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WO2024131372A1 - 靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用 - Google Patents

靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用 Download PDF

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WO2024131372A1
WO2024131372A1 PCT/CN2023/130780 CN2023130780W WO2024131372A1 WO 2024131372 A1 WO2024131372 A1 WO 2024131372A1 CN 2023130780 W CN2023130780 W CN 2023130780W WO 2024131372 A1 WO2024131372 A1 WO 2024131372A1
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tcr
seq
cell
cells
cmv
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PCT/CN2023/130780
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English (en)
French (fr)
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王鹏然
沈良华
张岩
宋献民
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上海市第一人民医院
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    • 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
    • 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
    • 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
    • C12N15/867—Retroviral 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the field of biotechnology, and specifically relates to a specific TCR that restricts HLA-A*2402 and targets cytomegalovirus pp65, a T cell expressing the same, and an application thereof.
  • Cytomegalovirus belongs to the ⁇ subfamily of the human herpesviridae family. It has obvious host species specificity and is the largest and most complex virus in the human herpesviridae family. CMV is widely infected in the population and is usually latently infected. Most infected people have no clinical symptoms, but under certain conditions, it can invade multiple organs and systems and cause serious diseases. In recent years, the diagnosis and treatment of CMV infection has made great progress, which has significantly reduced the incidence of CMV infection after transplantation.
  • CMV infection still maintains a high incidence in allogeneic stem cell transplantation (allo-HSCT) patients, and its indirect effects, such as secondary implantation dysfunction, GVHD, and all-cause mortality, still have a huge impact on the prognosis of patients.
  • Existing anti-CMV drugs also have many shortcomings, such as single dosage form, similar targets, and can cause granulocytopenia/renal damage.
  • Current antiviral drugs, such as acyclovir and ganciclovir often cause serious side effects, such as bone marrow suppression.
  • 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 that they initiate HCMV infection of host cells and viral replication, and also play an important role in escaping T lymphocyte-mediated cytotoxicity. Studies have shown that among the above three proteins, pp65 protein has the most serious effect in causing graft vasculopathy and rejection reactions. Immune damage caused by allo-HSCT can reactivate latent HCMV, leading to serious clinical complications.
  • TCR-T therapy is It is achieved by capturing specific TCRs targeting tumor antigens or specific viral antigens and transforming T cells using genetic engineering technology. After infusion into the body, it can achieve the purpose of treating tumors or clearing viral infections. After the TCR gene carrying high affinity for CMV-pp65 is infected with T cells by lentivirus, CMV-pp65-specific TCR-T cells can be prepared.
  • CMV-pp65-TCR-T cells can directly and quickly clear CMV viruses through precise targeting; on the other hand, they can indirectly play a long-term protective role in completely clearing the virus and preventing reinfection through TCR-T cell-mediated immune reconstruction.
  • This cell therapy has the potential to become one of the most effective treatments for CMV infection.
  • Allo-HSCT patients cannot effectively clear CMV due to low immune function, and are prone to primary CMV infection or latent CMV reactivation. After CMV infection/reactivation occurs in HSCT patients, it can cause CMV-related fever and a series of related diseases including organ involvement, which is closely related to disease recurrence and subject survival, and the current conventional drug treatment effect is poor. Therefore, the application of CMV-TCR-T cell therapy in the treatment and prevention of CMV infection after HSCT or other organ transplantation has great clinical value and application prospects.
  • the purpose of the present invention is to provide a specific TCR that restricts HHLA-A*2402 and targets cytomegalovirus pp65, a T cell expressing the same and an application thereof.
  • a T cell receptor comprising a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, and the amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is CARSNYGGSQGNLIF (SEQ ID NO.13); and/or
  • the amino acid sequence of CDR3 of the TCR ⁇ chain variable domain is CAISDPGSSFGGYTF (SEQ ID NO.21).
  • the TCR specifically binds to the QYDPVAALF-HLA-A*2402 complex.
  • the three complementarity determining regions (CDRs) of the TCR ⁇ chain variable domain are:
  • ⁇ -CDR1 SSNFYA (SEQ ID NO.9)
  • ⁇ -CDR2 MTLNGDE (SEQ ID NO.11),
  • ⁇ -CDR3 CARSNYGGSQGNLIF (SEQ ID NO.13).
  • the three complementary determining regions of the TCR ⁇ chain variable domain are:
  • ⁇ -CDR 1 ENHRY (SEQ ID NO. 17),
  • ⁇ -CDR 2 SYGVKD (SEQ ID NO.19),
  • the TCR comprises a TCR ⁇ chain variable domain and a TCR ⁇ chain variable domain, wherein the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO.7; and/or the TCR ⁇ chain variable domain is an amino acid sequence having at least 90% sequence identity with SEQ ID NO. ID NO. 15 has an amino acid sequence with at least 90% sequence identity.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence SEQ ID NO.7.
  • the TCR comprises the ⁇ chain variable domain amino acid sequence SEQ ID NO.15.
  • the TCR is an ⁇ heterodimer, which comprises the TCR ⁇ chain constant region TRAC*01 and the TCR ⁇ chain constant region TRBC1*01 or TRBC2*01.
  • the ⁇ chain amino acid sequence of the TCR is SEQ ID NO.3.
  • the ⁇ chain amino acid sequence of the TCR is SEQ ID NO.5.
  • the ⁇ chain and ⁇ chain of the TCR contain an artificial interchain disulfide bond.
  • 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 comprising at least two TCR molecules, wherein at least one TCR molecule is the TCR described in the first aspect of the present invention.
  • the third aspect of the present invention provides a nucleic acid molecule, which comprises a nucleic acid sequence encoding the TCR molecule described in the first aspect of the present invention or its complementary sequence.
  • the nucleic acid molecule contains the nucleotide sequence SEQ ID NO.8 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule contains the nucleotide sequence SEQ ID NO.16 encoding the variable domain of the TCR ⁇ chain.
  • the nucleic acid molecule contains the nucleotide sequence SEQ ID NO.4 encoding the TCR ⁇ chain.
