WO2021203608A1

WO2021203608A1 - Vegfr2-targeted metastatic cancer vaccine

Info

Publication number: WO2021203608A1
Application number: PCT/CN2020/109970
Authority: WO
Inventors: 齐海龙; 王晓芳; 严小娥; 李日勇; 罗天明; 谢皇帆; 刘德芳; 孙忠杰
Original assignee: 诺未科技（北京）有限公司
Priority date: 2020-04-09
Filing date: 2020-08-19
Publication date: 2021-10-14
Also published as: CN111440244B; CN111440244A

Abstract

Provided is a VEGFR2 (KDR)-targeted metastatic cancer vaccine. By means of fusion expression of VEGFR2 protein promoting generation of tumor angiogenesis with a DC cell ligand XCL1, the efficiency of cytophagy, processing and presentation of the VEGFR2 protein by DC cells is improved, and the effect of inhibiting tumor growth is improved. Experiments prove that the protein for nucleic acid vaccine expression can effectively bind the DC cells, induce specific T-cell reaction of VEGFR2 and significantly inhibit growth of tumor highly expressing VEGFR2 in various models.

Description

Metastatic cancer vaccine targeting VEGFR2

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office, the application number is 202010273819.0, and the invention title is "VEGFR2-targeted metastatic cancer vaccine" on April 9, 2020, the entire content of which is incorporated into this application by reference middle.

Technical field

The present invention relates to the field of cancer immunotherapy and prevention, in particular to a metastatic cancer vaccine targeting VEGFR2.

Background technique

Tumor metastasis is the main cause of cancer death. More than 90% of cancer death rates are caused by metastasis, and the formation of tumor blood vessels is essential for tumor growth and metastasis (Valastyan and Weinberg). Tumor blood vessels provide sufficient oxygen and nutrients for tumor growth and metastasis. The formation of new blood vessels is a necessary step for tumor blood vessel formation. The neovascularization theory believes that tumors induce the formation of new blood vessels from existing blood vessels. Based on this theory, researchers have developed a series of drugs that target tumor neovascularization. The current FDA-approved anti-angiogenesis drugs are mainly divided into three categories: monoclonal antibodies, receptor tyrosine kinase inhibitors, and fusion peptides. . For example, bevacizumab, sorafenib, and the fusion protein of the extracellular domain of the VEGFR1/2 protein used in the treatment of metastatic cancer can effectively improve the patient's progression-free survival (Cascone and Heymach). However, there are still many problems with anti-angiogenic drugs. Taking bevacizumab as an example, frequent parenteral administration, serious side effects, obvious drug resistance, and tumor rebound progression after metastasis are needed. These problems need to be resolved urgently.

Vascular endothelial growth factor (VEGF) is the most important regulator of neovascularization, and its function is mainly through the binding of three receptor tyrosine kinases VEGFR1/2/3. Studies have shown that VEGFR1 has high affinity for VEGF but low intracellular enzyme activity, which has a negative feedback inhibitory effect on the formation of new blood vessels. VEGFR3 is mainly expressed in adult lymphatic vessels, while VEGFR2 is the main mediator of VEGF-induced new blood vessels. Promoting factors (Dong et al.) that produce and increase endothelial cell permeability. VEGFR2 knockout mice developed to 8.5-9.5 days and died due to impaired angiogenesis (Shalaby et al.). In tumor tissues, VEGFR2 is highly expressed in tumor-associated endothelial cells in tumor tissues, and its expression is much higher than that of normal vascular cells. In addition to tumor-associated endothelial cells, VEGFR2 is also highly expressed in certain types of tumor cells, such as B cells Lymphoma, breast cancer, lung cancer, bladder cancer and melanoma, etc. (Decaussin et al., 1999; El-Obeid et al., 2004; Gakiopoulou-Givalou et al., 2003; Kranz et al., 1999), thus VEGFR2 is an ideal target for tumor therapy. The bevacizumab and sorafenib are anti-tumor drugs developed with VEGFR2 as the target.

Although vascular endothelial growth factor receptor 2 (VEGFR2) is highly expressed in some tumors and tumor-associated endothelial cells, because VEGFR2 is an embryonic tumor-associated antigen, it can be recognized and highly expressed during the body’s cellular immune development. Affinity-bound T cells have been eliminated by negative selection in the thymus (Houghton et al., 2007). Only T cells with low affinity for VEGFR2 survive the selection of the thymus, but these T cells cannot be activated by the body's own VEGFR2. Therefore, although tumor cells highly express VEGFR2, the body itself cannot produce effector T cells that can effectively recognize tumor cells with high expression of VEGFR2, thereby inhibiting angiogenesis and tumor growth. This phenomenon is called immune tolerance. Previous studies have shown that by optimizing certain epitopes in tumor-associated antigens such as VEGFR2 to increase their affinity with T cells, it can promote the production of T cells that do not recognize tumor-associated antigens such as VEGFR2, thereby breaking the VEGFR2 Immune tolerance (Guevara-Patino et al., 2006).

Previous studies have shown that the predicted and verified HLA-A2 and HLA-A24-restricted VEGFR2 immunogenic peptides (VEGFR2169-177: RFVPDGNRI and VEGFR2773-781: VLAMFFWLL, respectively) are used to immunize patients with advanced metastatic colorectal cancer and nerve fibers Tumor patients, conducted phase I/II clinical trials, the results showed that after stimulation with peptide vaccines, the increased proportion of VEGFR2-specific cytotoxic T cells (CTLs) can inhibit tumor angiogenesis, increase the number of CD8+ T cells in the tumor, and eliminate VEGFR2-positive tumor cells reduce tumor burden and improve the overall survival rate of patients (Hazama et al., 2014) and (Tamura et al., 2019). However, their research content is only applicable to people with the two subtypes of HLA-A2 and A24. Due to the HLA-restricted T cell immune response and polypeptide immunity generally cannot effectively cause antibody-mediated humoral immune responses, HLA- A2 and HLA-A24 restricted VEGFR immunogenic peptides may not have any effect on other HLA subtypes. Moreover, the immunological peptides used without epitope optimization can cause a limited degree of immune response, which may limit the effectiveness of this type of vaccine. In summary, it is necessary to design a preventive and therapeutic vaccine that targets VEGFR2 that can effectively break immune tolerance, antagonize angiogenesis and inhibit the formation of metastatic cancer foci through cell-specific immune responses.

