CN1884300A - Targeted function anti-liver cancer superantigen and its preparation method and use - Google Patents

Abstract

The invention discloses a super antigen target to liver cancer, and also discloses the method for preparing the same and its usage. Said super antigen comprises target part and super antigen part, and target part is genetic engineering antibody coming from Hab25 monoclonal antibody and combined with lover cancer. The preparing method comprises: construction of recombined carrier containing antibody and super antigen gene section, expression of super original fusion protein and purification. The recombined super antigen is characterized by double function of guide and killing liver cancer, and can be made to effective target medicine for treating tumor.

Description

A kind of anti-liver cancer superantigen with target function, Preparation Method And The Use

Technical field

The present invention relates to a kind of anti-liver cancer superantigen, relate in particular to a kind of target superantigen that contains the genetic engineering antibody that comes from anti-liver cancer Hab25 monoclonal antibody, also relate to the Preparation method and use of this target superantigen with target function.

Background technology

Hepatocellular carcinoma (HCC) be the most common, also be one of the most fatal malignant tumour, have the title of " cancer king ", be characterized in the morbidity concealment, shift early the case fatality rate height.The traditional treatment of liver cancer is based on excision and chemotherapy.Operation thoroughly excision provides best healing chance, is the prefered method for the treatment of liver cancer at present, yet for most tumors, too greatly or too disperse, can't excise more than 75% when detecting." X ray of gamma-rays or linear electron accelerator, energetic ray etc.; traditional route of administration has been improved in the chemotherapy aspect; for the primary hepatocarcinoma that can't excise is not very sensitive; it is to the destruction of body immune system in addition; treatment back patient's The average survival time only 4 months are seldom above 9 months though the radiotherapy aspect has adopted.

Topmost restriction for the patient that can not carry out surgical blanking is that general toxicity and levels of drugs do not reach effective concentration at traditional chemotherapy and radiation, and targeted therapy has good application prospects because of its highly selective and high-affinity for inoperable patient of treatment or the tumour that extensively shifts.At present the liver cancer targeted therapy is to utilize a kind ofly to have the antibody of special avidity to make carrier to liver cancer, make the commissure thing with the bullet that the killing tumor cells effect is arranged (superantigen, chemotherapeutics, biotoxin albumen radionuclide), reduce the purpose of infringement healthy tissues to reach more killing tumor cells.Usefulness such as Chen Zhinan ¹³¹The liver cancer monoclonal antibody Hepama-1 of I mark injects 23 routine patients by artery in the liver, has obtained curative effect preferably, and 75% (12/16) patient AFP value descends, and patient's knurl piece of 78% (18/23) dwindles.II phase clinical study is the result show: complete remission rate 4.1%, part complete remission rate are that survival rate is 46.2% more than 53.1%, two year.

Although antibody targeted treatment is most active one of the cancer strategy of controlling, some problems that exist have limited their practical application effect.Treatment antibody at liver cancer is mouse source property at present, is applied to human body, is prone to human antimouse antibody (HAMA), and in the therapeutic process of 131I-Hepama-1,43% (10/23) patient has produced the antibody at Hepama-1.Obviously, HAMA has limited multiple injection, and long-term treatment can't be carried out.The molecular weight of antibody is big in addition, and seepage force is poor, and being difficult for penetrating the tumour capillary vessel also is the major cause that causes liver cancer targeted therapy poor effect.Small molecular antibody have immunogenicity low, be easy to permeate destination organization, toxic side effect little, easily link to each other with effector molecule and constitute the advantages such as antibody molecule of new function, constantly carry out and constantly carry out the research work that single-chain antibody (scFv), disulfide linkage are stablized single-chain antibody (ds-scFv) and disulfide stabilized antibody antibody fragments such as (dsFv), these antibody fragments are compared with parental antibody, antigen-binding activity keeps, molecular weight reduces, molecular weight is generally about 30Kda, and is stronger to the noumenal tumour penetrativity.Liver cancer-resisting monoclone antibody Hab25 has been proved to be has good avidity and specificity to hepatocellular carcinoma.The big grade of domestic Sun Zhi is successfully transformed liver cancer-resisting monoclone antibody Hab25, has prepared the single-chain antibody and the Humanized single chain antibody of this monoclonal antibody.

