CN102516392A

CN102516392A - Cancer-targeted super antigen fusion protein, and preparation method and application thereof

Info

Publication number: CN102516392A
Application number: CN2011103787994A
Authority: CN
Inventors: 孙嘉琳
Original assignee: 孙嘉琳
Current assignee: Nanjing double bond targeted drug Technology Co., Ltd
Priority date: 2011-11-25
Filing date: 2011-11-25
Publication date: 2012-06-27
Anticipated expiration: 2031-11-25
Also published as: CN102516392B; WO2013075553A1

Abstract

The invention discloses a cancer-targeted super antigen fusion protein, and a preparation method and application thereof. The fusion protein contains: a) a ligand which promotes the growth of cancer cells and interacts with a cancer cell overexpression receptor; and b) super antigen Staphylococcus aureus enterotoxin E and mutants thereof which can cause anti-cancer immunologic reaction, wherein the Staphylococcus aureus enterotoxin E is abbreviated to SEE, and the mutants of the Staphylococcus aureus enterotoxin E are abbreviated to SEE-X, and X=1-3. Compared with SEA, the super antigen SEE disclosed by the invention is more stable; and SEE-1, SEE-2 and SEE-3, the mutants of SEE subjected to large-scale multi-point mutation reconstruction, reduce the interaction with MHC. Thus, the fusion protein composed of the SEE mutants and TGF-alpha, EGF, VEGF, GnRH or GRP indicates very high anti-cancer and anti-tumor biological activity.

Description

A kind of cancer-targeted super antigen fusion rotein and Preparation method and use

Technical field

The present invention relates to a kind of superantigen fusion rotein and Preparation method and use, particularly relate to a kind of cancer-targeted super antigen and mutant fusion protein thereof and Preparation method and use.

Background technology

The superantigen fusion rotein is applied to the drug research of cancer, for example cytokine and superantigen constructed fusion rotein (one Chinese patent application numbers 200310109829.7, " a kind of can in order to the super antigen fusion protein and the working method thereof of anticancer therapy "; One Chinese patent application numbers 201010118438.1, " cytokine-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine "; One Chinese patent application numbers 201010585818.6, " cytokine-superantigen fusion rotein is in the application of preparation cancer therapy drug "; PLoSOne 6; E16642; 2011), hormone and superantigen constructed fusion rotein (one Chinese patent application number 200510078775.1 " cancer-targeted super antigen fusion protein and working methods thereof ") and antibody and superantigen constructed fusion rotein (Proc Natl Acad Sci USA; 91,8945-8949,1994; Proc Natl Acad Sci USA; 92; 9791-9795; 1995), the hormone in cytokine in cytokine-superantigen fusion rotein and hormone-superantigen fusion rotein be through with cancer cells on great expression cytokine receptor and hormone receptor interaction and fusion rotein is navigated on the cancer cells and the CDCC of another part of fusion rotein to be superantigen can induce t cell responses of attacking cancer cells.

Above-mentioned superantigen is staphylococcus aureus toxin A (Staphylococcal-enterotoxin A; SEA); It can produce the spinoff of the lymphocytic reaction of general, and research (Proc Natl Acad Sci USA, 94 are arranged; 2489-2494,1997) it has been carried out the transformation of a point mutation.In addition, it is reported SEA itself also unstable (J Biol Chem, 275,1665-1672,2000).Improve the anticancer effect of superantigen fusion rotein, can select the new superantigen of thinking except SEA.

In addition; Activated have the material that utilize to be worth for example compound, nucleic acid, polypeptide etc. can improve its activity or utility value through the structure of transforming them; With the enzyme is example, can remove or means such as increase improve the catalytic activity of enzyme, heat-resisting, low temperature resistant, acidproof, alkaline-resisting, organic solvent-resistant etc. through the displacement of extensive multipoint mutation, fragment, amino acid.So, expect to create the higher superantigen of biological activity through to the large-scale point mutation transformation of superantigen.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, provide biological activity higher a kind of cancer-targeted super antigen fusion rotein.

Second purpose of the present invention provides a kind of preparation method of superantigen fusion rotein.

The 3rd purpose of the present invention provides a kind of purposes of superantigen fusion rotein.

Technical scheme of the present invention is summarized as follows:

A kind of cancer-targeted super antigen fusion rotein, this fusion rotein contains:

A) promote growth of cancer cells and with the part of cancer cells over-expresses acceptor interaction, said part is the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor;

B) can cause antitumor immune reaction the superantigen staphylococcus aureus enterotoxin E (Staphylococcal-enterotoxin E, SEE) and two mutants, said staphylococcus aureus enterotoxin E is represented with the SEE that abridges; The two mutants of said staphylococcus aureus enterotoxin E is represented said X=1-3 with abbreviation SEE-X.

Said cytokine is selected from transforminggrowthfactor-(the Transforming growth factor-α that is abbreviated as TGF-α; TGF-α), be abbreviated as Urogastron (the Epidermal growth factor of EGF; EGF), be abbreviated as VEGF vascular endothelial growth factor (Vascular endothelial cell growth factor, VEGF); Said hormone be selected from the gonadotropin releasing hormone of being abbreviated as GnRH (Gonadotropin-releaslng hormone, GnRH); The polypeptide of the non-hormone of the said non-antibody acellular factor be selected from the gastrin releasing peptide of being abbreviated as GRP (Gastrin-releasing peptide, GRP).

Said SEE is the aminoacid sequence shown in the SEQ ID No.2; Said SEE-1 is the aminoacid sequence shown in the SEQ ID No.4; Said SEE-2 is the aminoacid sequence shown in the SEQ ID No.6; Said SEE-3 is the aminoacid sequence shown in the SEQ ID No.8.

Said SEE-1 is that the one or more amino acid on 20,21,24,27,34,39,40,1,42,44,49,74,75,78,79,81,83,84,217,220,222,223,225,227 on the SEE are replaced.

Said SEE-2 is that the one or more amino acid on 20,21,24,27,34,35,39,40,41,42,44,45,46,49,74,75,78,79,81,83,84,188,190,217,219,220,222,223,224,225,227 on the SEE are replaced.

Said SEE-3 is that the one or more amino acid on 20,21,24,27,34,35,36,39,40,41,42,44,45,46,49,62,74,75,77,78,79,81,83,84,188,190,217,218,219,220,221,222,223,224,225,227 on the SEE are replaced.

