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WO2023274355A1 - 改造的间充质干细胞和免疫效应细胞联合治疗肿瘤 - Google Patents

改造的间充质干细胞和免疫效应细胞联合治疗肿瘤 Download PDF

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WO2023274355A1
WO2023274355A1 PCT/CN2022/102733 CN2022102733W WO2023274355A1 WO 2023274355 A1 WO2023274355 A1 WO 2023274355A1 CN 2022102733 W CN2022102733 W CN 2022102733W WO 2023274355 A1 WO2023274355 A1 WO 2023274355A1
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mesenchymal stem
cells
binding
molecule
pharmaceutical combination
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PCT/CN2022/102733
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English (en)
French (fr)
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杨选明
王媛
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上海交通大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464419—Receptors for interleukins [IL]
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule

Definitions

  • This application relates to the field of biomedicine, in particular to a modified mesenchymal stem cell and its use in combination with immune effector cells for the preparation of drugs for treating tumors.
  • Mesenchymal stem cells are a kind of pluripotent stem cells with self-renewal and multidirectional differentiation capabilities, and can differentiate into bone, cartilage, fat, etc. Clinical applications include repair of tissue damage, autoimmune diseases, gene therapy vectors, etc. Mesenchymal stem cells have the characteristics of tending to inflammatory tissues, and tumors are considered to be a chronic inflammatory tissue, so mesenchymal stem cells can chemotaxis and enrich in tumor tissues.
  • T cell adoptive cell therapy such as chimeric antigen receptor CAR-T cells, TCR-T, TIL, etc.
  • CAR-T or TCR-T etc. are a kind of tumor killer cells that have been artificially modified or expanded in vitro, through genetic modification (CAR-T or TCR-T etc.) to acquire the ability to target and kill tumors, or to enrich tumor-specific endogenous T cells through selective expansion (TIL, etc.), which is an important development direction in the field of tumor immunotherapy.
  • CAR-T has achieved significant clinical efficacy in the treatment of hematopoietic tumors, its efficacy in the treatment of solid tumors is not ideal.
  • the main reason is that there are various immunosuppressive mechanisms in the tumor microenvironment of solid tumors. Effectively provide T cell activation signals, so that the above-mentioned CAR-T, TCR-T, TIL, etc. cannot fully function, and overcome the immunosuppression of the solid tumor microenvironment.
  • the present application provides a modified mesenchymal stem cell and a drug combination comprising the modified mesenchymal stem cell and anti-tumor T cells.
  • the combined therapy of adoptive T cell therapy and mesenchymal stem cells solves the shortage of tumor-specific T cells, on the other hand, weakens the inhibition of the tumor microenvironment, provides signals for T cell activation, and improves the quantity and quality of adoptive T cells at the same time. Efficacy of T cell therapy against solid tumors.
  • the present application provides a mesenchymal stem cell, which expresses immune regulatory molecules, and the immune regulatory molecules include molecules involved in the first signal and/or the second signal of T cell activation.
  • the molecule involved in the first signal of T cell activation comprises a molecule involved in the process of antigen recognition.
  • the molecule involved in the first signal of T cell activation comprises a molecule capable of binding an antigen.
  • the molecule capable of binding an antigen comprises an antigen binding protein.
  • the molecule capable of binding an antigen comprises an antibody or fragment thereof.
  • the antigen comprises a tumor-associated antigen.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the antigen expressed by the mesenchymal stem cells includes CLDN18.2.
  • the molecule involved in the first signal of T cell activation comprises a molecule capable of binding CD3.
  • the CD3 molecule comprises a gamma chain, delta chain, epsilon chain, zeta chain and/or eta chain of CD3.
  • the molecule capable of binding CD3 comprises an anti-CD3 molecule.
  • the anti-CD3 molecule comprises an anti-CD3 antibody or fragment thereof.
  • the second signal of T cell activation comprises a co-stimulatory signal.
  • the molecule involved in the second signal of T cell activation comprises a co-stimulatory molecule or a molecule capable of binding a co-stimulatory molecule.
  • the co-stimulatory molecules include one or more co-stimulatory molecules selected from the CD28 family, the CD2 family and/or the TNFR family.
  • the co-stimulatory molecule comprises a polypeptide selected from the group consisting of CD28, OX40 and/or 4-1BB.
  • the molecule capable of binding a costimulatory molecule comprises a ligand for the costimulatory molecule.
  • the ligands capable of binding co-stimulatory molecules include: OX40L and/or 4-1BBL.
  • the molecule capable of binding a costimulatory molecule comprises a receptor for the costimulatory molecule.
  • the molecule capable of binding a co-stimulatory molecule comprises an anti-CD28 molecule.
  • the anti-CD28 molecule comprises an anti-CD28 antibody or fragment thereof.
  • the molecule capable of binding a co-stimulatory molecule comprises an anti-co-stimulatory molecule antibody or fragment thereof.
  • the mesenchymal stem cells also express molecules involved in a third signal of T cell activation.
  • the third signal of T cell activation comprises a cytokine signal.
  • the molecule involved in the third signal of T cell activation comprises a cytokine and/or a molecule capable of binding the cytokine.
  • said molecule capable of binding said cytokine comprises a receptor for said cytokine.
  • the cytokines include IL-12, IL-2, IL-5, IL7 and/or IL-15.
  • the cytokine comprises IL-7.
  • the cytokine comprises the amino acid sequence shown in SEQ ID NO: 10.
  • the cytokine comprises IL-2.
  • the cytokine comprises the amino acid sequence shown in SEQ ID NO: 12.
  • the mesenchymal stem cells further comprise molecules capable of improving the expansion ability of the mesenchymal stem cells in tumors.
  • the molecule capable of enhancing the ability of mesenchymal stem cells to expand in a tumor includes an enzyme.
  • the molecules associated with the expansion of mesenchymal stem cells in tumor cells include reduced coenzyme/quinone oxidoreductase (NQO1).
  • the molecule associated with the expansion of mesenchymal stem cells in tumor cells includes 5-methylcytosine dioxygenase (TET1).
  • the mesenchymal stem cells comprise a nucleic acid operably linked to a promoter comprising a hypoxia response element (HRE).
  • HRE hypoxia response element
  • the mesenchymal stem cells are allogeneic mesenchymal stem cells.
  • the mesenchymal stem cells are autologous mesenchymal stem cells.
  • the present application also provides a drug combination, which comprises the mesenchymal stem cells and immune effector cells.
  • the immune effector cells comprise T cells.
  • the T cells comprise CAR-T, TCR-T, TIL and/or T cells derived from pleural fluid, ascites fluid and/or PBMC.
  • the T cells comprise CD8+ T cells, CD4+ T cells, ⁇ - ⁇ T cells and/or T regulatory cells.
  • the immune effector cells include immune regulatory molecules, and the immune regulatory molecules can combine with the corresponding immune regulatory molecules expressed by the mesenchymal stem cells to provide the first signal for T cell activation, the second second signal and/or third signal.
  • the immune effector cells comprise molecules involved in the first signal of T cell activation.
  • the molecules involved in the first signal of T cell activation comprised by the immune effector cells comprise molecules involved in the process of antigen recognition.
  • said immune effector cells comprise a molecule capable of binding said antigen.
  • the antigen-binding molecules comprised by the immune effector cells target the same antigen as the antigen-binding molecules expressed by the mesenchymal stem cells.
  • the antigen may be selected from one or more of mesothelin, GPC-3, PSMA, EpCAM and GD2.
  • the molecule capable of binding an antigen comprises an antigen binding protein.
  • the antigen binding protein comprises an antibody or fragment thereof.
  • the antibody or fragment thereof comprises a scFv.
  • the immune effector cells comprise an anti-CLDN18.2 antibody or fragment thereof.
  • the immune effector cells comprise CD3 molecules.
  • the CD3 molecule comprises the amino acid sequence shown in SEQ ID NO:1.
  • the immune effector cells comprise a co-stimulatory molecule or a ligand capable of binding a co-stimulatory molecule.
  • the co-stimulatory molecule or the ligand capable of binding the co-stimulatory molecule contained in the immune effector cells is capable of binding to the co-stimulatory molecule ligand or co-stimulatory molecule contained in the mesenchymal stem cells.
  • the co-stimulatory molecules include one or more co-stimulatory molecules selected from the CD28 family, the CD2 family and/or the TNFR family.
  • the co-stimulatory molecule comprises a polypeptide selected from the group consisting of OX40 and/or 4-1BB.
  • the co-stimulatory molecule comprises the amino acid sequence shown in SEQ ID NO:6 and/or SEQ ID NO:7.
  • the immune effector cells comprise molecules involved in a third signal of T cell activation.
  • the immune effector cells comprise a cytokine or a molecule capable of binding a cytokine.
  • the molecule capable of binding a cytokine comprises a receptor for the cytokine.
  • the cytokine expressed by the immune effector cell or the receptor capable of binding the cytokine can bind to the cytokine expressed by the mesenchymal stem cell or the receptor capable of binding the cytokine.
  • the cytokines include IL-12, IL-2, and/or IL7.
  • the cytokine comprises the amino acid sequence shown in SEQ ID NO:10 and/or SEQ ID NO:12.
  • the receptors capable of binding cytokines include IL-12R, IL-2R and/or IL7R.
  • the receptor capable of binding cytokines comprises the amino acid sequence shown in SEQ ID NO: 11.
  • the immune effector cell comprises a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the antigen binding domain of the chimeric antigen receptor can target one or more of mesothelin, GPC-3, PSMA, EpCAM and GD2.
  • the antigen binding domain comprises an antigen binding protein capable of specifically binding CLDN18.2.
  • the antigen-binding protein capable of specifically binding CLDN18.2 comprises the amino acid sequence shown in SEQ ID NO:15.
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 17.
  • the co-stimulatory domain comprises a polypeptide selected from the group consisting of OX40 and/or 4-1BB.
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the intracellular signaling domain comprises a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the CAR further comprises a hinge region.
  • the hinge region comprises the amino acid sequence shown in SEQ ID NO:20.
  • the present application also provides a pharmaceutical composition, which comprises the mesenchymal stem cells or the pharmaceutical combination, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a method for preparing the mesenchymal stem cells or the drug combination.
  • the present application also provides the use of the mesenchymal stem cells or the drug combination in the preparation of medicines for preventing and/or treating tumors.
  • the tumor comprises a solid tumor.
  • the tumor comprises one or more selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, and prostate cancer.
  • Figure 1 shows the titer determination results of the CLDN18.2-targeting lentiviruses (Ab10Z-OX40-IL7R, Ab10Z-4-1BB-IL7R).
  • FIG. 2 shows the CAR (Ab10BBZ, Ab10Z-OX40-IL7R, Ab10Z-4-1BB-IL7R) expression analysis results of the CLDN8.2 CAR-T described in this application.
  • Figure 3 A-F shows the results of detecting the secreted expression of IL-2 and IL-7 using ELISA and CBA.
  • Figure 4 shows the tumor burden of mice treated with MSCs and CAR-T cells.
  • Figure 5 shows the tumor burden of mice treated with MSCs and CAR-T cells.
  • Figure 6 shows the detection of the proportion of CAR-T cells in peripheral blood of mice on the 10th day after receiving treatment.
  • Figure 7 shows the tumor burden of mice treated with MSCs and CAR-T cells.
  • Figure 8 shows the detection of the proportion of CAR-T cells in the peripheral blood of mice on the 10th day after receiving treatment.
  • Figure 9 shows the detection results of mRNA expression of TET1 and NQO1.
  • Figure 10 shows the detection of IL2 expression under normal culture and hypoxia-induced conditions.
  • T cell activation signal generally refers to a signal required for T cell activation, which includes the first signal, the second signal and/or the third signal.
  • the term "the first signal of T cell activation” can be used interchangeably with “antigen stimulation signal” and "TCR signal”, and generally refers to the TCR-CD3 complex on the surface of T cells combined with the antigen peptide-MHC molecule. T cell activation signal.
  • second signal of T cell activation can be used interchangeably with "co-stimulatory signal molecule”, which usually refers to some co-stimulatory molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB , CD40, etc., activate the second signal of immune cells by binding to their ligands.
  • co-stimulatory signal molecule usually refers to some co-stimulatory molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB , CD40, etc., activate the second signal of immune cells by binding to their ligands.
  • an antibody generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen.
  • an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), As well as antibody fragments (eg, Fab, Fab' and (Fab)2 fragments) that bind to the antigen.
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further distinguished into hypervariable regions called complementarity determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL may consist of three CDR and four FR regions, which may be arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • single-chain antibody generally refers to an antibody in which the variable region of the heavy chain and the variable region of the light chain are linked by a linker peptide.
  • an antigen binding molecule is used interchangeably with “antigen-binding molecule”, and generally refers to a molecule comprising an antigen-binding domain or antigen-binding portion capable of binding to a target antigen.
  • an antigen binding molecule can be a protein or a polypeptide.
  • tumor-associated antigen can be used interchangeably with “tumor antigen”, and generally refers to antigens associated with tumor cells, including tumor-associated antigens and tumor-specific antigens. The term includes antigens present in/on tumor cells and non-tumor cells.
  • the term "pharmaceutical combination” generally refers to a combination comprising at least two components.
  • the components in the drug combination can be used in combination. Wherein, using in combination may refer to using at the same time, or may refer to using separately.
  • the components of the combination can be mixed together or placed in separate containers.
  • the pharmaceutical combination comprises the mesenchymal stem cells and the immune effector cells.
  • the mesenchymal stem cells and the immune effector cells can be administered simultaneously or separately.
  • the mesenchymal stem cells can be mixed with the immune effector cells, or placed in different containers.
  • co-stimulatory domain generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for an effective response of lymphocytes to antigens.
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of OX40 and 4-1BB.
  • co-stimulatory molecule generally refers to a costimulatory ligand that specifically binds to mediate the costimulatory response of T cells, such as but not limited to a cognate binding partner on proliferating T cells.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Costimulatory molecules include, but are not limited to, OX40, CD28, CD40, 4-1BB (CD137).
