CN1966529A - Recombinant toxin agent containing gonadorelin and pyocyanin functional frament - Google Patents

Abstract

This invention relates to a gonadotropin-releasing hormone (GnRH) short peptide hormone as the guided agent, and chimeric toxin with reorganized pseudomonas exotoxin A functional fragment as the cytotoxic agent, as well as their preparation method and treatment in gonadal reactive or non-reactive tumor, wherein the guidance part of the chimeric toxin is based on GnRH.

Description

The reorganization toxic agents that contains gonadoliberin and pyocyanin function fragment

The present invention generally relates to the fused protein with target cell SC.More particularly, the present invention relates to the structure of the reorganization ETA function fragment (rPE38) that merges with gonadotropin releasing hormone, and said reconstitution cell toxicity fusion rotein is as antineoplastic agent, in the generation and the developmental application of the tumour that suppresses the steroid hormone stimulation.

Traditional tumor therapeuticing method is to use the intravital tumour cell of chemicals direct killing.Yet most of antitumor drugs also kill and wound normal cell in killing tumor cells.In order to improve the selectivity of antitumor drug to tumour cell, since the later stage seventies 20th century, people just attempt with some antitumor drug or lacked originally target optionally toxin (toxin that comprises bacterium or plant and animal material) chemical coupling to suitable guide molecule or claim on the identification molecule, with the antineoplastic agent with target-specific of preparation hybridization.Along with the development of Protocols in Molecular Biology, and then set up the method for preparing such fused protein with the DNA recombinant technology.

In the generation and growth course of tumour, many tumour cells are overexpression growth factor receptor protein matter in its surface all, for example EGF-R ELISA (EGF-R) and transforming growth factor-alpha acceptor (TGF α-R).Therefore, some cytotoxic agent (as Pseudomonas exotoxin (PE), diphtheria toxin, Toxins,exo-, cholera, staphylococcus intracellular toxin and Ricin etc.) can be connected on EGF, the TGF-α or bFGF molecule as directed agents, have the hybrid molecule of tumour cell guide function and cytotoxic activity with generation concurrently.These hybrid molecules are guided whole molecule target cell into and are killed and wounded target cell by its toxin moiety target tumour cell guidance capability by it.

More and the more deep cytotoxic agent of research is ETA (PEA) (referring to United States Patent (USP) 4,545,985) at present.The single chain polypeptide that PEA is made up of 613 amino acid.The X-ray crystallography research of PE molecule and mutation analysis are shown, the PE molecule comprise three with produce the relevant structural region of cytotoxicity: responsible and sensitive cells bonded N-terminal cell receptor land (I district); Be responsible for the middle transposition district (II district) of lps molecule transposition in cytosol; And responsible inactivating proteins and the C-terminal enzymatic activity district (III district) that finally causes necrocytosis.Wherein the I district comprises mediated cell bonded Ia district (amino acid/11-252) and at present not the Ib district of clear and definite its function (amino acid 365-399) as yet.Can use biological chemistry or recombinant DNA technology to modify the PE molecule, so that the PE fragment of the various modifications of one or more aminoacid deletion or replacement to be arranged in the preparation PE molecule, for example, generally will leave out Ia district in the PE molecule, only contain enzymatic and transposition district, the PE-protein that molecular weight is about 40KDa is called PE40.After having now found that the Ia and most of Ib district (amino acid 365-380) of deletion PE, i.e. PE38 lps molecule, this molecule still keeps its SC, but has reduced non-specific toxicity (for example referring to Hwang et al., Cell 48:129-136,1987; United States Patent (USP) 4,892,827 and European patent 0261671).

PE described by many prior art documents and various somatomedin, antibody, hormone or CD4 merge, optionally lead and kill and wound the have different epicyte proteins hybridization method of protein of target cell of (acceptor or antigen) to produce (referring to Pastan and Fitz.G., Science 254:1173-1177,1991).For example, people such as Chaudhary (PNAS USA 84:4538-4542,1987) have described the crossbred fused protein that forms between PE40 and TGF α (TGF α-PE40).The clinical practice of TGF α-PE40 depends on its combination and kills and wounds the ability of the cell with EGF-R ELISA.People such as Murphy (PNAS USA 83:8258-8262,1986) have described gene constructed, the expression and the melanoma selecting cell toxicity thereof of diphtheria toxin-α melanotropin (MSH) fused protein.United States Patent (USP) 5,428,143 disclose the hybridization protein of the cell that is used for selective killing HIV infection and have encoded that this hybridizes the structure of proteinic mosaic gene.Wherein said hybridization protein is made up of with the cytotoxic protein matter (PE38) that can kill the HIV infection the people CD4 that contains the HIV binding site.

