WO2006010308A1 - Preparation and application of therapeutic vaccines of tumour - Google Patents

Abstract

The present invention provides fusion proteins comprising human carcinoembryonic antigen and heat shock protein 70L1(CEA-Hsp 70L1 fusion protein), DNA sequences encoding the fusion proteins, vectors comprising the DNA sequences, host cells comprising the vectors, and recombinant methods for producing the fusion proteins as well as compositions containing the fusion proteins. The invention also provides a preparing method and application of CEA positive therapeutic vaccines of tumour. The vaccines were dendritic cells sensitized by the fusion proteins, when application in vivo, them can elicit immune responses for CEA antigenicity. The vaccines also can cure CEA positive tumours including colon carcinoma, nonsmall-cell lung cancer, gastric cancer and pancreatic cancer etc.

Description

 Preparation and application of tumor therapeutic vaccines

 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. Background technique

 Carcinoembryonic antigen (CEA) 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. At present, traditional treatments such as surgery, chemotherapy, and radiotherapy have certain curative effects on early CEA-positive tumors. On the one hand, chemotherapy and radiotherapy have large side effects. On the other hand, 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.

A variety of immunological and gene therapy protocols have been explored for 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. With the discovery of numerous T cell epitopes in the amino acid sequence of CEA and the ability to induce specific cellular immune responses in vitro, tumor vaccines based on these epitope peptides have received the attention of researchers, 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. However, it is generally believed that the body is immune-tolerant to CEA expressed by tumor cells in vivo. There is little evidence that 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.

 In recent years, the discovery of tumor antigens, tumor immune escape and the gradual clarification of anti-tumor immune response mechanisms have led to the recognition that how to effectively present tumor antigens and stimulate the body's ability to respond to tumor antigens is a key factor affecting efficacy. The deepening of dendritic cells (DC) research not only makes important progress in basic immunology research, but also brings hope to tumor immunotherapy. As the most powerful professional antigen-presenting cell (APC) in the body's immune system, DC can efficiently ingest, process and present antigens, and can significantly activate naive T cells to activate T cells. 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. After being stimulated by the antigen, 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

Confirmation The high expression of immune-related molecules such as molecules plays an important role in the function of DC. DCs sensitized with tumor antigens or some specific epitope peptides thereof have been confirmed in basic and clinical studies, and have been well established in tumors such as prostate cancer, melanoma, and malignant lymphoma. The efficacy is considered to be the most promising tumor immunotherapy program. However, the efficiency of sensitizing DCs in vitro using small-molecule antigen peptides is generally low. After ingesting small peptides, DCs often fail to effectively present antigens and stimulate specific CTLs. Therefore, how to enhance DC processing of antigens In order to stimulate tumor antigen-specific immune responses more effectively, it is still an urgent problem to be solved in tumor immunotherapy research. In recent years, attempts have been made to use 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. .

 With the advancement of immunology research, a new generation of adjuvants have been discovered, such as Heat Shock Protein (HSP), IFN-γ, IL-12, LPS, 11 1 (1-8 and 0 0, etc., where HSP With its unique biological effects, it has received more and more attention. When cells are under severe stress, HSP can be released from the cells and become an internal "danger signal" to send warning messages to APC. It can activate DC, and APC will further stimulate the immune response by chemotaxis and activation of HSP, triggering antigen uptake and presentation, and the resting Τ· 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. Based on the unique adjuvant effect of HSP, the use of adjuvants of HSP to induce anti-tumor and anti-infective immunity has become the hotspot of current immunotherapy. A new generation of vaccines based on HSP as an adjuvant has also entered clinical trials.

 However, to date, there has not been a very effective therapeutic vaccine for CEA-positive tumors. Therefore, there is an urgent need in the art to develop new and effective therapeutic vaccines for CEA-positive tumors and preparation methods thereof. Summary of the invention

 It is an object of the present invention to provide a novel substance having both biological activity of CEA and Hsp70L1, which is a fusion protein of CEA and Hsp70L1.

 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. In a first aspect of the invention, a fusion protein is provided which comprises -

(a) 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;

 (b) a heat shock protein element having an amino acid sequence of a human heat shock protein or an active fragment thereof;

(c) a 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).

 In another preferred embodiment, 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.

 Moreover, the linker peptide sequence contains 4-10 amino acids.

