd
Sph Biotherapeutics Shanghai Ltd
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 Sph Biotherapeutics Hk Ltd, Sph Biotherapeutics Shanghai Ltd filed Critical Sph Biotherapeutics Hk Ltd
Publication of WO2025148750A1 publication Critical patent/WO2025148750A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • 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 application relates to the field of biomedicine, and specifically to a fusion protein comprising IL-10 and a transmembrane domain and its application.
  • IL-10 Interleukin 10
  • IL-10-dependent CD8+T cell stimulation can induce tumor-specific immunity and mediate tumor regression.
  • T cell depletion limits anti-tumor immunity and response to immunotherapy, especially in solid tumors. The depletion of immune cells weakens the cytotoxic killing effect, which subsequently promotes tumor development and requires multiple treatments.
  • the present application provides a fusion protein comprising IL-10 and a transmembrane domain, which can be expressed on the cell membrane surface.
  • the expression of the fusion protein on the surface of cytotoxic immune cells can improve their activation efficacy and promote the secretion of IL-2 and IFN- ⁇ .
  • the fusion protein can also improve the cytotoxicity and sustainability of immune cells against tumor cells.
  • the fusion protein of the present application is co-expressed with ROR1 CAR, GPC3 CAR and NKG2D CAR in T cells, and can maintain a long-term effective cytotoxic effect on tumor cells.
  • the present application provides a fusion protein comprising IL-10 and a transmembrane domain, wherein the fusion protein is expressed on immune cells, and the immune cells do not include regulatory T cells.
  • the IL-10 is human IL-10.
  • amino acid sequence of IL-10 is as shown in SEQ ID NO:9.
  • the transmembrane domain is selected from the transmembrane domain of the following protein: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.
  • the transmembrane domain is the transmembrane domain of CD8.
  • amino acid sequence of the transmembrane domain is as shown in SEQ ID NO:11.
  • the fusion protein comprises IL-10 and a CD8 transmembrane domain.
  • the IL-10 and the transmembrane domain are indirectly linked.
  • the fusion protein further comprises a hinge region.
  • the IL-10 and transmembrane domain are connected by a hinge region.
  • the hinge region is selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 or LIGHT.
  • amino acid sequence of the hinge region is as shown in SEQ ID NO:11.
  • the fusion protein comprises IL-10, a CD8 hinge region, and a CD8 transmembrane domain.
  • the fusion protein is used to treat tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.
  • the fusion protein is used in adoptive cell therapy (ACT).
  • the ACT therapy is CAR-T therapy, TCR-T therapy, TIL therapy, NK therapy, iNKT therapy, CAR-NK therapy, CAR-iNKT therapy, ⁇ T therapy, DNT therapy, or CAR-DNT therapy.
  • the fusion protein is co-expressed with an antigen recognition receptor.
  • the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the fusion protein is co-expressed with CAR.
  • the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the antigen recognition receptor specifically binds a target antigen.
  • the modified immune cell comprises a fusion protein comprising IL-10, a CD8 hinge region and a CD8 transmembrane domain, and a chimeric antigen receptor comprising an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain.
  • the antigen recognition receptor specifically binds a target antigen.
  • the target antigen is an antigen associated with tumors, autoimmune diseases, severe infections, chronic aging, or cardiac fibrosis.
  • the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the antigen binding domain is an antibody or an antigen binding fragment thereof
  • the antigen-binding fragment is a Fab, (Fab)2, F(ab')2, scFv, di-scFv, Fv, VHH or dAb fragment.
  • the antigen binding domain is the extracellular domain (ECD) of NKG2D.
  • the target antigen is GPC3.
  • the antigen binding domain comprises scFv, and the amino acid sequence of the scFv is shown in SEQ ID NO:12.
  • the antigen binding domain comprises NKG2D ECD, and the amino acid sequence of the NKG2D ECD is shown in SEQ ID NO:14.
  • the transmembrane domain in the chimeric antigen receptor is the transmembrane domain of CD28.
  • the intracellular signaling domain is selected from the intracellular signaling domains of the following proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 or a domain containing at least one ITAM.
  • EBV Epstein-Barr virus
  • PBj14 Nef simian immunodeficiency virus
  • HSKV Kaposi's sarcoma herpes virus
  • DAP10 DAP-12 or a domain containing at least one ITAM.
  • the intracellular signaling domain is the intracellular signaling domain of CD3 ⁇ .
  • the chimeric antigen receptor further comprises an intracellular co-stimulatory domain.
  • the hinge region in the chimeric antigen receptor is selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the sequence encoding the self-cleavage peptide is located between the sequences encoding the fusion protein and the antigen recognition receptor.
  • the self-cleaving peptide comprises a 2A peptide.
  • the self-cleaving peptide is P2A.
