CN101463083B - Gene engineering monoclonal antibody combined with A-beta oligomer specificity - Google Patents
Gene engineering monoclonal antibody combined with A-beta oligomer specificity Download PDFInfo
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- CN101463083B CN101463083B CN2009100769910A CN200910076991A CN101463083B CN 101463083 B CN101463083 B CN 101463083B CN 2009100769910 A CN2009100769910 A CN 2009100769910A CN 200910076991 A CN200910076991 A CN 200910076991A CN 101463083 B CN101463083 B CN 101463083B
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Abstract
The invention relates to the technical field of engineered antibody and provides a monoclonal antibody. The variable region of heavy chain of the monoclonal antibody contains the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEO ID NO.3; the variable region of light chain of the monoclonal antibody contains the amino acid sequences shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The invention also specifically provides a humanization single-chain antibody generated by the recombinant strain of enterotoxigenic Escherichia coli with the accession number CGMCC No.2818 and the amino acid sequence of the humanization single-chain antibody is shown in SEQ ID NO.7. The antibody of the invention can be specifically bound with the A-beta oligomer, effectively inhibit the fibrosis aggregation of A-beta and obviously alleviate the toxic effect of the A-beta on cells. The invention also relates to a pharmaceutical composite containing the antibody. The antibody of the invention has strong activity, good specificity, easy preparation and wide prospect of experiment application and clinical application.
Description
Technical field
The invention belongs to the genetic engineering antibody technical field, be specifically related to and A-beta oligomer specificity bonded gene engineering monoclonal antibody, the pharmaceutical composition that the invention still further relates to this MONOCLONAL ANTIBODIES SPECIFIC FOR method and contain this monoclonal antibody.
Background technology
Studies show that the A Zihaimo disease (Alzheimer ' s Disease, be called for short AD), being commonly called as senile dementia is by avirulent amyloid-beta monomer molecule (β-Amyloid, A-beta 40/42 is hereinafter to be referred as A-beta, also can be write as A β) assemble forming the elderly that the oligomer of tool toxic action causes, mainly to form senile plaque with hypomnesis and brain be the nerve degenerative diseases (Selkoe etc. of feature, Science 1997,275,630-631; Koo etc., PNAS 1996,9989-9990).Medical statistics shows, in China and the American-European countries more than 60 years old the elderly have 5～6% to suffer from the A Zihaimo disease.This disease has been listed in and has caused dead the fourth-largest disease, is only second to heart trouble, cancer and apoplexy.China existing more than 60 years old population about 100,000,000, be 5% estimation according to population person in middle and old age dementia morbidity more than 60 years old, the whole nation has senile dementia patient about 5,000,000.This disease brings huge burden for family and society.
Experimental study in recent years proves that more and more condensing the intermediate product-A-beta oligomer that forms filament by the A-beta monomer is the main virulence factor that causes AD, rather than the previous thread aggregate (Haass that assert, C. etc., 2007, Nature reviews 8,101-112; Lesne, S, 2006, Nature 440,352-357; Shankar, G.M, 2008, Nature medicine 14,837-842).Yet, existing in the world at present detection and treatment AD antibody, be at the A-beta primary sequence mostly, be the monomeric antibody of A-beta, this antibody-like can with A-beta monomer, oligomer and filament combination simultaneously, in experimental study, pathology detection and clinical diagnosis, can not detect the real height that plays the oligomer level of pathogenic effects specifically.Simultaneously, this antibody-like easily causes severe side effect such as cerebral hemorrhage and meningoencephalitis in to the treatment of AD.
The Glabe laboratory on Science magazine in 2003, reported can with the antibody A 11 of oligomer specific combination (Kayed etc., 2003, Science 300,486-489).But this antibody is a kind of polyclonal antibody, and the amount at each antibody component of each antigenic determinant that contains in each production batch is uncontrollable, influences the comparability of detected result.And this antibody not only combines with the A-beta oligomer, and it also combines the specificity of no one-level sequence with multiple oligomer such as alpha-synuclein, Prion albumen, Regular Insulin, N,O-Diacetylmuramidases.
Single-chain antibody (single chain FV, ScFv) being an important function of gene engineered antibody, is the recombinant protein that utilizes gene engineering method that antibody heavy chain variable region (VH) gene and variable region of light chain (VL) the gene connection peptides by one section about 5～25 amino acid (linker) is connected to form on dna level.The C-terminal of single-chain antibody can be introduced structures such as halfcystine tail, casein kinase 2 enzyme substrates tail, E tail, help other molecule of mark and coupling, also can introduce calmodulin tail, c-myc tail, staphylococcal protein A,SPA tail, lipid label, Histidine tail etc., make expression product be easy to detect and purifying.
