CN102850454A - 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 reduces the infection level in the individual body that has 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 the 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, Virology 71:371-378,1975; Huang et al., J.Virol.43,1982).RSV is the common interstitial pneumonia of a kind of children's, multiplely is born in the infant.Because female generation that passes antibody can not preventing infection, birth young infant soon can be fallen ill, but the newborn infant is more rare.External idol has 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 finding and parainfluenza virus under Electronic Speculum is similar, the virion size is about 150nm, slightly little than parainfluenza virus, be RNA viruses, responsive to ether, without hemagglutination, form the distinctive born of the same parents (syncytium) that close in the cultivation of people's epithelium, viral in the endochylema internal breeding, visible intracytoplasmic inclusion.Syncytial virus only has a serotype, and the molecular biology method proof has 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 matter infiltrates.Main manifestations is alveolar septum broadening and oozes out take monocyte matter between main, comprising lymphocyte, plasmocyte and scavenger cell.Alveolar space is full of edematous fluid in addition, and visible hyalin membrane formation of lung.In some cases, the also lymphocytic infiltration of visible bronchiole wall.Oedema with the necrotic area occurs in pulmonary parenchyma, cause alveolar filling, consolidation and wither.The 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 the determination of immunofluorescence method serum IgG: Cord blood positive rate 93%, birth to 1 month is 89%, 1～6 month be all to reach more than 70% in 40%, 2 years old and 3 years old, 4 years old until be about 80% positive (complement is consistent therewith in conjunction with measuring) in 14 years old.
Because the fully generation of preventing infection of female biography antibody, whenever the syncytial virus pneumonia all may occur after birth.Be more common in below 3 years old, 1～6 month visible heavier case, the man is more than the woman.Northern China is more common in winter-spring season, and spring and summer then is more common in Guangdong.Because antibody can not be protected from infection fully, the again infection of syncytial virus is very common, and the someone observed 10 years, and infection rate is up to 65% again.The infectivity of syncytial virus is very strong, has the report kinsfolk in succession to infect, and when occuring within the family, older youngster and adult are generally upper respiratory tract infection.Secondary syncytial virus infection rate is up to 30%～50% in the bibliographical information institute.
From the whole world, comprising the U.S., Europe, Australia and Japanese, rsv infection all is a large problem for a long time.For premature infant, child and the elderly, rsv infection especially bothers, and also is like this for the grownup that those function of immune system descend.According to estimates, chronic severe infections can occur at least about nearly all children that 2/3,1-4 year is arranged in the children below 1 years old, 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 over past ten years, and its symptom and parainfluenza virus pneumonia, light disease Influenza Virus Pneumonia and light disease adenovirus pneumonia almost can't be distinguished clinically.Severe Influenza Virus Pneumonia and severe adenovirus pneumonia then high heat continue, and toxicity symptom and respiratory symptom are heavy, and clinical manifestation is serious far beyond the 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 the virus particle surface.The G albumen of the high glycosylation of 90KD is responsible for virion is attached to (Walsh et al., J.Gen.Virol.65:761-767,1984) on the 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 it is active that nucleoprotein N and envelope protein M2 have less protectiveness.Specific region (Walsh et al., Infect, Immun.43:756-758,1984 that monoclonal antibody also has been defined in F glycoprotein are merged in neutralization and inhibition; 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., Vaccine 9:231-7,1991).The monoclonal antibody of anti-G glycoprotein is than the monoclonal antibody of the anti-F glycoprotein virus that unlikely neutralizes, and do not have inhibition fusion-activity (Norrby et al., proc.Natl.Acad.Sci.USA 84:6572-6576,1987; 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 has about 90% conservative property (Toms, FEMSMicro-biol.Immunol.76:243-256,1991) infecting with the people between relevant RSV subgroup.
The past people concentrate on the live-virus vaccine of exploitation attenuation making great efforts.But because poor effect, fully attenuation or heredity are upper not unstable, and the people concentrate on RSV surface glycoprotein F and the G making great efforts recently.The anti-RSV monoclonal antibody of present unique listing is only ratified to infect RSV for the prevent premature youngster, is the antibody of anti-F albumen.The title of this antibody be palivizumab (
MedImmune makes), it acts on wide spectrum, but the dosage of at present each required palivizumab of administration is 15mg/kg (body weight), because the monoclonal antibody manufacturing technique requirent is high, and make costliness, so the commercially available prod is on the high side at present, is difficult in enormous quantities the popularization in China and produces.Thus, need a kind of effective ways that prevent or treat the RSV disease, and provide that specificity is higher, production technique is more economical, dosage is lower, and the cheaper product of price is to satisfy at present vast social demand.The present invention explores and satisfies this demand.
Summary of the invention
The technical problem that will solve required for the present invention has provided 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 defects that exists 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 purpose, the efficient monoclonal antibody of the low dosage that namely provides a class to be used for the treatment of the RSV disease, particularly can be used 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 basically pure form, does not contain other phylactic agent.
The invention provides a kind of anti-RSV human monoclonal antibodies, comprise people source constant region, its variable region of heavy chain is selected SEQ IDNO:1; 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.
Also provide the composition that contains this clone in the present invention, said composition contains this clone and can keep the nutritional medium of this clone.Suitable substratum contains carbon source, nitrogenous source, and if necessary, also contain VITAMIN and/or organic salt.
