CN102382193A

CN102382193A - Fusion protein capable of inducing and activating cancer-targeted T cells, preparation method and use thereof

Info

Publication number: CN102382193A
Application number: CN2011103387008A
Authority: CN
Inventors: 孙嘉琳
Original assignee: 孙嘉琳
Current assignee: Nanjing double bond targeted drug Technology Co., Ltd
Priority date: 2011-11-01
Filing date: 2011-11-01
Publication date: 2012-03-21
Anticipated expiration: 2031-11-01
Also published as: CN102382193B

Abstract

The present invention discloses a fusion protein capable of inducing and activating cancer-targeted T cells, a preparation method and use thereof. The protein comprises peptides which function on the cancer cells and costimulatory molecules B7.2. The peptides which function on the cancer cells are selected from transforming growth factor-alpha, epidernal growth factor, vascular endothelial growth factor, gonadotropin-releasing hormone or gastrin-releasing peptide. The fusion protein of the invention has a cancer targeting function. On one hand, the fusion protein can respectively function with the VEGFR, EGFR, GnRH-R or GRP-R; and on the other hand, the fusion protein interacts with corresponding receptors CD28 and CTLA-4 which are expressed on the T cell. Thus, the T cell is targetedly positioned to the periphery of cancer cells which express VEGFR, EGFR, GnRH-R or GRP-R in great numbers. Experiments prove that the fusion protein of the invention can restrain tumor growth and causes cancer cell apoptosis.

Description

Can induce and activate the fusion rotein and the Preparation method and use of cancer target T cell

Technical field

The present invention relates to a kind of fusion rotein and Preparation method and use, belong to biomedicine field.

Background technology

Urogastron (Epidermal growth factor; EGF), vascular endothelial growth factor (Vascularendothelial cell growth factor; VEGF), gonadotropin releasing hormone (Gonadotropin-releasinghormone, GnRH) and gastrin releasing peptide (gastrin-releasing peptide, the acceptor great expression in various tumor tissues that GRP) waits; For example the EGF acceptor in the intestinal mucosa tumour exceeds 300 times of (Gastroenterology than healthy tissues; 98,961-967,1990).So the EGF acceptor of unusual high expression level, vegf receptor, GnRH acceptor, GRP acceptor etc. become noticeable cancer treatment target spot.The acceptor of transforminggrowthfactor-(Transforming growth factor-α, TGF-α) is identical with the acceptor of EGF.

Above these cytokines and hormone and polypeptide etc. can interact with the corresponding acceptor (Receptor) on the cancer cells.So connect for example toxin protein and form fusion rotein of an effector in their back, just can target sexual assault cancer cells (J Biol Chem, 267,24034-24040,1992; Proc Natl Acad Sci USA, 99,7866-7871,2002; Cancer Res, 54,5154-5159,1994; J Biol Chem, 272,11597-11603,1997; Cancer Res, 57,290-294,1997).

The effective activation of T cell needs the participation of two signals, promptly needs antigen peptide-MHC mixture to combine to provide the 1st signal with TCR-CD3 on the T cell, and the 2nd signal that mediates of costimulatory molecules (Costimulatory molecules) B7.The corresponding acceptor (CD28 and CTLA-4) of expressing on B7.1 (CD80), B7.2 (CD86) molecule and the T cell of the most basic costimulatory signal by the antigen presenting cell expression provides (Amj Respir Crit Care Med, 162,5164-5168,2000; Immunol CellBiol, 77,304-311,1999; Clin Cancer Res, 13,5271-5279,2007; Trends Immunol, 24,313-318,2003).

B7.1 is expressed on activatory DC, B cell and the monocyte, and B7.2 is low expression level on B cell, T cell, DC and scavenger cell, but B7.2 can be induced quick rise, and B7.1 is induced up-regulated expression time ratio B7.2 evening.CD28 on the T cells combines with B7.1/B7.2 on the APC, can promote that IL-2 transcribes, and makes the IL-2 acceptor on the T cell, express increase simultaneously, thereby promotes T cell proliferation, can also make the T cell avoid apoptosis through the expression approach that increases Bcl-xl.

As the application of medicine, B7.1-Fc and B7.2-Fc can induce and activate anticancer T cell, demonstrate anti-tumor bioactivity (Cancer Res, 59,4964-4972,1999; Clin Cancer Res, 11,8492-8502,2005; JImmunol, 172,1347-1354,2004; Cancer Res, 62,5727-5735,2002).

