WO2021249549A1 - 一种嵌合抗原受体及其用途 - Google Patents

一种嵌合抗原受体及其用途 Download PDF

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WO2021249549A1
WO2021249549A1 PCT/CN2021/099797 CN2021099797W WO2021249549A1 WO 2021249549 A1 WO2021249549 A1 WO 2021249549A1 CN 2021099797 W CN2021099797 W CN 2021099797W WO 2021249549 A1 WO2021249549 A1 WO 2021249549A1
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car
cells
chimeric antigen
antigen receptor
cell
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French (fr)
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许琛琦
周秋萍
吴微
何星
孙赫
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Center for Excellence in Molecular Cell Science of CAS
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Center for Excellence in Molecular Cell Science of CAS
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Priority to JP2022576551A priority Critical patent/JP7542656B2/ja
Priority to KR1020237001456A priority patent/KR102935974B1/ko
Priority to US18/009,713 priority patent/US20240042026A1/en
Priority to EP21820851.0A priority patent/EP4166566A4/en
Publication of WO2021249549A1 publication Critical patent/WO2021249549A1/zh
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Definitions

  • the present invention relates to the field of chimeric antigen receptors, in particular to a chimeric antigen receptor and uses thereof.
  • CAR-T Chimeric antigen receptor
  • CAR-T T cells carrying CAR for specific tumor antigens
  • CAR-T therapy is widely used in the treatment of tumors, but there are still many limitations, such as the cytokine storm encountered in the clinical treatment of leukemia and solid tumors, neurotoxicity safety issues and problems that need to be improved, especially for solid tumors. Application of tumor immunotherapy.
  • cytokine storm CRS
  • NT neurotoxicity
  • Roche’s Actemra tocilizumab, IL-6R monoclonal antibody
  • tocilizumab was first approved by the US FDA.
  • Approved for the treatment of rheumatoid arthritis it can block the inflammatory response mediated by these cytokines.
  • the disadvantage is that it will increase the level of IL-6 in the patient's blood, and high concentrations of IL-6 can pass through the blood-brain barrier and induce neurotoxicity; 2IL-1 inhibitor: May 2018, from San Rafael Hospital, Italy- Two independent trials by the two research teams of the San Rafael Institute of Science and MSKCC (Memorial Sloan Kettering Cancer Center) have proved that CRS is triggered by the inflammatory molecule IL-1, and IL-1 is added to the treatment plan Receptor antagonist Kineret (anakinra, rheumatoid arthritis drug) can effectively control CRS by inhibiting IL-1 through competition.
  • Kineret anakinra, rheumatoid arthritis drug
  • the purpose of the present invention is to provide a chimeric antigen receptor and its use.
  • the first aspect of the present invention provides a chimeric antigen receptor.
  • a chimeric antigen receptor include:
  • the extracellular domain includes an antigen recognition region and a hinge region
  • the intracellular domain and the transmembrane domain are connected to the CD3 epsilon intracellular region at one end.
  • the second aspect of the present invention provides a polynucleotide sequence selected from:
  • the third aspect of the present invention provides a nucleic acid construct containing the aforementioned polynucleotide sequence
  • the nucleic acid construct is a vector
  • the nucleic acid construct is a lentiviral vector containing the aforementioned polynucleotide sequence of the replication initiation site, 3'LTR, and 5'LTR.
  • the fourth aspect of the present invention provides a lentiviral vector system, which contains the aforementioned nucleic acid construct and lentiviral vector auxiliary components.
  • the fifth aspect of the present invention provides a genetically modified T cell or a pharmaceutical composition containing the genetically modified T cell, characterized in that the cell contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with The aforementioned lentiviral vector system.
  • the sixth aspect of the present invention provides the application of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct or the aforementioned lentiviral vector system in the preparation of any one or more of the following products: (1) Preparation of T cells (2) Inhibit the secretion of cytokines IFN- ⁇ , IL-2 and TNF of T cells; (3) Inhibit T cell apoptosis; (5) Enhance T cell proliferation; (6) Improve T cell lethality; (7) ) Promote GrzB production; (8) Promote degranulation.
  • the seventh aspect of the present invention provides the application of the aforementioned chimeric antigen receptor, the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system or the aforementioned genetically modified T cell in the preparation of any one or more of the following products : (1) Treatment of tumors; (3) Inhibition of cytokine storms during tumor treatment; (4) Inhibition of neurotoxicity during tumor treatment;
  • the tumor is selected from one or more of leukemia or solid tumor.
  • the chimeric antigen receptor of the present invention and its use have the following beneficial effects:
  • the chimeric antigen receptor of the present invention can further improve the therapeutic effect of B-cell leukemia lymphoma in the treatment of B-cell leukemia lymphoma, and at the same time, it reduces the activation of macrophages and monocytes to produce inflammatory cytokines due to its down-regulation of its own cytokines. This can prevent the cytokine storm early and reduce the risk of neurotoxicity.
  • the therapeutic effect of solid tumors with high mesothelin expression is further improved, and at the same time, it can prevent cytokine storm early and reduce the risk of neurotoxicity.
  • Figure 1 a. Schematic diagram of the structure of targeting CD19 28Z CAR and E28Z CAR.
  • Figure 1 b. Anti-CD19 CAR on 28Z and E28Z CAR-T cell membrane surface level flow diagram.
  • Figure 1 Flow cytometry of the ratio of CD4 + and CD8 + cells in c.28Z and E28Z CAR-T.
  • Figure 1 d. Comparison of CD3 ⁇ phosphorylation levels of anti-CD19 28Z and E28Z CAR stimulated by CD19 antigen.
  • Figure 1 Changes in the CAR membrane surface level of e.anti-CD19 28Z and E28Z CAR stimulated by CD19 antigen.
  • Figure 1 Comparison of cytokine levels of f-h.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 The degranulation response of i.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 Granzyme release response of j.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 The toxic response of k.anti-CD19 28Z and E28Z CAR-T to CD19 + tumor cells.
  • Figure 1 1. The proliferation of anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 The apoptosis of m.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 The survival ability of n.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 The exhaustion state of o-p.anti-CD19 28Z and E28Z CAR-T cells stimulated by CD19 antigen.
  • Figure 1 q. CAR-T cell treatment of Raji subcutaneous xenograft tumor model flow chart.
  • Figure 1 Raji subcutaneous xenograft tumor growth after r.CAR-T cell therapy.
  • Figure 2 a. Schematic diagram of the structure of targeting mesothelin 28Z CAR and E28Z CAR.
  • Figure 2 b. Anti-mesothelin CAR on the surface of 28Z and E28Z CAR-T cell membrane horizontal flow cytometry.
  • Figure 2 Flow cytometry of the ratio of CD4 + and CD8 + cells in c.anti-mesothelin 28Z and E28Z CAR-T.
  • Figure 2 Comparison of cytokine levels of d-f.anti-mesothelin 28Z and E28Z CAR-T cells stimulated by mesothelin antigen.
  • Figure 2 The apoptosis of g.anti-mesothelin 28Z and E28Z CAR-T cells stimulated by mesothelin antigen.
  • Figure 2 The survival ability of h.anti-mesothelin 28Z and E28Z CAR-T cells stimulated by mesothelin antigen.
  • Figure 2 The cytotoxic response of i.anti-mesothelin 28Z and E28Z CAR-T to mesothelin + tumor cells.
