WO2020029774A1 - 一种嵌合t细胞受体star及其应用 - Google Patents

一种嵌合t细胞受体star及其应用 Download PDF

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WO2020029774A1
WO2020029774A1 PCT/CN2019/096820 CN2019096820W WO2020029774A1 WO 2020029774 A1 WO2020029774 A1 WO 2020029774A1 CN 2019096820 W CN2019096820 W CN 2019096820W WO 2020029774 A1 WO2020029774 A1 WO 2020029774A1
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cell receptor
cell
cancer
chain
chimeric
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French (fr)
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刘玥
王嘉盛
刘光娜
赵学强
林欣
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华夏英泰（北京）生物技术有限公司
清华大学
上海赛傲生物技术有限公司
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Definitions

  • the invention relates to the field of biomedicine, in particular to an antibody-T cell receptor chimeric receptor, a construction method and uses thereof, including treatment and diagnosis of diseases.
  • Chimeric antigen receptor T-cell therapy (CAR-T) and T-cell receptor therapy (TCR-T) are two leading-edge gene therapies that use patients' own T lymphocytes to treat cancer. They can express specific receptors and target specific cells such as tumor cells. They have received extensive attention and research, and they have transformed from basic immunological research to clinical applications. Recent advances in synthetic biology, immunology, and genetic engineering have made it possible to synthesize engineered T cells with enhanced specific functions.
  • CAR-T uses antibody fragments that can bind to specific antigens to identify antigens on the surface of tumor cells.
  • CD19 antigen-specific CAR-T cells have been used in clinical trials for the treatment of B-cell leukemia and lymphoma, and have shown continuous disease relief effects.
  • the chimeric antigen receptor (CAR) gives T cells the ability to recognize tumor antigens in an HLA-independent manner, which allows CAR-engineered T cells to recognize a wider range of targets than the natural T cell surface receptor TCR.
  • CAR-T technology has significant effects in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatment methods.
  • TCR T Cell Receptor
  • TCR T Cell Receptor
  • TCR is a molecule that specifically recognizes antigens on the surface of T cells and mediates immune responses.
  • TCR mainly recognizes antigen molecule polypeptides presented by histocompatibility complex molecules.
  • CAR-T cell therapy due to the great clinical success of CAR-T cell therapy, research on TCR therapy has been relatively less interesting.
  • TCR-T has obvious advantages over CAR-T. This is because the target antigen for CAR-T cell therapy is cell surface protein, and TCR-T recognizes MHC molecules, which can Presenting peptide chains obtained from cell surface and intracellular proteins, TCR-T is able to target a wider variety of antigens.
  • TAA tumor-associated antigens
  • TCRs T-cell receptors
  • the cloned TCR (or chimeric receptor) with high affinity for TAA can be transferred to T lymphocytes by transgenic technology, which can redirect the original T cells without tumor recognition ability to effectively recognize and kill tumor cells in vitro and in vivo.
  • TCR is a characteristic marker on the surface of all T cells. It binds to CD3 ( ⁇ , ⁇ , ⁇ , ⁇ ) with non-covalent bonds to form a TCR-CD3 complex.
  • TCR is a heterodimer composed of two different peptide chains. It consists of two peptide chains ⁇ and ⁇ . Each peptide chain can be divided into two parts: variable region (V region) and constant region (C region). The constant region includes three parts: the extracellular region, the transmembrane region, and the intracellular end; it is characterized by a short intracellular region.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen is specifically present in the V region. TCR is divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains of ⁇ and ⁇ , and TCR2 is composed of two chains of ⁇ and ⁇ . In peripheral blood, 90% -95% of T cells express TCR2; and any T cell expresses only one of TCR2 or TCR1.
  • Naturally occurring TCR is a membrane protein that is stabilized by its transmembrane region. Therefore, for the expression of soluble TCR in bacteria, obtaining a highly stable TCR that maintains the ability to specifically bind to its original ligand (i.e. pMHC)
  • pMHC original ligand
  • Some literatures describe truncated forms of TCR, which contain only extracellular regions or only extracellular and cytoplasmic regions. Although such TCRs can be recognized by TCR-specific antibodies, the yield is very low and cannot be achieved at low concentrations. Identification of the main histocompatibility complex-peptide complex indicates that it is easily denatured and not stable enough.
  • TCR-T technology which directly transforms T-cell-associated "probe" -TCR of tumor antigens, strengthens the specific recognition process of T cells against tumor cells, and improves the affinity of T lymphocytes for tumor cells, making the original tumor-free recognition Capable T cells effectively recognize and kill tumor cells in vitro and in vivo.
  • TCR-T cell therapy while increasing the number of T lymphocytes, at the same time increasing the lethality of T lymphocytes to tumor cells, thereby achieving a good tumor treatment effect.
  • TCR-T In the existing TCR-T therapy, it is generally necessary to isolate the endogenous TCR and engineer it to introduce new T cells and inject them back into the human body.
  • the number of T cells with the ability to target cancer cells will be large It is expected to identify and attack a variety of solid tumors and blood tumors.
  • exogenous TCR ⁇ and ⁇ chains and endogenous ⁇ and ⁇ chains will recognize each other and form a hybrid TCR.
  • This TCR mismatch phenomenon is thought to be widely present in TCR-T therapy, which may cause a decrease in TCR expression on the cell surface and a decrease in cell viability.
  • Mismatched TCR may also form a new TCR that can generate unknown specificity. This new TCR will bring on-target or off-target toxicity in TCR-T.
  • TCR expression in T cells, paired assembly, and affinity with pMHC are key factors that influence TCR gene therapy to fully exert its anti-tumor ability.
  • TCR gene therapy it can be seen that how to modify the TCR gene so that the TCR ⁇ chain and ⁇ chain can be correctly paired on the surface of T cells, and their expression efficiency and affinity are enhanced, while avoiding side effects and improving safety has become one of the hot spots of TCR gene therapy in recent years.
  • the main strategies include increasing the affinity of TCR in T cells, optimizing the pairing of TCR chains, and enhancing its surface expression efficiency.
  • TCR mismatches some researchers have adopted the following methods to reduce TCR mismatches: introducing disulfide bonds in the TCR chain; replacing the C region of the human TCR molecule with a mouse-derived conserved C region; and conserving the conserved domains at the TCR ⁇ and ⁇ chain junctions Amino acid residues were inverted; a single-chain TCR (scTCR) chimera was constructed; the C region of the TCR chain was fused with a CD3 molecule; a ⁇ TCR gene was transferred into ⁇ T cells, etc., but the above effects were not satisfactory. It can be seen that although modification of TCR-T has been proposed to optimize the matching strategy of exogenous TCR molecules, there are still many areas that need improvement.
  • the present invention solves the aforementioned technical problems in the prior art, and provides a chimeric T cell receptor (Synthetic T Cell Receptor and Antigen Receptor, STAR) that specifically binds to a target antigen.
  • the chimeric T cell receptor includes :
  • the antibody heavy chain variable region and the antibody light chain variable region specifically bind to an antigenic epitope of the target antigen.
  • the second subunit of the T cell receptor is a ⁇ chain; or (2) when the first subunit of the T cell receptor is a ⁇ chain, the second subunit of the T cell receptor is an ⁇ chain; or, (3) when the first subunit of the T cell receptor is When it is a ⁇ chain, the second subunit of the T cell receptor is a ⁇ chain; or, (4) When the first subunit of the T cell receptor is a ⁇ chain, the second subunit of the T cell receptor For the gamma chain.
  • first peptide chain and the second peptide chain are bound through disulfide bonds after being expressed in T cells.
  • the chimeric T cell receptor first subunit constant region and the T cell receptor second subunit constant region are derived from human or murine species, including different protein subtypes.
  • any chimeric T cell receptor can be subjected to any amino acid sequence modification, including but not limited to amino acid point mutation modification and polypeptide fragment substitution modification to reduce the mismatch with endogenously expressed T cell receptor .
  • amino acid sequence modification including but not limited to amino acid point mutation modification and polypeptide fragment substitution modification to reduce the mismatch with endogenously expressed T cell receptor .
  • the amino acid at position 48 of the constant region is mutated to cysteine, and the amino acid at position 57 of the constant region is mutated to cysteine, so that the first polypeptide and the second polypeptide pass through Disulfide linkage;
  • TCR ⁇ chain the 85th amino acid of the constant region is mutated to alanine, and the 88th amino acid of the constant region is mutated to glycine;
  • the first subunit of the T cell receptor is a TCR ⁇ chain, and the amino acid at position 48 of the constant region is mutated to cysteine; and / or, the second subunit of the T cell receptor is TCR ⁇ Chain, whose amino acid position 57 of the constant region is mutated to cysteine; or (2) the first subunit of the T cell receptor is a TCR ⁇ chain, whose amino acid position 85 of the constant region is mutated to alanine, and / or The second subunit of the T cell receptor is a TCR ⁇ chain, and the 88th amino acid of the constant region is mutated to glycine.
  • the target antigen is a tumor-specific antigen or a virus-specific antigen.
  • the specific target antigen is selected from CD19, CD20, EGFR, Her2, PSCA, CD123, CEA (Carcinoembryonic Antigen), FAP, CD133, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, VEGFR, CS1 , ROR1, EC, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL3, CD99, 5T4, CD22, CD30, CD33, CD138, CD171.
  • the antigen may be CD19, CD20, EGFR, Her2.
  • the antibody, antibody heavy chain variable region or antibody light chain variable region is from IMCC225 (cetuximab, Cetuximab / Cetux), rituximab (melova), Ofatumumab (OFA, Olfalimumab), CD19 monoclonal antibody (FMC63), Avastin (Bevacizumab), BEC2 (Atolimumab), Bexxar (Tosimab), Campath (Alendumab) (Antibody), Herceptin (trastuzumab), LymphoCide (epalizumab), MDX-210, Mylotarg (jemizumab ozomicin), mAb 17-1A (ejelizumab), Theragyn ( pemtumomab), Zamyl, Zevalin (teimumab) or high affinity antibodies obtained by screening.
  • the antibody comprises an antigen-binding fragment selected from the group consisting of Fab, F (a)
  • the target antigen-related disease is a cancer or viral infection-related disease.
  • the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, Head and neck cancer, kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, Sarcoma, stomach cancer, uterine cancer, and thyroid cancer; or, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), hepatitis B virus ( HBV), Kaposi's sarcoma-associated herpes
  • CMV
  • the first polypeptide and the second polypeptide form a complex with an endogenous CD3 subunit ( ⁇ , ⁇ , ⁇ , ⁇ ) of a T cell.
  • the antibody heavy chain and light chain variable regions are derived from IMCC225 (cetuximab, Cetuximab / Cetux), rituximab (melova), ofatumumab (ofamumab VH and VL of anti-CD19 monoclonal antibody FMC63.
  • nucleotide sequence of Cetux VH is SEQ ID NO: 3
  • amino acid sequence is SEQ ID NO: 13
  • nucleotide sequence of Cetux VL is SEQ ID NO: 4
  • amino acid sequence is SEQ ID NO: 14.
  • the nucleotide sequence of the FMC63-VH is SEQ ID NO: 5
  • the amino acid sequence is SEQ ID NO: 15
  • the nucleotide sequence of the FMC63-VL is SEQ ID NO: 6
  • the amino acid sequence is SEQ ID NO: 16 ;.
  • the OFA-VH nucleotide sequence is SEQ ID NO: 9
  • the amino acid sequence is SEQ ID NO: 19
  • the OFA-VL nucleotide sequence is SEQ ID NO: 10
  • the amino acid sequence is SEQ ID NO: 20.
  • the VH and VL derived from cetuximab, trastuzumab, and rituximab are fused with the TCR ⁇ chain or ⁇ chain, respectively, to obtain a VH-TCR ⁇ chain fusion or VL -TCR ⁇ chain fusion.
  • the VH and VL derived from IMCC225 (Cetuximab, Cetuximab / Cetux), Rituximab (Melohua), Ofatumumab (Ofalimumab), and CD19 monoclonal antibody FMC63 are respectively.
  • the TCR ⁇ chain constant region or ⁇ chain constant region is fused to obtain a VH-TCR ⁇ chain constant region fusion or a VL-TCR ⁇ chain constant region fusion.
  • the two different fusions are linked by a furin-p2A segment peptide sequence; preferably, the two different fusions are covalently bound through a disulfide bond after being expressed in a T cell. Furthermore, the two different fusions form a complex with CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) endogenous to T cells.
  • the present invention also provides a complex formed by a chimeric T cell receptor that specifically binds to a target antigen.
  • the chimeric T cell receptor and a CD3 subunit endogenously expressed by the T cell form complexes and can activate T cell-related signal transduction pathways when activated by target antigens.
  • the present invention also provides a nucleic acid encoding the chimeric T cell receptor of any one of the foregoing or the first polypeptide and the second polypeptide.
  • nucleic acid is as follows:
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • the T cell receptor (TCR) alpha or beta chain constant region is derived from a human TCR alpha or beta chain constant region, and may also be derived from a murine TCR alpha or beta chain constant region.
  • the ⁇ chain and / or ⁇ chain of the TCR can be modified with amino acid point mutations and polypeptide fragment substitutions to reduce mismatches with endogenously expressed T cell receptors.
  • the ⁇ and / or ⁇ chains of the TCR are cysteine-point mutated. Specifically, the 48th amino acid of the constant region of the TCR ⁇ chain is mutated to a cysteine, and the 57th amino acid of the constant region of the TCR ⁇ chain is mutated to a cysteine, so that an additional one is added between the first peptide chain and the second peptide chain. Disulfide bonding.