In anticancer target medicine, antibody is popular means very, but antibody just seals cancer cells, and is irrelevant with the T lymphocyte.Tumor tissues is rich in aorta and capillary vessel, and these blood vessels are not only carried nutrition to tumour, and a large amount of lymphocytes is arranged in the blood vessel, and wherein 70-80% is the T lymphocyte, but these T cells are not have the cancer cells specificity, are can not attack tumour.Utilizing these not have the specific T cell of cancer cells to come target sexual assault cancer cells, is the new strong means in a kind of anticancer field, and such medicine is different from antibody, does not report now.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, a kind of fusion rotein of inducing and activating cancer target T cell is provided.

Second purpose of the present invention provides a kind of recombinant vectors that contains the nucleotide sequence of the encoding fusion protein that can induce and activate cancer target T cell.

The 3rd purpose of the present invention provides a kind of preparation method who induces and activate the fusion rotein of cancer target T cell.

The 4th purpose of the present invention provides a kind of purposes of inducing and activating cancer target T cell.

Technical scheme of the present invention is summarized as follows:

A kind of fusion rotein of inducing and activating cancer target T cell; Comprise peptide and costimulatory molecules B7.2 (having another name called CD86) with the cancer cells effect; Said and the peptide cancer cells effect be selected from transforminggrowthfactor-, the Urogastron of being abbreviated as EGF, the vascular endothelial growth factor of being abbreviated as VEGF of being abbreviated as TGF-α, be abbreviated as the gonadotropin releasing hormone of GnRH or be abbreviated as the gastrin releasing peptide of GRP, and TGF-α-B7.2 is by shown in the SEQ ID No4; EGF-B7.2 is by shown in the SEQ ID No6; VEGF-B7.2 is by shown in the SEQ ID No8; GnRH-B7.2 is by shown in the SEQ ID No10; GRP-B7.2 is by shown in the SEQ IDNo12.

A kind of recombinant vectors contains the nucleotide sequence of said fusion rotein above the coding, and the nucleotides sequence of coding SEQ ID No4 is classified as shown in the SEQ ID No3; The nucleotides sequence of coding SEQ ID No6 is classified as shown in the SEQ ID No5; The nucleotides sequence of coding SEQ ID No8 is classified as shown in the SEQ ID No7; The nucleotides sequence of coding SEQ ID No10 is classified as shown in the SEQ ID No9; The nucleotides sequence of coding SEQ ID No12 is classified as shown in the SEQ ID No11.

A kind of varient or artificial mutant nucleotide sequence of transforming of inducing and activating the fusion rotein of cancer target T cell.

A kind of host cell, this host cell contains recombinant vectors recited above.

Can induce and activate the preparation method of the fusion rotein of cancer target T cell, said host cell above the cultivation is collected the corresponding fusion proteins TGF-α-B7.2 that expresses; EGF-B7.2; VEGF-B7.2; GnRH-B7.2; GRP-B7.2.

Above said fusion rotein and the related to cancer of inducing and activate cancer target T cell.

Above the said fusion rotein of inducing and activate cancer target T cell can induce and activated T cell.

Above the said fusion rotein of inducing and activate cancer target T cell in the application of the medicine of preparation treatment cancer and anti entity tumour.

Advantage of the present invention:

Fusion rotein of the present invention has the cancer targeting; On the one hand can be respectively and Receptor EGFR, the acceptor GnRH-R of GnRH or the acceptor GRP-R effect of GRP of acceptor VEGFR, EGF and the TGF-α of VEGF; On the other hand again with the T cell on the corresponding acceptor CD28 that expresses and CTLA-4 interact; So just with around the cell targeted cancer cells that navigates to great expression VEGFR, EGFR, GnRH-R or GRP-R of T, thereby inducing T cell is attacked cancer cells.But T cell excretory cytokine is interferon-(Interferon-γ for example; IFN-γ), pore-forming protein and granzyme and induce target cell to produce Fas etc. to cause cancer cell-apoptosis, so top fusion rotein has lethal effect clearly for noumenal tumour.

Experiment showed, that fusion rotein of the present invention can suppress tumor growth and the apoptosis that causes cancer cells.

Description of drawings

Fig. 1 representes that three kinds of fusion roteins of the present invention suppress sarcoma tumour Sarcoma (S180) growth, and what represent among the figure is the tumor weight of mouse.

