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WO2024245291A1 - Préparation et utilisation d'une structure car-t activée par métalloprotéinase - Google Patents

Préparation et utilisation d'une structure car-t activée par métalloprotéinase Download PDF

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Publication number
WO2024245291A1
WO2024245291A1 PCT/CN2024/096088 CN2024096088W WO2024245291A1 WO 2024245291 A1 WO2024245291 A1 WO 2024245291A1 CN 2024096088 W CN2024096088 W CN 2024096088W WO 2024245291 A1 WO2024245291 A1 WO 2024245291A1
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cells
car
tumor
cell
chimeric antigen
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程振国
王尧河
王丽虹
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Zhengzhou University
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Zhengzhou University
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the technical field of genetic engineering and molecular immunology, and in particular to the preparation and application of a metalloproteinase activated CAR-T structure.
  • Tumor cells can evade T cell recognition and attack due to loss/modulation of surface antigens, loss or reduced expression of major histocompatibility complex (MHC) class I molecules, resulting in immune escape.
  • Chimeric antigen receptor T cells, or CAR-T are a new type of cell therapy product that specifically recognizes and kills tumor cells after genetic engineering.
  • the antigen recognition mediated by CAR-T is not restricted by MHC and has become one of the most promising treatments for malignant tumors.
  • the classic CAR structure includes a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
  • CAR-T cells targeting CD19 and CD22 have been widely used in the treatment of hematological tumors such as B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, and have achieved significant therapeutic effects.
  • CAR-T cell products have been developed for the treatment of malignant solid tumors, and some have entered the clinical trial stage.
  • CAR-T cell therapy has become the most promising biological treatment, the toxic side effects of CAR-T therapy, such as off-target effects of CAR molecules, cytokine release syndrome, and CAR-T-related encephalopathy syndrome, have not yet been resolved. Therefore, while improving the efficacy of CAR-T, it is also crucial to further improve the safety of CAR-T cell products.
  • the purpose of the present invention is to provide a conditionally activated chimeric antigen receptor (CAR) and engineered immune cells.
  • CAR conditionally activated chimeric antigen receptor
  • Another object of the present invention is to provide a preparation method and application of conditionally activated chimeric antigen receptor (CAR) and engineered immune cells.
  • CAR conditionally activated chimeric antigen receptor
  • Another object of the present invention is to provide an immunotherapy that is less likely to go off-target, has lower toxic side effects, and is safer.
  • conditionally activated chimeric antigen receptor comprises:
  • the TSAR regulatory elements include: serum albumin binding peptide and matrix metalloproteinase cleavage sequence;
  • the antigen recognition domain targets tumor antigens.
  • the TSAR regulatory element has a nucleotide sequence selected from the following group:
  • nucleotide sequence having 80% or more homology to the sequence shown in SEQ ID NO:1 preferably having at least 85%, 90%, 95%, 99% or 100% homology
  • the TSAR regulatory element has a nucleotide sequence as shown in SEQ ID NO:1.
  • the TSAR regulatory element has an amino acid sequence selected from the group consisting of:
  • the TSAR regulatory element has an amino acid sequence as shown in SEQ ID NO:2.
  • the serum albumin binding peptide is a human serum albumin binding peptide.
  • the serum albumin binding peptide has a nucleotide sequence selected from the following group:
  • nucleotide sequence having 80% or more homology to the sequence shown in SEQ ID NO:3 preferably having at least 85%, 90%, 95%, 99% or 100% homology
  • the serum albumin binding peptide has a nucleotide sequence as shown in SEQ ID NO:3.
  • the serum albumin binding peptide has an amino acid sequence selected from the following group:
  • the serum albumin binding peptide has an amino acid sequence as shown in SEQ ID NO:4.
  • the matrix metalloproteinase cleavage sequence is a human matrix metalloproteinase cleavage sequence.
  • the matrix metalloproteinase cleavage sequence has a nucleotide sequence selected from the following group:
  • nucleotide sequence having 80% or more homology to the sequence shown in SEQ ID NO:5 (preferably having at least 85%, 90%, 95%, 99% or 100% homology); or
  • the matrix metalloproteinase cleavage sequence has a nucleotide sequence as shown in SEQ ID NO:5.
