WO2019101110A1 - 天花粉蛋白用于致敏和/激活树突状细胞中的应用 - Google Patents

天花粉蛋白用于致敏和/激活树突状细胞中的应用 Download PDF

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WO2019101110A1
WO2019101110A1 PCT/CN2018/116755 CN2018116755W WO2019101110A1 WO 2019101110 A1 WO2019101110 A1 WO 2019101110A1 CN 2018116755 W CN2018116755 W CN 2018116755W WO 2019101110 A1 WO2019101110 A1 WO 2019101110A1
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tumor
tcs
cells
protein
peptide
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French (fr)
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黄永焯
吴爱花
陈应之
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中国科学院上海药物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/50—Colon

Definitions

  • the present invention belongs to the field of biomedicine, and more particularly, relates to the use of trichosanthin protein in activating dendritic cells, and a fusion protein containing trichosanthin protein and tumor antigen prepared by using the same.
  • Tumor treatment usually includes surgical resection, chemotherapy, radiation therapy and immunotherapy.
  • Tumors are products of malignant transformation of normal cells in the body, which are characterized by constant proliferation and metastasis in the body. Therefore, the prominent immunological feature of tumor cells is the emergence of some new antigenic markers that are not visible in normal cells of the same type.
  • Tumor antigens that have been discovered successively include tumor-specific antigens and tumor-associated antigens. The former is unique to tumor cells; the latter mostly refers to embryonic antigens, which are shared by embryonic tissues and tumor tissues.
  • the body has a series of immune monitoring mechanisms, it is still difficult to prevent the occurrence and development of tumors. A small amount of tumor cells are not easy to cause the body to respond. When the tumor grows to a certain extent, beyond the ability of the body's immune response, the tumor cells can escape.
  • a trichosanthin or a gene encoding the same for the preparation of a dendritic cell activator and/or a sensitizer.
  • the use further comprises further preparing a tumor therapeutic vaccine.
  • the trichosanthin protein or its encoding gene can also be used in combination with other anti-tumor drugs.
  • the other anti-tumor drug comprises an anti-PD-1 antibody, an anti-PD-L1 antibody.
  • the anti-PD-1 antibody comprises: an antibody and a small molecule inhibitor of a PD-1/PD-L1 checkpoint, a CTLA4 antibody, or other antibodies acting on immune cells.
  • a method of activating and/or sensitizing dendritic cells in vitro comprising the steps of:
  • the trichosanthin protein and the tumor antigen are physically mixed or coupled to each other.
  • the trichosanthin protein and the tumor antigen are fusion proteins.
  • the fusion protein is further coupled to a transmembrane peptide.
  • the transmembrane peptide sequence comprises a polypeptide represented by R9 peptide (RRRRRRRRR), LMWP (VRRRRRRGGRRRR, SEQ ID NO.: 4), or TAT peptide (YGRKKRRQRRR, SEQ ID NO.: 5).
  • the fusion protein has the structure shown in Formula Ia or Formula Ib:
  • element T is a transmembrane peptide
  • element A is a tumor antigen
  • element P is a trichosanthin protein
  • E1 and/or E2 is an optional linker peptide, a tag sequence, a signal peptide and/or 1-3 amino acid residues
  • -" is a peptide bond.
  • the tumor comprises head and neck cancer, thyroid cancer, brain malignancy, lung cancer, skin cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, colorectal cancer, breast cancer, ovarian cancer. , cervical cancer, endometrial cancer, testicular cancer, bladder cancer, kidney cancer, or osteosarcoma.
  • the tumor antigen comprises a protein, a peptide and/or a DNA fragment.
  • the tumor antigen comprises an antigen that is overexpressed on the surface of a tumor cell, a differentiation antigen, or a virus-induced tumor antigen.
  • the tumor antigen comprises SEQ ID NO.: 6 (EDVTPENFLAVLR), SEQ ID NO.: 7 (TPENFLAVL), or SEQ ID NO.: 8 (HSVTYEHALRYLY).
  • the culture conditions include high glucose 1640 complete medium containing 10% fetal bovine serum, 1640 complete medium containing 10% fetal bovine serum, and complete culture with DMEM containing 10% fetal bovine serum.
  • Base 37 ° C, 5% CO 2 .
  • the mixture of (a) further includes other anti-tumor drugs.
  • the other anti-tumor drug comprises an anti-PD-1 antibody, an anti-PD-L1 antibody.
  • the anti-PD-1 antibody comprises: an antibody and a small molecule inhibitor of a PD-1/PD-L1 checkpoint, a CTLA4 antibody, or other antibodies acting on immune cells.
  • a dendritic cell activation and/or sensitization system comprising:
  • the trichosanthin protein and the tumor antigen in the system are physically mixed or coupled to each other.
  • the fusion protein is further coupled to a transmembrane peptide.
  • the dendritic cells are activated and/or sensitized after contact with the isolated dendritic cells in the system under suitable culture conditions.
  • an activated and/or sensitized dendritic cell prepared by the method of the second aspect of the invention.
  • the activated and/or sensitized dendritic cells have the following activities:
  • the expression level of CD80 is increased by at least 20%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 85%, 90%, 95% or 100%; and/or
  • the amount of IFN- ⁇ secreted by T cells is increased by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%. , 80%, 85%, 90%, 95% or 100%.
  • a fusion protein having a structure represented by Formula Ia or Formula Ib is provided:
  • element T is an optional transmembrane peptide
  • element A is a tumor antigen
  • element P is a trichosanthin protein
  • E1 and/or E2 is an optional linker peptide, a tag sequence, a signal peptide and/or 1-3 amino acid residues Base
  • "-" is a peptide bond.
  • sequence of the fusion protein is as shown in SEQ ID NO.: 1 (fusion protein containing transmembrane peptide and antigen 1) or 12 (fusion protein containing transmembrane peptide and antigen 2)
  • the signal peptide comprises an intein;
  • the tag sequence comprises a CBD tag sequence, a GST tag sequence, an MBP tag sequence, or a 6His tag sequence.
  • a polynucleotide comprising the fusion protein of the fifth aspect of the invention is provided.
  • sequence of the fusion protein is set forth in SEQ ID NO.: 2 (fusion protein without transmembrane peptide)
  • polynucleotide encoding the protein of SEQ ID NO.: 2 is represented by SEQ ID NO.: 3 (coding sequence)
  • a vector comprising the polynucleotide of the sixth aspect of the invention is provided.
  • a host cell comprising the vector of the seventh aspect of the present invention, or the gene of the host cell is integrated with the polynucleoside of the sixth aspect of the present invention acid.
  • a tumor vaccine composition comprising:
  • the pharmaceutically acceptable carrier comprises cholera toxin, CpG ODN, aluminum hydroxide, or a surfactant.
  • a method for the preparation of the fusion protein of the fifth aspect of the invention comprising the steps of:
  • the method may further comprise the steps of:
  • the isolation and purification comprises direct intein-mediated separation and purification.
  • the trichosanthin protein is derived from the dried root of the Trichosanthes kirilowii Maxim. or the Trichosan-thes rosthornii Herms.
  • the trichosanthin protein comprises a full length of the trichosanthin protein or a fragment thereof.
  • the trichosanthin sequence comprises the wild type full length sequence as set forth in SEQ ID NO.
  • the gene encoding the trichosanthin protein is the wild type protein sequence shown in SEQ ID NO.: 11.
  • the tumor vaccine composition further includes other anti-tumor drugs.
  • the other anti-tumor drug comprises an anti-PD-1 antibody, an anti-PD-L1 antibody.
  • the anti-PD-1 antibody comprises: an antibody and a small molecule inhibitor of a PD-1/PD-L1 checkpoint, a CTLA4 antibody, or other antibodies acting on immune cells.
  • a method for non-therapeutic inhibition of tumor cells in vitro comprises the steps of: adding a dendritic cell according to the fourth aspect of the present invention to a tumor cell culture, and the fifth aspect of the present invention The fusion protein or the tumor vaccine composition of the seventh aspect of the invention, thereby inhibiting tumor cells.
  • a method of treating a tumor comprising the steps of: administering to a subject in need thereof a safe and effective amount of the dendritic cell of the fourth aspect of the invention, the fusion of the fifth aspect of the invention.
  • the protein or the tumor vaccine composition of the ninth aspect of the invention comprising the steps of: administering to a subject in need thereof a safe and effective amount of the dendritic cell of the fourth aspect of the invention, the fusion of the fifth aspect of the invention.
  • the desired subject is a mammal, including a mouse, a rat, or a human, preferably a human.
  • the invention provides a pharmaceutical composition or formulation, comprising:
  • the (a) comprises the fusion protein of the fifth aspect of the invention.
  • the pharmaceutical composition or formulation comprises: a liposome obtained by loading (a) into a carrier liposome.
  • the liposome has a hydraulic diameter of from 50 to 200 nm, preferably from 80 to 150 nm, more preferably from 100 to 120 nm, most preferably 110 nm.
  • the (b) comprises: an antibody and a small molecule inhibitor of a PD-1/PD-L1 checkpoint, a CTLA4 antibody, or other effects on immune cells. antibody.
  • the total weight of the (a) and (b) is from 0.1 to 99.9% by weight, preferably 10%, based on the total weight of the pharmaceutical composition or formulation. 99.9 wt%, more preferably 70%-99.9 wt%.
  • the pharmaceutical composition is a liquid, solid, or semi-solid.
  • the pharmaceutical composition is in the form of an oral dosage form, an injection, or a topical pharmaceutical dosage form.
  • the pharmaceutical composition comprises a capsule, an oral solution, or an injection.
  • the pharmaceutical composition or formulation is a liquid formulation.
  • the composition is an oral preparation.
  • the carrier is selected from the group consisting of an infusion medium carrier and/or an injection vehicle carrier.
  • the carrier is one or more carriers selected from the group consisting of physiological saline, glucose. Saline, or a combination thereof.
  • composition or formulation may be used alone or in combination.
  • the combined use comprises: in combination with other drugs that treat and/or prevent tumors.
  • a method of treating and/or preventing a tumor comprising the step of administering a therapeutically effective amount of a subject to a subject in need of treatment and/or prevention of a tumor
  • the system according to the third aspect of the invention, the fusion protein according to the fifth aspect of the invention, the polynucleotide according to the sixth aspect of the invention, the vector according to the seventh aspect of the invention, the eighth aspect of the invention The host cell of aspect, the tumor vaccine composition of the ninth aspect of the invention, or the pharmaceutical composition or formulation of the thirteenth aspect of the invention.
  • the desired subject is a mammal, including a mouse, a rat, or a human, preferably a human.
  • Fig. 1 is an electrophoresis pattern after purification of a TCS-antigen peptide fusion protein of Preparation Example 1 according to the present invention.
  • Figure 2 is a chromatogram of the purification of the TCS-antigen peptide fusion protein of Example 1 according to the invention.
  • Figure 3 is an electrophoresis pattern after purification of the transmembrane peptide-TCS-antigen peptide fusion protein in Preparation Example 2 according to the present invention.
  • Figure 4 is a purification chromatogram of the transmembrane peptide-TCS-antigen peptide fusion protein of Preparation Example 2 according to the invention.
  • Figure 5 is an electrophoresis pattern of the purified TCS-antigen peptide, transmembrane peptide-TCS-antigen peptide prepared in Examples 1 and 2 according to the invention.
  • Fig. 6 is a graph showing the inhibitory effect of TCS and TCS-antigen peptide and transmembrane peptide-TCS-antigen peptide on proliferation of dendritic cells DC2.4 according to Experimental Example 1.
  • Figure 7 is a graph showing the fluorescence uptake of TCS and TCS-antigen peptides and transmembrane peptide-TCS-antigen peptides in dendritic cells DC2.4 cells according to Experimental Example 2.
  • Figure 8 is a flow chart showing the cellular uptake of TCS and TCS-antigen peptides and transmembrane peptide-TCS-antigen peptides in dendritic cells DC2.4 cells according to Experimental Example 2.
  • Figure 9 is a CD80 map of TCS and TCS-antigen peptide and transmembrane peptide-TCS-antigen peptide sensitizing BMDC in vitro according to Experimental Example 3 of the present invention.
  • Fig. 10 is a graph showing the induction of T cell proliferation in vitro by TCS and TCS-antigen peptides and transmembrane peptide-TCS-antigen peptides according to Experimental Example 4 of the present invention.
  • Figure 11 is a graph showing tumor volume increase in in vivo antitumor treatment of TCS and TCS-antigen peptides and transmembrane peptide-TCS-antigen peptides in a CT26 subcutaneous tumor-bearing mouse model according to Experimental Example 5.
  • Fig. 12 is a photograph showing the tumors of each treatment group in the in vivo antitumor treatment end point of the TCS and TCS-antigen peptides and the transmembrane peptide-TCS-antigen peptide in the CT26 subcutaneous tumor-bearing mouse model according to the present invention.