  • the nucleotide sequence of the human TCR ⁇ chain constant region cysteine mutant is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 11; the human TCR ⁇ chain constant region cysteine mutant nucleoside The acid sequence is SEQ ID NO: 2 and the amino acid sequence is SEQ ID NO: 12.
  • amino acid sequence of the constant region of the human TCR ⁇ chain is SEQ ID NO: 21
  • amino acid sequence of the constant region of the human TCR ⁇ chain is SEQ ID NO: 22
  • amino acid sequence of the constant region of the mouse TCR ⁇ chain is SEQ ID NO: 23
  • amino acid sequence of the mouse-derived TCR delta chain constant region is SEQ ID NO: 24.
  • the constant region cysteine mutant can also be selected from the polypeptides represented by the following sequences:
  • amino acid sequence is SEQ ID NO: 31, and the corresponding nucleotide sequence is SEQ ID NO: 25;
  • amino acid sequence is SEQ ID NO: 32, and the corresponding nucleotide sequence is SEQ ID NO: 26;
  • amino acid sequence is SEQ ID NO: 33, and the corresponding nucleotide sequence is SEQ ID NO: 27.
  • the murine TCR ⁇ constant region cysteine mutant nucleotide sequence is SEQ ID NO: 7, and the amino acid sequence is SEQ IDNO: 17; the murine TCR ⁇ constant region cysteine mutant nucleotide The sequence is SEQ ID NO: 8, and the amino acid sequence is SEQ ID NO: 18.
  • the first amino acid is Asn, which is the polypeptide shown in SEQ ID NO: 17, and the polypeptide whose first amino acid is Asp, His, or Tyr, so the mouse TCR ⁇ chain
  • the constant region cysteine mutant can also be selected from the polypeptides represented by the following sequences:
  • amino acid sequence is SEQ ID NO: 34, and the corresponding nucleotide sequence is SEQ ID NO: 28;
  • amino acid sequence is SEQ ID NO: 35, and the corresponding nucleotide sequence is SEQ ID NO: 29;
  • amino acid sequence is SEQ ID NO: 36, and the corresponding nucleotide sequence is SEQ ID NO: 30.
  • the T cell receptor (TCR) alpha chain can be replaced with a T cell receptor (TCR) gamma chain; the T cell receptor (TCR) beta chain can be replaced with a T cell receptor (TCR) delta chain.
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • TCR T cell receptor
  • the T cell receptor (TCR) delta chain may be derived from a human TCR gamma chain or a delta chain constant region, and may also be derived from a murine TCR gamma chain or a delta chain constant region.
  • the present invention also provides a vector comprising a nucleic acid encoding the chimeric T cell receptor of any one of the foregoing or the first polypeptide and the second polypeptide.
  • the vector is a plasmid. More preferably, the vector is selected from the group consisting of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus vector.
  • an effector cell that expresses any of the aforementioned chimeric T cell receptors or the aforementioned complexes on the cell surface thereof.
  • the effector cell comprises a nucleic acid encoding the chimeric T cell receptor.
  • the T cells are selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, and suppressor T cells.
  • a pharmaceutical composition comprising a chimeric T cell receptor according to any one of the chimeric T cell receptors described above and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition is provided comprising a nucleic acid encoding the chimeric T cell receptor according to any of the embodiments described above and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition is provided comprising an effector cell expressing any one of the chimeric T cell receptors described above and a pharmaceutically acceptable carrier.
  • the chimeric T cell receptor of the present invention and cells transfected with the chimeric T cell receptor of the present invention can be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.
  • the chimeric T cell receptors, chimeric T cell receptor complexes, and cells of the invention are typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be in any suitable form (depending on the desired method of administration to the patient). It can be provided in unit dosage form, usually in a sealed container, and can be provided as part of a kit. Such kits (but not required) include instructions for use. It may include a plurality of said unit dosage forms.
  • the chimeric T cell receptor of the present invention may be used alone, or may be bound or conjugated to a conjugate.
  • the conjugate includes a detectable label, a therapeutic agent, a PK (protein kinase) modified moiety, or a combination of any of these.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MIR (magnetic resonance imaging) or CT (electronic computed tomography) contrast agents, or capable of producing detectable products The enzyme.
  • the pharmaceutical composition may also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent.
  • the term refers to pharmaceutical carriers that do not themselves induce the production of antibodies that are harmful to the individual receiving the composition and are not excessively toxic after administration.
  • Such carriers include, but are not limited to, saline, buffers, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.
  • a pharmaceutically acceptable carrier in a therapeutic composition may contain liquids such as water, saline, glycerol, and ethanol.
  • the therapeutic composition can be prepared as an injectable, such as a liquid solution or suspension; it can also be prepared in a solid form suitable as a liquid carrier for injection into a solution or suspension before injection.
  • composition of the invention can be administered by conventional routes including, but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal or topical administration.
  • the object to be prevented or treated may be animals, especially humans.
  • compositions of the present invention When the pharmaceutical composition of the present invention is used for actual treatment, various different dosage forms of the pharmaceutical composition can be adopted according to the use situation.
  • an injection, an oral preparation and the like can be exemplified.
  • These pharmaceutical compositions can be formulated according to conventional methods by mixing, diluting or dissolving, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity Agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and co-solvents, and the formulation process can be performed in a conventional manner according to the dosage form.
  • suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicity Agents, preservatives, wetting agents, emulsifiers, dispersants, stabilizers, and co-solvents
  • the pharmaceutical composition of the present invention can also be administered in the form of a sustained release agent.
  • the polypeptide of the present invention can be incorporated into a pill or microcapsule with a sustained release polymer as a carrier, and then the pill or microcapsule is surgically implanted into a tissue to be treated.
  • the dosage of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient may be based on the weight, age, sex, and degree of symptoms of each patient to be treated And reasonably determined.
  • the chimeric T cell receptor of the present invention can be used as a drug or a diagnostic agent. Modifications or other modifications can be made to obtain characteristics that are more suitable for use as drugs or diagnostic agents.
  • a nucleic acid library comprising a sequence encoding a plurality of chimeric T cell receptors according to any of the chimeric T cell receptors described above.
  • a method of screening a sequence encoding a chimeric T cell receptor specific for a target antigen in a nucleic acid library comprising: a) applying The nucleic acid library is introduced into a plurality of cells such that the chimeric T cell receptor is expressed on the surface of the plurality of cells; b) the plurality of cells are warmed together with a ligand comprising the target antigen or one or more epitopes contained therein C) collecting cells that bind to the ligand; and d) isolating a sequence encoding a chimeric T cell receptor from the cells collected in step c), thereby identifying chimeric T cell receptors specific for the target antigen. body.
  • a chimeric T cell receptor of any one of the above chimeric T cell receptors in the preparation of a kit for treating or diagnosing a target antigen-related disease in a subject in need thereof .
  • a method for killing a target cell that presents a target antigen comprising associating the target cell with a chimeric T cell receptor expressing any of the chimeric T cell receptors described above. Effector cell contact in which a chimeric T cell receptor specifically binds to a target antigen.
  • a method for killing a target cell presenting a target antigen wherein the chimeric T cell receptor specifically binds to the target antigen.
  • the antibody heavy chain variable region and light chain variable region specifically bind to an antigen-binding module of the target antigen.
  • the contacting is in vivo. In some embodiments, the contacting is in vitro.
  • a method of treating a target antigen-related disease in an individual in need comprising administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising expressing a chimeric T cell receptor according to the above or The effector cell.
  • a method of treating a target antigen-related disease in an individual in need comprising administering to the individual an effective amount of a composition comprising effector T cells, the effector T cells comprising specifically binding to a target Antigen chimeric T cell receptor, comprising: a) a first polypeptide fused to an antibody heavy chain variable region; b) a second polypeptide fused to an antibody light chain variable region; wherein the antibody heavy chain may be The variable and light chain variable regions specifically bind to an antigen-binding module of the target antigen.
  • the chimeric T cell receptor is any one of the aforementioned chimeric T cell receptors.
  • a method for treating a T cell-mediated disorder due to cell, tissue, body part, or organ transplantation in a subject in need comprising the steps of: dividing a cell, tissue, body part Or an organ transplant into the subject; and administering two or more doses of a pharmaceutically acceptable amount of any one of the aforementioned chimeric T cell receptors to the subject.
  • the disease is cancer.
  • the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma , Head and neck cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer , Sarcoma, stomach cancer, uterine cancer and thyroid cancer.
  • the target antigen-related disease is a viral infection.
  • the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), hepatitis B virus (HBV), Kaposi Kaposi's Sarcoma associated herpes virus (KSHV), human papilloma virus (HPV), molluscum virus (MCV), human T cell leukemia virus 1 (HTLV-1), HIV (human immunodeficiency Virus) and hepatitis C virus (HCV).
  • CMV cytomegalovirus
  • EBV Epstein-Barr Virus
  • HBV hepatitis B virus
  • KSHV Kaposi Kaposi's Sarcoma associated herpes virus
  • HPV human papilloma virus
  • MMV molluscum virus
  • HTLV-1 human T cell leukemia virus 1
  • HIV human immunodeficiency Virus
  • HCV hepatitis C virus
  • the disease is adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, head and neck cancer, Kidney cancer, leukemia, lymphoma, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, sarcoma, stomach cancer , Uterine cancer and thyroid cancer.
  • a method of treating a target antigen-related disease in an individual in need comprising administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising a protein encoding a chimeric T cell receptor according to the above The nucleic acid of any one of the chimeric T cell receptors.
  • the polynucleotides of the present invention can be used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA technology. Generally there are the following steps: (1) using a polynucleotide (or variant) encoding the chimeric T cell receptor polypeptide of the present invention, or using a recombinant expression vector containing the polynucleotide to transform or transduce a suitable host cell (2) culturing host cells in a suitable medium; (3) isolating and purifying the chimeric T cell receptor polypeptide of the present invention from the medium or cells.
  • the present invention provides any one of the foregoing chimeric T cell receptors, the complexes, nucleic acids, vectors, or effector cells in the manufacture of a kit, formulation, or Use in a pharmaceutical composition.
  • the present invention provides a method for treating a target antigen-related disease or a cancer or viral infection-related disease in an individual in need thereof, comprising administering to the individual an effective amount of a pharmaceutical composition, the pharmaceutical composition comprising any of the foregoing Chimeric T cell receptor, said complex, nucleic acid, vector or effector cell.
  • the cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colorectal cancer, esophageal cancer, glioblastoma, glioma, hepatocellular carcinoma, Head and neck cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, mesothelioma, multiple myeloma, pancreatic cancer, pheochromocytoma, plasmacytoma, neuroblastoma, ovarian cancer, prostate cancer, Sarcoma, gastric cancer, uterine cancer, and thyroid cancer; or, the viral infection is caused by a virus selected from the group consisting of: cytomegalovirus (CMV), Epstein-Barr Virus (EBV), hepatitis B Virus (HBV), Kaposi's sarcoma-associated herpes virus (KSHV), human papilloma virus (HPV), mollus
  • Classical CAR molecules consist of single-chain antibody regions, hinge regions, transmembrane regions, and intracellular signal regions, and can be presented on the surface of T cells.
  • the single-chain antibody region contains the heavy chain variable region and light chain variable region of the antibody (in some cases, it also includes the IgG CH1 region, which plays a structural role), and the two are connected by a flexible linker peptide.
  • the intracellular signal region consists of a co-stimulatory signal molecule (4-1BB, CD28, etc.) and a signal molecule CD3 ⁇ connected in series with each other.
  • the chimeric T cell receptor constructed in the present application is expressed in cells by two polypeptide chains linked together via a disulfide bond covalent bond, wherein the first polypeptide is composed of an antibody heavy chain variable region (V H ) Fusion with TCR ⁇ constant region (C ⁇ ), the second polypeptide is made by fusion of antibody light chain variable region (V L ) with TCR ⁇ constant region (C ⁇ ); chimeric T cell receptor can be integrated with T cell
  • the source-expressed CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) form a complex to function.
  • the gene sequence of the chimeric T cell receptor is connected through the polypeptide fragment of the furin and p2A protease cleavage site.
  • Chimeric T cell receptors can have various combinations: the antibody heavy chain variable region (V H ) is fused with the TCR ⁇ constant region (C ⁇ ), and the antibody light chain variable region (V L ) is fused with the TCR ⁇ constant region (C ⁇ ) , Or the antibody light chain variable region (V L ) is fused to the TCR ⁇ constant region (C ⁇ ), the antibody heavy chain variable region (V H ) is fused to the TCR ⁇ constant region (C ⁇ ); and the exchange of the two is relatively furin and P2A. Before and after.
  • the light and heavy chain variable regions of an antibody can be replaced with a variety of specific antibody variable regions, such as anti-EGFR, CD19, CD20, and the like.
  • specific antibody variable regions such as anti-EGFR, CD19, CD20, and the like.
  • Various variants of the TCR ⁇ and ⁇ constant regions are also possible, including wild-type TCR ⁇ and ⁇ constant regions, cysteine single-point mutant TCR ⁇ and ⁇ constant regions, human-mouse chimeric TCR ⁇ and ⁇ constant regions, and half-containing Human and mouse chimeric TCR alpha and beta constant regions with cystine single point mutations.
  • the invention obtains T cell receptors with high stability and high specificity, which can be used for diagnosis and treatment of diseases.
  • the inventors creatively adopted several strategies.
  • the present application is performed by introducing a disulfide bond into the constant region of a TCR molecule. Theoretically, there are many sites capable of forming artificial interchain disulfide bonds in TCR, but finding suitable sites for forming artificial interchain disulfide bonds in TCR enables TCRs containing artificial interchain disulfide bonds to be successfully restored. It is very difficult to obtain TCR with high yield, high stability, and specific binding activity with its original ligand.
