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WO2019128994A1 - Cellule t car spécifique de muc1 exprimant de façon stable un anticorps pd-1, et son utilisation - Google Patents

Cellule t car spécifique de muc1 exprimant de façon stable un anticorps pd-1, et son utilisation Download PDF

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Publication number
WO2019128994A1
WO2019128994A1 PCT/CN2018/123527 CN2018123527W WO2019128994A1 WO 2019128994 A1 WO2019128994 A1 WO 2019128994A1 CN 2018123527 W CN2018123527 W CN 2018123527W WO 2019128994 A1 WO2019128994 A1 WO 2019128994A1
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antibody
seq
amino acid
cell
sequence
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Chinese (zh)
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钱其军
金华君
何周
李林芳
刘祥箴
王超
崔连振
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4256—Tumor associated carbohydrates
    • A61K40/4257—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • C07K16/2818—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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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 A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • 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 A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present invention relates to Mucl-specific CAR-T cells stably expressing a PD-1 antibody and uses thereof.
  • Chimeric antigen receptor T cell (CAR-T) treatment technology is undoubtedly a rising greed in the field of tumor immune cell therapy.
  • CAR-T technology uses genetic engineering technology to splicing the antibody variable region gene sequence of an antigen molecule with the intracellular region of the T lymphocyte immune receptor, and then by retrovirus or lentiviral vector, transposon or transfection.
  • the enzyme system or direct mRNA is transduced into lymphocytes and expresses the fusion protein on the cell surface, enabling T lymphocytes to recognize specific antigens in a non-MHC-restricted manner, enhancing their ability to recognize and kill tumors.
  • the structure of CAR has been proposed by the Eshhar research team in Israel since 1989. After nearly 30 years of development, it has been confirmed that T cells modified by CAR structure have a good effect in tumor immunotherapy.
  • the first generation of CAR receptors contained a single-chain variable fragment (scFv), and the intracellular activation signal was transmitted by the CD3 ⁇ signal chain.
  • scFv single-chain variable fragment
  • the first-generation CAR receptor lacks the costimulatory signal of T cells, which leads to T cells only exerting transient effects, short time in the body and less secretion of cytokines.
  • the second generation of CAR receptors increases the intracellular domain of costimulatory signaling molecules, including, for example, CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cells Co-stimulating agents (ICOS) and DNAX-activated protein 10 (DAP10) and other domains enhance T cell proliferation and cytokine secretion, IL-2, IFN- ⁇ and GM-CSF increase, thus breaking through the tumor microenvironment Immunosuppression, prolonged AICD (activation-induced cell death, AICD).
  • costimulatory signaling molecules including, for example, CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), inducible T cells Co-stimulating agents (ICOS) and DNAX-activated protein 10 (DAP10) and other domains enhance T cell proliferation and cytokine secretion, IL-2, IFN- ⁇ and GM
  • the third-generation CAR receptor recombines a secondary co-stimulatory molecule such as 4-1BB between the co-stimulatory structure CD28 and the ITAM signal chain, thus producing a triple-signal CAR receptor, a third-generation CAR receptor modification.
  • T cells have better effector function and survival time in vivo.
  • the commonly used classical CAR-T structure is the second-generation CAR receptor, and its structure can be divided into the following four parts: the antibody single-chain variable region (scFv), the hinge region, the transmembrane region, and the intracellular stimulation signal. Structure area. Among them, the CAR structure hinge region is responsible for forming a correct conformation and forming a dimer. The length of the hinge region and the amino acid sequence characteristics determine the spatial conformation of CAR and also determine its ability to bind to tumor cell surface antigens.
  • Solid tumors are highly heterogeneous, with high differences between different patients, different lesions in the same patient, and different tumor cells in the same lesion. This high degree of heterogeneity results in the lack of an ideal universal, broad-spectrum target for tumor-targeted therapy, limiting the efficacy of CAR-T cells in the treatment of solid tumors. Therefore, finding effective therapeutic targets for CAR-T cells has become a top priority for CAR-T cell therapy.
  • Muc1 (mucins, mucin) is a type of high molecular weight (>200kD) type I transmembrane glycoprotein (multiple O-glycosidic bond linked to Ser/Thr on the polypeptide backbone), normally expressed in a variety of tissues
  • the epithelial cells in the organs are near the lumen or glandular surface, showing a apical expression and a polar distribution.
  • Muc1 protein can be abnormally expressed on the surface of tumor cells, and the expression level is more than 100 times that of normal. Moreover, its polarity distribution on the cell surface is lost and can be evenly distributed throughout the cell surface.
  • PD-1 Programmed Death 1, reprogrammed cell death receptor 1
  • PD-1 and its ligand PD-L1/PD-L2 play important roles in the co-suppression and failure of T cells. Their interaction inhibits the proliferation of co-stimulatory T cells and the secretion of cytokines.
  • the expression of the anti-apoptotic molecule BCL-xl impairs the function of tumor-specific T cells, leading to the inability of some tumor patients to completely eliminate the tumor.
  • the PD-1 antibody competes with the PD-1 molecule of the tumor-specific T cell surface to compete for the binding of PD-1 to its ligand PD-L1/PD-L2, thereby alleviating PD-1 and PD-L1/PD-L2.
  • commercialized PD-1 antibodies are Nivolumab and Pidilizumab. These two monoclonal antibodies have been proven to have good clinical effects in solid tumors such as melanoma, colon cancer, prostate cancer, non-small cell lung cancer, and renal cell carcinoma, but PD-1 antibodies still have some inevitable clinical applications. The problem.
  • PD-1 monoclonal antibody is administered intravenously, most patients receiving PD-1 antibody blockade will have different degrees of drug side effects.
  • the production of PD-1 monoclonal antibody involves a complicated production preparation and purification process, which is costly and leads to expensive treatment.
  • CAR-T cells have the ability to kill tumor cells and can effectively enter the tumor tissue, but their activity is easily inhibited in the tumor microenvironment; and PD-1 antibody can reactivate the antitumor activity of T cells.
  • macromolecular antibodies have insufficient penetrating power to solid tumors, and systemic drugs have large toxic side effects, and the cost of drugs is high. Therefore, if the PD-1 antibody can be efficiently expressed by maintaining the killing toxicity of CAR-T cells, and the PD-1 antibody is expressed at a high level in the tumor by the tumor trait characteristic of the CAR-T cell, the CAR will be overcome at the same time.
  • -T cell therapy and PD-1 antibody treatment of each defect play a synergistic effect between the two to improve efficacy, while reducing treatment costs.
  • the present invention provides a T cell comprising: (1) a coding sequence comprising a chimeric antigen receptor recognizing a Muc1 antigen and a coding sequence of a PD-1 antibody; and/or (2) expression of a recognition Muc1 antigen Antigen receptor and PD-1 antibody.