Staphylotoxin superantigen (Superantigen, SAg) be one group of meta-bolites of streptococcus aureus, typical case's representative is staphyloentero-toxin (staphylococcal enterotoxin, SE) and staphylococcal intoxication shock toxin (staphylococcal toxic shock syndrome toxin-1, TSST-1).Because SAg inductive CTL (cytotoxic T lymphocytes) has powerful lethal effect (Kalland T to tumour cell, Dohlsten M, Abrahmsen L, et al.Targeting of superantigens.CellBiophys.1993 Jan-Jun; 22 (1-3): 147-164; Hansson J, Ohlsson L, Persson R, et al.Genetically engineered superantigens as tolerable antitumor agents.Proc Natl Acad SciUSA.1997 Mar 18; 94 (6): 2489-2494).Therefore the antineoplaston of using SAg has obtained people's fervent concern.But SAg also has toxicity to normal cell when tumour cell is killed and wounded, and can only be to MHCII ⁺The tumour molecule kill and wound.The Monoclonal Antibody technology is that the targeted therapy of tumour has brought hope.Antibody targeted superantigen treatment colorectal carcinoma, the renal cell carcinoma of external employing all obtained good result of treatment at animal experiment, and enter I/II clinical trial phase (Dohlsten M., Hedlund G., Akerblom E., et al.antibody-targeted superantigens:a different class ofanti-tumor agents.Proc Natl Acad Sci USA.1991; 88 (20): 9287-9291).But bibliographical information is not seen in the test of guiding superantigen treatment liver cancer as yet.In view of the grade malignancy of liver cancer is higher, it is still unknown whether the guiding superantigen has a therapeutic action to hepatocellular carcinoma.

Hab25 liver cancer-resisting monoclone antibody molecular weight is big, penetration power is poor, has limited the effect of its targeted therapy.And the appearance of small molecules genetic engineering antibody preferably resolves the problems referred to above, as single-chain antibody (ScFv), disulfide stabilized antibody (dsFv) (Rajagopal V, Pastan I, Kreitman RJ.A form ofanti-Tac (Fv) which is both single-chain and disulfide stabilized:comparison with itssingle-chain and disulfide-stabilized homologs.Protein Eng.1997Dec; 10 (12): 1453-1459.).The former connects VH and VL molecule by a Linker, being connected of latter VH and VL then is by a pair of interchain disulfide bond (Reiter Y, Brinkmann U, Jung SH, LeeB, Kasprzyk PG, King CR, Pastan I.Improved binding and antitumor activity of arecombinant anti-erbB2 immunotoxin by disulfide stabilization of the Fv fragment.JBiol Chem.1994 Jul 15; 269 (28): 18327-18331.).Yet of short duration separating causes the unstable of ScFv and then influenced the stability of connected toxin VH with VL; DsFv is more stable with respect to ScFv, but the low yield after the renaturation has limited its clinical application.And disulfide linkage is stablized a kind of novel gene engineered antibody that single-chain antibody sc-dsFv is recent report, be connected with a pair of disulfide linkage by a Linker simultaneously between its VH and the VL, have the advantage that ScFv output is high and dsFv stability is high concurrently, thereby have more advantage (Bera TK, Onda M, Brinkmann U, Pastan I.A bivalentdisulfide-stabilized Fv with improved antigen binding to erbB2.J Mol Biol.1998 Aug21; 281 (3): 475-483.).

Up to the present, Shang Weiyou comes from the genetic engineering antibody and the superantigen amalgamation and expression of Hab25 monoclonal antibody, the report of preparation target superantigen.