 In another preferred embodiment, the carcinoembryonic antigen element has the amino acid sequence of SEQ ID NO: 9, and the heat shock protein element has the amino acid sequence of SEQ ID NO: 10.

 In a second aspect of the invention, there is provided an isolated DNA molecule encoding the fusion protein of claim 1.

 In a third aspect of the invention, a vector comprising the above DNA molecule and a host cell comprising the above vector are provided. In a fourth aspect of the present invention, 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.

 In a fifth aspect of the invention, there is provided the use of a portion of said fusion protein for the preparation of a therapeutic vaccine for treating a CEA positive tumor or for sensitizing dendritic cells.

 In a sixth aspect of the invention, there is provided a dendritic cell which is a dendritic cell sensitized with the fusion protein of claim 1.

 In a seventh aspect of the invention, there is provided 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.

 In an eighth aspect of 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.

 In another preferred embodiment, the stimulating step comprises:

(1) Using 10-10 (^g/ml of the fusion protein of the present invention, incubated with 10 ⁵ - 10 ⁶ dendritic cells for 2-48 hours to collect sensitized dendritic cells And radioactively inactivated;

 (2) 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;

 Repeat steps (1) and (2) 3-10 times to collect proliferating T cells, which are CEA-positive tumor-specific T cells. DRAWINGS

 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. Among them, 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. Among them, 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-γ-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-γ 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

 As used herein, the term "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.

 As used herein, the terms "Carcinoembryonic antigen" and "CEA" are used interchangeably. As used herein, the term "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.

 Sequences of carcinoembryonic antigens and various cancer antigens are known in the art and are available on the Genbank database. For example, CEA's accession number is gil78676.

As used herein, the term "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.

 As used herein, 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.

 Taking 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.

 As used herein, 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. Furthermore, the fusion protein may or may not have an initial methionine or signal peptide.

 As used herein, the term "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).

 As used herein, the terms "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.

 As used herein, the terms "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. Typically, 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.

Examples of preferred 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 For the molecule, an active X factor cleavage site (IEGR) can be used as a linker. Similarly, 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. Fusion protein; the combination of the above three protocols can also be designed as a new amino acid linker. For example, the NVVVHQAHHHHHHEFTYK linker is a fusion of a protease cleavage site (Nla protease) and a metal affinity chromatography site of 6His.

 Further, 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.

 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.

 The term "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.

 In the present invention, various carriers known in the art such as commercially available carriers can be used. For example, 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.

 As used herein, "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. For example, if 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. In general, "operably connected" means adjacent, and for a leader sequence means adjacent in the reading frame.

 In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, more preferably a silkworm cell.

After obtaining the transformed host 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.

 In another aspect of the invention, a pharmaceutical composition is also provided. The pharmaceutical composition of the present 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. In the preparation of these compositions, 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. When 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. Thus, the composition may be in the form of tablets, pills, powders, solutions, syrups, sterile injectable solutions and the like. Examples of 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. For cell-containing compositions, the preferred form is a liquid dosage form.

 The 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.

When a pharmaceutical composition is used, 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 ^δ -10 ¹² cells/person/time. Of course, 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.

 In addition, 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. In summary, the main advantages of the present invention are as follows:

 (1) The 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.

(2) 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

 1. 1 person CEA cDNA clone:

 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:

 5' - CACGGTACCACCATGGAGTCTCCCTCGGCCCC- 3, (SEQ ID NO: 1)

 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:

 5' -CTCGTCGACTATCAGAGCAACCCCAACCAG-3' (SEQ ID NO: 2)

 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 μl, the primer concentration was 200 nM, the dNTP concentration was 200 μΜ, 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 results of restriction enzyme digestion showed that the restriction enzyme digestion (identified at both ends of the pGEM-T multiple cloning site) showed that the positive clone was cut into a 2109 bp insert (Fig. 1).

1. 2 CEA576- 669- Hsp70L1 recombinant expression plasmid construction:

 Using the full-length plasmid pGEM-T-CEA prepared above as a template, amplification was carried out using PCR primers at the 5' and 3' ends of the sequence below to obtain CEA576-669 DNA as an insert.

 The 5' oligonucleotide primer sequence used in the PCR reaction is:

 5'-ATTGAGCTCGTGAGTGCAAACCGCAGTGACC-3' (SEQ ID NO: 3). The primer contains a restriction endonuclease of the restriction endonuclease;

 The 3' primer sequence is:

 5' - TATGTCGACGACTATGGAATTATTGCGGCC-3' (SEQ ID NO: 4). 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.