  • the expression cassette of the chimeric antigen receptor recognizing the Mucl antigen and the expression cassette of the PD-1 antibody are integrated into the genome of the T cell.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a membrane protein signal peptide, a single-chain antibody against the Mucle proximal membrane, a hinge region of 50 amino acid residues or more, and a cross Membrane region, intracellular costimulatory signal domain and intracellular signal domain.
  • the signal peptide is a secreted signal peptide and a membrane-bound signal peptide, preferably a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide; more preferably a CD8 signal peptide; preferably, The amino acid sequence of the CD8 signal peptide is set forth in SEQ ID NO: 5.
  • amino acid sequence of the single chain antibody is set forth in SEQ ID NO: 7.
  • the hinge region longer than 50 amino acid residues is selected from the group consisting of a CD8 alpha hinge region, an IgD hinge region, an IgGl Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is CD8 ⁇ Hinge region or IgG4Fc CH2CH3 hinge region; more preferably, the amino acid sequence of the CD8 alpha hinge region is set forth in SEQ ID NO: 8; the amino acid sequence of the IgG4 Fc CH2CH3 hinge region is set forth in SEQ ID NO: 3.
  • the transmembrane region is a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region.
  • a CD28 transmembrane region preferably having an amino acid sequence as set forth in SEQ ID NO: 9.
  • the intracellular costimulatory signal domain comprises an intracellular domain of a costimulatory signaling molecule, including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase
  • a costimulatory signaling molecule including CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase
  • the amino acid sequence of the domain is set forth in SEQ ID NO: 10.
  • the intracellular signal domain is a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain is SEQ. ID NO: 12 stated.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-Muc1 single chain antibody, a CD8a hinge region or an IgG4 Fc CH2CH3 hinge region, a CD28 transmembrane region, a CD28 cell Internal domain and tyrosine activation motif of CD3 ⁇ .
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a CD8 signal peptide, an anti-Muc1 single chain antibody, an IgG4 Fc CH2CH3 hinge region, a CD28 transmembrane region, a CD28 intracellular domain, and Tyrosine activation motif of CD3 ⁇ .
  • the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-678 of SEQ ID NO: 13, or as set forth in SEQ ID NO: 13; preferably, The coding sequence of the chimeric antigen receptor is shown as bases 70 to 2034 of SEQ ID NO: 14, or as shown in SEQ ID NO: 14.
  • the amino acid sequence of the PD-1 antibody is as shown in amino acid residues 21-495 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1; preferably, shown
  • the coding sequence of the PD-1 antibody is shown as bases 61-1488 of SEQ ID NO: 2, or as shown in SEQ ID NO: 2.
  • the invention also provides a composition
  • a composition comprising: a vector comprising an expression cassette of a chimeric antigen receptor of the invention, the vector for integrating the expression cassette into the genome of a host cell; A vector comprising an expression cassette of a PD-1 antibody, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the PD-1 antibody is as shown in amino acid residues 21-495 of SEQ ID NO: 1, or as shown in SEQ ID NO: 1; preferably, said The coding sequence of the PD-1 antibody is shown as bases 63 to 1488 of SEQ ID NO: 2, or as shown in SEQ ID NO: 2.
  • the invention also provides a kit, the kit comprising:
  • a vector comprising an expression cassette of a chimeric antigen receptor of the present invention, which vector is used to integrate the expression cassette into the genome of a host cell;
  • a vector comprising an expression cassette of a PD-1 antibody, which vector is used to integrate the expression cassette into the genome of a host cell.
  • the amino acid sequence of the PD-1 antibody is as shown in amino acid residues 21-495 of SEQ ID NO: 1.
  • the present invention also provides a pharmaceutical composition comprising the T cell of the present invention or the T cell and the PD-1 antibody expressed thereby.
  • the cancer is a cancer in which the surface of the cancer cell abnormally expresses Mucl, preferably a cancer in which the expression level of Mucl is more than 100 times that on the surface of the cancer cell, and Mucl is uniformly distributed over the entire cell surface; preferably,
  • the cancer is selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreas Cancer or prostate cancer.
  • Figure 1 Expression cassette pattern of pNB328-Muc1CAR, pS328-Muc1CAR, pNB328-m279V, pS328-m279V-wt, pS328-m279V, pNB328-Muc1CAR-2A-m279V, pNB328-m279V-IRES-Muc1CAR.
  • 2A-2B The positive rate of expression of Muc1CAR gene and the expression of PD-1 antibody in PBMCs after activation and activation by different combinations of Muc1CAR gene and PD-1 antibody gene.
  • PBMCs were expressed by different ratios of pNB328-Muc1CAR and pS328-m279V plasmids to express Muc1CAR gene positive rate and PD-1 antibody expression.
  • Figure 4 Determination of the positive rate of Muc1CAR gene expression in T cells after activation of Muc1CAR gene and PD-1 antibody gene in PBMCs from different patient sources (from top to bottom, patient 1, patient 2, patient 3).
  • Figure 5 ELISA detection of PD-1 antibody gene expression after modification of PD-1 antibody gene by different patient-derived PBMCs.
  • FIGS 6A-6D Muc1CAR-anti PD1 pluripotent T cells enhance T cell killing activity in vitro.
  • A Flow-through detection of marker CD107 ⁇ , activation marker CD25 and depletion marker LAG3, indirectly reflecting T cell killing activity
  • B Flow-through detection of marker proteins CD45RO, CD62L and CCR7 reflecting the phenotype of T cell memory.
  • C Flow detection of the proportion of T cells CD3/CD4/CD8.
  • Muc1CAR-anti PD1 pluripotent T cells by multi-factor detection kit, Muc1CAR-T cells and Mock T cells stimulated by Muc1 antigen, IL-2, IL-4, IL-6, IL-10, TNF- ⁇ And changes in IFN- ⁇ cytokines.
  • Figure 7 Detection of killing effect of Muc1CAR-anti PD1 pluripotent T cells.
  • Figure 8 In vivo functional studies of Muc1 CAR T cells expressing PD-1 antibodies.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product (eg, CAR, single chain antibody, hinge region, and transmembrane region).
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • Fc fragment crystallizable (Fc) of an antibody
  • Fc fragment crystallizable
  • costimulatory molecule refers to a molecule that is present on the surface of an antigen presenting cell and that binds to a costimulatory molecule receptor on a Th cell to produce a costimulatory signal.
  • the proliferation of lymphocytes requires not only the binding of antigens, but also the signals of costimulatory molecules.
  • the costimulatory signal is transmitted to the T cells mainly by binding to the co-stimulatory molecule CD80 on the surface of the antigen presenting cells, and CD86 binds to the CD28 molecule on the surface of the T cell.
  • B cells receive a costimulatory signal that can pass through a common pathogen component such as LPS, or through a complement component, or through activated antigen-specific Th cell surface CD40L.
  • linker or hinge is a polypeptide fragment that links between different proteins or polypeptides for the purpose of maintaining the spatial conformation of the linked protein or polypeptide to maintain the function or activity of the protein or polypeptide.
