WO2011106981A1

WO2011106981A1 - Use of cytokine-superantigen fusion protein for preparing medicament against solid tumor

Info

Publication number: WO2011106981A1
Application number: PCT/CN2010/078854
Authority: WO
Inventors: 孙嘉琳
Original assignee: Sun Jialin
Priority date: 2010-03-05
Filing date: 2010-11-18
Publication date: 2011-09-09
Also published as: CN101829322A

Abstract

The invention discloses the use of a cytokine-superantigen fusion protein for preparing a medicament against solid tumor, wherein the cytokine is an epidermal growth factor or a vascular endothelial cell growth factor, and the superantigen is the superantigen of staphylococcus aureus enterotoxin A.

Description

Application of cytokine-superantigen fusion protein in preparing anti-solid tumor drugs

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

Receptors such as Epidermal growth factor (EGF) and Vascular endothelial growth factor (VEGF) are abundantly expressed in various tumor tissues, for example, EGF receptors in intestinal mucosal tumors are normal. The organization is 300 times higher (Gastroenterology, 98, 961-967, 1990). Therefore, abnormally high expression of EGF receptor and VEGF has become a compelling target for cancer therapy.

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 EGF gene was discovered in the early 1980s (Nucleic Acids Res., 10, 4467-4482, 1982; Nature, 303,

722-725, 1983), its mature form is a 53 amino acid polypeptide.

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.

The SEA gene was reported as early as the 1980s (J. Biol. Chem., 262, 7006-7013, 1987; J. Bacteriol., 170, 34-41, 1988). To alleviate the side effects of SEA, a point mutation was introduced at its position 227 (Proc. Natl. Acad. Sci. USA, 94, 2489-2494, 1997). In order to attenuate the serum response while retaining the biological activity of SEA, 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 νβ fragment of T cell receptor (TCR), which activates much more than the common antigen. Sputum cells (including CD4+, CD8+) release a large number of cytokines and produce potent cytotoxic effects on target cells. 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.

In addition, in order for an antibody to be a drug, it is necessary to genetically engineer a mouse antibody. Since antibiotics are used in large doses, often tens of milligrams per person per time are required, which requires an increase in the expression level of genetically engineered animal cells and the development of large-scale fermentation techniques. Therefore, the research and development cycle and investment cost of antibody drugs are very large.

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.

Chinese Patent Application No. 200310109829.7 "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

It is an object of the present invention to overcome the deficiencies in the prior art and to provide a cytokine-superantigen fusion protein for use in the preparation of an anti-solid tumor drug.

The technical solution of the present invention is summarized as follows:

The use of a cytokine-superantigen fusion protein for the preparation of an anti-solid tumor drug, the cytokine is epidermal growth factor or vascular endothelial growth factor, and 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-γ and tumor necrosis factor-α 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.

Advantages of the invention:

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. Experiments have shown that 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. On the surface of tumor cells, cytokines secreted by T cells, induction of target cells to produce Fas and perforin, and granzymes cause apoptosis of tumor cells. The cytokine-superantigen fusion protein has a significant killing effect on solid tumors. DRAWINGS

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. (In the figure: 4-1 is the control; 4-2 is SEA; 4-3 is EGF-SEA; 4-4 is VEGF-SEA)

Figure 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. (In the figure: 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. (In the figure: 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.

Figure 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)

Figure 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).

Figure 14 TNF-α 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-γ 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).

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).

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).

Figure 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 There are a large number of TU EL-positive S180 tumor cells, ie, dead cells, in the tumor tissues of SE-injected mice, and the cells showing shadows are dead cells).

Figure 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). This type of fusion protein can be specifically localized to cancer cells and cause an anti-cancer cytotoxic immune response around the cancer cells.

The invention is further illustrated by the following specific examples.

Example 1 Construction of EGF-SEA expression vector

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).

Example 1 Construction of VEGF-SEA expression vector

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. In the second step, 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).