  • Figure 2 J. anti-mesothelin CAR-T cell treatment of human PANC-1 pancreatic cancer cell line (high expression of mesothelin) xenograft subcutaneously xenograft flow chart.
  • Figure 2 The growth of Raji subcutaneous xenograft tumors after treatment with k.anti-mesothelin CAR-T cells.
  • the chimeric antigen receptor of the present invention includes:
  • the extracellular domain includes an antigen recognition region and a hinge region
  • the intracellular domain and the transmembrane domain are connected to the CD3 epsilon intracellular region at one end.
  • amino acid sequence of the intracellular region of CD3 ⁇ is shown in SEQ ID NO: 1; specifically: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI.
  • the intracellular domain includes an intracellular region of CD3 ⁇ , an intracellular region of a costimulatory signal transduction region, and an intracellular region of CD3 ⁇ which are connected in sequence.
  • the costimulatory signal transduction region is selected from one or more of the intracellular regions of CD27, CD28, CD134, 4-1BB or ICOS.
  • the costimulatory signal transduction region is selected from CD28. Stronger lethality.
  • the amino acid sequence of CD28 is shown in SEQ ID NO: 2, specifically RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS.
  • the amino acid sequence of the CD3 ⁇ intracellular region is shown in SEQ ID NO: 3. Specifically: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.
  • the antigen recognition region is selected from a single-chain antibody against a tumor surface antigen
  • the tumor surface antigen is selected from CD19, mesothelin, CD20, CD22, CD123, CD30, CD33, CD38
  • it is selected from CD19 or mesothelin. Good targeting specificity.
  • the transmembrane domain is selected from CD28TM, CD4, CD8 ⁇ , OX40 or H2-Kb.
  • the transmembrane domain is selected from CD28TM.
  • the amino acid sequence of the CD28TM is shown in SEQ ID NO: 4. Specifically, FWVLVVVGGVLACYSLLVTVAFIIFWV.
  • the single chain antibody contains a light chain variable region and a heavy chain variable region.
  • variable region of the light chain and the variable region of the heavy chain are connected by a linker sequence.
  • the single chain antibody is selected from FMC63 or SS1.
  • the single chain antibody contains the light chain variable region and the heavy chain variable region of the monoclonal antibody FMC63, and the light chain variable region and the heavy chain variable region are optionally connected by a linker.
  • the single chain antibody contains the light chain variable region and the heavy chain variable region of the monoclonal antibody SS1, and the light chain variable region and the heavy chain variable region are optionally connected by a linker.
  • the nucleotide sequence of the scFv of SS1 is shown in SEQ ID NO: 9. Specifically:
  • amino acid sequence of the scFv of SS1 is shown in SEQ ID NO: 10. Specifically:
  • the extracellular domain further includes a signal peptide to form a signal peptide-antigen recognition region-hinge region connected in sequence.
  • amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO: 5 (Anti-CD19 E28Z CAR) or SEQ ID NO: 6 (Anti-mesothelin E28Z CAR).
  • SEQ ID NO: 5 Anti-CD19 E28Z CAR:
  • SEQ ID NO: 5 Anti-CD19 E28Z CAR
  • SEQ ID NO: 7 Anti-CD19 E28Z CAR
  • SEQ ID NO: 6 The nucleotide sequence corresponding to SEQ ID NO: 6 (Anti-mesothelin E28Z CAR) is SEQ ID NO: 8 (Anti-mesothelin E28Z CAR, SEQ ID NO: 8 is as follows:
  • the above-mentioned parts forming the chimeric antigen receptor of the present invention may be directly connected to each other, or may be connected through a linker sequence.
  • the linker sequence may be a linker sequence suitable for antibodies known in the art, for example, a linker sequence containing G and S.
  • the linker contains one or more repeating motifs.
  • the motif can be GGGS, GGGGS, SSSSG, GGSSA, and GGSGG.
  • the motif is adjacent in the linker sequence, and no amino acid residues are inserted between the repeats.
  • the linker sequence can consist of 1, 2, 3, 4 or 5 repeating motifs.
  • the length of the linker can be 3-25 amino acid residues, for example, 3-15, 5-15, 10-20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, and is usually 2-20, such as 2-15, 2-10, 2-8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), arginine (R), glutamine (Q), etc.
  • the amino terminal or carboxy terminal of the fusion protein of the present invention may also contain one or more polypeptide fragments as protein tags.
  • Any suitable label can be used in this article.
  • the tags can be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • the polynucleotide sequence provided by the present invention is selected from:
  • the polynucleotide sequence of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the present invention also includes degenerate variants of the polynucleotide sequence encoding the fusion protein, that is, nucleotide sequences that encode the same amino acid sequence but have different nucleotide sequences.
  • the polynucleotide sequences described herein can usually be obtained by PCR amplification methods.
  • primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as a template.
  • the relevant sequence was obtained by amplification. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • the nucleic acid construct provided by the present invention contains the aforementioned polynucleotide sequence.
  • the nucleic acid construct also includes one or more regulatory sequences operably linked to the aforementioned polynucleotide sequence.
  • the CAR coding sequence of the present invention can be manipulated in various ways to ensure the expression of the protein.
  • the nucleic acid construct can be manipulated according to the different or requirements of the expression vector before inserting the nucleic acid construct into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the control sequence can be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutant, truncated and hybrid promoters, and can be derived from extracellular sequences that encode homology or heterologous to the host cell. Or the intracellular polypeptide gene is obtained.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operatively linked to the 3'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the control sequence can also be a suitable leader sequence, the untranslated region of the mRNA that is important for the host cell to translate.
  • the leader sequence is operably linked to the 5'end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the nucleic acid construct is a vector.
  • the expression of the polynucleotide sequence encoding the CAR is usually achieved by operably linking the polynucleotide sequence encoding the CAR to the promoter, and incorporating the construct into an expression vector.
  • the vector may be suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the polynucleotide sequence encoding the CAR of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector.
  • 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. Viruses that can be used as vectors include, but are not limited to, retrovirus, adenovirus, adeno-associated virus, herpes virus, and lentivirus.
  • a suitable vector contains an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
  • the nucleic acid construct is a lentiviral vector containing an initiation site of replication, 3'LTR, 5'LTR and the aforementioned polynucleotide sequence.
  • 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 to it.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse breast 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. Further, the use of inducible promoters can also be considered.
  • SV40 simian virus 40
  • MMTV mouse breast cancer virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • Epstein-Barr virus immediate early promoter Epstein-Barr virus immediate early promoter
  • Ruth's sarcoma virus promoter and human gene promoters,
  • an inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence operably linked to the inducible promoter during the period of expression, and turn off expression when the expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.
  • the expression vector introduced into the cell may also contain either or both of the selectable marker gene or the reporter gene, so as to facilitate the search for the cell population to be transfected or infected by the viral vector.
  • the selectable marker can be carried on a single piece of DNA and used in the co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences so that they can be expressed 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. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.
  • the vector can be easily introduced into host cells, for example, mammalian, bacterial, yeast, or insect cells, by any method in the art.
  • the expression vector can be transferred into the host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and so on.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids Plastid.
  • Biological methods for introducing polynucleotides into host cells include the use of viral vectors, especially lentiviral vectors, which has become the most widely used method for inserting genes into mammalian cells such as humans.
  • Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, adeno-associated virus, and so on.
  • Many virus-based systems have been developed for gene transfer into mammalian cells.
  • lentivirus provides a convenient platform for gene delivery systems.
  • the selected gene can be inserted into the vector and packaged into lentiviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to target cells in vivo or in vitro.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenovirus vectors are known in the art.
