WO2019096115A1 - Isolated t-cell receptor, cell modified by same, coding nucleic acids, expression vector, preparation method, pharmaceutical composition, and applications - Google Patents

Isolated t-cell receptor, cell modified by same, coding nucleic acids, expression vector, preparation method, pharmaceutical composition, and applications Download PDF

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WO2019096115A1
WO2019096115A1 PCT/CN2018/115191 CN2018115191W WO2019096115A1 WO 2019096115 A1 WO2019096115 A1 WO 2019096115A1 CN 2018115191 W CN2018115191 W CN 2018115191W WO 2019096115 A1 WO2019096115 A1 WO 2019096115A1
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cell
her2
cells
tcr
chain
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French (fr)
Chinese (zh)
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侯亚非
侯大炜
谭贤魁
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杭州康万达医药科技有限公司
合成免疫股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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

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  • the present invention is in the field of biotechnology, and in particular, relates to an isolated T cell receptor, a modified cell thereof, a nucleic acid encoding, an expression vector, a preparation method, a pharmaceutical composition, and an application.
  • Her2/neu is a transmembrane protein belonging to the EGFR family, which forms dimers with other family proteins and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signaling pathways (see literature “Growth Factors,” 2008;26:263", “Oncol Biol. Phys, 2004; 58: 903”).
  • Her2/neu protein is overexpressed in a variety of epithelial-derived cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc.
  • Herceptin targeting Her2/neu can significantly prolong the survival of Her2/neu-positive breast cancer patients (see the literature "N Engl J Med, 2001, 344: 783”), whereas Herceptin alone is used to treat Her2 positive
  • the clinical response rate for metastatic breast cancer is only 11% to 26% (see the literature "J Clin Oncol, 2002; 20: 7169”), indicating that Heceptin alone is not ideal for most Her2 high-expressing metastatic breast cancer.
  • Heceptin combined with chemotherapy increases clinical response rates, most Her2/neu overexpressing breast cancer patients develop resistance to Heceptin after one year (see the literature "J Clin Oncol, 2001; 19: 2587”).
  • Tumor vaccines targeting the Her2/neu 369-377 polypeptide antigen enter clinical trials, although clinical phase 1/2 shows that this vaccine can induce specific T killer cells against the Her2/neu 369-377 polypeptide antigen (see literature "Breast Care” , 2016; 11:116"), but the clinical phase III did not achieve the intended goal of extending patient survival (http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast- Cancer-halted-after-futility-review).
  • CARs chimeric antigen receptors
  • T-cell therapies adoptive transfer of in vitro cultured, chimeric antigen receptors (CARs)-based tumor-specific T-cell therapies, developed as the first CAR-T targeting Her2/neu antigens for solid tumors
  • CRS cytokine release syndrome
  • Severe cytokine storms and neurotoxicity are common toxicities in CAR-T therapy (see the literature “Blood, 2016; 127: 3321”), partly due to the unrestricted CAR, a non-native T cell receptor.
  • Cell activation is involved (see the literature "Nat Ned, 2015; 21: 581”) or with autocrine cytokine that does not require antigen stimulation (see the literature "Cancer Immunol Res, 2015; 3: 356”).
  • TCR-T therapy for adoptive transfer of T cells transfected with specific T cell receptor (ie TCR) genes is considered to be the most promising immune cell gene therapy for solid tumors (see the literature “Adv Immunol. 2016; 130:279 -94”).
  • clinical studies of TCR-T therapy targeting NY-ESO-1 antigen have shown encouraging clinical therapeutic effects (see the literature “Nat Med. 2015 Aug; 21(8): 914-921”).
  • the number of specific TCRs currently known to target tumor antigens and to efficiently recognize tumor cells is very limited, thus limiting the widespread use of TCR-T therapies.
  • TCR-T therapy does not exhibit the severe cytokine storm toxicity exhibited by CAR-T therapy
  • target antigen if the target antigen is derived from a self-protein, targeting a target antigen that is low in normal tissue cells may cause serious Autoimmune response, ie on target off tumor toxicity (Blood 2009; 114: 535-546).
  • a general strategy is to perform point mutations in the complementarity determing regions (CDRs) on the TCR in vitro, or by never undergoing a central tolerance mechanism. Induction was performed in a screened humanized mouse T cell pool (see the literature "Front Immunol. 2013; 4:363").
  • the present invention provides isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions, and uses.
  • the present invention provides:
  • An isolated T cell receptor comprising at least one of an ⁇ chain and a ⁇ chain, the ⁇ chain and the ⁇ chain each comprising a variable region and a constant region, characterized in that the T cell receptor
  • the antigen Her2/neu expressed by the tumor cell can be specifically recognized, and the amino acid sequence of the variable region of the ⁇ chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 19, the ⁇ The amino acid sequence of the variable region of the strand has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • T cell receptor (2) The T cell receptor according to (1), wherein the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule; Yes, the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • An isolated nucleic acid encoding a T cell receptor comprising a coding sequence of at least one of an ⁇ chain and a ⁇ chain of said T cell receptor, said ⁇ chain coding sequence and said ⁇ chain coding sequence Including a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing an antigen Her2/neu expressed by a tumor cell, and the amino acid sequence encoded by the ⁇ chain variable region coding sequence has At least 98% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the ⁇ chain variable region coding sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  • nucleic acid according to (6) wherein the nucleic acid is DNA or RNA.
  • the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule;
  • the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • nucleic acid according to any one of (6) to (11), wherein the ⁇ chain coding sequence and the ⁇ chain coding sequence are linked by a coding sequence of a cleavable linker polypeptide.
  • a recombinant expression vector comprising the nucleic acid according to any one of (6) to (14), and/or a complement thereof, which is operably linked to a promoter.
  • a T cell receptor-modified cell the surface of which is modified by the T cell receptor according to any one of (1) to (5), wherein the cell comprises a primitive T cell or a precursor thereof Cells, NKT cells, or T cell lines.
  • the method of transfection comprises: transfection with a viral vector, preferably, the viral vector comprises a ⁇ retroviral vector or a lentiviral vector;
  • the chemical means comprises a method of transfection with a liposome; physically, preferably, the physical means comprises an electrotransfection mode.
  • a pharmaceutical composition comprising the T cell receptor-modified cell according to (16) as an active ingredient, and a pharmaceutically acceptable excipient.
  • composition according to (24), wherein the pharmaceutical composition comprises the T cell having a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course per patient. Receptor modified cells.
  • composition according to (24), wherein the pharmaceutical composition is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
  • a method of treating a tumor and/or a cancer comprising administering a T cell receptor-modified cell according to (16) to a tumor and/or a cancer patient.
  • the invention has the following advantages and positive effects:
  • the present invention successfully induces T cell clones specific for HLA-A2-presented Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide) from HLA-A2-positive healthy donor peripheral blood.
  • a T cell clone carrying a native TCR that specifically recognizes a Her2/neu epitope polypeptide (such as Her2/neu 369-377 polypeptide) is screened therefrom, and the entire TCR sequence is obtained.
  • This TCR belongs to CD8 molecule-dependent, has a moderate affinity for Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide), and specifically recognizes HLA-A2 positive and expresses Her2/neu antigen-bearing tumor cells.
  • T cell clones carrying this TCR were screened by the central immune tolerance mechanism and entered the peripheral T cell bank.
  • the killer T cells carrying this TCR were present in normal human peripheral blood and did not cross-react to normal tissue cells that express Her2/neu protein in a small amount.
  • the present invention achieves a novel TCR that is capable of specifically recognizing tumor antigens while avoiding possible off-target toxic side effects.
  • the constant region of the TCR is also engineered (eg, by disulfide modification or murine transformation) to further reduce or avoid mismatching with the endogenous TCR when expressed on immune cells. occur.
  • Immunological cells modified with this TCR can specifically recognize a variety of HLA-A2 + and Her2/neu + tumor cell lines with significant anti-tumor effect.
  • the TCR used to modify immune cells does not cross-react to normal cells that express Her2/neu in a small amount. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.
  • the TCR-modified immune cells of the invention provide a new choice for treating HLA-A2 + and Her2/neu + tumor patients, and have good industrial application prospects.
  • FIG. 1 shows a table of Her2/neu 369-377 polypeptide (Her2-E75)-specific killer T cells induced from HLA-A2 + normal donor PBMC (specifically #2 PBMC) in Example 1 of the present invention.
  • Figure 1A shows the results of flow cytometry analysis of PBMC cells stained with CD8-APC antibody and Her2-E75 pentamer-PE after two rounds of Her2-E75 antigen polypeptide stimulation.
  • the right panel is stimulated by peptides.
  • the CD8 + pentamer + killer T cell population was subjected to FACS sorting to obtain T cell clones.
  • the left panel shows control cells without polypeptide stimulation.
  • FIG. 1B shows the phenotypic analysis of flow cytometry of CD8 + E75-tetramer + killer T cell clones stained with CD8-APC and Her2-E75 tetramer-PE.
  • the right panel shows CD8 + Her2 tetramer + T cell clone
  • Her2 CTL 1B5 is a purified Her2-E75 polypeptide-specific CTL cell clone.
  • the left panel shows control CTL cells without polypeptide stimulation.
  • the abscissa indicates the fluorescence intensity of CD8 molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • Figure 1C shows the results of T cell clonal function assay.
  • T cell clone Her2 CTL 1B5 slanted bar graph
  • control CTL cells without peptide stimulation point bar graph
  • the cell supernatant was taken for ELISA analysis of IFN- ⁇ , and each test group and control group were duplicated, and the results were shown as mean ⁇ SD.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of T cells secreting IFN- ⁇ .
  • Figure 2 shows two constructed lentiviral vectors carrying the Her2 TCR-1B5 TCR gene (shown as "pCDH-EF1 ⁇ -Her2 TCR-(PGK-GFP) vector” and "pCDH-EF1 ⁇ -Her2 TCR vector, respectively"
  • the main functional fragment The fragment shown above simultaneously expresses the TCR gene driven by the EF-1 ⁇ promoter and the GFP gene driven by the PKG promoter, and the fragment shown below expresses only the TCR gene.
  • the beta and alpha chains of each TCR are linked by the coding sequence of the cleavable linker polypeptide (furin-F2A).
  • FIG. 3 shows the results of phenotypic and functional assays of T cell lines transfected with the Her2 TCR-1B5 TCR gene.
  • J. RT3-T3.5 T cell line J. RT3
  • the GFP + Her2-E75 tetramer + cell population shown in Figure 3A is a cell expressing Her2 TCR-1B5 TCR, expressed as a percentage of each positive cell population to the total number of cells.
  • the TCR involved in the left panel adds a disulfide bond structure (Her2 TCR-1B5-dis) to the constant region of the ⁇ chain and the ⁇ chain
  • the TCR involved in the right panel is the constant region of the human ⁇ chain and ⁇ chain by the mouse constant region.
  • the homologous sequence was replaced (Her2 TCR-1B5-mC).
  • the abscissa indicates the fluorescence intensity of GFP molecule expression
  • the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • Figure 3B shows the expression of the constant regions of the TCR alpha and beta chains in different T cell variants after modification in different ways.
  • Her2 TCR-1B5-dis refers to a TCR in which the constant regions of the ⁇ chain and the ⁇ chain each add a disulfide bond structure;
  • Her2 TCR-1B5-mC means that the constant regions of the human ⁇ chain and the ⁇ chain are small.
  • the TCR replaced by the homologous sequence of the murine constant region.
  • the GFP + Her2-E75 tetramer + cells are positive cells expressing Her2 TCR-1B5 TCR, and the ordinate is the percentage of the TCR-positive cells to total GFP + cells.
  • the abscissa indicates different T cell line groups, wherein "Jurkat(TCR a+b+)" refers to Jurkat cells, both of which express both the ⁇ chain and the ⁇ chain, and "J.RT3(TCR a+b-)" refers to J .RT3-T3.5 cells, which are derived from Jurkat cells, and the ⁇ chain gene is deleted, and the ⁇ chain is still expressed.
  • Figure 3C shows that a T cell strain transfected with a lentiviral vector encoding the Her2 TCR-1B5 TCR gene can recognize the Her2-E75 polypeptide presented by T2 cells.
  • TCR alpha chain and the beta chain were modified in different ways, and TCR cells expressing TCR were mixed with T2 cells presented with different concentrations of Her2-E75 polypeptide for 16 hours, stained with anti-CD69-PE antibody, and subjected to flow cytometry analysis.
  • J.RT3-Her2-1B5-dis indicates J.RT3-T3.5 cells expressing Her2 TCR-1B5-dis
  • J.RT3-Her2-1B5-mC indicates expression of Her2 TCR-1B5-mC J.RT3-T3.5 cells
  • Jurkat-Her2-1B5-dis indicates Jurkat cells expressing Her2 TCR-1B5-dis
  • Jurkat-Her2-1B5-mC indicates Jurkat expressing Her2 TCR-1B5-mC cell.
  • the abscissa indicates the concentration of the Her2-E75 polypeptide presented by the T2 cells.
  • the ordinate is the percentage of CD69 + cells to total GFP + cells.
  • Figure 4 shows the phenotypic and functional test results of peripheral blood mononuclear cells (PBMC) transfected with the Her2 TCR-1B5-mC TCR gene.
  • Figure 4A shows the results of flow cytometric analysis of PBMCs from two different donors transfected with Her2 TCR-1B5-mC, stained with Her2-E75 tetramer-PE and anti-CD8-APC antibody. First, the lymphocyte population was separated according to the morphology and size of the cells, and the Her2-E75 tetramer + cell population was a cell expressing Her2 TCR-1B5 TCR.
  • the abscissa indicates the fluorescence intensity of CD8 molecule expression
  • the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer.
  • the percentages shown are the ratio of the number of lymphocytes to each positive cell population.
  • the left panel relates to a peripheral blood mononuclear cell (#1PBMC) provided by a donor, and the right panel relates to a different donor-provided PBMC (#2 PBMC).
  • CD8 + Her2-E75 tetramer + cells are killer T cells expressing Her2 TCR-1B5-mC.
  • the CD8 - Her2-E75 tetramer + cells may be CD4 + helper T cells expressing Her2 TCR-1B5-mC.
  • FIG. 4B shows that T cells expressing Her2 TCR-1B5-mC can recognize the Her2-E75 polypeptide presented by T2 cells.
  • Two different donor PBMCs transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP were mixed with T2 cells with different concentrations of Her2-E75 polypeptide for 16 hours, and the cell supernatant was taken for IFN- ⁇ .
  • ELISA analysis The target cells in the control group were T2 cells which presented the EBV virus antigen polypeptide LMP2 426-434 which binds to the HLA-A2 molecule.
  • T2+Her2-E75 0.1 ⁇ g/ml indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was presented
  • T2+Her2-E75 0.01 ⁇ g/ml indicates that Her2 was present at 0.01 ⁇ g/ml.
  • - T75 cell group of E75 polypeptide indicates a T2 cell group of 0.001 ⁇ g/ml of Her2-E75 polypeptide
  • T2+EBV-LMP 1 ⁇ g/ml indicates that 1 ⁇ g was presented.
  • FIG. 4C shows the results of the CD8 antibody blocking assay for T cell function.
  • the #2 PBMC transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP gene was co-cultured with the antigen polypeptide presented by T2 cells, and anti-human CD8 antibody was added to detect whether the function of T cell secreting IFN- ⁇ was detected. suppressed.
  • T2+Her2-E75 0.1 ⁇ g/ml indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was added without anti-human CD8 antibody
  • T2+Her2-E75 0.1 ⁇ g/ml+anti -CD8 indicates a T2 cell group in which 0.1 ⁇ g/ml of Her2-E75 polypeptide was added to the anti-human CD8 antibody.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • Each of the test groups and the control group in Figs. 4B and 4C was a duplicate well, and the results were shown as mean ⁇ SD.
  • FIG. 5 shows the results of functional assays of peripheral blood mononuclear fine (PBMC)-recognizing tumor cell lines transfected with the Her2 TCR-1B5-mC TCR gene.
  • Fig. 5A shows that the lentiviral vector encoding the Her2 TCR-1B5-mC TCR gene was transfected with #2 PBMC, and after mixing with different tumor cell lines for 16 hours, the cell supernatant was taken for ELISA analysis of IFN- ⁇ .
  • Each test group and control group were duplicate wells, and the results were shown as mean ⁇ SD.
  • the abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by T cells.
  • Figure 5B shows the addition of anti-CD8 antibody (shown as “colo205+anti-CD8”) or anti-HLA-ABC antibody to the culture well of the transfected #2 PBMC-targeted Colo205 cells, respectively (shown as "colo205+anti-HLA-ABC”), the function of the function blocking test results.
  • the abscissa indicates the different tumor cell line groups, and the ordinate indicates the concentration of IFN- ⁇ secreted by the T cells.
  • Colo205 and Coca-2 are HLA-A2 positive Her2/neu positive colon cancer cells
  • MAD-MB-231 is HLA-A2 positive Her2/neu positive breast cancer cells
  • H647 is HLA-A2 negative Her2/neu positive lung cancer cells
  • SK-OV-3 is an HLA-A2 negative Her2/neu positive ovarian cancer cell
  • Bjab is an HLA-A2 positive Her2/neu negative lymphoma cell.
  • the Her2/neu antigen belongs to the tumor-associated antigen, and the high-affinity T cells that recognize the Her2/neu antigen are mostly cleared by the central tolerance mechanism to avoid possible autoimmune responses (see the literature "Immunol Rev. 2016; 271(1) ): 127-40"). Therefore, it has become very difficult to induce a T cell clone having a Her2/neu antigen specifically recognized by a tumor cell from a peripheral blood T cell pool.
  • the Her2/neu 369-377 polypeptide naturally derived from the cell may be glycosylated to cause a difference in TCR recognition configuration (see the literature "Proc. Natl. Acad. Sci. USA 2003; 100: 15029 - 15034" ).
  • high-affinity T cell clones that can only recognize exogenously loaded antigenic peptides often obtain dominant expansion, and can specifically recognize the cell-producing
  • the growth of T cell clones of the endogenous Her2/neu antigen polypeptide is inhibited (see the literature "J Exp Med.
  • Screening for a high-affinity TCR T cells transfected with this high-affinity TCR recognize multiple tumor cells of HLA-A2 + Her2/neu + (see the literature "HUMAN GENE THERAPY 2014; 25: 730-739" ).
  • This TCR was not obtained from monoclonal T cells and therefore it was not possible to determine whether this TCR is a native TCR present in a T cell bank that has been screened for central tolerance.
  • Her2/neu protein is also expressed in small organs such as myocardium, lung, esophagus, kidney, and bladder (see “Oncogene. 1990 Jul; 5(7): 953-62"), so it is based on high affinity Her2/.
  • TCR-T therapy with neu antigen-specific TCR has a risk of off-target toxicity to normal tissues.
  • the tumor cells have high expression of Her2/neu protein, so the number of antigenic peptides presented by HLA on the cell surface will increase accordingly.
  • the difference in the number of HLA/antigen polypeptide complexes between tumor cells and normal cells can become normal T cells. And the window of the tumor tissue.
  • the present invention proposes to obtain the sequence of the native TCR from the auto-T cell repertoire, and then to express the TCR on the T cell in vitro, so that the obtained TCR-expressing T cells can recognize the increased expression of the tumor cells.
  • Her2/neu antigen is the key to the successful development of effective and low toxicity TCR-T therapy.
  • the present invention induces Her2/neu 369-377 presented to HLA-A2 from HLA-A2-positive healthy donor peripheral blood.
  • the polypeptide has a specific T cell clone and is screened for a T cell clone carrying a native TCR having a moderate affinity for the Her2/neu 369-377 polypeptide.
  • the present invention recognizes that specific T cell clones directed against the Her2/neu 369-377 polypeptide will proliferate after immunization with the Her2/neu 369-377 antigen polypeptide and thus cannot represent the natural presence of the in vivo T cell bank (repertoire).
  • a specific T cell population that recognizes the Her2/neu 369-377 polypeptide antigen presented by the target cell is a specific T cell population that recognizes the Her2/neu 369-377 polypeptide antigen presented by the target cell.
  • the present invention also does not employ a method by which other research groups induce polypeptide-specific T cells from HLA-A2 negative peripheral blood (see the document "The Journal of Immunology, 2010, 184: 1617 - 1629"), although it is easier to obtain from allogeneic PBMCs.
  • High-affinity allo-T cells recognizing the Her2/neu 369-377 polypeptide antigen are obtained, but this also increases the allogeneic response caused by T cell cross-recognition of other polypeptides presented by HLA-A2 molecules.
  • the present invention provides an isolated T cell receptor comprising at least one of an alpha chain and a beta chain, both of which comprise a variable region and a constant region, characterized in that The T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid sequence of the variable region of the ⁇ chain has at least 98%, preferably the amino acid sequence shown in SEQ ID NO: At least 98.5%, more preferably at least 99% identity, the amino acid sequence of the variable region of the beta chain having at least 98%, preferably at least 98.5%, more preferably at least the amino acid sequence set forth in SEQ ID NO: 99% consistency as long as it does not significantly affect the effects of the present invention. It is also preferred that the amino acid sequence of the variable region of the ⁇ chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the variable region of the ⁇ chain is set forth in SEQ ID NO: 20.
  • variable regions of the TCR alpha and beta chains are used to bind the antigenic polypeptide/major histocompatibility complex (MHC I), respectively comprising three hypervariable regions or complementarity determining regions (CDRs), ie, CDR1, CDR2, CDR3.
  • the CDR3 region is critical for the specific recognition of antigenic peptides presented by MHC molecules.
  • the TCR alpha chain is composed of different V and J gene segments, and the ⁇ chain is composed of different V, D and J gene segments.
  • the MHC class I molecule includes human HLA.
  • the HLA includes: HLA-A, B, C.
  • the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule.
  • the amino acid sequence of the antigen Her2/neu is shown in SEQ ID NO: 17.
  • the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  • the HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A*0201, 0202, 0203, 0204, 0205, 0206, and 0207.
  • the HLA-A2 molecule is preferably HLA-A*0201.
  • the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-11 amino acids in length. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
  • the T cell receptor recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0 to 7.0 ng/ml) ). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml.
  • maximum half-reactive polypeptide concentration refers to the concentration of the desired polypeptide that induces a T cell response to reach a maximum of 50%.
  • the maximum half-reactive polypeptide concentration of specific T cells against the cytomegalovirus (CMV) antigen CMV pp65 (495-503) polypeptide is reported to be between 0.1-1 ng/ml, and this TCR is considered to be high for CMV antigen polypeptides. Affinity (see the literature "Journal of Immunogical Methds 2007; 320: 119-131").
  • the T cell receptor has a moderate affinity for the Her2/neu antigen, thereby avoiding off-target toxicity which may be caused by high affinity.
  • the exogenous TCR ⁇ chain and ⁇ chain expressed by T cells may be mismatched with the ⁇ chain and ⁇ chain of TCR itself, not only diluting the expression level of the correct paired exogenous TCR, but also the antigen specificity of the mismatched TCR. It is clear that there is a potential danger of recognizing the autoantigen, and therefore it is preferred to modify the constant regions of the TCR alpha and beta chains to reduce or avoid mismatches.
  • the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the invention finds that the constant region of the alpha chain may be wholly or partially The ground is replaced by a homologous sequence derived from other species, and/or the constant region of the beta strand may be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse.
  • the replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
  • the constant region of the alpha chain may be modified with one or more disulfide bonds, and/or the constant region of the beta chain may be modified with one or more disulfide bonds, such as one or two.
  • TCR is modified in two different ways.
  • Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67 ( 8): 38.9-903., which is incorporated herein by reference in its entirety.
  • Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article.
  • amino acid sequence of the alpha chain is set forth in SEQ ID NOs: 2, 6, or 10
  • amino acid sequence of the beta strand is set forth in SEQ ID NOs: 4, 8, or 12.
  • the sequence is the original human sequence; for the amino acid sequence such as the ⁇ chain shown in SEQ ID NO: 6, it has one in the constant region. Sulfur bond; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 10, the constant region is replaced with a murine constant region.
  • the sequence is the original human sequence; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 8, it has a modification in the constant region. Sulfur bond; for the ⁇ chain of the amino acid sequence such as SEQ ID NO: 12, the constant region is replaced with a murine constant region.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 2, and the amino acid sequence of the beta strand is set forth in SEQ ID NO: 4.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 6
  • the amino acid sequence of the beta strand is set forth in SEQ ID NO: 8.
  • the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 10
  • the amino acid sequence of the beta strand is set forth in SEQ ID NO: 12.
  • the alpha chain of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10.
  • the alpha chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10.
  • the beta strand of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 4, 8, or 12.
  • the ⁇ chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 4, 8 or 12.
  • the alpha and/or beta strands of the TCRs of the invention may also bind to other functional sequences at the terminus (e.g., the C-terminus), such as the functional region sequences of the costimulatory signals CD28, 4-1BB, and/or CD3zeta.
  • the invention also provides an isolated T cell receptor-encoding nucleic acid comprising a coding sequence for at least one of an alpha chain and a beta chain of said T cell receptor, said alpha chain coding sequence and beta strand coding
  • the sequences each comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid encoded by the ⁇ chain variable region coding sequence
  • the sequence has at least 98%, preferably at least 98.5%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the ⁇ chain variable region coding sequence having SEQ ID NO:
  • the amino acid sequence shown at 20 has a consistency of at least 98%, preferably at least 98.5%, more preferably at least 99%, as long as the effects of the present invention are not significantly affected. It is also preferred that the ⁇ chain variable region coding sequence encodes the amino acid sequence set forth
  • the nucleic acid can be DNA or RNA.
  • the alpha chain variable region coding sequence is set forth in SEQ ID NO: 21, and the beta chain variable region coding sequence is set forth in SEQ ID NO: 22.
  • the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule.
  • the epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18).
  • the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-10 amino acids in length.
  • the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
  • the T cell receptor encoded by the nucleic acid recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0- Between 7.0ng/ml). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml. In this case, the T cell receptor has a moderate affinity for the Her2/neu antigen, and the off-target toxicity which may be caused by high affinity can be avoided.
  • the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the alpha chain constant region coding sequence is derived in whole or in part from other
  • the homologous sequence of the species is replaced, and/or the beta strand constant region coding sequence is replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse.
  • the replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
  • the alpha chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds, and/or the beta chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds.
  • the alpha chain coding sequence is set forth in SEQ ID NOs: 1, 5 or 9
  • the beta strand coding sequence is set forth in SEQ ID NOs: 3, 7 or 11.
  • the sequence is the original human sequence; and for the ⁇ chain of the coding sequence such as SEQ ID NO: 5, it has one in the constant region. Sulfur bond; for the ⁇ chain of the coding sequence as shown in SEQ ID NO: 9, the constant region is replaced with a murine constant region.
  • the sequence is the original human sequence; for the ⁇ chain of the coding sequence such as SEQ ID NO: 7, it has a modification in the constant region. Sulfur bond; for the ⁇ chain of the coding sequence as shown in SEQ ID NO: 11, the constant region is replaced with a murine constant region.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 1, and the coding sequence for the beta chain is set forth in SEQ ID NO: 3.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 5
  • the coding sequence for the beta chain is set forth in SEQ ID NO: 7.
  • the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 9, and the coding sequence for the beta chain is set forth in SEQ ID NO:11.
  • the alpha chain coding sequence and the beta strand coding sequence are joined by a coding sequence for a cleavable linker polypeptide, which increases the expression of the TCR in the cell.
  • cleavable linker polypeptide means that the polypeptide functions as a linker and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding the polypeptide is translated by ribosome skipping so that it is ligated The polypeptides are separated from one another.
  • cleavable linker polypeptides examples include F2A polypeptides, including but not limited to F2A polypeptides from picornaviruses, and class 2A sequences similar to other viruses.
  • the cleavable linker polypeptide also includes a standard canonical four amino acid motif, i.e., the R-X-[KR]-R amino acid sequence, which can be cleaved by the Furin enzyme.
  • the TCR encoded by this embodiment is a single-stranded chimeric T cell receptor, and after the expression of the single-stranded chimeric T cell receptor is completed, the cleavable linker polypeptide linking the ⁇ chain and the ⁇ chain is cleaved by a specific enzyme in the cell. Thereby forming equal amounts of free alpha and beta chains.
  • alpha and beta chains constituting the single-stranded chimeric TCR can also be replaced, as described above, in whole or in part by homologous sequences derived from other species, and/or modified (coding) one or more disulfide bonds.
  • sequence of the nucleic acid is set forth in SEQ ID NOs: 13, 15, or 23.
  • the nucleotide sequence of the nucleic acid is encoded sub-optimized to increase gene expression, protein translation efficiency, and protein expression, thereby enhancing the ability of the TCR to recognize antigen.
  • Encoding sub-optimization includes, but is not limited to, modification of the translation initiation region, alteration of mRNA structural fragments, and use of different codons encoding the same amino acid.
  • the sequence of the above TCR-encoding nucleic acid can be mutated, including the removal, insertion and/or substitution of one or more amino acid codons such that the expressed TCR recognizes the function of the Her2/neu antigen unchanged or enhanced.
  • conservative amino acid substitutions are made comprising substitution of one amino acid in the variable region of the TCR alpha chain and/or beta chain described above with another amino acid having similar structural and/or chemical properties.
  • similar amino acid refers to an amino acid residue having similar properties of polarity, electrical load, solubility, hydrophobicity, hydrophilicity, and the like.
  • the mutated TCR still has the biological activity of recognizing the above-described Her2/neu antigen polypeptide presented by the target cell.
  • the TCR maturation modification is performed, ie, comprising amino acids of the complementarity determining region 2 (CDR2) and/or CDR3 regions of the variable regions of the TCR alpha chain and/or beta chain described above. Removal, insertion and/or substitution alter the affinity of the TCR to bind to the Her2/neu antigen.
  • the invention also provides an isolated mRNA transcribed from the DNA according to the invention.
  • the invention also provides a recombinant expression vector comprising a nucleic acid (e.g., DNA) according to the invention operably linked to a promoter, and/or a complement thereof.
  • a nucleic acid e.g., DNA
  • the DNA of the invention is suitably operably linked to a promoter, enhancer, terminator and/or polyA signal sequence.
