CN102850454B - Anti-RSV (respiratory syncytial virus) human monoclonal antibody, and its preparation method - Google Patents
Anti-RSV (respiratory syncytial virus) human monoclonal antibody, and its preparation method Download PDFInfo
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Abstract
The invention relates to the biotechnical field, concretely relates to a monoclonal antibody of a virus, and more concretely relates to an anti-RSV human monoclonal antibody and its preparation method. The antibody can be used for enhancing the anti-RSV infection tolerance of human subjects, and reducing the infection level in infected individual bodies or mitigating symptoms caused by the RSV infection, and the administration dosage of the anti-RSV human monoclonal antibody is lower than that of current market products.
Description
Technical field
The present invention relates to biological technical field, specifically relate to monoclonal antibody of a kind of virus and preparation method thereof, more particularly relate to a kind of human monoclonal antibodies to anti respiratory syncytial virus RSV and preparation method thereof.Described antibody can be used for strengthening the tolerance of the anti-rsv infection of human subjects and reducing the infection level in the individual body having infected or alleviate the caused symptom of rsv infection.
Background technology
1, general introduction and mechanism of causing a disease
Respiratory syncytial virus pneumonia (respiratory syncytial virus pneumonia) is called for short syncytial virus pneumonia; it is a kind of tunicate minus-stranded rna virus; the Pneumovirinae (Pneumovirus) that belongs to Paramyxo virus (Paramyxo-viridae) section belongs to (Fenner; Virology71:371-378,1975; Huang et al., J.Virol.43,1982).RSV is the common interstitial pneumonia of a kind of children's, is multiplely born in infant.Because mother passes the generation that antibody can not preventing infection, birth young infant soon can be fallen ill, but newborn infant is more rare.External occasionally have ward infection to cause the report of maternity hospital's Neonatal Ward eruption and prevalence.
Respiratory syncytial virus (RSV, is called for short syncytial virus, also belongs to Paramyxoviridae) is to cause the modal cause of disease of infantile viral pneumonia, can cause interstitial pneumonia, and capillary bronchitis.In Beijing, 48% virus pneumonia and 58% capillary bronchitis system cause (1980～1984) by syncytial virus; In Guangzhou, 31.4% of infantile pneumonia and capillary bronchitis causes (1973～1986) by syncytial virus; In the U.S., 20%～25% infantile pneumonia and 50%～75% capillary bronchitis are caused by syncytial virus.
RSV under Electronic Speculum finding and parainfluenza virus similar, virion size is about 150nm, slightly little compared with parainfluenza virus, for RNA viruses, to ether sensitivity, without hemagglutination, cultivate and form the distinctive born of the same parents (syncytium) that close at people's epithelium, viral in endochylema internal breeding, visible intracytoplasmic inclusion.Syncytial virus only has a serotype, and molecular biology method proves to have two hypotypes recently.
Be 2～8 days (mostly being 4～6 days) latent period of syncytial virus infection.The typical finding of syncytial virus pneumonia is that monocytic interstitial infiltrates.Main manifestations is alveolar septum broadening and oozes out take monocyte as main interstitial, comprising lymphocyte, plasmocyte and scavenger cell.In addition alveolar space is full of edematous fluid, and visible hyalin membrane formation of lung.In some cases, the also lymphocytic infiltration of visible bronchiole wall.There is the oedema with necrotic area in pulmonary parenchyma, cause alveolar filling, consolidation and wither.Minority case is visible multinuclear fused cell in alveolar space, and form and measles giant cells are similar, but can not find intranuclear inclusion.
2, epidemiology
Syncytial virus infection is extremely wide.Resist full result (1978) in Beijing with determination of immunofluorescence method serum IgG: Cord blood positive rate 93%, birth was 89% to 1 month, within 1～6 month, be all to reach more than 70% for 40%, 2 years old and 3 years old, 4 years old until be the 80% left and right positive (complement is consistent therewith in conjunction with measuring) for 14 years old.
Because mother passes the fully generation of preventing infection of antibody, whenever syncytial virus pneumonia all may occur after birth.Be more common in below 3 years old, 1～6 month visible heavier case, man is more than female.Northern China is more common in winter-spring season, and spring and summer is more common in Guangdong.Because antibody can not be protected from infection completely, the infection again of syncytial virus is very common, and someone observes 10 years, then infection rate is up to 65%.The infectivity of syncytial virus is very strong, has report kinsfolk in succession to infect, and while generation within the family, older youngster and adult are generally upper respiratory tract infection.In bibliographical information institute, secondary syncytial virus infection rate is up to 30%～50%.
From the whole world, comprising the U.S., Europe, Australia and Japanese, rsv infection is all a large problem for a long time.For premature infant, child and the elderly, rsv infection especially bothers, and for the grownup of those function of immune system declines, is also like this.According to estimates, can there is chronic severe infections at least about the nearly all children that have 2/3,1-4 year in 1 years old following children, and this factor is considered to bring out children in the later stage and occurs stridulating and asthma-like symptom.
Syncytial virus pneumonia and capillary bronchitis account for first of China's Infant Viral Pneumonia nearly ten years, and its symptom and parainfluenza virus pneumonia, light disease Influenza Virus Pneumonia and light disease adenovirus pneumonia almost cannot be distinguished clinically.Severe Influenza Virus Pneumonia and severe adenovirus pneumonia high heat continue, toxicity symptom and respiratory symptom weight, and clinical manifestation is serious far beyond syncytial virus pneumonia.
