CN101829322A

CN101829322A - Application of superantigen fusion protein cell factor in preparation of anti-solid tumor medicament

Info

Publication number: CN101829322A
Application number: CN201010118438A
Authority: CN
Inventors: 孙嘉琳
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-03-05
Filing date: 2010-03-05
Publication date: 2010-09-15
Also published as: WO2011106981A1

Abstract

The invention discloses the application of a superantigen fusion protein cell factor in the preparation of an anti-solid tumor medicament. The cell factor may be an epidermal growth factor (EGF) or a vascular endothelial growth factor (VEGF), and the super-antigen is the super-antigen of staphylococcus-enterotoxin A. The superantigen fusion protein cell factor is actually a substitute for the antigen in the prior art, has a targeting function and is a new prospective medicament different from the antigen. Experiments show that the cell factor-superantigen fusion protein interacts with receptors of the EGFR and the VEGFR respectively and also interacts with T cell receptors (TCR) on T cells, so the T cells are positioned in a targeting manner on the surfaces of a great amount of tumor cells which express the EGFR or the EVGFR, and the cell factors secreted by the T cells induce the target cells to generate Fas, perforin, granzyme and the like to cause the apoptosis of the tumor cells. The cell factor-superantigen fusion protein has an obvious killing effect on the solid tumor.

Description

Cytokine-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine

Technical field

The present invention relates to a kind of purposes of fusion rotein, particularly relate to of the application of a kind of cytokine-superantigen fusion rotein at preparation solid tumor resisting medicine.

Background technology

Epidermal growth factor (Epidermal growth factor, EGF) and vascular endothelial cell growth factor (Vascularendothelial cell growth factor, VEGF) great expression in various tumor tissues such as receptor, for example the EGF receptor in the intestinal mucosa tumor exceeds 300 times of (Gastroenterology than normal structure, 98,961-967,1990).So the EGF receptor of unusual high expressed and VEGF become a noticeable cancer treatment target spot.

Epidermal growth factor is connected to alternative cancerous cell (Clin.Cancer Res., 11,329-334,2005 of killing and wounding high expressed EGF receptor on a kind of toxin protein or the RNA hydrolytic enzyme; Protein Eng., 11,1285-1292,1998), but this mode be not rely on immune system for example lymphocyte etc. attack tumor.

The EGF gene has been found (Nucleic Acids Res., 10,4467-4482,1982 in early days in the eighties; Nature, 303,722-725,1983), its mature form is 53 amino acid whose polypeptide.

The VEGF gene is to be found (Science, 246,1306-1309,1989 in the later stage eighties; Science, 246,1309-1312,1989) because the difference of mRNA is sheared, its mature form has various ways, length can be 189,165 and 121 amino acid whose polypeptide (J.Biol.Chem., 266,11947-11954,1991).

Cell receptor is a kind of albumen that is positioned at cell surface, make the interior a series of signal that takes place of cell conduct activities such as also causing gene regulation thus behind the signal of acceptance outside, and a large amount of receptors relevant with somatomedin are usually arranged on the cancerous cell, these receptors just become the target spot of antibody.If anti-these receptors of antibody promptly interact with them and just combine with them, thus the interaction of blocking-up cytokine and its receptor, thus reach the inhibition tumor growth.

The antibody receptor of sealing on the cancerous cell that can neutralize, thus ligand-mediated cell proliferation suppressed.Though antibody has antibody-dependent cytotoxicity effect (antibody dependent-cell mediated cytotoxicity), acts on not remarkable.(Staphylococcal-enterotoxin A, superantigen SEA) (Superantigen) can produce the cell toxicant of the cell mediated of reinforcement to the antibody by connecting a staphylococcus aureus toxin A.

The SEA gene has just been reported (J.Biol.Chem., 262,7006-7013,1987 as far back as the eighties; J.Bacteriol., 170,34-41,1988).In order to alleviate the side effect of SEA, on its 227th position, introduced a point mutation (Proc.Natl.Acad.Sci.USA, 94,2489-2494,1997).In order to weaken the biological activity that seroreaction keeps SEA simultaneously again, in SEA, introduce more point mutation (J.Mol.Biol., 333,893-905,2003).

SEA does not need the processed of antigen presenting cell, and mhc class ii molecule direct with complete protein form and on the cell membrane combines the formation complex, the V β fragment of identification T cell receptor (TCR), the T cell that activation is more much more than common antigen (comprises CD4+, CD8+), and discharge a large amount of cytokines, target cell is produced powerful cytotoxicity.The antibody superantigen fusion rotein of a group of Sweden shifts for the melanomatous lung of B16 and has carried out a lot of researchs (Proc.Natl.Acad.Sci.USA, 92,9791-9795,1995; Eur.Surg.Res., 35,457-463,2003), but how still unclear for the effect of solid tumor resisting.

In addition, make antibody become medicine, just must carry out humanized genetic engineering modified for Mus source antibody.Because the using dosage of antibody drug is very big, usually need tens of milligram/people/time, this just require to improve genetic engineering antibody zooblast expression and develop the large scale fermentation technology.So the cycle of the research and development of antibody drug and cost of investment are very huge.

Cancerous cell is changed by normal cell, and the antigen of cancerous cell is autoantigen, so cancerous cell can be escaped immune supervision.People are seeking the immunity that new anticancer method improves cancer patient always, particularly at the specific immunity of cancerous cell.Therefore, this area presses for a kind of special strong new cancer therapy drug at cancerous cell and is used for antitumor, particularly anti entity tumour.This medicine is different from antibody, does not also report now.