  • the term "intracellular signal transduction domain” generally refers to a domain capable of transducing signals located inside a cell.
  • the intracellular signaling domain can transduce signals into cells.
  • the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor.
  • the intracellular signaling domain may be selected from a CD3 ⁇ intracellular domain, a CD28 intracellular domain, a CD28 intracellular domain, a 4-1BB intracellular domain, and an OX40 intracellular domain.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • antigen for example, tumor-associated antigen (TAA)
  • TAA tumor-associated antigen
  • immune effector cells generally refers to immune cells involved in clearing foreign antigens and performing effector functions in an immune response.
  • the immune effector cells can be T cells.
  • hypoxia response element generally refers to a response element that responds to a hypoxia inducible factor (HIF).
  • transmembrane domain Transmembrane Domain
  • transmembrane domain generally refers to the domain in CAR that passes through the cell membrane and is connected to the intracellular signal transduction domain , plays the role of transmitting signals.
  • the term "self” refers to reference to any material from the same individual that is subsequently reintroduced into the individual.
  • the term "pharmaceutical composition” generally refers to a preparation in a form that allows the biological activity of the active ingredients to be effective, and which does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered .
  • these formulations may comprise the active ingredient of a drug together with a pharmaceutically acceptable carrier.
  • the drug product comprises a drug product for parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue.
  • the pharmaceutical product can be administered in different ways, for example intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally.
  • the term "neoplastic” generally refers to any new pathological growth of tissue. Tumors can be benign or malignant. In the present application, the tumor may be a solid tumor and/or a hematological tumor.
  • the term “combination” is also called “co-administration”, and generally refers to administration before, after, or at the same time as another pharmaceutical ingredient.
  • the two or more pharmaceutical ingredients used in combination may be administered by the same administration method, or may be administered by different administration methods, and may be administered simultaneously or sequentially.
  • one therapeutic agent used in combination may be the mesenchymal stem cells, and the second therapeutic agent may be the immune effector cells.
  • the combination can also include a third or even more therapeutic agents.
  • prevention generally refers to preventing the occurrence and onset, recurrence, and/or spread of a disease or one or more symptoms thereof by taking certain measures in advance.
  • treating generally refers to eliminating or ameliorating a disease, or one or more symptoms associated with a disease.
  • treatment generally refers to the administration of one or more drugs to a patient with the disease such that the disease is eliminated or remitted.
  • treatment may be administration of the drug combination and/or pharmaceutical composition in the presence or absence of other drugs after the onset of symptoms of a particular disease. For example, using the pharmaceutical combination and/or pharmaceutical composition described in the present application to prevent the occurrence, development, recurrence and/or metastasis of tumors.
  • homology generally refers to an amino acid sequence or a nucleic acid sequence having certain homology with the compared amino acid sequence and the compared nucleic acid sequence.
  • the term “homology” may be equated with sequence "identity”.
  • homologous sequences can include at least about 80%, at least about 85%, at least about 90%, at least about 99.1%, at least about 99.2%, at least about 99.3%, at least about 99.4%, at least about 99.5%, at least About 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9% identical amino acid sequences.
  • a homologue will comprise the same active site, etc., as the subject amino acid sequence.
  • Homology can be considered in terms of similarity (ie, amino acid residues having similar chemical properties/functions), or can be expressed in terms of sequence identity.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a mesenchymal stem cell capable of expressing immune regulatory molecules, and the immune regulatory molecules may include molecules involved in the first signal and/or the second signal of T cell activation.
  • the immune modulating molecule may be exogenous.
  • the molecules involved in the first signal of T cell activation may include molecules involved in the process of antigen recognition.
  • the molecules involved in the first signal of T cell activation may include molecules capable of binding antigens.
  • the molecule capable of binding an antigen may include an antigen-binding protein.
  • the antigen binding protein may comprise an antibody or a fragment thereof.
  • the fragments may include Fab, Fab', Fv fragments, F(ab') 2 , F(ab) 2 , scFv, di-scFv and/or dAb.
  • the antigens include tumor-associated antigens.
  • the tumor-associated antigen comprises a solid tumor.
  • the tumor-associated antigen comprises a hematoma.
  • the antigen comprises CLDN18.2.
  • the CLDN18.2 comprises human CLDN18.2.
  • the antigen binding protein may comprise an anti-CLDN18.2 molecule.
  • the anti-CLDN18.2 molecule comprises a CLDN18.2 antibody or fragment thereof.
  • the CLDN18.2 antibody or fragment thereof may comprise at least one CDR in a VH comprising the amino acid sequence shown in SEQ ID NO: 13.
  • the CLDN18.2 antibody or fragment thereof may comprise at least one CDR in a VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO: 14.
  • the CLDN18.2 antibody or fragment thereof may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may be divided into SEQ ID NO: 13 by any CDR division method known to those skilled in the art The amino acid sequence shown was obtained.
  • the CLDN18.2 antibody or fragment thereof may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 may be divided into SEQ ID NO: 14 by any CDR division method known to those skilled in the art The amino acid sequence shown was obtained.
  • the CLDN18.2 antibody or fragment thereof comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 13. In certain embodiments, the CLDN18.2 antibody or fragment thereof comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the anti-CLDN18.2 comprises a scFv comprising the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the anti-CLDN18.2 comprises a scFv comprising the amino acid sequence shown in SEQ ID NO:16.
  • the molecule involved in the first signal of T cell activation may be an exogenous molecule.
  • the molecule involved in the first signal of T cell activation may include CD3 molecule.
  • the molecules involved in the first signal of T cell activation may include molecules capable of binding to CD3.
  • the molecule capable of binding CD3 may include an anti-CD3 molecule.
  • the anti-CD3 molecule comprises an anti-CD3 antibody or fragment thereof.
  • the anti-CD3 antibody or fragment thereof comprises at least one CDR in a VH comprising the amino acid sequence set forth in SEQ ID NO:2.
  • the anti-CD3 antibody or fragment thereof may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2 and HCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 2 by any means known to those skilled in the art.
  • the anti-CD3 antibody or fragment thereof comprises at least one CDR in a VL comprising the amino acid sequence set forth in SEQ ID NO:3.
  • the anti-CD3 antibody or fragment thereof may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2 and LCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 3 by any means known to those skilled in the art.
  • the anti-CD3 antibody or fragment thereof comprises a VH comprising the amino acid sequence shown in SEQ ID NO:2.
  • the anti-CD3 antibody or fragment thereof comprises a VL comprising the amino acid sequence shown in SEQ ID NO:3.
  • the anti-CD3 antibody or fragment thereof comprises VH and VL, the VH comprising the amino acid sequence shown in SEQ ID NO:2, and the VL comprising the amino acid sequence shown in SEQ ID NO:3 .
  • the mesenchymal stem cells can express molecules involved in the second signal of T cell activation.
  • the molecule involved in the second signal of T cell activation may be an exogenous molecule.
  • the second signal includes a co-stimulatory signal.
  • the molecule involved in the second signal of T cell activation comprises a co-stimulatory molecule.
  • the co-stimulatory molecule comprises a polypeptide selected from the group consisting of CD28, OX40 and/or 4-1BB.
  • the molecule involved in the second signal of T cell activation comprises a molecule capable of binding a co-stimulatory molecule.
  • the molecule capable of binding a co-stimulatory molecule includes a ligand of the co-stimulatory molecule.
  • the ligand for the co-stimulatory molecule may include OX-40L and/or 4-1BBL.
  • the OX-40L comprises the amino acid sequence shown in SEQ ID NO:4.
  • the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO:5.
  • the molecule capable of binding a co-stimulatory molecule may comprise an anti-CD28 molecule.
  • the anti-CD28 molecule may comprise an anti-CD28 antibody or fragment thereof
  • the molecule capable of binding co-stimulatory molecules may include antibodies against co-stimulatory molecules.
  • the anti-co-stimulatory molecule antibodies can include anti-CD28 antibodies or fragments thereof.
  • the anti-CD28 antibody or fragment thereof comprises at least one CDR in a VH comprising the amino acid sequence shown in SEQ ID NO:8.
  • the anti-CD28 antibody or fragment thereof may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2 and HCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO:8 by any means known to those skilled in the art.
  • the anti-CD28 antibody comprises at least one CDR in a VL comprising the amino acid sequence set forth in SEQ ID NO:9.
  • the anti-CD28 antibody or fragment thereof may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2 and LCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 9 by any means known to those skilled in the art.
  • the anti-CD28 antibody comprises a VH comprising the amino acid sequence shown in SEQ ID NO:8.
  • the anti-CD28 antibody comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:9.
  • the anti-CD28 antibody comprises VH and VL
  • the VH comprises the amino acid sequence shown in SEQ ID NO:8
  • the VL comprises the amino acid sequence shown in SEQ ID NO:9.
  • the mesenchymal stem cells may also express molecules involved in the third signal of T cell activation.
  • the molecule involved in the third signal of T cell activation may be an exogenous molecule.
  • the third signal of T cell activation may include cytokine signal.
  • the molecules involved in the third signal of T cell activation may include cytokines.
  • the cytokines may include cytokines produced by lymphocytes.
  • the cytokines may include cytokines produced by non-lymphocytes.
  • the cytokines may include interleukins.
  • the cytokines may include IL-12, IL-2, IL-5, IL7, and/or IL-15.
  • the cytokine can be IL-7.
  • the IL-7 can be human IL-7.
  • the IL-7 may comprise the amino acid sequence shown in SEQ ID NO:10.
  • the cytokine can be IL-2.
  • the IL-2 can be human IL-2.
  • the IL-2 may comprise the amino acid sequence shown in SEQ ID NO: 12.
  • the mesenchymal stem cells may also contain other modifications.
  • the modification can improve the expansion ability of mesenchymal stem cells in tumors.
  • the modification capable of improving the expansion ability of mesenchymal stem cells in tumors includes expressing molecules in mesenchymal stem cells that can improve the ability of mesenchymal stem cells to expand in tumors.
  • the molecule capable of increasing the ability of mesenchymal stem cells to expand in tumors comprises an enzyme capable of improving the ability of mesenchymal stem cells to expand in tumors.
  • the molecule capable of enhancing the ability of mesenchymal stem cells to expand in a tumor includes an enzyme.
  • the enzyme may include reduced coenzyme/quinone oxidoreductase (NQO1).
  • NQO1 may comprise the amino acid sequence shown in SEQ ID NO:22.
  • the enzyme may include 5-methylcytosine dioxygenase (TET1).
  • TET1 may comprise the amino acid sequence shown in SEQ ID NO:23.
  • the mesenchymal stem cells also contain modifications that enable them to be expressed in normal tissues and to highly express corresponding immune regulatory molecules in a hypoxic tumor environment.
  • a hypoxia response element can be incorporated into the promoter.
  • HRE elements can be added to the promoter.
  • the HRE element can comprise the amino acid sequence shown in SEQ ID NO:24.
  • the mesenchymal stem cells may include autologous mesenchymal stem cells.
  • the mesenchymal stem cells may include allogeneic mesenchymal stem cells.
  • the present application provides a pharmaceutical combination, which comprises the mesenchymal stem cells and immune effector cells described in the present application.
  • the immune effector cells may comprise T cells.
  • the T cells may comprise CAR-T, TCR-T, TIL and/or T cells derived from pleural fluid, ascites fluid and/or PBMC.
  • the T cells may comprise CD8+ T cells, CD4+ T cells, ⁇ - ⁇ T cells and/or T regulatory cells.
  • the immune effector cells may comprise engineered immune effector cells.
  • the engineered immune effector cells can receive the first signal, the second signal and/or the third signal of T cell activation provided by the mesenchymal stem cells.
  • the immune effector cells contain immune regulatory molecules that can combine with the corresponding immune regulatory molecules expressed by the mesenchymal stem cells to provide the first signal and the second signal for T cell activation. and/or a third signal.
  • the mesenchymal stem cells express a molecule involved in the first signal of T cell activation, and the T cells are capable of expressing another molecule that, in combination with the corresponding molecule expressed by the mesenchymal stem cells, receives mesenchymal Stem cells provide the first signal.
  • the mesenchymal stem cells express anti-CD3 molecules, and the immune effector cells express CD3 molecules.
  • the immune effector cells express co-stimulatory molecules
  • the mesenchymal stem cells express the corresponding co-stimulatory molecule ligands.
  • the mesenchymal stem cells express cytokines and the immune effector cells express corresponding cytokine receptors.
  • the immune regulatory molecules contained in the immune cells may be exogenous molecules.
  • the immune effector cells may contain an antigen-binding protein that has the same target as the mesenchymal stem cells.
  • the target can be any target known in the art, for example, a tumor target.
  • the target can be selected from Mesothelin, GPC-3 (Glypican-3, Glypican 3), PSMA (Prostate Specific Membrane Antigen), EpCAM (Epithelial Cell Adhesion Molecule ) and GD2 (ganglioside).
  • the immune effector cells may comprise an antigen binding protein targeting one or more targets selected from the group consisting of mesothelin, GPC-3, PSMA, EpCAM and GD2.
  • the antigen-binding portion of the antigen-binding protein targeting mesothelin may comprise the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:30.
  • the antigen-binding portion of the GPC-3-targeting antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO: 25 or 26.
  • the antigen-binding portion of the PSMA-targeting antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:31.
  • the antigen-binding portion of the GD2-targeting antigen-binding protein may comprise the amino acid sequence shown in SEQ ID NO:27 or SEQ ID NO:28.
  • the immune effector cells may comprise an antigen-binding protein targeting CLDN18.2, and at the same time, the mesenchymal stem cells may also comprise an antigen-binding protein targeting CLDN18.2.
  • the immune effector cells may also contain the same molecules as the mesenchymal stem cells.
  • said immune effector cells comprise an antigen or a molecule capable of binding said antigen.
  • the antigen-binding molecules contained in the immune effector cells can target the same antigen as the antigen-binding molecules expressed by the mesenchymal stem cells.
  • the molecule capable of binding an antigen comprises an antigen binding protein.
  • the antigen binding protein comprises an antibody or fragment thereof.
