WO2018121605A1 - 一种多功能蛋白质 - Google Patents

一种多功能蛋白质 Download PDF

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WO2018121605A1
WO2018121605A1 PCT/CN2017/118984 CN2017118984W WO2018121605A1 WO 2018121605 A1 WO2018121605 A1 WO 2018121605A1 CN 2017118984 W CN2017118984 W CN 2017118984W WO 2018121605 A1 WO2018121605 A1 WO 2018121605A1
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sequence
antigen
peptide chain
cancer
protein
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PCT/CN2017/118984
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French (fr)
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张凤禹
高斌
王磊
吴亚松
魏卿
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天津天锐生物科技有限公司
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Priority to US16/475,060 priority Critical patent/US11530264B2/en
Priority to JP2019556411A priority patent/JP7221213B2/ja
Priority to EP17888600.8A priority patent/EP3578570A4/en
Priority to CN201780029217.7A priority patent/CN109153725B/zh
Publication of WO2018121605A1 publication Critical patent/WO2018121605A1/zh

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Definitions

  • the invention relates to the field of biology, and in particular to a multifunctional protein.
  • Bispecific antibodies also known as bifunctional antibodies or bivalent antibodies, can specifically bind to two different antigens simultaneously, with specificity and bifunctionality, in the fields of tumor immunotherapy and autoimmune diseases. with broadly application foreground.
  • Bispecific antibodies are not found in nature and can only be prepared by artificial methods. At present, methods for preparing bispecific antibodies mainly include chemical conjugation, hybridoma technology, and recombinant DNA technology.
  • Medarex has developed bispecific antibodies as early as 30 years ago and conducted phase III clinical trials in 2001. However, research in this field has been silent due to clinical trial failures and limited production problems. (Garber K, 2014) In 2009, the bispecific antibody Catumaxomab developed by Trion was approved by the European Union for the treatment of malignant ascites caused by EpCAM-positive tumors, but its high immunogenicity greatly limits its clinical application. (Spasevska I, 2014). In recent years, the rapid development of antibody engineering technology has brought new opportunities for the development of bispecific antibodies.
  • Lum Laboratories used commercial Herceptin and OKT3 to obtain Her2/CD3 bispecific antibodies by chemical coupling to recruit T cells to kill Her2-positive tumor cells (Msen et al., 2001). This clinically widely used antibody conjugate showed very good clinical safety and efficacy. Five of the 22 patients with metastatic breast cancer who participated in the clinical trial remained stable within 14.5 weeks after use. The condition (Lum et al., 2015).
  • OKT3 antibodies have also been used to chemically conjugate with other clinically used antibodies to kill tumors with corresponding targets: including EGFR-positive tumors (Reush U et al., 2006), (MAD et al., 2015) CD20 positive tumor line (Lum et al., 2013), B7-H3 positive tumor (MA et al., 2016) and the like.
  • the Her/CD3 antibody (Kiewe et al., 2006) developed on the same platform, anti-CD20/CD3 antibody for the treatment of relapsed B cell lymphoma has been approved for clinical trials and has a good safety record and efficacy (Buhmann et Al., 2009).
  • Anti-CD2/CD3 double antibodies have been used in experimental studies of melanoma (Ruf et al., 2004).
  • BiTE Bispecific T-cell engager
  • This novel small protein molecule is directly linked by the scFv of OKT3 and the anti-CD19 scFv via a linker peptide (Nagorsen D et al., 2012) (Patent No.: 201180063222.2; 201580009124.9), requiring only very low concentrations. It can inhibit the growth of non-Hodgkin's lymphoma (Bargou R et al., 2008).
  • TandAb links a pair of BiTE-like bispecific molecules with a linker peptide to form a tetrameric molecule called TandAb: 160kD has a molecular weight double that of BiTE, making it more compatible with CD3 and CD19 (Reusch U et al , 2015), at the same time, the pharmacokinetics of TandAb is also significantly improved compared to BiTE, the half-life in the blood reaches about 20h. It can mediate killing of non-Hodgkin's lymphoma and acute lymphoblastic leukemia.
  • DART is a combination of anti-CD19 and CD3 scFv through a disulfide-containing linker peptide, which can recruit T cells to kill tumors and has the advantages of stable and easy scale preparation (Johnson S et al, 2010; kuo SR et al ., 2012).
  • FcabFv fuses the antigen recognition portion of OKT3 with Fc (Wozniak G et al., 2010) which has a function of recognizing Her2 produced by CH3 mutation, and expresses a novel bispecific antibody highly similar to an ordinary antibody. It can effectively target Her2-positive tumors and inhibit tumor growth in animals (Wang L et al., 2013).
  • TriKE is inserted between the scFv of CD33 antibody and the scFv of CD16 antibody, and is linked by two linked peptides. It can effectively promote the activation and survival of NK cells in vivo while effectively targeting tumors. Adding an antigen that is beneficial for NK cells to treat myeloid malignancies or to target solid tumors. (Szun Szun Tay et al., 2016; Vallera DA. et al., 2016)
  • bispecific antibodies have become a new hotspot in the field of drug research, with at least 30 bispecific antibodies in clinical research (Garber K et al., 2014; Kontermann R E et al. 2015).
  • IL-15 is functionally similar to IL-2 and shares the same beta gamma receptor. Studies indicate that IL-2 or IL-15 is required for survival and proliferation of NK cells and CD8+ T cells (Boyman et al., 2007). . Although IL-15 and IL-2 share the same ⁇ receptor, each has a specific ⁇ receptor, and it was found that IL-15R ⁇ -sushi (the sushi domain of IL-15 receptor ⁇ ) is the super IL-15.
  • An agonist can greatly enhance the function of IL-15 (Han et al., 2011; Mortier et al., 2006) (Patent Application No.: 201280037114.2, 201510358540.1), a complex of IL-15 and IL-15R ⁇ -sushi It can completely replace the role of IL-2 in T/NK cells (Peter S. Kim1, 2016; Rosario et al., 2016), activate NK/CD8+ T cells and increase their cytotoxicity against tumors.
  • NIH National Institutes of Health
  • National Cancer Institute pointed out that IL-15 ranked first among the 12 immunotherapeutics for cancer treatment. By supporting the viability of CD8+ T cells, IL-15 has demonstrated great potential for long-term immune response in T cells.
  • IL-15 is more promising, more effective, less toxic in tumor treatment, and can stimulate the anti-tumor activity of T cells and NK cells. Fusion of IL-15 and IL-15R ⁇ -sushi complex or other functional cytokine and receptor complexes in bispecific molecules is a major direction to improve the efficacy of cellular immunotherapy.
  • PD-1 programmed death 1
  • PD-L1 receptors PD-L1, PD-L2
  • T cell activity Okazaki and Honjo, 2007
  • the binding of PD-1 on the surface of T cells to PD-L1/2 on the surface of other cells causes inhibition of T cells, which plays an important role in the process of avoiding autoimmune diseases and producing immune tolerance in humans, while tumor cells utilize PD1.
  • /PD-L1 checkpoint This immune system protects the self-regulating mechanism by expressing PD-L1/L2 in the tumor cell itself or in the tumor microenvironment, and PD1 binding on the surface of T cells, transmitting a negative signal, leading to a decline in T cell function. And depletion, to achieve the purpose of suppressing immune response, tumor escape.
  • the polypeptide chain X provided by the invention includes an antigen 1 binding domain R1, a co-peptide chain domain R2 and an antigen 2 binding domain R3;
  • the co-peptide linkage domain R2 is a cytokine binding domain in a cytokine or cytokine receptor.
  • the antigen 2 binding domain R3 is an antibody or molecule that recognizes T cell CD3.
  • the antigen 2 binding domain R3 is a receptor or antibody or other molecule that recognizes NK cell CD16.
  • the antigen to which the antigen 1 binding domain R1 binds is selected from any of the following cancer-related antigens: brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia. , lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin Lymphoma and uterine cancer.
  • cancer-related antigens brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia. , lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thy
  • the cancer-associated antigen is preferably any one of the following antigens: CD123, CD19, CD20, CD22, CD37, ROR1, mesothelin, CD33/IL3Ra, c-Met, BCMA, PSMA, EGFRvIII, GD-2, NY -ESO-1, MAGEA3, ⁇ -human chorionic gonadotropin, AFP, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), hsp70-2, M-CSF, PSA, PAP, LAGE-la, p53, Prostein, PSMA, Her2/neu, telomerase, PCTA-1, MAGE, ELF2M, IGF-I, IGF-II, IGF-I receptor, BCR-ABL, E2A- PRL, H4-RET, 1GH-IGK, MYL-RAR, GP100, Mart1, TSP-180, MAGE-4, MAGE-5, MAGE-6
  • the antigen in the complex of MHC and the short peptide of the antigen is any one of the above cancer-related antigens.
  • the cancer-associated antigen is preferably selected from any one of the following antigens: CD19, CD20, CD22, CD123, CD33/IL3Ra, Her2, PDL1, GP100, Mart1, BCMA, WT-1 and NY-ESO-1 or MHC A complex with a short peptide of the above antigen.
  • the antigen in the complex of the MHC and the short peptide of the antigen is preferably any one of the following antigens selected from the cancer-associated antigen.
  • the cancer-associated antigen is preferably selected from any one of the following antigens: CD19, CD20, CD22, Her2, PDL1, WT1, GP100, Mart1, BCMA, and NY-ESO-1 or a complex of MHC with the above-described antigen short peptide .
  • the antigen 1 binding domain R1 is an antigen-binding antibody, an antigen-binding ligand, an antigen-binding receptor or a polypeptide having antigen-binding function.
  • the antigen-binding antibody is an intact immunoglobulin, an antibody Fc, an antibody Fab, an antibody VH, an antibody VL or a full-length peptide chain or a partial peptide of an scFv.
  • the antigen-binding ligand or the antigen-binding receptor is a full-length peptide chain or a partial peptide.
  • the antigen 1 binding domain R1 is a TCR having an antigen recognition function.
  • the antigen 1 binding domain R1 is a TCR-like antibody or other molecule having an antigen recognition function.
  • Another object of the invention is to provide a protein.
  • the protein provided by the present invention which comprises the peptide chain X of claim 1 as a main peptide chain and a co-peptide chain Y to constitute a heterodimer;
  • the co-peptide chain Y comprises an antigen 3 binding domain R4 and a main peptide chain X linking domain R5,
  • co-peptide chain Y is the main peptide chain linking domain R5;
  • the main peptide chain linking domain R5 and the co-peptide chain linking domain R2 in the peptide chain X bind to each other.
  • polypeptide linkers are glycine- and/or serine-rich sequences or multiple copies of glycine- and/or serine-rich sequences, and polypeptide linkers typically include from 1 to 20 amino acid residues.
  • the main peptide chain domain R5 and the cytokine binding domain R2 in the cytokine or cytokine receptor are a pair of peptides having a mutual binding function.
  • co-peptide chain domain (R2) and main peptide chain domain (R5) are cytokines and receptor subunits.
  • a heterodimer is formed between the main peptide chain (X) and the co-peptide chain (Y) by a bond between the co-peptide chain link domain (R2) and the main peptide chain link domain (R5).
  • the antigen 3 to which the antigen 3 binding domain R4 binds is selected from any of the following cancer-related antigens: brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, Leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodge Golden lymphoma and uterine cancer.
  • cancer-related antigens brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, Leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymom
  • the cancer-associated antigen 3 is preferably one of the following antigens: CD123, CD19, CD20, CD22, CD37, ROR1, mesothelin, CD33/IL3Ra, c-Met, BCMA, PSMA, EGFRvIII, GD-2, NY-ESO-1, MAGEA3, ⁇ -human chorionic gonadotropin, AFP, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), hsp70-2, M-CSF , PSA, PAP, LAGE-la, p53, Prostein, PSMA, Her2/neu, PDL1, telomerase, PCTA-1, MAGE, ELF2M, IGF-I, IGF-II, IGF-I receptor, BCR-ABL , E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR, GP100, Mart1, TSP-180, MAGE-4, MAGE-5
  • the antigen in the complex of the MHC and the short peptide of the antigen is the cancer-associated antigen 3, preferably from any of the above antigens.
  • the cancer-associated antigen is preferably selected from any one of the following antigens: CD19, CD20, CD22, CD123, CD33/IL3Ra, Her2, PDL1, GP100, Mart1, BCMA, WT-1, NY_ESO-1 or MHC and A complex of the above short peptides of antigen.
  • the antigen in the complex of the above MHC and the short peptide of the antigen is any one of CD19, CD20, CD22, CD123, CD33/IL3Ra, Her2, PDL1, GP100, Mart1, BCMA, WT-1 and NY_ESO-1.
  • the cancer-associated antigen is preferably selected from any one of the following antigens: CD19, CD20, CD22, Her2, PDL1, WT1, GP100, Mart1, BCMA, NY_ESO-1 or a complex of MHC with the above-mentioned antigenic short peptide;
  • the antigen in the complex of MHC and the short peptide of the antigen is any one of CD19, CD20, CD22, Her2, PDL1, WT1, GP100, Mart1, BCMA, NY_ESO-1.
  • the positions of the antigen 1 binding domain R1 and the antigen 2 binding domain R3 are interchangeable.
  • the positions of the antigen 1 binding domain R1 and the antigen 3 binding domain R4 are interchangeable.
  • the positions of the antigen 2 binding domain R3 and the antigen 3 binding domain R4 are interchangeable.
  • the co-peptide chain-linking domain R2 and the main-peptide chain-linking domain R5 are a pair of peptides having a function of binding to each other.
  • a pair of ⁇ c cytokines and their receptor subunits can be bound to each other.
  • the optimal choices are IL15 and IL15R ⁇ and IL4 and IL4R ⁇ .
  • IL15 plays an indispensable role in maintaining the homeostasis and cell growth of T cells, NK cells and NKT cells, while providing additional B cells, dendritic cells (DCs), macrophages, and mast cells.
  • Physiological function. IL-15 can support the survival of CD8+ T cells, which is more promising and more effective than IL-2. It is less toxic in tumor treatment and can stimulate the anti-tumor activity of T cells and NK cells.
  • the co-peptide chain-linking domain R2 and the main-peptide chain-linking domain R5 are a pair of each other and can bind to each other with a cytokine and a receptor subunit thereof.
  • the cytokine is a ⁇ c family cytokine
  • the ⁇ c family cytokine is IL2, IL4, IL7, IL9, IL15 or IL21.
  • the cytokine and receptor subunits are optimally selected from the group consisting of IL15 and IL15R ⁇ or IL4 and IL4R ⁇ .
  • Each component of the main peptide chain of the polypeptide or protein or a component of the co-peptide chain is linked by a polypeptide linker consisting of 1-20 amino acid residues.
  • the polypeptide linker is rich in glycine and/or serine.
  • the antigen 1 binding domain R1 is an antiCD19-ScFv or AntiMHC/GP100-VHH or AntiMHC/Mart1-VHH or an extracellular region of anti MHC/WT1 or PD1 or AntiCD22-ScFv or antiCD3-ScFv or antiCD16-ScFv;
  • co-peptide linkage domain R2 is IL15R ⁇ sushi or IL4R ⁇ -N-FN3 or IL15 or IL4;
  • the antigen 2 binding domain R3 is an antiCD3-ScFv or antiCD16-ScFv or antiCD19-ScFv or AntiMHC/GP100-VHH or AntiMHC/Mart1-VHH or an extracellular region of antiWT1 or PD1 or AntiCD22-ScFv;
  • the antigen 3 binding domain R4 is the extracellular domain of PD1 or AntiMHC/GP100-VHH or AntiCD22-ScFv or antiCD19-ScFv or AntiMHC/Mart1-VHH or anti MHC/WT1 or antiCD3-ScFv or antiCD16-ScFv;
  • the main peptide chain joining domain R5 is IL15 or IL4 or IL15R ⁇ sushi or IL4R ⁇ -N-FN3.