Fig. 2 utilizes the detected T cell of routine immunization groupization, and brown point is the T cell.(among the figure: 2-1 is contrast; 2-2 is B7.2; 2-3 is TGF-α-B7.2; 2-4 is EGF-B7.2; 2-5 is VEGF-B7.2)

Fig. 3 utilizes the binding ability of 3 kinds of fusion roteins of the present invention of routine immunization group inspection and tumour.(among the figure: 3-1 is TGF-α-B7.2; 3-2 is EGF-B7.2; 3-3 is VEGF-B7.2)

Fig. 4 utilizes routine immunization group detection interferon-(IFN-γ), and brown part is exactly the IFN-γ of T emiocytosis.(among the figure: 4-1 is contrast; 4-2 is B7.2; 4-3 is TGF-α-B7.2; 4-4 is EGF-B7.2; 4-5 is VEGF-B7.2)

The cancer cells of being attacked by the T cell that Fig. 5 shows, maxicell is a cancer cells, minicell is the T cell.(among the figure: the 5-1 liver cancer cell; The 5-2 lung carcinoma cell)

What Fig. 6 showed is the cancer cells of apoptosis, and maxicell is a cancer cells, and minicell is the T cell, and saturate maxicell is that cancer cells is just at apoptosis.(among the figure: the 6-1 liver cancer cell; The 6-2 lung carcinoma cell)

Embodiment

Express a large amount of EGFR and VEGFR (Clin Cancer Res 13,2998-3005,2007 on a kind of S180 cancer cells in the mouse tumor; PLoS One 5, e15368,2010), so adopt mouse tumor S180 to be used for the mouse-borne tumor model experiment here.The cytokine of people and mouse for example VEGF, EGF and TGF-α structurally has very big similarity, and cross reaction can take place, and (Biochemistry 40,8930-8939,2001; J Control Release, 82,71-82,2002; J Biol Chem 270,4334-4340,1995; J Biol Chem 276,19166-19171,2001; Dev Dyn 204,228-239,1995).Equally, the B7.1 (CD80) in people source and B7.2 (CD86) also can in mouse, play a role (C1inCancer Res, 11,8492-8502,2005).So here can be in the mouse-borne tumor model biological activity of analysis fusioning protein, through specific embodiment the present invention is further described below.

The structure of embodiment 1 B7.2 (CD86) expression vector

Information (NM_175862 and L25259) according to the B7.2 gene (having another name called CD86) in the people of GenBank DB source; Entrust the synthetic nucleic acid sequence fragments of TAKARA company to comprise a connection peptides and a B7.2 gene (222 amino acid of Codocyte outside part) and in several bases of whole pulsating front HindIII and back XhoI limiting enzyme point; 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 HindIII and XhoI; Insert this fragment on the pET22b plasmid, so just produced expression vector pET22b-B7.2, can express B7.2 albumen.Sequence table (SEQ ID NO.2) only is a B7.2 albumen, visible another sequence table (SEQ IDNO.14) of the connection peptides of front.

The structure of embodiment 2 TGF-α-B7.2 expression vector

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 on the pET22b-B7.2 plasmid; So just produce expression vector pET22b-TGF-α-B7.2, can express TGF-α-B7.2 fusion rotein (seeing sequence table SEQ ID NO.4).

The structure of embodiment 3 EGF-B7.2 expression vectors

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 on the pET22b-B7.2 plasmid; So just produce expression vector pET22b-EGF-B7.2, can express EGF-B7.2 fusion rotein (seeing sequence table SEQ ID NO.6).

The structure of embodiment 4 VEGF-B7.2 expression vectors

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 on the pET22b-B7.2 plasmid; So just produced expression vector pET22b-VEGF-B7.2, but VEGF expression-B7.2 fusion rotein (seeing sequence table SEQ ID NO.8).

The structure of embodiment 5 GnRH-B7.2 expression vectors

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 on the pET22b-B7.2 plasmid; So just produce expression vector pET22b-GnRH-B7.2, can express GnRH-B7.2 fusion rotein (seeing sequence table SEQ ID NO.10).

The structure of embodiment 6 GRP-B7.2 expression vectors

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 on the pET22b-B7.2 plasmid; So just produce expression vector pET22b-GRP-B7.2, can express GRP-B7.2 fusion rotein (seeing sequence table SEQ ID NO.12).