  • the antibody or fragment thereof comprises a scFv.
  • the immune effector cells comprise an antigen-binding molecule comprising an anti-CLDN18.2 antibody or fragment thereof.
  • the anti-CLDN18.2 antibody or fragment thereof comprises at least one CDR in a VH comprising the amino acid sequence shown in SEQ ID NO: 13.
  • the CLDN18.2 antibody or fragment thereof may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2 and HCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 13 by any means known to those skilled in the art.
  • the anti-CLDN18.2 antibody or fragment thereof comprises at least one CDR in a VL comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the CLDN18.2 antibody or fragment thereof may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2 and LCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 14 by any means known to those skilled in the art.
  • the anti-CLDN18.2 antibody or fragment thereof comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 13.
  • the anti-CLDN18.2 antibody or fragment thereof comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 14.
  • the immune effector cells can express CD3 molecules.
  • the CD3 molecule may comprise the amino acid sequence shown in SEQ ID NO:1.
  • the immune effector cells may comprise costimulatory molecules or ligands capable of binding costimulatory molecules.
  • the co-stimulatory molecule or the ligand capable of binding to the costimulatory molecule contained in the immune effector cells can be combined with the ligand or costimulatory molecule expressed by the mesenchymal stem cell and capable of binding to the costimulatory molecule.
  • the costimulatory molecule is selected from one or more costimulatory molecules of the CD28 family, the CD2 family and/or the TNFR family.
  • the co-stimulatory molecules include polypeptides selected from the following proteins: OX40 and/or 4-1BB.
  • the OX40 may comprise the amino acid sequence shown in SEQ ID NO:6.
  • the 4-1BB can comprise the amino acid sequence shown in SEQ ID NO:7.
  • the ligands of costimulatory molecules include: OX40L and/or 4-1BBL.
  • OX40L may comprise the amino acid sequence shown in SEQ ID NO:4.
  • 4-1BBL can comprise the amino acid sequence shown in SEQ ID NO:5.
  • the immune effector cells may comprise receptors for the co-stimulatory molecules.
  • the immune effector cells may comprise molecules involved in the third signal of T cell activation.
  • the third signal of T cell activation may include a cytokine signal.
  • the immune effector cells may comprise cytokines or molecules capable of binding cytokines.
  • the cytokines may include cytokines produced by lymphocytes.
  • the cytokines may include cytokines produced by non-lymphocytes.
  • the cytokines may include interleukins.
  • the cytokines may include IL-12, IL-2, and/or IL7.
  • the molecule capable of binding a cytokine comprises a receptor for the cytokine.
  • the cytokine receptors may include IL-12R, IL-2R and/or IL7R.
  • the IL-7R may comprise the amino acid sequence shown in SEQ ID NO: 11.
  • the immune effector cells may comprise a chimeric antigen receptor (CAR), wherein the CAR may comprise an antigen binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signaling domain.
  • CAR chimeric antigen receptor
  • the antigen binding domain comprises an antigen binding protein.
  • the antigen binding protein may comprise an antibody or antigen binding fragment.
  • the antigen binding protein may comprise a scFv.
  • the scFv can comprise the amino acid sequence shown in SEQ ID NO: 15.
  • the antigen binding domain comprises an antigen binding protein capable of binding CLDN18.2.
  • the antigen binding protein capable of binding CLDN18.2 comprises at least one CDR in a VH comprising the amino acid sequence shown in SEQ ID NO:13.
  • the antigen binding protein capable of binding CLDN18.2 may comprise HCDR1, HCDR2 and HCDR3.
  • the HCDR1, HCDR2 and HCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 13 by any means known to those skilled in the art.
  • the antigen binding protein capable of binding CLDN18.2 comprises at least one CDR in a VL comprising the amino acid sequence shown in SEQ ID NO:14.
  • the antigen binding protein capable of binding CLDN18.2 may comprise LCDR1, LCDR2 and LCDR3.
  • the LCDR1, LCDR2 and LCDR3 can be obtained by dividing the amino acid sequence shown in SEQ ID NO: 14 by any means known to those skilled in the art.
  • the antigen-binding protein capable of binding CLDN18.2 comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:13.
  • the antigen binding protein capable of binding CLDN18.2 comprises a VL comprising the amino acid sequence shown in SEQ ID NO:14.
  • the transmembrane domain may comprise a transmembrane domain derived from a protein selected from the group consisting of ⁇ , ⁇ or zeta chains of T cell receptors, CD28, CD3e, CD45, CD4, CD5, CD8 ⁇ , CD9 , CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.
  • the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:17.
  • the co-stimulatory domain of the chimeric antigen receptor may comprise a polypeptide selected from the following proteins: CD28, 4-1BB, OX-40 and ICOS.
  • the co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 18.
  • the intracellular signaling domain of the chimeric antigen receptor may comprise a signaling domain derived from CD3 ⁇ .
  • the intracellular signaling domain may comprise the amino acid sequence shown in SEQ ID NO: 19.
  • the chimeric antigen receptor may further comprise a hinge region.
  • the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8A, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc ⁇ RI ⁇ , BTLA, GITR, DAP10, TIM1, SLAM, CD30, and LIGHT.
  • the hinge region may comprise the amino acid sequence shown in SEQ ID NO:20.
  • the chimeric antigen receptor may further comprise a signal peptide.
  • the C-terminus of the signal peptide is linked to the N-terminus of the targeting moiety.
  • the immune effector cells may include human cells.
  • the immune effector cells may include autologous immune effector cells.
  • the immune effector cells may include allogeneic immune effector cells.
  • the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain.
  • the antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 15
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO: 17
  • the The costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 18
  • the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO: 19.
  • the mesenchymal stem cells can be used in combination with immune effector cells expressing corresponding molecules.
  • MSC-4-1BBL and MSC-IL7 can be combined with Ab10Z-4-1BB-IL7R CAR-T cells.
  • MSC-OX40L and MSC-IL7 can be used in combination with the Ab10Z-OX40-IL7R CAR-T cells prepared in the examples of this application.
  • MSC-anti-CD3 and MSC-IL-2 can be used in combination with the CLDN18.2 CAR-T cells prepared in the examples of this application.
  • MSC-anti-CD3 and MSC-IL-2 can be used in combination with the CLDN18.2 CAR-T cells prepared in the examples of this application.
  • MSC-anti-CD3, MSC-IL-2 and MSC-anti-CD28 can be used in combination with the CLDN18.2 CAR-T cells prepared in the examples of this application.
  • the present application also provides a pharmaceutical composition, which comprises the mesenchymal stem cells described in the present application, and optionally a pharmaceutically acceptable carrier.
  • the present application also provides a pharmaceutical composition, which comprises the mesenchymal stem cells described in the present application, the immune effector cells described in the present application, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can be formulated for topical, parenteral, systemic, intracavity, intravenous, intraarterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal , intrathecal, oral or transdermal routes of administration, which may include injection or infusion.
  • Suitable formulations may contain virus and/or cells in sterile or isotonic medium.
  • Compositions, pharmaceutical compositions and kits can also be formulated as fluids, including gel forms. Fluid formulations can be formulated for administration to selected areas of the human or animal body by injection or infusion, eg, through a catheter.
  • the mesenchymal stem cells described in this application can be used in combination with the immune effector cells described in this application.
  • the mesenchymal stem cells can be administered simultaneously with the immune effector cells.
  • Simultaneous administration may mean that the components are mixed together or administered separately. It can be administered in the same way, such as in the same vein or other blood vessels, or in different ways, such as intravenous administration and intratumoral administration at the same time.
  • the mesenchymal stem cells may be administered sequentially with the immune effector cells.
  • the order of administration may be that the mesenchymal stem cells are administered first, and then the immune effector cells are administered; or the immune effector cells are administered first, and then the oncolytic virus is administered. It may be administered in the same manner or in a different manner.
  • Each component can be applied once or divided into several applications.
  • sequential administration can be at any time interval, including minutes, hours, days, weeks, months, or years.
  • sequential administration means at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months or 6 months apart application.
  • the dose administered may be a therapeutically effective amount.
  • the present application also provides a preparation method of the mesenchymal stem cells and/or the immune effector cells.
  • the preparation method of the mesenchymal stem cells and/or immune effector cells may include synthesizing a virus vector comprising the first signal, the second signal and/or the third signal molecule involved in T cell activation, preparing a virus, and transfecting After the cells are infected with mesenchymal stem cells and/or immune effector cells.
  • the present application also provides the use of the mesenchymal stem cells and/or the drug combination in the preparation of medicines for preventing and/or treating diseases and/or conditions.
  • the present application also provides a method for treating diseases and/or conditions, which comprises administering the mesenchymal stem cells and/or the drug combination described in the present application to a subject in need.
  • the present application also provides the mesenchymal stem cells and/or drug combination for treating diseases and/or conditions.
  • the diseases and/or conditions may include tumors.
  • the tumor comprises a solid tumor and/or a hematological tumor.
  • the tumor comprises one or more selected from the group consisting of pancreatic cancer, gastric cancer, lung cancer, and prostate cancer.
  • a CAR targeting CLDN18.2 is first prepared.
  • the following sequences were artificially synthesized: scFv Ab10 (SEQ ID NO:15), hinge region (SEQ ID NO:20), transmembrane region (SEQ ID NO:17), 4-1BB co-stimulatory factor (SEQ ID NO:18), CD3 ⁇ intracellular signaling domain (SEQ ID NO: 19).
  • the hinge region, transmembrane region, 4-1BB co-stimulatory factor and CD3 ⁇ intracellular signaling domain are connected end to end to obtain BBZ, and its sequence is shown in SEQ ID NO:21.
  • the scFv Ab10 (amino acid sequence SEQ ID NO: 15, nucleotide sequence SEQ ID NO: 16) and BBZ (SEQ ID NO: 21) that can specifically bind to CLDN18.2 are passed through overlap PCR, and XbaI and BamHI enzymes are added to both ends Cloning the pCDH-MSCVEF vector by cut site. Carry out PCR amplification, and use extended PCR to carry XbaI restriction site (including protective bases), hinge region, transmembrane region, 4-1BB costimulatory factor, CD3 ⁇ intracellular signaling domain, BamHI at the 5' end sequentially Restriction site, PCR amplification to obtain the CAR Ab10BBZ.
  • Clones with correct sequencing were extracted with NucleoBond Xtra Midi Plus EF kit without endotoxin, and co-transfected with lentiviral packaging plasmid (VSV-g, pMD Gag/Pol or RSV-REV) into 293X cells at 37°C, 5% CO 2 After culturing for 48 hours, the supernatant was collected, filtered at 0.45 ⁇ M, and then centrifuged at a speed of 25,000 RPM for 2 hours to concentrate the virus using a Beckman ultracentrifuge and SW28 rotor, which was pCDH-MSCVEF-Ab10BBZ virus (abbreviated as Ab10BBZ virus), For subsequent CAR-T cell production.
  • VSV-g lentiviral packaging plasmid
  • pMD Gag/Pol or RSV-REV lentiviral packaging plasmid
  • the obtained cells are Ab10BBZ, Ab10Z-OX40-IL7R, Ab10Z-4-1BB-IL7R CAR-T cells, passed Flow staining, for Ab10BBZ, Ab10Z-OX40-IL7R, Ab10Z-4-1BB-IL7R CAR-T cells with Alexa 647 AffiniPure F(ab') 2 Fragment Goat Anti-Mouse IgG, Fab fragment specific secondary antibody staining, the results are shown in Figure 2.
  • CLDN18.2, CLDN18.2-IL2, OX40L, 4-1BBL, IL-2, anti-CD3, anti-CD28 were synthesized, molecularly cloned into the pCDHEF-MSCV-puro vector, and the virus pCDH-MSCVEF- CLDN18.2, pCDH-MSCVEF-CLDN18.2-IL2, pCDH-MSCVEF-OX40L, pCDH-MSCVEF-4-1BBL, pCDH-MSCVEF-IL-2, pCDH-MSCVEF-anti-CD3, pCDH-MSCVEF-anti- CD28.
  • Example 293 cells were transfected to obtain virus supernatants, which were stored at -80°C for subsequent infection of mesenchymal stem cells.
  • Human umbilical cord-derived mesenchymal stem cells were cultured in a 6-well plate, and the virus supernatant obtained in Example 2.1 was added, and the virus infection enhancer polybrene 10 ⁇ g/ml was added. After 24 hours, fresh medium was changed, and puromycin was added for screening after 48 hours. Obtain mesenchymal stem cells stably expressing CLDN18.2, CLDN18.2-IL2, OX40L, 4-1BBL, IL-2, IL-7, anti-CD3, anti-CD28.
  • FIG. 3A-E shows MSCs described in this application (MSC-CLDN18.2, MSC-CLDN18.2-IL2, MSC-OX40L, MSC-4-1BBL, MSC-IL-2, MSC-anti-CD3, MSC -anti-CD28) the expression of the corresponding T cell activation molecule.
  • Figure 3F shows the secret expression of IL-7 detected by ELISA.
  • 2x10 6 CFPAC-1 tumor cells were subcutaneously inoculated into B-NDG mice, and 5x10 6 Ab10Z-OX40-IL7R, Ab10Z-4-1BB-IL7R CAR-T cells and 10 6 MSC-OX40L, MSC-4- 1BBL and MSC-IL7 cells were treated, and the tumor burden of the mice was measured, and the results are shown in Figure 4. It can be seen from the figure that the Ab10Z-OX40-IL7R/MSC-OX40L/MSC-IL7 treatment group and Ab10Z-4-1BB-IL7R/MSC-4-1BBL/MSC-IL7 cells can better control the tumor burden than the PBS group.
  • B-NDG mice were subcutaneously inoculated with 2x10 6 CFPAC-1 tumor cells, and treated with 5x10 6 Ab10BBZ CAR-T cells and 10 6 MSC, MSC-anti-CD3, MSC-IL-2 cells 6 days later. tumor burden.
  • the results are shown in Fig. 5 respectively. It can be seen from the figure that MSC-anti-CD3 and MSC-IL-2, compared with the control MSC, can cooperate with Ab10BBZ CAR-T cells to exert stronger anti-tumor effect.