Said this fusion rotein is: by the TGF-α-SEE shown in the SEQ ID No.10; By the EGF-SEE shown in the SEQ ID No.18; By the VEGF-SEE shown in the SEQ ID No.26; By the GnRH-SEE shown in the SEQ ID No.34; By the GRP-SEE shown in the SEQ ID No.42; By the TGF-α-SEE-1 shown in the SEQ ID No.12; By the EGF-SEE-1 shown in the SEQ ID No.20; By the VEGF-SEE-1 shown in the SEQ ID No.28; By the GnRH-SEE-1 shown in the SEQ ID No.36; By the GRP-SEE-1 shown in the SEQ ID No.44; By the TGF-α-SEE-2 shown in the SEQ ID No.14; By the EGF-SEE-2 shown in the SEQ ID No.22; By the VEGF-SEE-2 shown in the SEQ ID No.30; By the GnRH-SEE-2 shown in the SEQ ID No.38; By the GRP-SEE-2 shown in the SEQ ID No.46; By the TGF-α-SEE-3 shown in the SEQ ID No.16; By the EGF-SEE-3 shown in the SEQ ID No.24; By the VEGF-SEE-3 shown in the SEQ ID No.32; By the GnRH-SEE-3 shown in the SEQ ID No.40; Or by the GRP-SEE-3 shown in the SEQ ID No.48.

Recombinant vectors contains the nucleotide sequence of coding claim 7 said fusion rotein, and said nucleotides sequence is classified the nucleotide sequence SEQ ID No.9 of coding SEQ ID No.10 as; The nucleotide sequence SEQ ID No.11 of coding SEQ ID No.12; The nucleotide sequence SEQ ID No.13 of coding SEQ ID No.14; The nucleotide sequence SEQ ID No.15 of coding SEQ ID No.16; The nucleotide sequence SEQ ID No.17 of coding SEQ ID No.18; The nucleotide sequence SEQ ID No.19 of coding SEQ ID No.20; The nucleotide sequence SEQ ID No.21 of coding SEQ ID No.22; The nucleotide sequence SEQ ID No.23 of coding SEQ ID No.24; The nucleotide sequence SEQ ID No.25 of coding SEQ ID No.26; The nucleotide sequence SEQ ID No.27 of coding SEQ ID No.28; The nucleotide sequence SEQ ID No.29 of coding SEQ ID No.30; The nucleotide sequence SEQ ID No.31 of coding SEQ ID No.32; The nucleotide sequence SEQ ID No.33 of coding SEQ ID No.34; The nucleotide sequence SEQ ID No.35 of coding SEQ ID No.36; The nucleotide sequence SEQ ID No.37 of coding SEQ ID No.38; The nucleotide sequence SEQ ID No.39 of coding SEQ ID No.40; The nucleotide sequence SEQ ID No.41 of coding SEQ ID No.42; The nucleotide sequence SEQ ID No.43 of coding SEQ ID No.44; The nucleotide sequence SEQ ID No.45 of coding SEQ ID No.46; Or shown in the nucleotide sequence SEQ IDNo.47 of coding SEQ ID No.48.

A kind of preparation method of cancer-targeted super antigen fusion rotein cultivates the host cell that contains above-mentioned recombinant vectors, collects and expresses a kind of cancer-targeted super antigen fusion rotein.

A kind of cancer-targeted super antigen fusion rotein is in the application of the medicine of preparation treatment cancer and anti entity tumour.

Advantage of the present invention:

Superantigen SEE is than more stable (the J Biol Chem of SEA; 275; 1665-1672; 2000), and reduced the interaction with MHC, so the fusion rotein of being made up of SEE two mutants and TGF-α, EGF, VEGF, GnRH or GRP shows very high anticancer and anti-tumor bioactivity through improved SEE two mutants SEE-1, SEE-2 and the SEE-3 of extensive multipoint mutation.

Description of drawings

Fig. 1 is that TGF-α and SEE or SEE two mutants are formed fusion rotein and suppressed the S180 tumor growth, expression be tumor growth curve with the mouse dissection after tumor weight.

Fig. 2 is that EGF and SEE or SEE two mutants are formed fusion rotein and suppressed the S180 tumor growth, expression be tumor growth curve with the mouse dissection after tumor weight.

Fig. 3 is that VEGF and SEE or SEE two mutants are formed fusion rotein and suppressed the S180 tumor growth, expression be tumor growth curve with the mouse dissection after tumor weight.

Fig. 4 utilizes the detected T cell of immunohistochemical methods, and brown point is the T cell.(among the figure: 4-1 is TGF-α-SEE-3 administration group; 4-2 is an EGF-SEE-3 administration group; 4-3 is a VEGF-SEE-3 administration group)

Fig. 5 utilizes the detected interferon-by T emiocytosis of immunohistochemical methods (IFN-γ), and brown part is IFN-γ.(among the figure: 5-1 is TGF-α-SEE-3 administration group; 5-2 is an EGF-SEE-3 administration group; 5-3 is a VEGF-SEE-3 administration group)

Fig. 6 is that 6nRH and SEE or SEE two mutants are formed fusion rotein and killed and wounded Hep G2 liver cancer cell.

Fig. 7 is that GRP and SEE or SEE two mutants are formed fusion rotein and killed and wounded the A549 lung carcinoma cell.

The cancer cells of being attacked by the T cell that Fig. 8 shows, maxicell is a cancer cells, minicell is the T cell.(among the figure: 8-1 is the GnRH-SEE-3 group; 8-2 is the GRP-SEE-3 group)

Embodiment

Strategic thought of the present invention is following:

Though can improve the performance of SEA through a point mutation, seek one and then can increase substantially the biological activity of superantigen than better superantigen of SEA and the structure construction that carries out proteic multipoint mutation, obtain better cancer therapy effect.

The superantigen of streptococcus aureus origin has SEA, SEB, SEC, SED, SEF, SEG, SEH, SEI, SEJ etc., and they structurally have certain similarity (Infect Immun, 66; 3337-3348,1998), so the also reference each other of their space structure; SEA (EMBO J for example; 14,3292-3301,1995; J Biol Chem, 271,32212-32216,1996; J Mol Biol, 269,270-280,1997; Structure, 10,1619-1626,2002), SEB and SEC (Nature, 368,711-718,1994; Nature, 384,188-192,1996; J Mol Biol, 277,61-79,1998; Structure, 11,1151-1161,2003), SHE (EMBO J, 20,3306-3312,2001; J Mol Biol, 302,527-537,2000), and other relevant document (Nature, 346,471-473,1990; Infect Immun, 59,2126-2134,1991; J Exp Med, 175,415-424,1992; Proc Natl Acad Sci USA, 89,7727-7731,1992; J Exp Med, 177,175-184,1993; Infect Immun, 61,2059-2068,1993; J Biol Chem, 275,1665-1672,2000; Trends Microbiol, 8,369-375,2000; Immunity, 14,331-344,2001; Clin Exp Immunol, 133,299-306,2003; J Mol Biol, 333,893-905,2003; J Biol Chem, 278,50412-50421,2003; Scand J Immunol, 59,345-355,2004; Semin Immunol, 19,262-271,2007), superantigen comprise the 20-27 amino acid fragment among SEE and the SEA be considered to the T cell on TCR interact, thereby activated T cell; Amino acid fragments such as 34-49,74-84,187-190 and 217-227 are considered to interact with histocompatibility antigen MHC.Can seek a biological activity that improves it than the more effective superantigen of SEA and through large-scale multipoint mutation through these information.