Chinese patent application numbers 200310109829.7 " a kind of can in order to the super antigen fusion protein and the production method thereof of anticancer therapy " discloses a kind of method of the fusion rotein that makes up cytokine and superantigen and this fused protein method at expression in escherichia coli and purification, but the bioactive data of the fusion rotein solid tumor resisting of cytokine and superantigen openly not.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, the application of a kind of cytokine-superantigen fusion rotein at preparation solid tumor resisting medicine is provided.

Technical scheme of the present invention is summarized as follows:

Cytokine-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, and described cytokine is epidermal growth factor or vascular endothelial cell growth factor, and described superantigen is the superantigen of staphylococcus aureus toxin A.

Described cytokine-superantigen fusion rotein can be mobilized the T lymphocyte, and with described T lymphocyte targeting navigate to around the in-house tumor cell of entity tumor, described T lymphocyte has CD4 ⁺Or CD8 ⁺Feature and interacting with SEA.

Described cytokine-superantigen fusion rotein can be induced the in-house T lymphocytic emiocytosis of entity tumor cytokine interleukin II, interferon-and tumor necrosis factor-alpha.

Described cytokine-superantigen fusion rotein can the lip-deep apoptotic proteins Fas of inducing tumor cell high expressed.

Described cytokine-superantigen fusion rotein can be induced cytotoxic T lymphocyte secretion perforin and granzyme B.

Described cytokine-superantigen fusion rotein can suppress the growth of entity tumor in the mice body, causes the death of massive tumor cell in the tumor tissues.

Advantage of the present invention:

Cytokine-superantigen fusion rotein is actually the antibody that replaces prior art, also has targeting, is the new medicine of expecting that is different from antibody.Experimental results show that cytokine-superantigen fusion rotein had been that EGFR and VEGFR interact with the receptor of EGF and VEGF respectively both, again with the T cell on the TCR effect, so just with on the cell targeted tumor cell surface that navigates to great expression EGFR or VEGFR of T, by the cytokine of T emiocytosis, induce target cell to produce Fas and perforin and granzyme etc. cause apoptosis of tumor cells.Cytokine-superantigen fusion rotein has clearly lethal effect for entity tumor.

Description of drawings

Fig. 1 is the electrophoretogram that has only the highly purified VEGF-SEA fusion rotein of a band.(among the figure, 1: the rinsing liquid 2 in the purification: wash next VEGF-SEA) with eluent

Fig. 2 is highly purified SEA and EGF-SEA electrophoretogram.(1:SEA among the figure; 2:EGF-SEA)

Fig. 3 represents that EGF-SEA and VEGF-SEA fusion rotein suppress the tumor growth situation.

Fig. 4 represents the dissection of injected in mice EGF-SEA, VEGF-SEA, SEA and normal saline.(among the figure: 4-1 is contrast; 4-2 is SEA; 4-3 is EGF-SEA; 4-4 is VEGF-SEA)

Fig. 5 represents the heavy result of the tumor after the dissection of injected in mice EGF-SEA, VEGF-SEA, SEA and normal saline.

Fig. 6 utilizes the detected CD4 of routine immunization groupization ⁺The T cell.(among the figure: 6-1 is EGF-SEA; 6-2 is VEGF-SEA; 6-3 is SEA; 6-4 is contrast)

Fig. 7 is the CD4 on Fig. 6 tissue slice ⁺T reacting cells number is calculated.

Fig. 8 utilizes the detected CD8 of routine immunization groupization ⁺The T cell.(among the figure: 8-1 is EGF-SEA; 8-2 is VEGF-SEA; 8-3 is SEA; 8-4 is contrast)

Fig. 9 is the CD8 on Fig. 8 tissue slice ⁺T reacting cells number is calculated.

Figure 10 utilizes the detected and SEA reaction T cell of routine immunization groupization.(among the figure: 10-1 is EGF-SEA; 10-2 is VEGF-SEA; 10-3 is SEA; 10-4 is contrast)

Figure 11 is that the SEA reaction T number of cells on Figure 10 tissue slice is calculated.

Figure 12 is hatched the tumor tissues paraffin section with EGF-SEA and VEGF-SEA protein solution, earlier with anti-SEA antibody response, uses the 2nd antibody response of fluorescein-labeled anti-mice then, and dilution in 1: 50 is observed under fluorescence microscope at last.(among the figure: 12-1 is EGF-SEA; 12-2 is VEGF-SEA; 12-3 is SEA; 12-4 is contrast)

(among the figure: 13-1 is EGF-SEA in the IL-2 secretion in Figure 13 tumor tissues; 13-2 is VEGF-SEA; 13-3 is SEA; 13-4 is contrast, and 13-1,13-2 have shown the color development area that antibody response is arranged between the tumor cell).

(among the figure: 14-1 is EGF-SEA in TNF-α secretion in Figure 14 tumor tissues; 14-2 is VEGF-SEA; 14-3 is SEA; 14-4 is contrast, and 14-1,14-2 have shown the color development area that antibody response is arranged between the tumor cell).

(among the figure: 15-1 is EGF-SEA in IFN-γ secretion in Figure 15 tumor tissues; 15-2 is VEGF-SEA; 15-3 is SEA; 15-4 is contrast, and 15-1,15-2 have shown the color development area that antibody response is arranged between the tumor cell).

Reaction is the quantity that unit calculates cytokine secretion with the area to Figure 16 according to the antibody positive on the paraffin section of the immunohistochemical experiment of Figure 13-15.

Figure 17 utilizes enzyme-linked immunosorbent assay (ELISA) test kit (R ﹠amp; D Systems company) detects the interior cytokine secretion of tumor tissues.

Fas expression analysis on Figure 18 tumor cell checks that with anti-Fas antibody (Santa Cruz Biotechnolog company) (among the figure: 18-1 is EGF-SEA to tumor tissues; 18-2 is VEGF-SEA; 18-3 is SEA; 18-4 finds to express through the lot of F as on the mouse tumor S180 cell surface of EGF-SEA and VEGF-SEA injection for contrasting, and shaded portion is exactly a Fas albumen around the maxicell of demonstration antibody response).

