WO2023142683A1 - 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用 - Google Patents

靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用 Download PDF

Info

Publication number
WO2023142683A1
WO2023142683A1 PCT/CN2022/136297 CN2022136297W WO2023142683A1 WO 2023142683 A1 WO2023142683 A1 WO 2023142683A1 CN 2022136297 W CN2022136297 W CN 2022136297W WO 2023142683 A1 WO2023142683 A1 WO 2023142683A1
Authority
WO
WIPO (PCT)
Prior art keywords
tcr
cmv
cells
seq
cell
Prior art date
Application number
PCT/CN2022/136297
Other languages
English (en)
French (fr)
Inventor
王鹏然
沈良华
张岩
宋献民
Original Assignee
上海市第一人民医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海市第一人民医院 filed Critical 上海市第一人民医院
Publication of WO2023142683A1 publication Critical patent/WO2023142683A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2510/00—Genetically modified 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a TCR capable of recognizing antigenic peptides derived from cytomegalovirus, cytomegalovirus-specific T cells expressing the TCR, and their use in the prevention and treatment of cytomegalovirus-related diseases.
  • Cytomegalovirus belongs to the ⁇ -subfamily of the Human Herpesviridae family and has obvious host species specificity. It is the largest virus with the most complex structure in the Human Herpesviridae family. The infection rate in adults is more than 95%, and it is usually a recessive infection. Most infected people have no clinical symptoms, but under certain conditions, it can invade multiple organs and systems and cause serious diseases.
  • HCMV Human cytomegalovirus
  • ORF UL83 pp65 protein encoded by ORF UL83
  • phosphoprotein pp71 encoded by ORF UL82 phosphoprotein pp71 encoded by ORF UL82
  • pUL69 protein encoded by ORF UL69 The common feature of the above three proteins is to initiate HCMV infection of host cells and virus replication, and also play an important role in escaping T lymphocyte-mediated cytotoxicity. Studies have shown that among the above three proteins, pp65 protein is the most serious in causing graft vascular disease and rejection. Immunocompromised due to allo-HSCT can reactivate latent HCMV, leading to serious clinical complications.
  • TCR-T therapy is to capture specific TCR targeting tumor antigens or specific viral antigens, and apply genetic engineering technology to transform T cells, which can achieve the purpose of treating tumors or clearing viral infections after infusion.
  • Allo-HSCT patients are prone to primary CMV infection or reactivation of latent CMV because of low immune function and cannot effectively clear CMV.
  • CMV infection/reactivation in HSCT patients a series of related diseases that can cause CMV-associated fever to organ involvement are closely related to disease recurrence and the survival of subjects, and the current conventional drug treatment is less effective. Therefore, if CMV-TCR-T cell therapy can be applied to the field of treatment and prevention of CMV infection after HSCT or other organ transplantation, it will have great clinical value and application prospect.
  • the purpose of the present invention is to provide a HLA-A*0201-restricted specific TCR targeting cytomegalovirus epitope pp65, TCR-T cells expressing the TCR and applications thereof.
  • the first aspect of the present invention provides a T cell receptor (TCR), the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, the amino acid sequence of the CDR3 of the TCR ⁇ chain variable domain is CAFPYNNNDMRF( SEQ ID NO.13); and/or
  • the amino acid sequence of the CDR3 of the TCR ⁇ chain variable domain is CASSLEGYTEAFF (SEQ ID NO. 21).
  • the TCR specifically binds to the NLVPMVATV-HLA-A*0201 complex.
  • the three complementarity determining regions (CDRs) of the variable domain of the TCR ⁇ chain are:
  • ⁇ -CDR1 SSNFYA (SEQ ID NO.9)
  • ⁇ -CDR2 MTLNGDE (SEQ ID NO.11),
  • ⁇ -CDR3 CAFPYNNNDMRF (SEQ ID NO.13).
  • the three complementarity determining regions of the TCR ⁇ chain variable domain are:
  • ⁇ -CDR 1 MNHEY (SEQ ID NO.17),
  • ⁇ -CDR 2 SMNVEV (SEQ ID NO.19),
  • ⁇ -CDR 3 CASSLEGYTEAFF (SEQ ID NO.21).
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO.7
  • the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO.15.
  • the TCR comprises the amino acid sequence of the alpha chain variable domain SEQ ID NO.7.
  • the TCR comprises the amino acid sequence of the ⁇ -chain variable domain of SEQ ID NO.15.
  • the TCR is an ⁇ heterodimer, which comprises a TCR ⁇ chain constant region TRAC*01 and a TCR ⁇ chain constant region TRBC1*01 or TRBC2*01.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO.3.
  • amino acid sequence of the ⁇ chain of the TCR is SEQ ID NO.5.
  • an artificial interchain disulfide bond is contained between the ⁇ chain and ⁇ chain of the TCR.
  • amino acid sequence of the TCR is shown in SEQ ID NO.1.
  • the second aspect of the present invention provides a multivalent TCR complex, which comprises at least two TCR molecules, and at least one of the TCR molecules is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule comprising the nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or its complementary sequence.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO.8 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO.16 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO.4 encoding the TCR ⁇ chain.
  • the nucleic acid molecule comprises the nucleotide sequence SEQ ID NO.6 encoding the TCR beta chain.
  • the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO.2.
  • the fourth aspect of the present invention provides a vector, the vector contains the nucleic acid molecule described in the third aspect of the present invention; preferably, the vector is a viral vector; more preferably, the vector is a lentivirus Viral vector.
  • the fifth aspect of the present invention provides an isolated host cell containing the vector of the fourth aspect of the present invention or the exogenous nucleic acid molecule of the third aspect of the present invention integrated in the genome .
  • the sixth aspect of the present invention provides a cell transduced with the nucleic acid molecule described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention; preferably, the cell is a T cell or a stem cell .
  • the seventh aspect of the present invention provides a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and the TCR described in the first aspect of the present invention, the TCR complex described in the second aspect of the present invention, the The nucleic acid molecule of the third aspect of the invention, the vector of the fourth aspect of the invention, or the cell of the sixth aspect of the invention.
  • the eighth aspect of the present invention provides the T cell receptor described in the first aspect of the present invention, or the TCR complex described in the second aspect of the present invention, the nucleic acid molecule described in the third aspect of the present invention, the fourth aspect of the present invention.
  • the use of the carrier according to the aspect, or the cell according to the sixth aspect of the present invention, is used to prepare a medicine for treating cytomegalovirus-related diseases.
  • the ninth aspect of the present invention provides a method for treating diseases, comprising administering an appropriate amount of the T cell receptor described in the first aspect of the present invention or the TCR complex described in the second aspect of the present invention to a subject in need of treatment , the nucleic acid molecule of the third aspect of the present invention, the vector of the fourth aspect of the present invention, or the cell of the sixth aspect of the present invention, or the pharmaceutical composition of the seventh aspect of the present invention;
  • the disease is a disease related to cytomegalovirus infection (cytomegalovirus infection), such as CMV retinitis, CMV pneumonia, CMV gastroenteritis, and CMV encephalitis.
  • cytomegalovirus infection cytomegalovirus infection
  • CMV retinitis CMV pneumonia
  • CMV gastroenteritis CMV gastroenteritis
  • CMV encephalitis CMV encephalitis
  • Figure 1 The specific stimulation and screening process of CMV-pp65-TCR-T;
  • Figure 2 The results of in vitro specific expansion of CMV-pp65-TCR-T cells
  • FIG. 3 CMV-pp65-TCR-T carrier information
  • Figure 7 CMV-pp65-TCR-T in vivo killing verification - subcutaneous tumor model
  • Figure 8 CMV-pp65-TCR-T in vivo killing validation-transfer model.
  • the invention discloses a specific HLA-A*0201-restricted targeting cytomegalovirus (Cytomegalovirus, CMV) pp65 epitope NLVPMVATV specific T cell and its application.
  • the T cell receptor (T cell receptor) carried by the T cell , TCR) can specifically target CMV-pp65, and clear CMV virus accurately and rapidly.
  • CMV-pp65-specific TCR-T cells can be prepared by lentivirus infection of T cells with the high-affinity TCR gene of CMV-pp65.
  • CMV-pp65-TCR-T cells can quickly clear the CMV virus directly through precise targeting; on the other hand, they can also play a long-term role in the complete elimination of the virus and the prevention of reinfection through the immune reconstruction mediated by TCR-T cells.
  • the pp65-TCR-T cells specific for the pp65 antigen epitope of the present invention can specifically bind and kill target cells expressing the CMV pp65 antigen restricted by HLA-A*0201, and can be TCR-T cells for CMV-related diseases.
  • the treatment provides a new clinical program, and its application in the field of treatment and prevention of CMV infection after HSCT or other organ transplantation has great clinical value and application prospect.
  • MHC molecules are proteins of the immunoglobulin superfamily and can be class I or class II MHC molecules. Therefore, it is specific for the presentation of antigens, and different individuals have different MHCs, which can present different short peptides in a protein antigen to the surface of their respective APC cells.
  • Human MHC is often referred to as HLA genes or HLA complexes.
  • T cell receptor is the sole receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APCs antigen-presenting cells
  • T cells with different antigen specificities exert immune effects on their target cells.
  • TCR is a glycoprotein on the cell membrane surface that exists in the form of a heterodimer of ⁇ chain/ ⁇ chain or ⁇ chain/ ⁇ chain.
  • TCR heterodimers consist of alpha and beta chains in 95% of T cells, while 5% of T cells have TCRs consisting of gamma and delta chains.
  • the native ⁇ heterodimeric TCR has an ⁇ chain and a ⁇ chain, which constitute the subunits of the ⁇ heterodimeric TCR.
  • each chain of ⁇ and ⁇ contains a variable region, a connecting region, and a constant region, and the ⁇ chain usually also contains a short variable region between the variable region and the connecting region, but this variable region is often regarded as the connecting region a part of.
  • Each variable region comprises 3 CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, which are embedded in framework regions.
  • the CDR region determines the combination of the TCR and the pMHC complex, in which CDR3 is recombined from the variable region and the linker region, known as the hypervariable region.
  • the ⁇ and ⁇ chains of a TCR are generally regarded as having two "domains" each, namely a variable domain and a constant domain, the variable domains are composed of linked variable and linker regions.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT).
  • IMGT International Immunogenetics Information System
  • the constant domain sequence of the ⁇ chain of the TCR molecule is "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule is "TRBC1* 01" or "TRBC2*01”.
  • the ⁇ and ⁇ chains of TCR also contain transmembrane and cytoplasmic regions.
  • polypeptide of the present invention TCR of the present invention
  • T cell receptor of the present invention T cell receptor of the present invention
  • the first aspect of the present invention provides a TCR molecule capable of binding the NLVPMVATV-HLA-A*0201 complex.
  • said TCR molecule is isolated or purified.
  • the ⁇ and ⁇ chains of this TCR each have 3 complementarity determining regions (CDRs).
  • the ⁇ chain of the TCR comprises a CDR having the following amino acid sequence:
  • ⁇ -CDR1 SSNFYA (SEQ ID NO.9)
  • ⁇ -CDR2 MTLNGDE (SEQ ID NO.11),
  • ⁇ -CDR3 CAFPYNNNDMRF (SEQ ID NO.13).
  • the ⁇ chain of the TCR comprises a CDR having the following amino acid sequence:
  • ⁇ -CDR 1 MNHEY (SEQ ID NO.17),
  • ⁇ -CDR 2 SMNVEV (SEQ ID NO.19),
  • ⁇ -CDR 3 CASSLEGYTEAFF (SEQ ID NO.21).
  • Chimeric TCR can be prepared by embedding the amino acid sequence of the above CDR region of the present invention into any suitable framework structure.
  • the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned ⁇ and/or ⁇ chain CDR region sequence and any suitable framework structure.
  • the TCR ⁇ chain variable domain of the present invention is an amino acid sequence having at least 90%, preferably 95%, and more preferably 98% sequence identity with SEQ ID NO.7; and/or the TCR ⁇ chain variable domain of the present invention is an amino acid sequence identical to SEQ ID NO.7 NO. 15 has an amino acid sequence with at least 90%, preferably 95%, more preferably 98% sequence identity.
  • the TCR molecule of the present invention is a heterodimer composed of ⁇ and ⁇ chains.
  • the ⁇ chain of the heterodimeric TCR molecule comprises a variable domain and a constant domain
  • the amino acid sequence of the variable domain of the ⁇ chain comprises CDR1 (SEQ ID NO.9), CDR2 (SEQ ID NO.9) of the above ⁇ chain. ID NO.11) and CDR3 (SEQ ID NO.13).
  • the TCR molecule comprises the amino acid sequence of the alpha chain variable domain SEQ ID NO.7. More preferably, the amino acid sequence of the ⁇ -chain variable domain of the TCR molecule is SEQ ID NO.7.
  • the ⁇ chain of the heterogeneous dimerization TCR molecule comprises a variable domain and a constant domain
  • the amino acid sequence of the variable domain of the ⁇ chain comprises CDR1 (SEQ ID NO.17), CDR2 (SEQ ID NO.19) and CDR3 (SEQ ID NO.21).
  • the TCR molecule comprises the amino acid sequence of the ⁇ -chain variable domain of SEQ ID NO.15. More preferably, the amino acid sequence of the ⁇ chain variable domain of the TCR molecule is SEQ ID NO.15.
  • the TCR molecule of the present invention is a single-chain TCR molecule composed of part or all of the ⁇ chain and/or part or all of the ⁇ chain.
  • single-chain TCR molecules For the description of single-chain TCR molecules, reference can be made to Chung et al (1994) Proc. Natl. Acad. Sci. USA 91, 12654-12658. Those skilled in the art can easily construct single-chain TCR molecules comprising the CDRs regions of the present invention according to the literature.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably connected in order from N-terminus to C-terminus.
  • the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR molecule comprises CDR1 (SEQ ID NO.9), CDR2 (SEQ ID NO.11) and CDR3 (SEQ ID NO.13) of the above-mentioned ⁇ -chain.
  • the single-chain TCR molecule comprises the amino acid sequence of an ⁇ -chain variable domain of SEQ ID NO.7. More preferably, the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR molecule is SEQ ID NO.7.
  • the ⁇ -chain variable domain amino acid sequence of the single-chain TCR molecule comprises CDR1 (SEQ ID NO.17), CDR2 (SEQ ID NO.19) and CDR3 (SEQ ID NO.21) of the above-mentioned ⁇ -chain.
  • the single-chain TCR molecule comprises the amino acid sequence of the ⁇ -chain variable domain of SEQ ID NO.15. More preferably, the amino acid sequence of the ⁇ -chain variable domain of the single-chain TCR molecule is SEQ ID NO.15.
  • the constant domain of the TCR molecule of the present invention is a human or mouse constant domain.
  • the constant domain sequence of the ⁇ chain of the TCR molecule of the present invention can be "TRAC*01”
  • the constant domain sequence of the ⁇ chain of the TCR molecule can be "TRBC1*01” or "TRBC2*01”.
  • the amino acid sequence of the alpha chain of the TCR molecule of the present invention is SEQ ID NO.3, and/or the amino acid sequence of the beta chain is SEQ ID NO.5.
  • the TCRs of the invention may contain artificial disulfide bonds introduced between residues in the constant domains of their alpha and beta chains. It should be noted that the TCR of the present invention can contain both the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bond introduced between the constant domains as described above.
  • the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by native disulfide bonds present in the TCR.
  • the TCR constant regions of the invention can be engineered to prevent mismatches with endogenous TCR chains. For this reason, in the TCR of the present invention, artificial disulfide bonds are introduced between the residues of the constant domains of its ⁇ and ⁇ chains.
  • the TCRs of the invention may also be hybrid TCRs comprising sequences derived from more than one species.
  • murine TCRs are more efficiently expressed in human T cells than human TCRs.
  • a TCR of the invention may comprise a human variable domain and a murine constant domain.
  • a drawback of this approach is the potential for eliciting an immune response. Therefore, its use in adoptive T cell therapy should have regulatory protocols for immunosuppression to allow engraftment of murine-expressing T cells.
  • a second aspect of the present invention provides a nucleic acid molecule encoding a TCR molecule of the first aspect of the present invention or a portion thereof, which may be one or more CDRs, variable domains of ⁇ and/or ⁇ chains, and ⁇ chains and/or or beta strand.