  • amino acid sequence of the antiCD19-ScFv is sequence 1;
  • amino acid sequence of the IL15R ⁇ sushi is sequence 2;
  • amino acid sequence of the antiCD3-ScFv is sequence 3;
  • amino acid sequence of the extracellular region of PD1 is sequence 4.
  • amino acid sequence of IL15 is sequence 5;
  • the amino acid sequence of the AntiMHC/GP100-VHH is sequence 10;
  • the amino acid sequence of the AntiMHC/Mart1-VHH is sequence 11;
  • the amino acid sequence of the AntiMHC/WT1-VH is sequence 12;
  • the amino acid sequence of the IL4R ⁇ is the sequence 13;
  • the amino acid sequence of the AntiCD16-ScFv is sequence 14;
  • the amino acid sequence of the AntiCD22-ScFv is the sequence 15;
  • amino acid sequence of IL4 is sequence 16.
  • amino acid sequence of the polypeptide chain X is sequence 8;
  • amino acid sequence of the polypeptide chain X is sequence 17;
  • amino acid sequence of the polypeptide chain X is the sequence 19;
  • amino acid sequence of the polypeptide chain X is sequence 21;
  • amino acid sequence of the polypeptide chain X is the sequence 22;
  • amino acid sequence of the polypeptide chain X is sequence 23;
  • amino acid sequence of the polypeptide chain X is sequence 27;
  • amino acid sequence of the polypeptide chain X is sequence 29;
  • amino acid sequence of the polypeptide chain X is sequence 30;
  • amino acid sequence of the main peptide chain of the protein is sequence 8
  • amino acid sequence of the co-peptide chain is sequence 9
  • amino acid sequence of the main peptide chain of the protein is sequence 17, and the amino acid sequence of the co-peptide chain is sequence 9;
  • amino acid sequence of the main peptide chain of the protein is sequence 17, and the amino acid sequence of the co-peptide chain is sequence 18;
  • amino acid sequence of the main peptide chain of the protein is sequence 19, and the amino acid sequence of the co-peptide chain is sequence 9;
  • amino acid sequence of the main peptide chain of the protein is sequence 8
  • amino acid sequence of the co-peptide chain is sequence 20
  • amino acid sequence of the main peptide chain of the protein is sequence 21, and the amino acid sequence of the co-peptide chain is sequence 9;
  • amino acid sequence of the main peptide chain of the protein is sequence 22, and the amino acid sequence of the co-peptide chain is sequence 9;
  • amino acid sequence of the main peptide chain of the protein is sequence 23, and the amino acid sequence of the co-peptide chain is sequence 24;
  • amino acid sequence of the main peptide chain of the protein is sequence 25, and the amino acid sequence of the co-peptide chain is sequence 26;
  • amino acid sequence of the main peptide chain of the protein is sequence 27, and the amino acid sequence of the co-peptide chain is sequence 28;
  • amino acid sequence of the main peptide chain of the protein is sequence 29, and the amino acid sequence of the co-peptide chain is sequence 9.
  • amino acid sequence of the main peptide chain of the protein is sequence 30, and the amino acid sequence of the co-peptide chain is sequence 31;
  • amino acid sequence of the main peptide chain of the protein is sequence 21, and the amino acid sequence of the co-peptide chain is sequence 5;
  • amino acid sequence of the main peptide chain of the protein is sequence 8
  • amino acid sequence of the co-peptide chain is sequence 5.
  • a third object of the present invention is to provide a nucleic acid molecule encoding the above polypeptide or protein.
  • the present invention provides an encoding nucleic acid molecule of the above polypeptide or protein, comprising a nucleic acid molecule encoding the main peptide chain or a nucleic acid molecule encoding the main peptide chain and a nucleic acid molecule encoding the co-peptide chain.
  • a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to include the gene, or by utilizing a standard.
  • the technique is to isolate directly from cells and tissues containing the gene, or to synthesize polynucleotides by artificial synthesis.
  • Recombinant vectors, expression cassettes, recombinant bacteria, recombinant viruses or cells containing the above nucleic acid molecules are also within the scope of the present invention.
  • the recombinant vector is a nucleic acid molecule encoding the main peptide chain in the above nucleic acid molecule or a nucleic acid molecule encoding the main peptide chain and a nucleic acid molecule encoding the co-peptide chain inserted into an expression vector In the case, a vector expressing the above protein is obtained.
  • the above recombinant vector comprises the above polynucleotide sequence or combination.
  • a nucleic acid encoding a primary peptide chain (X) or a co-peptide chain (Y) can be ligated to a promoter, and the construct is incorporated into an expression vector to achieve encoding a primary peptide chain (X) or a co-peptide chain ( Expression of Y).
  • a typical cloning vector comprises a transcriptional and translational terminator, an initial sequence and a promoter that can be used to modulate expression of a desired nucleic acid sequence.
  • lentiviral vectors are a suitable tool for achieving long-term stable inheritance of genes because they allow long-term, stable integration of genes and their proliferation in daughter cells.
  • Lentiviral vectors have the added advantage of exceeding vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the multifunctional protein provided by the present invention comprises two peptide chains which can be expressed in the same cell by a known method, including but not limited to co-transfection of two encoding the main peptide chain (X) and the co-peptide chain (Y), respectively.
  • the nucleic acid sequence of Y) is ligated in tandem into an expression frame, and the 2A peptide is co-expressed by inserting a ribosome binding site between the nucleic acid sequences of the main peptide chain (X) and the co-peptide chain (Y).
  • the cell of interest is a prokaryotic cell, a yeast cell or a mammalian cell; wherein the mammalian cell is preferably a human cell.
  • the present invention also provides a kit comprising the above polypeptide chain X or the above protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, recombinant virus or cell.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for the preparation of an immunotherapeutic product is also within the scope of the present invention.
  • the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit are also used in immunocyte culture and/or promotion of immune cell expansion and/or immunoassay.
  • the scope of protection of the present invention is also used in immunocyte culture and/or promotion of immune cell expansion and/or immunoassay.
  • the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit in the preparation of immune cell culture and/or promotion of immune cell expansion and/or immunodetection products The application is also within the scope of the invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for stimulating T or NK cell proliferation is also within the scope of protection of the present invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for the preparation of a product for stimulating T or NK cell proliferation is also within the scope of protection of the present invention.
  • the above polypeptide or protein the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for mediating immune cell inhibition or killing of target cells expressing the antigen in the protein It is also the scope of protection of the present invention.
  • the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for preparing a target cell product which mediates immune cell inhibition or killing of an antigen expressing the protein The application is also within the scope of the invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for inhibiting or killing tumor cells is also within the scope of protection of the present invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for the preparation of a product for inhibiting or killing tumor cells is also within the scope of protection of the present invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for treating or detecting a tumor is also within the scope of protection of the present invention.
  • the use of the above polypeptide or protein, the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for the preparation of a therapeutic or detecting tumor product is also within the scope of the present invention.
  • the above polypeptide or protein the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for inhibiting or killing target cells expressing the antigen in the protein is also protected by the present invention The scope.
  • the above polypeptide or protein the above nucleic acid molecule or the above recombinant vector, expression cassette, recombinant strain, cell or recombinant virus or kit for the preparation of a target cell product for inhibiting or killing an antigen expressing the protein is also The scope of the invention is protected.
  • the immunotherapy is to inhibit or kill tumor cells by immune cells
  • the immune cell is a T cell or an NK cell or the like;
  • the antigen is a cancer associated antigen
  • the antigen is brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, Ovarian cancer, prostate cancer, pancreatic cancer, renal cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer-associated antigen, or any combination thereof;
  • the tumor is brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma.
  • the target cell is a prokaryotic cell, a yeast cell or a mammalian cell;
  • the mammalian cell is specifically a human cell
  • the human cell is specifically an immune cell
  • the immune cell is specifically a T cell or an NK cell.
  • the antigen-binding domain (R1/R3) of the main peptide chain and the co-peptide chain antigen binding domain (R4) have a function of binding antigen.
  • One of its antigen-binding domains is an antibody or molecule that recognizes CD3 of human T cells or a receptor or antibody or other molecule that recognizes NK cell CD16, and the other two are tumor-associated antigens selected from the group consisting of the following.
  • a tumor antigen to which an antigen binding domain binds is a protein produced by a tumor cell that elicits an immune response, particularly a T-cell mediated immune response.
  • the choice of the antigen binding domain of the invention will depend on the particular type of cancer being treated. Tumor antigens are well known in the art:
  • the tumor-associated antigen referred to in the present invention may also be a tumor-associated antigen selected from the group consisting of brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, and lymphoma. , leukemia, lung cancer, melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thymoma, sarcoma, non-Hodgkin's lymphoma, Huo Qijin lymphoma, uterine cancer, and any combination thereof.
  • the tumor antigens referred to in the present invention include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), ⁇ -human chorionic gonadotropin, ⁇ -fetoprotein (AFP), lectin- Reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostatic enzyme , prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate-cancer tumor antigen-1 (PCTA-1) ), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothe
  • the tumor antigen comprises one or more antigenic cancer epitopes associated with the malignancy.
  • Malignant tumors express many proteins that can be used as target antigens for immune challenge. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer.
  • Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2.
  • the other group of target antigens is a fetal cancer antigen such as carcinoembryonic antigen (CEA).
  • B-cell lymphoma the tumor-specific individual genotype immunoglobulin constitutes the only true tumor-specific immunoglobulin antigen that is unique to an individual's tumor.
  • B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.
  • Some of these antigens (CEA, HER-2, CD19, CD20, individual genotypes) have been used with limited success as targets for passive immunotherapy using monoclonal antibodies.
  • the tumor antigen referred to in the present invention may also be a tumor-specific antigen (TSA) or a tumor associated antigen (TAA).
  • TSA is unique to tumor cells and does not occur on other cells of the body.
  • the TAA-associated antigen is not unique to tumor cells, and conversely, it is also expressed on normal cells under conditions in which the immune tolerance state to the antigen cannot be induced.
  • Antigen expression on the tumor can occur under conditions that enable the immune system to respond to the antigen.
  • TAA may be an antigen expressed on normal cells when the immune system is immature and unable to respond during embryonic development, or they may be antigens that normally exist at very low levels on normal cells, but which are on tumor cells. Expressed at a higher level.
  • TSA or TAA antigens include the following: differentiation antigens such as MART-l/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specificity Lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-inhibiting genes such as p53, Ras, HER- 2/neu; unique tumor antigens produced by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-l/MelanA (MART-1), gp100 (Pmel
  • the tumor antigen referred to in the present invention may be a complex of MHC with the above-described antigenic peptide.
  • MHC antigenic peptide
  • HLA-GP100 complex HLA-Mart1 complex
  • HLA-WT1 complex HLA-WT1 complex
  • the antigen-binding domain (R1, R3) in the main peptide chain or the antigen-binding domain (R4) in the co-peptide chain is an antibody, a ligand, a receptor, and an antigen that can bind to an antigen. Binding ability polypeptide or any combination thereof.
  • the antibody may be an intact peptide chain or a partial peptide of Ig, Fab, scFv or any combination thereof.
  • the ligand or receptor can be its entire peptide chain, a partial peptide segment, or any combination thereof.
  • co-peptide chain domain (R2) and the main peptide chain domain (R5) are a pair of peptides having a mutual binding function.
  • the peptide having a mutual binding function may be a pair of receptors and ligands which can bind to each other or a pair of antibodies and antigens which can bind to each other.
  • a pair of ⁇ c cytokines and their receptor subunits can be bound to each other, and IL15 and IL15R ⁇ , IL4 and IL4R are optimally selected.
  • the functional domains of the main peptide chain and the co-peptide chain are linked by a polypeptide linker which is a glycine- and/or serine-rich sequence or a plurality of copy sequences rich in glycine and/or serine, and the polypeptide linker generally comprises 5 -20 amino acid residues.
  • FIG. 1 is a schematic diagram showing the molecular structure of a multifunctional protein.
  • A The multifunctional protein molecule is composed of a main peptide chain X and an auxiliary peptide chain Y, wherein the main peptide chain X includes an antigen binding domain R1, a co-peptide chain domain R2, an antigen binding domain R3, and a co-peptide chain Y includes an antigen binding domain R4.
  • the main peptide chain is connected to the domain R5;
  • B the multifunctional protein molecule is composed of a main peptide chain X and a co-peptide chain Y, wherein the main peptide chain X includes an antigen-binding domain R1, a co-peptide chain-linking domain R2, and an antigen-binding domain R3.
  • the peptide chain Y comprises a main peptide chain domain R5; the C: multifunctional protein molecule consists only of the main peptide chain X, wherein the main peptide chain X comprises an antigen binding domain R1, a co-peptide chain domain R2, and an antigen binding domain R3.
  • Figure 2 shows the gene expression framework of the multifunctional protein molecule.
  • Figure 3 is a diagram showing the SDS-PAGE expression of a multifunctional protein molecule polypeptide.
  • Lane 1 TiTE-1, main peptide chain about 65KD, co-peptide chain about 30KD
  • Lane 2 TiTE-6, main peptide chain about 65KD, co-peptide chain about 30KD
  • Lane 3 protein Marker, molecular weight from top to bottom For: 160KD, 120KD, 100KD, 70KD, 50KD, 40KD, 30KD, 25KD.
  • Figure 4 shows the killing results of TiTE-1, 15, 16, and 5 multifunctional protein molecules: A, negative control TiTE-6 protein kills malme-3M-CD19-luc; B, TiTE-1 protein kills malme-3M-CD19-luc ; C, TiTE-15 protein kills malme-3M-CD19-luc; D, TiTE-16 protein kills malme-3M-CD19-luc; E, TiTE-5 protein kills malme-3M-CD19-luc; F, TiTE- 5 protein kills malme-3M-CD22-luc. It is proved that the multi-functional proteins TiTE-1, 15, 16, and 5 provided by the present invention can kill tumor cells in vitro at a very low concentration, and the in vitro killing effect is best when the concentration is 0.5-5 ng/10E 6 cells.
  • Figure 5 shows the killing results of TiTE-6, 8, 9, 10, 11, 12, 13, and 14 multifunctional protein molecules: A, negative control TiTE-2 protein kills BV173-luc; B, TiTE-6 protein kills BV173-luc C, TiTE-8, 9, 10, 11, 12, 13, 14 protein kills BV173-luc. It was proved that the multifunctional proteins TiTE-6, 8, 9, 10, 11, 12, 13, and 14 provided by the present invention have a killing effect on WT1-positive tumor cells.
  • Figure 6 shows the killing results of TiTE-2, 3, 4 multifunctional protein molecules: A, negative control TiTE-6 protein kills malme-3M-luc; B, TiTE-2 protein kills malme-3M-luc; C, TiTE-3 The protein kills malme-3M-luc; D, TiTE-4 protein kills malme-3M-luc. It is proved that the multifunctional proteins TiTE-2, 3, and 4 provided by the present invention can kill tumor cells expressing the intracellular antigen in vitro.
  • Figure 7 shows the results of stimulation of NK cells by multifunctional protein molecules.
  • the multifunctional protein provided by the present invention can stimulate NK cell expansion, and is amplified about 140 times in 18 days.
