CN102443061A - High-functional antibody biological synthesis - Google Patents
High-functional antibody biological synthesis Download PDFInfo
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Abstract
The invention provides high-functional antibody biological synthesis, and particularly provides a novel screening method of a recombinant antibody. The method applies a repeated circulating process, namely gene bank diversification, protein expression and function screening, until a satisfied antibody is obtained. When the antibody is expressed, a multipurpose polypeptide sequence (Tag) is connected to the C end of the antibody; and by the polypeptide sequence, a dipolymer (scFv)2 can be formed by a single-chain antibody (scFv), and the affinity of the antibody is further enhanced. The polypeptide sequence can be used for marking, purifying and detecting the antibody. By adopting the method, certain anti-CEA (carcinoembryonic antigen) single-chain antibodies can be successfully obtained, and one antibody can be directly developed into a diagnosis and treatment reagent. The antibodies can provide a useful CDRs (complementarity determining region) bank, and the required high-activity CEA antibody is produced by virtue of sequence recombination.
Description
The application is that application number is " 200510113739.4 ", and denomination of invention is divided an application for the application for a patent for invention of " high-function antibody biosynthesizing ".
Technical field
The present invention relates to a kind of method for preparing antibody or other protein molecules.
Background technology
Research in recent years shows that antibody can be used as diagnosis and treats the various diseases that comprises cancer
[1]Through chemistry or bio-modification, antibody also can be used as detection, the diagnostic reagent of cancer
[1]Must possess following condition as medical diagnosis on disease, treatment reagent antibodies: the antibody of (1) diagnosis and treatment must have high-affinity, specificity, no cross reaction; (2) antibody as treatment reagent can not cause immunoreation in the body; (3) as intravital diagnosis and treatment antibody must be under 37 ℃ physiological condition non-inactivation, do not degrade, do not have precipitin reaction
[2]
Yet, from animal, obtain antibody and be difficult to reach above-mentioned standard.Then difficulty is higher to obtain human antibody through the animal approach, and process is very complicated.Biotechnology is through avoiding the use of animal at in-vitro screening antibody, and the antibody that makes effective acquisition reach above-mentioned standard becomes possibility.Recombinant DNA technology also can further be developed recombination sequence, thus produce animal the high function high reactivity molecule that can not produce.Owing to avoid the use of animal, human antibody also can be easy to obtain.In addition, biotechnology can design and control screening conditions external, thereby produces required antibody.For example, available high temperature screens constitutionally stable antibody.At present, external biological triage techniques commonly used comprises: phage shows to be held
[3], cell surface shows and to hold
[4,5]And rrna shows and to hold
[6]
The size of gene pool is that can decision produce one of principal element of high function high reactivity molecule.Gene pool is big more, and variety is big more, and the probability that then obtains high function high reactivity molecule is also big more
[7]The present apparent technology of holding is used the fixed gene pool usually, holds technology even if acellular rrna shows, and its gene pool maximum also can only reach 10
13-14Molecule.This gene pool size can only change 10 residues ((32) arbitrarily
10=10
14).Using fixedly, another shortcoming of gene pool is: if gene pool does not contain high function high reactivity molecule, which type of no matter uses show the technology of holding also be difficult to obtain satisfied antibody.The improvement way is that simulating nature is evolved, and sets up a dynamic gene storehouse, and effective voriability and functional screening are carried out simultaneously.
Summary of the invention
The invention provides a kind of method for preparing antibody or other protein molecules, may further comprise the steps:
(a) set up the DNA library;
(b) expressing said DNA library is solvable, free protein;
(c) based on said proteinic function, screening has the active mono-clonal bacterium of combination or group's cloning molecular, obtains the DNA of its encode functional protein matter;
(d) the different dna molecular sequence that obtains in above-mentioned (c) is recombinated, reset and suddenlys change to produce new dna sequence dna;
(e) from (d) the new new dna sequence dna that produces with mix from the different dna moleculars in (c);
Repeat (b) to (e), up to obtaining ideal activity protein.
Particularly, the present invention relates to
1. use following steps to obtain antibody (or other protein molecules):
A) set up the DNA library.
B) the expressible dna library is solvable, free protein.
