WO2023217192A1 - 基于msln前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用 - Google Patents

基于msln前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用 Download PDF

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WO2023217192A1
WO2023217192A1 PCT/CN2023/093318 CN2023093318W WO2023217192A1 WO 2023217192 A1 WO2023217192 A1 WO 2023217192A1 CN 2023093318 W CN2023093318 W CN 2023093318W WO 2023217192 A1 WO2023217192 A1 WO 2023217192A1
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car
cells
msln
cell
cancer
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French (fr)
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赵旭东
郭婧
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四川大学华西医院
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of immune cell therapy, and specifically relates to the preparation and application of chimeric antigen receptor immune cells constructed based on MSLN precursor protein.
  • MUC16 (CA125) is an important target for tumor treatment. It is usually expressed at low levels in normal tissues. Abnormally expressed MUC16 is often the cause of a variety of diseases. Studies have found that MUC16 is overexpressed in a variety of tumors, such as ovarian cancer, endometrial cancer, pancreatic cancer, colon cancer, breast cancer, and gastric cancer. In fact, as a type of high molecular weight, highly glycosylated protein, the expression of MUC16 in normal cells is affected by complex regulation, and its expression is usually restricted by epithelial cell polarity. However, during carcinogenesis, after cell polarity is lost, MUC16 is expressed on the surface of almost all epithelial cells, interacts with a variety of growth factors, regulates its downstream signaling pathways, and induces the development of cancer.
  • MUC16 monoclonal antibody drug Oregovomab has been used in patients with primary ovarian cancer.
  • an anti-MUC16 antibody drug (JCAR-020), CAR-T is in phase I clinical trials and is also used for targeted treatment of ovarian cancer.
  • JCAR-020 anti-MUC16 antibody drug
  • ADCs antibody drug conjugates
  • CAR-T cell therapy has achieved relatively successful clinical results in the field of hematological malignancies. Its function is to redirect T cells to recognize and eliminate cells expressing specific target antigens. The binding of CAR to target antigens expressed on the cell surface is independent of MHC receptors, resulting in potent T cell activation and potent anti-tumor responses.
  • Traditional CAR is most commonly composed of a single-chain antibody fragment (scFv), a transmembrane region, and a cytoplasmic signaling domain (usually derived from CD8, CD28, OX-40 or 4-1BB).
  • MSLN ovarian cancer antigen
  • MUC16 ovarian cancer antigen
  • the anti-MSLN antibody recognizes the CA125 junction Engage and block mesothelin-MUC16-dependent cell attachment on cancer cells.
  • 64-amino-acid region at the N-terminus of cell surface mesothelin is the smallest fragment with complete binding activity to MUC16 (CA125).
  • the purpose of the present invention is to provide a chimeric antigen receptor immune cell constructed based on MSLN precursor protein and targeting MSLN-binding proteins including MUC16 and its preparation and application methods.
  • a chimeric antigen receptor (CAR)
  • the CAR contains an extracellular binding domain
  • the extracellular binding domain includes an amino acid based on SEQ ID NO:1 the sequence of the MSLN precursor protein or the structure of a fragment thereof
  • the extracellular binding domain can specifically bind to MSLN-binding protein.
  • the binding is ligand-receptor binding.
  • the MSLN-binding protein includes MUC16.
  • the MSLN-binding protein includes MUC16 located on the cell membrane.
  • the extracellular binding domain has an amino acid sequence derived from MSLN precursor protein.
  • the extracellular binding domain includes MSLN precursor protein or a fragment thereof.
  • the extracellular binding domain contains a fragment of membrane-bound mature MSLN in the MSLN precursor protein.
  • amino acid sequence of the membrane-bound mature MSLN fragment is as shown in positions 296 to 598 (preferably positions 296 to 362) of the SEQ ID NO:1 sequence.
  • the extracellular binding domain further contains an N-terminal extension sequence fragment of membrane-bound mature MSLN in the MSLN precursor protein.
  • amino acid sequence of the N-terminal extension sequence fragment is shown in positions 287 to 295 (preferably positions 290 and 295) of the SEQ ID NO:1 sequence.
  • amino acid sequence of the extracellular binding domain is shown in positions 290 to 362 of SEQ ID NO:1.
  • the MSLN-binding protein is derived from humans or non-human mammals.
  • the non-human mammals include: rodents (such as rats, mice), primates (such as monkeys); preferably primates.
  • the extracellular binding domain of the CAR in addition to the first extracellular domain targeting MSLN binding protein, also includes a second extracellular domain targeting additional targets.
  • the additional target is a tumor-specific target.
  • the extracellular binding domain includes MSLN precursor protein or a fragment thereof, and the MSLN precursor protein or a fragment thereof has an amino acid sequence as shown in SEQ ID NO: 1, or has an amino acid sequence as shown in SEQ ID NO: 1 The amino acid sequence at positions 290 to 362 of the sequence shown in ID NO:1.
  • amino acid sequence of the extracellular binding domain is selected from the following group:
  • amino acid residues are replaced, deleted, changed or inserted, or at its N-terminus or C 1 to 30 amino acid residues are added to the end, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence is the same as SEQ ID NO :
  • the sequence shown at positions 290 to 362 of the sequence shown in 1 has ⁇ 85% (preferably ⁇ 90%, more preferably ⁇ 95%, such as ⁇ 96%, ⁇ 97%, ⁇ 98% or ⁇ 99%) Sequence identity; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).
  • the structure of the CAR is as shown in Formula I below: L-EB-H-TM-C-CD3 ⁇ -RP (I)
  • Each "-" is independently a connecting peptide or peptide bond
  • L is none or signal peptide sequence
  • EB is an extracellular binding domain that specifically binds to MSLN-binding protein
  • H is the null or hinge region
  • TM is the transmembrane domain
  • C is no or co-stimulatory signaling molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • RP is a null or reporter protein.
  • the reporter protein RP also includes a self-cleavage recognition site located at its N-terminus, preferably a T2A sequence.
  • the reporter protein RP is a fluorescent protein (such as green fluorescent protein, yellow fluorescent protein, red fluorescent protein).
  • the reporter protein RP is mKate2 red fluorescent protein.
  • amino acid sequence of the mKate2 red fluorescent protein is shown in SEQ ID NO: 2.
  • the L is a signal peptide selected from the following group of proteins: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • the L is a signal peptide derived from CD8.
  • amino acid sequence of L is shown in SEQ ID NO: 3.
  • the H is the hinge region of a protein selected from the following group: CD8, CD28, CD137, or a combination thereof.
  • the H is a hinge region derived from CD8.
  • amino acid sequence of H is shown in SEQ ID NO: 4.
  • the TM is the transmembrane region of a protein selected from the following group: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.
  • the TM is a transmembrane region derived from CD28.
  • amino acid sequence of the TM is shown in SEQ ID NO: 5.
  • the C is a costimulatory signal molecule selected from the following group of proteins: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1(CD11a/CD18), ICOS(CD278), NKG2D, GITR, TLR2, or combinations thereof.
  • the C is a costimulatory signal molecule derived from 4-1BB.
  • amino acid sequence of C is shown in SEQ ID NO: 6.
  • amino acid sequence of the cytoplasmic signaling sequence derived from CD3 ⁇ is shown in SEQ ID NO: 7.
  • amino acid sequence of the chimeric antigen receptor CAR is shown in SEQ ID NO: 8.
  • nucleic acid molecule encoding a chimeric antigen receptor as described in the first aspect of the invention is provided.
  • the nucleic acid molecule has the nucleotide sequence described in SEQ ID NO: 9.
  • a vector is provided, said vector containing the nucleic acid molecule according to the second aspect of the present invention.
  • the vector is selected from the following group: 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 is a pTomo lentiviral vector.
  • the vector also includes elements selected from the following group: promoter, transcription enhancing element WPRE, long terminal repeat sequence LTR, etc.
  • the vector includes the nucleotide sequence shown in SEQ ID NO: 9.
  • 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 the exogenous nucleic acid molecule as described in the second aspect of the present invention is integrated into the chromosome. Or express the CAR as described in the first aspect of the invention.
  • an engineered immune cell contains the vector as described in the third aspect of the present invention or the exogenous DNA as described in the second aspect of the present invention is integrated into the chromosome. Nucleic acid molecules or expressions of a CAR as described in the first aspect of the invention.
  • the engineered immune cells are T cells, NK cells, NKT cells, macrophages or combinations thereof.
  • the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).
  • the engineered immune cells are CAR-T cells.
  • a method for preparing engineered immune cells as described in the fifth aspect of the present invention comprising the following steps: converting the nucleic acid molecule as described in the second aspect of the present invention or the nucleic acid molecule as described in the second aspect of the present invention.
  • the vectors described in the three aspects are transduced into immune cells, thereby obtaining the engineered immune cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • a pharmaceutical composition which pharmaceutical composition contains the CAR as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, and the third aspect of the present invention.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation is an injection.
  • the concentration of the engineered immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml ml.
  • a CAR 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,
  • the host cells described in the fourth aspect, and/or the use of the engineered immune cells described in the fifth aspect of the present invention are used to prepare drugs or preparations for preventing and/or treating diseases with high expression of MSLN-binding protein.
  • the MSLN-binding protein includes MUC16.
  • the diseases associated with high expression of MSLN-binding protein include but are not limited to tumors, aging, obesity, cardiovascular disease, diabetes, neurodegenerative diseases, infectious diseases, etc.
  • the diseases associated with high expression of MSLN-binding protein include: tumors, aging, cardiovascular disease, obesity, etc.
  • the disease is a malignant tumor with high expression of MSLN-binding protein.
  • the high expression of the MSLN-binding protein means that the ratio of the expression level (F1) of the MSLN-binding protein to the expression level (F0) under normal physiological conditions (i.e., F1/F0) ⁇ 1.5, preferably ⁇ 2 , preferably ⁇ 2.5.
  • the tumors include solid tumors and hematological tumors.
  • the solid tumor is selected from the following group: pancreatic cancer, breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioblastoma , lung cancer, prostate cancer, nasopharyngeal cancer, or combinations thereof.
  • the blood tumor is selected from the following group: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse leukemia B-cell lymphoma (DLBCL), or combinations thereof.
  • AML acute myeloid leukemia
  • MM multiple myeloma
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • DLBCL diffuse leukemia B-cell lymphoma
  • an engineered immune cell as described in the fifth aspect of the present invention is provided. cells, or the use of the pharmaceutical composition according to the seventh aspect of the present invention, for preventing and/or treating cancer or tumors.
  • a method for treating diseases comprising administering to a subject in need of treatment an effective amount of engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention.
  • pharmaceutical compositions comprising administering to a subject in need of treatment an effective amount of engineered immune cells as described in the fifth aspect of the present invention, or as described in the seventh aspect of the present invention.
  • the disease is a disease with high expression of MSLN-binding protein.
  • the high expression of the MSLN-binding protein means that the expression level of the MSLN-binding protein is ⁇ 1.5 times the expression level under normal physiological conditions, preferably ⁇ 2 times, and more preferably ⁇ 2.5 times.
  • the disease is cancer or tumor.
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • the method can be used in combination with other treatment methods.
  • the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
  • FIG. 1 shows a schematic diagram of MSLN-CAR vector construction.
  • A is a schematic diagram of the MSLN precursor protein sequence, in which 1-34AA is the signal peptide, 34-596AA is the extracellular domain, 34-286AA is the megakaryocyte enhancer domain, and 296-598AA is the membrane-bound mature MSLN;
  • B is Schematic structural diagram of control plasmids MOCK-CAR and MSLN-CAR (the antigen-binding domain of MSLN-CAR is A Figure 290-362AA), in which the signal peptide, hinge region, and transmembrane region are all derived from human CD8 molecules, and 4-1BB is derived from For human CD137, CD3 ⁇ is derived from human CD3, and mKate2 is a fluorescent marker used to detect CAR expression;
  • C is the HindIII digestion identification of pTomo-MSLN-CAR vector.
