CN105886515A - Light and heavy chain variable region genes of monoclonal antibody H103 in specific binding with hypoxic liver cancer and applications of light and heavy chain variable region genes - Google Patents
Light and heavy chain variable region genes of monoclonal antibody H103 in specific binding with hypoxic liver cancer and applications of light and heavy chain variable region genes Download PDFInfo
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Abstract
The invention relates to light and heavy chain variable region genes of a monoclonal antibody H103 in specific binding with hypoxic liver cancer and applications of the light and heavy chain variable region genes, in particular to the light and heavy chain variable region genes of the monoclonal antibody H103 in specific binding with the human liver cancer anoxia phenotype and polypeptide coded by the genes, and the applications of the genes and the polypeptide in preparing medicines for realizing accurate diagnosis and targeted therapy on hypoxic liver tumor. According to the invention, the difference panning technology combining the normoxia negative deducting screen with the hypoxic positive selection is adopted by the inventor, and the light and heavy chain variable region genes of the antibody H103 in specific binding with hypoxic liver cancer cells is successfully cloned from the self-established human phage single-chain antibody library. The obtained light and heavy chain variable region genes can be used for coding the correct completely humanized antibody variable region. Based on the cloned light and heavy chain variable region genes of the monoclonal antibody H103 in specific binding with the human liver cancer anoxia phenotype, a variety of micromolecular genetically engineered antibodies, such as single-chain antibodies, chimeric antibodies and Fab antibodies, can be constructed and expressed by adopting the genetic engineering method, so that the purpose of accurate diagnosis and targeted therapy on the hypoxic phenotype liver tumor can be achieved.
Description
Technical field
The present invention relates to the monoclonal antibody H103 heavy chain of specific bond weary oxygen hepatoma carcinoma cell and chain variable region gene and by described gene code
Polypeptide, and described gene and polypeptide are in the application prepared in weary oxygen Diagnosis of Liver Neoplasm and targeted therapy.
Background technology
Hepatocarcinoma is China and the frequently-occurring disease of south east asia and commonly encountered diseases.Especially in China, there are about 130,000 people every year and die from this disease, account for full generation
The 42% of boundary's total death toll of annual hepatocarcinoma.In recent years, the trend that hepatocarcinoma is also gradually increased at the sickness rate of China.Hepatocarcinoma have onset concealment,
Incubation period length, high malignancy, the feature such as progress is fast, aggressive is strong, easy relapse and metastasis, poor prognosis, the early diagnosis of hepatocarcinoma and clinical treatment curative effect
It is not very good.
Tumor dormancy cell-stimulating is the main contributor causing liver cancer recurrence and transfer.Tumor dormancy phenomenon is the most universal, many dormancy
Primary carcinoma or transfer cancer often appear as cancerous tissue or cell weary oxygen feature in various degree.For liver cancer patient, many people are primary liver
In carcinectomy some months the most a few week, there is micro-/little metastasis in whole liver, lung or skeleton many places.The tumor cell of this dormancy is fast after being activated
Speed propagation, thus cause recurrence and transfer, the state of an illness to deteriorate rapidly.Therefore, during Clinical Processing primary hepatic carcinoma, should only not consider traditional diagnosis and control
Treatment scheme, also to consider precisely detection and targeted therapy to micro-/little metastasis dormancy hepatoma carcinoma cell from the feature of the weary oxygen of cancerous cell.The key of problem
Being, Tumor dormancy cell is that a group quantity seldom and is difficult to the akinete detected, and up to now, the most still lacks a species specific cell table
Tumor cell or the normal tissue cell of Tumor dormancy cell with fast breeding can be separated by face marker molecule.Due to Tumor dormancy cell usually position
In cancer nests weary oxygen microenvironment, the discovery of tumor hypoxia biomarker can qualitative and Tumor dormancy cell physical presence situation in antimer quantitatively,
But it finds prepared by the guide frequently relying on its specific binding antibody.Therefore, the present invention cultivates deduction screening by the normal oxygen of cell, in conjunction with weary oxygen
Cultivation forward screens, and from phage antibody library, preparation can the specific antibody of specific bond hepatocarcinoma weary oxygen rest cell.Utilize standby weary of this project system
Oxygen specific antibody, not only can resolve the molecular marker of hepatocarcinoma rest cell surface specific, also can be further combined with other routine clinical detection hands
Section, it is achieved precisely prediction micro-/little transfer of hepatocarcinoma, judging prognosis existence, assessment clinical treatment (such as immunization therapy) effect and diagnosis microresidual disease
(MRD)；It addition, be also expected the anti-relapse and metastasis treatment for carrying out accurate liver cancer targeting rest cell from now on to provide powerful.