At first the present invention selects SEE, and it is compared with SEA has very big similarity but than SEA more stable (J Biol Chem, 275,1665-1672,2000), in the aminoacid sequence of SEE, can introduce extensive multipoint mutation, and the foundation of point mutation is:

1. strengthen the binding ability of TCR, the effect of strengthening superantigen and TCR just can improve the ability of superantigen inducing T cell, can consider to be employed in the amino acid on the SEA same position here.

2. reduce the binding ability with histocompatibility antigen MHC, the interaction of superantigen SEE and histocompatibility antigen MHC such as synantibody are similar with the polypeptide antigen interaction, are the complementary interactions.The mode of its effect is:

1) interaction of polare Aminosaeren, i.e. the interaction of acidic amino acid in the superantigen and the basic aminoacids in the histocompatibility antigen, the perhaps interaction of basic aminoacids in the superantigen and the acidic amino acid in the histocompatibility antigen;

2) interaction of nonpolar hydrophilic amino acid, i.e. interaction between the nonpolar hydrophilic amino acid in superantigen and the histocompatibility antigen;

3) interaction of nonpolar hydrophobic amino acid, i.e. interaction between the nonpolar hydrophobic amino acid in superantigen and the histocompatibility antigen;

4) the amino acid whose interaction of 1-3 needs suitable space above, and promptly amino acid whose side chain lengths is suitable, the people for a change interactional amino acid whose length also can reduce these amino acid whose interactions.

So; Through changing the amino acid whose character of superantigen and histocompatibility antigen interaction zone, for example change in the superantigen and interactional amino acid whose polarity of histocompatibility antigen and length etc. can reduce or weaken the interaction between superantigen and the histocompatibility antigen.

Though disclosed the position of point mutation among the present invention embodiment 1 below, in fact can also on the position on these positions or its next door, introduce the amino acid of other similar physico-chemical property, also can obtain similar result.

As another part of modified version superantigen fusion rotein be selected from the cancer cells surface on the interactional part of acceptor (Receptor) (Ligand) of great expression; The polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor for example; In the present invention; Cytokine is TGF-α, EGF or VEGF, hormone GnRH, and the polypeptide of the non-hormone of the non-antibody acellular factor is GRP.

Superantigen is the superantigen SEE of staphylococcus aureus enterotoxin E, shown in SEQ ID No.2;

The SEE two mutants of superantigen for transforming through multipoint mutation, wherein two mutants SEE-1 is shown in the SEQ ID No.4, and two mutants SEE-2 is shown in the SEQ ID No.6, and two mutants SEE-3 is shown in the SEQ ID No.8;

Described multipoint mutation occurs in 20,21,24,27,34,35,36,39,40,41,42,44,45,46,49,62,74,75,77,78,79,81,83,84,188,190,217,218,219,220,221,222,223,224,225,227;

The amino acid of described multipoint mutation is, on 20, and Arg → Gly; On 21, Asn → Thr; On 24, Ser → Gly; On 27, Arg → Lys; On 34, Glu → Ser or Glu → Ala; On 35, Lys → Ser; On 36, Ala → Gly; On 39, Glu → Ser; On 40, Asn → Ser or Asn → Ala; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ala or Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Thr or Glu → Ser; On 62, Pro → Ser; On 74, Lys → Thr or Lys → Ser; On 75, Asp → Ala or Asp → Thr; On 77, Thr → Ser; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser or Lys → Thr; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Thr or Lys → Ser; On 218, Thr → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 221, Ser → Thr; On 222, Glu → Thr or Glu → Ser; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.

The part of utilization of the present invention and cancer cells acceptor interaction-modified version super antigen fusion protein; Wherein with the part of cancer cells acceptor interaction for example the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor can fused protein be navigated on the tumour cell; The modified version superantigen then causes more powerful antitumor immune reaction around tumour cell; Be the cell-mediated CDCC that relies on of superantigen (Superantigen-dependent-cellular-cytotoxicity, SDCC).Utilize this method just can such fused protein be navigated on the tumour cell and at tumour cell specifically and cause anticancer cell toxicant immunoreation on every side.

Through specific embodiment the present invention is further described below.

JEG-3 is used in experiment: as employed JEG-3 in the embodiment of the invention is murine sarcoma tumour Sarcoma (S180) tumour cell, human liver cancer cell Hep G2 and the human lung cancer cell A549 who is used to set up the mouse-borne tumor model, and these JEG-3s are available from ATCC.

EGF Receptor EGFR on the S180 cancer cells of existing report mouse and vegf receptor VEGFR can with EGF and VEGF interaction (PLoS One 6, e16642,2011 in people source; One Chinese patent application numbers 201010118438.1; " cytokine-superantigen fusion rotein is in the application of preparation solid tumor resisting medicine "); And the acceptor of TGF-α is identical with EGF; All be EGFR, so the S180 cancer cells can be used for the experiment that TGF, EGF or VEGF and SEE or SEE two mutants form fusion rotein.

Liver cancer cell is expressed acceptor GnRH-R (Endocrinology, 136,75-84,1995 of GnRH; Cancer Res, 60,3701-3705,2000), so Hep G2 cancer cells can be used for the experiment that GnRH and SEE or SEE two mutants form fusion rotein.

Lung carcinoma cell is expressed acceptor GRP-R (Oncogene, 20,1563-1569,2001 of GRP; Ann Oncol, 18,1457-1466,2007), so the A549 cancer cells can be used for the experiment that GRP and SEE or SEE two mutants form fusion rotein.

The two mutants of embodiment 1 design SEE

Because SEA and SEE have very big similarity (Infect Immun, 66,3337-3348,1998) on sequence, so the research of the space structure of SEA and other superantigen etc. can be used as reference (EMBO J, 14,3292-3301,1995 of SEE; J Biol Chem, 271,32212-32216,1996; J Mol Biol, 269,270-280,1997; Structure, 10,1619-1626,2002; Nature, 368,711-718,1994; Nature, 384,188-192,1996; J Mol Biol, 277,61-79,1998; Structure, 11,1151-1161,2003; EMBO J, 20,3306-3312,2001; J Mol Biol, 302,527-537,2000), in addition also with reference to relevant document (Nature, 346,471-473,1990; Infect Immun, 59,2126-2134,1991; J Exp Med, 175,415-424,1992; Proc Natl Acad Sci USA, 89,7727-7731,1992; J Exp Med, 177,175-184,1993; Infect Immun, 61,2059-2068,1993; J Biol Chem, 275,1665-1672,2000; Trends Microbiol, 8,369-375,2000; Immunity, 14,331-344,2001; Clin Exp Immunol, 133,299-306,2003; J Mol Biol, 333,893-905,2003; J Biol Chem, 278,50412-50421,2003; Scand J Immunol, 59,345-355,2004; Semin Immunol, 19,262-271,2007), through these information and SEE gene information (J Bacteriol, 170,2954-2960,1988; GenBank M21319) confirms the position of SEE point mutation, in the amino acid fragment of 20-27,34-49,74-84,187-190 and the 217-227 of the position of the point mutation that can consider in SEE, design as follows:

1. adopt among the SEA with TCR bonded amino acid segment because SEA has ability (Proc Natl Acad Sci USA, 91,8945-8949,1994 of very powerful inducing T cell; Proc Natl Acad Sci USA, 92,9791-9795,1995), designed 4 amino acid whose point mutation of the 20-27 position in the SEE sequence here: on 20, Arg → Gly; On 21, Asn → Thr; On 24, Ser → Gly; On 27, Arg → Lys.The point mutation of the two mutants of all SEE (SEE-1, SEE-2 and SEE-3) on this zone is all identical.