  • the present application provides a vector comprising the nucleic acid molecule.
  • the present application provides a cell comprising the nucleic acid molecule or the vector.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the present application provides a method for preparing the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition.
  • the present application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug for preventing and/or treating tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis.
  • the present application provides a method for preventing and/or treating tumors, autoimmune diseases, severe infections, chronic aging or cardiac fibrosis, which comprises administering the fusion protein, the modified immune cells, the nucleic acid molecule, the vector, the cells and/or the pharmaceutical composition to a subject in need.
  • the tumor is a solid tumor and/or a hematological tumor.
  • the tumor is a solid tumor and/or a hematological tumor.
  • Figure 1 shows the structural design of ROR1 VHH CAR, ROR1 VHH CAR-membrane-bound IL-10 and ROR1VHH CAR-secreted IL-10 described in this application.
  • FIG. 2 shows the flow cytometry experimental results of the CAR structure expression described in the present application.
  • Figure 3 shows the activation effect of the membrane-bound IL-10 described in the present application on ROR1 VHH-CAR-membrane-bound IL-10T cells.
  • FIG. 4 shows the killing effect of the membrane-bound IL-10 described in the present application on tumor target cells.
  • Figure 5 shows the activation effect of the membrane-bound IL-10 described in the present application on ROR1 VHH-CAR-membrane-bound IL-10T cells.
  • FIG. 7 shows the killing effect of the membrane-bound IL-10 described in the present application on HCC827 tumor target cells.
  • Figure 9 shows the structural design of GPC3 scfv CAR and GPC3 scfv CAR-membrane-bound IL-10 described in this application.
  • FIG. 13 shows the killing effect of the membrane-bound IL-10 described in the present application on Hep3b tumor target cells.
  • Figure 15 shows the structural design of NKG2D ECD CAR and NKG2D ECD CAR-membrane-bound IL-10 described in this application.
  • FIG. 16 shows the flow cytometry experimental results of the CAR structure expression described in the present application.
  • FIG. 18 shows the killing effect of the membrane-bound IL-10 described in the present application on MB231 tumor target cells.
  • FIG. 19 shows the killing effect of the membrane-bound IL-10 described in the present application on A375 tumor target cells.
  • FIG. 20 shows the killing effect of the membrane-bound IL-10 described in the present application on Huh7 tumor target cells.
  • the terms "IL-10” and “interleukin-10” are generally used interchangeably, and generally refer to an inflammatory and immunosuppressive factor.
  • the IL-10 may be a monomer or a polymer.
  • the IL-10 may be a dimer.
  • the IL-10 may be a homodimer.
  • the IL-10 may include IL-10 of any species.
  • the IL-10 may be human or non-human.
  • the IL-10 may be mouse.
  • the IL-10 may be human.
  • the IL-10 may be full-length IL-10 or truncated IL-10.
  • the IL-10 may be a functionally active fragment that retains the function of the full-length IL-10.
  • the IL-10 may be wild-type IL-10 or artificially modified IL-10.
  • the IL-10 is a modified IL-10.
  • transmembrane domain generally refers to a domain that can cross the plasma membrane of a cell, and these domains are anchored on the cell membrane.
  • the transmembrane domain may be a transmembrane domain of a peptide, polypeptide or protein.
  • the transmembrane domain can include a transmembrane domain of one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.
  • the transmembrane domain can be a transmembrane domain of CD8.
  • the transmembrane domain can be a transmembrane domain of CD28.
  • the transmembrane domain may be natural or artificially synthesized.
  • the transmembrane domain may be a transmembrane domain of any species.
  • the transmembrane domain may be derived from humans.
  • the transmembrane domain may be a transmembrane domain of human CD8.
  • the transmembrane domain may be a transmembrane domain of human CD28.
  • the term "hinge region” generally refers to a dimer molecule consisting of two polypeptides having the same amino acid sequence, generally including about 25 amino acid residues.
  • the hinge region is flexible.
  • the hinge region can be a hinge region of a peptide, a polypeptide or a protein.
  • the hinge region can include a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region can be derived from the hinge region of CD8.
  • the hinge region can be derived from the hinge region of CD28.
  • the hinge region can be derived from the hinge region of IgG4.
  • the hinge region may be natural or synthetic.
  • the hinge region may be a hinge region of any species.
  • the hinge region may be derived from humans.
  • the hinge region may be a hinge region of human CD8.
  • the hinge region may be a hinge region of human CD28.
  • the hinge region may be a hinge region of human IgG4.
  • fusion protein generally refers to a protein composed of two or more polypeptides, which are usually not bound in the natural state, and are formed by binding the amino and carboxyl termini of the protein together through peptide bonds to form a continuous polypeptide.