  • the matrix metalloproteinase cleavage sequence has an amino acid sequence selected from the group consisting of:
  • the matrix metalloproteinase cleavage sequence has an amino acid sequence as shown in SEQ ID NO:6.
  • the matrix metalloproteinase cleavage sequence is a site recognized and cleaved by matrix metalloproteinase (MMP).
  • the matrix metalloproteinase is selected from the following group: MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP22, MMP23, MMP24, MMP25, MMP26, or a combination thereof.
  • the matrix metalloproteinase is selected from the following group: MMP1, MMP3, MMP10, MMP12, MMP13, or a combination thereof.
  • the matrix metalloproteinase is MMP1.
  • conditionally activated chimeric antigen receptor has an amino acid sequence selected from the group consisting of List:
  • conditionally activated chimeric antigen receptor has an amino acid sequence as shown in SEQ ID NO:8.
  • the tumor antigen includes but is not limited to the following group:
  • PSMA PSMA, RANK, RORl, TNFRSF4, CD40, CD137, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, TCRa, TCRp, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, CD79b, Notch-1-4, or combination thereof.
  • the tumor antigen is B7-H3.
  • the tumor includes but is not limited to the following group:
  • Esophageal malignancy lung malignancy, gastric malignancy, skin malignancy, colorectal malignancy, liver malignancy, pancreatic malignancy, breast malignancy, ovarian malignancy, glioma, head and neck tumor, or a combination thereof.
  • the tumor is an esophageal malignant tumor (esophageal cancer).
  • the tumor is gastric cancer.
  • the tumor is colorectal cancer.
  • the tumor is liver cancer.
  • the tumor is pancreatic cancer.
  • the tumor is melanoma.
  • the tumor is lung cancer.
  • the tumor is ovarian cancer.
  • the tumor is breast cancer.
  • the tumor is a glioma.
  • the tumor is thyroid cancer.
  • the tumor is bladder cancer, prostate cancer, testicular cancer, cervical cancer, uterine cancer Endometrial cancer, or skin cancer.
  • the tumor includes diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, brain low-grade glioma, lung squamous cell carcinoma, gastric adenocarcinoma, testicular germ cell tumor, thymoma, uterine carcinosarcoma, or a combination thereof.
  • conditionally activated chimeric antigen receptor has a structure shown in Formula I: SP-TSAR-Binder-H-TM-C-CD3 ⁇ (I)
  • SP is none or signal peptide sequence
  • TSAR is the TSAR regulatory element
  • Binder is an antigen recognition domain that specifically binds to tumor antigens
  • H is no or hinge region
  • TM is the transmembrane domain
  • C is the co-stimulatory signaling domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ (including wild type or mutant/modified form thereof);
  • the "-" is a connecting peptide or a peptide bond.
  • the SP is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modified forms thereof, or a combination thereof.
  • the Binder is a B7-H3 single-chain antibody (B7-H3 scFv).
  • the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.
  • the H is the CD8 hinge region.
  • the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or their mutants/modified forms, or their combinations.
  • the TM is the CD8 transmembrane region.
  • the C is selected from the co-stimulatory signal domain of the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or its mutant/modified form, or a combination thereof.
  • proteins OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or its mutant/modified form, or a combination thereof.
  • the C is a CD137 (4-1BB) co-stimulatory signal domain, a CD28 co-stimulatory signal domain, or a combination thereof.
  • nucleic acid molecule encodes the conditionally activated chimeric antigen receptor as described in the first aspect of the present invention.
  • the nucleic acid molecule has a nucleotide sequence selected from the following group:
  • nucleotide sequence having 80% or more homology to the sequence shown in SEQ ID NO:7 preferably having at least 85%, 90%, 95%, 99% or 100% homology
  • the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:7.
  • a vector is provided, wherein the vector contains the nucleic acid molecule as described in the second aspect of the present invention.
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.
  • the vector is a lentiviral vector.
  • the vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.
  • the vector further includes a member selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.
  • a host cell in the fourth aspect of the present invention, contains the vector as described in the third aspect of the present invention, or an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into its chromosome, or the host cell expresses the conditionally activated chimeric antigen receptor as described in the first aspect of the present invention.