Antigen presenting cells refer to a type of cells that can process antigens and present antigen peptides to T cells in the form of antigen peptide-MHC molecular complexes, and play an important role in the body's immune recognition, immune response and immune regulation. Dendritic cell (DC) cell is a type of antigen-presenting cell in the body, and it is the only antigen-presenting cell in the body that can present antigen peptides to naive T cells and induce T cell activation and proliferation. , Is the initiator of the body's adaptive immune response. According to morphology and function, DC cells can be divided into two categories: classic DC and plasma cell-like DC. Plasma cell-like DCs mainly mediate the adaptive immune response after virus infection. Classical DCs have the strongest ability to process and present antigens, which mediate the elimination of dead cells in the body and the recognition of tumor cells (Carreno et al., 2017; Minetto et al., 2019). Therefore, more effective presentation of tumor antigens to classic DCs is the key to the success of the vaccine. Studies have found that classic DCs express a class of chemokine XCL1 receptors, so XCL1 molecules can efficiently bind to classic DC cells (Dorner et al., 2009) .

Summary of the invention

In view of this, the present invention provides a metastatic cancer vaccine targeting VEGFR2. The invention improves the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells by fusion expression of VEGFR2 protein which promotes tumor angiogenesis and DC cell ligand XCL1, and improves the effect of inhibiting tumor growth. Experiments have shown that the protein expressed by the nucleic acid vaccine of the present invention can effectively bind to DC cells, induce VEGFR2-specific T cell responses, and significantly inhibit the growth of melanomas that highly express VEGFR2 in a variety of models.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The fusion protein provided by the present invention includes the extracellular region of VEFGR2 that promotes tumor angiogenesis that can induce a specific CD8+T response or the extracellular region of the optimized form of binding epitope of MHC class I molecules; linkers; and specific binding with antigen crossover Human or mouse XCL1 protein of DC cells that present the ability;

in:

(I) The extracellular region of VEFGR2 that promotes tumor angiogenesis that can induce a specific CD8+T response or its MHC class I molecule binding epitope optimized form has the extracellular region as shown in SEQ ID No. 1 or 2. The amino acid sequence shown in amino acids 20 to 764; the human or mouse XCL1 that specifically binds to DC cells with antigen cross-presenting ability, as shown in the sequence of amino acids 1 to 114 in SEQ ID NO: 3 or 4;

or

(II) An amino acid sequence obtained by substituting, deleting or adding one or two amino acid residues to the amino acid sequence described in (I), and having the same or similar function as the amino acid sequence shown in (I);

or

(III) An amino acid sequence that has at least 90% sequence identity with the sequence described in (I) or (II), and is functionally identical or similar to the amino acid sequence shown in (I).

In some specific embodiments of the present invention, the fusion protein includes the sequence shown in amino acids 1 to 889 in SEQ ID Nos. 7 and 8.

On the basis of the above research, the present invention also provides a nucleotide encoding the fusion protein, which has

(Ⅰ) The nucleotide sequence shown in SEQ ID No. 11-16;

or

(II) The complementary nucleotide sequence of the nucleotide sequence arbitrarily shown in SEQ ID No. 11-16; or

(Ⅲ). A nucleotide sequence that encodes the same protein as the nucleotide sequence of (I) or (II), but is different from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code;

or

(IV), the nucleotide sequence obtained by substituting, deleting or adding one or two nucleotide sequences to the nucleotide sequence shown in (I), (II) or (III), and the nucleotide sequence with (I), The nucleotide sequence shown in (II) or (III) has the same or similar function;

or

(V), a nucleotide sequence that has at least 90% sequence identity with the nucleotide sequence described in (I), (II), (III) or (IV).

On the basis of the above research, the present invention also provides a recombinant expression vector, including a vector and the fusion protein or the nucleotide of the present invention.

In some specific embodiments of the present invention, the vector includes a mammalian cell expression vector or an insect rod-shaped cell expression vector; the vector includes pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP .

On the basis of the above research, the present invention also provides a recombinant strain or cell comprising the fusion protein or the nucleotide of the present invention.

On the basis of the above research, the present invention also provides a vaccine for preparing metastatic cancer or melanoma with high expression of VEGFR2 in the fusion protein, the nucleotide, the recombinant expression vector or the recombinant strain or cell. Or application in the preparation of medicines for preventing and/or treating metastatic cancer or melanoma with high expression of VEGFR2.

In some specific embodiments of the present invention, the metastatic cancer includes liver cancer, colorectal cancer or lung cancer.

On the basis of the above research, the present invention also provides a vaccine for metastatic cancer or melanoma with high expression of VEGFR2, including the fusion protein of the present invention, the nucleotide, the recombinant expression vector and the pharmaceutical Acceptable carriers, excipients and/or adjuvants.

On the basis of the above research, the present invention also provides drugs for the prevention and/or treatment of metastatic cancer or melanoma with high expression of VEGFR2, including the fusion protein of the present invention, the nucleotide, and the recombinant Expression vector and pharmaceutically acceptable excipients.

The vaccine provided by the present invention is composed of the extracellular region of the vascular endothelial cell growth factor receptor VEGFR2 that can cause a specific CD8+ T cell immune response and the extracellular region of the optimized form of MHC class I molecule binding epitope, and is expressed by fusion at its N-terminus The protein composed of the XCL1 molecule of DC that specifically binds to the antigen cross-presenting ability, contains the nucleic acid of the fusion protein and the carrier where the nucleic acid is located, and contains the fusion protein of the present invention. The nucleic acid and the carrier are used in the prevention and treatment of melanoma and metastatic cancer. Applications. The invention improves the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells by fusion expression of VEGFR2 protein which promotes tumor angiogenesis and DC cell ligand XCL1, and improves the effect of inhibiting tumor growth. Experiments have shown that the protein expressed by the nucleic acid vaccine of the present invention can effectively bind to DC cells, induce VEGFR2-specific T cell responses, and significantly inhibit the growth of melanomas that highly express VEGFR2 in a variety of models.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Figure 1 shows the vector map of the nucleotide encoding the fusion protein; Figure 1A shows the CMV promoter followed by the XCL1 sequence followed by the VEGFR2 wild-type extracellular region for the nucleic acid vaccine coding sequence; Figure 1B shows the CMV promoter followed by the XCL1 sequence Connecting VEGFR2MHCI molecules to optimize the mutant extracellular region nucleic acid vaccine coding sequence; Figure 1C shows the CMV promoter followed by the XCL1 secretion signal peptide sequence followed by the VEGFR2 extracellular region for nucleic acid vaccine coding sequence; Figure 1D shows pFASTbac-dual-MBP -XCL1-VEGFR2 wild-type fusion protein vaccine purification vector; Figure 1E shows the pFASTbac-dual-MBP-XCL1-VEGFR2 mutant fusion protein vaccine purification vector; Figure 1F shows the pFASTbac-dual-MBP-VEGFR2 wild-type protein vaccine purification vector;

Figure 2 shows the results of the three-dimensional structure prediction of the fusion protein; input the nucleotide sequence encoding the fusion protein into the website http://raptorx.uchicago.edu/ to predict the three-dimensional structure of the fusion protein, showing the XCL1-VEGFR2 fusion protein space Structure diagram