Single-chain antibody has a series of characteristics of self:
(1) only contains the V district of antibody, can keep more complete antigen binding site;
(2) lack the Fc section, do not combine, help being used for clinical as the drug targeting carrier with non-target cell;
(3) immunogenicity is low, is used for human body and is difficult for producing anti-foreign protein reaction;
(4) molecular weight is little, easily penetration rate of blood tube wall, tissue and solid tumor;
(5) the circulation of blood transformation period is short in the body, easily gets rid of from circulation of blood;
(6) 3 ' end at its gene connects the appropriate purpose molecule, as: enzyme, toxin, medicine etc. can make up bifunctional antibody;
(7) only combine not activating complement mediated cell immunity with antigen;
(8) be easy to genetic manipulation and genetically engineered mass production.
In recent years, single-chain antibody becomes one of focus in the genetic engineering antibody research field gradually, is demonstrating great potential aspect the clinical diagnosis of disease and the treatment.
At present, rarely seen in the world have a report about the single-chain antibody scFv of specific combination A-beta oligomer (Zameer A etc., 2008, J Mol Biol.384:917-928), and the avidity of this scFv and A-beta oligomer is lower.Though other has an antibody-like NU1, NU2 and NU4 to combine not obvious with the A-beta monomer, but they can combine with A-beta oligomer and filament simultaneously, thereby influence to the detection of A-beta oligomer in the sample (referring to Lambert etc., 2007, Journal ofneurochemistry 100,23-35).Up to the present, domesticly do not see the monoclonal antibody of specific combination A-beta oligomer or the report of single-chain antibody as yet.
Summary of the invention
The invention solves existing antibody while and A-beta monomer, oligomer and filament bonded technical problem, use phage display technology and successfully filter out and specially combine with the A-beta oligomer, and not with A-beta monomer and filament bonded monoclonal antibody.Monoclonal antibody of the present invention and A-beta oligomer specificity bonded characteristic and A-beta primary structure are irrelevant, and it plays a role by combining with the peculiar space multistory epitope of A-beta oligomer.
A-beta oligomer of the present invention is the oligomer that is formed by two to tens the A-beta monomer aggregation that do not wait, and intermolecularly mainly combines with hydrogen bond and hydrophobic bond, and molecular weight does not wait from 9kDa to 100kDa.Its space multistory epitope up to now as yet the someone inquire into clear.
On the one hand, the invention provides the monoclonal antibody of a specific specificity in conjunction with the A-beta oligomer, comprise variable region of heavy chain and variable region of light chain, variable region of heavy chain contains the aminoacid sequence shown in SEQ ID NO.1, SEQ ID N0.2 and the SEO IDNO.3, and variable region of light chain contains the aminoacid sequence shown in SEQ ID NO.4, SEQ ID NO.5 and the SEQID NO.6.
" antibody " of the present invention should be interpreted as containing any specificity binding factor with required specific binding domains.Thereby the function equivalent and the homologue of homologous antibody fragment, derivative, humanized antibody and antibody with it contained in this term, also comprises any polypeptide that contains the antigen binding domains, no matter is natural or synthetic the generation.The example of antibody is immunoglobulin (Ig) hypotype (as IgG, IgE, IgM, IgD and IgA) and hypotype subclass thereof; Also can be to comprise the fragment of antigen binding domains such as Fab, scFv, Fv, dAb, Fd; With double-stranded antibody (diabodies).Fusion to mosaic molecule or equivalent another polypeptide, that comprise the antigen binding domains is also included within wherein.The cloning and expression of chimeric antibody is described in EP.A-0120694 and EP.A.0125023.
Antibody can be modified by many modes, and available DNA recombinant technology produces other antibody or the chimeric molecule that keeps original antibodies specific.This technology can comprise that constant region or constant region that the DNA with the immune globulin variable region of encoding antibody or complementarity-determining region (CDRs) introduces different immunoglobulin (Ig)s add framework region.Referring to, EP.A.184187, GB 2188638A or .EP.A.239400.Can also carry out genetic mutation or other change to other cell of hybridoma or generation antibody, this can change or not change the binding specificity of the antibody that produces.
Monoclonal antibody provided by the invention can be humanized antibody, and it also contains the framework region of people's antibody.
Monoclonal antibody provided by the invention can also be a single-chain antibody.
In the specific embodiment of the present invention, the invention provides the single-chain antibody WC2 of specificity in conjunction with the A-beta oligomer, its aminoacid sequence is shown in SEQ ID NO.7, its variable region of heavy chain contains the aminoacid sequence shown in SEQ IDNO.1, SEQ ID N0.2 and the SEO ID NO.3, and variable region of light chain contains the aminoacid sequence shown in SEQID NO.4, SEQ ID NO.5 and the SEQ ID NO.6.