  • the nucleotide sequence encoding the CDR region of the alpha chain of the TCR molecule in the first aspect of the present invention is as follows:
  • the nucleotide sequence encoding the CDR region of the TCR molecule ⁇ chain in the first aspect of the present invention is as follows:
  • nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain of the present invention includes SEQ ID NO.10, SEQ ID NO.12 and SEQ ID NO.14, and/or the nucleic acid molecule of the present invention encoding the TCR ⁇ chain of the present invention Nucleotide sequences include SEQ ID NO.18, SEQ ID NO.20 and SEQ ID NO.22.
  • the nucleotide sequence of a nucleic acid molecule of the invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns.
  • the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but can encode the polypeptide of the present invention, for example, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable domain of the TCR ⁇ chain of the present invention includes SEQ ID NO.8 and /Or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR ⁇ chain variable domain of the present invention comprises SEQ ID NO.16.
  • nucleotide sequence of the nucleic acid molecule of the present invention comprises SEQ ID NO.4 and/or SEQ ID NO.6.
  • nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO.2.
  • nucleic acid sequence encoding the TCR of the present invention may be the same as the nucleic acid sequence shown in the figures of the present invention or a degenerate variant.
  • degenerate variant refers to a nucleic acid sequence that encodes a protein sequence with SEQ ID NO.1, but differs from the sequence of SEQ ID NO.2.
  • Nucleotide sequences may be codon optimized. Different cells use different codons, and the codons in the sequence can be changed to increase the expression according to the cell type. Codon usage tables for mammalian cells, as well as for a variety of other organisms, are well known to those skilled in the art.
  • the full-length sequence of the nucleic acid molecule of the present invention or its fragments can usually be obtained by, but not limited to, PCR amplification, recombination or artificial synthesis.
  • the DNA sequence encoding the TCR of the present invention (or its fragments, or its derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. DNA can be either the coding strand or the non-coding strand.
  • the invention also relates to vectors comprising the nucleic acid molecules of the invention, including expression vectors, ie constructs capable of expression in vivo or in vitro.
  • expression vectors include bacterial plasmids, bacteriophages, and animal and plant viruses.
  • Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpesviral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
  • AAV adeno-associated viral
  • the vector can transfer the nucleotide of the present invention into cells, such as T cells, so that the cells express antigen-specific TCR.
  • the vector should be consistently expressed at high levels in T cells.
  • the present invention also relates to host cells produced by genetic engineering with the vectors or coding sequences of the present invention.
  • the host cell contains the vector of the present invention or the nucleic acid molecule of the present invention is integrated in the chromosome.
  • the host cells are selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells, 293T cells and the like.
  • the present invention also includes isolated cells expressing the TCR of the present invention, especially T cells.
  • the T cells may be derived from T cells isolated from the subject, or may be part of a mixed cell population isolated from the subject, such as a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cells can be isolated from peripheral blood mononuclear cells (PBMC), and can be CD4 + helper T cells or CD8 + cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cells may be in a mixed population of CD4 + helper T cells/CD8 + cytotoxic T cells.
  • the cells of the invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSC).
  • stem cells such as hematopoietic stem cells (HSC).
  • HSCs hematopoietic stem cells
  • T cells expressing the TCR of the present invention can be used for adoptive immunotherapy.
  • Many suitable methods of performing adoptive therapy are known to those skilled in the art (eg, Rosenberg et al., (2008) Nat Rev Cancer 8(4):299-308).
  • CMV Cytomegalovirus
  • the present invention also relates to methods of treating and/or preventing CMV-related diseases in a subject, comprising the step of adoptively transferring CMV-specific T cells to the subject.
  • This CMV-specific T cell recognizes the major CMV matrix phosphoprotein pp65.
  • the CMV-specific T cells recognize the epitope NLVPMVATV.
  • CMV is a ubiquitous human herpesvirus that infects approximately 50% of normal individuals. In most cases, the immune response is able to control acute infection by recognizing CMV-derived antigens. The virus then exists in a latent state throughout the life of the host. Outgrowth is prevented by immune system effector mechanisms, including neutralizing antibodies against viral membrane proteins, HLA-restricted CMV-specific helper and cytotoxic T cells, and MHC-restricted effectors.
  • CMV infection is important for certain high-risk groups.
  • Major areas of infection risk include prenatal or postnatal infants, and immunocompromised individuals, such as organ transplant recipients, leukemia patients, or people infected with human immunodeficiency virus (HIV).
  • immunocompromised individuals such as organ transplant recipients, leukemia patients, or people infected with human immunodeficiency virus (HIV).
  • CMV infection Three clinical forms of CMV infection generally exist, including:
  • Neonatal CMV inclusion body disease which may range from asymptomatic to severe disease affecting the liver, spleen, and central nervous system with possible disability;
  • Acute acquired CMV infection similar to infectious mononucleosis, will manifest symptoms such as fever, malaise, and skeletal muscle pain;
  • the TCRs of the invention can be used to treat and/or prevent reactivation of latent CMV after allogeneic hematopoietic stem cell transplantation.
  • CMV disease in Allo-HSCT recipients is thought to arise primarily from reactivation of latent virus. Transmission of the virus can occur from donor bone marrow infusions or from allogeneic blood products. In immunocompromised bone marrow transplant recipients, viral reactivation often results in progressive CMV infection, which is a major cause of infectious morbidity and mortality in this patient population. Progressive CMV infection is a result of both immunosuppression and delayed immune recovery in these patients after transplantation.
  • Allo-HSCT recipients are subjected to adoptive immunotherapy, for example, using T cells expressing CMV-specific T cell receptors of the present invention.
  • prevention means avoiding, delaying, resisting or arresting the progression of a disease. For example, the likelihood of CMV infection and/or CMV reactivation can be prevented or reduced.
  • treatment means to relieve, cure or reduce the symptoms of a disease, or to reduce or arrest the progression of a disease.
  • Prevention or treatment can be carried out by isolating T cells from patients or volunteers suffering from related diseases, introducing the TCR of the present invention into the above T cells, and then returning these genetically modified cells to the patient.
  • the present invention provides a method for treating CMV-related diseases, comprising infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient.
  • CMV-related diseases comprising infusing isolated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient itself, into the patient.
  • T cells are derived from the patient itself, into the patient.
  • the number of cells to be isolated, transfected and reinfused can be determined by the physician.
  • TCR of the present invention can directly clear CMV virus quickly through precise targeting
  • the TCR of the present invention has high protein expression in vivo and has no mismatch with the endogenous TCR chain;
  • the T cells expressing the TCR of the present invention have strong lethality and specificity, and can be effectively used in the treatment of CMV infection in patients with hematopoietic stem cell transplantation (HSCT) or other transplantation patients.
  • HSCT hematopoietic stem cell transplantation
  • DC medium DC cell culture medium
  • GM-CSF and IL-4 1 ⁇ DC Differentiation Supplement
  • Mature DC cells can be harvested on the 7th day. Pipette vigorously and resuspend the cells at the bottom of the well plate to collect mature DC cells. Transfer the collected mature DC cells to a 15ml centrifuge tube, 350g, 5min After centrifugation, the supernatant was removed, and DCs were resuspended in DC medium at a density of 2 ⁇ 10 6 cells/ml for subsequent experiments.
  • CMV-PP65 target peptide was added 5 ⁇ g/ml to the above-mentioned mature DC cells (Mature DC).
  • the sealing mold (Parafilm) was sealed and placed in a rotator (MACSmix), and rotatably combined in a cell culture incubator for 4 hours.
  • MCSmix rotator
  • CMV-peptide CMV-PP65 target peptide-loaded DC cells
  • CMV-peptide-loaded DCs CMV-peptide-loaded DCs
  • the DC cells loaded with the CMV-PP65 target peptide were collected by centrifugation into a 15ml centrifuge tube.
  • CMV-PP65 target peptide CMV-PP65peptide
  • CD8+/Dextramer+ T cells were sorted by flow cytometry, and the TCR ⁇ and TCR ⁇ chain sequences of CD8+/Dextramer+ T cells were obtained by 10X Genomics single-cell sequencing technology.
  • Example 4 Infection with CMV-pp65-TCR lentivirus to activate T cells to produce CMV-pp65-TCR-T
  • CMV-pp65-TCR lentiviral particles infect activated CD8+ T cells, add 8 ⁇ g/ml polybrene, 500 ⁇ g, 90 min, 30° C. for centrifugal infection. After centrifugation, they were placed in a cell culture incubator at 37°C and 5% CO 2 for 3 days, followed by a second round of infection with CMV-pp65-TCR lentivirus.
  • K562-A:0201-GFP, K562-A:0201-GFP-mCherry-vector and K562-A:0201-GFP-pp65-mCherry cells were respectively prepared by lentivirus infection of K562 cells;
  • Example 6 HLA:A*0201-restricted CMV-pp65-TCR-T specifically kills pp65-HLA*A0201-K562 cells in vitro
  • K562-A:0201-GFP control cells and K562-A:0201-GFP-pp65-mCherry target cells were respectively prepared by lentivirus infection of K562 cells;
  • FACS detected the ratio of K562-A:0201-GFP or K562-A:0201-GFP-pp65-mCherry in the co-culture system to evaluate CMV-pp65-TCR-T and Reported-TCR -Whether TCR-T can specifically kill K562-A:0201-GFP-pp65-mCherry cells, and compare the killing efficiency of two TCR-Ts.
  • Example 7 HLA:A*0201-restricted CMV-pp65-TCR-T specifically kills pp65-HLA*A0201-K562 cells in vivo
  • pp65-TCR-T day3 group and CMV-pp65-TCR-T cell (pp65-TCR-T day3 group), and on day7, 5 ⁇ 10 6 CMV-pp65-TCR-T cell (pp65-TCR-T day37group) was injected into the lateral tail vein ;D0 (+4h), d7, d14, d21, d28, d35 after tumor cell injection, bioluminescent imaging was performed on mice to detect the expression of luciferase in tumor cells, and the photon intensity of luciferase was quantified to evaluate CMV - Whether pp65-TCR-T can inhibit the growth of K562-A:0201-GFP-pp65-luciferase target cells.
  • Mouse tail vein transfer model inject 1 ⁇ 10 6 target cells into the lateral tail vein of NOG mice on day 0, and inject 1 ⁇ 10 7 primary CD8 + T cells and CMV-pp65-TCR through the lateral tail vein on day 3 -T cell.
  • bioluminescent imaging was performed on mice to detect the expression of luciferase in tumor cells, and the photon intensity of luciferase was quantified to evaluate CMV- Inhibitory effect of pp65-TCR-T on target cell K562-A:0201-GFP-pp65-luciferase.
  • Fig. 1 is the specific stimulation and screening process of CMV-pp65-TCR-T of the present invention.
  • the whole test is carried out by CD8+ T cells of a healthy HLA-A:0201 donor through autologous mDCs loaded with pp65 polypeptide for two rounds of stimulation, and then used CMV-PP65-HLA*A:0201 Dextramer analyzes the specificity of CMV-pp65 antigen-specific TCR-T cells.
  • the CMV-pp65 antigen-specific TCR-T cells were sorted by flow cytometry using CMV-PP65-HLA*A:0201Dextramer and CD8a antibody, and the CMV-pp65 -TCR-T cells were subjected to single-cell sequencing by 10 ⁇ Genomics method to obtain the ⁇ and ⁇ chain sequences of CMV-pp65-TCR-T cells.
  • the T cell clone contains two ⁇ -chains, one of which is a redundant ⁇ -chain, which has been subsequently excluded by CMV-PP65-HLA*A:0201 Dextramer binding experiments in vitro.
  • C in the sequence is a cysteine residue introduced by artificial mutation
  • the codon-optimized TCR coding sequence is as follows:
  • the alpha chain and its coding sequence are as follows:
  • the ⁇ chain and its coding sequence are as follows:
  • the ⁇ -chain variable region and its coding sequence are as follows:
  • ⁇ -CDR1 SSNFYA (SEQ ID NO.9), the coding sequence is: TCCAGCAATTTCTACGCC (SEQ ID NO.10)
  • ⁇ -CDR2 MTLNGDE (SEQ ID NO.11), the coding sequence is: ATGACCCTCAACGGCGATGAA (SEQ ID NO.12)
  • ⁇ -CDR3 CAFPYNNNDMRF (SEQ ID NO.13), the coding sequence is: TGTGCTTTTTCCTTAACAACAACGATATGAGGTTC (SEQ ID NO.14).
  • the ⁇ -chain variable region and its coding sequence are as follows:
  • ⁇ -CDR 1 MNHEY (SEQ ID NO.17), the coding sequence is: ATGAACCATGAATAC (SEQ ID NO.18);
  • ⁇ -CDR 2 SMNVEV (SEQ ID NO.19), the coding sequence is: TCTATGAATGTGGAGGTG (SEQ ID NO.20);
  • ⁇ -CDR 3 CASSLEGYTEAFF (SEQ ID NO.21), the coding sequence is: TGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTT (SEQ ID NO.22).
  • control cells K562-A:0201-GFP in vitro vector control cells K562-A:0201-GFP-mCherry-vector, and target cells K562-A:0201-GFP-pp65-mCherry, by adding CMV-pp65-TCR -T effector cells were co-cultured with target cells or control cells (vehicle control and blank control) in vitro, and after intracellular and extracellular staining, the expression levels of CD137 and IFN ⁇ in CMV-pp65-TCR-T cells were detected by flow cytometry.
  • K562-A:0201-GFP-pp65-luciferase target cells and CMV-pp65-TCR-T effector cells were prepared in vitro, 1 ⁇ 10 6 target cells were subcutaneously injected into the right axilla of NOG mice on day 0, and passed through the side respectively on day 3.
  • mice 5 ⁇ 10 6 primary CD8 + T cells (CD8 + T day3group) and CMV-pp65-TCR-T cells (pp65-TCR-T day3group) were injected into the tail vein, and 5 ⁇ 10 6 CMV- pp65-TCR-T cell (pp65-TCR-T day7group) (A of Figure 7); the mice were detected by bioluminescent imaging in weeks 0-5 after tumor cell injection (B of Figure 7), and the mice The photon number intensity of luciferase is quantified.
  • CMV infection is very widespread in the population.
  • the infection rate of adults in China is over 95%. It is usually a recessive infection.
  • Most infected people have no clinical symptoms, but under certain conditions, it can invade multiple organs and systems and cause serious diseases.
  • CMV activation is common in hematopoietic stem cell transplantation (HSCT) patients, and some patients have poor drug treatment effects or low drug tolerance, and cannot complete the entire course of drug treatment. Due to the low immune function of patients, CMV cannot be effectively cleared, and a series of related diseases such as CMV-related fever and organ involvement can be caused, with a high mortality rate.
  • HSCT hematopoietic stem cell transplantation
  • Existing anti-CMV drugs also have many shortcomings, such as single dosage form, similar targets, and can cause agranulocytosis/kidney damage, etc.
  • the CMV-TCR-T cells of this project can precisely target and clear the target cells infected with CMV virus; on the other hand, they can play a long-term role in the complete elimination of the virus and the prevention of re-infection through the immune reconstruction mediated by TCR-T cells.
  • the CMV-pp65-TCR-T invented by this project has the following characteristics:
  • CMV-pp65-TCR-T can accurately target and quickly eliminate target cells expressing CMV antigens.
  • the protein expression in vivo is high and there is no mismatch with the endogenous TCR chain. It has strong lethality and specificity, etc. .
  • in vitro experiments show that the CMV-pp65-TCR-T of this project has a more significant killing effect on target cells than the Reported-TCR-T disclosed in CN102656188A, and can be effectively applied to the treatment of CMV infection in HSCT patients or other transplant patients.
  • this project provides ELISPOT (enzyme-linked immunospot) in vitro experimental evidence, which is recognized as the gold standard in the field of TCR-T, indicating that the TCR screened by this project can specifically recognize CMV antigens; at the same time,
  • ELISPOT enzyme-linked immunospot
  • the invention of this project also uses a subcutaneous tumorigenesis model that is closer to the clinical CMV infection situation, which also confirms the loading of this project The T cells obtained by screening the CMV-specific TCR can specifically eliminate target cells expressing CMV antigens.
  • the invention of this project provides complete in vivo and in vitro data that conforms to field standards and is closer to the clinical CMV infection situation, fully confirming the specificity and effectiveness of the CMV antigen-specific TCR obtained through screening.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Plant Pathology (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明提供了靶向巨细胞病毒抗原的TCR和表达其的T细胞及应用，具体的本发明公开了一种HLA-A*0201限制性靶向巨细胞病毒pp65抗原表位的特异性T细胞及其应用，该T细胞携带的T细胞受体可特异性靶向CMV-pp65，并精准且快速地清除CMV病毒。