Perforin analysis around Figure 19 tumor cell checks that with anti-perforin antibody (Santa Cruz Biotechnolog company) (among the figure: 19-1 is EGF-SEA to tumor tissues; 19-2 is VEGF-SEA; 19-3 is SEA; 19-4 is contrast, and finding has a large amount of perforin albumen through the mouse tumor S180 cell peripheral of EGF-SEA and VEGF-SEA injection, and the little strip portion of representing with arrow is exactly cumulative perforin albumen group).

Granzyme analysis around Figure 20 tumor cell checks that with particle-resistant enzyme antibody (Abcam company) (among the figure: 20-1 is EGF-SEA to tumor tissues; 20-2 is VEGF-SEA; 20-3 is SEA; 20-4 is contrast, and finding has a large amount of granzymes through the mouse tumor S180 cell peripheral of EGF-SEA and VEGF-SEA injection, as the maxicell surface of tumor cell and on every side shaded portion be exactly granzyme albumen).

Figure 21 tumor cell TUNEL dyeing, (among the figure: 21-1 is EGF-SEA; 21-2 is VEGF-SEA; 21-3 is SEA; 21-4 is contrast, and it is dead cell that the positive S180 tumor cell of a large amount of TUNEL is arranged in the mouse tumor tissue through TUNEL detection discovery EGF-SEA and VEGF-SEA injection, shows that the cell of shade is a dead cell).

The TUNEL positive rate of Figure 22 tumor cell calculates mortality rate according to the number that shows the shade dead cell on the painted tissue slice of TUNEL, and the death of neoplastic cells rate reaches 50-60%.

The specific embodiment

The present invention utilizes cytokine-super antigen fusion protein, the cytokine of promotion growth of cancer cells wherein can navigate to fused protein on the cancerous cell, superantigen then causes anticancer immunoreation around cancerous cell, be the cell-mediated cytotoxicity that relies on of superantigen (Superantigen-dependent-cellular-cytotoxicity, SDCC).Utilize the method just such fused protein can be navigated to specifically cancerous cell and cause anticancer cell toxicant immunoreation around the cancerous cell.

The present invention is further illustrated below by specific embodiment.

The structure of embodiment 1EGF-SEA expression vector

Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise 53 amino acid whose EGF of codified and connection peptides and increase several bases of restriction restriction enzyme site BamHI and EcoRI in the EGF front again, in the nucleotide sequence of the part of coding connection peptides, comprised the restriction restriction enzyme site of SalI and HindIII.Nucleic acid fragment that this is synthetic good inserts T carrier and carries out dna sequencing and identify, and then after promptly handling with BamHI and HindIII with the double digestion method, on this fragment insertion pET22b plasmid, has formed pET22b-EGF.Second step was the synthetic SEA nucleic acid sequence fragments that has Asp (227) → Ala point mutation, insert the T carrier earlier and carry out the dna sequencing evaluation, and then after promptly handling with the double digestion method with HindIII and XhoI, this fragment is inserted on the pET22b-EGF plasmid, so just produced expression vector pET22b-EGF-SEA, can express the EGF-SEA fusion rotein (seeing sequence table SEQ ID NO.2).

The structure of embodiment 2VEGF-SEA expression vector

Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise 121 amino acid whose VEGF of codified and connection peptides and increase several bases of restriction restriction enzyme site BamHI and EcoRI in the VEGF front again, in the nucleotide sequence of the part of coding connection peptides, comprised the restriction restriction enzyme site of SalI and HindIII.Nucleic acid fragment that this is synthetic good inserts T carrier and carries out dna sequencing and identify, and then after promptly handling with BamHI and HindIII with the double digestion method, on this fragment insertion pET22b plasmid, has formed pET22b-VEGF.Second step was to utilize to have synthesized SEA good and insertion T carrier among the embodiment 1, after promptly handling with the double digestion method with HindIII and XhoI, it is inserted on the pET22b-VEGF plasmid, but so just produced expression vector pET22b-VEGF-SEA VEGF expression-SEA fusion rotein (seeing sequence table SEQ ID NO.4).

Embodiment 3 various proteic expression, degeneration and renaturation and purification

With expression plasmid pET22b-EGF-SEA and pET22b-VEGF-SEA and in contrast the pET22b-SEA of usefulness change e. coli bl21 (DE3) over to electroporation respectively, utilize antibiotic Amp screening positive bacteria.The process of next various proteic expression, degeneration and renaturation and purification is roughly the same, operates as follows:

The e. coli bl21 (DE3) that contains expression plasmid carries out extensive 37 ℃ of cultivations earlier, and add IPTG then and make it concentration and reach 1mM and spend the night 30 ℃ and cultivate, thus abduction delivering albumen.The 2nd day centrifugation medium and collect thalline use the supercritical ultrasonics technology breaking cell wall, and centrifugal collection occlusion body precipitates, and the albumen here is to exist with the occlusion body form.The urea-denatured dissolving of 6M of occlusion body albumen, carry out the multistage dialysis then, dialysis solution is for example 3M, 2M and 1M of the carbamide that progressively dilutes, next be 0.5M carbamide, 0.4M the L-arginine, 375 μ M oxidized form of glutathione GSSG, 1.875mM reduced glutathion GSH, carry out centrifugally after the dialysis, the gained supernatant is exactly proteic renaturation solution.Carry out purification with HisBind Purification Kit test kit (Novagen company) for albumen, earlier with Binding Buffer flushing gel column, also in protein solution, add Binding Buffer simultaneously, with the protein sample upper prop, with Wash Buffer rinsing, use Elute Buffer eluting then, identify with protein electrophorese at last, so just obtained having only the highly purified albumen (Fig. 1 and Fig. 2) of a band.