  • the nucleic acid molecule contains the nucleotide sequence SEQ ID NO.6 encoding the TCR ⁇ chain.
  • the nucleic acid molecule contains the nucleotide sequence SEQ ID NO.2.
  • the fourth aspect of the present invention provides a vector, wherein 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 lentiviral vector.
  • the fifth aspect of the present invention provides an isolated host cell, wherein the host cell contains the vector described in the fourth aspect of the present invention or the exogenous nucleic acid molecule described in the third aspect of the present invention integrated into the genome.
  • the sixth aspect of the present invention provides a cell, which transduces 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, a PBMC cell (peripheral blood mononuclear cell) or a stem cell.
  • the cell is a T cell, a PBMC cell (peripheral blood mononuclear 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 nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the cell described in the sixth aspect of the present invention.
  • the eighth aspect of the present invention provides the use 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, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the cell described in the sixth aspect of the present invention for preparing a drug for treating cytomegalovirus-related diseases.
  • the ninth aspect of the present invention provides a method for treating a disease, comprising administering to a subject in need of treatment 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, the nucleic acid molecule described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, or the cell described in the sixth aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention;
  • the disease is a disease associated with cytomegalovirus infection (cytomegalovirus infection), such as CMV retinitis, CMV pneumonia, CMV gastroenteritis and CMV encephalitis.
  • cytomegalovirus infection such as CMV retinitis, CMV pneumonia, CMV gastroenteritis and CMV encephalitis.
  • FIG. 1 CMV-pp65-TCR-T in vitro stimulation and FACS analysis
  • FIG. 1 CMV-pp65-TCR-T vector information
  • the present invention discloses a specific T cell that targets the cytomegalovirus (CMV) pp65 antigen epitope restricted by HLA-A*2402 and its application.
  • the T cell receptor (T cell receptor, TCR) carried by the T cell can specifically target CMV-pp65 and can accurately and quickly eliminate the CMV virus.
  • the pp65-TCR-T cell specific to the pp65 antigen epitope (QYDPVAALF, SEQ ID NO.23) of the present invention can specifically bind to and kill the target cells that express the CMV pp65 antigen restricted by HLA-A*2402, and can provide a new solution for the clinical treatment of CMV-related diseases. It is applied to the field of treating and preventing CMV infection after HSCT or other organ transplantation, and has great clinical value and application prospects.
  • MHC molecules are proteins of the immunoglobulin superfamily and can be either class I or class II MHC molecules. Therefore, they are specific for antigen presentation. Different individuals have different MHCs that can present different short peptides in a protein antigen to the surface of their respective APC cells. Human MHC is usually called HLA gene or HLA complex.
  • T cell receptor is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • APC antigen-presenting cells
  • T cells with different antigen specificities can 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 are composed of ⁇ and ⁇ chains, while 5% of T cells have TCRs composed of ⁇ and ⁇ chains.
  • Natural ⁇ heterodimeric TCR has ⁇ chain and ⁇ chain, which constitute the subunits of ⁇ heterodimeric TCR.
  • each ⁇ and ⁇ chain contains a variable region, a connecting region and a constant region.
  • the ⁇ chain usually also contains a short variable region between the variable region and the connecting region, but the variable region is often regarded as part of the connecting region.
  • Each variable region contains three CDRs (complementarity determining regions), CDR1, CDR2 and CDR3, embedded in the framework structure (framework regions).
  • the CDR region determines the binding of TCR to the pMHC complex, among which CDR3 is recombined by the variable region and the connecting region and is called the hypervariable region.
  • the ⁇ and ⁇ chains of TCR are generally considered to have two "domains" each, namely the variable domain and the constant domain.
  • the variable domain is composed of a connected variable region and a connecting region.
  • the sequence of the TCR constant domain can be found in the public database of the International Immunogenetics Information System (IMGT), such as the constant domain sequence of the TCR molecule ⁇ chain is "TRAC*01", and the constant domain sequence of the TCR molecule ⁇ chain is "TRBC1*01" or "TRBC2*01".
  • IMGT International Immunogenetics Information System
  • the ⁇ and ⁇ chains of TCR also contain transmembrane regions 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
  • a first aspect of the present invention provides a TCR molecule that can bind to QYDPVAALF-HLA-A*2402.
  • the TCR molecule is isolated or purified.
  • the ⁇ and ⁇ chains of the TCR each have 3 complementary 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 CARSNYGGSQGNLIF (SEQ ID NO.13).
  • the ⁇ chain of the TCR comprises a CDR having the following amino acid sequence:
  • ⁇ -CDR 1 ENHRY (SEQ ID NO. 17),
  • ⁇ -CDR 2 SYGVKD (SEQ ID NO.19),
  • the CDR region amino acid sequence of the present invention can be embedded in any suitable framework structure to prepare a chimeric TCR.
  • the framework structure is compatible with the CDR region of the TCR of the present invention, a person skilled in the art can design or synthesize a TCR molecule 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.
  • variable domain of the TCR ⁇ chain 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 variable domain of the TCR ⁇ chain 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.15.
  • 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 ⁇ chain variable domain comprises CDR1 (SEQ ID NO.9), CDR2 (SEQ ID NO.11) and CDR3 (SEQ ID NO.13) of the above-mentioned ⁇ chain.
  • the TCR molecule comprises the amino acid sequence of the ⁇ 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 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.
  • the single-chain TCR molecule comprises V ⁇ , V ⁇ and C ⁇ , preferably connected in the order from N-terminus to C-terminus.
  • the amino acid sequence of the ⁇ chain variable domain of the single-chain TCR molecule comprises the 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 the ⁇ chain variable domain 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 amino acid sequence of the ⁇ chain variable domain of the single-chain TCR molecule comprises the 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 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 ⁇ chain of the TCR molecule of the present invention is SEQ ID NO.3, and/or the amino acid sequence of the ⁇ chain is SEQ ID NO.5.