  • Figure 2 shows the results of CAR transfection efficiency detection.
  • A is the fluorescence expression results of T cells infected with MOCK-CAR and MSLN-CAR for 72 hours, where BF is the bright field, mKate2 is the fluorescence expression of CAR; B is the fluorescence expression results of flow cytometry.
  • Figure 3 shows the killing effect of MSLN-CAR-T on different tumor cell lines. Among them, A shows the ovarian cancer results. B shows cervical cancer results. C shows breast cancer results.
  • Figure 4 shows the results of MUC16 expression detection in different ovarian cancer cell lines.
  • A shows the expression of MUC16 protein in different ovarian cancer cell lines detected by WB.
  • B shows qPCR detection of MUC16 mRNA levels.
  • C shows the expression level of MUC16 on the cell membrane detected by immunofluorescence.
  • Figure 5 shows the gradient killing results of MSLN-CAR-T on different ovarian cancer cell lines.
  • Figure 6 shows the IFN- ⁇ release results after MSLN-CAR-T kills different ovarian cancer cell lines.
  • FIG. 7 shows that overexpression of MUC16 enhances MSLN-CAR killing in the ovarian cancer cell line SKOV3. effect.
  • A shows the MUC16 protein expression detected by WB.
  • B shows MUC16 mRNA levels detected by qPCR.
  • C shows the expression level of MUC16 on the cell membrane detected by immunofluorescence.
  • D shows the killing assay of SKOV3-MUC16 by MSLN-CAR.
  • E is the test result of cytokine IFN- ⁇ release.
  • Figure 8 shows the killing results of MSLN-CAR-T on the normal cell line HEK-293T.
  • Figure 9 shows MSLN-CAR-T killing ovarian cancer stem cells.
  • a and B are respectively qPCR detection of the expression levels of tumor stem cell surface markers CD133, CD44, CD117 and CD24 in SKOV3-CSC and OVCAR3-CSC.
  • C is qPCR detection of MUC16 mRNA levels in tumor cells and stem cells.
  • D is immunofluorescence detection of the expression level of MUC16 on the cell membrane.
  • E is the detection of the killing of SKOV3-CSC and OVCAR3-CSC by MSLN-CAR-T and the release of cytokines IFN- ⁇ (F) and TNF- ⁇ (H).
  • Figure 10 shows that intraperitoneal and tail vein injection of MSLN-CAR-T cells inhibited the growth of OVCAR3 transplanted tumors in NCG mice.
  • A is the tumor burden monitored by small animal in vivo imaging at specific time points after MSLN-CAR-T intervention.
  • B is the weight change of mice in each group.
  • C is the statistics of tumor growth curve in mice.
  • D is the HE staining of important organs such as the heart, liver, lungs, spleen, kidneys, and brain of NCG mice after CAR-T intervention in two ways.
  • Figure 11 shows that intraperitoneal injection of MSLN-CAR-T cells significantly prolonged the survival of NCG tumor-bearing mice.
  • A is the tumor burden monitored by small animal in vivo imaging at specific time points after MSLN-CAR-T intervention.
  • B is the statistics of tumor growth curve in mice.
  • C is the Kaplan–Meier survival curve statistics from the time of injection of CAR-T cells until the mouse becomes paralyzed or dies.
  • the inventor developed a chimeric antigen receptor immune cell based on MSLN precursor protein for the first time.
  • Single-chain antibody scFv or endogenous receptor/ligand can be used as the target recognition region of CAR.
  • CAR target recognition and activation of intracellular signals are affected by many factors. Whether the obtained CAR works requires a large number of screening tests.
  • the inventors found through testing that using a specific fragment of the mesothelin precursor protein (i.e., amino acid sequence 290-362) as the extracellular binding domain of the CAR, the constructed CAR-T cells can specifically bind to MUC16-positive targets. cells (such as tumor cells), with strong killing ability and high safety. On this basis, the present invention was completed.
  • the term “contains” or “includes” can mean open, semi-closed, and closed. In other words, the term also includes “consisting essentially of” or “consisting of.”
  • Transduction refers to the process of delivering exogenous polynucleotides into host cells, transcribing and translating them to produce polypeptide products, including the use of plasmid molecules to convert exogenous polynucleotides into host cells.
  • the polynucleotide is introduced into a host cell (eg, E. coli).
  • Gene expression or “expression” refers to the process of gene transcription, translation, and post-translational modification to produce the gene's RNA or protein product.
  • Polynucleotide refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and the like. Polynucleotides may include modified nucleotides, such as methylated or capped nucleotides or nucleotide analogs. As used herein, the term polynucleotide refers to interchangeable single- and double-stranded molecules. Unless otherwise stated, polynucleotides in any embodiment described herein include double-stranded forms and two complementary single strands known or predicted to constitute the double-stranded form.
  • 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 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.
  • administering refers to the physical introduction of a 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 intravenously, intramuscularly, subcutaneously, intraperitoneally, spinally or Other routes of parenteral administration, such as by injection or infusion.
  • MSLN binding protein MUC16 (carbohydrate-associated antigen CA125)
  • MSLN-binding protein refers to a protein capable of binding MSLN (please review this definition), including but not limited to MUC16. Yes, the definition is correct
  • CA125 (also known as MUC16) is a highly glycosylated type I transmembrane protein discovered by Bast et al. in 1981 using the monoclonal antibody OC125 produced in mice immunized with human ovarian cancer cells.
  • the first cDNA clone was reported in 2001, with an average molecular weight between 2.5 million and 5 million Daltons, and was highly glycosylated with O-linked and N-linked oligosaccharides.
  • the peptide backbone of MUC16 consists of an N-terminal region, Ser/Thr/Pro-rich tandem repeats (TR) (156 amino acids, each with both N- and O-gly-cosylations) and a short cytoplasmic tail.
  • TR Ser/Thr/Pro-rich tandem repeats
  • the C-terminal region consists of.
  • the SEA domain with high levels of O-glycosylation in the TR repeats binds MSLN.
  • MUC16 is a tumor-specific antigen overexpressed in ovarian cancer. It is currently the most widely used clinically and an important serum biomarker for the diagnosis of ovarian cancer. Serum MUC16 in 90% of ovarian cancer patients is related to disease progression, so it is often used as a marker to monitor disease progression and recurrence. CA125 (MUC16) can inhibit the cytolytic reaction of ovarian cancer natural killer cells and inhibit the immune response against ovarian cancer cells.
  • MUC16 is also overexpressed in other tumors besides ovarian cancer, including cervical cancer, fallopian tube cancer, pancreatic cancer, colon cancer, peritoneal cancer, nasopharyngeal cancer, lung cancer, breast cancer, and gastric cancer. Therefore, it can be used as a target for the treatment of tumors, especially various solid tumors.
  • MSLN Mesothelin
  • the MSLN gene is located on chromosome 1p13.3, with a full length of 8kD.
  • the gene contains an open reading frame of 1884bp, encoding 17 exons and 628 amino acids.
  • the precursor protein of MSLN is a glycoprotein of about 69kD anchored on the cell membrane with glycosylphospholipid peptide inositol. It can be hydrolyzed into two parts by proteolytic enzymes.
  • the N-terminal is a soluble protein of 31kD, which has megakaryocyte stimulating activity.
  • MSLN megakaryocyte-potentiating factor
  • MSLN membrane-bound protein about 40kD long with cell adhesion
  • N-terminus Residues 296-359
  • MUC16-MSLN interaction plays a role in the adhesion process of cancer cells.
  • Anti-MSLN antibodies can eliminate the binding of MSLN to MUC16-expressing cells and block MUC16/mesothelin-dependent cell adhesion.
  • the present invention integrates MSLN precursor protein fragments into CAR vectors through genetic engineering for the first time, and modifies related immune cells, thereby achieving specific killing of MUC16-positive cells and can be used for the treatment of related diseases.
  • the present invention uses the N-terminal fragment of MSLN (SEQ ID NO. 1AA296-362) in the MSLN precursor protein and a fragment composed of 6 amino acids in the N-terminal region to construct the CAR.
  • the CAR of the present invention is constructed based on the MSLN precursor protein fragment and can bind to MSLN receptors including MUC16.
  • Chimeric antigen receptor consists of an extracellular antigen recognition region, It consists of a transmembrane region and an intracellular costimulatory signal region.
  • 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
  • CAR immune cells are isolated, activated and genetically modified to produce CAR immune cells, and then injected into the same patient. In this way, the probability of developing graft-versus-host disease is extremely low, 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. Such a high response rate has not been achieved by any previous treatment method, triggering an upsurge in clinical research around the world.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements.
  • the extracellular domain may be an scFv of an antibody based on the specific binding of an antigen-antibody, or a natural sequence or a derivative thereof based on the specific binding of a ligand-receptor.
  • the extracellular domain of the chimeric antigen receptor is an MSLN precursor protein or a fragment thereof that can specifically bind to the MUC16 target of the CAR of the present invention. More preferably, the extracellular binding domain of the chimeric antigen receptor of the present invention has the amino acid sequence at positions 290 to 362 of the sequence shown in SEQ ID NO:1.
  • the intracellular domain includes costimulatory signaling regions and zeta chain portions.
  • a costimulatory signaling domain refers to the portion of the intracellular domain that includes costimulatory molecules.
  • Costimulatory molecules are cell surface molecules that are required for effective lymphocyte response to antigen, rather than antigen receptors or their ligands.
  • Linkers can be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that serves to connect a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 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, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to fail to grow, be driven to death, or otherwise affected, and cause the patient's tumor burden to shrink or be eliminated.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a zeta chain.
  • the antigen binding domain is fused to the intracellular domain of a combination of the CD28 signaling domain and the CD3 ⁇ signaling domain.
  • the extracellular binding domain of the CAR of the present invention also includes sequence-based conservative variants, which means that compared with the amino acid sequence of positions 290 to 362 of SEQ ID NO: 1, there are at most 10, preferably At most 8, more preferably at most 5, most preferably at most 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide.
  • the number of added, deleted, modified and/or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, and more preferably 1-33%. More preferably, it is 5-30%, more preferably, it is 10-25%, and even more preferably, it is 15-20%.
  • the number of added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1-3, more preferably 1-2, most preferably 1.
  • the CAR can be designed to include the transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domains of the same or different surface membrane proteins, thus minimizing interaction with the receptor complex. Interactions with other members.
  • the intracellular domain in the CAR of the present invention includes the 4-1BB costimulatory domain and the signaling domain of CD3 ⁇ .
  • the CAR is a CAR that can specifically target MUC16.
  • a chimeric antigen receptor immune cell which contains the chimeric antigen receptor of the present invention that specifically targets MUC16.
  • the chimeric antigen receptor immune cells of the present invention can be CAR-T cells, CAR-NK cells, or CAR-macrophages.
  • the chimeric antigen receptor immune cells of the present invention are CAR-T cells.
  • CAR-T cell As used herein, the terms "CAR-T cell”, “CAR-T” and “CAR-T cell of the present invention” all refer to the CAR-T cell described in the fifth aspect of the present invention.
  • 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, targeting a certain Once the CAR gene construction of tumor markers 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) Using the patient’s autologous cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cells As used herein, the terms “CAR-NK cells”, “CAR-NK” and “CAR-NK cells of the present invention” all refer to the CAR-NK cells described in the fifth aspect of the present invention.