3, research and development of products and development trend
RSV has two main surface glycoproteins, F and G.Two glycoprotein (90KD and 68KD) are exposed to virus particle surface.The G albumen of the high glycosylation of 90KD is responsible for virion to be attached to (Walsh et al., J.Gen.Virol.65:761-767,1984) on target cell.The protein mediated viral tunicle of 68KD F and cytogamy and Syncytium formation (Walsh et al., J.Gen.Virol.66:409-415,1985).Described F and G surface glycoprotein are main protective antigen, and nucleoprotein N and envelope protein M2 have less protectiveness activity.Neutralization and inhibition fusion monoclonal antibody have also been defined in specific region (Walsh et al., Infect, Immun.43:756-758,1984 of F glycoprotein; Trudel et al., J.Gen.Virol.68:2273-80,1987; Beeler et al., J.Virol.63:2941-50,1989; Lopez et al., J.Virol.64:927-30,1990; Paradiso et al., Vaccine9:231-7,1991).The monoclonal antibody of anti-G glycoprotein unlikely neutralizes virus than the monoclonal antibody of anti-F glycoprotein, and does not have inhibition fusion-activity (Norrby et al., proc.Natl.Acad.Sci.USA84:6572-6576,1987; Garcia-Barreno et al., J.Virol.63:925-932,1989; Walsh et al., J.Gen.Virol.70:2953-2961,1989).The aminoacid sequence of F glycoprotein is infecting with people the conservative property (Toms, FEMS Micro-biol.Immunol.76:243-256,1991) that has about 90% between relevant RSV subgroup.
Past people will make great efforts to concentrate on the live-virus vaccine of exploitation attenuation.But due to poor effect, fully attenuation or heredity are upper not unstable, and the people concentrate on RSV surface glycoprotein F and G making great efforts recently.The anti-RSV monoclonal antibody of current unique listing is only ratified to infect RSV for prevent premature youngster, is the antibody of anti-F albumen.The title of this antibody be palivizumab (
, MedImmune manufactures), it acts on wide spectrum, but the dosage of at present each required palivizumab of administration is 15mg/kg(body weight), because monoclonal antibody manufacturing technique requirent is high, and make expensive, therefore commercially available prod is on the high side at present, is difficult in enormous quantities popularization in China and produces.Thus, need a kind of effective ways that prevent or treat RSV disease, and provide that specificity is higher, production technique is more economical, dosage is lower, and the cheaper product of price is to meet vast social demand at present.The present invention explores and meets this demand.
Summary of the invention
The technical problem that will solve required for the present invention has been to provide new human monoclonal antibodies to respiratory syncytial virus RSV fusion rotein and preparation method thereof, be a kind of new anti-RSV human monoclonal antibodies and preparation method thereof, the above-mentioned defect existing to overcome prior art.
That is to say, the present invention is directed to the deficiencies in the prior art, by practical studies and literature search and theoretical investigationes such as large number of biological technology experiments, the anti-RSV human monoclonal antibodies that provides a class avidity higher is provided one of object, the efficient monoclonal antibody of low dosage that provides a class to be used for the treatment of RSV disease, particularly can be for preventing and/or treating the human monoclonal antibodies of RSV disease, its specificity is for the F glycoprotein of RSV.Preferably, this monoclonal antibody is substantially pure form, does not contain other phylactic agent.
The invention provides a kind of anti-RSV human monoclonal antibodies, comprise people source constant region, SEQ ID NO:1 is selected in its variable region of heavy chain; Its variable region of light chain is selected from SEQ ID NO:2.Preferably, described monoclonal antibody and described sequence homology are 98% or 95%.
On the other hand, the invention provides the composition that contains one or more monoclonal antibodies and suitable carrier or thinner.
The composition that contains this clone is also provided in the present invention, and said composition contains this clone and can maintain the nutritional medium of this clone.Suitable substratum contains carbon source, nitrogenous source, and if need, also contain VITAMIN and/or organic salt.
Two of object of the present invention is to provide a kind of method that RSV infects in host for the treatment of or prevent, the method comprises to host uses the antibody of the present invention that is enough to reach treatment or prevents disease amount.
On the other hand, the present invention also comprises the pharmaceutical composition for preventing or treat RSV, comprising reducing inflammatory reaction, said composition comprises single antibody of the present invention or immunoreactivity fragment as promoting agent, or two antibody or the fragment of being no more than of the present invention is as promoting agent.
Other aspects of the present invention comprise the method that uses Antybody therapy human subjects rsv infection or induce these objects to tolerate RSV.
Three of object of the present invention is a kind of described human monoclonal antibodies or preparation methods of its conservative property mutant or its active fragments of containing.
Monoclonal antibody of the present invention can be utilized recombination method preparation, and therefore this invention also comprises for the preparation of the recombined material of monoclonal antibody and for the preparation of clone or the immortality cell of these antibody.
On the other hand, the invention provides a kind of method of breeding hybridoma cell line of the present invention, comprise this cell is cultivated in nutritional medium.Propagation method also represents that production can be from the method for the antibody of the present invention of substratum separation.Preferably, the breeding of hybridoma cell line of the present invention is external carrying out.Wherein this clone is cultivated in nutritional medium.If the suitable nutritional medium of cell of the present invention contains carbon source, nitrogenous source and needs, also contain VITAMIN and/or inorganic salt.As, can use and mend the RPMI1640 substratum that has 10% foetal calf serum (SIGMA).Another kind of suitable substratum is Sigma serum-free and without protein hybridoma substratum.
(1) technical conceive
Due to technical limitation, at present commercially available monoclonal antibodies medicine is always on the high side, limits it and uses and promote.Therefore, in conjunction with existing monoclonal antibody technique, carry out Improvement and perfection, particularly explore from aspects such as efficient anti-RSV human monoclonal antibodies of low dosage and preparation method thereof, have very important significance.
According to this idea and thinking, contriver, by experimental study and analysis and theory study repeatedly, successfully obtains the result of study of expection and the human monoclonal antibodies of the anti-RSV of application product.
(2) preparation method of monoclonal antibody
The preparation method of anti-RSV human monoclonal antibodies, comprises the steps:
1. the preparation of hybridoma, comprising:
A. mouse immune;
B. hybridoma is cultivated and screening;
2. monoclonal antibody ANC compares and screening;
3. the clone of monoclonal antibody and humanization;
4. the structure of monoclonal antibody expression plasmid;
5. the expression of monoclonal antibody and purifying.