  • the TCR of the present invention may contain artificial disulfide bonds introduced between residues of the constant domains of its ⁇ and ⁇ chains. It should be noted that the TCR of the present invention may contain a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the artificial disulfide bonds introduced as described above between the constant domains.
  • the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR may be linked by a natural disulfide bond present in the TCR.
  • the TCR constant region of the present invention can be modified to prevent mispairing with endogenous TCR chains.
  • artificial disulfide bonds are introduced between residues in the constant domains of the ⁇ and ⁇ chains of the TCR of the present invention.
  • the TCR of the present invention can also be a hybrid TCR containing sequences derived from more than one species.
  • studies have shown that murine TCRs can be expressed more effectively in human T cells than human TCRs. Therefore, the TCR of the present invention can contain human variable domains and mouse constant domains. The disadvantage of this method is that it may induce an immune response. Therefore, when it is used for adoptive T cell therapy, A conditioning regimen should be in place to provide immunosuppression to allow engraftment of murine T cells expressing T cells.
  • Each TCR chain of a specific TCR for targeting cytomegalovirus antigen epitope pp65 restricted by HLA-A*2402 disclosed in the present invention contains three hypervariable loops, which are called complementary determining regions CDR1-3.
  • CDR1 and CDR2 are encoded by V genes, while CDR3 is encoded by the connecting region between V and J or D and J, so CDR3 varies greatly. Since CDR3 is the TCR region that directly contacts the antigen, CDR3 plays a very important role in the interaction between TCR and peptide-MHC complex.
  • the CDR3 sequence has the greatest impact on the function of TCR. Even if different TCR-CDR3 sequences are highly similar, their TCR functions are very different even if there is only one amino acid difference.
  • the CMV-pp65-TCR-T of the present invention can be normally expressed and specifically activated in primary CD8+T cells of healthy donors, and in vitro killing experiments show that the CMV-pp65-TCR of this project has a significant killing effect on target cells. Therefore, the HLA-A*2402-restricted specific TCR targeting pp65 of the present invention can be specifically activated by target cells expressing CMV-pp65, and pp65-TCR-T cells can significantly kill target cells, and can subsequently be effectively used in the treatment of CMV infection in HSCT patients or other transplant patients.
  • amino acid names herein are represented by single letters or three letters in the internationally accepted English language, and the correspondence between single letters and three letters of the amino acid names is as follows: 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).
  • the second aspect of the invention provides a nucleic acid molecule encoding the TCR molecule of the first aspect of the invention or a portion thereof, wherein the portion may be one or more CDRs, a variable domain of an ⁇ and/or ⁇ chain, and an ⁇ chain and/or a ⁇ chain.
  • the nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule of the first aspect of the present invention is as follows:
  • the nucleotide sequence encoding the CDR region of the ⁇ chain of the TCR molecule of 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 nucleotide sequences of the nucleic acid molecules of the present invention encoding the TCR ⁇ chain of the present invention include SEQ ID NO.18, SEQ ID NO.20 and SEQ ID NO.22.
  • the nucleotide sequence of the nucleic acid molecule of the present 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 is capable of encoding 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 variable domain of the TCR ⁇ chain of the present invention includes SEQ ID NO.16.
  • nucleotide sequence of the nucleic acid molecule of the present invention includes 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 accompanying drawings of the present invention or a degenerate variant.
  • a "degenerate variant” refers to a nucleic acid sequence that encodes a protein sequence having SEQ ID NO.1, but is different from the sequence of SEQ ID NO.2.
  • the nucleotide sequence may be codon optimized. Different cells differ in the use of specific codons, and the codons in the sequence may be altered to increase expression depending on the cell type. Codon usage tables for mammalian cells and 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 fragment 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 fragment, or its derivative) can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to vectors comprising the nucleic acid molecules of the present invention, including expression vectors, i.e. constructs capable of in vivo or in vitro expression.
  • expression vectors i.e. constructs capable of in vivo or in vitro expression.
  • Commonly used 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, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.
  • AAV adeno-associated viral
  • the vector can transfer the nucleotide of the present invention into a cell, such as a T cell, so that the cell expresses an antigen-specific TCR.
  • a cell such as a T cell
  • the vector should be able to be expressed at a high level in a sustained manner in a T cell.
  • the present invention also relates to a host cell produced by genetic engineering using the vector or coding sequence of the present invention.
  • the nucleic acid molecule of the present invention is integrated into the vector or chromosome of the present invention.
  • the host cell is selected from: prokaryotic cells and eukaryotic cells, such as Escherichia coli, yeast cells, CHO cells, 293T cells, etc.
  • the present invention also includes cells expressing the separation of the TCR of the present invention, particularly T cells.
  • the T cell may be derived from a T cell separated from a subject, or may be a mixed cell population separated from a subject, such as a part of a peripheral blood lymphocyte (PBL) population.
  • PBL peripheral blood lymphocyte
  • the cell may be separated from peripheral blood mononuclear cells (PBMC), may be CD4+ helper T cells or CD8+ cytotoxic T cells.
  • PBMC peripheral blood mononuclear cells
  • the cell may be in a mixed population of CD4+ helper T cells/CD8+ cytotoxic T cells.
  • the cells of the present invention may also be or be derived from stem cells, such as hematopoietic stem cells (HSC).
  • stem cells such as hematopoietic stem cells (HSC).
  • HSC hematopoietic stem cells
  • T cells expressing the TCR of the present invention can be used for adoptive immunotherapy.
  • suitable methods for adoptive therapy e.g., Rosenberg et al., (2008) Nat Rev Cancer 8(4): 299-308.
  • CMV Cytomegalovirus
  • the present invention also relates to a method for treating and/or preventing CMV-related diseases in a subject, comprising the step of adoptively transferring CMV-specific T cells to the subject.
  • the CMV-specific T cells can recognize the major CMV matrix phosphoprotein pp65.
  • the CMV-specific T cells can recognize the epitopes NLVPMVATV.
