WO2011106981A1 - Utilisation d'une protéine de fusion cytokine-superantigène pour préparer un médicament contre une tumeur solide - Google Patents

Utilisation d'une protéine de fusion cytokine-superantigène pour préparer un médicament contre une tumeur solide Download PDF

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WO2011106981A1
WO2011106981A1 PCT/CN2010/078854 CN2010078854W WO2011106981A1 WO 2011106981 A1 WO2011106981 A1 WO 2011106981A1 CN 2010078854 W CN2010078854 W CN 2010078854W WO 2011106981 A1 WO2011106981 A1 WO 2011106981A1
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sea
cytokine
fusion protein
cells
tumor
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孙嘉琳
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Sun Jialin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/085—Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55522—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to the use of a fusion protein, and more particularly to the use of a cytokine-superantigen fusion protein for the preparation of an anti-solid tumor drug. Background technique
  • EGF Epidermal growth factor
  • VEGF Vascular endothelial growth factor
  • Epidermal growth factor is linked to a toxin protein or RA hydrolase to selectively kill cancer cells that highly express EGF receptors (Clin. Cancer Res., 11, 329-334, 2005; Protein Eng., 11, 1285-1292 , 1998), but this approach does not rely on the immune system such as lymphocytes to attack the tumor.
  • the VEGF gene was discovered in the late 1980s (Science, 246, 1306-1309, 1989; Science, 246, 1309-1312, 1989). Due to the different shearing of mR A, its mature form can take many forms, and the length can be It is a peptide of 189, 165 and 121 amino acids (J. Biol. Chem., 266, 11947-11954, 1991).
  • a cell receptor is a protein located on the surface of a cell. After receiving an external signal, a series of signals are transmitted inside the cell, thereby triggering activities such as gene regulation. Cancer cells often have a large number of growth factor-related receptors. The body becomes the target of the antibody. If an antibody interacts with, or binds to, these receptors, thereby blocking the interaction of the cytokine with its receptor, thereby inhibiting tumor growth.
  • the antibody neutralizes receptors on the blocked cancer cells, thereby inhibiting ligand-mediated cell proliferation. Although the antibody has antibody dependent-cell mediated cytotoxicity, the effect is not significant. Enhanced lymphocyte-mediated cytotoxicity can be produced by ligating a superantigen (Superantigen) of Staphylococcal-enterotoxin A (SEA) to the antibody.
  • Superantigen superantigen
  • SEA Staphylococcal-enterotoxin A
  • the SEA gene was reported as early as the 1980s (J. Biol. Chem., 262, 7006-7013, 1987; J. Bacteriol., 170, 34-41, 1988).
  • a point mutation was introduced at its position 227 (Proc. Natl. Acad. Sci. USA, 94, 2489-2494, 1997).
  • more point mutations were introduced in SEA (J. Mol. Biol" 333, 893-905, 2003).
  • SEA does not require the processing of antigen-presenting cells, but forms a complex with MHC class II molecules on the cell membrane in intact protein form, recognizing the ⁇ fragment of T cell receptor (TCR), which activates much more than the common antigen.
  • Sputum cells including CD4+, CD8+
  • a small group of antibody superantigen fusion proteins from Sweden have conducted many studies on lung metastasis of B16 melanoma (Proc. Natl. Acad. Sci. USA, 92, 9791-9795, 1995; Eur. Surg. Res., 35, 457 -463, 2003 ) , but right How the role of anti-solid tumors remains unclear.
  • Cancer cells are transformed from normal cells.
  • the antigens of cancer cells are autoantigens, so cancer cells can escape the surveillance of the epidemic system.
  • People are always looking for new anti-cancer methods to improve the immunity of cancer patients, especially the specific immunity against cancer cells. Therefore, there is an urgent need in the art for a powerful new anticancer drug specifically for cancer cells for use in anti-tumor, particularly against solid tumors. This drug is different from antibodies and has not been reported yet.
  • a superantigen fusion protein which can be used for anticancer treatment and a method for producing the same discloses a method for constructing a fusion protein of a cytokine and a superantigen, and expression and purification of the fusion protein in Escherichia coli The method, however, does not disclose data on the biological activity of fusion proteins of cytokines and superantigens against solid tumors. Summary of the invention
  • cytokine-superantigen fusion protein for the preparation of an anti-solid tumor drug
  • the cytokine is epidermal growth factor or vascular endothelial growth factor
  • the superantigen is a superantigen of S. aureus enterotoxin A.