  • the binding means that the two or more polypeptide components can be directly bound or indirectly bound.
  • the indirect binding can be through a linker.
  • the protein and/or amino acid sequence involved in the present application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein.
  • the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein.
  • the functional variant may include a protein or polypeptide that has been substituted, deleted and/or inserted with at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acids.
  • the functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition).
  • the functional variant may maintain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change.
  • the substitution may be a conservative substitution.
  • the homolog can be a protein or polypeptide having at least about 85% (for example, at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and/or the polypeptide.
  • the term "immune cell” generally refers to an immune cell that participates in an immune response and performs effector functions.
  • the immune cell can clear foreign antigens or promote immune effector responses.
  • the immune cell can be a natural immune cell or a modified immune cell.
  • the natural immune cell generally refers to a naturally occurring cell that participates in an immune response or is associated with an immune response.
  • a natural immune cell can be a T cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a ⁇ T cell, or a double negative T (DNT) cell.
  • the modified immune cell is also referred to as an engineered immune cell, and generally refers to an immune cell that is genetically modified by transcribing additional genetic material in the form of DNA or RNA.
  • the modified immune cell can express a chimeric antigen receptor (CAR), a T cell receptor (TCR), or an NKG2D CAR.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • NKG2D CAR NKG2D CAR
  • the modified immune cell can express a CAR.
  • the modified immune cell can be a CAR-T cell, a CAR-iNKT cell, or a CAR-DNT cell.
  • the modified immune cell may express TCR.
  • the modified immune cell may be a TCR-T cell.
  • the term "antigen recognition receptor” generally refers to a protein present on the surface of a cell that causes a response in the immune system by binding to a target antigen.
  • the antigen recognition receptor may be a B cell receptor, a T cell receptor or an NKG2D CAR.
  • the antigen recognition receptor may be natural or artificially synthesized.
  • the antigen recognition receptor may be a chimeric antigen receptor.
  • the term "chimeric antigen receptor” is generally used interchangeably with "CAR”, and generally refers to a recombinant polypeptide that can specifically bind to a corresponding antigen.
  • the chimeric antigen receptor may include an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain may include an antigen binding domain.
  • the extracellular domain may include a signal peptide.
  • the intracellular domain may include an intracellular signaling domain.
  • the intracellular domain may include an intracellular costimulatory domain.
  • a hinge region may be included between the extracellular domain and the transmembrane domain.
  • the target antigen can be a tumor antigen.
  • the target antigen can be a tumor-associated antigen.
  • the target antigen can be a tumor-specific antigen.
  • the target antigen may be ROR1, mesothelin, CD19, CD22, DLL3, GPC3, GPA33, B7-H3, EGFR, IL-13R ⁇ 2, FAP, TREM, Clauding 18.2, CDH17, B7H3, MUC1, GD2, CEA, CLL1, c-Met, HER2, NY-ESO-1, MAGEA4, HPV E6, HPV E7, KRAS G12C or KRAS G12D.
  • the target antigen may be ROR1.
  • the target antigen may be GPC3.
  • the target antigen may be a NKG2D ligand.
  • the term "pharmaceutically acceptable carrier” generally refers to a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient.
  • the pharmaceutically acceptable carrier includes a pharmaceutically acceptable carrier, excipient or stabilizer that is non-toxic to cells or mammals exposed thereto at the dose and concentration used.
  • a physiologically acceptable carrier can be water, salt, protein, polysaccharide, lipid or inactive virus particles.
  • amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 11.
  • amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 4.
  • CD8 CD80, CD81, CD86, CD9, CD94, CD97, CD99, CEA, CEACAM6, CLL1, CS1, DLL1, DLL3, EGFR, EGFR VIII, ErbB2, FGF19, GD2, GD3, HER3, IL3Ra, NCAM, NG2, NKG2A, NTBA, PD-1, PDL-1, PSGL1, PSMA, ROR1, VEGF, 5T4, AFP, BCMA, CTLA-4, ELF2M, FLT3, PSA, HPV-specific antigen, glioma-associated antigen, EBV-specific antigen, mesothelin, GPC3, GPA33, IL-11R ⁇ , IL-13R ⁇ 2, FAP or TREM.
  • the antigen binding domain can specifically bind to ROR1.
  • the antigen binding domain can comprise ROR1 VHH.
  • the antigen binding domain may include VHH, and the VHH may include HCDR1-3.
  • the VHH includes HCDR1, HCDR2 and HCDR3, the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 17, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 18, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 19.
  • the antigen binding domain may include VHH, and the amino acid sequence of the VHH may be shown in SEQ ID NO: 1.
  • the antigen binding domain may include the amino acid sequence shown in SEQ ID NO: 1.