2. reduce and the combining of histocompatibility antigen MHC, acidity or basic aminoacids are become polar neutral amino acid (Ser or Thr) or nonpolar hydrophobic amino acid (Ala) or change its polar character (basic aminoacids becomes acidic amino acid or acidic amino acid becomes basic aminoacids) perhaps change amino acid whose side chain lengths.

2.1.SEE-1 the design of two mutants

On 34, Glu → Ser; On 39, Glu → Ser; On 40, Asn → Ser; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ala; On 49, Glu → Thr; On 74, Lys → Thr; On 75, Asp → Ala; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser; On 217, Lys → Thr; On 220, Asn → Ser; On 222, Glu → Thr; On 223, Asn → Ser; On 225, His → Ser; On 227, Asp → Ser.Obtain the aminoacid sequence (SEQ ID NO.4) of SEE-1 at last.

2.2.SEE-2 the design of two mutants

On 34, Glu → Ser; On 35, Lys → Ser; On 39, Glu → Ser; On 40, Asn → Ser; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Thr; On 74, Lys → Thr; On 75, Asp → Ala; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Ser; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 222, Glu → Thr; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.Obtain the aminoacid sequence (SEQ ID NO.6) of SEE-2 at last.

2.3.SEE-3 the design of two mutants

On 34, Glu → Ala; On 35, Lys → Ser; On 36, Ala → Gly; On 39, Glu → Ser; On 40, Asn → Ala; On 41, Lys → Glu; On 42, Glu → Lys; On 44, Asp → Ser; On 45, Asp → Ala; On 46, Gln → Ser; On 49, Glu → Ser; On 62, Pro → Ser; On 74, Lys → Ser; On 75, Asp → Thr; On 77, Thr → Ser; On 78, Asn → Ser; On 79, Lys → Glu; On 81, Lys → Glu; On 83, Lys → Ser; On 84, Lys → Thr; On 188, Ser → Thr; On 190, Glu → Thr; On 217, Lys → Ser; On 218, Thr → Ser; On 219, Ile → Leu; On 220, Asn → Ser; On 221, Ser → Thr; On 222, Glu → Ser; On 223, Asn → Ser; On 224, Leu → Ile; On 225, His → Ser; On 227, Asp → Ser.Obtain the aminoacid sequence (SEQ ID NO.8) of SEE-3 at last.

The structure of embodiment 2 superantigens and two mutants expression vector thereof

The DNA nucleotide sequence of coding SEE derives from its gene information (J Bacteriol, 170,2954-2960,1988; The GenBank DB; M21319); And two mutants is the design according to embodiment 1; Entrust TAKARA company synthetic DNA fragment; It comprises sequence fragment and a connection peptides (shown in SEQ ID NO.50) of SEE gene or its two mutants and in several bases of whole pulsating front HindIII and back XhoI limiting enzyme point, synthetic good nucleic acid fragment is inserted the T carrier respectively and carries out dna sequencing and identify, and then with the double digestion method promptly with after HindIII and the XhoI processing; Insert fragment respectively on the pET22b plasmid, obtained to express carrier pET22b-SEE-1, pET22b-SEE-2 and the pET22b-SEE-3 of two mutants of carrier pET22b-SEE and the SEE of SEE gene.By the corresponding proteins sequence of these vector expressions, SEE is shown in the SEQ ID NO.2, and SEE-1 is shown in the SEQ ID NO.4, and SEE-2 is shown in the SEQ ID NO.6, and SEE-3 is shown in the SEQ ID NO.8.

The nucleotide sequence of coding SEE is shown in the SEQ ID NO.1, and the nucleotide sequence of coding SEE-1 is shown in the SEQ ID NO.3, and the nucleotide sequence of coding SEE-2 is shown in the SEQ ID NO.5, and the nucleotide sequence of coding SEE-3 is shown in the SEQ ID NO.7.

The nucleotide sequence of coding connection peptides is shown in the SEQ ID NO.49.

Embodiment 3TGF-α and SEE or SEE two mutants are formed the structure of Expression of Fusion Protein carrier

Information (NM_003236) according to the TGF-α gene in the people of GenBank DB source; Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise TGF-α gene and increase the several bases that limit restriction enzyme site BamHI and EcoRI again, comprised the restriction restriction enzyme site of SalI and HindIII in pulsating back in TGF-α front.This synthetic good nucleic acid fragment is inserted the T carrier and carries out the dna sequencing evaluation; And then after promptly handling with the double digestion method with BamHI and HindIII; Insert this fragment respectively the carrier of pET22b-SEE, pET22b-SEE-1, pET22b-SEE-2 and pET22b-SEE-3 among the embodiment 2; So just produce expression vector pET22b-TGF-α-SEE, pET22b-TGF-α-SEE-1, pET22b-TGF-α-SEE-2 and pET22b-TGF-α-SEE-3, can express TGF-α-SEE (SEQ ID NO.10), TGF-α-SEE-1 (SEQ ID NO.12), TGF-α-SEE-2 (SEQ ID NO.14) and TGF-α-SEE-3 (SEQ ID NO.16) fusion rotein.

Embodiment 4EGF and SEE or SEE two mutants are formed the structure of Expression of Fusion Protein carrier

Information (NM_001963 and NM_001178130) according to the EGF gene in the people of GenBank DB source; Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise the EGF gene and increase the several bases that limit restriction enzyme site BamHI and EcoRI again, comprised the restriction restriction enzyme site of SalI and HindIII in pulsating back in the EGF front.This synthetic good nucleic acid fragment is inserted the T carrier and carries out the dna sequencing evaluation; And then after promptly handling with the double digestion method with BamHI and HindIII; Insert this fragment respectively the carrier of pET22b-SEE, pET22b-SEE-1, pET22b-SEE-2 and pET22b-SEE-3 among the embodiment 2; So just produce expression vector pET22b-EGF-SEE, pET22b-EGF-SEE-1, pET22b-EGF-SEE-2 and pET22b-EGF-SEE-3, can express EGF-SEE (SEQ ID NO.18), EGF-SEE-1 (SEQ ID NO.20), EGF-SEE-2 (SEQ ID NO.22) and EGF-SEE-3 (SEQ ID NO.24) fusion rotein.