  • exemplary linkers include linkers containing G and/or S, as well as, for example, Furin 2A peptide.
  • an antibody that specifically binds to an antigen means that the antibody is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Affinity (KD) of 10 -8 M, 10 -9 M or 10 -10 M or less binds to the antigen.
  • KD Affinity
  • pharmaceutically acceptable excipient refers to carriers and/or excipients that are compatible pharmacologically and/or physiologically to the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers.
  • pH adjusting agents include, but are not limited to, phosphate buffers
  • surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include, but are not limited to, sodium chloride.
  • the term "effective amount” refers to a dose that can achieve a treatment, prevention, alleviation, and/or alleviation of a disease or condition described herein in a subject.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition described herein.
  • subject or “patient” may refer to a patient or other animal that receives the pharmaceutical composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog. , monkeys, cattle, horses, etc.
  • CAR chimeric antigen receptor
  • T cells immune cells
  • CAR typically comprises, in turn, an optional signal peptide, a polypeptide that binds to a tumor cell membrane antigen, such as a single chain antibody, a hinge region, a transmembrane region, and an intracellular signaling region.
  • a polypeptide that binds to a tumor cell membrane antigen is capable of binding to a membrane antigen that is widely expressed by tumor cells with moderate affinity.
  • the polypeptide that binds to the tumor cell membrane antigen may be a natural polypeptide or a synthetic polypeptide; preferably, the synthetic polypeptide is a single chain antibody or a Fab fragment.
  • single-chain antibody refers to an antibody fragment which is obtained by hinge-ligating an amino acid sequence of an antibody light chain variable region (VL region) and a heavy chain variable region (VH region), and having antigen-binding ability.
  • the single chain antibody of interest is from an antibody of interest.
  • Antibodies of interest may be human antibodies, including human murine chimeric antibodies and humanized antibodies.
  • the antibody may be secreted or membrane anchored; preferably a membrane anchored.
  • the IgG4 Fc fragment of the PD-1 antibody is easily phagocytosed by the monocyte/macrophage, and the PD-1 antibody of the PD-1 antibody IgG4Fc fragment is mutated to satisfy the T cell self-expression of the PD-1 antibody. It works well and does not cause ADCC reactions.
  • the present invention provides a PD-1 antibody comprising an anti-PD-1 single chain antibody and IgG4Fc.
  • the amino acid sequence of the IgG4 Fc is set forth in amino acid residues 267-495 of SEQ ID NO: 1; preferably, the coding sequence thereof is set at bases 799-1485 of SEQ ID NO: Shown.
  • the antibody light chain variable region (VL region) amino acid sequence of the anti-PD-1 single chain antibody is represented by amino acid residues 21 to 131 of SEQ ID NO: 1; The coding sequence thereof is shown in nucleotide sequences 61-393 of SEQ ID NO: 2.
  • the heavy chain variable region (VH region) amino acid sequence of the anti-PD-1 single chain antibody is set forth in amino acid sequence at positions 147-266 of SEQ ID NO: 1; preferably, the encoding The sequence is shown in nucleotide sequence 439-798 of SEQ ID NO: 2.
  • the amino acid sequence of the anti-PD-1 single chain antibody is represented by amino acid residues 21 to 266 of SEQ ID NO: 1; preferably, the coding sequence thereof is SEQ ID NO: 2, 61. -798 base sequence shown.
  • the PD-1 antibody further comprises a light chain signal peptide.
  • the PD-1 antibody comprises, in order from the N-terminus to the C-terminus, a light chain signal peptide, an anti-PD-1 single chain antibody, and an IgG4 Fc.
  • the amino acid sequence of the light chain signal peptide is represented by amino acid residues 1-20 of SEQ ID NO: 1; preferably, the coding sequence of the indicated light chain signal peptide is SEQ ID NO: 2 The base sequence of the 1-60th base is shown.
  • the amino acid sequence of the PD-1 antibody is set forth in amino acid sequence 21-495 of SEQ ID NO: 1, or as set forth in SEQ ID NO: 1.
  • the invention also encompasses a coding sequence for the PD-1 antibody or a complement thereof, the coding sequence comprising at least the coding sequence for an IgG4 Fc described herein or a complement thereof.
  • the coding sequence of the PD-1 antibody comprises the sequence set forth in bases 61-1495 of SEQ ID NO: 2, preferably the sequence set forth in SEQ ID NO: 2.
  • the present invention also encompasses a nucleic acid construct comprising the coding sequence of the PD-1 antibody of the present invention or a complement thereof.
  • the nucleic acid construct is an expression vector or an integration vector for integrating the coding sequence or its complement into a host cell.
  • the invention also provides a host cell comprising a nucleic acid construct as described herein.
  • the invention also provides the use of the PD-1 antibody, its coding sequence or complementary sequence, a nucleic acid construct, and a host cell for the preparation or treatment of a malignancy, particularly a tumor associated with PD-1, including but Not limited to the various malignancies described herein.
  • the present invention tests the Muc1CAR gene and the PD-1 antibody in various combinations, including linking the Muc1CAR gene, the PD-1 antibody and the PB with 2A.
  • Genes form a single plasmid, ligate the Muc1CAR gene with IRES, form a single plasmid with PD-1 antibody and PB gene, double plasmid combination of Muc1CAR gene plasmid carrying PB gene and PD-1 antibody plasmid, and PD-1 antibody plasmid carrying PB gene Combine with the double plasmid of the Muc1CAR gene plasmid.
  • the test showed that the Muc1CAR gene plasmid carrying the PB gene and the double plasmid combination of the PD-1 antibody plasmid can obtain stable Muc1 CAR gene and PD-1 antibody expression.
  • the present invention also provides a T cell which is modified by the Muc1CAR gene and can express the PD-1 antibody, and the T cell can stably express the Muc1CAR gene and the PD-1 antibody at a high level, and the exogenously expressed Muc1CAR gene can accurately target To Muc1 antigen, enhance the proliferation of T cells and the secretion of cytokines, enhance the killing of tumor cells by CAR-T cells, and exert anti-tumor effects by enhancing the immune response.
  • exogenously expressed PD-1 antibody can overcome the inhibition of immune microenvironment, promote the apoptosis of tumor cells, and exert an anti-tumor immune response.
  • the exogenous Muc1CAR gene and the PD-1 antibody gene can be integrated into the genome of T cells via the PB transposase system, thereby stably and continuously expressing in T cells.
  • the T cells of the present invention which stably express the Muc1CAR gene and the PD-1 antibody gene at a high level can be used for the treatment of various malignant tumors with high expression of Muc1.
  • the CAR of the invention typically contains an optional signal peptide sequence, an scFv that recognizes the Mucl antigen, a hinge region, a transmembrane region, an intracellular costimulatory signal domain, and an intracellular signal domain.