  • the antigen binding domain may comprise a scFv
  • the scFv may comprise VH and VL
  • the VH comprises HCDR1-3
  • the VL comprises LCDR1-3.
  • the amino acid sequence of the HCDR1 is shown in SEQ ID NO:24
  • the amino acid sequence of the HCDR2 is shown in SEQ ID NO:25
  • the amino acid sequence of the HCDR3 is shown in SEQ ID NO:26
  • the amino acid sequence of the LCDR1 is shown in SEQ ID NO:31
  • the amino acid sequence of the LCDR2 is shown in SEQ ID NO:32
  • the amino acid sequence of the LCDR3 is shown in SEQ ID NO:33.
  • the antigen binding domain may comprise a scFv, the scFv may comprise VH and VL, the amino acid sequence of the VH is shown in SEQ ID NO:30, and the amino acid sequence of the VL is shown in SEQ ID NO:38.
  • the antigen binding domain may comprise an scFv, and the amino acid sequence of the scFv may be as shown in SEQ ID NO: 12.
  • the antigen binding domain may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the antigen recognition receptor can be NKG2D CAR.
  • the fusion protein can be co-expressed with NKG2D CAR.
  • the NKG2D CAR can specifically bind to the target antigen.
  • the NKG2D CAR can recognize and bind to NKG2D ligands (NKG2DLs).
  • the ligands can include six cytomegalovirus UL16 binding proteins 1-6 (ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6) and MHC I chain-related molecules A and B (MICA, MICB).
  • the antigen binding domain may include NKG2D ECD.
  • the NKG2DCAR may include NKG2D ECD.
  • the amino acid sequence of the NKG2D ECD may be as shown in SEQ ID NO: 14.
  • the transmembrane domain in the chimeric antigen receptor can be selected from the transmembrane domain of the following group of proteins: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 or SLAM.
  • the transmembrane domain in the chimeric antigen receptor can be the transmembrane domain of CD8.
  • the transmembrane domain in the chimeric antigen receptor can be the transmembrane domain of CD28
  • amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 11.
  • amino acid sequence of the transmembrane domain may be as shown in SEQ ID NO: 4.
  • the intracellular signaling domain can be selected from the intracellular signaling domain of the following group of proteins: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpes virus (HSKV), DAP10, DAP-12 or a domain containing at least one ITAM.
  • the intracellular signaling domain can be the intracellular signaling domain of CD3 ⁇ .
  • amino acid sequence of the intracellular signaling domain can be as shown in SEQ ID NO:6.
  • the chimeric antigen receptor may also include an intracellular costimulatory domain.
  • the intracellular costimulatory domain may be selected from the intracellular costimulatory domain of the following group of proteins: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands of CD83, CD40 and MyD88.
  • the intracellular costimulatory domain may be the intracellular costimulatory domain of 4-1BB.
  • the intracellular costimulatory domain may be the intracellular costimulatory domain of CD28.
  • amino acid sequence of the intracellular co-stimulatory domain can be as shown in SEQ ID NO:5.
  • the chimeric antigen receptor may further include a hinge region.
  • the hinge region in the chimeric antigen receptor may be selected from the hinge region of the following group of proteins: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30 and LIGHT.
  • the hinge region in the chimeric antigen receptor may be the hinge region of CD8.
  • the hinge region in the chimeric antigen receptor may be the hinge region of CD28.
  • the hinge region in the chimeric antigen receptor may be the hinge region of IgG4.
  • amino acid sequence of the hinge region in the chimeric antigen receptor can be as shown in SEQ ID NO: 10.
  • amino acid sequence of the hinge region in the fusion protein can be as shown in SEQ ID NO: 3.
  • amino acid sequence of the hinge region can be as shown in SEQ ID NO: 13.
  • the fusion protein may comprise IL-10, a hinge region and a transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD28 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD28 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD28 hinge region and a CD28 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a hinge region and a transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a hinge region, a transmembrane domain, an intracellular co-stimulatory domain and an intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a CD28 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a CD8 hinge region, a CD28 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the chimeric antigen receptor may comprise an antigen binding domain, a CD28 hinge region, a CD8 transmembrane domain, a 4-1BB intracellular co-stimulatory domain and a CD3 ⁇ intracellular signaling domain.
  • the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain
  • the chimeric antigen receptor may comprise NKG2D ECD, IgG4 hinge region, CD28 transmembrane domain, 4-1BB intracellular co-stimulatory domain and CD3 ⁇ intracellular signaling domain.
  • the TRAV and/or TRBV may comprise three hypervariable regions CDR1, CDR2 and CDR3.
  • the TCR can specifically bind to the target antigen.