Description

靶向巨细胞病毒抗原的TCR和表达其的T细胞及应用 技术领域
本发明涉及能够识别源自巨细胞病毒抗原肽的TCR，表达上述TCR的巨细胞病毒特异性的T细胞，及他们在预防和治疗巨细胞病毒相关疾病中的用途。
背景技术
巨细胞病毒(cytomegalovirus,CMV)归属于人疱疹病毒科β亚科，具有明显的宿主种属特异性，是人疱疹病毒科中最大、结构也最复杂的病毒；CMV在人群中感染非常广泛，成人感染率达95％以上，通常呈隐性感染，多数感染者无临床症状，但在一定条件下侵袭多个器官和系统可产生严重疾病。近年来CMV感染的诊治取得了长足进步，使得移植后CMV感染的发病率有了明显下降，但CMV感染在异基因干细胞移植(allo-HSCT)患者中仍然保持了较高的发生率，其导致的间接效应，如继发性植入功能不良、GVHD、全因死亡率等对患者的预后仍产生巨大的影响。而现有抗CMV药物也存在诸多不足，如剂型单一、靶点相似、可导致粒细胞缺乏/肾损害等。当前抗病毒药物，如acyclovir，ganciclovir等常常引起严重的副作用，如骨髓抑制等。
人巨细胞病毒(HCMV)含有约20-25种蛋白质,其中在介导HCMV感染和病毒复制中有3种蛋白作用最为重要,即由ORF UL83编码的pp65蛋白、ORF UL82编码的磷蛋白pp71和ORF UL69编码的pUL69蛋白。上述3种蛋白共同的特点是启动HCMV对宿主细胞的感染和病毒的复制，同时在逃逸T淋巴细胞介导的细胞毒性作用中也具有重要作用。研究表明，在上述3种蛋白中又以pp65蛋白引起移植物血管病变和排斥反应的作用最为严重。因allo-HSCT引起的免疫受损，会使潜伏的HCMV再激活，从而导致严重的临床并发症。
近年来，以免疫检查点抑制剂、CAR-T(Chimeric Antibody Receptor Engineered T Cell)和以TCR-T(T cell Receptor Engineered T Cell)为代表的细胞免疫治疗在肿瘤治疗领域取得了重大进展。TCR-T治疗即是通过捕获针对肿瘤抗原或特定病毒抗原的特异性TCR，并应用基因工程技术改造T细胞，输注体内后可达到治疗肿瘤或清除病毒感染的目的。
allo-HSCT患者由于免疫功能低下，不能有效清除CMV，易发生原发性CMV感染或潜伏的CMV再激活。HSCT患者的CMV感染/再激活发生后，可引起CMV相关发热直至器官累及的系列相关性疾病，与疾病复发和受试者生存密切相关，且当前常规药物治疗效果较差。因此，如果能将CMV-TCR-T细胞疗法应用于治疗和预防HSCT或其他器官移植后的CMV感染领域，极具临床价值和应用前景。
发明内容
本发明的目的在于提供一种HLA-A*0201限制性靶向巨细胞病毒抗原表位pp65的特异性TCR、表达该TCR的TCR-T细胞及其应用。
本发明的第一方面，提供了一种T细胞受体(TCR)，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的CDR3的氨基酸序列为CAFPYNNNDMRF(SEQ ID NO.13)；和/或
所述TCRβ链可变域的CDR3的氨基酸序列为CASSLEGYTEAFF(SEQ ID NO. 21)。
在另一优选例中，所述TCR特异性结合NLVPMVATV-HLA-A*0201复合物。
在另一优选例中，所述TCRα链可变域的3个互补决定区(CDR)为：
α-CDR1：SSNFYA(SEQ ID NO.9)，
α-CDR2：MTLNGDE(SEQ ID NO.11)，
α-CDR3：CAFPYNNNDMRF(SEQ ID NO.13)。
在另一优选例中，所述TCRβ链可变域的3个互补决定区为：
β-CDR 1：MNHEY(SEQ ID NO.17)，
β-CDR 2：SMNVEV(SEQ ID NO.19)，
β-CDR 3：CASSLEGYTEAFF(SEQ ID NO.21)。
在另一优选例中，所述TCR包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域为与SEQ ID NO.7具有至少90％序列相同性的氨基酸序列；和/或所述TCRβ链可变域为与SEQ ID NO.15具有至少90％序列相同性的氨基酸序列。
在另一优选例中，所述TCR包含α链可变域氨基酸序列SEQ ID NO.7。
在另一优选例中，所述TCR包含β链可变域氨基酸序列SEQ ID NO.15。
在另一优选例中，所述TCR为αβ异质二聚体，其包含TCRα链恒定区TRAC*01和TCRβ链恒定区TRBC1*01或TRBC2*01。
在另一优选例中，所述TCR的α链氨基酸序列为SEQ ID NO.3。
在另一优选例中，所述TCR的β链氨基酸序列为SEQ ID NO.5。
在另一优选例中，所述TCR的α链与β链之间含有人工链间二硫键。
在另一优选例中，所述TCR的氨基酸序列如SEQ ID NO.1所示。
本发明的第二方面，提供了一种多价TCR复合物，其包含至少两个TCR分子，并且其中的至少一个TCR分子为本发明第一方面所述的TCR。
本发明的第三方面，提供了一种核酸分子，所述核酸分子包含编码本发明第一方面所述的TCR分子的核酸序列或其互补序列。
在另一优选例中，所述核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO.8。
在另一优选例中，所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO.16。
在另一优选例中，所述核酸分子包含编码TCRα链的核苷酸序列SEQ ID NO.4。
在另一优选例中，所述核酸分子包含编码TCRβ链的核苷酸序列SEQ ID NO.6。
在另一优选例中，所述核酸分子包含核苷酸序列SEQ ID NO.2。
本发明的第四方面，提供了一种载体，所述的载体含有本发明第三方面所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
本发明的第五方面，提供了一种分离的宿主细胞，所述的宿主细胞中含有本发明第四方面所述的载体或基因组中整合有外源的本发明第三方面所述的核酸分子。
本发明的第六方面，提供了一种细胞，所述细胞转导本发明第三方面所述 的核酸分子或本发明第四方面所述的载体；优选地，所述细胞为T细胞或干细胞。
本发明的第七方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的TCR、本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体、或本发明第六方面所述的细胞。
本发明的第八方面，提供了本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体、或本发明第六方面所述的细胞的用途，用于制备治疗巨细胞病毒相关疾病的药物。
本发明的第九方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的T细胞受体、或本发明第二方面所述的TCR复合物、本发明第三方面所述的核酸分子、本发明第四方面所述的载体、或本发明第六方面所述的细胞、或本发明第七方面所述的药物组合物；
优选地，所述的疾病为巨细胞病毒感染相关的疾病(巨细胞病毒感染症)，如CMV视网膜炎、CMV肺炎、CMV胃肠炎和CMV脑炎等。
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：CMV-pp65-TCR-T的特异性刺激及筛选流程；
图2：CMV-pp65-TCR-T细胞体外特异性扩增结果；
图3：CMV-pp65-TCR-T载体信息；
图4：CMV-pp65-TCR-T体外结合验证；
图5：CMV-pp65-TCR-T体外激活验证；
图6：CMV-pp65-TCR-T体外杀伤验证；
图7：CMV-pp65-TCR-T体内杀伤验证-皮下植瘤模型；
图8：CMV-pp65-TCR-T体内杀伤验证-转移模型。
具体实施方式
本发明公开了一种HLA-A*0201限制性靶向巨细胞病毒(Cytomegalovirus，CMV)pp65抗原表位NLVPMVATV的特异性T细胞及其应用，该T细胞携带的T细胞受体(T cell receptor，TCR)可特异性靶向CMV-pp65，并精准且快速地清除CMV病毒。CMV-pp65高亲和力的TCR基因经慢病毒感染T细胞后，可制备CMV-pp65特异性的TCR-T细胞。CMV-pp65-TCR-T细胞一方面可直接通过精准靶向作用快速清除CMV病毒；另一方面还可间接通过TCR-T细胞介导的免疫重建，对病毒的彻底清除和防止再感染发挥长期保护作用。因此，本发明的pp65抗原表位特异性的pp65-TCR-T细胞，能够特异性的结合并杀伤HLA-A*0201限制性表达CMV pp65抗原的靶细胞，可为CMV相关疾病的TCR-T治疗提供一种新的临床方案，将其应用于治疗和预防HSCT或其他器官移植后的CMV感染领域，极具临床价值和应用前景。
术语
MHC分子是免疫球蛋白超家族的蛋白质，可以是Ⅰ类或Ⅱ类MHC分子。因此，其对于抗原的呈递具有特异性，不同的个体有不同的MHC，能呈递一种蛋白抗原中不同的短肽到各自的APC细胞表面。人类的MHC通常称为HLA基因或HLA复合体。
T细胞受体(TCR)，是呈递在主组织相容性复合体(MHC)上的特异性抗原肽的唯一受体。在免疫系统中，通过抗原特异性的TCR与pMHC复合物的结合引发T细胞与抗原呈递细胞(APC)直接的物理接触，然后T细胞及APC两者的其他细胞膜表面分子就发生相互作用，这就引起了一系列后续的细胞信号传递和其他生理反应，从而使得不同抗原特异性的T细胞对其靶细胞发挥免疫效应。
TCR是由α链/β链或者γ链/δ链以异质二聚体形式存在的细胞膜表面的糖蛋白。在95％的T细胞中TCR异质二聚体由α和β链组成，而5％的T细胞具有由γ和δ链组成的TCR。天然αβ异质二聚TCR具有α链和β链，α链和β链构成αβ异源二聚TCR的亚单位。广义上讲，α和β各链包含可变区、连接区和恒定区，β链通常还在可变区和连接区之间含有短的多变区，但该多变区常视作连接区的一部分。各可变区包含嵌合在框架结构(framework regions)中的3个CDR(互补决定区)，CDR1、CDR2和CDR3。CDR区决定了TCR与pMHC复合物的结合，其中CDR3由可变区和连接区重组而成，被称为超变区。TCR的α和β链一般看作各有两个“结构域”即可变域和恒定域，可变域由连接的可变区和连接区构成。TCR恒定域的序列可以在国际免疫遗传学信息系统(IMGT)的公开数据库中找到，如TCR分子α链的恒定域序列为“TRAC*01”，TCR分子β链的恒定域序列为“TRBC1*01”或“TRBC2*01”。此外，TCR的α和β链还包含跨膜区和胞质区。
在本发明中，术语“本发明多肽”、“本发明的TCR”、“本发明的T细胞受体”可互换使用。
天然链间二硫键与人工链间二硫键
在天然TCR的近膜区Cα与Cβ链间存在一组二硫键，本发明中称为“天然链间二硫键”。在本发明中，将人工引入的，位置与天然链间二硫键的位置不同的链间共价二硫键称为“人工链间二硫键”。
发明详述
TCR分子
在抗原加工过程中，抗原在细胞内被降解，然后通过MHC分子携带至细胞表面。T细胞受体能够识别抗原呈递细胞表面的肽-MHC复合物。因此，本发明的第一方面提供了一种能够结合NLVPMVATV-HLA-A*0201复合物的TCR分子。优选地，所述TCR分子是分离的或纯化的。该TCR的α和β链各具有3个互补决定区(CDR)。
在本发明的一个优选地实施方式中，所述TCR的α链包含具有以下氨基酸序列的CDR：
α-CDR1：SSNFYA(SEQ ID NO.9)，
α-CDR2：MTLNGDE(SEQ ID NO.11)，
α-CDR3：CAFPYNNNDMRF(SEQ ID NO.13)。
在本发明的一个优选地实施方式中，所述TCR的β链包含具有以下氨基酸序列的CDR：
β-CDR 1：MNHEY(SEQ ID NO.17)，