  • Figure 13 is a graph showing changes in body weight of animals in each of the treatment groups in which the TCS and TCS-antigen peptides and the transmembrane peptide-TCS-antigen peptide were anti-tumor in vivo in the CT26 subcutaneous tumor-bearing mouse model according to the present invention.
  • Figure 14 is a graph showing the survival curves of the anti-tumor treatment groups of the TCS and TCS-antigen peptides and the transmembrane peptide-TCS-antigen peptide in the CT26 subcutaneous tumor-bearing mouse model according to the present invention.
  • Figure 15 is a graph showing tumor volume growth during administration according to Experimental Example 6 of the present invention.
  • Figure 16 is a graph showing changes in body weight of mice of each group during administration according to Experimental Example 6 of the present invention.
  • Figure 17 is a graph showing the survival curves of mice of each group during administration according to Experimental Example 6 of the present invention.
  • Figure 18 is a graph showing tumor volume growth during administration according to Experimental Example 7 of the present invention.
  • Figure 19 is a graph showing changes in body weight of mice of each group during administration according to Experimental Example 7 of the present invention.
  • Figure 20 is a graph showing changes in the number of CD8 + T cells in the spleen of each group of mice at the treatment end point according to Experimental Example 7 of the present invention.
  • Figure 21 is a graph showing changes in the number of CD8 + T cells in tumor tissues of each group of mice at the treatment end point according to Experimental Example 7 of the present invention.
  • Figure 22 is a particle size diagram of the prepared TCS-antigen peptide liposome.
  • Figure 23 is a diagram showing the SDS-PAGE electrophoresis of the prepared TCS-antigen peptide liposome after purification.
  • Figure 24 is a graph showing tumor volume growth during administration according to Example 8 of the present invention.
  • Figure 25 is a graph showing changes in body weight of mice of each group during administration according to Example 8 of the present invention.
  • Figure 26 is a survival curve of each group of mice according to Example 8 of the present invention.
  • Figure 27 is a graph showing changes in the number of CD8 + T cells in lymph nodes of each group of mice at the treatment end point according to Example 8 of the present invention.
  • Figure 28 is a graph showing changes in the number of CD8 + T cells in tumor tissues of each group of mice at the treatment end point according to Example 8 of the present invention.
  • trichosanthin can effectively activate and/or sensitize dendritic cells, thereby enhancing their antigen-presenting ability, and fusion proteins containing tumor antigens and trichosanthin can pass through. After processing and presentation of dendritic cells, it specifically activates the immune system against tumors in vivo, and inhibits tumor growth and spread, thereby becoming a therapeutic vaccine for tumors. Trichosanthin protein is highly safe and has few toxic and side effects, and is an ideal component of tumor therapeutic vaccine preparations. On the basis of this, the present invention has been completed.
  • Trichosanthin is a new anti-pregnancy drug discovered from the rich treasures of Chinese medicine. Trichosanthin is a protein extracted from the roots of the Cucurbitaceae plant. After more than 50 years of clinical practice, it has been proved that trichosanthin is a safe and effective mid-term abortion drug. Trichosanthin TCS is a type I ribosome inactivating protein with a molecular weight of 27 kDa. It has N-glycosidase activity and is capable of recognizing and deactivating ribosome large subunits of mammalian cells, thereby inhibiting cell protein synthesis and leading to cells. death. Because of its strong immunogenicity, its molecular weight is small, it is easy to be cleared by the kidney, and it lacks certain tumor targeting. These all limit its clinical application.
  • the trichosanthin protein in the present invention is an approved mid-term abortion drug, and the safety of human application has been confirmed. Therefore, it can be rationally and fully utilized as a new type of tumor vaccine component, which can not only ensure its safety and effectiveness, but also broaden the clinical application range of the protein.
  • the trichosanthin protein which can be used in the present invention is not particularly limited and may include wild-type and recombinant-type trichosanthin proteins and active fragments thereof as long as the protein or a fragment thereof has or has substantially (e.g., 70%, 75%, 80%, 85 is retained). The %, 90%, 95%, 99% or 100%) N-glycosidase activity is sufficient.
  • the trichosanthin protein useful in the present invention is as shown in SEQ ID NO.: 9 (wild type), and may also be a protein as shown in SEQ ID NO.: 10 (recombinant), wherein SEQ ID NO.: 9 is encoded.
  • the nucleotide sequence of the indicated protein is shown in SEQ ID NO.:11.
  • fusion protein protein of the present invention
  • fusion protein of the present invention fusion protein of the present invention
  • trichosanthin-tumor antigen fusion protein TSC-antigenic peptide
  • the fusion protein may further comprise a transmembrane element (T), and optionally a linker peptide, a tag sequence, a signal peptide and/or 1-3 amino acid residues.
  • T transmembrane element
  • the protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from a monomer.
  • the term also encompasses active fragments and derivatives of fusion proteins.
  • the fusion protein of the present invention is not particularly limited in terms of the ligation sequence, and the fusion protein of the present invention can be constructed from the N-terminal-C terminal or the C-terminal-N-terminal sequence; when the fusion protein contains a transmembrane peptide, it is preferred to penetrate the membrane.
  • the peptide and tumor antigen are constructed at both ends of the fusion protein, and the trichosanthin protein is constructed between the transmembrane peptide and the tumor antigen.
  • a preferred fusion protein is an isolated recombinant fusion protein sequence as set forth in SEQ ID NO.: 1, SEQ ID NO.: 2 and 12.
  • the fusion proteins of the invention have activated and/or sensitized dendritic cell (DC) cells and enhance their processing and presentation of tumor antigens.
  • the invention also includes active fragments, derivatives and analogs of the fusion proteins according to the invention.
  • fragment refers to a polypeptide that substantially retains its processing and presentation enhancing effect on a tumor antigen.
  • a polypeptide fragment, derivative or analog of the invention may be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) at one or more a polypeptide having a substituent group in one amino acid residue, or (iii) a polypeptide formed by fusing a fusion protein with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) an additional amino acid sequence
  • a polypeptide formed by fusion of the polypeptide sequence a fusion protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His).
  • a preferred class of reactive derivatives means that up to 3, preferably up to 2, and more preferably up to 1 amino acid are replaced by amino acids of similar or similar nature to the amino acid sequence of Formula Ia or Formula Ib. Peptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1, and preferably, the derivatives do not contain Cys.
  • substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln;His;Lys;Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala
  • the invention also provides analogs of the fusion proteins of the invention.
  • the difference between these analogs and the polypeptide represented by SEQ ID NO: 3. may be a difference in amino acid sequence, a difference in a modified form which does not affect the sequence, or a combination thereof.
  • Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptides of the invention may also be used in the form of a salt derived from a pharmaceutically or physiologically acceptable acid or base.
  • These salts include, but are not limited to, salts formed with hydrochloric acid, hydrobromic acid, sulfuric acid, citric acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, succinic acid, oxalic acid, fumaric acid, Malay. Acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, or isethionic acid.
  • Other salts include those formed with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, as well as esters, carbamates or other conventional "prodrugs".
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.
  • "isolated recombinant fusion protein&quot means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
  • the polynucleotide of the present invention encoding the fusion protein of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the full-length nucleotide sequence of the fusion protein or element thereof of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain a gene encoding a fusion protein of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
  • polynucleotide sequence of the present invention can be utilized to express or produce a recombinant protein by conventional recombinant DNA techniques. Generally there are the following steps:
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CH, NSO, COS7, or 293 cells, and the like.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and
  • the present invention also provides a precursor protein of the fusion protein, which utilizes an intein-mediated fusion protein acquisition method, and adds an intein-encoding gene to the gene construct of the fusion protein, and
  • the high purity fusion protein is directly obtained by label cleavage after expression of the protein, so that no further separation or purification steps are required.
  • the fusion protein provided by the present invention preferably contains a transmembrane peptide.
  • Penetrating peptides are a class of polypeptides that penetrate cell membrane activity, either found in nature or by artificial screening.
  • the transmembrane peptide which can be used in the present invention is not particularly limited, and may be any transmembrane peptide rich in basic amino acids and transmembrane peptide having amphiphilicity as long as it can promote TCS uptake by cells to their own lack of incorporation ability.
  • the transmembrane peptide of the present invention comprises low molecular weight protamine LMWP (VRRRRRRGGRRRR (SEQ ID NO.: 4)), TAT (YGRKKRRQRRR (SEQ ID NO.: 5)), R9 (RRRRRRRRR) transmembrane peptide of the present invention Not only can it enter the cell by itself, but also carry the TCS of the invention into cells, especially dendritic cells.
  • transmembrane peptide of the present invention can directly obtain a transmembrane peptide-containing fusion protein by introducing a transmembrane peptide gene into an expression vector expressing a fusion protein, or can also obtain a transmembrane peptide modification by intein-mediated mediated modification.
  • Method, preferred method includes:
  • a supernatant containing the C-terminal fusion intein and the CBD-tagged target protein is affinity-purified with a chitin column, and after washing away the heteroprotein, a buffer containing sodium 2-mercaptoethanesulfonate (MESNA) is used. Perform on-column cutting overnight and collect the eluate;
  • MESNA sodium 2-mercaptoethanesulfonate
  • DC Dendritic cells
  • APCs antigen presenting cells
  • Immature DCs have strong recognition.
  • phagocytic ability, mature DC can effectively activate the initial type of T cells, is at the center of initiation, regulation, and maintenance of immune response.
  • DCs in the human body are in an immature state, expressing low levels of costimulatory factors and adhesion factors, and the ability to stimulate the proliferation of the same mixed lymphocytes in vitro is low, but immature DCs have strong antigenic phagocytic ability and are ingested.
  • Antigens including in vitro processing
  • differentiated into mature DCs when stimulated by certain factors while mature DCs express high levels of costimulatory and adhesion factors.
  • the DC migrates from the peripheral tissues contacting the antigen into the secondary lymphoid organs, contacts the T cells and stimulates an immune response.
  • DC as the most powerful APC found at present, is capable of inducing the production of specific cytotoxic T lymphocytes (CTLs).
  • CTLs cytotoxic T lymphocytes
  • Co-culture of dendritic cells with the composition or fusion protein of the present invention can activate and/or sensitize immature dendritic cells to mature dendritic cells, thereby significantly enhancing antigen-presenting ability.
  • the specific performance is:
  • the expression level of CD80 is increased by at least 20-100%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, 85%, 90%, 95% or 100%; and/or
  • the amount of IFN- ⁇ secreted by the T cells is increased by at least 20-100%, preferably at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
  • the dendritic cells which can be used in the present invention are not particularly limited, and may be dendritic cells derived from the desired subject itself, which can be obtained by a conventional method or commercially available, and a preferred method of separation includes by a conventional method. Isolated from mammalian bone marrow.
  • Preferred dendritic cells are derived from mammals such as mice, rats, humans and the like.
  • the desired dendritic cells sufficient to activate the body's tumor immune system are approximately 1-5 x 10 5-6 per dose.
  • the tumor antigen which can be used in the present invention is not particularly limited, and includes obtaining a tumor gene mainly from a tumor cell, constructing a plasmid by genetic recombination, and transforming into expression vector (such as Escherichia coli), and the specific expression method is as those skilled in the art. Well known.
  • the fragment length is generally 10-50 bp.
  • the tumor antigen of the present invention is derived from the following tumors: including head and neck cancer, thyroid cancer, brain malignant tumor, lung cancer, skin cancer, melanoma, gastric cancer, pancreatic cancer, liver cancer, gallbladder cancer, colorectal cancer, breast cancer, ovary Cancer, cervical cancer, endometrial cancer, testicular cancer, bladder cancer, kidney cancer, or osteosarcoma.
  • the tumor antigen of the invention is derived from colorectal cancer, breast cancer, melanoma or lung cancer.
  • a preferred tumor antigen comprises the antigen as described in SEQ ID NO.: 6, 7 or 8, which is an antigen peptide constructed from a gene obtained from tumor cells induced by mouse lung epithelial TC-1 cells, and the fragments are of different lengths. It is 14-39 bp.
  • tumor antigens such as melanoma B16 cells and /B16-F10 cells, human ovarian cancer SKOV-3 cells and human ovarian cancer ES2 cells, colorectal cancer cells HCT116 and the like can be extracted from common tumor cell lines.
  • the invention also provides a composition for physically mixing trichosanthin and a tumor antigen for use as an activation and/or sensitization composition for activating and/or sensitizing DC cells.
  • the ratio of trichosanthin protein and tumor antigen is from 0.5:1 to 1:50, preferably from 1:1 to 1:10, and is usually physically mixed using an ice bath at a mixing condition of -20 ° C to 4 ° C. Save under the same conditions.
  • the compositions of the present invention are preferably in liquid form, and may also be formulated as injectables, such as liquid solutions or suspensions; they may also be in a solid form suitable as a liquid carrier in a solution or suspension prior to injection.
  • the composition may further contain a pharmaceutically acceptable carrier which maintains the function of the active ingredient.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, such as a composition of the invention.
  • the term refers to pharmaceutical carriers which do not themselves induce the production of antibodies harmful to the individual receiving the composition and which are not excessively toxic after administration.