Wherein, the method for described mouse immune is:
A. screen and obtain the cell strain of expressing anti-RSV F albumen by hybridoma technology.In the commercially available phosphoric acid buffer that contains RSV antigen (PBS) 120ul of Capricon company, add Freund's complete adjuvant (CFA) mixing, the emulsification of 120ul, make CFA RSV antigenic solution 240ul.Same method utilizes Freund's incomplete adjuvant (IFA) to make the IFA RSV antigenic solution of 240ul again;
B. carry out abdominal injection with the FCA RSV antigenic solution of 240ul to the female mouse of BALB/C in 7-8 age in week, carry out first immunisation.After first immunisation, carry out supplementary immunization every 2-3 week with FIA RSV antigenic solution.At separating Morr. cell intravenous injection 240ul RSV antigen (Capricon company system) before 10 days and 3 days.Take out spleen in immunity after the 42nd day, separating Morr. cell, merges splenocyte and SP2-0 rat bone marrow tumour cell by PEG method 1/1, prepares hybridoma.
Described hybridoma is cultivated and the method for screening is:
A. under aseptic condition, the outstanding HAT of using of hybridoma substratum is adjusted to concentration to 3 × 10
6/ ml, to the each hole packing bed board of 96 microwell plates (Croming company), 3 × 10
5/ hole, 100ul/ hole.96 microwell plates are placed in 37 ℃, the CO2gas incubator of 8%CO2 and leave standstill and cultivate, until hybridoma clone occurs;
B. prepare the antigen coated ELISA of RSV and screen 96 orifice plates: by RSV antigen (Capricon company) with PH7.0 and contain 0.1%(w/v) dilution of the phosphoric acid buffer (PBS) of NaN3, to concentration 0.5ug/ml, and point be filled to (NUNC company) in blank elisa plate, 100ul/ hole, leaves standstill overnight incubation under 2-8 ℃ of condition.The PBS damping fluid (abbreviation scavenging solution) of elisa plate containing 0.05%TWEEN20 of hatching after end cleans 3 times, and controls dry raffinate.Again by containing 1%(w/v) the PBS damping fluid (abbreviation coating buffer) of BSA adds in elisa plate, and 300ul/ hole, leaves standstill to hatch under 2-8 ℃ of condition and re-uses above for 6 hours, preserves as used to be placed under 2-8 ℃ of condition not in time;
C. before detecting, remove the coating buffer in elisa plate, prepare fresh coating buffer, then add 60ul in each hole.Take out hybridoma to be measured and cultivate 96 orifice plates, carry out mark and code at the each hole place that grows clone, under aseptic condition, draw the cells and supernatant in 40ul/ hole, add in the elisa plate being coated with, mix and carry out mark.Elisa plate rocks gently at ambient temperature and immune response was fully carried out in 1-2 hour.Remove reaction liquid, with scavenging solution washing elisa plate 3 times and control dry raffinate.The anti-mouse IgG polyclonal antibody (SANTA CRUZ) that dilutes horseradish peroxidase (POD) mark with the coating buffer of fresh preparation, extension rate is 1 × 10
4.In the every hole of elisa plate, add the detection antibody after 120ul dilution, room temperature lucifuge reaction 30 minutes.Remove reaction liquid termination reaction, with scavenging solution washing elisa plate 3 times and control dry raffinate, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, room temperature lucifuge reaction 15 minutes, then adds stop buffer termination reaction.Microplate reader for elisa plate (MD company) detects the light absorption value of 492nm wavelength;
D. screening obtain 3 hybridoma cell strains that expression is higher, be numbered BW01-4H13, BW01-7B05 and BW01-10E02, cell amplification cultivate after freezing be kept in liquid nitrogen stand-by.
The described comparison of monoclonal antibody ANC and screening method are:
The antigen neutralization ability of three kinds of anti-RSV monoclonal antibodies that screening hybridoma cell strain is obtained is confirmed.Buy three kinds of RSV virus strain from the biological product collecting center of USS (ATCC): B1wild-type strain (10[4.5] TCID (50)/ml), A2(10[5.5] TCID (50)/ml) and Long strain (10[6.7] TCID (50)/ml), the RSV of acquisition carries out 10 times of dilutions with the phosphate buffered saline buffer (abbreviation diluent) that contains bovine serum albumin (BSA).Prepare antigen extraction agent: the new base phenyl ether of 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol (DTT) and 0.1% polyoxyethylene.RSV virus and the antigen extraction agent of having diluted are carried out to equal-volume mixing, react and obtain antigen extraction liquid after 5-10 minute.Preparation has been coated with the sepharose (GE) of anti-mouse IgG polyclonal antibody (SANTA CRUZ), and colloid concentration is 15%(v/v) sepharose solution.And gelating soln is mixed with the antigen extracting, Dispersal risk titre detects mix reagent (detection liquid).The above-mentioned detection liquid mixing is added in 96 microwell plates (Croming company, be called for short Sptting plate), and each hole packing 50ul, carries out after mark stand-by.Simultaneously by BW01-4H13, BW01-7B05 and BW01-10E02 cell strain etc. Conditioned Media add respectively in each hole, 150ul/ hole.Setting up positive controls simultaneously, replace three kinds of antibody and detect liquid reaction with isopyknic scavenging solution, is respectively three kinds of positive controls.Then under room temperature condition, light shaking mixes and hatches 1-2 hour, and antigen and three kinds of antibody that three kinds of virus strain are extracted fully react.The day before yesterday is prepared positive detection elisa plate in experiment: at the upper coated goat-anti RSV polyclonal antibody (CHEMICON company) of blank elisa plate (NUNC company), antibody is diluted to 10ug/ml with coating buffer, 100ul/ hole is divided in elisa plate, leaves standstill overnight incubation under 2-8 ℃ of condition.Before using, clean positive detection elisa plate with scavenging solution, control dry raffinate.From Sptting plate, draw reaction mixture, and be transferred to successively in positive detection elisa plate, 100ul is drawn in every hole, strictly avoids gel sucking-off.Carry out after correspondence markings, positive detection elisa plate is constantly rocked and mixed gently, at room temperature reaction 1-2 hour, make coated antibody fully catch free antigen molecule.Set up negative control simultaneously, utilize scavenging solution and coated antibody response, as the background values detecting.Utilize three washings of scavenging solution, stop immune response.The anti-mouse IgG polyclonal antibody (SIGMA) that dilutes horseradish peroxidase (POD) mark with the coating buffer of fresh preparation, extension rate is 1 × 10
2.In the every hole of elisa plate, add the detection antibody after 100ul dilution, room temperature lucifuge reaction 30 minutes.Remove reaction liquid termination reaction, with scavenging solution washing elisa plate 3 times and control dry raffinate, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, room temperature lucifuge reaction 20 minutes, then adds stop buffer termination reaction.Microplate reader for elisa plate (MD company) detects the light absorption value of 492nm wavelength; Can draw from light absorption value result, except 4H13, all the other two kinds of antibody have certain neutralizing effect for three kinds of virus strain, and comparatively speaking the power of neutralising capacity is followed successively by 7B05 > 10E02 > 4H13.