 At the same time, HeLa cells (ATCC Number: CCL-2) which were induced by heat at 41 °C for 1 h were collected, and total RNA was prepared by using total RNA extraction reagent TrizoUlnvitrogen, and the first strand was synthesized by AMV reverse transcriptase (Promega). Using this as a template, amplification was carried out using PCR oligonucleotide primers of the 5' and 3' ends of the sequence below to obtain a DNA fragment encoding Hsp70L1.

 The 5' oligonucleotide primer sequence used in the PCR reaction is:

 5'-CTCGTCGACGCGGCCATCGGAGTTCACCTG-3' (SEQ ID NO: 5) The primer contains a restriction endonuclease of the Sal I restriction endonuclease;

 The 3' primer sequence is:

 5'-GGGGTACCAGATGCTATCTCAATAGAGATTGCT-3' (SEQ ID NO: 6) 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:

 5' - ATTGAGCTCGTGAGTGCAAACCGCAGTGACC-3' (SEQ ID NO: 7) The primer contains a restriction endonuclease of the Sal I restriction endonuclease;

 The 3' primer sequence is:

 5'-GGGGTACCAGATGCTATCTCAATAGAGATTGCT-3' (SEQ ID NO: 8) 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). It was confirmed by sequencing that the designed sequence of the correct CEA _576-669 - Hsp70Ll coding sequence was inserted, and the insertion position and the reading frame were all correct. The recombinant was recorded as pQE30-CEA _{57 69} -Hsp70Ll.

The results of restriction enzyme digestion showed that after electrophoresis by Sea I-Kpn I, the electrophoresis results showed that the positive clone was cut into a 1830 bp insert (Fig. 2). The amino acid sequences of CEA ₅₇₆ - ₆₆₉ and Hsp70L1 are shown in SEQ ID NOS: 9 and 10, respectively.

1. 3 Recombinant CEA ₅₇₆ — ₆₆₉ - Expression and purification of Hsp70L1 protein:

A single clone of M15 (pREP4) strain transformed with pQE30- CEA ₅₇₆ - ₆₆₉ - Hsp70L1 plasmid was picked and inoculated into LB containing 50μ _δ /ιη1 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. . Of about 1, 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.

SDS-PAGE electrophoresis showed that a significant recombinant protein expression band was observed in M15 (pREP4) strain of pQE30-CEA ₅₇₆ - ₆₆₉ _Hsp70L1 plasmid after 4 hours of ImM IPTG induction. The molecular weight was about 65kD, and the predicted His-CEA. The molecular weight of the -Hsp70Ll protein of interest is similar. This protein band was absent from the M15 strain transformed with the IPTG-induced vector pQE30 plasmid (Fig. 3).

The inclusion body after centrifugation was thoroughly washed with 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 results showed that the recombinant protein expressing CEA _576-e69 - Hsp70L1 was mainly present in inclusion bodies (Fig. 4).

 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. 02, and then After elution with pH 2.4, 0.1 mol/L glycine-HC1, the eluate was immediately transferred to pH 7.2, and dialyzed overnight in 0.15 MPBS. After purification of the target protein by HSP70L1 monoclonal antibody affinity chromatography, the ionic strength was reduced by dialysis, and further purified on a DEAE column with a gradient of 20-500 mM NaCl, and finally dissolved in PBS by dialysis. , kept at -80 ° C for use.

 The results showed that the inclusion bodies were fully washed and denatured, and subjected to HSP70L1 monoclonal antibody affinity chromatography.

Further purification by DEAE chromatography, the SDS-PAGE analysis showed that the recombinant CEA _{57 69} -Hsp70L1 protein appeared on the P1 peak, and the P0 and P2 peak proteins were impurity proteins (Fig. 5).

1.4 Purification of recombinant CEA ₅₇₆ - ₆₆₉ - Hsp70Ll Protein identification:

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.

 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Copper sulphate and 0. 5ml 2% potassium tartrate mixed with before use), shake, place at room temperature for 10 minutes; add phenol reagent 0. 25ml, shake and place at room temperature for 30 minutes, 650nm colorimetric. The protein concentration of the sample was calculated from the absorbance values of each tube and the standard curve.