  • the CAR-NK cells of the present invention can be used for tumors with high expression of MUC16.
  • Natural killer (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.
  • Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with 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 of the body; (2) they release very A small amount of cytokines thus reduces the risk of cytokine storm; (3) It is easy to amplify in vitro and develop into "off-the-shelf" products. Otherwise, it is similar to CAR-T cell therapy.
  • Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology to isolate directly from cells and tissues containing the gene.
  • interested in of genes can be produced synthetically.
  • the invention also provides vectors comprising the nucleic acid molecules of the invention.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia virus, in that 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 invention is typically operably linked to a promoter and incorporated into an expression vector.
  • This vector is suitable for replication and integration into eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences, and promoters that can be used to regulate 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 by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but 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, for example, in 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, herpesviruses, and lentiviruses.
  • a suitable vector will contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter elements can modulate the frequency with which transcription is initiated.
  • these are located in a region of 30-110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when the elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased by 50 bp before activity begins to decrease.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • 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 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, Mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter , Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the invention.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turning 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 contain either or both a selectable marker gene or a reporter gene to facilitate the identification of populations of cells that are transfected or infected by the viral vector. Identify and select expressing cells.
  • the selectable marker can be carried on a separate stretch of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at appropriate times after the DNA has been introduced into the recipient cell.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79 -82).
  • the reporter gene is a gene encoding mKate2 red fluorescent protein.
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.
  • the construct with a minimum of 5 flanking regions that shows the highest level of reporter gene expression is identified as the promoter.
  • Such promoter regions can be ligated to a reporter gene and used to evaluate the ability of an agent to regulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, eg, a mammalian, bacterial, yeast or insect cell, 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 of producing cells including 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). A preferred method of 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 especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, such as human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenovirus and adeno-associated virus, among others. See, for example, US 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 systems useful as delivery vehicles in vitro and in vivo are liposomes (eg, artificial membrane vesicles).
  • an exemplary delivery vehicle is liposomes.
  • lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • the nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of the liposomes, dispersed within the lipid bilayer of the liposomes, attached via linker molecules associated with both the liposomes and the oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, 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 lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any specific structure in solution.
  • Lipids are fatty substances, which may be naturally occurring or synthetic lipids.
  • lipids include lipid droplets that occur naturally in the cytoplasm as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.
  • the vector is a lentiviral vector.
  • the invention provides a method containing the chimeric antigen receptor CAR according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the vector according to the third aspect of the invention, or the fourth aspect of the invention.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • the invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LV) encoding expression cassettes of the invention.
  • T cells eg, T cells
  • LV lentiviral vectors
  • the transduced T cells can target the tumor cell marker MUC16, synergistically activate T cells, and induce immune cell immune responses, thus significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, comprising the steps of administering to the mammal a CAR-cell of the present invention.
  • the present invention includes a type of cell therapy in which a patient's autologous T cells (or allogeneic donors) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • This method has a very low probability of suffering from graft-versus-host disease, and the antigen is recognized by T cells in an MHC-free manner.
  • one CAR-T can treat all cancers that express this antigen.
  • CAR-T cells can Endoreplicate, resulting in long-term persistence that can lead to sustained tumor control.
  • CAR-T cells of the invention can undergo robust in vivo T cell expansion for an extended amount of time.
  • CAR-mediated immune responses can be part of an adoptive immunotherapy step, in which CAR-modified T cells induce an immune response specific for the antigen-binding domain in the CAR.
  • MUC16 CAR-T cells elicit a cell-specific immune response against MUC16.
  • Treatable cancers include tumors that are not vascularized or substantially unvascularized, as well as tumors that are vascularized.
  • Cancer includes non-solid tumors (such as hematological tumors, such as leukemias and lymphomas) and solid tumors.
  • Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas.
  • Hematologic cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic types , monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disorders, myelodysplastic syndromes, hairy cell leukemia, and myelodysplasia.
  • the CAR-modified T cells of the present invention may also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is human.
  • cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the invention provides methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-modified T cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • a pharmaceutical composition of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Mixtures such as EDTA or glutathione; adjunctive agents (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelates such as EDTA or glutathione
  • adjunctive agents e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • compositions of the present invention may be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount 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 - although appropriate dosages may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by the physician, It takes into account the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease. It may generally be stated that pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight (including all integers within those ranges). value) application. T cell compositions can also be administered multiple times at these dosages.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., before , simultaneously or subsequently) administered to a patient, such forms of treatment include, but are not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with specific tumors.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as for ARA-C) or natalizumab treatment in patients with MS or elfalizumab treatment in patients with psoriasis or other treatments in patients with specific tumors.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cellular compositions of the invention are administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, use of a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient of the treatment. Dosage proportions for human administration may be implemented in accordance with art-accepted practice. Generally, 1 ⁇ 10 6 to 1 ⁇ 10 10 CAR-T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, by intravenous infusion.
  • MUC16 is basically not expressed on the cell membrane of normal cells, but it is expressed in some tumors. Highly expressed on cell membranes.
  • the CAR immune cells of the present invention only target malignant cells with high expression of MUC16 on their cell membranes, and have little effect on other cells that do not express or lowly express MUC16.
  • the present invention uses the mode of action of combining ligands with receptors instead of scfv in the traditional sense.
  • the selectivity and affinity of receptor-ligand interactions are the result of long-term natural selection.
  • the conservation of receptor-ligand interactions determines that safety tests in animals, especially primates, are more reflective of their use in humans. security.
  • Table 2 shows the cell lines used in the examples.
  • the sequencing primers are universal sequencing primers. Both sequencing and enzyme digestion identification results showed that the CAR coding sequence was correctly inserted into the predetermined position of the plasmid ( Figure 1C).
  • All plasmids were extracted using QIAGEN's endotoxin-free large extraction kit, and the purified plasmids were transfected into HEK-293T cells using Beyotime lipo6000 for lentivirus packaging.
  • HEK-293T cells were cultured in 15 cm culture dishes for virus packaging.
  • 2ml OPTIMEM-dissolved plasmid mixture core plasmid 20 ⁇ g, pCMV ⁇ R8.9 10 ⁇ g, PMD2.G 4 ⁇ g
  • 2ml OPTIMEM and 68 ⁇ l of lipo6000 After standing at room temperature for 5 minutes, add the plasmid complex to the liposome complex and let stand at room temperature for 20 minutes. Add the above mixture dropwise to 293T cells, incubate at 37°C for 6 hours and then remove the medium. Re-add pre-warmed complete medium.
  • Example 4 Detection of positive rate of infected CAR-T cells by flow cytometry
  • CAR-T cells and NT cells (control group) 72 hours after virus infection were collected by centrifugation respectively. After washing once with PBS, the supernatant was discarded and the cells were resuspended in PBS containing 2% FBS. The positive rate was detected by flow cytometry.
  • OVCAR3, SKOV3, Hela, MDA-MB-468, MDA-MB-231, and HEK-293T cells were screened with Puromycin (1 ⁇ g/ml) for 2 weeks.
  • OVCAR3, SKOV3, Hela, MDA-MB-468, MDA-MB-231, HEK-293T-luciferase cells were screened with Puromycin (1 ⁇ g/ml) for 2 weeks.
  • the target cells used include: target cells with high expression of MUC16: OVCAR3; target cells with no or low expression of MUC16: SKOV3, Hela, MDA-MB-468, MDA-MB-231.
  • the cell density was adjusted to 2.5 ⁇ 10 4 /ml.
  • 100 ⁇ l of OVCAR3 and SKOV3-luciferase cells were seeded in a 96-well plate, and the cell density of CAR-T and NT cells was adjusted to 2.5 ⁇ 10 4 , 5 ⁇ 10 4 , 1 ⁇ 10 5 , and 2 ⁇ 10 5 /ml, according to E :T is 1:1, 2:1, 4:1, 8:1 and inoculated into a black 96-well plate, with 100 ⁇ l in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.
  • the cell supernatant was collected and stored at -80°C to detect IFN- ⁇ release (see Example 8).
  • Cell killing was detected using the promega fluorescence detection kit.
  • the cells were treated with 30 ⁇ l 1*PLB lysis buffer for 20 minutes. After adding 30 ⁇ l substrate to each well, the cells were immediately detected using a BioTek microplate reader.
  • % Cytotoxic Killing Cells (1-target cell fluorescence value when containing effector cells/target cell fluorescence value when there are no effector cells) ⁇ 100%
  • Results The results of the killing effect of MSLN-CAR-T on different tumor cell lines are shown in Figure 3.
  • the results in ovarian cancer cells (A), cervical cancer cells (B) and breast cancer cells (C) show that MSLN-CAR-T has good killing effect on a variety of tumor cell lines.
  • Example 7 Expression of MUC16 in ovarian cancer cells and CAR-T cell toxicity experiment
  • (1) Cell immunofluorescence Spread the target cells on a disc in a 24-well plate, fix the cells with 4% paraformaldehyde (PFA) for 20 minutes after 24 hours, wash three times with PBST, 5 minutes each time; use 10% goat serum Block for 1 hour at room temperature and incubate overnight with an antibody that specifically recognizes NUC16 at four degrees. The next day, wash three times with PBST for five minutes each time. Use CY3-labeled secondary antibody that specifically recognizes the primary antibody and incubate at room temperature for 1 hour. After washing three times with PBST, the nuclei were stained with DAPI. Confocal microscopy imaging.
  • PFA paraformaldehyde
  • Results The results of MUC16 expression detection in each cell line are shown in Figure 4.
  • the results consistently showed that OVCAR3 highly expressed MUC16 and SKOV3 expressed low MUC16.
  • Further immunofluorescence localization verified that MUC16 was highly expressed on the OVCAR3 cell membrane, while MUC16 was basically not expressed on the SKOV3 cell membrane ( Figure 4C).
  • Example 8 CAR-T targeting of ovarian cancer cells accompanied by release of cytokine IFN- ⁇
  • the release of cytokines when the CAR-T cells of the present invention are co-incubated with target cells is detected.
  • the cell supernatant incubated in the cell killing experiment was used for detection.
  • the method is as follows: Take the cell supernatant co-incubated with CAR-T cells of the present invention and OVCAR3 and SKOV3 target cells (E:T ratio is 4:1) in Example 7 and detect IFN- ⁇ according to IFN gamma Human ELISA Kit (life technology) .
  • Example 9 Effect of overexpressing MUC16 on MSLN-CAR-T tumor killing effect
  • the MUC16 overexpression plasmid (EX-Y1397-Lv183, ORF lentiviral expression clone) was purchased from Yijin Biotechnology to construct a SKOV3 stable overexpression MUC16 cell line.
  • Lenti-MUC16-EGFP-NeoR lentivirus packaging steps are the same as in Example 2. After the virus infected SKOV3 cells, they were screened with Neomycin (3 ⁇ g/ml) for 2 weeks, and SKOV3-MUC16 cells were successfully obtained. in the egg The overexpression efficiency was detected at the white and gene level, and MSLN-CAR-T killing assay was performed.
  • the cells of different ovarian cancer cell lines SKOV3-Vector and SKOV3-MUC16-luciferase were digested and counted and then the cell density was adjusted to 2.5 ⁇ 10 4 /ml. Inoculate 100 ⁇ l of luciferase cells in a 96-well plate, adjust the cell density of CAR-T/NT cells to 1 ⁇ 10 5 , and inoculate them into a black 96-well plate according to E:T ratio of 4:1, with 100 ⁇ l in each well. Mix the above target cells and T cells and incubate them in an incubator for 24 hours.