Monoclonal antibody of the present invention comprises heavy chain and light chain " variable region ", and some part of variable region is different on sequence in term used herein " variable " the expression antibody, and it has formed combination and the specificity of various specific antibodies to its specific antigen.Yet mutability is not evenly distributed in the whole antibody variable region.It concentrates on and is called complementary determining region in light chain and the variable region of heavy chain or highly becomes in three fragments in the district.Part conservative in the variable region is called framework region (FR).CDR in every chain closely is close together by the FR district and has formed the antigen-binding site of antibody with the CDR of another chain.
Hypervariable region CDR1, the CDR2 and CDR3 and catenation sequence of monoclonal antibody of the present invention in heavy chain and light chain, other is a framework region.Framework region can be replaced by other sequences under in conjunction with the impregnable condition of required three-dimensional structure, and the molecular basis of antibodies specific mainly comes from its hypervariable region CDR1, CDR2 and CDR3, and these zones are and antigen bonded key position.For keeping preferred binding characteristic, the sequence of CDR should keep as far as possible, yet, may need some amino acid changes to make the binding characteristic optimization, those skilled in the art can reach this purpose with standing procedure.
In the specific embodiment of the present invention, at random one or several amino acid of WC2 framework region being suddenlyd change respectively, lacks or adding one or several amino acid toward framework region does not influence antibody activity.As 81L is sported A, 177A is sported the antibody WC2-1 (its aminoacid sequence is shown in SEQ ID NO.8) that G forms, its activity is unaffected; Or with 119G and 213Q disappearance, or all do not influence antibody activity adding G behind the 43P or behind 244A, add A.
One or several amino acid in the WC2 hypervariable region suddenlyd change respectively, lacks or add one or several amino acid and do not influence antibody activity sometimes toward the hypervariable region.As 56T is sported A, 159A is sported the antibody WC2-2 (its aminoacid sequence is shown in SEQ ID NO.9) that T forms, its antibody activity is unaffected; Or with the 103K disappearance, or with the 223Q disappearance, or behind 66V, add A, or behind 162S, add G, all do not influence antibody activity.
Monoclonal antibody of the present invention is to separate to obtain from the phage antibody library of expressing more than one hundred million kinds of single-chain antibodies at phage surface respectively.Be about to the A-beta oligomer and be coated on the immune pipe, phage antibody library is added in the test tube, flush away is not attached to the phage on the immune pipe, breeds with the phage of pancreatin elution of bound and to it then, carries out 4 screenings of taking turns in the same way.Last is taken turns phage transfection Escherichia coli HB2151 under the wash-out, identify with the ELISA method and express the positive colony that produces soluble single-chain antibody.
The positive colony of the prepared single-chain antibody WC2 that the specific embodiment of the invention provides, now carry out preservation in China Committee for Culture Collection of Microorganisms common micro-organisms center (DSMZ of institute of microbiology of the Chinese Academy of Sciences), the address is the Datun Road, Chaoyang District, Beijing City, classification called after colon bacillus Escherichia coli, preservation date is on December 22nd, 2008, and deposit number is CGMCC No.2818.
Being used for the also available hybridoma method of monoclonal antibody of the present invention makes, because the dna sequence dna of code book invention humanized antibody can be used conventional means well known to those skilled in the art, as obtaining according to aminoacid sequence synthetic disclosed by the invention or with the amplification of PCR method, thereby also available recombinant DNA method, available the whole bag of tricks well known in the art is connected into this sequence in the suitable expression vector.At last, under the condition that is fit to antibody expression of the present invention, cultivate the host cell that transforms gained, those skilled in the art use the conventional separation and purification means purifying of knowing and obtain monoclonal antibody of the present invention then.
In the specific embodiment of the present invention, specificity provided by the invention is in conjunction with the antibody WC2 of A-beta oligomer, and molecular weight is about 29kDa.Binding constant k between WC2 and A-beta 42 oligomer
a=4.29 * 10
2M
-1s
-1, dissociation constant k
d=1.74 * 10
-5s
-1, affinity constant K
D=3.32 * 10
-7M.
With WC2 as identification antibody, horseradish peroxidase (HRP) mark can with the 9E10 of WC2 specific combination as second antibody, can detect the A-beta oligomer of 2.2nM in the sample or 5nM respectively with enzyme-linked immunosorbent assay (ELISA) or immunodotting (Dot-blot) experiment.Can specially detect the content of nmol level A-beta oligomer in the sample based on antibody of the present invention, those of ordinary skill in the art can predict, and described antibody can be used to detect the A-beta oligomer in scientific experiment and clinical detection.