C) based on protein function, screen mono-clonal bacterium or group's cloning molecular with combined function (Binding activity), obtain the DNA of its encode functional protein matter
D) reorganization, rearrangement and the sudden change the different dna molecular sequence that obtains in above-mentioned (c) carried out are to produce new dna sequence dna.
E) from (d) the new new dna sequence dna that produces with mix from the different dna moleculars in (c).
F) repeat (b) to (e), up to the active protein that obtains desirable (satisfaction).
2. according to aspect 1a (aspect 1 a item below adopts same rule), the DNA library can be a PCR segment or be inserted into expression plasmid, gene library coding intact proteins molecule or protein moiety zone independently.
3. according to aspect 1a and 2, the variety of gene library can be the mixing that derives from natural sequence, artificial sequence or both (nature and artificial sequence).
4. according to aspect 2 and 3, gene library can be coding single-chain antibody (scFv).Its variety is perhaps from natural library or from having given immunity library or both combinations.The gene pool resource can be from any animal, comprises mixing of people source or animal or human and animal.Preferential selection should be to mix with the CDRs of different sources and the gene pool for preparing with the human antibody framework.
5. according to aspect 1b, expression of gene is perhaps carried out in intestinal bacteria, perhaps in cell-free system, carries out.When adopting cell-free system to express, DNA (plasmid or PCR segment) can be that single molecule also can be group's molecule, for example: the 50-100 differing mol.
6. according to aspect 1c, when carrying out functional screening, can use bacterium colony filter membrane sieve method (colony filter screening), or ELI SA, or dot blotting (dot blotting), or protein chip (protein array), or these methods unite use.Active detection can be carried out under the condition of confirming in the screening, as: adopt and heat up, add denaturing agent, change incubation time, suppressor factor etc.
7. according to aspect 1d; When functional screening obtains to carry out in the dna molecular reorganization (recombination) of sequence and resets (shuffling), can pass through PCR, or the PCR correlation technique; Or other molecular biology methods, or the uniting to make and be used for accomplishing of these methods.Transgenation also can pass through rite-directed mutagenesis (site-directed) or random mutation (random mutation) is introduced.
8. according to aspect 1b, supplying in the gene expression system that this paper relates to can be E.coli, or cell-free system, or other heterologous hosts.
9. according to aspect 1b, expressed protein or monomer, or dimer (comprising the dimer that covalent linkage connects or non covalent bond connects).
10. according to aspect 1b, protein is recombinant antibodies.
11. according to aspect 9, the expressed protein portability supplies to detect the perhaps mark of purifying protein.
12. according to aspect 11, affinity tag can be a peptide species.In the expression process, affinity tag possibly form syzygy with protein.This polypeptide also can be by the chemical group mark.
13., form the dimer covalency and connect and to connect through the one or more cysteine residues that are positioned at protein N end or C-terminal or inside according to aspect 9.
14. according to aspect 9, it can be that non covalent bond through protein domain (domain) or protein sequence (sequence) is connected to form that dimer forms.
15. according to aspect 9, it can be to realize through cross-linking reagent that dimer forms.
16. according to aspect 11-12, its polypeptide marker sequence is (H) 7LGGC, preferential selection is connected in proteinic C-terminal.
17. the recombinant antibodies of anti-CEA (carcinoembryonic antigen), its avidity, contain give an example in the table 1 any heavy chain CDRs and light chain CDRs combination greater than 108M-1.
18. according to aspect 17, CEA is had high-affinity, narrow spectrum recombinant antibodies contains the CDR1 of any heavy chain of table 1, CDR2, CDR3 and light chain CDR1, CDR2, the independent assortment again of CDR3 (random recombination of CDRs).
19. according to aspect 17-18, CEA is had high-affinity, narrow spectrum recombinant antibodies, its framework derives from the human antibody framework.
20. according to aspect 17-19, CEA is had high-affinity, narrow spectrum recombinant antibodies only contains heavy chain.
21. according to aspect 17-20, CEA is had high-affinity, narrow spectrum recombinant antibodies comprises and occurs in the CDRs district or at any two mutants of antibody framework region.
22. according to aspect 17-21, CEA is had high-affinity, a large amount of preparations of narrow spectrum recombinant antibodies can be passed through any protein expression.
23. according to aspect 17-22, its recombinant antibodies can connect any anti-tumor agent comprising salmosin or detectable labelled reagent (like radio-labeling reagent, chemical illuminating reagent, fluorescent agent or enzyme marking reagent etc.).