  • Results The overexpression efficiency and the killing results of MSLN-CAR-T on the ovarian cancer cell line SKOV3 after overexpressing MUC16 are shown in Figure 7.
  • Figure 7-A shows WB detection of MUC16 protein expression.
  • Figure 7-B shows qPCR detection of MUC16mRNA levels.
  • Figure 7-C shows the expression level of MUC16 on the cell membrane detected by immunofluorescence. The results all showed that SKOV3 cells overexpressing MUC16 were successfully constructed.
  • Figure 7-D shows the killing effect of MSLN-CAR-T on SKOV3 after overexpressing MUC16.
  • Figure 7-E shows the release of IFN- ⁇ in the killing effect of MSLN-CAR-T on SKOV3 after overexpression of MUC16.
  • the results showed that compared with the control group SKOV3-Vector, the killing rate and IFN- ⁇ release of MSLN-CAR-T cells on SKOV3-MUC16 cells overexpressing MUC16 were significantly increased. This result shows that the killing effect of MSLN-CAR-T cells on MUC16 overexpressing tumor cells is significantly enhanced.
  • HEK-293T cells are a human embryonic kidney cell line. They are inoculated into a black 96-well plate at an effect-to-target ratio of 4:1. MSLN-CAR-T cells and HEK-293T-luciferase cells are co-incubated. MSLN-CAR-T cells are co-incubated with fluorescence value changes. CAR-T killing of HEK-293T cells.
  • Figure 8-A shows WB detection of MUC16 protein expression.
  • Figure 8-B shows qPCR detection of MUC16mRNA levels.
  • Figure 8-C shows the expression level of MUC16 on the cell membrane detected by immunofluorescence.
  • Figure 8-D shows the killing effect of MSLN-CAR-T on HEK-293T.
  • Figure 8-E shows the release of IFN- ⁇ from the killing effect of MSLN-CAR-T on HEK-293T.
  • Example 11 MSLN-CAR-T kills ovarian cancer stem cells
  • Figure 9-A/B shows the qPCR detection of the expression levels of cancer stem cell markers CD133, CD44, CD117, and CD24.
  • Figure 9-C shows qPCR detection of MUC16 mRNA levels in tumor cells and stem cells.
  • Figure 9-D shows the expression level of MUC16 on the cell membrane detected by immunofluorescence.
  • Figure 9-E shows the detection of the killing of SKOV3-CSC and OVCAR3-CSC by MSLN-CAR-T and the release of cytokines IFN- ⁇ (F) and TNF- ⁇ (H).
  • Example 12 Inhibitory effect of intraperitoneal and tail vein injection of MSLN-CAR-T cells on OVCAR3 transplanted tumors in NCG mice
  • OVCAR3-luciferase was digested and counted, and the cell density was adjusted to 5 ⁇ 10 6 /ml.
  • Six-week-old female NCG mice were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. and raised in the SPF animal room for about 1 week. They were inoculated intraperitoneally (IP) with 100 ⁇ l of tumor cell suspension.
  • IP intraperitoneally
  • MSLN-CAR-T IP intraperitoneal injection group
  • CD19- CAR-T IV
  • tail vein injection group MSLN-CAR-T
  • MSLN-CAR-T IV
  • tail vein injection group MSLN-CAR-T
  • CD19-CAR-T and MSLN-CAR-T were prepared as described above.
  • mice small animal in vivo imaging was performed every 7/14 days to monitor tumor burden and record changes in mouse body weight.
  • the main organs of the mice (heart, liver, spleen, lungs, kidneys, and brain) were collected and stained with HE.
  • Figure 10-A shows the in vivo imaging monitoring of tumor burden in small animals at specific time points after MSLN-CAR-T intervention.
  • Figure 10-B shows the changes in body weight of mice in each group.
  • Figure 10-C shows the statistics of tumor growth curves in mice.
  • Figure 10-D shows the HE staining of important organs such as the heart, liver, lungs, spleen, kidneys, and brain of NCG mice after CAR-T intervention in two ways.
  • Example 13 Intraperitoneal injection of MSLN-CAR-T cells prolongs the survival of NCG tumor-bearing mice
  • OVCAR3-luciferase was digested and counted, and the cell density was adjusted to 5 ⁇ 10 6 /ml.
  • Six-week-old female NCG mice were purchased from Nanjing Jicui Yaokang Biotechnology Co., Ltd. and raised in the SPF animal room for about 1 week. They were inoculated intraperitoneally (IP) with 100 ⁇ l of tumor cell suspension.
  • IP intraperitoneally
  • CD19-CAR-T and MSLN-CAR-T were prepared as described above.
  • CAR-T cells Count CAR-T cells and adjust the cell density to 1 ⁇ 10 8 /ml. Each mouse was injected with 200 ⁇ l of CAR-T cell suspension through the abdominal cavity or tail vein. liquid. Thereafter, small animal in vivo imaging was performed every 10 days and changes in tumor fluorescence signal were recorded. The survival conditions of the two groups of mice were recorded, and the survival period was calculated.
  • Figure 11-A shows the in vivo imaging monitoring of tumor burden in small animals at specific time points after MSLN-CAR-T intervention.
  • Figure 11-B shows the statistics of tumor growth curves in mice.
  • Figure 11-C shows Kaplan-Meier survival curve statistics from the time of injection of CAR-T cells until the mouse becomes paralyzed or dies.
  • Single-chain antibody scFv or endogenous receptor/ligand can be used as the target recognition region of CAR.
  • CAR target recognition and activation of intracellular signals are affected by many factors. Whether the obtained CAR works requires a lot of research work.
  • Previous studies have suggested that MSLN and MUC16 interact and can bind to each other.
  • the present invention uses a specific fragment of the MSLN precursor protein including the MSLN fragment and its N-terminal extension sequence (i.e., amino acid sequence 290-362) as Extracellular binding domain of CAR.
  • the inventor's research found that the CAR-T cells constructed using this fragment as the extracellular binding domain of the CAR can specifically bind to MUC16-positive target cells (such as tumor cells), with strong killing ability and high safety.

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Abstract

本发明提供了一种基于MSLN前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用。具体地，本发明提供了一种基于MSLN前体蛋白改造的嵌合抗原受体(CAR)，所述的CAR含有一胞外结合域，所述的胞外结合域能够特异性地靶向包括MUC16在内的MSLN结合蛋白。本发明的CAR免疫细胞具有高特异性和高效杀伤能力，且安全性高。