4 two kinds of cytokines of embodiment-superantigen fusion rotein suppresses the experiment of entity tumor

At first select male ICR mouse, in 4-5 week, 18-22g is divided into 4 groups, every group of 15 mices.With 2 * 10 ⁶Murine sarcoma cell sarcoma 180 (S180) is inoculated in the oxter, right side of mice, past every other day then intraperitoneal injection EGF-SEA and VEGF-SEA and SEA protein solution 0.2ml (250pmol)/only, and matched group injection equivalent normal saline (0.9%NaCl) was put to death mice after 9 days.The weight of observing the mouse tumor growing state and putting to death the back tumor.Fig. 3 represents that EGF-SEA and VEGF-SEA fusion rotein suppress the tumor growth situation.Fig. 4 represents the dissection of injected in mice EGF-SEA, VEGF-SEA, SEA and normal saline.Fig. 5 represents the heavy result of the tumor after the dissection of injected in mice EGF-SEA, VEGF-SEA, SEA and normal saline.Show that EGF-SEA and VEGF-SEA fusion rotein have very obvious suppression effect for entity tumor.

The lymphocytic detection of T in embodiment 5 tumor tissues

Two kinds of cytokine-superantigen fusion rotein and SEA albumen are handled and the mice S180 tumor tissues of matched group normal saline is cut into small pieces, and make paraffin section with paraffin embedding, carry out immunohistochemical assay then.In order to detect the CD4 in the tumor tissues ⁺And CD8 ⁺The T cell, the anti-CD4 of use Santa Cruz Biotechnolog company ⁺And CD8 ⁺Antibody, the anti-and avidin-biotin-perxidase complex (Zymed company) with two uses diaminobenzidine (DAB) colour developing at last then.

In order to detect the T cell with responding property of SEA, at first prepare anti-SEA serum multi-resistance, with the SEA protein immunization BALB/c mouse behind the purification, obtain to tire through immunity repeatedly and surpass 8000 antiserum, with Hitrap protein G-Sepharosecolumn (Amersham Biosciences company) purification antiserum, obtain the IgG part.Hatching the paraffin section of these tumor tissues in concentration is in the SEA solution of 0.8 μ g/ μ l, rinsing then, and earlier with anti-SEA antiserum IgG reaction, dilution in 1: 50, antibody is removed in rinsing, reuse two anti-reactions.

Fig. 6 utilizes the detected CD4 of routine immunization groupization ⁺The T cell.Fig. 7 is the CD4 on Fig. 6 tissue slice ⁺T reacting cells number is calculated.Fig. 8 utilizes the detected CD8 of routine immunization groupization ⁺The T cell.Fig. 9 is the CD8 on Fig. 8 tissue slice ⁺T reacting cells number is calculated.Figure 10 utilizes the detected and SEA reaction T cell of routine immunization groupization.Figure 11 is that the SEA reaction T number of cells on Figure 10 tissue slice is calculated.

In the mouse tumor tissue of EGF-SEA and VEGF-SEA injection, find to have a large amount of CD4 ⁺And CD8 ⁺And has a reactive T cell of SEA (point that 6-1,6-2 show among the figure is the T cell) (point that 8-1,8-2 show among the figure is the T cell) (point that 10-1,10-2 show among the figure is the T cell), this explanation is induced down at EGF-SEA and VEGF-SEA, has been full of wellability (infiltrating) T cell in the tumor tissues.And have only a spot of CD4 in the tumor of SEA processing group ⁺And CD8 ⁺And have the reactive T cell of SEA, and the matched group that normal saline is handled does not almost have any T cell.

SEA can illustrate that SEA combines with TCR on the T cell with t cell responses.

6 two kinds of cytokines of embodiment-superantigen fusion rotein is for the inspection of the binding ability of tumor cell

Hatch the tumor tissues paraffin section with EGF-SEA and VEGF-SEA protein solution, earlier with anti-SEA antibody response, use the 2nd antibody response of fluorescein-labeled anti-mice then, dilution in 1: 50 is observed under fluorescence microscope (Figure 12) at last.In the mouse tumor tissue through EGF-SEA and VEGF-SEA injection, can find that S180 tumor cell (maxicell) and T cell (minicell) all show fluorescence, show that EGF-SEA or VEGF-SEA can combine with Receptor EGFR or the vegf receptor VEGFR of EGF on the S180 cell, combine with TCR on the T cell again.

Just do not find T cell (minicell) in the tumor of the matched group of SEA processing and normal saline.

The detection of the embodiment 7 tumor tissues inner cell factors

Detect in the tumor tissues cytokine by T emiocytosis, reuse R ﹠amp with the anti-IL-2 antibody of Santa Cruz Biotechnolog company, anti-IFN-gamma antibodies and anti-TNF-Alpha antibodies; Enzyme-linkedimmunosorbent assay (ELISA) test kit of D Systems company detects the cytokine in the tumor.IL-2 secretion (13-1,13-2 have shown the color development area that antibody response is arranged between the tumor cell) in Figure 13 tumor tissues.TNF-α secretion (14-1,14-2 have shown the color development area that antibody response is arranged between the tumor cell) in Figure 14 tumor tissues.IFN-γ secretion (15-1,15-2 have shown the color development area that antibody response is arranged between the tumor cell) in Figure 15 tumor tissues.Reaction is the quantity that unit calculates cytokine secretion with the area to Figure 16 according to the antibody positive on the paraffin section of the immunohistochemical experiment of Figure 13-15.Figure 17 utilizes enzyme-linked immunosorbentassay (ELISA) test kit (R ﹠amp; D Systems company) detects the interior cytokine secretion of tumor tissues.