Figure 3 shows the detection of the expression of the nucleic acid encoding the fusion protein in HEK293T cells; after 48 hours of transfection of the plasmid vector carrying the nucleic acid encoding the fusion protein into HEK292T cells, Western blotting was used to detect the C-terminal with a Flag tag The expression of nucleotides encoding the fusion protein;

Figure 4 shows the detection of the fusion protein purified from insect rod-shaped cells SF9; Coomassie brilliant blue staining is used to detect the purification of XCL1-VEGFR2 secreted and expressed from insect rod-shaped cells;

Figure 5 shows that the fusion protein can effectively bind to MHCII+CD11c+CD8a+ antigen cross-presenting DC cells; the Flag-tagged SF9 cell-purified protein is incubated with the spleen cells enriched with CD11C magnetic beads, and Flag fluorescent antibody is used to detect the fusion protein and MHCII+CD11c+CD8a+ antigen cross-presenting DC cell binding situation; among them, Figure 5 (A) shows the fusion protein binding DC cell flow analysis diagram; Figure 5 (B) shows the statistics of the proportion of fusion protein binding DC cells in 5 (A) diagram;

Figure 6 shows the detection of the chemotactic effect of the fusion protein on MHCII+CD11c+CD8a+ antigen cross-presenting DC cells; using the Transwell test, spleen cells containing MHCII+CD11c+CD8a+ antigen cross-presenting DC cells are placed on the upper layer, and SF9 insect cells will be removed Place the purified fusion protein in the lower layer and observe the number of upper MHCII+CD11c+CD8a+ antigen cross-presenting DC cells entering the lower layer after 2 hours; the results show that the fusion protein has a chemotactic effect on MHCII+CD11c+CD8a+ antigen cross-presenting DC cells; , Figure 6(A) shows a flow cytometric analysis chart of fusion protein chemotactic DC cells; Figure 6(B) shows a statistical analysis chart of the proportion of 6(A) fusion protein chemotactic DC cells;

Figure 7 shows the detection of VEGFR2 (KDR) expression of melanoma cell line B16 cells after tumorigenesis; tumor cells are inoculated subcutaneously, after tumorigenesis, the tumor tissue and surrounding skin tissue are taken to extract total RNA, and real-time fluorescent quantitative PCR is used to expand The augmentation technique detects the expression of VEGFR2 in the tumor and surrounding skin, and the results show that the expression of VEGFR2 in the tumor tissue is significantly higher than that in the surrounding normal skin;

Figure 8 shows the growth of mouse melanoma after immunization with the fusion gene encoding XCL1-VEGFR2/XCL1-VEGFR2 mutant fusion protein; Figure 8A shows the fusion gene immunization method and the immunization process diagram; Figure 8B shows the melanoma after inoculation , A graph of tumor growth curve made by measuring the volume of the tumor; Figure 8C shows that the mice were sacrificed when the tumor in the ^{control group grew to the ethical end point of 2000 mm 3 and the tumor size comparison diagram was dissected;}

Figure 9 shows an analysis diagram of the proportion of granzyme B-positive CD8a T cells in lymphocytes infiltrating the mouse melanoma after immunization with a fusion gene encoding XCL1-VEGFR2 fusion protein;

Figure 10 shows the use of the fusion protein to immunize mice, and then the spleen cells are taken into the mice that were intravenously injected with melanoma cells to detect the formation of simulated melanoma metastatic nodules in the lungs; Figure 10A shows the fusion protein immunized by intravenous injection Method effect diagram; Figure 10B shows a diagram of mouse lung nodules after adoptive treatment with fusion protein immunization.

Detailed ways

The present invention discloses a metastatic cancer vaccine targeting VEGFR2. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The present invention uses the amino acid sequence of the extracellular region of VEGFR2 as an immunogen to facilitate the purification of soluble protein vaccines. The vaccine of the present invention can effectively induce specific T cell immune responses against VEGFR2 in humans or mice, breaking The immune tolerance of the original tumor-associated antigen VEGFR2 can effectively directly eliminate tumor cells expressing VEGFR2, and indirectly inhibit tumor growth and the formation of metastases by inhibiting tumor angiogenesis.

One aspect of the present invention relates to a fusion protein vaccine for metastatic cancer. The vaccine consists of 1) the extracellular region of vascular endothelial growth factor receptor 2 (VEFGR2) that promotes tumor angiogenesis and can induce a specific CD8+T response And its MHC class I molecule binding epitope optimized form extracellular region, and 2) N-terminal fusion expresses the human or mouse XCL1 protein composition that specifically binds to DC cells with antigen cross-presentation ability. For example, mouse VEGFR2, SEQ ID No. 1 and SEQ ID No. 2 are composed of amino acids 20-764, and the N-terminus is expressed through a linker fusion that specifically binds human or human DC cells with antigen cross-presentation ability. Mouse XCL1 is composed of the sequence shown in amino acids 1-114 of SEQ ID No. 3 or SEQ ID No. 4.

In some embodiments, the vaccine for metastatic cancer includes any sequence of amino acids 1-889 in SEQ ID No. 7 and SEQ ID No. 8.

Another aspect of the present invention relates to a nucleic acid encoding the fusion protein vaccine. The nucleic acid vaccine consists of 1) encoding the extracellular vascular endothelial growth factor receptor 2 (VEFGR2) that promotes tumor angiogenesis and can induce a specific CD8+T response. Region and the nucleotide sequence of the extracellular region of the optimized form of binding epitope of MHC class I molecules, and 2) the N-terminal fusion expressing the nucleoside of human or mouse XCL1 protein that specifically binds to DC cells with antigen cross-presenting ability through linker fusion Acid sequence composition. That is, it encodes the 20-764 amino acid sequence of SEQ ID No. 1 and SEQ ID No. 2 and the human or mouse XCL1 sequence that specifically binds to DC cells with antigen cross-presentation ability, that is, SEQ ID No. 3 or SEQ ID No. 4 in the amino acid sequence of positions 1-114.

In some embodiments, the sequence encoding VEFGR2 that promotes tumor angiogenesis and human or murine XCL1 that specifically binds to DC cells with antigen cross-presenting ability includes the nucleotides in SEQ ID No. 11-SEQ ID No. 16 sequence.

The present invention also provides a recombinant expression vector carrying the nucleic acid vaccine, which contains the fusion protein vaccine of the present invention and the nucleotide sequence and vector corresponding to the vaccine vaccine. The vector can be a mammalian cell expression vector or an insect rod-shaped cell expression vector. Specifically, the vector can be pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP.

The present invention finally provides a vaccine for metastatic cancer, including the fusion protein vaccine, nucleic acid vaccine and recombinant expression vector.