  • the combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.
  • the basic backbone of the recombinant expression vector can be any known expression vector, including plasmids or viruses, including but not limited to, for example, retroviral vectors (the virus prototype is Moloney murine leukemia virus (MMLV)) and lentiviruses. Vector (the virus prototype is human immunodeficiency type I virus (HIV)).
  • retroviral vectors the virus prototype is Moloney murine leukemia virus (MMLV)
  • Vector the virus prototype is human immunodeficiency type I virus (HIV)
  • a recombinant vector expressing a TCR of the present invention can be obtained by recombinant DNA techniques conventional in the art.
  • expression of the alpha and beta chain genes on the recombinant expression vector can be driven by two different promoters, including various known types, such as strongly expressed, weakly expressed, Inducible, tissue-specific, and differentiation-specific promoters.
  • the promoter may be of viral or non-viral origin, such as the CMV promoter, the promoter on the LTR of MSCV, the EF1-alpha promoter, and the PGK-1 promoter.
  • the driving directions of the two promoters can be either in the same direction or in the opposite direction.
  • expression of the alpha and beta chain genes on the recombinant expression vector can be driven by the same promoter, such as in the case of a single chain chimeric T cell receptor, the nucleotide sequence of the alpha chain and The nucleotide sequence of the beta strand is ligated by the Furin-F2A polypeptide coding sequence.
  • the recombinant expression vector may comprise coding sequences for other functional molecules in addition to the alpha and beta chain genes.
  • an autofluorescent protein such as GFP or other fluorescent protein
  • Another embodiment includes expressing an inducible suicide gene system, such as inducing expression of a herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing expression of a Caspase 9 (iCasp9) protein. Expression of these "safety-switches” can increase the safety of the cells modified by the TCR genes of the present invention for use in vivo.
  • HSV-TK herpes simplex virus-thymidine kinase
  • iCasp9 Caspase 9
  • Another embodiment includes expressing a human CD8 gene, comprising expressing the CD8 alpha chain and the beta chain, either alone or in combination, and the cells modified by the TCR gene of the present invention can enhance their ability to specifically recognize the Her2/neu antigen by expressing the CD8 molecule, or CD8-negative T cells (such as CD4 + T helper cells) acquire the ability to specifically recognize the Her2/neu antigen.
  • a human chemokine receptor gene such as CCR2, which binds to a corresponding chemokine ligand that is highly expressed in tumor tissue, thereby enhancing cells modified by the TCR gene of the present invention Homing in tumor tissue.
  • the present invention also provides a T cell receptor-modified cell whose surface is modified by a T cell receptor according to the present invention, wherein the cell comprises a primitive T cell or a precursor cell thereof, NKT cell, or T Cell line.
  • Modification in the "T cell receptor modification” means that a cell expresses a T cell receptor of the present invention by gene transfection, that is, the T cell receptor is anchored through a transmembrane region.
  • the modified cells are on the cell membrane and have the function of recognizing the antigenic polypeptide/MHC complex.
  • the invention also provides a method of preparing a T cell receptor modified cell according to the invention, comprising the steps of:
  • the cells of step 1) can be derived from mammals, including humans, dogs, mice, rats and their transgenic animals.
  • the cells can be derived from autologous or allogeneic. Allogeneic cells include cells from identical twins, allogeneic stem cells, genetically engineered allogeneic T cells.
  • the cells of step 1) include naive T cells or their precursor cells, NKT cells, or T cell strains.
  • naive T cell refers to mature T cells in peripheral blood that have not been activated by the corresponding antigen.
  • T cells can be isolated by methods known in the art.
  • T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, cord blood, and tumor tissue.
  • the T cells can be obtained from hematopoietic stem cells (HSCs), including bone marrow, peripheral blood, or cord blood, isolated by stem cell marker molecules such as CD34.
  • HSCs hematopoietic stem cells
  • the T cells can be derived from inducible pluripotent stem cells (iPS cells), including introducing a specific gene or a specific gene product into the somatic cells, transforming the somatic cells into stem cells, and inducing differentiation into T cells or precursors thereof in vitro. cell.
  • T cells can be obtained by a usual method such as density gradient centrifugation, and examples of density gradient centrifugation include Ficoll or Percoll density centrifugation.
  • One embodiment is the production of enriched T cells from peripheral blood using plasma apheresis or leukapheresis.
  • One embodiment is a method of separating a magnetic cell by labeling a specific cell population with an antibody (eg, Enriched CD8 + or CD4 + T cells were obtained by system (Miltenyi Biotec), or by flow cytometry.
  • the T cell precursor cells are hematopoietic stem cells.
  • the gene encoding the TCR of the present invention can be directly introduced into hematopoietic stem cells, and then transferred to the body to further differentiate into mature T cells; the coding gene can also be introduced into T cells which are differentiated and matured by hematopoietic stem cells under specific conditions in vitro.
  • the cells can be resuspended in a cryopreservation solution and stored in liquid nitrogen.
  • cryopreservation solutions include, but are not limited to, 10% (v/v) DMSO and 90% (v/v) human serum or fetal bovine serum.
  • the cells were frozen at -80 ° C at a temperature of 1 ° C per minute and then stored in the gas phase portion of the liquid nitrogen tank.
  • Other cryopreservation methods are to freeze the cells placed in the cryopreservation solution directly into -80 ° C or liquid nitrogen.
  • the nucleic acid of step 2) is a nucleic acid according to the invention, including the DNA and RNA.
  • the transfection includes physical, biological, and chemical means.
  • the physical method is to introduce the TCR gene into the cell in the form of DNA or RNA by calcium phosphate precipitation, liposome, microinjection, electroporation, gene gun, and the like.
  • Commercial instruments are currently available, including electrotransfers (eg Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), Multiporator (Eppendort, Germany)
  • the biological method is to introduce the TCR gene into a cell through a DNA or RNA vector, and a retroviral vector (such as a ⁇ retroviral vector) is a common tool for transfecting and inserting a foreign gene fragment into an animal cell (including a human cell).
  • viral vectors are derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, etc.
  • the chemical method is to introduce polynucleotides into cells, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, Microspheres, microbeads, micelles, and liposomes.
  • TCR gene is introduced into a cell
  • various assays are used to analyze whether the gene of interest is introduced into the target cell, including common molecular biology methods ( For example, Southern and Northern blots, RT-PCR and PCR, etc., or common biochemical methods (eg ELISA and Western) Trace), and methods of the present invention mentioned.
  • the transfection is carried out by a retroviral vector or a lentiviral vector.
  • T cells can be expanded in vitro by co-activation of a TCR/CD3 complex on the surface and a co-stimulatory molecule (such as CD28).
  • Stimulators that activate TCR, CD3, and CD28 can be adsorbed on the surface of a culture vessel, or on the surface of a co-culture (such as a magnetic bead), or can be directly cultured in a cell culture medium.
  • Another embodiment is to co-culture T cells with trophoblast cells that express helper stimulatory molecules or corresponding ligands including, but not limited to, HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • helper stimulatory molecules or corresponding ligands including, but not limited to, HLA-A2, ⁇ 2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
  • the T cell culture is cultured and expanded under appropriate culture conditions according to the usual method of in vitro culture of mammalian cells. For example, when the cells reach more than 70% confluence, they can be passaged, usually 2 to 3 days for fresh culture. When the cells reach a certain number, they are used directly or frozen as described above. The in vitro culture time can be within 24 hours or as long as 14 days or longer. After the frozen cells are thawed, the next step can be applied.
  • the cells can be cultured in vitro for hours to 14 days, or any number of hours in between.
  • T cell culture conditions include the use of basal culture fluids including, but not limited to, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo.
  • Conditions required for survival and proliferation of other cells include, but are not limited to, the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN- ⁇ , IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF- ⁇ and TNF-a, other culture additives (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormone, growth factors) .
  • the cells can be placed under suitable culture conditions, for example, the temperature can be at 37 ° C, 32 ° C, 30 ° C or room temperature, and the air conditions can be, for example, air containing 5% CO 2 .
  • the invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  • the tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  • a sample of a tumor and/or a cancer cell taken out from a host can be contacted with a T cell receptor-modified cell of the present invention at a concentration, and can be judged according to the degree of reaction between the two. Whether the tumor and/or cancer is HLA-A2 positive or HLA-A2 negative, and whether the antigen Her2/neu is expressed.
  • the present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises, as an active ingredient, a T cell receptor-modified cell according to the present invention, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition preferably comprises the T cell receptor modified cells in a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient, including any between the two endpoints The number of cells.
  • each course of treatment is 1-3 days, administered 1-3 times a day.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the pharmaceutically acceptable excipients include pharmaceutically or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives, including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, Preservatives, etc.
  • the pharmaceutical composition can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, and peritoneal Internal administration.
  • the invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a T cell receptor modified cell according to the invention.
  • the tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
  • the T cell receptor modified cells are preferably administered at a dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient.
  • each course of treatment is 1-3 days, administered 1-3 times a day.
  • the patient may be treated for one or more courses depending on the actual situation and needs.
  • the T cell receptor-modified cells can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
  • the T cell receptor-modified cells can eliminate the tumor cells expressing the Her2/neu antigen and/or alter the microenvironment of the tumor tissue to induce other anti-tumor immune responses after entering the therapeutic subject.
  • the present invention also provides the use of the isolated T cell receptor for detecting proliferation or survival of the TCR-T cell in a patient treated with the TCR-modified T cell (ie, TCR-T cell), thereby performing Drug metabolism studies, and understanding the efficacy and toxicity of this TCR-T cell.
  • the TCR sequence can be used as a primer to detect the number of TCR-T cells carrying the TCR in vivo by a PCR method. Compared to methods in which fluorescently labeled HLA/polypeptide complex multimers are stained and analyzed by flow cytometry, the amount of cells required for such applications is less and more sensitive.
  • the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
  • the cell line used to prepare the lentiviral particles was 293T cells (ATCC CRL-3216).
  • the T cell strains used to detect TCR phenotype and function were Jurkat cells (clone E6-1, ATCC TIB-152) and J. RT3-T3.5 cells (ATCC TIB-153).
  • the presenting cell strain used to present the antigenic polypeptide was T2 cells (174xCEM.T2, ATCC CRL-1992).
  • the tumor cell lines for detecting TCR function are human colon cancer colo205 cells (ATCC CCL-222) and Caco-2 cells (ATCC HTB-37), human breast cancer MDA-MB-231 cells (ATCC HTB-26), human ovary Cancer SK-OV-3 cells (ATCC HTB-77), human lymphoma cells Bjab (ACC-757-DSMZ), human lung cancer H647 cells (ATCC CRL-5824) and H1355 cells (ATCC CRL-5865).
  • the cell line was maintained in RPMI-1640 complete medium (Lonza, cat#12-115F), and 10% calf serum FBS (ATCC 30-2020) was added to RPMI-1640 complete medium, 2mmol/L L-glutamic acid. , 100 ⁇ g/ml penicillin and 100 ⁇ g/ml streptomycin.
  • Peripheral blood products from healthy donors used in the trial were from the Pacific Blood Center in San Francisco (#1PBMC and #2 PBMC are Trima Residual Cell Components #R32334 and #R33941 from the Apheresis Collection Kit, respectively).
  • CTL Her2/neu 369-377-specific killer T cells
  • Peripheral blood was centrifuged ( ⁇ 400 g) by Ficoll-Paque Premium (Sigma-Alorich, cat# GE-17-5442-02) for 30 minutes.
  • Mononuclear cells PBMC
  • HLA-A2 phenotype of the cells was detected by fluorescein FITC-labeled anti-HLA-A2 antibody (Biolegend, cat#343303).
  • the RNA of the positive cells was extracted, reverse-transcribed into cDNA and cloned into the vector. Then, HLA gene sequencing analysis was performed to determine the cell matching type as HLA-A*0201.
  • the HLA-A2-positive PBMC cells were cultured in a culture well of a 24-well culture plate, and the culture solution was the above RPMI-1640 complete medium. 2 ⁇ 10e6/ml PBMC per well was added with Her2/neu 369-377 polypeptide (Her2-E75, synthesized with Peptide 2.0, 10 ⁇ g/ml dissolved in DMSO) at a concentration of 1 ⁇ g/ml.
  • cytokines were added, human IL-2 (Peprtech, cat#200-02) 100 IU/ml, human IL-7 (Peprotech) , cat#200-07) 5ng/ml, human IL-15 (Peprotech, cat#200-15) 5ng/ml.
  • 10 to 14 days of culture, antigen re-stimulation of cultured T cells 10e6 culture cells were added to each well in a 24-well plate, and 2 ⁇ 10e6 25 ⁇ g/ml mitomycin C (Santa Cruz Biotechnology) was added.
  • the T cell phenotype expressing the Her2/neu 369-377 specific TCR was analyzed by flow cytometry.
  • the cells to be tested were collected in 1.5 ml tubes (the number of cells was approximately 10e5) using 1 ml of DPBS solution (2.7 mM KCl, 1.5 mM KH 2 PO 4 , 136.9 mM NaCl, 8.9 mM Na 2 HPO 4 ⁇ 7H 2 O, pH 7.4) Wash once and reset in 100 ⁇ l of DPBS containing 1% calf serum, add 5 ⁇ l of fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat #300912), and 10 ⁇ l of fluorescein PE-labeled Her2- E75/HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co, cat#T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer,
  • the flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and the result analysis was performed using Flowjo software (Flowjo Corporation). T cell clones were obtained by single cell separation using a flow cytometer (FACS sorter). PBMCs stimulated with Her2/neu369-377 polypeptide antigen were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75/HLA-A2 pentamer, followed by flow cytometry (Model: Sony cell sorter SH800) .
  • HLA-A2 positive autologous was treated by treatment with 25 ⁇ g/ml mitomycin C for 2 hours.
  • PBMC cells 10e5 cells per well, were incubated with 1 ⁇ g/ml Her2/neu 369-377 polypeptide overnight, and then added RPMI-1640 containing IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml. Complete medium.
  • the culture medium containing the cytokine was replaced every 3-4 days, and it was observed under the microscope whether or not T cell clones grew.
  • the proliferating T cells are collected, subjected to antigen re-stimulation as described above to obtain a sufficient number of cells, subjected to phenotypic or functional detection, and extracted RNA for cloning of the TCR gene.
  • T cell function assay The function of the T cell strain modified by Her2/neu 369-377 polypeptide-specific TCR is detected by the expression of CD69 on the surface of T cells. 10 e5 TCR cells transfected with TCR gene and 10e5 T2 cells were added to each well of a 96-well plate, and mixed culture was carried out in 100 ⁇ l/well of RPMI-1640 complete medium, and each test group was a duplicate well.
  • Her2/neu 369- The 377 polypeptide was then cultured overnight in an incubator at 5% CO 2 at 37 °C. The cells were collected and suspended in DPBS + 1% FBS solution, stained with APC-labeled anti-human CD69 antibody, and subjected to flow cytometry analysis. T cells expressing CD69 after stimulation with Her2/neu 369-377 antigen are thought to carry and express a TCR specific for Her2/neu 369-377.
  • Her2/neu 369-377-specific CTL clone and the function of primary T cells transfected with the TCR gene in PBMC were determined by detecting gamma interferon secreted in the supernatant of the cell after antigen stimulation.
  • Her2/neu 369-377-specific CTL clones or PBMC cells transfected with TCR gene were mixed with target cells in 96-well plates in 100 ⁇ l/well RPMI-1640 complete medium, and the target cells were added 10 times.
  • RNA from T cell clones using the Zymo Quick-RNA Microprep kit (Zymo Research, cat#R1050), using this as a template to obtain cDNA using the Smarter RACE 5'/3' kit (Takara, USA) Bio, cat#634858).
  • PCR was carried out using 5'-CDS primer and TCR ⁇ chain 3' primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO: 26) and ⁇ chain 3' primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 27) TCR ⁇ and ⁇ full sequence gene fragments were added and cloned into pRACE vector (Takara Bio, cat#634858, USA). The competent bacteria Stellar (Takara Bio, cat #636763, USA) was transformed and plasmids were obtained and sequenced.
  • the viral vector for expression of TCR is a replication-deficient lentiviral vector, including: GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP), available from System Biosciences (Cat# CD811A-1); and the vector pCDH-EF1 ⁇ -MCS which does not express GFP, was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by a conventional technique in the art.
  • GFP-expressing lentiviral vector pCDH-EF1 ⁇ -MCS-(PGK-GFP) available from System Biosciences (Cat# CD811A-1)
  • the vector pCDH-EF1 ⁇ -MCS which does not express GFP was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector by a conventional technique in the art.
  • the TCR ⁇ chain and the ⁇ chain and the entire gene sequence of the cleavable F2A sequence and the Furin fragment are synthesized, and linked to the multiple cloning site downstream of the EF-1 ⁇ promoter of the vector
  • the transcription sequence of the inserted TCR is TCR ⁇ chain (no stop codon), Furin fragment, F2A fragment, TCR ⁇ chain (see the literature “Gene Ther. 2008 Nov; 15(21): 1411–1423”).
  • the vector expressing GFP is driven by the inverted PGK promoter.
  • the vector that does not express GFP removes the PGK promoter as well as the GFP fragment.
  • TCR lentiviral particles were obtained by transfecting 293T cells with Lipofectaine 3000 transfection reagent (Thermo Fisher, cat# L3000001). Prepare 293T cells and transfection procedures according to the manufacturer's instructions. The transfection was carried out in a 96-well culture plate. First, a liposome solution of the transfected plasmid was prepared using Opti-MEM 1 culture solution (Thermo Fisher Co., cat #51985091), and P3000 reagent (P3000 reagent) was added to 250 ⁇ l of the culture solution according to the manufacturer's instructions.
  • Lipofectaine 3000 transfection reagent Thermo Fisher, cat# L3000001
  • P3000 reagent P3000 reagent
  • the TCR lentiviral vector plasmid and the viral packaging plasmid of the pCDH system SBI, cat#LV500A-1
  • another 250 ⁇ l of the culture solution was added with Lipofectaine 3000 reagent, and mixed for 15 minutes, and then added to the 293T cell culture well.
  • the cells were cultured for 16 hours at 37 ° C under 5% CO 2 , and changed to DMEM medium (Thermo Fisher Company, cat #11965092) containing 10% FBS. After 24 hours and 48 hours, the cell supernatants were collected and centrifuged at 2000 g.
  • the virus particles obtained by filtration with a 0.4 ⁇ m filter membrane were used to infect cells.
  • Recombinant TCR lentivirus was transfected into human T cells: cryopreserved primary PBMC cells were thawed and cultured in RPMI-1640 complete medium for 24 hours, and the dead cells were removed by Ficoll-Paque Premium density gradient centrifugation ( ⁇ 400 g) for 30 minutes.
  • Example 1 Induction of Her2/neu 369-377 polypeptide-specific killer T cells from HLA-A2 positive normal donor peripheral blood
  • polypeptide-specific killer T cells were induced from normal PBMC (#2 PBMC) by two rounds of in vitro stimulation with a low concentration of Her2/neu 369-377 polypeptide at 1 ⁇ g/ml, and flow cytometry and single cell separation were performed. .
  • the specific method is as described above. The results are as follows:
  • the right panel of Figure 1A shows that 0.013% of lymphocytes are CD8-positive killer T cells that bind to Her2/neu 369-377/HLA-A2 pentamer (ie, Her2-E75 pentamer), and there is no Her2 polypeptide in the left panel.
  • the stimulated control cells did not show CD8 positive pentamer positive cells.
  • the results indicate that the number of specific T cells recognizing the Her2/neu 369-377 antigen polypeptide is small in the natural T cell pool. Despite the small number, this group of T cells recognizing the Her2/neu 369-377 polypeptide can still be clearly distinguished.
  • high-affinity T cells and low-affinity T cells are further contained in the positive cells.
  • 453 CD8-positive pentameric positive cells were isolated by flow cytometry and subjected to monoclonal culture. After two rounds of antigen polypeptide re-stimulation and cytokine amplification, only one proliferation was obtained from the 453 isolated single T cells.
  • T cell clone Her2 CTL clone 1B5 (referred to as Her2 CTL 1B5).
  • FIG. 1B right panel shows that this purified CD8 + CTL clone binds to the Her2/neu 369-377/HLA-A2 tetramer (i.e., Her2-E75 tetramer).
  • Her2/neu 369-377 antigen polypeptide presented by 5x10 3 T2 cells (Her2/neu 369-377 antigen polypeptide was 10-fold diluted from 1 ⁇ g/ml to obtain After mixed cultures at a final concentration of 1 ⁇ g/ml, 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, and 0.001 ⁇ g/ml, the IFN- ⁇ secreted by T cells in the supernatant was detected to determine the specificity of the T cell clone.
  • FIG. 1C shows that T cell clone 1B5 can be activated by a minimum concentration of 1 ng/ml of antigenic polypeptide, and the recognition function of T cells is positively correlated with the concentration of antigenic polypeptide (dose-dependent), indicating the specificity of this T cell clone.
  • the Her2/neu 369-377 polypeptide presented by HLA-A2 was identified. More importantly, this T cell clone also recognized a colon cancer cell line colo205 of HLA-A2 + Her2/neu + . Therefore, this T cell clone Her2 TCR-1B5 not only recognizes the Her-E75 polypeptide, but also activates by colo205 cells to secrete IFN- ⁇ .
  • This example obtained directly from the Her2/neu 369-377 polypeptide-specific CTL clone obtained in Example 1 containing ⁇ and ⁇ chains which are matched (i.e., the two strands can together constitute a functional TCR of the recognition antigen polypeptide).
  • the complete TCR gene sequence, which encodes a TCR is called "Her2 TCR-1B5".
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 2
  • the coding sequence is shown in SEQ ID NO: 1
  • the amino acid sequence of the ⁇ chain of the TCR is shown in SEQ ID NO: 4
  • the coding sequence is SEQ. ID NO: 3 is shown.
  • This TCR is present in the peripheral T cell pool of HLA-A2 positive normal humans and does not cross-react to normal cells that express Her2/neu protein in a small amount, resulting in an autoimmune response.
  • the specific detection method is as described above. The results are as follows:
  • FIG. 2 shows a schematic representation of the constructed TCR lentiviral vector structural fragment.
  • the alpha and beta chains are linked by a resectable furin recognition fragment and an F2A polypeptide fragment.
  • the lentiviral vector shown as "pCDH-EF1 ⁇ -Her2 TCR-(PGK-GFP) vector
  • PGK-GFP reverse PGK promoter Driven
  • Another expression vector (shown as "pCDH-EF1[alpha]-Her2 TCR vector") (bottom of Figure 2) removes GFP and its promoter sequences. By reducing the length of the vector to increase the production of lentiviral particles, the interaction between the two promoters is avoided, thereby increasing the expression of TCR.
  • the following sequences were ligated to the above-described GFP-expressing vector and the GFP-expressing vector, respectively: i) a nucleotide sequence of a TCR ⁇ -chain and an ⁇ -chain which are linked by a cleavable linker polypeptide and which have a disulfide bond added in the constant region ( SEQ ID NO: 13) (corresponding TCR is Her2 TCR-1B5-dis, amino acid sequence is set forth in SEQ ID NO: 14); ii) constant region linked by a cleavable linker polypeptide is replaced by a human sequence to a murine The nucleotide sequence of the TCR ⁇ chain and the ⁇ chain of the source sequence (SEQ ID NO: 15) (corresponding TCR is Her2 TCR-1B5-mC, amino acid sequence is shown as SEQ ID NO: 16); iii) by cleavability Linking the nucleotide sequence of the original TCR ⁇ chain and the ⁇ chain linked by the polypeptid
  • Her2 TCR-1B5-dis recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-mC recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-mC w/o GFP recombinant lentiviral vector without carrying GFP.
  • Her2 TCR-1B5-wt recombinant lentiviral vector (carrying GFP);
  • Her2 TCR-1B5-wt w/o GFP recombinant lentiviral vector without GFP.
  • Her2 TCR gene fragment was amplified by PCR and cloned into the downstream of the EF1- ⁇ promoter of the two lentiviral vectors (ie, pCDH-EF1 ⁇ -MCS-(PGK-GFP) and pCDH-EF1 ⁇ -MCS): Her2 TCR
  • the TCR fragments of -1B5-dis and TCR-1B5-wt were both 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 28) and 3' primer 5'-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 29 ) amplified.
  • the TCR fragment of Her2 TCR-1B5-mC was amplified by 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 30) and 3' primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID NO: 31). .
  • the PCR was carried out using a Q5 high-fidelity PCR kit (NEB, cat#M0543S). After the reaction conditions were 98 ° C for 30 seconds, 25 cycles of 98 ° C for 10 seconds, 65 ° C for 10 seconds, and 72 ° C for 3 minutes were performed.
  • the obtained TCR fragment was cloned into the MCS region downstream of the EF1 ⁇ promoter of the pCDH-EF1 ⁇ -MCS-(PGK-GFP) vector or the pCDH-EF1 ⁇ -MCS vector.
  • the recombinant TCR lentiviral expression vectors constructed were prepared as described above to obtain respective recombinant TCR lentiviral particles.
  • Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67(8):3898 - 903. The entire disclosure is incorporated herein by reference.
  • Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article.
  • FIG. 3A shows a portion of a T cell line transfected with a lentivirus carrying the Her2TCR-1B5 TCR and GFP genes (i.e., the Her2 TCR-1B5-dis recombinant lentiviral vector and the Her2 TCR-1B5-mC recombinant lentiviral vector).
  • GFP + cells can bind to the Her2-E75 tetramer, indicating that these Lentiviral-transfected T cells recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule.
  • FIG. 3B shows the exogenous TCR (Her2 TCR) obtained by transfecting J. RT-T3.5 cells expressing only the beta chain or Jurkat cells expressing both the ⁇ chain and the ⁇ chain, and the constant region was replaced by the mouse constant region sequence.
  • the expression level of -1B5-mC) was significantly higher than that of TCR (Her2 TCR-1B5-dis) which added only one disulfide bond in the constant region.
  • FIG. 3C shows that the T cell line expressing exogenous Her2 TCR-1B5 can be activated by T2 cell-expressing Her2/neu 369-377 polypeptide to express CD69, indicating that this TCR has the function of recognizing the Her2/neu 369-377 polypeptide antigen.
  • the expression level of the exogenous TCR (Her2 TCR-1B5-mC) obtained by replacing the constant region with the mouse constant region sequence is not only increased, but also the specific recognition ability for the polypeptide antigen is significantly higher than that of the TCR modified by the additional disulfide bond ( Her2 TCR-1B5-dis) can be activated by lower concentrations of Her2/neu 369-377 polypeptide.
  • the minimum polypeptide concentration for activating Jurkat cells expressing Her2 TCR-1B5-mC was approximately 0.05 ⁇ g/ml, which was approximately 50-fold higher than the polypeptide concentration of the activated Her2 CTL 1B5 clone shown in Figure 1C. This indicates that the ability of the Her2 TCR-1B5 TCR recognition polypeptide antigen expressed by Jurkat cells is significantly lower than that of the Her2 TCR-1B5 TCR expressed on CD8 + CTL.
  • One possible reason is that Jurkat cells do not express CD8 molecules, and CD8 plays an important supporting role in the recognition function of Her2 TCR-1B5.
  • Example 3 Normal peripheral blood T cells are transfected with Her2 TCR-1B5-mC recombinant lentivirus and express a specific TCR that recognizes the Her2/neu 369-377 polypeptide.
  • TCR obtained by the present invention can be expressed in primary T cells and has the function of recognizing the Her2/neu antigen polypeptide
  • recombinant lentiviral particles carrying the Her2 TCR-1B5-mC gene (Her2 TCR-1B5-mC recombination slow)
  • Viral vectors were transfected with peripheral blood T cells from two different normal donors activated by CD3/CD28 antibody, and cells were harvested 7 days later for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows:
  • FIG. 4A shows that both peripheral blood mononuclear cells (#1PBMC and #2 PBMC, respectively) can bind to Her2-E75 tetramer, indicating that Her2 TCR-1B5-mC expressed in these cells can be specific.
  • the Her2/neu antigen polypeptide presented by HLA-A2 is sexually recognized.
  • the results also showed that in the Her2-E75 tetramer-positive cells (ie, expressing Her2 TCR-1B5-mC), most of the positive cells were CD8 + T killer cells, and a small number of positive cells were CD8-like lymphocytes, which is likely CD4 + T helper cells.
  • the fluorescence intensity of CD8 + CTL binding to Her2-E75 tetramer (#2 in Geom.Mean in #2 PBMC samples, 1404 in Geom.Mean in #1PBMC samples) was also significantly greater than CD4 + T cells (#2 PBMC samples)
  • the Geom Mean in the 560 is 560, and the Geom.Mean in the #1PBMC sample is 504).
  • transfection efficiency of lentivirus-infected CD8 + and CD4 + T cells is the same, it indicates that a part of the exogenous Her2/neu 369-377-specific TCR expressed on CD4 + cells does not bind to Her2-E75 tetramer, even if bound, Affinity is also lower than exogenously transfected TCR expressed on CD8 + T cells. This further demonstrates that the transfected TCR requires the auxiliary function of the CD8 molecule to effectively bind the Her2/HLA-A2 complex.
  • TCR-transfected PBMC cells were added to each well of a 96-well plate, and Her2/neu 369-377 antigenic peptide (Her2/neu 369-377 antigen) was presented at different concentrations in T2 cells (10e5 per well).
  • the polypeptide was 10-fold diluted from 0.1 ⁇ g/ml to obtain a final concentration of 0.1 ⁇ g/ml, 0.01 ⁇ g/ml, and 0.001 ⁇ g/ml.
  • the IFN- ⁇ secreted by T cells in the supernatant was detected. To determine the function of this TCR-expressing PBMC cell to specifically recognize the Her2/neu 369-377 polypeptide.
  • the target cells of the control group were T2 cells which presented 1 ⁇ g/ml of the EBV virus antigen polypeptide LMP2 426-434 which can bind to the HLA-A2 molecule.
  • Figure 4B shows that PBMC expressing Her2 TCR-1B5-mC can be activated by T2 cell-derived Her2/neu 369-377 antigen polypeptide to secrete IFN- ⁇ , indicating the expression of exogenous Her2 TCR-1B5-mC primary T
  • the cells can specifically recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule.