  • the cytokine-superantigen fusion protein is capable of mobilizing tau lymphocytes and targeting the T lymphocytes to surrounding tumor cells within solid tumor tissue, the T lymphocytes having CD4 + or CD8 + characteristics and Can interact with SEA.
  • the cytokine-superantigen fusion protein can induce secretion of the cytokines interleukin-2, interferon- ⁇ and tumor necrosis factor- ⁇ by T lymphocytes in solid tumor tissues.
  • the cytokine-superantigen fusion protein is capable of inducing high expression of the apoptotic protein Fas on the surface of tumor cells.
  • the cytokine-superantigen fusion protein is capable of inducing secretion of perforin and granzyme B by killer T lymphocytes.
  • the cytokine-superantigen fusion protein is capable of inhibiting the growth of solid tumors in vivo, resulting in the death of a large number of tumor cells in the tumor tissue.
  • the cytokine-superantigen fusion protein is actually a replacement for the antibody of the prior art and also has a targeting effect, and is a new and promising drug that is different from the antibody.
  • the cytokine-superantigen fusion protein interacts with the receptors of EGF and VEGF, namely EGFR and VEGFR, respectively, and with TCR on T cells, thus targeting T cells to a large number of EGFR or VEGFR.
  • EGF and VEGF namely EGFR and VEGFR
  • TCR TCR on T cells
  • Figure 1 is an electropherogram of a high purity VEGF-SEA fusion protein with only one band. (In the figure, 1: rinsing solution in purification 2: VEGF-SEA washed with eluent)
  • Figure 2 shows the high-purity SEA and EGF-SEA electropherograms.
  • Figure 1 SEA
  • 2 EGF-SEA
  • Figure 3 shows that EGF-SEA and VEGF-SEA fusion proteins inhibit tumor growth.
  • Figure 4 shows the anatomy of mice injected with EGF-SEA, VEGF-SEA SEA and saline.
  • 4-1 is the control; 4-2 is SEA; 4-3 is EGF-SEA; 4-4 is VEGF-SEA)
  • FIG. 5 shows the dissected tumor weight results of mice injected with EGF-SEA, VEGF-SEA SEA, and saline.
  • Figure 6 shows CD4+ T cells detected by conventional immunohistochemistry. (In the figure: 6-1 is EGF-SEA; 6-2 is
  • VEGF-SEA 6-3 for SEA; 6-4 for control
  • Figure 7 is a graph showing the number of CD4+ T reactive cells on the tissue section of Figure 6.
  • Figure 8 is a CD8+ T cell detected using conventional immunohistochemistry.
  • 8-1 is EGF-SEA
  • 8-2 is VEGF-SEA
  • 8-3 is SEA
  • 8-4 is control
  • Figure 9 is a graph showing the number of CD8+ T reactive cells on the tissue section of Figure 8.
  • Figure 10 is a T cell reacted with SEA detected by conventional immunohistochemistry.
  • 10-1 is EGF-SEA
  • 10-2 is VEGF-SEA
  • 10-3 is SEA
  • 10-4 is control
  • Figure 11 is a graph showing the number of T cells in the SEA reaction on the tissue section of Figure 10.
  • FIG. 12 Paraffin sections of tumor tissue were incubated with EGF-SEA and VEGF-SEA protein solution, first reacted with anti-SEA antibody, then reacted with fluorescein-labeled anti-mouse 2 antibody, diluted 1:50, and finally under a fluorescence microscope. Observed. (In the figure: 12-1 is EGF-SEA; 12-2 is VEGF-SEA; 12-3 is SEA; 12-4 is control)
  • FIG. 13 IL-2 secretion in tumor tissues (in the figure: 13-1 is EGF-SEA; 13-2 is VEGF-SEA; 13-3 is SEA; 13-4 is control, 13-1, 13-2 shows There is a color-developing region of antibody reaction between tumor cells).
  • FIG. 14 TNF- ⁇ secretion in tumor tissues (in the figure: 14-1 is EGF-SEA; 14-2 is VEGF-SEA; 14-3 is SEA; 14-4 is control, 14-1, 14-2 shows There is a color-developing region of antibody reaction between tumor cells).
  • Figure 15 IFN- ⁇ secretion in tumor tissues (in the figure: 15-1 is EGF-SEA; 15-2 is VEGF-SEA; 15-3 is SEA; 15-4 is control, 15-1, 15-2 shows There is a color-developing region of antibody reaction between tumor cells).