β-CDR 2：SMNVEV(SEQ ID NO.19)，
β-CDR 3：CASSLEGYTEAFF(SEQ ID NO.21)。
可以将上述本发明的CDR区氨基酸序列嵌入到任何适合的框架结构中来制备嵌合TCR。只要框架结构与本发明的TCR的CDR区兼容，本领域技术人员根据本发明公开的CDR区就能够设计或合成出具有相应功能的TCR分子。因此，本发明TCR分子是指包含上述α和/或β链CDR区序列及任何适合的框架结构的TCR分子。
本发明TCRα链可变域为与SEQ ID NO.7具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列；和/或本发明TCRβ链可变域为与SEQ ID NO.15具有至少90％，优选地95％，更优选地98％序列相同性的氨基酸序列。
在本发明的一个优选例中，本发明的TCR分子是由α与β链构成的异质二聚体。具体地，一方面所述异质二聚TCR分子的α链包含可变域和恒定域，所述α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO.9)、CDR2(SEQ ID NO.11)和CDR3(SEQ ID NO.13)。优选地，所述TCR分子包含α链可变域氨基酸序列SEQ ID NO.7。更优选地，所述TCR分子的α链可变域氨基酸序列为SEQ ID NO.7。另一方面，所述异质二聚TCR分子的β链包含可变域和恒定域，所述β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO.17)、CDR2(SEQ ID NO.19)和CDR3(SEQ ID NO.21)。优选地，所述TCR分子包含β链可变域氨基酸序列SEQ ID NO.15。更优选地，所述TCR分子的β链可变域氨基酸序列为SEQ ID NO.15。
在本发明的一个优选例中，本发明的TCR分子是由α链的部分或全部和/或β链的部分或全部组成的单链TCR分子。有关单链TCR分子的描述可以参考文献Chung et al(1994)Proc.Natl.Acad.Sci.USA 91,12654-12658。根据文献中所述，本领域技术人员能够容易地构建包含本发明CDRs区的单链TCR分子。具体地，所述单链TCR分子包含Vα、Vβ和Cβ，优选地按照从N端到C端的顺序连接。
所述单链TCR分子的α链可变域氨基酸序列包含上述α链的CDR1(SEQ ID NO.9)、CDR2(SEQ ID NO.11)和CDR3(SEQ ID NO.13)。优选地，所述单链TCR分子包含α链可变域氨基酸序列SEQ ID NO.7。更优选地，所述单链TCR分子的α链可变域氨基酸序列为SEQ ID NO.7。所述单链TCR分子的β链可变域氨基酸序列包含上述β链的CDR1(SEQ ID NO.17)、CDR2(SEQ ID NO.19)和CDR3(SEQ ID NO.21)。优选地，所述单链TCR分子包含β链可变域氨基酸序列SEQ ID NO.15。更优选地，所述单链TCR分子的β链可变域氨基酸序列为SEQ ID NO.15。
在本发明的一个优选例中，本发明的TCR分子的恒定域是人或鼠的恒定域。本领域技术人员知晓或可以通过查阅相关书籍或IMGT(国际免疫遗传学信息系统)的公开数据库来获得人的恒定域氨基酸序列。例如，本发明TCR分子α链的恒定域序列可以为“TRAC*01”，TCR分子β链的恒定域序列可以为“TRBC1*01”或“TRBC2*01”。优选地，本发明TCR分子α链的氨基酸序列为SEQ ID NO.3，和/或β链的氨基酸序列为SEQ ID NO.5。
本发明的TCR可以包含在其α和β链恒定域的残基间引入的人工二硫键。应注意，恒定域间含或不含上文所述的引入的人工二硫键，本发明的TCR均可含有TRAC恒定域序列和TRBC1或TRBC2恒定域序列。TCR的TRAC恒定域序列和TRBC1或TRBC2恒定域序列可通过存在于TCR中的天然二硫键连接。
可以对本发明的TCR恒定区进行改造，以阻止与内源性TCR链的错配。为此，本发明的TCR中，在其α和β链恒定域的残基间引入了人工二硫键。
另外，本发明的TCR还可以是包含衍生自超过一种物种序列的杂合TCR。 例如，有研究显示鼠科TCR在人T细胞中比人TCR能够更有效地表达。因此，本发明TCR可包含人可变域和鼠的恒定域。这一方法的缺陷是可能引发免疫应答。因此，在其用于过继性T细胞治疗时应当有调节方案来进行免疫抑制，以允许表达鼠科的T细胞的植入。
应理解，本文中氨基酸名称采用国际通用的单英文字母或三英文字母表示，氨基酸名称的单英文字母与三英文字母的对应关系如下：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、Ile(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
核酸分子
本发明的第二方面提供了编码本发明第一方面TCR分子或其部分的核酸分子，所述部分可以是一个或多个CDR，α和/或β链的可变域，以及α链和/或β链。
编码本发明第一方面TCR分子α链CDR区的核苷酸序列如下：
α CDR1-TCCAGCAATTTCTACGCC(SEQ ID NO.10)
α CDR2-ATGACCCTCAACGGCGATGAA(SEQ ID NO.12)
α CDR3-TGTGCTTTTCCTTATAACAACAACGATATGAGGTTC(SEQ ID NO.14)
编码本发明第一方面TCR分子β链CDR区的核苷酸序列如下：
β CDR1-ATGAACCATGAATAC(SEQ ID NO.18)
β CDR2-TCTATGAATGTGGAGGTG(SEQ ID NO.20)
β CDR3-TGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTT(SEQ ID NO.22)
因此，编码本发明TCRα链的本发明核酸分子的核苷酸序列包括SEQ ID NO.10、SEQ ID NO.12和SEQ ID NO.14，和/或编码本发明TCRβ链的本发明核酸分子的核苷酸序列包括SEQ ID NO.18、SEQ ID NO.20和SEQ ID NO.22。
本发明核酸分子的核苷酸序列可以是单链或双链的，该核酸分子可以是RNA或DNA，并且可以包含或不包含内含子。优选地，本发明核酸分子的核苷酸序列不包含内含子但能够编码本发明多肽，例如编码本发明TCRα链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO.8和/或编码本发明TCRβ链可变域的本发明核酸分子的核苷酸序列包括SEQ ID NO.16。更优选地，本发明核酸分子的核苷酸序列包含SEQ ID NO.4和/或SEQ ID NO.6。或者，本发明核酸分子的核苷酸序列为SEQ ID NO.2。
应理解，由于遗传密码的简并，不同的核苷酸序列可以编码相同的多肽。因此，编码本发明TCR的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。以本发明中的其中一个例子来说明，“简并的变异体”是指编码具有SEQ ID NO.1的蛋白序列，但与SEQ ID NO.2的序列有差别的核酸序列。
核苷酸序列可以是经密码子优化的。不同的细胞在具体密码子的利用上是不同的，可以根据细胞的类型，改变序列中的密码子来增加表达量。哺乳动物细胞以及多种其他生物的密码子选择表是本领域技术人员公知的。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明TCR(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。DNA可以是编码链或非编码链。
载体
本发明还涉及包含本发明的核酸分子的载体，包括表达载体，即能够在体内或体外表达的构建体。常用的载体包括细菌质粒、噬菌体和动植物病毒。
病毒递送系统包括但不限于腺病毒载体、腺相关病毒(AAV)载体、疱疹病毒载体、逆转录病毒载体、慢病毒载体、杆状病毒载体。
优选地，载体可以将本发明的核苷酸转移至细胞中，例如T细胞中，使得该细胞表达抗原特异性的TCR。理想的情况下，该载体应当能够在T细胞中持续高水平地表达。
细胞
本发明还涉及用本发明的载体或编码序列经基因工程产生的宿主细胞。所述宿主细胞中含有本发明的载体或染色体中整合有本发明的核酸分子。宿主细胞选自：原核细胞和真核细胞，例如大肠杆菌、酵母细胞、CHO细胞、293T细胞等。
另外，本发明还包括表达本发明的TCR的分离的细胞，特别是T细胞。该T细胞可衍生自从受试者分离的T细胞，或者可以是从受试者中分离的混合细胞群，诸如外周血淋巴细胞(PBL)群的一部分。如，该细胞可以分离自外周血单核细胞(PBMC)，可以是CD4 +辅助T细胞或CD8 +细胞毒性T细胞。该细胞可在CD4 +辅助T细胞/CD8 +细胞毒性T细胞的混合群中。
备选地，本发明的细胞还可以是或衍生自干细胞，如造血干细胞(HSC)。将基因转移至HSC不会导致在细胞表面表达TCR，因为干细胞表面不表达CD3分子。然而，当干细胞分化为迁移至胸腺的淋巴前体(lymphoid precursor)时，CD3分子的表达将启动在胸腺细胞的表面表达该引入的TCR分子。
有许多方法适合于用编码本发明TCR的DNA或RNA进行T细胞转染(如，Robbins等.，(2008)J.Immunol.180:6116-6131)。表达本发明TCR的T细胞可以用于过继免疫治疗。本领域技术人员能够知晓进行过继性治疗的许多合适方法(如，Rosenberg等.，(2008)Nat Rev Cancer8(4)：299-308)。
巨细胞病毒(cytomegalovirus,CMV)相关疾病
本发明还涉及在受试者中治疗和/或预防CMV相关疾病的方法，其包括过继性转移CMV特异性T细胞至该受试者的步骤。该CMV特异性T细胞可识别主要CMV基质磷蛋白pp65。该CMV特异性T细胞可识别表位NLVPMVATV。
CMV是遍在的人疱疹病毒，其感染约50％的正常个体。在多数病例中，免疫反应能够通过识别CMV衍生的抗原而控制急性感染。该病毒然后以潜伏状态一直存在于宿主的一生当中。外生长受到免疫系统效应因子机制的阻止，包括针对病毒膜蛋白的中和抗体、HLA限制性的CMV特异性辅助和细胞毒性T细胞以及MHC限制性的效应因子。
CMV感染对于某些高危群体是重要的。感染风险的主要范围包括产前或产后婴儿，以及免疫妥协个体，诸如器官移植接受者、白血病患者或感染人免疫缺陷病毒(HIV)的人群。
一般存在三种临床形式的CMV感染，包括：
(1)新生儿CMV包涵体病，其可能是从无症状至影响肝、脾和中枢神经系统、可能产生残疾的严重疾病；
(2)急性获得性CMV感染，其类似于传染性单核细胞增多症，会表现出发烧、不适、骨骼肌疼痛等症状；
(3)免疫妥协人员(例如，移植过器官的人或患有HIV的人)CMV感染，其具有CMV视网膜炎、CMV肺炎、CMV胃肠炎和CMV脑炎的风险。
本发明的TCR可用于治疗和/或预防同种异体造血干细胞移植后潜伏CMV 的再活化。
Allo-HSCT接受者的CMV疾病被认为主要产生自潜伏病毒的再活化。病毒的传播可发生自供体骨髓输注或产生自同种异体血液制品。在免疫妥协的骨髓移植接受者中，病毒再活化通常导致渐进的CMV感染，其为这一患者群体中感染性发病率和死亡率的主要原因。渐进CMV感染是这些患者在移植后的免疫抑制和延缓的免疫复原这两者的结果。
在本发明的方法中，例如应用本发明的表达CMV-特异性T细胞受体的T细胞对Allo-HSCT接受者进行过继性免疫治疗。
预防和治疗方法
术语“预防”是指避免、延缓、阻抗或阻碍疾病的进展。例如可以预防或降低CMV感染和/或CMV再活化的可能性。
本文所使用的“治疗”是指用于缓解、治愈或减少疾病的症状，或减少或阻止疾病的进展。
可以通过分离患有相关疾病的病人或志愿者的T细胞，并将本发明的TCR导入上述T细胞中，随后将这些基因工程修饰的细胞回输到病人体内来进行预防或治疗。