Description

基于MSLN前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用 技术领域
本发明属于免疫细胞治疗领域，具体涉及一种基于MSLN前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用。
背景技术
随着医疗水平的提高，肿瘤的防治取得了巨大的进展，但是仍然有多种肿瘤的诊治存在较大困难，例如由于早期卵巢癌通常没有明显的症状，因此常常在癌症扩散(例如扩散到肝脏或肺部)的晚期才诊断出该病，五年生存率较低，预后极差。如何快速精确地诊断早期肿瘤，为缺乏手术治疗指征的患者提供新的治疗方法一直是医学工作者的探索方向。随着影像学、分子生物学的快速进展，新的肿瘤细胞特异性靶点不断涌现，这些特异性靶点是肿瘤精确诊疗的基础。
MUC16(CA125)是肿瘤治疗的一个重要靶点，通常在正常组织中表达量较低，异常表达的MUC16往往是多种疾病的诱因。研究发现MUC16在多种肿瘤中过度表达，如卵巢癌、子宫内膜癌、胰腺癌、结肠癌、乳腺癌和胃癌等。事实上，作为一类高分子量、高度糖基化蛋白，MUC16在正常细胞中的表达受到复杂的调控影响，其表达通常被上皮细胞极性限制。然而，在癌变过程中，细胞极性丧失后，MUC16表达于几乎全部的上皮细胞表面，并与多种生长因子相互作用，调节其下游信号通路，诱导癌症的发展。
近期，MUC16单抗药物Oregovomab已用于原发性卵巢癌患者的用药。其次，一项抗MUC16抗体药物(JCAR-020)即CAR-T正处于一期临床试验，同样是用于卵巢癌靶向治疗。除此外，包括双特异性抗体(BiTE)及抗体偶联药物(ADC)等多项靶向MUC16的药物也正在进一步开发。
作为目前最受欢迎的靶向治疗方式之一，CAR-T细胞疗法已经在血液系统恶性肿瘤领域的取得较为成功的临床结果。其功能是重新引导T细胞识别和消除表达特定靶抗原的细胞。CAR与细胞表面上表达的靶抗原的结合不依赖于MHC受体，从而导致强有力的T细胞活化和强大的抗肿瘤反应。传统CAR最常见是由单链抗体片段(scFv)、跨膜区、细胞质信号域(通常来源于CD8、CD28、OX-40或4-1BB)组成。而近期研究发现，通过机体内自然选择的方式构建嵌合抗原受体T细胞，易于其区分恶性癌细胞(肿瘤细胞)与健康细胞(非肿瘤细胞)。通过改善嵌合抗原受体T细胞的持久性、存活度与增殖性，期望达到更好的抗肿瘤效果。例如内源配体-受体间的相互识别经过长期的自然选择，于是以内源受体-配体作为靶点识别区域也是CAR构建的重要选择。
肿瘤形成过程中，MSLN与卵巢癌抗原(MUC16)结合，促进癌细胞的黏附，从而促进肿瘤的胸腹膜种植和转移扩散。而抗MSLN抗体识别CA125结 合域并阻断癌细胞上的间皮素-MUC16依赖性的细胞附着。此外，研究证明，细胞表面间皮素N端由64个氨基酸组成的区域(残基296-359)是具有与MUC16(CA125)完全结合活性的最小片段。
因此，本领域需要开发以MUC16(CA125)为靶点的CAR-T疗法。
发明内容
本发明的目的就是提供一种基于MSLN前体蛋白构建的、以包括MUC16在内的MSLN结合蛋白为靶点的嵌合抗原受体免疫细胞及其制备和应用方法。
在本发明的第一方面，提供了一种嵌合抗原受体(CAR)，所述的CAR含有一胞外结合域，并且所述的胞外结合域包括基于SEQ ID NO:1所示氨基酸序列的MSLN前体蛋白或其片段的结构，
并且，所述的胞外结合域能够特异性地结合于MSLN结合蛋白。
在另一优选例中，所述的结合为配体受体结合。
在另一优选例中，所述的MSLN结合蛋白包括MUC16。
在另一优选例中，所述的MSLN结合蛋白包括位于细胞膜上的MUC16。
在另一优选例中，所述的胞外结合域具有来源于MSLN前体蛋白的氨基酸序列。
在另一优选例中，所述的胞外结合域包括MSLN前体蛋白或其片段。
在另一优选例中，所述的胞外结合域含有MSLN前体蛋白中膜结合成熟MSLN的片段。
在另一优选例中，所述膜结合成熟MSLN片段的氨基酸序列如SEQ ID NO:1序列的296至598位(优选地为296位至362位)所示。
在另一优选例中，所述的胞外结合域进一步含有MSLN前体蛋白中膜结合成熟MSLN的N端延伸序列片段。
在另一优选例中，所述的N端延伸序列片段的氨基酸序列如SEQ ID NO:1序列的287至295位(优选地为290位295位)所示。
在另一优选例中，所述的胞外结合域的氨基酸序列如SEQ ID NO:1序列的290至362位所示。
在另一优选例中，所述的MSLN结合蛋白来源于人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括：啮齿动物(如大鼠、小鼠)、灵长动物(如猴)；优选为灵长动物。
在另一优选例中，所述的CAR的胞外结合域除了含有针对MSLN结合蛋白的第一胞外结构域之外，还包括针对额外靶点的第二胞外结构域。
在另一优选例中，所述的额外靶点为肿瘤特异性靶点。
在另一优选例中，所述的胞外结合域包括MSLN前体蛋白或其片段，所述的MSLN前体蛋白或其片段具有如SEQ ID NO:1所示的氨基酸序列，或具有如SEQ ID NO:1所示序列的第290至362位的氨基酸序列。
在另一优选例中，所述的胞外结合域的氨基酸序列选自下组：
(i)如SEQ ID NO:1所示序列的第290至362位所示的序列；和
(ii)在如SEQ ID NO:1所示序列的第290至362位所示序列的基础上，进行一个或多个氨基酸残基的替换、缺失、改变或插入，或在其N端或C端添加1至30个氨基酸残基，较佳地1至10个氨基酸残基，更佳地1至5个氨基酸残基，从而获得的氨基酸序列；并且所述获得的氨基酸序列与如SEQ ID NO:1所示序列的第290至362位所示序列具有≥85％(优选地≥90％，更优选地≥95％，例如≥96％、≥97％、≥98％或≥99％)的序列同一性；并且所获得的氨基酸序列与(i)所示的序列具有相同或相似的功能。
在另一优选例中，所述CAR的结构如下式I所示：
L-EB-H-TM-C-CD3ζ-RP   (I)
式中，
各“-”独立地为连接肽或肽键；
L是无或信号肽序列；
EB是胞外结合域,所述胞外结合域特异性地结合于MSLN结合蛋白；
H是无或铰链区；
TM是跨膜结构域；
C是无或共刺激信号分子；
CD3ζ是源于CD3ζ的胞浆信号传导序列；
RP是无或报告蛋白。
在另一优选例中，所述的报告蛋白RP中还包括位于其N端的自剪切识别位点，优选地为T2A序列。
在另一优选例中，所述的报告蛋白RP为荧光蛋白(如绿色荧光蛋白、黄色荧光蛋白、红色荧光蛋白)。
在另一优选例中，所述的报告蛋白RP为mKate2红色荧光蛋白。
在另一优选例中，所述的mKate2红色荧光蛋白的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述的L是选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述的L是CD8来源的信号肽。
在另一优选例中，所述L的氨基酸序列如SEQ ID NO:3所示。
在另一优选例中，所述的H是选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的H是CD8来源的铰链区。
在另一优选例中，所述H的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述的TM是为选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的TM是CD28来源的跨膜区。
在另一优选例中，所述TM的氨基酸序列如SEQ ID NO:5所示。
在另一优选例中，所述的C是选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2，或其组合。
在另一优选例中，所述的C是4-1BB来源的共刺激信号分子。
在另一优选例中，所述C的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，所述的源于CD3ζ的胞浆信号传导序列的氨基酸序列如SEQ ID NO:7所示。
在另一优选例中，所述的嵌合抗原受体CAR的氨基酸序列如SEQ ID NO:8所示。
在本发明的第二方面，提供了一种核酸分子，所述核酸分子编码如本发明第一方面所述的嵌合抗原受体。
在另一优选例中，所述的核酸分子具有如SEQ ID NO:9所述的核苷酸序列。
在本发明的第三方面，提供了一种载体，所述的载体含有如本发明第二方面所述的核酸分子。
在另一优选例中，所述的载体选自下组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、逆转录病毒载体、转座子、或其组合。
在另一优选例中，所述载体为慢病毒载体。
在另一优选例中，所述载体选自下组：pTomo慢病毒载体、plenti、pLVTH、pLJM1、pHCMV、pLBS.CAG、pHR、pLV等。
在另一优选例中，所述的载体是pTomo慢病毒载体。
在另一优选例中，所述载体中还包括选自下组的元件：启动子、转录增强元件WPRE、长末端重复序列LTR等。
在另一优选例中，所述载体包含如SEQ ID NO:9所示的核苷酸序列。
在本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞含有如本发明第三方面所述的载体或染色体中整合有外源的如本发明第二方面所述的核酸分子或表达如本发明第一方面所述的CAR。
在本发明的第五方面，提供了一种工程化免疫细胞，所述的免疫细胞含有如本发明第三方面所述的载体或染色体中整合有外源的如本发明第二方面所述的核酸分子或表达如本发明第一方面所述的CAR。
在另一优选例中，所述的工程化免疫细胞为T细胞、NK细胞、NKT细胞、巨噬细胞或其组合。
在另一优选例中，所述的工程化的免疫细胞是嵌合抗原受体T细胞(CAR-T细胞)或嵌合抗原受体NK细胞(CAR-NK细胞)。
在另一优选例中，所述的工程化免疫细胞是CAR-T细胞。
在本发明的第六方面，提供了一种制备如本发明第五方面所述的工程化免疫细胞的方法，包括以下步骤：将如本发明第二方面所述的核酸分子或如本发明第三方面所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
在本发明的第七方面，提供了一种药物组合物，所述药物组合物含有如本发明第一方面所述的CAR、如本发明第二方面所述的核酸分子、如本发明第三方面所述的载体、如本发明第四方面所述的宿主细胞，和/或如本发明第五方面所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型为注射剂。
在另一优选例中，所述制剂中所述工程化的免疫细胞的浓度为1×103-1×108个细胞/ml，较佳地1×104-1×107个细胞/ml。
在本发明的第八方面，提供了一种如本发明第一方面所述的CAR、如本发明第二方面所述的核酸分子、如本发明第三方面所述的载体、如本发明第四方面所述的宿主细胞，和/或如本发明第五方面所述的工程化免疫细胞的用途，用于制备预防和/或治疗MSLN结合蛋白高表达的疾病的药物或制剂。
在另一优选例中，所述的MSLN结合蛋白包括MUC16。
在另一优选例中，所述的MSLN结合蛋白高表达相关的疾病包括但不限于肿瘤、衰老、肥胖、心血管疾病、糖尿病、神经退行性疾病、感染性疾病等。
在另一优选例中，所述的MSLN结合蛋白高表达相关的疾病包括：肿瘤、衰老、心血管疾病、肥胖等。
在另一优选例中，所述的疾病是MSLN结合蛋白高表达的恶性肿瘤。
在另一优选例中，所述MSLN结合蛋白高表达是指MSLN结合蛋白表达量(F1)与正常生理状况下表达量(F0)之比(即F1/F0)≥1.5，较佳地≥2，更佳地≥2.5。
在另一优选例中，所述肿瘤包括实体瘤和血液肿瘤。
在另一优选例中，所述的实体瘤选自下组：胰腺癌、乳腺癌、胃癌、肝胆癌、结直肠癌、膀胱癌、非小细胞肺癌、卵巢癌和食道癌、胶质细胞瘤、肺癌、前列腺癌、鼻咽癌或其组合。
在另一优选例中，所述血液肿瘤选自下组：急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)、弥漫性大B细胞淋巴瘤(DLBCL)，或其组合。
在本发明的第九方面，提供了一种如本发明第五方面所述的工程化免疫细 胞、或如本发明第七方面所述的药物组合物的用途，用于预防和/或治疗癌症或肿瘤。
在本发明的第十方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用有效量的如本发明第五方面所述的工程化免疫细胞、或如本发明第七方面所述的药物组合物。
在另一优选例中，所述疾病为MSLN结合蛋白高表达的疾病。
在另一优选例中，所述MSLN结合蛋白高表达是指MSLN结合蛋白表达量为正常生理状况下表达量的≥1.5倍，较佳地≥2倍，更佳地≥2.5倍。
在另一优选例中，所述疾病为癌症或肿瘤。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于所述对象的细胞(自体细胞)。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于健康个体的细胞(异体细胞)。
在另一优选例中，所述的方法可与其他治疗方法联合使用。