  • the ability to recognize antigenic polypeptides correlates with the amount of exogenous TCR expressed on T cells.
  • FIG. 4C shows that T cell secreting IFN- ⁇ is significantly inhibited by the addition of anti-human CD8 antibody when T cells are co-cultured with T2 cell-presenting antigen polypeptide (T2+Her2-E75, ie Her2/neu 369-377 polypeptide). .
  • T2+Her2-E75 T2 cell-presenting antigen polypeptide
  • Her2/neu 369-377 polypeptide T2 cell-presenting antigen polypeptide
  • Example 4 Her2/neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells transfected with Her2 TCR-1B5-mC recombinant lentivirus recognizes HLA-A2 + Her2/neu + tumor cells
  • 10e5 PBMCs transfected with Her2 TCR-1B5-mC w/o GFP recombinant lentivirus were added to each well of a 96-well plate (shown as #2 PBMC transfected with Her2 TCR-1B5), or 10e5 control #2 PBMCs that were not transfected with TCR were mixed with 10e5 different tumor cell lines, and then the secreted IFN- ⁇ was detected in the supernatant.
  • the specific method is as described above. The results are as follows:
  • FIG. 5A shows that T cells expressing Her2 TCR-1B5-mC can be activated by HLA-A2 + Her2/neu + tumor cell lines and secrete IFN- ⁇ .
  • Tumor cell lines include colon cancer Colo205 cells, breast cancer MDA- MB-231 cells, colon cancer Caco-2 cells, and lung cancer H1355 cells.
  • HLA-A2 - Her2/neu + ovarian cancer SK-OV3 cells, lung cancer H647 cells, and HLA-A2 + Her2/neu - lymphoma Bjab cells do not activate T cells transfected with Her2 TCR-1B5-mC TCR .
  • Her2 TCR-1B5-mC TCR can specifically recognize the Her2/neu antigen presented by HLA-A2 on the surface of tumor cells.
  • Control T cells from the same donor PBMC that were cultured in parallel but not transfected with Her2 TCR-1B5-mC were not activated by the listed tumor cell lines, indicating that the response to tumor cells was not non-specific.
  • Figure 5B shows that T cells expressing Her2 TCR-1B5-mC TCR (#2 PBMC transfected with Her2 TCR-1B5) showed significant responses to colo205 cells, and recognition functions were enhanced by anti-CD8 antibodies and anti-HLA molecules.
  • the antibody is almost completely blocked, which further indicates that the effector cells that recognize the Her2/neu antigen on the surface of tumor cells are CD8 + killer T cells, and their specific antigen recognition function depends on the auxiliary function of CD8, which is also related to the recognition of T2 cells.
  • the Her2/neu 369-377 polypeptide antigen was consistent in Her2 TCR-1B5-mC TCR.
  • the difference in sensitivity of different tumor cell lines to specific T cells may be related to the expression of different levels of Her2/neu antigen polypeptide/HLA-A2 complex in tumor cells, and may also be related to the inhibition of T cell function by tumor cells themselves.
  • the high-affinity TCR that specifically recognizes the Her2/neu 369-377 polypeptide can be obtained by in vitro induction of the Her2/neu 369-377 polypeptide, these high-affinity TCRs often fail to recognize the Her2/neu presented by the tumor cells.
  • Antigen Cancer Res. 1998; 58: 4902 - 4908. Cancer Immunol. Immunother. 2008; 57: 271 - 280).
  • Her2/neu 369-377 polypeptide binding HLA-A2 molecule differs from the configuration of the polypeptide/HLA complex presented in the cell (see the journal “Journal of Immunology, 2008, 180". :8135–8145”).
  • the Her2/neu 369-377 polypeptide acts as a mimotope antigen, and the specific TCR induced can recognize both Her2/neu 369-377 polypeptide and similar polypeptides presented by tumor cells.
  • the Her2/neu 373-382 polypeptide see the literature "J Immunol.
  • TCR Her2/neu 369-
  • the 377 polypeptide has high affinity but does not effectively recognize the corresponding mimotope polypeptide presented by the tumor cell.
  • the TCR specifically recognizing the Her2/neu 369-377 polypeptide of the present invention not only targets the Her2/neu 369-377 polypeptide proposed by the tumor cell, but also recognizes the Her2/ derived from the tumor cell.
  • Other mimotope polypeptides of neu are also recognized.
  • TCRs that naturally exist in the peripheral T cell pool that recognize the Her2/neu antigen are mostly low-intensity.
  • Another high-affinity TCR that recognizes CD8 function-independent high-affinity TCRs from tumor cells is paired by multiple alpha and beta chains of the Her2/neu 373-382 polypeptide-specific T cell population. (See the literature "HUMAN GENE THERAPY 2024, 25: 730-739"; WO/2017/133779). Since it is not directly obtained from specific monoclonal T cells, it is not possible to determine whether this TCR is present in the peripheral natural T cell pool.
  • TCRs high-affinity TCRs
  • function of these TCRs does not depend on the helper function of CD8, and thus the complementation of CD8 + killer T cell function can be obtained by transfecting CD4 + T cells.
  • CD8 function-dependent TCR the same purpose can be achieved by simultaneously expressing the TCR and the exogenous CD8 molecule by the expression vector.
  • the present invention provides a Her2/neu 373-382 polypeptide-specific TCR ⁇ chain and ⁇ chain full sequence induced from an in vitro pericellular T cell bank of HLA-A2 + , which is expressed and expressed after transfection.
  • Primary killer T cells of the region-modified TCR can recognize a variety of HLA-A2 + Her2/neu + tumor cells.
  • New methods and approaches have been provided for the development and clinical application of adoptive transfer of specific TCR-modified T cells to treat tumors.

Abstract

Provided are an isolated T-cell receptor, a cell modified by same, coding nucleic acids, an expression vector, a preparation method, a pharmaceutical composition, and applications. The isolated T-cell receptor (TCR) comprises at least one of an α chain and a β chain, both the α chain and the β chain comprising a variable region and a constant region, and is characterized in that the T cell receptor specifically identifies antigen Her2/neu expressed by tumor cells, the amino acid sequence of the variable region of the α chain has a consistency of at least 98% with the amino acid sequence as represented by SEQ ID NO: 19, and the amino acid sequence of the variable region of the β chain has a consistency of at least 98% with the amino acid sequence as represented by SEQ ID NO: 20. The TCR specifically identifies the tumor antigen and, at the same time, prevents any possible off-target toxicity reaction. The use of the TCR in modifying an immune cell provides significant antitumor effects.

Description

分离的T细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用Isolated T cell receptor, modified cell thereof, nucleic acid encoding vector, expression vector, preparation method, pharmaceutical composition and application 技术领域Technical field
本发明属于生物技术领域，具体而言，涉及分离的T细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用。The present invention is in the field of biotechnology, and in particular, relates to an isolated T cell receptor, a modified cell thereof, a nucleic acid encoding, an expression vector, a preparation method, a pharmaceutical composition, and an application.
背景技术Background technique
Her2/neu(ERBB2)是属于EGFR家族的一个跨膜蛋白，和家族其它蛋白形成二聚体并通过一系列细胞内信号传导途径来调控细胞增值，分化以及癌变等过程(参见文献“Growth Factors,2008；26:263”、“Oncol Biol.Phys,2004；58:903”)。Her2/neu蛋白在多种上皮来源的癌细胞中过度表达，如乳腺癌，胃癌，大肠癌，卵巢癌，胰腺癌，肺癌，食管癌，膀胱癌，肾癌等(参见文献“Trends in Molecular Med,2013；19:677”)，并且在原发灶和转移灶癌细胞中的表达相对均一(参见文献“J Clin Oncol,1998；8:103”)，因此，Her2/neu成为靶向治疗的适当靶点。Her2/neu (ERBB2) is a transmembrane protein belonging to the EGFR family, which forms dimers with other family proteins and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signaling pathways (see literature "Growth Factors," 2008;26:263", "Oncol Biol. Phys, 2004; 58: 903"). Her2/neu protein is overexpressed in a variety of epithelial-derived cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (See "Trends in Molecular Med" , 2013; 19:677"), and the expression in primary and metastatic cancer cells is relatively uniform (see the literature "J Clin Oncol, 1998; 8: 103"), therefore, Her2/neu becomes targeted therapy Appropriate target.
靶向Her2/neu的人源化单抗药物Herceptin可以显著延长Her2/neu阳性的乳腺癌患者生存期(参见文献“N Engl J Med,2001,344:783”)，然而单独使用Herceptin治疗Her2阳性的转移乳腺癌的临床反应率只有11％到26％(参见文献“J Clin Oncol,2002；20:7169”)，表明单用Heceptin对大部分Her2高表达的转移乳腺癌的疗效并不理想。尽管Heceptin联合化疗可提高临床应答率，但大多数Her2/neu过度表达的乳腺癌病人在一年后会对Heceptin产生抗性(参见文献“J Clin Oncol,2001；19:2587”)。The humanized monoclonal antibody Herceptin targeting Her2/neu can significantly prolong the survival of Her2/neu-positive breast cancer patients (see the literature "N Engl J Med, 2001, 344: 783"), whereas Herceptin alone is used to treat Her2 positive The clinical response rate for metastatic breast cancer is only 11% to 26% (see the literature "J Clin Oncol, 2002; 20: 7169"), indicating that Heceptin alone is not ideal for most Her2 high-expressing metastatic breast cancer. Although Heceptin combined with chemotherapy increases clinical response rates, most Her2/neu overexpressing breast cancer patients develop resistance to Heceptin after one year (see the literature "J Clin Oncol, 2001; 19: 2587").
Her2/neu阳性肿瘤患者体内会产生针对Her2/neu抗原的内源性抗体和T细胞反应(参见文献“Cancer Res,2005；65:650”)，因而，靶向Her2/neu抗原的特异性免疫治疗成为一种颇有前景的治疗手段。 特异性识别Her2/neu抗原表位多肽(epitope peptide)369-377的T细胞可以从Her2/neu高表达的卵巢癌腹水中成功分离(参见文献“J.Exp.Med.1995；181:2109–2117”)。靶向Her2/neu 369-377多肽抗原的肿瘤疫苗进入临床试验，尽管临床1/2期显示此疫苗可以诱导出针对Her2/neu 369-377多肽抗原的特异性T杀伤细胞(参见文献“Breast Care,2016；11:116”)，但临床三期却没达到延长病人生存期的预定目标(http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast-cancer-halted-after-futility-review)。过继传输经体外培养的，基于嵌合抗原受体(chimeric antigen receptors，CARs)的肿瘤特异性T细胞疗法被开发后，作为第一个针对实体瘤的，靶向Her2/neu抗原的CAR-T细胞疗法进入临床试验，但由于产生强烈的细胞因子风暴(cytokine release syndrome，CRS)导致病人死亡而被终止(参见文献“Nature Med,2016；22:26”)。严重的细胞因子风暴以及神经毒性是CAR-T治疗中常见的毒性反应(参见文献“Blood,2016；127:3321”)，部分原因可能和CAR这种非天然的T细胞受体不受限制的细胞活化有关(参见文献“Nat Ned,2015；21:581”)，或者与不需抗原刺激的细胞因子的自分泌有关(参见文献“Cancer Immunol Res,2015；3:356”)。Endogenous antibodies and T cell responses to Her2/neu antigens are produced in Her2/neu-positive tumor patients (see the literature "Cancer Res, 2005; 65:650"), thus, specific immunity against Her2/neu antigens Treatment has become a promising treatment. T cells that specifically recognize the Her2/neu epitope peptide 369-377 can be successfully isolated from the ovarian cancer ascites with high expression of Her2/neu (see the literature "J. Exp. Med. 1995; 181: 2109 - 2117"). Tumor vaccines targeting the Her2/neu 369-377 polypeptide antigen enter clinical trials, although clinical phase 1/2 shows that this vaccine can induce specific T killer cells against the Her2/neu 369-377 polypeptide antigen (see literature "Breast Care" , 2016; 11:116"), but the clinical phase III did not achieve the intended goal of extending patient survival (http://www.onclive.com/web-exclusives/phase-iii-nelipepimuts-study-in-breast- Cancer-halted-after-futility-review). Adoptive transfer of in vitro cultured, chimeric antigen receptors (CARs)-based tumor-specific T-cell therapies, developed as the first CAR-T targeting Her2/neu antigens for solid tumors Cell therapy enters clinical trials but is terminated by a strong cytokine release syndrome (CRS) leading to death (see "Nature Med, 2016; 22:26"). Severe cytokine storms and neurotoxicity are common toxicities in CAR-T therapy (see the literature "Blood, 2016; 127: 3321"), partly due to the unrestricted CAR, a non-native T cell receptor. Cell activation is involved (see the literature "Nat Ned, 2015; 21: 581") or with autocrine cytokine that does not require antigen stimulation (see the literature "Cancer Immunol Res, 2015; 3: 356").
过继转输经过特异性T细胞受体(即TCR)基因修饰的T细胞的TCR-T疗法被认为是针对实体瘤最具前景的免疫细胞基因疗法(参见文献“Adv Immunol.2016；130:279-94”)。其中，靶向NY-ESO-1抗原的TCR-T疗法的临床研究显示出令人鼓舞的临床治疗效果(参见文献“Nat Med.2015 Aug；21(8):914-921”)。然而，目前已知的靶向肿瘤抗原、并有效识别肿瘤细胞的特异性TCR的数量十分有限，因此限制了TCR-T疗法的广泛应用。另外，尽管TCR-T疗法没有出现CAR-T疗法中所表现出的严重的细胞因子风暴毒性，但如果靶抗原来源于自身蛋白，针对正常组织细胞中低表达的靶抗原有可能会导致严重的自身免疫反应，即转接脱靶(或称中靶脱瘤)(on target off tumor)毒性反应(Blood 2009；114:535-546)。另外，为了获得有效识别肿瘤细胞的高亲和性TCR，一般的策略是体外通过对TCR上的互补性决定区(complementarity determing regions,CDRs)进行基因点突变，或者通 过从未经过中枢耐受机制筛选的人源化小鼠T细胞库中进行诱导(参见文献“Front Immunol.2013；4:363”)。然而，这种高亲和性TCR可能会对正常自身蛋白产生交叉反应而导致对正常组织细胞的杀伤作用，即严重甚至致命的脱靶(off-target)毒性(参见文献“Curr Opin Immunol 2015；33:16–22”、参见文献“Sci Transl Med.2013；5(197):197ra103”)。因此，获得特异性靶向肿瘤抗原并有效识别肿瘤细胞，同时避免可能出现的脱靶毒副反应的新型TCR基因，是成功开发TCR-T免疫细胞基因疗法面临的主要挑战。TCR-T therapy for adoptive transfer of T cells transfected with specific T cell receptor (ie TCR) genes is considered to be the most promising immune cell gene therapy for solid tumors (see the literature "Adv Immunol. 2016; 130:279 -94"). Among them, clinical studies of TCR-T therapy targeting NY-ESO-1 antigen have shown encouraging clinical therapeutic effects (see the literature "Nat Med. 2015 Aug; 21(8): 914-921"). However, the number of specific TCRs currently known to target tumor antigens and to efficiently recognize tumor cells is very limited, thus limiting the widespread use of TCR-T therapies. In addition, although TCR-T therapy does not exhibit the severe cytokine storm toxicity exhibited by CAR-T therapy, if the target antigen is derived from a self-protein, targeting a target antigen that is low in normal tissue cells may cause serious Autoimmune response, ie on target off tumor toxicity (Blood 2009; 114: 535-546). In addition, in order to obtain a high-affinity TCR that effectively recognizes tumor cells, a general strategy is to perform point mutations in the complementarity determing regions (CDRs) on the TCR in vitro, or by never undergoing a central tolerance mechanism. Induction was performed in a screened humanized mouse T cell pool (see the literature "Front Immunol. 2013; 4:363"). However, this high-affinity TCR may cross-react with normal self-proteins leading to killing of normal tissue cells, ie severe or even fatal off-target toxicity (see the literature "Curr Opin Immunol 2015; 33" :16–22”, see the document “Sci Transl Med. 2013; 5(197): 197ra103”). Therefore, obtaining a novel TCR gene that specifically targets tumor antigens and effectively recognizes tumor cells while avoiding possible off-target toxic side effects is a major challenge in successfully developing TCR-T immune cell gene therapy.
发明内容Summary of the invention
为解决上述现有技术中所存在的问题，本发明提供了分离的T细胞受体、其修饰的细胞、编码核酸、表达载体、制备方法、药物组合物和应用。In order to solve the problems in the prior art described above, the present invention provides isolated T cell receptors, modified cells thereof, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions, and uses.
具体而言，本发明提供了：In particular, the present invention provides:
(1)一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％的一致性。(1) An isolated T cell receptor comprising at least one of an α chain and a β chain, the α chain and the β chain each comprising a variable region and a constant region, characterized in that the T cell receptor The antigen Her2/neu expressed by the tumor cell can be specifically recognized, and the amino acid sequence of the variable region of the α chain has at least 98% identity with the amino acid sequence shown in SEQ ID NO: 19, the β The amino acid sequence of the variable region of the strand has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
(2)根据(1)所述的T细胞受体，其中所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:18所示的Her2/neu 369-377。(2) The T cell receptor according to (1), wherein the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule; Yes, the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
(3)根据(1)所述的T细胞受体，其中所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。(3) The T cell receptor according to (1), wherein the constant region of the α chain and/or the constant region of the β chain is derived from a human; preferably, the α chain The constant region is replaced in whole or in part by homologous sequences derived from other species, and/or the constant region of the beta strand is replaced in whole or in part by homologous sequences derived from other species; more preferably The other species are mice.
(4)根据(1)所述的T细胞受体，其中所述α链的所述恒定区 修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区修饰有一个或多个二硫键。(4) The T cell receptor according to (1), wherein the constant region of the α chain is modified with one or more disulfide bonds, and/or the constant region modification of the β chain has one Or multiple disulfide bonds.
(5)根据(1)所述的T细胞受体，其中所述α链的氨基酸序列如SEQ ID NOs:2、6或10所示，所述β链的氨基酸序列如SEQ ID NOs:4、8或12所示。(5) The T cell receptor according to (1), wherein the amino acid sequence of the α chain is as shown in SEQ ID NOs: 2, 6, or 10, and the amino acid sequence of the β chain is SEQ ID NOs: 4, 8 or 12 is shown.
(6)一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％的一致性。(6) An isolated nucleic acid encoding a T cell receptor comprising a coding sequence of at least one of an α chain and a β chain of said T cell receptor, said α chain coding sequence and said β chain coding sequence Including a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing an antigen Her2/neu expressed by a tumor cell, and the amino acid sequence encoded by the α chain variable region coding sequence has At least 98% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the β chain variable region coding sequence having at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
(7)根据(6)所述的核酸，其中所述核酸为DNA或RNA。(7) The nucleic acid according to (6), wherein the nucleic acid is DNA or RNA.
(8)根据(6)所述的核酸，其中所述α链可变区编码序列如SEQ ID NO:21所示，所述β链可变区编码序列如SEQ ID NO:22所示。(8) The nucleic acid according to (6), wherein the α chain variable region coding sequence is represented by SEQ ID NO: 21, and the β chain variable region coding sequence is represented by SEQ ID NO: 22.
(9)根据(6)所述的核酸，其中被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:18所示的Her2/neu 369-377。(9) The nucleic acid according to (6), wherein the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule; Preferably, the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
(10)根据(6)所述的核酸，其中所述α链恒定区编码序列和/或所述β链恒定区编码序列来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。(10) The nucleic acid according to (6), wherein the α chain constant region coding sequence and/or the β chain constant region coding sequence is derived from a human; preferably, the α chain constant region coding sequence is wholly or partially The ground is replaced by a homologous sequence derived from other species, and/or the beta strand constant region coding sequence is replaced in whole or in part by homologous sequences derived from other species; more preferably, the other species are small mouse.
(11)根据(6)所述的核酸，其中所述α链恒定区编码序列包含一个或多个二硫键编码序列，并且/或者所述β链恒定区编码序列包含一个或多个二硫键编码序列。(11) The nucleic acid according to (6), wherein the α chain constant region coding sequence comprises one or more disulfide bond coding sequences, and/or the β chain constant region coding sequence comprises one or more disulfide Key coding sequence.
(12)根据(6)所述的核酸，其中所述α链编码序列如SEQ ID NOs:1、5或9所示，所述β链编码序列如SEQ ID NOs:3、7或11 所示。(12) The nucleic acid according to (6), wherein the α chain coding sequence is represented by SEQ ID NOs: 1, 5 or 9, and the β chain coding sequence is as shown in SEQ ID NOs: 3, 7 or .
(13)根据(6)-(11)中任一项所述的核酸，其中所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接。The nucleic acid according to any one of (6) to (11), wherein the α chain coding sequence and the β chain coding sequence are linked by a coding sequence of a cleavable linker polypeptide.
(14)根据(13)所述的核酸，其序列如SEQ ID NOs:13、15、或23所示。(14) The nucleic acid according to (13), which has the sequence shown in SEQ ID NOs: 13, 15, or 23.
(15)一种重组表达载体，其含有与启动子有效连接的、根据(6)-(14)中任一项所述的核酸，和/或其互补序列。(15) A recombinant expression vector comprising the nucleic acid according to any one of (6) to (14), and/or a complement thereof, which is operably linked to a promoter.
(16)一种T细胞受体修饰的细胞，该细胞的表面被(1)-(5)中任一项所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。(16) A T cell receptor-modified cell, the surface of which is modified by the T cell receptor according to any one of (1) to (5), wherein the cell comprises a primitive T cell or a precursor thereof Cells, NKT cells, or T cell lines.
(17)一种制备根据(16)所述的T细胞受体修饰的细胞的方法，包括以下步骤：(17) A method of producing a T cell receptor-modified cell according to (16), comprising the steps of:
1)提供细胞；1) providing cells;
2)提供编码根据(1)-(5)中任一项所述的T细胞受体的核酸；2) A nucleic acid encoding the T cell receptor according to any one of (1) to (5);
3)将所述核酸转染入所述细胞中。3) Transfecting the nucleic acid into the cell.
(18)根据(17)所述的方法，其中步骤1)所述的细胞来自自体或异体。(18) The method according to (17), wherein the cell of step 1) is derived from an autologous or allogeneic body.
(19)根据(17)所述的方法，其中所述转染的方式包括：采用病毒载体转染的方式，优选的是，所述病毒载体包括γ逆转录病毒载体或慢病毒载体；化学方式，优选的是，所述化学方式包括采用脂质体转染的方式；物理方式，优选的是，所述物理方式包括电转染方式。(19) The method according to (17), wherein the method of transfection comprises: transfection with a viral vector, preferably, the viral vector comprises a γ retroviral vector or a lentiviral vector; Preferably, the chemical means comprises a method of transfection with a liposome; physically, preferably, the physical means comprises an electrotransfection mode.
(20)根据(17)所述的方法，其中步骤2)所述的核酸为根据(6)-(14)中任一项所述的核酸。(20) The method of (17), wherein the nucleic acid according to any one of (6) to (14).
(21)根据(16)所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。(21) Use of the T cell receptor-modified cell according to (16) for the preparation of a medicament for treating or preventing a tumor and/or cancer.
(22)根据(21)所述的用途，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。(22) The use according to (21), wherein the tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive.
(23)根据(16)所述的T细胞受体修饰的细胞在制备用于检 测宿主的肿瘤和/或癌症的药物中的用途。(23) Use of the T cell receptor-modified cell according to (16) for the preparation of a medicament for detecting a tumor and/or cancer of a host.
(24)一种药物组合物，其中该药物组合物包括作为活性成分的根据(16)所述的T细胞受体修饰的细胞，及可药用辅料。(24) A pharmaceutical composition comprising the T cell receptor-modified cell according to (16) as an active ingredient, and a pharmaceutically acceptable excipient.
(25)根据(24)所述的药物组合物，其中所述药物组合物包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞。 (25) The pharmaceutical composition according to (24), wherein the pharmaceutical composition comprises the T cell having a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course per patient. Receptor modified cells.
(26)根据(24)所述的药物组合物，其中所述药物组合物适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。(26) The pharmaceutical composition according to (24), wherein the pharmaceutical composition is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
(27)一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据(16)所述的T细胞受体修饰的细胞。(27) A method of treating a tumor and/or a cancer comprising administering a T cell receptor-modified cell according to (16) to a tumor and/or a cancer patient.
(28)根据(27)所述的方法，其中所述T细胞受体修饰的细胞的施用剂量为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。 (28) The method according to (27), wherein the T cell receptor-modified cells are administered at a dose of 1 × 10 3 - 1 × 10 9 cells/kg body weight per patient per course of treatment. .
(29)根据(27)所述的方法，其中所述T细胞受体修饰的细胞通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。(29) The method according to (27), wherein the T cell receptor-modified cell passes through an artery, a vein, a subcutaneous, an intradermal, an intratumoral, a lymphatic vessel, a lymph node, a subarachnoid space, a bone marrow, Intramuscular and intraperitoneal administration.
(30)根据(27)所述的方法，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。(30) The method according to (27), wherein the tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive.
本发明与现有技术相比具有以下优点和积极效果：Compared with the prior art, the invention has the following advantages and positive effects:
本发明从HLA-A2阳性的健康供体外周血中成功诱导出对HLA-A2提呈的Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)有特异性的T细胞克隆，并从中筛选出携带有特异性识别Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)的天然TCR的T细胞克隆，进而获得了该TCR全序列。此TCR属于CD8分子依赖性，对Her2/neu抗原表位多肽(如Her2/neu 369-377多肽)具有中等亲和性，可特异性识别HLA-A2阳性并表达Her2/neu抗原的肿瘤细胞。另外，携带此TCR的T细胞克隆经过中枢免疫耐受机制筛选后进入外周T细胞库。携带此TCR的杀伤T细胞曾存在于正常人外周血，并未对 微量表达Her2/neu蛋白的正常组织细胞产生交叉反应。因此，本发明获得了能够特异性识别肿瘤抗原，同时能够避免可能出现的脱靶毒副反应的新型TCR。The present invention successfully induces T cell clones specific for HLA-A2-presented Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide) from HLA-A2-positive healthy donor peripheral blood. A T cell clone carrying a native TCR that specifically recognizes a Her2/neu epitope polypeptide (such as Her2/neu 369-377 polypeptide) is screened therefrom, and the entire TCR sequence is obtained. This TCR belongs to CD8 molecule-dependent, has a moderate affinity for Her2/neu epitope polypeptides (such as Her2/neu 369-377 polypeptide), and specifically recognizes HLA-A2 positive and expresses Her2/neu antigen-bearing tumor cells. In addition, T cell clones carrying this TCR were screened by the central immune tolerance mechanism and entered the peripheral T cell bank. The killer T cells carrying this TCR were present in normal human peripheral blood and did not cross-react to normal tissue cells that express Her2/neu protein in a small amount. Thus, the present invention achieves a novel TCR that is capable of specifically recognizing tumor antigens while avoiding possible off-target toxic side effects.
在进一步的发明中还对TCR的恒定区进行了改造(例如进行二硫键修饰或鼠源化改造)，以使得此TCR在免疫细胞上表达时能够进一步减少或避免与内源TCR错配的发生。In a further invention, the constant region of the TCR is also engineered (eg, by disulfide modification or murine transformation) to further reduce or avoid mismatching with the endogenous TCR when expressed on immune cells. occur.
用此TCR修饰的免疫细胞(例如原始T细胞、其前体细胞、NKT细胞、T细胞株)可特异性识别多种HLA-A2 +且Her2/neu +的肿瘤细胞株，具有显著的抗肿瘤效果。另一方面，用于修饰免疫细胞的TCR不会对微量表达Her2/neu的正常细胞产生交叉反应。因此，基于此TCR的TCR-T疗法可望治疗多种实体瘤。 Immunological cells modified with this TCR (eg, primitive T cells, precursor cells, NKT cells, T cell strains) can specifically recognize a variety of HLA-A2 + and Her2/neu + tumor cell lines with significant anti-tumor effect. On the other hand, the TCR used to modify immune cells does not cross-react to normal cells that express Her2/neu in a small amount. Therefore, TCR-T therapy based on this TCR is expected to treat a variety of solid tumors.
用本发明的TCR修饰的免疫细胞治疗肿瘤时，可有效避免采用CAR-T治疗时所引起的细胞因子风暴和免疫排斥反应。When treating tumors with the TCR-modified immune cells of the present invention, cytokine storms and immune rejection caused by CAR-T treatment can be effectively avoided.
本发明的TCR修饰的免疫细胞为治疗HLA-A2 +且Her2/neu +肿瘤患者提供了一种新的选择，具有良好的产业应用前景。 The TCR-modified immune cells of the invention provide a new choice for treating HLA-A2 + and Her2/neu + tumor patients, and have good industrial application prospects.