  • Figure 8 shows the flow results of TiTE-1, 6, 8, 9, 10, 11, 12 multifunctional protein molecules: A, T cell negative control; B, TiTE-1 T cell experimental group; C, BV173 negative control; , TiTE-1BV173 experimental group; E, BV173 negative control; F, TiTE-6BV173 experimental group; G, TiTE-8BV173 experimental group; H, TiTE-9BV173 experimental group; I, TiTE-10BV173 experimental group; J, TiTE-11BV173 Experimental group; K, TiTE-12BV173 experimental group; L, T cell negative control; M, TiTE-6T cell experimental group; N, TiTE-8T cell experimental group; O, TiTE-9T cell experimental group; P, TiTE-10T Cell experimental group; Q, TiTE-11T cell experimental group; R, TiTE-12T cell experimental group.
  • TiTE-1 binds well to CD3 antigen and CD19 antigen respectively; the antiMHC/WT1 and antiCD3 of TiTE-6, 8, 9, 10, 11, 12 have good binding function to WT1 intracellular antigen and CD3 antigen, respectively.
  • Figure 9 shows the flow results of TiTE-2, 3, and 4 multifunctional protein molecules: A, malme-3M negative control; B, TiTE-2malme-3M experimental group; C, TiTE-3malme-3M experimental group; D, TiTE -4malme-3M experimental group; E, T cell negative control; F, TiTE-2T cell experimental group; G, TiTE-3T cell experimental group; H, TiTE-4T cell experimental group.
  • A malme-3M negative control
  • B TiTE-2malme-3M experimental group
  • C TiTE-3malme-3M experimental group
  • D TiTE -4malme-3M experimental group
  • E T cell negative control
  • F TiTE-2T cell experimental group
  • G TiTE-3T cell experimental group
  • H TiTE-4T cell experimental group.
  • Figure 10 shows the flow results of TiTE-15, 16 multifunctional protein molecules: A, BV173 negative control; B, TiTE-15BV173 experimental group; C, TiTE-15BV173 experimental group; D, T cell negative control; E, TiTE- 15T cell experimental group; F, TiTE-16T cell experimental group. It can be seen from the figure that the multifunctional protein molecules TiTE-15, 16 bind well to the CD19 antigen and the CD3 antigen, respectively.
  • Figure 11 shows the flow results of TiTE-5 multifunctional protein molecule: A; malme-3M-CD19-Luc negative control; B, TiTE-5malme-3M-CD19-Luc experimental group; C, malme-3M-CD22-Luc Negative control; D, TiTE-5malme-3M-CD22-Luc experimental group; E, T cell negative control; F, TiTE-5T cell experimental group; It can be seen from the figure that the multifunctional protein molecule TiTE-5 binds well to the CD19 antigen, CD20 antigen and CD3 antigen, respectively.
  • Example 1 Construction of a multifunctional protein molecule vector
  • a novel multifunctional protein TiTE-1 to CD19-positive tumor cells which is polymerized by a main peptide chain X1 and a co-peptide chain Y1 to obtain a protein (Fig. 1);
  • the main peptide chain X1 includes an antigen 1 binding domain R1, a cytokine or cytokine receptor cytokine binding domain R2 and an antigen 2 binding domain R3;
  • the co-peptide chain Y1 includes an antigen 3 binding domain R4 and a main peptide chain X linking domain R5.
  • the antigen-binding domain (R1) of the main peptide chain (X1) was selected from antiCD19-ScFv (sequence 1)
  • the co-peptide chain domain (R2) was selected from IL15R ⁇ sushi (sequence 2)
  • the antigen-binding domain (R3) was selected from antiCD3-ScFv (sequence 3).
  • the antigen binding domain (R4) of the co-peptide chain (Y1) selects the extracellular domain of the receptor PD1 of PDL1 and PDL2 (sequence 4), and the primary peptide chain domain (R5) selects IL15 (sequence 5).
  • the signal peptide (amino acid sequence: MALPVTALLLPLALLLHAARP), the HindIII restriction site is added to the 5' end of the main peptide chain, the co-peptide chain domain of the main peptide chain (R2: L15R ⁇ sushi) and the antigen binding domain (R3: antiCD3-ScFv)
  • the linker peptide linked between them contains a BamHI restriction site; a P2A peptide is added to the 3' end of the main peptide chain and the 5' end of the co-peptide chain (amino acid sequence: GSGATNFSLLKQAGDVEENPGP); Xba I is added to the 3' end of the co-peptide chain Restriction sites.
  • the target fragment was recovered by gelatinization, and the three fragments recovered were ligated, and the single clone was selected and sequenced, and finally the target plasmid PCDNA3.1-TiTE-1 was obtained.
  • the recombinant vector PCDNA3.1-TiTE-1 is an expression cassette for the expression of the multifunctional protein-encoding gene TiTE-1 targeting CD19-positive tumor cells (the nucleotide sequence of the expression cassette is represented by the nucleotide sequence encoding the main peptide chain X1).
  • Sequence 6 and the nucleotide sequence sequence 7 encoding the co-peptide chain Y1, and the last nucleotide of the sequence 6 is immediately adjacent to the first nucleotide of the sequence 7) replaces the HindIII of the PCDNA3.1 vector (invitrogen product) And a fragment of the Xba I restriction site, the resulting recombinant vector expressing a functional multifunctional protein TiTE consisting of a main peptide chain X1 (amino acid sequence is sequence 8) and a co-peptide chain Y1 (amino acid sequence is sequence 9) -1.
  • the expression vector of TiTE-12 was constructed in a similar manner to the expression vectors of TiTE-13, TiTE-14, TiTE-15, and TiTE-16, and the domains thereof are shown in Table 1 below, and the expression framework is shown in Fig. 2.
  • Table 1 shows the structure of multifunctional proteins
  • amino acid sequence of AntiMHC/GP100-VHH is sequence 10;
  • the amino acid sequence of AntiMHC/Mart1-VHH is sequence 11;
  • the amino acid sequence of AntiMHC/WT1-VH is sequence 12;
  • amino acid sequence of IL4R ⁇ -N-FN3 is sequence 13;
  • the amino acid sequence of AntiCD16-ScFv is sequence 14;
  • the amino acid sequence of AntiCD22-ScFv is sequence 15;
  • amino acid sequence of IL4 is sequence 16;
  • the main peptide chain amino acid sequence of TiTE-2 is sequence 17, and the co-peptide chain amino acid sequence is sequence 9;
  • the main peptide chain amino acid sequence of TiTE-3 is sequence 17, and the co-peptide chain amino acid sequence is sequence 18;
  • the main peptide chain amino acid sequence of TiTE-4 is sequence 19, and the co-peptide chain amino acid sequence is sequence 9;
  • the main peptide chain amino acid sequence of TiTE-5 is sequence 8, and the co-peptide chain amino acid sequence is sequence 20;
  • the main peptide chain amino acid sequence of TiTE-6 is sequence 21, and the co-peptide chain amino acid sequence is sequence 9;
  • the main peptide chain amino acid sequence of TiTE-7 is sequence 22, and the co-peptide chain amino acid sequence is sequence 9;
  • the main peptide chain amino acid sequence of TiTE-8 is sequence 23, and the co-peptide chain amino acid sequence is sequence 24;
  • the main peptide chain amino acid sequence of TiTE-9 is sequence 25, and the co-peptide chain amino acid sequence is sequence 26;
  • the main peptide chain amino acid sequence of TiTE-10 is sequence 27, and the co-peptide chain amino acid sequence is sequence 28;
  • the main peptide chain amino acid sequence of TiTE-11 is sequence 29, and the co-peptide chain amino acid sequence is sequence 9.
  • the main peptide chain amino acid sequence of TiTE-12 is sequence 30, and the co-peptide chain amino acid sequence is sequence 31.
  • TiTE-13 is the main peptide chain, and the amino acid sequence of the main peptide chain is sequence 21.
  • the main peptide chain amino acid sequence of TiTE-14 is sequence 21, and the co-peptide chain amino acid sequence is sequence 5.
  • TiTE-15 is the main peptide chain, and the amino acid sequence of the main peptide chain is sequence 8.
  • the main peptide chain amino acid sequence of TiTE-16 is sequence 8, and the co-peptide chain amino acid sequence is sequence 5.
  • the encoding nucleic acid expressing TiTE-2 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 35, and the co-peptide chain encoding nucleic acid sequence is sequence 7;
  • the encoding nucleic acid expressing TiTE-3 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 35, and the co-peptide chain encoding nucleic acid sequence is sequence 36;
  • the encoding nucleic acid expressing TiTE-4 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last peptide of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 37, and the co-peptide chain encoding nucleic acid sequence is sequence 7;
  • the encoding nucleic acid expressing TiTE-5 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid Adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 6, and the co-peptide chain encoding nucleic acid sequence is sequence 38;
  • the encoding nucleic acid expressing TiTE-6 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid Adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 39, and the co-peptide chain encoding nucleic acid sequence is sequence 7;
  • the encoding nucleic acid expressing TiTE-7 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last peptide of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 40, and the co-peptide chain encoding nucleic acid sequence is sequence 7;
  • the encoding nucleic acid expressing TiTE-8 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 41, and the co-peptide chain encoding nucleic acid sequence is sequence 42;
  • the encoding nucleic acid expressing TiTE-9 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 43, and the co-peptide chain encoding nucleic acid sequence is sequence 44;
  • the encoding nucleic acid expressing TiTE-10 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 45, and the co-peptide chain encoding nucleic acid sequence is sequence 46;
  • the encoding nucleic acid expressing TiTE-11 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last peptide of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 47, and the co-peptide chain encoding nucleic acid sequence is sequence 7;
  • the encoding nucleic acid expressing TiTE-12 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 48, and the co-peptide chain encoding nucleic acid sequence is sequence 49;
  • the coding nucleic acid sequence expressing TiTE-13 is sequence 39;
  • the encoding nucleic acid expressing TiTE-14 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last base of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 39, and the co-peptide chain encoding nucleic acid sequence is sequence 50;
  • the nucleic acid sequence encoding TiTE-15 is sequence 6;
  • the encoding nucleic acid expressing TiTE-16 is composed of a main peptide chain encoding nucleic acid and a co-peptide chain encoding nucleic acid, and the last peptide of the 3' end of the main peptide chain encoding nucleic acid and the 1st base of the 5' end of the codon strand encoding nucleic acid a base adjacent; wherein the main peptide chain encoding nucleic acid sequence is sequence 6, the co-peptide chain encoding nucleic acid sequence is sequence 50;
  • the recombinant vector PCDNA3.1-TiTE-2 to the recombinant vector PCDNA3.1-TiTE-16 are HindIII and Xba I, respectively, which replace the encoding nucleic acid expressing TiTE-2 with the corresponding nucleic acid in the encoding nucleic acid expressing TiTE-16, replacing the PCDNA3.1 vector.
  • a fragment is obtained by cleavage of the fragment between the sites.
  • the vector PCDNA3.1-TiTE-1 constructed in Example 1 was transfected into the cells of the above 1 using PEI, and the transfection plasmid concentration was 1 mg/L, and the PEI concentration was 3 mg/L. Incubate for 5-6 days at 37 ° C, 8% CO 2 , 120 rpm.
  • the culture product of the above 2 was centrifuged at 4000 rpm, and the culture supernatant was collected, purified protein of Protein/capto L beads, eluted with 500 uL of pH 2.6-3.0 0.1 MGly-HCL eluate, and the eluate was collected.
  • the protein was collected by SDS-PAGE (see Fig. 3). It can be seen that the target protein of about 65KD and 30KD is obtained, which is the main peptide chain X and the co-peptide chain Y of the multifunctional protein molecule TiTE-1.
  • Example 3 Multi-functional protein molecule TiTE-1, 15, 16 mediated T cell killing CD19 + target cells in vitro
  • the 1*10E 4 target cell malme-3M-CD19-luc (transfected into the CD19 antigen (the nucleic acid sequence is sequence 32) and the Luc gene (the nucleic acid sequence is sequence 33) in the Malme-3M purchased by ATCC, The CD19 antigen and the Luc gene were expressed, and the cells malme-3M-CD19-luc) (50 uL) were plated in a 96-well plate, and cultured at 37 ° C, 5% CO 2 for 18-20 h.
  • the medium is aspirated and discarded, and 50 uL of fresh medium is added at 37 ° C, 5% CO 2 for 1-3 h.
  • stepwise dilution of the target protein TiTE-1 obtained in Example 2 to a concentration of 50, 5, 0.5, 0.05, 0.005 ng / uL;
  • T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50uL of 1*10E 5 T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50uL of 1*10E 5 T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50 5 0.5, 0.05, and 0.005 ng of the target protein obtained in Example 2 were incubated for 1-2 h at 126 ° C to obtain T cells incubated with the antibody.
  • Negative control group a bispecific control antibody (TiTE-6) with no killing effect on the target cells, adding 50, 5 , 0.5, 0.05, 0.005 ng of control antibody to 50 uL of 1*10E 5 T cells, respectively, at 37 ° C Incubate for 1-2 h.
  • killing efficiency ⁇ (negative control fluorescence value - experimental group fluorescence value) / negative control fluorescence value ⁇ * 100%.
  • FIG. 4B The result is shown in Figure 4B. It can be seen that the multi-function protein TiTE-1 provided by the present invention can kill CD19-positive tumor cells in vitro at a very low concentration compared with the control group in Fig. 4A, at a concentration of 0.5-5 ng/10E 6 cells. The best in vitro killing effect.
  • the TiTE-5 killing experiment was carried out, and the target cells were respectively treated with malme-3M-CD19-luc and malme-3M-CD22-luc (transfected into the CD22 antigen by the ATCC-purchased Malme-3M (the nucleic acid sequence thereof) It is sequence 34) and the Luc gene (the nucleic acid sequence is sequence 33).
  • the results are shown in Figures 4E and 4F. It can be seen that the multifunctional protein TiTE-5 provided by the present invention has killing function on CD19 and CD22 positive cells.
  • the tumor cells can be killed in vitro at a very low concentration, and the in vitro killing effect is best when the concentration is 0.5-5 ng/10E 6 cells.
  • Example 4 Multi-functional protein molecule TiTE-6, 8, 9, 10, 11, 12, 13, 14 mediates T cell killing of WT1 + target cells in vitro
  • stepwise dilution of the target protein TiTE-6 obtained in Example 2 to a concentration of 50, 5, 0.5, 0.05 ng / uL;
  • mice 50uL of 1*10E5 T cells (normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days) were added to 50, 5, respectively.
  • Negative control group a bispecific control antibody (TiTE-2) with no killing effect on the target cells, and 50, 5 , 0.5, and 0.05 ng of the control antibody were added to 50 uL of 1*10E 5 T cells, respectively. -2h.
  • killing efficiency ⁇ (negative control fluorescence value - experimental group fluorescence value) / negative control fluorescence value ⁇ * 100%.
  • the multifunctional protein TiTE-6 provided by the present invention can kill tumor cells using a very low concentration.
  • Example 5 Multi-functional protein molecule TiTE-2, 3, 4 mediates T cell killing target cells in vitro
  • the 1*10E 4 target cell malme-3M-luc (transfected into the Luc gene purchased by ATCC) (50 uL) was plated in a 96-well plate and cultured at 37 ° C, 5% CO 2 for 18-20 h.
  • the medium is aspirated and discarded, and 50 uL of fresh medium is added at 37 ° C, 5% CO 2 for 1-3 h.