Two of purpose of the present invention provides a kind of method that RSV infects for the treatment of or prevent in the host, the method comprises to the host uses the antibody of the present invention that is enough to reach the treatment or prevents the disease amount.
On the other hand, the present invention also comprises the pharmaceutical composition for prevention or treatment RSV, comprising reducing inflammatory reaction, said composition comprises single antibody of the present invention or immunoreactivity fragment as promoting agent, and perhaps 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 to be used Antybody therapy human subjects rsv infection or induces these objects to the method for RSV tolerance.
Three of purpose of the present invention is a kind of preparation methods that contain described human monoclonal antibodies or its conservative property mutant or its active fragments.
Monoclonal antibody of the present invention can be utilized recombination method preparation, so 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 the method for the antibody of the present invention that production can separate from substratum.Preferably, the breeding of hybridoma cell line of the present invention is external carrying out.Wherein this clone is cultivated in nutritional medium.The suitable nutritional medium of cell of the present invention contains carbon source, nitrogenous source and if necessary, also contains VITAMIN and/or inorganic salt.As, can use and mend RPMI 1640 substratum that 10% foetal calf serum (SIGMA) is arranged.Another kind of suitable medium is Sigma serum-free and without the protein hybridoma substratum.
(1) technical conceive
Because 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, experimental study and analysis and the theory study of contriver by repeatedly successfully obtains the result of study of expecting 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 comprises:
A. mouse immune;
B. hybridoma is cultivated and screening;
2. the monoclonal antibody ANC relatively reaches 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. obtain to express the cell strain of anti-RSV F albumen by the hybridoma technology screening.The Freund's complete adjuvant (CFA) that adds 120ul in the commercially available phosphoric acid buffer that contains RSV antigen (PBS) 120ul of Capricon company mixes, emulsification, makes CFARSV 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 the 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 with PEG method 1/1, the preparation hybridoma.
Described hybridoma is cultivated and the method for screening is:
A. under the aseptic condition that hybridoma is outstanding in HAT substratum adjustment concentration to 3 * 10
6/ ml is to each hole packing bed board of 96 microwell plates (Croming company), 3 * 10
5/ hole, the 100ul/ hole.96 microwell plates place 37 ℃, the CO2gas incubator of 8%CO2 to leave standstill cultivation, until the hybridoma clone occurs;
B. prepare the antigen coated ELISA of RSV and screen 96 orifice plates: with RSV antigen (Capricon company) with PH7.0 and contain phosphoric acid buffer (PBS) dilution of 0.1% (w/v) NaN3, to concentration 0.5ug/ml, and divide in the elisa plate that is filled to blank (NUNC company), overnight incubation is left standstill under the 2-8 ℃ of condition in the 100ul/ hole.The elisa plate of hatching after the end cleans 3 times with the PBS damping fluid (abbreviation scavenging solution) that contains 0.05%TWEEN 20, and controls dried raffinate.The PBS damping fluid (abbreviation coating buffer) that will contain again 1% (w/v) BSA adds in the elisa plate, and the 300ul/ hole is left standstill to hatch more than 6 hours under the 2-8 ℃ of condition and re-used, and then places under the 2-8 ℃ of condition such as untimely use and preserves;
C. remove the coating buffer in the elisa plate before detecting, prepare fresh coating buffer, in each hole, add 60ul again.Take out hybridoma to be measured and cultivate 96 orifice plates, carry out sign and code at each place, hole that grows the clone, draw the cells and supernatant in 40ul/ hole under the aseptic condition, add in the coated good elisa plate, mix and carry out sign.Elisa plate rocks gently at ambient temperature and immune response was fully carried out in 1-2 hour.Remove reaction liquid, wash elisa plate 3 times and control dried raffinate with scavenging solution.With the anti-mouse IgG polyclonal antibody (SANTA CRUZ) of the coating buffer of fresh preparation dilution horseradish peroxidase (POD) mark, extension rate is 1 * 10
4In the every hole of elisa plate, add the detection antibody after 120ul dilutes, room temperature lucifuge reaction 30 minutes.Remove the reaction liquid termination reaction, wash elisa plate 3 times and control dried raffinate with scavenging solution, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, then room temperature lucifuge reaction 15 minutes adds the stop buffer termination reaction.Elisa plate detects the light absorption value of 492nm wavelength with microplate reader (MD company);
D. screening obtains 3 hybridoma cell strains that expression is higher, is numbered BW01-4H13, BW01-7B05 and BW01-10E02, after cell amplification is cultivated freezing be kept in the liquid nitrogen stand-by.