  • a lentiviral vector is used.
  • the lentiviral vector system provided by the present invention contains the aforementioned nucleic acid construct and lentiviral vector auxiliary components.
  • the lentiviral auxiliary components include lentiviral packaging plasmids and cell lines.
  • the lentiviral vector system is formed by viral packaging of the aforementioned nucleic acid construct with the assistance of a lentiviral packaging plasmid and cell line.
  • the method for constructing the lentiviral vector system is a commonly used method in the field.
  • the present invention provides a method for activating T cells in vitro, which includes the step of infecting the T cells with the aforementioned lentivirus.
  • the CAR-T cells of the present invention can undergo stable in vivo T cell expansion and continue at high levels in the blood and bone marrow for a prolonged amount of time, and form specific memory T cells. After encountering and subsequently eliminating the target cell expressing the replacement antigen, the CAR-T cell of the present invention can differentiate into a central memory-like state in vivo.
  • the present invention also includes a type of cell therapy in which T cells are genetically modified to express the CAR described herein, and CAR-T cells are injected into recipients in need thereof.
  • the injected cells can kill the recipient's tumor cells.
  • CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the anti-tumor immune response caused by CAR-T cells can be an active or passive immune response.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.
  • the treatable cancer may be a non-solid tumor, such as a blood tumor, such as leukemia and lymphoma.
  • the diseases that can be treated with the CAR, its coding sequence, nucleic acid constructs, expression vectors, viruses and CAR-T cells of the present invention are preferably CD19-mediated diseases, especially CD19-mediated hematological tumors.
  • CD19-mediated diseases include but are not limited to leukemia and lymphoma, such as B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute Myeloid leukemia.
  • leukemia and lymphoma such as B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute Myeloid leukemia.
  • the genetically modified T cell or the pharmaceutical composition containing the genetically modified T cell provided by the present invention contains the aforementioned polynucleotide sequence, or contains the aforementioned nucleic acid construct, or is infected with the aforementioned lentiviral vector system.
  • the CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as related cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include CAR-T cells as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and preservatives.
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the number 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.
  • T cells described herein comprises pharmaceutical compositions may be 104 to 109 cells / kg body weight doses, dose is preferably 105 to 106 cells / kg body weight.
  • the T cell composition can also be administered multiple times at these doses.
  • the cells can be administered by using injection techniques known in immunotherapy.
  • the optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation.
  • the compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by intravenous injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes, or sites of infection.
  • the CAR-T cell of the present invention or a composition thereof can be combined with other therapies known in the art.
  • Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressive agents.
  • these radiotherapy preparations include: cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, fludarabine, rapamycin and mycophenol Sour etc.
  • the cell composition of the present invention is combined with bone marrow transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (E.g., before, at the same time, or after) and administered to the patient.
  • chemotherapeutic agents such as fludarabine, external beam radiotherapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH (E.g., before, at the same time, or after) and administered to the patient.
  • anti-tumor effect refers to a biological effect, which can be represented by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or the improvement of various physiological symptoms related to cancer.
  • Patient refers to a living organism that can cause an immune response, such as a mammal. Examples include, but are not limited to, humans, dogs, cats, mice, rats and their transgenic species.
  • aforementioned chimeric antigen receptor the aforementioned polynucleotide sequence, the aforementioned nucleic acid construct, the aforementioned lentiviral vector system or the aforementioned genetically modified T cell in the preparation of any one or more of the following products: (1) Tumor treatment (3) Inhibit the cytokine storm produced during tumor treatment; (4) Inhibit the neurotoxicity produced during tumor treatment.
  • the tumor is selected from one or more of leukemia or solid tumor.
  • the tumor is selected from B-cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. Conventional technology.
  • the anti-CD19 28Z CAR sequence refers to the sequence shared by Rosenberg and SA laboratory at NCBI. On this basis, we use gene synthesis technology to insert the full-length CD3 ⁇ intracellular segment behind the CD28TM transmembrane region, and construct a new CAR named E28Z .
  • sequence of anti-CD19 E28Z CAR is shown in SEQ ID NO: 7. Specifically:
  • the CAR was subcloned into the lentiviral expression plasmid pHAGE vector, the CAR expression plasmid and the packaging plasmids pSPAX2 and pMD2G were mixed at a ratio of 10:7.5:3.5, respectively, and then transferred into 293FT cells by calcium phosphate transfection to produce lentiviral particles.
  • T cell complete medium XIVO-15+1%PS+10ng/ml human IL-7+10ng/ml human IL-15+10mM+5% Human AB serum, 10mM neutralized NAC
  • Virus was removed one day after transfection and four days after antibody stimulation.
  • CAR-T cells with the same CAR expression were sorted out on the 5th day after antibody stimulation.
  • CAR-T is identified by a series of indicators, such as the level of CAR membrane, the ratio of CD4 and CD8 subgroups, the secretion of cytokines in vitro, the killing ability in vitro, the proliferation ability in vitro, the level of apoptosis in vitro, and the Sustained viability, and anti-tumor effect in the body, etc.
  • the pCD3 ⁇ band in the picture shows that the background pCD3 ⁇ phosphorylation level of E28Z at 0 min (that is, no CD19 antigen is encountered) is significantly higher than that of 28Z CAR-T cells, and after CD19 antigen-specific stimulation, The phosphorylation of pCD3 ⁇ increased first and then decreased, and the rate of decrease after phosphorylation of pCD3 ⁇ was significantly faster than that of 28Z CAR-T cells.
  • CAR-T cells and the CD19+ lymphoma cell line Raji were mixed in a 1.5ml EP tube at a ratio of 1:1, centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact, and cultured in a 37°C incubator with complete medium.
  • room temperature 400g, 1min
  • the CAR endocytosis reaction was terminated with cold PBS, the sample was placed on ice, washed once with cold PBS, and then stained with anti-mouse FM63 scFv coupled with Alexa Fluor647 antibody (4°C, 30min), and then flow cytometry 28Z CAR- The level of CAR on T and E28Z CAR-T cell membrane.
  • Anti-CD19 28Z and E28Z CAR-T are toxic to CD19+ tumor cells. Resuspend CD19 - K562 cells in 1640 serum-free medium, then pre-stained with 1x CellTraker deep red dye (37°water bath, 30min), then wash once with 1640 serum-free medium, resuspend in T cell complete medium and mix with CD19 + K562_CD19 (IRES mCherry) cells are mixed according to 1:1 as target cells. CAR-T cells and the mixed target cells were mixed and plated on a round-bottom 96-well plate in a ratio of 3:1, 1:1, and 1:3, mixed, and centrifuged at room temperature (400g, 1min) to promote cell-to-cell contact.
  • Figure 1: k which shows that anti-CD19 E28Z CAR-T is more toxic to CD19+ tumor cells than 28Z CAR-T cells.
  • 100,000 anti-mesothelin CAR-T cells and K562-Mesothelin cell line overexpressing mesothelin were seeded in a round-bottom 96-well plate at a ratio of 1:1, mixed, and centrifuged at room temperature (400g, 1min) to promote cell contact. Cultivation is based on incubating in a 37°C incubator and collecting samples within one day. Add 1x BFA to inhibit cytokine efflux 6 hours before receiving the sample.
  • mesothelin - K562 cells APC +, mCherry -
  • mesothelin + K562_mesothelin cells APC -, mCherry +
  • Figure 2 The results are shown in Figure 2: i, showing that anti-mesothelin E28Z CAR-T is more toxic to mesothelin + tumor cells than 28Z CAR-T cells.