  • stepwise dilution of the target protein TiTE-2, 3, 4 obtained in Example 2 to a concentration of 50, 5, 0.5, 0.05, 0.005 ng / uL;
  • T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50uL of 1*10E 5 T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50uL of 1*10E 5 T cells normal human peripheral blood density gradient centrifugation, mononuclear cell-rich white membrane layer, 50ng/mL OKT3, 300IU/mL IL2 stimulation for 14 days
  • 50, 5 0.5, 0.05, and 0.005 ng of the target protein obtained in Example 2 TiTE-2, 3, 4, and incubated at 37 ° C for 1-2 h to obtain T cells incubated with the antibody.
  • Negative control group a bispecific control antibody (TiTE-6) with no killing effect on the target cells, adding 50, 5 , 0.5, 0.05, 0.005 ng of control antibody to 50 uL of 1*10E 5 T cells, respectively, at 37 ° C Incubate for 1-2 h.
  • killing efficiency ⁇ (negative control fluorescence value - experimental group fluorescence value) / negative control fluorescence value ⁇ * 100%.
  • the cell growth curve is shown in Fig. 7. It can be seen that the multifunctional protein TiTE-1 provided by the present invention can stimulate NK cell expansion and has the function of IL15/IL15R ⁇ sushi.
  • the IL15/IL15R ⁇ sushi domains of TiTE2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, and 15 are identical to TiTE-1, and their functions are not significantly different.
  • Example 7 AntiCD19 and antiCD3 of the multifunctional protein molecule TiTE-1 and CD19 antigen and CD3 antigen and antiMHC/WT1 and antiCD3 of TiTE-6, 8, 9, 10, 11, 12, respectively, and WT1 intracellular antigen and CD3, respectively Flow verification of antigen binding function
  • T cell experimental group and BV173 cell experimental group 3*10E5 T cells respectively (normal human peripheral blood density gradient centrifugation, taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mLIL2 stimulation for 14 days) 5 ug of TiTE-1, 6, 8, 9, 10, 11, 12 multifunctional protein molecules were added to BV173 cells and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 uL of PBS. 2 uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 uL of PBS.
  • BD Cat. No. 558694
  • T cell negative control group and BV173 cell negative control group 3*10E5 T cells respectively (normal human peripheral blood density gradient centrifugation, taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mL IL2 stimulation 12- 2 uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to BV173 cells (ATCC) and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were resuspended in 200 uL of PBS as a negative control.
  • normal human peripheral blood density gradient centrifugation taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mL IL2 stimulation 12- 2 uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to BV173 cells (ATCC) and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re
  • TiTE-1 binds well to CD19 antigen and CD3 antigen, respectively; antiMHC of TiTE-6, 8, 9, 10, 11, and 12 /WT1 and antiCD3 function well with WT1 intracellular antigen and CD3 antigen, respectively.
  • Example 8 Flow cytometry of the binding function of antiMHC/Mart1 and antiCD3 antigens in anti-TEC/GP100 and antiCD3 and TiTE-4 in TiTE-2 and 3
  • T cell experimental group and malme-3M-Luc (transfected with Alex gene in Malme-3M purchased by ATCC) cell experimental group: 5ug TiTE was added to 3*10E5 T cells and malme-3M-Luc cells, respectively. -2, 3, 4 multifunctional protein molecules, incubating on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 uL of PBS. Add 2 uL of PE conjugated anti-hIL-15 (R&D, article number IC2471P) and incubate on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 uL of PBS.
  • PE conjugated anti-hIL-15 R&D, article number IC2471P
  • T cell negative control group and malme-3M-Luc cell negative control group 2uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to 3*10E5 T cells and malme-3M-Luc, respectively, and incubating on ice for 30 min. . The supernatant was removed by centrifugation, and the cells were resuspended in 200 uL of PBS as a negative control.
  • Flow cytometry As shown in Fig. 9, it can be seen from the figure that the multifunctional protein molecules TiTE-2 and 3 bind well to MHC/GP100 antigen and CD3 antigen, respectively, TiTE-4 and MHC/Mart1 antigen and CD3 antigen. The combination is good.
  • Example 9 Flow-through verification of the binding function of antiCD19 and antiCD3 of the multifunctional protein molecule TiTE-15/16 to CD19 antigen and CD3 antigen, respectively
  • T cell experimental group and BV173 cell experimental group 3*10E5 T cells respectively (normal human peripheral blood density gradient centrifugation, taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mLIL2 stimulation for 14 days) 5 ug of TiTE-15, 16 multi-function protein molecules were added to BV173 cells and incubated on ice for 30 min. The supernatant was removed by centrifugation and the cells were resuspended in 200 uL of PBS. 2 uL of FITC-Labeled Recombinant Protein L (ACRO Biosystem, Cat. No. RPL-PF141) was added and incubated on ice for 30 min. The supernatant was removed by centrifugation, washed twice in 500 uL PBS, and resuspended in 200 uL of PBS.
  • FITC-Labeled Recombinant Protein L ACRO Biosystem, Cat. No. RPL-PF141
  • T cell negative control group and BV173 cell negative control group 3*10E5 T cells respectively (normal human peripheral blood density gradient centrifugation, taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mL IL2 stimulation 12- 2 uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to BV173 cells (ATCC) and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were resuspended in 200 uL of PBS as a negative control.
  • normal human peripheral blood density gradient centrifugation taking monolayer of mononuclear cells, 50ng/mL OKT3, 300IU/mL IL2 stimulation 12- 2 uLAPC Mouse anti-Human CD279 (BD, Cat. No. 558694) was added to BV173 cells (ATCC) and incubated on ice for 30 min. The supernatant was removed by centrifugation, and the cells were re
  • Flow cytometry As shown in Fig. 10, it can be seen from the figure that the multifunctional protein molecules TiTE-15 and 16 bind well to the CD19 antigen and the CD3 antigen, respectively.
  • Example 10 Flow-through verification of the binding function of antiCD19, antiCD20 and antiCD3 to antigen in the multifunctional protein molecule TiTE-5
  • T cell experimental group malme-3M-CD19-Luc will ((transfecting the CD19 antigen gene and the Luc gene in the Malme-3M purchased by ATCC) and malme-3M-CD22-Luc ((Malme purchased by ATCC) -3M transfected CD22 antigen and Luc gene) cell experimental group: 5ug of TiTE-5 multi-function protein molecule was added to 3*10E5 T cells and malme-3M-Luc cells, respectively, and incubated on ice for 30 min. The supernatant was resuspended in 200 uL of PBS. 2 uL of PE conjugated anti-hIL-15 (R&D, Cat. No. IC2471P) was added and incubated on ice for 30 min. The supernatant was centrifuged and the cells were resuspended in 200 uL of PBS.
  • PE conjugated anti-hIL-15 R&D, Cat. No. IC2471P
  • PE conjugated anti-hIL-15 R&D, article number IC2471P
  • Flow cytometry As shown in Fig. 11, it can be seen from the figure that the multifunctional protein molecule TiTE-5 binds well to the CD19 antigen, CD20 antigen and CD3 antigen, respectively.
  • the experiments of the present invention prove that the multifunctional protein of the present invention can bind to different tumor antigens through the antigen binding domain of its two tumor-associated antigens, mediate specific cell killing, and improve the accuracy of targeting;
  • the antigen bound by the antigen binding domain is an immune screening point-associated antigen, which can block the immunosuppressive signal and improve the ability to kill tumor;
  • the multifunctional protein of the present invention introduces a cytokine and cytokine receptor complex, and they It can function as a cytokine.

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Abstract

一种多功能多肽链或蛋白质。一种多肽链X，包括抗原1结合域R1、辅肽链连接域R2和抗原2结合域R3；所述辅肽链连接域R2为细胞因子或细胞因子受体中的细胞因子结合域。一种蛋白质，由所述多肽链X作为主肽链与辅肽链Y组成异源二聚体。所述辅肽链Y包括抗原3结合域R4和主肽链X连接域R5，或所述辅肽链Y为主肽链连接域R5。多功能蛋白质通过其两个肿瘤相关抗原的抗原结合域与不同的肿瘤抗原结合，介导特异性的细胞杀伤；多功能蛋白质引入细胞因子与细胞因子受体复合物，可以发挥细胞因子的功能。

Description

一种多功能蛋白质 技术领域