The clone of described monoclonal antibody and humanized method are:
A. from 2 × 107BW01-7B05 hybridoma, extract total mRNA according to the operation instructions of RNesay test kit (Qiagen).Use widow-dT primer and reversed transcriptive enzyme to prepare strand cDNA, the aliquots containig of cDNA is used as to the initial substance of polymerase chain reaction (PCR) with the gene of amplification variable region;
B. to utilize the total mRNA of 1ug be template in the preparation of strand cDNA, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KClPH8.5, add the AMV reversed transcriptive enzyme of people's placenta Yeast Nucleic Acid inhibitor, 33uM random hexamer and 10 units of 1mM dithiothreitol (DTT) (DTT), 1mM dNTP, 25 units, reactive system is placed in 42 ℃ of reaction 1-2 hour.Primer P1(SEQ ID NO:1 for the variable region of heavy chain (VH) of 7B05 monoclonal antibody) and P2(SEQ ID NO:2) carry out polymerase chain reaction (PCR) amplification and obtain, variable region of light chain (VL) is with primer P3(SEQ ID NO:3) and P4(SEQ ID NO:4) carry out pcr amplification acquisition;
The pcr amplification of C.DNA fragment is take 1ug strand CRNA as template, in the buffer system of 10mM Tris-cl, 1.5mM Mg2Cl, 50mMKCl PH8.5, add the Taq enzyme (TAKARA) of 1mM dithiothreitol (DTT) (DTT), the corresponding primer of 0.5mM dNTPs, 1uM and 2.5 units, reactive system is placed in 1 minute, 55 ℃ primers of 94 ℃ of annealing of PCR instrument (THERMO) in conjunction with 2 minutes, 72 ℃ amplifications 2 minutes, 25 circulating reactions.The DNA fragmentation of amplification is with using axenic purification water dissolution, dilution after the extracting of phenol chloroform.DNA fragmentation is with being integrated into plasmid pUC18 (TAKARA) after EcoR I and BamH I (TAKARA) double digestion, and operation instructions is shown in pUC18 test kit.Utilize primer T7 to check order (Shanghai Mei Ji biotech company) to the product D NA of amplification, confirm the accuracy of its sequence;
D. submit to ncbi database to carry out sequence alignment the VH of 7B05 and VL sequence, and many people's that submitted to VH and VL sequence compare, select the highest sequence of similarity to carry out the humanization processing of sequence.By comparing, the VH sequence of 7B05 and people's IgG Cor sequence similarity degree is the highest, reaches 82%; VL sequence and people's IgG K102 sequence similarity degree is the highest, reaches 75%.Humanized result will improve humanized degree as far as possible and keep specificity and the high-affinity of antibody to antigen recognition site simultaneously as far as possible, therefore the sequence that retains mouse source 7B05 antibody at three hypervariable regions (CD1, CD2 and CD3) separately of VH and VL, all the other regions adopt people's IgG Cor sequence and K102 sequence.
The method of the structure of described monoclonal antibody expression plasmid is:
A.7B05 the constant region sequence of monoclonal antibody (CH and CL) adopts people's IgG Cor sequence fragment corresponding with K102 sequence.Wherein the amplification template of CH fragment is IgG Cor gene, and the amplification template of CL fragment is IgG K102 gene.CH and CL fragment adopt the mode of sequence assembly to obtain;
B. the synthetic employing primer of fragment is spelled overlapping extension PCR, will be as by fragment 1 and 2,3 and 4,5 and 6,7 and 8,9 and 10 pairing mixing respectively, carry out overlapping extension PCR, and experience annealing under conventional PCR condition, primer identification pairing and fragment amplification obtain 1-2,3-4,5-6,7-8,9-10 etc. overlapping and extend fragment.In like manner, by 1-2 and 3-4,5-6 and 7-8 splicing are extended, 9-10 fragment bye.After many wheel PCR, obtain 1-10 fragment.Finally through too much obtaining light heavy chain fragment after wheel splicing.Humanized heavy chain fragment is integrated into the MCSA interval of pIRES plasmid (Clontech, plasmid map as shown in Figure 1) between Nhe I and Mlu I, and light chain segments is integrated into the MCSB interval of pIRES plasmid between Not I and Sal I.Plasmid is after large scale culturing, frozen stand-by after plasmid extraction test kit (QIAGEN) preparation.