Described monoclonal antibody ANC relatively reaches screening method:
The antigen neutralization ability of three kinds of anti-RSV monoclonal antibodies that the screening hybridoma cell strain is obtained is confirmed.Buy three kinds of RSV virus strain from the biological product collecting center of USS (ATCC): B1 wild-type strain (10[4.5] TCID (50)/ml), A2 (10[5.5] and TCID (50)/ml) and Long strain (10[6.7] TCID (50)/ml), the phosphate buffered saline buffer (abbreviation diluent) that the RSV usefulness of acquisition contains bovine serum albumin (BSA) carries out 10 times of dilutions.Preparation antigen extraction agent: the new basic phenyl ether of 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol (DTT) and 0.1% polyoxyethylene.The RSV that diluted virus and antigen extraction agent are carried out the equal-volume mixing, react and namely obtain the 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 the sepharose solution of 15% (v/v).And the antigen that gelating soln is extracted together mixes Dispersal risk titre detection mix reagent (detection liquid).The above-mentioned detection liquid that mixes is added in 96 microwell plates (Croming company, be called for short Sptting plate), and each hole packing 50ul carries out behind the mark stand-by.Simultaneously with BW01-4H13, BW01-7B05 and BW01-10E02 cell strain etc. Conditioned Media add respectively in each hole the 150ul/ hole.Setting up simultaneously positive controls, namely replace three kinds of antibody and detect the liquid reaction with isopyknic scavenging solution, is respectively three kinds of positive controls.Then light shaking mixing and hatching 1-2 hour under the room temperature condition, antigen and three kinds of antibody that three kinds of virus strain are extracted fully react.Experiment prepares the positive detection elisa plate the day before yesterday: at the coated goat-anti RSV polyclonal antibody (CHEMICON company) of the elisa plate (NUNC company) of blank, antibody is diluted to 10ug/ml with coating buffer, the 100ul/ hole is divided in elisa plate, leaves standstill overnight incubation under the 2-8 ℃ of condition.Clean the positive detection elisa plate with scavenging solution before using, control dried raffinate.Draw reaction mixture from Sptting plate, and be transferred to successively in the positive detection elisa plate, 100ul is drawn in every hole, strictly avoids the gel sucking-off.After carrying out correspondence markings, the positive detection elisa plate is constantly rocked mixing gently, at room temperature reaction 1-2 hour, make coated antibody fully catch free antigen molecule.Set up simultaneously negative control, namely utilize scavenging solution and coated antibody response, as the background values that detects.Utilize three washings of scavenging solution, stop immune response.With the anti-mouse IgG polyclonal antibody (SIGMA) of the coating buffer of fresh preparation dilution horseradish peroxidase (POD) mark, extension rate is 1 * 10
2In the every hole of elisa plate, add the detection antibody after 100ul dilutes, room temperature lucifuge reaction 30 minutes.Remove the reaction liquid termination reaction, wash elisa plate 3 times and control dried raffinate with scavenging solution, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, then room temperature lucifuge reaction 20 minutes adds the stop buffer termination reaction.Elisa plate detects the light absorption value of 492nm wavelength with microplate reader (MD company); Can draw from the 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, with the aliquots containig of cDNA as the initial substance of polymerase chain reaction (PCR) gene with the amplification variable region;
B. the preparation of strand cDNA utilizes the total mRNA of 1ug to be template, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KClPH8.5, the AMV reversed transcriptive enzyme that adds people's placenta Yeast Nucleic Acid inhibitor, 33uM random hexamer and 10 units of 1mM dithiothreitol (DTT) (DTT), 1mM dNTP, 25 units, reactive system place 42 ℃ of reactions 1-2 hour.The variable region of heavy chain of 7B05 monoclonal antibody (VH) carries out polymerase chain reaction (PCR) amplification with primer P1 (SEQID NO:1) and P2 (SEQID NO:2) and obtains, and variable region of light chain (VL) carries out pcr amplification with primer P3 (SEQ ID NO:3) and P4 (SEQ ID NO:4) and obtains;
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, the Taq enzyme (TAKARA) that adds 1mM dithiothreitol (DTT) (DTT), the corresponding primer of 0.5mM dNTPs, 1uM and 2.5 units, reactive system places 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 used axenic purification water dissolution, dilution after using the extracting of phenol chloroform.Dna fragmentation is integrated into plasmid pUC18 (TAKARA) after with EcoR I and BamH I (TAKARA) double digestion, and operation instructions is seen the pUC18 test kit.Utilize primer T7 that the product D NA of amplification is checked order (Shanghai Mei Ji biotech company), 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, the humanization of selecting the highest sequence of similarity to carry out sequence is processed.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 simultaneously antibody to specificity and the high-affinity of antigen recognition site as far as possible, therefore keep the sequence of mouse source 7B05 antibody at separately three hypervariable regions (CD1, CD2 and CD3) of VH and VL, people's IgG Cor sequence and K102 sequence then adopted in all the other zones.