The result shows in the mouse tumor tissue of EGF-SEA and VEGF-SEA injection a large amount of IL-2, IFN-γ and TNF-α cytokine, and SEA handles and the tumor of the matched group of normal saline is interior with regard to cytokine seldom.

Embodiment 8 causes the analysis of the factor of apoptosis of tumor cells

Fas expression analysis on the tumor cell, (Santa Cruz Biotechnolog company) checks tumor tissues with anti-Fas antibody, find to express (Figure 18) through the lot of F as on the mouse tumor S180 cell surface of EGF-SEA and VEGF-SEA injection, the tumor cell of SEA group and matched group then seldom or does not almost have Fas to express.This may be by EGF-SEA and VEGF-SEA the secreted cytokine of the T cell in the inductive tumor tissues for example IFN-γ and TNF-α can make tumor cell high expressed Fas, and the FasL on the T cell can cause expressing the apoptosis of tumor cells of Fas, and this is the approach that killer T cell causes the target cell apoptosis.Fas expression analysis on Figure 18 tumor cell, (SantaCruz Biotechnolog company) checks tumor tissues with anti-Fas antibody, find to express through the lot of F as on the mouse tumor S180 cell surface of EGF-SEA and VEGF-SEA injection, shaded portion is exactly a Fas albumen around the maxicell of demonstration antibody response.

Perforin analysis around the tumor cell, (Santa Cruz Biotechnolog company) checks tumor tissues with anti-perforin antibody, finding has a large amount of perforin albumen (Figure 19 through the mouse tumor S180 cell peripheral of EGF-SEA and VEGF-SEA injection, 19-1 is EGF-SEA, 19-2 is VEGF-SEA, the little strip portion of representing with arrow is exactly cumulative perforin albumen group), and almost do not have perforin around the tumor cell of SEA group and matched group.Cytotoxic T lymphocyte can be secreted perforin makes the cell membrane of target cell form aperture, destroys the balance of the osmotic pressure of target cell, causes target cell death.

Granzyme analysis around the tumor cell, (Abcam company) checks tumor tissues with the particle-resistant enzyme antibody, finding has a large amount of granzymes (Figure 20) through the mouse tumor S180 cell peripheral of EGF-SEA and VEGF-SEA injection, granzyme analysis around Figure 20 tumor cell, (Abcam company) checks tumor tissues with the particle-resistant enzyme antibody, find mouse tumor S180 cell peripheral through EGF-SEA and VEGF-SEA injection have a large amount of granzymes (as the maxicell surface of tumor cell and on every side shaded portion be exactly granzyme albumen).

And almost do not have granzyme around the tumor cell of SEA group and matched group.Granzyme is can excretory a kind of proteolytic enzyme by cytotoxic T lymphocyte, destroy the cell membrane of target cell when perforin after, granzyme just can enter in the target cell, thereby degrades target cell.

Behind the cell death, chromosomal DNA will be degraded, and uses TUNEL test kit (Roche Applied Science company) can detect dna degradation in the nucleus, thereby can judge that these cells are dead.Under terminal deoxynucleotidyltransferase effect, have the 3-OH end that fluorescein-labeled nucleotide will be incorporated into apoptotic cell two strands or single stranded DNA, react with the anti-fluorescein antibody that has peroxidase labelling then.In the mouse tumor tissue through TUNEL detection discovery EGF-SEA and VEGF-SEA injection the positive S180 tumor cells of a large amount of TUNEL being arranged like this is dead cell.Figure 21 tumor cell TUNEL dyeing, it is dead cell (cell that shows shade is a dead cell) that the positive S180 tumor cell of a large amount of TUNEL is arranged in the mouse tumor tissue through TUNEL detection discovery EGF-SEA and VEGF-SEA injection.And the dead tumor cell in the tumor tissues of SEA group and matched group just seldom.

S180 tumor growth through the mice of EGF-SEA and VEGF-SEA administration is subjected to very big inhibition, finds to have a large amount of CD4 in the tumor tissues ⁺And CD8 ⁺And with the T lymphocyte of SEA reaction, by a large amount of cytokines of T emiocytosis in these tumor tissues for example IL-2, IFN-γ and TNF-α, wherein IFN-γ and TNF-α can cause apoptosis of tumor cells.The while killer T cell is CD8 for example ⁺T emiocytosis perforin and granzyme can tumoricidal cell membrane and degraded tumor cells around tumor cell.Detect by TUNEL, affirmed that the tumor group of EGF-SEA and VEGF-SEA group mice is woven with the massive tumor cell death.

Above a series of embodiment show that EGF-SEA and VEGF-SEA be that EGFR and VEGFR interact with the receptor of EGF and VEGF respectively both, again with the T cell on the TCR effect, so just with on the cell targeted tumor cell surface that navigates to great expression EGFR or VEGFR of T, by the cytokine of T emiocytosis, induce target cell to produce Fas and perforin and granzyme etc. cause apoptosis of tumor cells.It is to be noted that especially EGF-SEA and VEGF-SEA have clearly lethal effect for entity tumor.

The present invention has selected a brand-new strategy, superantigen is connected on the cytokine, produced novel cytokine-super antigen fusion protein like this, as a model, the present invention uses epidermal growth factor EGF and blood vessel endothelial cell growth factor VEGF to make up this novel fused protein with superantigen SEA respectively.

Though only select superantigen SEA at this, superantigen can also be selected from: the SEB of Staphylococcus aureus enterotoxin family, SEC, SED, SEE, the SPE-A of streptococcus toxin, SPE-B, SPE-C, shock syndrome toxin (Shock syndrome toxin), virus protein with and aminoacid sequence the nature of the homogeny more than 70% and artificial variant are arranged, also thought of the present invention can be described.The effect of superantigen SEA or other superantigen is the intravital immunoreation of exactor.