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Abstract

提供一种嵌合抗原受体，包括：依次连接的胞外结构域、跨膜结构域和胞内结构域；所述胞外结构域包括抗原识别区和铰链区；所述胞内结构域与跨膜结构域相连的一端连接有CD3ε胞内区。该嵌合抗原受体在B细胞白血病淋巴瘤治疗中可进一步提高B细胞白血病淋巴瘤的治疗效果，同时因其下调自身细胞因子来降低巨噬细胞单核细胞活化产生炎性细胞因子，从而可早期预防细胞因子风暴及降低神经毒性的风险。在mesothelin阳性的肿瘤治疗中进一步提高mesothelin高表达的实体瘤治疗效果，同时可早期预防细胞因子风暴及降低神经毒性的风险。

Description

一种嵌合抗原受体及其用途 技术领域
本发明涉及嵌合抗原受体领域，具体涉及一种嵌合抗原受体及其用途。
背景技术
嵌合抗原受体简称CAR(chimeric antigen receptor)，搭载针对特定肿瘤抗原CAR的T细胞称为CAR-T细胞。CAR-T疗法在肿瘤的治疗中应用广泛，但仍然存在诸多局限，例如在临床上治疗白血病和实体瘤所遇到的细胞因子风暴、神经毒性安全性问题和疗效有待提高的问题，尤其是实体瘤免疫治疗的应用。
目前解决细胞因子风暴(CRS)和神经毒性(NT)的方法均存在局限性：临床上目前主要是FDA批准的用罗氏的Actemra(tocilizumab，IL-6R单抗)管理CRS，tocilizumab首次被美国FDA批准用于治疗类风湿性关节炎治疗，可阻断这些细胞因子所介导的炎症反应。缺点是会使病人体内血液中的IL-6水平升高，高浓度IL-6可通过血脑屏障可以诱发神经毒性；②IL-1抑制剂：2018年5月，来自意大利圣拉菲尔医院-圣拉菲尔科学研究所和MSKCC(Memorial Sloan Kettering癌症中心)的两个研究团队的两项独立试验均证明了：CRS是由炎性分子IL-1引发的，在治疗方案中加入IL-1受体拮抗剂Kineret(anakinra，类风湿性关节炎药物)通过竞争抑制IL-1，能够有效控制CRS，因其具有血脑屏障穿透能力，也可控制神经毒性；局限性：tocilizumab和anakinra都不是从早期干预预防细胞CRS/NS,而是属于应急措施，发现补救不及时会存在致命的风险。而目前还不清楚早期干预发生副作用的最佳方案是什么。
设计一款针对CAR结构本身的改造是更优化的方案。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种嵌合抗原受体及其用途。
为实现上述目的及其他相关目的，本发明第一方面提供一种嵌合抗原受体。包括：
依次连接的胞外结构域、跨膜结构域和胞内结构域；
所述胞外结构域包括抗原识别区和铰链区；
所述胞内结构域与跨膜结构域相连的一端连接有CD3ε胞内区。
本发明第二方面提供一种多核苷酸序列，该多核苷酸序列选自：
(1)编码权利要求前述的嵌合抗原受体的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列；
本发明第三方面提供一种核酸构建物，所述核酸构建物含有前述多核苷酸序列；
优选的，所述核酸构建物为载体；
更优选的，所述核酸构建物为慢病毒载体含有复制起始位点、3’LTR、5’LTR前述的多核苷酸序列。
本发明第四方面提供慢病毒载体系统，所述慢病毒载体系统含有前述的核酸构建物及慢病毒载体辅助成分。
本发明第五方面提供一种基因修饰的T细胞或含该基因修饰的T细胞的药物组合物，其特征在于，所述细胞含有前述多核苷酸序列，或含有前述核酸构建物，或感染了前述慢病毒载体系统。
本发明第六方面提供前述嵌合抗原受体、前述多核苷酸序列、前述核酸构建物或前述慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)抑制T细胞的细胞因子IFN-γ、IL-2、TNF分泌；(3)抑制T细胞凋亡；(5)增强T细胞增殖能力；(6)提高T细胞杀伤力；(7)促进GrzB产生；(8)促进脱颗粒。
本发明第七方面提供前述嵌合抗原受体、前述多核苷酸序列、前述核酸构建物、前述慢病毒载体系统或前述基因修饰的T细胞在制备以下任一种或多种用途产品中的应用：(1)治疗肿瘤；(3)抑制肿瘤治疗时产生的细胞因子风暴；(4)抑制肿瘤治疗时产生的神经毒性；
优选的，所述肿瘤选自白血病或实体瘤中的一种或多种。
如上所述，本发明的嵌合抗原受体及其用途，具有以下有益效果：
本发明的嵌合抗原受体在B细胞白血病淋巴瘤治疗中可进一步提高B细胞白血病淋巴瘤的治疗效果，同时因其下调自身细胞因子来降低巨噬细胞单核细胞活化产生炎性细胞因子，从而可早期预防细胞因子风暴及降低神经毒性的风险。在mesothelin阳性的肿瘤治疗中进一步提高mesothelin高表达的实体瘤治疗效果，同时可早期预防细胞因子风暴及降低神经毒性的风险。
附图说明
图1:a.靶向CD19 28Z CAR和E28Z CAR的结构示意图。
图1:b.anti-CD19 CAR在28Z和E28Z CAR-T细胞膜表面水平流式图。
图1:c.28Z和E28Z CAR-T中CD4 +和CD8 +细胞比例的流式图。
图1:d.anti-CD19 28Z和E28Z CAR经CD19抗原刺激后的CD3ζ磷酸化水平比较。
图1:e.anti-CD19 28Z和E28Z CAR经CD19抗原刺激后的CAR膜表面水平变化。
图1:f-h.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后的细胞因子水平比较。
图1:i.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后的脱颗粒反应。
图1:j.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后的颗粒酶释放反应。
图1:k.anti-CD19 28Z和E28Z CAR-T对CD19 +肿瘤细胞毒性反应。
图1:l.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后增殖情况。
图1:m.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后凋亡情况。
图1:n.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后的存活能力。
图1:o-p.anti-CD19 28Z和E28Z CAR-T细胞经CD19抗原刺激后的exhaustion衰竭状态。图1:q.CAR-T细胞治疗Raji异种皮下移植瘤模型流程图。
图1:r.CAR-T细胞治疗后Raji异种皮下移植瘤生长情况。
图2:a.靶向mesothelin 28Z CAR和E28Z CAR的结构示意图。
图2:b.anti-mesothelin CAR在28Z和E28Z CAR-T细胞膜表面水平流式图。
图2:c.anti-mesothelin 28Z和E28Z CAR-T中CD4 +和CD8 +细胞比例的流式图。
图2:d-f.anti-mesothelin 28Z和E28Z CAR-T细胞经mesothelin抗原刺激后的细胞因子水平比较。
图2:g.anti-mesothelin 28Z和E28Z CAR-T细胞经mesothelin抗原刺激后凋亡情况。
图2:h.anti-mesothelin 28Z和E28Z CAR-T细胞经mesothelin抗原刺激后的存活能力。
图2:i.anti-mesothelin 28Z和E28Z CAR-T对mesothelin +肿瘤细胞毒性反应。
图2:j.anti-mesothelin CAR-T细胞治疗人源PANC-1胰腺癌细胞系(高表达mesothelin)异种皮下移植瘤模型流程图。
图2:k.anti-mesothelin CAR-T细胞治疗后Raji异种皮下移植瘤生长情况。
具体实施方式
本发明所述的嵌合抗原受体，包括：
依次连接的胞外结构域、跨膜结构域和胞内结构域；
所述胞外结构域包括抗原识别区和铰链区；
所述胞内结构域与跨膜结构域相连的一端连接有CD3ε胞内区。
进一步的，所述CD3ε胞内区的氨基酸序列如SEQ ID NO：1所示；具体的：KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI。
所述胞内结构域包括依次连接的CD3ε胞内区、共刺激信号传导区胞内区和CD3ζ胞内区。
在一种实施方式中，所述共刺激信号传导区选自CD27、CD28、CD134，4-1BB或ICOS的胞内区中的一种或多种。
在进一步优选的实施方式中，所述共刺激信号传导区选自CD28。杀伤能力更强。
CD28的氨基酸序列如SEQ ID NO：2所示，具体的，RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS。