  • the results showed that Ab10BBZ/MSC-IL2/MSC-anti-CD3 cell treatment reduced the tumor volume of mice by 90.8% (5.46 mm 3 to 0.5 mm 3 ) compared with the control Ab10BBZ/MSC.
  • Example 4 after tumor inoculation and the same treatment, peripheral blood was collected on the 10th day, and the proportion of CAR-T cells was detected by flow cytometry.
  • the results are shown in Figure 6, respectively. It can be seen from the figure that MSC-anti-CD3 and MSC-IL-2 can significantly enhance the proliferation of Ab10BBZ CAR-T cells in mice compared with the control MSC. As shown in Figure 6, compared with control MSCs, MSC-IL2/MSC-anti-CD3 cells enhanced the proliferation of Ab10BBZ CAR-T by 2.32-fold, from 1.08% to 2.51%.
  • Example 6 MSC-anti-CD3, MSC-IL-2, MSC-anti-CD28 enhance the proliferation ability of CLDN18.2 CART in vivo
  • Example 7 MSC-anti-CD3, MSC-IL-2, MSC-anti-CD28 enhance the proliferation ability of CLDN18.2 CART in vivo
  • mice were vaccinated and treated in the same manner as in Example 6, and peripheral blood was collected on the 10th day, and the proportion of CAR-T cells was detected by flow cytometry.
  • the results are shown in Fig. 8, respectively. It can be seen from the figure that MSC-anti-CD3, MSC-IL-2, and MSC-anti-CD28 can significantly enhance the proliferation of Ab10BBZ CAR-T cells in mice compared with the control MSC. As shown in Figure XX, MSC-anti-CD3, MSC-IL-2, MSC-anti-CD28 cells enhanced the proliferation of Ab10BBZ CAR-T compared with control MSCs.
  • Example 8 NQO1 and TET1 enhance the enrichment and continuous expansion of MSCs in tumors
  • Example 293 cells were transfected to obtain virus supernatant, which was stored at -80°C for subsequent infection of mesenchymal stem cells.
  • Human umbilical cord-derived mesenchymal stem cells were cultured in a 6-well plate, adding virus supernatant containing TET1, NQO1, TET1-2A-NQO1, virus infection enhancer polybrene 10 ⁇ g/ml, and changing the fresh medium after 24 hours, 48 Hours later, puromycin was added to screen to obtain mesenchymal stem cells stably expressing TET1, NQO1, and TET1-2A-NQO1.
  • the mRNA expressions of TET1 and NQO1 were detected by q-PCR. The result is shown in FIG. 9 .
  • Embodiment 9 HRE promoter selectively enhances the expression of IL-2 under hypoxic conditions
  • the synthesized IL2 molecule was cloned into the pCDH-HRE-puro vector to prepare the virus pCDH-HRE-IL2.
  • the virus supernatant was obtained by transfecting 293 cells and stored at -80°C for subsequent infection of mesenchymal stem cells.
  • Human umbilical cord-derived mesenchymal stem cells were cultured in a 6-well plate, added with viral supernatant containing HRE-IL2, virus infection enhancer polybrene 10 ⁇ g/ml, replaced with fresh medium after 24 hours, and obtained by adding puromycin for screening after 48 hours Mesenchymal stem cells stably expressing HRE-IL2.
  • the expression of IL2 was detected under normal culture and hypoxia-induced conditions, respectively. The result is shown in Figure 10. The results showed that the expression of IL-2 was significantly enhanced under hypoxic conditions compared with the normoxic group.

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Abstract

一种改造的间充质干细胞(MSC)，及其与抗肿瘤T细胞联合治疗肿瘤的方法，其中所述间充质干细胞提供T细胞活化的第一信号、第二信号、第三信号和/或抗原呈递细胞活化信号；其中所述T细胞可以是CAR-T，TCR-T，TIL、胸水腹水及PBMC来源的T细胞。

Description

改造的间充质干细胞和免疫效应细胞联合治疗肿瘤 技术领域
本申请涉及生物医药领域，具体的涉及一种改造的间充质干细胞，及其与免疫效应细胞联合用于制备治疗肿瘤的药物的用途。
背景技术
间充质干细胞是一种多能干细胞，具有自我更新和多向分化能力，可分化为骨骼、软骨、脂肪等。临床上应用包括组织损伤修复，自身免疫性疾病，基因治疗的载体等。间充质干细胞具有趋向炎性组织的特征，肿瘤被认为是一种慢性的炎性组织，因此间充质干细胞可以趋化并富集到肿瘤组织。
T细胞过继性细胞疗法中如嵌合抗原受体CAR-T细胞，TCR-T，TIL等是一种经人工修饰或体外扩增的肿瘤杀伤细胞，通过基因改造(CAR-T或TCR-T等)的方式使其获得靶向识别和杀伤肿瘤的能力，或通过选择性扩增方式(TIL等)富集肿瘤特异的内源性T细胞，是肿瘤免疫治疗领域的重要发展方向。然而，CAR-T虽然在对造血来源的肿瘤治疗取得了显著的临床疗效，但是其对实体肿瘤的治疗疗效尚不理想，其主要原因是实体肿瘤的肿瘤微环境存在多种免疫抑制机理，不能有效提供T细胞活化信号，使得上述CAR-T，TCR-T，TIL等不能完全发挥功能，克服实体肿瘤微环境的免疫抑制。
发明内容
本申请提供了一种经改造的间充质干细胞，及包含所述经改造的间充质干细胞和抗肿瘤T细胞的药物组合。过继性T细胞治疗和间充质干细胞的联合治疗，一方面解决肿瘤特异的T细胞数量的不足，一方面弱化肿瘤微环境的抑制，提供T细胞活化的信号，从数量和质量上同时提高过继性T细胞治疗抗实体肿瘤的疗效。
一方面，本申请提供了一种间充质干细胞，其表达免疫调控分子，所述免疫调控分子包括参与T细胞活化的第一信号和/或第二信号的分子。
在某些实施方式中，所述参与T细胞活化的第一信号的分子包括参与抗原识别过程的分子。
在某些实施方式中，所述参与T细胞活化的第一信号的分子包括能够结合抗原的分子。
在某些实施方式中，所述能够结合抗原的分子包括抗原结合蛋白。
在某些实施方式中，所述能够结合抗原的分子包含抗体或其片段。
在某些实施方式中，所述抗原包括肿瘤相关抗原。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述间充质干细胞表达的抗原包括CLDN18.2。
在某些实施方式中，所述参与T细胞活化的第一信号的分子包括能够结合CD3的分子。
在某些实施方式中，所述CD3分子包含CD3的γ链、δ链、ε链、ζ链和/或η链。
在某些实施方式中，所述能够结合CD3的分子包含anti-CD3分子。
在某些实施方式中，所述anti-CD3分子包含抗CD3抗体或其片段。
在某些实施方式中，所述T细胞活化的第二信号包括共刺激信号。
在某些实施方式中，所述参与T细胞活化的第二信号的分子包括共刺激分子或能够结合共刺激分子的分子。
在某些实施方式中，所述共刺激分子包括选自CD28家族、CD2家族和/或TNFR家族的一个或多个共刺激分子。
在某些实施方式中，所述共刺激分子包括选自下述蛋白的多肽：CD28、OX40和/或4-1BB。
在某些实施方式中，所述能够结合共刺激分子的分子包括所述共刺激分子的配体。
在某些实施方式中，所述能够结合共刺激分子的配体包括：OX40L和/或4-1BBL。
在某些实施方式中，所述能够结合共刺激分子的分子包括所述共刺激分子的受体。
在某些实施方式中，所述能够结合共刺激分子的分子包括anti-CD28分子。
在某些实施方式中，所述anti-CD28分子包含抗CD28抗体或其片段。
在某些实施方式中，所述能够结合共刺激分子的分子包括抗共刺激分子的抗体或其片段。
在某些实施方式中，所述间充质干细胞还表达参与T细胞活化的第三信号的分子。
在某些实施方式中，所述T细胞活化的第三信号包括细胞因子信号。
在某些实施方式中，所述参与T细胞活化的第三信号的分子包括细胞因子和/或能够结合所述细胞因子的分子。
在某些实施方式中，所述能够结合所述细胞因子的分子包括所述细胞因子的受体。
在某些实施方式中，所述细胞因子包括IL-12、IL-2、IL-5、IL7和/或IL-15。
在某些实施方式中，所述细胞因子包含IL-7。
在某些实施方式中，所述细胞因子包含SEQ ID NO:10所示的氨基酸序列。
在某些实施方式中，所述细胞因子包含IL-2。
在某些实施方式中，所述细胞因子包含SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述间充质干细胞还包含能够提高间充质干细胞在肿瘤内的扩增能力的分子。
在某些实施方式中，所述能够提高间充质干细胞在肿瘤内扩增能力的分子包括酶。
在某些实施方式中，所述与间充质干细胞在肿瘤细胞内扩增相关的分子包括还原型辅酶/醌氧化还原酶(NQO1)。
在某些实施方式中，所述与间充质干细胞在肿瘤细胞内扩增相关的分子包括5-甲基胞嘧啶双加氧酶(TET1)。
在某些实施方式中，所述间充质干细胞包含与启动子可操作地连接的核酸，所述启动子包含低氧响应元件(HRE)。
在某些实施方式中，所述间充质干细胞为异体间充质干细胞。
在某些实施方式中，所述间充质干细胞为自体间充质干细胞。
另一方面，本申请还提供了药物组合，其包含所述间充质干细胞和免疫效应细胞。
在某些实施方式中，所述免疫效应细胞包含T细胞。
在某些实施方式中，所述T细胞包含CAR-T，TCR-T，TIL和/或胸水、腹水和/或PBMC来源的T细胞。
在某些实施方式中，所述T细胞包含CD8+T细胞、CD4+T细胞、γ-δT细胞和/或T调节细胞。
在某些实施方式中，所述免疫效应细胞包含免疫调控分子，所述免疫调控分子能够与所述间充质干细胞表达的对应的免疫调控分子相结合，提供T细胞活化的第一信号、第二信号和/或第三信号。
在某些实施方式中，所述免疫效应细胞包含参与T细胞活化的第一信号的分子。
在某些实施方式中，所述免疫效应细胞包含的参与T细胞活化的第一信号的分子包含参与抗原识别过程的分子。
在某些实施方式中，所述免疫效应细胞包含能够结合所述抗原的分子。
在某些实施方式中，所述免疫效应细胞包含的能够结合抗原的分子与所述间充质干细胞表达的能够结合抗原的分子靶向相同的抗原。例如，所述抗原可选自间皮素，GPC-3，PSMA，EpCAM和GD2的一种或多种。
在某些实施方式中，所述能够结合抗原的分子包含抗原结合蛋白。
在某些实施方式中，所述抗原结合蛋白包含抗体或其片段。
在某些实施方式中，所述抗体或其片段包含scFv。
在某些实施方式中，所述免疫效应细胞包含抗CLDN18.2抗体或其片段。
在某些实施方式中，所述免疫效应细胞包含CD3分子。
在某些实施方式中，所述CD3分子包含SEQ ID NO:1所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包含共刺激分子或能够结合共刺激分子的配体。
在某些实施方式中，所述免疫效应细胞包含的共刺激分子或能够结合共刺激分子的配体能够与所述间充质干细胞中包含的共刺激分子的配体或共刺激分子相结合。
在某些实施方式中，所述共刺激分子包括选自CD28家族、CD2家族和/或TNFR家族的一个或多个共刺激分子。
在某些实施方式中，所述共刺激分子包括选自下述蛋白的多肽：OX40和/或4-1BB。
在某些实施方式中，所述共刺激分子包含SEQ ID NO:6和/或SEQ ID NO:7所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包含参与T细胞活化的第三信号的分子。
在某些实施方式中，所述免疫效应细胞包含细胞因子或能够结合细胞因子的分子。
在某些实施方式中，所述能够结合细胞因子的分子包含所述细胞因子的受体。
在某些实施方式中，所述免疫效应细胞表达的细胞因子或能够结合细胞因子的受体能够与所述间充质干细胞表达的细胞因子或能够结合细胞因子的受体相结合。