As the prior art more relevant with the present invention, international monopoly WO93/15751 discloses gonadotropin releasing hormone (GnRH) peptide directly has been coupled to the chimeric protein molecule of making on the Pseudomonas exotoxin molecule.It is said that such chimeric molecule that comes into operation can cause carrying in the pituitary gland destruction of the cell of GnRH acceptor, and with the reduction of sex hormone secretion, thereby the propagation that is expected to use it for the animal sterilization and suppresses the steroid hormone related neoplasms.In addition, International Patent Application WO 97/15325 has been described a kind of immunogenic carrier system that contains with the chemically combined GnRH of false unit cell extracellular toxin, can bring out the anti-GnRH antibody of generation high density with it in animal body as vaccine, thereby can be used for controlling behavior and the reactive tumour of treatment steroid hormone of becoming pregnant, reduce the reproductive hormone driving.

An object of the present invention is to provide a kind of chimeric toxin that forms by small peptide parahormone and the fusion of reorganization ETA function fragment, be characterised in that wherein said small peptide parahormone part is as directed agents, can with its surface on have the reactive or non-reacted tumour cell specific combination of periphery gonadal hormone of corresponding hormone receptor, and after internalization was in these tumour cells, said Pseudomonas exotoxin function fragment part can be killed and wounded these tumour target cells effectively as cytotoxic agent.

This purpose preferred embodiment according to the present invention, wherein said small peptide class trop(h)ic hormone is gonadotropin releasing hormone (GnRH).

This purpose preferred embodiment according to the present invention, the false unit cell exotoxin A of wherein said reorganization function fragment is PE38.

This purpose preferred embodiment according to the present invention, wherein said GnRH is connected to the position that is equivalent to the I district in the PE molecule with the gene fusion form.

Another object of the present invention provides the above-mentioned GnRH-PE38 chimeric toxin that contains the cytotoxicity significant quantity, and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle.

A further object of the present invention relates to the application of aforementioned pharmaceutical compositions in therapeutic glandular hormone reactivity or non-reacted tumour.

Fig. 1 demonstration is used to express the construction of recombinant plasmid of GnRH-PE38.

The influence of the protein synthesis of the GnRH-PE38 of Fig. 2 display part purifying reactive or non-reacted tissue-derived tumour cell and some healthy tissues primary cell culture to some sexual hormoue.Numerical value is with in the presence of different concns GnRH-PE38 (X-coordinate), and the specific chimeric toxin protein suppresses percentage (ordinate zou) expression that the malignant cell protein synthesis accounts for optimum cellular control unit.■: mammary cancer MCF-7 cell; ●: liver cancer HepG-2 cell; ▲: colorectal carcinoma HCT-8 cell; ※: cervical cancer Hela cell; Zero: the calf testis cell; △: mouse chest cell; : Turnover of Mouse Peritoneal Macrophages; ⊙ human cervical cancer 1 cell.

Term used herein " directed agents ", referring to can be only and the lip-deep acceptor of target cell to be killed and wounded or anti-The molecule of former specific bond or part. " directed agents " is also referred to as " identification molecule " or " ligand binding agent " sometimes. The example of such identification molecule is antibody or its fragment that can identify target cell, can with target cell on molecular specific In conjunction with growth factor, lymphokine, cell factor, antigen and hormone etc. According to preferred enforcement side of the present invention Case, said directed agents are little molecule small peptide parahormones (GnRH).

Term as used herein " GnRH " refer to can with its surface on have the cell knot of steroid hormone receptor Close, and when coming into operation in mammalian hosts with high dose, can cause natural GnRH's that anti-GnRH antibody produces Analog or derivative.

Natural GnRH is also referred to as luteinising hormone-releasing hormo (LHRH or LRH), and it is to have the lower decapeptide molecule that shows amino acid sequence:

pGlu-His-Trp-Ser-Tyr-Gly-Leu-rg-Pro-Gly-NH2

Used amino acid symbol is the general trigram dummy suffix notation in this area in the formula, and the representative of pGlu wherein Pyroglutamic acid. Applicable to GnRH analog of the present invention or derivative. The result of study demonstration, the 3rd in the molecule～6 amino acids are that to keep the GnRH activity necessary, and Ser⁴And Tyr⁵Then in receptors bind, play key work With. Show that on evidence gonad cell particularly GC surface has the GnRH acceptor. Proved mouse testis Expression of receptor relevant (Harwood, the J.P.et with the hormone in vivo level on cell (Legdig cell) surface Al., Endocrinology 107:407-413,1980). In addition, the somebody finds to have the parent on the rat endometrium The very high GnRH acceptor (Cao Yongqing etc., Chinese science B collects 1:32-37,1984) of making a concerted effort. Although at present to GnRH The outer mechanism of action of hypophysis still imperfectly understand, but certainly, the classes such as GnRH and sexual gland or endometrium are consolidated The combination of specific receptor is an important ring of the outer effect of GnRH performance hypophysis on alcohol hormone linked groups or the cell Joint. And shown, with the GnRH receptors bind that exists on target cell membrane and the cell endocrine granules basically be (Zhang Chongli etc., the Chinese applied physiology magazine 5 (1): 87-93,1988) that does not have species specificity. We Analysis of biological activity by the GnRH-PE38 fused protein of the inventive method preparation is clearly proved, GnRH-PE38 has with the tumour cell that comprises the multiple sexual organ of Hela cell source and has killing activity, and Normal cell to the Various Tissues source does not then have this specific killing activity (referring to embodiment 2).