The vaccine of the present invention provides a method for preventing recurrence or treatment for postoperative metastatic cancers such as liver cancer, colorectal cancer, lung cancer, and melanoma with high expression of VEGFR2. Including the administration of the vaccine of the present invention for other indications.

The present invention also provides the application of the vaccine, nucleic acid vaccine and vector in preparing liver cancer, colorectal cancer, lung cancer and other metastatic cancers after surgery and melanoma vaccines with high expression of VEGFR2.

The present invention also provides the vaccine, nucleic acid vaccine and carrier for treating melanoma.

The vaccine or drug of the present invention includes the fusion protein, the nucleic acid, the recombinant expression vector, and optionally pharmaceutically acceptable carriers, excipients and/or adjuvants.

In summary, the present invention provides a vaccine for metastatic cancer, the vaccine is optimized by the extracellular region of the vascular endothelial cell growth factor receptor VEGFR2 that can cause a specific CD8+ T cell immune response and MHC class I molecule binding epitopes. The extracellular region is fused at its N-terminus to express a protein composed of XCL1 molecules that specifically bind to the antigen cross-presenting ability, the nucleic acid containing the fusion protein and the vector in which the nucleic acid is located, including the fusion protein of the present invention, the nucleic acid and the vector Application in the prevention and treatment of melanoma and metastatic cancer. The invention improves the efficiency of phagocytosis, processing and presentation of the VEGFR2 protein by DC cells by fusion expression of VEGFR2 protein which promotes tumor angiogenesis and DC cell ligand XCL1, and improves the effect of inhibiting tumor growth. Experiments have shown that the protein expressed by the nucleic acid vaccine of the present invention can effectively bind to DC cells, induce VEGFR2-specific T cell responses, and significantly inhibit the growth of melanomas that highly express VEGFR2 in a variety of models.

The raw materials and reagents involved in the present invention can be purchased from the market.

The following examples further illustrate the present invention:

Example 1 The design scheme of fusion protein vaccine antigen and the construction and preparation of mammalian cell and insect rod-shaped cell expression plasmids

(1) Construction of fusion gene mammalian cell expression vector:

The fusion protein XCL1-VEGFR2 was constructed according to the amino acid sequence of mouse VEGFR2 (SEQ ID No.1 and SEQ ID No.2) and mouse XCL1 (SEQ ID No.4), in order to promote the translation of nucleic acid expressing the fusion protein in vivo The fusion protein can be effectively secreted outside the cell and chemotactic MHC-II+CD11c+CD8a+ antigen cross-presenting DC cells. We retained the secretion signal peptide of XCL1 protein (SEQ ID No.5), and at the same time, VEGFR2 mediates cell cross-presentation. The signal peptide for membrane positioning is removed (SEQ ID No. 6). The final amino acid sequence of the fusion protein (SEQ ID No. 7 and SEQ ID No. 8). For a single VEGFR2 protein, add the secretion signal peptide of the XCL1 protein to the N-terminus of the protein that removes its own signal peptide (SEQ ID No. 9 and SEQ ID No. 10) to ensure that the VEGFR2 protein expressed by the single VEGFR2 expression vector can also be the same Is secreted out of the cell. We optimized the nucleotide sequence corresponding to the amino acid sequence for mammalian cell expression preference codon optimization, synthesized it by Dynatech, and ligated it to pcDNA3.1/zeo(-) and pFastbac1-dual-MBP expression vector (SEQ ID No. 11 ~ SEQ ID No. 16).

(2) Amplification of fusion gene mammalian cell expression vector

For bacterial transformation, place competent bacteria frozen at -80°C on ice and thaw, add 100ng plasmid when it is almost completely thawed, mix gently, and place on ice for 30 minutes. Place the competent state in a 42°C water bath for 60 seconds, and immediately place it on ice for 2 minutes after taking it out. Add 500 μL of antibiotic-free LB medium to the tube, and shake culture in a shaker at 37°C for 1 hour. Centrifuge the bacteria at 4000 rpm for 2 minutes at room temperature, discard a portion of the supernatant (about 450 μL) and resuspend the bacteria, and apply an appropriate amount of the bacterial suspension to a petri dish containing the corresponding antibiotic. Place the petri dish face down and incubate overnight in a 37°C incubator. After the clone size is appropriate (about 16h), use a gun tip to pick the clones into antibiotic-added LB medium, and culture them in a shaker at 37°C until they become turbid. Take 15 mL of the bacterial solution cultured to a suitable concentration, centrifuge at 4000 rpm for 5 minutes at room temperature, and discard the supernatant. Bacterial DNA was extracted according to the instruction of "Plasmid Small Extraction Kit" (DP103) of Beijing Tiangen Company. First add 250μL of RNase-containing cell resuspension P1 to fully resuspend the bacterial pellet, and transfer the pellet to a 1.5mL EP tube. Add 250μL of alkaline cell lysate P2, gently invert and mix until the liquid is clear. Add 350μL of neutralization solution P3, and mix by inversion until flocculent sediment appears. The suspension was centrifuged at 12000rpm for 10mins at room temperature. Put the DNA adsorption column into the recovery tube, add 500 μL of balance solution to activate the adsorption membrane, centrifuge at 12000 rpm for 1 min, and discard the liquid. Transfer the supernatant obtained by centrifugation in step 4 to the DNA adsorption column, centrifuge at 12000 rpm for 1 min, and discard the liquid. Add 600 μL of cleaning solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the liquid, and repeat the washing once. Leave the recovery tube at 12000rpm for 2mins. Replace the collection tube with a new 1.5mL EP tube, and leave it at room temperature for 5 minutes to dry the adsorption column. Add 70μL of 65°C preheated elution buffer or high-pressure purified water, leave it at room temperature for 5mins to fully dissolve the DNA, centrifuge at 12000rpm for 3mins at room temperature, and collect the liquid. After the adsorption column is discarded, the concentration of the plasmid in the tube is determined and the plasmid name, concentration, and extraction date are marked.

(3) Construction of fusion insect rod cell expression vector

1) The basic principle of blue-white screening: the first component of this expression system is the pFastBac strain used to clone the target gene. Based on the selection of the pFastBac strain, the expression gene is controlled by the PH or p10 promoter, and the high-level expression expression frame in insect cells is surrounded by the left and right arms of Tn7, which contains a gentamicin resistance point, forming a miniature Tn7. The second component of this expression system is the DH10Bac strain of E. coli. DH10Bac cells contain viral plasmids with mini-attTn7 target sites and helper plasmids. As long as the pFastBac expression plasmid is transferred into DH10Bac cells, transformation will occur between the mini-Tn7 unit of the pFastBac strain and the target site of mini-attTn7, thereby producing a recombinant plasmid. The virus strain (bacmid) is amplified in the kana-resistant plasmid DH10Bac, and when the coloring substance X-gal and the inducer IPTG are present, it can supplement the lack of chromosome LacZ to form blue spots. The insertion of micro-Tn7 into the micro-attTn7 attachment site will interfere with the expression of LacZa peptide, so the clone containing the recombinant plasmid is white on the blue background, so choose the white spot analysis. The true leukoplakia clone is relatively large, so in order to avoid false positive selection, choose the largest and most independent leukoplakia.

