WO2006010308A1 - Preparation et application de vaccins therapeutiques a une tumeur - Google Patents

Preparation et application de vaccins therapeutiques a une tumeur Download PDF

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Publication number
WO2006010308A1
WO2006010308A1 PCT/CN2005/000763 CN2005000763W WO2006010308A1 WO 2006010308 A1 WO2006010308 A1 WO 2006010308A1 CN 2005000763 W CN2005000763 W CN 2005000763W WO 2006010308 A1 WO2006010308 A1 WO 2006010308A1
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cea
cells
fusion protein
protein
hsp70l1
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PCT/CN2005/000763
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English (en)
Chinese (zh)
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Xuetao Cao
Tao Wan
Yanfeng Wu
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Second Military Medical University
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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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • A61K39/464476—Heat shock proteins
    • 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
    • A61K39/46448—Cancer antigens from embryonic or fetal origin
    • A61K39/464482—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • 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
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the fields of biology and medicine. More specifically, the present invention relates to a fusion protein of human carcinoembryonic antigen (CEA) and human heat shock protein 70L1 (hereinafter referred to as Hsp70L1), a DNA sequence encoding the fusion protein, a vector containing the DNA sequence, and a host cell containing the vector , a method of genetically preparing the fusion protein, and the use of the fusion protein in the preparation of a CEA-positive tumor vaccine.
  • CEA human carcinoembryonic antigen
  • Hsp70L1 human heat shock protein 70L1
  • Carcinoembryonic antigen is a membrane-bound 180-200Kda intercellular adhesion glycoprotein that is highly expressed on a significant proportion of human tumor cell membranes, including more than 90% of colorectal cancer, gastric cancer, and pancreas. Cancer, 70% of non-small cell lung cancer and 50% of breast cancer, etc. These tumors are high-grade tumors, and their degree of malignancy is generally high and easy to metastasize and relapse.
  • traditional treatments such as surgery, chemotherapy, and radiotherapy have certain curative effects on early CEA-positive tumors.
  • chemotherapy and radiotherapy have large side effects.
  • many tumors are found late, often losing the best chance of surgical treatment. Therefore, immunization and gene therapy, which enhance the anti-tumor effect of the body's immune system to kill tumor cells and achieve the purpose of treating tumors, as a new model of tumor treatment, has become a hot spot in cancer therapy and immunology research.
  • CEA-positive tumors such as the construction of CEA recombinant virus vaccines, nucleic acid vaccines, application of suicide genes, development of anti-idiotypic antibodies, and application of cytokines.
  • Tsang will carry A recombinant vaccinia virus vaccine with HLA_A*0201-restricted nonapeptide epitope CAP-1 (CEA 6M - 613 , YLSGANLNL) is used to treat patients with CEA-positive tumors, and researchers have used a unique fowlpox virus-derived gold wire.
  • the vaccinia virus vector system ALVAC (canaryp 0X ) recombinant CAP-1 was used as a vaccine.
  • the body is immune-tolerant to CEA expressed by tumor cells in vivo.
  • a tumor patient has a strong cellular immune response against CEA, so the epitope contained in CEA is obtained in some ways. It can be effectively recognized by the body's immune system, which will cause the body's specific cellular immune response to tumors. Breaking the body's immune tolerance to CEA is the key point of CEA-positive tumor immunotherapy.
  • DC dendritic cells
  • APC antigen-presenting cell
  • the immune response is the initiator of the body's immune response and is at the center of initiation, regulation, and maintenance of the immune response.
  • DC migrates to the secondary lymphoid organs via the circulation or lymphatic system, and the processed antigen is presented to the T cells, in the process, MHCI, class II molecules, sticky one
  • auxiliary proteins such as KLH (keyhole limpet hemocyanin) to assist tumor-specific antigens to stimulate DCs, and as a result, enhance anti-tumor immune responses. Therefore, the discovery and application of highly efficient auxiliary molecules as an immunoadjuvant (Adjuvant) enhances the efficacy of tumor antigen peptide-sensitized DCs, thereby inducing stronger anti-tumor immunity, and is an important way to improve DC-based tumor immunotherapy. .
  • KLH keyhole limpet hemocyanin
  • HSP Heat Shock Protein
  • IFN- ⁇ IFN- ⁇
  • IL-12 IFN- ⁇
  • LPS Low-power protein
  • 11 1 1 (1-8 and 0 0, etc.