  • CMV is a ubiquitous human herpes virus that infects approximately 50% of normal individuals. In most cases, an immune response is able to control acute infection by recognizing CMV-derived antigens. The virus then remains in a latent state throughout the host's life. Outgrowth is prevented by immune system effector mechanisms, including neutralizing antibodies to 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.
  • the main areas of risk for infection 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 There are three general clinical forms of CMV infection, including:
  • Neonatal CMV inclusion disease which can range from asymptomatic to severe disease affecting the liver, spleen, and central nervous system, potentially causing disability;
  • CMV infection in immunocompromised persons e.g., those who have undergone organ transplantation or have HIV
  • immunocompromised persons who are at risk for CMV retinitis, CMV pneumonitis, CMV gastroenteritis, and CMV encephalitis.
  • the TCRs of the present invention can be used to treat and/or prevent the reactivation of latent CMV after allogeneic hematopoietic stem cell transplantation.
  • CMV disease in Allo HSCT recipients is thought to result 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 the leading 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 following transplantation.
  • the T cells expressing CMV-specific T cell receptors of the present invention are used to perform adoptive immunotherapy on Allo HSCT recipients.
  • prevention refers to avoiding, delaying, resisting or hindering the progression of a disease. For example, the possibility of CMV infection and/or CMV reactivation can be prevented or reduced.
  • treat refers to any treatment intended to alleviate, cure, or reduce the symptoms of a disease, or to reduce or prevent the progression of a disease.
  • Prevention or treatment can be carried out by isolating T cells from patients or volunteers suffering from relevant diseases, introducing the TCR of the present invention into the above T cells, and then returning these genetically engineered cells to the patient's body.
  • the present invention provides a method for treating CMV-related diseases, comprising: injecting separated T cells expressing the TCR of the present invention, preferably, the T cells are derived from the patient himself, into the patient. Generally, it comprises:
  • the number of cells isolated, transfected, and reinfused can be determined by the physician.
  • the TCR of the present invention can directly and rapidly eliminate CMV virus through precise targeting
  • TCR protein of the present invention is highly expressed in vivo and has no mismatch with endogenous TCR chains
  • Example 1 Induction of differentiation of monocytes into dendritic cells in vitro
  • PBMCs (or resuscitated frozen PBMCs) were obtained from the blood of healthy donors/patients by Lymphoprep density gradient centrifugation. The cells were resuspended in cytotoxic T lymphocyte medium and centrifuged at 350 g for 5 min. The supernatant was removed and an appropriate amount of DC medium was added to resuspend the PBMCs in a 6-well plate. After culturing in an incubator for 2 h, the medium and clumped non-adherent cells were removed by aspiration.
  • Mature DCs can be harvested on Day 7. Use a pipette to vigorously blow and resuspend the cells at the bottom of the well plate to collect the Mature DCs. Transfer the collected Mature DCs to a 15 ml centrifuge tube and centrifuge at 350g for 5 min. Remove the supernatant and use DC medium to resuspend the DCs at a density of 2 ⁇ 106 cells/ml for subsequent experiments.
  • PBMCs were obtained from the blood of healthy donors by Lymphoprep density gradient centrifugation.
  • CD8+ T cells in PBMCs were separated using EasySepTM Human CD8 Positive Selection Kit.
  • CMV-mRNA-loaded DCs and CD8+ T cells were added to 12-well plates and cultured at a DC/T ratio of 1:2.5.
  • IL-21 was added to the culture system at a final concentration of 30 ng/ml.
  • CD8+/Tetramer+ T cells were flow cytometry sorted and the TCR ⁇ and TCR ⁇ chain sequences of CD8+/Tetramer+ T cells were obtained using 10X Genomics single-cell sequencing technology.
  • the most frequently occurring TCR ⁇ / ⁇ chain sequence is optimized and its constant region is modified to avoid mismatch between the exogenous TCR and the endogenous TCR of the T cell; the expression sequence is codon optimized to increase the protein expression level; P2A and Furin-cleavage are used to enable TCR ⁇ and TCR ⁇ to be expressed simultaneously in one expression vector.
  • CD8+ T cells were isolated from PBMCs using EasySepTM Human CD8 Positive Selection Kit, and CD8+ T cells were activated in vitro using CD3/28 Dynabeads at a ratio of 1:3 CD8+ T cells:CD3/28 Dynabeads. The activated CD8+ T cells were stimulated at 37°C for 2 days.
  • CD8+ T cells were activated by infection with CMV-pp65-TCR lentiviral particles, and 8 ⁇ g/ml polybrene was added for centrifugation at 500 ⁇ g for 90 min at 30°C. After centrifugation, the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 3 days, followed by a second round of infection with CMV-pp65-TCR lentivirus.
  • CMV-pp65-TCR-T cells were aspirated, washed twice with FACS buffer, and then CMV-PP65-HLA*A:2402Tetramer-APC and CD8a-BV421 antibody were added for staining.
  • the CD8+/Tetramer+ (%) ratio was detected by FACS, and then CD8a+/Tetramer+ T cells were flow cytometry sorted for subsequent in vitro killing experiments.
  • K562 cells were infected with lentivirus to prepare K562-A:2402-GFP and K562-A:2402-GFP-pp65-mCherry cells;
  • K562 and CD8 + T cells were infected by lentiviral infection to prepare K562-A:2402-GFP-pp65-mCherry-luciferase target cells and CMV-pp65-TCR-T effector cells, respectively;
  • K562-A:2402-GFP-pp65-mCherry-luciferase target cells were injected into NOG mice through the tail vein to construct an in vivo CDX model. 4 h after the injection of target cells, tumor cells were traced and analyzed using a small animal imager to ensure the successful establishment of the CDX model. Then, on day 3, 1 ⁇ 10 7 saline, CD8 + T and CMV-pp65-TCR-T cells were reinfused into the tail vein.
  • D-luciferin potassium salt substrate was injected intraperitoneally on day 7, day 14, day 21, and day 28 after target cell injection, and then bioluminescence imaging was performed on each group of mice using a small animal imaging device to observe the killing of target cells in mice by CD8 + T cells and CMV-pp65-TCR-T cells.