在一种实施方式中，所述CD3ζ胞内区的氨基酸序列如SEQ ID NO：3所示。具体为：RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR。
在一种实施方式中，所述抗原识别区选自针对肿瘤表面抗原的单链抗体，所述肿瘤表面抗原选自CD19、间皮素(mesothelin)、CD20、CD22、CD123、CD30、CD33、CD38、CD138、BCMA、Fibroblast activation protein(FAP)、Glypican-3、CEA、EGFRvIII、PSMA、Her2、IL13Rα2、CD171和GD2中的一种或多种。
优选的，选自CD19或间皮素。靶向特异性好。
所述跨膜结构域选自CD28TM、CD4、CD8α、OX40或H2-Kb。
优选的，所述跨膜结构域选自CD28TM。具体的，所述CD28TM的氨基酸序列如SEQ ID NO：4所示。具体的，FWVLVVVGGVLACYSLLVTVAFIIFWV。
在一种实施方式中，所述单链抗体含有轻链可变区和重链可变区。
在一种实施方式中，所述轻链可变区与所述重链可变区通过接头序列相连。
在一种实施方式中，所述单链抗体选自FMC63或SS1。
FMC63可采用市售产品。
在一种实施方式中，所述单链抗体含有单克隆抗体FMC63的轻链可变区和重链可变区，所述轻链可变区和重链可变区任选地通过接头相连。
在一种实施方式中，所述单链抗体含有单克隆抗体SS1的轻链可变区和重链可变区，所述轻链可变区和重链可变区任选地通过接头相连。
SS1的scFv核苷酸序列如SEQ ID NO：9所示。具体为：
Figure PCTCN2021099797-appb-000001
Figure PCTCN2021099797-appb-000002
SS1的scFv氨基酸序列如SEQ ID NO：10所示。具体为：
Figure PCTCN2021099797-appb-000003
进一步的，所述胞外结构域还包括信号肽，形成依次连接的信号肽-抗原识别区-铰链区。
在一种实施方式中，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：5(Anti-CD19 E28Z CAR)或SEQ ID NO：6(Anti-mesothelin E28Z CAR)所示。
具体的，
SEQ ID NO：5(Anti-CD19 E28Z CAR)：
Figure PCTCN2021099797-appb-000004
SEQ ID NO：5(Anti-CD19 E28Z CAR)对应的核苷酸序列为SEQ ID NO：7(Anti-CD19 E28Z CAR)，SEQ ID NO：7如下：
Figure PCTCN2021099797-appb-000005
SEQ ID NO：6(Anti-mesothelin E28Z CAR)：
Figure PCTCN2021099797-appb-000006
SEQ ID NO：6(Anti-mesothelin E28Z CAR)对应的核苷酸序列为SEQ ID NO：8(Anti-mesothelin E28Z CAR，SEQ ID NO：8如下：
Figure PCTCN2021099797-appb-000007
Figure PCTCN2021099797-appb-000008
形成本发明的嵌合抗原受体的上述各个部分，相互之间可直接连接，或者可通过接头序列连接。接头序列可以是本领域周知的适用于抗体的接头序列，例如含G和S的接头序列。通常，接头含有一个或多个前后重复的基序。例如，该基序可以是GGGS、GGGGS、SSSSG、GSGSA和GGSGG。优选地，该基序在接头序列中是相邻的，在重复之间没有插入氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。接头的长度可以是3～25个氨基酸残基，例如3～15、5～15、10～20个氨基酸残基。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例 如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。因此，本发明的融合蛋白(即所述CAR)的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本文。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。这些标签可用于对蛋白进行纯化。
本发明提供的多核苷酸序列，该多核苷酸序列选自：
(1)编码权利前述的嵌合抗原受体的多核苷酸序列；和
(2)(1)所述多核苷酸序列的互补序列。
本发明的多核苷酸序列可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明也包括编码融合蛋白的多核苷酸序列的简并变异体，即编码相同的氨基酸序列但核苷酸序列有所不同的核苷酸序列。
本文所述的多核苷酸序列通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
本发明提供的核酸构建物，含有前述的多核苷酸序列。
所述核酸构建物还包括与前述的多核苷酸序列操作性连接的一个或多个调控序列。本发明所述的CAR的编码序列可以多种方式被操作以保证所述蛋白的表达。在将核酸构建物插入载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
调控序列也可以是合适的前导序列，对宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5′末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
优选的，所述核酸构建物为载体。
通常通过可操作地连接编码CAR的多核苷酸序列至启动子，并将构建体并入表达载体，实现编码CAR的多核苷酸序列的表达。该载体对于复制和整合真核细胞可为合适的。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、起始序列和启动子。
编码本发明CAR的多核苷酸序列可被克隆入许多类型的载体。例如，可被克隆入质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。进一步地，载体是表达载体。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。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。
更优选的，所述核酸构建物为慢病毒载体含有复制起始位点、3’LTR、5’LTR以及前述多核苷酸序列。
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。该启动子序列是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、EB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，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达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，而在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个，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。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荧光蛋白基因的基因。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。
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。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。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。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。
将多核苷酸引入宿主细胞的生物学方法包括使用病毒载体，特别是慢病毒载体，这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。已经开发了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，慢病毒提供了用于基因传递系统的方便的平台。可利用本领域中已知的技术将选择的基因插入载体并包装入慢病毒颗粒。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。许多反转录病毒系统在本领域中是已知的。在一些实施方案中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方案中，使用慢病毒载体。