Equally, as the positioning action that the epidermal growth factor EGF and the blood vessel endothelial cell growth factor VEGF of experiment material just utilizes their cancerous cell, adopt other cytokine that is closely related with cancerous cell also thought of the present invention can be described.

Such material has various chemotactic factors (Chemokine), Ephrin family, angiogenin (Angiopoietin, Ang), thrombopoietin (Thrombopoietin, TPO) and the blood plasma VII factor (Factor VII), urokinase type plasminogen activator (Urokinase-type plasminogen activator, uPA), gastrin releasing peptide (gastrin-releasing peptide, GRP), growth hormone releasing hormone (Growth hormone releasinghormone, GHRH), melanotropin α-MSH, prolactin antagonist (Prolactin, PRL), prolactin releasing hormone (PRH) (Prolactinreleasing hormone, PRLH), growth hormone (Growth hormone, GH), follicle stimulating hormone (Folliclestimulating hormone, FSH), placental lactogen hormone (Placental lactogen, PL), chorionic-gonadotropin hormone (Chorionic gonadotropin, CG) and corticotropin releasing hormone (Corticotropin releasinghormone, CRH), somatostatin (Somatostatin, SST), asialoglycoprotein glycoprotein (Asialoglycoprotein, ASGP), low density lipoprotein, LDL (Low density lipoprotein, LDL) and transferrins (Transferrin, Tf) etc., many tumor tissues are the receptor of these materials of overexpression all, thereby chemotactic factor, enzyme, peptide molecule parts such as hormone and other albumen formation fused protein that just can be as cytokine be connected with superantigen navigates to tumor tissues with superantigen.

Fused protein also can be connected the polypeptide fragment of cytokine and superantigen respectively by chemical reaction means such as chemical crosslink reactions, and for example covalent bond connects, thereby is built into fused protein.

Can carry out a part of polypeptide fragment of chemical modification, damaged fused protein and other polypeptide chain is connected on the first-class a series of transformation of these protein for fused protein.

Fused protein behind the purification can improve the space structure that it comprises disulfide bond by a series of proteinic degeneration and renaturation process, thereby improves its biological activity.

From bigger scope, the receptor of overexpression is actually interactional relation between a kind of part (Ligand) and the receptor on cytokine and its cancerous cell surface, utilizes the affinity of this part and receptor, and superantigen is navigated to tumor tissues.Except cytokine, other have with the corresponding peptide molecule of cancerous cell overexpression receptor be the specific localization that part also can be used for cancerous cell.

Except the corresponding part of top receptor said and on the cancerous cell, screen from phage display methods such as (phage display) with cancerous cell on receptor affinity is arranged and the artificial screening polypeptide of antagonism is arranged and other can be directly and the peptide molecule of cancerous cell surface interaction can form fused protein with superantigen.

As its dosage form of medicine can be emulsifying agent, liposome, dispersant, stabilizing agent etc. make together various injections, oral, apply ointment or plaster and the form of medication of medicine such as surgical procedure.

Except fused protein itself can be used as the medicine, the nucleotide fragments of encoding fusion protein matter or carrier can also be used as the gene therapy form.

The sequence explanation

The nucleotide sequence of SEQ ID NO:1 is a coding EGF-SEA fusion rotein;

SEQ ID NO:2 is the aminoacid sequence of EGF-SEA fusion rotein;

The nucleotide sequence of SEQ ID NO:3 is a coding VEGF-SEA fusion rotein;

SEQ ID NO:4 is the aminoacid sequence of VEGF-SEA fusion rotein;

SEQ ID NO:5 is the coded sequence of connection peptides;

SEQ ID NO:6 is the aminoacid sequence of connection peptides.

Sequence table

＜110〉Sun Jialin

＜120〉cytokine-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine

<160>6

<210>1

<211>885

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(885)

＜223〉coded sequence of EGF-SEA fusion rotein

<400>1

aattccgata?gcgagtgtcc?tctgagtcac?gatggttact?gtctacatga?cggcgtctgt 60

atgtatattg?aggctctaga?caagtacgcg?tgtaattgcg?ttgttggcta?catcggtgag 120

cgctgtcagt?atcgagatct?gaaatggtgg?gaacttagag?tcgacaagct?ttccggcgga 180

ggtggcagcg?agaaaagcga?agaaataaat?gaaaaagatt?tgcgaaaaaa?gtctgaattg 240

cagggaacag?ctttaggcaa?tcttaaacaa?atctattatt?acaatgaaaa?agctaaaact 300

gaaaataaag?agagtcacga?tcaattttta?cagcatacta?tattgtttaa?aggctttttt 360

acagatcatt?cgtggtataa?cgatttatta?gtagattttg?attcaaagga?tattgttgat 420

aaatataaag?ggaaaaaagt?agacttgtat?ggtgcttatt?atggttatca?atgtgcgggt 480

ggtacaccaa?acaaaacagc?ttgtatgtat?ggtggtgtaa?cgttacatga?taataatcga 540

ttgaccgaag?agaaaaaagt?gccgatcaat?ttatggctag?acggtaaaca?aaatacagta 600

cctttggaaa?cggttaaaac?gaataagaaa?aatgtaactg?ttcaggagtt?ggatcttcaa 660

gcaagacgtt?atttacagga?aaaatataat?ttatataact?ctgatgtttt?tgatgggaag 720

gttcagaggg?gattaatcgt?gtttcatact?tctacagaac?cttcggttaa?ttacgattta 780

tttggtgctc?aaggacagta?ttcaaataca?ctattaagaa?tatatagaga?taataaaacg 840

attaactctg?aaaacatgca?tattgcgata?tatttatata?caagt 885

<210>2

<211>295

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(295)