附图说明DRAWINGS
图1示出本发明实施例1中从HLA-A2 +正常供体PBMC(具体为#2 PBMC)中诱导出的Her2/neu 369-377多肽(Her2-E75)特异性杀伤性T细胞的表型和功能检测结果。图1A为经过两轮Her2-E75抗原多肽体外刺激后，PBMC细胞经CD8-APC抗体和Her2-E75五聚体-PE染色后进行流式细胞分析结果，右图是经多肽刺激的细胞，对CD8 +五聚体 +杀伤T细胞群进行FACS分选，以获得T细胞克隆。左图为没有多肽刺激的对照组细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75五聚体的荧光强度。图1B为CD8 +E75-tetramer +杀伤T细胞克隆经CD8-APC和Her2-E75四聚体-PE染色后流式细胞的表型分析，右图显示CD8 +Her2四聚体 +T细胞克隆Her2 CTL 1B5为纯化的Her2-E75多肽特异性CTL细胞克隆。左图为没有多肽刺激的对照组CTL细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75四聚体的荧光强度。图1C为T细胞克隆功能检测结 果，T细胞克隆Her2 CTL 1B5(斜线柱图)或者没有多肽刺激的对照CTL细胞(点柱图)与被T2细胞提呈的不同浓度Her-E75多肽共培养，或者与HLA-A2 +Her2/neu +的大肠癌细胞株colo205共培养后，取细胞上清进行IFN-γ的ELISA分析，各试验组和对照组均为复孔，结果显示为平均值±SD。横坐标表示不同实验组别，纵坐标表示T细胞分泌IFN-γ的浓度。 1 shows a table of Her2/neu 369-377 polypeptide (Her2-E75)-specific killer T cells induced from HLA-A2 + normal donor PBMC (specifically #2 PBMC) in Example 1 of the present invention. Type and function test results. Figure 1A shows the results of flow cytometry analysis of PBMC cells stained with CD8-APC antibody and Her2-E75 pentamer-PE after two rounds of Her2-E75 antigen polypeptide stimulation. The right panel is stimulated by peptides. The CD8 + pentamer + killer T cell population was subjected to FACS sorting to obtain T cell clones. The left panel shows control cells without polypeptide stimulation. The abscissa indicates the fluorescence intensity of CD8 molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 pentamer. Figure 1B shows the phenotypic analysis of flow cytometry of CD8 + E75-tetramer + killer T cell clones stained with CD8-APC and Her2-E75 tetramer-PE. The right panel shows CD8 + Her2 tetramer + T cell clone Her2 CTL 1B5 is a purified Her2-E75 polypeptide-specific CTL cell clone. The left panel shows control CTL cells without polypeptide stimulation. The abscissa indicates the fluorescence intensity of CD8 molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer. Figure 1C shows the results of T cell clonal function assay. T cell clone Her2 CTL 1B5 (slanted bar graph) or control CTL cells without peptide stimulation (point bar graph) were co-cultured with different concentrations of Her-E75 polypeptide presented by T2 cells. Or co-cultured with HLA-A2 + Her2/neu + colon cancer cell line colo205, the cell supernatant was taken for ELISA analysis of IFN-γ, and each test group and control group were duplicated, and the results were shown as mean ± SD. The abscissa indicates the different experimental groups, and the ordinate indicates the concentration of T cells secreting IFN-γ.
图2示出所构建的两种携带Her2 TCR-1B5 TCR基因的慢病毒载体(图中分别示为“pCDH-EF1α-Her2 TCR-(PGK-GFP)载体”、“pCDH-EF1α-Her2 TCR载体”)的主要功能片段。上方示出的片段同时表达EF-1α启动子所驱动的TCR基因和PKG启动子所驱动的GFP基因，下方示出的片段只表达TCR基因。各TCR的β链和α链均被可切割性连接多肽的编码序列(furin-F2A)所链接。Figure 2 shows two constructed lentiviral vectors carrying the Her2 TCR-1B5 TCR gene (shown as "pCDH-EF1α-Her2 TCR-(PGK-GFP) vector" and "pCDH-EF1α-Her2 TCR vector, respectively" The main functional fragment. The fragment shown above simultaneously expresses the TCR gene driven by the EF-1α promoter and the GFP gene driven by the PKG promoter, and the fragment shown below expresses only the TCR gene. The beta and alpha chains of each TCR are linked by the coding sequence of the cleavable linker polypeptide (furin-F2A).
图3示出经Her2 TCR-1B5 TCR基因转染的T细胞株的表型和功能检测结果。用编码Her2 TCR-1B5 TCR和GFP的慢病毒载体转染J.RT3-T3.5 T细胞株(J.RT3)，经Her2-E75四聚体-PE染色后进行流式细胞分析。图3A示出的GFP +Her2-E75四聚体 +细胞群为表达Her2 TCR-1B5 TCR的细胞，所示百分率为各阳性细胞群占总细胞数的比率。左图涉及的TCR为α链和β链的恒定区增加了一个二硫键结构(Her2 TCR-1B5-dis)，右图涉及的TCR为人α链和β链的恒定区被小鼠恒定区的同源序列所置换(Her2 TCR-1B5-mC)。横坐标表示GFP分子表达的荧光强度，纵坐标表示结合的Her2-E75四聚体的荧光强度。图3B示出TCRα链和β链的恒定区经不同方式修饰后在两种T细胞株的表达情况。图中“Her2 TCR-1B5-dis”指α链和β链的恒定区各增加了一个二硫键结构的TCR；“Her2 TCR-1B5-mC”指人α链和β链的恒定区被小鼠恒定区的同源序列所置换的TCR。GFP +Her2-E75四聚体 +细胞为表达Her2 TCR-1B5 TCR的阳性细胞，纵坐标为该TCR阳性细胞占总GFP +细胞的百分比。横坐标表示不同T细胞株组别，其中“Jurkat(TCR a+b+)”是指Jurkat细胞，其α链和β链均表达，而“J.RT3(TCR a+b-)”是指J.RT3-T3.5细胞，其来源于Jurkat细胞，并且β链基因缺失，α链仍有表达。图3C示出经编码Her2 TCR-1B5 TCR基因的慢病 毒载体转染的T细胞株可以识别被T2细胞所提呈的Her2-E75多肽。TCRα链和β链的恒定区经不同方式修饰，表达TCR的T细胞株与提呈不同浓度Her2-E75多肽的T2细胞混合培养16小时，用抗CD69-PE抗体染色后进行流式细胞分析。图中“J.RT3-Her2-1B5-dis”表示表达Her2 TCR-1B5-dis的J.RT3-T3.5细胞，“J.RT3-Her2-1B5-mC”表示表达Her2 TCR-1B5-mC的J.RT3-T3.5细胞，“Jurkat-Her2-1B5-dis”表示表达Her2 TCR-1B5-dis的Jurkat细胞，“Jurkat-Her2-1B5-mC”表示表达Her2 TCR-1B5-mC的Jurkat细胞。横坐标表示T2细胞所提呈的Her2-E75多肽的浓度。纵坐标为CD69 +细胞占总GFP +细胞的百分比。 Figure 3 shows the results of phenotypic and functional assays of T cell lines transfected with the Her2 TCR-1B5 TCR gene. J. RT3-T3.5 T cell line (J. RT3) was transfected with a lentiviral vector encoding Her2 TCR-1B5 TCR and GFP, and subjected to flow cytometry after staining with Her2-E75 tetramer-PE. The GFP + Her2-E75 tetramer + cell population shown in Figure 3A is a cell expressing Her2 TCR-1B5 TCR, expressed as a percentage of each positive cell population to the total number of cells. The TCR involved in the left panel adds a disulfide bond structure (Her2 TCR-1B5-dis) to the constant region of the α chain and the β chain, and the TCR involved in the right panel is the constant region of the human α chain and β chain by the mouse constant region. The homologous sequence was replaced (Her2 TCR-1B5-mC). The abscissa indicates the fluorescence intensity of GFP molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer. Figure 3B shows the expression of the constant regions of the TCR alpha and beta chains in different T cell variants after modification in different ways. In the figure, "Her2 TCR-1B5-dis" refers to a TCR in which the constant regions of the α chain and the β chain each add a disulfide bond structure; "Her2 TCR-1B5-mC" means that the constant regions of the human α chain and the β chain are small. The TCR replaced by the homologous sequence of the murine constant region. The GFP + Her2-E75 tetramer + cells are positive cells expressing Her2 TCR-1B5 TCR, and the ordinate is the percentage of the TCR-positive cells to total GFP + cells. The abscissa indicates different T cell line groups, wherein "Jurkat(TCR a+b+)" refers to Jurkat cells, both of which express both the α chain and the β chain, and "J.RT3(TCR a+b-)" refers to J .RT3-T3.5 cells, which are derived from Jurkat cells, and the β chain gene is deleted, and the α chain is still expressed. Figure 3C shows that a T cell strain transfected with a lentiviral vector encoding the Her2 TCR-1B5 TCR gene can recognize the Her2-E75 polypeptide presented by T2 cells. The constant regions of the TCR alpha chain and the beta chain were modified in different ways, and TCR cells expressing TCR were mixed with T2 cells presented with different concentrations of Her2-E75 polypeptide for 16 hours, stained with anti-CD69-PE antibody, and subjected to flow cytometry analysis. In the figure, "J.RT3-Her2-1B5-dis" indicates J.RT3-T3.5 cells expressing Her2 TCR-1B5-dis, and "J.RT3-Her2-1B5-mC" indicates expression of Her2 TCR-1B5-mC J.RT3-T3.5 cells, "Jurkat-Her2-1B5-dis" indicates Jurkat cells expressing Her2 TCR-1B5-dis, and "Jurkat-Her2-1B5-mC" indicates Jurkat expressing Her2 TCR-1B5-mC cell. The abscissa indicates the concentration of the Her2-E75 polypeptide presented by the T2 cells. The ordinate is the percentage of CD69 + cells to total GFP + cells.
图4示出经Her2 TCR-1B5-mC TCR基因转染的外周血单个核细胞(PBMC)的表型和功能检测结果。图4A为编码Her2 TCR-1B5-mC的慢病毒载体转染来自两个不同供体的PBMC，经Her2-E75四聚体-PE和抗CD8-APC抗体染色后进行流式细胞分析的结果。首先根据细胞形态和大小分出淋巴细胞群，Her2-E75四聚体 +细胞群为表达Her2 TCR-1B5 TCR的细胞。横坐标表示CD8分子表达的荧光强度，纵坐标表示结合的Her2-E75四聚体的荧光强度。所示百分率为各阳性细胞群占分出的淋巴细胞数的比率。左图涉及一个供体所提供的外周血单个核细胞(#1PBMC)，右图涉及另一个不同供体提供的PBMC(#2 PBMC)。CD8 +Her2-E75四聚体 +细胞为表达Her2 TCR-1B5-mC的杀伤性T细胞。CD8 -Her2-E75四聚体 +细胞可能为表达Her2 TCR-1B5-mC的CD4 +辅助T细胞。图4B示出表达Her2 TCR-1B5-mC的T细胞可以识别被T2细胞所提呈的Her2-E75多肽。经编码Her2 TCR-1B5-mC和GFP的慢病毒载体转染的两个不同供体PBMC分别与提呈不同浓度梯度Her2-E75多肽的T2细胞混合培养16小时，取细胞上清进行IFN-γ的ELISA分析。对照组中靶细胞为提呈可以结合HLA-A2分子的EBV病毒抗原多肽LMP2 426-434的T2细胞。图中“T2+Her2-E75 0.1μg/ml”表示提呈0.1μg/ml的Her2-E75多肽的T2细胞组，“T2+Her2-E75 0.01μg/ml”表示提呈0.01μg/ml的Her2-E75多肽的T2细胞组，“T2+Her2-E75 0.001μg/ml”表示提呈0.001μg/ml的Her2-E75多肽的T2细胞组，“T2+EBV-LMP 1μg/ml”表示提呈1μg/ml的EBV病毒抗原多肽LMP2  426-434的T2细胞组。横坐标表示不同供体的PBMC组别，纵坐标表示T细胞分泌的IFN-γ的浓度。图4C示出T细胞功能的CD8抗体阻断试验结果。其中，经编码Her2 TCR-1B5-mC和GFP基因的慢病毒载体转染的#2 PBMC与T2细胞提呈的抗原多肽共培养时加入抗人CD8抗体，检测T细胞分泌IFN-γ的功能是否被抑制。图中“T2+Her2-E75 0.1μg/ml”表示未加入抗人CD8抗体的、提呈0.1μg/ml的Her2-E75多肽的T2细胞组，“T2+Her2-E75 0.1μg/ml+anti-CD8”表示加入抗人CD8抗体的、提呈0.1μg/ml的Her2-E75多肽的T2细胞组。横坐标表示不同实验组别，纵坐标表示T细胞分泌的IFN-γ的浓度。图4B和4C中各试验组和对照组均为复孔，结果显示为平均值±SD。 Figure 4 shows the phenotypic and functional test results of peripheral blood mononuclear cells (PBMC) transfected with the Her2 TCR-1B5-mC TCR gene. Figure 4A shows the results of flow cytometric analysis of PBMCs from two different donors transfected with Her2 TCR-1B5-mC, stained with Her2-E75 tetramer-PE and anti-CD8-APC antibody. First, the lymphocyte population was separated according to the morphology and size of the cells, and the Her2-E75 tetramer + cell population was a cell expressing Her2 TCR-1B5 TCR. The abscissa indicates the fluorescence intensity of CD8 molecule expression, and the ordinate indicates the fluorescence intensity of the bound Her2-E75 tetramer. The percentages shown are the ratio of the number of lymphocytes to each positive cell population. The left panel relates to a peripheral blood mononuclear cell (#1PBMC) provided by a donor, and the right panel relates to a different donor-provided PBMC (#2 PBMC). CD8 + Her2-E75 tetramer + cells are killer T cells expressing Her2 TCR-1B5-mC. The CD8 - Her2-E75 tetramer + cells may be CD4 + helper T cells expressing Her2 TCR-1B5-mC. Figure 4B shows that T cells expressing Her2 TCR-1B5-mC can recognize the Her2-E75 polypeptide presented by T2 cells. Two different donor PBMCs transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP were mixed with T2 cells with different concentrations of Her2-E75 polypeptide for 16 hours, and the cell supernatant was taken for IFN-γ. ELISA analysis. The target cells in the control group were T2 cells which presented the EBV virus antigen polypeptide LMP2 426-434 which binds to the HLA-A2 molecule. In the figure, "T2+Her2-E75 0.1 μg/ml" indicates a T2 cell group in which 0.1 μg/ml of Her2-E75 polypeptide was presented, and "T2+Her2-E75 0.01 μg/ml" indicates that Her2 was present at 0.01 μg/ml. - T75 cell group of E75 polypeptide, "T2+Her2-E75 0.001 μg/ml" indicates a T2 cell group of 0.001 μg/ml of Her2-E75 polypeptide, and "T2+EBV-LMP 1 μg/ml" indicates that 1 μg was presented. /ml of the EBV virus antigen polypeptide LMP2 426-434 in the T2 cell group. The abscissa indicates the PBMC group of different donors, and the ordinate indicates the concentration of IFN-γ secreted by T cells. Figure 4C shows the results of the CD8 antibody blocking assay for T cell function. Among them, the #2 PBMC transfected with the lentiviral vector encoding Her2 TCR-1B5-mC and GFP gene was co-cultured with the antigen polypeptide presented by T2 cells, and anti-human CD8 antibody was added to detect whether the function of T cell secreting IFN-γ was detected. suppressed. In the figure, "T2+Her2-E75 0.1 μg/ml" indicates a T2 cell group in which 0.1 μg/ml of Her2-E75 polypeptide was added without anti-human CD8 antibody, "T2+Her2-E75 0.1 μg/ml+anti -CD8" indicates a T2 cell group in which 0.1 μg/ml of Her2-E75 polypeptide was added to the anti-human CD8 antibody. The abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN-γ secreted by T cells. Each of the test groups and the control group in Figs. 4B and 4C was a duplicate well, and the results were shown as mean ± SD.
图5示出经Her2 TCR-1B5-mC TCR基因转染的外周血单个核细(PBMC)识别肿瘤细胞株的功能检测结果。图5A示出编码Her2 TCR-1B5-mC TCR基因的慢病毒载体转染#2 PBMC，与不同肿瘤细胞株细胞混合培养16小时后，取细胞上清进行IFN-γ的ELISA分析结果。各试验组和对照组均为复孔，结果显示为平均值±SD。横坐标表示不同实验组别，纵坐标表示T细胞分泌的IFN-γ的浓度。图5B示出上述转染后的#2 PBMC靶向Colo205细胞的培养孔中分别加入抗CD8抗体(图中示为“colo205+anti-CD8”)或者抗HLA-ABC抗体后(图中示为“colo205+anti-HLA-ABC”)，进行的功能阻断试验结果。横坐标表示不同肿瘤细胞株组别，纵坐标表示T细胞分泌的IFN-γ的浓度。Colo205和Coca-2为HLA-A2阳性Her2/neu阳性结肠癌细胞，MAD-MB-231为HLA-A2阳性Her2/neu阳性乳腺癌细胞，H647为HLA-A2阴性Her2/neu阳性肺癌细胞，H1355为HLA-A2阳性Her2/neu阳性肺癌细胞，SK-OV-3为HLA-A2阴性Her2/neu阳性卵巢癌细胞，Bjab为HLA-A2阳性Her2/neu阴性淋巴瘤细胞。Figure 5 shows the results of functional assays of peripheral blood mononuclear fine (PBMC)-recognizing tumor cell lines transfected with the Her2 TCR-1B5-mC TCR gene. Fig. 5A shows that the lentiviral vector encoding the Her2 TCR-1B5-mC TCR gene was transfected with #2 PBMC, and after mixing with different tumor cell lines for 16 hours, the cell supernatant was taken for ELISA analysis of IFN-γ. Each test group and control group were duplicate wells, and the results were shown as mean ± SD. The abscissa indicates the different experimental groups, and the ordinate indicates the concentration of IFN-γ secreted by T cells. Figure 5B shows the addition of anti-CD8 antibody (shown as "colo205+anti-CD8") or anti-HLA-ABC antibody to the culture well of the transfected #2 PBMC-targeted Colo205 cells, respectively (shown as "colo205+anti-HLA-ABC"), the function of the function blocking test results. The abscissa indicates the different tumor cell line groups, and the ordinate indicates the concentration of IFN-γ secreted by the T cells. Colo205 and Coca-2 are HLA-A2 positive Her2/neu positive colon cancer cells, MAD-MB-231 is HLA-A2 positive Her2/neu positive breast cancer cells, and H647 is HLA-A2 negative Her2/neu positive lung cancer cells, H1355 For HLA-A2 positive Her2/neu positive lung cancer cells, SK-OV-3 is an HLA-A2 negative Her2/neu positive ovarian cancer cell, and Bjab is an HLA-A2 positive Her2/neu negative lymphoma cell.
具体实施方式Detailed ways
以下通过具体实施方式的描述并参照附图对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据本发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想， 均在本发明的范围之内。The invention is further described by the following description of the embodiments and with reference to the accompanying drawings, but this is not a limitation of the invention, and those skilled in the art can make various modifications or improvements according to the basic idea of the invention, but The basic idea of the invention is within the scope of the invention.
在本发明中，词语“肿瘤”、“癌症”、“肿瘤细胞”、“癌细胞”、“T细胞”、“T细胞受体”、“T细胞受体修饰”、“TCR可变区”、“TCR恒定区”、“抗原”、“抗原表位多肽”、“同源序列”、“编码”、“抗原提呈”、“重组DNA表达载体”、“启动子”、“互补序列”、“转染”、“自体”、“异体”、“特异性识别”、“TCR-T疗法”涵盖本领域通常认为的含义。In the present invention, the words "tumor", "cancer", "tumor cell", "cancer cell", "T cell", "T cell receptor", "T cell receptor modification", "TCR variable region" , "TCR constant region", "antigen", "epitope polypeptide", "homologous sequence", "coding", "antigen presentation", "recombinant DNA expression vector", "promoter", "complementary sequence" "Transfection", "autologous", "allogene", "specific recognition", "TCR-T therapy" encompasses what is generally considered in the art.
Her2/neu抗原属于肿瘤相关抗原，识别Her2/neu抗原的高亲和性T细胞多数被中枢耐受机制所清除，以避免导致可能的自身免疫反应(参见文献“Immunol Rev.2016；271(1):127-40”)。因此，从外周血T细胞库中诱导出具有特异性识别肿瘤细胞所表达的Her2/neu抗原的T细胞克隆变得十分困难。利用树突状细胞(Dendritic cell)提呈Her2/neu 369-377多肽抗原，进而从Her2/neu 369-377多肽疫苗免疫过的患者外周血中诱导出的高亲和性TCR尽管可以识别极低量外源所负载的(exogenously loaded)Her2/neu 369-377多肽，但不能识别肿瘤细胞内源提呈的(endogenously presented)抗原多肽(参见文献“Cancer Res.1998；58:4902–4908”)。这可能是由于外源负载的多肽/HLA复合物在构型(conformation)上与细胞内部自然提呈的HLA/多肽复合物有所不同而导致，或者由于Her2/neu 369-377多肽位于Her2蛋白高度糖化区，细胞内部自然提成的Her2/neu 369-377多肽可能被糖基化而导致TCR识别构型的差异(参见文献“Proc.Natl.Acad.Sci.USA 2003；100:15029–15034”)。体外通过Her2/neu 369-377抗原多肽诱导T细胞的过程中，仅能识别外源负载抗原多肽的高亲和性T细胞克隆往往获得优势生长(dominant expansion)，而能特异性识别被细胞提成的内源性Her2/neu抗原多肽的T细胞克隆生长受到抑制(参见文献“J Exp Med.2016 Nov 14；213(12):2811-2829”)，因而增加了获得可识别肿瘤细胞的功能性TCR的难度。经过中枢耐受机制筛选后，这些识别肿瘤细胞的TCR多属于中等亲和性，其功能往往依赖于CD8分子的辅助作用。有研究小组从HLA-A2阴性的外周血中诱导出异体T细胞(Allo–T cells)，可以特异性识别HLA-A2限制的Her2/neu 369-377抗原多肽， 用获得的TCR基因转染T细胞后，不仅可以识别肿瘤细胞提成的Her2/neu 369-377抗原多肽，也可交叉识别同家族的Her3以及Her4抗原表位(参见文献“Journal of Immunology,2008,180:8135–8145”)。然而，基于异体allo-TCR的TCR-T疗法存在产生针对其它正常自身蛋白抗原表位的异体反应(allo-reaction)的风险(参见文献“Int.J.Cancer 2009；125,649–655。Nat Immunol 2007；8:388–97”)。另外一个研究小组从Her2/neu 369-377多肽疫苗免疫过的肿瘤患者外周血中诱导出Her2/neu 369-377多肽特异性T细胞，并把来源于不同T细胞的alpha和beta链进行配对并筛选出一个高亲和性TCR，转染此高亲和性TCR的T细胞可识别HLA-A2 +Her2/neu +的多种肿瘤细胞(参见文献“HUMAN GENE THERAPY 2014；25:730–739”)。这个TCR不是从单克隆T细胞获得，因此不能确定此TCR是否是存在于经过中枢耐受筛选过的T细胞库的天然TCR。Her2/neu蛋白也在心肌、肺、食道、肾、膀胱这些重要脏器有微量表达(参见文献“Oncogene.1990 Jul；5(7):953-62”)，因此基于高亲和性Her2/neu抗原特异性TCR的TCR-T疗法有对正常组织产生脱靶毒性反应的风险。 The Her2/neu antigen belongs to the tumor-associated antigen, and the high-affinity T cells that recognize the Her2/neu antigen are mostly cleared by the central tolerance mechanism to avoid possible autoimmune responses (see the literature "Immunol Rev. 2016; 271(1) ): 127-40"). Therefore, it has become very difficult to induce a T cell clone having a Her2/neu antigen specifically recognized by a tumor cell from a peripheral blood T cell pool. High-affinity TCR induced by peripheral blood derived from Her2/neu 369-377 peptide vaccine in patients with Her2/neu 369-377 polypeptide antigen using dendritic cells, although it can be identified very low Exogenously loaded Her2/neu 369-377 polypeptide, but does not recognize endogenously presented antigenic peptides of tumor cells (see literature "Cancer Res. 1998; 58:4902–4908") . This may be due to the fact that the exogenously loaded polypeptide/HLA complex is different in conformation from the naturally present HLA/polypeptide complex present in the cell, or because the Her2/neu 369-377 polypeptide is located in the Her2 protein. In the highly glycosylated region, the Her2/neu 369-377 polypeptide naturally derived from the cell may be glycosylated to cause a difference in TCR recognition configuration (see the literature "Proc. Natl. Acad. Sci. USA 2003; 100: 15029 - 15034" ). In the process of inducing T cells by Her2/neu 369-377 antigen polypeptide in vitro, high-affinity T cell clones that can only recognize exogenously loaded antigenic peptides often obtain dominant expansion, and can specifically recognize the cell-producing The growth of T cell clones of the endogenous Her2/neu antigen polypeptide is inhibited (see the literature "J Exp Med. 2016 Nov 14; 213(12): 2811-2829"), thereby increasing the functionality to obtain identifiable tumor cells. The difficulty of TCR. After screening by the central tolerance mechanism, these TCRs that recognize tumor cells are mostly moderately affinitive, and their function often depends on the auxiliary role of CD8 molecules. A team of researchers induced allogeneic T cells (Allo–T cells) from HLA-A2-negative peripheral blood to specifically recognize HLA-A2-restricted Her2/neu 369-377 antigenic peptides and transfect T with the obtained TCR gene. After the cells, not only the Her2/neu 369-377 antigen polypeptide derived from the tumor cells but also the Her3 and Her4 antigenic epitopes of the same family can be recognized (see "Journal of Immunology, 2008, 180: 8135-8145"). However, allogeneic allo-TCR-based TCR-T therapies present a risk of producing allo-reactions against other normal self-protein epitopes (see the literature "Int. J. Cancer 2009; 125, 649-655. Nat Immunol 2007 ;8:388–97”). Another team induced Her2/neu 369-377 polypeptide-specific T cells from peripheral blood of tumor patients immunized with the Her2/neu 369-377 peptide vaccine, and paired alpha and beta chains derived from different T cells. Screening for a high-affinity TCR, T cells transfected with this high-affinity TCR recognize multiple tumor cells of HLA-A2 + Her2/neu + (see the literature "HUMAN GENE THERAPY 2014; 25: 730-739" ). This TCR was not obtained from monoclonal T cells and therefore it was not possible to determine whether this TCR is a native TCR present in a T cell bank that has been screened for central tolerance. Her2/neu protein is also expressed in small organs such as myocardium, lung, esophagus, kidney, and bladder (see "Oncogene. 1990 Jul; 5(7): 953-62"), so it is based on high affinity Her2/. TCR-T therapy with neu antigen-specific TCR has a risk of off-target toxicity to normal tissues.
肿瘤细胞高表达Her2/neu蛋白，因此细胞表面被HLA提呈的抗原多肽的数量也会相应增加，在肿瘤细胞和正常细胞上HLA/抗原多肽复合物数量的差异可成为特异性T细胞区分正常和肿瘤组织的窗口。本发明提出从自体T细胞库(auto-T cell repertoire)中获得天然TCR的序列，进而在体外使TCR表达在T细胞上，以使所得到的表达TCR的T细胞可识别肿瘤细胞增加表达的Her2/neu抗原，是成功开发有效低毒的TCR-T疗法的关键。The tumor cells have high expression of Her2/neu protein, so the number of antigenic peptides presented by HLA on the cell surface will increase accordingly. The difference in the number of HLA/antigen polypeptide complexes between tumor cells and normal cells can become normal T cells. And the window of the tumor tissue. The present invention proposes to obtain the sequence of the native TCR from the auto-T cell repertoire, and then to express the TCR on the T cell in vitro, so that the obtained TCR-expressing T cells can recognize the increased expression of the tumor cells. Her2/neu antigen is the key to the successful development of effective and low toxicity TCR-T therapy.
为了获得能够特异性识别肿瘤抗原，同时能够避免可能出现的脱靶毒副反应的TCR，本发明从HLA-A2阳性的健康供体外周血中诱导对HLA-A2提呈的Her2/neu 369-377多肽有特异性的T细胞克隆，并从中筛选出携带有对Her2/neu 369-377多肽具有中等亲和性的天然TCR的T细胞克隆。这不同于其他研究小组从经过Her2/neu 369-377多肽疫苗免疫过的肿瘤患者外周血诱导Her2/neu 369-377多肽特异性T细胞的策略(参见文献“HUMAN GENE THERAPY 2014，25:730– 739”)，本发明认为经过Her2/neu 369-377抗原多肽免疫后，针对Her2/neu 369-377多肽的特定T细胞克隆会优势增殖，因而不能代表体内T细胞库(repertoire)中自然存在的可识别靶细胞所提呈的Her2/neu 369-377多肽抗原的特异性T细胞群。本发明也没有采取其他研究小组从HLA-A2阴性外周血中诱导多肽特异性T细胞的方式(参见文献“The Journal of Immunology,2010,184:1617–1629”)，尽管从异体PBMC中更容易获得高亲和性的识别Her2/neu 369-377多肽抗原的allo-T细胞，但这也同时增加了T细胞交叉识别被HLA-A2分子提呈的其它多肽而导致的异体反应。In order to obtain a TCR capable of specifically recognizing a tumor antigen while avoiding possible off-target toxic side effects, the present invention induces Her2/neu 369-377 presented to HLA-A2 from HLA-A2-positive healthy donor peripheral blood. The polypeptide has a specific T cell clone and is screened for a T cell clone carrying a native TCR having a moderate affinity for the Her2/neu 369-377 polypeptide. This is in contrast to the strategy of other groups to induce Her2/neu 369-377 polypeptide-specific T cells from peripheral blood of tumor patients immunized with the Her2/neu 369-377 peptide vaccine (see the literature "HUMAN GENE THERAPY 2014, 25: 730– 739"), the present invention recognizes that specific T cell clones directed against the Her2/neu 369-377 polypeptide will proliferate after immunization with the Her2/neu 369-377 antigen polypeptide and thus cannot represent the natural presence of the in vivo T cell bank (repertoire). A specific T cell population that recognizes the Her2/neu 369-377 polypeptide antigen presented by the target cell. The present invention also does not employ a method by which other research groups induce polypeptide-specific T cells from HLA-A2 negative peripheral blood (see the document "The Journal of Immunology, 2010, 184: 1617 - 1629"), although it is easier to obtain from allogeneic PBMCs. High-affinity allo-T cells recognizing the Her2/neu 369-377 polypeptide antigen are obtained, but this also increases the allogeneic response caused by T cell cross-recognition of other polypeptides presented by HLA-A2 molecules.
基于上述构思，本发明提供了一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，只要不显著影响本发明的效果即可。还优选的是，所述α链的所述可变区的氨基酸序列如SEQ ID NO:19所示，所述β链的所述可变区的氨基酸序列如SEQ ID NO:20所示。Based on the above concept, the present invention provides an isolated T cell receptor comprising at least one of an alpha chain and a beta chain, both of which comprise a variable region and a constant region, characterized in that The T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid sequence of the variable region of the α chain has at least 98%, preferably the amino acid sequence shown in SEQ ID NO: At least 98.5%, more preferably at least 99% identity, the amino acid sequence of the variable region of the beta chain having at least 98%, preferably at least 98.5%, more preferably at least the amino acid sequence set forth in SEQ ID NO: 99% consistency as long as it does not significantly affect the effects of the present invention. It is also preferred that the amino acid sequence of the variable region of the α chain is as shown in SEQ ID NO: 19, and the amino acid sequence of the variable region of the β chain is set forth in SEQ ID NO: 20.