Term " ID used herein₅₀" refer to suppress that target cell protein is synthetic reaches the 50% required of control group The concentration of chimeric toxin. Application standard among the present invention³The H leucine mixes method and detects " ID₅₀" value.

The invention further relates to the method with recombinant DNA technology production and small-molecular peptides parahormone link coupled target-specific, cytotoxicity reorganization PE fused protein, this method comprises: (1) provides and carries the PE38 expression carrier; (2) nucleotide sequence of the coding small molecule peptide hormone that is connected in series of synthetic molecule, and it is cloned in the expression vector of linearizing step (1); (3) expression vector with step (2) transforms appropriate host cell; (4) be suitable for expressing cultivate under the condition of the said fused protein of forming by small-molecular peptides parahormone and Pseudomonas exotoxin said by transformed host cells; (5) from cell culture, reclaim and the said fused protein of purifying.

The PE molecule that is used to make up fused protein of the present invention can be the natural PE molecule that has lacked Ia district and most of Ib district, but also can and better be the PE38 molecule through modifying.For example, can be 1～4 halfcystine (Cys wherein through the PE38 molecule of modifying like this ²⁶⁵, Cys ²⁸⁷, Cys ³⁷²And Cys ³⁷⁹) PE38 deleted or that replaced by other amino acid such as L-Ala is (referring to European patent 0383599 and United States Patent (USP) 5,621,078), has amino acid 280～364 and 381～613 (aminoterminal 1～28 amino acids in the disappearance II district), and the reorganization PE38 molecule that 287 halfcystine is replaced by Serine is (referring to United States Patent (USP) 5,821,238), and can between the amino acid 600-613 of PE molecule C-terminal, insert first identification molecule respectively, and connect the reorganization Pseudomonas exotoxin of second identification molecule (referring to United States Patent (USP) 5 at the N-terminal of PE molecule, 705,163).In addition, can use site-specific mutagenesis technology well known to those skilled in the art or other technologies further to modify the PE molecule, to change it according to the application-specific purpose.Certainly, the prerequisite of these modifications is that the functional performance that is provided by the PE molecule is not provided in fact.

Owing to discern the molecular weight all less relatively (10～13 peptide) of the small-molecular peptides parahormone of molecule, can use general dna synthesizer on the 1000A solid phase carrier, to synthesize the nucleotide sequence of these hormones or its varient as directed agents in the antitumor chimeric toxin of the present invention or title.For the ease of being connected, can introducing suitable endonuclease (as NdeI) restriction enzyme site at 5 ' and/or 3 ' end of synthetic hormone gene sequence, and cause the sticky end that is suitable for connecting with free or the PE encoding sequence that is carried on the specific recombinant vectors.Can be according to recombinant technology well known by persons skilled in the art (for example referring to Sambrook et al., Molecular Cloning:A LaboratoryManual, Cold Spring Harbor laboratory, 1989), the gene of these synthetic small peptide parahormones of clones coding (or with its cDNA or genomic dna form), and be connected in the carrier of the linearizing PE38 of carrying as pKK-PE38 (Chandhary et al. at the position that is equivalent to deleted Ia district and most of Ib district or the suitable restriction enzyme site place in the III district, PNAS USA 84:4538-4542,1987) or among the PET-11b-PE38 (Novagen Co.).The recombinant vectors system that so obtains can be transformed into then in prokaryotic cell prokaryocyte such as the Bacillus coli cells to produce the GnRH-PE38 fused protein.In the DNA reorganization operation, generally use the required gene fragment of the pcr amplification technology amplification of standard and cause suitable restriction enzyme site.Use the Restriction Enzyme cutting method to identify the exactness of sequence closure and possible sudden change, at last with people's such as Sanger dideoxy chain termination carry out sequencing with further confirmation it.

Certainly, also can with known chemical process two parts be combined by suitable chemical coupling group.For example can use SPDP (N-succinimide-3-2-pyridine dithio)-third sulphur ester, glutaraldehyde, maleimide aminobenzoyl-N-succinic diamide ester, imino-sulfane etc. to carry the isodigeranyl functional cross-link agent of functional groups as " joint " that connect GnRH and PE38 molecule.

What should particularly point out here is, because with respect to cytotoxicity part PE38, the molecular weight of targeting part GnRH is much smaller, so form new secondary space conformation probably by the PE38 part in the fused protein of both generations, cause PE38 part to the curling of GnRH, influence the degree of the receptors bind of fused protein.Under the cysteine residues in the PE molecule not being replaced/deletes the situation of modifying, this possibility is bigger especially.In addition, as previously mentioned, because GnRH is a mammalian pituitary excretory one-level hormone, and corresponding Rd is lower on the tumor cell surface of relevant peripheral tissues, think increase these guide molecules and receptor in target cell combine probability and combination stability, best with the GnRH molecule as the targeting part in the chimeric toxin.