  • HSP Heat Shock Protein
  • APC APC will further stimulate the immune response by chemotaxis and activation of HSP, triggering antigen uptake and presentation, and the resting ⁇ cells are activated by the double stimulation of APC-presented antigen and dangerous signals, resulting in A series of immune processes such as antibody production, diseased cells and clearance of infectious agents.
  • Another object of the present invention is to provide a DNA encoding the fusion protein, a vector comprising the DNA sequence, and a host cell containing the vector.
  • Another object of the present invention is to provide a method for producing the CEA-Hsp70L1 fusion protein which is inexpensive and/or simple in steps.
  • Another object of the present invention is to provide a method for producing a CEA positive tumor therapeutic vaccine using the fusion protein.
  • a fusion protein is provided which comprises -
  • a carcinoembryonic antigen element having an amino acid sequence of a human carcinoembryonic antigen or an immunogenic fragment thereof; or a human cancer antigenic element;
  • linker peptide optionally located between (a) a carcinoembryonic antigen element or a cancer antigen element and (b) a heat shock protein element (more Preferably, the linker peptide is a linker peptide sequence of 0-20 amino acids).
  • the carcinoembryonic antigen element is selected from the group consisting of a full-length carcinoembryonic antigen, a fragment of the 576-669-containing carcinoembryonic antigen;
  • the heat shock protein element is selected from the group consisting of Hsp70 or Hsp70L1.
  • linker peptide sequence contains 4-10 amino acids.
  • the carcinoembryonic antigen element has the amino acid sequence of SEQ ID NO: 9
  • the heat shock protein element has the amino acid sequence of SEQ ID NO: 10.
  • a vector comprising the above DNA molecule and a host cell comprising the above vector are provided.
  • a method for producing a fusion protein of the present invention comprising the steps of: cultivating said host cell under conditions suitable for expression of said fusion protein, thereby expressing said fusion protein; And isolating the fusion protein.
  • a portion of said fusion protein for the preparation of a therapeutic vaccine for treating a CEA positive tumor or for sensitizing dendritic cells.
  • a dendritic cell which is a dendritic cell sensitized with the fusion protein of claim 1.
  • a CEA-positive tumor prophylactic or therapeutic vaccine comprising a safe and effective amount of dendritic cells according to the invention or T cells induced by dendritic cells or the present invention
  • the fusion protein described, as well as a pharmaceutically acceptable carrier comprising a safe and effective amount of dendritic cells according to the invention or T cells induced by dendritic cells or the present invention.
  • a method for inducing CEA-positive tumor-specific cellular immunity comprising the steps of: stimulating human peripheral blood lymphocytes in vitro using the fusion protein-sensitized dendritic cells of the present invention.
  • the stimulating step comprises:
  • the sensitized and inactivated dendritic cells are incubated with dendritic cells: T cells in a ratio of 1: 1-10000, and 10-100 U/ml of IL-2 is added to the complete medium. 10-lOOng/ml IL-7, 10-100 ng/ml IL-10, cultured for 7 ⁇ 2 days;
  • Figure 1 shows the restriction enzyme digestion of the recombinant pGEM-T-CEA plasmid. Among them, lane 1: 1 DNA/ECOT14I DNA molecular weight standard; Lane 2: pGEM-T-CEA plasmid digested with EcoRI.
  • Figure 2 shows the restriction enzyme digestion of the recombinant CEA 576 . 669 -Hsp70L1 plasmid.
  • lane 1 1 DNA/ECOT14I DNA molecular weight standard
  • Lane 2 pQE30-CEA 576 . 669 -Hsp70L1 plasmid digested with Scal-Kpnl
  • Figure 3 shows the induced expression of the recombinant CEA 576 669- Hsp70L1 fusion protein.
  • Lane 1 Protein molecular weight standard
  • Lane 2 IPTG-induced recombinant pQE30-CEA 576. 669- Hsp70Ll/M15
  • Lane 3 IPTG-induced pQE30/M15.
  • Figure 4 shows the expression of recombinant CEA 576 . 669 -Hsp70L1 protein in inclusion bodies.
  • Lane 1 protein molecular weight standard
  • Lane 2 IPTG-induced recombinant pQE30- CEA 5V6 . 669- Hsp70Ll/M15 ultrasound supernatant
  • Lane 3 IPTG-induced recombinant pQE30-CEA 576 . 669 -Hsp70Ll/M15 inclusion body
  • Figure 5 shows the DEAE column purification of recombinant CEA 576 . 669 -Hsp70L1 fusion protein.