24. according to aspect 17-23, its recombinant antibodies can be used for diagnosing in the body or a kind of basal component in the external CEA content reagent, the sample of detection comprises human sample or neoplasmic tissue sample.
25. according to aspect 17-24, its recombinant antibodies can be used as the medicine of treatment cancer.
26. according to aspect 24, its recombinant antibodies is used to diagnose body reagent to comprise (but being not limited to) following mode: ELISA, protein micro-arrays, dot blot, immuno-precipitation and in vivo detection.
27. according to aspect 24, its recombinant antibodies is used for the in-vivo diagnostic body and includes but is not limited to the level diagnosis of tumor tissues and be used for the surgical operation that antibody instructs.
28. according to aspect 24, its recombinant antibodies segment is used for diagnostic reagent and includes but is not limited to share with different lectins, patient behind detection diagnosis particular cancers patient or the cancer operation.
29. according to aspect 17-22, the recombinant antibodies of anti-CEA is used for gene diagnosis and treatment.
The accompanying drawing summary
Fig. 1 is the inventive method principle basic diagram.
Fig. 2 has shown ELISA result.It shows the combined function of the antibody of different coli strains expression to CEA.The different coli strains of numeral.PC representes that CEA antibody is over against photograph.
Fig. 3 has shown the ELISA result of recombinant antibodies 717.
Fig. 4 has shown the expression and the elisa assay of recombinant antibodies 636.(a) Western blot adopts horseradish peroxidase-anti-poly histidase len antibody to detect and from intestinal bacteria (E.coli), expresses and discharges recombinant antibodies 636.(b) ELISA shows the specificity of recombinant antibodies 636.
Fig. 5 has shown the dimeric formation of recombinant antibodies.Vestolen PP 7052 mills ammonia gel electrophoresis (SDS-PAGE) and Western engram analysis recombinant antibodies monomer or dimeric generation.(a) intercellular substance (periplasm) sample is handled with DTT; (b) intercellular substance (periplasm) sample DTT useless handles.Arrow is respectively monomer and dimer protein band.
Fig. 6 has shown the competitive ELISA analysis.Dotted line is represented the CEA concentration model (1ng/ml) of 50% inhibiting rate.The molecular weight of CEA is 180,000Da.
Fig. 7 has shown that the antigenic susceptibility of 96 couples of CEA of recombinant antibodies detects (ELISA).(a) antibody 96 concentration dilutions; (b) CEA antigen diluent.
Detailed Description Of The Invention
The present invention is based on dynamic gene storehouse and continuous function screening principle, described a new screening high-affinity, the method for specificity antibody.This method repeats " the continuous variation → protein expression of gene pool sequence → functional screening ", up to obtaining satisfied antibody.Specifically describe as follows.
We use antigens c EA, have obtained the recombinant antibodies of the anti-CEA of some high reactivities, have proved the feasibility and the validity (as follows) of above-mentioned approach.
The present invention and the existing technology of holding that shows have tangible difference: the first, do not use the fixed gene pool.All contain the molecule that reconfigures in each circulation gene pool, and the molecule that reconfigures derives from the particular sequence with specific antigen combined function.In other words, gene order is binned in each circulation all has generation, and the gene order source that produces reorganization comes from the active molecule that has that is screened in the circulation.The second, when carrying out functional screening, the antibody of generation is solvable, freely, combines with other carriers in any form.And the existing technology of holding that shows, antibody must directly link together with other carriers.
Setting up one of first-selected approach in initial gene storehouse (G1) is: end user source antibody framework
[9], on this framework, set up the CDRs storehouse.This CDRs storehouse can be the people source, also can be the mixture of other animals or people source and other animals.The CDRs storehouse also can be the animal (comprising the people source) that derives from after the immunity.Antibody expression generally uses intestinal bacteria, also can use the acellular albumen expression system.If use escherichia coli expression antibody, its functional screening uses membrane filtration bacterium colony screening method (seeing materials and methods).If use acellular expression system, then at first screen microcommunity molecule (for example, the 50-100 gene molecule), then, activated colony continues to separate (10-20 molecule), up to obtaining single molecule.