  • TCR containing artificial interchain disulfide bonds which can be well renatured, refolded, purified, has high stability, high renaturation yield, and can specifically bind to its original ligand.
  • the invention can modify the disulfide bond at a specific site to reduce the new reactivity of TCR-T cells and reduce the mismatch.
  • the invention introduces the fusion of the antibody antigen-binding fragment with the original TCR to form an antibody-T cell receptor-binding chimeric T cell receptor (STAR), which further improves the pairing of the alpha chain and the beta chain, and improves the binding of the TCR.
  • STAR antibody-T cell receptor-binding chimeric T cell receptor
  • the original TCR transformation in the body is less than CAR-T, which reduces the introduction of exogenous amino acids, reduces the risk of side effects, and improves safety.
  • the chimeric T cell receptor can mediate the activation of T cells after antigen stimulation and is compatible with the corresponding antibody-chimeric antigen receptor (CAR In comparison, the degree of antigen activation is comparable. What's more important is that in a resting state without antigen stimulation, the chimeric T cell receptor has no self-activation phenomenon, while CAR has a high self-activation phenomenon.
  • FIG. 1 Schematic diagram of the structure of STAR and CAR molecules.
  • STAR is expressed in cells by two polypeptide chains linked together via a disulfide bond covalent bond, where the first polypeptide chain is a fusion of the antibody heavy chain variable region (VH) and the TCR ⁇ constant region (C ⁇ ).
  • the second polypeptide chain is a fusion of the light chain variable region (VL) of the antibody and the TCR ⁇ constant region (C ⁇ );
  • STAR can form a complex with CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) endogenously expressed in T cells. Body functions.
  • FIG. 2 Schematic diagram of STAR and CAR molecular gene structural sequences.
  • the STAR gene sequence is connected by the polypeptide fragment of the furin and P2A protease cleavage site.
  • the two polypeptide chains will be transcribed and translated into proteins together, and then cut by the protease corresponding to furin and p2A into two independent proteins.
  • STAR can have multiple combinations: the antibody heavy chain variable region (VH) is fused to the TCR ⁇ constant region (C ⁇ ), the antibody light chain variable region (VL) is fused to the TCR ⁇ constant region (C ⁇ ), or the antibody light chain variable region (VL) is fused to TCR ⁇ constant region (C ⁇ ), antibody heavy chain variable region (VH) is fused to TCR ⁇ constant region (C ⁇ ); and the two exchange the relative order of furin and P2A.
  • FIG. 3 The upper membrane of EGFR-targeted STAR in human T cells.
  • the STAR gene was introduced into the human T cell line Jurkat Clone 5 (an endogenous TCR-deleted Jurkat subclone) using a lentiviral vector. Cells taken 3 days after infection were stained with anti-human TCR- ⁇ / ⁇ flow cytometry antibodies, and then subjected to flow cytometry. It can be found that STAR can be stained with antibodies against TCR- ⁇ / ⁇ , and the level of staining is comparable to that of natural E1-TCR compared to non-transgenic negative control cells. This result indicates that the STAR molecule can be coated, and its ⁇ chain and ⁇ chain can be paired.
  • FIG. 4 Ability of EGFR-targeted STARs to bind antigens on the surface of human T cell membranes.
  • the Jurkat Clone 5 cells 3 days after the introduction of the STAR gene were stained with the flow cytometry antibodies of the antigen proteins EGFR-His and anti-His-APC, and then subjected to flow cytometry. It was found that STAR showed stronger staining compared to the negative control cells of natural E1-TCR (specificity not specific to EGFR), and the staining level was comparable to the CAR of anti-EGFR.
  • FIG. 5 The ability of STAR to target EGFR to mediate T cell activation.
  • Jurkat Clone 5 cells were cultured in EGFR antigen-coated cell culture plates and co-incubated with tumor cells A549 (EGFR-positive human lung cancer cell line). After 24 hours, the cells were collected and stained with anti-human CD69-FITC flow antibody, and then subjected to flow detection.
  • the abscissa CD69 positive is a cell expressing CD69 molecule, a T cell activation marker. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • Figure 6 Function of EGFR-targeted STAR in human primary T cells.
  • Human peripheral blood cells were obtained, and CD4 + and CD8 + T cells were purified using a pan T cell isolation kit. T cells were then stimulated with anti-CD3 / CD28 antibodies for 72 hours, and then the STAR gene was transferred into T cells using a lentiviral vector. After virus infection, the cells were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU / mL IL-2. T cells were co-cultured with A431 cells, an EGFR-positive human skin cancer cell, to detect T cell activation and target cell death. After 8 hours of co-cultivation, T cells were taken for staining.
  • FIG. 7 The ability of STAR targeting CD19 to mediate T cell activation.
  • the STAR gene targeting CD19 was introduced into the human T cell line Jurkat Clone 5 using a lentiviral vector. T cells were taken 3 days after infection and co-incubated in tumor cells Raji, Mino, and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were collected and stained with anti-human CD69-FITC flow cytometry antibodies, followed by flow cytometry. CD69 positive cells are cells expressing T69 activation marker CD69 molecules. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • Figure 8 Function of STAR targeting CD19 in human primary T cells.
  • Human peripheral blood cells were obtained, and CD4 + and CD8 + T cells were purified using a pan T cell isolation kit. T cells were then stimulated with anti-CD3 / CD28 antibodies for 72 hours, and then the STAR gene targeting CD19 was transferred into T cells using a lentiviral vector. After virus infection, the cells were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU / mL IL-2. T cells were co-cultured with Raji and LY-1 cells to detect target cell death. After 8 hours of co-cultivation, T cells were taken for staining. It was found that both STAR and CAR can mediate T cell activation.
  • T-cell cytokine IFN- ⁇ From the level of T-cell cytokine IFN- ⁇ ( Figure 4), STAR can cause significant T-cell activation (its level is higher than the level of CAR). The expression level of IFN- ⁇ in T cells showed that the target cells had a significant activation effect on STAR-T cells.
  • FIG. 9 The ability of STAR to target CD20 to mediate T cell activation.
  • the STAR gene targeting CD20 was introduced into the human T cell line Jurkat Clone 5 using a lentiviral vector. T cells were taken 3 days after infection and co-incubated in tumor cells Raji, Mino, and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were collected and stained with anti-human CD69-FITC flow cytometry antibodies, followed by flow cytometry. CD69 positive cells are cells expressing T69 activation marker CD69 molecules. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • Figure 10 Function of STAR targeting CD20 in human primary T cells.
  • Human peripheral blood cells were obtained, and CD4 + and CD8 + T cells were purified using a pan T cell isolation kit. T cells were then stimulated with anti-CD3 / CD28 antibodies for 72 hours, and then the STAR gene targeting CD20 was transferred into T cells using a lentiviral vector. After virus infection, the cells were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU / mL IL-2. T cells were co-cultured with Raji and LY-1 cells to detect target cell death. After 8 hours of co-cultivation, T cells were taken for staining. It was found that both STAR and CAR can mediate T cell activation.
  • T-cell cytokine IFN- ⁇ From the level of T-cell cytokine IFN- ⁇ ( Figure 4), STAR can cause significant T-cell activation (its level is higher than the level of CAR). The expression level of IFN- ⁇ in T cells showed that the target cells had a significant activation effect on STAR-T cells.
  • the constant region (C region) of the ⁇ and ⁇ chains of TCR in STAR are derived from human peripheral blood T cell cDNA cloned by PCR molecules; based on the original TCR sequence, the ⁇ and ⁇ chains of the The 48th and 57th amino acid positions of the constant region were mutated to cysteine to help form an additional disulfide bond between the ⁇ and ⁇ chains, increase their mutual pairing efficiency, and named it E1-TCR.
  • the antibody heavy chain variable region (VH) and antibody light chain variable region (VL) were selected as Cetuximab (Cetux), which is used as an example to explain the content of the present invention. Other known antibodies can be substituted.
  • STAR contains two polypeptide chains. Cetux-VL and TCR ⁇ chain are fused into the first polypeptide chain, and Cetux-VH and ⁇ chain are fused into the second polypeptide chain.
  • the gene sequence of STAR is connected by the polypeptide fragment of the furin and p2A protease cleavage site.
  • the two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cut by proteases corresponding to furin and p2A into two independent protein subunits. These two subunits are covalently bound through disulfide bonds and form a complex with CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) endogenous to T cells (as shown in Figures 1 and 2).
  • the entire gene was inserted into the lentiviral expression vector pHAGE through restriction sites NheI and NotI.
  • This vector carries ampicillin resistance, the EF1 ⁇ promoter, and the IRES-RFP fluorescent reporter gene.
  • the nucleotide sequence of the E1TCR ⁇ chain constant region cysteine mutant is SEQ ID NO: 1;
  • the nucleotide sequence of the E1TCR ⁇ constant region cysteine mutant is SEQ ID NO: 2;
  • the nucleotide sequence of the Cetux VH is SEQ ID NO: 3;
  • the nucleotide sequence of the Cetux VL is SEQ ID NO: 4;
  • the Gibson Assembly product was transformed into the DH5 ⁇ strain and allowed to grow overnight on LB plates containing ampicillin. Monoclonal bacterial strains were selected for sequencing. The primers used for sequencing were seq-pHAGE-F and seq-pHAGE-R.
  • the bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Plasmids were extracted using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop. The final plasmid concentration was around 1000ng / ul, and the A260 / A280 value was greater than 1.8.
  • the pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were transfected into 293T cells in proportion (using PEI transfection). Collect the cell culture supernatants for 48 hours and 72 hours, mix the supernatant with PEG8000, leave it to stand overnight, and then centrifuge to obtain a virus pellet. Resuspend in a small volume of medium to achieve the effect of virus concentration.
  • Jurkat clone 5 cells endogenous TCR deleted Jurkat subclones
  • the concentrated lentivirus and the transgene Polybrene were added to the T cell culture medium and centrifuged at 1500 rpm for 2 hours at 32 ° C. After 3 days of infection, the fluorescent reporter gene can be observed and the expression of the target protein can be detected.
  • T cells 3 days after infection were taken, stained with anti-human TCR ⁇ / ⁇ -BV421 flow antibody, and then subjected to flow detection. It can be found (Figure 3) that compared to non-transgenic negative control cells, STAR can be stained with antibodies against TCR ⁇ / ⁇ , and the level of staining is comparable to natural E1-TCR. This result indicates that the STAR molecule can be coated, and its ⁇ chain and ⁇ chain can be paired.
  • T cells were taken 3 days after infection, stained with the flow cytometry antibodies of the antigen proteins EGFR-His and anti-His-APC, and then subjected to flow cytometry. It can be found (Figure 4) that STAR shows stronger staining compared to the native E1-TCR (specificity not specific to EGFR) negative control cells, and the staining level is comparable to the anti-EGFR CAR. This result indicates that STAR possesses antigen recognition and binding capabilities comparable to CAR molecules.
  • T cells were taken 3 days after infection, cultured in EGFR antigen-coated cell culture plates, and co-incubated with tumor cells A549 (EGFR positive human lung cancer cell line). After 24 hours, cells were collected and stained with anti-human CD69-FITC flow antibody, and then subjected to flow cytometry (Figure 5).
  • the abscissa CD69 positive is a cell expressing CD69 molecule, a T cell activation marker. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • T cells Human peripheral blood cells were obtained, and CD4 and CD8 T cells were purified from the whole T cell magnetic bead isolation kit. T cells were then stimulated and activated in a petri dish coated with anti-CD3 / CD28 antibodies for 48-72 hours. T cells became larger in volume, aggregated, and polarized in shape. At this time, the target gene was transferred into T cells using a lentiviral disease vector, and the infection method was centrifugation at 1500 rpm at 32 ° C for 2 hours. After virus infection, the cells were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU of IL-2.
  • T cells were co-cultured with A431 cells (an EGFR-positive human skin cancer cell) at a ratio of 1: 1 to 5: 1 to detect T cell activation and target cell death. After 8 hours of co-cultivation, T cells were taken for staining. It was found that both STAR and CAR can mediate T cell activation. Looking at the levels of the T cell marker CD69 ( Figure 6a) and the T cell cytokine IFN- ⁇ ( Figure 6b), STAR can cause significant T cell activation. After co-culture for 24 hours, the cell supernatant was taken to detect the level of lactate dehydrogenase (LDH) ( Figure 6c), which can reflect the death of target cells. The results show that STAR-T cells have a significant killing effect on target cells.
  • A431 cells an EGFR-positive human skin cancer cell
  • CDNA derived from human peripheral blood T cells or mouse spleen T cells was cloned by PCR; based on the original TCR sequence, the 48th and 57th amino acid positions of the constant region of the ⁇ chain and ⁇ chain were mutated, respectively. It is cysteine to help form an additional disulfide bond between the ⁇ and ⁇ chains and increase the efficiency of pairing with each other. It is named E1-TCR (human) or E11-TCR (rat).
  • the scFv fragment of the CD19-specific mouse monoclonal antibody (clone number FMC63) was selected as the antibody heavy chain variable region (VH) and antibody light chain variable region (VL).
  • VH antibody heavy chain variable region
  • VL antibody light chain variable region
  • STAR contains two polypeptide chains. FMC63-VL and TCR ⁇ chain are fused into the first polypeptide chain, and FMC63-VH and ⁇ chain are fused into the second polypeptide chain.
  • the gene sequence of STAR is connected by the polypeptide fragment of the furin and p2A protease cleavage site.