  • Such vectors are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).
  • Such carriers include, for example, water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers.
  • the immunological composition may also contain an immunological adjuvant.
  • the present invention provides an activation and/or sensitization system comprising trichosanthin, tumor antigens, and isolated dendritic cells.
  • the trichosanthin protein, tumor antigen may be present in a form of physical mixing (i.e., activation and/or sensitization compositions of the invention) or coupling to form a fusion protein.
  • the effective amount of dendritic cells usually contains 1-5 x 10 5-6 cells/dose, preferably 1-2 x 10 5-6 cells/dose, and the ratio to the composition of the present invention is 10 5 - 5 ⁇ 10 5 ; the ratio of the fusion protein to the present invention is 10 5 - 5 ⁇ 10 5 , and when the number of cells is 10 5 - 5 ⁇ 10 5 , the final concentration of the protein is 1 ⁇ M.
  • an effective amount refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject will depend on the size and health of the subject, the nature and extent of the condition, and the combination of therapeutic and/or therapeutic agents selected for administration.
  • composition and fusion protein of the present invention can be used for the culture of dendritic cells in vitro, for example, the composition of the present invention, the fusion protein and a certain amount of dendritic cells are co-cultured under suitable conditions, thereby activating and/or Sensitive dendritic cells, and antigens treated by dendritic cells are used for activation of T cells, thereby establishing cellular immunity against specific tumor types to achieve the effect of tumor therapeutic vaccines.
  • the prokaryotic expression vector pMXB10 was purchased from New England Biolabs, and the recombinant expression plasmid of TCS-antigen peptide fusion protein and transmembrane peptide-TCS-antigen peptide fusion protein was constructed by Shanghai Jierui Biotechnology Co., Ltd., and the flow antibody (Anti-Mouse CD80) And (Anti-Mouse CD8) were purchased from BD Bioscience.
  • Mouse colon cancer CT26, murine melanoma cells B16-F10, and murine dendritic cells DC2.4 were purchased from the Chinese Academy of Sciences cell bank. Materials used in other experiments were derived from conventional commercial products.
  • the plasmid TCS-antigen peptide of the construct was transformed into E. coli BL21 (DE3) competent cells.
  • the cells were collected by centrifugation at 4 ° C, 9,000 rpm for 3 min in a centrifuge.
  • HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5 Torr Tween 20, pH 8.5.
  • the cells were ultrasonically disrupted with a probe sonicator at 400 W for 35 min.
  • the supernatant containing the protein of interest was passed through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml/min. After the loading was completed, the non-specifically bound heteroprotein was washed away with 25 column volumes of HEPES buffer.
  • the plasmid transmembrane peptide-TCS-antigen peptide of the construct was transformed into E. coli BL21 (DE3) competent cells.
  • the cells were collected by centrifugation at 4 ° C, 9,000 rpm for 3 min in a pre-cooled centrifuge.
  • HEPES buffer containing 20 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5 Torr Tween 20, pH 8.5.
  • the cells were ultrasonically disrupted with a probe sonicator at 400 W for 35 min.
  • the supernatant of the target protein was passed through a chitin column pre-equilibrated with HEPES buffer at a flow rate of 1 ml/min. After the loading was completed, the non-specifically bound heteroprotein was washed away with 25 column volumes of HEPES buffer.
  • MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide blue, trade name: thiazole blue) method for the determination of recombinant protein drugs TCS, TCS-antigen peptide, Cytotoxicity of the Penetrating Peptide-TCS-Antigen Peptide
  • TCS recombinant protein drugs
  • TCS-antigen peptide Cytotoxicity of the Penetrating Peptide-TCS-Antigen Peptide
  • the murine dendritic cells DC2.4 cells in the logarithmic growth phase were digested and counted, diluted to a cell suspension with a density of 4 ⁇ 10 4 cells/mL, and transferred. To 96-well culture well plates, 100 ⁇ L of the cell suspension was added to each well, and cultured in DMEM complete medium containing 10% calf serum for 12 h (37 ° C, 5% CO 2 ).
  • TCS and TCS-antigenic peptides and transmembrane peptide-TCS-antigen peptide on the survival rate of dendritic cells DC2.4: the three had no significant effect on the survival rate of dendritic cells DC2.4, which proves
  • the antigenic peptide and/or TCS is not cytotoxic to dendritic cells (Fig. 6). And under the same low concentration protein conditions, the recombinant protein drug does not inhibit the proliferation of dendritic cells, so the drug combination is safe.
  • the fusion protein 1 (TCS-antigen peptide fusion protein), fusion protein 2 (penetrating peptide-TCS-antigen peptide fusion protein) obtained in Examples 1 and 2, and the recombinant trichosanthin protein TCS and our previously constructed recombinant ratio were prepared.
  • a multiple excess of Rhodamine B was mixed and allowed to react overnight at 4 ° C in the dark.
  • the excess rhodamine B fluorescein was removed using a desalting column.
  • the concentration of the protein was determined by the BCA method.
  • the mouse dendritic cells DC2.4 cells in the logarithmic growth phase were digested, the cells were counted, and the cell suspension was prepared at a concentration of 5 ⁇ 10 4 cells/mL, and inoculated into a 12-well cell culture plate, and each well was added. 1mL.
  • the uptake experiment was performed after the cells were grown to a suitable density. 1 ⁇ M of each protein drug labeled with Rhodamine B was added, and incubation was continued for 4 hours in an incubator.
  • the medium was discarded and washed three times with PBS, fixed with 4% paraformaldehyde for 12 minutes, washed three times with PBS, then DAPI stained for 10 minutes, washed three times with PBS, and photographed with a fluorescence microscope (Zeiss) (Fig. 7).
  • Another cell suspension of about 1 ⁇ 10 5 cells/mL was taken, and the uptake experiment was carried out according to the above method. After the experiment was washed three times with PBS, it was digested with 0.25% trypsin and then dispersed by FL1 using flow cytometry (BD Pharmingen). Channels were used to detect cellular uptake ( Figure 8).
  • TCS itself has fewer cells, and the TCS-antigen peptide fusion protein has a certain degree of incorporation, while the transmembrane peptide-TCS-antigen peptide fusion protein has enhanced fluorescence intensity.
  • the TCS-antigen peptide fusion protein can have a certain cell penetrating ability, the fusion of the transmembrane peptide can increase the cell-in efficiency of TCS, thereby making the intracellular drug fluorescence intensity stronger.
  • TCS and fusion protein 1 (TCS-antigen peptide) and fusion protein 2 (penetrating peptide-TCS-antigen peptide) in vitro sensitized antigen-presenting cells
  • BMDC bone marrow-derived dendritic cells
  • mice C57BL/6 mice, 6-8 weeks old, were taken as a source of bone marrow cells.
  • the instruments used were autoclaved before the experiment.
  • the mice were euthanized and immersed in 75% ethanol for 10 minutes.
  • the femur and tibia were exfoliated and immersed in 75% ethanol for 5 minutes, soaked in sterile PBS for 5 minutes and repeated twice.
  • the ends of the femur and tibia were cut off, and serum-free DMEM medium was aspirated using a 1 mL syringe.
  • the bone marrow was punched out, centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded.
  • BMDC induction medium containing 20 ng/mL GM-CSF + 10 ng/mL IL-4
  • BMDC induction medium containing 20 ng/mL GM-CSF + 10 ng/mL IL-4
  • BMDC dendritic cells
  • CD80 was significantly enhanced in each group of the experimental group, and the positive rate of the experimental group was increased by 43.7% compared with the control group. Since CD80 is an important costimulatory molecule expressed on antigen-presenting cells, it binds to the CD28 molecule on T cells, activates T lymphocytes, and exerts an immune effect. After the antigen-presenting cell BMDC was treated with a fusion protein drug such as TCS/antigen peptide (physical mixed composition), TCS-antigen peptide and transmembrane peptide-TCS-antigen peptide, CD80 was significantly up-regulated and expressed in flow mode.
  • TCS/antigen peptide physical mixed composition
  • results were as follows: after treatment with the drug, the first peak was weakened, the peak of the second peak was significantly increased, and the peak contrast was significantly increased, indicating an increase in the positive rate.
  • the results indicate that the recombinant fusion protein and the physical mixture can activate DC cells and improve the antigen-presenting ability, and the recombinant fusion protein is superior.
  • Recombinant protein TCS, TCS/antigen peptide mixture and TCS-antigenic peptide and transmembrane peptide-TCS-antigen peptide promote T cell proliferation in vitro
  • BMDC bone marrow-derived dendritic cells
  • mice C57BL/6 mice, 6-8 weeks old, were taken as a source of bone marrow cells.
  • the instruments used were autoclaved before the experiment.
  • the mice were euthanized and immersed in 75% ethanol for 10 minutes.
  • the femur and tibia were exfoliated and immersed in 75% ethanol for 5 minutes, soaked in sterile PBS for 5 minutes and repeated twice.
  • the ends of the femur and tibia were cut off, and the serum-free DMEM medium was aspirated using a 1 mL syringe.
  • the bone marrow was punched out, centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded.
  • BMDC medium containing 20 ng/mL GM-CSF + 10 ng/mL IL-4
  • BMDC medium containing 20 ng/mL GM-CSF + 10 ng/mL IL-4
  • the corresponding 1 ⁇ M of each recombinant protein was added, and the untreated BMDC cells were set as a blank control, and the culture was continued in the incubator for 12 to 24 hours.
  • mice C57BL/6 mice, 6-8 weeks old, were taken as a source of lymphocytes.
  • the instruments used in the operation were autoclaved before the experiment.
  • the mice were euthanized and immersed in 75% ethanol for 10 minutes, and the spleen was peeled off in a clean bench. This procedure pays attention to aseptic processing.
  • 4 mL of Mouse 1 ⁇ Lymphocyte Separation Solution was placed in a 35 mm Petri dish and ground.
  • the supernatant containing the spleen cells was immediately transferred to a 15 mL centrifuge tube and covered with 1 mL of RPMI 1640 medium. Centrifuge at 800 g for 30 minutes at room temperature. Note the slower acceleration and deceleration settings.
  • the lymphocyte layer was aspirated and 10 mL of RPMI 1640 medium was added and washed upside down.
  • the cells were collected by centrifugation at 250 g for 10 minutes at room temperature.
  • the supernatant was poured and resuspended in DMEM.
  • the cells were equally divided, added to the above culture plates, and cultured in the incubator for 3 days.
  • the supernatant was collected and the concentration of IFN- ⁇ was measured using an ELISA kit.
  • BMDC cells were co-incubated with T lymphocytes after treatment with each group of drugs, and the concentration of IFN- ⁇ in the experimental group was significantly increased compared with the control group, indicating that T lymphocytes were activated and Proliferation, indicating that trichosanthin protein can promote the antigen cross-presentation process between mature DC and T cells.
  • Figure 11 is a graph showing tumor volume growth during administration. As can be seen from the figure, the TCS group and the TCS/antigen peptide group have a certain inhibitory effect on tumor growth relative to the PBS group. The TCS-antigen peptide group and the transmembrane peptide-TCS-antigen peptide group have more significant antitumor effects.
  • Figure 12 is a visual representation of the tumor images of each group after administration. It can be seen from the figure that the tumor treated with PBS is significantly larger, and the TCS-antigen peptide and transmembrane peptide-TCS-antipeptide protein drug The treated mice had different degrees of tumor reduction.
  • Figure 13 is a graph showing changes in body weight of mice in each group during administration. It can be seen from the figure that the body weight of the experimental animals did not change significantly during the administration of each experimental group, which proved that the TCS-antigenic peptide and the transmembrane peptide-TCS-antigen peptide fusion protein constructed by the present invention have good organisms. Compatibility, system toxicity is low.
  • Figure 14 shows the survival curves of each group in the drug-administered group. It can be seen that the survival time of the PBS group is the shortest, and the survival time of the recombinant protein TCS-antigen peptide group is the longest. This demonstrates that the fusion protein TCS-antigen peptide constructed by the present invention has an effect of prolonging survival, and also demonstrates the antitumor effect of the recombinant protein TCS-antigen peptide.
  • mice On the 6th day after the tumor implantation, the experimental mice were randomly divided into 4 groups: trichosanthin group, anti-PD-1 group, trichosanthin+anti-PD-1 group, and PBS group as negative control. Trichosanthin and PBS were administered by intratumoral injection, and anti-PD-1 was administered by intraperitoneal injection. Trichosanthin was administered at a dose of 5 ⁇ g/day/4 days for a total of 3 doses; anti-PD-1 was administered at a dose of 250 ⁇ g/day/2 days for a total of 5 doses.
  • Figure 15 is a graph showing tumor volume growth during administration. It can be seen from the figure that the trichosanthin group and the anti-PD-1 group have significant inhibitory effects on tumor growth compared with the control group, while the trichosanthin + anti-PD-1 group has more significant tumor inhibition. effect. This proves that the recombinant trichosanthin protein constructed by the invention can cooperate with the PD-1/PD-L1 immunological checkpoint treatment, improve the therapeutic response rate of the PD-1/PD-L1 immunological checkpoint therapy, and improve the immunotherapy effect of the tumor.