In the specific embodiment of the present invention, the gathering of A-beta test shows that WC2 can effectively suppress the fibrosis gathering of A-beta.The neurocyte toxicity test shows that WC2 can obviously reduce the toxic action of A-beta to neurocyte, based on above-mentioned discovery, those of ordinary skill in the art can predict this monoclonal antibody or contain the pharmaceutical composition of this monoclonal antibody can be by specificity in conjunction with the A-beta oligomer, suppress the fibrosis gathering of A-beta and reduce the toxic action of A-beta, thereby realize the purpose of prevention and treatment senile dementia neurocyte.And can predict, use molecule, can avoid complete antibody serious side effects such as meningitis that patient AD brought, hematencephalons only for complete antibody 1/5 and when not containing segmental WC2 prevention of Fc and treatment senile dementia.
Pharmaceutical composition provided by the invention contains the pharmaceutically monoclonal antibody of the present invention and the pharmaceutically acceptable carrier of significant quantity.Term used herein " pharmaceutically acceptable " is meant that they can not produce disadvantageous, hypersensitive or other untoward reaction when carrier molecule body and composition suitably give the animal or human." pharmaceutically acceptable carrier " used herein should be compatible with monoclonal antibody of the present invention, can can not reduce the effect of pharmaceutical composition with its blend under normal conditions significantly, can be used as some materials of pharmaceutically acceptable carrier or its component.Object lesson can be carbohydrate, starch, Mierocrystalline cellulose and derivative thereof, stablizer, antioxidant, sanitas, apirogen water, etc. ooze salts solution and phosphate buffered saline buffer etc.
Pharmaceutical composition of the present invention can be made various formulations as required, and can by the doctor according to patient's kind, age, body weight and roughly factor such as disease condition, administering mode determine the useful dosage of patient is used.
Monoclonal antibody of the present invention has the following advantages:
1, specially combines, and do not combine with A-beta monomer and filament with the A-beta oligomer;
2, monoclonal antibody and antigen-binding specificity are good,
3, single-chain antibody lacks the Fc section, does not combine with non-target cell; Antibody molecule is less, less than 1/5 of complete antibody molecule, is easier to pass hemato encephalic barrier performance curative effect;
4, A-beta gathering test and neurocyte toxicity test show that described antibody can effectively suppress the fibrosis gathering of A-beta, and obviously reduce the toxic action of A-beta to neurocyte;
5, utilize phage expression antibody production height, active strong, specificity is good, is easy to screening.
Monoclonal antibody of the present invention has broad application prospects as the special preparation of A-beta oligomer and the immunological reagent for the treatment of as AD in clinical diagnosis, the experiment test sample.
Description of drawings
Fig. 1 detects WC2 and A-beta oligomer bonded ELISA experimental result picture;
The binding characteristic of WC2 and various form A-beta is compared in Fig. 2 Dot-Blot experiment, and mon represents the A-beta monomer, and oli represents the A-beta oligomer, and fib represents the A-beta filament;
Fig. 3 WC2 assembles the statistics figure that suppresses experiment to A-beta 42;
Fig. 4 WC2 suppresses the statistics figure of experiment to A-beta 42 cytotoxicities.
Embodiment
Before describing embodiments of the present invention in detail, be to be understood that application of the present invention is not limited to the following example, the present invention also can carry out with other embodiment.
The preparation of embodiment 1A-beta oligomer
A-beta monomer (available from the American Peptide company of the U.S.) is dissolved in HFIP (hexafluoroisopropanol is available from Sigma company), is prepared into the solution that concentration is 1mg/ml.Room-temperature water bath supersound process 10 minutes divides to install in the 1.5ml centrifuge tube, puts to allow HFIP volatilize fully in the vapor hood, and is standby in-20 ℃ of preservations.
The A-beta equilibrium at room temperature of above-mentioned processing after 10 minutes, is added DMSO (dimethyl sulfoxide (DMSO) is available from Sigma), A-beta is fully dissolved, and to make its final concentration be 1mg/ml.Get in the PBS damping fluid that a certain amount of A-beta joins pH 7.4, making A-beta concentration is 10 μ M.With this A-beta solution put 37 ℃ hatch 12 hours after, with 14000rpm centrifugal 20 minutes, abandon bottom settlings, promptly get the supernatant liquor that contains the A-beta oligomer.Confirm the formation situation of A-beta oligomer in the supernatant with atomic force microscope.