Summary of the invention

In order to overcome current liver cancer treatment weak effect, shortcoming that side effect is big, the invention provides a kind of anti-liver cancer superantigen that has target function efficiently.

Superantigen with target function of the present invention is made up of two portions, and targeting moiety is the genetic engineering antibody that comes from liver cancer-resisting monoclone antibody Hab25, adopts gene fusion to be connected between antibody and the superantigen part.

The genetic engineering antibody of Hab25 can be stablized single-chain antibody (ds-scFv) or disulfide stabilized antibody (dsFv) for single-chain antibody (scFv), disulfide linkage, and preferred disulfide linkage is stablized single-chain antibody (ds-scFv).Superantigen is common staphylococcus and the streptococcic toxin superantigen of coming from.The staphylococcus superantigen comprises staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), staphyloentero-toxin C (SEC), staphylococcal intoxication shock toxin (TSST-1) or its mutant, preferred staphylococcal enterotoxin A (SEA) or D227A mutant, staphylococcal intoxication shock toxin (TSST-1).The suis superantigen comprise SPE A, B, C (speA, speB, speC) etc.

In the fusion rotein, genetic engineering antibody both can be positioned at superantigen molecule front end, also can be positioned at the superantigen rear end, and the preferred gene engineered antibody is positioned at superantigen molecule front end.The two connection can be adopted connection peptides, also can be without connection peptides.Adopt the GGGSGGS connection peptides in the embodiment of the invention, it is all good that the liver cancer of antibody activates activity in conjunction with active and superantigen immunogene.

The present invention has also announced the preparation method of the superantigen of this target function, may further comprise the steps:

(1) makes up the recombinant expression vector that comprises Hab25 genetic engineering antibody and superantigen gene fragment;

(2) with the recombinant expression vector transformed into escherichia coli of above-mentioned structure, and abduction delivering;

(3) with expression product purifying and purifying, obtain fusion rotein.

In preparation fusion rotein process, can obtain the effect of solubility expression and insoluble expression by selecting different recombinant expression vectors for use.Select for use the expression vector such as the PTIG-Trx that express molecular chaperones (as Trx, GST) simultaneously can play usually, obtain soluble functional albumen expressing proteic short molten effect.But this method is not suitable for expressing the albumen that needs to form disulfide linkage, stablizes single-chain antibody (ds-scFv) or disulfide stabilized antibody (dsFv) as disulfide linkage.This antibody-like needs to form earlier inclusion body usually, and is external then by oxidation-reduction reaction formation disulfide linkage.And conventional expression of target superantigen is generally insoluble expression, thus can adopt conventional expression vector, as PET series.

Genetic engineering antibody is connected with the superantigen gene can pass through overlapping pcr, then with the PCR product cloning to prokaryotic expression carrier, as pET22b.Also can connect by restriction enzyme site.

The present invention adopts pet vector to prepare the ds-scFv25-SEAD227A fusion rotein, adopts the IPTG abduction delivering, obtains inclusion body, then the inclusion body of expressing is carried out sex change, renaturation, and renaturation product carries out preliminary purification, obtains the ds-scFv25-SEA fusion rotein.Cell ELISA measure antibody in this fusion rotein in conjunction with active with and stability, the result show the fusion rotein after the renaturation kept antibody in conjunction with activity and anti-tumor activity, and stability improves than single-chain antibody.

The present invention has made up the fusion rotein of HAB25 genetic engineering antibody and superantigen first, and purpose is to remedy the deficiency of superantigen in oncotherapy, reduces it to Normocellular injury when tumour cell is killed and wounded, to reach the ideal result of treatment.

In the research process of target superantigen in the past, it is difficult obtaining the good superantigen bifunctional molecule of antibody binding activity and anti-tumor activity simultaneously.In the fusion process of antibody and superantigen, the combination activity of tumour cell and superantigen immunogene activate characteristic and can partly even completely lose.The biologic activity of this research experiment showed, that the fusion rotein of preparation has good binding activity to liver cancer cell, confirm that it has possessed good tumor targeting effect, and activation T lymphocyte that can be successful kills and wounds to liver cancer.