The molecule that screening at last obtains can connect a peptide sequence HHHHHHHLGGC at its C end.Through the cysteine residues of C end, can let the single-chain antibody molecule form dimer.Dimeric formation can increase the ability of antibody conjugated antigen, also can make single H7-tag become double H7-tag (that is: HHHHHHHLGGC-CGGLHHHHHHH), thereby increases detection sensitivity.In addition, the halfcystine of C end also can be used as antibody labeling, makes that antibody becomes in the body, external diagnosis reagent (isotropic substance autography) comprises and be used for the surgical operation that antibody instructs, or become antibody drug reagent.
We adopt the present invention to obtain the single-chain antibody of some anti-CEA.Wherein an antibody can directly develop into diagnosis, treatment reagent.These antibody will provide the CDRs storehouse of usefulness, produce the required anti-CEA antibody of high reactivity through the sequence reorganization.
CEA is the abbreviation of Carcinoembryonic antigen (cancer tire antigen), and this antigen is confirmed as a kind of biomarker relevant with tumour by academia, can be used as the related antigen of diagnosing tumor and treatment
[10-15]Initial academia thinks that this antigen is that a kind of expression exists only in the albumen in the placenta tissue, but research has now confirmed that CEA can express, and comprises organs such as colon, stomach, tongue, esophagus, uterus, sweat gland and prostate gland in several kinds of normal adult face tissues.But in normal face tissue, CEA only appears at the front surface of cell, and seldom gets into blood circulation.For example, the CEA excretion is 50-70mg every day in the ight soil of health adult, and in the blood CEA content less than 10ng/ml.On the contrary, in tumour cell, CEA can be present in the whole tumour cell, and participates in blood circulation.So CEA content can be used as the cancer patient diagnosis index in the detection blood.
Embodiment
Materials and methods
PCR design of primers and mRNA
The required PCR primer in preparation amplification human antibody variable region is seen
[9]The required PCR primer in preparation amplification goat antibody variable region is seen
[16]The required PCR primer of preparation amplification rat antibody variable region is seen
[17]The have drawn from mRNA library of U.S. Clontech company, the basic storehouse of human antibody, people source CDRs (complementary decision territory) obtains through the amplification from the mRNA library of Clontech of RT-PCR (reverse transcriptase PCR) technology.The CDRs of goat obtains from the mRNA library of the goat of a CEACAMS (CEA) immunity through the RT-PCR technology.The CDRs of rat obtains from the mRNA library of the rat of CEA immunity through the RT-PCR technology.CEA antigen is buied from U.S. Scripps laboratory.
PCR and PCR Shuffling
Use above-mentioned PCR primer and amplification that PCR and RT-PCR technology through standard can realize antibody sequence, PCR Shuffling carries out, sees according to a kind of improved " dna fragmentation primer " method
[18]In brief, coding CDR1, CDR2, the multifarious PCR segment of CDR3 all can be used as the primer use that is connected with oligonucleotide one end.Through 30 take turns PCR after, through agarose electrophoretic analysis PCR product, target P CR gel band is cut and wash-out.
The screening of membrane filtration bacterium colony
[19]
The PCR segment is connected with expression vector and changes in the Bacillus coli cells.After the inoculation, agar plate is 37 ℃ of following incubated overnight.Put down hydrophilic first pvdf membrane gently bacterial colony is arranged planar surface then, bacterial colony is transferred on the pvdf membrane (microporous type GVWP, aperture 0.22um) in long.Then with second hydrophobicity PVDF transfer film (Immobilion P; Milli-pore Corp) encapsulates with 3ug/ml capture antigen solution earlier, immerse after blockading with the PBS solution that contains 3%BSA again (this substratum contains 1mM IPTG and 50-100ug/ml ammonia Bian Qing enzyme is plain) in the 2xTY liquid nutrient medium.After the film after wetting is positioned over the plain fresh agar plate surface of the ammonia Bian Qing enzyme that contains 1mMIPTG and 100ug/ml with second, again first pvdf membrane that contains bacterial colony is positioned over the surface of second film, make bacterium colony towards last.This folded film is an incubated overnight under the 25-30 degrees celsius in envrionment temperature.After the cultivation, first film that has bacterium colony moved on the fresh Agar Plating, is positioned over 4 degrees centigrade of storages, as subsequent use bacterium.Second film shifted out from agar plate surface, with 3 times (each 10 minutes) of PBS damping fluid washing that contain 0.1% polysorbas20, detect recombinant antibodies with enzyme len antibody detection method then immediately.Promptly use the anti-poly Histidine antibody of this film and horseradish peroxidase (enzyme couplet) (HRP-coupled anti-(His) 6tag antibody, Sigma, Britain).During use, diluted incubation 1 hour with the PBS solution that contains 1%BSA with 1: 2500 earlier.After 3 cleanings, this film detects through a kind of chemical illuminating reagent (Piece, Britain).