The screening of embodiment 2 positive colonies
(PBS pH=7.4) dilutes embodiment 1 gained A-beta oligomer to 10～100 μ g/mL, gets 4mL and joins in the immune pipe, and 4 ℃ of bags are spent the night to be cushioned liquid with bag.Abandon supernatant, wash pipe rapidly 3 times with PBS.Fill with the immunity pipe with 3%BSA, room temperature is vertically sealed 2h.Abandon supernatant, wash pipe rapidly 3 times with PBS.Be suspended in phage antibody library (available from the MRC center of Britain) among the 4mL 3%BSA and join immunity and manage, after room temperature is put upside down and hatched 1h, vertically hatch 1h.With the PBS of 0.1%Twenn-20 washing 10 times (second take turns screening and with after scouring 20 times).After PBS blotted, add 500 μ L pancreatin-PBS eluant solution phage, room temperature is put upside down and is hatched 10min.In the e. coli tg1 (available from the MRC center of Britain) of 1.75mL OD600=0.4, add the phage that 250 μ L elute, hatch 30min, do not shake for 37 ℃.4 ℃ of preservations of phage that residue elutes.Get 10 respectively
4, 10
6Times diluent is applied on 2 * TY (available from Sigma company) flat board 37 ℃ of overnight incubation.With 4 ℃ of centrifugal 5min of 11600g of remaining TG1 nutrient solution,, be applied on 2 * TY flat board 37 ℃ of overnight incubation with the resuspended precipitation of 100 μ L substratum.
In the flat board that covers with bacterial clone, add 2mL 2 * TY substratum, and scrape with glass stick and to get bacterium, collect the thalline suspension, get 50 μ L and join 50mL 2 * TY substratum, adding final concentration in the substratum is the Amp of 100 μ g/mL, 1% glucose (purchase give birth in Shanghai worker's biotechnology company limited), 37 ℃ are shaken to OD600=0.4 and (need 1～2h) approximately.Residue bacterium liquid adds 15% glycerine ,-70 ℃ of preservations.
In the 10mL culture, add 5 * 10
10Helper phage M13K07 (available from the MRC center of Britain) 10 μ L.Hatch for 37 ℃ and leave standstill 30min.With 4 ℃ of centrifugal 10min of bacterium liquid 3000g, resuspended being deposited in 50mL 2 * TY substratum, adding final concentration is 100 μ g/mL Amp, 50 μ g/mL kantlex (purchase give birth in Shanghai worker's biotechnology company limited) and 0.1% glucose, 30 ℃ of shaking table overnight incubation.With overnight culture 3300g, 4 ℃ of centrifugal 15min collect supernatant, add the PEG (polyoxyethylene glycol) (20%) of 1/4 volume and the mixing solutions of NaCl (2.5M), place more than 1 hour for 4 ℃ behind the abundant mixing.4 ℃ of centrifugal 30min of 3300g fully abandon clean supernatant.The resuspended 2mL PBS that is deposited in, 4 ℃ of centrifugal 10min of 11600g.Obtain the phage antibody display libraries after supernatant is the enrichment first round.Get 1 μ L phage supernatant and measure titre.
Above enrichment screening process is repeated 3 times, promptly carry out second and third, the screening of four-wheel.
Be coated onto on the flat board through the above-mentioned 4 phage-infect intestinal bacteria HB2151 (available from the MRC center of Britain) that take turns the enrichment screening, the picking mono-clonal is to 96 porocyte culture plates, add 2 * TY substratum, 200 μ L in every hole, contain the glucose of 100 μ g/mL Amp and 1% in the substratum.37 ℃ of shaking tables (300r/min) overnight incubation.2 μ L bacterium liquid are drawn in every hole, join in the other 96 new hole microbial culture plates, and 2 * TY substratum, 200 μ L are added in every hole, contain the glucose of 100 μ g/mL Amp and 1% in the substratum, and 37 ℃ of shaking tables are cultivated 3h, and OD600 reaches 0.9.Add certain volume glycerine again in first block of plate, the glycerine final concentration is 15% ,-70 ℃ of preservations.Every hole adds IPTG to final concentration 1mmol/L in second 96 orifice plate, 30 ℃ of shaking table overnight incubation.
With the antigenic solution 50 μ Ls of concentration with volume, 4 ℃ of bags were spent the night when every hole added with screening in elisa plate, and it is PBS that bag is cushioned liquid, pH=7.4.Wash plate 3 times with PBS, 37 ℃ of sealings of 3%BSA 2h, 200 μ L/ holes.Wash plate 3 times with PBS, with 4 ℃ of centrifugal 10min of 800g of above-mentioned culture, every hole is got 50 μ L and is joined in the elisa plate, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 three times.Every hole adds the 9E10 (available from U.S. Santa cruz company) of the HRP mark after the 50 μ L dilution, and Dilution ratio is 1: 5000, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds substrate solution TMB (available from U.S. Amresco company) 50 μ L, places 37 ℃ of 20min, and every then hole adds 1mmol/L sulfuric acid 50 μ L termination reactions, puts enzyme mark detector and measures OD value (wavelength 490nm).The OD value is higher than does not wrap the tentative positive clone by the clone of A-beta oligomer negative control more than 2 times, and measures its dna sequence dna and do further checking.