Small-molecular peptides parahormone as ligand binding agent can be inserted in the PE38I district, preferably be equivalent on the position in Ia district of natural PE molecule (referring to Heimbrook et al., PNAS USA 87:4697-4701,1990).But also can be inserted on the position of PE molecule C-terminal amino acid 605～613.

Can in intestinal bacteria or other prokaryotic cell prokaryocytes, express the fused protein that comprises PE molecule and small-molecular peptides parahormone.The recombinant protein plasmagene will operably be connected on the suitable expression control sequenc, as be connected to the T that is suitable for using in intestinal bacteria ₇, on trp or λ promotor, ribosome bind site and the transcription termination signal.Can use known method for transformation, as the electroporation that is suitable for the calcium chloride facture of prokaryotic cell prokaryocyte or is suitable for mammalian cell with recombinant plasmid transformed of the present invention in the host cell of selecting.Can select the positive cell that transformed based on the antibiotics resistance that contained antibiotics resistance gene on the plasmid is given.In case expressed required fused protein, can separate and this fused protein of purifying according to methods known in the art.For example, can be from fermenting culture centrifugal collection somatic cells and with N,O-Diacetylmuramidase or Guanidinium hydrochloride cracking it, ultracentrifugation and dialysis repeatedly in low phosphorus hydrochlorate (about 20mM) solution then.In mixture, add urea then and forgive intravital protein with dissolved cell.After recentrifuge is removed insolubles, successively through ion exchange chromatography (IEC) and the required GnRH-PE38 reorganization hybridization protein of volume-exclusion chromatography (SEC) purifying.In addition, also can use saltout, methods such as affinity chromatography and the gel electrophoresis of preparation property carry out purifying (referring to R.Scopes, ProteinPurificafion, Springer-Verlag, N, Y., 1982).

In general, use polyacrylamide gel with SDS-PAGE electrophoretic method (Laemmli, Nature227:680-689,1970) analyze each column chromatography wash-out part, and use anti-PE antiserum(antisera) of multi-clone rabbit and Vectastain test kit (Vector Labs, Buvlingame Calif.) monitors it with immunoblotting.The recombinant fusion protein purified product is carried out protein synthesis inhibition test (Prior et al., Cell 64:1017-1029,1991), to detect the cytotoxicity (ID of fused protein ₅₀).For the GnRH-PE38 fused protein, then mainly detect its external killing activity, and use the different target cells that comprise tumour or normal cell ³The leucine of the H mark method of mixing detects fusion rotein to the isocellular protein synthesis restraining effect of HeLa, Swiss 3T3.In addition, can use the wheat germ extract that is rich in elongation factor 2 to detect the ADP-ribosylation activity of protein example by Colliev and the described method of Kondel (J.Biol.Chem.246:1496-1503,1971).

Can be with GnRH-PE38 fused protein of the present invention as the primary activity composition, and add one or more pharmaceutically acceptable carrier or vehicle, make the pharmaceutical composition that is suitable for clinical application.Said carrier or vehicle comprise but be not only limited to phosphate buffered saline (PBS), physiological saline, etc. ooze glucose solution, dextran, dextran etc.According to the difference of the disease of being treated, can add in pharmaceutical composition of the present invention that one or more and fused protein of the present invention have that auxiliary or synergistic other are natural, the active compound of synthetic or reorganization.In addition, can in pharmaceutical composition of the present invention, add and be selected from human serum albumin, low molecular weight peptide, glycine or Methionin and metallic cation (as Zn ²⁺, Mn ²⁺, Mg ²⁺And Ca ²⁺) protein protectant, and the stablizer that is selected from polyoxyethylene glycol, carboxymethyl cellulose, polyglycine, gsh.

Can be by the outer approach of conventional route of administration, the particularly gi tract pharmaceutical composition of the present invention that comes into operation, for example by administration in intravenously, intraperitoneal, intramuscular, intracutaneous, the subcutaneous or mucous membrane.The effective dosage ranges of pharmaceutical composition of the present invention can be from several nanogram(ng)s to tens milligram/kg body weight/sky, but at the concrete dosage of each given patient will be according to age of the character of disease to be treated or pathological state and severity, patient, body weight, factors such as the response capacity of medicine and administering mode are decided.

What should particularly point out is, although the more deep mechanism of action is still indeterminate, our laboratory has proved promptly that as far back as calendar year 2001 GnRH-PE38 protein of the present invention all has tangible specific combination activity and cytotoxicity to comprising tumor cell lines such as colorectal carcinoma HT-29 cell, ovarian cancer OVCAR3 cell, adenocarcinoma of cervix HeLa cell and liver cancer HepG-2 cell.

Therefore as seen, GnRH-PE38 protein of the present invention not only has obvious external lethal effect to the reactive tumour of the so-called steroid hormone that is subjected to hypothalamic pituitary gonadal axis control, and the adenocarcinoma cell of certain non-sexual gland organ origin such as S-180 sarcoma cell and myeloma cell are also had similar identical specificity in conjunction with, internalization and cytotoxic activity.