  • Figure 6 shows SDS-PAGE identification of purified recombinant CEA 576 . 669 -Hsp70L1 fusion protein.
  • Figure 7 shows that CEA-specific CTLs were induced in vitro by CEA 576 . 669 -Hsp70L1 fusion protein-sensitized colon cancer patients (CEA + , HLA-A*0201 + ) DC.
  • Figure 8 shows the results of ELISP0T detection of antigen-specific IFN- ⁇ -depleted sputum cells.
  • Figure 9 shows that mouse spleen cells of each immunized group secrete IL-2 after being stimulated by different antigens in vitro.
  • Figure 10 shows the results of IFN- ⁇ ELISP0T after spleen cells of each immunized group were stimulated with different antigens in vitro.
  • Figure 11 shows the results of detection of CEA-specific CTLs by DC immunization sensitized by CEA 576 . 669 - Hsp70L1 fusion protein.
  • Figure 12 shows the growth of SW480 tumors in nude mice of each adoptive return group.
  • Figure 13 shows the survival curves of tumor-bearing nude mice. detailed description
  • the inventors have extensively and intensively studied to fuse the CEA gene and the heat shock protein (Hsp70L1) gene to produce a fusion protein of CEA-Hsp70L1 linked by a suitable amino acid linker.
  • the fusion protein has the biological functions of both, and has the immunogenicity of the tumor antigen CEA, and the tumor antigen CEA can be effectively sensitized to the dendritic cells by the heat shock protein, thereby producing a CEA-positive tumor vaccine. Therefore, the CEA-Hsp70L1 fusion protein obtained by the present invention can enhance the antitumor effect under the synergy of both, and provide a novel compound for the treatment of antitumor and the like.
  • the present invention has been completed on this basis. Definition
  • CEA positive tumor includes a tumor that highly expresses a CEA antigen on a tumor cell membrane, and representative examples include, but are not limited to: malignant colon cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, and the like. Tumor.
  • cancer antigen refers to a polypeptide or protein that is expressed (or highly expressed) in cancer cells but not expressed or underexpressed in normal cells. Examples of cancer antigens include, but are not limited to, HER2/neu, PSA. HPVE7.
  • CEA's accession number is gil78676.
  • heat shock protein includes heat shock proteins and heat shock protein-like proteins, representative examples Subunits include, but are not limited to, Hsp70, Hsp70Ll (Hsp70-like protein 1) and other heat shock proteins. The sequences of these heat shock proteins are available on the Genbank database.
  • the term "element” refers to a segment of a peptide that constitutes a fusion protein derived from the amino acid sequences of carcinoembryonic antigen, cancer antigen, heat shock protein. At the DNA level, a component refers to the corresponding coding sequence.
  • Hsp70L1 (Hsp70-like protein 1) as an example, it is a new molecule discovered by large-scale random sequencing from a human peripheral blood mononuclear-derived DC cDNA library (GenBank accession number AF143723), bioinformatics analysis. This indicates that the molecule has the characteristics of a typical HSP70 family and has high homology with HSP70 family members.
  • the results of the present inventors suggest that Hsp70L1 has an adjuvant-like effect similar to HSP70, which induces DC-producing cytokines, which has the characteristic of promoting the immune response to shift to the TM-type immune response, and in vivo experiments also show that Hsp70L1 cross-links with antigenic peptide.
  • the complex immunity induces an antigen-specific Th1 type immune response.
  • the terms "progeny of carcinoembryonic antigen and heat shock protein 70L1", "CEA-Hsp70L1 fusion protein” and the like are used interchangeably to refer to the amino acid sequence of a carcinoembryonic antigen element and the amino acid of the heat shock protein 70L1 element.
  • a sequence-fused protein in which a peptide sequence may or may not be linked.
  • the fusion protein may or may not have an initial methionine or signal peptide.
  • carcinoembryonic antigen element in a fusion protein refers to a portion of an amino acid sequence in the fusion protein that is substantially identical to a native or variant full length carcinoembryonic antigen or immunogenic fragment thereof.
  • the amino acid sequence has substantially the same biological activity as the native carcinoembryonic antigen.
  • a preferred carcinoembryonic antigen element is a human carcinoembryonic antigen or an immunogenic fragment thereof, more preferably a full length human carcinoembryonic antigen (e.g., SEQ ID NO: 9) or an immunogenic fragment thereof (e.g., containing 576-669) Fragment of the CEA amino acid sequence).