Embodiment 5VEGF and SEE or SEE two mutants are formed the structure of Expression of Fusion Protein carrier

Information (NM_003376) and reference papers (J.Biol.Chem. according to the VEGF gene in the people of GenBank DB source; 266; 11947-11954; 1991), entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise VEGF gene (121 amino acid) and increase the several bases that limit restriction enzyme site BamHI and EcoRI again, comprised the restriction restriction enzyme site of SalI and HindIII in pulsating back in the VEGF front.This synthetic good nucleic acid fragment is inserted the T carrier and carries out the dna sequencing evaluation; And then after promptly handling with the double digestion method with BamHI and HindIII; Insert this fragment respectively the carrier of pET22b-SEE, pET22b-SEE-1, pET22b-SEE-2 and pET22b-SEE-3 among the embodiment 2; So just produced expression vector pET22b-VEGF-SEE, pET22b-VEGF-SEE-1, pET22b-VEGF-SEE-2 and pET22b-VEGF-SEE-3, but VEGF expression-SEE (SEQ ID NO.26), VEGF-SEE-1 (SEQ ID NO.28), VEGF-SEE-2 (SEQ ID NO.30) and VEGF-SEE-3 (SEQ ID NO.32) fusion rotein.

Embodiment 6GnRH and SEE or SEE two mutants are formed the structure of Expression of Fusion Protein carrier

Information (NM_021081 and NM_000825 etc.) according to the GnRH gene in the people of GenBank DB source; Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise the GnRH gene and increase the several bases that limit restriction enzyme site BamHI and EcoRI again, comprised the restriction restriction enzyme site of SalI and HindIII in pulsating back in the GnRH front.This synthetic good nucleic acid fragment is inserted the T carrier and carries out the dna sequencing evaluation; And then after promptly handling with the double digestion method with BamHI and HindIII; Insert this fragment respectively the carrier of pET22b-SEE, pET22b-SEE-1, pET22b-SEE-2 and pET22b-SEE-3 among the embodiment 2; So just produce expression vector pET22b-GnRH-SEE, pET22b-GnRH-SEE-1, pET22b-GnRH-SEE-2 and pET22b-GnRH-SEE-3, can express GnRH-SEE (SEQ ID NO.34), GnRH-SEE-1 (SEQ ID NO.36), GnRH-SEE-2 (SEQ ID NO.38) and GnRH-SEE-3 (SEQ ID NO.40) fusion rotein.

Embodiment 7GRP and SEE or SEE two mutants are formed the structure of Expression of Fusion Protein carrier

Information (NM_002091, NM_001012512 and NM_001012513 etc.) according to the GRP gene in the people of GenBank DB source; Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise the GnRH gene and increase the several bases that limit restriction enzyme site BamHI and EcoRI again, comprised the restriction restriction enzyme site of SalI and HindIII in pulsating back in the GnRH front.This synthetic good nucleic acid fragment is inserted the T carrier and carries out the dna sequencing evaluation; And then after promptly handling with the double digestion method with BamHI and HindIII; Insert this fragment respectively the carrier of pET22b-SEE, pET22b-SEE-1, pET22b-SEE-2 and pET22b-SEE-3 among the embodiment 2; So just produce expression vector pET22b-GRP-SEE, pET22b-GRP-SEE-1, pET22b-GRP-SEE-2 and pET22b-GRP-SEE-3, can express GRP-SEE (SEQ ID NO.42), GRP-SEE-1 (SEQ ID NO.44), GRP-SEE-2 (SEQ ID NO.46) and GRP-SEE-3 (SEQ ID NO.48) fusion rotein.

Embodiment 8 various Expression of Fusion Protein, sex change and renaturation and purifying

Change various expression plasmid pET22b-TGF-α-SEE, pET22b-TGF-α-SEE-1, pET22b-TGF-α-SEE-2, pET22b-TGF-α-SEE-3, pET22b-EGF-SEE, pET22b-EGF-SEE-1, pET22b-EGF-SEE-2, pET22b-EGF-SEE-3, pET22b-VEGF-SEE, pET22b-VEGF-SEE-1, pET22b-VEGF-SEE-2, pET22b-VEGF-SEE-3, pET22b-GnRH-SEE, pET22b-GnRH-SEE-1, pET22b-GnRH-SEE-2, pET22b-GnRH-SEE-3, pET22b-GRP-SEE, pET22b-GRP-SEE-1, pET22b-GRP-SEE-2 and pET22b-GRP-SEE-3 over to e. coli bl21 (DE3) with electroporation respectively, utilize microbiotic Amp (Ampicillin) screening positive bacteria.The process of next various proteic expression, sex change and renaturation and purifying is roughly the same, operates as follows:

The e. coli bl21 (DE3) that contains expression plasmid carries out extensive 37 ℃ of cultivations earlier, add then IPTG (Isopropylthio-β-D-galactoside) makes it concentration and reaches 1mM and spend the night 30 ℃ and cultivate, thus abduction delivering albumen.The 2nd day centrifugation medium with collect thalline, use the supersonic method breaking cell wall, centrifugal collection inclusion body precipitates, the albumen here is to exist with the inclusion body form.Inclusion body albumen is with the urea-denatured dissolving of 6M, carries out the multistage dialysis then, and dialysis solution is for example 3M, 2M and 1M of the urea that progressively dilutes; Next be 0.5M urea; 0.4M the L-l-arginine, 375 μ M Sleep-promoting factor B GSSG, 1.875mM reduced glutathion GSH; Carry out centrifugally after the dialysis, the gained supernatant is exactly proteic renaturation solution.Carry out purifying with HisBind Purification Kit test kit (Novagen company) for albumen; With Binding Buffer flushing gel column, also in protein solution, add Binding Buffer simultaneously, earlier with the protein sample upper prop; With Wash Buffer rinsing; Using Elute Buffer wash-out then, identify with protein electrophorese, is that the albumen of a band is used for later experiment with purity.Obtain various high purity fusion rotein TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, TGF-α-SEE-3, EGF-SEE, EGF-SEE-1, EGF-SEE-2, EGF-SEE-3, VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2, VEGF-SEE-3, GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 like this.

Embodiment 9 mouse tumors form experiment

At first select 450 of male ICR mouses, in 4-5 week, 18-22g, murine sarcoma cell S180 buy from ATCC, carry out vitro culture earlier, are expelled to the ICR mouse peritoneal again, carry out large scale culturing in the body, and be last, takes out Intraabdominal S180 cell, with 2x10 ⁶Murine sarcoma cell S180 is inoculated in the oxter, right side of ICR mouse.

Embodiment 10TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 fusion rotein suppresses the mouse tumor experiment

Get 150 tumor-bearing mices among the embodiment 9; Be divided into 5 groups; Every group 30, injected TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 fusion rotein respectively in the 2nd, 4,6,8 day behind the inoculated tumour cell, dosage is 100pmol; A control group injecting normal saline killed mouse on the 9th day.The result shows that TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2 and TGF-α-SEE-3 have restraining effect to growth of tumor; Wherein, TGF-α-SEE-2 and TGF-α-SEE-3 have shown better effect; Tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 1) appears in TGF-α-SEE-3 medication group the 7th talent.