  • an engineered immune cell wherein the engineered immune cell contains the vector as described in the third aspect of the present invention, or an exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into its chromosome, or it expresses the conditionally activated chimeric antigen receptor as described in the first aspect of the present invention, and the engineered immune cell is a T cell, a NK cell, a NKT cell, or a macrophage.
  • the engineered immune cells include autologous or allogeneic ⁇ T cells, ⁇ T cells, NKT cells, NK cells, or a combination thereof.
  • the engineered immune cells are CAR-T cells or CAR-NK cells.
  • a method for preparing the engineered immune cells as described in the fifth aspect of the present invention comprising the following steps: transducing the nucleic acid molecule as described in the second aspect of the present invention or the vector as described in the third aspect of the present invention into the immune cells, thereby obtaining the engineered immune cells.
  • the method further comprises the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a pharmaceutical composition comprising:
  • (Z2) a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is in the form of an injection.
  • the concentration of the engineered immune cells in the pharmaceutical composition is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the eighth aspect of the present invention there is provided a use of the conditionally activated chimeric antigen receptor as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, and/or the engineered immune cell as described in the fifth aspect of the present invention for preparing a drug or preparation for preventing and/or treating cancer or tumors.
  • the tumor is a tumor that highly expresses matrix metalloproteinases.
  • the tumor includes but is not limited to the following group:
  • Esophageal malignancy lung malignancy, gastric malignancy, skin malignancy, colorectal malignancy, liver malignancy, pancreatic malignancy, breast malignancy, ovarian malignancy, glioma, head and neck tumor, or a combination thereof.
  • the tumor is an esophageal malignant tumor (esophageal cancer).
  • the tumor is gastric cancer.
  • the tumor is colorectal cancer.
  • the tumor is liver cancer.
  • the tumor is pancreatic cancer.
  • the tumor is melanoma.
  • the tumor is lung cancer.
  • the tumor is ovarian cancer.
  • the tumor is breast cancer.
  • the tumor is a glioma.
  • the tumor is thyroid cancer.
  • the tumor is bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, or skin cancer.
  • the tumors include diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, brain low-grade glioma, lung squamous cell carcinoma, gastric Adenocarcinoma, testicular germ cell tumor, thymoma, uterine carcinosarcoma, or a combination thereof.
  • the ninth aspect of the present invention there is provided a use of the engineered immune cell as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention, for preventing and/or treating cancer or tumors.
  • a method for modifying a chimeric antigen receptor comprising the steps of:
  • kits for preparing the engineered immune cells as described in the fifth aspect of the present invention comprising a container, and a nucleic acid molecule as described in the second aspect of the present invention, or a vector as described in the third aspect of the present invention, located in the container.
  • a method for treating cancer or tumors comprising administering an effective amount of the engineered immune cells as described in the fifth aspect of the present invention, or the pharmaceutical composition as described in the seventh aspect of the present invention to a subject in need of treatment.
  • the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • Figure 1 shows a schematic diagram of the TSAR.CAR molecular structure.
  • FIG2 shows the anti-tumor pattern of TSAR.CAR.
  • FIG3 shows flow cytometry analysis of B7-H3 expression levels on the surface of malignant esophageal tumor cell lines.
  • Figure 4 shows the Western Blot detection of B7-H3 expression levels in malignant esophageal tumor cell lines.
  • FIG5 shows the protein expression level of B7-H3 in different tissues analyzed by protein database.
  • FIG6 shows the analysis of the RNA expression level of B7-H3 in different tumor tissues using the GEPIA database.
  • Figure 7 shows the protein expression level of B7-H3 in different tumor tissues analyzed by protein database (HPA00928 antibody).
  • FIG8 shows the RNA expression levels of different MMPs in 120 pairs of esophageal squamous cell carcinoma tissues.
  • Figure 9 shows the Western blot detection of the expression of MMP1 and MMP11 in 10 esophageal squamous cell carcinoma cell lines.
  • Figure 10 shows the expression levels of KYSE150, KYSE150-hMMP1 full length, and KYSE150-hMMP1 activated length detected by Western blot.
  • FIG. 11 shows flow cytometric analysis to detect the binding of HSA to HSABP.
  • FIG. 12 shows flow cytometric analysis of the cleavage of B7-H3.TSAR molecules by MMP1 treatment.