TCRα链和β链的可变区用于结合抗原多肽/主要组织相容性复合体(MHC I)，分别包括三个超变区或称为互补决定区(complementarity determining regions，CDRs)，即，CDR1、CDR2、CDR3。其中CDR3区域对特异性识别被MHC分子提呈的抗原多肽至关重要。TCRα链是不同的V和J基因片段重组而成，β链则是不同的V、D和J基因片段重组而成。特定基因片段重组结合所形成的相应CDR3区域，以及结合区域回文以及随机插入的核苷酸(palindromic and random nucleotide additions)形成了TCR对抗原多肽识别的特异性(参见文献“Immunobiology:The immune system in health and disese.5 th editin,Chapter 4,The generation of Lymphocyte antigen receptors”)。所述MHC I类分子包括人HLA。所述HLA包 括：HLA-A、B、C。 The variable regions of the TCR alpha and beta chains are used to bind the antigenic polypeptide/major histocompatibility complex (MHC I), respectively comprising three hypervariable regions or complementarity determining regions (CDRs), ie, CDR1, CDR2, CDR3. The CDR3 region is critical for the specific recognition of antigenic peptides presented by MHC molecules. The TCR alpha chain is composed of different V and J gene segments, and the β chain is composed of different V, D and J gene segments. The corresponding CDR3 regions formed by recombinant binding of specific gene fragments, as well as the binding regions and palindromic and random nucleotide additions, form the specificity of TCR recognition for antigenic polypeptides (see "Immunobiology: The immune system" In health and disese.5 th editin, Chapter 4, The generation of Lymphocyte antigen receptors"). The MHC class I molecule includes human HLA. The HLA includes: HLA-A, B, C.
进一步具体地，所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽。抗原Her2/neu的氨基酸序列如SEQ ID NO:17所示。优选的是，所述抗原表位多肽包括如SEQ ID NO:18所示的Her2/neu 369-377。HLA-A2阳性细胞表达的HLA-A2等位基因包括HLA-A*0201、0202、0203、0204、0205、0206和0207。优选的是，所述HLA-A2分子优选为HLA-A*0201。More specifically, the T cell receptor is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule. The amino acid sequence of the antigen Her2/neu is shown in SEQ ID NO: 17. Preferably, the epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18. The HLA-A2 alleles expressed by HLA-A2 positive cells include HLA-A*0201, 0202, 0203, 0204, 0205, 0206, and 0207. Preferably, the HLA-A2 molecule is preferably HLA-A*0201.
在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu 369-377多肽(SEQ ID NO:18)。在其它实施方案中，所述抗原Her2/neu的抗原表位多肽为与Her2/neu 369-377多肽具有4-9个连续的相同氨基酸(例如，4、5、6、7、8或9个连续的相同氨基酸)的抗原表位多肽，并且这些多肽的长度为8-11个氨基酸。例如，在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu 373-382多肽(SEQ ID NO:25)。In one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-11 amino acids in length. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
优选地，所述T细胞受体识别Her2/neu 369-377多肽的最大半反应多肽浓度在1.0-10ng/ml之间(例如，在3.0-8.0ng/ml、5.0-7.0ng/ml之间)。在本发明的一个实施方案中，所述最大半反应多肽浓度约为6.9ng/ml。术语“最大半反应多肽浓度”是指诱导T细胞反应达到最大值的50％所需多肽的浓度。据报道，针对巨细胞病毒(CMV)抗原CMV pp65(495-503)多肽的特异性T细胞的最大半反应多肽浓度在0.1-1ng/ml之间，而此TCR对CMV抗原多肽被认为具有高亲和性(参见文献“Journal of Immunogical Methds 2007；320:119-131”)。在本发明中，所述T细胞受体对Her2/neu抗原具有中等亲和性，从而可避免高亲和性可能带来的脱靶毒性。Preferably, the T cell receptor recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0 to 7.0 ng/ml) ). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml. The term "maximum half-reactive polypeptide concentration" refers to the concentration of the desired polypeptide that induces a T cell response to reach a maximum of 50%. The maximum half-reactive polypeptide concentration of specific T cells against the cytomegalovirus (CMV) antigen CMV pp65 (495-503) polypeptide is reported to be between 0.1-1 ng/ml, and this TCR is considered to be high for CMV antigen polypeptides. Affinity (see the literature "Journal of Immunogical Methds 2007; 320: 119-131"). In the present invention, the T cell receptor has a moderate affinity for the Her2/neu antigen, thereby avoiding off-target toxicity which may be caused by high affinity.
T细胞表达的外源TCRα链和β链有可能和T细胞本身TCR的α链和β链发生错配，不仅会稀释正确配对的外源TCR的表达量，错配TCR的抗原特异性也不明确，因而有识别自身抗原的潜在危险，因此优选将TCRα链和β链的恒定区修饰以减少或避免错配。The exogenous TCR α chain and β chain expressed by T cells may be mismatched with the α chain and β chain of TCR itself, not only diluting the expression level of the correct paired exogenous TCR, but also the antigen specificity of the mismatched TCR. It is clear that there is a potential danger of recognizing the autoantigen, and therefore it is preferred to modify the constant regions of the TCR alpha and beta chains to reduce or avoid mismatches.
在一个实施方案中，所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，本发明发现所述α链的所述恒定区可以 全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区可以全部或部分地被来源于其它物种的同源序列所替换。更优选地，所述其它物种为小鼠。所述替换可以增加细胞中TCR的表达量，并且可以进一步提高被该TCR修饰的细胞对Her2/neu抗原的特异性。In one embodiment, the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the invention finds that the constant region of the alpha chain may be wholly or partially The ground is replaced by a homologous sequence derived from other species, and/or the constant region of the beta strand may be replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse. The replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
所述α链的所述恒定区可以修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区可以修饰有一个或多个二硫键，例如1个或2个。The constant region of the alpha chain may be modified with one or more disulfide bonds, and/or the constant region of the beta chain may be modified with one or more disulfide bonds, such as one or two.
在具体的实施方式中，制备了两种不同方式修饰的TCR，Her2 TCR-1B5-dis是通过点突变在TCR恒定区增加一个二硫键，方法在文献“Cancer Res.2007 Apr 15；67(8):3898-903.”中描述，其全文通过引用方式并入本文。Her2 TCR-1B5-mC是用小鼠TCR恒定区序列置换相应的人TCR恒定区序列，方法在文献“Eur.J.Immunol.2006 36:3052–3059”中描述，其全文通过引用方式并入本文。In a specific embodiment, TCR is modified in two different ways. Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67 ( 8): 38.9-903., which is incorporated herein by reference in its entirety. Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article.
在具体的实施方案中，所述α链的氨基酸序列如SEQ ID NOs:2、6或10所示，所述β链的氨基酸序列如SEQ ID NOs:4、8或12所示。In a specific embodiment, the amino acid sequence of the alpha chain is set forth in SEQ ID NOs: 2, 6, or 10, and the amino acid sequence of the beta strand is set forth in SEQ ID NOs: 4, 8, or 12.
其中，对于氨基酸序列如SEQ ID NO:2所示的α链，其序列为原始的人源序列；对于氨基酸序列如SEQ ID NO:6所示的α链，其在恒定区修饰有1个二硫键；对于氨基酸序列如SEQ ID NO:10所示的α链，其恒定区替换为鼠源恒定区。Wherein, for the amino acid sequence such as the α chain shown in SEQ ID NO: 2, the sequence is the original human sequence; for the amino acid sequence such as the α chain shown in SEQ ID NO: 6, it has one in the constant region. Sulfur bond; for the α chain of the amino acid sequence such as SEQ ID NO: 10, the constant region is replaced with a murine constant region.
其中，对于氨基酸序列如SEQ ID NO:4所示的β链，其序列为原始的人源序列；对于氨基酸序列如SEQ ID NO:8所示的β链，其在恒定区修饰有1个二硫键；对于氨基酸序列如SEQ ID NO:12所示的β链，其恒定区替换为鼠源恒定区。Wherein, for the β chain of the amino acid sequence such as SEQ ID NO: 4, the sequence is the original human sequence; for the β chain of the amino acid sequence such as SEQ ID NO: 8, it has a modification in the constant region. Sulfur bond; for the β chain of the amino acid sequence such as SEQ ID NO: 12, the constant region is replaced with a murine constant region.
在一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:2所示，β链的氨基酸序列如SEQ ID NO:4所示。在另一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:6所示，β链的氨基酸序列如SEQ ID NO:8所示。在又一个具体实施方案中，所述TCR的α链的氨基酸序列如SEQ ID NO:10所示，β链的氨基酸序列如SEQ ID NO:12所示。In a specific embodiment, the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 2, and the amino acid sequence of the beta strand is set forth in SEQ ID NO: 4. In another specific embodiment, the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 6, and the amino acid sequence of the beta strand is set forth in SEQ ID NO: 8. In still another specific embodiment, the amino acid sequence of the alpha chain of the TCR is set forth in SEQ ID NO: 10, and the amino acid sequence of the beta strand is set forth in SEQ ID NO: 12.
在本发明其它具体的实施方案中，所述TCR的α链具有在SEQ ID NOs:2、6或10所示氨基酸序列中替换、删除、和/或添加一个或多个氨基酸而得到的氨基酸序列；例如，所述α链具有与SEQ ID NOs:2、6或10所示氨基酸序列至少90％、优选至少95％、更优选至少99％的一致性。In other specific embodiments of the invention, the alpha chain of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10. For example, the alpha chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 2, 6, or 10.
在本发明其它具体的实施方案中，所述TCR的β链具有在SEQ ID NOs:4、8或12所示氨基酸序列中替换、删除、和/或添加一个或多个氨基酸而得到的氨基酸序列；例如，所述β链具有与SEQ ID NOs:4、8或12所示氨基酸序列至少90％、优选至少95％、更优选至少99％的一致性。In other specific embodiments of the invention, the beta strand of the TCR has an amino acid sequence obtained by replacing, deleting, and/or adding one or more amino acids in the amino acid sequence set forth in SEQ ID NOs: 4, 8, or 12. For example, the β chain has at least 90%, preferably at least 95%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NOs: 4, 8 or 12.
本发明的TCR的α链和/或β链还可以在末端(例如C末端)结合其它功能性序列，例如共刺激信号CD28、4-1BB和/或CD3zeta的功能区序列。The alpha and/or beta strands of the TCRs of the invention may also bind to other functional sequences at the terminus (e.g., the C-terminus), such as the functional region sequences of the costimulatory signals CD28, 4-1BB, and/or CD3zeta.
本发明还提供了一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％、优选至少98.5％、更优选至少99％的一致性，只要不显著影响本发明的效果即可。还优选的是，所述α链可变区编码序列编码如SEQ ID NO:19所示的氨基酸序列，所述β链可变区编码序列编码如SEQ ID NO:20所示的氨基酸序列。The invention also provides an isolated T cell receptor-encoding nucleic acid comprising a coding sequence for at least one of an alpha chain and a beta chain of said T cell receptor, said alpha chain coding sequence and beta strand coding The sequences each comprise a variable region coding sequence and a constant region coding sequence, wherein the T cell receptor is capable of specifically recognizing the antigen Her2/neu expressed by the tumor cell, and the amino acid encoded by the α chain variable region coding sequence The sequence has at least 98%, preferably at least 98.5%, more preferably at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 19, the amino acid sequence encoded by the β chain variable region coding sequence having SEQ ID NO: The amino acid sequence shown at 20 has a consistency of at least 98%, preferably at least 98.5%, more preferably at least 99%, as long as the effects of the present invention are not significantly affected. It is also preferred that the α chain variable region coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 19, and the β chain variable region coding sequence encodes the amino acid sequence set forth in SEQ ID NO: 20.
所述核酸可以为DNA或RNA。The nucleic acid can be DNA or RNA.
优选地，所述α链可变区编码序列如SEQ ID NO:21所示，所述β链可变区编码序列如SEQ ID NO:22所示。Preferably, the alpha chain variable region coding sequence is set forth in SEQ ID NO: 21, and the beta chain variable region coding sequence is set forth in SEQ ID NO: 22.
进一步具体地，被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽。More specifically, the T cell receptor encoded by the nucleic acid is capable of specifically recognizing an epitope polypeptide of the antigen Her2/neu presented by an HLA-A2 molecule.
在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为 Her2/neu 369-377多肽(SEQ ID NO:18)。在其它实施方案中，所述抗原Her2/neu的抗原表位多肽为与Her2/neu 369-377多肽具有4-9个连续的相同氨基酸(例如，4、5、6、7、8或9个连续的相同氨基酸)的抗原表位多肽，并且这些多肽的长度为8-10个氨基酸。例如，在一个实施方案中，所述抗原Her2/neu的抗原表位多肽为Her2/neu 373-382多肽(SEQ ID NO:25)。In one embodiment, the epitope polypeptide of the antigen Her2/neu is a Her2/neu 369-377 polypeptide (SEQ ID NO: 18). In other embodiments, the antigenic epitope polypeptide of the antigen Her2/neu is 4-9 consecutive identical amino acids to the Her2/neu 369-377 polypeptide (eg, 4, 5, 6, 7, 8, or 9) Epitope polypeptides of consecutive identical amino acids), and these polypeptides are 8-10 amino acids in length. For example, in one embodiment, the antigenic epitope polypeptide of the antigen Her2/neu is a Her2/neu 373-382 polypeptide (SEQ ID NO: 25).
优选地，被所述核酸编码的所述T细胞受体识别Her2/neu 369-377多肽的最大半反应多肽浓度在1.0-10ng/ml之间(例如，在3.0-8.0ng/ml、5.0-7.0ng/ml之间)。在本发明的一个实施方案中，所述最大半反应多肽浓度约为6.9ng/ml。在此情况下，所述T细胞受体对Her2/neu抗原具有中等亲和性，可避免高亲和性可能带来的脱靶毒性。Preferably, the T cell receptor encoded by the nucleic acid recognizes a maximum half-reactive polypeptide concentration of the Her2/neu 369-377 polypeptide between 1.0 and 10 ng/ml (eg, between 3.0 and 8.0 ng/ml, 5.0- Between 7.0ng/ml). In one embodiment of the invention, the maximum half-reactive polypeptide concentration is about 6.9 ng/ml. In this case, the T cell receptor has a moderate affinity for the Her2/neu antigen, and the off-target toxicity which may be caused by high affinity can be avoided.
在一个实施方案中，所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换。更优选地，所述其它物种为小鼠。所述替换可以增加细胞中TCR的表达量，并且可以进一步提高被该TCR修饰的细胞对Her2/neu抗原的特异性。In one embodiment, the constant region of the alpha chain and/or the constant region of the beta chain is derived from a human; preferably, the alpha chain constant region coding sequence is derived in whole or in part from other The homologous sequence of the species is replaced, and/or the beta strand constant region coding sequence is replaced in whole or in part by homologous sequences derived from other species. More preferably, the other species is a mouse. The replacement can increase the expression level of TCR in the cells, and can further increase the specificity of the cells modified by the TCR to the Her2/neu antigen.
所述α链恒定区编码序列可以包含一个或多个二硫键的编码序列，并且/或者所述β链恒定区编码序列可以包含一个或多个二硫键的编码序列。The alpha chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds, and/or the beta chain constant region coding sequence may comprise a coding sequence for one or more disulfide bonds.
在具体的实施方案中，所述α链编码序列如SEQ ID NOs:1、5或9所示，所述β链编码序列如SEQ ID NOs:3、7或11所示。In a specific embodiment, the alpha chain coding sequence is set forth in SEQ ID NOs: 1, 5 or 9, and the beta strand coding sequence is set forth in SEQ ID NOs: 3, 7 or 11.
其中，对于编码序列如SEQ ID NO:1所示的α链，其序列为原始的人源序列；对于编码序列如SEQ ID NO:5所示的α链，其在恒定区修饰有1个二硫键；对于编码序列如SEQ ID NO:9所示的α链，其恒定区替换为鼠源恒定区。Wherein, for the α chain of the coding sequence as shown in SEQ ID NO: 1, the sequence is the original human sequence; and for the α chain of the coding sequence such as SEQ ID NO: 5, it has one in the constant region. Sulfur bond; for the α chain of the coding sequence as shown in SEQ ID NO: 9, the constant region is replaced with a murine constant region.
其中，对于编码序列如SEQ ID NO:3所示的β链，其序列为原始的人源序列；对于编码序列如SEQ ID NO:7所示的β链，其在恒定区修饰有1个二硫键；对于编码序列如SEQ ID NO:11所示的β 链，其恒定区替换为鼠源恒定区。Wherein, for the β chain of the coding sequence as shown in SEQ ID NO: 3, the sequence is the original human sequence; for the β chain of the coding sequence such as SEQ ID NO: 7, it has a modification in the constant region. Sulfur bond; for the β chain of the coding sequence as shown in SEQ ID NO: 11, the constant region is replaced with a murine constant region.
在一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:1所示，β链的编码序列如SEQ ID NO:3所示。在另一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:5所示，β链的编码序列如SEQ ID NO:7所示。在又一个具体实施方案中，所述TCR的α链的编码序列如SEQ ID NO:9所示，β链的编码序列如SEQ ID NO:11所示。In a specific embodiment, the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 1, and the coding sequence for the beta chain is set forth in SEQ ID NO: 3. In another specific embodiment, the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 5, and the coding sequence for the beta chain is set forth in SEQ ID NO: 7. In yet another specific embodiment, the coding sequence for the alpha chain of the TCR is set forth in SEQ ID NO: 9, and the coding sequence for the beta chain is set forth in SEQ ID NO:11.
在另外的实施方案中，所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接，这样可以增加TCR在细胞内的表达。术语“可切割性连接多肽”是指该多肽起到连接作用，并且可以被特定的酶切割，或者编码此多肽的核酸序列通过核糖体跳跃方式(ribosome skipping)进行翻译，从而使被其连接的多肽彼此分离。可切割性连接多肽的例子是本领域已知的，例如F2A多肽，F2A多肽序列包括但不限于来自微小核糖核酸病毒的F2A多肽、以及来自其它病毒相似的2A类序列。另外，可切割性连接多肽也包括可被Furin酶切割的标准的四氨基酸基序(canonical four amino acid motif)，即R-X-[KR]-R氨基酸序列。该实施方案所编码的TCR为单链嵌合T细胞受体，该单链嵌合T细胞受体表达完成后，连接α链和β链的可切割性连接多肽会被细胞中的特定酶切割，从而形成等量游离的α链和β链。In additional embodiments, the alpha chain coding sequence and the beta strand coding sequence are joined by a coding sequence for a cleavable linker polypeptide, which increases the expression of the TCR in the cell. The term "cleavable linker polypeptide" means that the polypeptide functions as a linker and can be cleaved by a specific enzyme, or the nucleic acid sequence encoding the polypeptide is translated by ribosome skipping so that it is ligated The polypeptides are separated from one another. Examples of cleavable linker polypeptides are known in the art, such as F2A polypeptides, including but not limited to F2A polypeptides from picornaviruses, and class 2A sequences similar to other viruses. In addition, the cleavable linker polypeptide also includes a standard canonical four amino acid motif, i.e., the R-X-[KR]-R amino acid sequence, which can be cleaved by the Furin enzyme. The TCR encoded by this embodiment is a single-stranded chimeric T cell receptor, and after the expression of the single-stranded chimeric T cell receptor is completed, the cleavable linker polypeptide linking the α chain and the β chain is cleaved by a specific enzyme in the cell. Thereby forming equal amounts of free alpha and beta chains.
组成单链嵌合TCR的α链和β链也可如上文所述，恒定区(及其相应的编码序列)全部或部分地被来源于其它物种的同源序列所替换，并且/或者修饰有(编码)一个或多个二硫键。The alpha and beta chains constituting the single-stranded chimeric TCR can also be replaced, as described above, in whole or in part by homologous sequences derived from other species, and/or modified (coding) one or more disulfide bonds.
在具体的实施方案中，所述核酸的序列如SEQ ID NOs:13、15、或23所示。In a specific embodiment, the sequence of the nucleic acid is set forth in SEQ ID NOs: 13, 15, or 23.
优选地，对所述核酸的核苷酸序列进行编码子优化以增加基因表达、蛋白翻译效率以及蛋白表达，从而增强TCR识别抗原的能力。编码子优化包括但不限于翻译启动区域的修饰、改变mRNA结构片段、以及使用编码同一氨基酸的不同密码子。Preferably, the nucleotide sequence of the nucleic acid is encoded sub-optimized to increase gene expression, protein translation efficiency, and protein expression, thereby enhancing the ability of the TCR to recognize antigen. Encoding sub-optimization includes, but is not limited to, modification of the translation initiation region, alteration of mRNA structural fragments, and use of different codons encoding the same amino acid.
在其它的实施方案中，可以对上述TCR编码核酸的序列进行突变， 包括去除、插入和/或置换一个或多个氨基酸密码子，使得所表达的TCR识别Her2/neu抗原的功能不变或者增强。例如，在一个实施方案中，进行保守氨基酸置换，包括对上述TCRα链和/或β链的可变区中的一个氨基酸用结构和/或化学属性相似的另一个氨基酸进行置换。术语“相似的氨基酸”是指具有相似的极性、电负荷、可溶性、疏水性、亲水性等属性的氨基酸残基。突变后的TCR仍具有识别上述被靶细胞提呈的Her2/neu抗原多肽的生物活性。在另一个实施方案中，进行TCR成熟性(TCR maturation)修饰，即，包括对上述TCRα链和/或β链的可变区中的互补决定区2(CDR2)和/或CDR3区域的氨基酸进行去除、插入和/或置换，从而改变TCR结合Her2/neu抗原的亲和性。In other embodiments, the sequence of the above TCR-encoding nucleic acid can be mutated, including the removal, insertion and/or substitution of one or more amino acid codons such that the expressed TCR recognizes the function of the Her2/neu antigen unchanged or enhanced. . For example, in one embodiment, conservative amino acid substitutions are made comprising substitution of one amino acid in the variable region of the TCR alpha chain and/or beta chain described above with another amino acid having similar structural and/or chemical properties. The term "similar amino acid" refers to an amino acid residue having similar properties of polarity, electrical load, solubility, hydrophobicity, hydrophilicity, and the like. The mutated TCR still has the biological activity of recognizing the above-described Her2/neu antigen polypeptide presented by the target cell. In another embodiment, the TCR maturation modification is performed, ie, comprising amino acids of the complementarity determining region 2 (CDR2) and/or CDR3 regions of the variable regions of the TCR alpha chain and/or beta chain described above. Removal, insertion and/or substitution alter the affinity of the TCR to bind to the Her2/neu antigen.
本发明还提供了一种分离的、由根据本发明所述的DNA转录的mRNA。The invention also provides an isolated mRNA transcribed from the DNA according to the invention.
本发明还提供了一种重组表达载体，其含有与启动子有效连接的根据本发明所述的核酸(例如DNA)，和/或其互补序列。The invention also provides a recombinant expression vector comprising a nucleic acid (e.g., DNA) according to the invention operably linked to a promoter, and/or a complement thereof.
优选地，在所述重组表达载体中，本发明所述的DNA合适地与启动子、增强子、终止子和/或polyA信号序列有效连接。Preferably, in the recombinant expression vector, the DNA of the invention is suitably operably linked to a promoter, enhancer, terminator and/or polyA signal sequence.
本发明的重组表达载体的上述作用元件的组合能够促进DNA的转录和翻译，并增强mRNA的稳定性。The combination of the above-described functional elements of the recombinant expression vector of the present invention can promote transcription and translation of DNA and enhance the stability of mRNA.
重组表达载体的基本骨架可以是任何已知的表达载体，包括质粒或病毒，病毒载体包括但不限于(例如)逆转录病毒载体(病毒原型为莫洛尼鼠白血病病毒(MMLV))和慢病毒载体(病毒原型为人类免疫缺陷I型病毒(HIV))。表达本发明所述TCR的重组载体可以通过本领域常规的重组DNA技术来获得。The basic backbone of the recombinant expression vector can be any known expression vector, including plasmids or viruses, including but not limited to, for example, retroviral vectors (the virus prototype is Moloney murine leukemia virus (MMLV)) and lentiviruses. Vector (the virus prototype is human immunodeficiency type I virus (HIV)). A recombinant vector expressing a TCR of the present invention can be obtained by recombinant DNA techniques conventional in the art.
在一个实施方案中，重组表达载体上的α链和β链基因的表达可以由两个不同的启动子所驱动，启动子包括各种已知的类型，例如强表达的、弱表达的、可诱导的、组织特异性的、和分化特异性的启动子。启动子可以是病毒来源的或者非病毒来源的，例如CMV启动子、MSCV的LTR上的启动子、EF1-α启动子、和PGK-1启动子。两个启动子的驱动方向可以是同向也可以是反向的。In one embodiment, expression of the alpha and beta chain genes on the recombinant expression vector can be driven by two different promoters, including various known types, such as strongly expressed, weakly expressed, Inducible, tissue-specific, and differentiation-specific promoters. The promoter may be of viral or non-viral origin, such as the CMV promoter, the promoter on the LTR of MSCV, the EF1-alpha promoter, and the PGK-1 promoter. The driving directions of the two promoters can be either in the same direction or in the opposite direction.
在另一个实施方案中，重组表达载体上的α链和β链基因的表 达可以由同一个启动子所驱动，例如编码单链嵌合T细胞受体的情况，α链的核苷酸序列和β链的核苷酸序列由Furin-F2A多肽编码序列相连接。In another embodiment, expression of the alpha and beta chain genes on the recombinant expression vector can be driven by the same promoter, such as in the case of a single chain chimeric T cell receptor, the nucleotide sequence of the alpha chain and The nucleotide sequence of the beta strand is ligated by the Furin-F2A polypeptide coding sequence.
在另一些实施方案中，重组表达载体除了包含α链和β链基因外，还可以包含其它功能分子的编码序列。一个实施方案包括表达自发荧光蛋白(如GFP或其它荧光蛋白)以用于体内追踪成像。另一个实施方案包括表达可诱导的自杀基因系统，例如诱导表达单纯疱疹病毒-胸腺嘧啶核苷激酶(HSV-TK)蛋白，或者诱导表达Caspase 9(iCasp9)蛋白。表达这些“安全转换分子”(safety-switch)可以增加经本发明所述TCR基因修饰的细胞在体内使用的安全性。另一个实施方案包括表达人CD8基因，包括单独或联合表达CD8α链和β链，经本发明所述TCR基因修饰的细胞表达CD8分子后可以增强其特异性识别Her2/neu抗原的能力，或使CD8阴性T细胞(如CD4 +T辅助细胞)获得特异性识别Her2/neu抗原的能力。另一个实施方案包括表达人趋化因子受体基因，例如CCR2，这些趋化因子受体可结合肿瘤组织中高表达的相应趋化因子配体，从而可以增加经本发明所述TCR基因修饰的细胞在肿瘤组织中的归巢。 In other embodiments, the recombinant expression vector may comprise coding sequences for other functional molecules in addition to the alpha and beta chain genes. One embodiment includes expression of an autofluorescent protein (such as GFP or other fluorescent protein) for in vivo tracking imaging. Another embodiment includes expressing an inducible suicide gene system, such as inducing expression of a herpes simplex virus-thymidine kinase (HSV-TK) protein, or inducing expression of a Caspase 9 (iCasp9) protein. Expression of these "safety-switches" can increase the safety of the cells modified by the TCR genes of the present invention for use in vivo. Another embodiment includes expressing a human CD8 gene, comprising expressing the CD8 alpha chain and the beta chain, either alone or in combination, and the cells modified by the TCR gene of the present invention can enhance their ability to specifically recognize the Her2/neu antigen by expressing the CD8 molecule, or CD8-negative T cells (such as CD4 + T helper cells) acquire the ability to specifically recognize the Her2/neu antigen. Another embodiment includes expressing a human chemokine receptor gene, such as CCR2, which binds to a corresponding chemokine ligand that is highly expressed in tumor tissue, thereby enhancing cells modified by the TCR gene of the present invention Homing in tumor tissue.
本发明还提供了一种T细胞受体修饰的细胞，该细胞的表面被本发明所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。The present invention also provides a T cell receptor-modified cell whose surface is modified by a T cell receptor according to the present invention, wherein the cell comprises a primitive T cell or a precursor cell thereof, NKT cell, or T Cell line.
所述“T细胞受体修饰”中的“修饰”是指，通过基因转染使细胞表达有本发明所述的T细胞受体，即，所述T细胞受体通过跨膜区锚定在所修饰的细胞的细胞膜上，并具有识别抗原多肽/MHC复合物的功能。"Modification" in the "T cell receptor modification" means that a cell expresses a T cell receptor of the present invention by gene transfection, that is, the T cell receptor is anchored through a transmembrane region. The modified cells are on the cell membrane and have the function of recognizing the antigenic polypeptide/MHC complex.
本发明还提供了一种制备根据本发明所述的T细胞受体修饰的细胞的方法，包括以下步骤：The invention also provides a method of preparing a T cell receptor modified cell according to the invention, comprising the steps of:
1)提供细胞；1) providing cells;
2)提供编码本发明T细胞受体的核酸；2) providing a nucleic acid encoding a T cell receptor of the invention;
3)将所述核酸转染入所述细胞中。3) Transfecting the nucleic acid into the cell.
步骤1)所述的细胞可以来源于哺乳动物，包括人、犬、小鼠、 大鼠及其转基因动物。所述细胞可以来自自体或异体。异体细胞包括来自同卵双胞胎的细胞、异体干细胞、经基因改造的异体T细胞。The cells of step 1) can be derived from mammals, including humans, dogs, mice, rats and their transgenic animals. The cells can be derived from autologous or allogeneic. Allogeneic cells include cells from identical twins, allogeneic stem cells, genetically engineered allogeneic T cells.