  • Figure 16 shows the amount of cytokine secretion in units of area based on antibody positive reactions on paraffin sections of the immunohistochemistry experiments of Figures 13-15.
  • Figure 17 and I used an enzyme-linked immunosorbent assay (ELISA) to test the secretion of cytokines in tumor tissues using a U-box (R & D Systems).
  • ELISA enzyme-linked immunosorbent assay
  • Fig. 18 Analysis of Fas expression on tumor cells, and anti-Fas antibody (Santa Cruz Biotechnolog) was used to examine tumor tissues (in the figure: 18-1 is EGF-SEA; 18-2 is VEGF-SEA; 18-3 is SEA; As a control, 18-4 was found to express a large amount of Fas on the surface of mouse S180 cells injected with EGF-SEA and VEGF-SEA, and it was shown that the shaded portion around the large cells of the antibody reaction was Fas protein.
  • Figure 19 Perforin analysis around tumor cells, tumor tissue was examined with anti-perforin antibody (Santa Cruz Biotechnolog) (in the figure: 19-1 is EGF-SEA; 19-2 is VEGF-SEA; 19-3 is SEA; 19-4 is the control, It was found that mice injected with EGF-SEA and VEGF-SEA had a large amount of perforin protein around the tumor S180 cells, and the small band portion indicated by the arrow was the accumulated perforin protein group).
  • anti-perforin antibody Santa Cruz Biotechnolog
  • Fig. 20 Analysis of granzymes around tumor cells, using anti-granzyme antibody (Abeam) to examine tumor tissues (in the figure: 20-1 is EGF-SEA; 20-2 is VEGF-SEA; 20-3 is SEA; 20- 4 For the control, it was found that there were a large amount of granzymes around the tumor S180 cells injected with EGF-SEA and VEGF-SEA, and the shaded part of the large cell surface of the tumor cells and its surroundings was the granzyme protein).
  • Abeam anti-granzyme antibody
  • FIG. 21 Tumor cell TU EL staining, (in the figure: 21-1 is EGF-SEA; 21-2 is VEGF-SEA; 21-3 is SEA; 21-4 is control, EGF-SEA and VEGF are found by TU EL detection.
  • 21-1 is EGF-SEA
  • 21-2 is VEGF-SEA
  • 21-3 is SEA
  • 21-4 is control
  • EGF-SEA and VEGF are found by TU EL detection
  • FIG. 22 TUNEL positive rate of tumor cells. Mortality was calculated based on the number of shadow dead cells on the TUNEL-stained tissue sections, and the tumor cell death rate reached 50-60%. detailed description
  • the present invention utilizes a cytokine-superantigen fusion protein in which a cytokine that promotes cancer cell growth can localize a fusion protein to a cancer cell, and a superantigen causes an anticancer immune response around the cancer cell, that is, superantigen-dependent Superantigen-dependent-cellular-cytotoxicity (SDCC).
  • SDCC superantigen-dependent Superantigen-dependent-cellular-cytotoxicity
  • Entrusted TAKARA to synthesize a nucleic acid sequence fragment comprising EGF and a linker peptide encoding 53 amino acids and a few bases in addition to the restriction endonuclease sites BamHI and EcoRI in front of EGF, in a nucleic acid sequence encoding a portion of the linker peptide Contains restriction endonuclease sites for Sail and Hindlll.
  • This synthetic nucleic acid fragment was inserted into the T vector and identified by DNA sequencing, and then treated with BamHI and Hindlll by double digestion, and this fragment was inserted into the pET22b plasmid to form pET22b-EGF.
  • the second step is to synthesize a fragment of the SEA nucleic acid sequence with Asp (227) ⁇ Ala point mutation, first insert the T vector and identify it by DNA sequencing, and then use the double enzyme digestion method to treat the fragment with Hindlll and B Xhol. Insertion into the pET22b-EGF plasmid resulted in the expression vector pET22b-EGF-SEA, which expresses the EGF-SEA fusion protein (see Sequence Listing SEQ ID N0.2).
  • Entrusted TAKARA to synthesize a nucleic acid sequence fragment comprising a VEGF and a linker peptide encoding a 121 amino acid and a few bases of the restriction endonuclease sites BamHI and EcoRI in front of the VEGF, in a nucleic acid sequence encoding a portion of the linker peptide Contains restriction endonuclease sites for Sail and Hindlll.