因此，本发明提供了一种治疗CMV相关疾病的方法，包括将分离的表达本发明TCR的T细胞，优选地，该T细胞来源于病人本身，输入到病人体内。一般地，包括：
(1)分离病人的T细胞；
(2)用本发明核酸分子或能够编码本发明TCR分子的核酸分子体外转导T细胞；
(3)将基因工程修饰的T细胞输入到病人体内。
分离、转染及回输的细胞的数量可以由医师决定。
本发明的主要优点在于：
(1)本发明的TCR可直接通过精准靶向作用快速清除CMV病毒；
(2)本发明的TCR体内蛋白表达量高且与内源性TCR链无错配；
(3)表达本发明TCR的T细胞杀伤力能力强，特异性强等，可以有效用于造血干细胞移植(HSCT)患者或其他移植患者CMV感染的治疗。
下面的具体实施例，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和Russell等人，分子克隆：实验室手册(Molecular Cloning-A Laboratory Manual)(第三版)(2001)CSHL出版社)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，否则百分比和份数按重量计算。除非另外说明，否则百分比和份数按重量计算。以下实施例中所用的实验材料和试剂如无特别说明均可从市售渠道获得。
实施例1 体外诱导单核细胞(Monocytes)分化至树突细胞(DC细胞)
(1)使用淋巴细胞分离液(Lymphoprep)密度梯度离心法获取健康供者/患者血液中PBMCs(或复苏已冻存的PBMCs)，使用细胞毒性T淋巴细胞培养基(cytotoxic T lymphocyte medium)重悬后，350g，5min离心后去除上清，加入适量DC细胞培养基重悬PBMCs至6孔板中，培养箱中培养2h后，吸除培养基和结块的非贴壁细胞。
(2)每孔沿侧壁轻轻加入3ml含终浓度为1×DC Differentiation Supplement(GM-CSF与IL-4)的DC细胞培养基(DC medium)，将6孔板放置于37℃，5％CO 2培养箱中培养5d。
(3)第5天时，在不更换培养基的情况下，每孔加入30μl 100×DC Maturation Supplement至培养基中(DC Maturation Supplement终浓度为1×)。将6孔板放置于37℃，5％CO 2培养箱中诱导成熟2d。
(4)第7天时可收获成熟DC细胞(Mature DC)，移液器大力吹打和重悬孔板底部细胞以收取成熟DC细胞，将收集的成熟DC细胞转移至15毫升离心管，350g，5min离心后去除上清，按2×10 6cells/ml密度使用DC细胞培养基(DC medium)重悬DC供后续实验。
实施例2 人CMV-pp65抗原特异性T细胞的体外刺激和扩增
(1)向上述成熟DC细胞(Mature DC)中加入5μg/ml的CMV-PP65目标肽。封口模(Parafilm)密封后放入旋转器(MACSmix)中，于细胞培养箱中旋转结合4h。mDCs与CMV-PP65目标肽(CMV-peptide)孵育完成后，使用辐照仪以40Gy剂量辐照负载CMV-PP65目标肽的DC细胞(CMV-peptide-loaded DCs)。辐照结束后，离心收集负载CMV-PP65目标肽的DC细胞至15ml离心管中。
(2)使用淋巴细胞分离液(Lymphoprep)密度梯度离心法获取健康供者血液中PBMCs，使用试剂盒(EasySep TM Human CD8Positive Selection Kit)分离PBMCs中的CD8+T细胞，以DC/T为1:2.5的比例，将负载CMV-PP65目标肽的DC细胞(CMV-peptide-loaded DCs)与CD8+T细胞加至12孔板中培养，培养体系中加入终浓度为30ng/ml的IL-21，以及5μg/ml的CMV-PP65目标肽(CMV-PP65peptide)。
(3)将12孔板放置在37℃，5％CO 2细胞培养箱中孵育过夜。第一天时，向每个孔中加入10ng/ml的IL-2、IL-7、IL-15，随后置于细胞培养箱中，37℃，5％CO 2培养10天。培养过程中每2-3天进行半量换液。并在第5天及第10天时，往各培养体系中加入终浓度为5μg/ml的CMV-PP65目标肽(CMV-PP65peptide)。
(4)第11天时，参考上述步骤，制备辐照的负载CMV-PP65目标肽的DC细胞(Irradiated CMV-peptide-loaded DCs)与各培养体系中的细胞进行第二轮体外刺激，继续刺激7天。
(5)第18天时，吸取孔板中每孔各500μl细胞，经缓冲液(FACS buffer)洗涤两次后，加入CMV-PP65-HLA:A:0201Dextramer-FITC与CD8a-PE，混匀后4℃避光孵育20min，孵育结束后FACS buffer洗涤3次后，400μl含1×DAPI工作液的缓冲液(FACS buffer)重悬细胞后检测CD8+/Dextramer+(％)比例检测。
实施例3 单细胞测序获取HLA:A:0201限制型CMV-pp65抗原特异性TCR
(1)流式分选CD8+/Dextramer+T细胞，10X Genomics单细胞测序技术获取CD8+/Dextramer+T细胞的TCRα与TCRβ链序列。
(2)经单细胞测序得到TCR序列后，对出现频率最高的TCRα/β链序列 进行序列优化，对其恒定区进行改造，以避免外源TCR与T细胞内源TCR发生错配；表达序列进行密码子优化，提高蛋白表达量；利用P2A及弗林蛋白酶酶切位点(Furin-cleavage)使得TCRα与TCRβ能在一个表达载体中同时表达，而不产生多余的脯氨酸尾巴。
(3)构建慢病毒表达载体，将上述TCRα与TCRβ序列插入到同一慢病毒表达载体，通过293T细胞进行病毒包装，生产CMV-pp65抗原特异性TCR特异的病毒颗粒。
实施例4感染CMV-pp65-TCR慢病毒至活化T细胞产生CMV-pp65-TCR-T
(1)使用试剂盒(EasySep TM Human CD8Positive Selection Kit)分离PBMCs中的CD8+T细胞，以1:3 CD8+ T cell:CD3/28免疫磁珠(Dynabeads)比例，体外使用CD3/28免疫磁珠(Dynabeads)活化CD8+T细胞，37℃刺激活化CD8+T细胞2d。
(2)CMV-pp65-TCR慢病毒颗粒感染活化CD8+T细胞，加入8μg/ml聚凝胺(polybrene)，500×g，90min，30℃进行离心感染。离心结束后置于细胞培养箱中，37℃，5％CO 2培养3天，随后进行CMV-pp65-TCR慢病毒的二轮感染。
(3)CMV-pp65-TCR慢病毒二轮感染3天后，吸取CMV-pp65-TCR-T细胞，经缓冲液(FACS buffer)洗涤两次后，加入CMV-PP65-HLA*A:0201Dextramer-FITC与CD8a-PE抗体染色，FACS检测CD8+/Dextramer+(％)比例，随后流式分选CD8a+/Dextramer+T细胞用于后续体外杀伤实验。
实施例5 HLA:A*0201限制型CMV-pp65-TCR-T体外激活实验
A.FACS检测TCR-T细胞CD137、IFNγ及TNF-α的表达水平
(1)通过慢病毒感染K562细胞分别制备K562-A:0201-GFP，K562-A:0201-GFP-mCherry-vector与K562-A:0201-GFP-pp65-mCherry细胞；
(2)取实施例4中制备的CMV-pp65-TCR-T/Reported-TCR-T细胞，分别按效靶比例1:1，分别接种TCR-T细胞与K562细胞至圆底96孔板中培养。
(3)于共培养24h后进行胞外染色，固定破膜，胞内染色后，FACS检测共培养体系中CD8+T细胞的CD137,TNFα及IFNγ的表达水平，以评估K562-A:0201-GFP-pp65-mCherry靶细胞是否可在体外特异性激活CMV-pp65-TCR-T细胞。
B.Elispot检测TCR-T细胞IFNγ的释放水平
(1)使用无菌的pH 7.4的无钙镁离子的1×DPBS稀释包被抗体(MabtechIFNγ,1-D1K：1mg/ml)至15μg/ml(1.5μg/100μl)。每孔加入100μl稀释的1-D1K抗体溶液，4～8℃孵育过夜。
(2)去除plate中的抗体，无菌DPBS清洗后，以效靶比1:1比例铺板细胞，每孔加入100μl 1640完全培养基，3×10 4CMV-pp65-TCR-T cells或K562细胞，轻拍板的边缘使得细胞均匀铺开，放入培养箱，37℃，5％CO 2培养20h。
(3)20h后去除细胞，DPBS清洗4～6遍以完全去除细胞，然后每孔加入100μl，生物素标记的检测抗体7-B6-1-biotin 37℃孵育2h。孵育结 束后，DPBS清洗plate 5遍，最后一遍弃掉液体后，轻轻在无菌纸上扣干。随后每孔加入200μl Streptavidin-HRP，37℃孵育1h。
(4)孵育结束后，DPBS清洗plate 5遍，每孔加入100μl底物TMB，直至清晰的spots出现。最后加入去离子水来终止显色反应。Elispots-reader仪器扫描平板后进行统计分析。
实施例6 HLA:A*0201限制型CMV-pp65-TCR-T体外特异杀伤pp65-HLA*A0201-K562细胞
(1)通过慢病毒感染K562细胞分别制备K562-A:0201-GFP对照细胞与K562-A:0201-GFP-pp65-mCherry靶细胞；
(2)取实施例4中制备的CMV-pp65-TCR-T/Reported-TCR-T细胞，分别按1:1及5:1的效靶比例，接种T细胞与K562细胞至圆底96孔板中培养。
(3)于共培养24h后，FACS检测共培养体系中K562-A:0201-GFP或K562-A:0201-GFP-pp65-mCherry的比例，以评估CMV-pp65-TCR-T及Reported-TCR-T是否可特异杀伤K562-A:0201-GFP-pp65-mCherry细胞，并比较两种TCR-T的杀伤效率。
实施例7 HLA:A*0201限制型CMV-pp65-TCR-T体内特异杀伤pp65-HLA*A0201-K562细胞
(1)通过慢病毒感染K562细胞制备K562-A:0201-GFP-pp65-luciferase靶细胞；通过慢病毒感染CD8 +T细胞制备CMV-pp65-TCR-T效应细胞
(2)小鼠皮下植瘤模型：day0时于NOG小鼠右腋下处皮下注射1×10 6靶细胞，day3时分别通过侧尾静脉注射5×10 6primary CD8 +T cell(CD8 +T day3 group)与CMV-pp65-TCR-T cell(pp65-TCR-T day3 group)，另于day7时侧尾静脉注射5×10 6CMV-pp65-TCR-T cell(pp65-TCR-T day37group)；肿瘤细胞注射后的d0(+4h)，d7，d14，d21，d28，d35对小鼠进行生物发光成像检测肿瘤细胞中luciferase的表达，并对其luciferase的光子数强度进行定量，以评估CMV-pp65-TCR-T是否可抑制K562-A:0201-GFP-pp65-luciferase靶细胞的生长。
(3)小鼠尾静脉转移模型：day0时于NOG小鼠侧尾静脉注射1×10 6靶细胞，day3时分别通过侧尾静脉注射1×10 7primary CD8 +T cell与CMV-pp65-TCR-T cell。肿瘤细胞注射后的d0(+4h)，d28，d35，d42，d49，d56对小鼠进行生物发光成像检测肿瘤细胞中luciferase的表达，并对其luciferase的光子数强度进行定量，以评估CMV-pp65-TCR-T对靶细胞K562-A:0201-GFP-pp65-luciferase的抑制作用。
实验结果：
CMV-pp65-TCR-T筛选流程：