Use the positive colony that phage display technology screens by above-mentioned steps, now carry out preservation in China Committee for Culture Collection of Microorganisms common micro-organisms center (DSMZ of institute of microbiology of the Chinese Academy of Sciences), classification called after colon bacillus Escherichia coli, preservation date is on December 22nd, 2008, and deposit number is CGMCC No.2818.
The evaluation of embodiment 3 antibody
Abeta monomer, oligomer and fibrous body (the Abeta monomer is hatched more than 4 days for 37 ℃, and verifies through atomic force microscope) are put NC (nitrocellulose) film 3 μ L respectively.Film with after the 5%BSA sealing, is added scFv WC2 and hatched 1 hour, wash film 3 times with PBS, each 5 minutes.The Protein A (available from U.S. Santa cruz company) that adds dilution in 1: 5000 was again hatched 1 hour, washed film 3 times with PBST (Tween-20 concentration is 0.1%), each 5 minutes, developed the color with DAB.Only demonstrate spot at A-beta oligomer place, the clone of display dot just is not needed clone in A-beta monomer and fibrous body.
With the evaluation of checking order of above-mentioned positive colony, the clone who meets antibody basic structure in the antibody library then is complete strand genetic engineering antibody.Sequencing primer is:
LMB3:5 '-CAG GAA ACA GCT ATG AC-3 ' (its nucleotide sequence is shown in SEQ ID NO.10)
PHEN seq:5 '-CTA TGC GGC CCC ATT CA-3 ' (its nucleotide sequence is shown in SEQ ID NO.11)
Preparation and the purifying of embodiment 4 antibody WC2
(1) spawn culture
Substratum consists of 2 * YT substratum, 100 μ g/mL Amp (penbritin) (giving birth to worker's biotechnology company limited available from Shanghai), 1% glucose.The bacterial classification access 5mL culture medium culturing of Screening and Identification is spent the night, change the 500mL substratum again over to, cultivate 3h for 37 ℃, adding IPTG (isopropylthiogalactoside) (giving birth to worker's biotechnology company limited available from Shanghai) is 1mM to final concentration, 30 ℃ of overnight incubation.
(2) purifying antibody
With 4 ℃ of cultures, the centrifugal 45min of 12000g with the filtering membrane vacuum filtration of supernatant with 0.45 μ m and 0.22 μ m, fully removes bacterial debris and other impurity, concentrates supernatant to 150mL with VivaFlow50 film bag.RProtein A affinity column with PH 7.4,0.15M PBS balance GE company carries out wash-out with 0.3M pH 3.0 sodium-acetates as elutriant behind the sample upper prop, and every collection tube is collected 0.5-1ml.Eluted product is 4 ℃ of dialysed overnight in the PBS of pH 7.4.Carry out purity detecting (the about 29KD of antibody molecule amount) with sds gel electrophoresis commonly used then, with the protein concentration of antibody behind BCA test kit or other common method mensuration purifying.The present productive rate of this antibody is 40mg/L.With antibody with the small volume packing, and in-20 ℃ of preservations.
Embodiment 5 antibody affinity costant k
DWith binding constant k
aMensuration
Utilize surface plasma resonance method (SPR) antagonist k
DMeasure.Go up fixedly A-beta oligomer in detection chip (CM5) (available from Sweden Biacore company), and, WC2 is diluted to the different concns gradient with the HEPES damping fluid with HEPES damping fluid equilibrate overnight.Applied sample amount is 35 μ L during detection, and flow velocity is 5 μ L/min, and data retardation time is 120 seconds behind the last sample, and detects the protein binding signal, at last with BIAcore company analysis software reading of data and calculate the K of each antibody
DValue.Record binding constant WC2 between WC2 and the A-beta oligomer and the binding constant k between A-beta 42 oligomer
a=4.29 * 10
2M
-1s
-1, dissociation constant k
d=1.74 * 10
-5s
-1, affinity constant K
D=3.32 * 10
-7M.
Embodiment 6ELISA detection WC2 combines with the A-beta oligomer.
