CN106554413A - Full human monoclonal antibody and application for H10N8 subtype avian influenza virus - Google Patents
Full human monoclonal antibody and application for H10N8 subtype avian influenza virus Download PDFInfo
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Abstract
The invention belongs to biological technical field, it is related to full human monoclonal antibody for H10N8 subtype avian influenza virus, its Fab, and their application.The antibody its be mainly characterized by what is determined due to complementary determining region (CDR) specific gene sequences that are present in light chain of antibody and heavy chain gene variable region, and the antibody of specific binding H10N8 subtype avian influenza virus hemagglutinins (HA) of effective expression is obtained in protokaryon and eukaryotic cell；Using the antibody CDR region or part or full genome, the genetic engineering antibody of multi-form can be transformed and is produced in protokaryon and eukaryotic cell and any expression system, be further used for preparing the medicine for clinically preventing or treating H10N8 subtype avian influenza virus relevant diseases.
Description
Technical field
The invention belongs to biological technical field, be related to for H10N8 subtype avian influenza virus monoclonal antibody and
Using, more particularly to the full human monoclonal antibody for H10N8 subtype avian influenza virus, its antigen binding
Fragment, and their application.
Background technology
Prior art discloses bird flu viruss is the influenza virus for infecting birds, does not generally infect the mankind.However,
The human infection's bird flu case of China's Hong Kong first report in 1997.This high pathogenic avian influenza viruss are
H5N1 hypotypes, cut-off on December 10th, 2013 have resulted in 15, whole world countries and regions totally 648 cases
Report, wherein 384 dead.Subsequently, multiple avian influenza virus subtypes such as H9N2, H7N9 are also sent out in succession
Now there is the ability of the infection mankind.It should be noted that in December, 2013, China Jiangxi finder infection is newly
Type H10N8 bird flu case, this is to find first in the world to infect the H10N8 bird flu viruss of people,
Two cases are made a definite diagnosis, wherein 1 dead.Research shows that the main immunogenic antigen of influenza virus is disease
The transmembrane glycoprotein hemagglutinin (HA) and neuraminidase protein (NA) on malicious cyst membrane surface, wherein, HA
Identification target cell surface receptors are simultaneously combined with receptor, with host cell membrane fusion activity, machine can be induced
Body produces protectiveness neutralizing antibody；The HA antigenic variation caused due to HA gene mutation frequently can lead to exempt from
Epidemic disease failure and variation strain are popular, and the frequent and lasting generation antigenic variation of influenza virus there is presently no
Effectively for the vaccine of the new bird flu viruss such as H10N8, do not have specific medicine yet.Therefore,
R＆D work of this area researcher concern about the biological engineering preparation of novel specific viral infection resisting, its
In, antibody plays important role in terms of anti-infective therapy and urgent passive immunity prevention always, especially
Antibody of the specificity for H10N8 subtype avian influenza virus, which will be effective for urgent prevention and treatment
The generation of H10N8 subtype avian influenzas and development.
It is reported that, diversified specific murine source, Ren Yuan can be obtained by the restructuring of antibody molecule gene level
Change and human antibody, make to have the research of monoclonal antibody breakthrough and increasingly show which is important
Meaning and actual application prospect.The phage antibody gene bank technology that the beginning of the nineties at the end of the eighties in last century rises
Rise and whole gene engineered antibody technical field of research development, make the full human monoclonal antibody in the world today and
The developmental research of new construction antibody obtains remarkable progress and has stepped into substantive applied research by phase of basic research
And the development phase.People source containing specific antibody or animal serum immunoglobulin are to prevent and treat infection
Disease is with a long history, and the development of full human monoclonal antibody, bring to the biological product development in this field
New hope and long-range prospect.The extracorporeal antivirus effect neutralization activity of monoclonal antibody and in vivo protection body opposing disease
Poison attack obtained many it is demonstrated experimentally that as anti-hepatitis A virus (HAV), Hantaan virus, RSV viruses, SARS virus,
The monoclonal antibodies such as prosperous Nipah virus, Ebola virus 100% can protect animal from virus attack in vivo.