图1为本发明CMV-pp65-TCR-T的特异性刺激及筛选流程，整个试验由健康HLA-A:0201供者的CD8+T细胞经自体的mDCs负载pp65多肽进行两轮刺激后，使用CMV-PP65-HLA*A:0201Dextramer对CMV-pp65抗原特异性的TCR-T细胞进行特异性的分析。
流式结果(图2)显示，经过两轮mDCs-pp65多肽刺激后，健康供者D6的CD8a +/Dextramer +T比例由0.16％提高至81.3％，表明CMV-pp65特异的TCR-T细胞在体外进行特异性的扩增。
单细胞测序获取CMV-pp65-TCR序列：
经过自体mDCs负载pp65多肽进行两轮刺激后，使用CMV-PP65-HLA*A:0201Dextramer及CD8a抗体对CMV-pp65抗原特异性的TCR-T细胞进行流式分选，分选所得的CMV-pp65-TCR-T细胞经10×Genomics法进行单细胞测序，以获取CMV-pp65-TCR-T细胞的α与βchain序列。
经单细胞测序后，将测序所得的频次前10名进行排序。单细胞测序结果显示分选后CD8a +/Dextramer +T细胞中99.64％的T细胞克隆由一种TCR-T组成，证明此次实验(Antigen-specific T cell priming)特异性良好。该T细胞克隆包含两条α链，其中一条为冗余α链，后续已通过CMV-PP65-HLA*A:0201Dextramer体外结合实验将其排除。
最终获得的TCR序列如下：
CMV-pp65-TCR：
Figure PCTCN2022136297-appb-000001
Figure PCTCN2022136297-appb-000002
序列中的 C为经人工突变引入的半胱氨酸残基；
经密码子优化后的TCR编码序列如下：
Figure PCTCN2022136297-appb-000003
Figure PCTCN2022136297-appb-000004
其中，
α链及其编码序列如下：
Figure PCTCN2022136297-appb-000005
β链及其编码序列如下：
Figure PCTCN2022136297-appb-000006
Figure PCTCN2022136297-appb-000007
α链可变区及其编码序列如下：
Figure PCTCN2022136297-appb-000008
其包含的3个CDR序列如下：
α-CDR1：SSNFYA(SEQ ID NO.9)，编码序列为：TCCAGCAATTTCTACGCC(SEQ ID NO.10)
α-CDR2：MTLNGDE(SEQ ID NO.11)，编码序列为：ATGACCCTCAACGGCGATGAA(SEQ ID NO.12)
α-CDR3：CAFPYNNNDMRF(SEQ ID NO.13)，编码序列为：TGTGCTTTTCCTTATAACAACAACGATATGAGGTTC(SEQ ID NO.14)。
β链可变区及其编码序列如下：
Figure PCTCN2022136297-appb-000009
Figure PCTCN2022136297-appb-000010
其包含的3个CDR序列如下：
β-CDR 1：MNHEY(SEQ ID NO.17)，编码序列为：ATGAACCATGAATAC(SEQ ID NO.18)；
β-CDR 2：SMNVEV(SEQ ID NO.19)，编码序列为：TCTATGAATGTGGAGGTG(SEQ ID NO.20)；
β-CDR 3：CASSLEGYTEAFF(SEQ ID NO.21)，编码序列为：TGCGCTTCCTCCCTCGAGGGGTACACCGAGGCATTTTTT(SEQ ID NO.22)。
CMV-pp65-TCR-T载体构建及序列优化：
经测序得到pp65-TCR(SEQ ID NO.1)序列后，对其进行序列优化，具体包括：
(1)对其恒定区进行改造，阻止与内源性TCR链的错配；
(2)表达序列进行人源密码子优化，提高TCR蛋白表达量；
(3)利用P2A及Furin-cleavage使得alpha链和beta链能在一个质粒上同时表达，而不产生因单独使用P2A而产生的末端脯氨酸残留。
随后，将优化完成的pp65-TCR序列(图3)克隆至慢病毒表达载体中。
CMV-pp65-TCR-T体外结合实验：
利用293T细胞进行TCR慢病毒的包装，CMV-pp65-TCR慢病毒两轮感染健康HLA-A:0201供者的PBMC或jurkat T后进行CMV-pp65-HLA*A:0201 Dextramer染色，流式结果显示Tetramer +/mTCRβC +jurkatT(图4的子图A)及Dextramer +/primary CD8 +T(图4的子图B)细胞分群明显，该结果表明本项目筛选并鉴定的CMV-pp65-TCR-T在健康供者的jurkatT及primary CD8 +T细胞中正常表达，并可与CMV-PP65-HLA*A:0201 Tetramer/Dextramer结合。
CMV-pp65-TCR-T体外激活实验：
体外制备对照细胞K562-A:0201-GFP，载体对照组细胞K562-A:0201-GFP-mCherry-vector，以及靶细胞K562-A:0201-GFP-pp65-mCherry，通过将CMV-pp65-TCR-T效应细胞与靶细胞或对照细胞(载体对照及空白对照)进行体外共培养，胞内外染色后，流式检测CMV-pp65-TCR-T细胞的CD137及IFNγ表达的水平。
流式实验结果(图5)表明K562-A:0201-GFP-pp65-mCherry靶细胞可在体外特异性激活CMV-pp65-TCR-T细胞，激活后CMV-pp65-TCR-T的CD137及IFNγ的表达水平显著增高，而载体对照组K562细胞与对照K562细胞对CMV-pp65-TCR-T细胞无明显激活作用。
此外，IFNγ-Elispot实验结果同样表明K562-A:0201-GFP-pp65-mCherry靶细胞可在体外特异性激活CMV-pp65-TCR-T细胞，激活后CMV-pp65-TCR-T的IFNγ表达水平显著增高，而对照细胞K562-A:0201-GFP对CMV-pp65-TCR-T无激活作用。
CMV-pp65-TCR-T体外杀伤实验：
体外制备靶细胞K562-A:0201-GFP-pp65-mCherry，CN102656188A公开的Reported TCR-T以及本项目的CMV-pp65-TCR-T。
通过将CMV-pp65-TCR-T或Reported TCR-T效应细胞与靶细胞按效靶比1:1及5:1，体外共培养24h后，流式检测GFP+靶细胞的比例，流式实验结果(图6的A、B)表明CMV-pp65-TCR-T与Reported TCR-T细胞均可在体外杀伤表达pp65的K562-A:0201-GFP-pp65-mCherry细胞，但本项目公开的CMV-pp65-TCR-T在杀伤效果方面优于Reported TCR-T。
随后通过将CMV-pp65-TCR-T与Reported TCR-T效应细胞与靶细胞按效靶比1:1进行体外共培养，24h后进行胞内染色，随后流式检测细胞因子TNF-α的表达，以评估CMV-pp65-TCR-T与Reported TCR-T的细胞因子释放情况。流式实验结果表明本项目筛选并鉴定的CMV-pp65-TCR-T细胞较Reported TCR-T可在体外释放更高水平的TNF-α，特异性的杀伤表达pp65的K562-A:0201-GFP-pp65-mCherry细胞(图6的C)，而CMV-pp65-TCR-T与K562-A0201对照组细胞共培养后并无TNF-α及IFN-γ细胞因子的释放，显示其特异性良好。
CMV-pp65-TCR-T体内杀伤实验-皮下植瘤模型：
体外制备K562-A:0201-GFP-pp65-luciferase靶细胞及CMV-pp65-TCR-T效应细胞，day0时于NOG小鼠右腋下处皮下注射1×10 6靶细胞，day3时分别通过侧尾静脉注射5×10 6primary CD8 +T cell(CD8 +T day3group)与CMV-pp65-TCR-T cell(pp65-TCR-T day3group)，另于day7时侧尾静脉注射5×10 6CMV-pp65-TCR-T cell(pp65-TCR-T day7group)(图7的A)；肿瘤细胞注射后的第0-5周对小鼠进行生物发光成像检测(图7的B)，并对小鼠的luciferase的光子数强度进行定量。体内实验结果表明本项目的CMV-pp65-TCR-T细胞可在体内特异性的杀伤K562-A:0201-GFP-pp65-luciferase靶细胞(图7的B、C)，day3或day7注射CMV-pp65-TCR-T均可显著抑制肿瘤细胞K562-A:0201-GFP-pp65-luciferase的生长，并显著延长小鼠的生存时间(图7的D)，其对靶细胞的杀伤效果显著优于未进行pp65-TCR转导的CD8 +T对照组细胞。
CMV-pp65-TCR-T体内杀伤实验-转移模型：
体外制备K562-A:0201-GFP-pp65-luciferase靶细胞及CMV-pp65-TCR-T效应细胞，day0时于NOG小鼠尾静脉注射1×10 6靶细胞，day3时分别通过侧尾静脉注射1×10 7primary CD8 +T cell与CMV-pp65-TCR-T cell(图8的A)；肿瘤细胞注射后的第4-8周对小鼠进行生物发光成像检测(图8的B)，并对小鼠的luciferase的光子数强度进行定量。
体内实验结果表明本项目的CMV-pp65-TCR-T细胞可在体内特异性的杀伤K562-A:0201-GFP-pp65-luciferase靶细胞(图8的B和C)，day3注射CMV-pp65-TCR-T可显著抑制肿瘤细胞K562-A:0201-GFP-pp65-luciferase的生长，其抑制效果显著优于未进行pp65-TCR转导的CD8 +T对照组细胞。
技术效果
CMV在人群中感染非常广泛，中国成人感染率达95％以上，通常呈隐性感染，多数感染者无临床症状，但在一定条件下侵袭多个器官和系统可产生严重疾病。CMV激活在造血干细胞移植(HSCT)患者中较为常见，部分患者药物治疗效果较差或药物耐受性低，无法完成整个药物治疗疗程。患者由于免疫功能低下，不能有效清除CMV，可引起CMV相关发热直至器官累及的系列相关性疾病，具有很高的病死率。现有抗CMV药物也存在诸多不足，如剂型单一、靶点相似、可导致粒细胞缺乏/肾损害等。因此亟需有新的临床治疗方案用于HSCT患者的CMV感染的治疗。本项目的CMV-TCR-T细胞一方面可精准靶向清除感染CMV病毒的靶细胞；另一方面可间接通过TCR-T细胞介导的免疫重建，对病毒的彻底清除和防止再感染发挥长期保护作用。
本项目发明的CMV-pp65-TCR-T具有以下几个特点：
体内外实验表明CMV-pp65-TCR-T可精准靶向并快速清除表达CMV抗原的靶细胞，体内蛋白表达量高且与内源性TCR链无错配，杀伤力能力强，特异性强等。且体外实验表明本项目的CMV-pp65-TCR-T较CN102656188A公开的Reported-TCR-T对靶细胞的杀伤作用更为显著，可以有效应用于HSCT患者或其他移植患者CMV感染的治疗。
此外，相较于现有相关公开发明，本项目提供了TCR-T领域内公认金标准的ELISPOT(酶联免疫斑点)体外实验证据，说明本项目筛选获得的TCR能够特异识别CMV抗原；同时，本项目发明除了利用尾静脉模型证实装载CMV特异性TCR的T细胞可以特异清除表达CMV抗原的靶细胞外，还利用了更贴近临床CMV感染情形的皮下成瘤模型，同样也证实了装载本项目筛选获得的CMV特异性TCR的T细胞可以特异清除表达CMV抗原的靶细胞。
总体而言，本项目发明提供了完整的、符合领域标准的且更贴近临床CMV感染情形的体内外数据，充分证实筛选获得的CMV抗原特异TCR的特异性、有效性。
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 (14)