The method of the expression of described monoclonal antibody and the structure of purifying is:
A.7B05 the acquisition of monoclonal antibody, by transfection CHO-K1 cell (ATCC), obtains the cell strain of stably express antibody protein in the situation that of drug screening;
B. transfection adopts the lipofectamineTM2000 lipofectamine box that Invitrogen company produces, to specifications operation.The blank of transfection experiment adopts pIRES carrier transfection CHO-K1 cell.In substratum, add G418(200uM) combine pressurization screening, cell is placed under 8%CO2,37 ℃ of conditions and cultivates, and within 3 days, changes a subculture, removes dead floating cell.After cultured continuously 20 days, in Tissue Culture Dish, there is being dispersed into the cell clone of simple community, cell is removed after substratum and use trypsin INVITROGEN) digestion is transferred to 96 orifice plates by limiting dilution assay after separating and cultivates and screen, and guarantees every Kong Zhongwei single cell as far as possible.96 orifice plates are placed in to cultivate under 8%CO2,37 ℃ of conditions and after 1 week, get culture supernatant and carry out ELISA and measure expressing quantity;
C. mouse-anti-human T NFR primary antibodie (SIGMA) with PH7.0 and contain 0.1%(w/v) the PBS(diluent of NaN3) dilution, extension rate 1 × 103.Antibody after dilution adds treats coated elisa plate (CORING), 100ul/ hole, and 2-8 ℃ is spent the night.Elisa plate cleans three times with the PBS damping fluid (abbreviation scavenging solution) containing 0.05%TWEEN20, every hole adds 100ul5%(w/v) BSA phosphate buffered saline buffer room temperature effect 1-2 hour, cells and supernatant to be detected adds in elisa plate, 100ul/ hole, and room temperature standing and reacting is hatched for 1-2 hour.Discard reaction solution, with scavenging solution cleaning three times, add the mouse-anti human IgG-Fc/HRP bis-anti-(SIGMA) of 1000 times of diluted, 150ul/ hole, room temperature leaves standstill hatches TMB reagent (PERCE) colour developing after 1-2 hour, and 450nm surveys OD value.With the negative contrast of fresh culture;
D. select the most much higher clonal expansion to 24 orifice plate of expression level, cultivate and detect protein expression after 3 days, detect and adopt above-mentioned ELISA to detect.Select high 10 clones of expression amount to continue amplification cultivation, keep cell screening condition constant (G418(200uM) and MSX(50ug/ml)); 6 clonal expansions that after many wheel amplifications, selection expression level is the highest, to T75 culturing bottle, are set up cell bank cryopreservation by cell strain.Clone BW-7B05-3 the highest expression level is inoculated in 2L rolling bottle simultaneously, with EX-CELL302 substratum (SIGMA) cultivation containing 5% calf serum (INVITROGEN), in the time that cell covers with bottle wall, be changed to serum free medium EX-CELL302, receive every other day liquid, receive liquid 3-5 time continuously;
E. utilize the specific adsorption effect of albumin A to IgG, adopt separating medium rProtein A Sepharose4Fast Flow(GE) cells and supernatant of results is carried out to affinity chromatography, purifying expressing protein.Working method is referring to the description of product.After purifying, measure A260 and A280 value with ultraviolet spectrophotometer.Protein quantification formula: protein content (mg/ml)=OD280 value × 1.45-OD260 value × 0.74.
(3) main application and technology speciality
The present invention provides a kind of new anti-RSV human monoclonal antibodies and preparation method thereof for industries such as medicine, thereby has expanded the application of the new treatment of respiratory syncytial virus pneumonia or prevention product.
The anti-RSV human monoclonal antibodies of the present invention is safe, can be used in scale operation and the application of the industry such as medical treatment, diagnosis and industry.
Compared with existing anti-RSV disease product, the anti-RSV human monoclonal antibodies of the present invention, at aspects such as practical applications, can reduce unit dosage greatly, reduces the distinguishing features such as production cost.
Therefore, the successful exploitation of the method for the invention has great significance for the detection of this active principle of epitope in the research and development of vaccine, the method, also for control, early screening, the diagnostic work of RSV disease provide accurate, easy, effective monitoring means, is with a wide range of applications and social demand simultaneously.
In a word, active adaption of the present invention the need of work in field and the needs of human nature service such as modern biology, prevention and health care, clinical diagnosis, the present invention is this anti-RSV human monoclonal antibodies of research and development, to improving existing treatment or preventing RSV disease product to have important value.
Accompanying drawing explanation
Fig. 1 is the gene mapping of pIRES expression vector.
Fig. 2 is multiple clone site and the enzyme point of contact collection of illustrative plates of PIRES expression vector.
Fig. 3 is the absolute number result of the plaque that every microgram humanization 7B05 monoclonal antibody and Synagis antibody are corresponding.
Fig. 4 is the comparison diagram of humanization 7B05 monoclonal antibody and the neutralizing effect of Synagis to RSV-B1 virus strain.
Embodiment
The present invention has studied existing RSV disease treatment or prevention method, and the human monoclonal antibodies of a kind of new anti-RSV is provided, and is convenient to the safe handling in the fields such as modern biology, prevention and health care, clinical diagnosis.
The present invention finally need to be prepared into anti-RSV human monoclonal antibodies and apply, and will enumerate embodiment below and be further described.If you have questions, can contact directly with contriver.In above-mentioned some experimental datas that provide and the following example, provide several anti-RSV human monoclonal antibodies and preparation method thereof and some experimental study contents by aforementioned summary of the invention, but the research contents that list in the place that should be appreciated that the present invention is not limited to this, should also be appreciated that term as used herein is only for describing specific embodiment, and be not limitation of the invention.
That is to say; in the present invention; the embodiment of above-described embodiment and the following stated is all in order to set forth better the present invention; particularly these embodiment are only to exemplary description of the present invention, can not explain for the restriction to the application or be used for limiting the scope of the invention.
In order to be illustrated more clearly in the present invention, below in conjunction with following embodiment, the present invention is described in detail.
The preparation of embodiment 1 hybridoma
1. mouse immune
Screen and obtain the cell strain of expressing anti-RSV F albumen by hybridoma technology.In the commercially available phosphoric acid buffer that contains RSV antigen (PBS) 120ul of Capricon company, add Freund's complete adjuvant (CFA) mixing, the emulsification of 120ul, make CFARSV antigenic solution 240ul.Same method utilizes Freund's incomplete adjuvant (IFA) to make the IFA RSV antigenic solution of 240ul again.
Carry out abdominal injection to the female mouse of BALB/C in 7-8 age in week with the FCA RSV antigenic solution of 240ul, carry out first immunisation.After first immunisation, carry out supplementary immunization every 2-3 week with FIA RSV antigenic solution.At separating Morr. cell intravenous injection 240ul RSV antigen (Capricon company system) before 10 days and 3 days.Take out spleen in immunity after the 42nd day, separating Morr. cell, merges splenocyte and SP2-0 rat bone marrow tumour cell by PEG method 1/1, prepares hybridoma.
2. hybridoma is cultivated and screening
Under aseptic condition, the outstanding HAT of using of hybridoma substratum is adjusted to concentration to 3 × 10
6/ ml, to the each hole packing bed board of 96 microwell plates (Croming company), 3 × 10
5/ hole, 100ul/ hole.96 microwell plates are placed in 37 ℃, the CO2gas incubator of 8%CO2 and leave standstill and cultivate, until hybridoma clone occurs.