Summary of the invention
It is an object of the present invention to provide heavy chain and the chain variable region gene of monoclonal antibody H103 of a kind of weary oxygen specific bond, in order to
Specific recognition can be given expression to after both restructuring and combine the antibody activity fragment of weary oxygen hepatoma carcinoma cell.
Another object of the present invention is to described gene and polypeptide preparation answering in precisely diagnosis and targeted therapy weary oxygen liver neoplasm medicine
With.
Another object of the present invention is to described gene and polypeptide preparation answering in precisely diagnosis and targeted therapy weary oxygen liver neoplasm medicine
With.
According to an aspect of the present invention, the monoclonal antibody H103 heavy chain and the light chain that the present invention relates to a kind of specific bond weary oxygen hepatoma carcinoma cell can
Become district's gene (sequence has been filed on GeneBank, serial number: KP347981.1).The hepatocarcinoma weary oxygen specific antibodies H103 heavy chain of the present invention is with light
Chain variable region gene is clone from the full Large human naive scFv phage library oneself built.Wherein said heavy chain variable region gene total length is 399bp,
Its nucleotide sequence is as shown in sequence table<400>1, and the aminoacid sequence of its coding is as shown in sequence table<400>3.Described light chain variable
District's full length gene is 366bp, its nucleotide sequence as shown in sequence table<400>2, its coding aminoacid sequence such as sequence table in<400>4
Shown in, can be used for expression specificity identification after two gene recombinaton and combine the antibody activity fragment of weary oxygen liver tumor cells.
According to another aspect of the present invention, the invention still further relates to described gene and polypeptide in preparation for precisely diagnosis and targeted therapy weary oxygen liver
Application in tumour medicine.
The present inventor collects and is extracted 9 example Patients with Primary PMBC, utilizes designed, designed primer queue Successful amplification to go out a complete set of people antibody VH
With VL variable region gene be assembled into scFv Antibody geometric mean titer.Turn through 6 batch electricity, it is established that humanized's immunity phage antibody library,
Storage capacity is 2 × 107, it is 70% that Random clones plasmid enzyme restriction shows that its scFv gene is correctly inserted into rate, and Random clones order-checking shows scFv in original storehouse
Antibody VH with VL corresponding homology germline gene multiformity is preferable；Titre after saving the original antibodies storehouse built reaches as high as 6 × 1010pfu L-1,
His-tag Western shows that it has preferable scFv antibody and presents ability to express.After redemption, phage presents antibody library to take turns normal oxygen through 4 negative
After deduction screening combines the naughty sieve of difference that the weary oxygen positive selects, the combination accumulation rate of positive colony weary oxygen hepatoma carcinoma cell adds about 460 times, scFv
Insertion rate last take turns and increase to 98%.Flow cytometry examination finally determines can tie with HCCLM3 cell (high metastatic potential hepatoma cell strain) weary oxygen
The positive candidate clone closed has 7.Wherein, H103 clone strain presents strong positive combination.The HB2151 bacterium expression product of H103 clone strain gene
Show that through SDS-PAGE electrophoresis the pericentral siphon chamber product mainly protein band Han 27kD of induction accounts for total protein 60%.Western blot analyzes card
This band real is the single-chain antibody with His-tag label.Through HPLC after purification, target antibody concentration is H103 antibody cloning expression product
0.16mg/mL.Flow cytometry Immunofluorescence test shows the various kinds of cell strains such as hepatoma carcinoma cell HCCLM3 that H103 single-chain antibody can cultivate with weary oxygen
Strong positive specific bond, and only have the faint combination of trace with normal oxygen cultivation hepatoma carcinoma cell and non-tumor cell.Liver cancer tissue dyeing is shown by SABC,
H103 single-chain antibody is specifically combined in the Fa Yang cancer nests district of liver cancer tissue, and it combines distribution mainly at the tumor tissue that blood capillary is deficient.