  • a signal peptide is a short peptide chain (5-30 amino acids in length) that directs the transfer of newly synthesized proteins to the secretory pathway, often referred to as the N-terminal amino acid sequence in the newly synthesized polypeptide chain that directs transmembrane transfer (localization) of the protein. (Sometimes not necessarily at the N-terminus), it is responsible for directing proteins into subcellular organelles with different membrane structures.
  • the signal peptide can be a secreted signal peptide or a membrane-bound signal peptide.
  • the signal peptide is a CD8 signal peptide, a CD28 signal peptide or a CD4 signal peptide; more preferably a CD8 signal peptide.
  • the amino acid sequence of the CD8 signal peptide can be as set forth in SEQ ID NO: 5; in certain embodiments, the coding sequence is set forth in bases 1-66 of SEQ ID NO: 14.
  • the scFv recognizing the Mucl antigen described herein may be a single chain antibody directed against the Mucl antigen as known in the art.
  • the light chain variable region amino acid sequence and the heavy chain variable region amino acid sequence of the single chain antibody are derived from an antibody directed against the proximal membrane end amino acid sequence of Mucl.
  • the Mucl1 proximal membrane amino acid sequence is set forth in SEQ ID NO: 6.
  • An exemplary anti-Muc1 single chain antibody has the amino acid sequence set forth in SEQ ID NO: 7, and an exemplary coding sequence thereof is set forth in bases 67-807 of SEQ ID NO: 14.
  • the hinge region refers to the region between the functional regions of the immunoglobulin heavy chain CH1 and CH2, which is rich in proline, does not form an alpha helix, is prone to stretching and is somewhat distorted, and is beneficial to the antigen binding site of the antibody. Complementary binding between epitopes.
  • the hinge region suitable for use herein may be selected from any one or more of the extracellular hinge region of CD8a, the IgG1 Fc CH2CH3 hinge region, the IgD hinge region, the extracellular hinge region of CD28, the IgG4 Fc CH2CH3 hinge region, and the extracellular hinge region of CD4. .
  • the hinge region is preferably a hinge region that is longer than 50 amino acid residues, more preferably 80 amino acids or longer.
  • a CD8 alpha hinge region or an IgG4 Fc CH2CH3 hinge region is used herein.
  • the amino acid sequence of an exemplary CD8 alpha hinge region is set forth in SEQ ID NO: 8.
  • the amino acid sequence of the exemplary IgG4 FcCH2CH3 hinge region is set forth in SEQ ID NO: 3
  • the coding sequence for the exemplary IgG4 FcCH2CH3 hinge region is set forth in SEQ ID NO: 4 or as shown in SEQ ID NO: 14 positions 806-1491.
  • the transmembrane region may be one of a CD28 transmembrane region, a CD8 transmembrane region, a CD3 ⁇ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region, and a DAP10 transmembrane region; preferably a CD28 transmembrane region
  • the amino acid sequence thereof is set forth in SEQ ID NO: 9; in certain embodiments, the coding sequence is set forth in bases 1492-1575 of SEQ ID NO: 14.
  • the intracellular co-stimulatory signal domain including the intracellular domain of the costimulatory signaling molecule may be selected from the group consisting of CD28, CD134/OX40, CD137/4-1BB, lymphocyte-specific protein tyrosine kinase (LCK), and inducible T cell costimulation. Intracellular domain of factor (ICOS) and DNAX activator protein 10 (DAP10).
  • the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD28, preferably the amino acid sequence thereof is set forth in SEQ ID NO: 10, and the exemplary coding sequence is set forth in SEQ ID NO: 14. Bases 1576-1698 are shown.
  • the intracellular domain of the costimulatory signaling molecule is the intracellular domain of CD137/4-1BB; preferably, the amino acid sequence of the CD137/4-1BB is set forth in SEQ ID NO:11 Show.
  • the intracellular signal domain is preferably an immunoreceptor tyrosine activation motif, which may be a CD3 sputum intracellular signal domain or an Fc ⁇ RI gamma intracellular signal domain; preferably a CD3 sputum intracellular signal domain, preferably the amino acid sequence of the CD3 sputum intracellular signal domain As set forth in SEQ ID NO: 12; in certain embodiments, the coding sequence is set forth in bases 1699-2034 of SEQ ID NO: 10.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus: an optional CD signal peptide, an scFv, an IgG4 Fc CH2CH3 hinge region, a CD28 transmembrane region, an intracellular domain of CD28, and a CD3 ⁇ Intracellular signal domain; preferably, the amino acid sequence of the chimeric antigen receptor is as shown in amino acid residues 23-678 of SEQ ID NO: 13.
  • the chimeric antigen receptor further comprises a signal peptide, preferably the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 13.
  • the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus: an optional CD signal peptide, an scFv, a CD8 alpha hinge region, a CD28 transmembrane region, an intracellular domain of CD28, and a CD3 cell. Internal signal domain.
  • the invention also encompasses chimeric antibody receptors and coding sequences thereof as described herein.
  • the above-described various portions of the chimeric antigen receptors can be directly connected to each other or can be connected by a linker sequence.
  • the linker sequence can be a linker sequence suitable for use in antibodies well known in the art, such as linker sequences comprising G and S.
  • the linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, and 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the amount of glycine in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, and 2 to 8.
  • the linker may also contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), styrene Amino acid (F), arginine (R), glutamine (Q), and the like.
  • a suitable cleavage site which necessarily introduces one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest.
  • promote expression of a recombinant protein obtain a recombinant protein that is automatically secreted outside the host cell, or facilitate purification of the recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein.
  • Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like.
  • the amino or carboxy terminus of a CAR herein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • polynucleotide sequences encoding the chimeric antigen receptor.
  • the polynucleotide sequence herein may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotide sequences described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, and the relevant sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.
  • the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the polynucleotide sequence encoding a fusion protein described herein is set forth in SEQ ID NO: 14.
  • nucleic acid constructs comprising a polynucleotide sequence encoding the chimeric antigen receptor described herein or a polynucleotide sequence encoding the PD-1 antibody, and one or operably linked to the sequences Multiple regulatory sequences.
  • the nucleic acid construct is an expression cassette.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is typically operably linked to the coding sequence of the protein to be expressed.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and may be derived from an extracellular or heterologous source encoding the host cell. Or the gene of the intracellular polypeptide is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used herein.
  • the nucleic acid construct is a vector.
  • the coding sequence of the CAR or the coding sequence of the PD-1 antibody can be cloned into many types of vectors, for example, but not limited to plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • the vector can be an expression vector.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • suitable vectors comprise an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism.
  • the invention employs a retroviral vector containing a replication initiation site, a 3'LTR, a 5' LTR, a coding sequence for a CAR described herein, or a PD-1 antibody A coding sequence, and optionally a selectable marker.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter sequences.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme Promoter and creatine kinase promoter. Further, an inducible promoter can also be considered.