＜223〉EGF-SEA fusion rotein

<400>2

Asn?Ser?Asp?Ser?Glu?Cys?Pro?Leu?Ser?His?Asp?Gly?Tyr?Cys?Leu?His

1 5 10 15

Asp?Gly?Val?Cys?Met?Tyr?Ile?Glu?Ala?Leu?Asp?Lys?Tyr?Ala?Cys?Asn

20 25 30

Cys?Val?Val?Gly?Tyr?Ile?Gly?Glu?Arg?Cys?Gln?Tyr?Arg?Asp?Leu?Lys

35 40 45

Trp?Trp?Glu?Leu?Arg?Val?Asp?Lys?Leu?Ser?Gly?Gly?Gly?Gly?Ser?Glu

50 55 60

Lys?Ser?Glu?Glu?Ile?Asn?Glu?Lys?Asp?Leu?Arg?Lys?Lys?Ser?Glu?Leu

65 70 75 80

Gln?Gly?Thr?Ala?Leu?Gly?Asn?Leu?Lys?Gln?Ile?Tyr?Tyr?Tyr?Asn?Glu

85 90 95

Lys?Ala?Lys?Thr?Glu?Asn?Lys?Glu?Ser?His?Asp?Gln?Phe?Leu?Gln?His

100 105 110

Thr?Ile?Leu?Phe?Lys?Gly?Phe?Phe?Thr?Asp?His?Ser?Trp?Tyr?Asn?Asp

115 120 125

Leu?Leu?Val?Asp?Phe?Asp?Ser?Lys?Asp?Ile?Val?Asp?Lys?Tyr?Lys?Gly

130 135 140

Lys?Lys?Val?Asp?Leu?Tyr?Gly?Ala?Tyr?Tyr?Gly?Tyr?Gln?Cys?Ala?Gly

145 150 155 160

Gly?Thr?Pro?Asn?Lys?Thr?Ala?Cys?Met?Tyr?Gly?Gly?Val?Thr?Leu?His

165 170 175

Asp?Asn?Asn?Arg?Leu?Thr?Glu?Glu?Lys?Lys?Val?Pro?Ile?Asn?Leu?Trp

180 185 190

Leu?Asp?Gly?Lys?Gln?Asn?Thr?Val?Pro?Leu?Glu?Thr?Val?Lys?Thr?Asn

195 200 205

Lys?Lys?Asn?Val?Thr?Val?Gln?Glu?Leu?Asp?Leu?Gln?Ala?Arg?Arg?Tyr

210 215 220

Leu?Gln?Glu?Lys?Tyr?Asn?Leu?Tyr?Asn?Ser?Asp?Val?Phe?Asp?Gly?Lys

225 230 235 240

Val?Gln?Arg?Gly?Leu?Ile?Val?Phe?His?Thr?Ser?Thr?Glu?Pro?Ser?Val

245 250 255

Asn?Tyr?Asp?Leu?Phe?Gly?Ala?Gln?Gly?Gln?Tyr?Ser?Asn?Thr?Leu?Leu

260 265 270

Arg?Ile?Tyr?Arg?Asp?Asn?Lys?Thr?Ile?Asn?Ser?Glu?Asn?Met?His?Ile

275 280 285

Ala?Ile?Tyr?Leu?Tyr?Thr?Ser

290 295

<210>3

<211>1089

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(1089)

＜223〉coded sequence of VEGF-SEA fusion rotein

<400>3

gcacccatgg?cagaaggagg?agggcagaat?catcacgaag?tggtgaagtt?catggatgtc 60

tatcagcgca?gctactgcca?tccaatcgag?accctggtgg?acatcttcca?ggagtaccct 120

gatgagatcg?agtacatctt?caagccatcc?tgtgtgcccc?tgatgcgatg?cgggggctgc 180

tgcaatgacg?agggcctgga?gtgtgtgccc?actgaggagt?ccaacatcac?catgcagatt 240

atgcggatca?aacctcacca?aggccagcac?ataggagaga?tgagcttcct?acagcacaac 300

aaatgtgaat?gcagaccaaa?gaaagataga?gcaagacaag?aaaaatgtga?caagecgagg 360

cgggtcgaca?agctttccgg?cggaggtggc?agcgagaaaa?gcgaagaaat?aaatgaaaaa 420

gatttgcgaa?aaaagtctga?attgcaggga?acagctttag?gcaatcttaa?acaaatctat 480

tattacaatg?aaaaagctaa?aactgaaaat?aaagagagtc?acgatcaatt?tttacagcat 540

actatattgt?ttaaaggctt?ttttacagat?cattcgtggt?ataacgattt?attagtagat 600

tttgattcaa?aggatattgt?tgataaatat?aaagggaaaa?aagtagactt?gtatggtgct 660

tattatggtt?atcaatgtgc?gggtggtaca?ccaaacaaaa?cagcttgtat?gtatggtggt 720

gtaacgttac?atgataataa?tcgattgacc?gaagagaaaa?aagtgccgat?caatttatgg 780

ctagacggta?aacaaaatac?agtacctttg?gaaacggtta?aaacgaataa?gaaaaatgta 840

actgttcagg?agttggatct?tcaagcaaga?cgttatttac?aggaaaaata?taatttatat 900

aactctgatg?tttttgatgg?gaaggttcag?aggggattaa?tcgtgtttca?tacttctaca 960

gaaccttcgg?ttaattacga?tttatttggt?gctcaaggac?agtattcaaa?tacactatta 1020

agaatatata?gagataataa?aacgattaac?tctgaaaaca?tgcatattgc?gatatattta 1080

tatacaagt 1089

<210>4

<211>363

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(363)