2) Preparation of blue and white spots: construction of recombinant plasmid pFastBac HTB: use the above-mentioned experimental method for primer design (SEQ ID No. 17-20), agarose gel recovery, enzyme digestion reaction, ligation reaction, transformation and plasmid extraction, etc. The experiment finally obtained the recombinant plasmid that cloned the target gene into pFastBac HTB; the recombinant plasmid pFastBac HTB transformed into DH10Bac competent cells: Pick the target plaque (white spot): 48 hours later, until the blue and white spots can be clearly distinguished, choose the largest The most independent leukoplakia 2 to 3, use 5ml LB medium (50μg/ml kanamycin, 10μg/ml tetracycline, 7μg/ml gentamicin) shake at 37℃, 220rpm for 8-12 hours, Until the bacterial liquid is turbid, then use this bacterial liquid to extract Bacmid.

Table 1

Example 2 Three-dimensional structure prediction of fusion protein

According to the nucleotide sequence of the fusion protein, we used the website http://raptorx.uchicago.edu/ to predict the three-dimensional structure of the fusion protein of XCL1 and VEGFR2 extracellular domain. XCL1 is a chemokine, and the normal function of its chemotactic function needs to maintain a complete spatial structure. In order to ensure that XCL1 still retains its own spatial structure after the fusion of XCL1 and the extracellular domain of VEGFR2, we predicted the three-dimensional structure of the fused amino acid sequence on the website http://raptorx.uchicago.edu/, and the results are shown in Figure 2. After the fusion of XCL1 and VEGFR2, the two still retain their original spatial structure, which does not affect the chemotactic function of XCL1.

Example 3 Detection of expression effect of mammalian cell expression vector encoding XCL1-VEGFR2 fusion protein

24 hours before transfection, inoculate 1*10 ⁶ HEK293T cells in a 6-well cell culture plate, and start the transfection experiment when the cell density reaches 70%-80%. Before transfection, preheat the cell culture medium and serum-free Opti-MEM medium in a 37° water bath in advance. During transfection, add 5 micrograms of empty vector (Vector), VEGFR2 expression vector alone, XCL1-VEGFR2 wild-type and mutant fusion gene plasmids, and 20 μL of PEI transfection reagent into 200 μL of serum-free Opti-MEM, and mix them evenly. Let stand at room temperature for 10 minutes. Replace the cells to be transfected with fresh medium, gently add the above transfection system, and shake gently. Put the cells back into the cell incubator and culture for 6 hours, then change the medium. After 48 hours of transfection, the cells were collected and Western Blot was used to detect the expression effect of XCL1-VEGFR2 fusion gene plasmid HEK293T cells.

In order to facilitate the detection of the expression effect of the fusion gene, we attached a 5-amino acid Flag tag composed of DDDDK to the C-terminus of the fusion protein, so that the Flag tag antibody can be used to detect the expression of the fusion protein. Collect the cells and add 60 μL of 0.5% NP40 lysis buffer containing PMSF or Cocktail protease inhibitor. Fully resuspend the cells and spin at 4°C to lyse the cells for 30 minutes. Centrifuge the lysate at 12000 rpm and 4°C for 10 minutes, collect the supernatant into a new 1.5 mL EP tube, and discard the precipitate. Add 5×SDS-PAGE protein loading buffer according to the actual volume of the sample. After mixing, heat the sample in an air bath at 100°C for 10 minutes, and then perform Western blot immediately. Use Flag tag antibody (Sigma, F3165) for detection. Figure 3 shows that the empty vector (Vector) has no protein expression, and the VEGFR2 expression vector alone, XCL1-VEGFR2 wild-type and mutant fusion gene plasmids can be effectively expressed and the expression amount is equivalent.

Example 4 Expression of insect rod cell expression vector encoding XCL1-VEGFR2 fusion protein and protein purification

Selection of cells and culture medium: The cell expression used in this experiment is insect cell SF9 (Spodoptera frugiperda). This cell can be cultured in attachment or suspension. Generally, SF9 cells are cultured in suspension to express a large amount of the target protein. The medium is a serum-free medium, and the Gibico medium should be used for adherent culture during the virus coating stage of this experiment. For the mass preparation stage of recombinant protein, the suspension culture of Yiqiao Shenzhou SIM SF medium is used.

(1) Transfection of insect cells

Prepare the cells: use a polystyrene 6-well culture plate, the total number of cells is 0.9×10 ⁶ cells/well, move gently up and down and left and right to make the cells evenly spread on the culture plate, and let it stand for 30 minutes;

(2) Preparation of DNA/lipid mixture:

1) Add 100μl Gibco medium to a 1.5ml EP tube, then add 1μl Genejuice, this is solution 1;

2) Add 100μl Gibco medium to another 1.5ml EP tube, and then add 1μg Bacmid, this is solution 2;

3) Mix Solution 1 and Solution 2 thoroughly, and incubate for 30 minutes at room temperature;

(3) Transfection: Add 800μl of Gibco medium to the DNA/lipid mixture, blot the cell culture medium in the culture plate, and quickly transfer 1ml of the DNA/lipid mixture to the cell monolayer. Shake gently, 27°C Let stand for 5 hours;

(4) Cultivation: After 5 hours, remove the transfection medium, add 2ml Gibco medium, and cultivate at 27°C;

(5) Virus infection can be observed after 72 hours of incubation, and the P0 virus strain is extracted on the 5th to 6th day.

(6) Preparation, amplification and identification of virus strains

1) Preparation of virus strains: Once the transfected cells are obtained and late-stage transfection occurs, the virus-containing cells can be collected from the wells in the 6-well plate and transferred to a sterile 2.0 ml centrifuge tube. Centrifuge for 5 min to remove cells and debris. Then transfer the supernatant to a new 2.0ml centrifuge tube, which is the P0 virus strain, add 10% FBS, and store at 4°C in the dark.

2) Amplify the virus strain: add 100～200μl of P0 virus to ^{a medium with a total cell number of 18～20×10 6} and a total volume of 15ml, and use T75 for virus amplification; collect the virus after culturing for 4～5 days And transferred to a sterile 15ml centrifuge tube. Centrifuge at 500×g for 5 min, and collect the supernatant and precipitate separately

(7) Mass production of recombinant protein

1) Virus infection: Use insect cells SF9 to express the target protein, expand the cells to the appropriate cell density for infection, and add the corresponding amount of virus. After about 65 hours, the cell suspension was collected.