  • Figure 13 shows the difference in the killing ability of B7-H3.TSAR.CAR-T and B7-H3.CAR-T against KYSE150 and KYSE150 overexpressing human MMP1 analyzed by in vitro cell experiments.
  • Figure 14 shows an in vivo animal experiment comparing the killing abilities of B7-H3.TSAR.CAR-T and B7-H3.CAR-T.
  • Figure 15 shows the in vitro experimental analysis of the anti-tumor activity of B7-H3.TSAR.CAR-T against colon cancer, liver cancer, gastric cancer, pancreatic cancer, lung cancer, melanoma, ovarian cancer, and breast cancer cell lines.
  • TSAR.CAR Tuour-Selectively Activating Reaction CAR
  • TSAR regulatory element a conditionally activated regulatory element
  • HABP human serum albumin binding peptide
  • MMP human matrix metalloproteinase
  • the CAR and engineered immune cells of the present invention utilize the characteristics of high expression of matrix metalloproteinases in the tumor microenvironment to achieve conditional activation based on matrix metalloproteinases, thereby reducing the toxic and side effects caused by off-target effects during immunotherapy, and improving the safety of engineered immune cell products.
  • the present invention was completed on this basis.
  • administering refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.
  • the terms “comprise” or “include” may be open-ended, semi-closed, In other words, the term also includes “consisting essentially of” or “consisting of.”
  • Transduction refers to the process of transferring exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to introduce exogenous polynucleotides into host cells.
  • Gene expression or “expression” refers to the process of gene transcription, translation and post-translational modification to produce the RNA or protein product of the gene.
  • the potential substituted amino acids are within one or more of the following groups: glycine, alanine; and valine, isoleucine, leucine and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine lysine, arginine and histidine; and/or phenylalanine, tryptophan and tyrosine; methionine and cysteine.
  • the present invention also provides non-conservative amino acid substitutions that allow amino acid substitutions from different groups.
  • the synNotch system first recognizes the first target antigen through a synthetic Notch receptor, and after activation, it can induce the expression of CAR molecules targeting the second target antigen.
  • the dual-receptor CAR molecule recognizes two target antigens on the cell surface at the same time to activate CAR-T cells.
  • the CAR and engineered immune cells of the present invention utilize the high expression of matrix metalloproteinases in the tumor microenvironment to achieve conditional activation based on metalloproteinases, thereby reducing toxic and side effects caused by off-target effects during immunotherapy and improving the safety of engineered immune cell products.
  • conditionally activated CAR molecule of the present invention contains a TSAR (tumor-selectively activating reaction) regulatory element.
  • TSAR tumor-selectively activating reaction
  • the TSAR regulatory element of the present invention has a nucleotide sequence as shown in SEQ ID NO: 1 and an amino acid sequence as shown in SEQ ID NO: 2.
  • the TSAR regulatory element consists of two parts: a serum albumin binding peptide and a matrix metalloproteinase cleavage sequence.
  • the serum albumin binding peptide of the present invention is a human serum albumin binding peptide (human serum albumin binding peptide, HSABP);
  • the matrix metalloproteinase cleavage sequence (matrix metalloproteinase cleavage sequence, MMPCS) of the present invention is a human matrix metalloproteinase cleavage sequence.
  • the serum albumin binding peptide can bind to serum albumin (e.g., human serum albumin (HSA)), and interfere with the binding of the single-chain antibody of the CAR molecule to the tumor cell surface antigen through steric hindrance, so that the genetically engineered cells such as CAR-T cells are in a low/inactivated state.
  • serum albumin e.g., human serum albumin (HSA)
  • the serum albumin binding peptide of the present invention preferably has a nucleotide sequence as shown in SEQ ID NO:3 and an amino acid sequence as shown in SEQ ID NO:4.
  • the matrix metalloproteinase cleavage sequence can be specifically cleaved by matrix metalloproteinases (MMPs), thereby relieving the steric hindrance effect mediated by HSABP and activating genetically engineered cells such as CAR-T cells.
  • MMPs matrix metalloproteinases
  • the matrix metalloproteinase cleavage sequence of the present invention preferably has a nucleotide sequence as shown in SEQ ID NO: 5 and an amino acid sequence as shown in SEQ ID NO: 6.