Monoclonal antibody H103 based on the above-mentioned specific bond being cloned into weary oxygen human liver cancer cell is light, heavy chain variable region gene, can use
Gene engineering method, builds and expresses multiple little molecular gene engineered antibody, such as single-chain antibody, chimeric antibody, Fab antibody etc., in order to for liver
Precisely diagnosis and the targeted therapy purpose of the weary oxygen of cancer.
Accompanying drawing explanation
Fig. 1 is to utilize the deduction screening of normal oxygen feminine gender to combine the difference naughty sieve principle signal that the weary oxygen positive selects from phage antibody library.
Fig. 2 is to identify that self-built human single chain variable fragments antibody phage presents storehouse.Wherein, A is to utilize Random clones in sequencing primer PCR amplification bank
Agar grain results of weak current, B is light, the heavy chain variable region gene multiformity of antibody library constructed by IMGT people's online sequence alignment analysis of IG germline gene.
Fig. 3 is by identifying the expression of H103 single-chain antibody screened, purification and weary oxygen binding characteristic.Wherein, A is Coomassie brilliant blue dyeing
H103 single-chain antibody is prokaryotic expression product and the result of affine female purification thereof in BL21 (DE3) bacterium, and B is that flow cytometry compares H103 strand
The antibody normal oxygen on different hepatoma cell strains and weary oxygen association index.
Fig. 4 is that laser co-focusing analyzes the H103 single-chain antibody weary oxygen binding specificity to hepatoma carcinoma cell.
Fig. 5 is the immunohistochemical staining analysis on dissimilar liver cancer tissue of H103 single-chain antibody.Wherein, A is Isotype control single-chain antibody E4B7S
On normal liver tissue dye, B, C, D, E, F be followed successively by H103 single-chain antibody normal liver tissue, low differentiation, differentiated, middle differentiation and in
Differentiation non-tumor tissue coloration result around.
Fig. 6 is the immunohistochemical staining analysis in liver cancer tissue different blood vessel distributed areas of the H103 single-chain antibody.A and B is low differentiation, C and D
For middle differentiation, E and F differentiated；A, C and E are that blood capillary enriches district, and B, D and F are without blood capillary district.
Fig. 7 is the Sequencing chromatogram of weary oxygen specific bond single-chain antibody H103 chain variable region gene.
Fig. 8 is the Sequencing chromatogram of weary oxygen specific bond single-chain antibody H103 heavy chain variable region gene.
Detailed description of the invention
Anti-human liver cancer weary oxygen specific bond monoclonal antibody H103 is light, the screening of heavy chain variable region gene and clone.
Institute's employment single-chain antibody library is that the antibody diversity that Nankai University's medical college Zhang Sihe laboratory uses traditional banking process to set up is good,
The full people source immunologic pattern phage antibody library that storage capacity is high, in storehouse, contained antibody molecule hypotype is mainly IgG and IgM.
Large Copacity people source scFv antibody library builds:
Patients with Primary (non-row surgical resection or transplantation immunity suppression) fresh peripheral blood PMBC is extracted with lymphocyte separation medium,
Trizol cell lysis, chloroform-isopropanol-dehydrated alcohol method extracts total serum IgE.With the cDNA of reverse transcription as template, design synthesis human IgG resists
Body germline variable region gene complete set primer also introduces Sfi I, Not I restriction enzyme site, and PCR expands human IgG VH, VL gene bank.Enzyme action gel
It is assembled into ScFv gene with (Gly4Ser) 3-linker genetic fragment through SOE-PCR after purification and is recombined into pCANTAB 5his plasmid.Glycogen sinks
Forming sediment and link product, electricity is coated with SOB-GT flat board after turning X11-Blue competence bacteria and cultivates the people source scFv phage antibody library i.e. obtaining bacterial versions.