  • the two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cut by the protease corresponding to furin and p2A into two independent protein subunits.
  • the two subunits are covalently bound through disulfide bonds and form a complex with the endogenous CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) of T cells.
  • the entire gene was inserted into the lentiviral expression vector pHAGE through restriction sites NheI and NotI.
  • This vector carries ampicillin resistance, the EF1 ⁇ promoter, and the IRES-RFP fluorescent reporter gene.
  • the nucleotide sequence of the E1TCR ⁇ chain constant region cysteine mutant is SEQ ID NO: 1;
  • the nucleotide sequence of the E1TCR ⁇ constant region cysteine mutant is SEQ ID NO: 2;
  • the nucleotide sequence of the FMC63-VH is SEQ ID NO: 5;
  • the nucleotide sequence of the FMC63-VL is SEQ ID NO: 6;
  • the Gibson Assembly product was transformed into the DH5 ⁇ strain and allowed to grow overnight on LB plates containing ampicillin. Monoclonal bacterial strains were selected for sequencing. The primers used for sequencing were seq-pHAGE-F and seq-pHAGE-R.
  • the bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Plasmids were extracted using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop. The final plasmid concentration was around 1000ng / ul, and the A260 / A280 value was greater than 1.8.
  • the pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were transfected into 293T cells in proportion (using PEI transfection). Collect the cell culture supernatants for 48 hours and 72 hours, mix the supernatant with PEG8000, leave it to stand overnight, and then centrifuge to obtain a virus pellet. Resuspend in a small volume of medium to achieve the effect of virus concentration.
  • Jurkat clone 5 cells endogenous TCR deleted Jurkat subclones
  • the concentrated lentivirus and the transgene Polybrene were added to the T cell culture medium and centrifuged at 1500 rpm for 2 hours at 32 ° C. After 3 days of infection, the fluorescent reporter gene can be observed and the expression of the target protein can be detected.
  • T cells were taken 3 days after infection and co-incubated in tumor cells Raji, Mino, and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, the cells were collected and stained with anti-human CD69-FITC flow antibody, followed by flow detection ( Figure 7).
  • CD69 positive cells are cells expressing T69 activation marker CD69 molecules. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • T cells Human peripheral blood cells were obtained, and CD3 + T cells were purified from the whole T cell magnetic bead isolation kit. T cells were then stimulated and activated in a petri dish coated with anti-CD3 / CD28 antibodies for 48-72 hours. T cells became larger in volume, aggregated, and polarized in shape. At this time, the target gene was transferred into T cells using a lentiviral disease vector, and the infection method was centrifugation at 1500 rpm at 32 ° C for 2 hours. After virus infection, they were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU / mL IL-2.
  • T cells were co-cultured with Raji and LY-1 cells at a ratio of 1: 1 to 5: 1 to detect T cell activation and target cell death. After 8 hours of co-cultivation, T cells were taken for staining. It was found that both STAR and CAR can mediate T cell activation. From the level of T cell cytokine IFN- ⁇ ( Figure 8), STAR can cause significant T cell activation. The expression level of IFN- ⁇ in T cells showed that the target cells had a significant activation effect on STAR-T cells.
  • the scFv fragment of the antibody heavy chain variable region (VH) and antibody light chain variable region (VL) was selected from the CD20-specific antibody Ofatumumab (Ofalimumab, OFA), which is used as an example to explain the content of the present invention. Antibodies can be replaced.
  • STAR contains two polypeptide chains.
  • OFA-VL and TCR ⁇ chain are fused into a first polypeptide segment, and OFA-VH and ⁇ chain are fused into a second polypeptide segment.
  • the gene sequence of STAR is connected by the polypeptide fragment of the furin and p2A protease cleavage site.
  • the two polypeptide chains will be transcribed and translated into a fusion polypeptide, and then cut by the protease corresponding to furin and p2A into two independent protein subunits.
  • the two subunits are covalently bound through disulfide bonds and form a complex with the endogenous CD3 subunits ( ⁇ , ⁇ , ⁇ , ⁇ ) of T cells.
  • the entire gene was inserted into the lentiviral expression vector pHAGE through restriction sites NheI and NotI.
  • This vector carries ampicillin resistance, the EF1 ⁇ promoter, and the IRES-RFP fluorescent reporter gene.
  • the nucleotide sequence of the E11 TCR ⁇ chain constant region cysteine mutant is SEQ ID NO: 7;
  • the nucleotide sequence of the E11 TCR ⁇ chain constant region cysteine mutant is SEQ ID NO: 8;
  • the nucleotide sequence of the OFA-VH is SEQ ID NO: 9;
  • the nucleotide sequence of the OFA-VL is SEQ ID NO: 10;
  • the Gibson Assembly product was transformed into the DH5 ⁇ strain and allowed to grow overnight on LB plates containing ampicillin. Monoclonal bacterial strains were selected for sequencing. The primers used for sequencing were seq-pHAGE-F and seq-pHAGE-R.
  • the bacteria with correct sequencing results were inoculated in LB liquid medium and cultured overnight. Plasmids were extracted using an endotoxin-removing kit. The plasmid concentration was measured by Nanodrop. The final plasmid concentration was around 1000ng / ul, and the A260 / A280 value was greater than 1.8.
  • the pHAGE vector carrying the target gene and the packaging plasmids pMD2.G and psPAX2 were transfected into 293T cells in proportion (using PEI transfection). Collect the cell culture supernatants for 48 hours and 72 hours, mix the supernatant with PEG8000, leave it to stand overnight, and then centrifuge to obtain a virus pellet. Resuspend in a small volume of medium to achieve the effect of virus concentration.
  • Jurkat clone 5 cells endogenous TCR deleted Jurkat subclones
  • the concentrated lentivirus and the transgene Polybrene were added to the T cell culture medium and centrifuged at 1500 rpm for 2 hours at 32 ° C. After 3 days of infection, the fluorescent reporter gene can be observed and the expression of the target protein can be detected.
  • T cells were taken 3 days after infection and co-incubated in tumor cells Raji, Mino, and LY-1 (CD19 and CD20 positive human lymphoma cell lines). After 24 hours, cells were collected and stained with anti-human CD69-FITC flow antibody, followed by flow cytometry ( Figure 9).
  • CD69 positive cells are cells expressing T69 activation marker CD69 molecules. It can be found that STAR can cause T cells to express CD69 activation markers under antigen stimulation, that is, STAR can mediate T cell activation after antigen stimulation, and the degree of activation is comparable to CAR. At the same time, it can be found that in the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a higher level of self-activation.