  • Figure 16 is a graph showing changes in body weight of mice in each group during administration. It can be seen from the figure that the body weight of the experimental animals did not change significantly during the administration of each experimental group, which proved that the recombinant trichosanthin protein constructed by the present invention has good biocompatibility and low systemic toxicity.
  • Figure 17 shows the survival curves of the mice in each group. It can be seen that the survival time of the control group is the shortest, and the mice with trichosanthin + anti-PD-1 have the highest survival rate and the longest survival time. This proves that the trichosanthin protein constructed by the invention can cooperate with the PD-1/PD-L1 immunological checkpoint treatment, improve the therapeutic response rate of the PD-1/PD-L1 immunological checkpoint therapy, and improve the immunotherapy effect of the tumor.
  • mice On the 6th day after the tumor implantation, the experimental mice were randomly divided into 4 groups: TCS-antigen peptide group, anti-PD-1 group, TCS-antigen peptide + anti-PD-1 group, and the PBS group was used as a negative control.
  • TCS-antigen peptide and PBS were administered by intratumoral injection, and anti-PD-1 was administered by intraperitoneal injection.
  • the TCS-antigen peptide was administered at a dose of 2 ⁇ g/d/2 days for a total of 3 times; the anti-PD-1 was administered at a dose of 200 ⁇ g/d/2 days for a total of 5 doses.
  • the tumor volume of the PBS group reached 2000 mm 3 , it was determined as the end point of death.
  • the rats were euthanized, the tumor was removed, and the blood and the capsule on the surface of the tumor were carefully removed and weighed.
  • FIG. 18 is a graph showing tumor volume growth during administration.
  • the TCS-antigen peptide group has a certain inhibitory effect on tumor growth compared with the control group, while the anti-PD-1 group and the TCS-antigenic peptide + anti-PD-1 group have significant effects.
  • the tumor suppressing effect, and the TCS-antigen peptide + anti-PD-1 group showed the best effect of inhibiting tumor growth.
  • the TCS-antigen peptide constructed by the invention can cooperate with the PD-1/PD-L1 immunological checkpoint treatment, improve the therapeutic response rate of the PD-1/PD-L1 immunological checkpoint therapy, and improve the immunotherapy effect of the tumor.
  • Figure 19 is a graph showing changes in body weight of mice in each group during administration. It can be seen from the figure that the body weight of the experimental animals did not change significantly during the administration of each experimental group, which proved that the recombinant TCS-antigenic peptide constructed by the present invention has good biocompatibility and low systemic toxicity.
  • Figure 20 shows the changes in the number of CD8 + T cells in the spleen of each group of mice at the end of the treatment. It can be seen that the TCS-antigen peptide + anti-PD-1 group can significantly increase the proportion of CD8 + T cells in the spleen.
  • Figure 21 shows the changes in the number of CD8 + T cells in tumor tissues of each group of mice at the end of treatment. It can be seen that the TCS-antigen peptide + anti-PD-1 group can significantly increase the proportion of CD8 + T cells in tumor tissues. .
  • TCS-antigenic peptide constructed by the present invention can be treated in combination with the PD-1/PD-L1 immunological checkpoint to improve the immunotherapy effect of the tumor by increasing the number of CD8 + T cells in the spleen and tumor tissues.
  • the incorporation of recombinant TCS-antigen peptide into liposomes can further improve the efficacy of tumor immunotherapy of recombinant TCS-antigen peptides.
  • Phospholipids and cholesterol were dissolved in 10 ml of dichloromethane, and the TCS-antigen peptide was injected into the lipid solution under ultrasonic bath, and probed for 3 min (300 W, 1.5 s, 1.5 s).
  • the dichloromethane was removed by rotary evaporation, hydrated by adding 9 ml of ultrapure water, and the probe was sonicated for 3 min (300 W, 1.5 s, 1.5 s).
  • the TCS-antigen peptide liposome was repeatedly pushed by an extruder.
  • a 1 ml liposome solution was taken and the hydraulic diameter of the TCS-antigen peptide liposome was measured using a dynamic light scattering instrument.
  • the TCS-antigen peptide liposome was transferred to an ultrafiltration tube, and the unencapsulated TCS-antigen peptide was removed by centrifugal ultrafiltration. After the purified TCS-antigen peptide liposome was demulsified, the entrapment of the TCS-antigen peptide was characterized by SDS-PAGE electrophoresis.
  • Figure 22 is a particle size diagram of the prepared TCS-antigen peptide liposome. As can be seen from the figure, the liposome has a hydraulic diameter of 110 nm and has a good dispersion.
  • Figure 23 is a diagram showing the SDS-PAGE electrophoresis of the prepared TCS-antigen peptide liposome after purification. As can be seen from the figure, the prepared liposome can effectively entrap the TCS-antigen peptide. It indicates that the preparation of TCS-antigen peptide liposome was successful.
  • Figure 24 is a graph showing tumor volume growth during administration.
  • the recombinant TCS group and the antigenic peptide group have a certain inhibitory effect on tumor growth compared with the control group, while the TCS-antigen peptide group and the TCS-antigen peptide liposome group have significant tumor inhibition.
  • the effect, and the TCS-antigen peptide liposome group showed the best effect of inhibiting tumor growth. This demonstrates that the TCS-antigen peptide liposome constructed by the present invention can improve the immunotherapeutic effect of the TCS-antigen peptide on tumors.
  • Figure 25 is a graph showing changes in body weight of mice in each group during administration. It can be seen from the figure that the body weight of the experimental animals did not change significantly during the administration of each experimental group, and it was confirmed that the recombinant TCS-antigenic peptide and the TCS-antigenic peptide liposome constructed by the present invention have good biological phases. Capacitive, systemic toxicity is low.