Estimating antibody library storage capacity by gradient dilution, Sfi I+Not I double digestion identifies scFv gene recombinaton insertion rate, utilizes after Random clones order-checking
Ku Zhong antibody gene family distribution multiformity analyzed by V-BASE data base tool bag.
Often the screening of oxygen deduction and weary oxygen forward screen:
Antibody library take normal oxygen deduction screening and weary oxygen forward screen alternately: each take turns wash in a pan sieve time, be initially charged " input " scFv of redemption
Storehouse is cultivated to normal oxygen and is deducted screening on HCCLM3 cell, then the HCCLM3 hepatoma carcinoma cell that unconjugated secondary storehouse adds the cultivation of weary oxygen is just being carried out
To screening.Determine each take turns wash in a pan sieve for effectively enrichment on the basis of, repeatedly wash in a pan and be sieved to finally combine enrichment times more than more than 200 times.Whole naughty sieve
After end, 100 single bacterium colonies of random picking are carried out saving, expanding and prepare supernatant by preceding method.Weary oxygen is cultivated HCCLM3 cell use
2XPBS/EDTA gentleness digests, and it is resuspended that PBS/0.5%BSA rinses closing, carries out adding biotinylated after low temperature concussion combines with saving supernatant
Anti-M13 phage envelope protein antibody and Streptavidin-Alexa Fluor 488 4 DEG C are hatched, and up flow type analysis determines the positive of candidate
In conjunction with clone.
Weary oxygen specific bond scFv antibody-soluble is expressed and CHARACTERISTICS IDENTIFICATION:
Strong positive combining clone's H103 ScFv gene cloning and enters pET28a Plastid transformation BL21 (DE3) bacterium, line is transferred 30 DEG C after choosing clone
Abduction delivering 14h.Extract bacteria periplasm chamber expression product, HiTrap Anti-E-Tag post affinity purification specificity scFv.Use HiTrap again
After Desalting post will change buffer, scFv antibody prepared by SDS-PAGE purification Identification, its purity of gel imaging scanning analysis, Bradford
Method measures antibody protein concentration.The HCCLM3 hepatoma carcinoma cell finally cultivated with the fixing normal oxygen of 4%PFA 4 DEG C and weary oxygen, with the scFv antibody of purification
Be one resist, mouse-anti E-Tag be two resist, FITC-goat anti-mouse IgG be three resist, DAPI redyes karyon and does Immunofluorescence test, laser co-focusing
Basis of microscopic observation is taken pictures.It is that an anti-ABC method is carried out that immunohistochemical staining uses based on single-chain antibody, the wherein H103 antibody thinner ratio of purification
Being 1: 10, the anti-his tag antibody thinner ratio of biotin crosslinking is 1: 50, and avidin DH and biotin HRP is from Vectastain Elite
ABC kit, concrete operations illustratively step is carried out.
Weary oxygen specific bond H103 monoclonal antibody is light, heavy chain variable region gene diagnoses and targeted therapy weary oxygen liver neoplasm for accurate in preparation
In application:
The research of Hepatoma m Abs application aspect is concentrated mainly on the application in hepatocarcinoma cell HepG2 is studied of (1) Hepatoma m Abs: monoclonal antibody
Development provides one and finds new tumor associated antigen or the effective means of tumor antigen novel site.These antigens can be used for clinical serum and check, tool
There is certain diagnosis and evaluate the meaning of therapeutic effect.(2) Hepatoma m Abs targeted drug research: the radio immuno imaging diagnosis with monoclonal antibody as carrier, guiding
Chemotherapy and guiding radiotherapy achieve substantial progress in recent years.Mainly have: 1. chemicals-monoclonal antibody cross-linking agent；2. cytotoxin-monoclonal antibody cross-linking agent；
3. radionuclide-monoclonal antibody cross-linking agent etc..