ELISA
With the antigen coated ELISA micro titer plate well of 100ul CEA, remove Special Circumstances, CEA concentration is generally 0.5ug/ml.With titer plate be positioned over 4 degrees centigrade spend the night encapsulate after, blockade with the PBS solution of 1%BSA.The antibody (scFv) of the anti-CEA that adding is extracted from colibacillus periplasm and made through Ni+ column chromatography (Qiagen, Britain) purifying, room temperature or 37 ℃ cultivate 1 hour down after; PBS washings with containing 0.05%Tween20 is washed plate 3 times; Horseradish peroxidase-anti-poly histidase len antibody (Sigma, Britain) after the dilution in 1: 2500 is added in the titration plate hole, and room temperature is after 1 hour; The ELISA developer (Sigma, Britain) that adds 100ul.Reading under 450nm emission spectral filter.Being at war with property ELISA when reaction, with different concns free CEA as suppressor factor, suppress recombinant antibodies and with micro plate on the association reaction that carries out of the CEA antigen of embedding.
Embodiment
The screening of the recombinant antibodies of embodiment 1 anti-CEA
The basic boom that employing is made up of 3 people source heavy chains (VH1, VH3, VH5) and goat heavy chain VH makes up the initial pcr gene library (G1) of recombinant antibodies variable region, and these heavy chain frameworks are connected as combining unit with people's light chain V3 again.The CDRs storehouse in different animals source then is to introduce through PCR shuffling technology.Be cloned in the expression vector of the poly histidine mark that carries a PelB leader sequence and design voluntarily in this pcr gene storehouse.Then this carrier is changed among the intestinal bacteria E.coli and places on the agar plate and grow, then with the E.coli colony lift to the PVDF filter membrane.Use membrane filtration bacterium colony screening method, detect the recombinant antibodies that from intestinal bacteria E.coli, discharges.Select positive bacterium colony, extract its DNA and prepare the CDRs storehouse as next step pcr amplification.After the three-wheel circulation, be contrast, adopt CEA antigen bonded ELISA methods analyst positive with milk powder.Fig. 2 shows that some recombinant antibodies and CEA have specificity to combine.All and CEA bonded clone bacterium are checked order.CDRs preface example result sees table 1.Wherein the antigen of several recombinant antibodies knot character has also been carried out elisa assay, confirms the antigenic single-minded combination of its CEA, and the result sees Fig. 3 and Fig. 4.
Recombinant antibodies dimer (scFv) in colibacillus periplasm for ease
2Formation, we introduce cysteine residues at the antibody C-terminal.Simultaneously, in the generation that detects recombinant antibodies, poly Histidine (His) 6 also is introduced into for ease.We detect expression and the generation form (be monomer or dimer) of institute's recombinant antibodies at E.coli with western blotting method.Recombinant antibodies (96scFv) extracts its intercellular substance (periplasm) behind expression in escherichia coli, handle the pericentral siphon extract respectively with the point sample damping fluid (loading buffer) that contains or do not contain WR 34678 (DTT) then.Sample is transferred to after Vestolen PP 7052 mills ammonia gel (SDS-PAGE) electrophoresis on the pvdf membrane (Immobilion P), and now adopts horseradish peroxidase-anti-poly histidase len antibody to detect.Analysis revealed after sample is handled with DTT, only shows an expection monomer band, and the sample of not handled by DTT then produces two protein belts, is respectively monomer and dimer protein band (see figure 5).This result shows: recombinant antibodies scFv is strong through cysteine residues formation covalency two sulphur of C-end, produces antibody dimer (scFv)