本发明涉及生物领域，尤其涉及一种多功能蛋白质。
背景技术
双特异性抗体(bispecific antibody)，又称为双功能抗体或双价抗体，可以同时特异性结合2个不同的抗原，具有特异性和双功能性，在肿瘤免疫治疗及自身免疫病等领域中具有广阔的应用前景。
双特异性抗体在自然状态下不存在，只能通过人工方法制备。目前制备双特异性抗体方法主要有化学缀合、杂交瘤技术、重组DNA技术等。
早在30年以前Medarex公司就已经开发出了双特异性抗体，并且在2001年进行了III期临床试验。但是由于临床试验失败并且受限于生产问题，该领域的研究归于沉寂。(Garber K，2014)2009年，Trion公司开发的双特异性抗体Catumaxomab被欧盟批准上市用于治疗EpCAM阳性肿瘤所引起的恶性腹水，但由于鼠源抗体的高免疫原性大大限制了其临床应用(Spasevska I，2014)。近些年，抗体工程技术迅猛发展，为双特异性抗体的开发带来了新的契机。
1、化学偶联合成双特异抗体。
Lum实验室使用商业化Herceptin和OKT3通过化学偶联获得Her2/CD3双特异性抗体可招募T细胞杀伤Her2阳性肿瘤细胞(Msen et al.,2001)。这种基于临床上广泛使用的抗体偶联物，显示出非常好的临床安全性及疗效,22名参与临床试验的转移性乳腺癌的病人中有5位在使用后的14.5星期内仍然维持稳定的病情(Lum et al.,2015)。
OKT3抗体还被用来和其他临床上使用的抗体通过化学偶联，用于杀伤具有相应靶点的肿瘤：包括EGFR阳性肿瘤(Reush U et al.,2006)，(MAD et al.,2015)CD20阳性肿瘤系(Lum et al.,2013),B7-H3阳性肿瘤(MA et al.,2016)等。
2、杂交瘤技术生产双特异抗体
Lindnorer等用抗CD3抗体的大鼠杂交瘤与分泌抗EpCAM小鼠杂交瘤融合制备出可分泌多达11种由不同重链和轻链组成的抗体(lindnoer et  al.，1995)，这种采用异源大鼠及小鼠杂交瘤制备出的双抗，不可避免地产生人抗鼠(HAMA)交叉反应。令人惊奇的是，这种抗药物反应反而与病人与该抗体反应效果呈正比，(OTT MG et al.,2012)其机制有待进一步探讨。2009年已经被批准在临床上使用(Carberk.，2014)。用同样平台开发出的Her/CD3抗体(Kiewe et al.,2006)，用于治疗复发B细胞淋巴瘤的抗CD20/CD3抗体已获准进入临床试验，并获得良好的安全记录和疗效(Buhmann et al.,2009)。抗CD2/CD3双抗已用于治疗黑色素瘤的实验研究(Ruf et al.,2004)。
3、重组表达双特异分子
Genentech科学家Shalaby等将人源化抗CD3抗体UCHT1的Fab片段与抗HER2抗体4D5通过接头肽连接，在大肠杆菌表达系统成功表达。这种双特异性抗体可特异性识别Her2高表达的乳腺癌细胞SK-BR-3，并可介导人外周血T细胞对该种肿瘤细胞系的杀伤(Shalaby et al.，1992)。随着合成生物分子及蛋白质重组技术的发展,通过基因工程制备出的招募T细胞靶向肿瘤的蛋白质分子后来居上,已成为这类药物的主流。
BiTE 2014年12月，FDA批准了一种新型工程化CD3靶向双特异性抗体分子-BiTE(Bispecific T-cell engager)用于治疗急性淋巴白血病。这种新型的小蛋白分子直接由OKT3的scFv与抗CD19的scFv通过一连接肽连接而成(Nagorsen D et al.，2012)(专利号申请：201180063222.2；201580009124.9)，仅需极低的浓度既可抑制非霍奇金淋巴瘤生长(Bargou R et al.,2008)。由于这种分子高效的招募T细胞杀伤靶细胞的能力，更多的基于BiTE平台的靶向不同肿瘤的产品已进入临床试验阶段。其中包括识别EpCAM，CEA，DSMA等多种BiTE分子(Thakur A et al.,2016)
TandAb将一对类似BiTE分子的双特异性分子用连接肽连接，形成称作TandAb的四聚体分子：160kD的分子量是BiTE的双倍，使得与CD3和CD19的亲和力更高(Reusch U et al.,2015)，同时，TandAb的药代动力学较之BiTE也显著改善，在血液中的半衰期达到20h左右。可介导杀伤非霍奇金淋巴瘤及急性淋巴白血病。
DART是将抗CD19与CD3的scFv通过含二硫键的连接肽连接而成，可招募T细胞杀伤肿瘤，并具有稳定及容易规模化制备的优点(Johnson S  et al,2010；kuo SR et al.,2012)。
FcabFv是将OKT3的抗原识别部分,与经过CH3突变产生的具有识别Her2的功能的Fc(Wozniak G et al.,2010)相融合，表达出一种和普通抗体高度相似的新颖双特异性抗体，能有效地靶向Her2阳性肿瘤，并在动物体内抑制肿瘤生长(Wang L et al.,2013)。
TriKE是将IL15插入到CD33抗体的scFv和CD16抗体的scFv之间表达，中间通过两个连接肽连接，在有效地靶向肿瘤的同时，可促进体内NK细胞持久性的激活和生存，IL15的加入有利于于NK细胞治疗髓系恶性肿瘤或靶向实体瘤的抗原。(Szun Szun Tay et al.，2016；Vallera DA.et al.，2016)
目前，双特异性抗体已经成为药物研究领域中的新热点，至少有30多种双特异性抗体处于临床研究阶段(Garber K et al.，2014；Kontermann R E et al.2015)。
T细胞和NK细胞的培养基中都必须添加一定量的IL-2(Bodnar et al.,2008；Grund et al.,2005)。IL-15与IL-2功能类似，而且共用相同的βγ受体，研究表示IL-2或IL-15对于NK细胞和CD8+T细胞的存活和增殖是必须的(Boyman et al.,2007)。IL-15与IL-2虽然共用相同的βγ受体，但各自又有特异的α受体，研究发现IL-15Rα-sushi(IL-15受体α的sushi结构域)是IL-15的超级激动剂，可以极大的提高IL-15的功能(Han et al.,2011；Mortier et al.,2006)(专利申请号：201280037114.2，201510358540.1)，IL-15与IL-15Rα-sushi的复合体可以完全替代IL-2在T/NK细胞中的作用(Peter S.Kim1,2016；Rosario et al.,2016)，激活NK/CD8+T细胞并提高其杀伤肿瘤的细胞毒性。美国国立卫生研究院(NIH)国家癌症研究所指出，在治疗癌症的12种免疫治疗药物中，IL-15位列第一位。通过支持CD8+T细胞的生存能力，IL-15在T细胞长期免疫应答方面展现了很大的潜能。相比较于IL-2，IL-15更有前途，更具疗效，在肿瘤治疗中毒性更低，更可以刺激T细胞、NK细胞的抗肿瘤活性。在双特异分子中融合IL-15与IL-15Rα-sushi的复合体或其他有功能的细胞因子与受体复合物是提高细胞免疫治疗疗效的一大方向。
PD-1(programmed death 1)和它的受体PD-L1，PD-L2是T细胞活性的重要调节因子(Okazaki and Honjo，2007)。T细胞表面的PD-1与其他细胞表面的PD-L1/2的结合造成T细胞的抑制，这在人体避免自体免疫病和产生免疫耐受的过程中起到重要作用，而肿瘤细胞利用PD1/PD-L1检查点这种免疫系统保护自我的调节机制，通过在肿瘤细胞自身或肿瘤微环境表达PD-L1/L2，于T细胞表面表达的PD1结合，传递负信号，导致T细胞功能衰退及耗竭，达到抑制免疫应答，肿瘤逃逸的目的。(Freeman et al.，2000；Keir et al.，2008；Parry et al.，2005)。因此研究者探索用PD-1或PD-L1的抗体去结合抗原，在T细胞靶向肿瘤的同时，阻断PD1检查点通路，结果显示此方法能够显著提高T细胞活性，增强机体对病原微生物和癌症的抵抗能力(Topalian et al.，2012；Yanan Guo et al.，2016)。已有多项临床试验证明了PD-1/PD-L1抗体对于黑色素瘤(Cho et al.，2016；Hamid et al.，2013)、多发性骨髓瘤(Badros et al.，2015)、白血病(Porkka et al.，2014)等癌症都有很好的治疗效果(专利申请号：200380109929.8，201310258289.2，201180019629.5)。
发明公开
本发明的一个目的是提供一种多肽链X。
本发明提供的多肽链X，包括抗原1结合域R1、辅肽链连接域R2和抗原2结合域R3；
所述辅肽链连接域R2为细胞因子或细胞因子受体中的细胞因子结合域。
上述的多肽链X中，
所述抗原2结合域R3为识别T细胞CD3的抗体或分子。
上述的多肽链X中，
所述抗原2结合域R3为识别NK细胞CD16的受体或抗体或其他分子。
上述的多肽链X中，
所述抗原1结合域R1所结合的抗原选自下述的癌症相关的抗原中任一种：脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋 巴瘤、霍奇金淋巴瘤和子宫癌。
上述的多肽链X中，
所述癌症相关抗原优选自下述抗原中任一种:CD123，CD19、CD20、CD22、CD37、ROR1、间皮素、CD33/IL3Ra、c-Met、BCMA、PSMA、EGFRvIII、GD-2、NY-ESO-1、MAGEA3、β-人绒毛膜促性腺素、AFP、RAGE-1、MN-CAIX、人端粒酶反转录酶、RU1、RU2(AS)、hsp70-2、M-CSF、PSA、PAP、LAGE-la、p53、Prostein、PSMA、Her2/neu、端粒酶、PCTA-1、MAGE、ELF2M、IGF-I、IGF-II、IGF-I受体、BCR-ABL、E2A-PRL、H4-RET、1GH-IGK、MYL-RAR、GP100、Mart1、TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、p185erbB2、p180erbB-3、c-met、nm-23H1、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p15、p16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、WT1、CD68、FGF-5、G250、EpCAM、MA-50、MG7-Ag、MOV 18、NB/70K、RCAS1、SDCCAG16、TA-90、TAAL6、TAG72、TLP、p53、Ras、TPS、Epstein Barr病毒抗原EBVA、人乳头瘤病毒(HPV)抗原E6、人乳头瘤病毒(HPV)抗原E7或MHC与上述抗原的短肽的复合物。
MHC与所述抗原的短肽的复合物中的所述抗原为上述癌症相关抗原中任一种。
上述的多肽链X中，
所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、CD123，CD33/IL3Ra、Her2、PDL1、GP100、Mart1，BCMA，WT-1和NY-ESO-1或MHC与上述抗原的短肽的复合物。
上述MHC与所述抗原的短肽的复合物中的所述抗原为所述癌症相关抗原优选的选自下述抗原中任一种。
上述的多肽链X中，
所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、Her2、PDL1、WT1、GP100、Mart1、BCMA和NY-ESO-1或MHC与上述抗原短肽的复合物。
上述的多肽链X中，
所述抗原1结合域R1为结合抗原的抗体、结合抗原的配体、结合抗 原的受体或具有抗原结合功能的多肽。
上述的多肽链X中，
所述结合抗原的抗体是完整免疫球蛋白，抗体的Fc、抗体的Fab、抗体的VH、抗体的VL或scFv的全长肽链或部分肽段。
上述的多肽链X中，
所述结合抗原的配体或所述结合抗原的受体是全长肽链或部分肽段。
上述的多肽链X中，
所述抗原1结合域R1为具有抗原识别功能的TCR。
上述的多肽链X中，
所述抗原1结合域R1为具有抗原识别功能的TCR样抗体或其他分子。
本发明另一个目的是提供一种蛋白质。
本发明提供的蛋白质，其为由权利要求1所述的肽链X作为主肽链与辅肽链Y组成异源二聚体；
所述辅肽链Y包括抗原3结合域R4和主肽链X连接域R5，
或所述辅肽链Y为主肽链连接域R5；
所述主肽链连接域R5与所述肽链X中的辅肽链连接域R2相互结合。
上述主肽链和辅肽链的各功能域之间通过多肽接头连接。这些多肽接头为富含甘氨酸和/或丝氨酸的序列或富含甘氨酸和/或丝氨酸的多个拷贝序列，多肽接头一般包括1-20个氨基酸残基。
主肽链连接域R5和细胞因子或细胞因子受体中的细胞因子结合域R2是一对具有相互结合功能的肽段。
上述辅肽链连接域(R2)和主肽链连接域(R5)互为细胞因子和受体亚基。主肽链(X)和辅肽链(Y)之间通过辅肽链连接域(R2)和主肽链连接域(R5)之间的结合形成异源二聚体。
上述蛋白中，
所述抗原3结合域R4所结合的抗原3选自下述的癌症相关的抗原中任一种：脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和子宫癌。
上述蛋白中，
所述癌症相关抗原3优选自下述抗原中任一种:CD123，CD19、CD20、CD22、CD37、ROR1、间皮素、CD33/IL3Ra、c-Met、BCMA、PSMA、EGFRvIII、GD-2、NY-ESO-1、MAGEA3、β-人绒毛膜促性腺素、AFP、RAGE-1、MN-CAIX、人端粒酶反转录酶、RU1、RU2(AS)、hsp70-2、M-CSF、PSA、PAP、LAGE-la、p53、Prostein、PSMA、Her2/neu、PDL1、端粒酶、PCTA-1、MAGE、ELF2M、IGF-I、IGF-II、IGF-I受体、BCR-ABL、E2A-PRL、H4-RET、1GH-IGK、MYL-RAR、GP100、Mart1、TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、p185erbB2、p180erbB-3、c-met、nm-23H1、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p15、p16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、WT1、CD68、FGF-5、G250、EpCAM、M344、MA-50、MG7-Ag、MOV18、NB/70K、RCAS1、SDCCAG16、TA-90、TAAL6、TAG72、TLP、p53、Ras、TPS、Epstein Barr病毒抗原EBVA、人乳头瘤病毒(HPV)抗原E6、人乳头瘤病毒(HPV)抗原E7或MHC与上述抗原短肽的复合物。
上述MHC与所述抗原的短肽的复合物中的所述抗原为所述癌症相关抗原3优选自上述抗原中任一种。
上述蛋白中，
所述的癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、CD123，CD33/IL3Ra、Her2、PDL1、GP100、Mart1，BCMA，WT-1、NY_ESO-1或MHC与上述抗原短肽的复合物。
上述MHC与所述抗原的短肽的复合物中的所述抗原为CD19、CD20、CD22、CD123，CD33/IL3Ra、Her2、PDL1、GP100、Mart1，BCMA，WT-1和NY_ESO-1中任一种。
上述蛋白中，
所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、Her2、PDL1、WT1、GP100、Mart1、BCMA、NY_ESO-1或MHC与上述抗原短肽的复合物；上述MHC与所述抗原的短肽的复合物中的所述抗原为CD19、CD20、CD22、Her2、PDL1、WT1、GP100、Mart1、BCMA、NY_ESO-1中任一种。
上述中，
所述抗原1结合域R1和所述抗原2结合域R3的位置能互换。
上述中，
所述抗原1结合域R1和所述抗原3结合域R4的位置能互换。
上述中，
所述抗原2结合域R3和所述抗原3结合域R4的位置能互换。
上述中，
所述辅肽链连接域R2和所述主肽链连接域R5是一对具有相互结合功能的肽段。优选为一对可以相互结合γc细胞因子及其受体亚基。其最优的选择为IL15与IL15Rα和IL4与IL4Rα。IL15在维持T细胞、NK细胞和NKT细胞的稳态，细胞生长中起着不可或缺的作用，同时又能对B细胞、树突状细胞(DC)、巨噬细胞、肥大细胞提供额外的生理功能。IL-15可支持CD8+T细胞的生存能力，较于IL-2更有前途，更具疗效，在肿瘤治疗中毒性更低，更可以刺激T细胞、NK细胞的抗肿瘤活性。
上述中，
所述辅肽链连接域R2和所述主肽链连接域R5是互为一对可以相互结合细胞因子及其受体亚基。
上述中，
所述细胞因子为γc家族细胞因子，
所述γc家族细胞因子为IL2、IL4、IL7、IL9、IL15或IL21。
上述中，
所述细胞因子和受体亚基最优选自IL15与IL15Rα或IL4与IL4Rα。
上述中，
所述多肽或蛋白的主肽链中各组分或辅肽链中各组分通过由1-20个氨基酸残基组成的多肽接头连接。
上述中，
所述多肽接头中富含甘氨酸和/或丝氨酸。
上述中，
所述抗原1结合域R1为antiCD19-ScFv或AntiMHC/GP100-VHH或AntiMHC/Mart1-VHH或anti MHC/WT1或PD1的胞外区或AntiCD22-ScFv或 antiCD3-ScFv或antiCD16-ScFv；
或，所述辅肽链连接域R2为IL15Rαsushi或IL4Rα-N-FN3或IL15或IL4；
或，所述抗原2结合域R3为antiCD3-ScFv或antiCD16-ScFv或antiCD19-ScFv或AntiMHC/GP100-VHH或AntiMHC/Mart1-VHH或antiWT1或PD1的胞外区或AntiCD22-ScFv；
或，所述抗原3结合域R4为PD1的胞外区或AntiMHC/GP100-VHH或AntiCD22-ScFv或antiCD19-ScFv或AntiMHC/Mart1-VHH或anti MHC/WT1或antiCD3-ScFv或antiCD16-ScFv；
或，所述主肽链连接域R5为IL15或IL4或IL15Rαsushi或IL4Rα-N-FN3。
上述中，
所述antiCD19-ScFv的氨基酸序列为序列1；
所述IL15Rαsushi的氨基酸序列为序列2；
所述antiCD3-ScFv的氨基酸序列为序列3；
所述PD1的胞外区的氨基酸序列为序列4；
所述IL15的氨基酸序列为序列5；
所述AntiMHC/GP100-VHH的氨基酸序列为序列10；
所述AntiMHC/Mart1-VHH的氨基酸序列为序列11；
所述AntiMHC/WT1-VH的氨基酸序列为序列12；
所述IL4Rα的氨基酸序列为序列13；
所述AntiCD16-ScFv的氨基酸序列为序列14；
所述AntiCD22-ScFv的氨基酸序列为序列15；
所述IL4的氨基酸序列为序列16。
上述中，
所述多肽链X的氨基酸序列为序列8；
或所述多肽链X的氨基酸序列为序列17；
或所述多肽链X的氨基酸序列为序列19；
或所述多肽链X的氨基酸序列为序列21；