  • This synthetic nucleic acid fragment was inserted into the T vector and identified by DNA sequencing, and then treated with BamHI and Hindlll by double digestion, and this fragment was inserted into the pET22b plasmid to form pET22b-VEGF.
  • the SEA which has been synthesized and inserted into the T vector in Example 1 was treated with Hindpl and Xhol by double digestion, and then inserted into the pET22b-VEGF plasmid, thereby producing the expression vector pET22b-VEGF.
  • -SEA can express VEGF-SEA fusion protein (see preface List SEQ ID NO. 4).
  • the expression plasmids pET22b-EGF-SEA and pET22b-VEGF-SEA and pET22b-SEA as a control were separately electroporated into E. coli BL21 (DE3), and positive bacteria were screened by antibiotic Amp.
  • the process of expression, denaturation and renaturation and purification of the various proteins is roughly the same, as follows:
  • Escherichia coli BL21 (DE3) containing the expression plasmid was first cultured at a large-scale 37 ° C, and then IPTG was added thereto to a concentration of 1 mM and cultured at 30 ° C overnight to induce expression of the protein. On the second day, the culture solution was centrifuged and the cells were collected, the cell wall was broken by an ultrasonic method, and the inclusion body precipitate was collected by centrifugation, and the protein here was present as an inclusion body.
  • the inclusion body protein is denatured with 6M urea and then subjected to multi-stage dialysis.
  • the dialysis solution is gradually diluted urea such as 3M, 2M and 1M, followed by 0.5M urea, 0.4M L-arginine, 375 ⁇ oxidized glutathione.
  • mice Male ICR mice were first selected, 4-5 weeks, 18-22 g, divided into 4 groups of 15 mice each. 2x10 6 mouse sarcoma cell sarcoma 180 (S180) was inoculated into the right side of the mouse, and then intraperitoneally injected with EGF-SEA and VEGF-SEA and SEA protein solution 0.2 ml (250 pmol)/day. The control group was injected with the same amount of normal saline (0.9% NaCl), and the mice were sacrificed 9 days later. The tumor growth of the mice and the weight of the tumor after sacrifice were observed. Figure 3 shows that EGF-SEA and VEGF-SEA fusion proteins inhibit tumor growth.
  • Figure 4 shows the anatomy of mice injected with EGF-SEA, VEGF-SEA SEA and saline.
  • Figure 5 shows the dissected tumor weight results of mice injected with EGF-SEA, VEGF-SEA SEA, and saline. It indicates that EGF-SEA and VEGF-SEA fusion proteins have a very significant inhibitory effect on solid tumors.
  • cytokine-superantigen fusion proteins and SEA protein treatments and mice in the control group of saline S180 tumor tissues were cut into small pieces, embedded in paraffin, and then subjected to immunohistochemistry.
  • To detect CD4+ and CD8+ T cells in tumor tissues Santa Cruz Biotechnolog anti-CD4+ and CD8+ antibodies were used, followed by secondary antibodies and avidin-biotin-perxidase complex (Zymed), and finally diaminobenzi dine (DAB). .
  • anti-SEA serum polyclonal antibody was first prepared, and the purified SEA protein was immunized to BALB/c mice. After multiple immunizations, antiserum with a titer of more than 8000 was obtained, using Hitrap protein G- Sepharose column (Amersham Biosciences) Purified antiserum to obtain an IgG fraction. Paraffin sections of these tumor tissues were incubated in a concentration of 0.8 ⁇ ⁇ / ⁇ 1 in SEA solution, then rinsed, first reacted with anti-SEA antiserum IgG, diluted 1:50, rinsed to remove antibodies, and then reacted with secondary antibody.
  • Figure 6 shows CD4+ T cells detected using conventional immunohistochemistry.
  • Figure 7 is a CD4+T reaction on the tissue section of Figure 6. The number of cells is calculated.
  • Figure 8 is a CD8+ T cell detected using conventional immunohistochemistry.
  • Figure 9 is a graph showing the number of CD8+ T reactive cells on the tissue section of Figure 8.
  • Figure 10 is a T cell reactive with SEA detected by conventional immunohistochemistry.
  • Figure 11 is a calculation of the number of SEA response T cells on the tissue section of Figure 10.
  • CD4 + and CD8 + and SEA-reactive T cells were found in the tumor tissues of mice injected with EGF-SEA and VEGF-SEA (the small spots shown in 6-1 and 6-2 are T cells).
  • the small dots shown in 8-1 and 8-2 are T cells) (the small dots shown in Figures 10-1 and 10-2 are T cells), which indicates that the tumor is induced by EGF-SEA and VEGF-SEA.