  1. 一种T细胞受体(TCR)，其特征在于，所述T细胞受体包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域的CDR3的氨基酸序列为CAFPYNNNDMRF(SEQ ID NO.13)；和/或
    所述TCRβ链可变域的CDR3的氨基酸序列为CASSLEGYTEAFF(SEQ ID NO.21)。
  2. 如权利要求1所述的T细胞受体(TCR)，其特征在于，所述TCRα链可变域的3个互补决定区(CDR)为：
    α-CDR1：SSNFYA(SEQ ID NO.9)，
    α-CDR2：MTLNGDE(SEQ ID NO.11)，
    α-CDR3：CAFPYNNNDMRF(SEQ ID NO.13)；和/或
    所述TCRβ链可变域的3个互补决定区为：
    β-CDR 1：MNHEY(SEQ ID NO.17)，
    β-CDR 2：SMNVEV(SEQ ID NO.19)，
    β-CDR 3：CASSLEGYTEAFF(SEQ ID NO.21)。
  3. 如权利要求1所述的TCR，其特征在于，其包含TCRα链可变域和TCRβ链可变域，所述TCRα链可变域为与SEQ ID NO.7具有至少90％序列相同性的氨基酸序列；和/或，所述TCRβ链可变域为与SEQ ID NO.15具有至少90％序列相同性的氨基酸序列。
  4. 如权利要求1所述的TCR，其特征在于，所述TCR的氨基酸序列如SEQ ID NO.1所示。
  5. 一种多价TCR复合物，其特征在于，包含至少两个TCR分子，并且其中的至少一个TCR分子为上述权利要求中任一项所述的TCR。
  6. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1所述的TCR分子的核酸序列或其互补序列。
  7. 如权利要求6所述的核酸分子，其特征在于，所述的核酸分子包含编码TCRα链可变域的核苷酸序列SEQ ID NO.8；和/或
    所述的核酸分子包含编码TCRβ链可变域的核苷酸序列SEQ ID NO.16。
  8. 一种载体，其特征在于，所述的载体含有权利要求6所述的核酸分子；优选地，所述的载体为病毒载体；更优选地，所述的载体为慢病毒载体。
  9. 一种分离的宿主细胞，其特征在于，所述的宿主细胞中含有权利要求8中所述的载体或染色体中整合有外源的权利要求6所述的核酸分子。
  10. 一种细胞，其特征在于，所述细胞转导权利要求6所述的核酸分子或权利要求8中所述载体；优选地，所述细胞为T细胞或干细胞。
  11. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1-4中任一项所述的TCR、权利要求5中所述的TCR复合物、权利要求6所述的核酸分子、或权利要求10中所述的细胞。
  12. 权利要求1-4中任一项所述的T细胞受体、或权利要求5中所述的TCR复合物或权利要求10中所述的细胞的用途，其特征在于，用于制备治疗巨细胞病毒感染症的药物。
  13. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-4中任一项所述的TCR、权利要求5中所述的TCR复合物、权利要求6所述的核酸分子、或权利要求10中所述的细胞或权利要求11所述的药物组合物。
  14. 如权利要求13所述的方法，其特征在于，所述的疾病为巨细胞病毒感 染相关的疾病(巨细胞病毒感染症)，如CMV视网膜炎、CMV肺炎、CMV胃肠炎和CMV脑炎等。
PCT/CN2022/136297 2022-01-27 2022-12-02 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用 WO2023142683A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210099611.0 2022-01-27
CN202210099611 2022-01-27