  • Figure 26 shows the survival curves of the mice in each group. It can be seen that the survival time of the control group is the shortest, and the survival time of the TCS-antigen peptide liposome group is the longest. This demonstrates that the TCS-antigen peptide liposome constructed by the present invention has an effect of prolonging survival, and also proves that the TCS-antigen peptide liposome has a stronger antitumor effect than the TCS-antigenic peptide.
  • Figure 27 shows the changes in the number of CD8 + T cells in the lymph nodes of each group of mice at the end of the treatment. It can be seen that the TCS-antigen peptide liposome group can significantly increase the proportion of CD8 + T cells in the lymph nodes.
  • Figure 28 shows the changes in the number of CD8 + T cells in tumor tissues of each group of mice at the end of treatment. It can be seen that the TCS-antigen peptide liposome group can effectively increase the proportion of CD8 + T cells in tumor tissues. This demonstrates that the TCS-antigen peptide liposome constructed by the present invention can improve the antitumor effect of the TCS-antigen peptide by increasing the number of CD8 + T cells in lymph nodes and tumor tissues.

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Abstract

一种天花粉蛋白或其编码基因的用途，用于制备树突状细胞激活剂和/或致敏剂。天花粉蛋白可以激活和/或致敏树突状细胞，加强其抗原提呈能力；含有肿瘤抗原和天花粉蛋白的融合蛋白经过树突状细胞的加工以及提呈后，特异性激活体内针对肿瘤的免疫系统，达到抑制肿瘤生长以及扩散的作用，成为肿瘤的治疗性疫苗。天花粉蛋白安全性高，毒副作用小。

Description

天花粉蛋白用于致敏和/激活树突状细胞中的应用 技术领域
本发明属于生物医药领域，更具体而言，本发明涉及天花粉蛋白在激活树突状细胞中的应用，以及利用该特性制备的含天花粉蛋白以及肿瘤抗原的融合蛋白。
背景技术
恶性肿瘤严重威胁着人类的健康。随着人们生活习惯的改变，肿瘤的发病率与病死率呈上升趋势。肿瘤的治疗手段通常包括手术切除，化学治疗，放射治疗和免疫治疗等。
尽管近些年癌症药物治疗已经取得了较大的进展，但是大分子药物在癌症治疗中的应用较少。随着生物技术的发展，大分子药物，尤其是蛋白类药物，受到越来越多的关注。
肿瘤是机体正常细胞恶变的产物，其特点是不断增殖并在体内转移。因此肿瘤细胞在免疫学上的突出特点是出现某些在同类正常细胞中看不到的新的抗原标志。现已陆续发现的肿瘤抗原包括肿瘤特异性抗原和肿瘤相关抗原。前者为肿瘤细胞所独有；后者大多指胚胎性抗原，为胚胎组织与肿瘤组织所共有。尽管机体内具有一系列的免疫监视机制，但仍难以阻止肿瘤的发生和发展。少量肿瘤细胞不易引起机体应答，待肿瘤生长至一定程度，超越了机体免疫应答的能力，肿瘤细胞即得以逃逸。
因此，如何在机体的免疫系统中建立抗肿瘤的机制，尤其是利用蛋白类药物对肿瘤进行免疫治疗，成了目前热门的话题。然而事实上，由于肿瘤抗原免疫原性不足、抗原提呈体系效率低下，如何有效建立机体对肿瘤的免疫性，高效、特异性地提高机体对肿瘤的识别并杀死肿瘤细胞等仍然是本领域中亟待解决的问题。
发明内容
本发明第一方面，提供了一种天花粉蛋白(trichosanthin，TCS)或其编码基因的用途，用于制备树突状细胞激活剂和/或致敏剂。
在另一优选例中，所述用途还包括进一步制备肿瘤治疗性疫苗。
在另一优选例中，所述天花粉蛋白或其编码基因还可与其他抗肿瘤药物联用。
在另一优选例中，所述其他抗肿瘤药物包括抗PD-1抗体、抗PD-L1抗体。
在另一优选例中，所述抗PD-1抗体包括：PD-1/PD-L1检查点的抗体和小分子抑制剂，CTLA4抗体，或其他作用于免疫细胞的抗体。
本发明第二方面，提供了一种体外激活和/或致敏树突状细胞的方法，包括步骤：
(a)提供一种含有天花粉蛋白和肿瘤抗原的混合物；
(b)在(a)所述混合物的存在下，培养树突状细胞，从而激活和/或致敏树突状细胞。
在另一优选例中，(a)中所述的混合物中，天花粉蛋白和肿瘤抗原经物理混合或互相偶联。
在另一优选例中，所述天花粉蛋白和肿瘤抗原为融合蛋白。
在另一优选例中，所述的融合蛋白还偶联有穿膜肽。
在另一优选例中，所述穿膜肽序列包括R9肽(RRRRRRRRR)、LMWP(VRRRRRRGGRRRR，SEQ ID NO.:4)、或TAT肽(YGRKKRRQRRR，SEQ ID NO.:5)所示的多肽。
在另一优选例中，所述的融合蛋白具有式Ia或式Ib所示的结构：
T-E1-P-E2-A 式Ia，
A-E1-P-E2-T 式Ib；
其中，元件T为穿膜肽，元件A为肿瘤抗原，元件P为天花粉蛋白；E1和/或E2为任选的连接肽、标签序列、信号肽和/或1-3个氨基酸残基，“-”为肽键。
在另一优选例中，所述的肿瘤包括头颈癌、甲状腺癌、脑部恶性肿瘤、肺癌、皮肤癌、黑色素瘤、胃癌、胰腺癌、肝癌、胆囊癌、结直肠癌、乳腺癌、卵巢癌、子宫颈癌、子宫内膜癌、睾丸癌、膀胱癌、肾癌、或骨肉瘤。
在另一优选例中，所述的肿瘤抗原包括蛋白、肽段和/或DNA片段。
在另一优选例中，所述的肿瘤抗原包括在肿瘤细胞表面过度表达的抗原、分化抗原、或病毒诱发的肿瘤抗原。
在另一优选例中，所述的肿瘤抗原包括SEQ ID NO.:6(EDVTPENFLAVLR)、SEQ ID NO.:7(TPENFLAVL)、或SEQ ID NO.:8(HSVTYEHALRYLY)。
在另一优选例中，所述的培养条件包括在含10％胎牛血清的高糖1640完全 培养基，含10％胎牛血清的1640完全培养基，含10％胎牛血清的DMEM完全培养基，37℃，5％CO 2。
在另一优选例中，所述(a)的混合物中，还包括其他抗肿瘤药物。
在另一优选例中，所述其他抗肿瘤药物包括抗PD-1抗体、抗PD-L1抗体。
在另一优选例中，所述抗PD-1抗体包括：PD-1/PD-L1检查点的抗体和小分子抑制剂，CTLA4抗体，或其他作用于免疫细胞的抗体。
本发明第三方面，提供了一种树突状细胞激活和/或致敏体系，所述的体系包括：
(i)天花粉蛋白；
(ii)肿瘤抗原；和
(iii)分离的树突状细胞。
在另一优选例中，所述体系中的天花粉蛋白和肿瘤抗原经物理混合或互相偶联。
在另一优选例中，所述的融合蛋白还偶联有穿膜肽。
在另一优选例中，在合适的培养条件下，所述体系中天花粉蛋白和肿瘤抗原与所述分离的树突状细胞进行接触后，所述树突状细胞被激活和/或致敏。
本发明第四方面，提供了一种激活和/或致敏的树突状细胞，是由本发明第二方面所述方法制备的。
在另一优选例中，所述被激活和/或致敏的树突状细胞具有以下活性：
(1)CD80的表达量上升了至少20％，优选至少为25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％；和/或
(2)刺激T细胞分泌IFN-γ的量上升了至少20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。
本发明第五方面，提供了一种融合蛋白，所述的融合蛋白具有式Ia或式Ib所示的结构：
T-E1-P-E2-A 式Ia，
A-E1-P-E2-T 式Ib；
其中，元件T为任选的穿膜肽，元件A为肿瘤抗原，元件P为天花粉蛋白；E1和/或E2为任选的连接肽、标签序列、信号肽和/或1-3个氨基酸残基，“-”为肽键。
在另一优选例中，所述的融合蛋白的序列如SEQ ID NO.:1(融合蛋白含 穿膜肽和抗原1)或12所示(融合蛋白含穿膜肽和抗原2)
(RRRRRRGGRRRR(穿膜肽)DVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYADETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIALLLNRNNMA(天花粉蛋白)GGGG(连接肽)EDVTPENFLAVLR(肿瘤抗原))
SEQ ID NO.:12
Figure PCTCN2018116755-appb-000001
在另一优选例中，所述信号肽包括内含肽；所述标签序列包括CBD标签序列、GST标签序列、MBP标签序列、或6His标签序列。
本发明第六方面，提供了一种多核苷酸，所述的多核苷酸编码本发明第五方面所述的融合蛋白。
在另一优选例中，所述的融合蛋白的序列如SEQ ID NO.:2(不含穿膜肽的融合蛋白)所示
Figure PCTCN2018116755-appb-000002
在另一优选例中，编码SEQ ID NO.:2所示蛋白的多核苷酸如SEQ ID NO.:3所示(编码序列)
Figure PCTCN2018116755-appb-000003
Figure PCTCN2018116755-appb-000004
本发明第七方面，提供了一种载体，所述的载体含有本发明第六方面所述的多核苷酸。
本发明第八方面，提供了一种宿主细胞，所述的宿主细胞含有本发明第七方面所述的载体，或所述的宿主细胞的基因中整合有本发明第六方面所述的多核苷酸。
本发明第九方面，提供了一种肿瘤疫苗组合物，所述肿瘤疫苗组合物含有：
A)本发明第五方面所述的融合蛋白；
B)疫苗学上可接受的载体。
在另一优选例中，所述疫苗学上可接受的载体包括霍乱毒素、CpG ODN、氢氧化铝、或表面活性剂。
本发明第十方面，提供了一种制备本发明第五方面所述融合蛋白的方法，包括步骤：
(I)在合适的条件下，培养本发明第八方面所述的宿主细胞，从而获得表达所述的融合蛋白。
在另一优选例中，所述的方法还可包括步骤：
(II)对获得的融合蛋白进行分离和纯化。
在另一优选例中，所述的分离和纯化包括直接采用内含肽介导的分离和纯化。
在另一优选例中，所述的天花粉蛋白来源于天花粉葫芦科植物栝楼Trichosanthes kirilowii Maxim.或双边栝楼Trichosan-thes rosthornii Herms的干燥根
在另一优选例中，所述的天花粉蛋白包括天花粉蛋白全长或其片段。
在另一优选例中，所述的天花粉蛋白序列包括如SEQ ID NO.:9所示的野生型全长序列
(MDVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYADETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYERLQTAAGKIRENIPLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIALLLNRNNMAGGGGEDVTPENFLAVLR)、或如SEQ ID NO.:10所示的重组型天花粉蛋白
Figure PCTCN2018116755-appb-000005
在另一优选例中，所述的天花粉蛋白的编码基因如SEQ ID NO.:11所示野生型蛋白序列。
Figure PCTCN2018116755-appb-000006
在另一优选例中，所述的肿瘤疫苗组合物还包括其他抗肿瘤药物。
在另一优选例中，所述其他抗肿瘤药物包括抗PD-1抗体、抗PD-L1抗体。
在另一优选例中，所述抗PD-1抗体包括：PD-1/PD-L1检查点的抗体和小分子抑制剂，CTLA4抗体，或其他作用于免疫细胞的抗体。
本发明第十一方面，提供了一种体外非治疗性抑制肿瘤细胞的方法，包括步骤：向肿瘤细胞培养物中加入本发明第四方面所述的树突状细胞、本发明第五方面所述的融合蛋白或本发明第七方面所述的肿瘤疫苗组合物，从而抑制肿瘤细胞。
本发明第十二方面，提供了一种治疗肿瘤的方法，包括步骤：向需要的对象施用安全有效量的本发明第四方面所述的树突状细胞、本发明第五方面所述的融合蛋白或本发明第九方面所述的肿瘤疫苗组合物。
在另一优选例中，所述所需要的对象为哺乳动物，包括小鼠、大鼠、或人，优选为人。
本发明第十三方面，提供了一种药物组合物或制剂，其特征在于，包括：
(a)第一活性成分天花粉蛋白或其编码基因；
(b)第二活性成分抗PD-1抗体；和
(c)药学上可接受的载体。
在另一优选例中，所述药物组合物或制剂中，所述(a)包括如本发明第五方面所述的融合蛋白。
在另一优选例中，所述药物组合物或制剂包括：将(a)载入载体脂质体，而获得的脂质体。
在另一优选例中，所述脂质体的水力直径为50-200nm，较佳地80-150nm，更佳地100-120nm，最佳地110nm。
在另一优选例中，所述药物组合物或制剂中，所述(b)包括：PD-1/PD-L1检查点的抗体和小分子抑制剂，CTLA4抗体，或其他作用于免疫细胞的抗体。