The results showed that the purified recombinant CEA _576-669- Hsp70L1 fusion protein was subjected to SDS-PAGE electrophoresis, slow silver staining, showing a single protein band, molecular weight of about 65KD, gel scanning analysis, purified recombinant CEA ₅₇₆ - ₆₆₉ -Hsp70Ll The purity of the fusion protein was >95%. The endotoxin content is less than 0. lEU / g. The protein concentration was determined to be 0.45 mg/ml (Fig. 6). Example 2 Preparation of a therapeutic dendritic cell vaccine for a new CEA positive tumor

 2. 1 Isolation and culture of DC and T lymphocytes:

Peripheral blood leukocytes from freshly isolated colon cancer patients (CEA ⁺ , HLA-Α*020 ) 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.

2. 2 DC sensitization:

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 2χ10 ⁵ cells/ml, and 1 ml/well was divided into 24-well plates, and 10 μ _§ /πι1 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.

Purified T lymphocytes were co-cultured with sensitized DCs at 37° (5% C0 ₂ (T _: DC = 10: 1), and added to 20 U/ml -2 on the fifth day (31 _§ !^) 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. Mi ltenyi Biotec) Positive selection of CD8+ T cells was performed in strict accordance with the manufacturer's instructions. Example 3 CEA5T6-669-Hsp70L1 fusion protein-sensitized dendritic cells induce CEA-specific T lymphocytes in vitro

 3. 1 CTL killing test:

The CTL killing ability was measured by a 4-hour ⁵¹ Cr release method. SW480 cells (CEA+, HLA_A*0201+), LoVo cells (CEA+, HLA-Α*020Γ), and Τ2 cells were used as target cells, respectively. A portion of Τ2 cells were suspended in 200 μl of RPMI 1640 medium without fetal bovine serum, first with 10 μ /πι 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. Add 20 (^Ci ^5l Cr (Amer sham Pharmacia), mark in a 37 ° C water bath for 90 minutes, lightly mix once every 15 minutes, then thoroughly wash away the residual ⁵¹ Cr with the medium, used as a target cell for killing According to 10:1, 5:1, 2. 5:1, three different effect targets were added to the corresponding prepared effect cells (sorted CD8+ T cells) for 4 hours, and the supernatants of each well were collected by centrifugation. The cp counter was used to detect the cpm value. The maximal release group was treated with a single target cell plus ΙΟΟμΙ 0.1% NP40; the spontaneous release group was a separate target cell plus complete medium. Each group was provided with three duplicate wells.

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 。. In Fig. 7 , 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. Significant killing activity (0.05). There was no obvious killing effect on the effector cells of LoVo cells (Fig. 7B), CEA _576-669 _Hsp70Ll group and CEA _57B - ₆₆₉ group, indicating that the killing effect of the effector cells on the target cells of the above two groups is HLA-A*0201 restricted. There is no killing effect on target cells that surface-present non-HLA-A*0201 restricted epitopes. For T2 cells loaded with CAP-1 (CEA ₆ o5-ei3, YLSGANLNL), the results (Fig. 7C) showed that they could be killed by effector cells in the CEA ₅₇₆ - ₆₆₉ - Hsp70L1 group and the CEA ₅₇₆ - ₆₆₉ group, and the killing effect of the former Significantly higher than the latter (0.05), and the effector cells of the Hsp70L1 control group had no significant killing activity (0.05). Figure 7D shows that all of the effector cells of the experimental group were unable to kill T2 cells that did not express the CEA antigen but expressed HLA-A*0201 molecule. For incorporating an irrelevant peptide Tyr ₃₆₈ - T2 cell _376, and found that effector cells in all experimental groups did not show its significant killing activity (Figure 7E), show that the killing activity of effector cells only for CEA antigen. This result demonstrates CEA ₅₇₆ - ₆₆₉ _Hsp70Ll cancer patients sensitized fusion protein source of DC in vitro peripheral blood mononuclear cells induce an HLA-A * 0201 restricted CEA-specific CTL.

3. 2 human IFN-γ ELISP0T detection:

 CD8+ T cells sorted by immunomagnetic beads are used as reaction cells and directly transferred to coated with anti-IFN-γ antibody.

ELISP0T test board. SW480 (CEA ⁺ , HLA- A*0201+); LoVo (CEA+, HLA- Α*020Γ); Τ2 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-γ were detected by the method described in the IFN-γ ELISP0T assay kit. Each of the above tests has three duplicate holes.