在某些实施方式中，所述细胞因子包括IL-12、IL-2、和/或IL7。
在某些实施方式中，所述细胞因子包含SEQ ID NO:10和/或SEQ ID NO:12所示的氨基酸序列。
在某些实施方式中，所述能够结合细胞因子的受体包括IL-12R、IL-2R和/或IL7R。
在某些实施方式中，所述能够结合细胞因子的受体包含SEQ ID NO:11所示的氨基酸序列。
在某些实施方式中，所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述嵌合抗原受体包含抗原结合结构域、跨膜域、共刺激域和胞内信号传导域。例如，所述嵌合抗原受体的抗原结合结构域可靶向间皮素，GPC-3，PSMA，EpCAM和GD2的一种或多种。
在某些实施方式中，所述抗原结合结构域包含能够特异性结合CLDN18.2的抗原结合蛋白。
在某些实施方式中，所述能够特异性结合CLDN18.2的抗原结合蛋白包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述跨膜域包含SEQ ID NO:17所示的氨基酸序列。
在某些实施方式中，所述共刺激域包含选自下述蛋白的多肽：OX40和/或4-1BB。
在某些实施方式中，所述共刺激域包含SEQ ID NO:18所示的氨基酸序列。
在某些实施方式中，所述胞内信号传导域包含源自CD3ζ的信号传导结构域。
在某些实施方式中，所述胞内信号传导域包含SEQ ID NO:19所示的氨基酸序列。
在某些实施方式中，所述CAR还包含铰链区。
在某些实施方式中，所述铰链区包含SEQ ID NO:20所示的氨基酸序列。
另一方面，本申请还提供了药物组合物，其包含所述间充质干细胞或所述药物组合，以及任选地药学上可接受的载剂。
另一方面，本申请还提供了制备所述间充质干细胞或所述药物组合的方法。
另一方面，本申请还提供了所述间充质干细胞或所述药物组合在制备药物中的用途，所述药物用于预防和/或治疗肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤。
在某些实施方式中，所述肿瘤包括选自下组的一种或多种：胰腺癌、胃癌、肺癌和前列腺癌。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：
图1显示的是所述靶向CLDN18.2的慢病毒(Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R)的滴度测定结果。
图2显示的是本申请所述CLDN8.2 CAR-T的CAR(Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R)表达分析检测结果。
图3 A-F显示的是使用ELISA和CBA检测IL-2和IL-7的分泌表达结果。
图4显示的是使用MSC和CAR-T细胞治疗的小鼠的肿瘤负荷。
图5显示的是使用MSC和CAR-T细胞治疗的小鼠的肿瘤负荷。
图6显示的是接受治疗后第10天，小鼠外周血中CAR-T细胞的比例检测。
图7显示的是使用MSC和CAR-T细胞治疗后的小鼠的肿瘤负荷。
图8显示的是接受治疗后第10天，小鼠外周血中CAR-T细胞的比例检测。
图9显示的是TET1，NQO1的mRNA表达检测结果。
图10显示的是在正常培养和低氧诱导条件下检测IL2的表达。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，本领域技术人员可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“T细胞活化信号”通常指T细胞活化所需要的信号，其包含第一信号、第二信号和/或第三信号。
在本申请中，术语“T细胞活化的第一信号”可以与“抗原刺激信号”、“TCR信号”互换使用，通常指T细胞表面TCR-CD3复合体与抗原肽-MHC分子结合提供的T细胞活化信号。
在本申请中，术语“T细胞活化的第二信号”可以与“共刺激信号分子”互换使用，通常指免疫细胞表面的一些共刺激分子，如CD28、CD134/OX40、CD137/4-1BB、CD40等，通过与其配体结合，激活免疫细胞的第二信号。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab)2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH)和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
在本申请中，术语“单链抗体(scFv)”通常指由所述重链可变区和所述轻链可变区通过连接肽连接而成的抗体。
在本申请中，术语“能够结合抗原的分子”可以与“抗原结合分子”互换使用，通常指包含能够与靶抗原结合的抗原结合区或抗原结合部分的分子。例如，抗原结合分子可为蛋白质或多肽。
在本申请中，术语“肿瘤相关抗原”可以与“肿瘤抗原”互换使用，通常指与肿瘤细胞相关的抗原，其包括肿瘤相关抗原和肿瘤特异性抗原。该术语包括存在于肿瘤细胞和非肿瘤细胞中/上的抗原。
在本申请中，术语“药物组合”通常指包含至少两种组分的组合。药物组合中的各组分可以搭配使用。其中搭配使用可以指同时使用，也可以指分别使用。组合中的各组分可以混合在一起，也可以放置在不同容器中。例如，所述药物组合包含所述间充质干细胞和所述免疫效应细胞。所述间充质干细胞可以和所述免疫效应细胞同时施用，也可以分别施用。所述间充质干细胞可以和所述免疫效应细胞混合在一起，也可以分别放置在不同容器中。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如OX40和4-1BB的共刺激结构域。
在本申请中，术语“共刺激分子”通常是指与共刺激配体特异性结合从而介导T细胞的共刺激应答，例如但不限于增殖的T细胞上的同源结合配偶体。共刺激分子是有效免疫应答所需的除抗原受体或其配体之外的细胞表面分子。共刺激分子包括但不限于OX40，CD28，CD40，4-1BB(CD137)。
在本申请中，术语“胞内信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。例如，所述胞内信号传导结构域是所述嵌合抗原受体的胞内信号传导结构域。例如，在某些实施方式中，所述胞内信号传导结构域可选自CD3ζ胞内域，CD28胞内域，CD28胞内域，4-1BB胞内域和OX40胞内域。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor，CAR)通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括抗原(例如，肿瘤相关抗原(tumor-associated antigen，TAA))结合区、跨膜结构域、共刺激结构域和胞内信号结构域。
在本申请中，术语“免疫效应细胞”通常是指在免疫应答中参与清除异物抗原和行使效应功能的免疫细胞。例如，在某些实施方式中，所述免疫效应细胞可以为T细胞。
在本申请中，术语“低氧响应元件(HRE)”通常是指响应低氧诱导因子(HIF)的应答元件。
在本申请中，术语“跨膜域”(Transmembrane Domain)可以与“跨膜结构域”互换使用，通常是指CAR中穿过细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。
在本申请中，术语“自体“是指引用来自相同个体的任何材料，其随后被再次引入个体。
在本申请中，术语“药物组合物”通常指以允许活性成分的生物学活性有效的形式存在的制剂，并且其不含有对所述制剂待施用的受试者有不可接受的毒性的另外成分。在某些实施方案中，这些制剂可以包含药物的活性组分以及药学上可接受的载剂。在某些实施方式中，所述药物产品包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中的药物产品。所述药物产品可以通过不同方式给药，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。
在本申请中，术语“肿瘤”通常是指任何新的病理性的组织增生。肿瘤可能是良性的，也可能是恶性的。在本申请中，所述肿瘤可以是实体瘤和/或血液瘤。
在本申请中，术语“联用”又称“共同施用”，通常指在另一个药物成分之前，或之后，或同时给药。联用的两个或多个药物成分可以是通过相同的给药方式给药，也可以是通过不同给药方式给药，可以是同时施用，也可以是先后施用。例如，联用的一种治疗剂可以是所述间充质干细胞，第二种治疗剂可以是所述免疫效应细胞。联用也可以包含第三种甚至更多种治疗剂。
在本申请中，术语“预防”通常是指通过预先采取某些措施而防止疾病或其一种或多种症状的产生和发作，复发，和/或扩散。在本申请中，术语“治疗”通常指消除或改善疾病，或与疾病相关的一种或多种症状。在某些实施方案中，治疗通常指向患有这种疾病的患者施用一种或多种药物使得疾病消除或缓解。在某些实施方案中，“治疗”可以是在特定疾病的症状发作后，在其他药物存在或不存在的情况下施用所述药物组合和/或药物组合物。例如，使用本申请所述药物组合和/或药物组合物防止肿瘤的产生，发展，复发和/或转移。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“同源性”通常是指与比较的氨基酸序列和比较的核酸序列具有一定同源性的氨基酸序列或核酸序列。术语“同源性”可以等同于序列“同一性”。同源序列可以 包括与主题序列是至少约80％、至少约85％、至少约90％、至少约99.1％、至少约99.2％、至少约99.3％、至少约99.4％、至少约99.5％、至少约99.6％、至少约99.7％、至少约99.8％或至少约99.9％相同的氨基酸序列。通常，同源物将包含与主题氨基酸序列相同的活性位点等。同源性可以根据相似性(即具有相似化学性质/功能的氨基酸残基)来考虑，也可以在序列同一性方面表达同源性。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
发明详述
间充质干细胞
一方面，本申请提供一种间充质干细胞，其能够表达免疫调控分子，所述免疫调控分子可以包括参与T细胞活化的第一信号和/或第二信号的分子。
在本申请中，所述免疫调控分子可以是外源性的。
在本申请中，所述参与T细胞活化的第一信号的分子可以包括参与抗原识别过程的分子。
在本申请中，所述参与T细胞活化的第一信号的分子可以包括能够结合抗原的分子。
在本申请中，所述能够结合抗原的分子可以包括抗原结合蛋白。
在本申请中，所述抗原结合蛋白可以包括抗体或其片段。例如，所述片段可以包括Fab，Fab’，Fv片段，F(ab’) 2，F(ab) 2，scFv，di-scFv和/或dAb。
在本申请中，所述抗原包括肿瘤相关抗原。在某些实施方式中，所述肿瘤相关抗原包括实体瘤。在某些实施方式中，所述肿瘤相关抗原包括血液瘤。在某些实施方式中，所述抗原包括CLDN18.2。在某些实施方式中，所述CLDN18.2包括人CLDN18.2。在某些实施方式中，所述抗原结合蛋白可以包含anti-CLDN18.2分子。在某些实施方式中，所述anti-CLDN18.2分子包含CLDN18.2抗体或其片段。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含VH中的至少一个CDR，所述VH包含SEQ ID NO:13所示的氨基酸序列。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含VL中的至少一个CDR，所述VL可以包含SEQ ID NO:14所示的氨基酸序列。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含HCDR1，HCDR2和HCDR3，所述HCDR1，HCDR2和HCDR3可以通过任何本领域技术人员已知的CDR划分方式划分SEQ ID NO:13所示的氨基酸序列得到。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含LCDR1，LCDR2和LCDR3，所述LCDR1，LCDR2和LCDR3可以通过任何本领域技术人员已知的CDR划分方式划分SEQ ID NO:14所示的氨基 酸序列得到。在某些实施方式中，所述CLDN18.2抗体或其片段包含VH，所述VH包含SEQ ID NO:13所示的氨基酸序列。在某些实施方式中，所述CLDN18.2抗体或其片段包含VL，所述VL包含SEQ ID NO:14所示的氨基酸序列。
在某些实施方式中，所述anti-CLDN18.2包含scFv，所述scFv包含SEQ ID NO:15所示的氨基酸序列。在某些实施方式中，所述anti-CLDN18.2包含scFv，所述scFv包含SEQ ID NO:16所示的氨基酸序列。
在本申请中，所述参与T细胞活化第一信号的分子可以为外源性分子。
在本申请中，所述参与T细胞活化的第一信号的分子可以包括CD3分子。
在本申请中，所述参与T细胞活化的第一信号的分子可以包括能够结合CD3的分子。在本申请中，所述能够结合CD3的分子可以包括anti-CD3分子。
在某些实施方式中，所述anti-CD3的分子包含抗CD3抗体或其片段。
在某些实施方式中，所述抗CD3抗体或其片段包含VH中的至少一个CDR，所述VH包含SEQ ID NO:2所示的氨基酸序列。在某些实施方式中，所述抗CD3抗体或其片段可以包含HCDR1，HCDR2和HCDR3。在某些实施方式中，所述HCDR1，HCDR2和HCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:2所示的氨基酸序列得到。
在某些实施方式中，所述抗CD3抗体或其片段包含VL中的至少一个CDR，所述VL包含SEQ ID NO:3所示的氨基酸序列。在某些实施方式中，所述抗CD3抗体或其片段可以包含LCDR1，LCDR2和LCDR3。在某些实施方式中，所述LCDR1，LCDR2和LCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:3所示的氨基酸序列得到。
在某些实施方式中，所述抗CD3抗体或其片段包含VH，所述VH包含SEQ ID NO:2所示的氨基酸序列。
在某些实施方式中，所述抗CD3抗体或其片段包含VL，所述VL包含SEQ ID NO:3所示的氨基酸序列。
在某些实施方式中，所述抗CD3抗体或其片段包含VH和VL，所述VH包含SEQ ID NO:2所示的氨基酸序列，且所述VL包含SEQ ID NO:3所示的氨基酸序列。
在本申请中，所述间充质干细胞能够表达参与T细胞活化的第二信号的分子。
在本申请中，所述参与T细胞活化的第二信号的分子可以为外源性分子。
在本申请中，所述第二信号包括共刺激信号。
在某些实施方式中，所述参与T细胞活化的第二信号的分子包括共刺激分子。例如，所述共刺激分子包括选自下述蛋白的多肽：CD28，OX40和/或4-1BB。
在某些实施方式中，所述参与T细胞活化的第二信号的分子包括能够结合共刺激分子的分子。
在本申请中，所述能够结合共刺激分子的分子包括所述共刺激分子的配体。例如，所述共刺激分子的配体可以包括OX-40L和/或4-1BBL。在某些实施方式中，所述OX-40L包含SEQ ID NO:4所示的氨基酸序列。在某些实施方式中，所述4-1BBL包含SEQ ID NO:5所示的氨基酸序列。
在某些实施方式中，所述能够结合共刺激分子的分子可以包括anti-CD28分子。