  • CMV-pp65-HLA*A:2402 Tetramer and CD8a antibody were used to flow sort CMV-pp65-TCR-T cells, and the sorted CMV-pp65-TCR-T cells were sequenced by 10 ⁇ Genomics method to obtain the ⁇ and ⁇ chain sequences of CMV-pp65-TCR-T cells.
  • the top 10 frequencies obtained by sequencing were ranked. The single-cell sequencing results showed that after sorting, the proportion of 1#TCR clone was 74.17%, proving that the antigen-specific T cell priming experiment had good specificity.
  • the alpha chain and beta chain can be expressed simultaneously on the same plasmid.
  • the optimized pp65-TCR sequence ( FIG. 2 ) was cloned into a lentiviral expression vector.
  • the gene sequence after codon optimization is as follows:
  • ⁇ -chain sequence is:
  • the gene sequence is as follows:
  • the ⁇ -chain sequence is:
  • the gene sequence is as follows:
  • variable region sequence of the ⁇ -chain is:
  • the gene sequence is as follows:
  • the three CDR sequences of the ⁇ chain are as follows:
  • ⁇ -CDR2 MTLNGDE (SEQ ID NO.11), coding sequence: ATGACATTGAATGGTGATGAA (SEQ ID NO.12)
  • ⁇ -CDR3 CARSNYGGSQGNLIF (SEQ ID NO.13), encoding sequence: TGCGCACGATCAAACTACGGCGGGTCCCAGGGCAACCTGATTTTC (SEQ ID NO.14)
  • variable region sequence of the ⁇ -chain is:
  • the gene sequence is as follows:
  • the three CDR sequences of the ⁇ chain are as follows:
  • ⁇ -CDR1 ENHRY (SEQ ID NO.17), coding sequence: GAGAATCATAGATAC (SEQ ID NO.18)
  • ⁇ -CDR2 SYGVKD (SEQ ID NO.19), coding sequence: AGCTACGGCGTGAAAGAC (SEQ ID NO.20)
  • ⁇ -CDR3 CAISDPGSSFGGYTF (SEQ ID NO.21), coding sequence: TGTGCTATCTCTGACCCCGGATCTTCATTCGGCGGCTATACCTTT (SEQ ID NO.22).
  • TCR lentivirus was packaged using 293T cells. After two rounds of infection of PBMCs or Jurkat 76 cell lines from healthy HLA-A:2402 donors with CMV-pp65-TCR lentivirus, CMV-pp65-HLA*A:2402 Tetramer staining was performed. Flow cytometry results showed that Tetramer+/mTCR ⁇ C+jurkatT and Tetramer+/primary CD8+T ( Figure 3) cell clusters were obvious. This result indicated that the CMV-pp65-TCR-T screened and identified in this project was normally expressed in primary CD8+T cells and Jurkat 76 cells from healthy donors, and could bind to CMV-PP65-HLA*A:2402 tetramers.
  • Effector cells and target cells were prepared separately by lentivirus, and then the effector cells and target cells were co-cultured in vitro at an effector-target ratio of 5:1 for 4 hours, and the proportion of target cell apoptosis (Violet+/eF780+) was detected by flow cytometry.
  • the experimental results show that CMV-pp65-TCR-T cells can kill K562-A:2402-GFP-pp65-mCherry target cells in vitro, with a killing efficiency of about 65% in 4 hours, while for K562-A:2402-GFP control cells that do not express pp65, the killing efficiency is only about 10%.
  • This result shows that the CMV-pp65-TCR-T cells screened and identified in this project can specifically kill K562-A:2402-GFP-pp65-mCherry cells expressing pp65 in vitro.
  • CMV-pp65-TCR-T effector cells and K562 cells were prepared by lentiviral infection of primary CD8+T cells and K562 cells.
  • K562-A:2402-GFP-pp65-mCherry-luciferase target cells, target cells and effector cells were injected into NOG mice by tail vein at the time points shown in Figure 6A, and bioluminescence imaging of mice was performed on days 0, 7, 14, 21, and 28 after intravenous injection of target cells.
  • the experimental results ( Figure 6B) showed that the saline group and the primary CD8 + T cell treatment group could not effectively inhibit the growth of tumor cells. In contrast, 3 mice in the CMV-pp65-TCR-T group achieved tumor cell clearance.
  • CMV infection is very common in the population, with an infection rate of more than 95% in Chinese adults. It is usually latent infection, and 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 more common in hematopoietic stem cell transplantation (HSCT) patients. Some patients have poor drug treatment effects or low drug tolerance and cannot complete the entire course of drug treatment. Due to low immune function, patients cannot effectively eliminate CMV, which can cause CMV-related fever and a series of related diseases involving organs, with a high mortality rate.
  • HSCT hematopoietic stem cell transplantation
  • Existing anti-CMV drugs also have many shortcomings, such as a single dosage form, similar targets, and can cause granulocytopenia/renal damage.
  • the CMV-TCR-T cells of the present invention can accurately target and eliminate target cells infected with CMV virus; on the other hand, they can indirectly play a long-term protective role in the complete elimination of the virus and prevention of reinfection through TCR-T cell-mediated immune reconstruction.
  • the HLA*A:2402-restricted CMV-pp65-TCR-T invented in this project has the following characteristics: in vitro experiments show that CMV-pp65-TCR-T can accurately target and quickly eliminate target cells infected with CMV virus, the protein expression level in vivo is high and there is no mismatch with the endogenous TCR chain, it has strong lethality and specificity, etc., and can be effectively used in the treatment of CMV infection in HSCT patients or other transplant patients.