  • heat shock protein 70L1 element or “Hsp70L1 element” are used interchangeably to refer to a portion of the amino acid sequence in the fusion protein that is associated with a native or variant full length heat shock protein. 70L1 or an active fragment thereof has substantially the same amino acid sequence and has substantially the same biological activity as the native heat shock protein 70L1.
  • a preferred Hsp70L1 element is human heat shock protein 70L1, more preferably full length heat shock protein 70L1 (e.g., SEQ ID NO: 10) or an active fragment thereof.
  • the sequences of carcinoembryonic antigen and heat shock protein 70L1 may be derived from humans or from non-human animals. However, the natural sequence of humans is preferred.
  • linker peptide or “amino acid linker” are used interchangeably and refer to a short peptide that acts as a link between the amino acid sequence of a carcinoembryonic antigen element and the amino acid sequence of a heat shock protein element.
  • the linker peptide is usually from 1 to 20 amino acids in length, preferably from 3 to 10 amino acids, and most preferably from 4 to 6 amino acids.
  • the skilled person can follow conventional methods in the art (see, for example, PNAS 1998; 95: 5929-5934; Protein Eng, 2000; 13(5): 309-312; Protein Eng, 2003; 15(11): 871-879, etc.) Design the linker peptide.
  • the linker peptide does not affect or severely affect the amino acid sequence of the carcinoembryonic antigen element and the amino acid sequence of the Hsp70L1 element to form the correct folding and spatial conformation.
  • linker peptides include, but are not limited to, in order to facilitate protein folding into mutually independent domains, it is suitable to use a sequence such as SGGGGSGGGG as a tether; to facilitate CEA-Hsp70L1 to cleave two independent proteins
  • IEGR active X factor cleavage site
  • chymotrypsin 1 The cleavage site of papain, plasmin, fibrin, trypsin, etc. can also be designed as an amino acid linker; in order to facilitate purification, 6His can be used as a linker to purify CEA-Hsp70Ll by metal affinity chromatography.
  • the Fusion protein can also be designed as a new amino acid linker.
  • the NVVVHQAHHHHHHEFTYK linker is a fusion of a protease cleavage site (Nla protease) and a metal affinity chromatography site of 6His.
  • Another preferred mode is to directly connect the carcinoembryonic antigen element and the heat shock protein element without any linker peptide.
  • the DNA sequence encoding the fusion protein of the present invention can be fully synthesized.
  • the coding sequence of carcinoembryonic antigen and or heat shock protein 70L1 can also be obtained by PCR amplification or synthesis, and then spliced together to form a DNA sequence encoding the fusion protein of the present invention.
  • the DNA sequence encoding the novel fusion protein of the present invention After obtaining the DNA sequence encoding the novel fusion protein of the present invention, it is ligated into a suitable expression vector and transferred to a suitable host cell. Finally, the transformed host cells are cultured, and the novel fusion protein of the present invention is obtained by isolation and purification.
  • vector as used herein includes plasmids, cosmids, expression vectors, cloning vectors, viral vectors and the like.
  • Representative states include, but are not limited to, vectors that can be expressed in eukaryotic cells such as CHO, COS series, eukaryotic cells, vectors that can be expressed in Saccharomyces cerevisiae or Pichia pastoris, insect cells that can be found in silkworms, etc.
  • a vector expressed in and a prokaryotic expression vector include eukaryotic cells such as CHO, COS series, eukaryotic cells, vectors that can be expressed in Saccharomyces cerevisiae or Pichia pastoris, insect cells that can be found in silkworms, etc.
  • a vector expressed in and a prokaryotic expression vector include eukaryotic cells such as CHO, COS series, eukaryotic cells, vectors that can be expressed in Saccharomyces cerevisiae or Pichia pastoris, insect cells that can
  • various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then a nucleotide sequence encoding a novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence.
  • the signal peptide DNA is expressed as a precursor and is involved in the secretion of the polypeptide, then the signal peptide (secretion leader sequence) DNA is operably linked to the polypeptide DNA; if the promoter controls the transcription of the sequence, then it is operably linked to A coding sequence; if the ribosome binding site is placed at a position that enables translation, then it is operably linked to the coding sequence.
  • “operably connected” means adjacent, and for a leader sequence means adjacent in the reading frame.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the host cell is a eukaryotic cell, more preferably a silkworm cell.
  • the cell can be cultured under conditions suitable for expression of the fusion protein of the present invention to express the fusion protein.
  • the recombinant 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, ultra-treatment, ultra-centrifugation, 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.