The present invention has made up the disulfide linkage of Hab25 first and has stablized single-chain antibody ds-scFv, and has carried out amalgamation and expression efficiently with SEA (D227A) gene by prokaryotic expression carrier on this basis.Experimental results show that fusion rotein has good stability, be better than the stability of the single-chain antibody of bibliographical information, be more conducive to clinical use.

The anti-hepatoma-targeting superantigen that comes from Hab25 that the present invention obtained has possessed the dual-use function of guiding and killing tumor cell, might develop into effective immunotherapy of tumors targeted drug.

Description of drawings

Fig. 1 cuts the evaluation collection of illustrative plates for the enzyme of recombinant plasmid pET22b-scdsFv-SEA (D227A).Wherein

1: cut with the NdeI/BamHI enzyme; 2: cut with the BamHI/SalI enzyme; 3: cut with the NdeI/SalI. enzyme.

Fig. 2 is that the SDS-PAGE of recombinant protein analyzes collection of illustrative plates.Wherein

1: induce the full bacterium lysate of E.coli, 2: induce the E.coli inclusion body; 3:Q Sepharose HP purifying protein; 4: low molecular weight of albumen standard (94kDr; 67kDr; 43kDr; 31kDr); 5:Hiprep26/60Sephacryl S-200HR purifying protein.

Fig. 3 is that the Western blot of recombinant protein analyzes collection of illustrative plates, wherein

The scdsFv-SEA albumen (lane 1) of 1:Q sepharose HP purifying; 2: inclusion body; M: low molecular weight of albumen standard.

Fig. 4 analyzes collection of illustrative plates for the recombinant protein disulfide linkage

Fig. 5 be recombinant protein in conjunction with activity and Detection of Stability

(A) specific activity of recombinant protein and SCFV, DSFV; (B) 37 ℃ of combinations of hatching different time of recombinant protein are active; (C) recombinant protein is hatched 30 minutes combination activity in differing temps; (D) combination of 37 ℃ of different urea concentration effects in 30 minutes of recombinant protein is active;

Fig. 6: scdsFv-SEA (D227A) and SEA are to the killing activity of liver cancer cell SMMC-7721, and initial concentration is 10 μ g/ml, imitate target than 8: 1.

Embodiment

Embodiment strand disulfide stabilized antibody target superantigen SEA (D227A)

The structure of molecule and active the detection

1 material and method

1.1 material hepatoma cell strain SMMC-7721, SEA (D227A) and HAb18 dsFv plasmid, BL21plusS host bacterium and pET22b expression vector are this chamber and make up preservation.NdeI, BamHI, SalI,, Ex ^TMArchaeal dna polymerase, T4 dna ligase, IPTG are all available from Takara company.E.Z.N.A ^Gel extraction kit is available from Omega company.Q Sepharose HP, Hiprep26/60 Sephacryl S-200 HR are available from Amersham Pharmacia company.Sulfydryl alkanisation reagent A MS (4-acetamido-4 '-maleimidylstilbene-2,2 '-disulfonic acid) available from Molecular Probes company.The BCA protein assay reagent is available from Pierce company.PCR primer (table 1) is given birth to worker company by Shanghai and is synthesized.