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Abstract

提供一种靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用。提供一种HLA-A*2402限制性靶向巨细胞病毒pp65抗原表位的特异性T细胞，该T细胞携带的T细胞受体可特异性靶向CMV-pp65，可精准且快速地清除CMV病毒。

Description

靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用
相关申请交叉引用
本专利申请要求于2022年12月23日提交的、申请号为2022116658275、发明名称为“靶向巨细胞病毒pp65的TCR和表达其的T细胞及应用”的中国专利申请的优先权，上述申请的全文以引用的方式并入本文中。
技术领域
本发明属于生物技术领域，具体地本发明涉及HLA-A*2402限制性靶向巨细胞病毒pp65的特异性TCR、表达其的T细胞及应用。
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，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，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。携带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。该细胞疗法有希望成为CMV感染最有效的疗法之一。
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，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HHLA-A*2402限制性靶向巨细胞病毒pp65的特异性TCR、表达其的T细胞及应用。
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CARSNYGGSQGNLIF(SEQ ID NO.13)；和/或
所述TCRβ链可变域的CDR3的氨基酸序列为CAISDPGSSFGGYTF(SEQ ID NO.21)。
在另一优选例中，所述TCR特异性结合QYDPVAALF-HLA-A*2402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：CARSNYGGSQGNLIF(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：ENHRY(SEQ ID NO.17)，
β-CDR 2：SYGVKD(SEQ ID NO.19)，
β-CDR 3：CAISDPGSSFGGYTF(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、PBMC细胞(外周血单个核细胞)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体外刺激及FACS分析；
图2：CMV-pp65-TCR-T载体信息；
图3：CMV-pp65-TCR-T体外结合验证；
图4：CMV-pp65-TCR-T体外激活验证；
图5：CMV-pp65-TCR-T体外杀伤验证；
图6：CMV-pp65-TCR-T体内杀伤验证。
具体实施方式
本发明公开了一种HLA-A*2402限制性靶向巨细胞病毒(Cytomegalovirus，CMV)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(T cell receptor，TCR)可特异性靶向CMV-pp65，可精准且快速地清除CMV病毒。本发明的pp65抗原表位(QYDPVAALF，SEQ ID NO.23)特异性的pp65-TCR-T细胞，能够特异性的结合并杀伤HLA-A*2402限制性表达CMV pp65抗原的靶细胞，可为CMV相关疾病的临床治疗提供一种新的方案，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QYDPVAALF-HLA-A*2402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：CARSNYGGSQGNLIF(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：ENHRY(SEQ ID NO.17)，
β-CDR 2：SYGVKD(SEQ ID NO.19)，
β-CDR 3：CAISDPGSSFGGYTF(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。
本发明公开的一种HLA-A*2402限制性靶向巨细胞病毒抗原表位pp65的特异性TCR的每个TCR链包含三个高可变环区(hypervariable loops)，称之为互补决定区CDR1-3。CDR1和CDR2由V基因编码，而CDR3由V和J或D和J之间连接区编码，因此CDR3变化程度较大。由于CDR3是与抗原直接接触的TCR区域，因此CDR3在TCR与肽-MHC复合物的相互作用中起到了十分重要的作用，CDR3序列对TCR的功能影响最大，即便不同的TCR-CDR3序列高度相似，但即便只有一个氨基酸的区别，其TCR功能也大不相同。体外结合及激活实验表明本发明的CMV-pp65-TCR-T可在健康供者的初级CD8+T细胞(primary CD8+T细胞)中正常表达并特异性激活，体外杀伤实验表明本项目的CMV-pp65-TCR对靶细胞的杀伤作用显著。因此，本发明的HLA-A*2402限制性靶向pp65的特异性TCR，能够特异性地被表达CMV-pp65的靶细胞激活，pp65-TCR-T细胞可显著地杀伤靶细胞，后续可有效应用于HSCT患者或其他移植患者CMV感染的治疗。
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：
αCDR1-AGCTCCAATTTTTATGCC(SEQ ID NO.10)
αCDR2-ATGACATTGAATGGTGATGAA(SEQ ID NO.12)
αCDR3-TGCGCACGATCAAACTACGGCGGGTCCCAGGGCAACCTGATTTTC(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：
βCDR1-GAGAATCATAGATAC(SEQ ID NO.18)
βCDR2-AGCTACGGCGTGAAAGAC(SEQ ID NO.20)
βCDR3-TGTGCTATCTCTGACCCCGGATCTTCATTCGGCGGCTATACCTTT(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
(1)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)，cytotoxic T lymphocyte medium重悬后，350g，5min离心后去除上清，加入适量DC medium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 medium，将6孔板放置于37℃，5％CO2培养箱中培养5d。
(3)Day 5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％CO2培养箱中诱导成熟2d。
(4)Day 7时可收获Mature DC，移液器大力吹打和重悬孔板底部细胞以收取Mature DC，将收集的Mature DC转移至15毫升离心管，350g，5min离心后去除上清，按2×106cells/ml密度使用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Mature DC中加入5μg的CMV-PP65-mRNA，使用Bio-Rad细胞电转仪以250V，800us，4times，0.6sinterval，2mm cuvette进行DC细胞的电转。电转结束后，收集CMV-mRNA-loaded DCs至15ml离心管中。
(2)Lymphoprep密度梯度离心法获取健康供者血液中PBMCs，使用EasySepTM Human CD8 Positive Selection Kit分离PBMCs中的CD8+T细胞，以DC/T为1:2.5的比例，将CMV-mRNA-loaded DCs与CD8+T cells加至12孔板中培养，培养体系中加入终浓度为30ng/ml的IL-21。
(3)将12孔板放置在37℃，5％CO2细胞培养箱中孵育过夜。Day 1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％CO2培养10天。培养过程中每2-3天进行半量换液。