  • MMPs Matrix metalloproteinases
  • MPCS matrix metalloproteinase cleavage sequences
  • the tumor microenvironment is the internal environment in which tumor cells grow, and it is also an important reason for the poor effect of CAR-T cells in treating malignant solid tumors.
  • the tumor microenvironment is mainly composed of tumor cells and their surrounding immune cells, inflammatory cells, nearby interstitial tissues, blood vessels, and various cytokines, chemokines, and metalloproteinases (MMPs).
  • MMPs are a family of zinc- and calcium-dependent proteases that can degrade the extracellular matrix and basement membrane to promote the invasion and metastasis of tumor cells.
  • MMPs are widely present in the tumor microenvironment of lung cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, rectal cancer, bladder cancer, etc., and their increased expression is closely related to the poor prognosis of patients.
  • Esophageal malignant tumor tissues highly express MMP1, MMP3, MMP10, MMP12, and MMP13. Among them, the high expression of MMP1 can not only promote tumor cell proliferation, but also has a direct relationship with the poor effect of neoadjuvant chemotherapy. Patients with malignant esophageal cancer with high expression of MMP1 have a poor prognosis.
  • Matrix metalloproteinase cleavage sequence is a protein that is specifically recognized and cleaved by matrix metalloproteinases.
  • the cleavage site can relieve the steric hindrance effect mediated by HSABP and activate genetically engineered cells such as CAR-T cells.
  • the matrix metalloproteinase cleavage sequence of the present invention preferably has a nucleotide sequence as shown in SEQ ID NO:5 and an amino acid sequence as shown in SEQ ID NO:6.
  • TAR.CAR TAR.CAR
  • Chimeric antigen receptor is composed of an extracellular antigen recognition region, a transmembrane region, and an intracellular co-stimulatory signal region.
  • the design of CAR has gone through the following process:
  • the first generation of CAR has only one intracellular signal component CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy.
  • the second generation of CAR introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40, and ICOS based on the original structure. Compared with the first generation of CAR, the function is greatly improved, further enhancing the persistence of CAR-T cells and the ability to kill tumor cells.
  • some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and developed into the third and fourth generation of CAR.
  • the extracellular segment of CAR can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines, thereby eliminating target cells.
  • the patient's autologous cells or allogeneic donors
  • This method has a very low probability of graft-versus-host disease, and the antigen is recognized by immune cells in a non-MHC restricted manner.
  • CAR-immune cell therapy has achieved a very high clinical response rate in the treatment of hematological malignancies. This high response rate cannot be achieved by any previous treatment method and has triggered a wave of clinical research around the world.
  • the chimeric antigen receptor of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes a target-specific binding element.
  • the extracellular domain can be an scFv of an antibody based on antigen-antibody specific binding, or a natural sequence or a derivative thereof based on ligand-receptor specific binding.
  • the intracellular domain includes a co-stimulatory signaling region and a zeta chain portion.
  • the co-stimulatory signaling region refers to a portion of the intracellular domain that includes co-stimulatory molecules.
  • Co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.
  • joint generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain.
  • the joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the CAR of the present invention when expressed in T cells, can recognize antigens based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to not grow, be caused to die or otherwise be affected, and causes the patient's tumor load to shrink or be eliminated.
  • the antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ⁇ chain.
  • the CAR of the present invention has the structure shown in Formula I: SP-TSAR-Binder-H-TM-C-CD3 ⁇ (I)
  • the CAR of the present invention can be applied to a variety of tumor antigens and a variety of tumor types.
  • the CAR of the present invention can target the tumor antigens and tumor cells that the Binder specifically binds to accordingly.
  • the CAR of the present invention has a nucleotide sequence as shown in SEQ ID NO:7 and an amino acid sequence as shown in SEQ ID NO:8.
  • HSABP in the TSAR regulatory element binds to the highly abundant HSA in the body, and masks the binding of B7-H3 scFv to the antigen through the steric hindrance effect, so that CAR-T is in a low/unactivated state, with weak or no killing function, which can effectively avoid off-target effects.