在另一优选例中，所述药物组合物或制剂中，所述(a)和(b)的总重量占所述药物组合物或制剂总重量的0.1-99.9wt％,较佳地10-99.9wt％，更佳地70％-99.9wt％。
在另一优选例中，所述药物组合物为液态、固体、或半固体。
在另一优选例中，所述的药物组合物的剂型为口服剂型、注射剂、或外用药物剂型。
在另一优选例中，所述药物组合物的剂型包括胶囊、口服液、或注射剂。
在另一优选例中，所述药物组合物或制剂为液态制剂。
在另一优选例中，所述组合物为口服制剂。
在另一优选例中，所述的载体选自下组：输液剂载体和/或注射剂载体，较佳地，所述的载体是选自下组的一种或多种载体：生理盐水、葡萄糖盐水、或其组合。
在另一优选例中，所述组合物或制剂可单独使用，或联合使用。
在另一优选例中，所述的联合使用包括：与其它治疗和/或预防肿瘤的药物联合使用。
本发明的第十四方面，提供了一种治疗和/或预防肿瘤的方法，其特征在于，所述方法包括步骤：向需要治疗和/或预防肿瘤的受试者施用治疗有效量的如本发明第三方面所述的体系、如本发明第五方面所述的融合蛋白、如本发明第六方面所述的多核苷酸、如本发明第七方面所述的载体、如本发明第八方面所述的宿主细胞、如本发明第九方面所述的肿瘤疫苗组合物，或如本发明第十三方面所述的药物组合物或制剂。
在另一优选例中，所述所需要的对象为哺乳动物，包括小鼠、大鼠、或人，优选为人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为根据发明制备实施例1的TCS-抗原肽融合蛋白纯化后的电泳图。
图2为根据发明制备实施例1中TCS-抗原肽融合蛋白的纯化色谱图。
图3为根据发明制备实施例2中穿膜肽-TCS-抗原肽融合蛋白纯化后的电泳图。
图4为根据发明制备实施例2中穿膜肽-TCS-抗原肽融合蛋白的纯化色谱图。
图5为根据发明制备实施例1和2中制备所得纯化后的TCS-抗原肽、穿膜肽-TCS-抗原肽的电泳图。
图6为根据实验实施例1中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽对树突状细胞DC2.4的增殖的抑制效果。
图7为根据实验实施例2中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在树突状细胞DC2.4细胞的细胞摄取情况荧光图。
图8为根据实验实施例2中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在树突状细胞DC2.4细胞的细胞摄取情况的流式分析图。
图9为根据本发明实验实施例3中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在体外致敏BMDC的CD80图。
图10为根据本发明实验实施例4中TCS和TCS-抗原肽及穿膜肽-TCS-抗原 肽在体外诱导T细胞增殖图。
图11为根据实验实施例5中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在CT26皮下荷瘤小白鼠模型中体内抗肿瘤治疗中的肿瘤体积增长曲线图。
图12为根据本发明实验实施例5中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在CT26皮下瘤荷瘤小白鼠模型中体内抗肿瘤治疗终点时各治疗组肿瘤照片。
图13为根据本发明实验实施例5中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在CT26皮下荷瘤小白鼠模型中体内抗肿瘤的各治疗组的动物体重变化曲线图。
图14为根据本发明实验实施例5中TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽在CT26皮下荷瘤小白鼠模型中体内抗肿瘤的各治疗组的动物生存曲线图。
图15为根据本发明实验实施例6的给药过程中的肿瘤体积增长图。
图16为根据本发明实验实施例6的给药过程中各组别小鼠的体重变化图。
图17为根据本发明实验实施例6的给药过程中各组别小鼠的生存曲线。
图18为根据本发明实验实施例7的给药过程中的肿瘤体积增长图。
图19为根据本发明实验实施例7的给药过程中各组别小鼠的体重变化图。
图20为根据本发明实验实施例7的治疗终点各组别小鼠的脾脏中CD8 +T细胞数量的变化。
图21为根据本发明实验实施例7的治疗终点各组别小鼠的肿瘤组织中CD8 +T细胞数量的变化。图22为制备的TCS-抗原肽脂质体的粒径图。
图23为制备的TCS-抗原肽脂质体纯化后的SDS-PAGE电泳图。
图24为根据本发明实施例8的给药过程中的肿瘤体积增长图。
图25为根据本发明实施例8的给药过程中各组别小鼠的体重变化图。
图26为根据本发明实施例8的各组别小鼠的生存曲线。
图27为根据本发明实施例8的治疗终点各组别小鼠的淋巴结中CD8 +T细胞数量的变化。
图28为根据本发明实施例8的治疗终点各组别小鼠的肿瘤组织中CD8 +T细胞数量的变化。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，天花粉蛋白可以有效激活和/或致敏树突状细胞，从而加强其抗原提呈能力，而含有肿瘤抗原和天 花粉蛋白的融合蛋白能够经过树突状细胞的加工以及提呈后，特异性激活体内针对肿瘤的免疫系统，达到抑制肿瘤生长以及扩散的作用，从而成为肿瘤的治疗性疫苗。而天花粉蛋白安全性高，毒副作用小，是理想的肿瘤治疗性疫苗制剂的组成部分。在此基础上，完成了本发明。
天花粉蛋白
天花粉蛋白是从中华民族丰富的医药宝库中发掘出来的抗孕的新药。而天花粉蛋白是从葫芦科植物栝楼的根提取出来的一种蛋白。且经过50多年临床实践，证明了天花粉蛋白是安全有效的中期引产药。天花粉蛋白TCS是分子量为27kDa的I型核糖体失活蛋白，它具有N-糖苷酶活性，能够识别哺乳动物细胞的核糖体大亚基并使其脱嘌呤，从而抑制细胞的蛋白质合成，导致细胞死亡。因其具有较强的免疫原性，同时，其分子量较小，容易被肾清除，缺乏一定的肿瘤靶向性。这些都限制了它在临床上的应用。
本发明中的天花粉蛋白为已批准上市的中期引产药物，其人体应用的安全性已得到确证。故可以合理充分利用，将其作为一种新型的肿瘤疫苗组分，既可以保证其安全性和有效性，又可以拓宽该蛋白的临床应用范围。
可用于本发明的天花粉蛋白没有特殊限制，可以包括野生型和重组型的天花粉蛋白及其活性片段，只要该蛋白或其片段具有或基本具有(如保留了70％、75％、80％、85％、90％、95％、99％或100％)N-糖苷酶活性即可。
优选地，可用于本发明的天花粉蛋白如SEQ ID NO.:9所示(野生型)，也可以是如SEQ ID NO.:10所示蛋白(重组型)，其中编码SEQ ID NO.:9所示蛋白的核苷酸序列如SEQ ID NO.:11所示。
融合蛋白及其制备
在本发明中，“融合蛋白”、“本发明蛋白”、“本发明融合蛋白”、“天花粉蛋白-肿瘤抗原融合蛋白”、“TSC-抗原肽”可互换使用，指具有式Ia或式Ib所述结构，
T-E1-P-E2-A 式Ia，
A-E1-P-E2-T 式Ib；
即含有包括天花粉蛋白元件和肿瘤抗原相偶联的融合蛋白，任选地，所述的融合蛋白还可以含穿膜元件(T)、和任选的连接肽、标签序列、信号肽和/或 1-3个氨基酸残基。本发明蛋白可以是单体或由单体形成的多聚体(如二聚体)。此外，应理解，所述术语还包括融合蛋白的活性片段和衍生物。
就连接顺序而言，本发明融合蛋白没有特殊限制，可以从N端-C端或C端-N端的顺序构建本发明融合蛋白；当所述融合蛋白中含有穿膜肽时，优选将穿膜肽和肿瘤抗原构建于所述融合蛋白的两端，而天花粉蛋白构建于穿膜肽与肿瘤抗原之间。
一种优选的融合蛋白为分离的重组融合蛋白序列如SEQ ID NO.:1、SEQ ID NO.:2和12所示。
本发明融合蛋白具有激活和/或致敏树突状细胞(DC)细胞，并增强其对肿瘤抗原的加工与提呈的作用。本发明还包括根据本发明融合蛋白的活性片段、衍生物和类似物。如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持其对肿瘤抗原的加工与提呈增强作用的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或几个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)融合蛋白与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合于此多肽序列而形成的多肽(与前导序列、分泌序列或6His等标签序列融合而形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
一类优选的活性衍生物指与式Ia或式Ib的氨基酸序列相比，有至多3个，较佳地至多2个，更佳地至多1个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表1进行氨基酸替换而产生，优选地，所述的衍生物不含有Cys。
表1
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供本发明融合蛋白的类似物。这些类似物与SEQ ID NO:.3所示的多肽的差别可以是氨基酸序列上的差异，也可以是不影响序列的修饰形式上的差异，或者兼而有之。类似物还包括具有不同于天然L-氨基酸的残基(如D-氨基酸)的类似物，以及具有非天然存在的或合成的氨基酸(如β、γ-氨基酸)的类似物。应理解，本发明的多肽并不限于上述例举的代表性的多肽。
修饰(通常不改变一级结构)形式包括：体内或体外的多肽的化学衍生形式如乙酰化或羧基化。修饰还包括糖基化，如那些在多肽的合成和加工中或进一步加工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。修饰形式还包括具有磷酸化氨基酸残基(如磷酸酪氨酸，磷酸丝氨酸，磷酸苏氨酸)的序列。还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明多肽还可以以由药学上或生理学可接受的酸或碱衍生的盐形式使用。这些盐包括(但不限于)与如下酸形成的盐：氢氯酸、氢溴酸、硫酸、柠檬酸、酒石酸、磷酸、乳酸、丙酮酸、乙酸、琥珀酸、草酸、富马酸、马来酸、草酰乙酸、甲磺酸、乙磺酸、苯磺酸、或羟乙磺酸。其他盐包括：与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以及以酯、氨基甲酸酯或其他常规的“前体药物”的形式。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的重组融合蛋白”是指重组融合蛋白基本上不含天然 与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化重组融合蛋白。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的主带。
编码多核苷酸、载体、宿主及融合蛋白的合成
编码本发明融合蛋白的本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码多肽的功能。
本发明融合蛋白或其元件的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明融合蛋白的编码基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋 白质的方法。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列来表达或生产重组蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CH、NSO、COS7、或293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明还提供了所述融合蛋白的前体蛋白，所述前体蛋白利用了内含肽介导的融合蛋白获得方式，在融合蛋白的基因构建物中加入了内含肽的编码基因，并在表达蛋白后通过标签剪切，直接获得了高纯度的融合蛋白，从而无需进一步采用其它分离或纯化步骤。
穿膜肽
本发明提供的融合蛋白中，优选含有穿膜肽。穿膜肽是从自然中发现或通过人工筛选得到的一类具有穿透细胞膜活性的多肽。可用于本发明的穿膜肽没有特殊限制，可以为任何富含碱性氨基酸的穿膜肽和具有两亲性的穿膜肽，只要可以促进细胞对本身缺乏入胞能力的TCS摄取。优选地，本发明的穿膜肽包括低分子量鱼精蛋白LMWP(VRRRRRRGGRRRR(SEQ ID NO.:4))、TAT(YGRKKRRQRRR(SEQ ID NO.:5))、R9(RRRRRRRRR)本发明穿膜肽不仅能够自己入胞，还能携带本发明TCS进入细胞，尤其是树突状细胞。此外，本发明穿膜肽可通过在表达融合蛋白的表达载体中导入穿膜肽的基因直接获得含穿膜肽的融合蛋白，或也可以通过内含肽介导而获得有穿膜肽修饰的方法，优选的方法包括：
(a)将重组质粒转化大肠杆菌BL21(DE3)感受态细胞；
(b)将含有重组质粒的菌株用LB培养基培养至对数生长期，加入异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导目的蛋白表达；
(c)离心收集菌体并超声破碎；
(d)将含有C端融合内含肽与CBD标签的目的蛋白的上清液用几丁质柱亲和纯化，洗去杂蛋白后，用含有2-巯基乙烷磺酸纳(MESNA)的缓冲液进行柱上切割过夜，收集洗脱液；
(e)超滤浓缩后，加入上述穿膜肽，反应过夜，形成TCS-穿膜肽连接物，其中蛋白与多肽的摩尔比为1:20；用脱盐柱除去溶液中多余的多肽，从步骤(e) 中得到的TCS-穿膜肽连接物产率为90％-95％。
树突状细胞
树突状细胞(Dendritic cells,DC)是机体功能最强的专职抗原递呈细胞(Antigen presenting cells,APC)，它能高效地摄取、加工处理和递呈抗原，未成熟DC具有较强的识别和吞噬能力，成熟DC能有效激活初始型T细胞，处于启动、调控、并维持免疫应答的中心环节。