(4)Day 11时，参考实施方式4.1，制备CMV-mRNA-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)Day 18时，吸取孔板中每孔各500μl细胞，经FACS buffer洗涤两次后，加入CMV-PP65-HLA:A:2402Tetramer-APC与CD8a-BV421，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FACS buffer重悬细胞后检测CD8+/Tetramer+(％)比例检测。
实施例3.单细胞测序获取HLA-A*2402限制型CMV-pp65抗原特异性TCR
(1)流式分选CD8+/Tetramer+T细胞，10X Genomics单细胞测序技术获取CD8+/Tetramer+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/βchain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Furin-cleavage使得TCRα与TCRβ能在一个表达载体中同时表达。
(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细胞构建CMV-pp65-TCR-T
(1)使用EasySepTM Human CD8 Positive Selection Kit分离PBMCs中的CD8+T细胞，以1:3 CD8+T cell:CD3/28 Dynabeads比例，体外使用CD3/28 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 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％CO2培养3d，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3d后，吸取CMV-pp65-TCR-T细胞，经FACS buffer洗涤两次后，加入CMV-PP65-HLA*A:2402Tetramer-APC与CD8a-BV421抗体染色，FACS检测CD8+/Tetramer+(％)比例，随后流式分选CD8a+/Tetramer+T细胞用于后续体外杀伤实验。
实施例5.流式检测CMV-pp65-TCR-T体外激活实验：
(1)通过慢病毒感染K562细胞分别制备K562-A:2402-GFP与K562-A:2402-GFP-pp65-mCherry细胞；
(2)通过慢病毒制备CMV-pp65-TCR-T细胞，分别按效靶比例E:T＝5:1，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后收集孔内细胞进行染色，FACS检测共培养体系中CD8+T细胞的CD25,CD69的表达水平，以评估K562-A:2402-GFP-pp65-mCherry靶细胞是否可在体外特异性激活CMV-pp65-TCR-T细胞。
实施例6.流式检测CMV-pp65-TCR-T体外杀伤实验：
(1)取实施例5中制备的CMV-pp65-TCR-T细胞以及K562-A:2402-GFP与K562-A:2402-GFP-pp65-mCherry细胞，分别按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:2402-GFP或K562-A:2402-GFP-pp65-mCherry的比例，以评估CMV-pp65-TCR-T对靶细胞及对照细胞的的杀伤效率差异。
实施例7.CDX模型检测CMV-pp65-TCR-T体内杀伤实验：
(1)通过慢病毒感染法感染K562及CD8+T细胞分别制备K562-A:2402-GFP-pp65-mCherry-luciferase靶细胞及CMV-pp65-TCR-T效应细胞；
(2)day0时尾静脉注射3×106K562-A:2402-GFP-pp65-mCherry-luciferase靶细胞至NOG小鼠构建体内CDX模型，在靶细胞注射后4h使用小动物成像仪对肿瘤细胞进行示踪分析以确保CDX模型的成功建立，随后在day3时分别尾静脉回输1×107saline，CD8+T及CMV-pp65-TCR-T细胞。
(3)分别在靶细胞注射后day7，day14，day21，day28时腹腔注射D-荧光素钾盐底物，随后使用小动物成像仪对各组小鼠进行生物发光成像，观察CD8+T及CMV-pp65-TCR-T细胞对小鼠体内靶细胞的杀伤情况。
实验结果
CMV-pp65-TCR-T筛选流程：
整个试验由健康HLA-A:2402供者的CD8+T细胞经自体的mDCs负载pp65peptides进行两轮刺激后，使用CMV-PP65-HLA*A:2402Tetramer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1)显示，经过两轮mDCs-pp65peptides刺激后，健康供者的CD8a+/Tetramer+T比例由0％提高至0.74％，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 mRNA进行两轮刺激后，使用CMV-PP65-HLA*A:2402 Tetramer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，1#TCR克隆占比为74.17％，证明此次antigen-specific T cell priming实验特异性良好。
CMV-pp65-TCR-T载体构建及序列优化：
经测序得到pp65-TCR(1#TCR)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。
随后，将优化完成的pp65-TCR序列(图2)克隆至慢病毒表达载体中。
最终获得的pp65-TCR序列信息如下：
经密码子优化后的基因序列如下：

其中，α链(α-chain)序列为：
其基因序列如下：

β链(β-chain)序列为：
其基因序列如下：
α链(α-chain)可变区序列为：
其基因序列如下：
α链的3个CDR序列如下：
α-CDR1：SSNFYA(SEQ ID NO.9)，编码序列为：AGCTCCAATTTTTATGCC(SEQ ID NO.10)
α-CDR2：MTLNGDE(SEQ ID NO.11)，编码序列为：ATGACATTGAATGGTGATGAA(SEQ ID NO.12)
α-CDR3：CARSNYGGSQGNLIF(SEQ ID NO.13)，编码序列为：TGCGCACGATCAAACTACGGCGGGTCCCAGGGCAACCTGATTTTC(SEQ ID NO.14)
β链(β-chain)可变区序列为：
其基因序列如下：
β链的3个CDR序列如下：
β-CDR1：ENHRY(SEQ ID NO.17)，编码序列为：GAGAATCATAGATAC(SEQ ID NO.18)
β-CDR2：SYGVKD(SEQ ID NO.19)，编码序列为：AGCTACGGCGTGAAAGAC(SEQ ID NO.20)
β-CDR3：CAISDPGSSFGGYTF(SEQ ID NO.21)，编码序列为：TGTGCTATCTCTGACCCCGGATCTTCATTCGGCGGCTATACCTTT(SEQ ID NO.22)。
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:2402供者的PBMC或jurkat 76细胞株后进行CMV-pp65-HLA*A:2402Tetramer染色，流式结果显示Tetramer+/mTCRβC+jurkatT及Tetramer+/primary CD8+T(图3)细胞分群明显，该结果表明本项目筛选并鉴定的CMV-pp65-TCR-T在健康供者的及primary CD8+T细胞及jurkat 76细胞中正常表达，并可与CMV-PP65-HLA*A:2402四聚体结合。
CMV-pp65-TCR-T体外激活实验：
通过慢病毒制备CMV-pp65-TCR-T效应细胞，K562-A:2402-GFP-pp65-mCherry靶细胞与K562-A:2402-GFP对照细胞，分别按效靶比例E:T＝5:1，分别接种TCR-T细胞与K562细胞至圆底96孔板中培养。于共培养24h后收集孔内细胞进行流式检测。流式实验结果(图4)表明K562-A:2402-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CD25及CD69的表达水平显著增高，而对照组K562-A:2402-GFP细胞对CMV-pp65-TCR-T细胞无明显激活作用。
CMV-pp65-TCR-T体外杀伤实验：
通过慢病毒分别制备效应细胞与靶细胞，随后将效应细胞与靶细胞按效靶比5:1进行外共培养4h后，流式检测靶细胞凋亡的比例(Violet+/eF780+)。实验结果(图5)表明CMV-pp65-TCR-T细胞可在体外杀伤K562-A:2402-GFP-pp65-mCherry靶细胞，4h的杀伤效率约为65％，而对不表达pp65的K562-A:2402-GFP对照细胞，其杀伤效率仅约10％。该结果表明本项目筛选并鉴定的CMV-pp65-TCR-T细胞可在体外特异性的杀伤表达pp65的K562-A:2402-GFP-pp65-mCherry细胞。
