WO2023274355A1 - Combinaison de cellules souches mésenchymateuses modifiées et de cellules effectrices immunitaires pour le traitement de tumeurs - Google Patents

Combinaison de cellules souches mésenchymateuses modifiées et de cellules effectrices immunitaires pour le traitement de tumeurs 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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Chinese (zh)
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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

La présente invention concerne une cellule souche mésenchymateuse (MSC) modifiée, et une méthode de traitement des tumeurs par combinaison de MSC avec des cellules T anti-tumorales. Les MSC fournissent un premier signal, un deuxième signal et un troisième signal pour l'activation des cellules T et/ou un signal d'activation des cellules présentant un antigène ; et les cellules T peuvent être des cellules CAR-T, TCR-T, TIL, et des cellules T dérivées du liquide pleural, des ascites et des PBMC.
PCT/CN2022/102733 2021-07-02 2022-06-30 Combinaison de cellules souches mésenchymateuses modifiées et de cellules effectrices immunitaires pour le traitement de tumeurs WO2023274355A1 (fr)

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