人体内大部分DC处于非成熟状态，表达低水平的共刺激因子和粘附因子，体外激发同种混合淋巴细胞增殖反应的能力较低，但未成熟DC具有极强的抗原吞噬能力，在摄取抗原(包括体外加工)或受到某些因素刺激时即分化为成熟DC，而成熟的DC表达高水平的共刺激因子和粘附因子。DC在成熟的过程中，由接触抗原的外周组织迁移进入次级淋巴器官，与T细胞接触并激发免疫应答。DC作为目前发现的功能最强的APC，能够诱导特异性的细胞毒性T淋巴细胞(cytotoxic T lymphocyte，CTL)生成。近年来研究表明,应用肿瘤相关抗原或抗原多肽体外冲击致敏DC，回输或免疫接种于载瘤宿主，可诱发特异性CTL的抗肿瘤免疫反应。
而采用本发明组合物或融合蛋白对树突状细胞进行共培养，能够激活和/或致敏未成熟状态的树突状细胞成为成熟状态的树突状细胞，使其抗原提呈能力显著增强，具体表现为：
(1)CD80的表达量上升了至少20-100％，优选至少为25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％；和/或
(2)刺激T细胞分泌IFN-γ的量上升了至少20-100％，优选至少为25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。
可用于本发明的树突状细胞没有特别限制，可以为来源于所需治疗对象本身的树突状细胞，其可采用常规方法获得或经市售购买，一种优选的分离方式包括通过常规方法从哺乳动物的骨髓中分离筛选获得。
优选的树突状细胞来源于哺乳动物，例如小鼠、大鼠、人类等。
通常，足以激活机体肿瘤免疫系统的所需树突状细胞大约为1-5×10 5-6个/剂。
肿瘤抗原
通常，可用于本发明的肿瘤抗原没有特别限制，包括主要从肿瘤细胞获取肿瘤基因，通过基因重组方式构建质粒并转化至表达载体中(如大肠杆菌)表达，具体的表达方法如本领域技术人员所熟知。片段长度一般为10-50bp。
优选地，本发明肿瘤抗原来源于以下肿瘤：包括头颈癌、甲状腺癌、脑部恶性肿瘤、肺癌、皮肤癌、黑色素瘤、胃癌、胰腺癌、肝癌、胆囊癌、结直肠癌、乳腺癌、卵巢癌、子宫颈癌、子宫内膜癌、睾丸癌、膀胱癌、肾癌、或骨肉瘤。优选地，本发明的肿瘤抗原来源于结直肠癌、乳腺癌、黑色素瘤或肺癌。
一种优选的肿瘤抗原包括如SEQ ID NO.:6、7或8所述的抗原，为从小鼠肺上皮TC-1细胞诱导的肿瘤细胞中获取的基因所构建的抗原肽，片段长短不一为14-39bp。
此外，还可以从常见的肿瘤细胞系中提取肿瘤抗原，例如黑色素瘤B16细胞和/B16-F10细胞，人卵巢癌SKOV-3细胞和人卵巢癌ES2细胞，结直肠癌细胞HCT116等。
激活和/或致敏组合物
本发明还提供了一种将天花粉蛋白以及肿瘤抗原进行物理混合后的组合物，用于作为激活和/或致敏DC细胞的激活和/或致敏组合物。
在本发明组合物中，天花粉蛋白以及肿瘤抗原的比例为0.5:1-1:50，优选地为1:1-1：10，通常采用冰浴物理混合，混合条件-20℃～4℃并在相同条件下保存。本发明组合物优选为液体形式，也可以制成注射剂，例如液体溶液或悬液；还可制成在注射前适合配入溶液或悬液中、液体载体的固体形式。
此外，除了作为活性成分的天花粉蛋白以及肿瘤抗原以外，该组合物还可以含有维持活性成分功能的药学上可接受的载体。术语“药学上可接受的载体”指用于治疗剂(例如本发明组合物)给药的载体。该术语指这样一些药剂载体：它们本身不诱导产生对接受该组合物的个体有害的抗体，且给药后没有过分的毒性。这些载体是本领域普通技术人员所熟知的。在Remington's Pharmaceutical Sciences(Mack Pub.Co.，N.J.1991)中可找到关于药学上可接受的载体的充分讨论。这类载体包括如水、盐水、甘油和乙醇。另外，这些载体中还可能存在辅助性的物质，如润湿剂或乳化剂、pH缓冲物质等。此外，免疫组合物中还可以含有免疫佐剂。
树突状细胞激活和/或致敏体
本发明提供了一种含有天花粉蛋白、肿瘤抗原以及分离的树突状细胞的激活和/或致敏体系。
在该体系中，所述的天花粉蛋白、肿瘤抗原可以以物理混合(即本发明激活和/或致敏组合物)或偶联形成融合蛋白的形式存在。其中，有效量的树突状细胞通常含有1-5×10 5-6个细胞/剂，优选为1-2×10 5-6个细胞/剂，而其与本发明组合物的比例为10 5-5×10 5；与本发明融合蛋白的比例10 5-5×10 5，当细胞数量为10 5-5×10 5时，蛋白的终浓度为1μM。
本文所用的术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。
应用
本发明组合物、融合蛋白可用于体外对树突状细胞的培养，例如将本发明组合物、融合蛋白与一定量的树突状细胞在合适的条件下进行共培养，从而激活和/或致敏树突状细胞，并将近树突状细胞处理的抗原用于T细胞的激活，从而建立针对特定肿瘤类型的细胞免疫，以达到肿瘤治疗性疫苗的效果。
通用方法：
试剂和药品
原核表达载体pMXB10购自New England Biolabs,TCS-抗原肽融合蛋白及穿膜肽-TCS-抗原肽融合蛋白的重组表达质粒由上海捷瑞生物科技有限公司代为构建,流式抗体(Anti-Mouse CD80)和(Anti-Mouse CD8)购自BD Bioscience。鼠源结肠癌CT26，鼠源黑色素瘤细胞B16-F10，鼠源树突状细胞DC2.4均购自中国科学院细胞库。其它实验所用材料均来源于常规市售产品。
制备实施例1
融合蛋白1(TCS-抗原肽蛋白)的制备
TCS-抗原肽蛋白的原核表达和纯化
a：代构的质粒TCS-抗原肽转化至大肠杆菌BL21(DE3)感受态细胞中。
b：将含有重组质粒TCS-抗原肽的菌株转至含有100μg/ml Amp的LB培养基中，于37℃恒温摇床中220rpm培养至对数生长期(600nm吸光值为0.6-0.8)，加入终浓度为1mM的IPTG，于25℃，150rpm表达过夜(16h)。
c：用离心机4℃，9,000rpm离心3min，收集菌体。
d:用HEPES缓冲液(含20mM HEPES，150mM NaCl，1mM EDTA，0.5‰吐温20，pH 8.5)将菌体重悬。
e:用探头超声破碎仪以400W功率超声破碎细胞35min。
f:12,000rpm，4℃离心30min，收集上清液。
g：将含有目的蛋白的上清液过用HEPES缓冲液预平衡的几丁质柱，流速为1ml/min。上样完成后，用25倍柱体积的HEPES缓冲液洗去非特异性结合的杂蛋白。
h：用3倍柱体积的含有50mM半胱氨酸的HEPES缓冲液流经柱子，并保留少量缓冲液，关闭柱子出口，在柱上切割过夜(16h)。
i：打开柱子出口，收集流出的含有目的蛋白的缓冲液，并继续加入3倍柱体积的HEPES缓冲液，继续将目的蛋白洗脱下来。
J：将收集到的蛋白洗脱液用截留分子量为10,000的超滤管进行浓缩，并用脱盐柱除去溶液中多余的半胱氨酸，得到目的重组蛋白TCS-抗原肽。(图1为SDS-PAGE电泳图、图2为经脱盐柱纯化图。)
制备实施例2
融合蛋白2(穿膜肽-TCS-抗原肽蛋白)的制备
穿膜肽-TCS-抗原肽蛋白的原核表达和纯化
a：将代构的质粒穿膜肽-TCS-抗原肽转化至大肠杆菌BL21(DE3)感受态细胞中。
b：将含有重组质粒穿膜肽-TCS-抗原肽的菌株转至含有100μg/ml Amp的LB培养基中，于37℃恒温摇床中220rpm培养至对数生长期(600nm吸光值为0.6-0.8)，加入终浓度为1mM的IPTG，于37℃，220rpm表达过夜(16h)。
c：用预冷过的离心机4℃，9,000rpm离心3min，收集菌体。
d：用HEPES缓冲液(含20mM HEPES，150mM NaCl，1mM EDTA，0.5‰吐 温20，pH 8.5)将菌体重悬。
e：用探头超声破碎仪以400W功率超声破碎细胞35min。
f：12,000rpm，4℃离心30min，收集上清液。
g：将有目的蛋白的上清液过用HEPES缓冲液预平衡的几丁质柱，流速为1ml/min。上样完成后，用25倍柱体积的HEPES缓冲液洗去非特异性结合的杂蛋白。
h：用3倍柱体积的含有50mM半胱氨酸的HEPES缓冲液流经柱子，并保留少量缓冲液，关闭柱子出口，在柱上切割过夜(16h)。
i：打开柱子出口，收集流出的含有目的蛋白的缓冲液，并继续加入3倍柱体积的HEPES缓冲液，继续将目的蛋白洗脱下来。
J：将收集到的蛋白洗脱液用截留分子量为10,000的超滤管进行浓缩，并用脱盐柱除去溶液中多余的半胱氨酸，得到目的重组蛋白TCS-抗原肽。(图3为SDS-PAGE电泳图、图4为经脱盐柱纯化图。)
实验实施例1
MTT(3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴蓝，商品名：噻唑蓝)法分别测定重组蛋白类药物TCS、TCS-抗原肽、穿膜肽-TCS-抗原肽的细胞毒作用将对数生长期的鼠源树突状细胞DC2.4细胞消化并计数，稀释成密度为4×10 4个细胞/mL的细胞悬液，转移到96细胞培养孔板中，每孔加入100μL细胞悬液，用含10％小牛血清的DMEM完全培养基培养12h(37℃，5％CO 2)。
通过预实验确定最佳药物浓度范围，加入不同浓度的溶液，每个浓度做6个复孔。培养48小时，加入MTT(5mg/ml，购自美国Sigma-Aldrich公司)20μl，培养4_h，小心吸取培养上清，每孔加入DMSO 200μL，轻摇震荡使结晶物充分溶解混匀。用酶标仪(Thermo Scientific)测定各组的OD值，主波长为490nm，参考波长为570nm。计算各组的细胞存活率：细胞存活率(％)＝实验组平均OD值/对照组平均OD值×100％。
结果如下：
TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽对树突状细胞DC2.4的存活率的影响：三者对树突状细胞DC2.4的生存率均没有明显的影响，这证明了抗原肽和/或TCS对树突状细胞的细胞毒性不大(图6)。且在相同的低浓度蛋白条件下，此重组蛋白药物不会抑制树突状细胞的增殖，因此该药物组合是安全的。
实验实施例2
TCS和TCS-抗原肽及穿膜肽-TCS-抗原肽融合蛋白在树突状细胞DC2.4细胞中的细胞摄取情况
1.蛋白药物的罗丹明(Rhodamine B)荧光标记
将制备实施例1、2中得到的融合蛋白1(TCS-抗原肽融合蛋白)、融合蛋白2(穿膜肽-TCS-抗原肽融合蛋白)及我们之前构建的重组天花粉蛋白TCS与摩尔比3倍过量的罗丹明B混合，于4℃避光反应过夜。用脱盐柱除去多余的罗丹明B荧光素。用BCA法测定蛋白的浓度。
2.树突状细胞DC2.4中的细胞摄取实验
将对数生长期的鼠源树突状细胞DC2.4细胞消化，细胞计数，调整浓度制备成5×10 4个细胞/mL的细胞悬液，接种至12孔细胞培养板内，每孔加入1mL。待细胞长到适宜密度后进行摄取实验。加入罗丹明B标记的各蛋白药物1μM，继续在培养箱中培养4小时。弃去培养基，并用PBS洗涤三次，加4％多聚甲醛固定12分钟，PBS洗涤三次，然后DAPI染色10分钟，PBS洗涤三次，用荧光显微镜(Zeiss)进行拍摄(图7)。另取细胞约1×10 5个细胞/mL的细胞悬液，按照上述方法进行摄取实验，实验终点PBS洗三次后，用0.25％胰酶消化分散后用流式细胞仪(BD Pharmingen)的FL2通道检测细胞摄取情况(图8)。
结果如下：
从图7、图8中可见，TCS本身入胞较少，TCS-抗原肽融合蛋白有一定程度的入胞，而穿膜肽-TCS-抗原肽融合蛋白的荧光强度有所增强。这证明了虽然TCS-抗原肽融合蛋白能够具有一定的细胞穿透能力，但融合了穿膜肽后能提高TCS的入胞效率，从而使胞内药物荧光强度更强。
实验实施例3
重组蛋白TCS和融合蛋白1(TCS-抗原肽)及融合蛋白2(穿膜肽-TCS-抗原肽)在体外致敏抗原提呈细胞实验
1.骨髓来源的树突状细胞(BMDC)的提取
取6-8周大的C57BL/6小鼠作为骨髓细胞来源。实验前将所用器械经高温高压灭菌处理。小鼠安乐死后浸泡于75％的乙醇中10分钟,剥离出股骨和胫骨，并浸泡于75％的乙醇中5分钟,无菌PBS中浸泡5分钟，重复2次。剪去股骨 和胫骨的两端，使用1mL注射器吸取无血清DMEM培养基，将骨髓冲出，1800rpm离心5分钟，弃上清。加入3至5mL的红细胞裂解液，室温静置3分钟，离心，去上清。用BMDC诱导培养基(含20ng/mL GM-CSF+10ng/mL IL-4)将细胞重新分散，转移至12孔板中，并于37℃、5％二氧化碳培养箱中培养。培养三天后更换新的培养基，共培养7天。
2.重组蛋白TCS、TCS/抗原肽(物理混合组合物)和TCS-抗原肽融合蛋白及穿膜肽-TCS-抗原肽融合蛋白致敏BMDC实验
经培养后的第5至7天可看到聚集体生成，即为树突状细胞(BMDC)。当观察到有足够多的聚集体生成时，分别加入1μM的各重组蛋白，并且以LPS为阳性对照，继续在培养箱中培养16至24小时。收集非贴壁、非增殖、成熟的BMDC，3,000rpm,离心5分钟，弃上清，并用流式抗体CD80进行标记，冰上孵育0.5至1小时，用流式细胞仪(BD Pharmingen)进行检测。
结果如下：
从图9可见实验组的每一组中CD80均明显地增强，实验组的阳性率较对照组的阳性率提升了43.7％。由于CD80是表达在抗原提呈细胞上的重要的共刺激分子，与T细胞上的CD28分子结合，激活T淋巴细胞，发挥免疫效应。用TCS/抗原肽(物理混合组合物)、TCS-抗原肽及穿膜肽-TCS-抗原肽等融合蛋白药物对抗原提呈细胞BMDC进行处理后，CD80均有明显的上调，表现在流式结果上为：用药物处理后，第一个峰值减弱，第二个峰的峰值显著增加，且两峰对比显著增强，表明阳性率增加。该结果表明所构建的重组融合蛋白以及物理混合物均可以较好地激活DC细胞，提高其抗原提呈能力，其中，重组融合蛋白更优。
实验实施例4
重组蛋白TCS、TCS/抗原肽混合物和TCS-抗原肽及穿膜肽-TCS-抗原肽在体外促进T细胞增殖实验
1.骨髓来源的树突状细胞(BMDC)的提取
取6-8周大的C57BL/6小鼠作为骨髓细胞来源。实验前将所用器械经高温高压灭菌处理。小鼠安乐死后浸泡于75％的乙醇中10分钟,剥离出股骨和胫骨，并浸泡于75％的乙醇中5分钟,无菌PBS中浸泡5分钟，重复2次。剪去股骨和胫骨的两端，使用1mL注射器吸取无血清DMEM培养基，将骨髓冲出，1800 rpm离心5分钟，弃上清。加入3至5mL的红细胞裂解液，室温静置3分钟,离心，去上清。用BMDC培养基(含20ng/mL GM-CSF+10ng/mL IL-4)将细胞重新分散，转移至12孔板中，并于37℃、5％二氧化碳培养箱中培养。培养三天后更换新的培养基，共培养5天。
2.重组蛋白TCS、TSC/抗原肽物理混合组合物和TCS-抗原肽及穿膜肽-TCS-抗原肽对T细胞的增殖实验
在培养后的第5天加入相对应的1μM的各重组蛋白，并设置不处理的BMDC细胞为空白对照，继续于培养箱中培养12至24小时。