  • a large number of matrix metalloproteinases (such as MMP1) in the malignant tumor tissue recognize the MMPCS in the TSAR regulatory element, remove the HSABP and its bound HSA at the front end of the B7-H3 scFv, and eliminate the steric hindrance effect mediated by HSABP, so that B7-H3 scFv can smoothly and efficiently bind to the target antigen on the surface of the tumor cell, transforming into an activated CAR-T cell, and exerting the killing function of specific tumor cells. Therefore, based on the design of the TSAR regulatory element, conditional activation mediated by matrix metalloproteinases is achieved, which improves the safety of CAR products.
  • MMP1 matrix metalloproteinases
  • the chimeric antigen receptor immune cell of the present invention can be a T cell, a NK cell, a NKT cell, or a macrophage.
  • CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor markers, once the CAR gene construction targeting a certain tumor marker is completed, it can be widely used; (3) CAR can use both tumor protein markers and glycolipid non-protein markers, expanding the target range of tumor markers; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • NK cells are a major type of immune effector cells that protect the body from viral infection and tumor cell invasion through non-antigen specific pathways. NK cells may acquire new functions through engineering (gene modification), including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cell Cytotoxic effect.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzyme, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are very easy to expand in vitro and develop into "ready-made” products. Other than that, it is similar to CAR-T cell therapy.
  • the nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques.
  • the gene of interest can be produced synthetically.
  • the present invention also provides vectors comprising nucleic acid molecules of the present invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration into eukaryotic cells.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety.
  • the present invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.
  • the expression vector can be provided to the cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector comprises a replication origin that works in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene is inserted into a vector and packaged into retroviral particles.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro.
  • retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can work cooperatively or independently to start transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • Epstein-Barr Epstein-Barr
  • Rous sarcoma virus promoter Rous sarcoma virus promoter
  • human gene promoters such as but not limited to
  • an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector.
  • selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells.
  • Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.
  • Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences.
  • reporter gene is following gene: it is not present in or is expressed by receptor organism or tissue, and its coded polypeptide, the expression of which is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter gene is measured at the appropriate time.
  • Suitable reporter gene can include the gene of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei etc., 2000FEBS Letters 479:79-82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate the ability of agents to regulate promoter-driven transcription.
  • vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to introduce a nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid may be associated with a lipid.
  • Nucleic acids associated with lipids may be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, attached to a liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or complexed with micelles, or otherwise associated with lipids.
  • the lipids, lipid/DNA or lipid/expression vectors associated with the composition are not limited to any specific structure in the solution.
  • a lipid is a fatty substance, which may be a naturally occurring or synthetic lipid.
  • lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a pharmaceutical composition, which contains: a conditionally activated chimeric antigen receptor as described in the first aspect of the present invention, a nucleic acid molecule as described in the second aspect of the present invention, a vector as described in the third aspect of the present invention, a host cell as described in the fourth aspect of the present invention, or an engineered immune cell as described in the fifth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition is a liquid preparation.
  • the pharmaceutical composition is an injection.
  • the concentration of the engineered immune cells in the pharmaceutical composition is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.
  • the pharmaceutical composition of the present invention is preferably formulated for intravenous administration.
  • the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient.
  • This approach makes the probability of graft-versus-host reaction extremely low, and the antigen is recognized by the T cell in an MHC-free manner.
  • one CAR-T can treat all cancers that express the antigen.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition and engineered immune cells of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or can be determined by clinical trials.
  • the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition.
  • the pharmaceutical composition comprising the T cells described herein can be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight (including all integer values within the range).
  • the T cell composition can also be administered multiple times at these doses.
  • the cells can be administered using injection techniques known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • immunotherapy see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988.
  • the optimal dosage and treatment regimen can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation.
  • the compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by intravenous injection.
  • the composition of the T cell can be directly injected into the tumor, lymph node or infection site.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents.
  • the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after).
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after).
  • the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of the expanded immune cells of the invention following transplantation.
  • the expanded cells are administered prior to or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to practices accepted in the art.
  • 1 ⁇ 10 5 to 1 ⁇ 10 10 modified T cells of the present invention can be administered to the patient, for example, by intravenous reinfusion, per treatment or per course of treatment.
  • conditionally activated CAR and engineered immune cells of the present invention can effectively avoid off-target effects, thereby reducing toxic side effects and improving safety.