2) Collection of cell supernatant: The target protein is a secreted protein, so the target protein is in the supernatant. The cell suspension expressing the target protein was centrifuged at 8000 rpm/4°C for 30 min in a high-speed centrifuge to separate the cells from the supernatant, and then the supernatant was taken, and then directly subjected to in vitro affinity chromatography for protein purification.

3) Protein purification constructed by affinity chromatography using polyhistidine tag (His-tag) has a His-tag composed of six consecutive histidines on the N-terminus of the target protein, histidine side chain The imidazole group can bind metal ions (nickel or cobalt). If the nickel or cobalt ions are immobilized on the chromatography medium, when the mixed solution containing the His-tag fusion protein flows through the chromatography column, the His-tag fusion protein is adsorbed on the column, and all the miscellaneous proteins flow out; then use a higher concentration The imidazole solution (such as 200 mM) is eluted, and the imidazole will compete with the imidazole ring of His-tag, and finally the His-tag fusion protein will be eluted. The histidine tag is very small and hardly affects the function, activity or structure of the target protein, so it is widely used. The main experimental steps are as follows:

1) Binding: add the cell supernatant obtained after centrifugation to the nickel column, combine at 4°C, and slowly flow the supernatant through the Ni Beads to make the target protein with the His tag fully bind to the Ni Beads;

2) Wash the contaminated protein: first wash with a PBS solution added with 20mM imidazole, and use Coomassie brilliant blue staining solution to check whether the contaminated protein is completely washed during the washing process;

3) Elution of the target protein: The target protein is eluted with the PBS elution solution added with 200 mM imidazole, and the Coomassie brilliant blue staining solution is used to check whether the washing is complete during the elution process;

4) Concentrate the target protein solution eluted in step 3 with a 4°C centrifuge to a final volume of 500 μl.

5) Separation and purification of gel filtration chromatography column: apply the concentrated solution obtained in step 4 to the gel filtration chromatography column Superdex 200 that has been pre-balanced with PBS, collect the target protein peak, and use Western Blot to identify whether it is the purpose Protein, and the purity of the protein was identified by 12% SDS-PAGE electrophoresis, as shown in Figure 4;

6) Concentration and cryopreservation: According to the results of 12% SDS-PAGE electrophoresis identification, the protein with the required purity is concentrated, and the protein concentration is measured by a micro-spectrophotometer, and then aliquoted, and stored in liquid nitrogen quick freezing at -80°C.

Example 5: Detection of the ability of XCL1-VEGFR2 fusion protein to bind MHC-II+CD11c+CD8a+ antigen to cross-present DC cells

After obtaining the purified fusion protein, the present invention first verifies whether the protein obtained from insect cells can effectively bind to MHCII+CD11c+CD8a+ antigen cross-presenting DC cells.

(1) Isolation and purification of CD11c+DC cells

Using mouse whole spleen as the source of CD11c+DC cells, first take mouse spleen and use 70um pore size cell mesh to grind into single cells in 1640 medium. After centrifugation at 200g for 10 minutes, discard the supernatant and use R&D company red blood cell lysate ( Catalog No. WL2000) spleen cells were treated with split red. Specifically, the lysis solution A was diluted 10 times with distilled water to prepare a working solution, and each spleen was resuspended by adding 2 mL of working solution, and placed at room temperature for 10 minutes, during which the neutralization solution B was diluted with distilled water 10 times to prepare a working solution. Ten minutes later, add 10 mL of neutralization solution B to the lysis solution and centrifuge at 200 g for 10 minutes after neutralization. The cell pellet was washed once with PBS buffer containing 1% inactivated FBS and counted. Take a total of 1*10 ⁸ cells and resuspend them in 400 μL of PBS buffer containing 1% inactivated FBS, add 100 μ LCD11c magnetic beads (Miltenyi Biotec: 130-108-338), and place them in the dark at 4° for 20 minutes. During this period, 1% FBS-inactivated PBS buffer was used to equilibrate the column that adsorbed the magnetic beads. After 20 minutes, the cells and the magnetic bead mixture were transferred to the magnetic bead adsorption column together, and the column was placed on the magnetic stand. After the cells were completely inserted into the column, the cells were washed three times with 3 mL of 1% inactivated FBS PBS buffer. Afterwards, the column was removed from the magnetic stand and placed on the top of the 15 mL centrifuge tube. 5 mL of 1% FBS-inactivated PBS buffer was added and quickly pushed to elute the CD11c-positive DC cells, counted and centrifuged at 200 g for 10 min. The finally obtained CD11c-positive DC cells were resuspended to 1*10 ⁶ per mL with serum-free 1640. Spread in a 24-well plate, 1mL per well. The cells were divided into BSA protein control group, VEGFR2 protein control group and XCL1-VEGFR2 wild-type protein experimental group. The total amount of protein in each group was 50μg, mixed well, and incubated in a 37° carbon dioxide incubator for 40 minutes. The cells were collected and centrifuged at 500g. , And washed twice with 1% inactivated FBS PBS buffer. And perform flow dyeing: MHC-II APC, Flag-dye light 488, CD8α-Percp/Cy5.5. Use BD LSRII instrument to collect cells at medium speed, and analyze the ratio of MHC-II+Flag-dye light 488+CD8α+ cells between different groups. Repeat 3 times for each group.

(2) The results show that the number and proportion of MHC-II+CD11c+CD8a+ bound to the VEGFR2 protein without XCL1 and the BSA control protein are similar, while the VEGFR2 protein fused with XCL1 binds to the MHC-II+CD11c+CD8a+ antigen more strongly On cross-presenting DC cells, the binding capacity is more than 20 times higher than that of the control group, as shown in Figures 5A and 5B.