Based on the Research foundation of prior art, present inventor intends providing for H10N8 subtype avian influenzas
The full human monoclonal antibody of virus, its Fab, and they are to people's infection H10N8 hypotypes fowl stream
The application of the good prevention and treatment of sense.
Prior art related to the present invention has：
1.Chen, H.et al.Clinical and epidemiological characteristics of a fatal case of avian influenza A
H10N8 virus infection：A descriptive study.Lancet 383,714-721 (2014).
2.Dimitrov, D.S.Therapeutic antibodies, vaccines and antibodyomes.MAbs 2,347-56 (2010).
3.Ying, T.et al.Exceptionally potent neutralization of Middle East respiratory syndrome
Coronavirus by human monoclonal antibodies.J Virol 88,7796-805 (2014).
The content of the invention
It is an object of the invention to provide full human monoclonal antibody for H10N8 subtype avian influenza virus and should
With, more particularly to the full human monoclonal antibody for H10N8 subtype avian influenza virus, its antigen binding fragment
Section, and their application.
The invention discloses the full people source Dan Ke of specific binding H10N8 subtype avian influenza virus hemagglutinins
Grand antibody.The full human monoclonal antibody of the present invention includes cloning m813 and clone m819.The present invention also public affairs
The Fab of these antibody, bi-specific antibody, and the conjugate with effector molecule are opened.
In the present invention, described antibody is determined by the complementation being present in light chain of antibody and heavy chain gene variable region
What area (CDR) specific gene sequences were determined, and the special of effective expression is obtained in protokaryon and eukaryotic cell
Property with reference to H10N8 subtype avian influenza virus hemagglutinins (HA) antibody.Using this antibody CDR region or
Part or full genome, can transform and produce the base of multi-form in protokaryon and eukaryotic cell and any expression system
Because of engineered antibody；Described antibody and combinations thereof can be used for, such as, people infection H10N8 hypotype fowl
The prevention and treatment of influenza.
The invention also discloses the described monoclonal antibody of coding, Fab, bi-specific antibody, and
The nucleic acid of conjugate.In some embodiments of the invention, the carrier comprising these nucleic acid is disclosed, and is included
The host cell of these carriers.In further embodiment, disclose comprising these monoclonal antibodies, resist
The Pharmaceutical composition of former binding fragment, bi-specific antibody, conjugate, nucleic acid, and carrier；In other enforcements
In scheme, these monoclonal antibodies, Fab, bi-specific antibody, conjugate, nucleic acid, and carry
Body is used for the preparation or medicine for preparing detection, prevention, or treatment H10N8 subtype avian influenzas；In the present invention
Some embodiments in, monoclonal antibody, or the aminoacid sequence of the light chain variable district of Fab and
/ or weight chain variable district aminoacid sequence, comprising at least one (such as three) clone m813 or clone m819
Light chain and/or weight chain variable district complementary determining region (CDR).
More specifically, a kind of full human monoclonal antibody of the invention, or Fab, comprising：
The light chain variable district of antibody contains SEQ ID NO：1 27-33 positions, 51-53 positions, and/or 90-99
Amino acids, and the weight chain variable district of antibody contains SEQ ID NO：2 26-33 positions, 51-57 positions, and/
Or 96-109 amino acids；Or
The light chain variable district of antibody contains SEQ ID NO：9 27-32 positions, 50-52 positions, and/or 89-97
Amino acids residue, and the weight chain variable district of antibody contains SEQ ID NO：10 26-33 positions, 51-57 positions,
And/or 96-109 amino acids residues,
Wherein monoclonal antibody or Fab specifically bind H10N8 subtype avian influenza virus；
Or,
Described monoclonal antibody or Fab, wherein：
The aminoacid sequence of light chain variable district and SEQ ID NO：1 has at least 90% homogeny, and weight chain variable
The aminoacid sequence in area and SEQ ID NO：2 with least 90% homogeny；Or
The aminoacid sequence of light chain variable district and SEQ ID NO：9 have at least 90% homogeny, and weight chain variable
The aminoacid sequence in area and SEQ ID NO：10 with least 90% homogeny.