  • conditionally activated CAR and engineered immune cells of the present invention exert their killing function, the extracellular conditionally activated regulatory elements have been cut off. Therefore, the presence of the conditionally activated regulatory elements of the present invention will not affect the killing function of the engineered immune cells themselves.
  • a recombinant B7-H3.TSAR.CAR molecule (as shown in FIG. 1 ) was constructed and T cells expressing the CAR molecule were prepared, and the steps were as follows:
  • FIG. 2 it is a schematic diagram of the anti-tumor effect of TSAR.CAR-T cells of the present invention: Since the human body contains a large amount of human albumin, when TSAR.CAR-T enters the non-tumor area of the human body, HSABP can immediately bind to HSA, causing the conformation of the extracellular structure of CAR-T cells to change. Due to the steric hindrance effect, scFv cannot smoothly bind to the B7-H3 protein, and B7-H3.TSAR.CAR-T is in an inactive state.
  • B7-H3.TSAR.CAR-T When B7-H3.TSAR.CAR-T enters the tumor area, MMP1 expressed in large quantities in solid tumor tissues can cut B7-H3.TSAR.CAR-T at the MMPCS site, eliminating the steric hindrance effect, and B7-H3.TSAR.CAR-T becomes a classic activated B7-H3.CAR-T.
  • scFv binds to the tumor antigen binding region and the target antigen B7-H3 on the surface of the tumor cell to exert a tumor killing function.
  • B7-H3 was positively expressed in all esophageal squamous cell carcinoma cells.
  • 3Western blot (electrophoresis, membrane transfer, blocking, application of primary antibody, TBST washing, application of secondary antibody, TBST washing, exposure) was used to detect the expression of B7-H3 protein content.
  • Proteinatlas database https://www.proteinatlas.org was used to analyze the protein expression of B7-H3 in normal tissues.
  • B7-H3 was positively expressed in various tissues, especially in the choroid plexus, adrenal gland, salivary gland, lung, stomach, gallbladder, pancreas, urinary bladder, prostate, endometrium, placenta, etc.
  • the GEPIA database http://gepia2.cancer-pku.cn was used to analyze the RNA expression of B7-H3 in different tumor tissues.
  • B7-H3 is expressed in COAD (colon cancer), DLBC (diffuse large B-cell lymphoma), ESCA (esophageal cancer), GBM (diffuse large B-cell lymphoma), HNSC (head and neck squamous cell carcinoma), KIRC (clear cell renal carcinoma), KIRP (renal papillary cell carcinoma), LGG (low-grade glioma of the brain), LUSC (lung squamous cell carcinoma), PAAD (pancreatic cancer), SKCM (skin melanoma), STAD (gastric adenocarcinoma), TCGT (testicular germ cell tumor), THYM (thymoma), UCS (uterine carcinosarcoma) was significantly higher than that of adjacent cancer tissues.
  • the Proteinatlas database https://www.proteinatlas.org was used to analyze the protein expression of B7-H3 in different tumor tissues, and the antibody number was HPA00928 antibody.
  • CD276 protein is expressed in glioma, thyroid cancer, lung cancer, colorectal cancer, head and neck tumors, gastric cancer, liver cancer, pancreatic cancer, bladder cancer, prostate cancer, testicular cancer, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, melanoma, and skin cancer.
  • Example 7 RNA sequencing analysis of RNA expression of MMPs in 120 pairs of esophageal squamous cell carcinoma tissues
  • This example involves the expression of MMPs family members in esophageal squamous cell carcinoma tissue, and the specific steps are as follows:
  • RNA expression of 17 MMPs was analyzed by bioinformatics, and relevant heat maps were drawn.
  • Example 8 Expression of MMP1 and MMP11 in ten esophageal squamous cell carcinoma cell lines
  • This example involves verification of MMP1 protein expression in tumor cells, and the specific steps are as follows:
  • ESCC cells not only express the full-length MMP1 (MMP1 OL) protein, but also express the active segment (22/25kD, MMP1 AL) and inactive segment (27kD) proteins to varying degrees.