在某些实施方式中，所述anti-CD28分子可以包含抗CD28抗体或其片段
在本申请中，所述能够结合共刺激分子的分子可以包括抗共刺激分子的抗体。例如，所述抗共刺激分子的抗体可以包括抗CD28抗体或其片段。
在某些实施方式中，所述抗CD28抗体或其片段包含VH中的至少一个CDR，所述VH包含SEQ ID NO:8所示的氨基酸序列。在某些实施方式中，所述抗CD28抗体或其片段可以包含HCDR1，HCDR2和HCDR3。在某些实施方式中，所述HCDR1，HCDR2和HCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:8所示的氨基酸序列得到。
在某些实施方式中，所述抗CD28抗体包含VL中的至少一个CDR，所述VL包含SEQ ID NO:9所示的氨基酸序列。在某些实施方式中，所述抗CD28抗体或其片段可以包含LCDR1，LCDR2和LCDR3。在某些实施方式中，所述LCDR1，LCDR2和LCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:9所示的氨基酸序列得到。
在某些实施方式中，所述抗CD28抗体包含VH，所述VH包含SEQ ID NO:8所示的氨基酸序列。
在某些实施方式中，所述抗CD28抗体包VL，所述VL包含SEQ ID NO:9所示的氨基酸序列。
在某些实施方式中，所述抗CD28抗体包含VH和VL，所述VH包含SEQ ID NO:8所示的氨基酸序列，且所述VL包含SEQ ID NO:9所示的氨基酸序列。
在本申请中，所述间充质干细胞还可以表达参与T细胞活化的第三信号的分子。
在本申请中，所述参与T细胞活化的第三信号的分子可以为外源性分子。
在本申请中，所述T细胞活化的第三信号可以包括细胞因子信号。
在本申请中，所述参与T细胞活化的第三信号的分子可以包括细胞因子。
在本申请中，所述细胞因子可以包括由淋巴细胞产生的细胞因子。
在本申请中，所述细胞因子可以包括由非淋巴细胞产生的细胞因子。
在本申请中，所述细胞因子可以包括白细胞介素。
在某些实施方式中，所述细胞因子可以包括IL-12、IL-2、IL-5、IL7和/或IL-15。
例如，所述细胞因子可以为IL-7。
例如，所述IL-7可以为人IL-7。
例如，所述IL-7可以包含SEQ ID NO:10所示的氨基酸序列。
例如，所述细胞因子可以为IL-2。
例如，所述IL-2可以为人IL-2。
例如，所述IL-2可以包含SEQ ID NO:12所示的氨基酸序列。
在本申请中，所述间充质干细胞还可以包含其他改造。例如，所述改造能够提高间充质干细胞在肿瘤内的扩增能力。
在本申请中，所述能够提高间充质干细胞在肿瘤内的扩增能力的改造包含在间充质干细胞中表达能够提高间充质干细胞在肿瘤内扩增能力的分子。
在某些实施方式中，所述能够提高间充质干细胞在肿瘤内扩增能力的分子包含能够提高间充质干细胞在肿瘤内的扩增能力的酶。
在某些实施方式中，所述能够提高间充质干细胞在肿瘤内扩增能力的分子包括酶。例如，所述酶可以包括还原型辅酶/醌氧化还原酶(NQO1)。例如，所述NQO1可以包含SEQ ID NO:22所示的氨基酸序列。例如，所述酶可以包括5-甲基胞嘧啶双加氧酶(TET1)。例如，所述TET1可以包含SEQ ID NO:23所示的氨基酸序列。
在某些实施方式中，所述间充质干细胞还包含能够使得其在正常组织地表达，在低氧的肿瘤环境中高表达相应的免疫调控分子的改造。例如，可以在启动子中加入低氧响应元件。例如，可以在启动子中加入HRE元件。例如，所述HRE元件可以包含SEQ ID NO:24所示的氨基酸序列。
在本申请中，所述间充质干细胞可以包含自体间充质干细胞。
在本申请中，所述间充质干细胞可以包含异体间充质干细胞。
间充质干细胞和免疫效应细胞联合
另一方面，本申请提供了一种药物组合，所述药物组合包含本申请所述的间充质干细胞和免疫效应细胞。
在本申请中，所述免疫效应细胞可以包含T细胞。例如，所述T细胞可以包含CAR-T，TCR-T，TIL和/或胸水、腹水和/或PBMC来源的T细胞。例如，所述T细胞可以包含CD8+T细胞、CD4+T细胞、γ-δT细胞和/或T调节细胞。
在本申请中，所述免疫效应细胞可以包含经改造的免疫效应细胞。
在本申请中，所述经改造的免疫效应细胞可以接收所述间充质干细胞提供的T细胞活化的第一信号、第二信号和/或第三信号。
在本申请中，所述免疫效应细胞包含免疫调控分子，所述免疫调控分子能够与所述间充质干细胞表达的对应的免疫调控分子相结合，提供T细胞活化的第一信号、第二信号和/或第三信号。例如，所述间充质干细胞表达参与T细胞活化的第一信号的分子，所述T细胞能够表达另一种分子，与所述间充质干细胞表达的相应的分子相结合，接收间充质干细胞提供的第一信号。例如，所述间充质干细胞表达anti-CD3分子，所述免疫效应细胞表达CD3分子。例如，所述免疫效应细胞表达共刺激分子，所述间充质干细胞表达相应的共刺激分子的配体。例如，所述间充质干细胞表达细胞因子，所述免疫效应细胞表达相应的细胞因子受体。
在本申请中，所述免疫细胞包含的免疫调控分子可以为外源分子。
在本申请中，所述免疫效应细胞可以包含与所述间充质干细胞具有相同靶点的抗原结合蛋白。在本申请中，所述靶点可以为本领域任何已知的靶点，例如，肿瘤靶点。例如，所述靶点可以选自间皮素(Mesothelin)，GPC-3(Glypican-3，磷脂酰肌醇蛋白聚糖3)，PSMA(前列腺特异性膜抗原)，EpCAM(上皮细胞粘附分子)和GD2(神经节苷脂)的一种或多种。
例如，所述免疫效应细胞可以包含靶向选自下组的一种或多种靶点的抗原结合蛋白：间皮素，GPC-3，PSMA，EpCAM和GD2。例如，所述靶向间皮素的抗原结合蛋白的抗原结合部分可包含SEQ ID NO:29或SEQ ID NO:30所示的氨基酸序列。例如，所述靶向GPC-3的抗原结合蛋白的抗原结合部分可包含SEQ ID NO:25或26所示的氨基酸序列。例如，所述靶向PSMA的抗原结合蛋白的抗原结合部分可包含SEQ ID NO:31所示的氨基酸序列。例如，所述靶向GD2的抗原结合蛋白的抗原结合部分可包含SEQ ID NO:27或SEQ ID NO:28所示的氨基酸序列。
例如，所述免疫效应细胞可以包含靶向CLDN18.2的抗原结合蛋白，同时，所述间充质干细胞也可以包含靶向CLDN18.2的抗原结合蛋白。在本申请中，所述免疫效应细胞还可以包含与所述间充质干细胞相同的分子。
在本申请中，所述免疫效应细胞包含抗原或能够结合所述抗原的分子。在本申请中，所述免疫效应细胞包含的能够结合抗原的分子能够与所述间充质干细胞表达的能够结合抗原的分子靶向相同的抗原。在某些实施方式中，所述能够结合抗原的分子包含抗原结合蛋白。在某些实施方式中，所述抗原结合蛋白包含抗体或其片段。在某些实施方式中，所述抗体或其片段包含scFv。
在某些实施方式中，所述免疫效应细胞包含的能够结合抗原的分子包含抗CLDN18.2抗体或其片段。
在某些实施方式中，所述抗CLDN18.2抗体或其片段包含VH中的至少一个CDR，所述VH包含SEQ ID NO:13所示的氨基酸序列。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含HCDR1，HCDR2和HCDR3。在某些实施方式中，所述HCDR1，HCDR2和HCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:13所示的氨基酸序列得到。
在某些实施方式中，所述抗CLDN18.2抗体或其片段包含VL中的至少一个CDR，所述VL包含SEQ ID NO:14所示的氨基酸序列。在某些实施方式中，所述CLDN18.2抗体或其片段可以包含LCDR1，LCDR2和LCDR3。在某些实施方式中，所述LCDR1，LCDR2和LCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:14所示的氨基酸序列得到。
在某些实施方式中，所述抗CLDN18.2抗体或其片段包含VH，所述VH包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述抗CLDN18.2抗体或其片段包含VL，所述VL包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述免疫效应细胞可以表达CD3分子。例如，所述CD3分子可以包含SEQ ID NO:1所示的氨基酸序列。
在本申请中，所述免疫效应细胞可以包含共刺激分子或能够结合共刺激分子的配体。
在本申请中，所述免疫效应细胞包含的共刺激分子或能够结合共刺激分子的配体能够与所述间充质干细胞表达的能够结合共刺激分子的配体或共刺激分子相结合。在某些实施方式中，所述共刺激分子选自CD28家族、CD2家族和/或TNFR家族的一种或多种共刺激分子。所述共刺激分子包括选自下述蛋白的多肽：OX40和/或4-1BB。例如，所述OX40可以包含SEQ ID NO:6所示的氨基酸序列。例如，所述4-1BB可以包含SEQ ID NO:7所示的氨基酸序列。所述共刺激分子的配体包括：OX40L和/或4-1BBL。例如，所述OX40L可以包含SEQ ID NO:4所示的氨基酸序列。例如，所述4-1BBL可以包含SEQ ID NO:5所示的氨基酸序列。
在本申请中，所述免疫效应细胞可以包含所述共刺激分子的受体。
在本申请中，所述免疫效应细胞可以包含参与T细胞活化的第三信号的分子。在本申请中，所述T细胞活化的第三信号可以包含细胞因子信号。
在本申请中，所述免疫效应细胞可以包含细胞因子或能够结合细胞因子的分子。
在本申请中，所述细胞因子可以包括由淋巴细胞产生的细胞因子。
在本申请中，所述细胞因子可以包括由非淋巴细胞产生的细胞因子。
在本申请中，所述细胞因子可以包括白细胞介素。
在某些实施方式中，所述细胞因子可以包括IL-12、IL-2、和/或IL7。在某些实施方式中，所述能够结合细胞因子的分子，包括所述细胞因子的受体。例如，所述细胞因子的受体可以包括IL-12R、IL-2R和/或IL7R。例如，所述IL-7R可以包含SEQ ID NO:11所示的氨基酸序列。
在本申请中，所述免疫效应细胞可以包含嵌合抗原受体(CAR)，其中所述CAR可以包含抗原结合结构域、跨膜域、共刺激域和胞内信号传导域。
在某些实施方式中，所述抗原结合结构域包含抗原结合蛋白。在某些实施方式中，所述抗原结合蛋白可以包含抗体或抗原结合片段。例如，所述抗原结合蛋白可以包含scFv。例如，所述scFv可以包含SEQ ID NO:15所示的氨基酸序列。
在某些实施方式中，所述抗原结合结构域包含能够结合CLDN18.2的抗原结合蛋白。在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白包含VH中的至少一个CDR，所述VH包含SEQ ID NO:13所示的氨基酸序列。在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白可以包含HCDR1，HCDR2和HCDR3。在某些实施方式中，所述HCDR1，HCDR2和HCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:13所示的氨基酸序列得到。
在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白包含VL中的至少一个CDR，所述VL包含SEQ ID NO:14所示的氨基酸序列。在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白可以包含LCDR1，LCDR2和LCDR3。在某些实施方式中，所述LCDR1，LCDR2和LCDR3可以通过任何本领域技术人员已知的方式划分SEQ ID NO:14所示的氨基酸序列得到。
在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白包含VH，所述VH包含SEQ ID NO:13所示的氨基酸序列。
在某些实施方式中，所述能够结合CLDN18.2的抗原结合蛋白包含VL，所述VL包含SEQ ID NO:14所示的氨基酸序列。
在本申请中，所述跨膜域可以包含源自选自下述蛋白的跨膜结构域：T细胞受体的α，β或ζ链、CD28、CD3e、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
在本申请中，所述跨膜域可以包含SEQ ID NO:17所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的共刺激域可以包含选自下述蛋白的多肽：CD28、4-1BB、 OX-40和ICOS。在某些实施方式中，所述共刺激域可以包含SEQ ID NO:18所示的氨基酸序列。
在本申请中，所述嵌合抗原受体的胞内信号传导域可以包含源自CD3ζ的信号传导结构域。在某些实施方式中，所述胞内信号传导域可以包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可以包含铰链区。例如，所述铰链区可以包含源自选自下组中的一种或多种蛋白的铰链区：CD28、IgG1、IgG4、IgD、4-1BB、CD4、CD27、CD7、CD8A、PD-1、ICOS、OX40、NKG2D、NKG2C、FcεRIγ、BTLA、GITR、DAP10、TIM1、SLAM、CD30和LIGHT。例如，所述铰链区可以包含SEQ ID NO:20所示的氨基酸序列。
在本申请中，所述嵌合抗原受体还可以包含信号肽。在某些实施方式中，所述信号肽的C端与所述靶向部分的N端连接。
在本申请中，所述免疫效应细胞可以包括人细胞。
在本申请中，所述免疫效应细胞可以包括自体免疫效应细胞。
在本申请中，所述免疫效应细胞可以包括异体免疫效应细胞。
在某些实施方式中，所述嵌合抗原受体包含抗原结合结构域、跨膜域、共刺激域和胞内信号传导域。在某些实施方式中，所述嵌合抗原受体的抗原结合结构域包含SEQ ID NO:15所示的氨基酸序列，所述跨膜域包含SEQ ID NO:17所示的氨基酸序列，所述共刺激域包含SEQ ID NO:18所示的氨基酸序列，且所述胞内信号传导域包含SEQ ID NO:19所示的氨基酸序列。
在本申请中，所述间充质干细胞可以和表达相应分子的免疫效应细胞联用。例如，MSC-4-1BBL和MSC-IL7可以和Ab10Z-4-1BB-IL7R CAR-T细胞联用。例如，MSC-0X40L和MSC-IL7可以和本申请实施例中制备的Ab10Z-OX40-IL7R CAR-T细胞联用。例如，MSC-anti-CD3和MSC-IL-2可以和本申请实施例中制备的CLDN18.2 CAR-T细胞联用。例如，MSC-anti-CD3和MSC-IL-2可以和本申请实施例中制备的CLDN18.2 CAR-T细胞联用。例如，MSC-anti-CD3，MSC-IL-2和MSC-anti-CD28可以和本申请实施例中制备的CLDN18.2 CAR-T细胞联用。
药物组合物、制备方法及用途
另一方面，本申请还提供了一种药物组合物，其包含本申请所述的间充质干细胞，及任选地药学上可接受的载剂。
另一方面，本申请还提供了一种药物组合物，其包含本申请所述的间充质干细胞，以及本申请所述的免疫效应细胞，及任选地药学上可接受的载剂。
在本申请中，所述药物组合物可以被配制用于局部、肠胃外、全身、腔内、静脉内、动脉内、肌肉内、鞘内、眼内、结膜内、瘤内、皮下、皮内、鞘内、口服或透皮给药途径，其可包括注射或输注。合适的制剂可以在无菌或等渗培养基中包含病毒和/或细胞。组合物、药物组合物和试剂盒还可以配制成流体，包括凝胶形式。可配制流体制剂用于通过注射或输注(例如通过导管)施用于人体或动物体的选定区域。
