CN1241574A - Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody - Google Patents
Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody Download PDFInfo
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- CN1241574A CN1241574A CN 98112905 CN98112905A CN1241574A CN 1241574 A CN1241574 A CN 1241574A CN 98112905 CN98112905 CN 98112905 CN 98112905 A CN98112905 A CN 98112905A CN 1241574 A CN1241574 A CN 1241574A
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Abstract
The present invention relates to gene engineering technology. Technologically,the present invention features that hybrid tumor technology is first utilized to prepare mouse-originating monoclone antibody HAb25 with specific combination activity to human liver cancer cells; and HAb25 is re-constructed through gene engineering process into liver cancer resisting single stranded antibody scFv25 and liver cancer resisting bifunctional antibody pBV220-scFv25-PE40 and pGEX4T-1-scFv25-TNFa. The liver cancer resisting gene engineering antibody has the features of small molecular weight, strong penetration capacity, high molecule stability, low toxic side effect, high efficiency,easy mass production, etc., and thus has great latent clinical application capability.
Description
The present invention relates generally to genetically engineered.
According to the statistics of the World Health Organization, the dead and liver cancer of ten thousand patients surplus the whole world just has 100 every year, and the sickness rate of China's liver cancer accounts for global 40%.At present external Shouval and Dunk etc. and domestic Xie Hong etc. have reported liver cancer-resisting monoclone antibody, and the report of anti-ferritin monoclonal antibody, some monoclonal anti body and functions are wherein also arranged simultaneously
131Behind the I mark, carried out effective clinical liver cancer radio-immuno-image.Yet for genetic engineering antibody, though existing reports such as the genetically engineered of tumours such as resistive connection intestinal cancer, mammary cancer mouse/people's chimeric antibody, single-chain antibodies, the genetic engineering antibody of anti-liver cancer has not yet to see report both at home and abroad.
Monoclonal antibody is used for tumor treatment research already, but along with going deep into of studying, mouse monoclonal antibody exposes many weak points, mainly is: 1. the monoclonal antibody molecule amount is big, and penetrativity is low, is difficult for penetrating the tumour capillary vessel; 2. mouse monoclonal antibody immunogenicity height, human body are used and can be produced human antimouse antibody (HAMA), have limited its repeated use.
The objective of the invention is to utilize genetically engineered that monoclonal antibody is transformed into single-chain antibody, be used to overcome above-mentioned shortcoming, further make up bifunctional antibody and given antibody new function again, make it have the guiding killing activity, this makes has pushed ahead again a step the diagnosis of liver cancer and treatment research.
Technical scheme of the invention process
One. the preparation of liver cancer-resisting monoclone antibody HAb 25:
Fresh primary hepatocyte hepatocarcinoma sample preparations liver cancer cell suspension with surgical resection is the immunogen immune BALB/C mice, adopts the peritoneal immunity approach, has selected peritoneal macrophage or thymocyte as feeder cell.With mouse boosting cell after the immunity and SP2/0 cytogamy, obtain hybridoma, carry out the immunohistochemical method screening with paraffin section, all and normal liver cell paraffin section does not react and cuts into slices being kept of reaction with liver cancer, obtain the liver cancer-resisting monoclone antibody HAb 25 hybridoma cell strain of sustainable secretory antibody, the HAb25 hybridoma cell strain is inoculated in mouse peritoneal, collect ascites, albumen obtains the HAb25 activated protein through ammonium sulphate precipitation and cation normal-pressure liquid chromatography purifying in the ascites.HAb25 proves to have stronger specificity and higher avidity after testing, is in particular in:
1. hepatocellular carcinoma paraffin section positive reaction rate is 84% (79/94), with all livers of cancer, hepatitis, hepatic hemangioma, hepatic cyst and hepatic metastases knurl no cross reaction, has only 5.7% (3/53) cross reaction with liver cirrhosis.
2. behind colloid gold label, at 4 ℃ down and liver cancer cell effect after 1 hour, cultivate down after 0,5,10,20,30,60,120 minute for 37 ℃, electricity under Electronic Speculum, observe antibody by pinocytosis, lining cave in, fine hair fracture engulf, glycocalyx caves in and directly the diffusion path internalization to liver cancer cell
3.HAb25 warp
131Behind the I mark, inject lotus people liver cancer nude mice, 72 hours can be in the clear video picture of tumor locus (ECT), and the T/N value is 6.84.
4.HAb25 warp
131Injecting 5 with 3-5mci/ people behind the I mark, to doubt be the patient of liver cancer, but wherein 4 examples video picture under ECT.The immunoconjugate of HAb25 and medicine has certain therapeutic action to lotus people liver cancer nude mice.