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 and the corresponding fragment of 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 pieced together overlapping extension PCR, will be as with fragment 1 and 2,3 and 4,5 and 6,7 and 8, overlapping extension PCR is carried out in 9 and 10 respectively pairing mixing, and experience annealing under conventional PCR condition, primer identification pairing and fragment amplification obtain 1-2,3-4,5-6,7-8, the overlapping and extension fragment such as 9-10.In like manner with 1-2 and 3-4,9-10 fragment bye is extended in 5-6 and 7-8 splicing.Behind many wheel PCR, obtain the 1-10 fragment.Finally through too much obtaining weight chain fragment after the wheel splicing.Humanized heavy chain fragment is integrated into the MCSA interval of pIRES plasmid (Clontech, plasmid map are as shown in Figure 1) between Nhe I and Mlu I, light chain segments is integrated into the MCSB interval of pIRES plasmid between Not I and Sal I.Plasmid is after large scale culturing, and is 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 obtains the cell strain of stably express antibody protein by transfection CHO-K1 cell (ATCC) in the situation of drug screening;
B. the lipofectamineTM 2000 lipofectamine boxes that Invitrogen company produces are adopted in transfection, to specifications operation.The blank of transfection experiment adopts pIRES carrier transfection CHO-K1 cell.Add G418 (200uM) and unite the pressurization screening in substratum, cell places under 8%CO2,37 ℃ of conditions and cultivates, and changes a subculture in 3 days, removes dead floating cell.After the cultured continuously 20 days, the cell clone of simple community appears in the Tissue Culture Dish being dispersed into, cell use trypsin INVITROGEN after removing substratum) 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 place to cultivate under 8%CO2, the 37 ℃ of conditions and get culture supernatant after 1 week and carry out ELISA and measure expressing quantity;
C. mouse-anti-human T NFR primary antibodie (SIGMA) is with PH7.0 and contain PBS (diluent) dilution of 0.1% (w/v) NaN3, extension rate 1 * 103.Antibody after the dilution adds elisa plate to be coated with (CORING), the 100ul/ hole, and 2-8 ℃ is spent the night.Elisa plate cleans three times with the PBS damping fluid (abbreviation scavenging solution) that contains 0.05%TWEEN 20, every hole adds 100ul 5% (w/v) BSA phosphate buffered saline buffer room temperature effect 1-2 hour, cells and supernatant to be detected adds in the elisa plate, the 100ul/ hole, the room temperature standing and reacting was hatched in 1-2 hour.Discard reaction solution, clean three times with scavenging solution, add the mouse-anti human IgG-Fc/HRP two anti-(SIGMA) of 1000 times of diluted, the 150ul/ hole, room temperature leaves standstill hatches TMB reagent (PERCE) colour developing after 1-2 hour, and 450nm surveys the 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 10 high clones of expression amount to continue amplification cultivation, keep cell screening condition constant (G418 (200uM) and MSX (50ug/ml)); Select 6 the highest clonal expansions of expression level to the T75 culturing bottle after many wheel amplifications, cell strain is set up cell bank and cryopreservation.The clone BW-7B05-3 that expression level is the highest is inoculated in the 2L rolling bottle simultaneously, cultivate with EX-CELL 302 substratum (SIGMA) that contain 5% calf serum (INVITROGEN), when treating that cell covers with bottle wall, be changed to serum free medium EX-CELL 302, receive every other day liquid, receive liquid 3-5 time continuously;
E. utilize albumin A to the specific adsorption effect of IgG, adopt separating medium rProteinA Sepharose 4Fast Flow (GE) that the cells and supernatant of results is carried out affinity chromatography, the purifying expressing protein.Working method is referring to the description of product.Behind the 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.
Compare with existing anti-RSV disease product, the anti-RSV human monoclonal antibodies of the present invention can reduce unit dosage, the distinguishing features such as Decrease production cost greatly at aspects such as practical applications.
Therefore, the develop 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 is with a wide range of applications and social demand also for control, early screening, the diagnostic work of RSV disease provide accurate, easy, effective monitoring means simultaneously.
In a word, active adaption of the present invention the need of work in the fields such as modern biology, prevention and health care, clinical diagnosis and the needs of human nature service, the present invention is this anti-RSV human monoclonal antibodies of research and development, to improving and improving existing treatment or prevent RSV disease product to have important value.
Description of drawings
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 plaque corresponding to every microgram humanization 7B05 monoclonal antibody and Synagis antibody.
Fig. 4 is that humanization 7B05 monoclonal antibody and Synagis are to the comparison diagram of the neutralizing effect of 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 anti-RSV newly 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 use, and the below will enumerate embodiment and further specify.If any problem, can contact directly with the contriver.Provide several anti-RSV human monoclonal antibodies and preparation method thereof and some experimental study contents by aforementioned summary of the invention in above-mentioned some experimental datas that provide and the following example, 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 only is used 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 all is in order to set forth better the present invention; particularly these embodiment only are to exemplary description of the present invention, can not explain for to the application's restriction or be used for limiting protection scope of the present 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
Obtain to express the cell strain of anti-RSV F albumen by the hybridoma technology screening.The Freund's complete adjuvant (CFA) that adds 120ul in the commercially available phosphoric acid buffer that contains RSV antigen (PBS) 120ul of Capricon company mixes, emulsification, makes CFARSV antigenic solution 240ul.Same method utilizes Freund's incomplete adjuvant (IFA) to make the IFARSV antigenic solution of 240ul again.
Carry out abdominal injection with the FCARSV antigenic solution of 240ul to the female mouse of BALB/C in 7-8 age in week, carry out first immunisation.After the first immunisation, carry out supplementary immunization every 2-3 week with the FIARSV 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 with PEG method 1/1, the preparation hybridoma.
2. hybridoma is cultivated and screening
Under the aseptic condition that hybridoma is outstanding in HAT substratum adjustment concentration to 3 * 10
6/ ml is to each hole packing bed board of 96 microwell plates (Croming company), 3 * 10
5/ hole, the 100ul/ hole.96 microwell plates place 37 ℃, the CO2gas incubator of 8%CO2 to leave standstill cultivation, until the hybridoma clone occurs.
Preparation RSV antigen coated ELISA screens 96 orifice plates: with RSV antigen (Capricon company) with PH7.0 and contain phosphoric acid buffer (PBS) dilution of 0.1% (w/v) NaN3, to concentration 0.5ug/ml, and divide in the elisa plate that is filled to blank (NUNC company), overnight incubation is left standstill under the 2-8 ℃ of condition in the 100ul/ hole.The elisa plate of hatching after the end cleans 3 times with the PBS damping fluid (abbreviation scavenging solution) that contains 0.05%TWEEN 20, and controls dried raffinate.The PBS damping fluid (abbreviation coating buffer) that will contain again 1% (w/v) BSA adds in the elisa plate, and the 300ul/ hole is left standstill to hatch more than 6 hours under the 2-8 ℃ of condition and re-used, and then places under the 2-8 ℃ of condition such as untimely use and preserves.