The A-beta oligomer bag of different concns by the enzyme linked immunological microwell plate, is put 37 ℃ of 2h, with 3%BSA37 ℃ of sealing 2h, 200 μ L/ holes.Wash plate 3 times with PBS, behind adding WC2 (5 μ g/ml) the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 three times.Every hole adds the ELIAS secondary antibody (HRP-9E10) after the 100 μ L dilution, and Dilution ratio is 1: 5000, behind the incubated at room 1h, with the PBS washing that contains 0.1%Tween-20 3 times.Every hole adds substrate solution TMB 100 μ L, places 37 20min ℃, and every then hole adds 1mmol/L sulfuric acid termination reaction, measures OD value (wavelength 490nm) (* P＜0.01) on enzyme-linked immunosorbent assay instrument.The result shows that WC2 can detect the A-beta oligomer of (being equivalent to every hole 6.25ng) of 2.2nM in the sample as shown in Figure 1.
Embodiment 7Dot-blot detects WC2 specific combination A-beta oligomer
1) will contain concentration be the sample 2 μ l points of A-beta oligomer of 5nM to nitrocellulose membrane, put in the air and dry.
2), put room temperature 2 hours with 5% skim-milk closing membrane.
3) film is put among the PBS solution that contains scFv WC2 (1 μ g/ml) or the control antibodies 4G8 (available from U.S. Signet company) and is incubated bath, put 37 ℃ 1 hour.
4) wash film 3 times, each 5 minutes with the PBS that contains 0.05%Tween-20.
5) film is joined the 9E10 (available from U.S. Santa cruz company) of HRP mark of dilution in 1: 1000, or the Protein A (1: 5000) of HRP mark put 37 ℃ 1 hour
6) washing is with 4.
7) film is joined in the DAB solution colour developing several minutes.
The result as shown in Figure 2, control antibodies 4G8 has can be in conjunction with the monomeric characteristic of A-beta, after A-beta42 was gathered into oligomer and filament, 4G8 still combined with them, and WC2 can only combine with the A-beta oligomer.
Embodiment 8WC2 is to A-beta42 accumulative restraining effect.
The 10 μ M A-beta42 that add or do not add WC2 antibody are hatched 28h in 37 ℃.180 μ L ThT (thioflavin) solution (with the preparation of pH6.5 PB damping fluid) are added to 96 hole black microwell plates, and add the above-mentioned sample of 20 μ L, mixing, is excitation wavelength with 450nm on multi-functional microplate reader again, and 480nm is an emission wavelength working sample fluorescence intensity.Fluorescence intensity is represented the aggregation extent of sample, and promptly high more its aggregation extent of fluorescence intensity is big more.The result is shown in Figure 3, compares with the A-beta42 that does not add antibody, and the A-beta42 fluorescence intensity behind the adding 0.25 and 1 μ M WC2 obviously reduces (* P＜0.01), shows that WC2 has the obvious suppression effect to the gathering of A-beta42.
Embodiment 9WC2 is to the Cytotoxic restraining effect of A-beta42.
With the substratum that contains 10% foetal calf serum (MEM) neuroblastoma (SH-SY5Y) is made into the individual cells suspension, with 7000 cell inoculations in every hole to 96 porocyte culture plates, every pore volume 100 μ L.Cell was in 37 ℃ of cultivations 24 hours, and incubator CO2 concentration is 5%.Every hole adds sample, is divided into three groups, is respectively: the mixture of A-beta42 and scFv WC2, A-beta42 organize and the cell that do not add sample as control group, the proteic final concentration of A-beta42 is 1 μ mol/L, the final concentration of scFv WC2 also is 1 μ mol/L.
Cell continues to cultivate after 48 hours, and every hole adds MTT solution (5mg/mL) 10 μ L, hatches 3 hours for 37 ℃, stop cultivating, every hole adds 100mol/L MTT lysate (the 0.01N HCl that contains 10%SDS and 5% isopropylcarbinol), and 37 ℃ of overnight incubation are fully dissolved crystallisate.Select the 450nm wavelength, on enzyme-linked immunosorbent assay instrument, measure each hole absorbance value.Is ordinate zou with each absorbance value of adding behind the sample divided by the absorbance value that does not add sample, statistics as shown in Figure 4, the adding pair cell of A-beta42 produces tangible toxicity, and WC2 can suppress this cytotoxicity (* P＜0.05).
Antibody WC2-1, WC2-2 activity experiment that embodiment 10 antibody WC2 sudden change forms
According to the previous experiments method, the antibody WC2-1 (its aminoacid sequence is shown in SEQ ID NO.8), the WC2-2 (its aminoacid sequence is shown in SEQID NO.9) that antibody WC2 are replaced with its indivedual amino acid mutations and form, carry out gathering test and the neurocyte toxicity test of A β, the result shows that WC2-1, WC2-2 can effectively suppress the fibrosis gathering of A β equally and obviously reduce the toxic action of A β to neuroblastoma cell SH-SY5Y.With WC2-1, WC2-2 as identification antibody, with enzyme-linked immunosorbent assay (ELISA) or immunodotting (Dot-blot) as experimental technique, with horseradish peroxidase (HRP) mark can with the 9E10 of WC2 specific combination as second antibody, can detect the A beta oligomers of nM level in the sample equally respectively.