  • the TCR can specifically bind to NY-ESO-1, MAGEA1, MAGEA3, MAGEA4, MAGEA10, HPV E6, HPV E7, KRAS G12C, KRAS G12D, AFP, HBsAg, mesothelin, PD-1, MART-1, CD19, CD28, CD8, CT83, F8, GPC3, H3 K27M, HLA-A, LMP-2, MC2R or PRAME.
  • the fusion protein may comprise IL-10, a hinge region and a transmembrane domain
  • the T cell receptor may comprise an ⁇ chain and a ⁇ chain
  • the ⁇ chain may comprise TRAV and TRAC
  • the ⁇ chain may comprise TRBV and TRBC.
  • the fusion protein may comprise IL-10, a CD28 hinge region and a CD8 transmembrane domain
  • the T cell receptor may comprise an ⁇ chain and a ⁇ chain
  • the ⁇ chain may comprise TRAV and TRAC
  • the ⁇ chain may comprise TRBV and TRBC.
  • the fusion protein may comprise IL-10, CD8 hinge region and CD8 transmembrane domain
  • the T cell receptor may comprise an ⁇ chain and a ⁇ chain
  • the ⁇ chain may comprise TRAV and mTRAC
  • the ⁇ chain may comprise TRBV and mTRBC.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the T cell receptor may comprise an ⁇ chain and a ⁇ chain
  • the ⁇ chain may comprise TRAV and TRAC targeting NY-ESO-1
  • the ⁇ chain may comprise TRBV and TRBC targeting NY-ESO-1.
  • the fusion protein may comprise IL-10, a CD8 hinge region and a CD8 transmembrane domain
  • the T cell receptor may comprise an ⁇ chain and a ⁇ chain
  • the ⁇ chain may comprise TRAV and hTRAC targeting NY-ESO-1
  • the ⁇ chain may comprise TRBV and hTRBC targeting NY-ESO-1.
  • the immune cells can promote an immune response.
  • the immune cell may be a T cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, a TIL cell, an iNKT cell, a CIK cell, a ⁇ T cell or a DNT cell.
  • the immune cells are T cells.
  • the immune cell may be a T cell.
  • the present application provides an isolated nucleic acid molecule encoding the fusion protein.
  • the isolated nucleic acid molecule can encode a complete fusion protein or a portion thereof.
  • the present application provides an isolated nucleic acid molecule encoding the fusion protein and the antigen recognition receptor.
  • the isolated nucleic acid molecule can encode a complete fusion protein and/or an antigen recognition receptor, or a portion thereof.
  • the isolated nucleic acid molecule can encode a fusion protein and an antigen recognition receptor, respectively.
  • the isolated nucleic acid molecule can encode a fusion protein and an antigen recognition receptor simultaneously.
  • the nucleic acid molecule encoding the fusion protein can be located before the nucleic acid molecule encoding the antigen recognition receptor.
  • the nucleic acid molecule encoding the fusion protein can be located after the nucleic acid molecule encoding the antigen recognition receptor.
  • the nucleic acid molecule may comprise a sequence encoding a self-cleaving peptide.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the antigen recognition receptor.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the CAR.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the ROR1 CAR.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the GPC3 CAR.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the NKG2D CAR.
  • the sequence encoding the self-cleaving peptide may be located between the sequence encoding the fusion protein and the TCR.
  • sequence encoding the self-cleavage peptide may be located between the sequences encoding the TCR ⁇ chain and ⁇ chain.
  • the self-cleaving peptide may include a 2A peptide.
  • the self-cleaving peptide may be P2A, T2A, E2A or F2A.
  • the self-cleaving peptide may be T2A.
  • the self-cleaving peptide may be GSGP2A.
  • the nucleic acid molecule may comprise a sequence encoding a signal peptide.
  • the signal peptide may be a CD8a signal peptide.
  • the signal peptide may be an IL-2 signal peptide.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding a hinge region, and a gene encoding a transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences in sequence from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD8 hinge region, and a gene encoding the CD8 transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD8 hinge region, and a gene encoding the CD28 transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD28 hinge region, and a gene encoding the CD8 transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end: a gene encoding IL-10, a gene encoding the CD28 hinge region, and a gene encoding the CD28 transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end in sequence: a gene encoding a signal peptide, a gene encoding an antigen recognition receptor, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding an intracellular co-stimulatory domain, a gene encoding an intracellular signaling domain, a gene encoding a self-cleavage peptide, a gene encoding a signal peptide, a gene encoding IL-10, a gene encoding a hinge region, and a gene encoding a transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end in sequence: a gene encoding a signal peptide, a gene encoding IL-10, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding a self-cleavage peptide, a gene encoding a signal peptide, a gene encoding an antigen recognition receptor, a gene encoding a hinge region, a gene encoding a transmembrane domain, a gene encoding an intracellular co-stimulatory domain, and a gene encoding an intracellular signaling domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end in sequence: a gene encoding a CD8a signal peptide, a gene encoding a GPC3 scFv, a gene encoding a CD8 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding 4-1BB, a gene encoding CD3 ⁇ , a gene encoding a P2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain.