  • the tissue is filled with infiltrating T cells.
  • the SEA treatment group had only a small amount of CD4 + and CD8 + and SEA-reactive T cells in the tumor, while the saline-treated control group had almost no T cells.
  • T cells small cells
  • Figure 16 shows the amount of cytokine secretion in units of area based on antibody positive reactions on paraffin sections of the immunohistochemistry experiments of Figures 13-15.
  • Figure 17 and the enzyme-linked immunosorbent assay (ELISA) were used to test the secretion of cytokines in tumor tissues using the Ll box (R & D Systems).
  • Fas expression analysis on tumor cells and anti-Fas antibody (Santa Cruz Biotechnolog) was used to examine tumor tissues, and a large amount of Fas expression on the surface of mouse S180 cells injected with EGF-SEA and VEGF-SEA was found (Fig. 18).
  • Tumor cells in the SEA group and the control group showed little or no Fas expression. This may be caused by EGF-SEA and VEGF-SEA-induced cytokines secreted by T cells in tumor tissues such as IFN- ⁇ and TNF- ⁇ , which can make tumor cells highly express Fas, while FasL on T cells can cause expression.
  • Fas tumor cell apoptosis, which is a pathway for killing T cells to cause apoptosis in target cells.
  • Figure 18 Fas tumor cell apoptosis, which is a pathway for killing T cells to cause apoptosis in target cells.
  • Fas protein Analysis of Fas expression on tumor cells, using anti-Fas The antibody (Santa Cruz Biotechnolog) was used to examine tumor tissues, and it was found that a large amount of Fas expression on the surface of mouse S180 cells injected with EGF-SEA and VEGF-SEA showed that the shaded portion around the large cells of the antibody reaction was Fas protein.
  • Perforin analysis around tumor cells, and anti-perforin antibody was used to examine tumor tissues, and it was found that a large amount of perforin protein was surrounded by mouse S180 cells injected with EGF-SEA and VEGF-SEA (Fig. 19, 19).
  • EGF-SEA EGF-SEA
  • VEGF-SEA VEGF-SEA
  • the small band indicated by the arrow is the accumulated perforin protein group
  • Killer T lymphocytes secrete perforin to form pores in the cell membrane of the target cell, disrupting the balance of the osmotic pressure of the target cells, leading to death of the target cells.
  • the granzymes around the tumor cells were analyzed with anti-granzyme antibody (Abeam) to find a large amount of granzymes around the tumor S180 cells injected with EGF-SEA and VEGF-SEA (Fig. 20).
  • anti-granzyme antibody Abeam
  • Granzyme is a proteolytic enzyme secreted by killer T lymphocytes. When perforin destroys the cell membrane of the target cell, the granzyme can enter the target cell, thereby degrading the target cell.
  • the chromosomal DNA is degraded, and the TU EL kit (Roche Applied Science) can be used to detect DNA degradation in the nucleus, thereby judging that these cells have died.
  • the TU EL kit (Roche Applied Science) can be used to detect DNA degradation in the nucleus, thereby judging that these cells have died.
  • fluorescein-labeled nucleotides are incorporated into the 3-OH end of apoptotic cell double-stranded or single-stranded DNA, followed by a peroxidase-labeled anti-fluorescein antibody. Carry out the reaction.
  • FIG. 22 TUNEL positive rate of tumor cells. Mortality was calculated based on the number of shadow dead cells on the TUNEL-stained tissue sections, and the tumor cell death rate reached 50-60%.
  • EGF-SEA and B VEGF-SEA are both receptors for EGF and B VEGF, respectively.
  • EGFR interacts with VEGFR and interacts with TCR on T cells, thus targeting T cells to large
  • cytokines secreted by T cells In particular, EGF-SEA and VEGF-SEA have a significant killing effect on solid tumors.
  • the present invention selects a novel strategy for attaching superantigens to cytokines, thus producing a novel cytokine-superantigen fusion protein.
  • the present invention uses epidermal growth factor EGF and vascular endothelial growth factor VEGF, respectively.
  • This novel fusion protein was constructed with superantigen SEA.
  • the superantigen may also be selected from the group consisting of: SEB, SEC, SED, SEE of the Staphylococcus aureus enterotoxin family, SPE-A, SPE-B, SPE-C of streptococcal toxin, shock synthesis
  • SEB SEB
  • SEC Staphylococcus aureus enterotoxin family
  • SPE-A SPE-B
  • SPE-C of streptococcal toxin
  • shock synthesis The concept of the present invention can also be explained by the natural and artificial variants of Shock syndrome toxin, viral proteins and amino acid sequences having more than 70% identity.