Example 6 Detection of the chemotaxis ability of XCL1-VEGFR2 fusion protein on MHCII+CD11c+CD8a+ antigen cross-presenting DC cells

(1) In view of the fact that XCL1-VEGFR2 fusion protein can effectively bind to MHCII+CD11c+CD8a+ antigen cross-presenting DC cells, based on previous studies that XCL1 chemokine can effectively recruit MHCII+CD11c+CD8a+ antigen cross-presenting DC cells, we designed Tanswell Experiments verify that the fusion protein can effectively chemotactic and recruit DC cells. The Transwell test uses a double-layered cell culture plate with upper and lower layers, the lower layer is added with chemokines, and the upper layer is covered with target cells. In this experiment, it is DC cells enriched with CD11c magnetic beads. The bottom of the upper culture chamber is a membrane with a specific pore size. In this experiment, a 24-well Transwell membrane with a diameter of 6.5 mm and a pore size of 5 μm was selected according to previous research reports. Transmembrane enters the lower layer. The number of cells entering the lower layer is directly proportional to the chemotactic ability of the chemokine. We first resuspend the DC cells enriched with CD11c magnetic beads to 1* ¹⁰⁷ per mL, and add 100μL of cells to the upper chamber, namely 1*10 ⁶ cells. In the lower layer, 700μL of the control medium containing 70ng of BSA protein, 70ng of VEGFR2 protein, and 70ng of XCL1-VEGFR2 wild-type and mutant proteins of the experimental group were added to ensure that the liquid level of the upper and lower media was consistent and prevent the upper cells from The liquid level is different and enters the lower layer due to pressure. Place the culture plate in a carbon dioxide incubator for 5 hours. Remove the upper chamber to collect the cells in the lower culture medium, centrifuge at 500 g, and wash twice with 1% FBS-inactivated PBS buffer. And perform flow staining: MHC-II APC, CD11c-PE, CD8α-Percp/Cy5.5. Use BD Canto II instrument to collect cells at medium speed, and analyze the ratio of MHC-II+CD11c-PE+CD8α+ cells between different groups. Repeat 3 times for each group.

(2) The results show that the number and ratio of MHC-II+CD11c+CD8a+ chemotaxis of VEGFR2 protein without XCL1 and BSA control protein are similar, while VEGFR2 protein fused with XCL1 can chemoattract both wild-type and mutants. More MHC-II+CD11c+CD8a+ antigen cross-presenting DC cells have statistically significant differences. The statistical method is Unpaired test, as shown in Figures 6A and 6B.

Example 7 Intervention effect of fusion gene on the occurrence of B16 melanoma cell allograft tumor

In view of the normal expression of the fusion gene in mammalian cells, the fusion protein can also effectively chemoattract and combine with MHC-II+CD11c+CD8a+ antigen to cross-present DC cells. We extracted separate VEGFR2 and XCL1-VEGFR2 wild-type and mutant plasmids, and used Wealtec's gene gun (GDS-80) to inject immune plasmids into mice. And after allotransplantation of B16 melanoma cells, the inhibition of the growth of B16 melanoma cells by fusion gene immunization was observed.

(1) B16 melanoma cell culture and detection of VEGFR2 gene expression after tumorigenesis Resuscitate B16 cells: pre-warm cell culture medium (cell culture medium is prepared by adding 10% FBS to Corning's DMEM medium). Take out the frozen cells to be resuscitated from liquid nitrogen or -80°C refrigerator, and quickly melt them in a 37°C water bath. After the cells were completely lysed, centrifuged at 1500 rpm for 3 min, and at the same time added 8 mL of pre-warmed cell culture medium to the petri dish. After centrifugation, the supernatant was discarded, and 1 mL of pre-warmed cell culture medium was added. The cells were resuspended and transferred to a prepared petri dish. The cell name, date, and number of passages were marked. The cells were cultured in a 37°C, 5% CO2 incubator. Passage the next day, pre-warm the cell culture medium, trypsin, and PBS buffer. Use a vacuum pump to suck out the old medium in the petri dish. Add 7mL PBS buffer to rinse the cells once and aspirate the PBS. Add 1mL trypsin and place it in a 37°C incubator for proper time for digestion. Add 3mL culture medium to terminate the digestion, pipette the cells to a single cell suspension with an electric pipette and transfer to a 15mL centrifuge tube, centrifuge at 1500rpm for 3min, and prepare a new culture dish at the same time, add 8mL culture medium to each. After centrifugation, discard the supernatant, add appropriate amount of medium according to the required passage ratio to resuspend the cells, pipette to a single cell suspension, take 1 mL of the single cell suspension into a new culture dish, and put it back into the 37°C incubator with 5% CO2 Continue to cultivate. When the cells grow to 80%-90% density, digest the cell count according to the above method and resuspend it in serum-free DMEM medium. According to our previous experience of tumor formation, adjust the cell concentration to 1*10 ⁶ cells per mL, each 100 μL of cells were subcutaneously inoculated into the armpits of the mice. Two weeks later, the formed melanoma and surrounding normal skin were taken, and total RNA was extracted. Collect an appropriate amount of mouse tissue and add 1 mL Trizol (Invitrogen). Lysis with a tissue homogenizer for 2mins, and rotate for 30mins at 4°C for complete lysis. Add 200μL of chloroform to each 1mL of Trizol, shake vigorously for 15s, and let stand at room temperature for 15mins. Pre-cooled centrifuge at 4℃, 12000rpm, centrifugation for 10mins. After centrifugation, transfer the upper aqueous phase to a new 1.5mL EP tube, add 500μL of isopropanol, mix upside down and centrifuge at 12000rpm, 4°C for 10mins. A white precipitate can be seen after centrifugation, the supernatant is discarded, and the RNA precipitate is washed with 75% ethanol in DEPC water, and centrifuged at 7500 rpm for 5 mins at room temperature. Discard the supernatant, and place the dried RNA at room temperature until the precipitate is translucent. Take an appropriate volume of DEPC water to dissolve RNA. Mix the dissolved RNA, use Nanodrop to detect the concentration and purity of the RNA, and mark the RNA species type, concentration, and extraction time on the EP tube wall and lid. The primers (SEQ ID No. 21-24) shown in the table below were used for real-time PCR detection. The results showed that compared with surrounding normal skin, the expression level of VEGFR2 in the formed melanoma was significantly increased. As shown in Figure 7, it is reasonable to use B16 cells to evaluate the fusion gene vaccine.

Table 2

(2) The preventive effect of fusion gene immunization on the occurrence of B16 melanoma cell allograft tumors

After confirming that the B16 cell tumorigenesis model is available, we injected the mice with gene gun plasmids according to the immunization strategy marked on the time axis in Figure 8A. C57B6 (purchased from Tonglihua) week-old male mice were divided into three groups injected with separate VEGFR2 and XCL1-VEGFR2 wild-type and mutant plasmids, five in each group, and depilatory cream was used to carry out the right side of the mouse near the groin Hair removal treatment at the lymph nodes. After that, use a gene gun to inject the plasmid into the hair removal site, each 50μg, once a week, a total of four injections, one week after the last injection, the B16 melanoma cells with good tumorigenic conditions were explored, and the tumor formation time was observed every two days. Measure the long axis a and short axis b of the tumor, calculate the tumor volume according to a*b*b/2, and draw the tumor growth curve. The results are shown in Figures 8A and 8B. Both the wild-type and mutant-type plasmids of XCL1-VEGFR2 can effectively delay the onset and growth rate of tumors. It shows that the therapeutic effect of fusion gene immunization is significantly effective.