另一方面，本申请所述的间充质干细胞可以和本申请所述的免疫效应细胞可以联合使用。在某些实施方式中，所述间充质干细胞可以和所述免疫效应细胞同时施用。同时施用可以是各组分混合在一起施用，也可以是分开施用。可以以相同方式施用，例如施用于同一条静脉或其他血管，也可以是不同方式施用，例如静脉内施用和瘤内施用同时进行。
在某些实施方式中，所述间充质干细胞可以和所述免疫效应细胞先后施用。施用的顺序可以是先施用所述间充质干细胞，再施用所述免疫效应细胞；也可以是先施用所述免疫效应细胞，再施用所述溶瘤病毒。可以以相同方式施用，也可以以不同方式施用。各组分可以一次施用，也可以分多次施用。在某些实施方案中，先后施用可以是以任何时间间隔施用，包括分钟、小时、天、周、月或年。在某些实施方案中，先后施用是指以至少2分钟、5分钟、10分钟、、30分钟、1小时、6小时、8小时、12小时、24小时、36小时、48小时、3天、4天、5天、6天、1周、2周、3周、1个月、6周、2个月、3个月、4个月、5个月或6个月之一的时间间隔分开的施用。
在某些实施方式中，施用的剂量可以为治疗有效量。
另一方面，本申请还提供了所述间充质干细胞和/或所述免疫效应细胞的制备方法。其中所述间充质干细胞和/或免疫效应细胞的制备方法可以包括合成包含所述参与T细胞活化的第一信号、第二信号和/或第三信号分子的病毒载体、制备病毒，转染细胞后，感染间充质干细胞和/或免疫效应细胞。
另一方面，本申请还提供所述间充质干细胞和/或药物组合在制备药物中的用途，所述药物用于预防和/或治疗疾病和/或病症。
另一方面，本申请还提供了一种治疗疾病和/或病症的方法，其包括向有需要的受试者施用本申请所述的间充质干细胞和/或药物组合。
另一方面，本申请还提供了所述间充质干细胞和/或药物组合，其用于治疗疾病和/或病症。
在本申请中，所述疾病和/或病症可以包括肿瘤。
在某些实施方式中，所述肿瘤包括实体瘤和/或血液瘤。
在某些实施方式中，所述肿瘤包括选自下组的一种或多种：胰腺癌、胃癌、肺癌和前列 腺癌。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请发明的各个技术方案，而不用于限制本申请发明的范围。
实施例
实施例1 抗CLDN18.2 CAR-T细胞(Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R CAR-T细胞)的制备
1.1慢病毒载体pCDH-MSCVEF-Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R的构建及病毒生产
为构建本申请所述的CAR-T细胞，首先制备靶向CLDN18.2的CAR。人工合成以下序列：scFv Ab10(SEQ ID NO:15)，铰链区(SEQ ID NO:20)，跨膜区(SEQ ID NO:17)，4-1BB共刺激因子(SEQ ID NO:18)，CD3ζ胞内信号传导结构域(SEQ ID NO:19)。其中，将铰链区，跨膜区，4-1BB共刺激因子和CD3ζ胞内信号传导结构域首尾相连可获得BBZ，其序列如SEQ ID NO:21所示。
将可特异性结合CLDN18.2的scFv Ab10(氨基酸序列SEQ ID NO:15，核苷酸序列SEQ ID NO:16)和BBZ(SEQ ID NO:21)通过overlap PCR，两端加入XbaI和BamHI酶切位点克隆pCDH-MSCVEF载体。进行PCR扩增，并用延伸PCR在5’端依次带上XbaI酶切位点(含保护碱基)、铰链区、跨膜区、4-1BB共刺激因子、CD3ζ胞内信号传导结构域、BamHI酶切位点，PCR扩增得到所述CAR Ab10BBZ。测序正确的克隆用NucleoBond Xtra Midi Plus EF试剂盒无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol或RSV-REV)共同转染293X细胞，37℃、5％CO 2培养48小时后收取上清，0.45μM过滤后，使用贝克曼超速离心机和SW28转头，以25000RPM的速度离心2小时以浓缩病毒，即为pCDH-MSCVEF-Ab10BBZ病毒(简称为Ab10BBZ病毒)，用于后续CAR-T细胞生产。
同时用与制备Ab10BBZ病毒相同的方法生产pCDH-MSCVEF-Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R病毒(简称Ab10Z-OX40-IL7R和Ab10Z-4-1BB-IL7R病毒)，将所得病毒感染293细胞，使用anti-mouse Fab抗体(Jackson ImmunoResearch#115-605-006)用流式检测的方法测病毒滴度，如图1所示。图1中显示的是在添加1μL、3μL、9μL的所述病毒的时候的流式检测结果，以不添加病毒作为空白对照。
结果显示，随着所加病毒剂量的增加，所述CAR Ab10Z-OX40-IL7R和Ab10Z-4-1BB-IL7R的表达量也随之增加。
1.2 Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒(购自stem cell Catlog#19671纯化后，接种到anti-hCD3(购自Bioxcell#BE0001-2)和anti-hCD28(购自Bioxcell#BE0248)包被的96孔培养板，2天后，按照MOI＝10-20感染本实施例步骤1.1制备的Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R病毒，1天后换液继续细胞培养，培养基为含10％FBS的RPMI完全培养基，IL2(50IU/ml)，IL21(4ng/ml)，按照每6天使用人工抗原呈递细胞(X射线100Gray辐照后的Raji-CLDN18.2细胞)或anti-hCD3(0.1μg/ml)或anti-hCD28(0.25μg/ml)刺激，经过2轮刺激后，所得细胞即为Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R CAR-T细胞，通过流式染色，对Ab10BBZ，Ab10Z-OX40-IL7R，Ab10Z-4-1BB-IL7R CAR-T细胞用Alexa
Figure PCTCN2022102733-appb-000001
647 AffiniPure F(ab') 2Fragment Goat Anti-Mouse IgG,Fab fragment specific二抗染色，其结果如图2所示。
结果显示，所得细胞均为CAR阳性。
实施例2 MSC-CLDN18.2，MSC-CLDN18.2-IL2，MSC-OX40L，MSC-4-1BBL，MSC-IL-2，MSC-anti-CD3，MSC-anti-CD28细胞的制备
2.1.CLDN18.2，CLDN18.2-IL2,OX40L,4-1BBL,IL-2,anti-CD3,anti-CD28病毒的生产
参考实施例1，合成CLDN18.2，CLDN18.2-IL2，OX40L，4-1BBL，IL-2，anti-CD3，anti-CD28，分子克隆到pCDHEF-MSCV-puro载体，制备病毒pCDH-MSCVEF-CLDN18.2，pCDH-MSCVEF-CLDN18.2-IL2,pCDH-MSCVEF-OX40L,pCDH-MSCVEF-4-1BBL,pCDH-MSCVEF-IL-2,pCDH-MSCVEF-anti-CD3,pCDH-MSCVEF-anti-CD28。
利用与实施例1中的方法，转染293细胞获得病毒上清，保存于-80度，用于后续间充质干细胞的感染。
2.2 CLDN18.2，CLDN18.2-IL2,OX40L,4-1BBL,IL-2,IL-7，anti-CD3,anti-CD28间充质干细胞CAR-T细胞的制备
将人脐带来源的间充质干细胞，培养于6孔板，加入实施例2.1中所得的病毒上清，病毒感染增强剂polybrene 10μg/ml，24小时后换新鲜培养基，48小时后加入puromycin筛选获得稳定表达CLDN18.2，CLDN18.2-IL2，OX40L，4-1BBL，IL-2，IL-7，anti-CD3，anti-CD28的间充质干细胞。通过流式染色，用CLDN18.2，OX40L，4-1BBL，或Alexa
Figure PCTCN2022102733-appb-000002
647 AffiniPure F(ab') 2Fragment Goat Anti-Mouse IgG，Fab fragment specific二抗染色检测CLDN18.2，OX40L，4-1BBL，anti-CD3，anti-CD28分子的膜表面表达，通过ELISA和CBA检测IL-2和IL-7的分泌表达。其结果如图3A-F所示。图3A-E显示的是本申请所述MSCs(MSC-CLDN18.2， MSC-CLDN18.2-IL2，MSC-OX40L，MSC-4-1BBL，MSC-IL-2，MSC-anti-CD3，MSC-anti-CD28)的对应T细胞活化分子的表达。图3F显示的是ELISA检测IL-7的分泌表达。
实施例3 MSC-OX40L，MSC-4-1BBL，MSC-IL-7和CLDN18.2 CART联合治疗小鼠肿瘤
将2x10 6CFPAC-1肿瘤细胞皮下接种到B-NDG小鼠，6天后给予5x10 6Ab10Z-OX40-IL7R,Ab10Z-4-1BB-IL7R CAR-T细胞和10 6MSC-OX40L，MSC-4-1BBL和MSC-IL7细胞治疗，测量小鼠的肿瘤负荷，其结果如图4所示。由图可知，Ab10Z-OX40-IL7R/MSC-OX40L/MSC-IL7治疗组和Ab10Z-4-1BB-IL7R/MSC-4-1BBL/MSC-IL7细胞相对于PBS组能够更好的控制肿瘤负荷。结果显示，与对照PBS相比，Ab10Z-OX40-IL7R/MSC-OX40L/MSC-IL7治疗组和Ab10Z-4-1BB-IL7R/MSC-4-1BBL/MSC-IL7治疗使得小鼠肿瘤体积减少了80.7％和80.8％(201.55mm 3到38.75mm 3和38.55mm 3)。
实施例4 MSC-anti-CD3，MSC-IL-2和CLDN18.2 CART联合治疗小鼠肿瘤
将2x10 6CFPAC-1肿瘤细胞皮下接种到B-NDG小鼠，6天后给予5x10 6Ab10BBZ CAR-T细胞和10 6MSC，MSC-anti-CD3，MSC-IL-2细胞治疗，测量小鼠的肿瘤负荷。其结果分别如图5所示。由图可知，MSC-anti-CD3，MSC-IL-2相对对照MSC，能够和Ab10BBZ CAR-T细胞协同作用，发挥更强的抗肿瘤作用。结果显示，与对照Ab10BBZ/MSC相比，Ab10BBZ/MSC-IL2/MSC-anti-CD3细胞治疗使得小鼠肿瘤体积减少了90.8％(5.46mm 3到0.5mm 3)。
实施例5 MSC-anti-CD3，MSC-IL-2和增强CLDN18.2 CART体内增殖能力
参照实施例4，接种肿瘤并进行同样的治疗后，第10天取外周血，流式细胞仪检测其中CAR-T细胞的比例。其结果分别如图6所示。由图可知，MSC-anti-CD3，MSC-IL-2相对对照MSC，能够显著增强Ab10BBZ CAR-T细胞在小鼠体内的增值。如图6所示，与对照MSC相比，MSC-IL2/MSC-anti-CD3细胞增强了Ab10BBZ CAR-T的增值2.32倍，从1.08％增加到2.51％。
实施例6 MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28增强CLDN18.2 CART体内增殖能力
将2x10 6CFPAC-1肿瘤细胞皮下接种到B-NDG小鼠，6天后给予5x10 6Ab10BBZ CAR-T细胞和10 6MSC，MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28，MSC-IL-2-HRE细胞治疗， 测量小鼠的肿瘤负荷。其结果如图7所示。由图可知，MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28相对对照MSC，能够和Ab10BBZ CAR-T细胞协同作用，发挥更强的抗肿瘤作用。结果显示，与对照Ab10BBZ/MSC相比，Ab10BBZ/MSC-IL2，Ab10BBZ/MSC-anti-CD3，Ab10BBZ/MSC-anti-CD28，Ab10BBZ/MSC-IL2-HRE，细胞治疗使得小鼠肿瘤减少了22.7％，32.9％，41.5％，41.7％(1.42mm 3到1.09mm 3，0.95mm 3，0.83mm 3，0.75mm 3)。
实施例7 MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28增强CLDN18.2 CART体内增殖能力
和实施例6同样地接种和治疗小鼠，第10天取外周血，流式细胞仪检测其中CAR-T细胞的比例。其结果分别如图8所示。由图可知，MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28相对对照MSC，能够显著增强Ab10BBZ CAR-T细胞在小鼠体内的增值。如图XX所示，与对照MSC相比，MSC-anti-CD3，MSC-IL-2，MSC-anti-CD28细胞增强了Ab10BBZ CAR-T的增殖。
实施例8 NQO1和TET1增强MSC在肿瘤内富集及持续扩增能力
参考实施例2的方法，合成TET1，NQO1，TET1-2A-NQO1，分子克隆到pCDHEF-MSCV-puro载体，制备病毒pCDH-MSCVEF-TET1，pCDH-MSCVEF-NQO1,pCDH-MSCVEF-TET1-2A-NQO1。
采用实施例2中的方法，转染293细胞获得病毒上清，保存于-80度，用于后续间充质干细胞的感染。将人脐带来源的间充质干细胞，培养于6孔板，加入包含TET1，NQO1，TET1-2A-NQO1的病毒上清，病毒感染增强剂polybrene 10μg/ml，24小时后换新鲜培养基，48小时后加入puromycin筛选获得稳定表达TET1，NQO1，TET1-2A-NQO1的间充质干细胞。通过q-PCR，检测TET1，NQO1的mRNA表达。其结果如图9所示。
实施例9 HRE启动子选择性增强IL-2在低氧条件下的表达
参考实施例2的方法，合成IL2分子克隆到pCDH-HRE-puro载体，制备病毒pCDH-HRE-IL2。
参考实施例2的方法，转染293细胞获得病毒上清，保存于-80度，用于后续间充质干细胞的感染。将人脐带来源的间充质干细胞，培养于6孔板，加入包含HRE-IL2的病毒上清，病毒感染增强剂polybrene 10μg/ml，24小时后换新鲜培养基，48小时后加入puromycin筛选获得稳定表达HRE-IL2的间充质干细胞。分别在正常培养和低氧诱导条件下检测IL2的表达。 其结果如图10所示。结果显示，与常氧组相比，IL-2的表达在低氧条件下显著增强。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方式的范围内。

Claims (82)

  1. 间充质干细胞，其表达免疫调控分子，所述免疫调控分子包括参与T细胞活化的第一信号和/或第二信号的分子。
  2. 根据权利要求1所述的间充质干细胞，其中所述参与T细胞活化的第一信号的分子包括参与抗原识别过程的分子。