Two, anti-liver cancer single-chain antibody (scFv
25) preparation:
Extract total RNA from the HAb25 hybridoma, reverse transcription synthesize cDNA, and the RT-PCR method amplifies light, the heavy chain variable region gene of monoclone antibody HAb 25, and (VL VH), is the IgV district gene of rearrangement by analysis.VL and VH are coupled together by the polypeptide Linker of synthetic, form the active antibody molecule that a molecular weight has only whole antibody 1/6.Specify as follows:
1. get the strain of HAb25 hybrid cell, extract total RNA, be the synthetic cDNA of primer reverse transcription through Oligo (dT), add light, heavy chain primer and go out VL and VH through pcr amplification, be cloned into the pUC19 plasmid vector and carry out sequential analysis, compare, higher homology is arranged with known IgV sequence, and the definitive variation of new antibodies arranged, successful acquisition light, heavy chain variable region gene VL, the VH of HAb25.
2. the structure of single-chain antibody: the small peptide Linker with synthetic is cloned into the pUC19 cloning vector earlier, be built into pUC19-Linker, further VL is cloned into pUC19-Linker, be built into pUC19-Linker-VL, again VH is cloned into pUC19-Linker-VL, be built into pUC19-VH-Linker-VL, anti-liver cancer single-chain antibody cloning vector pUC19-scFv promptly recombinates
25, then with scFv
25Subclone is gone into pET15b, is built into the recombinant prokaryotic expression vector pET15b-scFv of anti-liver cancer single-chain antibody
25
3.scFv
25Abduction delivering: the reorganization pET15b-scFv
25Prokaryotic expression carrier host bacterium is through IPTG (isopropylthio-) abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → be further purified obtains anti-liver cancer single-chain antibody scFv
25Activated protein.
4.scFv
25Determination of activity: scFv
25With add parental antibody HAb25 behind the SMMC-7721 liver cancer cell effect blocking antigen, add the sheep anti-mouse igg of FITC mark after the effect, compare with parental antibody self and irrelevant antibody.The cells were tested by flow cytometry result shows, scFv
25Can seal the affine activity of parental antibody 53%.
Advantage: the single-chain antibody molecular weight is little, penetrativity is strong, immunogenicity is low, the transformation period is short, circulation of blood and whole body are cleaned up advantages such as fast and easy genetically engineered mass production, is a kind of genetic engineering antibody that has very much the clinical application potentiality.
Three, the preparation of anti-liver cancer single-chain bifunctional antibody:
Utilize genetic engineering means will have liver-cancer cell specific in conjunction with active HAb25 single-chain antibody and PE40 (false pseudomonas bacillus extracellular toxin) or huamn tumor necrosis factory alpha (TNF α, the tumor cytotoxicity factor of the immune species specificity that produces by monocyte and scavenger cell) gene coupling, make up anti-liver cancer bifunctional antibody, and be cloned into prokaryotic expression carrier and express.
1.pBV220-scFv
25The structure of the anti-liver cancer monochain immunotoxin of-PE40 is expressed and cytotoxicity
To keep parental antibody HAb25 to liver-cancer cell specific in conjunction with active scFv
25Gene excises the coupling of the amino acid whose muton PE40 of 4-252 gene with PE, makes up anti-liver cancer monochain immunotoxin scFv
25-PE40 is cloned into prokaryotic expression carrier pBV220 then, is built into reorganization pBV220-scFv
25The anti-liver cancer monochain immunotoxin of-PE40 prokaryotic expression carrier.Its concrete implementation step is as follows:
(1) structure of anti-liver cancer monochain immunotoxin
Be built into pBV220scFv
25, and then the PE40 gene clone gone into pBV220scFv
25In, make the PE40 gene be next to scFv
253 ' end, be built into pBV220scFv
25-PE40 prokaryotic expression carrier.
(2) pBV220scFv
25The abduction delivering of-PE40 and purifying
Reorganization pBV220scFv
25-PE40 expression vector is expressed through 42 ℃ of thermal inductions, collects thalline and extracts inclusion body, and again through sex change, renaturation is further purified, and obtains purity and reaches electrophoretically pure anti-liver cancer monochain immunotoxin scFv
25-PE40.
(3) anti-liver cancer monochain immunotoxin scFv
25The immunocompetence of-PE40 is measured
With scFv
25-PE40 is one anti-, is two anti-with mouse-anti PE40 antibody, is three anti-with the sheep anti-mouse igg of FITC mark, and HCC-9204 liver cancer cell smear is carried out indirect IF staining, and the result shows, scFv
25-PE40 can combine with liver cancer cell specificity.