Publications (1)

Publication Number Publication Date
WO2023142683A1 true WO2023142683A1 (zh) 2023-08-03

Family

ID=87470343

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/136297 WO2023142683A1 (zh) 2022-01-27 2022-12-02 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用

Country Status (1)

Country Link
WO (1) WO2023142683A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102656188A (zh) * 2009-09-29 2012-09-05 Ucl商务股份有限公司 能够识别来自巨细胞病毒的抗原的t细胞受体
WO2018057823A1 (en) * 2016-09-23 2018-03-29 Memorial Sloan Kettering Cancer Center Generation and use in adoptive immunotherapy of stem cell-like memory t cells
CN110357953A (zh) * 2019-07-17 2019-10-22 深圳市因诺转化医学研究院 识别人巨细胞病毒pp65抗原的TCR
CN113121676A (zh) * 2020-01-15 2021-07-16 深圳华大生命科学研究院 一种靶向巨细胞病毒抗原的特异性t细胞受体及其应用
CN113150111A (zh) * 2021-02-01 2021-07-23 上海木夕生物科技有限公司 一种HLA-A*0201限制性CMVpp65特异性T细胞受体及其应用
CN113461803A (zh) * 2020-11-04 2021-10-01 北京可瑞生物科技有限公司 一种特异性识别巨细胞病毒的t细胞受体及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102656188A (zh) * 2009-09-29 2012-09-05 Ucl商务股份有限公司 能够识别来自巨细胞病毒的抗原的t细胞受体
WO2018057823A1 (en) * 2016-09-23 2018-03-29 Memorial Sloan Kettering Cancer Center Generation and use in adoptive immunotherapy of stem cell-like memory t cells
CN110357953A (zh) * 2019-07-17 2019-10-22 深圳市因诺转化医学研究院 识别人巨细胞病毒pp65抗原的TCR
CN113121676A (zh) * 2020-01-15 2021-07-16 深圳华大生命科学研究院 一种靶向巨细胞病毒抗原的特异性t细胞受体及其应用
CN113461803A (zh) * 2020-11-04 2021-10-01 北京可瑞生物科技有限公司 一种特异性识别巨细胞病毒的t细胞受体及其应用
CN113150111A (zh) * 2021-02-01 2021-07-23 上海木夕生物科技有限公司 一种HLA-A*0201限制性CMVpp65特异性T细胞受体及其应用

Also Published As

Publication number Publication date
CN117106061A (zh) 2023-11-24

Similar Documents

Publication Publication Date Title
CA2906587C (en) Compositions and methods for use of recombinant t cell receptors for direct recognition of tumor antigen
JP2018183175A (ja) Ｗｔ−１に特異的なｔ細胞免疫治療
CN109320615B (zh) 靶向新型bcma的嵌合抗原受体及其用途
CN107001444B (zh) 识别eb病毒短肽的t细胞受体
JP2020517308A (ja) Ｔｃｒ及びペプチド
CN110857319B (zh) 一种分离的t细胞受体、其修饰的细胞、编码核酸及其应用
WO2019091347A1 (zh) 识别乙肝病毒(hbv)表面抗原s183-91表位的tcr及其用途
JP6580579B2 (ja) Ｔ細胞受容体を発現する細胞を生産する方法および組成物
JP2013505734A (ja) Ｔ細胞受容体
CN112004924A (zh) 增强treg细胞抑制特性的方法
CN109776671B (zh) 分离的t细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用
CN112204133A (zh) Car nk细胞
KR20220086650A (ko) Hla에 특이적인 키메라 항원 수용체
TW202144399A (zh) 一種辨識hpv抗原的t細胞受體及其編碼序列
JP2022533092A (ja) アセチルコリン受容体キメラ自己抗体受容体細胞の組成物および方法
CN117736300A (zh) 靶向巨细胞病毒pp65的T细胞受体和表达其的T细胞及应用
CN109517798B (zh) 一种嵌合cea抗原受体的nk细胞及其制备方法与应用
CN112940108B (zh) 识别ebv抗原的t细胞受体以及该t细胞受体的应用
WO2023217145A1 (zh) 抗原短肽用于筛选治疗与hpv相关的疾病的药物中的用途及其筛选的tcr
KR101794304B1 (ko) Ｅｂｖ 항원 특이 ｔ―세포 수용체 및 이의 용도
CN112940109B (zh) 识别ebv抗原的t细胞受体及其应用
WO2023142683A1 (zh) 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用
CN111171137A (zh) 一种识别afp抗原短肽的t细胞受体及其编码序列
CN112279908B (zh) 识别ebv抗原短肽的t细胞受体及其应用
CN117106061B (zh) 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22923474

Country of ref document: EP

Kind code of ref document: A1