＜223〉VEGF-SEA fusion rotein

<400>4

Ala?Pro?Met?Ala?Glu?Gly?Gly?Gly?Gln?Asn?His?His?Glu?Val?Val?Lys

1 5 10 15

Phe?Met?Asp?Val?Tyr?Gln?Arg?Ser?Tyr?Cys?His?Pro?Ile?Glu?Thr?Leu

20 25 30

Val?Asp?Ile?Phe?Gln?Glu?Tyr?Pro?Asp?Glu?Ile?Glu?Tyr?Ile?Phe?Lys

35 40 45

Pro?Ser?Cys?Val?Pro?Leu?Met?Arg?Cys?Gly?Gly?Cys?Cys?Asn?Asp?Glu

50 55 60

Gly?Leu?Glu?Cys?Val?Pro?Thr?Glu?Glu?Ser?Asn?Ile?Thr?Met?Gln?Ile

65 70 75 80

Met?Arg?Ile?Lys?Pro?His?Gln?Gly?Gln?His?Ile?Gly?Glu?Met?Ser?Phe

85 90 95

Leu?Gln?His?Asn?Lys?Cys?Glu?Cys?Arg?Pro?Lys?Lys?Asp?Arg?Ala?Arg

100 105 110

Gln?Glu?Lys?Cys?Asp?Lys?Pro?Arg?Arg?Val?Asp?Lys?Leu?Ser?Gly?Gly

115 120 125

Gly?Gly?Ser?Glu?Lys?Ser?Glu?Glu?Ile?Asn?Glu?Lys?Asp?Leu?Arg?Lys

130 135 140

Lys?Ser?Glu?Leu?Gln?Gly?Thr?Ala?Leu?Gly?Asa?Leu?Lys?Gln?Ile?Tyr

145 150 155 160

Tyr?Tyr?Asn?Glu?Lys?Ala?Lys?Thr?Glu?Asn?Lys?Glu?Ser?His?Asp?Gln

165 170 175

Phe?Leu?Gln?His?Thr?Ile?Leu?Phe?Lys?Gly?Phe?Phe?Thr?Asp?His?Ser

180 185 190

Trp?Tyr?Asn?Asp?Leu?Leu?Val?Asp?Phe?Asp?Ser?Lys?Asp?Ile?Val?Asp

195 200 205

Lys?Tyr?Lys?Gly?Lys?Lys?Val?Asp?Leu?Tyr?Gly?Ala?Tyr?Tyr?Gly?Tyr

210 215 220

Gln?Cys?Ala?Gly?Gly?Thr?Pro?Asn?Lys?Thr?Ala?Cys?Met?Tyr?Gly?Gly

225 230 235 240

Val?Thr?Leu?His?Asp?Asn?Asn?Arg?Leu?Thr?Glu?Glu?Lys?Lys?Val?Pro

245 250 255

Ile?Asn?Leu?Trp?Leu?Asp?Gly?Lys?Gln?Asn?Thr?Val?Pro?Leu?Glu?Thr

260 265 270

Val?Lys?Thr?Asn?Lys?Lys?Asn?Val?Thr?Val?Gln?Glu?Leu?Asp?Leu?Gln

275 280 285

Ala?Arg?Arg?Tyr?Leu?Gln?Glu?Lys?Tyr?Asn?Leu?Tyr?Asn?Ser?Asp?Val

290 295 300

Phe?Asp?Gly?Lys?Val?Gln?Arg?Gly?Leu?Ile?Val?Phe?His?Thr?Ser?Thr

305 310 315 320

Glu?Pro?Ser?Val?Asn?Tyr?Asp?Leu?Phe?Gly?Ala?Gln?Gly?Gln?Tyr?Ser

325 330 335

Asn?Thr?Leu?Leu?Arg?Ile?Tyr?Arg?Asp?Asn?Lys?Thr?Ile?Asn?Ser?Glu

340 345 350

Asn?Met?His?Ile?Ala?Ile?Tyr?Leu?Tyr?Thr?Ser

355 360

<210>5

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉coded sequence of connection peptides

<400>5

gtcgacaagc?tttccggcgg?aggtggc 27

<210>6

<211>9

<212>PRT

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(9)

＜223〉connection peptides

<400>6

Val?Asp?Lys?Leu?Ser?Gly?Gly?Gly?Gly

1 5

Claims

1. cytokine-superantigen fusion rotein is characterized in that in the application of preparation solid tumor resisting medicine described cytokine is epidermal growth factor or vascular endothelial cell growth factor, and described superantigen is the superantigen of staphylococcus aureus toxin A.

2. cytokine according to claim 1-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, it is characterized in that described cytokine-superantigen fusion rotein can mobilize the T lymphocyte, and with described T lymphocyte targeting navigate to around the in-house tumor cell of entity tumor, described T lymphocyte has CD4 ⁺Or CD8 ⁺Feature and interacting with SEA.

3. cytokine according to claim 1-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, it is characterized in that described cytokine-superantigen fusion rotein can induce the in-house T lymphocytic emiocytosis of entity tumor cytokine interleukin II, interferon-and tumor necrosis factor-alpha.

4. cytokine according to claim 1-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, it is characterized in that the high expressed that described cytokine-superantigen fusion rotein can the lip-deep apoptotic proteins Fas of inducing tumor cell.

5. cytokine according to claim 1-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, it is characterized in that described cytokine-superantigen fusion rotein can induce cytotoxic T lymphocyte secretion perforin and granzyme B.

6. cytokine according to claim 1-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine, it is characterized in that described cytokine-superantigen fusion rotein can suppress the growth of entity tumor in the mice body, causes the death of massive tumor cell in the tumor tissues.