  • SV40 prion 40
  • MMTV mouse breast cancer virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • EB virus immediate early promoter EB virus immediate early promoter
  • Russ sarcoma virus promoter avian leukemia virus promoter
  • an inducible promoter can also be considered.
  • an inducible promoter provides a molecular switch that is capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter upon expression of the term, and shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • various promoter sequences disclosed in CN201510021408.1 can be used, including but not limited to the CCEF promoter containing the mCMV enhancer, the hCMV enhancer, and the EF1 ⁇ promoter shown in SEQ ID NO: 5 of the application.
  • the selectable marker includes either or both of the selectable marker genes or reporter genes to facilitate identification and selection of the expressed cells from the population of cells infected by the viral vector.
  • Useful selectable marker genes include, for example, antibiotic resistance genes such as neo and the like.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • the coding sequence of the chimeric antigen receptor described herein and the coding sequence of the PD-1 antibody are separately cloned into a vector for integration of the nucleic acid sequence of interest into the genome of the host cell (also referred to as integration).
  • vectors especially transposon vectors.
  • the transposon vector is a eukaryotic expression vector comprising a transposable element selected from the group consisting of piggybac, sleeping beauty, frog prince, Tn5 or Ty.
  • Such transposon vectors contain the 5' inverted terminal repeat (5' LTR) of the corresponding transposon and the 3' inverted terminal repeat (3' LTR) of the corresponding transposon.
  • the transposase can be a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system.
  • the sequences of the 5'LTR and 3'LTR in the vector are also correspondingly changed to sequences adapted to the transposition system, which can be readily determined by those skilled in the art.
  • LTR is the expression cassette for the CAR or antibody of the invention, including the corresponding promoter sequence, the coding sequence for the CAR or antibody, and the polyA tailing signal sequence.
  • the transposase is a transposase from a piggybac transposition system.
  • the 5' inverted terminal repeat and the 3' inverted terminal repeat of the transposon are the 5' inverted terminal repeat and the 3' inverted terminal repeat of the piggybac transposon, respectively.
  • the transposon 5' inverted terminal repeat is SEQ ID NO: 1 as described in CN 201510638974.7, the disclosure of which is incorporated herein by reference.
  • the transposon 3' inverted terminal repeat is as shown in CN 201510638974.7 SEQ ID NO: 4.
  • the piggybac transposase is a transposase comprising a c-myc nuclear localization signal coding sequence.
  • the coding sequence for the piggybac transposase is as shown in CN 201510638974.7 SEQ ID NO: 5.
  • the promoter of the transposase coding sequence can be a variety of promoters known in the art for controlling expression of the transposase coding sequence.
  • the expression of the transposase coding sequence is controlled using a CMV promoter.
  • the sequence of the CMV promoter can be as shown in CN 201510638974.7 SEQ ID NO: 6.
  • the vector of the present invention comprising a coding sequence for a chimeric antigen receptor is the pNB328 vector disclosed in CN 201510638974.7.
  • the coding sequence of the chimeric antigen receptor of the present invention can be prepared by a method conventional in the art and cloned into a suitable vector.
  • the vector for integrating a gene of interest into the genome of a host cell does not contain a transposase coding sequence.
  • such vectors can be obtained by removing the transposase coding sequence based on the pNB328 vector.
  • such vectors are used to integrate the coding sequence for the PD-1 antibody and the signal peptide coding sequence (e.g., the coding sequence for the light chain signal peptide) into the genome of the host cell.
  • the amino acid sequence of an exemplary light chain signal peptide is shown in amino acid residues 1-20 of SEQ ID NO: 1, and the coding sequence of an exemplary light chain signal peptide is SEQ ID NO: 2 bases 1-60. Shown.
  • a T cell modified by the MuclCAR gene and capable of expressing a PD-1 antibody described herein can be transferred:
  • a vector containing a transposase coding sequence for integration into a chimeric antigen receptor expression cassette in a T cell genome, and an expression cassette for integration of a PD-1 antibody described herein in a T cell genome a vector comprising a transposase coding sequence
  • a vector containing a transposase coding sequence for integration into a chimeric antigen receptor expression cassette in a T cell genome, and an expression cassette for integration of a PD-1 antibody described herein in a T cell genome a vector that does not contain a transposase coding sequence;
  • the T cell is transformed into a vector comprising a transposase coding sequence for integration into a chimeric antigen receptor expression cassette in the T cell genome, and for integration into a T cell genome as described herein.
  • the expression cassette of the PD-1 antibody contains no vector for the transposase coding sequence.
  • the T cell is transformed into a vector containing a chimeric antigen receptor coding sequence constructed using the pNB328 vector as a backbone vector and constructed as a backbone vector using the pS328 vector (with no transposase coding sequence compared to pNB328).
  • a vector containing a PD-1 antibody expression cassette is containing a PD-1 antibody expression cassette.
  • the coding sequence of the chimeric antigen receptor is set forth in SEQ ID NO: 14; the coding sequence of the PD-1 antibody is as set at bases 61-1488 of SEQ ID NO: 2.
  • the signal peptide of the PD-1 antibody in the vector comprising the coding sequence for the PD-1 antibody, is a light chain signal peptide.
  • the amino acid sequence of an exemplary light chain signal peptide can be as shown in amino acid residues 1-20 of SEQ ID NO: 1.
  • the vector comprising a transposase coding sequence that incorporates a chimeric antigen receptor coding sequence in the T cell genome in turn contains a 5' LTR, a promoter, and a CD8 signal peptide encoding.
  • Sequence coding sequence of scFv recognizing Muc1 antigen, coding sequence of IgG4Fc CH2CH3 hinge region, coding sequence of CD28 transmembrane region, coding sequence of CD28 intracellular domain, coding sequence of CD3 intracellular signal domain, polyA tailing signal sequence a coding sequence for a 3' LTR and a transposase and a promoter thereof; said vector comprising a transposase coding sequence encoding a coding sequence for a PD-1 antibody described herein in a T cell genome at 5' LTR
  • the coding sequence of the promoter, the light chain signal peptide, the coding sequence of the PD-1 antibody, and the polyA tailing signal sequence are sequentially contained between the 3' and the L'LTR.
  • the mass ratio of the vector containing the chimeric antigen receptor coding sequence to the vector containing the PD-1 antibody coding sequence is from 1 to 7:1 to 7, preferably from 1 to 3:1 to 3, preferably 1 : 1 to 3, more preferably 1:1 to 2, still more preferably 1:1.
  • Methods of transfection are routine methods in the art including, but not limited to, viral transduction, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • the vector is transfected into a cell of interest using electroporation.
  • the cells of interest may be various T cells well known in the art including, but not limited to, peripheral blood T lymphocytes, cytotoxic killer T cells (CTLs), helper T cells, suppressor/regulatory T cells, y ⁇ T cells, and cytokines.