本发明提供的慢病毒载体系统，所述慢病毒载体系统含有前述的核酸构建物及慢病毒载体辅助成分。
所述慢病毒辅助成分包括慢病毒包装质粒和细胞系。
所述慢病毒载体系统是前述核酸构建物在慢病毒包装质粒、细胞系的辅助下，经过病毒包装而成。所述慢病毒载体系统构建方法为本领域常用方法。
本发明提供的一种离体活化T细胞的方法，所述方法包括使用前述慢病毒感染所述T细胞的步骤。
本发明的CAR-T细胞可经历稳固的体内T细胞扩展并在血液和骨髓中以高水平持续延长的时间量，并形成特异性记忆T细胞。在遇到并随后消除表达替代抗原的靶细胞后，本发明的CAR-T细胞可体内分化成中心记忆样状态。
本发明还包括一类细胞疗法，其中T细胞被基因修饰以表达本文所述的CAR，和CAR-T细胞被注入需要其的接受者中。注入的细胞能够杀死接受者的肿瘤细胞。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。
由CAR-T细胞引起的抗肿瘤免疫应答可为主动或被动免疫应答。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-T细胞诱导对CAR中的抗原结合部分特异性的免疫应答。
可治疗的癌症可以是非实体瘤，如血液肿瘤，例如白血病和淋巴瘤。尤其是，可采用本发明的CAR、其编码序列、核酸构建物、表达载体、病毒以及CAR-T细胞治疗的疾病优选为CD19介导的疾病，尤其是CD19介导的血液肿瘤。
具体而言，本文中，“CD19介导的疾病”包括但不限于白血病和淋巴瘤，例如B细胞淋巴瘤、套细胞淋巴瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、多毛细胞白血病、和急性髓性白血病。
本发明提供的基因修饰的T细胞或含该基因修饰的T细胞的药物组合物，所述细胞含有前述多核苷酸序列，或含有前述核酸构建物，或感染了前述慢病毒载体系统。
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/或与其他组分诸如相关的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CAR-T细胞，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。
当指出“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10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量。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、通过静脉内注射或腹膜内施用给患者。在一个实施方案中，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。在另一个实施方案中，本发明的T细胞组合物优选通过静脉注射施用。T细胞的组合物可被直接注入肿瘤、淋巴结或感染位置。
在本发明的一些实施方案中，本发明的CAR-T细胞或其组合物可与本领域已知的其它疗法结合。所述疗法包括但不限于化疗、放疗和免疫抑制剂。例如，可结合各种放疗制剂进行 治疗，这些放疗制剂包括：环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯、FK506、氟达拉滨、雷帕霉素和麦考酚酸等。在进一步的实施方案中，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)、环磷酰胺或抗体诸如OKT3或CAMPATH的T细胞烧蚀疗法结合(例如，之前、同时或之后)而施用给患者。
本文中，“抗肿瘤效应”指一种生物学效应，其可由肿瘤体积的减少、肿瘤细胞数的减少、转移数的减少、预期寿命的增加或与癌相关的各种生理症状的改善表示。
“患者”、“对象”、“个体”等等在本文中可交换使用，指可引起免疫应答的活有机体，如哺乳动物。例子包括但不限于人、狗、猫、小鼠、大鼠和其转基因物种。
前述嵌合抗原受体、前述多核苷酸序列、前述核酸构建物、前述慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)抑制T细胞的细胞因子IFN-γ、IL-2、TNF分泌；(3)抑制T细胞凋亡；(5)增强T细胞增殖能力；(6)提高T细胞杀伤力；(7)促进GrzB产生；(8)促进脱颗粒。
前述嵌合抗原受体、前述多核苷酸序列、前述核酸构建物、前述慢病毒载体系统或前述基因修饰的T细胞在制备以下任一种或多种用途产品中的应用：(1)治疗肿瘤；(3)抑制肿瘤治疗时产生的细胞因子风暴；(4)抑制肿瘤治疗时产生的神经毒性。
优选的，所述肿瘤选自白血病或实体瘤中的一种或多种。
更优选的，所述肿瘤选自B细胞淋巴瘤、套细胞淋巴瘤、急性淋巴细胞白血病、慢性淋巴细胞白血病、多毛细胞白血病、和急性髓性白血病。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例 中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。
实施例1
anti-CD19 28Z CAR序列参考Rosenberg,S.A.实验室于NCBI共享的序列，我们在此基础上，利用基因合成技术，CD3ε全长胞内段插在CD28TM跨膜区后面，构建出来新型CAR命名为E28Z。
anti-CD19 E28Z CAR的序列如SEQ ID NO：7所示。具体为：
Figure PCTCN2021099797-appb-000009
Figure PCTCN2021099797-appb-000010
将CAR亚克隆至慢病毒表达质粒pHAGE载体，将CAR表达质粒与包装质粒pSPAX2和pMD2G分别按照10：7.5：3.5的比例混合后用磷酸钙转染的方法转入293FT细胞以生产慢病毒颗粒。用超高速离心方法分别表达28Z CAR或E28Z CAR慢病毒经浓缩为小体积高滴度病毒(～10^8/ml)，然后分别转染经αCD3/αCD28 Ab-beads活化一天的原代T细胞(MOI＝10)。原代T细胞用T细胞完全培养基(XIVO-15+1％P.S.+10ng/ml human IL-7+10ng/ml human IL-15+10mM+5％Human AB serum,10mM neutralized NAC)培养期间，病毒转染一天后则除去，抗体刺激四天后也除去，抗体刺激后的第5天分选出CAR表达量相同的CAR-T细胞。经过一段时间扩增后，对CAR-T进行一系列的指标鉴定，如CAR上膜水平，CD4和CD8亚群比例，体外细胞因子分泌，体外杀伤能力、体外增殖能力，体外凋亡水平，体外持续生存能力，以及体内抗肿瘤效果等。
靶向CD19 28Z CAR和E28Z CAR的结构如图1：a所示。
用anti-mouse FM63 scFv偶联Alexa Fluor647抗体流式检测28Z CAR-T和E28Z CAR-T细胞膜表面的CAR水平，流式图结果如图1：b所示，显示两者CAR阳性率和表达量(MFI)没差异。
用anti-human CD4-APC和anti-human CD8-PE-Cy7抗体流式检测28Z CAR-T和E28Z CAR-T细胞，流式图结果如图1：c所示，显示两者的CD4 +CAR-T细胞和CD8 +CAR-T细胞的比例相近。
CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例混合，4℃离心促细胞间接触，37℃水浴计时，在一系列时间点用LDS sample buffer终止反应，然后用anti-pCD3ζ(p142)抗体western blotting检测。结果如图1：d所示，图片pCD3ζ条带显示E28Z在0min(即未遇到CD19抗原)时本底的pCD3ζ磷酸化水平明显高于28Z CAR-T细胞，而经过CD19抗原特异刺激之后，pCD3ζ磷酸化先升后降，pCD3ζ磷酸化后面的下降速度明显快于28Z CAR-T细胞。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例在1.5ml EP管混合后，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养，在一系列时间点用冷PBS终止CAR内吞反应，收样品置冰，冷PBS洗一次，然后用anti-mouse FM63 scFv偶联Alexa Fluor647抗体染色(4℃，30min)，后流式检测28Z CAR-T和E28Z CAR-T细胞膜上CAR的水平。结果如图1：e所示，表明，28Z和E28Z CAR上膜水平是先急剧下降，而后慢慢回复上升，其中28Z CAR膜水平比E28Z CAR下降的快，后面回复上升也是明显快于E28Z CAR。