Preparation RSV antigen coated ELISA screens 96 orifice plates: by RSV antigen (Capricon company) with PH7.0 and contain 0.1%(w/v) phosphoric acid buffer (PBS) dilution of NaN3, to concentration 0.5ug/ml, and point be filled to (NUNC company) in blank elisa plate, 100ul/ hole, leaves standstill overnight incubation under 2-8 ℃ of condition.The PBS damping fluid (abbreviation scavenging solution) of elisa plate containing 0.05%TWEEN20 of hatching after end cleans 3 times, and controls dry raffinate.Again by containing 1%(w/v) the PBS damping fluid (abbreviation coating buffer) of BSA adds in elisa plate, and 300ul/ hole, leaves standstill to hatch under 2-8 ℃ of condition and re-uses above for 6 hours, preserves as used to be placed under 2-8 ℃ of condition not in time.
Before detecting, remove the coating buffer in elisa plate, prepare fresh coating buffer, then add 60ul in each hole.Take out hybridoma to be measured and cultivate 96 orifice plates, carry out mark and code at the each hole place that grows clone, under aseptic condition, draw the cells and supernatant in 40ul/ hole, add in the elisa plate being coated with, mix and carry out mark.Elisa plate rocks gently at ambient temperature and immune response was fully carried out in 1-2 hour.Remove reaction liquid, with scavenging solution washing elisa plate 3 times and control dry raffinate.The anti-mouse IgG polyclonal antibody (SANTA CRUZ) that dilutes horseradish peroxidase (POD) mark with the coating buffer of fresh preparation, extension rate is 1 × 10
4.In the every hole of elisa plate, add the detection antibody after 120ul dilution, room temperature lucifuge reaction 30 minutes.Remove reaction liquid termination reaction, with scavenging solution washing elisa plate 3 times and control dry raffinate, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, room temperature lucifuge reaction 15 minutes, then adds stop buffer termination reaction.Microplate reader for elisa plate (MD company) detects the light absorption value of 492nm wavelength.
Screening obtain 3 hybridoma cell strains that expression is higher, be numbered BW01-4H13, BW01-7B05 and BW01-10E02, cell amplification cultivate after freezing be kept in liquid nitrogen stand-by.
Embodiment 2 monoclonal antibody ANC comparisons
The antigen neutralization ability of three kinds of anti-RSV monoclonal antibodies that screening hybridoma cell strain is obtained is confirmed.Buy three kinds of RSV virus strain from the biological product collecting center of USS (ATCC): B1wild-type strain (10[4.5] TCID (50)/ml), A2(10[5.5] TCID (50)/ml) and Long strain (10[6.7] TCID (50)/ml), the RSV of acquisition carries out 10 times of dilutions with the phosphate buffered saline buffer (abbreviation diluent) that contains bovine serum albumin (BSA).
Prepare antigen extraction agent: the new base phenyl ether of 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol (DTT) and 0.1% polyoxyethylene.RSV virus and the antigen extraction agent of having diluted are carried out to equal-volume mixing, react and obtain antigen extraction liquid after 5-10 minute.
Preparation has been coated with the sepharose (GE) of anti-mouse IgG polyclonal antibody (SANTA CRUZ), and colloid concentration is 15%(v/v) sepharose solution.And gelating soln is mixed with the antigen extracting, Dispersal risk titre detects mix reagent (detection liquid).For concentration and the virus titer of antigen extraction liquid, mitigation scheme (table one) as shown in the table:
The each antigen extraction liquid of table one relaxes scheme
| Virus strain | Antigen extraction liquid (ml) | Diluent (ml) | Sepharose solution (ml) |
| B1wild-type strain | 0.3 | 1.2 | 1.5 |
| A2 | 0.09 | 1.41 | 1.5 |
| Long strain | 0.7 | 0.8 | 1.5 |
The above-mentioned detection liquid mixing is added in 96 microwell plates (Croming company, be called for short Sptting plate), and each hole packing 50ul, carries out after mark stand-by.Simultaneously by BW01-4H13, BW01-7B05 and BW01-10E02 cell strain etc. Conditioned Media add respectively in each hole, 150ul/ hole.Setting up positive controls simultaneously, replace three kinds of antibody and detect liquid reaction with isopyknic scavenging solution, is respectively three kinds of positive controls.Then under room temperature condition, light shaking mixes and hatches 1-2 hour, and antigen and three kinds of antibody that three kinds of virus strain are extracted fully react.
The day before yesterday is prepared positive detection elisa plate in experiment: at the upper coated goat-anti RSV polyclonal antibody (CHEMICON company) of blank elisa plate (NUNC company), antibody is diluted to 10ug/ml with coating buffer, 100ul/ hole is divided in elisa plate, leaves standstill overnight incubation under 2-8 ℃ of condition.
Before using, clean positive detection elisa plate with scavenging solution, control dry raffinate.From Sptting plate, draw reaction mixture, and be transferred to successively in positive detection elisa plate, 100ul is drawn in every hole, strictly avoids gel sucking-off.Carry out after correspondence markings, positive detection elisa plate is constantly rocked and mixed gently, at room temperature reaction 1-2 hour, make coated antibody fully catch free antigen molecule.Set up negative control simultaneously, utilize scavenging solution and coated antibody response, as the background values detecting.Utilize three washings of scavenging solution, stop immune response.
The anti-mouse IgG polyclonal antibody (SIGMA) that dilutes horseradish peroxidase (POD) mark with the coating buffer of fresh preparation, extension rate is 1 × 10
2.In the every hole of elisa plate, add the detection antibody after 100ul dilution, room temperature lucifuge reaction 30 minutes.Remove reaction liquid termination reaction, with scavenging solution washing elisa plate 3 times and control dry raffinate, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, room temperature lucifuge reaction 20 minutes, then adds stop buffer termination reaction.Microplate reader for elisa plate (MD company) detects the light absorption value of 492nm wavelength.