In the present invention, monoclonal antibody or Fab are full people sources.
In the present invention preferably, monoclonal antibody is IgG；Fab is scFv, Fv, Fab or F (ab)2。
In the present invention, described monoclonal antibody or Fab are bi-specific antibody, and itself and one kind are imitated
Molecule coupling labeled is answered, effector molecule therein is detectable label, such as fluorescent labeling, radioactive label, Avidin,
Biotin, or enzyme；The conjugate effector molecule therein for being obtained is toxin or chemotherapeutics, especially described toxin
It is Pseudomonas Exotoxin.
Also disclose in the present invention, a kind of nucleic acid molecules, which encodes above-mentioned monoclonal antibody or antigen binding fragment
Section, bi-specific antibody, or described conjugate；Described nucleic acid molecules, may be operably coupled to promoter.
A kind of plasmid is also disclosed in the present invention, which contains above-mentioned nucleic acid molecules；Plasmid of the present invention is outstanding
Which is a kind of virus particle.
The host cell containing above-mentioned plasmid is also disclosed in the present invention.
The present invention further discloses one kind detects H10N8 subtype avian influenza virus hemagglutinins in biological sample
The method of albumen, which includes：In the biological sample from the mankind or birdss, and described monoclonal antibody or
Fab, bi-specific antibody, or described conjugate under conditions of it be enough to form immune complex,
There is H10N8 in representing biological sample in the presence or absence of detection immune complex, the presence of wherein immune complex
Subtype avian influenza virus hemagglutinin.
The present invention further discloses a kind of detection experimenter whether method with H10N8 subtype avian influenzas, its
Including：Biological sample of the detection from the experimenter of suspected infection H10N8 subtype avian influenzas, and effective agent
The combination of the monoclonal antibody of amount, Fab, bi-specific antibody, or conjugate, wherein monoclonal
The combination of antibody, Fab, bi-specific antibody, or conjugate is compared negative control increase expression and is received
Examination person suffers from H10N8 subtype avian influenzas；Wherein negative control is a reference standard, or monoclonal antibody,
The combination of Fab, bi-specific antibody, or conjugate and the sample from health volunteer.
In the detection method of the present invention, the sample is blood, serum, blood plasma, sputum, or biopsy samples.
The present invention further discloses a kind of Pharmaceutical composition, which contains the described monoclonal of effective preventive dose
Antibody or Fab, described bi-specific antibody, described conjugate, described nucleic acid, or institute
The plasmid stated, and a kind of pharmaceutical acceptable carrier.
The present invention obtains anti-for full people's resource monoclonal of H10N8 subtype avian influenza virus in the world first
Body, and its Fab.Using this antibody CDR region or part or full genome, can be in protokaryon and eucaryon
The genetic engineering antibody of multi-form is transformed and is produced in cell and any expression system, can be used to prepare in clinic
Upper prevention or the medicine for the treatment of H10N8 subtype avian influenza virus relevant diseases.
In order to make it easy to understand, will be described in detail to the present invention by specific drawings and Examples below.
It is important to note that instantiation and accompanying drawing are merely to explanation, it is clear that one of ordinary skill in the art
Various amendments and change can be made within the scope of the invention to the present invention according to illustrating herein, this
A little amendments and change are also included in the scope of the present invention.In addition, the present invention refer to open source literature, these documents
It is that, in order to more clearly describe the present invention, their entire contents are included and referred to herein, just looks like them
Full text repeated description is excessively herein.
Description of the drawings
Fig. 1. using ELISA detections specific antigen binding fragment (Fab) and H10N8 subtype avian influenza virus
The binding ability of HA.
Detections of Fig. 2 .Fab to H10N8 subtype avian influenza virus neutralising capacities.