SEQUENCE LISTING
＜110〉Tsing-Hua University
＜120〉with A-beta oligomer specificity bonded gene engineering monoclonal antibody
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165 170 175
Ala Pro Lys Leu Leu Ile Tyr Leu Gly Ser His Leu Ile Ser Gly Val
180 185 190
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
195 200 205
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
210 215 220
Asn Gln Lys Arg Pro Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
225 230 235 240
Lys Arg Ala Ala
<210>10
<211>17
<212>DNA
＜213〉primer LMB3
<400>10
caggaaacag ctatgac 17
<210>11
<211>17
<212>DNA
＜213〉primer pHEN seq
<400>11
ctatgcggcc ccattca 17
Claims (12)
1. monoclonal antibody, it is characterized in that, its variable region of heavy chain contains CDR1, CDR2, CDR3, the aminoacid sequence of CDR1, CDR2, CDR3 is respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEO ID NO.3, variable region of light chain contains CDR1, CDR2, CDR3, and the aminoacid sequence of CDR1, CDR2, CDR3 is respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
2. monoclonal antibody according to claim 1 is characterized in that, is humanized antibody.
3. single-chain antibody, it is characterized in that, its variable region of heavy chain contains CDR1, CDR2, CDR3, the aminoacid sequence of CDR1, CDR2, CDR3 is respectively shown in SEQ ID NO.1, SEQ ID NO.2 and SEO ID NO.3, variable region of light chain contains CDR1, CDR2, CDR3, and the aminoacid sequence of CDR1, CDR2, CDR3 is respectively shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
4. single-chain antibody according to claim 3, its aminoacid sequence is shown in SEQ ID NO.7.
5. single-chain antibody according to claim 3, its aminoacid sequence is shown in SEQ ID NO.8.
6. single-chain antibody according to claim 3, its aminoacid sequence is shown in SEQ ID NO.9.
7. single-chain antibody according to claim 4 is characterized in that, is the coli strain generation of CGMCCNo.2818 by deposit number.
8. claim 1 or the 2 described monoclonal antibodies application in preparation detection A-beta oligomer reagent.
9. the application of each described single-chain antibody of claim 3 to 7 in preparation detection A-beta oligomer reagent.
10. claim 1 or 2 described monoclonal antibodies are preparing the anti-ageing year purposes in the dementia medicine.
11. each described single-chain antibody of claim 3 to 7 is preparing the anti-ageing year purposes in the dementia medicine.
12. a pharmaceutical composition is characterized in that, contains claim 1 or 2 described monoclonal antibodies or each the described single-chain antibody of claim 3-7 and the pharmaceutically acceptable carrier of significant quantity.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1649623A (en) * | 2002-02-28 | 2005-08-03 | 明德赛特生物制药美国公司 | Specific antibodies to amyloid beta peptide, pharmaceutical compositions and methods of use thereof |
| CN1798766A (en) * | 2003-05-30 | 2006-07-05 | 神经实验室有限公司 | Humanized antibodies that recognize beta amyloid peptide |
| CN101325972A (en) * | 2005-12-12 | 2008-12-17 | Ac免疫有限公司 | A beta 1-42 specific monoclonal antibodies with therapeutic properties |
-
2009
- 2009-01-16 CN CN2009100769910A patent/CN101463083B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1649623A (en) * | 2002-02-28 | 2005-08-03 | 明德赛特生物制药美国公司 | Specific antibodies to amyloid beta peptide, pharmaceutical compositions and methods of use thereof |
| CN1798766A (en) * | 2003-05-30 | 2006-07-05 | 神经实验室有限公司 | Humanized antibodies that recognize beta amyloid peptide |
| CN101325972A (en) * | 2005-12-12 | 2008-12-17 | Ac免疫有限公司 | A beta 1-42 specific monoclonal antibodies with therapeutic properties |
Non-Patent Citations (2)
| Title |
|---|
| Andleeb Zameer et al..Anti-oligomeric Aβ Single-chain Variable Domain Antibody Blocks Aβ-induced Toxicity Against Human Neuroblastoma Cells.《J. Mol. Biol.》.2008,第384卷 * |
| Mary P. Lambert et al..Monoclonal antibodies that target pathological assemblies of Aβ.《Journal of Neurochemistry》.2007,第100卷 * |
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| CN101463083A (en) | 2009-06-24 |
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