Example 3 Expression, Denaturation and Refolding of Various Proteins and Purification

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, 375μΜ oxidized glutathione. The peptide GSSG, 1.875 mM reduced glutathione GSH, was dialyzed and dialyzed, and the resulting supernatant was a renaturation solution of the protein. Purify the protein with the H Bind Purification Kit (Novagen). First rinse the gel column with Binding Buffer. Also add Binding Buffer to the protein solution, apply the protein sample to the column, rinse with Wash Buffer, and then use Elute Buffer. Elution, and finally identification by protein electrophoresis, resulted in a high-purity protein with only one band (Figures 1 and 2).

Example 4 Experiment of two cytokine-superantigen fusion proteins inhibiting solid tumors

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.

Example 5 Detection of T lymphocytes in tumor tissues

Two 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). .

In order to detect T cells reactive with SEA, 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 μ _§ / μ1 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.

A large number of 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). In the figure, 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.

The ability of SEA to react with T cells suggests that SEA is combined with TCR on T cells.

Example 6 Examination of binding ability of two cytokine-superantigen fusion proteins to tumor cells

Tumor tissue paraffin sections were incubated with EGF-SEA and VEGF-SEA protein solution, first reacted with anti-SEA antibody, then fluorescein-labeled anti-mouse 2 antibody, diluted 1:50, and finally observed under a fluorescence microscope ( Figure 12). In the tumor tissues of mice injected with EGF-SEA and VEGF-SEA, it was found that S180 tumor cells (large cells) and BT cells (small cells) showed fluorescence, indicating that EGF-SEA or VEGF-SEA can be associated with S180 cells. EGF receptor EGFR or VEGF receptor VEGFR binds to TCR on T cells.

Only T cells (small cells) were found in the tumors of the SEA-treated and saline-controlled groups.

Example 7 Detection of Cytokines in Tumor Tissues

Santa Cruz Biotechnolog anti-IL-2 antibody, anti-IFN-γ antibody and anti-TNF-α antibody were used to detect cytokines secreted by T cells in tumor tissues, and then R&D Systems' enzyme-linked immunosorbent assay ( ELISA) kit to detect cytokines in tumors. Figure 13 IL-2 secretion in tumor tissues (13-1, 13-2 show chromogenic regions with antibody responses between tumor cells). Figure 14 TNF-a secretion in tumor tissues (14-1, 14-2 show the color-developing region of antibody reaction between tumor cells). Figure 15 IFN-γ secretion in tumor tissues (15-1, 15-2 shows a color-developing region with antibody response 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 the enzyme-linked immunosorbent assay (ELISA) were used to test the secretion of cytokines in tumor tissues using the Ll box (R & D Systems).

The results showed that there were a large number of IL-2, IFN-y and TNF-a cytokines in the tumor tissues of mice injected with EGF-SEA and VEGF-SEA, while few cytokines were found in the tumors of the SEA-treated and saline-controlled groups.

Example 8 Analysis of Factors Affecting Tumor Cell Apoptosis

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-γ and TNF-α, 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. 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 (Santa Cmz Biotechnolog) 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). -1 is EGF-SEA, 19-2 is VEGF-SEA, and the small band indicated by the arrow is the accumulated perforin protein group), while there is almost no perforin around the tumor cells of the SEA group and the control 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). Peripheral granzyme analysis, anti-granzyme antibody (Abeam) was used to examine tumor tissues, and it was found that there were a large number of granzymes around the tumor S180 cells injected with EGF-SEA and VEGF-SEA (as the large cell surface of tumor cells and The shaded part around it is the granzyme protein).

There were almost no granzymes around the tumor cells in the SEA group and the control group. 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.

After the cell dies, 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. Under the action of terminal deoxynucleotidyl transferase, 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. Thus, after TU EL detection, a large number of TU EL-positive S180 tumor cells, ie, dead cells, were found in the tumor tissues of mice injected with EGF-SEA and VEGF-SEA. Figure 21 TUNEL staining of tumor cells. TUNEL assay showed that a large number of TUNEL-positive S180 tumor cells, ie, dead cells, were found in the tumor tissues of mice injected with EGF-SEA and VEGF-SEA (cells showing negative cells were dead cells).

There were few dead tumor cells in the tumor tissues of the SEA group and the control group.

Figure 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%.