Specific embodiment
Embodiment 1
Prepare H10N8 subtype avian influenza virus HA albumen
The H10N8 HA Gene of H 9 Subtype AIV of synthesis is inserted in expression plasmid, by expression plasmid wink
When transfect 293 FREESTYLE cells, expression trimer HA albumen simultaneously carries out purification；
Screening H10N8 subtype avian influenza virus specific antigen binding fragment (Fab)
Resisted using the large-scale phage display that peripheral blood lymphocytes cDNA using 40 healthy volunteers builds
Body library, for the HA protein screening antibody of biotin labeling, 1012The Fab of individual phage display is in room temperature
Under in 1, respectively with 5,3,3,1 micrograms antigens are incubated two hours 2,3,4 wheels；HA albumen is coated on into ELISA
On plate, with the enrichment of polyclone Phage-ELISA detection antibody；1st, 2,3,4 wheel phage with it is coated
Albumen is incubated, and couples the combination of antibody test phage and albumen with phage-resistant HRP；According to polyclone
Phage-ELISA result, obtains the enrichment of highly significant after the 4th wheel screening；Therefore, from the 4th wheel sieve
The phage that choosing is obtained, infection TG1 cells random choose are cloned and carry out monoclonal phage ELISA, enter one
Step carries out the Fab of sequencing identification enrichment；
Detection specific antigen binding fragment (Fab) and the binding ability of H10N8 subtype avian influenza virus HA
Ten clones have selected according to sequencing result, m810-m819 is named as, their soluble expression product
Preparation carry out by known references substantially, specially：Plasmid containing clone m810-m819 is proceeded to into HB2151
Competent cell, from picking single bacterium colony in the ammonia benzyl plate of overnight growth, is inoculated with SB inoculums,
10-12 hours, results antibacterial therefrom extraction purification Fab, by H10N8 are expressed under 30 degree of IPTG inductive conditions
Subtype avian influenza virus HA albumen is coated on elisa plate, adds the m810-m819 Fab of gradient concentration dilution
Incubation, detects the binding ability of Fab and H10N8 subtype avian influenza virus HA with anti-FLAG tag antibodies；
ELISA results show, have four combination HA that can be very strong in ten Fab clones, be m811, m813,
M815, m819 (as shown in Figure 2)；
Detection antibody is to H10N8 subtype avian influenza virus neutralising capacities
By H10N8 subtype avian influenza virus HA albumen, NA albumen, and expressing luciferase are used as report base
Because of the HIV-1 genes of defect, common transfection 293T cells are packed H10N8 subtype avian influenza pseudoviruss, are used
Similar condition, packs H1N1, H7N9 pseudoviruss, by m811, m813, m815, m817, m819
Five Fab infect HEK-293 cells respectively together with pseudoviruss, and cell pyrolysis liquid luciferase is detected after incubation；
PRELIMINARY RESULTS as shown in Fig. 2 two kinds of antibody of m813 and m819 can be very strong specificity in and H10N8
Subtype avian influenza pseudoviruss.
Sequence according to the present invention and mark point row are as follows：
SEQ ID NO：1 is the aminoacid sequence of m813 light chain variable districts；
SEQ ID NO：2 is the aminoacid sequence of m813 weight chain variable districts；
SEQ ID NO：3 is the aminoacid sequence of m813 light chain variable districts CDR1；
SEQ ID NO：4 is the aminoacid sequence of m813 light chain variable districts CDR2；
SEQ ID NO：5 is the aminoacid sequence of m813 light chain variable districts CDR3；
SEQ ID NO：6 is the aminoacid sequence of m813 weight chain variable districts CDR1；
SEQ ID NO：7 is the aminoacid sequence of m813 weight chain variable districts CDR2；
SEQ ID NO：8 is the aminoacid sequence of m813 weight chain variable districts CDR3；
SEQ ID NO：9 is the aminoacid sequence of m819 light chain variable districts；
SEQ ID NO：10 is the aminoacid sequence of m819 weight chain variable districts；
SEQ ID NO：11 is the aminoacid sequence of m819 light chain variable districts CDR1；
SEQ ID NO：12 is the aminoacid sequence of m819 light chain variable districts CDR2；
SEQ ID NO：13 is the aminoacid sequence of m819 light chain variable districts CDR3；
SEQ ID NO：14 is the aminoacid sequence of m819 weight chain variable districts CDR1；
SEQ ID NO：15 is the aminoacid sequence of m819 weight chain variable districts CDR2；
SEQ ID NO：16 is the aminoacid sequence of m819 weight chain variable districts CDR3.