  • T cells Human peripheral blood cells were obtained, and CD3 + T cells were purified from the whole T cell magnetic bead isolation kit. T cells were then stimulated and activated in a petri dish coated with anti-CD3 / CD28 antibodies for 48-72 hours. T cells became larger in volume, aggregated, and polarized in shape. At this time, the target gene was transferred into T cells using a lentiviral disease vector, and the infection method was centrifugation at 1500 rpm at 32 ° C for 2 hours. After virus infection, they were cultured to a sufficient amount in RPMI 1640 medium containing 20% serum and 200 IU / mL IL-2.
  • T cells were co-cultured with Raji and LY-1 cells at a ratio of 1: 1 to 5: 1 to detect T cell activation and target cell death. After 8 hours of co-cultivation, T cells were taken for staining. It was found that both STAR and CAR can mediate T cell activation. From the level of T cell cytokine IFN- ⁇ ( Figure 10), STAR can cause significant T cell activation. The expression level of IFN- ⁇ in T cells showed that the target cells had a significant activation effect on STAR-T cells.
  • the present invention has successfully constructed multiple target STARs, and successfully verified that the antibody-T cell chimeric receptor involved in this application can interact with the CD3 subunits ( ⁇ , ⁇ , ⁇ ) endogenously expressed by T cells. , ⁇ ) to form a complex to function, which can mediate the activation of T cells after antigen stimulation, and the degree of antigen activation is comparable to the corresponding antibody-chimeric antigen receptor (CAR) prepared, and more importantly, In the resting state without antigen stimulation, STAR has no self-activation phenomenon, while CAR has a high self-activation phenomenon. Due to space limitations, the present invention lists exemplary STARs, which are sufficient to support the successful construction and outstanding technical effects of the STARs of the present invention.

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Abstract

涉及一种嵌合T细胞受体(Synthetic T Cell Receptor and Antigen Receptor，STAR)，以及相关制剂、药物、或其在制备细胞药物中的用途，还涉及所述制剂或药物组合物用于治疗相应的疾病，例如肿瘤或感染性疾病等。

Description

一种嵌合T细胞受体STAR及其应用 技术领域
本发明涉及生物医药领域，具体地涉及一种抗体-T细胞受体嵌合受体，构建方法及其用途，包括治疗及诊断疾病。
背景技术
嵌合抗原受体T细胞疗法(CAR-T)和T细胞受体疗法(TCR-T)都是利用患者自身的T淋巴细胞治疗癌症的两类前沿基因疗法。它们能够表达特异性受体，靶向识别特异性的细胞如肿瘤细胞，受到广泛的关注和研究，从最开始的基础免疫研究转变为临床应用。基于合成生物学、免疫学和遗传改造技术的最新进展，使得合成改造的特异性功能加强的T细胞成为可能。
CAR-T是利用能够与特定抗原结合的抗体片段来识别肿瘤细胞表面的抗原。近年来，CD19抗原特异性CAR-T细胞用于治疗B细胞白血病和淋巴瘤临床试验中，显示出持续的疾病缓解效果。嵌合抗原受体(CAR)赋予T细胞HLA非依赖的方式识别肿瘤抗原的能力，这使得经过CAR改造的T细胞相对于天然T细胞表面受体TCR能够识别更广泛的目标。目前，CAR-T技术在急性白血病和非霍奇金淋巴瘤的治疗上有着显著的疗效，被认为是最有前景的肿瘤治疗方式之一。
与CAR-T不同，TCR(T cell receptor)是T细胞表面特异性识别抗原和介导免疫应答的分子。TCR主要识别由组织相容复合物分子提呈的抗原分子多肽。近几年，由于CAR-T细胞疗法在临床上取得巨大的成功，TCR疗法的研究相对不那么引人关注。然而，在治疗实体瘤方面，TCR-T与CAR-T相比有着明显的优势，这是因为CAR-T细胞治疗的靶点抗原为细胞表面蛋白，而TCR-T识别MHC分子，MHC分子能够呈递从细胞表面和细胞内蛋白中获得的肽链，因此TCR-T能够靶向更多种抗原。
众所周知，大多数肿瘤相关抗原(TAA)是自身抗原，由于胸腺的选择机制和耐受机制，机体针对这些抗原产生的T淋巴细胞的大多数T细胞受体(TCR)的亲和力都比较低，从而限制了其肿瘤识别及杀伤效果。将克隆的高亲和力识别TAA的TCR(或嵌合受体)通过转基因技术转给T淋巴细胞，可以使重定向的原来无肿瘤识别能力的T细胞在体外和体内有效地识别并杀伤肿瘤细胞。TCR为所有T细胞表面的特征性标志，以非共价键与CD3(ε,δ,γ,ζ)结合，形成TCR-CD3复合体。
TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)和恒定区(C区)两部分，其中恒定区又包括胞外区、跨膜区和胞内末端三部分；其特点是胞内区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。外周血中，90％-95％的T细胞表达TCR2；而且任一T细胞只表达TCR2或TCR1之一。
天然存在的TCR是一种膜蛋白，通过其跨膜区得以稳定，因此对于在细菌中表达可溶性TCR而言，获得保持与其原配体(即pMHC)特异性结合能力的高稳定性TCR是一件非常困难的事情，如专利文献WO99/18129中所述。有些文献描述了截短形式的TCR，它仅仅包含胞外区或者仅仅包含胞外和胞质区，尽管这样的TCR可以被TCR特异性的抗体识别，但是产率很低，并且低浓度时不能识别主组织相容性复合体-肽复合体，说明其很容易变性，不够稳定。
TCR-T技术优势在于：传统的免疫过继治疗，只是增加了效应细胞的数量，对于效应细胞的特异性并没有提高，而且效应细胞即使能和肿瘤细胞结合，其亲和力也比较低。TCR-T技术，直接改造T细胞的结合肿瘤抗原的“探头”-TCR，加强了T细胞针对肿瘤细胞的特 异性识别过程，而且提高了T淋巴细胞对于肿瘤细胞的亲和力，使得原来无肿瘤识别能力的T细胞在体外和体内有效地识别并杀伤肿瘤细胞。总之，TCR-T细胞疗法，一边增加了T淋巴细胞的数量，一边提高了T淋巴细胞对于肿瘤细胞的杀伤性，从而达到了很好的肿瘤治疗效果。
现有TCR-T疗法中，一般需要分离出内源的TCR，并加以工程化改造，将其引入全新的T细胞，并输注回人体，带有靶向癌细胞能力的T细胞数量将大增，有望对多种实体肿瘤与血液肿瘤进行识别与攻击。然而外源性的TCRα和β链与内源性的α和β链之间会相互识别配对形成杂合TCR。这种TCR错配现象被认为广泛存在于TCR-T疗法中，可能引发细胞表面的TCR表达量下降、细胞活性降低。错配TCR还可能形成一种能产生未知特异性的新的TCR，这种新的TCR会在TCR-T中带来自身免疫性(on-target)或交叉反应(off-target)毒性。虽然目前TCR错配引起的自身反应疾病还没有临床证据，但是由杂合TCR所带来的自身免疫风险却不容忽视。因此，TCR在T细胞的表达和配对组装以及与pMHC的亲和力都是影响TCR基因治疗充分发挥抗肿瘤能力的关键因素。
可见，如何改造修饰TCR基因使得TCRα链和β链能在T细胞表面正确配对，并且其表达效率和亲和力都增强，同时避免产生副作用、提高安全性成为近年来TCR基因治疗的一个热点之一。主要的策略包括提高TCR在T细胞中的亲和力、优化TCR链的配对以及增强其表面表达效率。比如有研究者采用以下方法来减少TCR的错配：在TCR链中引入二硫键；采用鼠源性保守C区替换人TCR分子的C区；将TCRα链和β链链接口处保守结构域的氨基酸残基翻转；构建单链TCR(scTCR)嵌合体；将TCR链的C区与CD3分子融合；将aβTCR基因转入γδT细胞中等，但上述效果均不尽如人意。可见，目前虽然提出了对TCR-T进行修饰以优化外源TCR分子的配对策略，但仍然有很多需要改进的地方。
发明内容
本发明解决现有技术中存在的上述技术问题，提供一种特异性结合至靶抗原的嵌合T细胞受体(Synthetic T Cell Receptor and Antigen Receptor，STAR)，所述嵌合T细胞受体包含：
a)抗体重链可变区与T细胞受体(TCR)第一亚基恒定区融合得到的第一肽链；和，
b)抗体轻链可变区与T细胞受体第二亚基恒定区融合得到的第二肽链；
其中，所述抗体重链可变区与抗体轻链可变区特异性结合至所述靶抗原的抗原表位。
在一些实施方案中，根据前述的嵌合T细胞受体，(1)当所述T细胞受体第一亚基为α链时，所述T细胞受体第二亚基为β链；或，(2)当所述T细胞受体第一亚基为β链时，所述T细胞受体第二亚基为α链；或，(3)当所述T细胞受体第一亚基为γ链时，所述T细胞受体第二亚基为δ链；或，(4)当所述T细胞受体第一亚基为δ链时，所述T细胞受体第二亚基为γ链。
具体的，所述第一肽链和第二肽链在T细胞中表达后通过二硫键结合。
在一些实施方案中，嵌合T细胞受体第一亚基恒定区和T细胞受体第二亚基恒定区，其种属来源为人源或鼠源，包括不同的蛋白亚型。
在一些实施方案中，对所述的嵌合T细胞受体可进行任何氨基酸序列改造，包括但不限于氨基酸点突变修饰、多肽片段替换修饰，以降低与内源表达的T细胞受体错配。例如：TCRα链，其恒定区第48位氨基酸突变为半胱氨酸，且，其恒定区第57位氨基酸突变为半胱氨酸，使得所述第一多肽和第二多肽之间通过二硫键连接；再例如：TCRα链，其恒定区第85位氨基酸突变为丙氨酸，且，其恒定区第88位氨基酸突变为甘氨酸；
具体的，(1)所述T细胞受体第一亚基是TCRα链，其恒定区第48位氨基酸突变为半胱氨酸，和/或，所述T细胞受体第二亚基是TCRβ链，其恒定区第57位氨基酸突变为半胱氨酸；或(2)所述T细胞受体第一亚基是TCRα链，其恒定区第85位氨基酸突变为丙 氨酸，和/或，所述T细胞受体第二亚基是TCRβ链，恒定区第88位氨基酸突变为甘氨酸。
在一些实施方案中，所述的靶抗原为肿瘤特异性抗原或病毒特异性抗原。具体的所述靶抗原选自CD19，CD20，EGFR，Her2，PSCA，CD123，CEA(癌胚抗原)，FAP，CD133，EGFRVIII，BCMA，PSMA，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171。优选的，所述抗原可以为CD19、CD20，EGFR，Her2。
具体的，在一些实施方案中，所述的抗体、抗体重链可变区或抗体轻链可变区来自IMCC225(西妥昔单抗、Cetuximab/Cetux)、利妥昔单抗(美罗华)、Ofatumumab(OFA，奥法木单抗)、CD19单克隆抗体(FMC63)、Avastin(贝伐单抗)、BEC2(阿妥莫单抗)、Bexxar(托西莫单抗)、Campath(阿仑单抗)、Herceptin(曲妥单抗)、LymphoCide(依帕珠单抗)、MDX-210、Mylotarg(吉姆单抗奥佐米星)、单抗17-1A(依决洛单抗)、Theragyn(pemtumomab)、Zamyl、Zevalin(替伊莫单抗)或筛选获得的高亲和力抗体。优选的，其所述抗体包括选自Fab、F(ab’)2、Fab’、scFv、Fv、VH、VL的抗原结合片段。
在一些实施方案中，所述靶抗原相关疾病为癌症或病毒感染相关疾病。具体的，所述癌症选自下组：肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、白血病、淋巴瘤、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌；或，所述病毒感染是由选自以下病毒引起：巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-BarrVirus；EBV)、B型肝炎病毒(HBV)、卡波西氏肉瘤相关疱疹病毒(KSHV)、人类乳头状瘤病毒(HPV)、传染性软疣病毒(MCV)、人类T细胞白血病病毒1(HTLV-1)、HIV(人类免疫缺陷病毒)及C型肝炎病毒(HCV)。
本发明的嵌合T细胞受体，所述第一多肽和第二多肽与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体。
在一些实施方案中，所述抗体重链可变区和轻链可变区来源于IMCC225(西妥昔单抗、Cetuximab/Cetux)、利妥昔单抗(美罗华)、Ofatumumab(奥法木单抗)、CD19单克隆抗体FMC63的VH和VL。
优选的，所述Cetux VH的核苷酸序列为SEQ ID NO:3，氨基酸序列为SEQ ID NO:13所述Cetux VL的核苷酸序列为SEQ ID NO:4，氨基酸序列为SEQ ID NO:14。
优选的，所述FMC63-VH的核苷酸序列为SEQ ID NO:5，氨基酸序列为SEQ ID NO:15，所述FMC63-VL的核苷酸序列为SEQ ID NO:6，氨基酸序列为SEQ ID NO:16；。
优选的，所述OFA-VH的核苷酸序列为SEQ ID NO:9，氨基酸序列为SEQ ID NO:19，所述OFA-VL的核苷酸序列为SEQ ID NO:10，氨基酸序列为SEQ ID NO:20。
在一些实施方案中，所述来源于西妥昔单抗、曲妥昔单抗、利妥昔单抗的VH和VL分别与所述TCRα链或β链融合获得VH-TCRα链融合体或VL-TCRβ链融合体。
进一步优选的，所述来源于IMCC225(西妥昔单抗、Cetuximab/Cetux)、利妥昔单抗(美罗华)、Ofatumumab(奥法木单抗)、CD19单克隆抗体FMC63的VH和VL分别与所述TCRα链恒定区或β链恒定区融合获得VH-TCRα链恒定区融合体或VL-TCRβ链恒定区融合体。
优选的，所述两个不同的融合体通过furin-p2A段肽序列相连；优选的，所述两个不同的融合体在T细胞中表达后通过二硫键共价结合。更进一步的，所述两个不同的融合体与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体。
另外，本发明还提供了一种特异性结合至靶抗原的由嵌合T细胞受体形成的复合体，由前述任一的嵌合T细胞受体与T细胞内源表达的CD3亚基(ε,δ,γ,ζ)形成复合体，且被靶抗 原激活后可介导T细胞相关信号转导通路。
在一些实施方案中，本发明还提供了一种核酸，其编码前述任一项的嵌合T细胞受体或所述第一多肽及所述第二多肽。