Embodiment 11EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 fusion rotein suppress the mouse tumor experiment

Get 150 tumor-bearing mices among the embodiment 9; Be divided into 5 groups; Every group 30, injected EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 fusion rotein respectively in the 2nd, 4,6,8 day behind the inoculated tumour cell, dosage is 100pmol; A control group injecting normal saline killed mouse on the 9th day.The result shows that EGF-SEE, EGF-SEE-1, EGF-SEE-2 and EGF-SEE-3 have restraining effect to growth of tumor; Wherein, EGF-SEE-2 and EGF-SEE-3 have shown better effect; Tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 2) appears in EGF-SEE-3 medication group the 7th talent.

Embodiment 12VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 fusion rotein suppress the mouse tumor experiment

Get 150 tumor-bearing mices among the embodiment 9; Be divided into 5 groups; Every group 30, the 2nd, 4,6,8 day difference injection of VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 fusion rotein behind the inoculated tumour cell, dosage is 100pmol; A control group injecting normal saline killed mouse on the 9th day.The result shows that VEGF-SEE, VEGF-SEE-1, VEGF-SEE-2 and VEGF-SEE-3 have restraining effect to growth of tumor; Wherein, VEGF-SEE-2 and VEGF-SEE-3 have shown better effect; Tumour occurs organizing late 1-2 days than other, and very little tumour (Fig. 3) appears in VEGF-SEE-3 medication group the 7th talent.

The lymphocytic detection of T in embodiment 13 tumor tissues

The S180 tumor tissues of the various fusion rotein administration of process among embodiment 10-12 mouse and the mouse S180 tumor tissues of control group saline water are cut into small pieces, process paraffin section, carry out immunohistochemical assay then with paraffin embedding.In order to detect the T cell in the tumor tissues, use the anti-cd 3 antibodies of Santa Cruz Biotechnolog company, the anti-and avidin-biotin-perxidase complex (Zymed company) with two uses Diaminobenzidine (DAB) colour developing at last then.(among the figure: 4-1 is TGF-α-SEE-3 administration group to Fig. 4; 4-2 is an EGF-SEE-3 administration group; 4-3 is a VEGF-SEE-3 administration group) experimental result of the fusion rotein just formed by SEE-3, utilize the detected T cell of immunohistochemical methods method, brown point is the T cell.

Through testing: also all found the existence of T cell in the mouse tumor tissue of the administration group of TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, EGF-SEE, EGF-SEE-1, EGF-SEE-2, VEGF-SEE, VEGF-SEE-1 and VEGF-SEE-2 fusion rotein.

The detection of embodiment 14 tumor tissues internal interference element-γ (IFN-γ)

Adopt with embodiment 13 similar immunohistochemical methods and detect the interior cytokine IFN-γ by T emiocytosis of tumor tissues among the embodiment 10-12, antibody is the anti-IFN-gamma antibodies of Santa Cruz Biotechnolog company.(among the figure: 5-1 is TGF-α-SEE-3 administration group to Fig. 5; 5-2 is an EGF-SEE-3 administration group; 5-3 is a VEGF-SEE-3 administration group) utilize the detected interferon-of immunohistochemical methods (IFN-γ) by T emiocytosis, brown part is IFN-γ.

Through testing: also all found existence in the mouse tumor tissue of the administration group of TGF-α-SEE, TGF-α-SEE-1, TGF-α-SEE-2, EGF-SEE, EGF-SEE-1, EGF-SEE-2, VEGF-SEE, VEGF-SEE-1 and VEGF-SEE-2 fusion rotein by the IFN-γ of induced t cell.

Embodiment 15 calf serums and fusion rotein hatch experiment

The calf serum dilution that the GnRH-SEE of employed high density, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein are all cultivated with cancer cells in the cancer cells culture experiment; Under 37 ℃, hatched 5 hours then, employed these fusion roteins all pass through the processing of hatching of calf serum in the experiment of all cancer cells later on.The experiment of hatching here is that whether the biological activity of fusion rotein changes under the situation for the existence of the basic antibody (IgM and IgG etc.) of checking opposing proteasome degradation and the anti-superantigen in thermally-stabilised etc. and the serum in calf serum.

The inducer T lymphocyte of embodiment 16GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein is active

The human peripheral blood cell obtains T lymphocyte (J Clin Invest, 91 available from the Tianjin Blood Center with Ficoll density centrifugation and nylon hair column method; 1490-1498; 1993), these T cells are used the DMEM culture medium culturing on 6 orifice plates, and add 10% calf serum.There is 5x10 in each hole ⁵Individual T cell; The GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and the GRP-SEE-3 fusion rotein that add the 5pmol amount then; These albumen all pass through hatching of calf serum, use the IFN-γ test kit (R&D Systems company) of enzyme-linked immunosorbent assay (ELISA) to detect the content of the IFN-γ in the substratum then.

Table 1 is for analyzing GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein and stimulate the ability of T cell, detection be cytokine IFN-γ.

That table 1 detects is cytokine IFN-γ (pg/ml)

The result shows the vigor that GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein all have stimulates the T cell, and wherein, GnRH-SEE-2 and GnRH-SEE-3 fusion rotein have shown the vigor of stronger stimulation T cell.

Table 2 is for analyzing GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein and stimulate the ability of T cell, detection be cytokine IFN-γ.

That table 2 detects is cytokine IFN-γ (pg/ml)

The result shows the vigor that GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein all have stimulates the T cell, and wherein, GRP-SEE-2 and GRP-SEE-3 fusion rotein have shown the vigor of stronger stimulation T cell.

Embodiment 17GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 fusion rotein kill and wound cancer cells

With the liver cancer cell Hep G2 and the lung cell A549 in DMEM culture medium culturing human cancer cell source, the cancer cells number is 5x10 on 6 orifice plates ⁵Individual cancer cells/hole adds the T cell then, is that 10 times of cancer cells are 5x10 ⁶Individual T cells/well; GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3, GRP-SEE, GRP-SEE-1, GRP-SEE-2 and the GRP-SEE-3 fusion rotein that calf serum is hatched that pass through that adds various dosage more respectively; Wherein, GnRH superantigen fusion rotein joins liver cancer cell Hep G2 culture hole, and GRP superantigen fusion rotein joins the lung cell A549 culture hole.

CDCC or cancer cells fragmentation effect adopt MTT (Methabenzthiazuron) method to measure (Immunology, 82,117-125,1994), and the cell growth-inhibiting is with 100-[(A _Test-A _b)/(A _c-A _b)] calculating of x100 formula, A _TestRefer to the growth of cancer cells under the adding of T cell, A _bRefer to and have only substratum, A in the hole _cRefer to growth of cancer cells.The number of times of every kind of cancer cells killing experiments is all more than 20 later on.

Fig. 6 is an experimental result, shows that GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GnRH-SEE-3 have the ability of killing hepatoma cell Hep G2, and wherein GnRH-SEE-2 and GnRH-SEE-3 just have higher cancer cells lethal effect under very low dosage.

Fig. 7 is an experimental result, shows that GRP-SEE, GRP-SEE-1, GRP-SEE-2 and GRP-SEE-3 have the ability of killing and wounding lung cell A549, and wherein GRP-SEE-2 and GRP-SEE-3 just have higher cancer cells lethal effect under very low dosage.