在另一优选例中，所述的其他治疗方法包括化疗、放疗、靶向治疗等方法。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了MSLN-CAR载体构建示意图。其中，A为MSLN前体蛋白序列示意图，其中1-34AA为信号肽、34-596AA为胞外结构域、34-286AA为巨核细胞增强因子结构域、296-598AA为膜结合成熟MSLN；B为对照组质粒MOCK-CAR及MSLN-CAR结构示意图(其中MSLN-CAR的抗原结合域为A图290-362AA)，其中信号肽、铰链区、跨膜区均来源于人CD8分子，4-1BB来自于人CD137，CD3ζ来源于人CD3，mKate2为荧光标记，用于检测CAR表达；C为pTomo-MSLN-CAR载体HindIII酶切鉴定。
图2显示了CAR转染效率检测结果。其中，A为MOCK-CAR及MSLN-CAR感染T细胞72小时后细胞荧光表达结果，其中BF为明场，mKate2为CAR荧光表达；B为流式检测荧光表达结果。
图3显示了MSLN-CAR-T对不同肿瘤细胞系的杀伤作用。其中，A显示了卵巢癌结果。B显示了宫颈癌结果。C显示了乳腺癌结果。
图4显示了不同卵巢癌细胞系MUC16表达检测的结果。其中，A显示了WB检测MUC16蛋白在不同卵巢癌细胞系的表达。B显示了qPCR检测MUC16mRNA水平。C显示了免疫荧光检测MUC16在细胞膜上的表达水平。
图5显示了MSLN-CAR-T对不同卵巢癌细胞系的梯度杀伤结果。
图6显示了MSLN-CAR-T对不同卵巢癌细胞系杀伤后的IFN-γ释放结果。
图7显示了在卵巢癌细胞系SKOV3中过表达MUC16增强MSLN-CAR杀伤 作用。其中，A显示了WB检测的MUC16蛋白表达。B显示了qPCR检测的MUC16mRNA水平。C显示了免疫荧光检测MUC16在细胞膜上的表达水平。D显示了MSLN-CAR对SKOV3-MUC16的杀伤检测。E为细胞因子IFN-γ释放检测结果。
图8显示了MSLN-CAR-T对正常细胞系HEK-293T的杀伤结果。
图9显示了MSLN-CAR-T杀伤卵巢癌干细胞。其中，A、B分别为qPCR检测SKOV3-CSC、OVCAR3-CSC肿瘤干细胞表面标志物CD133、CD44、CD117、CD24的表达水平。C为qPCR检测肿瘤细胞与干细胞MUC16mRNA水平。D为免疫荧光检测MUC16在细胞膜上的表达水平。E为MSLN-CAR-T对SKOV3-CSC及OVCAR3-CSC的杀伤及细胞因子IFN-γ(F)、TNF-α(H)释放检测。
图10显示了腹腔及尾静脉注射MSLN-CAR-T细胞均抑制NCG小鼠中OVCAR3移植瘤的生长。其中，A为MSLN-CAR-T干预后特定时间点小动物活体成像监测肿瘤负荷。B为各组小鼠体重变化。C为小鼠体内的肿瘤生长曲线统计。D为CAR-T经两种方式干预后NCG小鼠心脏、肝脏、肺脏、脾脏、肾脏、脑等重要脏器的HE染色。
图11显示了腹腔注射MSLN-CAR-T细胞显著延长NCG荷瘤小鼠生存期。其中，A为MSLN-CAR-T干预后特定时间点小动物活体成像监测肿瘤负荷。B为小鼠体内的肿瘤生长曲线统计。C为从注射CAR-T细胞起至小鼠瘫痪或者死亡为止Kaplan–Meier生存曲线统计。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种基于MSLN前体蛋白构建的嵌合抗原受体免疫细胞。单链抗体scFv或内源受体/配体可作为CAR的靶点识别区域，但CAR识别靶点和激活胞内信号受多种因素影响，所获得的CAR是否工作需要进行大量筛选测试。本发明人通过测试发现，将间皮素前体蛋白的特定片段(即290-362位氨基酸序列)作为CAR的胞外结合域，所构建的CAR-T细胞能够特异性地结合MUC16阳性的靶细胞(如肿瘤细胞)，杀伤能力强且安全性高。在此基础上完成了本发明。
术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。在描述本发明之前，应当理解本发明不限于所述的具体方法和实验条件，因为这类方法和条件可以变动。还应当理解本文所用的术语其目的仅在于描述具体实施方案，并且不意图是限制性的，本发明的范围将仅由所附的权利要求书限制。
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、 99.3、99.4等)。
如本文所用，术语“任选”或“任选地”意味着随后所描述的事件或情况可以发生但不是必须发生。
如本文所用，术语“含有”或“包括(包含)”可以使开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”或“由…构成”。
“转导”、“转染”、“转化”或本文用到的术语指的是将外源多核苷酸传递导至宿主细胞，转录和翻译产生多肽产物的过程，包括利用质粒分子将外源多核苷酸引入宿主细胞(例如大肠杆菌)。
“基因表达”或“表达”指的是基因转录，翻译和翻译后修饰产生基因的RNA或蛋白产物的过程。
“多核苷酸”指的是任意长度的核苷酸的聚合形式，包括脱氧核苷酸(DNA)，核糖核苷酸(RNA)，其杂合序列和类似物。多核苷酸可包括修饰的核苷酸，比如甲基化或加帽的核苷酸或核苷酸类似物。本文使用的术语多核苷酸指可互换的单链和双链分子。除非另有说明，本文描述的任意实施例里的多核苷酸包括双链的形式和已知的或可预测的构成双链形式的两条互补的单链。
保守氨基酸的取代是本领域已知的。在一些实施例中，潜在的取代氨基酸在以下组的一个或多个内：甘氨酸，丙氨酸；和缬氨酸，异亮氨酸，亮氨酸和脯氨酸；天冬氨酸，谷氨酸；天冬酰胺，谷氨酰胺；丝氨酸，苏氨酸赖氨酸，精氨酸和组氨酸；和/或苯丙氨酸，色氨酸和酪氨酸；蛋氨酸和半胱氨酸。此外，本发明还提供了允许来自不同基团的氨基酸取代的非保守的氨基酸取代。
本领域技术人员将容易理解本文所述的所有参数，尺寸，材料和构造的含义。实际参数，尺寸，材料和/或配置取决于使用本发明说明的特定应用。本领域技术人员能够理解，实施例或权利要求仅是通过示例的方式给出的，并且在等效物或权利要求的范围内，本发明的实施例可涵盖的范围不限于具体描述和要求的范围。
本文的定义和使用的所有定义应被理解为超过词典定义或通过引用并入的文档中的定义。
本文所发明的所有参考文献，专利和专利申请都相对于其所引用的主题通过引用并入，在某些情况下可能包含整个文档。
应当理解，对于本文所述的包括一个以上步骤的任何方法，步骤的顺序不一定限于这些实施例中描述的顺序。
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内、肌内、皮下、腹膜内、脊髓或其它肠胃外给药途径，例如通过注射或输注。
MSLN结合蛋白MUC16(糖类相关抗原CA125)
如本发明所用，术语“MSLN结合蛋白”指能够结合MSLN的蛋白(？请复核该定义)，包括但不限于MUC16。是的，定义无误
CA125(也称为MUC16)是1981年由Bast等在用人卵巢癌细胞免疫的小鼠产生的单克隆抗体OC125发现的一种高度糖基化的Ⅰ型跨膜蛋白。2001年报道了第一个cDNA克隆，平均分子量在250万～500万道尔顿之间,还与O-连接和N-连接寡糖发生高度糖基化。MUC16的肽骨架由N末端区域、富含Ser/Thr/Pro-的串联重复序列(TR)(156个氨基酸，每个氨基酸同时具有N-和O-gly-cosylations)和具有短胞质尾的C末端区域组成。TR重复序列中具有高水平O-糖基化的SEA结构域可与MSLN结合。
MUC16是卵巢癌中过度表达的一种肿瘤特异性抗原，目前是临床上应用最为广泛的，诊断卵巢癌的重要血清生物标志物。90％卵巢癌患者血清MUC16与病程进展有关,所以也通常用作监测疾病进展和复发的标志物。CA125(MUC16)可抑制卵巢癌自然杀伤细胞的细胞溶解反应,可抑制针对卵巢癌细胞的免疫应答。
MUC16在除卵巢癌的其它肿瘤中也存在过度表达，包括宫颈癌、输卵管癌、胰腺癌、结肠癌、腹膜癌、鼻咽癌、肺癌、乳腺癌和胃癌等。因此，能够作为治疗肿瘤，尤其是多种实体瘤的靶点。
间皮素(Mesothelin,MSLN)及MSLN前体蛋白
MSLN基因位于染色体1p13.3，全长8kD。该基因包含有1884bp的开放阅读框，编码17个外显子，628个氨基酸。MSLN的前体蛋白是长约69kD的以糖基磷脂肽肌醇锚定在细胞膜上的糖蛋白，可以被蛋白水解酶水解成2部分，其中N端为31kD的可溶性蛋白，具有巨核细胞刺激活性，被称为巨核细胞强化因子(megakaryocyte-potentiating factor MPF)；而C端为长约40kD的膜结合蛋白，具有细胞黏附性，被称为MSLN，其N末端(残基296-359)可与CA125结合。MUC16-MSLN相互作用在癌细胞粘附过程中发挥作用，抗MSLN抗体可消除MSLN与表达MUC16阳性细胞的结合、阻断MUC16/间皮素依赖性细胞附着。
基于此，本发明首次将MSLN前体蛋白片段通过基因工程方式整合入CAR载体中，并修饰了相关免疫细胞，从而实现对MUC16阳性的细胞特异杀伤，可用于相关疾病的治疗。本发明使用了MSLN前体蛋白中，MSLN的N末端片段(SEQ ID NO.1AA296-362)及其N端区域6个氨基酸构成的片段构建CAR。本发明的CAR基于MSLN前体蛋白片段构建，能够结合包括MUC16的MSLN受体。
本发明嵌合抗原受体(CAR)
嵌合免疫抗原受体(Chimeric antigen receptor，CAR)由胞外抗原识别区域、 跨膜区以及胞内共刺激信号区域组成。
CAR的胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子，进而清除靶细胞。首先分离病人自体细胞(或者异源供体)，激活并进行基因改造产生CAR的免疫细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被免疫细胞以非MHC限制方式识别。
CAR-免疫细胞治疗在血液恶性肿瘤治疗中取得了非常高的临床反应率，这样的高反应率是以往任何一种治疗手段都无法达到的，在世界各引发了临床研究的热潮。
具体地，本发明的嵌合抗原受体(CAR)包括胞外结构域、跨膜结构域、和胞内结构域。
胞外结构域包括靶-特异性结合元件。所述的胞外结构域可以是基于抗原-抗体的特异性结合的抗体的scFv，也可以是基于配体-受体的特异性结合的天然序列或其衍生物。
在本发明中，所述嵌合抗原受体的胞外结构域是一种可特异性结合本发明CAR的MUC16靶点的MSLN前体蛋白或其片段。更优选地，本发明嵌合抗原受体的胞外结合域具有如SEQ ID NO:1所示序列的第290至362位的氨基酸序列。
细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与CD28信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
在本发明中，本发明CAR的胞外结合域还包括基于序列的保守性变异体，指与SEQ ID NO:1的第290至362位的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量，优选为不超过初始氨基酸序列总氨基酸数量的40％，更优选为不超过35％，更优选为1-33％，更优选为5-30％，更优选为10-25％，更优选为15-20％。
在本发明中，所述添加、缺失、修饰和/或取代的氨基酸数量通常是1、2、 3、4或5个，较佳地为1-3个，更佳地为1-2个，最佳地为1个。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括4-1BB共刺激结构域和CD3ζ的信号传导结构域。
在本发明的一个实施方式中，所述的CAR是可以特异性靶向MUC16的CAR。
嵌合抗原受体免疫细胞(CAR-免疫细胞)
在本发明中，提供了一种嵌合抗原受体免疫细胞，其包含本发明的具有特异性靶向MUC16的嵌合抗原受体。
本发明的嵌合抗原受体免疫细胞可以是CAR-T细胞，也可以是CAR-NK细胞，CAR-巨噬细胞。优选地，本发明的嵌合抗原受体免疫细胞是CAR-T细胞。
如本文所用，术语“CAR-T细胞”、“CAR-T”、“本发明CAR-T细胞”均指本发明第五方面所述的CAR-T细胞。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤标志物，针对某一种肿瘤标志物的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质标志物，又可利用糖脂类非蛋白质标志物，扩大了肿瘤标志物的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“本发明CAR-NK细胞”均指本发明第五方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于MUC16高表达的肿瘤。
自然杀伤(NK)细胞是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与CAR-T细胞相比，CAR-NK细胞还具有一下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣 的基因可被合成生产。
本发明也提供了包含本发明的核酸分子的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、 小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。在本发明的一个实施方式中，报告基因是编码mKate2红色荧光蛋白的基因。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、 纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选的实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的嵌合抗原受体CAR、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面的宿主细胞或本发明第五方面所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×103-1×108个细胞/ml，更优地1×104-1×107个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物MUC16，协同激活T细胞，引起免疫细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体 内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，MUC16的CAR-T细胞引起抗MUC16的细胞特异性免疫应答。