The results showed that T cells induced by DCs sensitized with CEA ₅₇₆ - ₆₆₉ -Hsp70L1, when the stimulating cells were

When SW480 (CEA ⁺ , HLA_A*0201 ⁺ ) or T2 (CEA ⁺ , HLA- Α *02θΓ) cells loaded with CAP-1, the number of cells secreting IFN-γ was significantly higher than that of stimulating cells as LoVo (CEA+, HLA). -A*020D or group of T2 cells not loaded with antigenic peptide (0.05), SW480 and T2 cells loaded with CAP-1 have no significant difference in response to the reaction cells. 05); CEA ₅₇₆ - _6M sensitization The number of cells secreting IFN-γ induced by DCs in _SW cells or T2 cells loaded with CAP-1 was significantly lower than that of T cells induced by CEA _576-669- Hsp70L1 sensitized DCs ( 0. 05); For the Hsp70L1 group, the induction of several stimulator cells failed to observe a significant increase in the number of IFN-γ-secreting sputum cells; when the stimulator cells were loaded with unrelated antigenic peptide Τ2 cells, the responses of all experimental groups None of the cells showed an increase in the frequency of Τ cells releasing IFN-γ. This result indicates that T cells induced by DCs sensitized with CEA _57e - ₆₆₉ -Hsp70L1 fusion protein are CEA antigen-specific and have HLA-A*0201 restriction (Fig. 8). Example 4 CEA576-669-Hsp70L1 fusion protein-sensitized DC induces antigen-specific antitumor effect in vivo

 4. 1 Mouse bone marrow dendritic cells (BMDC) isolated:

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 10μ _§ / ηι1) 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;

4. 2 Protein sensitization in vitro BMDC:

The mouse DCs cultured to the sixth day were added with 1 (^g/ml CEA ₅₇₆ - ₆₆₉ - Hsp70L1 fusion protein, control recombinant CEAw protein and control recombinant Hs _P 70L1 protein, and unstimulated DC was set as a blank control group. After 4 hours of incubation, the cells were collected and used to immunize mice.

4. 3 Immunization of HLA-A*0201/K ^b transgenic mice:

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 μ _§ /πι1 protein or BMDC of syngeneic mice sensitized without any factors. Each mouse was immunized three times, one week apart.

4. Preparation of 4 spleen cells from immunized mice and IL- 2 ELISA, IFN-γ ELISP0T detection:

Seven days after the last immunization, the mouse spleens were aseptically removed, and the red blood cells were dissolved to prepare a single cell suspension. Portion of CEA576 CAP-1 peptide, Con A , an irrelevant peptide Tyr ₃₆₈ - ₃₇₆ or PBS and then 24 hours of stimulation, cell supernatants were collected for ELISA detection of IL-2 in the supernatant, and the cells for the detection of secreted IFN- ELISP0T Gamma spleen cell colonies. Each of the above tests has three duplicate holes.

The results _show, CEA 576-669 - IL-2 levels and the number of IFN- γ 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-γ secreting cells was not increased, and the irrelevant antigen peptide Tyr _3e8 - _37e could not significantly induce IL-2 and IFN-γ stimulation of mouse spleen cells of each immunized group (Fig. 9 and Fig. 10). .

4. 5 CTL killing test:

Each group of immunized mice spleen cells above was 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.