Table 1 amplification scdsFv-SEA (D227A) gene the primer

Primer sequence
	P 1 5’-ccgCATATGgaggtacagctggttgaatctgg P 2 5’-cccccgccaccagagccgccttcaccgagctctgaggagacggt P 3 5’-gaaggcggctctggtggcgggggatcaggagggggcggttctgagctcgtgatgacccag P 4 5’-cgcGGATCCacctccacgtttgatctcgacctt P 5 5’-attGGATCCggaggttcaagcgagaaaagcgaagaaata P 6 5’-cacGTCGACttaacttgtatataaatatatagcaatatgcatg

1.2 method

1.2.1 P1, P2 and two pairs of primers of P3, P4 are used in the structure of scdsFv-SEA (D227A) recombinant expression plasmid and evaluation, VH and VL with the dsFv plasmid is that template is carried out pcr amplification respectively, amplified production is analyzed through the agarose gel electrophoresis of 20g/L, obtains VH-linker, linker-VL gene respectively.According to the indication of marker, downcut the gel that contains target gene fragment from sepharose, the operation steps purifying that reclaims test kit according to the DNA fast purifying reclaims amplified production.Equivalent PCR product with recovery is a template, uses P1, P4 primer, and overlapping extension PCR method amplification obtains the scdsFv gene.Using P5, P6 primer, is that template amplification obtains connector-SEA (D227A) gene with SEA (D227A).After above-mentioned two PCR products are purified, carry out double digestion with NdeI, BamHI and BamHI, SalI respectively, with be connected through the pET22b carrier of NdeI, SalI double digestion ratio with 3: 3: 1, be built into recombinant expression plasmid pET22b-scdsFv-SEA (D227A).With this recombinant plasmid transformed e. coli bl21 plusS, bacterium colony PCR screening and cloning, and through the enzyme evaluation of cutting and check order.

1.2.2 the abduction delivering of recombinant protein and evaluation will contain the e. coli bl21 plusS of recombinant protein gene expression plasmid, inoculum size inoculation Superbroth substratum by 1%, 37 ℃ of shaking culture to A600 values are about 1, IPTG (final concentration 1mmol/L) induces 5h for 37 ℃, centrifugal collection bacterium carries out SDS-PAGE and Western blot and analyzes.

1.2.3 behind the change renaturation collection bacterium of recombinant protein, with 50mmol/L Tris, 20mmol/L EDTA, pH 8.0,2% Triton X-100, the resuspended thalline of 0.5mol/L NaCl solution carries out carrying out ultrasonic bacteria breaking.Centrifugal, collection inclusion body, washings (50mmol/L Tris, 20mmol/L EDTA, pH 8.0) repeatedly after the washing, precipitation is dissolved in sex change liquid (7mol/L Guanidinium hydrochloride, 0.1mol/L Tris-HCl, pH 8.0,2mmol/L EDTA, 10mg/ml DTE), more than the room temperature sex change 4h, centrifugal collection supernatant, be diluted in renaturation solution (2mol/L urea, 10mmol/L Tris-HCl, 0.9mmol/L GSSG at 1: 100, pH 10.0) in, behind 10 ℃ of renaturation 36-48h, add GSSG to 2mmol/L, 10 ℃ are continued to hatch 12h.

1.2.4 the purification renaturation liquid of recombinant protein behind 0.45 μ m membrane filtration, is splined on the Q Sepharose HP chromatography column after A liquid (20mmol/L Tris-HCl, pH 9.6) balance.Last sample finishes, and with 3-5 column volume of A liquid flushing, uses B liquid (the A liquid that contains 1mol/L NaCl) wash-out then again, collects elution peak.The elution peak that will contain target protein is splined on Hiprep 26/60 Sephacryl S-200HR molecular sieve column and is further purified, and balance liquid is 25mmol/L Tris-HCl (pH 7.0), 0.15mol/LNaCl.

Get four identical duplicate samples 1.2.5 the recombinant protein disulfide linkage is identified, be divided into two groups, one group adds DTT (final concentration 100mmol/L), 100 ℃ of heating 10min, and another group is not handled.10% trichoroacetic acid(TCA) (TCA) post precipitation, get a alkanisation solution (the 150mmol/L Tris-HCl (pH7.5) that adds in two groups respectively, 2% SDS, 15mmol/L AMS), remain two parts resuspended with solution (150mmol/L Tris-HCl (pH7.5), 2% SDS), behind the incubated at room 1h, all add 2 * not sample buffers that contain DTT, the SDS-PAGE electrophoresis is migration situation relatively.