尽管本文公开的数据具体公开了包括MSLN前体蛋白或其片段、铰链和跨膜区、和4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)和实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐 剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“有效量”、“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量，优选105至106个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，New Eng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对具体肿瘤患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×106个至1×1010个本发明的CAR-T细胞，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
(a)靶点特异：MUC16在正常细胞的细胞膜上基本不表达，但在部分肿瘤 细胞膜上高表达。本发明的CAR免疫细胞仅针对细胞膜高表达MUC16的恶性细胞，而对其他不表达或低表达MUC16的细胞作用小。
(b)本发明利用配体与受体相结合作用方式，而非传统意义上的scfv。受体-配体相互作用的选择性和亲和力是经过长期自然选择的，受体-配体相互作用的保守性决定了在动物特别是灵长类动物中的安全性试验更能反应其在人体的安全性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
本申请实施例中的试剂、质粒、和细胞，除非另外说明，均为可市售获得的。表1总结了本发明的序列。
表1本发明涉及的序列总结

表2显示了实施例中使用的细胞系。
表2细胞系
实施例1：制备MSLN-CAR载体
基于MSLN的核苷酸序列(NC_000016)、人CD8信号肽、人CD8α铰链区、人CD8跨膜区、人4-1BB胞内区以及人CD3ζ胞内区基因序列信息，通过人工合成方法或PCR法获得相应的核苷酸序列。合成CD8信号肽及MSLN胞外区域，并通过AgeI(Thermo)和NheI(Thermo)双酶切该CAR分子的核苷酸序列，经T4DNA连接酶(NEB)连接插入已将CD8跨膜区、4-1BB共刺激结构域、CD3ζ信号传导区插入的慢病毒载体pTomo中。转化感受态大肠杆菌(Stbl3)。
将重组质粒进行测序，比对测序结果以确认质粒是否正确，测序引物为通用测序引物。测序和酶切鉴定结果均表明，CAR的编码序列正确地插入了质粒的预定位置(图1C)。
所有质粒均用QIAGEN公司的无内毒素大抽试剂盒抽提，纯化质粒用碧云天lipo6000转染HEK-293T细胞进行慢病毒包装。
实施例2：病毒包装
在15cm培养皿中培养HEK-293T细胞用于病毒包装。待HEK-293T细胞汇合度在80％-90％左右进行转染，准备2ml OPTIMEM溶解的质粒混合物(核心质粒20μg、pCMVΔR8.9 10μg、PMD2.G 4μg)；在另一离心管中2ml OPTIMEM以及68μl的lipo6000。室温静置5min后，将质粒复合物加入脂质体复合物中，室温静置20min。将上述混合物滴加入293T细胞中，37℃孵育6小时后去除培养基。重新加入预热的完全培养基。收集48小时和72小时病毒上清后于4℃3000rpm离心20分钟后用0.45μm滤膜过滤后于25000rpm 4℃离心2.5小时进行病毒浓缩。浓缩的病毒用30μl病毒溶解液过夜溶解后，病毒滴度用QPCR检测。结果显示，病毒滴度达到要求。
实施例3：CAR-T细胞制备
用Ficool分离液从人外周血中分离单核细胞，由RosetteSep Human T Cell Enrichment Cocktail(Stemcell technologies)获得纯化的CD3+T细胞。T细胞用CD3/CD28磁珠进行活化(Life technology)，再加入终浓度200U/ml的IL2(PeproTech)，刺激培养48小时后进行病毒感染。慢病毒在lentiboost存在时按照MOI＝20感染T细胞制备CAR-T细胞。感染一天后更换培养基。
实施例4：流式细胞仪检测感染CAR-T细胞的阳性率
分别离心收集病毒感染72小时后的CAR-T细胞和NT细胞(对照组)，PBS洗涤一次后弃上清，用含有2％FBS的PBS重悬细胞，流式检测阳性率。
结果：转染效率的结果如图2所示。
如图2A所示，CAR-T细胞表达的CAR-T2A-mKate2融合蛋白经切割后，形成的mKate2蛋白在胞内表现出红色荧光。
如图2B所示，采用流式细胞术进行检测，表明CAR或mKate2CAR-T的阳性表达率为约60％。
实施例5：携带luciferase的靶细胞构建
pTomo-CMV-Luciferase-IRES-Puro慢病毒包装步骤与实施例2中相同。
病毒感染OVCAR3、SKOV3、Hela、MDA-MB-468、MDA-MB-231、HEK-293T细胞后用Puromycin(1μg/ml)筛选2周，成功获得OVCAR3、SKOV3、Hela、MDA-MB-468、MDA-MB-231、HEK-293T-luciferase细胞。
实施例6：CAR-T细胞杀伤
在本实施例中，检测本发明CAR-T细胞对不同靶细胞的杀伤能力。采用的靶细胞包括：高表达MUC16的靶细胞：OVCAR3；不表达或低表达MUC16的靶细胞：SKOV3、Hela、MDA-MB-468、MDA-MB-231。
将OVCAR3、SKOV3、Hela、MDA-MB-468、MDA-MB-231、HEK-293T-luciferase细胞消化计数后调整细胞密度为2.5×104/ml。将100μl luciferase细胞接种于96孔板中，将CAR-T和NT细胞调整细胞密度为1×105/ml,按照效靶比E:T为4:1接种至黑色96孔板中，每孔接种100μl。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
将OVCAR3、SKOV3-luciferase细胞消化计数后调整细胞密度为2.5×104/ml。将100μl OVCAR3、SKOV3-luciferase细胞接种于96孔板中，将CAR-T和NT细胞调整细胞密度为2.5×104、5×104、1×105、2×105/ml,按照E:T为1:1、2:1、4:1、8:1接种至黑色96孔板中，每孔接种100μl。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
收集细胞上清保存于-80℃检测IFN-γ释放量(见实施例8)。细胞杀伤用promega荧光检测试剂盒检测，首先细胞用30μl 1*PLB裂解液处理细胞20分钟，每孔加入30μl底物后立即用BioTek酶标仪检测。
细胞毒性杀伤细胞％＝(1-含效应细胞时靶细胞荧光值/无效应细胞时靶细胞荧
光值)×100％
结果：MSLN-CAR-T对不同肿瘤细胞系的杀伤作用结果如图3所示。在卵巢癌细胞(A)、宫颈癌细胞(B)和乳腺癌细胞(C)中的结果表明MSLN-CAR-T对多种肿瘤细胞系均有良好的杀伤效果。
实施例7：卵巢癌细胞MUC16的表达和CAR-T细胞毒性实验
(1)细胞免疫荧光：将靶细胞铺于24孔板的圆片上，24小时后用4％多聚甲醛(PFA)固定细胞20分钟，PBST洗三次，每次5分钟；用10％山羊血清室温封闭1小时，用特异性识别NUC16的抗体四度孵育过夜。第二天用PBST洗三次，每次五分钟。用CY3标记的特异性识别一抗的二抗,室温孵育1小时。PBST洗三次后，DAPI染核。共聚焦显微镜成像。
(2)免疫印迹：收集6cm培养皿细胞，5500r/min，4℃离心5min；去上清后，根据细胞数目加入含蛋白酶抑制剂PMSF的RIPA细胞裂解液，冰上裂解20min，14,000r/min，4℃离心30min，收集上清溶液测浓度；蛋白质样品上样量50μg，进行蛋白电泳，检测靶细胞MUC16的表达。
(3)qPCR：收集6孔板细胞，去除培养基，加入1ml Trizol裂解细胞，室温静置5min，加入200μl/1ml Trizol的氯仿，颠倒混匀6-8次，室温静置5min；12000g，4℃离心15分钟，吸取上层清液到另一离心管中；加入等体积的异丙醇，颠倒混合，室温放置10min；12000g，4℃离心10分钟，弃上清；加入1ml 70％乙醇(用RNase free H2O配制)洗涤，7500g，室温离心5min；弃上清，室温放置10min干燥RNA，加30μl RNase free水溶解RNA；Nandrop 2000测定RNA的浓度，并用1％琼脂糖凝 胶电泳检测RNA的完整性及定量的准确性。按RevertAidTM First Strand cDNA Synthesis Kit(Thermo Scientific)的说明书合成cDNA，并进行mRNA水平的检测。
结果：各细胞系MUC16表达检测的结果见图4。通过WB(图4A)、qPCR(图4B)检测靶细胞MUC16的表达，结果一致表明OVCAR3高表达MUC16,SKOV3低表达MUC16。进一步通过免疫荧光定位验证了OVCAR3细胞膜上MUC16高表达，SKOV3细胞膜上MUC16基本不表达(图4C)。
MSLN-CAR-T对不同卵巢癌细胞系的梯度杀伤结果如图5所示。(A)MSLN-CAR-T对SKOV3的梯度杀伤作用；(B)MSLN-CAR-T对OVCAR3的梯度杀伤作用。结果表明，MSLN-CAR-T细胞对MUC16高表达肿瘤细胞的杀伤作用随着效靶比(E:T)升高而逐渐增强。
实施例8：CAR-T靶向卵巢癌细胞伴随细胞因子IFN-γ释放
在本实施例中，检测本发明CAR-T细胞与靶细胞共孵育情况下的细胞因子的释放情况。采用细胞杀伤实验中共孵育的细胞上清进行检测。
方法如下：取实施例7中本发明CAR-T细胞与OVCAR3、SKOV3靶细胞(E：T比为4：1)共孵育的细胞上清按照IFN gamma Human ELISA Kit(life technology)检测IFN-γ。
用Standard Dilution Buffer溶解标准品，并进行梯度稀释成1000pg/ml、500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.2pg/ml、15.6pg/ml、0pg/ml的标准品。
每孔中加入50μl Incubation buffer、50μl检测样本、50μl IFN-γbiotin conjugated solution，混匀后室温静置90分钟。
然后依次按照以下步骤进行操作：
(1)用1*Wash Buffer洗孔4次，每次停留1分钟。
(2)每孔加入100μl 1*Streptavidin-HRP solution，室温静置45分钟。
(3)用1*Wash Buffer洗孔4次，每次停留1分钟。
(4)加入100μl Stabilized chromogen，室温静置30分钟.
(5)每孔加入100μl Stop solution后混匀。
(6)450nm处检测吸光值。
结果如图6所示。MSLN-CAR-T杀伤OVCAR3后的细胞因子明显增加，SKOV3无明显变化。结果表明，MSLN-CAR-T细胞对肿瘤细胞的杀伤作用伴随着IFN-γ释放，提示该杀伤作用与IFN-γ释放有关。
实施例9：过表达MUC16后对MSLN-CAR-T肿瘤杀伤作用影响
根据MUC16的CDS区序列，从易锦生物购买MUC16过表达质粒(EX-Y1397-Lv183，ORF lentiviral expression clone)，构建SKOV3稳定过表达MUC16细胞株。
Lenti-MUC16-EGFP-NeoR慢病毒包装步骤与实施例2中相同。病毒感染SKOV3细胞后用Neomycin(3μg/ml)筛选2周，成功获得SKOV3-MUC16细胞。在蛋 白及基因水平上检测过表达效率，并进行MSLN-CAR-T杀伤检测。
将不同卵巢癌细胞系SKOV3-Vector、SKOV3-MUC16-luciferase细胞消化计数后调整细胞密度为2.5×104/ml。将100μl luciferase细胞接种于96孔板中，将CAR-T/NT细胞调整细胞密度为1×105,按照E:T为4:1接种至黑色96孔板中，每孔接种100μl。将上述靶细胞和T细胞混匀后至于培养箱孵育24小时。
结果：过表达效率及MSLN-CAR-T对卵巢癌细胞系SKOV3过表达MUC16后的杀伤结果如图7所示。图7-A为WB检测MUC16蛋白表达。图7-B为qPCR检测MUC16mRNA水平。图7-C为免疫荧光检测MUC16在细胞膜上的表达水平。结果均显示，过表达MUC16的SKOV3细胞构建成功。
图7-D为MSLN-CAR-T对SKOV3过表达MUC16后的杀伤作用。图7-E为MSLN-CAR-T对SKOV3过表达MUC16后杀伤作用的IFN-γ释放。结果显示，相较于对比组SKOV3-Vector，MSLN-CAR-T细胞对过表达MUC16的SKOV3-MUC16细胞的杀伤率和IFN-γ释放量均明显上升。该结果表明MSLN-CAR-T细胞对MUC16过表达肿瘤细胞的杀伤作用明显增强。