  • the CEA-Hsp70L1 fusion protein of the present invention has the function of inhibiting the growth of tumor neovascular endothelial cells by CEA, and the effect of inducing apoptosis of tumor cells by Hsp70L1.
  • a pharmaceutical composition in another aspect of the invention, comprises an effective amount of the novel CEA-Hsp70L1 fusion protein of the present invention, or dendritic cells sensitized with the fusion protein or T cells induced by dendritic cells, and at least one pharmaceutically acceptable Accepted carrier, diluent or excipient.
  • the active ingredient is usually mixed with excipients, or diluted with excipients, or enclosed in a carrier in the form of a capsule or sachet.
  • the excipient acts as a diluent, it can be a solid, semi-solid or liquid material as a vehicle for the excipient, carrier or active ingredient.
  • the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like.
  • suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like.
  • the preparation may also include a wetting agent, an emulsifier, a preservative (e.g., methyl and propyl hydroxybenzoate), a sweetener, and the like.
  • the preferred form is a liquid dosage form.
  • composition can be formulated in unit or multi-dose form.
  • Each dosage form contains a predetermined amount of active material calculated to produce the desired therapeutic effect, as well as suitable pharmaceutical excipients.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • a safe and effective amount of a fusion protein of the invention or a corresponding dendritic cell or tau cell is administered to a human, wherein the safe and effective amount is usually at least about 1 microgram of fusion protein per kilogram of body weight, and in most In the case of no more than about 8 mg of fusion protein per kilogram of body weight, preferably the dose is about 1 microgram to 1 milligram of fusion protein per kilogram of body weight; alternatively, usually 10 5 to 10 14 cells per person per time, more preferably 10 ⁇ -10 12 cells/person/time.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the fusion protein of the present invention may also be combined with other therapeutic agents, including but not limited to: various cytokines such as IFN, TNF, IL-2, etc.; various tumor chemotherapy drugs, such as 5-Fu , drugs such as methotrexate that affect nucleic acid biosynthesis, alkylating agents such as nitrogen mustard and cyclophosphamide, drugs such as doxorubicin and actinomycin D that interfere with transcriptional processes to prevent RNA synthesis, vincristine, hi-tree Alkali-like drugs that affect protein synthesis and certain hormones and other drugs.
  • various cytokines such as IFN, TNF, IL-2, etc.
  • various tumor chemotherapy drugs such as 5-Fu
  • drugs such as methotrexate that affect nucleic acid biosynthesis
  • alkylating agents such as nitrogen mustard and cyclophosphamide
  • drugs such as doxorubicin and actinomycin D that interfere with transcriptional processes to prevent RNA synthesis, vincristine, hi-
  • CEA-Hsp70L1 fusion protein which has both the immunogenicity of CEA and the immunoadjuvant effect of Hsp70L1, is a novel drug with dual advantages for treating tumors, and thus can more specifically induce antigen-specificity. Sexual T cell immune response.
  • the fusion protein of the present invention sensitizes DC, and can effectively recognize, process, and process the CEA-specific antigen peptide, and the T cells presented to the body stimulate a stronger CEA-specific T cell immune response.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions, such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, The conditions stated in 1989), or in accordance with the conditions recommended by the manufacturer. 669 - - Hs P 70Ll Protein Expression and Purification of Recombinant CEA 576 cases of Embodiment 1
  • the CEA-positive human colon cancer cell line LS 174T (ATCC Number: CL-188) was collected, and the total RNA was prepared by using the total RNA extraction reagent Trizol (Invitrogen), and the first strand was synthesized by AMV reverse transcriptase (Promega). This is a template and amplified with PCR oligonucleotide primers at the 5' and 3' ends of the sequence to obtain a DNA fragment encoding CEA.
  • the 5' oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a cleavage site of a Kpn I restriction endonuclease, and after the cleavage site is a partial coding sequence of a translation initiator and CEA;
  • the 3' primer sequence is:
  • the primer contains a restriction endonuclease of the Sal I restriction endonuclease, a translation terminator and a partial coding sequence of CEA.
  • the reaction parameters were: 95 ° C for 30 seconds, 62. 5 ° C for 30 seconds, 72 ° C for 50 seconds, 28 cycles and 72 ° C for 10 minutes, and the expected product size was 2109 bp.
  • the reaction volume was 50 ⁇ l, the primer concentration was 200 nM, the dNTP concentration was 200 ⁇ , and the magnesium ion concentration was 1.5 mM.
  • the amplified product was analyzed by 0.8% agarose gel electrophoresis.