Remove the coating buffer in the elisa plate before detecting, prepare fresh coating buffer, in each hole, add 60ul again.Take out hybridoma to be measured and cultivate 96 orifice plates, carry out sign and code at each place, hole that grows the clone, draw the cells and supernatant in 40ul/ hole under the aseptic condition, add in the coated good elisa plate, mix and carry out sign.Elisa plate rocks gently at ambient temperature and immune response was fully carried out in 1-2 hour.Remove reaction liquid, wash elisa plate 3 times and control dried raffinate with scavenging solution.With the anti-mouse IgG polyclonal antibody (SANTA CRUZ) of the coating buffer of fresh preparation dilution horseradish peroxidase (POD) mark, extension rate is 1 * 10
4In the every hole of elisa plate, add the detection antibody after 120ul dilutes, room temperature lucifuge reaction 30 minutes.Remove the reaction liquid termination reaction, wash elisa plate 3 times and control dried raffinate with scavenging solution, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, then room temperature lucifuge reaction 15 minutes adds the stop buffer termination reaction.Elisa plate detects the light absorption value of 492nm wavelength with microplate reader (MD company).
Screening obtains 3 hybridoma cell strains that expression is higher, is numbered BW01-4H13, BW01-7B05 and BW01-10E02, after cell amplification is cultivated freezing be kept in the liquid nitrogen stand-by.
Embodiment 2 monoclonal antibody ANCs relatively
The antigen neutralization ability of three kinds of anti-RSV monoclonal antibodies that the screening hybridoma cell strain is obtained is confirmed.Buy three kinds of RSV virus strain from the biological product collecting center of USS (ATCC): B1 wild-type strain (10[4.5] TCID (50)/ml), A2 (10[5.5] and TCID (50)/ml) and Long strain (10[6.7] TCID (50)/ml), the phosphate buffered saline buffer (abbreviation diluent) that the RSV usefulness of acquisition contains bovine serum albumin (BSA) carries out 10 times of dilutions.
Preparation antigen extraction agent: the new basic phenyl ether of 0.4M NaCl, 0.1M citric acid, 10mM dithiothreitol (DTT) and 0.1% polyoxyethylene.The RSV that diluted virus and antigen extraction agent are carried out the equal-volume mixing, react and namely obtain the 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 the sepharose solution of 15% (v/v).And the antigen that gelating soln is extracted together mixes Dispersal risk titre detection mix reagent (detection liquid).For concentration and the virus titer of antigen extraction liquid, mitigation scheme (table one) as shown in the table:
Each antigen extraction liquid of table one relaxes scheme
| Virus strain | Antigen extraction liquid (ml) | Diluent (ml) | Sepharose solution (ml) |
| The B1wild-type strain | 0.3 | 1.2 | 1.5 |
| A2 | 0.09 | 1.41 | 1.5 |
| The Long strain | 0.7 | 0.8 | 1.5 |
The above-mentioned detection liquid that mixes is added in 96 microwell plates (Croming company, be called for short Sptting plate), and each hole packing 50ul carries out behind the mark stand-by.Simultaneously with BW01-4H13, BW01-7B05 and BW01-10E02 cell strain etc. Conditioned Media add respectively in each hole the 150ul/ hole.Setting up simultaneously positive controls, namely replace three kinds of antibody and detect the liquid reaction with isopyknic scavenging solution, is respectively three kinds of positive controls.Then light shaking mixing and hatching 1-2 hour under the room temperature condition, antigen and three kinds of antibody that three kinds of virus strain are extracted fully react.
Experiment prepares the positive detection elisa plate the day before yesterday: at the coated goat-anti RSV polyclonal antibody (CHEMICON company) of the elisa plate (NUNC company) of blank, antibody is diluted to 10ug/ml with coating buffer, the 100ul/ hole is divided in elisa plate, leaves standstill overnight incubation under the 2-8 ℃ of condition.
Clean the positive detection elisa plate with scavenging solution before using, control dried raffinate.Draw reaction mixture from Sptting plate, and be transferred to successively in the positive detection elisa plate, 100ul is drawn in every hole, strictly avoids the gel sucking-off.After carrying out correspondence markings, the positive detection elisa plate is constantly rocked mixing gently, at room temperature reaction 1-2 hour, make coated antibody fully catch free antigen molecule.Set up simultaneously negative control, namely utilize scavenging solution and coated antibody response, as the background values that detects.Utilize three washings of scavenging solution, stop immune response.
With the anti-mouse IgG polyclonal antibody (SIGMA) of the coating buffer of fresh preparation dilution horseradish peroxidase (POD) mark, extension rate is 1 * 10
2In the every hole of elisa plate, add the detection antibody after 100ul dilutes, room temperature lucifuge reaction 30 minutes.Remove the reaction liquid termination reaction, wash elisa plate 3 times and control dried raffinate with scavenging solution, add 100ul reaction substrate O-Phenylene Diamine (OPD) in every hole, then room temperature lucifuge reaction 20 minutes adds the stop buffer termination reaction.Elisa plate detects the light absorption value of 492nm wavelength with microplate reader (MD company).