  • T cells of mixed cell populations such as induced killer cells (CIK) and tumor infiltrating lymphocytes (TIL).
  • CIK induced killer cells
  • TIL tumor infiltrating lymphocytes
  • the T cell can be derived from a PBMC of a B cell malignancy patient.
  • the T cell is a primary cultured T cell.
  • the invention also provides a composition comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette for a PD-1 antibody described herein.
  • Suitable agents may also be included in the compositions including, but not limited to, transfection reagents.
  • the invention also provides a kit comprising a vector comprising a chimeric antigen receptor expression cassette as described herein and a vector comprising an expression cassette of a PD-1 antibody described herein, or a composition described herein .
  • a reagent or instrument for transferring the vector into a cell can also be provided in the kit.
  • the expression cassettes described herein contain, in addition to the coding sequences for the CARs or antibodies described herein, at least the appropriate promoter and polyA tailing signal sequences.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a T cell or a T cell as described herein and a PD-1 antibody expressed thereby.
  • Suitable pharmaceutically acceptable carriers or excipients may be included in the pharmaceutical compositions.
  • the pharmaceutical composition contains a therapeutically or prophylactically effective amount of T cells.
  • the therapeutically or prophylactically effective amount of the T cell can be determined according to factors such as the condition of the patient.
  • the invention also provides the use of a T cell or a pharmaceutical composition thereof described herein or or a T cell and an PD-1 antibody thereof, for the manufacture of a medicament for the treatment or prevention of a malignancy.
  • the invention also provides a method of treating or preventing a malignant tumor, the method comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a T cell of the invention.
  • the cancer suitable for the treatment or prevention of the T cells described herein is preferably a Mucl-positive cancer, specifically including a cancer having abnormal expression of Muc1 on the surface of the cancer cell, such as more than 100 times when the expression level of Muc1 on the surface of the cancer cell is normal, and Muc1 is A cancer that is evenly distributed across the cell surface.
  • cancers may be selected from the group consisting of: adenocarcinoma, lung cancer, colon cancer, colon cancer, breast cancer, ovarian cancer, melanoma, non-small cell lung cancer, renal cell carcinoma/cervical cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer. , esophageal cancer, pancreatic cancer or prostate cancer.
  • Example 1 Recombinant plasmids pNB328-Muc1CAR, pS328-Muc1CAR, pNB328-m279V, pS328-m279V-wt, pS328-m279V, pNB328-Muc1CAR-2A-m279V, Construction of pNB328-m279V-IRES-Muc1CAR and acquisition of various types of pluripotent T cells.
  • pS328 lacks the PB transposon sequence compared to pNB328, and constitutes a recombinant plasmid, which is named pNB328-Muc1CAR and pS328-Muc1CAR, respectively.
  • the coding sequence of the wild-type PD-1 antibody is the nucleotide sequence of 61-1488 of SEQ ID NO: 16; the amino acid sequence of the light chain signal peptide is shown by amino acid residues 1 to 20 of SEQ ID NO: 15, wild The amino acid sequence of the type PD-1 antibody is as shown in amino acid residues 21 to 495 of SEQ ID NO: 15) and the foreign gene of the mutant PD-1 antibody (including the coding sequence of the light chain signal peptide; light chain signal peptide
  • the coding sequence is shown in nucleotide sequence 1-60 of SEQ ID NO: 2, and the coding sequence of the mutant PD-1 antibody is as shown in SEQ ID NO: 2, bases 61-1488; amino acids of light chain
  • a polyclonal cleavage site (BglII-XbaI-EcoRI-BamHI) was inserted downstream of the restriction site (SalI-NheI-HindIII-SpeI) and loaded into EcoR1+Sal
  • recombinant plasmids were constructed, which were named pNB328-m279V, pS328-m279V-wt, and pS328-m279V, respectively.
  • the aforementioned foreign gene of the Muc1CAR gene and the foreign gene of the mutant PD-1 antibody are ligated with 2A (SEQ ID NO: 17, amino acid sequence as shown in SEQ ID NO: 18), and a polyclonal enzyme is introduced upstream thereof.
  • a site (BglII-XbaI-EcoRI-BamHI) was inserted downstream of the restriction enzyme site (SalI-NheI-HindIII-SpeI), and inserted into the pNB328 vector digested with EcoR1+SalI to construct a recombinant plasmid. Named pNB328-Muc1CAR-2A-m279V.
  • the foreign gene of the previously synthesized mutant PD-1 antibody was ligated with the Muc1CAR foreign gene as IRES (SEQ ID NO: 19), and a polyclonal cleavage site (BglII-XbaI-EcoRI-BamHI) was introduced upstream thereof.
  • the restriction enzyme cleavage site (SalI-NheI-HindIII-SpeI) was inserted downstream, and inserted into the pNB328 vector digested with EcoR1+SalI to construct a recombinant plasmid designated as pNB328-m279V-IRES-Muc1CAR.
  • each recombinant plasmid obtained was shown in Fig. 1.
  • the promoter sequence and the polyA tailing signal sequence are not shown in the respective structural pattern diagrams, which are located between the 5'LTR and the signal peptide sequence and before the 3' LTR, respectively.
  • PBMCs Peripheral blood mononuclear cells
  • the PBMCs were cultured for 2-4 h, and the unattached suspension cells were the initial T cells.
  • the suspension cells were collected into 15 ml centrifuge tubes, centrifuged at 1200 rpm for 3 min, the supernatant was discarded, physiological saline was added, and centrifuged at 1200 rpm for 3 min. Saline, and repeat this step;
  • Plasmid pNB328-m279V (mass ratio 1:1)
  • b tube was added 4ug recombinant plasmid pNB328-Muc1CAR and 4ug recombinant plasmid pS328-m279V (mass ratio 1:1)
  • c tube was added 8ug pNB328-Muc1CAR-2A-m279V plasmid
  • d 8ug pNB328-m279V-IRES-Muc1CAR was added to the tube
  • 8ug pNB328 vector plasmid was added to the e tube
  • 4ug recombinant plasmid pNB328-Muc1CAR and 4ug recombinant plasmid pS328-m279V-wt were added to the tube
  • 4ug recombinant plasmid pNB328-Muc1CAR was added to the tube.
  • the micro-pipette in the kit transfers the electrically-transferred cell suspension to a six-well plate with a culture medium (AIM-V medium containing 2% FBS), mixes and is placed at 37 ° C, 5% CO2 incubator After 6 hours, the stimulating factors IL-2 and Muc1 antigen and CD28 antibody were added, and cultured at 37 ° C, 5% CO 2 for 3 to 4 days to observe the growth of T cells, and the corresponding T cells were obtained, which were named pS328-Muc1CAR+.
  • a culture medium AIM-V medium containing 2% FBS
  • pNB328-antiPD1 T cells pNB328-Muc1CAR+pS328-antiPD1 T cells
  • Muc1 CAR-2A-antiPD1 T cells antiPD1-IRES-Muc1 CAR T cells
  • pNB328-Muc1CAR+pS328-antiPD1-wt T cells and Muc1 CAR T cells.