2
The mensuration of embodiment 3 recombinant antibodies avidity, specificity and stability
From the recombinant antibodies 96 of E.coli intercellular substance through Ni
+Behind the affinity chromatography purifying, be suppressor factor, adopt the competitive ELISA analytical procedure to detect this antibody the antigenic avidity of CEA with free CEA antigen.If represent the affinity of antibody height with the CEA concentration of 50% inhibiting rate, this inhibition curve shows that recombinant antibodies 96 has the apparent avidity (10 less than 0.001nM concentration
-12M) (Fig. 6).We also (see Fig. 7 a) through 96scFv antibody and the antigenic binding curve of CEA that western blotting (western blotting) method has been measured known content.This result shows that also recombinant antibodies 96 and the antigenic avidity of CEA are less than 0.01nM.The specificity of recombinant antibodies 96 also confirms with the combination situation of a series of different usual proteins through detecting antibody 96, as with the albumen of BSA, milk powder, KLH etc. as contrast.The result shows: antibody 96 and the equal no cross reaction of above-mentioned control protein.The stability experiment of antibody 96 is to be employed in the ELISA method that improves under the temperature condition to measure, and the result shows: this antibody does not have obvious bonding force to descend under 37 ℃ of conditions at least in 3 hours.Moreover we also observe: when suitably solvable but non-activity antibody (inclusion bodies) dilutes to urea with PBS solution, can be observed antibody 96 and recover or be folded into to have the active antibody of the CEA of combination very soon.This easy renaturation fact has also proved the stability of this antibody.
The antigenic susceptibility of 96 couples of CEA of embodiment 4 recombinant antibodies detects
Adopt the ELISA method to measure the avidity of CEA (50ng, 25ng, 12.5ng, 6ng, 3ng) with the 96ScFv antibody of different concns, detection shows that 96scFv antibody recognition content is lower than 3ngCEA antigen (seeing Fig. 7 b).Show that this antibody detects very sensitive to the concentration of CEA.
Table 1. different zones CDRs sequence
Heavy chain (V
H)
| The clone | | CDR2 | CDR3 | |
| 3 | | WINPNSGGTNYAQKFQG | SVNGDSVPY | |
| 15 | | WINPNSGGTNYAQKFQG | GLWDYYY | |
| 32 | | WINPNSGGTNYAQKFQG | DLNNWNYYYYY | |
| 96 | | GGVSSGALTAYNTALQS | SFITIFGVVIIHYYY | |
| 168 | | WINPNSGGRNYAQKFQG | DLNNWNYYYYY | |
| 717 | | WINPNSGGTNYAQKFQG | DLNNWNYYYY | |
| 520 | | VIWYDGSNKYYADSVKG | EIAG | |
| 291 | | IIYPGDSDTRYSPSFQG | QSSGWY | |
| 774 | | IIYPGDSDTRYSPSFQG | QSSGWY | |
| 636 | YSFTSY | IIYPGDSDTQY | QAGMLSP |
Light chain (V
L)
| The clone | | CDR2 | CDR3 | |
| 3 | | DDSDRPS | QVWDSSSD | |
| 15 | | DDSDRPS | QPFDSSLNG | |
| 32 | | DDSDRPQ | KVWDSSSD | |
| 96 | | GDSDRPS | ASYQSTYSG | |
| 168 | | ATTDRAS | QVWDSSSD | |
| 717 | | GNSNRPS | QSYDSSLSG | |
| 520 | | GDSDRPS | AAWDDTLHG | |
| 291 | | GNSNRPS | QSYDSSLSG | |
| 774 | TGSSSDVGAGHDVH | | QPFDS SLNG | |
| 636 | TGSSSTTGAGYDVH | GNNNRPS | QSYDNTLSG |
Reference
1.Carter，P.Improving the efficacy of antibody-based cancer therapies.Nature Review Cancer.1，118-129(2001).
2.
A，Plückthun A.Stability engineering of antibody single-chain Fv fragments.J.Mol.Biol.305，989-1010(2001).
3.McCafferty J，Griffiths AD，Winter G，Chiswell DJ.Phageantibodies：filamentous phage displaying antibody variable domains.Nature 348，552-554(1990)
4.Shusta EV，VanAntwerp J，Wittrup，KD.Biosynthetic polypeptide libraries.Curr Opin Biotechnol.10，117-122(1999).
5.Georgiou G，Stathopoulos，C，Daugherty，PS，et al.Display of heterologous proteins on the surface ofmicroorganisms：from the screening of combinatorial libraries to live recombinant vaccines.Nat Biotechnol.15，29-34(1997).