具体的，所述核酸的结构如下：
(1)依次为抗体重链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体轻链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端；或，
(2)依次为抗体重链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体轻链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端；或，
(3)依次为抗体轻链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体重链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端；或，
(4)依次为抗体轻链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体重链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端；或，
(5)将(1)-(4)中的T细胞受体(TCR)α链替换为T细胞受体(TCR)γ链，T细胞受体(TCR)β链替换为T细胞受体(TCR)δ链对应的核酸。
在一些具体实施方案中，所述T细胞受体(TCR)α链或β链恒定区来源于人TCRα链或β链恒定区，还可以来源于鼠TCRα链或β链恒定区。
在一些具体实施方案中，TCR的α链和/或β链可进行氨基酸点突变修饰、多肽片段替换修饰，以降低与内源表达的T细胞受体错配。优选的，TCR的α链和/或β链进行半胱氨酸点突变。具体的，TCRα链恒定区第48位氨基酸突变为半胱氨酸，TCRβ链恒定区第57位氨基酸突变为半胱氨酸，使得所述第一肽链和第二肽链之间通过增加一个二硫键的方式连接。
更优选的，人源TCRα链恒定区半胱氨酸突变体核苷酸序列为SEQ ID NO:1，氨基酸序列为SEQ ID NO:11；人源TCRβ链恒定区半胱氨酸突变体核苷酸序列为SEQ ID NO:2，氨基酸序列为SEQ ID NO:12。
在另一个实施方案中，人源TCRγ链恒定区的氨基酸序列为SEQ ID NO:21，人源TCRδ链恒定区的氨基酸序列为SEQ ID NO:22，鼠源TCRγ链恒定区的氨基酸序列为SEQ ID NO:23，鼠源TCRδ链恒定区的氨基酸序列为SEQ ID NO:24。
进一步的，天然人源TCRα链恒定区首位氨基酸存在四种形式；首位氨基酸为Asp，即SEQ ID NO:11所示的多肽，以及首位氨基酸为Asn、His或Tyr的多肽，因此人源TCRα链恒定区半胱氨酸突变体还可以选自以下序列表示的多肽：
①氨基酸序列为SEQ ID NO:31，对应的核苷酸序列为SEQ ID NO:25；
②氨基酸序列为SEQ ID NO:32，对应的核苷酸序列为SEQ ID NO:26；
③氨基酸序列为SEQ ID NO:33，对应的核苷酸序列为SEQ ID NO:27。
更优选的，鼠源TCRα链恒定区半胱氨酸突变体核苷酸序列为SEQ ID NO:7，氨基酸序列为SEQ ID NO:17；鼠源TCRβ恒定区半胱氨酸突变体核苷酸序列为SEQ ID NO:8，氨基酸序列为SEQ ID NO:18。
进一步的，天然鼠源TCRα链恒定区首位氨基酸存在四种形式；首位氨基酸为Asn，即SEQ ID NO:17所示的多肽，以及首位氨基酸为Asp、His或Tyr的多肽，因此鼠源TCRα链恒定区半胱氨酸突变体还可以选自以下序列表示的多肽：
①氨基酸序列为SEQ ID NO:34，对应的核苷酸序列为SEQ ID NO:28；
②氨基酸序列为SEQ ID NO:35，对应的核苷酸序列为SEQ ID NO:29；
③氨基酸序列为SEQ ID NO:36，对应的核苷酸序列为SEQ ID NO:30。
在一些实施方式中，T细胞受体(TCR)α链可替换为T细胞受体(TCR)γ链；T细胞受体(TCR)β链可替换为T细胞受体(TCR)δ链。优选的，T细胞受体(TCR)γ链和T细胞受体(TCR)δ链可来源于人TCRγ链或δ链恒定区，还可以来源于鼠TCRγ链或δ链恒定区。
在一些实施方案中，本发明还提供了一种载体，其包含编码前述任一项的嵌合T细胞受体或所述第一多肽及所述第二多肽的核酸。优选的，所述载体是质粒。更优选的，所述载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。
在一些实施方案中，根据以上所述嵌合T细胞受体中的任一者，提供一种效应细胞，其表达前述任一的嵌合T细胞受体或前述复合体于其细胞表面上。在一些实施方案中，效应细胞包含编码所述嵌合T细胞受体的核酸。在一些实施方案中，T细胞选自下组：细胞毒性T细胞、辅助T细胞、自然杀伤T细胞及抑制T细胞。
在一些实施方案中，提供一种药物组合物，其包含根据以上所述嵌合T细胞受体中的任一者的嵌合T细胞受体及药物上可接受的载剂。在一些实施方案中，提供一种药物组合物，其包含编码根据以上所述实施方案中的任一者的所述嵌合T细胞受体的核酸及药物上可接受的载剂。在一些实施方案中，提供一种药物组合物，其包含表达根据以上所述嵌合T细胞受体的任一者的效应细胞及药物上可接受的载剂。
本发明的嵌合T细胞受体和本发明嵌合T细胞受体转染的细胞可与药学上可接受的载体一起在药物组合物中提供。本发明的嵌合T细胞受体、嵌合T细胞受体的复合体和细胞通常作为无菌药物组合物的一部分提供，所述组合物通常包括药学上可接受的载体。该药物组合物可以是任何合适的形式(取决于给予患者的所需方法)。其可采用单位剂型提供，通常在密封的容器中提供，可作为试剂盒的一部分提供。此类试剂盒(但非必需)包括使用说明书。其可包括多个所述单位剂型。
本发明的嵌合T细胞受体可以单独使用，也可与偶联物结合或偶联。所述偶联物包括可检测标记物、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合。
用于诊断目的的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MIR(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
药物组合物还可含有药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。这类载体包括但并不限于：盐水、缓冲液、葡萄糖、水、甘油、乙醇、佐剂及其组合。治疗性组合物中药学上可接受的载体可含有液体，如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。通常，可将治疗性组合物制成可注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
一旦配成本发明的组合物，可将其通过常规途径进行给药，其中包括(但并不限于)：眼内、肌内、静脉内、皮下、皮内或局部给药。待预防或治疗的对象可以是动物，尤其是人。
当本发明的药物组合物被用于实际治疗时，可根据使用情况而采用各种不同剂型的药物组合物。较佳地，可以例举的有针剂、口服剂等。这些药物组合物可根据常规方法通过混合、稀释或溶解而进行配制，并且偶尔添加合适的药物添加剂，如赋形剂、崩解剂、粘合剂、润滑剂、稀释剂、缓冲剂、等渗剂、防腐剂、润湿剂、乳化剂、分散剂、稳定剂和助溶剂，而且该配制过程可根据剂型用惯常方式进行。
本发明的药物组合物还可以缓释剂形式给药。例如，本发明多肽可被掺入以缓释聚合物 为载体的药丸或微囊中，然后将该药丸或微囊通过手术植入待治疗的组织。
当本发明的药物组合物被用于实际治疗时，作为活性成分的本发明多肤或其药学上可接受的盐的剂量，可根据待治疗的每个病人的体重、年龄、性别、症状程度而合理地加以确定。
本发明的嵌合T细胞受体可用作药物或诊断试剂。可通过修饰或其他改进以使其获得更适于作为药物或诊断试剂使用的特征。
在一些实施方案中，提供一种核酸文库，其包含编码多个根据以上所述嵌合T细胞受体任一者的嵌合T细胞受体的序列。
在一些实施方案中，提供一种筛选根据以上所述实施方案中的任一者的核酸文库中编码对靶抗原具有特异性的嵌合T细胞受体的序列的方法，其包含：a)将核酸文库引入至多个细胞中，使得嵌合T细胞受体表达于多个细胞的表面上；b)将多个细胞与包含靶抗原或其中所含的一或多个表位的配体一起温育；c)收集结合至配体的细胞；及d)自步骤c)中所收集的细胞中分离编码嵌合T细胞受体的序列，从而鉴别对靶抗原具有特异性的嵌合T细胞受体。
还提供本文所述构建体中的任一者的制造方法、制品及适合于本文所述方法的试剂盒。
在一些实施方案中，还提供一种以上所述嵌合T细胞受体中的任一者的嵌合T细胞受体在制备治疗或诊断有需要个体的靶抗原相关疾病的试剂盒中的用途。
在一些实施方案中，提供一种杀伤递呈靶抗原的靶细胞的方法，其包含使靶细胞与表达根据以上所述嵌合T细胞受体中的任一者的嵌合T细胞受体的效应细胞接触，其中嵌合T细胞受体特异性结合至靶抗原。
在一些实施方案中，提供一种杀伤递呈靶抗原的靶细胞的方法，其中所述的嵌合T细胞受体特异性结合至靶抗原。其中，所述抗体重链可变区和轻链可变区特异性结合至所述靶抗原的抗原结合模块。
在一些实施方案中，根据以上所述的靶细胞杀伤方法中的任一者，该接触为体内的。在一些实施方案中，该接触为体外的。
在一些实施方案中，提供一种治疗有需要个体的靶抗原相关疾病的方法，其包含向个体施用有效量的药物组合物，该药物组合物包含表达根据以上所述嵌合T细胞受体或所述效应细胞。
在一些实施方案中，提供一种治疗有需要个体的靶抗原相关疾病的方法，其包含向个体施用有效量的组合物，该组合物包含效应T细胞，该效应T细胞包含特异性结合至靶抗原的嵌合T细胞受体，其包含：a)抗体重链可变区融合的第一多肽；b)抗体轻链可变区融合的第二多肽；其中，所述抗体重链可变区和轻链可变区特异性结合至所述靶抗原的抗原结合模块。优选的，所述嵌合T细胞受体为前述任一嵌合T细胞受体。
在一些实施方案中，提供一种在有需要的受试者中治疗由于细胞、组织、机体部分或器官移植发生的T细胞介导的病症的方法，其包括步骤：将细胞、组织、机体部分或器官移植入所述受试者；并施用两次或多次剂量的药物上可接受量的前述任意一个嵌合T细胞受体至所述受试者。
在一些实施方案中，根据以上的方法中的任一者，所述疾病为癌症。在一些实施方案中，癌症选自下组：肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、淋巴瘤、白血病、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌。在一些实施方案中，靶抗原相关疾病为病毒感染。在一些实施方案中，病毒感染是由选自以下的病毒引起：巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-Barr Virus；EBV)、B型肝炎病毒(HBV)、卡波西氏肉瘤相关疱疹病毒(Kaposi's Sarcoma associated herpes virus；KSHV)、人类乳头状瘤病毒(HPV)、传染性软疣病毒(MCV)、 人类T细胞白血病病毒1(HTLV-1)、HIV(人类免疫缺陷病毒)及C型肝炎病毒(HCV)。
更优选的，所述疾病为肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、白血病、淋巴瘤、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌等。
在一些实施方案中，提供一种治疗有需要个体的靶抗原相关疾病的方法，其包含向个体施用有效量的药物组合物，该药物组合物包含编码根据以上所述嵌合T细胞受体中的任一者的嵌合T细胞受体的核酸。
通过常规的重组DNA技术，可利用本发明的多核苷酸来表达或生产重组的本发明多肽。一般来说有以下步骤：(1)用编码本发明嵌合T细胞受体多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；(2)在合适的培养基中培养宿主细胞；(3)从培养基或细胞中分离、纯化出本发明的嵌合T细胞受体多肽。
在一些实施方案中，本发明提供前述任一的嵌合T细胞受体、所述复合体、核酸、载体或效应细胞在制备治疗或诊断有需要个体的靶抗原相关疾病的试剂盒、制剂或药物组合物中的用途。
在一些实施方案中，本发明提供一种治疗有需要个体的靶抗原相关疾病或癌症或病毒感染相关疾病的方法，其包含向个体施用有效量的药物组合物，该药物组合物包含前述任一的嵌合T细胞受体、所述复合体、核酸、载体或效应细胞。
具体的，所述癌症选自下组：肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、淋巴瘤、白血病、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌；或者，所述病毒感染是由选自以下的病毒引起：巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-Barr Virus；EBV)、B型肝炎病毒(HBV)、卡波西氏肉瘤相关疱疹病毒(KSHV)、人类乳头状瘤病毒(HPV)、传染性软疣病毒(MCV)、人类T细胞白血病病毒1(HTLV-1)、HIV(人类免疫缺陷病毒)及C型肝炎病毒(HCV)。
经典的CAR分子由单链抗体区、铰链区、跨膜区以及胞内信号区组成，可被呈递在T细胞表面。其中，单链抗体区包含抗体的重链可变区和轻链可变区(在某些情况下还会包括IgG CH1区域，起到结构上的作用)，两者通过柔性连接肽相连。胞内信号区由共刺激信号分子(4-1BB、CD28等)以及信号分子CD3ζ相互串联组成。
而本申请所构建的嵌合T细胞受体在细胞中表达后由两条多肽链经由二硫键共价键连接在一起，其中第一多肽是由抗体重链可变区(V H)与TCRα恒定区(Cα)融合而成，第二多肽是由抗体轻链可变区(V L)与TCRβ恒定区(C β)融合而成；嵌合T细胞受体可与T细胞内源表达的CD3亚基(ε、δ、λ、ζ)形成复合体发挥功能。嵌合T细胞受体的基因序列通过furin和p2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译表达成蛋白质，之后再被furin和p2A对应的蛋白酶切割成独立的两个蛋白质。嵌合T细胞受体可以有多种组合：抗体重链可变区(V H)与TCRα恒定区(Cα)融合、抗体轻链可变区(V L)与TCRβ恒定区(C β)融合，或者抗体轻链可变区(V L)与TCRα恒定区(Cα)融合、抗体重链可变区(V H)与TCRβ恒定区(C β)融合；以及两者交换相对furin和P2A的前后顺序。抗体的轻链和重链可变区可替换成多种特异性的抗体可变区，如抗EGFR、CD19、CD20等。TCRα和β恒定区也可存在多种变体，包括野生型TCRα和β恒定区、半胱氨酸单点突变型TCRα和β恒定区、人鼠嵌合型TCRα和β恒定区，以及含半胱氨酸单点突变的人鼠嵌合型TCRα和β恒定区。
本发明获得了高稳定性、高特异性的T细胞受体，可用于疾病的诊断和治疗。在TCR 设计过程中，发明人创造性地采用了多个策略。本申请通过在TCR分子的恒定区引入二硫键的改造。理论上在TCR中能够形成人工链间二硫键的位点非常多，但找到在TCR中形成人工链间二硫键的合适位点使得含有人工链间二硫键的TCR都能够被成功复性、重折叠来得到高产量、高稳定的，并且具有与其原配体特异性结合活性的TCR是非常困难的。本领域技术人员致力于开发含有人工链间二硫键的，能够被很好地复性、重折叠、纯化且具有高稳定性、复性收率高同时能够与其原配体特异性结合的TCR。本发明在特定位点进行二硫键的改造能够减少TCR-T细胞的新反应性，降低错配。
CAR-T疗法虽然捷报频频，但安全性问题是业界以及FDA重点关注的问题，避免自激活降低副反应是目前亟待解决的问题。本发明引入抗体抗原结合片段与原始TCR进行融合，形成抗体-T细胞受体结合的嵌合T细胞受体(STAR)，更进一步提高了α链和β链的配对，且提高了TCR的结合活性，更重要的是，对体内的原始TCR改造相对CAR-T要少，减少了外源氨基酸的引入，降低了副作用的发生风险，并提高了安全性。经多个靶点的嵌合T细胞受体的验证，结果表明嵌合T细胞受体可以介导T细胞在抗原刺激后的激活，且与制备获得的相应抗体-嵌合抗原受体(CAR)相比，抗原激活的程度相当，更重要的是在无抗原刺激的静息状态下，嵌合T细胞受体无自激活现象，而CAR有很高的自激活现象。