  • Example 2 PBMCs were modified and expressed by different combinations of Muc1CAR gene and PD-1 antibody gene. Muc1CAR gene positive rate and PD-1 antibody expression assay.
  • the different types of different patient-derived activated T cells constructed above were precipitated at 1 ⁇ 10 6 cells on the 12th day after electroporation, the cell pellet was washed twice with PBS, 2.5 ul of Biotin-Muc1 antibody was added, and incubation was carried out for 30 min at 4 °C. The cell pellet was washed twice with PBS, 2 ul of PE-streptomycin secondary antibody was added, and the cell pellet was washed twice with PBS, and the cells were transferred to a flow tube by adding 400 ul of PBS, and detected by a machine.
  • Double-antibody sandwich ELISA using a human PD-1 recombinant protein-coated ELISA plate, HRP-labeled mouse anti-human IgG4 mAb, commercialized PD-1 antibody as a standard, 50-fold dilution of the sample to be tested The amount of PD-1 antibody expression in the T cells after gene modification was quantitatively detected.
  • Example 3 PBMCs modified by pNB328-Muc1CAR and pS328-m279V plasmids with different mass ratios Determination of Muc1CAR gene positive rate and PD-1 antibody expression
  • the recombinant plasmids pNB328-Muc1CAR and pS328-m279V were electroporated into PBMCs cells (1:1, 3:5, 1:3, 1:7) at different mass ratios as described in Example 1, to obtain multiple simultaneous expression of Muc1CAR gene and PD.
  • T cells of the -1 antibody Culture after transfection of these electrically day 12 the number of T cells by 2 ⁇ 10 6 cells were collected press 2 ⁇ 10 6 cells cells / well plated in 6-well plates plus 3ml AIM-V culture solution, placed in 37 °C, The cells were cultured in a 5% CO2 incubator, and the cell supernatant was collected after 24 hours of culture, and stored at -20 ° C until use.
  • Double-antibody sandwich ELISA using a human PD-1 recombinant protein-coated ELISA plate, HRP-labeled mouse anti-human IgG4 mAb, commercialized PD-1 antibody as a standard, 50-fold dilution of the sample to be tested
  • the amount of PD-1 antibody in the T cells after gene modification was quantitatively detected.
  • 1 ⁇ 10 6 cell pellet was collected, and the cell pellet was washed twice with PBS, 2.5 ul of Biotin-Muc1 antibody was added, and incubated at 4° C. for 30 min, PBS.
  • the cell pellet was washed twice, 2 ul of PE-streptomycin secondary antibody was added, and the cell pellet was washed twice with PBS.
  • the cells were transferred to a flow tube by adding 400 ul of PBS, and detected by a machine.
  • T cells obtained by electroporation of the pNB328-Muc1CAR plasmid and the pS328-m279V plasmid at a 1:1 mass ratio have higher Muc1 CAR gene and PD-1 antibody secretion as shown in Figs. 3A, 3B and the following table.
  • Example 4 Different patient-derived PBMCs were activated by Muc1CAR gene and PD-1 antibody gene modification. Cell expression of Muc1CAR gene positive rate was determined.
  • Mock T cells, Muc1 CAR T cells, and Muc1 CAR-antiPD1 T cells transfected with pNB328-Muc1CAR and pS328-m279V plasmids were cultured in Example 1, and cell pellets were collected on day 12 at a number of 1 ⁇ 10 6 cells, PBS. Wash the cell pellet 2 times, add 2.5 ul of Biotin-Muc1 antibody, incubate at 4 °C for 30 min, wash the cell pellet 2 times with PBS, add 2 ul of PE-streptomycin secondary antibody, wash the cell pellet 2 times with PBS, and transfer the cells by adding 400 ul PBS. In the flow tube, the machine is detected.
  • the Muc1CAR recombinant protein can be stably expressed on the surface of T cells.
  • Example 5 Different patient-derived pBMCs were activated by Muc1CAR gene and PD-1 antibody gene modification. The cells express quantitative detection of the expression level of PD-1 antibody.
  • Mock T cells, Muc1 CAR T cells and Muc1 CAR-antiPD1 T cells transfected with pNB328-Muc1CAR and pS328-m279V plasmids were cultured in Example 1, and cells were collected at 2 ⁇ 10 6 cell numbers on day 12 and pressed 2 ⁇ 10 6 cells/well, placed in a 6-well plate supplemented with 3 ml of AIM-V medium, placed in a 37 ° C, 5% CO 2 incubator, and collected for 24 h after culture, and stored at -20 ° C. .
  • Double antibody sandwich ELISA method (using human PD-1 recombinant protein coated ELISA plate, HRP-labeled mouse anti-human IgG4 mAb, commercial anti-PD-1 antibody as standard, 50 times sample to be tested) After dilution, the expression level of PD-1 antibody in T cells modified by Muc1CAR gene and PD-1 antibody gene was quantitatively detected.
  • Muc1 CAR T cells modified with the PD-1 antibody gene were able to stably express PD-1 antibodies at a high level.
  • Example 6 Different patient-derived PBMCs cells were modified with Muc1CAR gene and PD-1 antibody gene. Detection of Muc1CAR genome expression levels in the cellular genome.
  • the Mock T cells obtained in Example 1 and the Muc1 CAR T cells and the genomic DNA of the Muc1 CAR-antiPD1 T cells into which the pNB328-Muc1CAR and the pS328-m279V plasmid were transferred were extracted (kit method), and the experimental procedure was carried out by referring to the instructions attached to the kit.
  • the DNA concentrations of Mock T cells, Muc1 CAR T cells and Muc1 CAR-antiPD1 T cells were determined.
  • the expression level of Muc1CAR genome was detected by real-time fluorescent quantitative PCR.
  • the reaction procedure was: 50 ° C, 2 min ⁇ 95 ° C, 10 min ⁇ 95 ° C, 15 s. ⁇ 60 ° C, 1 min, 40 cycles.
  • the CT value of the obtained Muc1CAR genome and the CT value of Actin were calculated according to the corresponding formula to determine the absolute copy number content.
  • Muc1CAR genome was integrated into the T cell genome via the PB transposase system, as shown in the following table:
  • Example 7 Flow cytometry detection of Mock T cells, Muc1 CAR T cells and Muc1 CAR-antiPD1 T cells The difference between phenotype and cytokine secretion.
  • Example 1 The suspended Mock T cells obtained in Example 1 were collected, Muc1CAR T cells and Muc1CAR-antiPD1 T cells transfected with pNB328-Muc1CAR and pS328-m279V plasmids were washed twice in PBS, centrifuged at 1200 rpm for 5 min, and 2 ul of the same type were added.