  • Superantigen The role of SEA or other superantigens is to stimulate immune responses in the body.
  • EGF and vascular endothelial growth factor VEGF which are experimental materials, only utilize the localization of their cancer cells, and other cytokines closely related to cancer cells can also explain the various trends of the present invention.
  • the fusion protein can also be linked to a polypeptide fragment of a cytokine and a superantigen by a chemical reaction means such as a chemical crosslinking reaction, for example, a covalent bond, thereby constructing a fusion protein.
  • a chemical reaction means such as a chemical crosslinking reaction, for example, a covalent bond
  • a series of modifications can be made to the fusion protein by chemical modification, deletion of a portion of the polypeptide fragment of the fusion protein, and attachment of other polypeptides to these proteins.
  • the purified fusion protein can improve its biological activity by refining the steric structure of the disulfide bond through a series of denaturation and renaturation processes.
  • cytokines On a larger scale, the receptors overexpressed by cytokines and their cancer cells are actually a relationship between a ligand (Ligand) and a receptor, and the affinity of this ligand and receptor is utilized. , localize the superantigen to the tumor tissue.
  • ligands other polypeptide molecules that correspond to cancer cell overexpression receptors, ie, ligands, can also be used for specific localization of cancer cells.
  • ligands corresponding to receptors on cancer cells
  • artificially screened polypeptides having affinity and antagonistic effects on receptors on cancer cells screened by phage display and the like can be Polypeptide molecules that interact directly with the surface of cancer cells can form fusion proteins with superantigens.
  • the dosage form of the drug may be an emulsifier, a liposome, a dispersing agent, a stabilizer, or the like, which is prepared into a form of administration of various drugs such as injection, oral administration, application, and surgical treatment.
  • nucleotide fragment or vector encoding a fusion protein can also be used as a gene therapy form.
  • the cytokine-superantigen fusion protein is capable of inhibiting the growth of a solid tumor in a mouse, resulting in the death of a large number of tumor cells in the tumor tissue; and in the light of the present invention, those skilled in the art should also It can be understood that the cytokine-superantigen fusion protein is capable of inhibiting the growth of solid tumors in vivo, and is not limited to mice. It is to be noted that all obvious changes and similar inventions having equivalent substitutions are included in the scope of the present invention as long as they do not depart from the spirit of the invention. Sequence description
  • the nucleotide sequence of SEQ ID NO: 1 is an EGF-SEA fusion protein
  • SEQ ID NO: 2 is the amino acid sequence of the EGF-SEA fusion protein
  • the nucleotide sequence of SEQ ID NO: 3 is a VEGF-SEA fusion protein
  • SEQ ID NO: 4 is the amino acid sequence of a VEGF-SEA fusion protein
  • SEQ ID NO: 5 is the coding sequence of a linker peptide
  • SEQ ID NO: 6 is the amino acid sequence of the linker peptide.

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Abstract

L'invention porte sur l'utilisation d'une protéine de fusion cytokine-superantigène pour préparer un médicament contre une tumeur solide, la cytokine étant un facteur de croissance épidermique ou un facteur de croissance de cellule endothéliale vasculaire, et le superantigène est le superantigène de l'entérotoxine A du staphylocoque doré.
PCT/CN2010/078854 2010-03-05 2010-11-18 Utilisation d'une protéine de fusion cytokine-superantigène pour préparer un médicament contre une tumeur solide WO2011106981A1 (fr)

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CN201010118438.1 2010-03-05
CN201010118438A CN101829322A (zh) 2010-03-05 2010-03-05 细胞因子-超抗原融合蛋白在制备抗实体瘤药物的应用

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CN101829322A (zh) * 2010-03-05 2010-09-15 孙嘉琳 细胞因子-超抗原融合蛋白在制备抗实体瘤药物的应用
CN102114239A (zh) * 2010-12-14 2011-07-06 孙嘉琳 细胞因子-超抗原融合蛋白在制备抗癌药物的应用
CN102516392B (zh) * 2011-11-25 2014-05-28 孙嘉琳 一种癌靶向超抗原融合蛋白及制备方法及用途
CN111093689A (zh) * 2017-07-03 2020-05-01 转矩医疗股份有限公司 免疫刺激融合分子及其用途

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