步骤1)所述的细胞包括原始T细胞或其前体细胞、NKT细胞、或T细胞株。术语“原始T细胞(naive T cell)”是指外周血中尚未被相应抗原活化的成熟T细胞。这些细胞可以通过本领域已知的方法分离得到。例如，T细胞可以从不同组织器官获得，包括外周血、骨髓、淋巴组织、脾脏、脐带血、肿瘤组织。一个实施方案中，T细胞可以来自造血干细胞(HSCs)，包括来自骨髓、外周血或者脐带血，通过干细胞标记分子例如CD34而分离获得。一个实施方案中，T细胞可以来自诱导性多功能干细胞(iPS cells)，包括把特定基因或特定基因产物导入体细胞，使该体细胞转化为干细胞后，体外诱导分化成T细胞或其前体细胞。T细胞可以通过常用方法如密度梯度离心法而获得，密度梯度离心法的例子包括Ficoll或者Percoll密度离心。一个实施方案是利用血浆分离置换法(apheresis)或白细胞去除法(leukapheresis)从外周血获得富集的T细胞的产物。一个实施方案是用抗体标记特定细胞群后，通过磁珠分离的方式(如
Figure PCTCN2018115191-appb-000001
系统(Miltenyi Biotec))、或流式细胞分离的方式获得富集的CD8 +或CD4 +T细胞。 The cells of step 1) include naive T cells or their precursor cells, NKT cells, or T cell strains. The term "naive T cell" refers to mature T cells in peripheral blood that have not been activated by the corresponding antigen. These cells can be isolated by methods known in the art. For example, T cells can be obtained from different tissues and organs, including peripheral blood, bone marrow, lymphoid tissue, spleen, cord blood, and tumor tissue. In one embodiment, the T cells can be obtained from hematopoietic stem cells (HSCs), including bone marrow, peripheral blood, or cord blood, isolated by stem cell marker molecules such as CD34. In one embodiment, the T cells can be derived from inducible pluripotent stem cells (iPS cells), including introducing a specific gene or a specific gene product into the somatic cells, transforming the somatic cells into stem cells, and inducing differentiation into T cells or precursors thereof in vitro. cell. T cells can be obtained by a usual method such as density gradient centrifugation, and examples of density gradient centrifugation include Ficoll or Percoll density centrifugation. One embodiment is the production of enriched T cells from peripheral blood using plasma apheresis or leukapheresis. One embodiment is a method of separating a magnetic cell by labeling a specific cell population with an antibody (eg,
Figure PCTCN2018115191-appb-000001
Enriched CD8 + or CD4 + T cells were obtained by system (Miltenyi Biotec), or by flow cytometry.
优选地，所述T细胞前体细胞为造血干细胞。可以将本发明所述TCR的编码基因直接引入造血干细胞，然后转输到体内，进一步分化成为成熟T细胞；也可以将编码基因引入在体外特定条件下由造血干细胞分化成熟的T细胞中。Preferably, the T cell precursor cells are hematopoietic stem cells. The gene encoding the TCR of the present invention can be directly introduced into hematopoietic stem cells, and then transferred to the body to further differentiate into mature T cells; the coding gene can also be introduced into T cells which are differentiated and matured by hematopoietic stem cells under specific conditions in vitro.
所述细胞可以被重悬于冻存溶液里置于液氮中保存。常用冻存溶液包括但不限包含10％(v/v)DMSO和90％(v/v)人血清或胎牛血清。细胞以每分钟降低温度1℃的条件冻存于-80℃，然后保存于液氮罐的气相部分。其它冻存方法是把置于冻存液的细胞直接放入-80℃或液氮中进行冻存。The cells can be resuspended in a cryopreservation solution and stored in liquid nitrogen. Common cryopreservation solutions include, but are not limited to, 10% (v/v) DMSO and 90% (v/v) human serum or fetal bovine serum. The cells were frozen at -80 ° C at a temperature of 1 ° C per minute and then stored in the gas phase portion of the liquid nitrogen tank. Other cryopreservation methods are to freeze the cells placed in the cryopreservation solution directly into -80 ° C or liquid nitrogen.
步骤2)所述的核酸为根据本发明所述的核酸，包括所述DNA和RNA。The nucleic acid of step 2) is a nucleic acid according to the invention, including the DNA and RNA.
所述转染包括物理方式、生物方式和化学方式。物理方式是通 过磷酸钙沉淀、脂质体、微注射、电穿孔、基因枪等途径把TCR基因以DNA或RNA的形式导入细胞内。目前已有商业化的仪器，包括电转移仪(例如Amaxa Nucleofector-II(德国Amaxa Biosystems公司)、ECM 830(BTX)(美国Harvard Instruments)、Gene Pulser II(美国BioRad公司)、Multiporator(德国Eppendort公司)。生物方式是通过DNA或RNA载体把TCR基因引入细胞内，逆转录病毒载体(例如γ逆转录病毒载体)是转染并插入外源基因片段到动物细胞(包括人细胞)的常用工具，其它病毒载体来源于慢病毒、痘病毒、单纯疱疹病毒、腺病毒以及腺病毒相关病毒等。化学方式是把多核苷酸引入细胞内，包括胶态分散系统，比如大分子复合物、纳米胶囊、微球体、微珠、微团和脂质体。无论以什么方式把TCR基因引入细胞，要用各种检测方法分析目的基因是否引入靶细胞内，所述检测方法包括常见的分子生物学方法(例如Southern印迹和Northern印迹、RT-PCR和PCR等)，或者常见的生物化学方法(例如ELISA和Western印迹)，以及本发明所提及的方法。The transfection includes physical, biological, and chemical means. The physical method is to introduce the TCR gene into the cell in the form of DNA or RNA by calcium phosphate precipitation, liposome, microinjection, electroporation, gene gun, and the like. Commercial instruments are currently available, including electrotransfers (eg Amaxa Nucleofector-II (Amaxa Biosystems, Germany), ECM 830 (BTX) (Harvard Instruments, USA), Gene Pulser II (BioRad, USA), Multiporator (Eppendort, Germany) The biological method is to introduce the TCR gene into a cell through a DNA or RNA vector, and a retroviral vector (such as a γ retroviral vector) is a common tool for transfecting and inserting a foreign gene fragment into an animal cell (including a human cell). Other viral vectors are derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, etc. The chemical method is to introduce polynucleotides into cells, including colloidal dispersion systems, such as macromolecular complexes, nanocapsules, Microspheres, microbeads, micelles, and liposomes. Regardless of the manner in which the TCR gene is introduced into a cell, various assays are used to analyze whether the gene of interest is introduced into the target cell, including common molecular biology methods ( For example, Southern and Northern blots, RT-PCR and PCR, etc., or common biochemical methods (eg ELISA and Western) Trace), and methods of the present invention mentioned.
优选地，所述转染通过逆转录病毒载体或慢病毒载体进行。Preferably, the transfection is carried out by a retroviral vector or a lentiviral vector.
转染后所述细胞的培养可以根据实际应用通过其各自的常规方法和条件进行。例如，T细胞通过表面的TCR/CD3复合体，以及辅助刺激分子(如CD28)共同激活后，可获得体外扩增。激活TCR、CD3和CD28的刺激物(如抗TCR、CD3或CD28的抗体)可以吸附在培养容器表面，或者共培养物(比如磁珠)表面，也可以直接加入细胞培养液中共同培养。另一个实施方案是将T细胞与滋养细胞共同培养，所述滋养细胞表达辅助刺激分子或者相应的配体，包括但不限于HLA-A2、β2-微球蛋白、CD40、CD83、CD86、CD127、4-1BB。The culture of the cells after transfection can be carried out according to the actual application by their respective conventional methods and conditions. For example, T cells can be expanded in vitro by co-activation of a TCR/CD3 complex on the surface and a co-stimulatory molecule (such as CD28). Stimulators that activate TCR, CD3, and CD28 (such as antibodies against TCR, CD3, or CD28) can be adsorbed on the surface of a culture vessel, or on the surface of a co-culture (such as a magnetic bead), or can be directly cultured in a cell culture medium. Another embodiment is to co-culture T cells with trophoblast cells that express helper stimulatory molecules or corresponding ligands including, but not limited to, HLA-A2, β2-microglobulin, CD40, CD83, CD86, CD127, 4-1BB.
依照通常的哺乳动物细胞体外培养的方法，T细胞培在适当培养条件下进行培养和扩增。例如，细胞达到70％以上融合状态(confluence)时可进行传代，一般2到3天换新鲜培养液。当细胞达到一定数目时直接使用，或按上述描述进行冻存。体外培养的时间可以是24小时之内，也可以长达14天或更长。冻存细胞解冻后可进行下一步应用。The T cell culture is cultured and expanded under appropriate culture conditions according to the usual method of in vitro culture of mammalian cells. For example, when the cells reach more than 70% confluence, they can be passaged, usually 2 to 3 days for fresh culture. When the cells reach a certain number, they are used directly or frozen as described above. The in vitro culture time can be within 24 hours or as long as 14 days or longer. After the frozen cells are thawed, the next step can be applied.
在一个实施方案中，细胞可以在体外培养数小时到14天，或者之 间任何小时数。T细胞培养条件包括使用基础培养液，包括但不限于RPMI 1640、AIM-V、DMEM、MEM、a-MEM、F-12、X-Vivo 15和X-Vivo。其它细胞生存和增殖所需要的条件包括但不限于使用血清(人或胎牛血清)、白介素-2(IL-2)、胰岛素、IFN-γ、IL-4、IL-7、GM-CSF、IL-10、IL-12、IL-15、IL-21、TGF-β和TNF-a，其它培养添加物(包括氨基酸、丙酮酸钠、维生素C、2-巯基乙醇、生长激素、生长因子)。细胞可置于适当的培养条件，例如，温度可处于37℃、32℃、30℃或者室温，并且空气条件可为(例如)含5％CO 2的空气。 In one embodiment, the cells can be cultured in vitro for hours to 14 days, or any number of hours in between. T cell culture conditions include the use of basal culture fluids including, but not limited to, RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo. Conditions required for survival and proliferation of other cells include, but are not limited to, the use of serum (human or fetal bovine serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-21, TGF-β and TNF-a, other culture additives (including amino acids, sodium pyruvate, vitamin C, 2-mercaptoethanol, growth hormone, growth factors) . The cells can be placed under suitable culture conditions, for example, the temperature can be at 37 ° C, 32 ° C, 30 ° C or room temperature, and the air conditions can be, for example, air containing 5% CO 2 .
本发明还提供了根据本发明所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。The invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的，包括但不限于乳腺癌、卵巢癌、胃癌、食管癌、肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌、子宫颈癌、子宫内膜癌、唾液腺癌、皮肤癌、肺癌、骨癌以及脑癌。The tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
本发明还提供了根据本发明所述的T细胞受体修饰的细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。The invention also provides the use of a T cell receptor modified cell according to the invention for the preparation of a medicament for detecting a tumor and/or cancer of a host.
在本发明的一个实施方案中，可将从宿主取出的肿瘤和/或癌症细胞的样本与本发明所述的T细胞受体修饰的细胞以一定浓度进行接触，根据二者的反应程度可以判断所述肿瘤和/或癌症是HLA-A2阳性的还是HLA-A2阴性的，以及是否表达抗原Her2/neu。In one embodiment of the present invention, a sample of a tumor and/or a cancer cell taken out from a host can be contacted with a T cell receptor-modified cell of the present invention at a concentration, and can be judged according to the degree of reaction between the two. Whether the tumor and/or cancer is HLA-A2 positive or HLA-A2 negative, and whether the antigen Her2/neu is expressed.
本发明还提供了一种药物组合物，其中该药物组合物包括作为活性成分的根据本发明所述的T细胞受体修饰的细胞，及可药用辅料。The present invention also provides a pharmaceutical composition, wherein the pharmaceutical composition comprises, as an active ingredient, a T cell receptor-modified cell according to the present invention, and a pharmaceutically acceptable excipient.
所述药物组合物优选包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞，包括两个端点之间的任何数量的细胞。优选的是，每个疗程1-3天，每天施用1-3次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。 The pharmaceutical composition preferably comprises the T cell receptor modified cells in a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient, including any between the two endpoints The number of cells. Preferably, each course of treatment is 1-3 days, administered 1-3 times a day. The patient may be treated for one or more courses depending on the actual situation and needs.
所述可药用辅料包括药用或生理载体、赋形剂、稀释剂(包括生理盐水、PBS溶液)、以及各种添加剂，包括糖类、脂类、多肽、 氨基酸、抗氧化剂、佐剂、保鲜剂等。The pharmaceutically acceptable excipients include pharmaceutically or physiological carriers, excipients, diluents (including physiological saline, PBS solution), and various additives, including sugars, lipids, polypeptides, amino acids, antioxidants, adjuvants, Preservatives, etc.
所述药物组合物可通过合适的给药途径给药，其适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。The pharmaceutical composition can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, and peritoneal Internal administration.
本发明还提供了一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据本发明所述的T细胞受体修饰的细胞。The invention also provides a method of treating a tumor and/or cancer comprising administering to a tumor and/or cancer patient a T cell receptor modified cell according to the invention.
所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的，包括但不限于乳腺癌、卵巢癌、胃癌、食管癌、肠癌、胰腺癌、膀胱癌、肾癌、前列腺癌、子宫颈癌、子宫内膜癌、唾液腺癌、皮肤癌、肺癌、骨癌以及脑癌。The tumor and/or cancer is antigenic Her2/neu positive and is HLA-A2 positive, including but not limited to breast cancer, ovarian cancer, gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, bladder cancer, kidney cancer, Prostate cancer, cervical cancer, endometrial cancer, salivary gland cancer, skin cancer, lung cancer, bone cancer, and brain cancer.
所述T细胞受体修饰的细胞的施用剂量优选为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。优选的是，每个疗程1-3天，每天施用1-3次。可以根据实际情况和需要对患者进行一个或多个疗程的治疗。 The T cell receptor modified cells are preferably administered at a dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient. Preferably, each course of treatment is 1-3 days, administered 1-3 times a day. The patient may be treated for one or more courses depending on the actual situation and needs.
所述T细胞受体修饰的细胞可通过合适的给药途径给药，其适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。The T cell receptor-modified cells can be administered by a suitable administration route, which is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, Intramuscular and intraperitoneal administration.
所述T细胞受体修饰的细胞进入治疗对象体内后可以消除表达Her2/neu抗原的肿瘤细胞，和/或改变肿瘤组织的微环境而诱发其它抗肿瘤免疫反应。The T cell receptor-modified cells can eliminate the tumor cells expressing the Her2/neu antigen and/or alter the microenvironment of the tumor tissue to induce other anti-tumor immune responses after entering the therapeutic subject.
本发明还提供了所述分离的T细胞受体用于检测接受该TCR修饰的T细胞(即TCR-T细胞)治疗的患者体内的该TCR-T细胞的增殖或生存情况的应用，从而进行药物代谢研究，和了解该TCR-T细胞的疗效和毒性。具体而言，TCR序列可作为引物，通过PCR方法检测体内携带此TCR的TCR-T细胞的数量。与荧光标记的HLA/多肽复合物多聚体染色后用流式细胞法进行分析的方法相比，所述应用所需要的细胞量少，也更敏感。The present invention also provides the use of the isolated T cell receptor for detecting proliferation or survival of the TCR-T cell in a patient treated with the TCR-modified T cell (ie, TCR-T cell), thereby performing Drug metabolism studies, and understanding the efficacy and toxicity of this TCR-T cell. Specifically, the TCR sequence can be used as a primer to detect the number of TCR-T cells carrying the TCR in vivo by a PCR method. Compared to methods in which fluorescently labeled HLA/polypeptide complex multimers are stained and analyzed by flow cytometry, the amount of cells required for such applications is less and more sensitive.
以下通过例子的方式进一步解释或说明本发明的内容，但这些例子不应被理解为对本发明的保护范围的限制。The contents of the present invention are further explained or illustrated by way of examples, but the examples are not to be construed as limiting the scope of the invention.
例子example
以下除非特别说明，否则以下例子中所用实验方法均使用生物工程领域的常规实验流程、操作、材料和条件进行。Unless otherwise stated, the experimental methods used in the following examples were carried out using conventional experimental procedures, procedures, materials and conditions in the field of bioengineering.
以下除非特别说明，否则各试剂的百分浓度(％)均指该试剂的体积百分浓度(％(v/v))。Unless otherwise indicated, the percent concentration (%) of each reagent refers to the volume percent concentration (% (v/v)) of the reagent.
材料和方法Materials and Method
细胞株：用于制备慢病毒颗粒的细胞株为293T细胞(ATCC CRL-3216)。用于检测TCR表型和功能的T细胞株为Jurkat细胞(clone E6-1，ATCC TIB-152)和J.RT3-T3.5细胞(ATCC TIB-153)。用于提呈抗原多肽的提呈细胞株为T2细胞(174xCEM.T2，ATCC CRL-1992)。用于检测TCR功能的肿瘤细胞株为人结肠癌colo205细胞(ATCC CCL-222)和Caco-2细胞(ATCC HTB-37)、人乳腺癌MDA-MB-231细胞(ATCC HTB-26)、人卵巢癌SK-OV-3细胞(ATCC HTB-77)、人淋巴瘤细胞Bjab(ACC-757-DSMZ)、人肺癌H647细胞(ATCC CRL-5824)和H1355细胞(ATCC CRL-5865)。细胞株用RPMI-1640完全培养基(Lonza，cat#12-115F)维持培养，RPMI-1640完全培养基中加入10％小牛血清FBS(ATCC 30-2020)，2mmol/L L-谷氨酸，100μg/ml青霉素和100μg/ml链霉素。Cell line: The cell line used to prepare the lentiviral particles was 293T cells (ATCC CRL-3216). The T cell strains used to detect TCR phenotype and function were Jurkat cells (clone E6-1, ATCC TIB-152) and J. RT3-T3.5 cells (ATCC TIB-153). The presenting cell strain used to present the antigenic polypeptide was T2 cells (174xCEM.T2, ATCC CRL-1992). The tumor cell lines for detecting TCR function are human colon cancer colo205 cells (ATCC CCL-222) and Caco-2 cells (ATCC HTB-37), human breast cancer MDA-MB-231 cells (ATCC HTB-26), human ovary Cancer SK-OV-3 cells (ATCC HTB-77), human lymphoma cells Bjab (ACC-757-DSMZ), human lung cancer H647 cells (ATCC CRL-5824) and H1355 cells (ATCC CRL-5865). The cell line was maintained in RPMI-1640 complete medium (Lonza, cat#12-115F), and 10% calf serum FBS (ATCC 30-2020) was added to RPMI-1640 complete medium, 2mmol/L L-glutamic acid. , 100 μg/ml penicillin and 100 μg/ml streptomycin.
外周血：试验所用健康供者的人外周血制品均来自位于旧金山的Pacific血液中心(#1PBMC和#2 PBMC分别为来自Apheresis法收集试剂盒的Trima残留细胞组分#R32334和#R33941)。Peripheral blood: Peripheral blood products from healthy donors used in the trial were from the Pacific Blood Center in San Francisco (#1PBMC and #2 PBMC are Trima Residual Cell Components #R32334 and #R33941 from the Apheresis Collection Kit, respectively).
体外诱导Her2/neu 369-377特异性杀伤T细胞(CTL)：外周血经Ficoll-Paque Premium(Sigma-Alorich公司，cat#GE-17-5442-02)密度梯度离心(×400g)30分钟后获得单个核细胞(PBMC)。首先用荧光素FITC标记的抗HLA-A2抗体(Biolegend公司，cat#343303)染色检测细胞的HLA-A2表型，流式细胞分析后提取阳性细胞的RNA，逆转录为cDNA并克隆到载体上，之后进行HLA基因测序分析，确定细胞配型为HLA-A*0201。HLA-A2阳性的PBMC细胞培养在24-孔培养板的培养孔，培养液为上述RPMI-1640完全培养基。每孔2×10e6/ml PBMC，加入Her2/neu 369-377多肽(Her2-E75，用Peptide2.0合成， 10μg/ml溶于DMSO)，浓度为1μg/ml。置于5％CO 2、37℃条件下的培养箱培养16-24小时后加入以下细胞因子，人IL-2(Peprtech公司，cat#200-02)100IU/ml，人IL-7(Peprotech公司，cat#200-07)5ng/ml，人IL-15(Peprotech公司，cat#200-15)5ng/ml。培养10到14天，对培养的T细胞进行抗原再刺激：在24-孔板中每孔加入10e6个培养细胞，同时加入2×10e6个经25μg/ml丝裂霉素C(Santa Cruz Biotechnology公司，cat#SC-3514)处理2小时的HLA-A2阳性的自体PBMC细胞作为滋养细胞，每孔加入终浓度为1μg/ml的Her2/neu 369-377多肽，培养过夜后加入IL-2 100IU/ml，IL-7 5ng/ml，IL-15 5ng/ml。经两轮上述抗原刺激和再刺激后，收集扩增的T细胞进行表型分析以及T细胞克隆。 Induction of Her2/neu 369-377-specific killer T cells (CTL) in vitro: Peripheral blood was centrifuged (×400 g) by Ficoll-Paque Premium (Sigma-Alorich, cat# GE-17-5442-02) for 30 minutes. Mononuclear cells (PBMC) were obtained. First, the HLA-A2 phenotype of the cells was detected by fluorescein FITC-labeled anti-HLA-A2 antibody (Biolegend, cat#343303). After flow cytometry, the RNA of the positive cells was extracted, reverse-transcribed into cDNA and cloned into the vector. Then, HLA gene sequencing analysis was performed to determine the cell matching type as HLA-A*0201. The HLA-A2-positive PBMC cells were cultured in a culture well of a 24-well culture plate, and the culture solution was the above RPMI-1640 complete medium. 2×10e6/ml PBMC per well was added with Her2/neu 369-377 polypeptide (Her2-E75, synthesized with Peptide 2.0, 10 μg/ml dissolved in DMSO) at a concentration of 1 μg/ml. After incubation for 16-24 hours in an incubator at 5% CO 2 at 37 ° C, the following cytokines were added, human IL-2 (Peprtech, cat#200-02) 100 IU/ml, human IL-7 (Peprotech) , cat#200-07) 5ng/ml, human IL-15 (Peprotech, cat#200-15) 5ng/ml. 10 to 14 days of culture, antigen re-stimulation of cultured T cells: 10e6 culture cells were added to each well in a 24-well plate, and 2×10e6 25 μg/ml mitomycin C (Santa Cruz Biotechnology) was added. , cat#SC-3514) Treated 2 hours of HLA-A2-positive autologous PBMC cells as trophoblast cells, adding Her2/neu 369-377 polypeptide at a final concentration of 1 μg/ml per well, and adding IL-2 100 IU after overnight incubation. Ml, IL-7 5 ng/ml, IL-15 5 ng/ml. After two rounds of stimulation and restimulation of the above antigen, the expanded T cells were collected for phenotypic analysis and T cell cloning.
流式细胞分析及单细胞分离：表达Her2/neu 369-377特异性TCR的T细胞表型是通过流式细胞来分析的。收集被检测的细胞置于1.5ml管(细胞数目约为10e5个)，用1ml DPBS溶液(2.7mM KCl，1.5mM KH 2PO 4，136.9mM NaCl,8.9mM Na 2HPO 4·7H 2O，pH 7.4)洗一遍，并重置于100μl含有1％小牛血清的DPBS中，加入5μl荧光素APC标记的抗人CD8抗体(Biolegend公司，cat#300912)，以及10μl荧光素PE标记的Her2-E75/HLA-A2四聚体(Her2-E75四聚体，MBL International Co公司，cat#T01014)或者Her2-E75/HLA-A2五聚体(Her2-E75五聚体，Proimmune公司，cat#F214-2A-D)，冰上孵育30分钟后用DPBS溶液洗两遍，重悬于100μl PBS溶液进行流式细胞分析。流式细胞仪为MACSQuant Analyzer 10(Miltenyi Biotec公司)，用Flowjo软件(Flowjo公司)进行结果分析。T细胞克隆是利用流式细胞分离仪(FACS sorter)进行单细胞分离后培养获得。对Her2/neu369-377多肽抗原刺激过的PBMC用APC标记的抗人CD8抗体和PE标记的Her2-E75/HLA-A2五聚体染色，然后进行流式细胞分离(型号：Sony cell sorter SH800)。单个CD8 +Her2-E75/HLA-A2五聚体 +细胞被分选到96-孔培养板的单个培养孔后，加入经25μg/ml丝裂霉素C处理2小时的HLA-A2阳性的自体PBMC细胞，每孔10e5个细胞，加入1μg/ml Her2/neu 369-377多肽培养过夜后，加入含有IL-2  100IU/ml、IL-7 5ng/ml、IL-15 5ng/ml的RPMI-1640完全培养液。每3-4天换新鲜含有所述细胞因子的培养液，显微镜下观察是否有T细胞克隆生长。收集增殖的T细胞，按上述方法进行抗原再刺激以获得足够数量的细胞，进行表型或功能检测，以及提取RNA进行TCR基因的克隆。 Flow cytometry and single cell isolation: The T cell phenotype expressing the Her2/neu 369-377 specific TCR was analyzed by flow cytometry. The cells to be tested were collected in 1.5 ml tubes (the number of cells was approximately 10e5) using 1 ml of DPBS solution (2.7 mM KCl, 1.5 mM KH 2 PO 4 , 136.9 mM NaCl, 8.9 mM Na 2 HPO 4 ·7H 2 O, pH 7.4) Wash once and reset in 100 μl of DPBS containing 1% calf serum, add 5 μl of fluorescein APC-labeled anti-human CD8 antibody (Biolegend, cat #300912), and 10 μl of fluorescein PE-labeled Her2- E75/HLA-A2 tetramer (Her2-E75 tetramer, MBL International Co, cat#T01014) or Her2-E75/HLA-A2 pentamer (Her2-E75 pentamer, Proimmune, cat#F214 -2A-D), incubated on ice for 30 minutes, washed twice with DPBS solution, and resuspended in 100 μl of PBS solution for flow cytometry analysis. The flow cytometer was MACSQuant Analyzer 10 (Miltenyi Biotec), and the result analysis was performed using Flowjo software (Flowjo Corporation). T cell clones were obtained by single cell separation using a flow cytometer (FACS sorter). PBMCs stimulated with Her2/neu369-377 polypeptide antigen were stained with APC-labeled anti-human CD8 antibody and PE-labeled Her2-E75/HLA-A2 pentamer, followed by flow cytometry (Model: Sony cell sorter SH800) . After a single CD8 + Her2-E75/HLA-A2 pentamer + cell was sorted into a single culture well of a 96-well culture plate, HLA-A2 positive autologous was treated by treatment with 25 μg/ml mitomycin C for 2 hours. PBMC cells, 10e5 cells per well, were incubated with 1μg/ml Her2/neu 369-377 polypeptide overnight, and then added RPMI-1640 containing IL-2 100IU/ml, IL-7 5ng/ml, IL-15 5ng/ml. Complete medium. The culture medium containing the cytokine was replaced every 3-4 days, and it was observed under the microscope whether or not T cell clones grew. The proliferating T cells are collected, subjected to antigen re-stimulation as described above to obtain a sufficient number of cells, subjected to phenotypic or functional detection, and extracted RNA for cloning of the TCR gene.
T细胞功能检测：经Her2/neu 369-377多肽特异性TCR修饰的T细胞株的功能是通过T细胞表面CD69的表达来检测的。在96-孔板的每孔中加入10e5个转染TCR基因的T细胞以及10e5个T2细胞，在100μl/每孔RPMI-1640完全培养基中进行混合培养，各试验组为复孔。再加入不同浓度(分别为1μg/ml、0.5μg/ml、0.1μg/ml、0.05μg/ml、0.01μg/ml、0.005μg/ml、0.001μg/ml和0μg/ml)Her2/neu 369-377多肽后置于5％CO 2、37℃条件下的孵育箱过夜培养。收集细胞并悬浮于DPBS+1％FBS溶液中，用APC标记的抗人CD69抗体进行染色后进行流式细胞分析。经Her2/neu 369-377抗原刺激后表达CD69的T细胞被认为其携带并表达Her2/neu 369-377特异性的TCR。Her2/neu 369-377特异性CTL克隆以及PBMC中的转染TCR基因的原代T细胞的功能，是通过检测抗原刺激后细胞上清中分泌的γ干扰素来确定的。Her2/neu 369-377特异性CTL克隆或者转染TCR基因的PBMC细胞分别与靶细胞在96孔板中，在100μl/每孔RPMI-1640完全培养基中进行混合培养，靶细胞是加入10倍稀释浓度的Her2/neu 369-377多肽的T2细胞，或者各种肿瘤细胞株细胞，各试验组为复孔。抗体功能阻断试验中，细胞培养孔中同时加入10μg/ml抗人CD8抗体(Biolegend公司，cat#300912)，或者抗HLA-ABC抗体(w6/32克隆，Biolegend公司，cat#311402)，细胞置于5％CO 2、37℃条件下的孵育箱过夜培养。18-24小时收集细胞上清，并用人IFN-γELISA Read-set-Go试剂盒(eBioscience公司，cat#88-7316)对上清中的IFN-γ进行检测。 T cell function assay: The function of the T cell strain modified by Her2/neu 369-377 polypeptide-specific TCR is detected by the expression of CD69 on the surface of T cells. 10 e5 TCR cells transfected with TCR gene and 10e5 T2 cells were added to each well of a 96-well plate, and mixed culture was carried out in 100 μl/well of RPMI-1640 complete medium, and each test group was a duplicate well. Add different concentrations (1μg/ml, 0.5μg/ml, 0.1μg/ml, 0.05μg/ml, 0.01μg/ml, 0.005μg/ml, 0.001μg/ml and 0μg/ml) Her2/neu 369- The 377 polypeptide was then cultured overnight in an incubator at 5% CO 2 at 37 °C. The cells were collected and suspended in DPBS + 1% FBS solution, stained with APC-labeled anti-human CD69 antibody, and subjected to flow cytometry analysis. T cells expressing CD69 after stimulation with Her2/neu 369-377 antigen are thought to carry and express a TCR specific for Her2/neu 369-377. The Her2/neu 369-377-specific CTL clone and the function of primary T cells transfected with the TCR gene in PBMC were determined by detecting gamma interferon secreted in the supernatant of the cell after antigen stimulation. Her2/neu 369-377-specific CTL clones or PBMC cells transfected with TCR gene were mixed with target cells in 96-well plates in 100 μl/well RPMI-1640 complete medium, and the target cells were added 10 times. T2 cells of a diluted concentration of Her2/neu 369-377 polypeptide, or cells of various tumor cell lines, each of which was a duplicate well. In the antibody function blocking assay, 10 μg/ml anti-human CD8 antibody (Biolegend, cat#300912) or anti-HLA-ABC antibody (w6/32 clone, Biolegend, cat#311402) was added to the cell culture well. Incubate overnight in an incubator at 5% CO 2 at 37 °C. The cell supernatant was collected for 18-24 hours, and IFN-γ in the supernatant was detected using a human IFN-γ ELISA Read-set-Go kit (eBioscience, cat#88-7316).