或所述多肽链X的氨基酸序列为序列22；
或所述多肽链X的氨基酸序列为序列23；
或所述多肽链X的氨基酸序列为序列27；
或所述多肽链X的氨基酸序列为序列29；
或所述多肽链X的氨基酸序列为序列30；
或所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列9；
或所述蛋白的主肽链的氨基酸序列为序列17，辅肽链的氨基酸序列为序列9；
或所述蛋白的主肽链的氨基酸序列为序列17，辅肽链的氨基酸序列为序列18；
或所述蛋白的主肽链的氨基酸序列为序列19，辅肽链的氨基酸序列为序列9；
或所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列20；
或所述蛋白的主肽链的氨基酸序列为序列21，辅肽链的氨基酸序列为序列9；
或所述蛋白的主肽链的氨基酸序列为序列22，辅肽链的氨基酸序列为序列9；
或所述蛋白的主肽链的氨基酸序列为序列23，辅肽链的氨基酸序列为序列24；
或所述蛋白的主肽链的氨基酸序列为序列25，辅肽链的氨基酸序列为序列26；
或所述蛋白的主肽链的氨基酸序列为序列27，辅肽链的氨基酸序列为序列28；
或所述蛋白的主肽链的氨基酸序列为序列29，辅肽链的氨基酸序列为序列9。
或所述蛋白的主肽链的氨基酸序列为序列30，辅肽链的氨基酸序列为序列31；
或所述蛋白的主肽链的氨基酸序列为序列21，辅肽链的氨基酸序列为序列5；
或所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列5。
本发明第三个目的是提供上述多肽或蛋白的编码核酸分子。
本发明提供上述多肽或蛋白的编码核酸分子，由编码所述主肽链的核酸分子或编码所述主肽链的核酸分子和编码所述辅肽链的核酸分子组成。
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如，例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离，或者通过人工合成的方式合成多核苷酸。
含有上述的核酸分子的重组载体、表达盒、重组菌、重组病毒或细胞也是本发明保护的范围。
上述重组载体中，所述重组载体为将上述的核酸分子中的编码所述主肽链的核酸分子或编码所述主肽链的核酸分子和编码所述辅肽链的核酸分子菌插入表达载体中，得到表达上述的蛋白的载体。
上述重组载体包含上述多核苷酸序列或组合。在一个实施方式中，可连接编码主肽链(X)或辅肽链(Y)的核酸至启动子，并将构建体并入表达载体，实现编码主肽链(X)或辅肽链(Y)的表达。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。例如慢病毒载体是实现基因长期稳定遗传的适宜工具，因为它们可以将基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌反转录病毒诸如鼠科白血病病毒的载体的额外优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的额外优点。本发明提供的多功能蛋白质包含两条肽链，可通过公知的方式使其表达于同一细胞中，包括但不限于共转染分别编码主肽链(X)和辅肽链(Y)的两种表达载体，或将编码主肽链(X)和辅肽链(Y)的核酸序列克隆至含有两套表达框架的的表达载体中，或者将编码主肽链(X)和辅肽链(Y)的核酸序列串联克隆至一个表达框架中，通过在主肽链(X)和辅肽链(Y)的核酸序列中间插入核糖体结合位点，2A肽实现两种肽链的共表达。
上述细胞中，所述目的细胞为原核细胞、酵母细胞或哺乳动物细胞；其中哺乳动物细胞优选为人类细胞。
本发明还提供了一种试剂盒，其包括上述多肽链X或上述的蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、重组病毒或细胞。
上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在免疫治疗中的应用也是本发明保护的范围；
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备免疫治疗产品中的应用也是本发明保护的范围。
上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在免疫细胞培养和/或促进免疫细胞扩增和/或免疫检测中的应用也是本发明保护的范围；
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备免疫细胞培养和/或促进免疫细胞扩增和/或免疫检测产品中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在刺激T或NK细胞增殖中的应用也是本发明保护的范围；
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备刺激T或NK细胞增殖产品中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在介导免疫细胞抑制或杀伤表达所述蛋白中抗原的靶细胞中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备介导免疫细胞抑制或杀伤表达所述蛋白中抗原的靶细胞产品中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在抑制或杀伤肿瘤细胞中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、 重组菌、细胞或重组病毒或试剂盒在制备抑制或杀伤肿瘤细胞产品中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在治疗或检测肿瘤中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备治疗或检测肿瘤产品中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在抑制或杀伤表达所述蛋白中抗原的靶细胞中的应用也是本发明保护的范围。
或上述的多肽或蛋白、上述的核酸分子或上述的重组载体、表达盒、重组菌、细胞或重组病毒或试剂盒在制备抑制或杀伤表达所述蛋白中抗原的靶细胞产品中的应用也是本发明保护的范围。
上述中，所述免疫治疗为通过免疫细胞抑制或杀伤肿瘤细胞；
或，所述免疫细胞为T细胞或NK细胞等；
或，所述抗原为癌症相关抗原；
或所述抗原为脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、子宫癌相关抗原或其任何组合；
或，所述肿瘤为脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、子宫癌中任一种或任何组合；
或，所述靶细胞为原核细胞、酵母细胞或哺乳动物细胞；
或，所述哺乳动物细胞具体为人类细胞；
或，所述人类细胞具体为免疫细胞，
或，所述免疫细胞具体为T细胞或NK细胞。
上述多肽或蛋白中，所述主肽链的抗原结合域(R1/R3)、辅肽链抗 原结合域(R4)具有结合抗原的功能。其抗原结合域其中一个为识别人T细胞的CD3的抗体或分子或识别NK细胞CD16的受体或抗体或其他分子，其他两个是选自下述的肿瘤相关的抗原。其抗原结合域所结合的肿瘤抗原是由引起免疫应答特别是T-细胞介导的免疫应答的肿瘤细胞产生的蛋白质。本发明的抗原结合结构域的选择将取决于待治疗癌症的具体类型。肿瘤抗原在本领域中是公知的：
在一个实施方式中，本发明提及的肿瘤相关抗原也可以是选自下述的肿瘤相关的抗原：脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、子宫癌和其任何组合。
具体而言：
在一个实施方式中，本发明提及的肿瘤抗原包括例如神经胶质瘤相关的抗原、癌胚抗原(CEA)、β-人绒毛膜促性腺素、α-胎蛋白(AFP)、凝集素-反应的AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶反转录酶、RU1、RU2(AS)、肠羧酸酯酶、mut hsp70-2、M-CSF、前列腺酶、前列腺-特异性抗原(PSA)、PAP、NY-ESO-1、LAGE-la、p53、prostein、PSMA、Her2/neu、存活素和端粒酶、前列腺-癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性白细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF)-I、IGF-II、IGF-I受体和间皮素。
在一个实施方式中，肿瘤抗原包括与恶性肿瘤相关的一个或多个抗原癌症表位。恶性肿瘤表达可用作免疫攻击的靶抗原的许多蛋白。这些分子包括但不限于组织-特异性抗原诸如MART-1、黑素瘤中的酪氨酸酶和GP 100、和前列腺癌中的前列腺酸性磷酸酶(PAP)和前列腺-特异性抗原(PSA)。其他靶分子属于转化相关分子诸如致癌基因HER-2/Neu/ErbB-2的组。而另一组的靶抗原为胎性癌抗原诸如癌胚抗原(CEA)。在B-细胞淋巴瘤中，肿瘤-特异性个体基因型免疫球蛋白构成对个体肿瘤唯一的真正的肿瘤-特异性免疫球蛋白抗原。B-细胞分化抗原诸如CD19、CD20和CD37是B-细胞淋巴瘤中靶抗原的其他候选物。这些抗原中的一些(CEA、HER-2、CD19、CD20、个体基因型)已经有限 成功地用作利用单克隆抗体的被动免疫疗法的靶标。
在一个实施方式中本发明中提及的肿瘤抗原也可为肿瘤-特异性抗原(TSA)或肿瘤相关抗原(TAA)。TSA为对肿瘤细胞唯一的，并不发生在身体的其他细胞上。TAA相关的抗原不是对肿瘤细胞唯一的，并且相反，其在不能诱导对抗原的免疫耐受状态的病症下，也在正常细胞上进行表达。肿瘤上的抗原表达可在使免疫系统能够响应抗原的病症下发生。TAA可为在胚胎发育期间，当免疫系统不成熟并且不能响应时，在正常细胞上表达的抗原，或它们可为在正常细胞上以极低的水平正常存在的抗原，但其在肿瘤细胞上以更高的水平进行表达。
TSA或TAA抗原的非限制性例子包括以下：分化抗原诸如MART-l/MelanA(MART-1)、gp100(Pmel 17)、酪氨酸酶、TRP-1、TRP-2和肿瘤-特异性多谱系抗原诸如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15；过表达的胚胎抗原诸如CEA；过表达的致癌基因和突变的肿瘤-抑制基因诸如p53、Ras、HER-2/neu；由染色体易位产生的独特的肿瘤抗原诸如BCR-ABL、E2A-PRL、H4-RET、1GH-IGK、MYL-RAR；和病毒抗原，诸如Epstein Barr病毒抗原EBVA和人乳头瘤病毒(HPV)抗原E6和E7。其他大的、基于蛋白的抗原包括TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、NY-ESO、p185erbB2、p180erbB-3、c-met、nm-23H1、PSA、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、α-胎蛋白、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV 18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TA-90\Mac-2结合蛋白\亲环蛋白C相关蛋白、TAAL6、TAG72、TLP和TPS。
在一个实施方式中本发明中提及的肿瘤抗原可以使MHC与上述抗原肽段的复合物。包括但不限于HLA-GP100复合物，HLA-Mart1复合物，HLA-WT1复合物。
在一个实施方式中，其中所述主肽链中的抗原结合域(R1、R3)或辅肽链中的抗原结合域(R4)是可以与抗原结合的抗体、配体、受体、具有抗原结合能力的多肽或其任意组合。
抗体可以是Ig，Fab，scFv的完整肽链或部分肽段或其任意组合。配体或受体可以是其完整肽链、部分肽段或其任意组合。
其中所述辅肽链连接域(R2)和主肽链连接域(R5)是一对具有相互结合功能的肽段。
其中具有相互结合功能的肽段可以是一对可以相互结合的受体与配体或是一对可以相互结合的抗体与抗原。优选为一对可以相互结合γc细胞因子及其受体亚基，最优选择为IL15与IL15Rα，IL4与IL4R。
主肽链和辅肽链的各功能域之间通过多肽接头连接，这些多肽接头为富含甘氨酸和/或丝氨酸的序列或富含甘氨酸和/或丝氨酸的多个拷贝序列，多肽接头一般包括5-20个氨基酸残基。
附图说明
图1为多功能蛋白分子结构示意图。A：多功能蛋白分子由主肽链X和辅肽链Y组成，其中主肽链X包括抗原结合域R1、辅肽链连接域R2、抗原结合域R3，辅肽链Y包括抗原结合域R4、主肽链连接域R5；B：多功能蛋白分子由主肽链X和辅肽链Y组成，其中主肽链X包括抗原结合域R1、辅肽链连接域R2、抗原结合域R3，辅肽链Y包括主肽链连接域R5；C：多功能蛋白分子仅由主肽链X，其中主肽链X包括抗原结合域R1、辅肽链连接域R2、抗原结合域R3。
图2为多功能蛋白分子基因表达框架。
图3为多功能蛋白质分子多肽表达纯化SDS-PAGE图。泳道1：TiTE-1，主肽链约65KD，辅肽链约30KD；泳道2：TiTE-6，主肽链约65KD，辅肽链约30KD；泳道3：蛋白Marker，分子量自上至下依次为：160KD、120KD、100KD、70KD、50KD、40KD、30KD、25KD。
图4为TiTE-1、15、16、5多功能蛋白分子杀伤结果：A，阴性对照TiTE-6蛋白杀伤malme-3M-CD19-luc；B，TiTE-1蛋白杀伤malme-3M-CD19-luc；C，TiTE-15蛋白杀伤malme-3M-CD19-luc；D，TiTE-16蛋白杀伤malme-3M-CD19-luc；E，TiTE-5蛋白杀伤malme-3M-CD19-luc；F，TiTE-5蛋白杀伤malme-3M-CD22-luc。证明本发明所提供的多功能蛋白TiTE-1、15、16、5使用极低的浓度即可对肿瘤细胞进行体外杀伤，浓度为0.5-5ng/10E 6个细胞时体外杀伤效果最好。
图5为TiTE-6、8、9、10、11、12、13、14多功能蛋白分子杀伤结果：A，阴性对照TiTE-2蛋白杀伤BV173-luc；B，TiTE-6蛋白杀伤BV173-luc；C，TiTE-8、9、10、11、12、13、14蛋白杀伤BV173-luc。证明本发明所提供的多功能蛋白TiTE-6、8、9、10、11、12、13、14对WT1阳性肿瘤细胞具有杀伤效果。
图6为TiTE-2、3、4多功能蛋白分子杀伤结果：A，阴性对照TiTE-6蛋白杀伤malme-3M-luc；B，TiTE-2蛋白杀伤malme-3M-luc；C，TiTE-3蛋白杀伤malme-3M-luc；D，TiTE-4蛋白杀伤malme-3M-luc。证明本发明所提供的多功能蛋白TiTE-2、3、4可对表达形影胞内抗原的肿瘤细胞进行体外杀伤
图7为多功能蛋白分子刺激NK细胞扩增结果。
Figure PCTCN2017118984-appb-000001
为不加任何白介素因子刺激NK细胞扩增结果，5天后细胞几乎全部死亡；
Figure PCTCN2017118984-appb-000002
为多功能蛋白分子刺激NK细胞扩增结果,本发明所提供的多功能蛋白可刺激NK细胞扩增，18天约扩增140倍。
图8为TiTE-1、6、8、9、10、11、12多功能蛋白分子的流式结果：A，T细胞阴性对照；B，TiTE-1T细胞实验组；C，BV173阴性对照；D，TiTE-1BV173实验组；E，BV173阴性对照；F，TiTE-6BV173实验组；G，TiTE-8BV173实验组；H，TiTE-9BV173实验组；I，TiTE-10BV173实验组；J，TiTE-11BV173实验组；K，TiTE-12BV173实验组；L，T细胞阴性对照；M，TiTE-6T细胞实验组；N，TiTE-8T细胞实验组；O，TiTE-9T细胞实验组；P，TiTE-10T细胞实验组；Q，TiTE-11T细胞实验组；R，TiTE-12T细胞实验组。实验证明多功能蛋白分子TiTE-1分别与CD3抗原和CD19抗原结合良好；TiTE-6、8、9、10、11、12的antiMHC/WT1和antiCD3分别与WT1胞内抗原和CD3抗原结合功能良好。
图9为TiTE-2、3、4多功能蛋白分子的流式结果：A，malme-3M阴性对照；B，TiTE-2malme-3M实验组；C，TiTE-3malme-3M实验组；D，TiTE-4malme-3M实验组；E，T细胞阴性对照；F，TiTE-2T细胞实验组；G，TiTE-3T细胞实验组；H，TiTE-4T细胞实验组。从图中可看出多功能蛋白分子TiTE-2、3分别与MHC/GP100抗原和CD3抗原结合良好，TiTE-4与MHC/Mart1抗原和CD3抗原结合良好。
图10为TiTE-15、16多功能蛋白分子的流式结果：A，BV173阴性对照；B，TiTE-15BV173实验组；C，TiTE-15BV173实验组；D，T细胞阴性对照；E，TiTE-15T细胞实验组；F，TiTE-16T细胞实验组。从图中可看出多功能蛋白分子TiTE-15、16分别与CD19抗原和CD3抗原结合良好。
图11为TiTE-5多功能蛋白分子的流式结果：A；malme-3M-CD19-Luc阴性对照；B，TiTE-5malme-3M-CD19-Luc实验组；C，malme-3M-CD22-Luc阴性对照；D，TiTE-5malme-3M-CD22-Luc实验组；E，T细胞阴性对照；F，TiTE-5T细胞实验组；。从图中可看出多功能蛋白分子TiTE-5分别与CD19抗原、CD20抗原和CD3抗原结合良好。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、表达多功能蛋白分子载体的构建
1、构建靶向CD19阳性肿瘤细胞的新型多功能蛋白质TiTE-1
向CD19阳性肿瘤细胞的新型多功能蛋白质TiTE-1，其由主肽链X1和辅肽链Y1聚合得到蛋白(图1)；
主肽链X1包括抗原1结合域R1、细胞因子或细胞因子受体中的细胞因子结合域R2和抗原2结合域R3；
辅肽链Y1包括抗原3结合域R4和主肽链X连接域R5。
主肽链(X1)的抗原结合域(R1)选用antiCD19-ScFv(序列1)、辅肽链连接域(R2)选用IL15Rαsushi(序列2)、抗原结合域(R3)选用antiCD3-ScFv(序列3)；
辅肽链(Y1)的抗原结合域(R4)(a)选用PDL1与PDL2的受体PD1的胞外区(序列4)，主肽链连接域(R5)选用IL15(序列5)。
2、信号肽(氨基酸序列为：MALPVTALLLPLALLLHAARP)，主肽链5’端添加HindIII酶切位点，主肽链的辅肽链连接域(R2：L15Rαsushi)和抗原结合域(R3：antiCD3-ScFv)之间连接的接头肽上含有BamHI酶切位点；在主肽链的3’端与辅肽链5’端添加P2A肽段(氨基酸序列为： GSGATNFSLLKQAGDVEENPGP)；辅肽链3’端添加Xba I酶切位点。