The result is shown in FIG. Effector cells from the CEA ₅₇₆ - ₆₆₉ - Hsp70L1 and CEA _57e - ₆₆₉ groups showed significant killing of SW480 cells (Fig. 11A), and the effector cells of the CEA _{57 e9} - Hs _P 70L1 group were significantly more potent than CEA. _{The 576-669} group (0.05), while the other two control groups including the Hsp70L1 group, the unsensitized DC immunized group had no significant killing activity against the target cell SW480. For the T2 cells loaded with CAP-1, the results (Fig. 11B) revealed that the effector cells of the CEA _676- as ₉ -Hsp70L1 group and the group had cytotoxic activity against CAP-1 sensitized T2 cells, and the killing effect of the former was significantly higher than that of the T2 cells. (0. 05), while the other two control groups included the Hsp70L1 group, the effect of the unsensitized DC immunization group The cells had no significant killing activity against T2 cells loaded with CAP-1. For LoVo cells, the results (Fig. 11C) showed that the effector cells of the CEA _- Hsp70L1 group and the CEA _576-e69 group alone had no significant killing effect on LoVo cells, indicating that the killing of the target cells by the effector cells of the above two immunized groups was HLA. - A*0201 is restricted and has no killing effect on target cells surface-presenting non-HLA-A*0201 restricted epitopes. Figure 11D shows that all of the effector cells of the experimental group were unable to kill T2 cells. For T2 cells bound with the irrelevant peptide Tyr _3e8 - ₃₇₆ , the results (Fig. 11E) showed that all the effector cells of the experimental group did not show significant killing activity, indicating that the killing activity of the effector cells was only for the CEA antigen, indicating CEA ₅₇₆ - ₆₆₉ - Hsp70L1 fusion protein-sensitized DC-immunized HLA-A*0201/K ^b transgenic mice induced CEA-specific CTLs (Fig. 11). 4. 6 Observation of the therapeutic effect of tumor-bearing and adoptive reinfusion in nude mice:

Female nude mice 6-8 weeks old (purchased from Shanghai Experimental Animal Center of Chinese Academy of Sciences) were inoculated subcutaneously in the right abdomen to in vitro cultured expanded SW480 tumor cells. Three days later, the treatment was followed up. 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. The second day after the reinfusion, IL-2 2000U was intraperitoneally injected, and the same method was injected every other day. The same dose of IL-2, the second return 7 days after the first return, the same method. Tumor appearance and growth were observed every two days after inoculation of tumor cells. The time of tumor appearance in each mouse was recorded, the diameter of the tumor was measured, and the survival of each group of mice was observed.

The results showed that CEA ₅₇₆ - _6e9 - Hsp70L1 fusion protein-sensitized DC-immunized HLA-A*0201/K ^b transgenic mouse spleen cells were successively reinfused to nude mice bearing SW480 tumors, which significantly inhibited tumors in nude mice. Growth, and the survival rate of the mice was improved. 12, reinfusion CEA ₅₇₆ - _{669 -Hsp70Ll} splenocytes nude mice, tumors several other time significantly later than the control group (0.05). On the 27th day, the average tumor diameter of the mice in which tumor growth occurred in this group was significantly lower than that of the control mice (0.05). As can be seen from Fig. 13, all the control nude mice died between the 27th day and the 44th day of tumor-bearing, and the survival rate of the nude mice in the CEA _57e -6e9_Hsp70L1 group was _gradually returned on the 44th day. 62. 5%, the survival rate of the mice in this group was still 37.5% observed on the 60th day, and no tumor growth was observed in the surviving mice. All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art after the above-described teachings of the present invention.

Claims

Rights request A fusion protein characterized in that it comprises:  (a) a carcinoembryonic antigen element having an amino acid sequence of a human carcinoembryonic antigen or an immunogenic fragment thereof; or a cancer antigen element;  (b) a heat shock protein element having an amino acid sequence of a human heat shock protein or an active fragment thereof;  (c) a linker peptide sequence of 0-20 amino acids located between (a) a carcinoembryonic antigen element or a cancer antigen element and (b) a heat shock protein element.  The fusion protein according to claim 1, wherein 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.  Moreover, the linker peptide sequence contains 4-10 amino acids.  The fusion protein according to claim 1, wherein the carcinoembryonic antigen element has the amino acid sequence of SEQ ID NO: 9, and the heat shock protein element has SEQ ID NO: Amino acid sequence.  4. An isolated DNA molecule characterized in that it encodes the fusion protein of claim 1. A carrier comprising the DNA molecule of claim 4.  6. A host cell, characterized in that it comprises the vector of claim 5.  7. A method of producing the fusion protein of claim 1, comprising the steps of: cultivating the host cell of claim 6 under conditions suitable for expression of the fusion protein, thereby expressing the Fusion protein; and  The fusion protein is isolated.  8. Use of a fusion protein according to claim 1 for the preparation of a therapeutic vaccine for the treatment of CEA positive tumors.  A dendritic cell characterized in that it is a dendritic cell sensitized with the fusion protein of claim 1.  A therapeutic vaccine for CEA-positive tumors, which comprises a safe and effective amount of dendritic cells according to claim 9 or T cells induced by dendritic cells or the fusion protein of claim 1. And a pharmaceutically acceptable carrier.