1.2.6 the detecting in conjunction with active and Detection of Stability recombinant protein of recombinant protein in conjunction with the active ELISA method that adopts.Concrete grammar is, with SMMC-7721 liver cancer cell coated cell plate, is one anti-with the anti-SEA antibody of rabbit, and the goat anti-rabbit igg of horseradish oxydase mark is two anti-, and DAB is a chromogenic substrate.Use said system, detect respectively recombinant protein in 37 ℃ of PBS in the different time with its stability in the 30min under different urea concentrations, differing temps.

1.2.7 the cell killing activity of recombinant protein detects the guiding killing activity that adopts MTS colorimetric method for determining recombinant protein, is with reference to product with SEA, is target cell with the SMMC-7721 liver cancer cell, is the effector cell with the human peripheral lymphocyte.Concrete grammar is: every hole adds 5 * 10 respectively in the 96 porocyte plates ³Individual SMMC-7721,4 * 10 ⁴The recombinant protein of individual lymphocyte and doubling dilution, cumulative volume are 200 μ l.After 37 ℃, 5%CO2 incubator are hatched 3-5 days, serum-free RPMI 1640 washes plate three times, add 100 μ l and contain the RPMI RPMI-1640 and the 20 μ l MTS/phenazinemethosulfate solution of 10% calf serum, 37 ℃, 5%CO2 incubator continue to cultivate the light absorption value of measuring 490nm behind the 1-4h.

2 results

2.1 the structure of scdsFv-SEA (D227A) plasmid and identify with the amplification VH-linker, linker-VL be template, P1, P4 are that primer carries out pcr amplification, occur the specific amplified band about 750bp, the size conform to expected result.The positive colony of PCR Screening and Identification, its plasmid be respectively through NdeI and BamHI, and BamHI and SalI, NdeI and SalI double digestion identify, respectively at 750bp, and 720bp, specific band appears in the 1500bp place, with expected results conform to (Fig. 1).Sequencing result shows that the scdsFv-SEA that recombinant plasmid carries (D227A) nucleotide sequence and amino acid sequence coded thereof are identical with expection.

2.2 the abduction delivering of recombinant protein and evaluation promptly obtain engineering bacteria with recombinant plasmid transformed e. coli bl21 plusS.Engineering bacteria is behind Superbroth culture medium culturing and IPTG abduction delivering, and the SDS-PAGE electrophoresis is found, is about the 55000 appearance one new bands of expressing at Mr, and size estimates that together the result conforms to.Its expression amount accounts for about 30% of bacterial protein, exists with the form of inclusion body.With the anti-SEA antibody of rabbit is one anti-, and horseradish oxydase mark goat anti-rabbit igg is two anti-, and Western blot analyzes and shows that the new band of expressing is target protein (Fig. 3).

2.3 after the thalline ultrasonication after the renaturation of recombinant protein and purifying are induced, centrifugal collection inclusion body is after washing, the inclusion body electrophoresis purity can reach about 60%, after the abundant sex change of sex change liquid, and the rapid dilution renaturation, in 10 ℃ hatch 36h after, add GSSG and further form disulfide linkage.After renaturation solution passed through Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR purifying in succession, purity was up to (Fig. 2) more than 95%.Albumen is quantitative through BCA protein quantification test kit, and every liter of inductor can obtain the target protein of about 60mg, much larger than the output (about 4-7mg) of disulfide linkage target superantigen.