  • the nucleic acid molecule may contain the following nucleotide sequences from the 5' end to the 3' end in sequence: a gene encoding a CD8a signal peptide, a gene encoding a NKG2D ECD, a gene encoding an IgG4 hinge region, a gene encoding a CD28 transmembrane domain, a gene encoding 4-1BB, a gene encoding CD3 ⁇ , a gene encoding a P2A self-cleavage peptide, a gene encoding a CD8a signal peptide, a gene encoding IL-10, a gene encoding a CD8 hinge region, and a gene encoding a CD8 transmembrane domain.
  • the nucleic acid molecule can be produced or synthesized by the following methods: (i) amplified in vitro, such as produced by polymerase chain reaction (PCR) amplification, (ii) produced by cloning and recombination, (iii) purified, such as by enzyme digestion and gel electrophoresis fractionation, or (iv) synthesized, such as by chemical synthesis.
  • the nucleic acid molecule can be DNA and/or RNA.
  • the nucleic acid molecule can be an artificially synthesized nucleic acid analog.
  • the nucleic acid molecule can be a modified nucleic acid molecule.
  • the present application provides a vector comprising the nucleic acid molecule.
  • the vector may contain one or more of the nucleic acid molecules. In the present application, the vector may contain one or more of the nucleic acid molecules. In the present application, the vector may be an expression vector or a cloning vector. In the present application, the vector may be a viral vector or a non-viral vector. In the present application, the vector may be a viral vector, a plasmid vector, a phage vector or other vectors commonly used in, for example, genetic engineering.
  • the viral vector may be an adenovirus, an adeno-associated virus, a retrovirus (including a lentivirus). In the present application, the vector may be a fusion vector or a non-fusion vector.
  • the vector may further comprise other genes.
  • the other genes may be marker genes.
  • the present application provides a method for preparing the fusion protein, the method comprising transfecting or transducing the nucleic acid molecule into a cell.
  • the DNA sequence of the fusion protein is cloned into the vector and then transfected into an immune cell for expression.
  • the present application provides a method for preparing the fusion protein and the antigen recognition receptor, the method comprising transfecting or transducing the nucleic acid molecule into a cell.
  • the DNA sequences of the fusion protein and the antigen recognition receptor are cloned into the vector and then transfected into immune cells for expression.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein, the modified immune cell, the nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or suitable preparations of preservatives.
  • the acceptable ingredients of the composition are preferably nontoxic to the recipient at the dosage and concentration used.
  • the pharmaceutical composition of the present invention may include liquid, frozen and lyophilized compositions.
  • the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents that are compatible with pharmaceutical administration, are generally safe, non-toxic, and neither biologically nor otherwise undesirable.
  • the pharmaceutical composition can include parenteral, percutaneous, intracavitary, intra-arterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical composition can be administered to a patient or subject by infusion or injection.
  • the administration of the pharmaceutical composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the present application provides a method for preparing the fusion protein.
  • the present application provides a method for preparing the modified immune cells.
  • the present application provides a method for preparing the pharmaceutical composition.
  • the present application provides the use of the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition in the preparation of a drug for preventing and/or treating a disease.
  • the present application provides a method for preventing and/or treating a disease, comprising administering the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition to a subject in need thereof.
  • the present application provides the fusion protein, the modified immune cell, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition, which are used for preventing and/or treating a disease.
  • the prevention and/or treatment may be preventing the onset of the disease, slowing down or reversing the progression of the disease, preventing or slowing down the onset of one or more symptoms associated with the disease, reducing or alleviating one or more symptoms associated with the disease, reducing the severity and duration of the disease and any symptoms associated therewith, or preventing further increase in the severity of the disease and any symptoms associated therewith.
  • the disease may be a tumor, an autoimmune disease, a severe infection, chronic aging or cardiac fibrosis.
  • the tumor may be a solid tumor. In the present application, the tumor may be a hematological tumor.
  • the tumor can be a ROR1 positive tumor.
  • the tumor can be a GPC3 positive tumor.
  • the tumor can be a tumor expressing NKG2D ligand.
  • the tumor can be lung cancer, breast cancer, ovarian cancer, cervical cancer, uterine cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer, brain cancer or skin cancer.
  • PBMCs peripheral blood mononuclear cells
  • ROR1 VHH CAR The structure of ROR1 VHH CAR is: CD8a signal peptide (SEQ ID NO:2), ROR1 VHH (SEQ ID NO:1), CD28 hinge region (SEQ ID NO:3), CD28 transmembrane domain (SEQ ID NO:4), 4-1BB (SEQ ID NO:5) and CD3 ⁇ (SEQ ID NO:6).