6.He M，Taussig M.J.Ribosome display：Cell-free protein display technology.Briefings in Functional Genomics and Proteomics.1，204-212(2002).
7.Ling MM Large antibody display libraries for isolation of high affinity antibodies.Comb.Chem.High Throughput Screen.6，421-432(2003)
8.Goldenberg D M.(1992)Cancer imaging with CEAantibodies：historical and current perspectives.TheInternational Journal of Biological Markers 7，183-188.
9.Knappik A，Ge L，Honegger A et al.，(2000)Fully synthetic human combinatorial antibody libraries(HuCAL)based on modular consensus frameworks and CDRs randomized withtrinucleotides.J Mol Biol.296，57-86.
10.Ledermann J A，Recent RHJ，Massof C，Kelly AMB，Adam T& Bagshawe K D(1991)A phase--I study of repeated therapy with radiolabelled antibody to carcinoembryonic antigen using intermittent or continuous administration of cyclosporin A tosuppress the immune response，Int.J.Cancer.47，659-664.
11.Pedley R B，Dale R，Boden J A，Begent R H J，Keep P AGreen A J(1989).The effect of second antibody clearance on the distribution and dosimetry of radiolabelled anti-CEA antibody in a human colonic tumour xenograft model.Int.J.Cancer.43，713-318.
12.Pedley R B，Boden J A，Boden R，Dale R，Begent R H(1993)Comparative radioimmunotherapy using intact or F(ab′)2fragment s of
131Ianti-CEA antibody in a colonic xenograft model.Br.J.Cancer.68，69-73.
13.Ledermann J A，Begent R H J，Bagshawe K D，Riggs S J，Searle F，Glaser M G，Green A J，Dale R G.(1988)Repeated antitumour antibody therapy in man with suppression of the host response by Cyclosporin A.Br.J.Cancer 58，654.
14.Boxer GM，Begent R H J，Kelly AM B，Southall P J，Blair S B，Theodoron N A，Dawson P M，Ledermann J A.(1992)Factors influencing variability of localisation of antibodies to carcinoembryonic antigen(CEA)in patients with colorectal carcinoma--implications for radiommunotherapy.Br.J.Cancer.65，825-831.
15.Blair S D，Theodorou N A，Begent R H J et al(1990).Comparison of anti-fetal colonic microvillus and anti-CEAantibodies in peroperative radioimmunolocalisation of colorectal cancer，Br.J.Cancer，61，891.
16.Dufour，V.，Nau，F(1997)Genomic organisation of the sheep immunoglobulin J
H segments and their contribution to heavy chain variable region diversity.Immunogenetics 46，283-292
17.Kabat，E.A.，Wu，T.T.，Perry，H.M.，Gottesman，K.S.，Foeller，C(1991).Sequences of proteins of immunologicalinterest.5th ed.US Public Health Service，National Institute of Health，Bethesda，MD
18.Sharker，G andSommer，S.S(1990)The‘megaprimer’method of site-directed mutagenesis.Biotechniques 8，404-407
19.Skerra，A.，Dreher，ML and Winter，G(1991)Filter screening of antibody Fab fragment secreted from individual bacterial colonies.Anal.Biochem 196，151-155
Claims (13)
1. the recombinant antibodies of anti-CEA, its avidity is higher than 10
-8M, and contain following any heavy chain CDRs and light chain CDRs combination
Heavy chain
CDR1 CDR2 CDR3
TFTGYY WINPNSGGTNYAQKFQG SVNGDSVPY
TFTGYY WINPNSGGTNYAQKFQG GLWDYYY
TFTGYY WINPNSGGTNYAQKFQG DLNNWNYYYYY
TFTGHY GGVSSGALTAYNTALQS SFITIFGVVII
HYYY
TFTGYY WINPNSGGRNYAQKFQG DLNNWNYYYYY
FSLTKY WINPNSGGTNYAQKFQG DLNNWNYYYY
AFSTYG VIWYDGSNKYYADSVKG EIAG
SFTTSW IIYPGDSDTRYSPSFQG QSSGWY
SFTTSW IIYPGDSDTRYSPSFQG QSSGWY
YSFTSY IIYPGDSDTQY QAGMLSP
Light chain
CDR1 CDR2 CDR3
GGNNIGSKSVH DDSDRPS QVWDSSSD
GGNNIGSKSVH DDSDRPS QPFDSSLNG
GGNNIGSKSVH DDSDRPQ KVWDSSSD
GGHDIGSKSVH GDSDRPS ASYQSTYSG
GGNNIGSKSVH ATTDRAS QVWDSSSD
TGSSSNIGAGHDVH GNSNRPS QSYDSSLSG
SGSSSNIGGNAYVG GDSDRPS AAWDDTLHG
TGSSSNIGAGHDVH GNSNRPS QSYDSSLSG
TGSSSDVGAGHDVH GNSNRPS QPFDSSLNG
TGSSSTTGAGYDVH GNNNRPS QSYDNTLSG
2. according to the antibody of claim 1, said antibody has high-affinity to CEA, and contains the independent assortment again of any heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2, CDR3.