  3. 根据权利要求1-2中任一项所述的间充质干细胞，其中所述参与T细胞活化的第一信号的分子包括能够结合抗原的分子。
  4. 根据权利要求3所述的间充质干细胞，其中所述能够结合抗原的分子包括抗原结合蛋白。
  5. 根据权利要求3-4中任一项所述的间充质干细胞，其中所述能够结合抗原的分子包含抗体或其片段。
  6. 根据权利要求3-5中任一项所述的间充质干细胞，其中所述抗原包括肿瘤相关抗原。
  7. 根据权利要求6所述的间充质干细胞，其中所述肿瘤包括实体瘤和/或血液瘤。
  8. 根据权利要求3-7中任一项所述的间充质干细胞，其中所述抗原包括CLDN18.2。
  9. 根据权利要求1-8中任一项所述的间充质干细胞，其中所述参与T细胞活化的第一信号的分子包括能够结合CD3的分子。
  10. 根据权利要求9所述的间充质干细胞，其中所述CD3分子包含CD3的γ链、δ链、ε链、ζ链和/或η链。
  11. 根据权利要求9-10中任一项所述的间充质干细胞，其中所述能够结合CD3的分子包含anti-CD3分子。
  12. 根据权利要求11所述的间充质干细胞，其中所述anti-CD3分子包含抗CD3抗体或其片段。
  13. 根据权利要求1-12中任一项所述的间充质干细胞，其中所述T细胞活化的第二信号包括共刺激信号。
  14. 根据权利要求1-13中任一项所述的间充质干细胞，其中所述参与T细胞活化的第二信号的分子包括共刺激分子或能够结合共刺激分子的分子。
  15. 根据权利要求14所述的间充质干细胞，其中所述共刺激分子包括选自CD28家族、CD2家族和/或TNFR家族的一个或多个共刺激分子。
  16. 根据权利要求14-15中任一项所述的间充质干细胞，其中所述共刺激分子包括选自下述蛋白的多肽：CD28、OX40和/或4-1BB。
  17. 根据权利要求14-16中任一项所述的间充质干细胞，其中所述能够结合共刺激分子的分子包括所述共刺激分子的配体。
  18. 根据权利要求17所述的间充质干细胞，其中所述能够结合共刺激分子的配体包括：OX40L和/或4-1BBL。
  19. 根据权利要求14-18中任一项所述的间充质干细胞，其中所述能够结合共刺激分子的分子包括所述共刺激分子的受体。
  20. 根据权利要求14-19中任一项所述的间充质干细胞，其中所述能够结合共刺激分子的分子包括anti-CD28分子。
  21. 根据权利要求20所述的间充质干细胞，其中所述anti-CD28分子包含抗CD28的抗体或其片段。
  22. 根据权利要求14-21中任一项所述的间充质干细胞，其中所述能够结合共刺激分子的分子包括抗共刺激分子的抗体或其片段。
  23. 根据权利要求1-22中任一项所述的间充质干细胞，其还表达参与T细胞活化的第三信号的分子。
  24. 根据权利要求23所述的间充质干细胞，其中所述T细胞活化的第三信号包括细胞因子信号。
  25. 根据权利要求23-24中任一项所述的间充质干细胞，其中所述参与T细胞活化的第三信号的分子包括细胞因子和/或能够结合所述细胞因子的分子。
  26. 根据权利要求25所述的间充质干细胞，其中所述能够结合所述细胞因子的分子包括所述细胞因子的受体。
  27. 根据权利要求25-26中任一项所述的间充质干细胞，其中所述细胞因子包括IL-12、IL-2、IL-5、IL7和/或IL-15。
  28. 根据权利要求25-27中任一项所述的间充质干细胞，其中所述细胞因子包含IL-7。
  29. 根据权利要求28所述的间充质干细胞，其中所述细胞因子包含SEQ ID NO:10所示的氨基酸序列。
  30. 根据权利要求25-29中任一项所述的间充质干细胞，其中所述细胞因子包含IL-2。
  31. 根据权利要求30所述的间充质干细胞，其中所述细胞因子包含SEQ ID NO:12所示的氨基酸序列。
  32. 根据权利要求1-31中任一项所述的间充质干细胞，其还包含能够提高间充质干细胞在肿瘤内的扩增能力的分子。
  33. 根据权利要求32所述的间充质干细胞，其中所述能够提高间充质干细胞在肿瘤内扩增能力的分子包括酶。
  34. 根据权利要求32-33中任一项所述的间充质干细胞，其中所述与间充质干细胞在肿瘤细 胞内扩增相关的分子包括还原型辅酶/醌氧化还原酶(NQO1)。
  35. 根据权利要求32-34中任一项所述的间充质干细胞，其中所述与间充质干细胞在肿瘤细胞内扩增相关的酶包括5-甲基胞嘧啶双加氧酶(TET1)。
  36. 根据权利要求1-35中任一项所述的间充质干细胞，其包含与启动子可操作地连接的核酸，所述启动子包含低氧响应元件(HRE)。
  37. 根据权利要求1-36中任一项所述的间充质干细胞，其为异体间充质干细胞。
  38. 根据权利要求1-37中任一项所述的间充质干细胞，其为自体间充质干细胞。
  39. 药物组合，其包含权利要求1-38中任一项所述的间充质干细胞和免疫效应细胞。
  40. 根据权利要求39所述的药物组合，其中所述免疫效应细胞包含T细胞。
  41. 根据权利要求40所述的药物组合，其中所述T细胞包含CAR-T，TCR-T，TIL和/或胸水、腹水和/或PBMC来源的T细胞。
  42. 根据权利要求40-41中任一项所述的药物组合，其中所述T细胞包含CD8+T细胞、CD4+T细胞、γ-δT细胞和/或T调节细胞。
  43. 根据权利要求39-42中任一项所述的药物组合，其中所述免疫效应细胞包含免疫调控分子，所述免疫调控分子能够与所述间充质干细胞表达的对应的免疫调控分子相结合，提供T细胞活化的第一信号、第二信号和/或第三信号。
  44. 根据权利要求39-43中任一项所述的药物组合，其中所述免疫效应细胞包含参与T细胞活化的第一信号的分子。
  45. 根据权利要求44所述的药物组合，其中所述参与T细胞活化的第一信号的分子包含参与抗原识别过程的分子。
  46. 根据权利要求39-45中任一项所述的药物组合，其中所述免疫效应细胞包含能够结合所述抗原的分子。
  47. 根据权利要求46所述的药物组合，其中所述免疫效应细胞包含的能够结合抗原的分子与所述间充质干细胞表达的能够结合抗原的分子所靶向的抗原相同。
  48. 根据权利要求46-47中任一项所述的药物组合，其中所述能够结合抗原的分子包含抗原结合蛋白。
  49. 根据权利要求48所述的药物组合，其中所述抗原结合蛋白包含抗体或其片段。
  50. 根据权利要求49所述的药物组合，其中所述抗体或其片段包含scFv。
  51. 根据权利要求39-50中任一项所述的药物组合，其中所述免疫效应细胞包含抗CLDN18.2抗体或其片段。
  52. 根据权利要求39-51中任一项所述的药物组合，其中所述免疫效应细胞包含CD3分子。
  53. 根据权利要求52所述的药物组合，其中所述CD3分子包含SEQ ID NO:1所示的氨基酸序列。
  54. 根据权利要求39-53中任一项所述的药物组合，其中所述免疫效应细胞包含共刺激分子或能够结合共刺激分子的配体。
  55. 根据权利要求54所述的药物组合，其中所述免疫效应细胞包含的共刺激分子或能够结合共刺激分子的配体能够与所述间充质干细胞中包含的共刺激分子的配体或共刺激分子相结合。
  56. 根据权利要求54-55中任一项所述的药物组合，其中所述共刺激分子包括选自CD28家族、CD2家族和/或TNFR家族的一个或多个共刺激分子。
  57. 根据权利要求56所述的药物组合，其中所述共刺激分子包括选自下述蛋白的多肽：OX40和/或4-1BB。
  58. 根据权利要求54-57中任一项所述的药物组合，其中所述共刺激分子包含SEQ ID NO:6和/或SEQ ID NO:7所示的氨基酸序列。
  59. 根据权利要求39-58中任一项所述的药物组合，其中所述免疫效应细胞包含参与T细胞活化的第三信号的分子。
  60. 根据权利要求59所述的药物组合，其中所述T细胞活化的第三信号包含细胞因子信号。
  61. 根据权利要求39-60中任一项所述的药物组合，其中所述免疫效应细胞包含细胞因子或能够结合细胞因子的分子。
  62. 根据权利要求61所述的药物组合，其中所述能够结合细胞因子的分子包含所述细胞因子的受体。
  63. 根据权利要求60-62中任一项所述的药物组合，其中所述免疫效应细胞表达的细胞因子或能够结合细胞因子的受体能够与所述间充质干细胞表达的细胞因子或能够结合细胞因子的受体相结合。
  64. 根据权利要求61-63中任一项所述的药物组合，其中所述细胞因子包括IL-12、IL-2、和/或IL7。
  65. 根据权利要求64所述的药物组合，其中所述细胞因子包含SEQ ID NO:12和/或SEQ ID NO:10所示的氨基酸序列。
  66. 根据权利要求62-65中任一项所述的药物组合，其中所述能够结合细胞因子的受体包括IL-12R、IL-2R和/或IL7R。
  67. 根据权利要求62-66中任一项所述的药物组合，其中所述能够结合细胞因子的受体包含 SEQ ID NO:11所示的氨基酸序列。
  68. 根据权利要求39-67中任一项所述的药物组合，其中所述免疫效应细胞包含嵌合抗原受体(CAR)，其中所述嵌合抗原受体包含抗原结合结构域、跨膜域、共刺激域和胞内信号传导域。
  69. 根据权利要求68所述的药物组合，其中所述抗原结合结构域包含能够特异性结合CLDN18.2的抗原结合蛋白。
  70. 根据权利要求69所述的药物组合，其中所述抗原结合蛋白包含SEQ ID NO:15所示的氨基酸序列。
  71. 根据权利要求68-70中任一项所述的药物组合，其中所述跨膜域包含SEQ ID NO:17所示的氨基酸序列。
  72. 根据权利要求68-71中任一项所述的药物组合，其中所述共刺激域包含选自下述蛋白的多肽：OX40和/或4-1BB。
  73. 根据权利要求72所述的药物组合，其中所述共刺激域包含SEQ ID NO:18所示的氨基酸序列。
  74. 根据权利要求68-73中任一项所述的药物组合，其中所述胞内信号传导域包含源自CD3ζ的信号传导结构域。
  75. 根据权利要求74所述的药物组合，其中所述胞内信号传导域包含SEQ ID NO:19所示的氨基酸序列。
  76. 根据权利要求68-75中任一项所述的药物组合，其中所述CAR还包含铰链区。
  77. 根据权利要求76所述的药物组合，其中所述铰链区包含SEQ ID NO:20所示的氨基酸序列。
  78. 药物组合物，其包含权利要求1-38中任一项所述的间充质干细胞或权利要求39-77中任一项所述的药物组合，以及任选地药学上可接受的载剂。
  79. 制备权利要求1-38中任一项所述的间充质干细胞或权利要求39-77中任一项所述的药物组合的方法。
  80. 根据权利要求1-38中任一项所述的间充质干细胞或权利要求39-77中任一项所述的药物组合在制备药物中的用途，所述药物用于预防和/或治疗肿瘤。
  81. 根据权利要求80所述的用途，其中所述肿瘤包括实体瘤。
  82. 根据权利要求80-81中任一项所述的用途，其中所述肿瘤包括选自下组的一种或多种：胰腺癌、胃癌、肺癌和前列腺癌。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106659742A (zh) * 2014-08-18 2017-05-10 埃普塞斯有限责任两合公司 表达免疫应答刺激细胞因子以吸引和/或激活免疫细胞的基因修饰间充质干细胞
WO2019242505A1 (zh) * 2018-06-17 2019-12-26 上海健信生物医药科技有限公司 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
CN110615843A (zh) * 2018-06-20 2019-12-27 上海隆耀生物科技有限公司 一种包含第三信号受体的嵌合抗原受体及其应用
CN111386123A (zh) * 2017-11-22 2020-07-07 布里格姆及妇女医院股份有限公司 Msc表达的免疫调节剂与car-t结合用于癌症治疗

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106659742A (zh) * 2014-08-18 2017-05-10 埃普塞斯有限责任两合公司 表达免疫应答刺激细胞因子以吸引和/或激活免疫细胞的基因修饰间充质干细胞
CN111386123A (zh) * 2017-11-22 2020-07-07 布里格姆及妇女医院股份有限公司 Msc表达的免疫调节剂与car-t结合用于癌症治疗
WO2019242505A1 (zh) * 2018-06-17 2019-12-26 上海健信生物医药科技有限公司 靶向cldn18.2的抗体、双特异性抗体、adc和car及其应用
CN110615843A (zh) * 2018-06-20 2019-12-27 上海隆耀生物科技有限公司 一种包含第三信号受体的嵌合抗原受体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALIJANI, N. ET AL.: "A review on transcriptional regulation responses to hypoxia in mesenchymal stem cells", CELL BIOLOGY INTERNATIONAL, vol. 44, 28 August 2019 (2019-08-28), XP071983371, DOI: 10.1002/cbin.11211 *
HOMBACH ANDREAS A., GEUMANN ULF, GÜNTHER CHRISTINE, HERMANN FELIX G., ABKEN HINRICH: "IL7-IL12 Engineered Mesenchymal Stem Cells (MSCs) Improve A CAR T Cell Attack Against Colorectal Cancer Cells", CELLS, vol. 9, no. 4, 3 March 2020 (2020-03-03), pages 873, XP093017839, DOI: 10.3390/cells9040873 *
MAHAIRA LOUISA G., KATSARA OLGA, PAPPOU EFTHIMIA, ILIOPOULOU ELENI G., FORTIS SOTIRIOS, ANTSAKLIS ARISTIDIS, FOTINOPOULOS PANAGIOT: "IGF2BP1 Expression in Human Mesenchymal Stem Cells Significantly Affects Their Proliferation and Is Under the Epigenetic Control of TET1/2 Demethylases", STEM CELLS AND DEVELOPMENT, MARY ANN LIEBERT, INC. PUBLISHERS, US, vol. 23, no. 20, 15 October 2014 (2014-10-15), US , pages 2501 - 2512, XP093017838, ISSN: 1547-3287, DOI: 10.1089/scd.2013.0604 *

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