Utilize following formula to calculate three kinds of antibody (being called for short 4H13,7B05 and 10E02) to viral capture ability (neutralization ratio):
Neutralization ratio %=[1-(light absorption value-negative control)/(positive control-negative control)] 100%
Three kinds of antibody to viral neutralization ratio evaluation as shown in following table (table two).Wherein " +++ " represents that neutralization ratio is more than 80%, and " ++ " is illustrated in more than 60%, and "+" is illustrated in more than 30%, and "-" is illustrated in below 30%.
Three kinds of antibody of table two are to RSV virus neutralization ratio
Can find out from upper table result, except 4H13, all the other two kinds of antibody have certain neutralizing effect for three kinds of virus strain, and comparatively speaking the power of neutralising capacity is followed successively by 7B05 > 10E02 > 4H13.
Clone and the humanization of embodiment 37B05 monoclonal antibody
According to the operation instructions of RNesay test kit (Qiagen) from 2 × 10
7in BW01-7B05 hybridoma, extract total mRNA.Use widow-dT primer and reversed transcriptive enzyme to prepare strand cDNA, the aliquots containig of cDNA is used as to the initial substance of polymerase chain reaction (PCR) with the gene of amplification variable region.
It is template that the preparation of strand cDNA utilizes the total mRNA of 1ug, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KCl PH8.5, add the AMV reversed transcriptive enzyme of people's placenta Yeast Nucleic Acid inhibitor, 33uM random hexamer and 10 units of 1mM dithiothreitol (DTT) (DTT), 1mM dNTP, 25 units, reactive system is placed in 42 ℃ of reaction 1-2 hour.Primer P1(SEQ ID NO:1 for the variable region of heavy chain (VH) of 7B05 monoclonal antibody) and P2(SEQ ID NO:2) carry out polymerase chain reaction (PCR) amplification and obtain, variable region of light chain (VL) is with primer P3(SEQ ID NO:3) and P4(SEQ ID NO:4) carry out pcr amplification acquisition.Primer is synthetic by Shanghai Mei Ji biotech company, and primer sequence is as follows:
SEQ?ID?NO:1:AGCGGATCCA?GGGGCCAGTG?GATAGAC
SEQ?ID?NO:2:TGGATGGTGG?GAAGATG
SEQ?ID?NO:3:GGCCAGTGGA?TAGAC
SEQ?ID?NO:4:TACAGTTGGT?GCAGCA
The pcr amplification of DNA fragmentation is take 1ug strand CRNA as template, in the buffer system of 10mM Tris-cl, 1.5mM Mg2Cl, 50mM KClPH8.5, add the Taq enzyme (TAKARA) of 1mM dithiothreitol (DTT) (DTT), the corresponding primer of 0.5mM dNTPs, 1uM and 2.5 units, reactive system is placed in 1 minute, 55 ℃ primers of 94 ℃ of annealing of PCR instrument (THERMO) in conjunction with 2 minutes, 72 ℃ amplifications 2 minutes, 25 circulating reactions.The DNA fragmentation of amplification is with using axenic purification water dissolution, dilution after the extracting of phenol chloroform.DNA fragmentation is with being integrated into plasmid pUC18 (TAKARA) after EcoR I and BamH I (TAKARA) double digestion, and operation instructions is shown in pUC18 test kit.Utilize primer T7 to check order (Shanghai Mei Ji biotech company) to the product D NA of amplification, confirm the accuracy of its sequence.
Submit to ncbi database to carry out sequence alignment the VH of 7B05 and VL sequence, and many people's that submitted to VH and VL sequence compare, select the highest sequence of similarity to carry out the humanization processing of sequence.By comparing, the VH sequence of 7B05 and people's IgG Cor sequence similarity degree is the highest, reaches 82%; VL sequence and people's IgG K102 sequence similarity degree is the highest, reaches 75%.Humanized result will improve humanized degree as far as possible and keep specificity and the high-affinity of antibody to antigen recognition site simultaneously as far as possible, therefore the sequence that retains mouse source 7B05 antibody at three hypervariable regions (CD1, CD2 and CD3) separately of VH and VL, all the other regions adopt people's IgG Cor sequence and K102 sequence.People's IgG Cor sequence and the comparison of K102 sequence and the VH of humanization 7B05 and the result of VL are as follows:
The VH of 7B05 after humanization
The structure of embodiment 47B05 monoclonal antibody expression plasmid
The constant region sequence (CH and CL) of 7B05 monoclonal antibody adopts people's IgG Cor sequence fragment corresponding with K102 sequence.Wherein the amplification template of CH fragment is IgG Cor gene, and the amplification template of CL fragment is IgG K102 gene.CH and CL fragment adopt the mode of sequence assembly to obtain, and design primer is as follows:
CH synthetic primer fragment
CH synthetic primer fragment
The synthetic employing primer of fragment is spelled overlapping extension PCR, will be as by fragment 1 and 2,3 and 4,5 and 6,7 and 8,9 and 10 pairing mixing respectively, carry out overlapping extension PCR, and experience annealing under conventional PCR condition, primer identification pairing and fragment amplification obtain 1-2,3-4,5-6,7-8,9-10 etc. overlapping and extend fragment.In like manner, by 1-2 and 3-4,5-6 and 7-8 splicing are extended, 9-10 fragment bye.After many wheel PCR, obtain 1-10 fragment.Finally through too much obtaining light heavy chain fragment after wheel splicing.Humanized heavy chain fragment is integrated into the MCSA interval of pIRES plasmid (Clontech, plasmid map is as shown in Fig. 2) between Nhe I and Mlu I, and light chain segments is integrated into the MCSB interval of pIRES plasmid between Not I and Sal I.Plasmid is after large scale culturing, frozen stand-by after plasmid extraction test kit (QIAGEN) preparation.
Expression and the purifying of embodiment 57B05 monoclonal antibody
The acquisition of 7B05 monoclonal antibody, by transfection CHO-K1 cell (ATCC), obtains the cell strain of stably express antibody protein in the situation that of drug screening.