Utilize following formula to calculate three kinds of antibody (being called for short 4H13,7B05 and 10E02) to the capture ability (neutralization ratio) of virus:
Neutralization ratio %=[1-(light absorption value-negative control)/(positive control-negative control)] 100%
Three kinds of antibody to the neutralization ratio evaluation of virus shown in following table (table two).Wherein " +++" the expression neutralization ratio is more than 80%, " ++ " 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 that from upper table result except 4H13, all the other two kinds of antibody have certain neutralizing effect for three kinds of virus strain, 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
7Extract total mRNA in the BW01-7B05 hybridoma.Use widow-dT primer and reversed transcriptive enzyme to prepare strand cDNA, with the aliquots containig of cDNA as the initial substance of polymerase chain reaction (PCR) gene with the amplification variable region.
The preparation of strand cDNA utilizes the total mRNA of 1ug to be template, in the buffer system of 50mM Tris-cl, 8mM Mg2Cl, 30mM KCl PH8.5, the AMV reversed transcriptive enzyme that adds people's placenta Yeast Nucleic Acid inhibitor, 33uM random hexamer and 10 units of 1mM dithiothreitol (DTT) (DTT), 1mM dNTP, 25 units, reactive system place 42 ℃ of reactions 1-2 hour.The variable region of heavy chain of 7B05 monoclonal antibody (VH) carries out polymerase chain reaction (PCR) amplification with primer P1 (SEQ ID NO:1) and P2 (SEQ ID NO:2) and obtains, and variable region of light chain (VL) carries out pcr amplification with primer P3 (SEQ ID NO:3) and P4 (SEQ ID NO:4) and obtains.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 10mMTris-cl, 1.5mMMg2Cl, 50mM KClPH8.5, the Taq enzyme (TAKARA) that adds 1mM dithiothreitol (DTT) (DTT), the corresponding primer of 0.5mM dNTPs, 1uM and 2.5 units, reactive system places 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 used axenic purification water dissolution, dilution after using the extracting of phenol chloroform.Dna fragmentation is integrated into plasmid pUC18 (TAKARA) after with EcoR I and BamH I (TAKARA) double digestion, and operation instructions is seen the pUC18 test kit.Utilize primer T7 that the product D NA of amplification is checked order (Shanghai Mei Ji biotech company), 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, the humanization of selecting the highest sequence of similarity to carry out sequence is processed.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 simultaneously antibody to specificity and the high-affinity of antigen recognition site as far as possible, therefore keep the sequence of mouse source 7B05 antibody at separately three hypervariable regions (CD1, CD2 and CD3) of VH and VL, people's IgG Cor sequence and K102 sequence then adopted in all the other zones.The VH of mouse source 7B05 and VL are as follows with comparison and the humanization result of people's IgG Cor sequence and K102 sequence:
The VH of 7B05 behind the humanization
1 5 10 15
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly human IgG VH (Cor)
The VH of Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly humanization 7B05
The VH of Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly mouse source 7B05
20 25 30
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys
Ala Leu Val Lys Leu Ser Cys Lys Ala Ser Gly Phe Asn Ile Lys
35 40 45
Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Asp Tyr Tyr Ile Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Asp Tyr Tyr Ile Tyr Trp Val Lys Gln Arg Pro Glu Gln Gly Leu
50 55 60
Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Val Phe
Glu Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly Asn Thr Val Phe
65 70 75
Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Asp Pro Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser
Asp Pro Lys Phe Gln Gly Lys Ala Ser Ile Thr Ser Asp Thr Ser
80 85 90
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp
95 100 105
Thr Ala Val Tyr Tyr Cys Ala
Thr Ala Val Tyr Tyr Cys Ala Tyr Tyr Gly Thr Ser Ser Phe Asp
Thr Ala Val Tyr Tyr Cys Ala Tyr Tyr Gly Thr Ser Ser Phe Asp
110 115
The VH of Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser humanization 7B05
The VH of Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser mouse source 7B05
The VL of 7B05 behind the humanization
1 5 10 15
Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val human IgG VL (K102)
The VL of Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val humanization 7B05
The VL of Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Met Tyr Val Ser Leu mouse source 7B05
20 25 30
Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser
Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn
Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn
35 40 45
Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Arg Tyr Leu Asn Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys
50 55 60
Leu Leu Ile Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser
Leu Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser
Thr Leu Ile His Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser
65 70 75
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Arg Phe Ser Gly Ser Gly Ser Gly Gln Glu Tyr Ser Leu Thr Ile
80 85 90
Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
Phe His Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
Phe His Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu
107
The VL of Ile Lys humanization 7B05
The VL of Ile Lys mouse source 7B05
The structure of embodiment 47B05 monoclonal antibody expression plasmid
The constant region sequence of 7B05 monoclonal antibody (CH and CL) adopts people's IgG Cor sequence and the corresponding fragment of 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 the design primer is as follows:
The synthetic employing primer of fragment is pieced together overlapping extension PCR, will be as with fragment 1 and 2,3 and 4,5 and 6,7 and 8, overlapping extension PCR is carried out in 9 and 10 respectively pairing mixing, and experience annealing under conventional PCR condition, primer identification pairing and fragment amplification obtain 1-2,3-4,5-6,7-8, the overlapping and extension fragment such as 9-10.In like manner with 1-2 and 3-4,9-10 fragment bye is extended in 5-6 and 7-8 splicing.Behind many wheel PCR, obtain the 1-10 fragment.Finally through too much obtaining weight chain fragment after the wheel splicing.Humanized heavy chain fragment is integrated into the MCSA interval of pIRES plasmid (Clontech, plasmid map is shown in Fig. 2) between Nhe I and Mlu I, light chain segments is integrated into the MCSB interval of pIRES plasmid between Not I and Sal I.Plasmid is after large scale culturing, and is frozen stand-by after plasmid extraction test kit (QIAGEN) preparation.