Embodiment 7 various proteic expression, sex change and renaturation and purifying

Change various expression plasmid pET22b-B7.2, pET22b-TGF-α-B7.2, pET22b-EGF-B7.2, pET22b-VEGF-B7.2, pET22b-GnRH-B7.2 and pET22b-GRP-B7.2 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 His.Bind 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 6 kinds of high purity protein B7.2, TGF-α-B7.2, EGF-B7.2, VEGF-B7.2, GnRH-B7.2 and GRP-B7.2 like this, wherein B7.2 is used for control experiment.

Embodiment 8 suppresses tumor experiment

At first select male ICR mouse, in 4-5 week, 18-22g is divided into 5 groups, every group of 30 mouse.Murine sarcoma cell S180 buys from ATCC, carries out vitro culture earlier, is expelled to the ICR mouse peritoneal again, carries out large scale culturing in the body, and is last, takes out Intraabdominal S180 cell, with 2x10 ⁶Murine sarcoma cell S180 is inoculated in the oxter, right side of other five groups ICR mouse; Inject B7.2, TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 protein solution 0.2ml (250pmol)/only respectively toward intraperitoneal every other day then; And control group injection equivalent saline water (0.9%NaCl) was put to death mouse after 9 days.The weight of observing the mouse tumor growing state and putting to death the back tumour.Fig. 1 representes respectively to organize the weight of the tumour after mouse is dissected, and the result shows that TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 fusion rotein suppress tumor growth very effectively, and the effect of injection B7.2 is not so good.

The lymphocytic detection of T in embodiment 9 tumor tissues

TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 fusion rotein and B7.2 albumen are handled and the mouse S180 tumor tissues of control group saline water is cut into small pieces, and process paraffin section with paraffin embedding, carry out immunohistochemical assay then.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.In the mouse tumor tissue of TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 injection, find that a large amount of T cells is arranged (the brown point that Fig. 2 shows is the T cell); This explanation is induced down at TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2, has been full of wellability (Infiltrating) T cell in the tumor tissues.And have only a spot of T cell in the tumour of B7.2 processing group, and the control group that saline water is handled does not almost have any T cell.The result here show that fusion rotein can target property with the organization internal of T cell enrichment to noumenal tumour.

10 3 kinds of fusion roteins of embodiment are for the inspection of the binding ability of cancer cells

Owing to the label of 6 Histidines is arranged, on the pET22b carrier so the C-terminal of fusion rotein of the present invention all has this label, so available anti-6 Histidine antibody (Santa Cruz Biotechnolog company) detect the existence of fusion rotein.Hatch the tumor tissues paraffin section with TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 fusion rotein solution, detect with anti-6 Histidine antibody then.Find that TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 fusion rotein can combine (the brown part among Fig. 3) with the S180 cancer cells, show that TGF-α, EGF and the VEGF in the people source in the fusion rotein can successively act on EGFR and the VEGFR on the mouse cancer cells.

The detection of embodiment 11 tumor tissues internal interference element-γ (IFN-γ)

Detect in the tumor tissues by the cytokine IFN-γ of T emiocytosis with immunohistochemical method, antibody is the anti-IFN-gamma antibodies of SantaCruz Biotechnolog company.Fig. 4 is the result who measures; Being illustrated in TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2 induces down; T emiocytosis in the tumor tissues a large amount of IFN-γ (among Fig. 4: the brown part among 4-3,4-4 and the 4-5); And the B7.2 group has only positive reaction (Fig. 4-2) seldom, saline water group (Fig. 4-1) not to have what IFN-γ secretion.

Embodiment 12 GnRH-B7.2 and GRP-B7.2 inducer T lymphocyte are active

PBC is obtained T lymphocyte (JClin Invest, 91,1490-1498,1993) available from the Tianjin Blood Center with Ficoll density centrifugation and nylon hair column method, and these T cells are used the DMEM culture medium culturing.Add B7.2, GnRH-B7.2 and GRP-B7.2 then respectively.

On 6 orifice plates with DMEM culture medium culturing T cell; 3 kinds of albumen that add 5pmol amount then 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 B7.2, GnRH-B7.2 and GRP-B7.2 albumen and stimulate the ability of T cell, detection be cytokine IFN-γ.