Below further illustrate the present invention by embodiment, but it will be appreciated by those of skill in the art that these embodiment do not constitute the await the reply restriction of claim scope to the present invention.

The preparation of embodiment 1:GnRH-PE38 fused protein

A. the structure of recombinant expression plasmid and evaluation

A. the amplification of initial plasmid DNA and linearizing: be in T with carrying ₇Amino acid/11～3 of the coding PE molecule under promotor and SD sequence and the terminator control and amino acid 253～613 (are removed wherein Ib district major part, be △ 365-380) plasmid pVC (the Chaudhary et al. of nucleotide sequence, PNAS USA 84:4538-4542,1987) transformed competence colibacillus intestinal bacteria HB101 cell, and will be incubated in the LB substratum that contains penbritin (50 μ g/ml), by cell transformed with amplified plasmid dna.After cultivation is finished, smudge cells, centrifugal collection plasmid also extracts plasmid DNA with phenol/chloroform.With NdeI plasmid DNA is cut into linearity, and handles so that its terminal dephosphorylation with calf intestinal alkaline phosphatase.

B. the synthetic that contains the GnRH-gene:, use the gene order (36bp) of the synthetic coding GnRH shown in SEQ ID NO.1 of dna synthesizer (Applied Biosystems Inc.AB1394 type) according to the aminoacid sequence of people GnRH-.

C. the structure of recombinant plasmid pVC8-GnRH-PE38 and screening: at T ₄Dna ligase exists down, and according to about 1: the molar ratio of 2-4 links together the double-stranded GnRH-nucleotide coding sequence of linearizing and terminal dephosphorylized pVC8-PE38 and the processing of process terminal phosphate.Because 3 ' end leaves the AT protuberance when NdeI digestion pVC8-PE38, and the synthetic GnRH-of institute encoding sequence gene has the TA base at 5 ' end respectively, thereby is easy to realize under the situation of only using single enzyme to cut correct connection of GnRH gene order of modifying and the pVC8 plasmid vector (pVC8-PE38) that carries PE38 after the annealing.Figure I has shown the structure of recombinant plasmid pVC8-GnRH-PE38.

Use resulting ligation mixture transformed into escherichia coli HB101 then.The inoculation that will be transformed is selected the amicillin resistance bacterium colony in containing on penbritin (50 μ g/ml) the LB agar plate after the cultivation, and on nitrocellulose filter with radiolabeled probe (γ- ³²P-GnRH) (A chain) in situ hybridization (65 ℃ of prehybridizations 2 hours; Hybridized 12 hours for 55 ℃).From in situ hybridization male transformant, in a small amount extract plasmid DNA, carry out dot hybridization with above-mentioned probe with identical method once more, with empty pVC8 carrier sequence as negative control.

Picked at random hybridization male plasmid is cut identification method and sepharose (2%) electrophoretic method is identified with enzyme.Select correctly to be connected with the recombinant plasmid of single GnRH encoding sequence and it named to be pVC8-GnRH-PE38.

In general, as in ligation, increasing the molar ratio of GnRH encoding sequence, should wherein be connected in series with the recombinant plasmid of a plurality of GnRH encoding sequences in theory to pVC8-PE38.After the SDS-PAGE electrophoretic analysis, the recombinant plasmid of only selecting to be connected with single (GnRH) nucleotide coding sequence with forward carries out dna sequence analysis.SEQID NO:2 has shown the nucleotide sequence of measured GnRH-PE38 hybrid gene.SEQ ID NO:3 then shows the aminoacid sequence of the fused protein of being inferred by GnRH-PE38.

The expression of B.GnRH-PE38 fused protein and the purifying of product:

To (contain T with carrying the recombinant plasmid pVC8-PE38-GnRH of GnRH-PE38 hybrid gene and not containing the e. coli bl21 (DE3) that the initial plasmid pVC8-PE38 (in contrast) of GnRH gene order transforms respectively ₇Rna polymerase gene) cultivate in the LB substratum that contains penbritin (50 μ g/ml) (Studier, F.W.and Moffatt, B.A., J.Mol, Biol, 189:113-130,1986).Work as A ₆₀₀Reach at about 0.8～1.2 o'clock and add 1mM isopropylthio-(IPTG) (final concentration 1mM/L), 37 ℃ are continued to cultivate 3 hours, to induce the expression of purpose product.Centrifugal collecting cell and will be suspended in lysis buffer (50mM Tris-HCl behind the cell precipitation thing multigelation then, pH7.5,5mM EDTA and 0.2mg/ml N,O-Diacetylmuramidase) in, the centrifugal supernatant (soluble fractions) of removing, and with resuspended thing ultrasonic (3 * 20 seconds) processing, with smudge cells.Recentrifuge (25,000g, 20 minutes) collecting precipitation thing (insoluble part) back adds ice-cold sex change damping fluid (100mM Tris-HCl, pH7.5,1mM EDTA 15mM 3-mercaptoethanol, 7M Guanidinium hydrochloride).Stir recentrifuge after 2 hours under the room temperature, and the gained protein soln is diluted to refolding damping fluid (the 50mM Tris-HCl of 200 times of volumes, pH7.8,1mMEDTA, 0.1M NaCl and 10mM 3-mercaptoethanol) in, placed 24 hours for 4 ℃, with (PBS) (pH7.4) the thoroughly dialysis (changing liquid 4 times) in the 10mM phosphate buffered saline (PBS) of folding again protein soln, centrifugal then (20,000g, 30 minutes), supernatant is the GnRH-PE38 crude extract.