  • the PCR product was recombined in a conventional manner with a commercially available pGEM-T vector (purchased from Promega) and transformed into competent E. coli BL21, and the positive clone was picked and identified, purified and sequenced (ABI's Model 377 sequencer, BigDye Terminator reagent). Box, PE company). Perform fully automated DNA sequencing. It was confirmed by sequencing that the designed sequence correct CEA coding sequence has been inserted. The recombinant was recorded as pGEM-T-CEA.
  • the 5' oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a restriction endonuclease of the restriction endonuclease
  • the 3' primer sequence is:
  • This primer contains a cleavage site for the Sal I restriction endonuclease.
  • Reaction parameters 95 ° C for 15 seconds, 56. 5 ° C for 30 seconds, 72. C45 seconds, extended at 72 ° C for 10 minutes after 29 cycles, product The expected size was 276 bp, and the PCR product was purified and then digested with Sail, and then the digested product was purified.
  • HeLa cells ATCC Number: CCL-2
  • total RNA was prepared by using total RNA extraction reagent TrizoUlnvitrogen, and the first strand was synthesized by AMV reverse transcriptase (Promega).
  • TrizoUlnvitrogen total RNA extraction reagent TrizoUlnvitrogen
  • AMV reverse transcriptase Promega
  • the 5' oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a restriction endonuclease of the Sal I restriction endonuclease
  • the 3' primer sequence is:
  • This primer contains a cleavage site for Kpnl restriction endonuclease.
  • Reaction parameters 95 ° C for 15 seconds, 53 ° C for 30 seconds, 72 ° C for 40 seconds, after 29 cycles, 72 ° C extension for 10 minutes, the expected product size is 1527 bp, the PCR product was purified and then digested with Sal I, the product was digested. Purification was carried out using a kit.
  • the purified product of CEA 57M69 and Hsp70L1 was ligated overnight under a 16 ° C water bath under the action of T4 DNA ligase (TaKaRa), and the resulting ligation product was diluted 1:500 as a template, using 5' and 3' sequences as follows.
  • the PCR primer of the end is amplified to obtain a DNA fragment encoding CEA 57 M -Hsp70L1.
  • the 5' oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a restriction endonuclease of the Sal I restriction endonuclease
  • the 3' primer sequence is:
  • This primer contains a cleavage site for Kpnl restriction endonuclease.
  • the reaction parameters were: 95 ° C for 30 seconds, 64. 9 ° C for 30 seconds, 72 ° C for 45 seconds, 30 cycles of 72 ° C for 10 minutes, and the expected product size was 1830 bp.
  • the obtained PCR product was purified, digested with Seal-Kpnl, and recombined with plasmid P QE30 (purchased from Qiagen) according to a conventional method and transformed into competent E. coli host strain M15 (pREP4) (purchased from Qiagen), and picked. Positive clones were identified and purified by Seal-Kpnl digestion (ABI 377 sequencer, BigDye Terminator kit, PE).
  • a single clone of M15 (pREP4) strain transformed with pQE30- CEA 576 - 669 - Hsp70L1 plasmid was picked and inoculated into LB containing 50 ⁇ ⁇ / ⁇ 1 ampicillin and 25 g/ml kanamycin, 37 °C shaker Overnight growth, inoculated to 1: 100 In 2YT medium of ampicillin and kanamycin, the shaker was grown to 0D 6 at 37 °C. .
  • IPTG was added to a final concentration of ImM, induce the expression of four hours, the cells were harvested by centrifugation row SDS- PAGE electrophoresis was observed 576 recombinant CEA - 669 - Hsp70Ll protein expression.
  • inclusion body washing solution A (2M urea, 0.1% Triton X-100), and then washed with B lOOmM NaH 2 P0 4 , 10 mM Tris. CI, pH 8. 0) Wash 1-2 times.
  • the washed inclusion bodies were fully dissolved with BufferA (100 mM Na P0 4 , 10 mM Tris. Cl, 8 M urea, pH 8.0), adjusted to a protein concentration of about 5 mg/ml, and filtered.
  • the HSP70L1 monoclonal antibody (expressing HSP70L1 by a conventional method, and then preparing the obtained monoclonal antibody) is cross-linked with CNBr-activated Sepharose 4B to form an affinity chromatography column, and the filtered protein sample is pH 7. 2, 0 After dialysis on a 15M PBS overnight, the affinity chromatography column equilibrated with pH 7.2, 0.150. 15M PBS, flow rate of 0.5 ml / min, and then washed with a large amount of PBS to the effluent OD2800.