(among the figure: 8-1 is the GnRH-SEE-3 group to Fig. 8; 8-2 is a GRP-SEE-3 group) cancer cells of being attacked by the T cell that shows, maxicell is a cancer cells, minicell is the T cell.

In experiment: GnRH-SEE, GnRH-SEE-1, GnRH-SEE-2, GRP-SEE, GRP-SEE-1 and GRP-SEE-2 group, also observed the phenomenon of the cancer cells of T cell attack.

The present invention has selected a kind of superantigen SEE and two mutants thereof, makes the SEE two mutants than SEE more powerful induced t cell ability arranged, and more can resist the antibody neutralizing effect in the blood.TGF-α-the SEE, EGF-SEE, VEGF-SEE, GnRH-SEE and the GRP-SEE that form by SEE; TGF-α-SEE-2, EGF-SEE-2, VEGF-SEE-2, GnRH-SEE-2 and GRP-SEE-2 and the TGF-α-SEE-3 that is made up of SEE-3, EGF-SEE-3, VEGF-SEE-3, GnRH-SEE-3 and the GRP-SEE-3 fusion rotein be made up of SEE-2 all have antitumous effect; Wherein, TGF-α-SEE-2, EGF-SEE-2, VEGF-SEE-2, GnRH-SEE-2, GRP-SEE-2TGF-α-SEE-3, EGF-SEE-3, VEGF-SEE-3, GnRH-SEE-3 and GRP-SEE-3 fusion rotein have shown stronger cancer cells and the anti-tumor bioactivity of killing and wounding.

Two mutants SEE-1, SEE-2 and the SEE-3 of the superantigen SEE here is selected from SEE and introduced extensive a plurality of point mutation; Because SEA and SEE are at the homogeny that has on the aminoacid sequence more than 80%, so can also can obtain the two mutants of SEE from SEA through point mutation.Through the molecular biology working method of range gene point mutation, on existing SEE two mutants, introduce point mutation and obtain the higher superantigen of biological activity in addition.

Determined point mutation position from embodiment 1, the amino acid that on these positions, can introduce other similar physico-chemical property also can obtain the biological activity identical or higher with the SEE two mutants.

From bigger scope; Other superantigen is the SEB, SEC, SED, SEF, SEG, SEH, SEI, SEJ etc. of Staphylococcus aureus enterotoxin family for example; The SPE-A of streptococcus toxin, SPE-B, SPE-C; Shock syndrome toxin (Shock syndrome toxin), the superantigen in viral protein source can be strengthened the T cytositimulation ability of superantigen, the stability of superantigen and the neutralizing effect of resisting the superantigen of antibody in the blood through the multipoint mutation means.

Can select and the interactional peptide molecule of cancer cells surface receptor as another part of SEE and mutant fusion protein thereof; Such polypeptide has Prostatropin (Basic fibroblast growth factor; BFGF) and FGF family, interleukin-family (Interleukin) for example interleukin II, interleukin 3, interleukin 4, interleukin-6, interleukin 8, interleukin 11, IL-13, rHuGM-CSF (Granulocyte-macrophage colony-stimulating factor; GM-CSF), heparin combines EGF like growth factor (Heparin-binding EGF-like growth factor; HB-EGF), rhIGF-1 (Insulin-like growth factor; IGF), pHGF (Hepatocyte growth factor; HGF), Thr6 PDGF BB (Platelet-derived growth factor; PDGF), NGFF (Nerve growth factor; NGF), PlGF (Placental growth factor; PGF), STEMCELLFACTOR (Stem cell factor; SCF), Heregulin, erbB part, various chemokine (Chemokine) and family, Ephrin family, angiogenin (Angiopoietin), TSF (Thrombopoietin) and the blood plasma VII factor (Factor VII), urokinase type plasminogen activator (Urokinase-type plasminogen activator), growth hormone releasing hormone (Growth hormone releasing hormone), melanotropin α-MSH, prolactin antagonist (Prolactin), prolactin releasing hormone (PRH) (Prolactin releasing hormone), tethelin (Growth hormone), follicle stimulating hormone (Follicle stimulating hormone), placental lactogen hormone (Placental lactogen), chorionic-gonadotropin hormone (Chorionic gonadotropin) and corticotropin releasing hormone (Corticotropin releasing hormone), Somatostatin (Somatostatin), asialoglycoprotein gp (Asialoglycoprotein), low-density lipoprotein (Low density lipoprotein) and Transferrins,iron complexes (Transferrin) etc., and these proteic aminoacid sequences have the nature of the homogeny more than 70% and artificial varient etc.Many tumor tissues are these material acceptors of overexpression all; Thereby peptide molecule parts such as chemokine, enzyme, hormone and other albumen just can be connected to form fused protein with costimulatory molecules as cytokine, costimulatory molecules is navigated in the tumor tissues.

Top fused protein can also be connected polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor etc. respectively through means such as chemical crosslink reactions with the protein fragments of superantigen, for example covalent linkage connects and makes up.Can carry out a part of polypeptide fragment of chemically modified, 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 purifying can improve the space structure that it comprises disulfide linkage through a series of proteinic sex change and renaturation process, improves its biological activity.

From bigger scope; The acceptor of over-expresses is actually interactional relation between a kind of part and the acceptor on the polypeptide of cytokine, hormone or the non-hormone of the non-antibody acellular factor that is adopted in the experiment here and its corresponding cancer cells surface; Utilize the avidity of this part and acceptor, superantigen is navigated to tumor tissues.Except cytokine, hormone or polypeptide, other is every to be the specific localization that part also can be used for cancer cells with corresponding protein of the acceptor of cancer cells over-expresses or peptide molecule.The corresponding part of acceptor on top said and cancer cells, screen from phage display methods such as (Phage display) with cancer cells on acceptor avidity is arranged and have antagonistic action artificial screening protein or polypeptide and with other method screen can be directly and the protein of cancer cells surface interaction or peptide molecule can form fused protein with costimulatory molecules.

As its formulation of medicine can be emulsifying agent, liposome, dispersion agent, stablizer etc. process 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.