(4) cytotoxicity experiment:
Carry out anti-liver cancer scFv by mtt assay
25-PE40 is to the toxicity test of HCC-9204 cell, and the result shows that anti-liver cancer scFv-PE40 has clear and definite selective killing effect to liver cancer cell.
2. pGEX 4T-1-scFv recombinates
25The structure of the anti-liver cancer single-chain bifunctional of-TNF α antibody is expressed and the targeted therapy effect
(1) pGEX 4T-1-scFv is gone in TNF α gene clone
25, make up reorganization pGEX 4T-1-scFv
25-TNF α prokaryotic expression carrier.
(2) anti-liver cancer scFv
25The purifying of-TNF α: reorganization pGEX 4T-1-scFv
25-TNF α host bacterium is through the IPTG abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → GST affinitive layer purification → zymoplasm digests and is further purified, and obtains anti-liver cancer bifunctional antibody scFv
25-TNF alpha active albumen.
(3) the indirect IF staining result shows, scFv
25-TNF α has kept the specific binding activity of parental antibody preferably; MTT tests demonstration, scFv
25-TNF α is obvious to the cytotoxicity of liver cancer cell SMMC-7721; The targeted therapy experiment of carrying out in lotus people liver cancer nude mouse shows scFv
25-TNF α can make the liver cancer enclosed mass of 2mm diameter alleviate fully, so scFv
25-TNF α has bigger clinical application potentiality.
Anti cancer gene engineering recombinant immunotoxin has following characteristics: molecular weight is little, is easy to penetrate blood vessel and enters tumor center and play a role, and anti-knurl is renderd a service higher; Exist with the fusion rotein form, stability of molecule is good; Owing to got receptor binding domains except that toxin, only kept the bio-toxicity part, effectively reduce toxic side effect; Recombinant immunotoxin also has easily by the mass-produced characteristics of genetically engineered.Above-mentioned work is that further fundamental research and clinical application are laid a good foundation.
Claims (1)
1. liver cancer-resisting monoclone antibody HAb 25 and single-chain antibody thereof and bifunctional antibody is characterized in that:
(1) liver cancer-resisting monoclone antibody HAb 25: the liver cancer cell suspension with the fresh primary hepatocyte hepatocarcinoma sample preparations of surgical resection is the immunogen immune BALB/C mice.With mouse boosting cell after the immunity and SP2/0 cytogamy, obtain hybridoma, hybridoma screens with immunohistochemical method, obtain the liver cancer-resisting monoclone antibody HAb 25 hybridoma cell strain of sustainable secretory antibody, this cell strain is inoculated in mouse peritoneal, collect ascites, albumen obtains the HAb25 activated protein through ammonium sulphate precipitation and cation normal-pressure liquid chromatography purifying in the ascites.
(2). anti-liver cancer single-chain antibody (scFv
25):
1. light, the heavy chain variable region gene of HAb25 (VL, clone VH): extract total RNA from above-mentioned hybridoma, reverse transcription synthesize cDNA, the RT-PCR method amplifies light, the heavy chain variable region gene of monoclone antibody HAb 25, is the IgV district gene of rearrangement by analysis.
2. anti-liver cancer single-chain antibody scFv
25Structure: earlier the small peptide Linker with synthetic is cloned into the pUC19 cloning vector, be built into pUC19-Linker, further VL is cloned into pUC19-Linker, be built into pUC19-Linker-VL, again VH is cloned into pUC19-Linker-VL, be built into pUC19-VH-Linker-VL, anti-liver cancer single-chain antibody cloning vector pUC19-scFv promptly recombinates
25, then with scFv
25Subclone is gone into pET15b, is built into the recombinant prokaryotic expression vector pET15b-scFv that molecular weight has only the anti-liver cancer single-chain antibody of whole antibody 1/6
25
3. pET15b-scFv
25Abduction delivering: the reorganization pET15b-scFv
25Prokaryotic expression carrier host bacterium is through IPTG (isopropylthio-) abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → be further purified obtains anti-liver cancer single-chain antibody scFv
25Activated protein.
(3) anti-liver cancer bifunctional antibody:
1. anti-liver cancer monochain immunotoxin (scFv
25-structure PE40), expression: pUC19-scFv is gone in the PE40 gene clone
25In, be built into pUC19-scFv
25-PE40 anti-liver cancer monochain immunotoxin the cloning vector of recombinating, and then with scFv
25-PE40 subclone is gone among the prokaryotic expression carrier pBV220, is built into pBV220-scFv
25-PE40, anti-liver cancer monochain immunotoxin prokaryotic expression carrier pBV220-scFv promptly recombinates
25-PE40.Above-mentioned carrier host bacterium is through 42 ℃ of abduction deliverings, and collection thalline → extraction inclusion body → sex change → renaturation → be further purified obtains anti-liver cancer monochain immunotoxin scFv
25-PE40 activated protein.