获得单克隆TCR基因：利用Zymo Quick-RNA Microprep试剂盒(Zymo Research公司，cat#R1050)从T细胞克隆提纯总RNA，以此为模板利用Smarter RACE 5’/3’试剂盒获得cDNA(美国Takara Bio公司，cat#634858)。用5’-CDS引物和TCRβ链3’引物5’- GCCTCTGGAATCCTTTCTCTTG-3’(SEQ ID NO:26)以及α链3’引物5’-TCAGCTGGACCACAGCCGCAG-3’(SEQ ID NO:27)进行PCR，扩增出TCRα和β全序列基因片段，并分别克隆到pRACE载体(美国Takara Bio，cat#634858)上。转化感受态细菌Stellar(美国Takara Bio公司，cat#636763)并获得质粒后进行测序。Obtaining the monoclonal TCR gene: Purification of total RNA from T cell clones using the Zymo Quick-RNA Microprep kit (Zymo Research, cat#R1050), using this as a template to obtain cDNA using the Smarter RACE 5'/3' kit (Takara, USA) Bio, cat#634858). PCR was carried out using 5'-CDS primer and TCRβ chain 3' primer 5'-GCCTCTGGAATCCTTTCTCTTG-3' (SEQ ID NO: 26) and α chain 3' primer 5'-TCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 27) TCRα and β full sequence gene fragments were added and cloned into pRACE vector (Takara Bio, cat#634858, USA). The competent bacteria Stellar (Takara Bio, cat #636763, USA) was transformed and plasmids were obtained and sequenced.
重组TCR慢病毒表达载体的制备：用于表达TCR的病毒载体为复制缺陷型慢病毒载体，包括：表达GFP的慢病毒载体pCDH-EF1α-MCS-(PGK-GFP)，可购自System Biosciences公司(Cat#CD811A-1)；以及不表达GFP的载体pCDH-EF1α-MCS，通过采用本领域常规技术去除pCDH-EF1α-MCS-(PGK-GFP)载体上的PGK启动子及GFP基因而得到。根据所获得的TCR基因序列，合成TCRβ链和α链以及之间可切割的F2A序列和Furin酶切片段的全基因序列，并链接到所述载体的EF-1α启动子下游的多克隆位点，插入TCR的转录顺序依次为TCRβ链(无终止密码子)，Furin酶切片段，F2A片段，TCRα链(方法参见文献“Gene Ther.2008Nov；15(21):1411–1423”)。表达GFP的载体是被反向的PGK启动子驱动的。不表达GFP的载体则是去除了PGK启动子以及GFP片段。Preparation of recombinant TCR lentiviral expression vector: The viral vector for expression of TCR is a replication-deficient lentiviral vector, including: GFP-expressing lentiviral vector pCDH-EF1α-MCS-(PGK-GFP), available from System Biosciences (Cat# CD811A-1); and the vector pCDH-EF1α-MCS which does not express GFP, was obtained by removing the PGK promoter and GFP gene on the pCDH-EF1α-MCS-(PGK-GFP) vector by a conventional technique in the art. According to the obtained TCR gene sequence, the TCRβ chain and the α chain and the entire gene sequence of the cleavable F2A sequence and the Furin fragment are synthesized, and linked to the multiple cloning site downstream of the EF-1α promoter of the vector The transcription sequence of the inserted TCR is TCR β chain (no stop codon), Furin fragment, F2A fragment, TCR α chain (see the literature “Gene Ther. 2008 Nov; 15(21): 1411–1423”). The vector expressing GFP is driven by the inverted PGK promoter. The vector that does not express GFP removes the PGK promoter as well as the GFP fragment.
重组TCR慢病毒颗粒的制备：TCR慢病毒颗粒是通过Lipofectaine 3000转染试剂(Thermo Fisher公司，cat#L3000001)转染293T细胞而获得的。依照厂家说明书准备293T细胞以及转染流程。转染在96孔培养板进行，首先用Opti-MEM 1培养液(Thermo Fisher公司，cat#51985091)制备转染质粒的脂质体溶液，依照厂家说明在250μl培养液中加入P3000试剂(P3000 reagent)、以及TCR慢病毒载体质粒和pCDH系统的病毒包装质粒(SBI公司，cat#LV500A-1)，另外250μl培养液中加入Lipofectaine 3000试剂，混合孵育15分钟后加入293T细胞培养孔。5％CO 2、37℃条件下培养16小时，换含10％FBS的DMEM培养液(Thermo Fisher公司，cat#11965092)，继续培养24小时和48小时后分别收集细胞上清，2000g离心后，用0.4μm过滤膜过滤后得到的病毒颗粒用于感染细胞。 Preparation of recombinant TCR lentiviral particles: TCR lentiviral particles were obtained by transfecting 293T cells with Lipofectaine 3000 transfection reagent (Thermo Fisher, cat# L3000001). Prepare 293T cells and transfection procedures according to the manufacturer's instructions. The transfection was carried out in a 96-well culture plate. First, a liposome solution of the transfected plasmid was prepared using Opti-MEM 1 culture solution (Thermo Fisher Co., cat #51985091), and P3000 reagent (P3000 reagent) was added to 250 μl of the culture solution according to the manufacturer's instructions. ), and the TCR lentiviral vector plasmid and the viral packaging plasmid of the pCDH system (SBI, cat#LV500A-1), and another 250 μl of the culture solution was added with Lipofectaine 3000 reagent, and mixed for 15 minutes, and then added to the 293T cell culture well. The cells were cultured for 16 hours at 37 ° C under 5% CO 2 , and changed to DMEM medium (Thermo Fisher Company, cat #11965092) containing 10% FBS. After 24 hours and 48 hours, the cell supernatants were collected and centrifuged at 2000 g. The virus particles obtained by filtration with a 0.4 μm filter membrane were used to infect cells.
重组TCR慢病毒转染人T细胞：冻存的原代PBMC细胞解冻后 在RPMI-1640完全培养液中培养24小时，经Ficoll-Paque Premium密度梯度离心(×400g)30分钟去除死细胞，置于用2μg/ml抗人CD3抗体(Biolegend公司，OKT3克隆cat#317303)和2μg/ml抗人CD28抗体(Biolegend公司，cat#302914)处理(其中每孔加入100μl含有上述CD3抗体和CD28抗体的DPBS溶液)24小时的24孔板培养孔中，细胞浓度为2×10e6/ml，培养48小时后收集细胞，重悬于新鲜的重组TCR慢病毒颗粒上清液中置于24孔板的孔中，加入4ng/ml聚凝胺(Santa Cruz biotechnology公司，cat#sc-134220)后，于32℃，1000g离心2小时，用含有IL-2 100IU/ml、IL-7 5ng/ml、IL-15 5ng/ml的RPMI-1640完全培养液置换一半病毒上清后继续培养，每3天换新鲜含有上述细胞因子的培养液。一般72小时后可进行表型和功能检测。转染T细胞株也依照上述步骤进行，如果病毒载体上带有GFP标记，一般转染后48小时即可在荧光显微镜下观察到GFP阳性细胞。Recombinant TCR lentivirus was transfected into human T cells: cryopreserved primary PBMC cells were thawed and cultured in RPMI-1640 complete medium for 24 hours, and the dead cells were removed by Ficoll-Paque Premium density gradient centrifugation (×400 g) for 30 minutes. Treatment with 2 μg/ml anti-human CD3 antibody (Biolegend, OKT3 clone cat #317303) and 2 μg/ml anti-human CD28 antibody (Biolegend, cat #302914) (wherein 100 μl of the above-mentioned CD3 antibody and CD28 antibody were added per well) DPBS solution) in a 24-hour 24-well plate culture well, the cell concentration was 2×10e6/ml, and after 48 hours of culture, the cells were collected and resuspended in fresh recombinant TCR lentiviral particle supernatant in a well of a 24-well plate. After adding 4 ng/ml polybrene (Santa Cruz Biotechnology, cat#sc-134220), it was centrifuged at 1000 g for 2 hours at 32 ° C for 1 hour, containing IL-2 100 IU/ml, IL-7 5 ng/ml, IL- 15 5 ng/ml of RPMI-1640 complete medium was used to replace half of the virus supernatant, and the culture was continued, and the culture medium containing the above cytokines was replaced every 3 days. Phenotypic and functional tests are typically performed after 72 hours. The transfected T cell line was also carried out according to the above procedure. If the viral vector carries the GFP label, GFP-positive cells can be observed under a fluorescence microscope 48 hours after the transfection.
实施例1：从HLA-A2阳性的正常供体外周血诱导Her2/neu 369-377多肽特异性杀伤T细胞Example 1: Induction of Her2/neu 369-377 polypeptide-specific killer T cells from HLA-A2 positive normal donor peripheral blood
本实施例用1μg/ml的低浓度Her2/neu 369-377多肽经过两轮体外刺激从正常PBMC(#2 PBMC)中诱导出多肽特异性杀伤T细胞，并进行流式细胞分析及单细胞分离。具体方法如上文所述。结果如下：In this example, polypeptide-specific killer T cells were induced from normal PBMC (#2 PBMC) by two rounds of in vitro stimulation with a low concentration of Her2/neu 369-377 polypeptide at 1 μg/ml, and flow cytometry and single cell separation were performed. . The specific method is as described above. The results are as follows:
图1A右图显示0.013％的淋巴细胞为可结合Her2/neu 369-377/HLA-A2五聚体(即Her2-E75五聚体)的CD8阳性杀伤性T细胞，左图中没有经Her2多肽刺激的对照细胞没有出现CD8阳性五聚体阳性细胞。结果说明在自然T细胞库中，识别Her2/neu 369-377抗原多肽的特异性T细胞数量很少。尽管数量少，这群可识别Her2/neu 369-377多肽的T细胞仍可被清晰地区分出来。另外根据结合Her2-E75五聚体的荧光强度，阳性细胞中又包含高亲和性T细胞和低亲和性T细胞。通过流式细胞分离出453个CD8阳性五聚体阳性细胞后进行单克隆培养，经过两轮抗原多肽再刺激以及细胞因子扩增，从这453个分离出的单个T细胞中仅获得一个增殖的T细胞克隆Her2 CTL克隆1B5(称为Her2 CTL 1B5)。图1B右图显示此纯化的CD8 +CTL克隆 可结合Her2/neu 369-377/HLA-A2四聚体(即Her2-E75四聚体)。每孔5x10 3个Her2 CTL 1B5细胞与被5x10 3个T2细胞提呈的不同浓度Her2/neu 369-377抗原多肽(Her2/neu 369-377抗原多肽从1μg/ml开始进行10倍稀释，从而得到终浓度为1μg/ml、0.1μg/ml、0.01μg/ml、0.001μg/ml的不同组)混合培养后，检测上清中T细胞分泌的IFN-γ，用以确定此T细胞克隆特异性识别Her2/neu 369-377多肽的功能。肿瘤细胞培养孔中加入5x10 3个colo205细胞进行混合培养后，检测上清中T细胞分泌的IFN-γ。图1C结果显示，T细胞克隆1B5可以被最低浓度为1ng/ml的抗原多肽所激活，而且T细胞的识别功能与抗原多肽的浓度成正相关(具有剂量依赖性)，说明此T细胞克隆特异性识别被HLA-A2所提呈的Her2/neu 369-377多肽。更重要的是，此T细胞克隆还识别一株HLA-A2 +Her2/neu +的结肠癌细胞株colo205。因此，此T细胞克隆Her2 TCR-1B5不仅可识别Her-E75多肽，还可以被colo205细胞激活而分泌IFN-γ。 The right panel of Figure 1A shows that 0.013% of lymphocytes are CD8-positive killer T cells that bind to Her2/neu 369-377/HLA-A2 pentamer (ie, Her2-E75 pentamer), and there is no Her2 polypeptide in the left panel. The stimulated control cells did not show CD8 positive pentamer positive cells. The results indicate that the number of specific T cells recognizing the Her2/neu 369-377 antigen polypeptide is small in the natural T cell pool. Despite the small number, this group of T cells recognizing the Her2/neu 369-377 polypeptide can still be clearly distinguished. Further, according to the fluorescence intensity of the Her2-E75 pentamer, high-affinity T cells and low-affinity T cells are further contained in the positive cells. 453 CD8-positive pentameric positive cells were isolated by flow cytometry and subjected to monoclonal culture. After two rounds of antigen polypeptide re-stimulation and cytokine amplification, only one proliferation was obtained from the 453 isolated single T cells. T cell clone Her2 CTL clone 1B5 (referred to as Her2 CTL 1B5). Figure 1B right panel shows that this purified CD8 + CTL clone binds to the Her2/neu 369-377/HLA-A2 tetramer (i.e., Her2-E75 tetramer). 5x10 3 Her2 CTL 1B5 cells per well and different concentrations of Her2/neu 369-377 antigen polypeptide presented by 5x10 3 T2 cells (Her2/neu 369-377 antigen polypeptide was 10-fold diluted from 1 μg/ml to obtain After mixed cultures at a final concentration of 1 μg/ml, 0.1 μg/ml, 0.01 μg/ml, and 0.001 μg/ml, the IFN-γ secreted by T cells in the supernatant was detected to determine the specificity of the T cell clone. Identify the function of the Her2/neu 369-377 polypeptide. After 5×10 3 colo205 cells were added to the tumor cell culture well for mixed culture, the IFN-γ secreted by the T cells in the supernatant was detected. Figure 1C shows that T cell clone 1B5 can be activated by a minimum concentration of 1 ng/ml of antigenic polypeptide, and the recognition function of T cells is positively correlated with the concentration of antigenic polypeptide (dose-dependent), indicating the specificity of this T cell clone. The Her2/neu 369-377 polypeptide presented by HLA-A2 was identified. More importantly, this T cell clone also recognized a colon cancer cell line colo205 of HLA-A2 + Her2/neu + . Therefore, this T cell clone Her2 TCR-1B5 not only recognizes the Her-E75 polypeptide, but also activates by colo205 cells to secrete IFN-γ.
实施例2：Her2/neu 369-377多肽特异性TCR全序列的获得和鉴定Example 2: Acquisition and identification of the complete TCR specific sequence of Her2/neu 369-377 polypeptide
本实施例直接从由实施例1得到的Her2/neu 369-377多肽特异性CTL克隆中获得包含相匹配(即，两条链可共同组成识别抗原多肽的功能性TCR)的α和β链的完整TCR基因序列，其编码的TCR称为“Her2 TCR-1B5”。该TCR的α链的氨基酸序列如SEQ ID NO:2所示，编码序列如SEQ ID NO:1所示，并且该TCR的β链的氨基酸序列如SEQ ID NO:4所示，编码序列如SEQ ID NO:3所示。此TCR存在于HLA-A2阳性正常人的外周T细胞库中，不会对微量表达Her2/neu蛋白的正常细胞产生交叉反应而导致自身免疫反应。具体检测方法如上文所述。结果如下：This example obtained directly from the Her2/neu 369-377 polypeptide-specific CTL clone obtained in Example 1 containing α and β chains which are matched (i.e., the two strands can together constitute a functional TCR of the recognition antigen polypeptide). The complete TCR gene sequence, which encodes a TCR, is called "Her2 TCR-1B5". The amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 2, the coding sequence is shown in SEQ ID NO: 1, and the amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 4, and the coding sequence is SEQ. ID NO: 3 is shown. This TCR is present in the peripheral T cell pool of HLA-A2 positive normal humans and does not cross-react to normal cells that express Her2/neu protein in a small amount, resulting in an autoimmune response. The specific detection method is as described above. The results are as follows:
为了检测所获TCR的抗原特异性及其功能，TCRα链和β链序列被克隆到复制缺陷型慢病毒表达载体中。图2显示所构建的TCR慢病毒载体结构片段示意图。α链和β链由可切除的furin识别片段和F2A多肽片段相联结。为了追踪被TCR慢病毒载体转染的T细胞，该慢病 毒载体(图中示为“pCDH-EF1α-Her2 TCR-(PGK-GFP)载体”)可同时表达GFP，由反向的PGK启动子所驱动(图2上方)。另一种表达载体(图中示为“pCDH-EF1α-Her2 TCR载体”)(图2下方)去除了GFP及其启动子序列。通过减小载体长度以增加慢病毒颗粒的产出，又避免了两个启动子之间的相互影响，从而增加TCR的表达量。To detect the antigen specificity and function of the obtained TCR, the TCR alpha chain and beta chain sequences were cloned into a replication-defective lentiviral expression vector. Figure 2 shows a schematic representation of the constructed TCR lentiviral vector structural fragment. The alpha and beta chains are linked by a resectable furin recognition fragment and an F2A polypeptide fragment. To track T cells transfected with TCR lentiviral vector, the lentiviral vector (shown as "pCDH-EF1α-Her2 TCR-(PGK-GFP) vector") can simultaneously express GFP, by reverse PGK promoter Driven (above Figure 2). Another expression vector (shown as "pCDH-EF1[alpha]-Her2 TCR vector") (bottom of Figure 2) removes GFP and its promoter sequences. By reducing the length of the vector to increase the production of lentiviral particles, the interaction between the two promoters is avoided, thereby increasing the expression of TCR.
将以下序列分别连接至上述表达GFP的载体和不表达GFP的载体：i)由可切割性连接多肽链接的、在恒定区增加1个二硫键的TCRβ链和α链的核苷酸序列(SEQ ID NO:13)(对应的TCR为Her2 TCR-1B5-dis，氨基酸序列如SEQ ID NO:14所示)；ii)由可切割性连接多肽链接的、恒定区由人源序列替换为鼠源序列的TCRβ链和α链的核苷酸序列(SEQ ID NO:15)(对应的TCR为Her2 TCR-1B5-mC，氨基酸序列如SEQ ID NO:16所示)；iii)由可切割性连接多肽链接的原始TCRβ链和α链的核苷酸序列(SEQ ID NO:23)(对应的TCR为Her2 TCR-1B5-wt，编码的氨基酸序列如SEQ ID NO:24所示)；从而得到6种重组慢病毒载体：The following sequences were ligated to the above-described GFP-expressing vector and the GFP-expressing vector, respectively: i) a nucleotide sequence of a TCR β-chain and an α-chain which are linked by a cleavable linker polypeptide and which have a disulfide bond added in the constant region ( SEQ ID NO: 13) (corresponding TCR is Her2 TCR-1B5-dis, amino acid sequence is set forth in SEQ ID NO: 14); ii) constant region linked by a cleavable linker polypeptide is replaced by a human sequence to a murine The nucleotide sequence of the TCR β chain and the α chain of the source sequence (SEQ ID NO: 15) (corresponding TCR is Her2 TCR-1B5-mC, amino acid sequence is shown as SEQ ID NO: 16); iii) by cleavability Linking the nucleotide sequence of the original TCR β chain and the α chain linked by the polypeptide (SEQ ID NO: 23) (corresponding TCR is Her2 TCR-1B5-wt, the encoded amino acid sequence is shown as SEQ ID NO: 24); 6 recombinant lentiviral vectors:
1)Her2 TCR-1B5-dis重组慢病毒载体(携带GFP)；1) Her2 TCR-1B5-dis recombinant lentiviral vector (carrying GFP);
2)Her2 TCR-1B5-dis w/o GFP重组慢病毒载体(不携带GFP)；2) Her2 TCR-1B5-dis w/o GFP recombinant lentiviral vector (without carrying GFP);
3)Her2 TCR-1B5-mC重组慢病毒载体(携带GFP)；3) Her2 TCR-1B5-mC recombinant lentiviral vector (carrying GFP);
4)Her2 TCR-1B5-mC w/o GFP重组慢病毒载体(不携带GFP)。4) Her2 TCR-1B5-mC w/o GFP recombinant lentiviral vector (without carrying GFP).
5)Her2 TCR-1B5-wt重组慢病毒载体(携带GFP)；5) Her2 TCR-1B5-wt recombinant lentiviral vector (carrying GFP);
6)Her2 TCR-1B5-wt w/o GFP重组慢病毒载体(不携带GFP)。6) Her2 TCR-1B5-wt w/o GFP recombinant lentiviral vector (without GFP).
各Her2 TCR基因片段通过PCR扩增后，分别克隆到上述两种慢病毒载体(即pCDH-EF1α-MCS-(PGK-GFP)和pCDH-EF1α-MCS)的EF1-α启动子下游：Her2 TCR-1B5-dis和TCR-1B5-wt的TCR片段均是用5’引物5’-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3’(SEQ ID NO:28)和3’引物5’-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3’(SEQ ID NO:29)扩增而得。Her2 TCR-1B5-mC的TCR片段是用5’引物 5’-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3’(SEQ ID NO:30)和3’引物5’-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3’(SEQ ID NO:31)扩增而得。PCR使用Q5高保真PCR试剂盒(NEB,cat#M0543S)，反应条件为98℃30秒后，进行25个循环98℃10秒，65℃10秒，72℃3分钟。获得的TCR片段克隆到pCDH-EF1α-MCS-(PGK-GFP)载体或pCDH-EF1α-MCS载体的EF1α启动子下游的MCS区域。Each Her2 TCR gene fragment was amplified by PCR and cloned into the downstream of the EF1-α promoter of the two lentiviral vectors (ie, pCDH-EF1α-MCS-(PGK-GFP) and pCDH-EF1α-MCS): Her2 TCR The TCR fragments of -1B5-dis and TCR-1B5-wt were both 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 28) and 3' primer 5'-GTTGATTGTCGACGCCCTCAGCTGGACCACAGCCGCAG-3' (SEQ ID NO: 29 ) amplified. The TCR fragment of Her2 TCR-1B5-mC was amplified by 5' primer 5'-AGAGCTAGCGAATTCAACATGGATACCTGGCTCGTATG-3' (SEQ ID NO: 30) and 3' primer 5'-GTTGATTGTCGACGCCCTCAACTGGACCACAGCCT-3' (SEQ ID NO: 31). . The PCR was carried out using a Q5 high-fidelity PCR kit (NEB, cat#M0543S). After the reaction conditions were 98 ° C for 30 seconds, 25 cycles of 98 ° C for 10 seconds, 65 ° C for 10 seconds, and 72 ° C for 3 minutes were performed. The obtained TCR fragment was cloned into the MCS region downstream of the EF1α promoter of the pCDH-EF1α-MCS-(PGK-GFP) vector or the pCDH-EF1α-MCS vector.
将构建得到的重组TCR慢病毒表达载体按前述方法制备得到各自的重组TCR慢病毒颗粒。The recombinant TCR lentiviral expression vectors constructed were prepared as described above to obtain respective recombinant TCR lentiviral particles.
按照上述方式能够得到两种不同方式修饰的TCR，Her2 TCR-1B5-dis是通过点突变在TCR恒定区增加一个二硫键，方法在文献“Cancer Res.2007 Apr 15；67(8):3898-903.”中描述，其全文通过引用方式并入本文。Her2 TCR-1B5-mC是用小鼠TCR恒定区序列置换相应的人TCR恒定区序列，方法在文献“Eur.J.Immunol.2006 36:3052–3059”中描述，其全文通过引用方式并入本文。图3A显示，用携带Her2TCR-1B5 TCR和GFP基因的慢病毒(即，上述Her2 TCR-1B5-dis重组慢病毒载体和Her2 TCR-1B5-mC重组慢病毒载体)转染T细胞株后，一部分GFP +细胞可以结合Her2-E75四聚体，说明这些被慢病毒转染的T细胞可以识别被HLA-A2分子提呈的Her2/neu 369-377多肽。由所得数据计算，如果TCR恒定区增加一个二硫键，37.5％(13.2/(13.2+22)×100％)被转染的T细胞(GFP +)所表达的TCR可以识别Her2/neu多肽抗原(图3A左图)。而TCR恒定区被小鼠TCR相应的恒定区所置换的，则有60％(34.6/(34.6+23)×100％)被转染的T细胞(GFP +)所表达的TCR可以识别Her2/neu多肽抗原(图3A右图)。与外源TCRα链和β链经二硫键修饰后得到的TCR相比，外源TCR恒定区被相应小鼠TCR恒定区所置换而形成的杂交TCR可以进一步减少与内源TCR的错配几率。图3B显示，不论转染只表达beta链的J.RT-T3.5细胞还是同时表达α链和β链的Jurkat细胞，恒定区被小鼠恒定区序列置换后得到的外源TCR(Her2 TCR-1B5-mC)的表达量明 显高于只在恒定区增加一个二硫键的TCR(Her2 TCR-1B5-dis)。图3C显示，表达外源Her2 TCR-1B5的T细胞株可以被T2细胞提呈的Her2/neu 369-377多肽所激活而表达CD69，说明此TCR具有识别Her2/neu 369-377多肽抗原的功能。恒定区被小鼠恒定区序列置换后得到的外源TCR(Her2 TCR-1B5-mC)的表达量不仅增加，对多肽抗原的特异性识别能力也明显高于经额外二硫键修饰的TCR(Her2 TCR-1B5-dis)，可以被更低浓度的Her2/neu 369-377多肽所激活。然而，激活表达Her2 TCR-1B5-mC的Jurkat细胞的最低多肽浓度约为0.05μg/ml，比图1C显示的激活Her2 CTL 1B5克隆的多肽浓度高约50倍。这说明Jurkat细胞表达的Her2 TCR-1B5 TCR识别多肽抗原的能力(avidity)要明显低于CD8 +CTL上表达的Her2 TCR-1B5 TCR。可能的一个原因是Jurkat细胞不表达CD8分子，而CD8对Her2 TCR-1B5的识别功能起着重要的辅助作用。 Two different ways of modifying the TCR can be obtained as described above. Her2 TCR-1B5-dis is a disulfide bond added to the TCR constant region by point mutation in the literature "Cancer Res. 2007 Apr 15; 67(8):3898 - 903. The entire disclosure is incorporated herein by reference. Her2 TCR-1B5-mC replaces the corresponding human TCR constant region sequence with a mouse TCR constant region sequence as described in the literature "Eur. J. Immunol. 2006 36: 3052-3059", which is incorporated by reference in its entirety by reference. This article. Figure 3A shows a portion of a T cell line transfected with a lentivirus carrying the Her2TCR-1B5 TCR and GFP genes (i.e., the Her2 TCR-1B5-dis recombinant lentiviral vector and the Her2 TCR-1B5-mC recombinant lentiviral vector). GFP + cells can bind to the Her2-E75 tetramer, indicating that these Lentiviral-transfected T cells recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule. From the data obtained, if the TCR constant region is increased by a disulfide bond, 37.5% (13.2/(13.2+22)×100%) of the TCR expressed by the transfected T cell (GFP + ) can recognize the Her2/neu polypeptide antigen. (Figure 3A left). While the TCR constant region was replaced by the corresponding constant region of the mouse TCR, 60% (34.6/(34.6+23)×100%) of the TCR expressed by the transfected T cell (GFP + ) recognized Her2/ Neu polypeptide antigen (Figure 3A right panel). Compared with the TCR obtained by the disulfide bond modification of the exogenous TCR α chain and the β chain, the hybrid TCR formed by replacing the foreign TCR constant region by the corresponding mouse TCR constant region can further reduce the mismatch probability with the endogenous TCR. . Figure 3B shows the exogenous TCR (Her2 TCR) obtained by transfecting J. RT-T3.5 cells expressing only the beta chain or Jurkat cells expressing both the α chain and the β chain, and the constant region was replaced by the mouse constant region sequence. The expression level of -1B5-mC) was significantly higher than that of TCR (Her2 TCR-1B5-dis) which added only one disulfide bond in the constant region. Figure 3C shows that the T cell line expressing exogenous Her2 TCR-1B5 can be activated by T2 cell-expressing Her2/neu 369-377 polypeptide to express CD69, indicating that this TCR has the function of recognizing the Her2/neu 369-377 polypeptide antigen. . The expression level of the exogenous TCR (Her2 TCR-1B5-mC) obtained by replacing the constant region with the mouse constant region sequence is not only increased, but also the specific recognition ability for the polypeptide antigen is significantly higher than that of the TCR modified by the additional disulfide bond ( Her2 TCR-1B5-dis) can be activated by lower concentrations of Her2/neu 369-377 polypeptide. However, the minimum polypeptide concentration for activating Jurkat cells expressing Her2 TCR-1B5-mC was approximately 0.05 μg/ml, which was approximately 50-fold higher than the polypeptide concentration of the activated Her2 CTL 1B5 clone shown in Figure 1C. This indicates that the ability of the Her2 TCR-1B5 TCR recognition polypeptide antigen expressed by Jurkat cells is significantly lower than that of the Her2 TCR-1B5 TCR expressed on CD8 + CTL. One possible reason is that Jurkat cells do not express CD8 molecules, and CD8 plays an important supporting role in the recognition function of Her2 TCR-1B5.
实施例3：正常外周血T细胞经Her2 TCR-1B5-mC重组慢病毒转染后表达可识别Her2/neu 369-377多肽的特异性TCRExample 3: Normal peripheral blood T cells are transfected with Her2 TCR-1B5-mC recombinant lentivirus and express a specific TCR that recognizes the Her2/neu 369-377 polypeptide.