The lipofectamine that transfection adopts Invitrogen company to produce
tM2000 lipofectamine boxes, to specifications operation.The blank of transfection experiment adopts pIRES carrier transfection CHO-K1 cell.In substratum, add G418(200uM) combine pressurization screening, cell is placed under 8%CO2,37 ℃ of conditions and cultivates, and within 3 days, changes a subculture, removes dead floating cell.After cultured continuously 20 days, in Tissue Culture Dish, there is being dispersed into the cell clone of simple community, cell is removed after substratum and use trypsin INVITROGEN) digestion is transferred to 96 orifice plates by limiting dilution assay after separating and cultivates and screen, and guarantees every Kong Zhongwei single cell as far as possible.96 orifice plates are placed in to cultivate under 8%CO2,37 ℃ of conditions and after 1 week, get culture supernatant and carry out ELISA and measure expressing quantity.
Mouse-anti-human T NFR primary antibodie (SIGMA) is with PH7.0 and contain 0.1%(w/v) the PBS(diluent of NaN3) dilution, extension rate 1 × 10
3.Antibody after dilution adds treats coated elisa plate (CORING), 100ul/ hole, and 2-8 ℃ is spent the night.Elisa plate cleans three times with the PBS damping fluid (abbreviation scavenging solution) containing 0.05%TWEEN20, every hole adds 100ul5%(w/v) BSA phosphate buffered saline buffer room temperature effect 1-2 hour, cells and supernatant to be detected adds in elisa plate, 100ul/ hole, and room temperature standing and reacting is hatched for 1-2 hour.Discard reaction solution, with scavenging solution cleaning three times, add the mouse-anti human IgG-Fc/HRP bis-anti-(SIGMA) of 1000 times of diluted, 150ul/ hole, room temperature leaves standstill hatches TMB reagent (PERCE) colour developing after 1-2 hour, and 450nm surveys OD value.With the negative contrast of fresh culture.
Select the most much higher clonal expansion to 24 orifice plate of expression level, cultivate and detect protein expression after 3 days, detect and adopt above-mentioned ELISA to detect.Select high 10 clones of expression amount to continue amplification cultivation, keep cell screening condition constant (G418(200uM) and MSX(50ug/ml)); 6 clonal expansions that after many wheel amplifications, selection expression level is the highest, to T75 culturing bottle, are set up cell bank cryopreservation by cell strain.Clone BW-7B05-3 the highest expression level is inoculated in 2L rolling bottle simultaneously, with EX-CELL302 substratum (SIGMA) cultivation containing 5% calf serum (INVITROGEN), in the time that cell covers with bottle wall, be changed to serum free medium EX-CELL302, receive every other day liquid, receive liquid 3-5 time continuously.
Utilize the specific adsorption effect of albumin A to IgG, adopt separating medium rProtein A Sepharose4Fast Flow(GE) cells and supernatant of results is carried out to affinity chromatography, purifying expressing protein.Working method is referring to its description of product.After purifying, measure A260 and A280 value with ultraviolet spectrophotometer.Protein quantification formula: protein content (mg/ml)=OD280 value × 1.45-OD260 value × 0.74.
In the RSV of embodiment 67B05 monoclonal antibody and experiment
The external virus neutralizing cpaacity of 7B05 monoclonal antibody is measured with the experiment of standard plaque, has adopted commercially available RSV antibody as parallel control in experiment.Hep2 cell is regulated to concentration and is inoculated in 12 orifice plates (CRONING) with the RPMI1640 substratum (SIGMA) containing 10% foetal calf serum (SIGMA), and inoculum density is 2 × 10
5/ hole, 5%CO2,37 ℃ of overnight incubation are stand-by.7B05 monoclonal antibody is carried out to concentration gradient dilution with substratum, and the RSV-B1 in about 200PFU/ hole is added in the antibody-solutions having diluted, add rabbit complement serum (SIGMA), incubated at room 1-2 hour simultaneously.Antibody-viral the mixing solutions that adds 200ul/ hole in the substratum of Hep2 cell, incubated at room 2-3 hour, makes virus infected cell.Remove all substratum, add and contain 1%(w/v) fresh culture of methylcellulose gum (MERCK), culture dish is hatched 6 days under 35 ℃ of conditions, carries out the fixing dyeing of cell.
Remove the substratum that contains methylcellulose gum in culture dish, the fixed cell 30 minutes at ambient temperature of the methyl alcohol with 100%, then with scavenging solution cleaning culture dish three times.Add primary antibodie---the goat-anti RSV polyclonal antibody (CHEMICON company) with 1000 times of dilutions of diluent, 100ul/ hole, room temperature reaction 1-2 hour, adds scavenging solution to clean three times, termination reaction.Add and use two of 800 times of dilutions of diluent to resist---the anti-sheep polyclonal antibody of rabbit (MERCK) of horseradish peroxidase-labeled, 100ul/ hole, room temperature reaction 1-2 hour, adds scavenging solution to clean three times, termination reaction.Add chlorinated naphthalene phenol reagent (PERCE) 200ul/ hole, room temperature reaction 10 minutes, clear water rinses removes reagent, plaque number in dry rear statistics single hole.
What Fig. 3 showed is the result of the absolute number of the plaque that every microgram humanization 7B05 antibody is corresponding, wherein also comprises the result of Synagis antibody.Result shows that the IC50 of 7B05 is 15ng/ml, and corresponding avidity is 100pM, and the IC50 of Synagis antibody is 300ng/ml, and corresponding avidity is 2nM.
What Fig. 4 showed is the neutralizing effect that 7B05 antibody and Synagis compare RSV-B1 virus strain.In and data (% of contrast) be to calculate according to the 160-180 of each experiment plaque absolute number.External avidity is 1pM to the EC50 value of the antibody of the present invention of 5nM between 10-100ng/ml.
Claims (3)
1. an anti-RSV human monoclonal antibodies, comprises people source constant region, it is characterized in that: its variable region of heavy chain is selected from SEQ ID NO:1; Its variable region of light chain is selected from SEQ ID NO:2.
2. anti-RSV human monoclonal antibodies as claimed in claim 1, is characterized in that: described anti-RSV human monoclonal antibodies is the antibody of specificity for RSV F albumen.
3. anti-RSV human monoclonal antibodies as claimed in claim 1, is characterized in that: described anti-RSV human monoclonal antibodies is substantially pure form, does not contain other phylactic agent.
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