Table 1 IFN-γ (pg/ml)

The result shows that GnRH-B7.2 and GRP-B7.2 fusion rotein are the same with B7.2 can activated T cell, makes T emiocytosis cytokine IFN-γ.

Simultaneously, in not adding proteic control group (having only cancer cells and T cell), then do not detect IFN-γ.

Embodiment 13 GnRH-B7.2 and GRP-B7.2 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, concentration is 2x10 on 6 orifice plates ⁵Individual/hole, add the T cell then, add B7.2, GnRH-B7.2 or GRP-B7.2 more respectively, proteic amount all is 5pmol.

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.

Imitate the target ratio and be meant the ratio of T cell and tumour cell quantity; Under 5: 1,15: 1 situation, add B7.2, GnRH-B7.2 or the GRP-B7.2 of 5pmol amount respectively, the result table 2 (below); Increase along with the T cell quantity; The tumor cytotoxicity effect also increases, and under T cell and 15: 1 condition of tumour cell, after cultivating in 70 hours, measures; GnRH-B7.2 and GRP-B7.2 kill rate or CDCC reach 60 (%) above (table 2), and the effect of B7.2 is lower than GnRH-B7.2 and GRP-B7.2.Do not adding under the situation that albumen only adds the T cell in addition, CDCC is found below 1-4 (%).

Table 2 is the T cell and the common cultivation of the ratio of cancer cells different quantities, induces down at B7.2, GnRH-B7.2 or GRP-B7.2, and the T cell is a lethal effect to the CDCC of tumour cell.

Fig. 5 and Fig. 6 are that the microscopic examination cancer cells is by the effect of killing and wounding through GnRH-B7.2 or GRP-B7.2 inductive T cell.

The cancer cells of being attacked by the T cell that Fig. 5 shows is (among the figure: the 5-1 liver cancer cell; The 5-2 lung carcinoma cell), big cell is a cancer cells, and minicell on every side is the T cell, shows that the T cell attacking cancer cells.

Fig. 6 shows is that the cancer cells of apoptosis is (among the figure: the 6-1 liver cancer cell; The 6-2 lung carcinoma cell), big cell is a cancer cells, and minicell on every side is the T cell, and these T cells are being attacked cancer cells, and saturate maxicell is that cancer cells is just at apoptosis.

The experiment here shows that GnRH-B7.2 and GRP-B7.2 can distinguish killing hepatoma cell Hep G2 and lung cell A549, and lethal effect is more much bigger than B7.2.

The present invention has selected a brand-new strategy; The costimulatory molecules B7.2 (CD86) of T cell (for example: gastrin releasing peptide), produced novel cytokine-costimulatory molecules fused protein, hormone-costimulatory molecules fused protein and polypeptide-costimulatory molecules fused protein like this is connected to cytokine, hormone and polypeptide.

The cytokine here, hormone and polypeptide can adopt indivedual amino acid whose point mutation and not influence biological activity; For example first amino acid of GnRH is that the amino acid on the gene order (NM_021081 and NM_000825) can be Gln (FEBS Lett; 472,241-246,2000; Cancer Res, 64,2090-2095,2004), be all Gln (seeing sequence table SEQ ID NO.10) with first amino acid of GnRH in the GnRH-B7.2 fusion rotein here; Also can be Glu (J Biol Chem, 272,11597-11603,1997; Cancer Res, 60,3701-3705,2000).

As a model; Though only select costimulatory molecules B7.2 (CD86) at this; Costimulatory molecules can also be selected from other B7 family member for example B7.1 (CD80), B7-H1 (PD-L1), B7-H2 (B7RP-1 or ICOS-L or B7h or GL-50), B7-H3 (B7RP-2), B7-H4 (B7x or B7S1), B7-DC (PD-L2) etc.; And these proteic aminoacid sequences have the nature of the homogeny more than 70% and artificial varient etc., also thought of the present invention can be described.The effect of costimulatory molecules B7.2 (CD86) or other costimulatory molecules is the intravital immunoreation of exciter.Further, except the B7 family member, other the molecule that can stimulate the T cell can be as an integral part in the fusion rotein among the present invention program.

Equally; As TGF-α, EGF, VEGF, GnRH and the GRP in TGF-α-B7.2, EGF-B7.2, VEGF-B7.2, GnRH-B7.2 and the GRP-B7.2 fusion rotein of experiment material; Just utilize the positioning action of their tumour cells; If adopt other cytokine, hormone, enzyme or the polypeptide etc. that are closely related with tumour cell that thought of the present invention also can be described, because a large amount of acceptors is arranged with the corresponding cancer cells of these cytokines, hormone, enzyme or polypeptide surface.