10万CAR-T细胞与CD19 +淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一天即收样。收样前6小时加1x BFA抑制细胞因子外排，收样后将样品PBS洗一次，然后4％PFA室温固定5min，0.1％TritonX-100室温打孔5min,接着按照常规操作胞内染色检测IL-2、IFN-γ、TNF-α。结果如图1：f-h所示，表明E28Z CAR-T细胞经CD19 +细胞特异刺激后产生的细胞因子IL-2、IFN-γ、TNF-α都明显少于28Z CAR-T细胞。
结合图1d和图1e，说明E28Z CAR pCD3ζ磷酸化水平下降较快不是由于CAR膜水平下调引起的，因为CD19特异刺激后28Z CAR膜水平反而比E28Z CAR下降的快。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养2h、4h，其中完全培养基中还加有anti-human CD107a-eFluor660抗体和1x Monensin(防止anti-human CD107a抗体内吞降解)，收样时用冷PBS终止反应，PBS洗一次后流式检测。结果如图1：i所示，表明E28Z CAR-T细胞经CD19抗原特异刺激后的脱颗粒反应速率明显快于28Z CAR-T细胞。
10万CAR-T细胞与CD19 +淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一天即收样。收样前6小时加1x BFA抑制颗粒酶外排，收样后将样品PBS洗一次，然后4％PFA室温固定5min，0.1％TritonX-100室温打孔5min,接着按照常规操作胞内染色检测颗粒酶Granzyme B。结果如图1：j所示，表明E28Z CAR-T细胞经CD19 +细胞特异刺激后产生的颗粒酶Granzyme B都明显多于28Z CAR-T细胞。
anti-CD19 28Z和E28Z CAR-T对CD19 +肿瘤细胞毒性反应。将CD19 -K562细胞重悬于1640无血清培养基，然后预染1x CellTraker deep red dye(37°水浴，30min)，接着1640无血清培养基洗一次，用T细胞完全培养基重悬后与CD19 +K562_CD19(IRES mCherry)细胞按照1：1 混合为靶细胞。CAR-T细胞再与该混合型靶细胞按照3：1，1：1，1：3比例混合铺板于圆底96孔板，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一天即收样，置冰，流式检测CD19 -K562细胞(APC +,mCherry -)百分比与CD19 +K562_CD19细胞(APC -,mCherry +)百分比变化，实验组的“CD19 -K562细胞(APC +,mCherry -)百分比/CD19 +K562_CD19细胞(APC -,mCherry +)百分比”记为N。N值以未加CAR-T细胞的混合型靶细胞样品孔的“N 0＝CD19 -K562细胞(APC +,mCherry -)百分比/CD19 +K562_CD19细胞(APC -,mCherry +)百分比”标准化后，计得存活比率N/N 0，则杀伤率公式为“Lysis(％)＝1-N/N 0”。结果如图1：k所示，显示，anti-CD19 E28Z CAR-T对CD19 +肿瘤细胞毒性比28Z CAR-T细胞较大。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一段时间。于不同时间点收样后将样品PBS洗一次，按照Foxp3/Transcription Factor Staining Buffer Set试剂盒说明书操作，用anti-human Ki67-APC抗体染色后流式检测Ki67的表达量。结果如图1：l表示，anti-CD19 E28Z CAR-T经CD19 +Raji细胞刺激后持续一段时间一直比28Z CAR-T增殖快。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一段时间。于不同时间点收样后将样品PBS洗一次，按照Annexin V Apoptosis Detection Kit APC试剂盒说明书染色好后流式检测CAR-T细胞Annexin V的表达水平。结果如图1：m，表示，anti-CD19 E28Z CAR-T经CD19 +Raji细胞刺激后的凋亡水平持续一段时间一直比28Z CAR-T明显低。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一段时间。于不同时间点收样后将样品PBS洗一次，用anti-human CD4-APC和anti-human CD8-PE-Cy7抗体染色检测CAR +T细胞存活数量。结果如图1：n，表示，anti-CD19 E28Z CAR-T经CD19 +Raji细胞刺激后的存活数量比28Z CAR-T显著增多。
10万CAR-T细胞与CD19+淋巴瘤细胞系Raji按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一段时间。于不同时间点收样后将样品PBS洗一次，用anti-human PD-1-APC和anti-human TIM3-PE抗体染色检测CAR +T细胞的PD-1和TIM3表达情况。结果图1:o-p，表示，anti-CD19 E28Z CAR-T经CD19 +Raji细胞刺激后的PD-1和TIM3表达情况于第7天后比28Z CAR-T低，说明E28Z CAR不会引起T细胞的exhaustion状态，即使有本底较强的pCD3ζ磷酸化水平(图1d)。
无菌操作准备3x10^7/ml Raji细胞-PBS重悬液，在每只6周龄B-NDG雌鼠的左侧背部皮下接种100ul Raji细胞3百万，时间点记为day0。6天后，Raji皮下瘤长至直径7-8mm时，将小鼠随机分组，然后对每只小鼠尾静脉注射100ul 8百万CAR-T细胞，模型见图1：q，之后定期用游标卡尺测量肿瘤的大小，记录数据，肿瘤面积＝长x宽。结果如图1:r.，结果表明，vector对照组的小鼠的肿瘤生长最快，其次是28Z组，治疗效果最好的是E28Z组，E28Z组治疗效果明显高于28Z组。
实施例2
如图2：a所示，将靶向CD19 28Z CAR和E28Z CAR中的FMC63 scFv(anti-CD19)序列依次换成SS1 scFv(anti-mesothelin)即分别为靶向mesothelin 28Z CAR和E28Z CAR。
用一抗Rat anti-HA和二抗goat anti-Rat IgG-Alexa Fluor647抗体流式检测28Z和E28Z CAR-T细胞膜表面CAR水平，流式图结果如图2:b.所示，显示两者CAR阳性率和表达量(MFI)没差异。
用anti-human CD4-APC和anti-human CD8-PE-Cy7抗体流式检测anti-mesothelin 28Z CAR-T和E28Z CAR-T细胞，流式图结果如图2:c，显示两者的CD4 +CAR-T细胞和CD8 +CAR-T细胞的比例相近。
10万anti-mesothelin CAR-T细胞与过表达mesothelin的K562-Mesothelin细胞系按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一天即收样。收样前6小时加1x BFA抑制细胞因子外排，收样后将样品PBS洗一次，然后4％PFA室温固定5min，0.1％TritonX-100室温打孔5min,接着按照常规操作胞内染色检测IL-2、IFN-γ、TNF-α。结果图2:d-f.所示，表明anti-mesothelin E28Z CAR-T细胞经K562-Mesothelin细胞特异刺激后产生的细胞因子IL-2、IFN-γ、TNF-α都明显少于anti-mesothelin 28Z CAR-T细胞。
10万CAR-T细胞与过表达mesothelin的K562-Mesothelin细胞系按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一段时间。于不同时间点收样后将样品PBS洗一次，按照Annexin V Apoptosis Detection Kit APC试剂盒说明书染色好后流式检测CAR-T细胞Annexin V的表达水平。结果如图2:g，表示，anti-mesothelin E28Z CAR-T经K562-Mesothelin细胞系刺激后的凋亡水平持续一段时间一直比anti-mesothelin 28Z CAR-T明显低。