3、PCR扩增antiCD19-IL15Rαsushi片段、antiCD3片段和P2A-PD1-IL15片段，进行核酸凝胶电泳；重叠PCR扩增antiCD3-P2A-PD1-IL15片段，进行核酸凝胶电泳；使用HindIII和BamHI切割antiCD19-IL15Rαsushi片段，使用BamHI和Xba I切割antiCD3-P2A-PD1-IL15；使用HindIII和Xba I切割载体PCDNA3.1(Invitrogen)。
4、切胶回收目的片段，将回收的三个片段进行连接，转化挑去单克隆并测序鉴定，最后得到目的质粒PCDNA3.1-TiTE-1。
重组载体PCDNA3.1-TiTE-1为将表达靶向CD19阳性肿瘤细胞的多功能蛋白质编码基因TiTE-1的表达盒(该表达盒的核苷酸序列由编码主肽链X1的核苷酸序列序列6和编码辅肽链Y1的核苷酸序列序列7组成，且序列6的最后一位核苷酸与序列7的第一位核苷酸紧邻)替换PCDNA3.1载体(invitrogen产品)的HindIII和Xba I酶切位点间片段，得到的重组载体，该载体表达由主肽链X1(氨基酸序列为序列8)和辅肽链Y1(氨基酸序列为序列9)组成的功能性多功能蛋白质TiTE-1。
5、按照上述步骤分别构建多功能蛋白质TiTE-2、TiTE-3、TiTE-4、TiTE-5、TiTE-6、TiTE-7、TiTE-8、TiTE-9、TiTE-10、TiTE-11、TiTE-12的表达载体，用类似的方法构建TiTE-13、TiTE-14、TiTE-15、TiTE-16的表达载体，其结构域如下表1所示，其表达框架见图2。
表1 为多功能蛋白质组成结构
Figure PCTCN2017118984-appb-000003
Figure PCTCN2017118984-appb-000004
其中，AntiMHC/GP100-VHH的氨基酸序列为序列10；
AntiMHC/Mart1-VHH的氨基酸序列为序列11；
AntiMHC/WT1-VH的氨基酸序列为序列12；
IL4Rα-N-FN3的氨基酸序列为序列13；
AntiCD16-ScFv的氨基酸序列为序列14；
AntiCD22-ScFv的氨基酸序列为序列15；
IL4的氨基酸序列为序列16；
TiTE-2的主肽链氨基酸序列为序列17，辅肽链氨基酸序列为序列9；
TiTE-3的主肽链氨基酸序列为序列17，辅肽链氨基酸序列为序列18；
TiTE-4的主肽链氨基酸序列为序列19，辅肽链氨基酸序列为序列9；
TiTE-5的主肽链氨基酸序列为序列8，辅肽链氨基酸序列为序列20；
TiTE-6的主肽链氨基酸序列为序列21，辅肽链氨基酸序列为序列9；
TiTE-7的主肽链氨基酸序列为序列22，辅肽链氨基酸序列为序列9；
TiTE-8的主肽链氨基酸序列为序列23，辅肽链氨基酸序列为序列24；
TiTE-9的主肽链氨基酸序列为序列25，辅肽链氨基酸序列为序列26；
TiTE-10的主肽链氨基酸序列为序列27，辅肽链氨基酸序列为序列28；
TiTE-11的主肽链氨基酸序列为序列29，辅肽链氨基酸序列为序列9。
TiTE-12的主肽链氨基酸序列为序列30，辅肽链氨基酸序列为序列31。
TiTE-13为主肽链，该主肽链的氨基酸序列为序列21。
TiTE-14的主肽链氨基酸序列为序列21，辅肽链氨基酸序列为序列5。
TiTE-15为主肽链，该主肽链的氨基酸序列为序列8。
TiTE-16的主肽链氨基酸序列为序列8，辅肽链氨基酸序列为序列5。
表达TiTE-2的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列35，辅肽链编码核酸序列为序列7；
表达TiTE-3的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列35，辅肽链编码核酸序列为序列36；
表达TiTE-4的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列37，辅肽链编码核酸序列为序列7；
表达TiTE-5的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列6，辅肽链编码核酸序列为序列38；
表达TiTE-6的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列39，辅肽链编码核酸序列为序列7；
表达TiTE-7的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列40，辅肽链编码核酸序列为序列7；
表达TiTE-8的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列41，辅肽链编码核酸序列 为序列42；
表达TiTE-9的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列43，辅肽链编码核酸序列为序列44；
表达TiTE-10的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列45，辅肽链编码核酸序列为序列46；
表达TiTE-11的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列47，辅肽链编码核酸序列为序列7；
表达TiTE-12的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列48，辅肽链编码核酸序列为序列49；
表达TiTE-13的编码核酸序列为序列39；
表达TiTE-14的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列39，辅肽链编码核酸序列为序列50；
表达TiTE-15的编码核酸序列为序列6；
表达TiTE-16的编码核酸由主肽链编码核酸和辅肽链编码核酸组成，且主肽链编码核酸的3'端末端最后一位碱基与辅肽链编码核酸5'末端第1位碱基相邻；其中，主肽链编码核酸序列为序列6，辅肽链编码核酸序列为序列50；
重组载体PCDNA3.1-TiTE-2至重组载体PCDNA3.1-TiTE-16分别为将表达TiTE-2的编码核酸至表达TiTE-16的编码核酸中对应核酸替换PCDNA3.1载体的HindIII和Xba I酶切位点间片段，得到的重组载体。
实施例2、多靶向功能性多功能蛋白质分子多肽表达纯化
1、37℃、8％CO2、120rpm培养293F(invitrogen)至细胞密度为1*10E 6Cell/ml。
2、将实施例1中构建的载体PCDNA3.1-TiTE-1使用PEI转染到上述1的细胞中，转染质粒浓度为1mg/L，PEI浓度为3mg/L。37℃，8％CO2、120rpm培养5-6天。
3、4000rpm离心上述2的培养产物，收集培养基上清液，Protein/capto L beads纯化蛋白，500uLpH2.6-3.0 0.1MGly-HCL洗脱液洗脱，收集洗脱液。
4、SDS-PAGE检测收集蛋白(见图3)，可以看出，得到约65KD和30KD的目的蛋白，即为多功能蛋白分子TiTE-1的主肽链X和辅肽链Y。
5、使用相同的方法表达纯化TiTE-2、TiTE-3、TiTE-4、TiTE-5、TiTE-7、TiTE-8、TiTE-9、TiTE-10、TiTE-11、TiTE-12等多功能蛋白质。
实施例3、多功能蛋白分子TiTE-1、15、16介导T细胞体外杀伤CD19 +靶细胞实验验证
1、将1*10E 4靶细胞malme-3M-CD19-luc(将ATCC购买的Malme-3M中转染CD19抗原(其核酸序列为序列32)与Luc基因(其核酸序列为序列33)，使其表达CD19抗原与Luc基因，得到细胞malme-3M-CD19-luc)(50uL)铺中于96孔板内，37℃、5％CO2培养18-20h。
2、待细胞贴壁后吸取并弃去培养基，加入50uL新鲜培养基37℃、5％CO2培养1-3h。
3、逐级稀释实施例2得到的目的蛋白TiTE-1至浓度为50、5、0.5、0.05、0.005ng/uL；
实验组：分别向50uL的1*10E 5的T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激14天)中加入50、5、0.5、0.05、0.005ng实施例2得到的目的蛋白TiTE-137℃孵育1-2h，得到与抗体孵育好的T细胞。
阴性对照组：对该靶细胞无杀伤作用的双特异性对照抗体(TiTE-6)，向50uL的1*10E 5的T细胞中分别加入50、5、0.5、0.05、0.005ng对照抗体37℃孵育1-2h。
4、将与抗体孵育好的T细胞50uL加入铺有靶细胞的96孔板中，37℃、5％CO2培养22-24h。
5、每孔加入100uL1％Triton裂解液，反复吹匀，静置3-5min，使细胞完全裂解，取50uL裂解液置于黑色96孔板中，加入50uL底物(300ug/mL Luc水溶液与2mg/mL ATP水溶液按照体积比为 3:1混和)吹吸混匀，迅速测其荧光值。
6.计算杀伤效率：杀伤效率＝{(阴性对照荧光值-实验组荧光值)/阴性对照荧光值}*100％。
结果如图4B所示。可以看出，与图4A中对照组相比本发明所提供的多功能蛋白TiTE-1使用极低的浓度即可对CD19阳性肿瘤细胞进行体外杀伤，浓度为0.5-5ng/10E 6个细胞时体外杀伤效果最好。
7、用相同的方式，进行TiTE-15、16杀伤实验，验证使用极低的浓度的多功能蛋白质即可对肿瘤细胞进行杀伤。
结果如图4C、4D所示，可以看出，本发明所提供的多功能蛋白TiTE-15、16使用极低的浓度即可对肿瘤细胞进行体外杀伤，浓度为0.5-5ng/10E 6个细胞时体外杀伤效果最好。
8、用相同的方式，进行TiTE-5杀伤实验，靶细胞分别使用malme-3M-CD19-luc和malme-3M-CD22-luc(将ATCC购买的Malme-3M中转染CD22抗原(其核酸序列为序列34)与Luc基因(其核酸序列为序列33)。结果如图4E、4F所示。可以看出，本发明所提供的多功能蛋白TiTE-5对CD19和CD22阳性细胞均有杀伤功能，且使用极低的浓度即可对肿瘤细胞进行体外杀伤，浓度为0.5-5ng/10E 6个细胞时体外杀伤效果最好。
实施例4、多功能蛋白分子TiTE-6、8、9、10、11、12、13、14介导T细胞体外杀伤WT1 +靶细胞实验验证
1、将1*10E 4靶细胞BV173(50uL)(ATCC购买BV173转染Luc基因(其核酸序列为序列33))铺中于96孔板内，37℃、5％CO2培养1-2h。
2、逐级稀释实施例2得到的目的蛋白TiTE-6至浓度为50、5、0.5、0.05ng/uL；
实验组：分别向50uL的1*10E5的T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激14天) 中加入50、5、0.5、0.05ng实施例2得到的目的蛋白TiTE-6 37℃孵育1-2h。
阴性对照组：对该靶细胞无杀伤作用的双特异性对照抗体(TiTE-2)，向50uL的1*10E 5的T细胞中分别加入50、5、0.5、0.05ng对照抗体37℃孵育1-2h。
3、将与抗体孵育好的T细胞50uL加入铺有靶细胞的96孔板中，37℃、5％CO2培养22-24h。
4、每孔加入100uL1％Triton裂解液，反复吹匀，静置3-5min，使细胞完全裂解，取50uL裂解液置于黑色96孔板中，加入50uL底物(300ug/mL Luc水溶液与2mg/mL ATP水溶液按照体积比为3:1混和)吹吸混匀，迅速测其荧光值。
5、计算杀伤效率：杀伤效率＝{(阴性对照荧光值-实验组荧光值)/阴性对照荧光值}*100％。
结果如图5B所示，可以看出，本发明所提供的多功能蛋白TiTE-6使用极低的浓度即可对肿瘤细胞进行杀伤。
6、用相同的方法，进行TiTE-8、9、10、11、12、13、14杀伤实验，效靶比为10:1，抗体浓度为每个体系中加入5ng相应抗体。实验结果如图5C所示，可以看出，本发明所提供的多功能蛋白WT1阳性肿瘤细胞进行杀伤。
实施例5、多功能蛋白分子TiTE-2、3、4介导T细胞体外杀伤靶细胞实验
1、将1*10E 4靶细胞malme-3M-luc(将ATCC购买的Malme-3M中转染Luc基因)(50uL)铺中于96孔板内，37℃、5％CO2培养18-20h。
2、待细胞贴壁后吸取并弃去培养基，加入50uL新鲜培养基37℃、5％CO2培养1-3h。
3、逐级稀释实施例2得到的目的蛋白TiTE-2、3、4至浓度为50、5、0.5、0.05、0.005ng/uL；
实验组：分别向50uL的1*10E 5的T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激14天)中加入50、5、0.5、0.05、0.005ng实施例2得到的目的蛋白TiTE-2、3、 4，37℃孵育1-2h，得到与抗体孵育好的T细胞。
阴性对照组：对该靶细胞无杀伤作用的双特异性对照抗体(TiTE-6)，向50uL的1*10E 5的T细胞中分别加入50、5、0.5、0.05、0.005ng对照抗体37℃孵育1-2h。
4、将与抗体孵育好的T细胞50uL加入铺有靶细胞的96孔板中，37℃、5％CO2培养22-24h。
5、每孔加入100uL1％Triton裂解液，反复吹匀，静置3-5min，使细胞完全裂解，取50uL裂解液置于黑色96孔板中，加入50uL底物(300ug/mL Luc水溶液与2mg/mL ATP水溶液按照体积比为 3:1混和)吹吸混匀，迅速测其荧光值。
6.计算杀伤效率：杀伤效率＝{(阴性对照荧光值-实验组荧光值)/阴性对照荧光值}*100％。
结果如图6所示。可以看出，本发明所提供的多功能蛋白TiTE-2、3、4使用极低的浓度即可对肿瘤细胞进行体外杀伤，浓度为0.5-5ng/10E 6个细胞时体外杀伤效果最好。
实施例6、多功能蛋白分子TiTE-1刺激NK细胞扩增
1、将6*10E5的NK92细胞(中国典型培养物保藏中心)接种于2ML培养基(基础培养基Alpha medium，12.5％马血清，12.5％FBS，0.2mM肌醇，0.1mM巯基乙醇，0.02mM叶酸)中，添加40ng/mL的实施例2得到的TiTE-1多功能蛋白分子，37℃、5％CO2培养。
2、培养2-3d后，计细胞总数，传代继续培养至18d，每次传代调整细胞密度为3*10E5个细胞/mL，添加40ng/mL的TiTE-1多功能蛋白分子。
细胞生长曲线如图7所示，可以看出，本发明所提供的多功能蛋白TiTE-1能够刺激NK细胞扩增，具有IL15/IL15Rαsushi的功能。
TiTE2、3、4、5、6、7、9、10、11、12、13、15的IL15/IL15Rαsushi结构域与TiTE-1相同，其功能无显著差异
实施例7、多功能蛋白分子TiTE-1的antiCD19和antiCD3分别与CD19抗原和CD3抗原及TiTE-6、8、9、10、11、12的antiMHC/WT1和antiCD3分别与WT1胞内抗原和CD3抗原结合功能的流式验证
1、T细胞实验组和BV173细胞实验组：分别向3*10E5个T细胞(正 常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激14天)和BV173细胞中加入5ug的TiTE-1、6、8、9、10、11、12多功能蛋白分子，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。加入2uLAPC Mouse anti-Human CD279(BD，货号558694)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。
T细胞阴性对照组和BV173细胞阴性对照组：分别向3*10E5个T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激12-14天)和BV173细胞(ATCC)中加入2uLAPC Mouse anti-Human CD279(BD，货号558694)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞，作为阴性对照。
2、进行流式分析如图8所示，从图中可看出多功能蛋白分子TiTE-1分别与CD19抗原和CD3抗原结合良好；TiTE-6、8、9、10、11、12的antiMHC/WT1和antiCD3分别与WT1胞内抗原和CD3抗原结合功能良好。
实施例8、多功能蛋白分子TiTE-2、3中antiMHC/GP100和antiCD3与TiTE-4中antiMHC/Mart1和antiCD3抗原结合功能的流式验证
1、T细胞实验组和malme-3M-Luc将(ATCC购买的Malme-3M中转染Luc基因)细胞实验组：分别向3*10E5个T细胞和malme-3M-Luc细胞中加入5ug的TiTE-2、3、4多功能蛋白分子，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。加入2uL PE conjugated anti-hIL-15(R&D，货号IC2471P)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。
T细胞阴性对照组和malme-3M-Luc细胞阴性对照组：分别向3*10E5个T细胞和malme-3M-Luc中加入2uLAPC Mouse anti-Human CD279(BD，货号558694)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞，作为阴性对照。
2、进行流式分析如图9所示，从图中可看出多功能蛋白分子TiTE-2、3分别与MHC/GP100抗原和CD3抗原结合良好，TiTE-4与MHC/Mart1抗原和CD3抗原结合良好。
实施例9、多功能蛋白分子TiTE-15/16的antiCD19和antiCD3分别与CD19抗原和CD3抗原结合功能的流式验证
1、T细胞实验组和BV173细胞实验组：分别向3*10E5个T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激14天)和BV173细胞中加入5ug的TiTE-15、16多功能蛋白分子，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。加入2uL FITC-Labeled Recombinant Protein L(ACRO Biosystem，货号RPL-PF141)，冰上孵浴30min。离心去上清，500uLPBS洗涤两遍使用200uL的PBS重悬细胞。
T细胞阴性对照组和BV173细胞阴性对照组：分别向3*10E5个T细胞(正常人外周血密度梯度离心，取富含单个核细胞的白膜层，50ng/mLOKT3，300IU/mLIL2刺激12-14天)和BV173细胞(ATCC)中加入2uLAPC Mouse anti-Human CD279(BD，货号558694)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞，作为阴性对照。
2、进行流式分析如图10所示，从图中可看出多功能蛋白分子TiTE-15、16分别与CD19抗原和CD3抗原结合良好。
实施例10、多功能蛋白分子TiTE-5中antiCD19、antiCD20和antiCD3与抗原结合功能的流式验证