Material has Prostatropin (Basic fibroblast growth factor like this; 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-likegrowth 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 (Growthhormone releasing hormone), melanotropin α-MSH, prolactin antagonist (Prolactin), prolactin releasing hormone (PRH) (Prolactin releasing hormone), tethelin (Growth hormone), follicle stimulating hormone (Folliclestimulating hormone), placental lactogen hormone (Placental lactogen), chorionic-gonadotropin hormone (Chorionicgonadotropin) and corticotropin releasing hormone (Corticotropin releasing hormone), Somatostatin (Somatostatin), asialoglycoprotein gp (Asialoglycoprotein), low-density lipoprotein (Low densitylipoprotein) 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.

These fused proteins can also be connected cytokine, hormone or polypeptide etc. respectively through means such as chemical crosslink reactions with the protein fragments of costimulatory molecules, 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.

For structure and the function that the fusion rotein among the present invention is described better; It here is example with EGF-B7.2; Connection peptides in the fusion rotein can adopt all lengths and component, though connection peptides has various variations, its effect is the same with described in the embodiment here; Be that activation with the T cell of the cancer cells targeting of EGF and B7.2 combines, promptly reach simultaneously with ground (in a place) to play a role.Even can also make up the Fc segment that comprises antibody, and EGF-B7.2-Fc for example, thus the dimer of fusion rotein formed, this is actually bimolecular fusion rotein, and its essence is to be the same with scheme described in the embodiment here.

From bigger scope; Here the acceptor of over-expresses is actually interactional relation between a kind of part (Ligand) and the acceptor on cytokine, hormone or the polypeptide that is adopted in the experiment and its corresponding cancer cells surface; Utilize the avidity of this part and acceptor, costimulatory molecules 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; Said cytokine-costimulatory molecules fusion rotein is TGF-α-B7.2, EGF-B7.2 and VEGF-B7.2; Hormone-costimulatory molecules fusion rotein is GnRH-B7.2; Polypeptide-costimulatory molecules fusion rotein is GRP-B7.2; They can induce and stimulate the T lymphocyte to come anti-respectively sarcoma S180, Hep G2 liver cancer and A549 lung cancer, still, 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. the fusion rotein that can induce and activate cancer target T cell; It is characterized in that comprising peptide and costimulatory molecules B7.2 with the cancer cells effect; Said and the peptide cancer cells effect be selected from transforminggrowthfactor-, the Urogastron of being abbreviated as EGF, the vascular endothelial growth factor of being abbreviated as VEGF of being abbreviated as TGF-α, be abbreviated as the gonadotropin releasing hormone of GnRH or be abbreviated as the gastrin releasing peptide of GRP, and TGF-α-B7.2 is by shown in the SEQ ID No4; EGF-B7.2 is by shown in the SEQ ID No6; VEGF-B7.2 is by shown in the SEQ ID No8; GnRH-B7.2 is by shown in the SEQ ID No10; GRP-B7.2 is by shown in the SEQ ID No12.

2. recombinant vectors is characterized in that containing the nucleotide sequence of the said fusion rotein of coding claim 1, and the nucleotides sequence of coding SEQ IDNo4 is classified as shown in the SEQ ID No3; The nucleotides sequence of coding SEQ ID No6 is classified as shown in the SEQ ID No5; The nucleotides sequence of coding SEQ ID No8 is classified as shown in the SEQ ID No7; The nucleotides sequence of coding SEQ ID No10 is classified as shown in the SEQ IDNo9; The nucleotides sequence of coding SEQ ID No12 is classified as shown in the SEQ ID No11.

3. the varient of the said a kind of fusion rotein of inducing and activate cancer target T cell of claim 1 or the artificial mutant nucleotide sequence of transforming.

4. a host cell is characterized in that this host cell contains the described recombinant vectors of claim 2.

5. the said preparation method who induces and activate the fusion rotein of cancer target T cell of claim 1 is characterized in that cultivating the described host cell of claim 4, collects the described fusion rotein of claim 1 of expressing.

6. described fusion rotein of claim 1 and related to cancer.

7. the described fusion rotein of claim 1 can be induced and activated T cell.

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