3.脾脏内淋巴细胞的提取
取6-8周大的C57BL/6小鼠作为淋巴细胞来源。实验前将手术所用器械经高温高压灭菌处理。小鼠安乐死后浸泡于75％的乙醇中10分钟,于超净台中剥离出脾脏。此过程注意无菌操作。在35mm培养皿中放入4mL Mouse 1×淋巴细胞分离液，研磨。把悬有脾脏细胞的分离液立即转移到15mL离心管中，覆盖1mL的RPMI 1640培养基。室温，800g离心30分钟。注意设置较慢的加速度和减速度。吸出淋巴细胞层，再加入10mL RPMI 1640培养基，颠倒洗涤。室温，250g离心10分钟收集细胞。倾倒上清液，用DMEM重悬。细胞均分，加入上述培养板里，继续于培养箱中培养3天。收集上清，用ELISA试剂盒检测IFN-γ的浓度。
结果如下：
从图10中看出，BMDC细胞经各组药物处理后，与T淋巴细胞共孵育，相较于对照组，实验组的IFN-γ的浓度都有明显地提高，表明T淋巴细胞被激活并增殖，说明天花粉蛋白能够促进成熟DC与T细胞之间的抗原交叉呈递过程。
实验实施例5
重组蛋白TCS、TSC/抗原肽物理混合组合物和TCS-抗原肽及穿膜肽-TCS-抗原肽在皮下瘤模型中的微针经皮给药的体内药效情况
1.CT26皮下荷瘤小鼠模型的建立
将对数生长期的CT26细胞经0.25％胰酶消化分散后，细胞计数调整浓度制备4×10 6个细胞/mL的细胞悬液。取体质量为18-22g的Balb/c白鼠(购自上海斯莱科实验动物有限公司)25只，在白鼠背部皮下注射细胞悬液100μL/只。观察CT26细胞在Balb/c体内的生长和成瘤情况。
2.融合蛋白的抗肿瘤效应
在种瘤后第五天将实验小鼠随机分成5组：TCS组、TCS/抗原肽组、TCS-抗原肽组、穿膜肽-TCS-抗原肽组，以PBS组为阴性对照。各组均采用微针经皮方式给药。给药前，用微针预处理。给药剂量均为10mg/kg。每三天给一次药，给药次数为四次。给药期间每天监视白鼠体重变化，并测定瘤的长径(a)、短径(b)，计算肿瘤体积：V＝a×b 2/2。待PBS组的肿瘤体积到达2000mm 3时判定为死亡终点，将白鼠安乐死，取下肿瘤，小心去除瘤表面的血迹及包膜，称重。
结果如下：
图11为给药过程中的肿瘤体积增长图。从图中可以看到，相对于PBS组,TCS组和TCS/抗原肽组对肿瘤的生长有一定的抑制作用。而TCS-抗原肽组和穿膜肽-TCS-抗原肽组有更为显著的抑瘤效果。图12则直观地显示了给药完成后各组的肿瘤照片，从图中可见，经PBS处理的小鼠肿瘤明显较大，而经TCS-抗原肽及穿膜肽-TCS-抗原肽蛋白药物处理的小鼠肿瘤有不同程度的减小。
图13为给药过程中各组别小鼠的体重变化图。从图中可见，各实验组在给药过程中，实验动物的体重并未发生明显的变化，证明本发明所构建的TCS-抗原肽及穿膜肽-TCS-抗原肽融合蛋白具有良好的生物相容性，系统毒性较低。
图14为给药组各组的生存曲线，可以看出，PBS组的生存期最短，重组蛋白TCS-抗原肽组小鼠的生存期最长。这证明了本发明所构建的融合蛋白TCS-抗原肽具有延长生存期的作用，同时也证明了重组蛋白TCS-抗原肽的抗肿瘤作用。
实验实施例6
重组天花粉蛋白协同anti-PD-1抗体在皮下瘤模型中的体内药效情况
1.CT26皮下荷瘤小鼠模型的建立
将对数生长期的CT26细胞经0.25％胰酶消化分散后，细胞计数调整浓度制备4×10 6个细胞/mL的细胞悬液。取体质量为18-22g的Balb/c白鼠(购自上海斯莱科实验动物有限公司)20只，在白鼠背部皮下注射细胞悬液100μL/只。观察CT26细胞在Balb/c体内的生长和成瘤情况。
2.天花粉蛋白协同anti-PD-1抗体的抗肿瘤效应
在种瘤后的第6天将实验小鼠随机分成4组：天花粉蛋白组、anti-PD-1 组、天花粉蛋白+anti-PD-1组，以PBS组为阴性对照。天花粉蛋白和PBS采用瘤旁注射方式给药，anti-PD-1采用腹腔注射方式给药。天花粉蛋白的给药剂量为5μg/只/4天，共给药3次；anti-PD-1的给药剂量为250μg/只/2天，共给药5次。给药期间每天监视白鼠体重变化，并测定瘤的长径(a)、短径(b)，计算肿瘤体积：V＝a×b 2/2。待肿瘤体积到达2000mm 3时判定为死亡终点，将白鼠安乐死，取下肿瘤，小心去除瘤表面的血迹及包膜，称重。
结果如下：
图15为给药过程中的肿瘤体积增长图。从图中可以看到，相对于对照组,天花粉蛋白组和anti-PD-1组对肿瘤的生长均有明显的抑制作用，而天花粉蛋白+anti-PD-1组具有更为显著的抑瘤效果。这证明了本发明所构建重组天花粉蛋白可以协同PD-1/PD-L1免疫检查点治疗,提高PD-1/PD-L1免疫检查点疗法的治疗响应率，改善肿瘤的免疫治疗效果。
图16为给药过程中各组别小鼠的体重变化图。从图中可见，各实验组在给药过程中，实验动物的体重并未发生明显的变化，证明本发明所构建的重组天花粉蛋白具有良好的生物相容性，系统毒性较低。
图17为各组别小鼠的生存曲线，可以看出，对照组的生存期最短，天花粉蛋白+anti-PD-1组小鼠的生存率最高、生存期最长。这证明了本发明所构建天花粉蛋白可以协同PD-1/PD-L1免疫检查点治疗,提高PD-1/PD-L1免疫检查点疗法的治疗响应率，改善肿瘤的免疫治疗效果。
实验实施例7
重组TCS-抗原肽协同anti-PD-1抗体在皮下瘤模型中的体内药效情况
1.CT26皮下荷瘤小鼠模型的建立
将对数生长期的CT26细胞经0.25％胰酶消化分散后，细胞计数调整浓度制备4×10 6个细胞/mL的细胞悬液。取体质量为18-22g的Balb/c白鼠(购自上海斯莱科实验动物有限公司)20只，在白鼠背部皮下注射细胞悬液100μL/只。观察CT26细胞在Balb/c体内的生长和成瘤情况。
2.TCS-抗原肽协同anti-PD-1的抗肿瘤效应
在种瘤后的第6天将实验小鼠随机分成4组：TCS-抗原肽组、anti-PD-1组、TCS-抗原肽+anti-PD-1组，以PBS组为阴性对照。TCS-抗原肽和PBS采用瘤旁注射方式给药，anti-PD-1采用腹腔注射方式给药。TCS-抗原肽的给药 剂量为2μg/只/2天，共给药3次；anti-PD-1的给药剂量为200μg/只/2天，共给药5次。给药期间每天监视白鼠体重变化，并测定瘤的长径(a)、短径(b)，计算肿瘤体积：V＝a×b 2/2。待PBS组的肿瘤体积到达2000mm 3时判定为死亡终点，将白鼠安乐死，取下肿瘤，小心去除瘤表面的血迹及包膜，称重。
结果如下：
图18为给药过程中的肿瘤体积增长图。从图中可以看到，相对于对照组,TCS-抗原肽组对肿瘤的生长有一定的抑制作用，而anti-PD-1组和TCS-抗原肽+anti-PD-1组均具有显著的抑瘤效果，且TCS-抗原肽+anti-PD-1组表现出最好的抑制肿瘤生长的效果。这证明了本发明所构建TCS-抗原肽可以协同PD-1/PD-L1免疫检查点治疗,提高PD-1/PD-L1免疫检查点疗法的治疗响应率，改善肿瘤的免疫治疗效果。
图19为给药过程中各组别小鼠的体重变化图。从图中可见，各实验组在给药过程中，实验动物的体重并未发生明显的变化，证明本发明所构建的重组TCS-抗原肽具有良好的生物相容性，系统毒性较低。
图20为治疗终点各组别小鼠的脾脏中CD8 +T细胞数量的变化，可以看出，TCS-抗原肽+anti-PD-1组可以明显提高脾脏中CD8 +T细胞的比例。图21为治疗终点各组别小鼠的肿瘤组织中CD8 +T细胞数量的变化，可以看出，TCS-抗原肽+anti-PD-1组可以明显提高肿瘤组织中的CD8 +T细胞的比例。这证明了本发明所构建TCS-抗原肽可以协同PD-1/PD-L1免疫检查点治疗，通过提高脾脏和肿瘤组织中CD8 +T细胞的数量来改善肿瘤的免疫治疗效果。
实验实施例8
将重组TCS-抗原肽载入脂质体可以进一步改善重组TCS-抗原肽的肿瘤免疫治疗的疗效。
重组TCS、TCS-抗原肽、抗原肽和TCS-抗原肽脂质体在皮下瘤模型中的体内药效情况
1.TCS-抗原肽脂质体的制备
将磷脂和胆固醇溶于10ml二氯甲烷中，在水浴超声下将TCS-抗原肽注入脂质溶液中，改用探头超声3min(300W,超1.5s,停1.5s)。旋蒸除去二氯甲烷，加入9ml超纯水水化，探头超声3min(300W,超1.5s,停1.5s)。用挤压仪反复推挤得到TCS-抗原肽脂质体。
2.TCS-抗原肽脂质体的粒径表征
取1ml脂质体溶液，采用动态光散射仪测定TCS-抗原肽脂质体的水力直径。
3.TCS-抗原肽脂质体的载药量和包封率表征
将TCS-抗原肽脂质体转入超滤管，通过离心超滤除去未被包载的TCS-抗原肽。纯化后的TCS-抗原肽脂质体破乳后，采用SDS-PAGE电泳表征TCS-抗原肽的包载情况。
4.B16-F10皮下荷瘤小鼠模型的建立
将对数生长期的B16-F10细胞经0.25％胰酶消化分散后，细胞计数调整浓度制备2×10 6个细胞/mL的细胞悬液。取体质量为18-22g C57BL/6黑鼠(购自上海斯莱科实验动物有限公司)25只，在黑鼠背部皮下注射细胞悬液100μL/只。观察B16-F10细胞在C57BL/6黑鼠体内的生长和成瘤情况。
5.TCS-抗原肽脂质体的抗肿瘤效应
在种瘤后的第10天将实验小鼠随机分成5组：重组TCS组、TCS-抗原肽组、抗原肽组、TCS-抗原肽脂质体组，以PBS组为阴性对照。各组均采用皮下注射方式给药。给药剂量为5μg TCS/只/3天，共给药4次。给药期间每天监视黑鼠体重变化，并测定瘤的长径(a)、短径(b)，计算肿瘤体积：V＝a×b 2/2。待肿瘤体积到达2000mm 3时判定为死亡终点，将黑鼠安乐死，取下肿瘤，小心去除瘤表面的血迹及包膜，称重。
结果如下：
图22为制备的TCS-抗原肽脂质体的粒径图。从图中可以看到，脂质体的水力直径为110nm，具有较好的分散度。
图23为制备的TCS-抗原肽脂质体纯化后的SDS-PAGE电泳图。从图中可以看到，制备的脂质体可以有效包载TCS-抗原肽。表明TCS-抗原肽脂质体的制备成功。
图24为给药过程中的肿瘤体积增长图。从图中可以看到，相对于对照组,重组TCS组和抗原肽组对肿瘤的生长有一定的抑制作用，而TCS-抗原肽组和TCS-抗原肽脂质体组均具有显著的抑瘤效果，且TCS-抗原肽脂质体组表现出最好的抑制肿瘤生长的效果。这证明了本发明所构建TCS-抗原肽脂质体可以改善TCS-抗原肽对肿瘤的免疫治疗效果。
图25为给药过程中各组别小鼠的体重变化图。从图中可见，各实验组在 给药过程中，实验动物的体重并未发生明显的变化，证明本发明所构建的重组TCS-抗原肽和TCS-抗原肽脂质体均具有良好的生物相容性，系统毒性较低。
图26为各组别小鼠的生存曲线，可以看出，对照组的生存期最短，TCS-抗原肽脂质体组小鼠的生存期最长。这证明了本发明所构建的TCS-抗原肽脂质体具有延长生存期的作用，同时也证明了TCS-抗原肽脂质体较TCS-抗原肽具有更强的抗肿瘤作用。
图27为治疗终点各组别小鼠的淋巴结中CD8 +T细胞数量的变化，可以看出，TCS-抗原肽脂质体组可以明显提高淋巴结中CD8 +T细胞的比例。
图28为治疗终点各组别小鼠的肿瘤组织中CD8 +T细胞数量的变化，可以看出，TCS-抗原肽脂质体组可以有效提高肿瘤组织中的CD8 +T细胞的比例。这证明了本发明所构建TCS-抗原肽脂质体可以通过提高淋巴结和肿瘤组织中CD8 +T细胞的数量来改善TCS-抗原肽的抗肿瘤效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种天花粉蛋白(trichosanthin，TCS)或其编码基因的用途，其特征在于，用于制备树突状细胞激活剂和/或致敏剂。
  2. 一种体外激活和/或致敏树突状细胞的方法，其特征在于，包括步骤：
    (a)提供一种含有天花粉蛋白和肿瘤抗原的混合物；
    (b)在(a)所述混合物的存在下，培养树突状细胞，从而激活和/或致敏树突状细胞。
  3. 一种树突状细胞激活和/或致敏体系，其特征在于，所述的体系包括：
    (i)天花粉蛋白；
    (ii)肿瘤抗原；和
    (iii)分离的树突状细胞。
  4. 如权利要求3所述的体系，其特征在于，所述体系中的天花粉蛋白和肿瘤抗原经物理混合或互相偶联。
  5. 一种激活和/或致敏的树突状细胞，其特征在于，是由权利要求2所述方法制备的。
  6. 一种融合蛋白，其特征在于，所述的融合蛋白具有式Ia或式Ib所示的结构：
    T-E1-P-E2-A式Ia，
    A-E1-P-E2-T式Ib；
    其中，元件T为任选的穿膜肽，元件A为肿瘤抗原，元件P为天花粉蛋白；E1和/或E2为任选的连接肽、标签序列、信号肽和/或1-3个氨基酸残基，“-”为肽键。
  7. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求6所述的融合蛋白。
  8. 一种载体，其特征在于，所述的载体含有权利要求7所述的多核苷酸。
  9. 一种宿主细胞，其特征在于，所述的宿主细胞含有权利要求8所述的载体，或所述的宿主细胞的基因中整合有权利要求7所述的多核苷酸。
  10. 一种肿瘤疫苗组合物，其特征在于，所述肿瘤疫苗组合物含有：
    A)权利要求6所述的融合蛋白；
    B)疫苗学上可接受的载体。
  11. 一种制备权利要求6所述融合蛋白的方法，其特征在于，包括步骤：
    (I)在合适的条件下，培养权利要求9所述的宿主细胞，从而获得表达所述的融合蛋白。
  12. 一种体外非治疗性抑制肿瘤细胞的方法，其特征在于，包括步骤：向肿瘤细胞培养物中加入权利要求5所述的树突状细胞、权利要求6所述的融合蛋白或权利要求10所述的肿瘤疫苗组合物，从而抑制肿瘤细胞。
  13. 一种药物组合物或制剂，其特征在于，包括：
    (a)第一活性成分天花粉蛋白或其编码基因；
    (b)第二活性成分抗PD-1抗体；和
    (c)药学上可接受的载体。
  14. 如权利要求13所述的药物组合物或制剂，其特征在于，所述药物组合物或制剂包括：将(a)载入载体脂质体，而获得的脂质体。
  15. 一种治疗和/或预防肿瘤的方法，其特征在于，所述方法包括步骤：向需要治疗和/或预防肿瘤的受试者施用治疗有效量的如权利要求3所述的体系、如权利要求6所述的融合蛋白、如权利要求7所述的多核苷酸、如权利要求8所述的载体、如权利要求9所述的宿主细胞、如权利要求10所述的肿瘤疫苗组合物、如权利要求13或14所述的药物组合物或制剂。
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