With light, protein drug that heavy chain variable region gene reconstructs certain forms of the present invention, can be directly used for the accurate diagnosis of liver neoplasm
And targeted therapy.It addition, using light, the polypeptide of heavy chain variable region gene coding of the present invention and derivant thereof as carrier, cell toxicant in crosslinking
Property medicine, toxin, radionuclide, enzyme and biological response modifier etc. as targeted drug it can also be used to weary oxygen has related disorders to include but not limited to
The diagnosis of liver neoplasm and treatment.
From light, the polypeptide of heavy chain variable region gene coding of the present invention, the novel antibodies of the various ways that can reassemble into, mainly include
But it is not limited to following several form: (1) small molecular antibody.Mainly by being formed Fab antibody by VH-CH1 and VL-C1, with a polypeptide (GLy4Ser)
3 joints connect VH gene and the single-chain antibody of VL gene, are formed Fv fragment antibody, by VH or VL by VH and VL with non-covalent bond combination
The single domain antibody of one functional domain composition, the atom etc. being made up of single CDR.(2) multivalence miniantibody.Double-strand is mainly had to resist
Body, (ScFv)2, Flex miniantibody, LD miniantibody, F (ab ')2、(scFv)4Deng.Owing to there being polyvalent antigen binding site, affine
Power is high, and molecular size is moderate, can penetrate tumor tissues, feature that also clearance rate in kidney is slower and there is higher clinical value.
(3) bi-specific antibody.It is the class antibody with dual specificity and dual-use function, also known as bifunctional antibody.(4) recombinant antibodies merges egg
In vain.Genetic fragments such as Fab or Fv, it is connected having of being formed with other protein gene such as the toxin of non-antibody or enzyme and specific biological activity is led
To a kind of recombiant protein of target site.(5) recombinant immunotoxin.Encoding antibody and the gene recombinaton of toxin being produced, feature is efficient, non-spy
Different toxicity is low, internal good stability, easily penetrates tumor and internal use is safer.
With the above-mentioned polypeptide restructuring by light, heavy chain variable region gene coding or derivative antibody molecule as carrier, various anticancer effectors in crosslinking
Matter and the immune conjugate that formed or targeted drug mainly have following several form: (1) antibody-nucleic conjugate.This conjugate can be effective by nucleic
Ground guides tumor tissues local, thus decreases the normal tissue injury caused because of radiotherapy external exposure and relevant untoward reaction, and can carry out tumor
Level diagnosis and targeted therapy.This method is radio-immuno-image and radioimmunotherapy.Have with the conventional nucleic of monoclonal antibody coupling125I,131I,111In,90Y,99Tcm,188Re and186Re etc..(2) Antibody-chemotherapeutic drug conjugate.This conjugate can guide tumor specifically, can lower and align
The often damage of tissue, reduces the toxic and side effects of chemotherapeutics.The chemotherapeutics of often coupling is such as: the phosphoamide in alkylating agent；Ammonia first in antimetabolic
Petrin, 5-fluorouracil；Antibiotics amycin, epirubicin, daunorubicin；Plant vincristine, mitomycin etc..(3) antibody-
Toxin conjugated thing.This conjugate is also known as immunotoxin.It kills cytosis and by force and does not relies on biological auxiliary mechanism.It kills tumor mechanism and puts
Treating, chemotherapy is different, so the tumor of general radiotherapy, chemotherapy effect difference can be used, has a extensive future.Conventional toxin has ricin, diphtheria poison
Element, abrin, saporin, pseudomonas extracellular toxin, streptolysin, perforin etc..(4) antibody-biological response modifier (BRM)
Conjugate.BRM individually regulates the immunocompetence of body and killing tumor cell achieved with preferable curative effect.But due to the internal rear only part of its input
Arriving tumor target position, its lethal effect is not in full use and toxic side effect.By BRM and monoclonal antibody coupling, carry it by monoclonal antibody and arrive target
Position and play kill tumor effect, the effect making these factors is higher, and more single-minded.Conventional BRM has INF and IL-2 etc..(5)
Antibody targeted enzymolysis prodrug.The conjugate of antibody with drug specificity activating enzymes is injected in vivo, after separated in time, injects prodrug so that it is swollen
Tumor position changes into high concentration active anticancer medicine with killing tumor cell.At present, can as prodrug have benzoic acid hydrogen mustard Glutamine Derivatives,
Phosphoric acid Rhizoma Dysosmae Versipellis ethylidene ring, phosphoric acid mitomycin, glucosiduronic acid daunorubicin, amycin, 5-flurocytosine and cephalosporin chlormethine etc..Activating enzymes have
Carboxypeptidase G2, alkali phosphatase, penicillin amidase, beta-lactamase, cytosine deaminase, β-glucosyl enzym and amino peptidase etc..(6)
Immunoliposome or Nano medication complex.MAb is coupled to guidance quality and fat that MAb can be fully combined by the surface of liposome with antigenic specificity
Plastid can wrap up the characteristic of high amount of drug and combine together, the upper multiple Nano medication of embedding, then can improve target-oriented drug and curative effect.