1、T细胞实验组、malme-3M-CD19-Luc将((将ATCC购买的Malme-3M中转染CD19抗原基因与Luc基因)及malme-3M-CD22-Luc将((将ATCC购买的Malme-3M中转染CD22抗原与Luc基因)细胞实验组：分别向3*10E5个T细胞和malme-3M-Luc细胞中加入5ug的TiTE-5多功能蛋白分子，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。加入2uL PE conjugated anti-hIL-15(R&D，货号IC2471P)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞。
T细胞阴性对照组和malme-3M-CD19-Luc、malme-3M-CD22-Luc细胞阴性对照组：分别向3*10E5个T细胞和malme-3M-CD19-Luc、malme-3M-CD22-Luc中加入2uL PE conjugated anti-hIL-15(R&D，货号IC2471P)，冰上孵浴30min。离心去上清，使用200uL的PBS重悬细胞，作为阴性对照。
2、进行流式分析如图11所示，从图中可看出多功能蛋白分子TiTE-5分别与CD19抗原、CD20抗原和CD3抗原结合良好。
工业应用
本发明的实验证明，本发明的多功能蛋白质可通过其两个肿瘤相关抗原的抗原结合域与不同的肿瘤抗原结合，介导特异性的细胞杀伤，提高了靶向的精确性；若其中一个抗原结合域所结合的抗原为免疫筛查点相关抗原，可以将阻断免疫抑制信号，提高了杀伤肿瘤的能力；本发明的多功能蛋白质中引入了细胞因子与细胞因子受体复合物，它们可以发挥细胞因子的功能。

Claims (35)

  1. 一种多肽链X，包括抗原1结合域R1、辅肽链连接域R2和抗原2结合域R3；
    所述辅肽链连接域R2为细胞因子或细胞因子受体中的细胞因子结合域。
  2. 根据权利要求1所述的多肽链X，其特征在于：
    所述抗原2结合域R3为识别T细胞CD3的受体或抗体或其他分子。
  3. 根据权利要求1或2所述的多肽链X，其特征在于：
    所述抗原2结合域R3为识别NK细胞CD16的受体或抗体或其他分子。
  4. 根据权利要求1-3中任一所述的多肽链X，其特征在于：
    所述抗原1结合域R1所结合的抗原选自下述的癌症相关的抗原中任一种：脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和子宫癌。
  5. 根据权利要求4所述的多肽链X，其特征在于：
    所述癌症相关抗原优选自下述抗原中任一种:CD123，CD19、CD20、CD22、CD37、ROR1、间皮素、CD33/IL3Ra、c-Met、BCMA、PSMA、EGFRvIII、GD-2、NY-ESO-1、MAGEA3、β-人绒毛膜促性腺素、AFP、RAGE-1、MN-CA IX、人端粒酶反转录酶、RU1、RU2(AS)、hsp70-2、M-CSF、PSA、PAP、LAGE-la、p53、Prostein、PSMA、Her2/neu、端粒酶、PCTA-1、MAGE、ELF2M、IGF-I、IGF-II、IGF-I受体、BCR-ABL、E2A-PRL、H4-RET、1GH-IGK、MYL-RAR、GP100、Mart1、TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、p185erbB2、p180erbB-3、c-met、nm-23H1、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p15、p16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、WT1、CD68、FGF-5、G250、EpCAM、MA-50、MG7-Ag、MOV 18、NB/70K、RCAS1、SDCCAG16、TA-90、TAAL6、TAG72、TLP、p53、Ras、TPS、Epstein Barr病毒抗原EBVA、人乳头瘤病毒(HPV)抗原E6、人乳头瘤病毒(HPV)抗原E7或MHC与上述抗原的短肽的复合物。
  6. 根据权利要求4或5所述的多肽链X，其特征在于：
    所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、CD123，CD33/IL3Ra、Her2、PDL1、GP100、Mart1、BCMA、WT-1、NY-ESO-1或MHC与上述抗原的短肽的复合物。
  7. 根据权利要求6所述的多肽链X，其特征在于：
    所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、Her2、PDL1、WT1、GP100、Mart1、BCMA、NY-ESO-1或MHC与上述抗原的短肽的复合物。
  8. 根据权利要求1-7中任一所述的多肽链X，其特征在于：
    所述抗原1结合域R1为结合抗原的抗体、结合抗原的配体、结合抗原的受体或具有抗原结合功能的多肽。
  9. 根据权利要求8所述的多肽链X，其特征在于：
    所述结合抗原的抗体是完整免疫球蛋白，抗体的Fc、抗体的Fab、抗体的VH或VHH、抗体的VL或scFv的全长肽链或部分肽段。
  10. 根据权利要求8或9所述的多肽链X，其特征在于：所述结合抗原的配体或所述结合抗原的受体是受体或配体的全长肽链或部分肽段。
  11. 根据权利要求10所述的多肽链X，其特征在于：所述抗原1结合域R1为具有抗原识别功能的TCR。
  12. 根据权利要求10所述的多肽链X，其特征在于：所述抗原结合域R1为具有抗原识别功能的TCR样抗体或其他分子。13、一种蛋白质，其为由权利要求1所述的肽链X作为主肽链与辅肽链Y组成异源二聚体；
    所述辅肽链Y包括抗原3结合域R4和主肽链X连接域R5，
    或所述辅肽链Y为主肽链连接域R5；
    所述主肽链连接域R5与所述肽链X中辅肽链连接域R2相互结合。
  13. 根据权利要求13所述的蛋白，其特征在于：
    所述抗原3结合域R4所结合的抗原3选自下述的癌症相关的抗原中任一种：脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和子宫癌。
  14. 根据权利要求13或14所述的蛋白，其特征在于：
    所述癌症相关抗原3优选自下述抗原中任一种:CD123，CD19、CD20、CD22、CD37、ROR1、间皮素、CD33/IL3Ra、c-Met、BCMA、PSMA、EGFRvIII、GD-2、NY-ESO-1、MAGEA3、β-人绒毛膜促性腺素、AFP、RAGE-1、MN-CA IX、人端粒酶反转录酶、RU1、RU2(AS)、hsp70-2、M-CSF、PSA、PAP、LAGE-la、p53、Prostein、PSMA、Her2/neu、PDL1、端粒酶、PCTA-1、MAGE、ELF2M、IGF-I、IGF-II、IGF-I受体、BCR-ABL、E2A-PRL、H4-RET、1GH-IGK、MYL-RAR、GP100、Mart1、TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、p185erbB2、p180erbB-3、c-met、nm-23H1、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p15、p16、43-9F、5T4、791Tgp72、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、WT1、CD68、FGF-5、G250、EpCAM、M344、MA-50、MG7-Ag、MOV18、NB/70K、RCAS1、SDCCAG16、TA-90、TAAL6、TAG72、TLP、p53、Ras、TPS、Epstein Barr病毒抗原EBVA、人乳头瘤病毒(HPV)抗原E6、人乳头瘤病毒(HPV)抗原E7或MHC与上述抗原的短肽的复合物。
  15. 根据权利要求13-15中任一所述的蛋白，其特征在于：
    所述的癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、CD123，CD33/IL3Ra、Her2、PDL1、GP100、Mart1、BCMA、WT-1、NY_ESO-1或MHC与上述抗原的短肽的复合物。
  16. 根据权利要求13-16中任一所述的蛋白，其特征在于：
    所述癌症相关抗原优选的选自下述抗原中任一种：CD19、CD20、CD22、Her2、PDL1、WT1、GP100、Mart1、BCMA、NY_ESO-1或MHC与上述抗原的短肽的复合物。
  17. 根据权利要求1-12中任一所述多肽链X或权利要求13-17中任一所述的蛋白，其特征在于：所述抗原1结合域R1和所述抗原2结合域R3的位置能互换。
  18. 根据权利要求13-17中任一所述的蛋白，其特征在于：所述抗原1结合域R1和所述抗原3结合域R4的位置能互换。
  19. 根据权利要求13-17中任一所述的蛋白，其特征在于：所述抗原2结合域R3和所述抗原3结合域R4的位置能互换。
  20. 根据权利要求13-20中任一所述的蛋白，其特征在于：
    所述辅肽链连接域R2和所述主肽链连接域R5是一对具有相互结合功能的肽段。
  21. 根据权利要求13-21中任一所述的蛋白，其特征在于：
    所述辅肽链连接域R2和所述主肽链连接域R5是互为一对可以相互结合细胞因子及其受体亚基。
  22. 根据权利要求21或22所述的蛋白，其特征在于：
    所述细胞因子为γc家族细胞因子，
    所述γc家族细胞因子为IL2、IL4、IL7、IL9、IL15或IL21。
  23. 根据权利要求21-23任一所述的蛋白，其特征在于：
    所述细胞因子及其受体亚基选自IL15与IL15Rα或IL4与IL4Rα。
  24. 根据权利要求1-12中任一所述多肽链X或权利要求13-24中任一所述的蛋白，其特征在于：
    所述多肽链X中各组分或所述蛋白的主肽链中各组分或辅肽链中各组分通过由1-20个氨基酸残基组成的多肽接头连接。
  25. 根据权利要求25所述的蛋白，其特征在于：
    所述多肽接头中富含甘氨酸和/或丝氨酸。
  26. 根据权利要求1-12中任一所述多肽链X或权利要求13-26中任一所述的蛋白，其特征在于：
    所述抗原1结合域R1为antiCD19-ScFv或AntiMHC/GP100-VHH或AntiMHC/Mart1-VHH或anti MHC/WT1或PD1的胞外区或AntiCD22-ScFv或antiCD3-ScFv或antiCD16-ScFv；
    或，所述辅肽链连接域R2为IL15Rαsushi或IL4Rα-N-FN3或IL15或IL4；
    或，所述抗原2结合域R3为antiCD3-ScFv或antiCD16-ScFv或antiCD19-ScFv或AntiMHC/GP100-VHH或AntiMHC/Mart1-VHH或antiWT1或PD1的胞外区或AntiCD22-ScFv；
    或，所述抗原3结合域R4为PD1的胞外区或AntiMHC/GP100-VHH或AntiCD22-ScFv或antiCD19-ScFv或AntiMHC/Mart1-VHH或anti MHC/WT1或antiCD3-ScFv或antiCD16-ScFv；
    或，所述主肽链连接域R5为IL15或IL4或IL15Rαsushi或IL4Rα-N-FN3。
  27. 根据权利要求27所述的多肽链X或蛋白，其特征在于：
    所述antiCD19-ScFv的氨基酸序列为序列1；
    所述IL15Rαsushi的氨基酸序列为序列2；
    所述antiCD3-ScFv的氨基酸序列为序列3；
    所述PD1的胞外区的氨基酸序列为序列4；
    所述IL15的氨基酸序列为序列5；
    所述AntiMHC/GP100-VHH的氨基酸序列为序列10；
    所述AntiMHC/Mart1-VHH的氨基酸序列为序列11；
    所述AntiMHC/WT1-VH的氨基酸序列为序列12；
    所述IL4Rα-N-FN3的氨基酸序列为序列13；
    所述AntiCD16-ScFv的氨基酸序列为序列14；
    所述AntiCD22-ScFv的氨基酸序列为序列15；
    所述IL4的氨基酸序列为序列16。
  28. 根据权利要求27或28所述的多肽链X或蛋白，其特征在于：
    所述多肽链X的氨基酸序列为序列8；
    或所述多肽链X的氨基酸序列为序列17；
    或所述多肽链X的氨基酸序列为序列19；
    或所述多肽链X的氨基酸序列为序列21；
    或所述多肽链X的氨基酸序列为序列22；
    或所述多肽链X的氨基酸序列为序列23；
    或所述多肽链X的氨基酸序列为序列27；
    或所述多肽链X的氨基酸序列为序列29；
    或所述多肽链X的氨基酸序列为序列30；
    或，所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列17，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列17，辅肽链的氨基酸序列为 序列18；
    或所述蛋白的主肽链的氨基酸序列为序列19，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列20；
    或所述蛋白的主肽链的氨基酸序列为序列21，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列22，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列23，辅肽链的氨基酸序列为序列24；
    或所述蛋白的主肽链的氨基酸序列为序列25，辅肽链的氨基酸序列为序列26；
    或所述蛋白的主肽链的氨基酸序列为序列27，辅肽链的氨基酸序列为序列28；
    或所述蛋白的主肽链的氨基酸序列为序列29，辅肽链的氨基酸序列为序列9；
    或所述蛋白的主肽链的氨基酸序列为序列30，辅肽链的氨基酸序列为序列31；
    或所述蛋白的主肽链的氨基酸序列为序列21，辅肽链的氨基酸序列为序列5。
    或所述蛋白的主肽链的氨基酸序列为序列8，辅肽链的氨基酸序列为序列5。
  29. 权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白的编码核酸分子，由编码所述主肽链的核酸分子或编码所述主肽链的核酸分子和编码所述辅肽链的核酸分子组成。
  30. 含有权利要求30所述的核酸分子的重组载体、表达盒、重组菌、重组病毒或细胞。
  31. 根据权利要求31所述的重组载体，其特征在于：所述重组载体为将权利要求30所述的核酸分子中的编码所述主肽链的核酸分子或编码 所述主肽链的核酸分子和编码所述辅肽链的核酸分子菌插入表达载体中，得到表达权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白的载体。
  32. 根据权利要求31所述的细胞，其特征在于：所述目的细胞为原核细胞、酵母细胞或哺乳动物细胞；
  33. 一种试剂盒，其包括权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、重组病毒或细胞。
  34. 权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在免疫治疗中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备免疫治疗产品中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在免疫细胞培养和/或促进免疫细胞扩增和/或免疫检测中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备免疫细胞培养和/或促进免疫细胞扩增和/或免疫检测产品中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在抑制或杀伤表达所述蛋白中抗原的靶细胞中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表 达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备抑制或杀伤表达所述蛋白中抗原的靶细胞产品中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在介导免疫细胞抑制或杀伤表达所述蛋白中抗原的靶细胞中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备介导免疫细胞抑制或杀伤表达所述蛋白中抗原的靶细胞产品中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在治疗或杀伤肿瘤细胞中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备抑制或杀伤肿瘤细胞产品中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在治疗或检测肿瘤中的应用；
    或权利要求1-12中任一所述多肽链X或权利要求13-29中任一所述的蛋白、权利要求30所述的核酸分子或权利要求31所述的重组载体、表达盒、重组菌、细胞或重组病毒或权利要求34所述的试剂盒在制备治疗或检测肿瘤产品中的应用。
  35. 根据权利要求35所述的应用，其特征在于：
    所述免疫治疗为通过免疫细胞抑制或杀伤肿瘤细胞；
    或，所述免疫细胞为T细胞或NK细胞等；
    或，所述抗原为癌症相关抗原；
    或所述抗原为脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、子宫癌相关抗原或其任何组合；
    或，所述肿瘤为脑癌、膀胱癌、乳腺癌、宫颈癌、结直肠癌、肝癌、肾脏癌、淋巴瘤、白血病、肺癌、黑色素瘤、转移性黑色素瘤、间皮瘤、神经母细胞瘤、卵巢癌、前列腺癌、胰腺癌、肾癌、皮肤癌、胸腺瘤、肉瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤、子宫癌中任一种或任何组合；
    或，所述靶细胞为原核细胞、酵母细胞或哺乳动物细胞；
    或，所述哺乳动物细胞具体为人类细胞；
    或，所述人类细胞具体为免疫细胞，
    或，所述免疫细胞具体为T细胞或NK细胞。
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US11530264B2 (en) 2022-12-20
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JP2020504628A (ja) 2020-02-13
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