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。下面结合实施例进一步详述本发明。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1STAR与CAR分子的结构示意图。STAR在细胞中表达后由两条多肽链经由二硫键共价键连接在一起，其中第一多肽链是由抗体重链可变区(VH)与TCRα恒定区(Cα)融合而成，第二多肽链是由抗体轻链可变区(VL)与TCRβ恒定区(Cβ)融合而成；STAR可与T细胞内源表达的CD3亚基(ε、δ、λ、ζ)形成复合体发挥功能。
图2STAR与CAR分子基因结构序列示意图。STAR基因序列通过furin和P2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译表达成蛋白质，之后再被furin和p2A对应的蛋白酶切割成独立的两个蛋白质。STAR可以有多种组合：抗体重链可变区(VH)与TCRα恒定区(Cα)融合、抗体轻链可变区(VL)与TCRβ恒定区(Cβ)融合，或者抗体轻链可变区(VL)与TCRα恒定区(Cα)融合、抗体重链可变区(VH)与TCRβ恒定区(Cβ)融合；以及两者交换相对furin和P2A的前后顺序。
图3靶向EGFR的STAR在人类T细胞中的上膜情况。利用慢病毒载体将STAR基因导入人类T细胞系Jurkat Clone 5(内源性TCR缺失的Jurkat亚克隆)中。取感染3天后的细胞，用anti-human TCR-α/β的流式抗体染色，之后进行流式检测。可发现，与未转基因的阴性对照细胞相比，STAR可以被抗TCR-α/β的抗体染色，并且染色水平与天然的E1-TCR相当。这一结果表明STAR分子可以上膜，并且其α链和β链可以配对。
图4靶向EGFR的STAR在人类T细胞膜表面时结合抗原的能力。上述STAR基因导入3天后的Jurkat Clone 5细胞，用抗原蛋白EGFR-His以及anti-His-APC的流式抗体染色，之后进行流式检测。可发现，与天然的E1-TCR(特异性不针对于EGFR)的阴性对照细胞相比，STAR显示较强的染色，并且染色水平与anti-EGFR的CAR相当。
图5靶向EGFR的STAR介导T细胞激活的能力。上述STAR基因导入3天后的Jurkat Clone 5细胞，分别在EGFR抗原包被的细胞培养板中培养、以及与肿瘤细胞A549(EGFR阳性的人肺癌细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式 抗体染色，之后进行流式检测。横坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
图6靶向EGFR的STAR在人原代T细胞中的功能。取得人外周血细胞，使用pan T细胞分离试剂盒将其中CD4+和CD8+T细胞纯化出来。然后将T细胞用抗CD3/CD28的抗体刺激激活72小时后，用慢病毒载体将STAR基因转入T细胞。病毒感染后，在含20％血清和200IU/mL IL-2的RPMI 1640培养基中培养至足够数量。将T细胞与A431细胞(一种EGFR高阳性的人皮肤癌细胞)共培养，检测T细胞激活和靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞标志物CD69(图6a)和T细胞细胞因子IFN-γ(图6b)水平看，STAR可引起的显著的T细胞激活。共培养24小时后，取细胞上清检测乳酸盐脱氢酶(LDH)的水平(图6c)，可反映靶细胞死亡情况。结果表明，STAR-T与CAR-T细胞对靶细胞都有明显的杀伤作用。
图7靶向CD19的STAR介导T细胞激活的能力。利用慢病毒载体将靶向CD19的STAR基因导入人类T细胞系Jurkat Clone 5中。取感染3天后的T细胞，分别在肿瘤细胞Raji、Mino、LY-1(CD19和CD20阳性的人淋巴瘤细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式抗体染色，之后进行流式检测。纵坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
图8靶向CD19的STAR在人原代T细胞中的功能。取得人外周血细胞，使用pan T细胞分离试剂盒将其中CD4+和CD8+T细胞纯化出来。然后将T细胞用抗CD3/CD28的抗体刺激激活72小时后，用慢病毒载体将靶向CD19的STAR基因转入T细胞。病毒感染后，在含20％血清和200IU/mL IL-2的RPMI 1640培养基中培养至足够数量。将T细胞与Raji和LY-1细胞共培养，检测靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞细胞因子IFN-γ(图4)水平看，STAR可引起的显著的T细胞激活(其水平高于CAR的水平)。T细胞内IFN-γ表达水平结果表明，靶细胞对STAR-T细胞有明显的激活作用。
图9靶向CD20的STAR介导T细胞激活的能力。利用慢病毒载体将靶向CD20的STAR基因导入人类T细胞系Jurkat Clone 5中。取感染3天后的T细胞，分别在肿瘤细胞Raji、Mino、LY-1(CD19和CD20阳性的人淋巴瘤细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式抗体染色，之后进行流式检测。纵坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
图10靶向CD20的STAR在人原代T细胞中的功能。取得人外周血细胞，使用pan T细胞分离试剂盒将其中CD4+和CD8+T细胞纯化出来。然后将T细胞用抗CD3/CD28的抗体刺激激活72小时后，用慢病毒载体将靶向CD20的STAR基因转入T细胞。病毒感染后，在含20％血清和200IU/mL IL-2的RPMI 1640培养基中培养至足够数量。将T细胞与Raji和LY-1细胞共培养，检测靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞细胞因子IFN-γ(图4)水平看，STAR可引起的显著的T细胞激活(其水平高于CAR的水平)。T细胞内IFN-γ表达水平结果表明， 靶细胞对STAR-T细胞有明显的激活作用。
具体实施方式
除非另有说明，本发明的实施采用分子生物学、微生物学、细胞生物学、生物化学和免疫学的常规技术，其均在本领域技术人员知晓的范围内。
下面详细描述本发明的实施例，以便为本领域普通技术人员提供如何进行和利用本发明的试验、筛选和治疗方法的充分公开和说明，需要说明的是，这些实施例仅是说明性的，而不能理解为对本发明的限制。
实施例1、靶向EGFR的STAR的构建
具体构建方法如下：
1、TCR恒定区序列确定
STAR中的TCR的α链和β链的恒定区(C区)均来源于人外周血T细胞的cDNA经PCR分子克隆而得；在原始TCR序列的基础上，分别把α链和β链的恒定区的第48和57个氨基酸位点突变为半胱氨酸，以帮助α链和β链之间形成额外的一个二硫键，增加其相互配对效率，并命名为E1-TCR。
2、靶向EGFR的抗体序列确定
抗体重链可变区(VH)和抗体轻链可变区(VL)选择西妥昔单抗(Cetuximab，简称Cetux)，仅作为实例解释本发明的内容，其他已知抗体均可进行替换。
3、靶向EGFR的STAR构建
STAR含有两条多肽链，将Cetux-VL与TCRβ链融合为第一多肽链，将Cetux-VH与α链融合为第二多肽链。STAR的基因序列通过furin和p2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译成一条融合多肽，之后再被furin和p2A对应的蛋白酶切割成为两个独立的蛋白亚基，这两个亚基间通过二硫键共价结合，并与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体(如图1和图2所示)。
整个基因通过限制性内切酶切位点NheI和NotI插入进慢病毒表达载体pHAGE中。该载体携带氨苄抗性、EF1α启动子以及IRES-RFP荧光报告基因。
4、基因片段的克隆与组装
获得的四个片段“Cetux VL”，“TCRβ-C”，“Cetux-VH”，“TCRα-C”分别从pHAGE-Cetux-28zCAR载体以及pHAGE-E1-TCR载体上克隆获得。每对引物上都带有与前后同源的25bp碱基，四个片段通过Gibson Assembly的方法一步重组连接到慢病毒载体中。从而获得STAR。
所述E1TCRα链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:1；
所述E1TCRβ链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:2；
所述Cetux VH的核苷酸序列为SEQ ID NO:3；
所述Cetux VL的核苷酸序列为SEQ ID NO:4；
5.载体转化和测序
Gibson Assembly的产物被转化入了DH5α菌株，使其在含氨苄的LB平板上过夜生长。挑单克隆隆菌进行测序，测序引物选用pHAGE载体上的引物seq-pHAGE-F和seq-pHAGE-R。
6.质粒提取
将测序结果正确的菌接种在LB液体培养基中，过夜培养。使用具有去内毒素功能的试剂盒提取质粒。质粒浓度用Nanodrop测量，质粒终浓度在1000ng/ul左右，A260/A280值大于1.8。
实施例2、靶向EGFR的STAR功能验证
1.慢病毒包装
将携带目标基因的pHAGE载体与包装质粒pMD2.G与psPAX2按比例转染进293T细胞(使用PEI转染)。收集48小时和72小时的细胞培养基上清，并将上清与PEG8000混合，静置过夜后离心，可得病毒沉淀。用小体积的培养基重悬，起到病毒浓缩的效果。
2.慢病毒感染人类T细胞系
将携带目标基因的慢病毒感染Jurkat clone 5细胞(内源性TCR缺失的Jurkat亚克隆)。将浓缩后的慢病毒和助转剂Polybrene一同加入到T细胞培养基中，在32℃下1500rpm离心感染2小时。感染3天后，可观察荧光报告基因，并可以检测目标蛋白表达。
3.靶向EGFR的STAR上膜情况及抗原结合能力检测
取感染3天后的T细胞，用anti-human TCRα/β-BV421的流式抗体染色，之后进行流式检测。可发现(图3)，与未转基因的阴性对照细胞相比，STAR可以被抗TCRα/β的抗体染色，并且染色水平与天然的E1-TCR相当。这一结果表明STAR分子可以上膜，并且其α链和β链可以配对。
取感染3天后的T细胞，用抗原蛋白EGFR-His以及anti-His-APC的流式抗体染色，之后进行流式检测。可发现(图4)，与天然的E1-TCR(特异性不针对于EGFR)的阴性对照细胞相比，STAR显示较强的染色，并且染色水平与anti-EGFR的CAR相当。这一结果表明，STAR具有和CAR分子相当的抗原识别和结合能力。
4.靶向EGFR的STAR-T细胞与靶细胞共孵育及其介导T细胞激活能力检测
取感染3天后的T细胞，分别在EGFR抗原包被的细胞培养板中培养、以及与肿瘤细胞A549(EGFR阳性的人肺癌细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式抗体染色，之后进行流式检测(图5)。横坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
5.人类原代T细胞的分离、培养和慢病毒感染
取得人外周血细胞，使用全T细胞磁珠分离试剂盒将其中CD4和CD8T细胞纯化出来。然后将T细胞在用抗CD3/CD28的抗体包被的培养皿中刺激激活48-72小时后，可观察到T细胞体积变大、聚团生长以及形状发生极化等现象。此时，用慢病毒病载体将目标基因转入T细胞，感染方法为32℃下1500rpm离心感染2小时。病毒感染后，在含20％血清和200IU IL-2的RPMI 1640培养基中培养至足够数量。
6.靶向EGFR的STAR在人原代T细胞中的功能验证
将T细胞与A431细胞(一种EGFR高阳性的人皮肤癌细胞)按照1:1至5:1的数量比例进行共培养，检测T细胞激活和靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞标志物CD69(图6a)和T细胞细胞因子IFN-γ(图6b)水平看，STAR可引起的显著的T细胞激活。共培养24小时后，取细胞上清检测乳酸盐脱氢酶(LDH)的水平(图6c)，可反映靶细胞死亡情况。结果表明，STAR-T细胞对靶细胞有明显的杀伤作用。
实施例3、靶向CD19的STAR的构建
具体构建方法如下：
1、TCR恒定区序列确定
来源于人外周血T细胞或小鼠脾脏T细胞的cDNA经PCR分子克隆而得；在原始TCR序列的基础上，分别把α链和β链的恒定区的第48和57个氨基酸位点突变为半胱氨酸，以帮助α链和β链之间形成额外的一个二硫键，增加其相互配对效率，并分别命名为E1-TCR (人源)或E11-TCR(鼠源)。
2、靶向CD19的抗体序列确定
抗体重链可变区(VH)和抗体轻链可变区(VL)选择CD19特异性鼠单克隆抗体(克隆号FMC63)的scFv片段，仅作为实例解释本发明的内容，其他已知抗体均可进行替换。
3、靶向CD19的STAR构建
STAR含有两条多肽链，将FMC63-VL与TCRβ链融合为第一多肽链，将FMC63-VH与α链融合为第二多肽链。STAR的基因序列通过furin和p2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译成一条融合多肽，之后再被furin和p2A对应的蛋白酶切割成为两个独立的蛋白亚基，这两个亚基间通过二硫键共价结合，并与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体。
整个基因通过限制性内切酶切位点NheI和NotI插入进慢病毒表达载体pHAGE中。该载体携带氨苄抗性、EF1α启动子以及IRES-RFP荧光报告基因。
4、基因片段的克隆与组装
获得的四个片段“FMC63-VL”，“TCRβ-C”，“FMC63-VH”，“TCRα-C”分别从pHAGE-FMC63-41BBzCAR载体以及pHAGE-E1-TCR载体上克隆获得。每对引物上都带有与前后同源的25bp碱基，四个片段通过Gibson Assembly的方法一步重组连接到慢病毒载体中。从而获得STAR。
所述E1TCRα链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:1；
所述E1TCRβ链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:2；
所述FMC63-VH的核苷酸序列为SEQ ID NO:5；
所述FMC63-VL的核苷酸序列为SEQ ID NO:6；
5.载体转化和测序
Gibson Assembly的产物被转化入了DH5α菌株，使其在含氨苄的LB平板上过夜生长。挑单克隆隆菌进行测序，测序引物选用pHAGE载体上的引物seq-pHAGE-F和seq-pHAGE-R。
6.质粒提取
将测序结果正确的菌接种在LB液体培养基中，过夜培养。使用具有去内毒素功能的试剂盒提取质粒。质粒浓度用Nanodrop测量，质粒终浓度在1000ng/ul左右，A260/A280值大于1.8。
实施例4、靶向CD19的STAR的功能验证
1.慢病毒包装
将携带目标基因的pHAGE载体与包装质粒pMD2.G与psPAX2按比例转染进293T细胞(使用PEI转染)。收集48小时和72小时的细胞培养基上清，并将上清与PEG8000混合，静置过夜后离心，可得病毒沉淀。用小体积的培养基重悬，起到病毒浓缩的效果。
2.慢病毒感染人类T细胞系
将携带目标基因的慢病毒感染Jurkat clone 5细胞(内源性TCR缺失的Jurkat亚克隆)。将浓缩后的慢病毒和助转剂Polybrene一同加入到T细胞培养基中，在32℃下1500rpm离心感染2小时。感染3天后，可观察荧光报告基因，并可以检测目标蛋白表达。
3.FMC63-STAR-T细胞与靶细胞共孵育及其介导T细胞激活能力检测
取感染3天后的T细胞，分别在肿瘤细胞Raji、Mino、LY-1(CD19和CD20阳性的人淋巴瘤细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式抗体染色，之后进行流式检测(图7)。纵坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原 刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
4.人类原代T细胞的分离、培养和慢病毒感染