Claims (5)
1. a heavy chain variable region gene for monoclonal antibody H103 of specific bond weary oxygen hepatoma carcinoma cell, it has the sequence of sequence table<400>1.
2., by the polypeptide product of claim 1 gene code, it has the sequence of sequence table<400>3.
3. a chain variable region gene for monoclonal antibody H103 of specific bond weary oxygen hepatoma carcinoma cell, it has the sequence of sequence table<400>2.
4., by the polypeptide product of claim 3 gene code, it has the sequence of sequence table<400>4.
5. the gene described in claim 1 or 3 diagnoses and the application in targeted therapy weary oxygen liver neoplasm for accurate in preparation.
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| CN201610169372.6A CN105886515A (en) | 2016-03-24 | 2016-03-24 | Light and heavy chain variable region genes of monoclonal antibody H103 in specific binding with hypoxic liver cancer and applications of light and heavy chain variable region genes |
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| CN201610169372.6A CN105886515A (en) | 2016-03-24 | 2016-03-24 | Light and heavy chain variable region genes of monoclonal antibody H103 in specific binding with hypoxic liver cancer and applications of light and heavy chain variable region genes |
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Citations (3)
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|---|---|---|---|---|
| US20020161211A1 (en) * | 2000-10-19 | 2002-10-31 | Volkhard Lindner | Compositions, methods and kits relating to REMODELIN |
| CN1381461A (en) * | 2002-03-15 | 2002-11-27 | 陈志南 | Light chain or heavy chain variable region gene of human liver cancer monoclonal antibody HAb18 and its application |
| CN1887901A (en) * | 2006-06-23 | 2007-01-03 | 深圳市安群生物工程有限公司 | Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit |
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|---|---|---|---|---|
| US20020161211A1 (en) * | 2000-10-19 | 2002-10-31 | Volkhard Lindner | Compositions, methods and kits relating to REMODELIN |
| CN1381461A (en) * | 2002-03-15 | 2002-11-27 | 陈志南 | Light chain or heavy chain variable region gene of human liver cancer monoclonal antibody HAb18 and its application |
| CN1887901A (en) * | 2006-06-23 | 2007-01-03 | 深圳市安群生物工程有限公司 | Human tumor M2-type pyruvate kinase antigen determinant polypeptide and antibody and their application in diagnositic kit |
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| STEFAN KRESS ET AL.: "Expression of hypoxia-inducible genes in tumor cells", 《J CANCER RES CLIN ONCOL》 * |
| ZHANG,S.-H.: "Synthetic construct anti-PKM2 scFv anitbody 103 gene，partial cds，ACCESSION：KP347981", 《GENBANK》 * |
| 师敬飞: "M2型丙酮酸激酶(PKM2)的相关研究——TCRγδ配体或肿瘤生物标志物的鉴定", 《中国学位论文全文数据库》 * |
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