In the specific embodiment of the present invention; TGF-α-SEE, EGF-SEE and VEGF-SEE that said cytokine-superantigen fusion rotein is made up of SEE; TGF-α-the SEE-1, EGF-SEE-1 and the VEGF-SEE-1 that form by the two mutants SEE-1 of SEE; TGF-α-the SEE-2, EGF-SEE-2 and the VEGF-SEE-2 that form by the two mutants SEE-2 of SEE, and the TGF-α-SEE-3, EGF-SEE-3 and the VEGF-SEE-3 that form by the two mutants SEE-3 of SEE; The GnRH-SEE that said hormone-the superantigen fusion rotein is made up of SEE, the GnRH-SEE-1 that forms by the two mutants SEE-1 of SEE, the GnRH-SEE-2 that forms by the two mutants SEE-2 of SEE, and the GnRH-SEE-3 that forms by the two mutants SEE-3 of SEE; The GRP-SEE that polypeptide-the superantigen fusion rotein is made up of SEE of the non-hormone of the said non-antibody acellular factor; The GRP-SEE-1 that forms by the two mutants SEE-1 of SEE; The GRP-SEE-2 that forms by the two mutants SEE-2 of SEE, and the GRP-SEE-3 that forms by the two mutants SEE-3 of SEE.These fusion roteins can induce and stimulate the T lymphocyte to come anti-respectively sarcoma S180, Hep G2 liver cancer and A549 lung cancer; But; As long as the cancer cells (cell in various internal organs, the blood, bone, brain, intestines and skin etc.) of other kind is gone up the acceptor of expressing TGF-α, EGF, VEGF, GnRH and GRP; Can adopt the fusion rotein here to come killing tumor cells, thereby be applied to the treatment of various cancers or malignant tumour.

The description of above embodiment and preferred implementation belongs to the schematic illustration of the present invention that claim is limited, rather than restriction the present invention.What need particularly point out is, only otherwise break away from aim of the present invention, all conspicuous changes and the similar invention with equivalent substitution all are included within protection scope of the present invention.

Claims

1. cancer-targeted super antigen fusion rotein is characterized in that this fusion rotein contains:

B) can cause superantigen staphylococcus aureus enterotoxin E and the two mutants thereof that antitumor immune reacts, said staphylococcus aureus enterotoxin E is represented with abbreviation SEE; The two mutants of said staphylococcus aureus enterotoxin E is represented said X=1-3 with abbreviation SEE-X.

2. a kind of cancer-targeted super antigen fusion rotein according to claim 1 is characterized in that said cytokine is selected from transforminggrowthfactor-, the Urogastron of being abbreviated as EGF of being abbreviated as TGF-α, the vascular endothelial growth factor of being abbreviated as VEGF; Said hormone is selected from the gonadotropin releasing hormone of being abbreviated as GnRH; The polypeptide of the non-hormone of the said non-antibody acellular factor is selected from the gastrin releasing peptide of being abbreviated as GRP.

3. a kind of cancer-targeted super antigen fusion rotein according to claim 1 is characterized in that said SEE is the aminoacid sequence shown in the SEQ ID No.2; Said SEE-1 is the aminoacid sequence shown in the SEQ ID No.4; Said SEE-2 is the aminoacid sequence shown in the SEQ ID No.6; Said SEE-3 is the aminoacid sequence shown in the SEQ ID No.8.

4. a kind of cancer-targeted super antigen fusion rotein according to claim 3 is characterized in that said SEE-1 is that one or more amino acid on 20,21,24,27,34,39,40,1,42,44,49,74,75,78,79,81,83,84,217,220,222,223,225,227 on the SEE are replaced.

5. a kind of cancer-targeted super antigen fusion rotein according to claim 3 is characterized in that said SEE-2 is that one or more amino acid on 20,21,24,27,34,35,39,40,41,42,44,45,46,49,74,75,78,79,81,83,84,188,190,217,219,220,222,223,224,225,227 on the SEE are replaced.

6. a kind of cancer-targeted super antigen fusion rotein according to claim 3 is characterized in that said SEE-3 is that one or more amino acid on 20,21,24,27,34,35,36,39,40,41,42,44,45,46,49,62,74,75,77,78,79,81,83,84,188,190,217,218,219,220,221,222,223,224,225,227 on the SEE are replaced.

7. a kind of cancer-targeted super antigen fusion rotein according to claim 1 is characterized in that said this fusion rotein is:

By the TGF-α-SEE shown in the SEQ ID No.10;

By the EGF-SEE shown in the SEQ ID No.18;

By the VEGF-SEE shown in the SEQ ID No.26;

By the GnRH-SEE shown in the SEQ ID No.34;

By the GRP-SEE shown in the SEQ ID No.42;

By the TGF-α-SEE-1 shown in the SEQ ID No.12;

By the EGF-SEE-1 shown in the SEQ ID No.20;

By the VEGF-SEE-1 shown in the SEQ ID No.28;

By the GnRH-SEE-1 shown in the SEQ ID No.36;

By the GRP-SEE-1 shown in the SEQ ID No.44;

By the TGF-α-SEE-2 shown in the SEQ ID No.14;

By the EGF-SEE-2 shown in the SEQ ID No.22;

By the VEGF-SEE-2 shown in the SEQ ID No.30;

By the GnRH-SEE-2 shown in the SEQ ID No.38;

By the GRP-SEE-2 shown in the SEQ ID No.46;

By the TGF-α-SEE-3 shown in the SEQ ID No.16;

By the EGF-SEE-3 shown in the SEQ ID No.24;

By the VEGF-SEE-3 shown in the SEQ ID No.32;

By the GnRH-SEE-3 shown in the SEQ ID No.40;

Or by the GRP-SEE-3 shown in the SEQ ID No.48.

8. recombinant vectors is characterized in that containing the nucleotide sequence of the said fusion rotein of coding claim 7, and said nucleotides sequence is classified the nucleotide sequence SEQ ID No.9 of coding SEQ ID No.10 as;

The nucleotide sequence SEQ ID No.11 of coding SEQ ID No.12;

The nucleotide sequence SEQ ID No.13 of coding SEQ ID No.14;

The nucleotide sequence SEQ ID No.15 of coding SEQ ID No.16;

The nucleotide sequence SEQ ID No.17 of coding SEQ ID No.18;

The nucleotide sequence SEQ ID No.19 of coding SEQ ID No.20;

The nucleotide sequence SEQ ID No.21 of coding SEQ ID No.22;

The nucleotide sequence SEQ ID No.23 of coding SEQ ID No.24;

The nucleotide sequence SEQ ID No.25 of coding SEQ ID No.26;

The nucleotide sequence SEQ ID No.27 of coding SEQ ID No.28;

The nucleotide sequence SEQ ID No.29 of coding SEQ ID No.30;

The nucleotide sequence SEQ ID No.31 of coding SEQ ID No.32;

The nucleotide sequence SEQ ID No.33 of coding SEQ ID No.34;

The nucleotide sequence SEQ ID No.35 of coding SEQ ID No.36;

The nucleotide sequence SEQ ID No.37 of coding SEQ ID No.38;

The nucleotide sequence SEQ ID No.39 of coding SEQ ID No.40;

The nucleotide sequence SEQ ID No.41 of coding SEQ ID No.42;

The nucleotide sequence SEQ ID No.43 of coding SEQ ID No.44;

The nucleotide sequence SEQ ID No.45 of coding SEQ ID No.46;

Or shown in the nucleotide sequence SEQ ID No.47 of coding SEQ ID No.48.

9. the preparation method of the said a kind of cancer-targeted super antigen fusion rotein of claim 1 is characterized in that cultivating the host cell that contains the said recombinant vectors of claim 8, collects and expresses the described fusion rotein of claim 1.

10. the described fusion rotein of claim 1 is in the application of the medicine of preparation treatment cancer and anti entity tumour.