实施例10：MSLN-CAR-T对非肿瘤细胞的杀伤作用
HEK-293T细胞是人胚肾细胞系，按照效靶比4:1接种至黑色96孔板中，将MSLN-CAR-T细胞与HEK-293T-luciferase细胞共孵育，通过荧光值变化检测MSLN-CAR-T对HEK-293T细胞的杀伤。
结果如图8所示。图8-A为WB检测MUC16蛋白表达。图8-B为qPCR检测MUC16mRNA水平。图8-C为免疫荧光检测MUC16在细胞膜上的表达水平。图8-D为MSLN-CAR-T对HEK-293T的杀伤作用。图8-E为MSLN-CAR-T对HEK-293T杀伤作用的IFN-γ释放。
上述结果表明，非肿瘤细胞HEK-293T中MUC16表达水平低，MSLN-CAR-T对HEK-293T没有显著的杀伤作用。
实施例11：MSLN-CAR-T对卵巢癌肿瘤干细胞杀伤
肿瘤细胞系SKOV3、OVCAR3-luciferase细胞系用选择性培养基DMEM/F12+EGF(20ng/ml)+Bfgf(20ng/ml)+B27(1×)+1％PS，待肿瘤干细胞成球后一方面免疫荧光检测MUC16表达，另一方面提取RNA并反转录为cDNA，检测肿瘤干细胞分子标记表达CD133、CD44、CD117、CD24等。MSLN-CAR-T对肿瘤干细胞的杀伤及细胞因子检测如前所述。
实验结果如图9所示，图9-A/B为qPCR检测肿瘤干细胞标志物CD133、CD44、CD117、CD24的表达水平。图9-C为qPCR检测肿瘤细胞与干细胞MUC16mRNA水平。图9-D为免疫荧光检测MUC16在细胞膜上的表达水平。图9-E为MSLN-CAR-T对SKOV3-CSC及OVCAR3-CSC的杀伤及细胞因子IFN-γ(F)、TNF-α(H)释放检测。
上述结果表明，与SKOV3、OVCAR3细胞相比，SKOV3-CSC、OVCAR3-CSC的干细胞标志物CD133、CD44、CD117及CD24表达均显著提高。且 OVCAR3-CSC为MUC16阳性表达，而SKOV3-CSC为阴性表达，MSLN-CAR-T对OVCAR3-CSC细胞有显著杀伤，而对SKOV3-CSC则没有杀伤，且杀伤作用与细胞因子IFN-γ及TNF-α释放呈正相关。
实施例12：腹腔及尾静脉注射MSLN-CAR-T细胞对NCG小鼠OVCAR3移植瘤的抑制作用
将OVCAR3-luciferase消化并计数，调整细胞密度为5×106/ml。6周龄雌性NCG小鼠购自南京集萃药康生物科技股份有限公司并在SPF动物房饲养1周左右，腹腔(IP)接种100μl肿瘤细胞悬液。待肿瘤接种5天后进行小动物活体成像并分组如下：CD19-CAR-T(IP)腹腔注射组(n＝4)、MSLN-CAR-T(IP)腹腔注射组(n＝4)、CD19-CAR-T(IV)尾静脉注射组(n＝4)、MSLN-CAR-T(IV)尾静脉注射组(n＝4)。CD19-CAR-T和MSLN-CAR-T制备如前所述，计数CAR-T细胞并调细胞密度为1×108/ml，每只小鼠经腹腔或尾静脉注射200μl CAR-T细胞悬液。其后每隔7/14天进行小动物活体成像监测肿瘤负荷并记录小鼠体重变化。待6周左右结束实验，对小鼠主要脏器取材(心脏、肝脏、脾脏、肺脏、肾脏、大脑)，进行HE染色。
实验结果如图10所示，图10-A为MSLN-CAR-T干预后特定时间点小动物活体成像监测肿瘤负荷。图10-B为各组小鼠体重变化。图10-C为小鼠体内的肿瘤生长曲线统计。图10-D为CAR-T经两种方式干预后NCG小鼠心脏、肝脏、肺脏、脾脏、肾脏、脑等重要脏器的HE染色。
上述结果表明，与CD19-CAR-T组相比，腹腔或尾静脉注射MSLN-CAR-T细胞均能显著抑制肿瘤的生长。并且与尾静脉注射组相比，MSLN-CAR-T腹腔注射组效果最佳。HE染色结果显示无论腹腔或者尾静脉注射CD19-CAR-T和MSLN-CAR-T组均没有引起主要脏器的损伤。
实施例13：腹腔注射MSLN-CAR-T细胞延长NCG荷瘤小鼠生存期
将OVCAR3-luciferase消化并计数，调整细胞密度为5×106/ml。6周龄雌性NCG小鼠购自南京集萃药康生物科技股份有限公司并在SPF动物房饲养1周左右，腹腔(IP)接种100μl肿瘤细胞悬液。待肿瘤接种5天后进行小动物活体成像并分组如下：CD19-CAR-T(IP)腹腔注射组(n＝6)和MSLN-CAR-T(IP)腹腔注射组(n＝6)。CD19-CAR-T和MSLN-CAR-T制备如前所述，计数CAR-T细胞并调细胞密度为1×108/ml，每只小鼠经腹腔或尾静脉注射200μl CAR-T细胞悬液。其后每隔10天进行小动物活体成像并记录肿瘤荧光信号变化。记录两组小鼠生存情况，统计生存期。
实验结果如图11所示，图11-A为MSLN-CAR-T干预后特定时间点小动物活体成像监测肿瘤负荷。图11-B为小鼠体内的肿瘤生长曲线统计。图11-C为从注射CAR-T细胞起至小鼠瘫痪或者死亡为止Kaplan-Meier生存曲线统计。
上述结果表明，腹腔注射MSLN-CAR-T细胞显著抑制了OVCAR3移植瘤的生长，并且延长荷瘤小鼠的生存期。
讨论
单链抗体scFv或内源受体/配体可作为CAR的靶点识别区域，但CAR识别靶点和激活胞内信号受多种因素影响，所获得的CAR是否工作需要大量做研究工作。既往研究提示了MSLN与MUC16间存在相互作用，能够互相结合，本发明使用了MSLN前体蛋白中，包含MSLN片段及其N端部分延伸序列的一个特定片段(即290-362位氨基酸序列)作为CAR的胞外结合域。本发明人研究发现，使用该片段作为CAR的胞外结合域所构建的CAR-T细胞能够特异性地结合MUC16阳性的靶细胞(如肿瘤细胞)，杀伤能力强且安全性高。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述的CAR含有一胞外结合域，并且所述的胞外结合域包括基于SEQ ID NO:1所示氨基酸序列的MSLN前体蛋白或其片段的结构，
    并且，所述的胞外结合域能够特异性地结合于MSLN结合蛋白。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的胞外结合域包括MSLN前体蛋白或其片段，所述的MSLN前体蛋白或其片段具有如SEQ ID NO:1所示的氨基酸序列，或具有如SEQ ID NO:1所示序列的第290至362位的氨基酸序列。
  3. 如权利要求1所述的嵌合抗原受体，其特征在于，所述CAR的结构如下式I所示：
    L-EB-H-TM-C-CD3ζ-RP     (I)
    式中，
    各“-”独立地为连接肽或肽键；
    L是无或信号肽序列；
    EB是胞外结合域；
    H是无或铰链区；
    TM是跨膜结构域；
    C是无或共刺激信号分子；
    CD3ζ是源于CD3ζ的胞浆信号传导序列；
    RP是无或报告蛋白。
  4. 如权利要求1所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体CAR的氨基酸序列如SEQ ID NO:8所示。
  5. 一种核酸分子，其特征在于，所述核酸分子编码如权利要求1所述的嵌合抗原受体。
  6. 如权利要求5所述的核酸分子，其特征在于，所述的核酸分子具有如SEQ ID NO:9所述的核苷酸序列。
  7. 一种载体，其特征在于，所述的载体含有如权利要求5所述的核酸分子。
  8. 一种宿主细胞，其特征在于，所述的宿主细胞含有如权利要求7所述的载体或染色体中整合有外源的如权利要求5所述的核酸分子或表达如权利要求1所述的CAR。
  9. 一种工程化免疫细胞，其特征在于，所述的免疫细胞含有如权利要求7所述的载体或染色体中整合有外源的如权利要求5所述的核酸分子或表达如权利要求1所述的CAR。
  10. 如权利要求9所述的工程化免疫细胞，其特征在于，所述的工程化的免疫细胞是嵌合抗原受体T细胞(CAR-T细胞)或嵌合抗原受体NK细胞(CAR-NK细胞)。
  11. 一种制备如权利要求9所述的工程化免疫细胞的方法，其特征在于，包括 以下步骤：将如权利要求5所述的核酸分子或如权利要求7所述的载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
  12. 一种药物组合物，其特征在于，所述药物组合物含有如权利要求1所述的CAR、如权利要求5所述的核酸分子、如权利要求7所述的载体、如权利要求8所述的宿主细胞，和/或如权利要求9所述的工程化免疫细胞，以及药学上可接受的载体、稀释剂或赋形剂。
  13. 一种如权利要求1所述的CAR、如权利要求5所述的核酸分子、如权利要求7所述的载体、如权利要求8所述的宿主细胞，和/或如权利要求9所述的工程化免疫细胞的用途，其特征在于，用于制备预防和/或治疗MSLN结合蛋白高表达的疾病的药物或制剂。
  14. 如权利要求13所述的用途，其特征在于，所述的MSLN结合蛋白高表达的疾病选自下组：肿瘤、衰老、肥胖、心血管疾病、糖尿病、神经退行性疾病和感染性疾病。
  15. 如权利要求14所述的用途，其特征在于，所述的肿瘤选自下组：胰腺癌、乳腺癌、胃癌、肝胆癌、结直肠癌、膀胱癌、非小细胞肺癌、卵巢癌、食道癌、胶质细胞瘤、肺癌、前列腺癌、鼻咽癌、急性髓细胞白血病(AML)、多发性骨髓瘤(MM)、慢性淋巴细胞白血病(CLL)、急性淋巴白血病(ALL)和弥漫性大B细胞淋巴瘤(DLBCL)。
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110236385A1 (en) * 2008-12-05 2011-09-29 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Blocking mesothelin peptide fragments
CN106480097A (zh) * 2016-10-13 2017-03-08 南京凯地生物科技有限公司 利用CRISPR/Cas9技术敲除人PD‑1基因构建可靶向MSLN新型CAR‑T细胞的方法及其应用
CN107557336A (zh) * 2017-09-15 2018-01-09 山东兴瑞生物科技有限公司 一种抗muc16安全型嵌合抗原受体修饰的免疫细胞及其应用
CN110527667A (zh) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 一种靶向muc16的嵌合抗原受体和嵌合抗原受体t细胞及其制备方法和应用
CN111925990A (zh) * 2020-09-02 2020-11-13 北京立康生命科技有限公司 一种针对卵巢癌的Anti-MUC16 CAR-T细胞及其制备方法
WO2021155034A1 (en) * 2020-01-29 2021-08-05 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using muc16 specific fusion proteins
CN114269791A (zh) * 2019-08-08 2022-04-01 纳夫罗根公司 用于治疗体液免疫抑制疾病的体液免疫抑制拮抗剂的组合物和用途
CN115819613A (zh) * 2022-05-10 2023-03-21 四川大学华西医院 基于msln前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019236577A2 (en) * 2018-06-04 2019-12-12 Intrexon Corporation Muc16 specific chimeric antigen receptors and uses thereof

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110236385A1 (en) * 2008-12-05 2011-09-29 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Blocking mesothelin peptide fragments
CN106480097A (zh) * 2016-10-13 2017-03-08 南京凯地生物科技有限公司 利用CRISPR/Cas9技术敲除人PD‑1基因构建可靶向MSLN新型CAR‑T细胞的方法及其应用
CN107557336A (zh) * 2017-09-15 2018-01-09 山东兴瑞生物科技有限公司 一种抗muc16安全型嵌合抗原受体修饰的免疫细胞及其应用
CN110527667A (zh) * 2018-05-25 2019-12-03 深圳宾德生物技术有限公司 一种靶向muc16的嵌合抗原受体和嵌合抗原受体t细胞及其制备方法和应用
CN114269791A (zh) * 2019-08-08 2022-04-01 纳夫罗根公司 用于治疗体液免疫抑制疾病的体液免疫抑制拮抗剂的组合物和用途
WO2021155034A1 (en) * 2020-01-29 2021-08-05 TCR2 Therapeutics Inc. Compositions and methods for tcr reprogramming using muc16 specific fusion proteins
CN111925990A (zh) * 2020-09-02 2020-11-13 北京立康生命科技有限公司 一种针对卵巢癌的Anti-MUC16 CAR-T细胞及其制备方法
CN115819613A (zh) * 2022-05-10 2023-03-21 四川大学华西医院 基于msln前体蛋白构建的嵌合抗原受体免疫细胞制备及其应用

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