The clarifying supernatant liquor that as above obtains is added on the DEAE-Sepharose Fast Flow post of crossing with above-mentioned refolding damping fluid balance (Pharmacia), with TE damping fluid (the 20mM Tris-HCl that contains 0.05～0.5M NaCl, pH8.0,1mM EDTA) stepwise elution, and collect protein active peak part.After the Amicon ultra-fine filter that the YM-30 film is equipped with in use concentrates, make the 2.6 * 100cm Sephacryl S-200 post (Pharmacia) of enriched material by crossing with above-mentioned refolding damping fluid balance, and with the TE damping fluid that contains 0.15M NaCl (20mM TrisHCl, pH8.0,1mM EDTA) wash-out.Collect active peak part and cross 2.0 * 20cmQ-Sepharose post (Pharmacia) once more, with collecting protein peak value (A behind the NaCl gradient elution ₂₈₀) part and thoroughly dialysis in 20mM PBS, the dialysis back stores for future use under-20 ℃.Lipidated protein＞96% of purifying like this.

According to the method for manufacturer recommendation, use Vectastain ABC test kit (Vector LaboratoriesInc.) and the anti-PE antiserum(antisera) of multi-clone rabbit that the GnRH-PE38 protein of purifying is identified.

The target-specific of embodiment 2:GnRH-PE38 fused protein and biologic activity analysis

A. protein synthesis inhibition test: detect the verify restraining effect of protein synthesis of the tumour selected or normal cell system of GnRH-PE38 fusion rotein of the present invention according to the described method of people such as Prior (Cell 64:1017-1023,1991).

The target cell of using in the experiment comprises the normal cell culture of former generation such as calf testis cell, mouse chest cell, mammary gland cell and peritoneal macrophage of knurls such as colorectal carcinoma HT-29 cell, ovarian cancer OVCAR cell, adenocarcinoma of cervix Hela cell and liver cancer HepG-2 cell system and pre-preparation.

With every hole 2 * 10 ⁴The density of individual cell with various tested cell inoculations in 96 hole microtest plates, at 5%CO ₂The following 37 ℃ of insulations of environment add the GnRH-PE38 of different concns and the equivalent PE38 of sample in contrast in each hole after 24 hours, final volume is 200 μ l/ holes.All all are diluted in 0.2% bovine serum albumin-phosphate buffered saline (PBS) (BSA-PBS) by test agent.37 ℃ of insulations add after 24 hours [ ³H] (Amershanm, Corp.) (5 μ Ci/ holes are diluted among the BSA-PBS) continues insulation 12 hours for the leucine of mark.-70 ℃ freezing and melt fast after with cell harvesting to glass fiber filter, detect the radioactivity be incorporated in the cell protein with the β counter then.Do not contact toxin protein and only add the results are shown among Fig. 2 that the percentage incorporation of the control cells of BSA-PBS represents to account for.

From result shown in Figure 2 as can be seen, the partially purified GnRH-PE38 fusion rotein of the present invention (the peak value part behind the DEAE-Sepharose post) can kill the tumour cell (ID in the reactive organ mammary gland of steroid hormone, uterine cervix source in dosage dependence mode ₅₀Value is about 15-40 μ g/ml).And find that toxin has also showed significant cytotoxicity (ID to colorectal carcinoma and liver cancer cell ₅₀Value is about 55-80 μ g/ml).

In addition, can find out further that from Fig. 2 GnRH-PE38 of the present invention does not then show detectable cytotoxic activity for the primary cell culture in the normal sexual gland related tissue (calf testis cell and human cervical cancer 1 cell) that derives from healthy donors, Lymphoid tissue (mouse chest cell) and epithelium (rat skin or fibrocyte) source.

The specificity of B.GnRH-PE38 is in conjunction with test

Basically according to Qayum, the described method of people such as A. (Br.J.Cancer 62:96-99,1990) is carried out GnRH-PE38 pair and is combined it with Hela cell cytosol membrane portions ¹²⁵The specificity competition of I-GnRH and replacement research.