  • the CEA 576 - e6g- Hsp70Ll fusion protein gel was obtained by slow silver staining by SDS-PAGE, the protein gel was scanned by DTS scanner, and the purity and molecular weight of the fusion protein were calculated by IraageMster software.
  • the endotoxin content of the recombinant CEA 57M69 -Hsp70L1 protein was detected using a sputum kit according to the instructions.
  • the sample was diluted with water without LPS, and the LPS standard was used as a reference.
  • Peripheral blood leukocytes from freshly isolated colon cancer patients were isolated and peripheral blood mononuclear cells were isolated by density gradient centrifugation using a lymphocyte separation solution (Ficoll-Histopaque 1. 077) (Sigma). Incubate at 37° (:, 5% C0 2 for 2 hours, adherent monocytes containing human recombinant GM-CSF (500 U/ml) (Serpentine) and human recombinant IL-4 (10 ng/ M ml) (Promega) was cultured in complete medium at 37 ° C, 5% CO 2 to obtain peripheral blood mononuclear cell-derived DCs. Non-adherent cells were suspended in RPMI 1640 medium with 5% fetal bovine serum. A nylon hair column (incubated at 37 ° C for 1 hour) was used to obtain purified T lymphocytes.
  • the DCs from peripheral blood mononuclear cells of CEA-positive colon cancer patients cultured to the sixth day were collected, adjusted to a concentration of 2 ⁇ 10 5 cells/ml, and 1 ml/well was divided into 24-well plates, and 10 ⁇ ⁇ / ⁇ 1 was added to CEA.
  • 576 - 669 -Hsp70Ll fusion protein, control recombinant CEA 576 - e69 Hsp70Ll recombinant proteins and control proteins were collected after 4 hours of incubation cells were washed suspended in complete medium for stimulating T lymphocytes with the body.
  • the lymphocytes were collected after 7 days of culture, and co-cultured with freshly prepared 2 ⁇ 10 5 /ml protein-sensitized DCs in a ratio of 10:1 for a second round of stimulation, co-culture for 7 days, and The third round of stimulation was performed, and 20 U/ml rhIL-2 was added every 3 days during the culture, and fresh medium was replaced in 2-3 days.
  • the cells were collected 7 days after the last round of stimulation, and immunomagnetic beads were used.
  • Example 3 CEA5T6-669-Hsp70L1 fusion protein-sensitized dendritic cells induce CEA-specific T lymphocytes in vitro
  • the CTL killing ability was measured by a 4-hour 51 Cr release method.
  • SW480 cells CEA+, HLA_A*0201+
  • LoVo cells CEA+, HLA- ⁇ *020 ⁇
  • ⁇ 2 cells were used as target cells, respectively.
  • a portion of ⁇ 2 cells were suspended in 200 ⁇ l of RPMI 1640 medium without fetal bovine serum, first with 10 ⁇ / ⁇ 1 CAP-1 (CEA 6. 5-613 , YLSGANLNL, one HLA-A contained in CEA 576-669 ) * 0201 restricted epitope) or an irrelevant peptide Tyr 368 - 37e were incubated for 60 min.
  • Killing rate (%) (experimental group cpm-spontaneous release group cpm) / (maximum release group cpm-spontaneous release group cpm) X 100.
  • the results are shown in Figure ⁇ .
  • the effector cells of the CEA 576-6 _Hsp70L1 group showed cytotoxic activity against SW480 cells, and the killing ability was significantly higher than that of the CEA 576-669 group (0.05), while the effector cells of the control group Hsp70L1 did not show.
  • CD8+ T cells sorted by immunomagnetic beads are used as reaction cells and directly transferred to coated with anti-IFN- ⁇ antibody.
  • ELISP0T test board SW480 (CEA + , HLA- A*0201+); LoVo (CEA+, HLA- ⁇ *020 ⁇ ); ⁇ 2 cells Three cells were irradiated with 4000 rad cobalt M to stimulate cells, and some T2 cells bound CAP-1 antigen. Peptide or unrelated peptide Tyr 3e8-376 . The stimulated cells are separately added to the detection wells containing the reaction cells. T cell colonies secreting IFN- ⁇ were detected by the method described in the IFN- ⁇ ELISP0T assay kit. Each of the above tests has three duplicate holes.