Expression and the purifying of embodiment 5 7B05 monoclonal antibodies
The acquisition of 7B05 monoclonal antibody obtains the cell strain of stably express antibody protein by transfection CHO-K1 cell (ATCC) in the situation 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.Add G418 (200uM) and unite the pressurization screening in substratum, cell places under 8%CO2,37 ℃ of conditions and cultivates, and changes a subculture in 3 days, removes dead floating cell.After the cultured continuously 20 days, the cell clone of simple community appears in the Tissue Culture Dish being dispersed into, cell use trypsin INVITROGEN after removing substratum) 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 place to cultivate under 8%CO2, the 37 ℃ of conditions and get culture supernatant after 1 week and carry out ELISA and measure expressing quantity.
Mouse-anti-human T NFR primary antibodie (SIGMA) is with PH7.0 and contain PBS (diluent) dilution of 0.1% (w/v) NaN3, extension rate 1 * 10
3Antibody after the dilution adds elisa plate to be coated with (CORING), the 100ul/ hole, and 2-8 ℃ is spent the night.Elisa plate cleans three times with the PBS damping fluid (abbreviation scavenging solution) that contains 0.05%TWEEN 20, every hole adds 100ul 5% (w/v) BSA phosphate buffered saline buffer room temperature effect 1-2 hour, cells and supernatant to be detected adds in the elisa plate, the 100ul/ hole, the room temperature standing and reacting was hatched in 1-2 hour.Discard reaction solution, clean three times with scavenging solution, add the mouse-anti human IgG-Fc/HRP two anti-(SIGMA) of 1000 times of diluted, the 150ul/ hole, room temperature leaves standstill hatches TMB reagent (PERCE) colour developing after 1-2 hour, and 450nm surveys the 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 10 high clones of expression amount to continue amplification cultivation, keep cell screening condition constant (G418 (200uM) and MSX (50ug/ml)); Select 6 the highest clonal expansions of expression level to the T75 culturing bottle after many wheel amplifications, cell strain is set up cell bank and cryopreservation.The clone BW-7B05-3 that expression level is the highest is inoculated in the 2L rolling bottle simultaneously, cultivate with EX-CELL 302 substratum (SIGMA) that contain 5% calf serum (INVITROGEN), when treating that cell covers with bottle wall, be changed to serum free medium EX-CELL 302, receive every other day liquid, receive liquid 3-5 time continuously.
Utilize albumin A to the specific adsorption effect of IgG, adopt separating medium rProtein A Sepharose 4 Fast Flow (GE) that the cells and supernatant of results is carried out affinity chromatography, the purifying expressing protein.Working method is referring to its description of product.Behind the purifying, measure A260 and A280 value with ultraviolet spectrophotometer.Protein quantification formula: protein content (mg/ml)=OD280 value * 1.45-OD260 value * 0.74.
Among the RSV of embodiment 6 7B05 monoclonal antibodies and the 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 the experiment.Hep 2 cells are regulated concentration with RPMI 1640 substratum (SIGMA) that contain 10% foetal calf serum (SIGMA) and be inoculated in 12 orifice plates (CRONING), inoculum density is 2 * 10
5/ hole, 5%CO2,37 ℃ of overnight incubation are stand-by.7B05 monoclonal anti body and function substratum is carried out the concentration gradient dilution, and the RSV-B1 in about 200PFU/ hole is added in the good antibody-solutions of dilution, add simultaneously rabbit complement serum (SIGMA), incubated at room 1-2 hour.The antibody in adding 200ul/ hole-viral mixing solutions incubated at room 2-3 hour, makes virus infected cell in the substratum of Hep 2 cells.Remove all substratum, add the fresh culture that contains 1% (w/v) methylcellulose gum (MERCK), culture dish was hatched 6 days under 35 ℃ of conditions, carried out the fixing dyeing of cell.
Remove the substratum that contains methylcellulose gum in the culture dish, the methyl alcohol with 100% is fixed cell 30 minutes at ambient temperature, 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, the 100ul/ hole room temperature reaction 1-2 hour, adds scavenging solution and cleans termination reaction three times.Adding with two of 800 times of dilutions of diluent and resist---the anti-sheep polyclonal antibody of rabbit (MERCK) of horseradish peroxidase-labeled, the 100ul/ hole room temperature reaction 1-2 hour, adds scavenging solution and cleans termination reaction three times.Add chlorinated naphthalene phenol reagent (PERCE) 200ul/ hole, room temperature reaction 10 minutes, flushing with clean water is removed reagent, plaque number in the dry rear statistics single hole.