2.4 the analysis of recombinant protein disulfide linkage is (Kobayashi according to the literature, T, Kishigami S, Sone M, etal.Respiratory chain is required to maintain oxidized states of the DsbA-DsbB disulfide bondformation system in aerobically growing Escherichia coli cells[J] .Proc Natl Acad SciUSA, 1997,94:11857-11862.), contain disulfide bond protein and be slower than it in the electrophoretic speed of irreducibility SDS-PAGE in the electrophoretic travelling speed of reductibility SDS-PAGE.And AMS can be in conjunction with sulfydryl freely, increase proteic molecular weight, therefore can judge albumen sulfydryl and disulfide linkage situation (1.Jurado P by albumen electrophoretic mobility of its SDS-PAGE before and after the AMS alkanisation, Ritz D, Beckwith J, et al.Production of functional single-chain Fv antibodies in the cytoplasm of Escherichia coli[J] .JMol Biol, 2002,320:1-10.2.Masaaki H, Nobuyuki T, Hideo O, et al.Analyses of intramoleculardisulfide bonds in proteins by polyacrylamide gel electrophoresis following two-stepalkylation[J] .Anal Biochem, 1988,168:193-201.).As Fig. 4, and under the irreducibility situation (DTT-, lane 2 and lane 4), the recombinant protein mobility is close, and no AMS combination is described; Have situation (lane 1 and lane 4) at AMS, the travelling speed of reductibility is slower, and the AMS combination has been described.Comprehensive heavier histone can be concluded through becoming renaturation in activity identification non-reduced and reduction electrophoretic migration situation of SDS-PAGE and recombinant protein, form disulfide linkage at light, variable region of heavy chain.

, recombinant protein shows that than single-chain antibody and disulfide linkage antibody target superantigen molecule, recombinant protein is to the combination active unaffected (Fig. 5 A) of liver cancer cell SMMC-7721 2.5 detecting in conjunction with active and Detection of Stability ELISA method.Aspect stable, recombinant protein can be in 37 ℃ of PBS 9 days in conjunction with active unaffected (Fig. 5 B), and it can also be in 5mol/L urea concentration and 56 ℃ of water-baths, preserve in the 30min 50% in conjunction with active (Fig. 5 C, D).

Show that recombinant protein is still preserved higher cell killing activity 2.6 the MTS method that detects the recombinant protein cell killing activity detects, its 50% tumor suppression concentration is (IC ₅₀) be 5.86 * 10 ^-10Mol/L is a little more than natural superantigen SEA (IC ₅₀1.169 * 10 ^-10Mol/L), with document report similar (Dohlsten M, lando PA, Bjork P, et al.Immunotherapy of human colon cancer by antibody-targetedsuperantigens[J] .Cancer Immunol Immunoher, 1995,41:162-168.).Illustrate, do not influence the activity of the superantigen of its fusion with strand disulfide linkage antibody formation as targeted molecular.

Claims (6)

1. superantigen with target function, comprise targeting moiety and superantigen part, it is characterized in that targeting moiety is the genetic engineering antibody that can come from the Hab25 monoclonal antibody with the hepatocellular carcinoma bonded, partly for coming from staphylococcus and streptococcic toxin superantigen, the two adopts gene fusion to be connected to superantigen.

2. the superantigen with target function according to claim 1 is characterized in that described genetic engineering antibody is that single-chain antibody scFv, disulfide linkage are stablized single-chain antibody ds-scFv or disulfide stabilized antibody dsFv.

3. the superantigen with target function according to claim 1 is characterized in that described superantigen is staphylococcal enterotoxin A, staphylococcal enterotoxin B, staphyloentero-toxin C, staphylococcal intoxication shock toxin or its mutant.

4. according to each described superantigen of claim 1-3, it is characterized in that described genetic engineering antibody fusion is positioned at the superantigen front end, adopts the GGGSGGS connection peptides in the middle of the two connects with target function.

5. the described preparation method with superantigen of target function of claim 1 may further comprise the steps:

(1) makes up the recombinant expression vector that comprises Hab25 genetic engineering antibody and superantigen gene fragment;

(2) with the recombinant expression vector transformed into escherichia coli of above-mentioned structure, and abduction delivering;

(3) with the expression product purifying, obtain fusion rotein.

6. each described superantigen application in the anti-liver cancer immunotherapy medicaments of preparation of claim 1-3 with target function.

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