  • BMDC Mouse bone marrow dendritic cells
  • Cervical dislocation method killed HLA-A*0201/K b transgenic mice (The Jackson Laboratory), aseptically took the femur, washed out the bone marrow cells in serum-free medium, dissolved red blood cells, and added anti-Ia, B220, CD4, CD8 mAb (final concentration For 10 ⁇ ⁇ / ⁇ 1) and complement (10: 1 dilution), sputum, sputum and Ia+ cells were dissolved in a 37 °C water bath for 45 minutes, followed by 10 ng/ml raGM-CSF (Promega), lng/ml mIL-4 ( After 3 days of culture, the adherent cells were re-added to fresh complete medium and mGM-CSF, mIL-4 for 3 days, and then boiled to collect loosely adherent proliferating cell aggregates and cultured in new bottles. After 3 days, the suspension cells were collected. It is the enriched BMDC;
  • a protein in vitro sensitized HLA- A * 0201 / K b transgenic mice transfected BMDC of syngeneic mice were immunized. Each mouse was subcutaneously injected with BMDC of lxlO 6 homologous mice sensitized with 10 ⁇ ⁇ / ⁇ 1 protein or BMDC of syngeneic mice sensitized without any factors. Each mouse was immunized three times, one week apart.
  • CEA 576-669 - IL-2 levels and the number of IFN- ⁇ secreting cells was significantly higher than Hsp70Ll CEA 576 - 669 group (0.05); the same group Hsp70Ll mouse splenocytes and secretion of IL- 2
  • the number of IFN- ⁇ secreting cells was not increased, and the irrelevant antigen peptide Tyr 3e8 - 37e could not significantly induce IL-2 and IFN- ⁇ stimulation of mouse spleen cells of each immunized group (Fig. 9 and Fig. 10). .
  • mice spleen cells were added 5 g / ml recombinant CEA 576 - 669 protein as the antigen restimulation, supplemented with 25 U / ml IL-2, cultured 7-9 days, as tested for CTL killing of target The ability of cells.
  • the target cells were the same as in Example 6.
  • the C 1 killing ability of the 5 1 Cr release method was the same as in Example 6.
  • Example immunization packet with HLA 6 - A * 0201 / K b transgenic mice by intraperitoneal injection of additional separately provided control of IL-2.
  • the spleen cells of the immunized mice after 7-9 days of restimulation by the in vitro antigen were collected and returned to the tumor-bearing nude mice.

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Abstract

L'invention concerne des protéines de fusion comprenant un antigène carcino-embryonnaire humain et la protéine de choc thermique 70L1 (protéine de fusion CEA-Hsp 70L1), des séquences d'ADN codant pour les protéines de fusion, des vecteurs comprenant les séquences d'ADN, des cellules hôtes comprenant les vecteurs et des méthodes de recombinaison permettant de produire les protéines de fusion ainsi que des compositions contenant lesdites protéines de fusion. L'invention concerne également un procédé de préparation et l'application de vaccins CEA thérapeutiques positifs à une tumeur. Lesdits vaccins qui sont des cellules dendritiques sensibilisées par les protéines de fusion, lors de leur application in vivo, peuvent susciter des réponses immunitaires au pouvoir antigénique de CEA. Ces vaccins peuvent également soigner des tumeurs CEA positives à savoir le carcinome du colon, le cancer bronchopulmonaire «non à petites cellules», le cancer gastrique et le cancer pancréatique, etc.
PCT/CN2005/000763 2004-07-30 2005-05-31 Preparation et application de vaccins therapeutiques a une tumeur WO2006010308A1 (fr)

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CN1865281B (zh) * 2006-06-02 2012-08-22 北京大学人民医院 卵巢癌抗独特型抗体6b11 t细胞表位肽及其应用
CN100391980C (zh) * 2006-08-02 2008-06-04 中国人民解放军军事医学科学院基础医学研究所 抗肿瘤多靶点复合抗原及其编码基因与应用
CN102154223A (zh) * 2010-02-11 2011-08-17 中国人民解放军第二军医大学 携带融合蛋白基因的重组腺病毒及其制备方法和应用
CN105693862B (zh) * 2016-03-07 2019-06-18 中国人民解放军第二军医大学 抗融合蛋白cea576-669hsp70l1单克隆抗体4e8及其用途
CN105693863B (zh) * 2016-03-07 2019-06-18 中国人民解放军第二军医大学 抗融合蛋白cea576-669hsp70l1单克隆抗体3c1及其用途
CN106749673B (zh) * 2016-11-22 2021-07-02 张震宇 一种融合蛋白、制备方法及其应用

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