  • Control antibody IgG1-PE fluorescent flow antibody anti-CD25-PE, anti-LAG3-PE, anti-PD1-PE; isotype control antibody IgG1-PC5, fluorescent flow antibody anti-CD107 ⁇ -PC5; isotype control antibody IgG1FITC, Fluorescent flow antibody anti-CD62L-FITC; isotype control antibody IgG1-PC5, fluorescent flow antibody anti-CD45RO-PC5; isotype control antibody IgG1-PE, fluorescent flow antibody anti-CCR7-PE, light-elastic precipitation to mix Uniform, incubate at room temperature for 30 min in the dark, wash once with PBS, transfer the cells to a flow tube by adding 400 ul PBS, and check on the machine.
  • Muc1CAR-antiPD1 T cells can block the PD-1 protein on the surface of T cells well, and Muc1CAR T cells and Muc1CAR-antiPD1 T cells have obvious killing activity in vitro.
  • the activation marker CD25 was significantly higher than Mock T cells and the depletion marker LAG3 of Muc1CAR-antiPD1 T cells was significantly lower than Mock T cells and Muc1 CAR T cells, as shown in Figures 6A and 6B.
  • a 24-well plate was coated with 5 ug/ml of Muc1 antigen, coated at 4 ° C overnight, washed 3 times with PBS, and 3 ⁇ 10 5 Mock T cells obtained in Example 1, Muc1 CAR T cells and transferred to pNB328- were added.
  • the Muc1CAR-antiPD1 T cells of Muc1CAR and pS328-m279V plasmid were cultured for 24 hours, and the cell supernatant was collected. Detecting Muc1CAR T cells and Muc1CAR-antiPD1 T cell cytokine secretion after antigen stimulation with Muc1 by BD TM CBA Human Th1 / Th2 Cytokine Kit II:
  • Th1/Th2 cytokine standard double dilution 5000pg/ml, 2500pg/ml, 1250pg/ml, 625pg/ml, 312.5pg/ml, 156pg/ml, 80pg/ml, 40pg/ml) , 20pg/ml, 0pg/ml) and 50ul of the sample to be tested (diluted by dilution 2 times);
  • Example 8 Mock T cells, Muc1 CAR T cells and Muc1 CAR-antiPD1 T cells were cultured in vitro Killing experiments of tumor cells.
  • RTCA real-time label-free cell function analyzer
  • Target cell plating cervical cancer cell Hela, liver cancer cell HCC-LM3, lung cancer cell A549 (purchased from the American Type Culture Collection ATCC), placed in a plate containing the detection electrode at 10 4 cells/50 ⁇ l per well, placed After a few minutes, wait for the cells to stabilize, then put them into the instrument and start step 2 to culture the cells;
  • step 2 After the target cells were cultured for 24 hours, the step 2 was suspended, and effector cells (Mock T cells obtained in Example 1, Muc1 CAR T cells, and Muc1CAR-antiPD1 T transfected with pNB328-Muc1CAR and pS328-m279V plasmids were added. Cells), 50 ⁇ l per well, the target ratio was set to 4:1, and the untransformed Mock T cells were used as a control. Step 3 was started, and after 24 hours of co-culture, a cell proliferation curve was obtained.
  • Example 9 In vivo functional studies of Muc1 CAR T cells expressing PD-1 antibodies.
  • the first step 15 to 6 weeks old NSG completely immunodeficient mice, with an average weight of 22 ⁇ 27g, provided by Beijing Weitongda Biotechnology Co., Ltd., SPF animal laboratory.
  • the second step in vitro culture of human cervical cancer Hela, take the logarithmic growth phase adherent growth cells, 0.25% trypsin digestion, centrifugation, collect the cells, resuspend with PBS solution, centrifuge at room temperature for 2 minutes at 3000g, discard the supernatant, and then After resuspending in PBS, the cells were collected by centrifugation, and the cell suspension concentration was adjusted to 5 ⁇ 10 7 /ml.
  • the third step subcutaneous inoculation of HeLa-luc cells in the right flank of the mouse, 0.1 ml / only. After inoculation for about 10 days, the size of the tumor was observed by a living imager. NSG immunodeficient mice were randomly divided into 4 groups, 5 mice in each group, and PBS (100 ul) and Mock T cells obtained in Example 1 and Muc1CAR were respectively administered.
  • Muc1CAR-antiPD1-wt T cells into which pNB328-Muc1CAR and pS328-m279V-wt were transferred, and Muc1CAR-antiPD1T cells (1 ⁇ 10 7 cells/only) into which pNB328-Muc1CAR and pS328-m279V plasmid were transferred.
  • the route of administration is tail vein injection.
  • Step 4 The living state of the mice was observed daily and the tumor changes of the mice were observed by a living imager every 10 days.

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Abstract

L'invention concerne une cellule T CAR à ciblage spécifique de l'antigène Muc1, exprimant de manière stable un anticorps PD-1 à un niveau élevé, ainsi que son utilisation. En particulier, l'invention concerne une cellule T qui contient une séquence codante qui exprime un récepteur antigénique chimérique qui reconnaît l'antigène Muc1 et une séquence codante d'un anticorps PD-1, et/ou qui exprime un récepteur antigénique chimérique qui reconnaît l'antigène Muc1 et un anticorps PD-1. Le récepteur antigénique chimérique comprend, de l'extrémité N-terminale à l'extrémité C-terminale, un peptide signal de protéine membranaire, un anticorps monocaténaire proximal membranaire anti-Muc1, une région charnière ayant au moins 50 résidus d'acides aminés de longueur, une région transmembranaire, un domaine intracellulaire de signal costimulant et un motif d'activation de tyrosine de récepteur immunitaire. La cellule T selon l'invention permet de contrer une inhibition immunitaire microenvironnementale, de favoriser l'apoptose de cellules tumorales, d'exercer une réponse immunitaire antitumorale, et peut donc être utilisée pour traiter une variété de tumeurs malignes positives quant à Muc1.
PCT/CN2018/123527 2017-12-28 2018-12-25 Cellule t car spécifique de muc1 exprimant de façon stable un anticorps pd-1, et son utilisation Ceased WO2019128994A1 (fr)

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EP3769816A1 (fr) * 2019-07-25 2021-01-27 Ospedale Pediatrico Bambino Gesù Vecteur car-cd123 et ses utilisations
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CN113773393B (zh) * 2020-06-10 2023-11-28 广州长峰生物技术有限公司 多特异性单链抗体及其应用
CN111944850B (zh) * 2020-08-28 2023-03-31 澳门大学 表达抗cd22嵌合抗原受体和pd-l1阻断蛋白的细胞的制备方法、表达载体及应用
CN113527494B (zh) * 2020-11-26 2022-08-02 四川大学华西医院 一种新型的抗肿瘤转换受体t细胞
CN113336848B (zh) * 2021-02-03 2022-08-19 上海莱馥医疗科技有限公司 一种抗pd-1抗体及其用途

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