CMV-pp65-TCR-T体内杀伤实验：
通过慢病毒感染原代CD8+T以及K562细胞，分别制备CMV-pp65-TCR-T效应细胞与 K562-A:2402-GFP-pp65-mCherry-luciferase靶细胞，如图6A所示时间点将靶细胞及效应细胞尾静脉注射至NOG小鼠中，分别于靶细胞静脉注射后的第0，7,14,21,28天进行小鼠的生物发光成像，实验结果(图6B)表明，saline组以及原代的CD8+T细胞治疗组无法有效抑制肿瘤细胞的生长，对比而言CMV-pp65-TCR-T组有3只小鼠达到肿瘤细胞清除的状态。该结果表明回输CMV-pp65-TCR-T细胞可在体内杀伤K562-A:2402-GFP-pp65-mCherry-Luciferase细胞，证明本项目筛选并鉴定的CMV-pp65-TCR-T细胞可在体内特异性的杀伤表达pp65的HLA-A2402限制性的靶细胞。
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。本发明的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。总体而言，本项目发明的HLA*A:2402限制性的CMV-pp65-TCR-T具有以下几个特点：体外实验表明CMV-pp65-TCR-T可精准靶向并快速清除感染CMV病毒的靶细胞，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，可以有效应用于于HSCT患者或其他移植患者CMV感染的治疗。
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 (10)

  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CARSNYGGSQGNLIF(SEQ ID NO.13)；和/或
    所述TCRβ链可变域的CDR3的氨基酸序列为CAISDPGSSFGGYTF(SEQ ID NO.21)；
    更优选地，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：CARSNYGGSQGNLIF(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：ENHRY(SEQ ID NO.17)，
    β-CDR 2：SYGVKD(SEQ ID NO.19)，
    β-CDR 3：CAISDPGSSFGGYTF(SEQ ID NO.21)。
  2. 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。
  3. 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。
  4. 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。
  5. 一种核酸分子，其特征在于，所述核酸分子包含编码上述任一权利要求所述的TCR分子的核酸序列或其互补序列；
    优选地，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。
  6. 一种载体，其特征在于，所述的载体含有权利要求5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。
  7. 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6中所述的载体或染色体中整合有外源的权利要求5所述的核酸分子。
  8. 一种细胞，其特征在于，所述细胞转导权利要求5所述的核酸分子或权利要求6中所 述载体；优选地，所述细胞为T细胞或干细胞。
  9. 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1-3中任一项所述的TCR、权利要求4中所述的TCR复合物、权利要求5所述的核酸分子、或权利要求8中所述的细胞。
  10. 权利要求1-3中任一项所述的T细胞受体、或权利要求4中所述的TCR复合物或权利要求8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。
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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
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
CN110951690A (zh) * 2010-09-20 2020-04-03 生物技术细胞和基因治疗公司 抗原特异性t细胞受体和t细胞表位
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
CN113881680A (zh) * 2020-07-01 2022-01-04 华夏英泰（北京）生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用
WO2023142683A1 (zh) * 2022-01-27 2023-08-03 上海市第一人民医院 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102668219B1 (ko) * 2015-03-16 2024-05-28 막스-델부뤽-센트럼 퓌어 몰레쿨라레 메디친 인 데어 헬름홀츠-게마인샤프트 Mhc 세포 라이브러리를 이용하여 신규한 면역원성 t 세포 에피토프를 검출하고 신규한 항원-특이적 t 세포 수용체를 단리하는 방법
SG11202002635RA (en) * 2017-09-29 2020-04-29 The United States Of America As Represented By The Secretary Methods of isolating t cells having antigenic specificity for a p53 cancer-specific mutation
US20220144916A1 (en) * 2019-02-12 2022-05-12 Board Of Regents, The University Of Texas System High affinity engineered t-cell receptors targeting cmv infected cells
KR102555328B1 (ko) * 2020-02-06 2023-07-17 아주대학교산학협력단 CMV pp65 펩타이드/HLA-A*02 복합체에 특이적인 TCR-유사 항체 및 이의 용도
WO2024139780A1 (zh) * 2022-12-26 2024-07-04 上海市第一人民医院 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

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
CN110951690A (zh) * 2010-09-20 2020-04-03 生物技术细胞和基因治疗公司 抗原特异性t细胞受体和t细胞表位
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
CN113881680A (zh) * 2020-07-01 2022-01-04 华夏英泰（北京）生物技术有限公司 T细胞抗原受体、其多聚体复合物及其制备方法和应用
WO2023142683A1 (zh) * 2022-01-27 2023-08-03 上海市第一人民医院 靶向巨细胞病毒抗原的tcr和表达其的t细胞及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HONG CHEOL-HWA, PYO HONG-SEON, BAEK IN-CHEOL, KIM TAI-GYU: "Rapid identification of CMV-specific TCRs via reverse TCR cloning system based on bulk TCR repertoire data", FRONTIERS IN IMMUNOLOGY, FRONTIERS MEDIA, LAUSANNE, CH, vol. 13, 18 November 2022 (2022-11-18), Lausanne, CH , XP093184460, ISSN: 1664-3224, DOI: 10.3389/fimmu.2022.1021067 *

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