2. anti-liver cancer bifunctional antibody (scFv
25-TNF α) structure, expression: pUC19-scFv is gone in TNF α gene clone
25In, be built into pUC19-scFv
25-TNF α anti-liver cancer bifunctional antibody the cloning vector of recombinating, and then with scFv
25-TNF α subclone is gone among the prokaryotic expression carrier pGEX 4T-1, is built into pGEX4T-1-scFv
25-TNF α, anti-liver cancer single-chain bifunctional antibody prokaryotic expression carrier pGEX 4T-1-scFv promptly recombinates
25-TNF α.Above-mentioned carrier host bacterium is through the IPTG abduction delivering, and collection thalline → extraction inclusion body → sex change → renaturation → GST affinitive layer purification → zymoplasm digests and is further purified, and obtains anti-liver cancer bifunctional antibody scFv
25-TNF alpha active albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98112905 CN1241574A (en) | 1998-07-02 | 1998-07-02 | Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98112905 CN1241574A (en) | 1998-07-02 | 1998-07-02 | Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody |
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|---|---|
| CN1241574A true CN1241574A (en) | 2000-01-19 |
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|---|---|---|---|
| CN 98112905 Pending CN1241574A (en) | 1998-07-02 | 1998-07-02 | Liver cancer-resisting monoclone antibody HAb25 and its single stranded antibody and bifunctional antibody |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1694700A1 (en) * | 2003-11-11 | 2006-08-30 | National Cancer Center | Neutralizable epitope of hgf and neutralizing antibody binding to the same |
| CN100410274C (en) * | 2006-04-21 | 2008-08-13 | 暨南大学 | Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses |
| CN101537180B (en) * | 2002-07-18 | 2016-02-10 | 莫鲁斯有限公司 | The recombinant production of mixtures of antibodies |
| CN107001481A (en) * | 2014-07-22 | 2017-08-01 | 奥隆制药 | By making method that the antibody with completely immune globulin form is positioned in cytoplasm by antibody penetration cell film and application thereof |
| US10787487B2 (en) | 2018-06-21 | 2020-09-29 | Orum Therapeutics Inc. | Cell/tissue-specific cell-penetrating antibodies |
| US10851177B2 (en) | 2014-07-22 | 2020-12-01 | Orum Therapeutics Inc. | Method for inhibiting intracellular activated RAS using intact immunoglobulin-type antibody having cytosol-penetrating ability and use thereof |
| US11155641B2 (en) | 2016-05-27 | 2021-10-26 | Orum Therapeutics Inc. | Cytosol-penetrating antibody and use thereof |
-
1998
- 1998-07-02 CN CN 98112905 patent/CN1241574A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101537180B (en) * | 2002-07-18 | 2016-02-10 | 莫鲁斯有限公司 | The recombinant production of mixtures of antibodies |
| EP1694700A1 (en) * | 2003-11-11 | 2006-08-30 | National Cancer Center | Neutralizable epitope of hgf and neutralizing antibody binding to the same |
| EP1694700A4 (en) * | 2003-11-11 | 2007-10-31 | Nat Cancer Ct | Neutralizable epitope of hgf and neutralizing antibody binding to the same |
| AU2004287743B2 (en) * | 2003-11-11 | 2008-10-09 | National Cancer Center | Neutralizable epitope of HGF and neutralizing antibody binding to the same |
| CN100410274C (en) * | 2006-04-21 | 2008-08-13 | 暨南大学 | Specific anti-human liver cancer phage single-chain antibody HscFv4-16 gene and its uses |
| CN107001481A (en) * | 2014-07-22 | 2017-08-01 | 奥隆制药 | By making method that the antibody with completely immune globulin form is positioned in cytoplasm by antibody penetration cell film and application thereof |
| US10844136B2 (en) | 2014-07-22 | 2020-11-24 | Orum Therapeutics Inc. | Method for positioning, in cytoplasm, antibody having complete immunoglobulin form by penetrating antibody through cell membrane, and use for same |
| US10851177B2 (en) | 2014-07-22 | 2020-12-01 | Orum Therapeutics Inc. | Method for inhibiting intracellular activated RAS using intact immunoglobulin-type antibody having cytosol-penetrating ability and use thereof |
| US11155641B2 (en) | 2016-05-27 | 2021-10-26 | Orum Therapeutics Inc. | Cytosol-penetrating antibody and use thereof |
| US10787487B2 (en) | 2018-06-21 | 2020-09-29 | Orum Therapeutics Inc. | Cell/tissue-specific cell-penetrating antibodies |
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