为了进一步验证本发明所获得的TCR能否在原代T细胞表达并具有识别Her2/neu抗原多肽的功能，用携带Her2 TCR-1B5-mC基因的重组慢病毒颗粒(Her2 TCR-1B5-mC重组慢病毒载体)转染经CD3/CD28抗体活化的、来自两个不同正常供体的外周血T细胞，7天后收集细胞进行Her2-E75四聚体染色。具体方法如上文所述。结果如下：To further verify whether the TCR obtained by the present invention can be expressed in primary T cells and has the function of recognizing the Her2/neu antigen polypeptide, recombinant lentiviral particles carrying the Her2 TCR-1B5-mC gene (Her2 TCR-1B5-mC recombination slow) Viral vectors were transfected with peripheral blood T cells from two different normal donors activated by CD3/CD28 antibody, and cells were harvested 7 days later for Her2-E75 tetramer staining. The specific method is as described above. The results are as follows:
图4A显示，两个供体外周血单个核细胞(分别为#1PBMC和#2 PBMC)中均有淋巴细胞可以结合Her2-E75四聚体，说明这些细胞表达的Her2 TCR-1B5-mC可以特异性识别被HLA-A2提呈的Her2/neu抗原多肽。结果还显示，Her2-E75四聚体阳性细胞(即表达Her2 TCR-1B5-mC)中，大多数阳性细胞为CD8 +T杀伤细胞，小部分阳性细胞为CD8-的淋巴细胞，这很可能是CD4 +的T辅助细胞。CD8 +CTL结合Her2-E75四聚体的荧光强度(#2 PBMC样本中的Geom.Mean为1469，#1PBMC样本中的Geom.Mean为1404)，也明显大于CD4 +T细胞(#2 PBMC样本中的Geom Mean为560，#1PBMC样本中的 Geom.Mean为504)。如果慢病毒感染CD8 +和CD4 +T细胞的转染效率一样，说明一部分表达在CD4 +细胞上的外源Her2/neu 369-377特异性TCR并不能结合Her2-E75四聚体，即使结合，亲和力也低于表达在CD8 +T细胞上的外源转染TCR。这也进一步说明转染的TCR需要CD8分子的辅助功能才能有效结合Her2/HLA-A2复合物。 Figure 4A shows that both peripheral blood mononuclear cells (#1PBMC and #2 PBMC, respectively) can bind to Her2-E75 tetramer, indicating that Her2 TCR-1B5-mC expressed in these cells can be specific. The Her2/neu antigen polypeptide presented by HLA-A2 is sexually recognized. The results also showed that in the Her2-E75 tetramer-positive cells (ie, expressing Her2 TCR-1B5-mC), most of the positive cells were CD8 + T killer cells, and a small number of positive cells were CD8-like lymphocytes, which is likely CD4 + T helper cells. The fluorescence intensity of CD8 + CTL binding to Her2-E75 tetramer (#2 in Geom.Mean in #2 PBMC samples, 1404 in Geom.Mean in #1PBMC samples) was also significantly greater than CD4 + T cells (#2 PBMC samples) The Geom Mean in the 560 is 560, and the Geom.Mean in the #1PBMC sample is 504). If the transfection efficiency of lentivirus-infected CD8 + and CD4 + T cells is the same, it indicates that a part of the exogenous Her2/neu 369-377-specific TCR expressed on CD4 + cells does not bind to Her2-E75 tetramer, even if bound, Affinity is also lower than exogenously transfected TCR expressed on CD8 + T cells. This further demonstrates that the transfected TCR requires the auxiliary function of the CD8 molecule to effectively bind the Her2/HLA-A2 complex.
在96-孔板的每孔中加入10e5个转染TCR的PBMC细胞，与不同浓度被T2细胞(每孔10e5个)提呈的Her2/neu 369-377抗原多肽(Her2/neu 369-377抗原多肽从0.1μg/ml开始进行10倍稀释，从而得到终浓度为0.1μg/ml、0.01μg/ml、0.001μg/ml的不同组)混合培养后，检测上清中T细胞分泌的IFN-γ，用以确定此表达TCR的PBMC细胞特异性识别Her2/neu 369-377多肽的功能。对照组靶细胞为提呈1μg/ml的可以结合HLA-A2分子的EBV病毒抗原多肽LMP2 426-434的T2细胞。图4B显示，表达Her2 TCR-1B5-mC的PBMC可以被T2细胞提呈的Her2/neu 369-377抗原多肽所激活而分泌IFN-γ，说明表达外源Her2 TCR-1B5-mC的原代T细胞可以特异性识别被HLA-A2分子提呈的Her2/neu 369-377多肽。识别抗原多肽的能力与外源TCR在T细胞上的表达量相关。其识别抗原多肽的最大半反应(half-maximum reaction，EC 50)多肽浓度经曲线拟合推算为约6.9ng/ml(IC 50Tool Kit，http://www.ic50.tk/)，尽管此反应敏感度低于识别病毒抗原等外源抗原的高亲和性TCR的EC 50(EC 50约10e-10M)(参见文献“CANCER RESEARCH 1998,58.4902-4908”和“HUMAN GENE THERAPY 2014,25:730–739”)，但仍处于可识别常见肿瘤相关抗原的TCR亲和力范围之内(如文献“Eur J Immunol(2012)42:3174–9”所述)。 10e5 TCR-transfected PBMC cells were added to each well of a 96-well plate, and Her2/neu 369-377 antigenic peptide (Her2/neu 369-377 antigen) was presented at different concentrations in T2 cells (10e5 per well). The polypeptide was 10-fold diluted from 0.1 μg/ml to obtain a final concentration of 0.1 μg/ml, 0.01 μg/ml, and 0.001 μg/ml. After mixed culture, the IFN-γ secreted by T cells in the supernatant was detected. To determine the function of this TCR-expressing PBMC cell to specifically recognize the Her2/neu 369-377 polypeptide. The target cells of the control group were T2 cells which presented 1 μg/ml of the EBV virus antigen polypeptide LMP2 426-434 which can bind to the HLA-A2 molecule. Figure 4B shows that PBMC expressing Her2 TCR-1B5-mC can be activated by T2 cell-derived Her2/neu 369-377 antigen polypeptide to secrete IFN-γ, indicating the expression of exogenous Her2 TCR-1B5-mC primary T The cells can specifically recognize the Her2/neu 369-377 polypeptide presented by the HLA-A2 molecule. The ability to recognize antigenic polypeptides correlates with the amount of exogenous TCR expressed on T cells. Which recognizes the polypeptide antigen reaction half maximum (half-maximum reaction, EC 50 ) polypeptide concentration was estimated curve fit of about 6.9ng / ml (IC 50 Tool Kit , http: //www.ic50.tk/), although this The reaction sensitivity is lower than the EC 50 (EC 50 of about 10e-10M) of the high affinity TCR recognizing the foreign antigen such as the viral antigen (see the literature "CANCER RESEARCH 1998, 58.4902-4908" and "HUMAN GENE THERAPY 2014, 25: 730–739”), but still within the range of TCR affinity that recognizes common tumor-associated antigens (as described in the literature “Eur J Immunol (2012) 42: 3174–9).
图4C显示T细胞与T2细胞提呈的抗原多肽(T2+Her2-E75，即Her2/neu 369-377多肽)共培养时加入抗人CD8抗体后，T细胞分泌IFN-γ的功能被显著抑制。这说明CD8分子的辅助作用对于外源TCR识别Her2/neu 369-377抗原多肽至关重要，也显示本发明所述的Her2 TCR-1B5是CD8功能依赖型TCR。Figure 4C shows that T cell secreting IFN-γ is significantly inhibited by the addition of anti-human CD8 antibody when T cells are co-cultured with T2 cell-presenting antigen polypeptide (T2+Her2-E75, ie Her2/neu 369-377 polypeptide). . This indicates that the helper effect of the CD8 molecule is essential for the recognition of the Her2/neu 369-377 antigen polypeptide by the exogenous TCR, and also shows that the Her2 TCR-1B5 of the present invention is a CD8 function-dependent TCR.
实施例4：正常外周血T细胞经Her2 TCR-1B5-mC重组慢病毒转 染后表达的Her2/neu 369-377多肽特异性TCR可识别HLA-A2 +Her2/neu +肿瘤细胞 Example 4: Her2/neu 369-377 polypeptide-specific TCR expressed by normal peripheral blood T cells transfected with Her2 TCR-1B5-mC recombinant lentivirus recognizes HLA-A2 + Her2/neu + tumor cells
在96-孔板的每孔中加入10e5个经Her2 TCR-1B5-mC w/o GFP重组慢病毒转染后的PBMC(图中所示为转染Her2 TCR-1B5的#2 PBMC)，或者10e5个不转染TCR的对照#2 PBMC，与10e5个不同肿瘤细胞株混合培养，之后检测上清液中分泌的IFN-γ。具体方法如上文所述。结果如下：10e5 PBMCs transfected with Her2 TCR-1B5-mC w/o GFP recombinant lentivirus were added to each well of a 96-well plate (shown as #2 PBMC transfected with Her2 TCR-1B5), or 10e5 control #2 PBMCs that were not transfected with TCR were mixed with 10e5 different tumor cell lines, and then the secreted IFN-γ was detected in the supernatant. The specific method is as described above. The results are as follows:
图5A显示，表达Her2 TCR-1B5-mC的T细胞均可被HLA-A2 +Her2/neu +的肿瘤细胞株所激活并分泌IFN-γ，肿瘤细胞株包括结肠癌Colo205细胞、乳腺癌MDA-MB-231细胞、结肠癌Caco-2细胞、以及肺癌H1355细胞。而HLA-A2 -Her2/neu +的卵巢癌SK-OV3细胞、肺癌H647细胞、以及HLA-A2 +Her2/neu -的淋巴瘤Bjab细胞却不能激活转染Her2 TCR-1B5-mC TCR的T细胞。说明Her2 TCR-1B5-mC TCR可以特异性识别肿瘤细胞表面被HLA-A2提呈的Her2/neu抗原。来源同一供体PBMC，平行培养但没有转染Her2 TCR-1B5-mC的对照组T细胞不能被所列肿瘤细胞株所激活，说明对肿瘤细胞的反应不是非特异性的。图5B显示，表达Her2 TCR-1B5-mC TCR的T细胞(转染Her2 TCR-1B5的#2 PBMC)对colo205细胞表现出明显的反应，而且识别功能可以被抗CD8抗体以及抗HLA一类分子的抗体几乎完全阻断，这进一步说明识别肿瘤细胞表面Her2/neu抗原的效应细胞为CD8 +杀伤性T细胞，其特异性抗原识别功能依赖于CD8的辅助功能，这也和识别T2细胞提呈的her2/neu 369-377多肽抗原时Her2 TCR-1B5-mC TCR的表现是一致的。 Figure 5A shows that T cells expressing Her2 TCR-1B5-mC can be activated by HLA-A2 + Her2/neu + tumor cell lines and secrete IFN-γ. Tumor cell lines include colon cancer Colo205 cells, breast cancer MDA- MB-231 cells, colon cancer Caco-2 cells, and lung cancer H1355 cells. HLA-A2 - Her2/neu + ovarian cancer SK-OV3 cells, lung cancer H647 cells, and HLA-A2 + Her2/neu - lymphoma Bjab cells do not activate T cells transfected with Her2 TCR-1B5-mC TCR . This indicates that Her2 TCR-1B5-mC TCR can specifically recognize the Her2/neu antigen presented by HLA-A2 on the surface of tumor cells. Control T cells from the same donor PBMC that were cultured in parallel but not transfected with Her2 TCR-1B5-mC were not activated by the listed tumor cell lines, indicating that the response to tumor cells was not non-specific. Figure 5B shows that T cells expressing Her2 TCR-1B5-mC TCR (#2 PBMC transfected with Her2 TCR-1B5) showed significant responses to colo205 cells, and recognition functions were enhanced by anti-CD8 antibodies and anti-HLA molecules. The antibody is almost completely blocked, which further indicates that the effector cells that recognize the Her2/neu antigen on the surface of tumor cells are CD8 + killer T cells, and their specific antigen recognition function depends on the auxiliary function of CD8, which is also related to the recognition of T2 cells. The Her2/neu 369-377 polypeptide antigen was consistent in Her2 TCR-1B5-mC TCR.
讨论discuss
不同肿瘤细胞株对特异性T细胞的反应敏感性差异可能与肿瘤细胞表达不同水平Her2/neu抗原多肽/HLA-A2复合体有关，也可能与肿瘤细胞本身对T细胞功能不同的抑制作用有关。尽管特异性识别Her2/neu 369-377多肽的高亲和性TCR可以通过Her2/neu 369-377多肽体外诱导而获得，但这些高亲和性TCR往往不能识别肿瘤细胞所提呈 的Her2/neu抗原(Cancer Res.1998；58:4902–4908.Cancer Immunol.Immunother.2008；57:271–280)。一个原因可能是外源Her2/neu 369-377多肽结合HLA-A2分子的构型与细胞内所提呈的多肽/HLA复合物的构型有所不同(参见文献“Journal of Immunology,2008,180:8135–8145”)。另一个可能原因为，Her2/neu 369-377多肽作为模拟表位(mimotope)抗原，所诱导的特异性TCR既可识别Her2/neu 369-377多肽，也可识别被肿瘤细胞提呈的相似多肽，例如Her2/neu 373-382多肽(参见文献“J Immunol.2013 Jan 1；190(1):479–488”)，然而高亲和性TCR虽然对HLA-A2提呈的Her2/neu 369-377多肽具有高亲和力，却不能有效识别相应的被肿瘤细胞提呈的模拟表位多肽。本发明所述的特异性识别Her2/neu 369-377多肽的TCR，不仅靶向肿瘤细胞所提呈的Her2/neu 369-377多肽，也可能同时识别被肿瘤细胞所提呈的来源于Her2/neu的其它模拟表位多肽。The difference in sensitivity of different tumor cell lines to specific T cells may be related to the expression of different levels of Her2/neu antigen polypeptide/HLA-A2 complex in tumor cells, and may also be related to the inhibition of T cell function by tumor cells themselves. Although the high-affinity TCR that specifically recognizes the Her2/neu 369-377 polypeptide can be obtained by in vitro induction of the Her2/neu 369-377 polypeptide, these high-affinity TCRs often fail to recognize the Her2/neu presented by the tumor cells. Antigen (Cancer Res. 1998; 58: 4902 - 4908. Cancer Immunol. Immunother. 2008; 57: 271 - 280). One reason may be that the configuration of the exogenous Her2/neu 369-377 polypeptide binding HLA-A2 molecule differs from the configuration of the polypeptide/HLA complex presented in the cell (see the journal "Journal of Immunology, 2008, 180". :8135–8145”). Another possible reason is that the Her2/neu 369-377 polypeptide acts as a mimotope antigen, and the specific TCR induced can recognize both Her2/neu 369-377 polypeptide and similar polypeptides presented by tumor cells. For example, the Her2/neu 373-382 polypeptide (see the literature "J Immunol. 2013 Jan 1; 190(1): 479-488"), whereas the high affinity TCR, although presented to HLA-A2, is Her2/neu 369- The 377 polypeptide has high affinity but does not effectively recognize the corresponding mimotope polypeptide presented by the tumor cell. The TCR specifically recognizing the Her2/neu 369-377 polypeptide of the present invention not only targets the Her2/neu 369-377 polypeptide proposed by the tumor cell, but also recognizes the Her2/ derived from the tumor cell. Other mimotope polypeptides of neu.
由于识别自身抗原的高亲和性T细胞大多被中枢耐受机制所清除，外周T细胞库中自然存在的可以识别Her2/neu抗原的TCR大多为中低亲和性。另外一个可以识别肿瘤细胞的CD8功能非依赖型的高亲和性TCR是来自经Her2/neu 373-382多肽特异性T细胞群的多个α链和β链进行配对后，通过功能检测筛选而出(参见文献“HUMAN GENE THERAPY 2024，25:730–739”；WO/2016/133779)。由于不是从特异性的单克隆T细胞直接获得，不能确定此TCR是否存在于外周自然T细胞库。一般认为，高亲和性T细胞的过继转输治疗的疗效要优于靶向同一抗原的低亲和性T细胞(参见文献“Clin Exp Immunol(2015)180:255–70”)。然而，高亲和性TCR本身容易产生识别自身抗原的自身免疫性反应(参见文献“Blood(2009)114:535–46”)，没有经过中枢耐受机制筛选的TCR也会识别抗原低表达的正常组织，或者针对其它类似的自身抗原表位产生交叉反应的脱靶毒性(参见文献“Sci Transl Med(2013)5:197ra103.Blood(2013)122:863–71”)。选择高亲和性TCR的另一个原因是这些TCR的功能不依赖CD8的辅助功能，因而可以通过转染CD4 +T细胞而获得对CD8 +杀伤T细胞功能的辅助作用。对于CD8功能依赖型TCR，通过表达载体同时表达TCR和外 源CD8分子也可以达到同样的目的。 Since most of the high-affinity T cells recognizing autoantigens are cleared by the central tolerance mechanism, the TCRs that naturally exist in the peripheral T cell pool that recognize the Her2/neu antigen are mostly low-intensity. Another high-affinity TCR that recognizes CD8 function-independent high-affinity TCRs from tumor cells is paired by multiple alpha and beta chains of the Her2/neu 373-382 polypeptide-specific T cell population. (See the literature "HUMAN GENE THERAPY 2024, 25: 730-739"; WO/2016/133779). Since it is not directly obtained from specific monoclonal T cells, it is not possible to determine whether this TCR is present in the peripheral natural T cell pool. It is generally believed that adoptive transfusion therapy of high-affinity T cells is superior to low-affinity T cells that target the same antigen (see the literature "Clin Exp Immunol (2015) 180: 255-70"). However, high-affinity TCRs themselves are prone to autoimmune responses that recognize autoantigens (see the literature "Blood (2009) 114: 535–46"), and TCRs that have not been screened by central tolerance mechanisms also recognize low antigen expression. Normal tissue, or off-target toxicity that produces cross-reactivity against other similar autoantigen epitopes (see the literature "Sci Transl Med (2013) 5:197ra103. Blood (2013) 122:863-71"). Another reason for the selection of high-affinity TCRs is that the function of these TCRs does not depend on the helper function of CD8, and thus the complementation of CD8 + killer T cell function can be obtained by transfecting CD4 + T cells. For the CD8 function-dependent TCR, the same purpose can be achieved by simultaneously expressing the TCR and the exogenous CD8 molecule by the expression vector.
总之，本发明提供了一种从HLA-A2 +的自体外周T细胞库中诱导而来的Her2/neu 373-382多肽特异性TCRα链和β链全序列，经转染后表达此TCR及恒定区被修饰的TCR的原代杀伤性T细胞可以识别多种HLA-A2 +Her2/neu +的肿瘤细胞。为开发和临床应用过继转输经特异性TCR修饰的T细胞来治疗肿瘤提供了新的方法和途径。 In summary, the present invention provides a Her2/neu 373-382 polypeptide-specific TCR α chain and β chain full sequence induced from an in vitro pericellular T cell bank of HLA-A2 + , which is expressed and expressed after transfection. Primary killer T cells of the region-modified TCR can recognize a variety of HLA-A2 + Her2/neu + tumor cells. New methods and approaches have been provided for the development and clinical application of adoptive transfer of specific TCR-modified T cells to treat tumors.

Claims (30)

  1. 一种分离的T细胞受体，包括α链和β链中的至少一者，所述α链和β链均包含可变区和恒定区，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞所表达的抗原Her2/neu，并且所述α链的所述可变区的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％的一致性，所述β链的所述可变区的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％的一致性。An isolated T cell receptor comprising at least one of an alpha chain and a beta chain, each of said alpha and beta chains comprising a variable region and a constant region, characterized in that said T cell receptor is capable of specificity Identifying the antigen Her2/neu expressed by the tumor cell, and the amino acid sequence of the variable region of the alpha chain has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 19, the The amino acid sequence of the variable region has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  2. 根据权利要求1所述的T细胞受体，其中所述的T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:18所示的Her2/neu 369-377。The T cell receptor according to claim 1, wherein said T cell receptor is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by an HLA-A2 molecule; preferably, The epitope polypeptide includes Her2/neu 369-377 as shown in SEQ ID NO: 18.
  3. 根据权利要求1所述的T细胞受体，其中所述α链的所述恒定区和/或所述β链的所述恒定区来源于人；优选地，所述α链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链的所述恒定区全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。The T cell receptor according to claim 1, wherein said constant region of said α chain and/or said constant region of said β chain is derived from a human; preferably, said constant region of said α chain Fully or partially replaced by homologous sequences derived from other species, and/or said constant regions of said beta strand are replaced in whole or in part by homologous sequences derived from other species; more preferably, said Other species are mice.
  4. 根据权利要求1所述的T细胞受体，其中所述α链的所述恒定区修饰有一个或多个二硫键，并且/或者所述β链的所述恒定区修饰有一个或多个二硫键。The T cell receptor according to claim 1, wherein said constant region of said α chain is modified with one or more disulfide bonds, and/or said constant region modification of said β chain has one or more Disulfide bond.
  5. 根据权利要求1所述的T细胞受体，其中所述α链的氨基酸序列如SEQ ID NOs:2、6或10所示，所述β链的氨基酸序列如SEQ ID NOs:4、8或12所示。The T cell receptor according to claim 1, wherein the amino acid sequence of the α chain is as shown in SEQ ID NOs: 2, 6, or 10, and the amino acid sequence of the β chain is SEQ ID NOs: 4, 8, or 12. Shown.
  6. 一种分离的、编码T细胞受体的核酸，包含所述T细胞受体的α链和β链中的至少一者的编码序列，所述α链编码序列和β链编 码序列均包含可变区编码序列和恒定区编码序列，其特征在于，所述T细胞受体能够特异性识别肿瘤细胞表达的抗原Her2/neu，并且所述α链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:19所示的氨基酸序列至少98％的一致性，所述β链可变区编码序列编码的氨基酸序列具有与SEQ ID NO:20所示的氨基酸序列至少98％的一致性。An isolated nucleic acid encoding a T cell receptor comprising a coding sequence for at least one of an alpha chain and a beta chain of said T cell receptor, said alpha chain coding sequence and said beta chain coding sequence each comprising a variable a region coding sequence and a constant region coding sequence, characterized in that the T cell receptor is capable of specifically recognizing an antigen Her2/neu expressed by a tumor cell, and the amino acid sequence encoded by the α chain variable region coding sequence has the same SEQ ID The amino acid sequence represented by NO: 19 is at least 98% identical, and the amino acid sequence encoded by the β chain variable region coding sequence has at least 98% identity to the amino acid sequence set forth in SEQ ID NO: 20.
  7. 根据权利要求6所述的核酸，其中所述核酸为DNA或RNA。The nucleic acid according to claim 6, wherein the nucleic acid is DNA or RNA.
  8. 根据权利要求6所述的核酸，其中所述α链可变区编码序列如SEQ ID NO:21所示，所述β链可变区编码序列如SEQ ID NO:22所示。The nucleic acid according to claim 6, wherein the α chain variable region coding sequence is represented by SEQ ID NO: 21, and the β chain variable region coding sequence is set forth in SEQ ID NO: 22.
  9. 根据权利要求6所述的核酸，其中被所述核酸编码的所述T细胞受体能够特异性识别被HLA-A2分子所提呈的所述抗原Her2/neu的抗原表位多肽；优选的是，所述抗原表位多肽包括如SEQ ID NO:18所示的Her2/neu 369-377。The nucleic acid according to claim 6, wherein said T cell receptor encoded by said nucleic acid is capable of specifically recognizing an epitope polypeptide of said antigen Her2/neu presented by an HLA-A2 molecule; preferably The epitope polypeptide comprises Her2/neu 369-377 as set forth in SEQ ID NO: 18.
  10. 根据权利要求6所述的核酸，其中所述α链恒定区编码序列和/或所述β链恒定区编码序列来源于人；优选地，所述α链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换，并且/或者所述β链恒定区编码序列全部或部分地被来源于其它物种的同源序列所替换；更优选地，所述其它物种为小鼠。The nucleic acid according to claim 6, wherein said α chain constant region coding sequence and/or said β chain constant region coding sequence are derived from a human; preferably, said α chain constant region coding sequence is wholly or partially derived from a source The homologous sequences of other species are replaced, and/or the beta strand constant region coding sequence is replaced in whole or in part by homologous sequences derived from other species; more preferably, the other species are mice.
  11. 根据权利要求6所述的核酸，其中所述α链恒定区编码序列包含一个或多个二硫键编码序列，并且/或者所述β链恒定区编码序列包含一个或多个二硫键编码序列。The nucleic acid according to claim 6, wherein said alpha chain constant region coding sequence comprises one or more disulfide bond coding sequences, and/or said beta chain constant region coding sequence comprises one or more disulfide bond coding sequences .
  12. 根据权利要求6所述的核酸，其中所述α链编码序列如SEQ ID NOs:1、5或9所示，所述β链编码序列如SEQ ID NOs:3、7或11所示。The nucleic acid according to claim 6, wherein the alpha chain coding sequence is set forth in SEQ ID NOs: 1, 5 or 9, and the beta strand coding sequence is set forth in SEQ ID NOs: 3, 7 or 11.
  13. 根据权利要求6-11中任一项所述的核酸，其中所述α链编码序列和所述β链编码序列之间由可切割性连接多肽的编码序列连接。The nucleic acid according to any one of claims 6 to 11, wherein the alpha chain coding sequence and the beta strand coding sequence are joined by a coding sequence of a cleavable linker polypeptide.
  14. 根据权利要求13所述的核酸，其序列如SEQ ID NOs:13、15、或23所示。The nucleic acid according to claim 13, which has the sequence shown as SEQ ID NOs: 13, 15, or 23.
  15. 一种重组表达载体，其含有与启动子有效连接的、根据权利要求6-14中任一项所述的核酸，和/或其互补序列。A recombinant expression vector comprising a nucleic acid according to any one of claims 6-14, and/or a complement thereof, operably linked to a promoter.
  16. 一种T细胞受体修饰的细胞，该细胞的表面被权利要求1-5中任一项所述的T细胞受体修饰，其中所述细胞包括原始T细胞或其前体细胞，NKT细胞，或T细胞株。A T cell receptor-modified cell, the surface of which is modified by the T cell receptor according to any one of claims 1 to 5, wherein the cell comprises a primitive T cell or a precursor cell thereof, NKT cell, Or T cell strain.
  17. 一种制备根据权利要求16所述的T细胞受体修饰的细胞的方法，包括以下步骤：A method of preparing a T cell receptor modified cell according to claim 16, comprising the steps of:
    1)提供细胞；1) providing cells;
    2)提供编码根据权利要求1-5中任一项所述的T细胞受体的核酸；2) providing a nucleic acid encoding the T cell receptor according to any one of claims 1 to 5;
    3)将所述核酸转染入所述细胞中。3) Transfecting the nucleic acid into the cell.
  18. 根据权利要求17所述的方法，其中步骤1)所述的细胞来自自体或异体。The method of claim 17 wherein the cells of step 1) are derived from autologous or allogeneic.
  19. 根据权利要求17所述的方法，其中所述转染的方式包括：采用病毒载体转染的方式，优选的是，所述病毒载体包括γ逆转录病毒载体或慢病毒载体；化学方式，优选的是，所述化学方式包括采用脂质体转染的方式；物理方式，优选的是，所述物理方式包括电转染方式。The method according to claim 17, wherein said means for transfecting comprises: transfecting with a viral vector, preferably said viral vector comprises a gamma retroviral vector or a lentiviral vector; chemically, preferably Yes, the chemical means includes the manner of transfection with liposomes; physically, preferably, the physical means include electroporation.
  20. 根据权利要求17所述的方法，其中步骤2)所述的核酸为根据权利要求6-14中任一项所述的核酸。The method according to claim 17, wherein the nucleic acid of step 2) is the nucleic acid according to any one of claims 6-14.
  21. 根据权利要求16所述的T细胞受体修饰的细胞在制备用于治疗或预防肿瘤和/或癌症的药物中的用途。Use of a T cell receptor modified cell according to claim 16 for the preparation of a medicament for the treatment or prevention of a tumor and/or cancer.
  22. 根据权利要求21所述的用途，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。The use according to claim 21, wherein the tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive.
  23. 根据权利要求16所述的T细胞受体修饰的细胞在制备用于检测宿主的肿瘤和/或癌症的药物中的用途。The use of a T cell receptor-modified cell according to claim 16 for the preparation of a medicament for detecting a tumor and/or cancer of a host.
  24. 一种药物组合物，其中该药物组合物包括作为活性成分的根据权利要求16所述的T细胞受体修饰的细胞，及可药用辅料。A pharmaceutical composition comprising the T cell receptor-modified cell according to claim 16 as an active ingredient, and a pharmaceutically acceptable excipient.
  25. 根据权利要求24所述的药物组合物，其中所述药物组合物包含每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重的所述T细胞受体修饰的细胞。 The pharmaceutical composition according to claim 24, wherein said pharmaceutical composition comprises said T cell receptor modification in a total dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course per patient. Cell.
  26. 根据权利要求24所述的药物组合物，其中所述药物组合物适于经动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。The pharmaceutical composition according to claim 24, wherein said pharmaceutical composition is suitable for transarterial, intravenous, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, and Administered intraperitoneally.
  27. 一种治疗肿瘤和/或癌症的方法，包括对肿瘤和/或癌症患者施用根据权利要求16所述的T细胞受体修饰的细胞。A method of treating a tumor and/or cancer comprising administering a T cell receptor modified cell according to claim 16 to a tumor and/or a cancer patient.
  28. 根据权利要求27所述的方法，其中所述T细胞受体修饰的细胞的施用剂量为每个患者每个疗程总剂量范围为1×10 3-1×10 9个细胞/Kg体重。 The method according to claim 27, wherein said T cell receptor-modified cells are administered at a dose ranging from 1 x 10 3 to 1 x 10 9 cells/kg body weight per course of treatment per patient.
  29. 根据权利要求27所述的方法，其中所述T细胞受体修饰的细胞通过动脉、静脉、皮下、皮内、瘤内、淋巴管内、淋巴结内、蛛网膜下腔内、骨髓内、肌肉内和腹膜内给药。The method according to claim 27, wherein said T cell receptor-modified cells are passed through arteries, veins, subcutaneous, intradermal, intratumoral, intralymphatic, intralymphatic, subarachnoid, intramedullary, intramuscular, and intramuscular. Administered intraperitoneally.
  30. 根据权利要求27所述的方法，其中所述肿瘤和/或癌症是抗原Her2/neu阳性的，并且是HLA-A2阳性的。The method of claim 27, wherein the tumor and/or cancer is antigen Her2/neu positive and HLA-A2 positive.
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