That Fig. 3 shows is the result of the absolute number of plaque corresponding to every microgram humanization 7B05 antibody, wherein also comprises the result of Synagis antibody.The 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 the RSV-B1 virus strain.In and data (% of contrast) be that 160-180 plaque absolute number according to each experiment calculates.External avidity is 1pM to the EC50 value of the antibody of the present invention of 5nM between 10-100ng/ml.
Claims (22)
1. an anti-RSV human monoclonal antibodies or its conservative property mutant or its active fragments comprise people source constant region, and its variable region of heavy chain is selected SEQ ID NO:1; Its variable region of light chain is selected from SEQ ID NO:2.
2. monoclonal antibody as claimed in claim 1 or its conservative property mutant or its active fragments, itself and sequence homology claimed in claim 1 are 98% or 95%.
3. monoclonal antibody as claimed in claim 1 or its conservative property mutant or its active fragments are that specificity is for the antibody of RSV F albumen.
4. anti-RSV human monoclonal antibodies as claimed in claim 1 is basically pure form, does not contain other phylactic agent.
5. dna molecular, its weight chain variabl area sequence SEQ ID NO:1 claimed in claim 1 that encodes.
6. dna molecular, its light chain variable region sequence SEQ ID NO:2 claimed in claim 1 that encodes.
7. the composition of a carrier or thinner comprises one or more monoclonal antibodies as claimed in claim 1 or its conservative property mutant or its active fragments.
8. a hybridoma cell line can obtain monoclonal antibody as claimed in claim 1 or its conservative property mutant or its active fragments.
9. clone as claimed in claim 8 also contains this clone and can keep the nutritional medium of this clone.
10. clone as claimed in claim 9, its described nutritional medium can be carbon source or nitrogenous source.
11. clone as claimed in claim 8, its described nutritional medium also contains VITAMIN and/or organic salt.
12. treat or prevent the method that RSV infects for one kind in the host, the method comprises to the host uses antibody as claimed in claim 1 or its conservative property mutant or its active fragments that is enough to reach the treatment or prevents the disease amount.
13. a prevention or the pharmaceutical composition for the treatment of RSV, said composition single antibody as claimed in claim 1 or immunoreactivity fragment are as promoting agent.
14. composition as claimed in claim 13 also comprises being no more than two antibody as claimed in claim 1 or fragment as promoting agent.
15. composition as claimed in claim 13 its role is to reduce inflammatory reaction.
16. preparation method who contains human monoclonal antibodies claimed in claim 1 or its conservative property mutant or its active fragments.
17. method as claimed in claim 16 is for using gene recombination method.
18. the method for the hybridoma cell line of breeding this claim 8 comprises this clone is cultivated in nutritional medium.
19. method as claimed in claim 18 comprises from the antibody claimed in claim 1 of substratum separation or the method for its conservative property mutant or its active fragments.
20. such as the method for claim 18, the breeding of described hybridoma cell line is external carrying out.
21. such as the method for claim 18, described nutritional medium contains carbon source, nitrogenous source.
22. such as the method for claim 18, described nutritional medium also contains VITAMIN and/or inorganic salt.
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| CN105050622A (en) * | 2013-03-14 | 2015-11-11 | 瑞泽恩制药公司 | Human antibodies to respiratory syncytial virus f protein and methods of use thereof |
| CN107207583A (en) * | 2014-10-29 | 2017-09-26 | 阿瑞迪思医药品股份有限责任公司 | There are specific human monoclonal antibodies to the F protein of respiratory syncytial virus (RSV) (RSV) |
| WO2022268120A1 (en) * | 2021-06-22 | 2022-12-29 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Anti-rsv antibodies and uses thereof |
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| WO2005079479A2 (en) * | 2004-02-17 | 2005-09-01 | Absalus, Inc. | Super-humanized antibodies against respiratory syncytial virus |
| CN1680447A (en) * | 1995-06-07 | 2005-10-12 | 生物基因Idec公司 | Neutralizing high affinity human monoclonal antibodies specific to respiratory syncytial virus f-protein |
| CN1986569A (en) * | 2005-12-23 | 2007-06-27 | 首都儿科研究所 | Humanized respiratory syncytial virus-resisting neutralizing gentically engineered Fab antibody |
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| CN1680447A (en) * | 1995-06-07 | 2005-10-12 | 生物基因Idec公司 | Neutralizing high affinity human monoclonal antibodies specific to respiratory syncytial virus f-protein |
| WO2005079479A2 (en) * | 2004-02-17 | 2005-09-01 | Absalus, Inc. | Super-humanized antibodies against respiratory syncytial virus |
| CN1986569A (en) * | 2005-12-23 | 2007-06-27 | 首都儿科研究所 | Humanized respiratory syncytial virus-resisting neutralizing gentically engineered Fab antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105050622A (en) * | 2013-03-14 | 2015-11-11 | 瑞泽恩制药公司 | Human antibodies to respiratory syncytial virus f protein and methods of use thereof |
| CN105050622B (en) * | 2013-03-14 | 2018-10-23 | 瑞泽恩制药公司 | The human antibodies and its application method of virus F protein matter are merged for respiratory tract |
| CN107207583A (en) * | 2014-10-29 | 2017-09-26 | 阿瑞迪思医药品股份有限责任公司 | There are specific human monoclonal antibodies to the F protein of respiratory syncytial virus (RSV) (RSV) |
| WO2022268120A1 (en) * | 2021-06-22 | 2022-12-29 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Anti-rsv antibodies and uses thereof |
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