  • ROR1 VHH CAR-secretory IL-10 The structure of ROR1 VHH CAR-secretory IL-10 is as follows: the IL-2 signal peptide (SEQ ID NO:8) sequence is connected to GSGP2A (SEQ ID NO:7) after ROR1 VHH CAR to induce expression, and then the IL-10 (SEQ ID NO:9) sequence is connected.
  • ROR1 VHH CAR-membrane-bound IL-10 The structure of ROR1 VHH CAR-membrane-bound IL-10 is as follows: after ROR1 VHH CAR, the CD8a signal peptide sequence is connected through GSGP2A to induce expression, followed by the IL-10 sequence, and finally the CD8 hinge region (SEQ ID NO: 10) and the CD8 transmembrane domain (SEQ ID NO: 11) are connected to localize the expression of IL-10 on the cell membrane.
  • All lentiviruses were prepared from HKE293T. After freshly resuspended HEK293T cells were washed in ice-cold PBS buffer, the cells were planted in a 10 cm culture dish with a cell density of 90%-95% of the culture dish area. The mixed lentiviral packaging plasmid and transfection plasmid were mixed with Lipofectamine-2000/3000 and transiently transfected into the planted HEK293T cells. After 48 hours of culture, the supernatant was collected and filtered using a 0.45 ⁇ m filter, and the samples were aliquoted into 1 ml per tube and stored in a -80 ° C refrigerator.
  • the frozen PBMC was revived and added to a 24-well plate, and CD3 and CD28 coupled magnetic beads and IL-2 (500IU/ml) were added for 24 hours of activation.
  • the activated PBMC cells were distributed to multiple wells of a 24-well plate, the corresponding lentivirus and polybrene (8g/ml) were added, centrifuged at 2000g for 2 hours in a desktop centrifuge, and then transferred to a 37°C and 5% CO2 incubator for culture.
  • the transfected cells were further cultured in X-VIVO-15 cell culture medium containing IL-2 (500IU/ml) for 48 hours for subsequent experiments and analysis.
  • Flow cytometry was performed using a flow cytometer (Beckman Coulter) in plate mode, and data were analyzed using FlowJo software.
  • Cells were washed once with FACS buffer (PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0), resuspended to 5 ⁇ 10 7 cells/mL, and placed on ice before staining. Fluorescently conjugated antibody staining was added and distributed into the wells of a 96-well round-bottom tissue culture plate. After incubation at 4°C for 45 minutes, 150 ⁇ L of FACS buffer was added to each well for washing the cells. After centrifugation at 300xg for 3 minutes, the supernatant was removed. After washing and suspension with FACS buffer, the expression of cell ROR1 CAR was analyzed by flow cytometry.
  • FACS buffer PBS containing 0.5% BSA, 0.1% NaN3, 2mM EDTA, pH 7.0
  • the flow cytometry validation results are shown in Figure 2A.
  • the positive rates of the ROR1 VHH CAR T cell group were 29.8%, the ROR1 VHH CAR-secretory IL-10 group were 15.5%, and the ROR1 VHH CAR-membrane-bound IL-10 group were 20.7%, compared with the positive rate of the non-transduced virus T cell group of 2.21%.
  • Example 2 Membrane-bound IL-10 can improve the activation efficacy of ROR1-CAR-T cells
  • ROR1 VHH CAR T cells The activation efficacy of ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells was determined by co-culturing them with tumor cells.
  • Lentivirally transduced PBMC (1 ⁇ 10 4 ) were co-cultured with negative expressing cells (MEC (ROR1-), 10 ⁇ 10 4 ), or with target tumor cells (MEC-ROR1 (expressing ROR1+), MB231 (expressing ROR1+), 10 ⁇ 10 4 ) in a round-bottom 96-well tissue culture plate, with a ratio of effector cells to target cells of 1:10, and IMDM culture medium was added to a final volume of 200 ⁇ L per well. After centrifugation at 300xg for 5 minutes, the tissue culture plate was incubated at 37°C and 5% CO 2 for 24 hours. 100 ⁇ L of supernatant was transferred to a new round-bottom 96-well tissue culture plate. PBMCs not transduced with lentivirus were co-cultured with target tumor cells as the negative control group.
  • the IFN- ⁇ (Biolegend, CAT#430104) and IL-2 concentrations (Biolegend, CAT#431804) in the culture supernatant were determined using the EILSA kit. The experimental method was carried out according to the instructions provided by the reagent manufacturer. In brief, after incubation at 37°C for 1 hour using assay diluent buffer, 20 ⁇ l of culture supernatant (1:5 dilution concentration) or different concentrations of IFN- ⁇ /IL-2 standard solution were mixed and added to the ELISA plate coated with capture antibody. After incubation with streptavidin-conjugated peroxidase, TMB substrate and stop solution, the absorbance at 450 nm was measured using a microplate reader. The concentration of IFN- ⁇ /IL-2 in the culture supernatant was calculated based on the standard curve of known standards.