取得人外周血细胞，使用全T细胞磁珠分离试剂盒将其中CD3 +T细胞纯化出来。然后将T细胞在用抗CD3/CD28的抗体包被的培养皿中刺激激活48-72小时后，可观察到T细胞体积变大、聚团生长以及形状发生极化等现象。此时，用慢病毒病载体将目标基因转入T细胞，感染方法为32℃下1500rpm离心感染2小时。病毒感染后，在含20％血清和200IU/mL IL-2的RPMI 1640培养基中培养至足够数量。
5.靶向CD19的STAR在人原代T细胞中的功能验证
将T细胞与Raji和LY-1细胞按照1:1至5:1的数量比例进行共培养，检测T细胞激活和靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞细胞因子IFN-γ(图8)水平看，STAR可引起的显著的T细胞激活。T细胞内IFN-γ表达水平结果表明，靶细胞对STAR-T细胞有明显的激活作用。
实施例5、靶向CD20的STAR的构建
具体构建方法如下：
1、靶向CD20的抗体序列确定
抗体重链可变区(VH)和抗体轻链可变区(VL)选择CD20特异性抗体Ofatumumab(奥法木单抗，OFA)的scFv片段，仅作为实例解释本发明的内容，其他已知抗体均可进行替换。
2、靶向CD20的STAR构建
STAR含有两条多肽链，将OFA-VL与TCRβ链融合为第一多肽段，将OFA-VH与α链融合为第二多肽段。STAR的基因序列通过furin和p2A蛋白酶切位点多肽段相连，两条多肽链将一同被转录并翻译成一条融合多肽，之后再被furin和p2A对应的蛋白酶切割成为两个独立的蛋白亚基，这两个亚基间通过二硫键共价结合，并与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体。
整个基因通过限制性内切酶切位点NheI和NotI插入进慢病毒表达载体pHAGE中。该载体携带氨苄抗性、EF1α启动子以及IRES-RFP荧光报告基因。
3、基因片段的克隆与组装
获得的四个片段“OFA-VL”，“TCRβ-C”，“OFA-VH”，“TCRα-C”分别从pHAGE-OFA-41BBzCAR载体以及pHAGE-E11-TCR载体上克隆获得。每对引物上都带有与前后同源的25bp碱基，四个片段通过Gibson Assembly的方法一步重组连接到慢病毒载体中。从而获得STAR。
所述E11 TCRα链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:7；
所述E11 TCRβ链恒定区半胱氨酸突变体的核苷酸序列为SEQ ID NO:8；
所述OFA-VH的核苷酸序列为SEQ ID NO:9；
所述OFA-VL的核苷酸序列为SEQ ID NO:10；
4.载体转化和测序
Gibson Assembly的产物被转化入了DH5α菌株，使其在含氨苄的LB平板上过夜生长。挑单克隆隆菌进行测序，测序引物选用pHAGE载体上的引物seq-pHAGE-F和seq-pHAGE-R。
5.质粒提取
将测序结果正确的菌接种在LB液体培养基中，过夜培养。使用具有去内毒素功能的试剂盒提取质粒。质粒浓度用Nanodrop测量，质粒终浓度在1000ng/ul左右，A260/A280值大于1.8。
实施例6、靶向CD20的STAR的功能验证
1.慢病毒包装
将携带目标基因的pHAGE载体与包装质粒pMD2.G与psPAX2按比例转染进293T细胞(使用PEI转染)。收集48小时和72小时的细胞培养基上清，并将上清与PEG8000混合，静置过夜后离心，可得病毒沉淀。用小体积的培养基重悬，起到病毒浓缩的效果。
2.慢病毒感染人类T细胞系
将携带目标基因的慢病毒感染Jurkat clone 5细胞(内源性TCR缺失的Jurkat亚克隆)。将浓缩后的慢病毒和助转剂Polybrene一同加入到T细胞培养基中，在32℃下1500rpm离心感染2小时。感染3天后，可观察荧光报告基因，并可以检测目标蛋白表达。
3.OFA-STAR-T细胞与靶细胞共孵育及其介导T细胞激活能力检测
取感染3天后的T细胞，分别在肿瘤细胞Raji、Mino、LY-1(CD19和CD20阳性的人淋巴瘤细胞系)共孵育。24小时后，收集细胞，并用anti-human CD69-FITC的流式抗体染色，之后进行流式检测(图9)。纵坐标CD69阳性的为表达T细胞激活标志物CD69分子的细胞。可发现STAR在抗原刺激下可以使得T细胞表达CD69的激活标志物，即STAR可以介导T细胞在抗原刺激后的激活，并且激活程度与CAR相当。同时可以发现，在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有较高的自激活水平。
4.人类原代T细胞的分离、培养和慢病毒感染
取得人外周血细胞，使用全T细胞磁珠分离试剂盒将其中CD3 +T细胞纯化出来。然后将T细胞在用抗CD3/CD28的抗体包被的培养皿中刺激激活48-72小时后，可观察到T细胞体积变大、聚团生长以及形状发生极化等现象。此时，用慢病毒病载体将目标基因转入T细胞，感染方法为32℃下1500rpm离心感染2小时。病毒感染后，在含20％血清和200IU/mL IL-2的RPMI 1640培养基中培养至足够数量。
5.靶向CD20的STAR在人原代T细胞中的功能验证
将T细胞与Raji和LY-1细胞按照1:1至5:1的数量比例进行共培养，检测T细胞激活和靶细胞死亡情况。共培养8小时后，取T细胞进行染色，可发现STAR和CAR都可以介导T细胞激活。从T细胞细胞因子IFN-γ(图10)水平看，STAR可引起的显著的T细胞激活。T细胞内IFN-γ表达水平结果表明，靶细胞对STAR-T细胞有明显的激活作用。
由此可见，本发明已经成功构建了多个靶点的STAR，成功验证了本申请所涉及的抗体-T细胞嵌合受体能够与T细胞内源表达的CD3亚基(ε、δ、λ、ζ)形成复合体发挥功能，可以介导T细胞在抗原刺激后的激活，且与制备获得的相应抗体-嵌合抗原受体(CAR)相比，抗原激活的程度相当，更重要的是在无抗原刺激的静息状态下，STAR无自激活现象，而CAR有很高的自激活现象。限于篇幅，本发明列出了示例性的STAR，已经足够支持本发明的STAR的成功构建和突出的技术效果。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。
Figure PCTCN2019096820-appb-000001
Figure PCTCN2019096820-appb-000002
Figure PCTCN2019096820-appb-000003
Figure PCTCN2019096820-appb-000004
Figure PCTCN2019096820-appb-000005
Figure PCTCN2019096820-appb-000006
Figure PCTCN2019096820-appb-000007
Figure PCTCN2019096820-appb-000008
Figure PCTCN2019096820-appb-000009
Figure PCTCN2019096820-appb-000010
Figure PCTCN2019096820-appb-000011
Figure PCTCN2019096820-appb-000012
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Figure PCTCN2019096820-appb-000017

Claims (25)

  1. 一种特异性结合至靶抗原的嵌合T细胞受体(STAR)，所述嵌合T细胞受体包含：
    a)抗体重链可变区与T细胞受体(TCR)第一亚基恒定区融合得到的第一肽链；和，
    b)抗体轻链可变区与T细胞受体第二亚基恒定区融合得到的第二肽链；
    其中，所述抗体重链可变区与抗体轻链可变区特异性结合至所述靶抗原的抗原表位。
  2. 根据权利要求1所述的嵌合T细胞受体，其特征在于：
    (1)当所述T细胞受体第一亚基为α链时，所述T细胞受体第二亚基为β链；或，
    (2)当所述T细胞受体第一亚基为β链时，所述T细胞受体第二亚基为α链；或，
    (3)当所述T细胞受体第一亚基为γ链时，所述T细胞受体第二亚基为δ链；或，
    (4)当所述T细胞受体第一亚基为δ链时，所述T细胞受体第二亚基为γ链。
  3. 根据权利要求1-2任一项所述的嵌合T细胞受体，其特征在于，所述第一肽链和第二肽链在T细胞中表达后通过二硫键结合。
  4. 根据权利要求1-3任一项所述的嵌合T细胞受体，其特征在于：所述T细胞受体第一亚基恒定区和T细胞受体第二亚基恒定区的种属来源为人源或鼠源，包括不同的蛋白亚型。
  5. 根据权利要求1-4任一项所述的嵌合T细胞受体，其特征在于：对所述的嵌合T细胞受体进行氨基酸序列改造以降低与内源表达的T细胞受体错配，所述修改包括但不限于氨基酸点突变修饰、多肽片段替换修饰。
  6. 根据权利要求1-5任一项所述的嵌合T细胞受体，其特征在于：
    (1)所述T细胞受体第一亚基是TCRα链，其恒定区第48位氨基酸突变为半胱氨酸，且，所述T细胞受体第二亚基是TCRβ链，其恒定区第57位氨基酸突变为半胱氨酸；或
    (2)所述T细胞受体第一亚基是TCRα链，其恒定区第85位氨基酸突变为丙氨酸，且，所述T细胞受体第二亚基是TCRβ链，恒定区第88位氨基酸突变为甘氨酸。
  7. 根据前述任一权利要求所述的嵌合T细胞受体，其特征在于，所述的靶抗原为肿瘤特异性抗原或病毒特异性抗原。
  8. 根据权利要求1-7任一项所述的嵌合T细胞受体，其特征在于，所述靶抗原选自CD19，CD20，EGFR，Her2，PSCA，CD123，CEA(癌胚抗原)，FAP，CD133，EGFRVIII，BCMA，PSMA，CA125，EphA2，C-met，L1CAM，VEGFR，CS1，ROR1，EC，NY-ESO-1，MUC1，MUC16，mesothelin，LewisY，GPC3，GD2，EPG，DLL3，CD99，5T4，CD22，CD30，CD33，CD138，CD171。
  9. 根据前述任一权利要求所述的嵌合T细胞受体，其特征在于，所述抗体、抗体重链可变区或抗体轻链可变区来自IMCC225(西妥昔单抗、Cetuximab/Cetux)、Ofatumumab(奥法木单抗)、CD19单克隆抗体FMC63、利妥昔单抗(美罗华)、Avastin(贝伐单抗)、BEC2(阿妥莫单抗)、Bexxar(托西莫单抗)、Campath(阿仑单抗)、Herceptin(曲妥单抗)、LymphoCide(依帕珠单抗)、MDX-210、Mylotarg(吉姆单抗奥佐米星)、单抗17-1A(依决洛单抗)、Theragyn(pemtumomab)、Zamyl、Zevalin(替伊莫单抗)或筛选获得的高亲和力抗体。
  10. 根据前述任一权利要求所述的嵌合T细胞受体，其特征在于，所述靶抗原相关疾 病为癌症或病毒感染相关疾病。
  11. 根据权利要求10所述的嵌合T细胞受体，其特征在于，其中所述癌症选自下组：肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、白血病、淋巴瘤、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌；或，所述病毒感染是由选自以下病毒引起：巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-BarrVirus；EBV)、B型肝炎病毒(HBV)、卡波西氏肉瘤相关疱疹病毒(KSHV)、人类乳头状瘤病毒(HPV)、传染性软疣病毒(MCV)、人类T细胞白血病病毒1(HTLV-1)、HIV(人类免疫缺陷病毒)及C型肝炎病毒(HCV)。
  12. 根据前述任一权利要求所述的嵌合T细胞受体，其特征在于，所述第一肽链和第二肽链与T细胞内源的CD3亚基(ε,δ,γ,ζ)形成复合体。
  13. 一种特异性结合至靶抗原的由嵌合T细胞受体形成的复合体，其特征在于，由前述任一权利要求所述的嵌合T细胞受体与T细胞内源表达的CD3亚基(ε,δ,γ,ζ)形成复合体，且被靶抗原激活后可介导T细胞相关信号转导通路。
  14. 一种核酸，其特征在于，其编码权利要求1-12任一项的嵌合T细胞受体或权利要求13所述复合体中的所述第一肽链和所述第二肽链。
  15. 一种核酸，其特征在于，包括：
    (1)依次为抗体重链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体轻链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端；或，
    (2)依次为抗体重链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体轻链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端；或，
    (3)依次为抗体轻链可变区，T细胞受体(TCR)α链恒定胞外段、跨膜区和胞内末端，链接子(linker)，抗体重链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端；或，
    (4)依次为抗体轻链可变区，T细胞受体(TCR)β链恒定区胞外段、跨膜区和胞内末端，链接子(linker)，抗体重链可变区，T细胞受体(TCR)α链恒定区胞外段、跨膜区和胞内末端；或，
    (5)将(1)-(4)中的T细胞受体(TCR)α链替换为T细胞受体(TCR)γ链，T细胞受体(TCR)β链替换为T细胞受体(TCR)δ链对应的核酸。
  16. 一种载体，其特征在于，其包含编码权利要求1-12任一项的嵌合T细胞受体或权利要求13所述复合体中的所述第一肽链和所述第二肽链的核酸序列，或，包含权利要求14-15所述的核酸。
  17. 根据权利要求16所述的载体，其特征在于，所述载体选自逆转录病毒载体、慢病毒载体、腺病毒载体和腺相关病毒载体。
  18. 一种效应细胞，其特征在于，其表达权利要求1-12任一项的嵌合T细胞受体或权 利要求13所述复合体于其细胞表面上。
  19. 根据权利要求18所述的效应细胞，其特征在于，效应细胞为T细胞，优选的，所述效应细胞为细胞毒性T细胞、辅助T细胞、自然杀伤T细胞及抑制T细胞。
  20. 一种药物组合物，其包含权利要求1-12任一项所述的嵌合T细胞受体、权利要求13所述的复合体、权利要求任一项14-15所述的核酸、权利要求16-17任一项所述的载体或权利要求18-19任一项所述的效应细胞，以及药物上可接受的载剂。
  21. 一种包含权利要求1-12任一项所述的嵌合T细胞受体、权利要求13所述的复合体、权利要求14-15任一项所述的核酸、权利要求16-17任一项所述的载体或权利要求18-19任一项所述的效应细胞、权利要求20所述的药物组合物的试剂盒。
  22. 一种包含权利要求1-12任一项所述的嵌合T细胞受体、权利要求13所述的复合体、权利要求14-15任一项所述的核酸、权利要求16-17任一项所述的载体或权利要求18-19任一项所述的效应细胞在制备治疗或诊断有需要个体的靶抗原相关疾病的试剂盒、制剂或药物组合物中的用途。
  23. 一种杀伤递呈靶抗原的靶细胞的方法，其包含使靶细胞与权利要求18-19任一项所述的效应细胞相接触，其中所述的嵌合T细胞受体特异性结合至靶抗原。
  24. 一种治疗有需要个体的靶抗原相关疾病或癌症或病毒感染相关疾病的方法，其包含向个体施用有效量的药物组合物，该药物组合物包含权利要求1-12任一项所述的嵌合T细胞受体、权利要求13所述的复合体、权利要求14-15任一项所述的核酸、权利要求16-17任一项所述的载体或权利要求18-19任一项所述效应细胞、权利要求20所述的药物组合物。
  25. 根据权利要求24所述的方法，其特征在于，所述癌症选自下组：肾上腺皮质癌、膀胱癌、乳腺癌、子宫颈癌、胆管癌、结肠直肠癌、食道癌、神经胶母细胞瘤、神经胶质瘤、肝细胞癌、头颈癌、肾癌、淋巴瘤、白血病、肺癌、黑素瘤、间皮瘤、多发性骨髓瘤、胰脏癌、嗜铬细胞瘤、浆细胞瘤、神经母细胞瘤、卵巢癌、前列腺癌、肉瘤、胃癌、子宫癌及甲状腺癌；或者，所述病毒感染是由选自以下的病毒引起：巨细胞病毒(CMV)、埃-巴二氏病毒(Epstein-BarrVirus；EBV)、B型肝炎病毒(HBV)、卡波西氏肉瘤相关疱疹病毒(KSHV)、人类乳头状瘤病毒(HPV)、传染性软疣病毒(MCV)、人类T细胞白血病病毒1(HTLV-1)、HIV(人类免疫缺陷病毒)及C型肝炎病毒(HCV)。
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