10万CAR-T细胞与过表达mesothelin的K562-Mesothelin细胞系按照1：1比例接种于圆底96孔板中，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱 中培养一段时间。于不同时间点收样后将样品PBS洗一次，用anti-human CD4-APC和anti-human CD8-PE-Cy7抗体染色检测CAR +T细胞存活数量。结果如图2:h，表示，anti-mesothelin E28Z CAR-T经K562-Mesothelin细胞系刺激后的存活数量比28Z CAR-T明显增多。
将mesothelin -K562细胞重悬于1640无血清培养基，然后预染1x CellTraker deep red dye(37°水浴，30min)，接着1640无血清培养基洗一次，用T细胞完全培养基重悬后与mesothelin +K562_mesothelin(IRES mCherry)细胞按照1：1混合为靶细胞。CAR-T细胞再与该混合型靶细胞按照3：1，1：1，1：3比例混合铺板于圆底96孔板，混匀，室温离心(400g,1min)促细胞间接触，用完全培养基于37℃培养箱中培养一天即收样，置冰，流式检测mesothelin -K562细胞(APC +,mCherry -)百分比与mesothelin +K562_mesothelin细胞(APC -,mCherry +)百分比变化，实验组的“mesothelin -K562细胞(APC +,mCherry -)百分比/mesothelin +K562_mesothelin细胞(APC -,mCherry +)百分比”记为N。N值以未加CAR-T细胞的混合型靶细胞样品孔的“N 0＝mesothelin -K562细胞(APC +,mCherry -)百分比/mesothelin +K562_mesothelin细胞(APC -,mCherry +)百分比”标准化后，计得存活比率N/N 0，则杀伤率公式为“Lysis(％)＝1-N/N 0”。结果如图2:i，显示，anti-mesothelin E28Z CAR-T对mesothelin +肿瘤细胞毒性比28Z CAR-T细胞较大。
无菌操作准备2x10^7/ml PANC-1细胞-PBS重悬液，在每只6周龄B-NDG雌鼠的左侧背部皮下接种100ul PANC-1细胞2百万，时间点记为day0。7天后，PANC-1皮下瘤长至直径7-8mm时，将小鼠随机分组，然后对每只小鼠尾静脉注射100ul 6百万anti-mesothelin CAR-T细胞，模型如图2:j所示，之后定期用游标卡尺测量肿瘤的大小，记录数据，肿瘤面积＝长x宽。结果如图2:k.表明，vector对照组与实验组28Z组的小鼠的肿瘤生长最快，两者没有差异，而E28Z组治疗效果比较好，统计有差异。
实验结果：
(1)、E28Z与28Z CAR-T细胞的上膜水平以及CD4和CD8比例相当；
(2)、CAR-T细胞经过Raji细胞体外特异性刺激后，E28Z分泌的细胞因子(IFN-γ、IL-2、TNF)明显比28Z低，而产生的GrzB明显比28Z高；
(3)、CAR-T细胞经过Raji细胞体外特异性刺激后，起初短时间内28Z的表面CAR(％)下降速率比E28Z快，然后表面CAR(％)回复速率也明显比E28Z快。
(4)、CAR-T细胞经过Raji细胞体外特异性刺激扩增一段时间后，于不同时间点采样检测凋亡水平，E28Z与28Z相比，先是两者相当，而后是E28Z凋亡水平明显低于28Z；
(5)、CAR-T细胞经过Raji细胞体外特异性刺激扩增一段时间后，于不同时间点采样检测增殖能力(即Ki67表达水平)，E28Z增殖能力明显高于28Z；
(7)、CAR-T细胞经过Raji细胞特异性刺激扩增一段时间后，于不同时间点采样检测扩增的CAR+T细胞数目，随着时间的推移，E28Z数量越明显高于28Z；
(8)、CAR-T细胞与CD19+K562:CD19-K562共孵育的体外杀伤实验中，较高剂量的E28Z的杀伤能力可明显高于28Z。
(9)、小鼠体内抗肿瘤实验中，E28Z的疗效可明显高于28Z。
以上所述，仅为本发明的较佳实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。凡熟悉本专业的技术人员，在不脱离本发明的精神和范围的情况下，当可利用以上所揭示的技术内容而做出的些许更动、修饰与演变的等同变化，均为本发明的等效实施例；同时，凡依据本发明的实质技术对上述实施例所作的任何等同变化的更动、修饰与演变，均仍属于本发明的技术方案的范围内。

Claims (14)

  1. 一种嵌合抗原受体，包括：
    依次连接的胞外结构域、跨膜结构域和胞内结构域；
    所述胞外结构域包括抗原识别区和铰链区；
    所述胞内结构域与跨膜结构域相连的一端连接有CD3ε胞内区。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，还包括以下特征中的一项或多项：
    (1)所述CD3ε胞内区的氨基酸序列如SEQ ID NO：1所示；
    (2)所述胞内结构域包括依次连接的CD3ε胞内区、共刺激信号传导区和CD3ζ胞内区。
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，所述共刺激信号传导区选自CD27、CD28、CD134、4-1BB和ICOS的胞内区中的一种或多种。
  4. 如权利要求3所述的嵌合抗原受体，其特征在于，还包括以下特征中的一项或多项：
    (1)所述CD28胞内区的氨基酸序列如SEQ ID NO：2所示；
    (2)所述CD3ζ胞内区的氨基酸序列如SEQ ID NO：3所示。
  5. 如权利要求1所述的嵌合抗原受体，其特征在于，还包括以下特征中的一项或多项：
    a.所述抗原识别区选自针对肿瘤表面抗原的单链抗体，所述肿瘤表面抗原选自CD19、间皮素、CD20、CD22、CD123、CD30、CD33、CD38、CD138、BCMA、Fibroblast activation protein、Glypican-3、CEA、EGFRvIII、PSMA、Her2、IL13Rα2、CD171和GD2中的一种或多种；
    b.所述跨膜结构域选自CD28TM、CD4、CD8α、OX40或H2-Kb。
  6. 如权利要求5所述的嵌合抗原受体，其特征在于，还包括以下特征中的一项或多项：
    c.所述单链抗体选自FMC63或SS1；
    d.所述CD28 TM的氨基酸序列如SEQ ID NO：4所示。
  7. 如权利要求1-6任一所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的氨基酸序列如SEQ ID NO：5或SEQ ID NO：6所示。
  8. 一种多核苷酸序列，该多核苷酸序列选自：
    (1)编码权利要求1-7任一所述的嵌合抗原受体的多核苷酸序列；和
    (2)(1)所述多核苷酸序列的互补序列。
  9. 如权利要求8所述的多核苷酸序列，其特征在于，所述多核苷酸序列如SEQ ID NO：7或SEQ ID NO：8所示。
  10. 一种核酸构建物，所述核酸构建物含有权利要求8-9任一所述的多核苷酸序列；
    优选的，所述核酸构建物为载体；
    更优选的，所述核酸构建物为慢病毒载体，含有复制起始位点、3’LTR、5’LTR以及权利要求8-9任一所述的多核苷酸序列。
  11. 一种慢病毒载体系统，所述慢病毒载体系统含有权利要求10所述的核酸构建物及慢病毒载体辅助成分。
  12. 一种基因修饰的T细胞，其特征在于，所述细胞含有权利要求8-9任一所述的多核苷酸序列，或含有权利要求10所述的核酸构建物，或感染了权利要求11所述的慢病毒载体系统。
  13. 权利要求1－7中任一项所述的嵌合抗原受体、权利要求8-9任一所述的多核苷酸序列，或含有权利要求10所述的核酸构建物，或权利要求11所述的慢病毒载体系统在制备以下任一种或多种用途产品中的应用：(1)制备T细胞；(2)抑制T细胞的细胞因子IFN-γ、IL-2、TNF分泌；(3)抑制T细胞凋亡；(5)增强T细胞增殖能力；(6)提高T细胞杀伤力；(7)促进GrzB产生；(8)促进脱颗粒。
  14. 权利要求1－7中任一项所述的嵌合抗原受体、权利要求8-9任一所述的多核苷酸序列，或含有权利要求10所述的核酸构建物，或权利要求11所述的慢病毒载体系统在或权利要求12所述的基因修饰的T细胞在制备以下任一种或多种用途产品中的应用：(1)治疗肿瘤；(3)抑制肿瘤治疗时产生的细胞因子风暴；(4)抑制肿瘤治疗时产生的神经毒性；
    优选的，所述肿瘤选自白血病或实体瘤中的一种或多种。
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