  • FIG. 3A The results of the enzyme-linked immunosorbent assay are shown in Figure 3.
  • the concentration of IL-2 secreted by PBMCs in the cell culture medium increased after the lentiviral-transduced PBMCs were co-cultured with target tumor cells (CAR T cells + MEC-1-ROR1-GFP/MB231-GFP).
  • the secretion concentration of IL-2 was much higher than that of the ROR1 VHH CAR T cell group and the ROR1 VHH CAR-secreting IL-10 T cell group.
  • the concentration of IFN- ⁇ secreted by PBMC in the cell culture medium increased after the lentivirus-transduced PBMCs were co-cultured with target tumor cells (CAR T cells + MEC-1-ROR1-GFP/MB231-GFP).
  • target tumor cells CAR T cells + MEC-1-ROR1-GFP/MB231-GFP.
  • the secretion concentration of IFN- ⁇ was much higher than that of the ROR1 VHH CAR T cell group and the ROR1 VHH CAR-secreting IL-10 T cell group.
  • Example 3 Membrane-expressed IL-10ROR1 CAR T cells have the strongest cell-killing effect
  • the tumor killing efficacy of ROR1 VHH CAR T cells, ROR1 VHH CAR-secreting IL-10 T cells, and ROR1 VHH CAR-membrane-bound IL-10 T cells was determined by co-culturing them with tumor cells.
  • Lentivirus-transduced PBMCs (1 ⁇ 10 4 ) were co-cultured with negative expressing cells (MEC-1 (ROR1-), 10 ⁇ 10 4 ), or with target tumor cells (MEC-1-ROR1 (expressing ROR1+), MB231 (expressing ROR1+), 10 ⁇ 10 4 ) in flat-bottom 96-well tissue culture plates at a ratio of 1:10 for effector cells to target cells, and IMDM culture medium was added to a final volume of 200 ⁇ L per well.
  • MEC-1 negative expressing cells
  • MEC-1-ROR1 target tumor cells
  • MB231 expressing ROR1+
  • Incucyte Instrument Test Insertion In the SX1 Live-Cell Analysis System, after incubation at 37°C and 5% CO2 for 48 hours, T cells were challenged after drug resistance. The cell culture plate was centrifuged at 300g for 5 minutes, 100 ⁇ l of supernatant was taken, and 50k tumor cells were added to the final volume of 200 ⁇ l. The cells were incubated for 24 hours in the SX1 Live-Cell Analysis System. The killing effect of lentiviral-transfected PBMC on target tumor cells was analyzed using Incucyte 2022A Rev1 software.
  • NKG2D ECD CAR-T cells NKG2D ECD CAR-membrane-bound IL-10 was determined by co-culturing tumor cells.
  • Lentivirus-transduced PBMC 0.3 ⁇ 10 4
  • PBMC negative expressing cells
  • target tumor cells M231-GFP (expressing Huh7 (MIC A/B+), A375-GFP (expressing Huh7 (MIC A/B-), 1.5 ⁇ 10 4
  • the ratio of effector cells to target cells was 1:5, and IMDM culture medium was added to a final volume of 200 ⁇ L per well.
  • T cells were challenged after drug resistance.
  • the cell culture plate was centrifuged at 300g for 5 minutes, 100 ⁇ l of supernatant was taken, and 5k tumor cells were added to a final volume of 200 ⁇ l.
  • the cells were incubated for 24 hours in the SX1 Live-Cell Analysis System.
  • the killing effect of lentiviral-transfected PBMC on target tumor cells was analyzed using Incucyte 2022A Rev1 software.

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Abstract

L'invention concerne une protéine de fusion, comprenant de l'IL-10 et un domaine transmembranaire, la protéine de fusion étant exprimée sur une cellule immunitaire, et la cellule immunitaire ne comprenant pas de cellule T régulatrice. L'invention concerne également une cellule immunitaire modifiée, comprenant une protéine de fusion et un récepteur de reconnaissance d'antigène, la protéine de fusion comprenant de l'IL-10 et un domaine transmembranaire, et la cellule immunitaire ne comprenant pas de cellule T régulatrice. L'invention concerne également une utilisation de la protéine de fusion et de la cellule immunitaire modifiée dans la prévention et/ou le traitement de maladies.
PCT/CN2024/144113 2024-01-08 2024-12-31 Il-10 exprimé par membrane et son utilisation Pending WO2025148750A1 (fr)

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