(3) Explore whether the fusion gene immunization induces a stronger cell-specific T cell response. When the tumor in the control group grew to the ethical endpoint of 2000 mm ³ , the mice were sacrificed and the tumors loaded by the mice in each group were taken off and photographed. Grind the cells into a single cell suspension with a 100μm mesh screen. After 800g centrifugation, resuspend the cells with 80% percoll solution and ^{add 4mL per 1000mm 3} to resuspend. Carefully add 40% pecoll solution to the upper layer. Centrifuge at 800g for 20 minutes and discard. The uppermost tumor cells are left with the middle layer of lymphocytes, washed and counted, and finally the cells are resuspended in 1640 medium containing 10% heat-inactivated FBS at a concentration of 1*10 ⁶ cells per mL, and the cells are plated in a 24-well plate. 1 mL per well was stimulated by adding VEGFR2 epitope polypeptide, and Golgi stop (Biolegend) was added to inhibit the secretion of intracellular factors. After 12 hours, the collected cells were washed twice with 1% FBS-inactivated PBS buffer, and subjected to flow cytometry: Gran B-PE, CD8α-Percp/Cy5.5. The results are shown in Figure 9. The mouse tumors immunized with XCL1-VEGFR2 wild-type and mutant plasmids have significantly more VEGFR2-specific T cells compared with the VEGFR2 immunization group alone, indicating that XCL1-VEGFR2 wild-type and mutant types Plasmid immunization did induce specific CD8a cytotoxic T lymphocytes.

Example 8: Therapeutic effect of T cell adoptive therapy on melanoma after immunization with fusion protein

In view of the successful induction of specific cytotoxic T lymphocytes against VEGFR2 in mice after immunization with the fusion gene, the present invention uses the fusion protein purified from insect rod cells to immunize the T cell donor mouse. Induce specific cytotoxic T lymphocytes against VEGFR2 in mice, and adopt them to recipient mice with B16 tumor cells to see if it can effectively inhibit the occurrence and development of tumors in recipient mice.

(1) Fusion protein immunization of adoptive T cell donor mice

In order to enable the fusion protein purified from insect rod cells to effectively induce specific cytotoxic T lymphocytes against VEGFR2, the team of the present invention explored and optimized the route of administration based on the characteristics of XCL1 targeting DC cells in the previous study. First, The results of intravenous administration are shown in Figure 10A. Intravenous injection of KDR protein plus LPS adjuvant does not produce anti-tumor effects. Finally, it was determined that the fusion protein 40 micrograms dose plus Poly: IC 30 micrograms plus incomplete Freund’s adjuvant emulsified and subcutaneously immunized, once a week, twice co-immunization can effectively induce the production of specific cytotoxic T lymphocytes against VEGFR2. After subsequent treatment, the B16 melanoma cell allograft has an inhibitory effect.

(2) The therapeutic effect of adoptive T cell therapy on melanoma

The mice were sacrificed one week after the second subcutaneous protein immunization, and the mouse spleens were taken and ground according to the method in Example 5, split red and enriched with CD8a magnetic beads for cytotoxic T lymphocytes, and finally the obtained cells were resuspended In the serum-free 1640 medium, the concentration is 2* ¹⁰⁷ ^{cells per mL. For mice that were inoculated with 3*10 5} B16 melanoma cells via the tail vein two days in advance, the T cells obtained by adopting the tail vein again each had a total of 200 μL. 2*10 ⁶ cells. Two weeks later, the mice were dissected and the lung tissues of the mice were fixed in 10% paraformaldehyde. The photos were taken the next day. The results are shown in Figure 10B. After cytotoxic T lymphocytes, the lung nodules of the recipient mice were significantly reduced. It shows that mice immunized with XCL1-VEGFR2 wild-type and mutant proteins produce cytotoxic T lymphocytes against VEGFR2, and can effectively kill melanoma cells expressing VEGFR2 in vivo.

The metastatic cancer vaccine targeting VEGFR2 provided by the present invention has been introduced in detail above. This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above examples is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

The fusion protein is characterized in that it includes the extracellular region of VEFGR2 (KDR) that promotes tumor angiogenesis and can induce a specific CD8+T response or its MHC class I molecule binding epitope-optimized extracellular region; linker; and specificity Human or mouse XCL1 protein that binds to DC cells with antigen cross-presentation ability;

in:

The amino acid sequence of the extracellular region of VEFGR2 that can induce a specific CD8+T response or its MHC class I molecule binding epitope optimized form of the extracellular region of VEFGR2 that promotes tumor angiogenesis is as shown in SEQ ID No. 1 or 2 from 20 to 2 The amino acid at position 764 is shown; the amino acid sequence of the human or murine XCL1 protein that specifically binds to DC cells with antigen cross-presenting ability is shown in amino acids 1 to 114 of SEQ ID No. 3 or 4.
The fusion protein according to claim 1, wherein the fusion protein comprises a sequence as shown in amino acids 1 to 889 in SEQ ID Nos. 7 and 8.
The nucleotide encoding the fusion protein of claim 1 or 2, wherein the sequence of the nucleotide:

(Ⅰ), as shown in SEQ ID No. 11-16;

or

(II), as shown in the complementary nucleotide sequence of the nucleotide sequence arbitrarily shown in SEQ ID No. 11-16; or

(Ⅲ). For example, a nucleotide sequence that encodes the same protein as the nucleotide sequence of (I) or (II), but is different from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code Shown.
The recombinant expression vector is characterized by comprising a vector and the fusion protein according to claim 1 or 2 or the nucleotide according to claim 3.
The recombinant expression vector of claim 4, wherein the vector comprises pcDNA3.1(+), pcDNA3.1(-), pFastbac1-dual-MBP.
A recombinant strain or cell comprising the fusion protein according to claim 1 or 2 or the nucleotide according to claim 3.
The fusion protein as claimed in claim 1 or 2, the nucleotide as claimed in claim 3, the recombinant expression vector as claimed in claim 4 or 5, or the recombinant strain or cell as claimed in claim 6 in preparation and transfer The application of a vaccine for a melanoma with high expression of VEGFR2 or a vaccine or a medicine for the prevention and/or treatment of a metastatic cancer or a melanoma with a high expression of VEGFR2.
The use according to claim 7, wherein the metastatic cancer comprises liver cancer, colorectal cancer or lung cancer.
A vaccine for metastatic cancer or melanoma with high expression of VEGFR2, characterized by comprising the fusion protein according to claim 1 or 2, the nucleotide according to claim 3, and the vaccine according to claim 4 or 5. Recombinant expression vectors and pharmaceutically acceptable carriers, excipients and/or adjuvants.
A drug for preventing and/or treating metastatic cancer or melanoma with high expression of VEGFR2, characterized by comprising the fusion protein according to claim 1 or 2, the nucleotide according to claim 3, and the fusion protein according to claim 4 Or the recombinant expression vector described in 5 and pharmaceutically acceptable excipients.