The Hela cell monolayer of cultivating is separated to contains 10mM Tris-HCl, pH7.5,1mM EDTA, with the preparation cell homogenates, and centrifugal (500g, 15 minutes) remove big fragment in the damping fluid of 1mM DTT and 1mg/ml bovine serum albumin.With 25,000g obtained the endochylema membrane portions in centrifugal 30 minutes with 4 ℃ of supernatant parts, and it is suspended in the cold above-mentioned damping fluid (0.5mg protein/ml).Final volume with every hole 100 μ l is added on this endochylema film suspension (100 μ g/ hole) in each hole of 24 well culture plates.Respectively add 5 μ M (0.25 μ Ci) then ¹²⁵I-GnRH-and 37 ℃ of temperature were bathed 1 hour.Add unlabelled GnRH (0.01-1000 μ M) or GnRH-PE38 chimeric toxin of the present invention (0.1-500mM) after 1 hour more respectively and continue insulation 24 hours.The insulation back is washed sample and is detected the bonded part with the γ calculating instrument with above-mentioned damping fluid.Adding partially purified GnRH-PE38 chimeric toxin with cumulative concentration can replace and Hela cytolemma bonded gradually along with the increase of GnRH-PE38 concentration ¹²⁵I-GnRH.

Though be not limited to theory, but we infer that this higher receptor-specific affinity of GnRH-PE38 may be not have the space extensibility of the guide molecule of halfcystine owing to prolonged this, and then have increased guide molecule and associated receptor or the antigenic chance that combines.In addition, this experimental result prompting, as if GnRH of the present invention be suitable for being developed to the antineoplastic agent that can use as the chimeric toxin of targeting part in clinical practice.

Sequence table

(1) general information

(I) applicant: the high section in Shenzhen bio tech ltd

(II) denomination of invention: the gonadotropin releasing hormone and the Pseudomonas aeruginosa exotoxin A toxicity function fragment (PE38) that contain guiding

The reorganization toxic agents

(III) sequence number: 3

(IV) address:

(A) contact person: Wang Zhimin

(B) street: the biological brooder building 2-101A.B in High-Tech Road, high and new technology industrial development zone, Shenzhen

(C) city: Shenzhen

(D) country: the People's Republic of China (PRC)

(E) postcode: 518057

(V) computer-reader form:

(A) amboceptor type: 3.5 inches floppy disks

(B) computer: IBM PC

(C) operating system: WINDOWS98

(D) software: WORD2000

(VI) telecommunication information:

(A) phone: 13510077369

(B) fax: 0755-26031096

(2) information of SEQ ID No:1

(I) sequence signature:

(A) length: 36 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: SEQ ID No:1

TATGCAGCAC?TGGTCCTATG?GACTGCGCCC?TGGACA

(2) information of SEQ ID No:2

(I) sequence signature:

(A) length: 1086 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: cDNA

(III) sequence description: SEQ ID NO:2

ATGCAGCACT?GGTCCTATGG?ACTGCGCCCT?GGACATATGG?CCGAAGAGGG?CGGCAGCCTG

GCCGCGCTGA?CCGCGCACCA?GGCTTGCCAC?CTGCCGCTGG?AGACTTTCAC?CCGTCATCGC

CAGCCGCGCG?GCTGGGAACA?ACTGGAGCAG?TGCGGCTATC?CGGTGCAGCG?GCTGGTCGCC

CTCTACCTGG?CGGCGCGGCT?GTCGTGGAAC?CAGGTCGACC?AGGTGATCCG?CAACGCCCTG

GCCAGCCCCG?GCAGCGGCGG?CGACCTGGGC?GAAGCGATCC?GCGAGCAGCC?GGAGCAGGCC

CGTCTGGCCC?TGACCCTGGC?CGCCGCCGAG?AGCGAGCGCT?TCGTCCGGCA?GGGCACCGGC

AACGACGAGG?CCGGCGCGGC?CAACGGCCCG?GCGGACAGCG?GCGACGCCCT?GCTGGAGCGC

AACTATCCCA?CTGGCGCGGA?GTTCCTCGGC?GACGGCGGCG?ACGTCAGCTT?CAGCACCCGC

GGCACGCAGA?ACTGGACGGT?GGAGCGGCTG?CTCCAGGCGC?ACCGCCAACT?GGAGGAGCGC

GGCTATGTGT?TCGTCGGCTA?CCACGGCACC?TTCCTCGAAG?CGGCGCAAAG?CATCGTCTTC

GGCGGGGTGC?GCGCGCGCAG?CCAGGACCTC?GACGCGATCT?GGCGCGGTTT?CTATATCGCC

GGCGATCCGG?CGCTGGCCTA?CGGCTACGCC?CAGGACCAGG?AACCCGACGC?ACGCGGCCGG

ATCCGCAACG?GTGCCCTGCT?GCGGGTCTAT?GTGCCGCGCT?CGAGCCTGCC?GGGCTTCTAC

CGCACCAGCC?TGACCCTGGC?CGCGCCGGAG?GCGGCGGGCG?AGGTCGAACG?GCTGATCGGC

CATCCGCTGC?CGCTGCGCCT?GGACGCCATC?ACCGGCCCCG?AGGAGGAAGG?CGGGCGCCTG

GAGACCATTC?TCGGCTGGCC?GCTGGCCGAG?CGCACCGTGG?TGATTCCCTC?GGCGATCCCC

ACCGACCCGC?GCAACGTCGG?CGGCGACCTC?GACCCGTCCA?GCATCCCCGA?CAAGGAACAG

GCGATCAGCG?CCCTGCCGGA?CTACGCCAGC?CAGCCCGGCA?AACCGCCGCG?CGAGGACCTG

AAGTAA

(2) information of SEQ ID NO:3

(I) sequence signature:

(A) length: 359 amino acid

(B) type: amino acid

(C) chain: strand

(D) topological framework: linearity

(II) molecule type: protein

(III) sequence description: SEQ ID NO:3

Met-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-His-Met-Ala-Glu-Glu-Gly-Gly-Ser-Leu-Ala-Ala-Leu-Thr-Ala-His-Gln-Ala-Cys-His-Leu-Pro-Leu-Glu-Thr-Phe-Thr-Arg-His-Arg-Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu-Gly-Gln-Cys-Gly-Tyr-Pro-Val-Gln-Arg-Leu-Val-Ala-Leu-Tyr-Leu-Ala-Ala-Arg-Leu-Ser-Trp-Asp-Gln-Val-Asp-Gln-Val-Ile-Arg-Asn-Ala-Leu-Ala-Ser-Pro-Gly-Ser-Gly-Gly-Asp-Leu-Gly-Glu-Ala-Ile-Arg-Glu-Gln-Pro-Gln-Gln-Ala-Arg-Leu-Ala-Leu-Thr-Leu-Ala-Ala-Ala-Glu-Ser-Glu-Arg-Phe-Val-Arg-Gln-Gly-Thr-Gly-Asn-Asp-Gln-Ala-Gly-Ala-Ala-Ala-Gly-Pro-Ala-Asp-Gly-Asp-Ala-Len-Len-Glu-Arg-Asn-Tyr-Pro-Thr-Gly-Ala-Glu-Phe-Len-Gly-Asp-Gly-Gly-Asp-Val-Ser-Phe-Ser-Thr-Arg-Gly-Thr-Gln-Asn-Trp-Thr-Val-Gln-Arg-Leu-Leu-Gln-Ala-His-Arg-Gln-Leu-Gln-Gln-Arg-Gly-Tyr-Val-Phe-Val-Gly-Tyr-His-Gly-Thr-Phe-Len-Gln-Ala-Ala-Gln-Ser-Ile-Val-Phe-Gly-Gly-Arg-Ala-Arg-Ser-Gln-Gln-Asp-Leu-Asp-Asp-Ala-Ile-Trp-Arg-Gly-Phe-Tyr-Ile-Ala-Gly-Asp-Pro-Ala-Len-Ala-Tyr-Gly-Tyr-Ala-Gln-Asp-Gln-Gln-Pro-Asp-Ala-Arg-Gly-Arg-Ile-Arg-Asp-Gly-Ala-Len-Len-Arg-Val-Tyr-Val-Pro-Arg-Ser-Ser-Len-Pro-Gly-Phe-Tyr-Arg-Thr-Ser-Len-Thr-Len-Ala-Ala-Pro-Gln-Ala-Ala-Gly-Gln-Val-Gln-Arg-Len-Ile-Gly-His-Pro-Len-Pro-Len-Arg-Len-Asp-Ala-Ile-Thr-Gly-Pro-Gln-Gln-Gly-Gly-Arg-Len-Gln-Thr-Ile-Len-Gly-Trp-Pro-Len-Ala-Gln-Arg-Thr-Val-Val-Ile-Pro-Ser-Ala-Ile-Pro-Thr-Asp-Pro-Arg-Asn-Val-Gly-Gly-Asp-Len-Asp-Pro-Ser-Ser-Ile-Pro-Asp-Lys-Gln-Gln-Ala-Ile-Ser-Ala-Len-Pro-Asp-Tyr-Ala-Ser-Gln-Pro-Gly-Lys-Pro-Arg-Gln-Asp-Len-Lys-end.

Claims (5)

1. merge the chimeric toxin that forms by small peptide hormone and reorganization ETA function fragment, be characterised in that wherein said small peptide parahormone part is as directed agents, can have the reactive or non-reacted tumour cell of the gonadal hormone of corresponding hormone receptor or other parts with its surface combines specifically, and after internalization was in these tumour cells, said reorganization ETA function fragment part can be killed and wounded these tumour target cells effectively as cytotoxic agent.

2. according to the chimeric toxin of claim 1, wherein said Pseudomonas exotoxin is PE38.

3. according to the chimeric toxin of claim 1, wherein said small peptide parahormone is GnRH.

4. contain the chimeric toxin of any one and the pharmaceutical composition of one or more pharmaceutically acceptable carriers or vehicle in the with good grounds claim 1～3.

5. the application of or non-reacted tumour reactive at the therapeutic glandular hormone according to the pharmaceutical composition of claim 4.

Images