  • MMP1 OL full-length MMP1
  • AL active segment
  • 27kD inactive segment
  • This example involves the construction of MMP1 overexpressing cells and protein expression verification, and the specific steps are as follows:
  • 2Use 293T cells to package and produce lentivirus containing the full-length and active segments of MMP1;
  • 5Western blot (electrophoresis, membrane transfer, blocking, application of primary antibody, TBST washing, application of secondary antibody, TBST washing, exposure) was used to detect the expression of MMP1 protein content.
  • flow cytometry analysis is performed to analyze the effective binding of B7-H3.TSAR.CAR-T to HSA through HSABP.
  • the specific steps are as follows:
  • B7-H3.CAR-T B7-H3.TSAR.CAR-T were starved for 24 h (cultured in X-VIVO medium without human serum albumin and IL-2 at 37°C for 24 h);
  • the starved cells were incubated with HSA-FITC at room temperature or 4°C for 10 min, 20 min, and 30 min respectively;
  • Example 11 Flow cytometric analysis of whether T cells expressing B7-H3.TSAR.CAR molecules can be MMP1 activation
  • flow cytometry analysis is performed to determine whether T cells expressing B7-H3.TSAR.CAR molecules can be activated by MMP1.
  • the specific steps are as follows:
  • HSABP-MMPCS-scFv-FITC Some of the cells after incubation were incubated with HSABP-MMPCS-scFv-FITC at 37°C for 1 hour, and the other part of the cells were incubated with activated MMP1 protein (activated by APMA at 37°C for 2 hours) and HSABP-MMPCS-scFv-FITC at 37°C for 1 hour;
  • the cell group containing activated MMP1 protein was able to better bind to the fluorescently labeled HSABP-MMPCS-scFv recombinant protein, proving that MMP1 cleavage can relieve the blocking effect of HSA on the single-chain antibody.
  • the killing ability of CAR-T cells was detected by cell LDH release experiment for 24 hours.
  • the inventors found that the killing effect of B7-H3.CAR-T cells on KYSE150 cells with low expression of MMP1 was significantly higher than that of B7-H3.TSAR.CAR-T.
  • B7-H3.TSAR.CAR-T and B7-H3.CAR-T had the same killing effect (as shown in Figure 13), proving that MMP1 can activate B7-H3.TSAR.CAR-T cells and exert anti-tumor activity.
  • Example 13 In vivo animal experiment comparing the killing effects of B7-H3.TSAR.CAR-T and B7-H3.CAR-T ability
  • the anti-tumor activity of B7-H3.TSAR.CAR T cells is determined by luciferase method, and the specific steps are as follows:
  • blasticidin at a final concentration of 10 ⁇ g/mL for resistance screening for 3-5 days;
  • the anti-tumor activity of B7-H3.TSAR.CAR-T against colorectal cancer Colo205 cells, liver cancer HuH7 cells, gastric cancer SGC7901 cells, pancreatic cancer Panc1 cells, lung cancer NCI-H460 cells, melanoma A375 cells, and ovarian cancer SKOV3 cells was significantly higher than that of the control cells, and with the increase of the effector-target ratio, the anti-tumor activity of B7-H3.TSAR.CAR-T gradually increased.

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Abstract

Préparation et utilisation d'une structure CAR-T activée par métalloprotéinase. La présente invention concerne spécifiquement un récepteur antigénique chimérique activé de manière conditionnelle comprenant un élément régulateur TSAR, une cellule immunitaire modifiée, son procédé de préparation et son utilisation. En utilisant la caractéristique d'expression élevée de métalloprotéinase matricielle dans un microenvironnement tumoral, un produit de cellule immunitaire modifié basé sur l'activation conditionnelle, peut permettre une immunothérapie qui ne se détourne pas facilement de la cible et présente des effets secondaires toxiques moindres et une sécurité plus élevée.
PCT/CN2024/096088 2023-05-31 2024-05-29 Préparation et utilisation d'une structure car-t activée par métalloprotéinase Pending WO2024245291A1 (fr)

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US20220054544A1 (en) * 2018-09-21 2022-02-24 Harpoon Therapeutics, Inc. Conditionally active receptors
US20220281936A1 (en) * 2019-07-25 2022-09-08 The University Of Chicago Compositions and methods comprising protease-activated therapeutic agents
CN116761816A (zh) * 2020-12-21 2023-09-15 艾洛基治疗公司 蛋白酶活化cd45门控car
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