3. according to the antibody of claim 1 or 2, said antibody has high-affinity to CEA, narrow spectrum recombinant antibodies, and its framework derives from the human antibody framework.
4. according to each antibody of claim 1-3, said antibody has high-affinity to CEA, and narrow spectrum recombinant antibodies only contains heavy chain.
5. according to each antibody of claim 1-4, said antibody has high-affinity to CEA, and narrow spectrum recombinant antibodies comprises and occurs in the CDRs district or at any two mutants of antibody framework region.
6. according to each antibody of claim 1-5, said antibody has high-affinity to CEA, and a large amount of preparations of narrow spectrum recombinant antibodies are carried out through any protein expression.
7. according to each antibody of claim 1-6, wherein recombinant antibodies can connect any anti-tumor agent comprising salmosin or detectable labelled reagent.
8. according to each antibody of claim 1-7, wherein recombinant antibodies can be used for diagnosing in the body or a kind of basal component in the external CEA content reagent, and the sample of detection comprises human sample or neoplasmic tissue sample.
9. according to each antibody of claim 1-8, wherein recombinant antibodies can be used as the medicine of treatment cancer.
10. according to Claim 8 antibody, wherein recombinant antibodies is used for detecting in ELISA, dot blotting, protein chip, immunoprecipitation and the body.
11. antibody according to Claim 8, wherein recombinant antibodies is used for the level diagnosis of tumor tissues and is used for the surgical operation that antibody instructs.
12. antibody according to Claim 8, wherein the recombinant antibodies segment is used for share with different lectins, patient behind detection diagnosis particular cancers patient or the cancer operation.
13. according to each antibody of claim 1-6, wherein the recombinant antibodies of anti-CEA is used for gene diagnosis and treatment.
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|---|---|---|---|
| CN2011102605113A CN102443061A (en) | 2005-10-14 | 2005-10-14 | High-functional antibody biological synthesis |
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| CN 200510113739 Division CN1948476A (en) | 2005-10-14 | 2005-10-14 | High-function antibody biosynthesis |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4853464A (en) * | 1987-03-05 | 1989-08-01 | Peralta Cancer Research Institute | Hybridoma cell line XMMBR-B14 and monoclonal antibody which is specific for a non-cross reactive epitope of CEA |
| CN1634990A (en) * | 2004-10-14 | 2005-07-06 | 上海润龙生物科技有限公司 | High affinity immune globulin binding molecule and method for preparation |
-
2005
- 2005-10-14 CN CN2011102605113A patent/CN102443061A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4853464A (en) * | 1987-03-05 | 1989-08-01 | Peralta Cancer Research Institute | Hybridoma cell line XMMBR-B14 and monoclonal antibody which is specific for a non-cross reactive epitope of CEA |
| CN1634990A (en) * | 2004-10-14 | 2005-07-06 | 上海润龙生物科技有限公司 | High affinity immune globulin binding molecule and method for preparation |
Non-Patent Citations (2)
| Title |
|---|
| KARL BAUER ET AL.: "Diversification of Ig heavy chain genes in human preterm neonates prematurely exposed to environmental antigens", 《THE JOURNAL OF IMMUNOLOGY》 * |
| S.D. BLAIR ET AL.: "Comparison of anti-fetal colonic microvillus and anti-CEA antibodies in peroperative radioimmunolocalisation of colorectal cancer", 《BR. J. CANCER》 * |
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