S180 tumor growth in mice administered with EGF-SEA and VEGF-SEA was greatly inhibited, and a large amount of CD4 ⁺ and CD8 ⁺ and T lymphocytes reactive with SEA were found in tumor tissues, and T cells in these tumor tissues were observed. A large number of cytokines such as IL-2, IFN-γ and TNF-α are secreted, wherein IFN-γ and TNF-a cause tumor cell apoptosis. At the same time, killer T cells such as CD8+ T cells secrete perforin and granzymes around the tumor cells, which can destroy the cell membrane of the tumor cells and degrade the tumor cells. By TUNEL assay, a large number of tumor cell deaths were confirmed in the tumor tissues of the EGF-SEA and VEGF-SEA mice.

The above series of examples show that 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 On the surface of tumor cells expressing EGFR or VEGFR, cytokines secreted by T cells, induction of target cells to produce Fas and perforin and granzymes, etc., cause tumor cell apoptosis. 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. As a model, the present invention uses epidermal growth factor EGF and vascular endothelial growth factor VEGF, respectively. This novel fusion protein was constructed with superantigen SEA.

Although only the superantigen SEA is selected, 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 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.

Similarly, epidermal growth factor 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. Chemokine, Ephrin family, Angiopoietin (Ang), Thrombopoietin (TPO) and Plasma Factor VII, Urokinase-type plasminogen Activator , uPA ), gastrin-releasing peptide (GRP), growth hormone releasing hormone (GHRH), melatonin a-MSH, prolactin (PRL), prolactin Prolactin releasing hormone (PRLH), growth hormone (GH), Follicle stimulating hormone (FSH), Placental lactogen (PL), Chorionic gonadotropin (Chorionic gonadotropin, CG) and corticotropin release Corticotropin releasing hormone (CRH), Somatostatin (SST), Asialoglycoprotein (ASGP), Low density lipoprotein (LDL), and Transferrin (Tf) Many tumor tissues overexpress the receptors of these substances, so that polypeptide ligands such as chemokines, enzymes, hormones and other proteins can be linked to superantigens like cytokines to form fusion proteins, and superantigens can be localized to tumors. organization.

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 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.

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. In addition to cytokines, other polypeptide molecules that correspond to cancer cell overexpression receptors, ie, ligands, can also be used for specific localization of cancer cells. In addition to the above-mentioned 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.

In addition to the fusion protein itself as a drug, a nucleotide fragment or vector encoding a fusion protein can also be used as a gene therapy form.

The above description of the embodiments and the preferred embodiments are intended to be illustrative of the invention as defined by the appended claims. In a specific embodiment of the present invention, 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.

Claims

1 . Use of a cytokine-superantigen fusion protein for preparing an anti-solid tumor drug, characterized in that the cytokine is epidermal growth factor or vascular endothelial growth factor, and the superantigen is super-staphylococcus aureus enterotoxin A antigen.

2. The use of a cytokine-superantigen fusion protein according to claim 1 for the preparation of an anti-solid tumor drug, characterized in that said cytokine-superantigen fusion protein is capable of mobilizing tau lymphocytes and said T lymphocytes Targeted around tumor cells within solid tumor tissue, the T lymphocytes have CD4 ⁺ or CD8 ⁺ characteristics and are capable of interacting with SEA.

3. The use of the cytokine-superantigen fusion protein according to claim 1 for the preparation of an anti-solid tumor drug, characterized in that the cytokine-superantigen fusion protein induces secretion of cytokines by T lymphocytes in solid tumor tissues. Interleukin-2, interferon-gamma and tumor necrosis factor-alpha.

The use of the cytokine-superantigen fusion protein according to claim 1 for the preparation of an anti-solid tumor drug, characterized in that the cytokine-superantigen fusion protein is capable of inducing a high level of the apoptotic protein Fas on the surface of a tumor cell. expression.

The use of the cytokine-superantigen fusion protein according to claim 1, in the preparation of an anti-solid tumor drug, characterized in that the cytokine-superantigen fusion protein is capable of inducing secretion of perforin and granzyme by killer T lymphocytes. B.

The use of the cytokine-superantigen fusion protein according to claim 1, in the preparation of an anti-solid tumor drug, characterized in that the cytokine-superantigen fusion protein is capable of inhibiting the growth of solid tumors in vivo, resulting in a large amount of tumor tissue. The death of tumor cells.