Claims (22)
1. for the full human monoclonal antibody of H10N8 subtype avian influenza virus, or Fab, it is characterised in that
Which includes：
The light chain variable district of antibody contains SEQ ID NO：1 27-33 positions, 51-53 positions, and/or 90-99 amino acids,
And the weight chain variable district of antibody contains SEQ ID NO：2 26-33 positions, 51-57 positions, and/or 96-109 amino acids；
Or
The light chain variable district of antibody contains SEQ ID NO：9 27-32 positions, 50-52 positions, and/or 89-97 amino acids are residual
Base, and the weight chain variable district of antibody contains SEQ ID NO：10 26-33 positions, 51-57 positions, and/or 96-109 positions
Amino acid residue,
Wherein monoclonal antibody or Fab specifically bind H10N8 subtype avian influenza virus.
2. the full human monoclonal antibody for H10N8 subtype avian influenza virus as described in claim 1, or antigen binding
Fragment, it is characterised in that described monoclonal antibody or Fab, wherein：
The aminoacid sequence of light chain variable district and SEQ ID NO：1 has at least 90% homogeny, and the ammonia of weight chain variable district
Base acid sequence and SEQ ID NO：2 with least 90% homogeny；Or
The aminoacid sequence of light chain variable district and SEQ ID NO：9 have at least 90% homogeny, and the ammonia of weight chain variable district
Base acid sequence and SEQ ID NO：10 with least 90% homogeny.
3. the monoclonal antibody or Fab for H10N8 subtype avian influenza virus as described in claim 1 or 2,
Characterized in that, monoclonal antibody therein or Fab are full people sources.
4. the monoclonal antibody or antigen for H10N8 subtype avian influenza virus as any one of claim 1-3 is tied
Close fragment, it is characterised in that monoclonal antibody therein is IgG.
5. the monoclonal antibody or antigen for H10N8 subtype avian influenza virus as any one of claim 1-3 is tied
Close fragment, it is characterised in that wherein Fab is scFv, Fv, Fab or F (ab)2。
6. a kind of bi-specific antibody, containing monoclonal antibody or Fab any one of claim 1-5.
7. the monoclonal antibody or Fab any one of claim 1-5, or it is double described in claim 6
Specific antibody, is coupled into conjugate with a kind of effector molecule.
8. the conjugate described in claim 7, effector molecule therein is detectable label.
9. the conjugate as described in claim 8, detectable label therein is fluorescent labeling, radioactive label, Avidin,
Biotin, or enzyme.
10. the conjugate as described in claim 7, effector molecule therein is toxin or chemotherapeutics.
11. conjugates as described in claim 10, toxin therein is Pseudomonas Exotoxin.
A kind of 12. nucleic acid molecules, its monoclonal antibody or Fab any one of coding claim 1-5,
Bi-specific antibody described in claim 6, or the conjugate any one of claim 7-11.
13. nucleic acid molecules as described in claim 12, which may be operably coupled to promoter.
14. a kind of plasmids, containing the nucleic acid molecules described in claim 12 or claim 13.
Plasmid described in 15. claim 15, wherein plasmid are a kind of virus particles.
16. a kind of host cells, containing the plasmid described in claim 14 or claim 15.
A kind of 17. methods that H10N8 subtype avian influenza virus hemagglutinins are detected in biological sample, it is characterised in that its
Including：
Containing biological sample, and the monoclonal antibody or Fab any one of claim 1-5, right
The bi-specific antibody described in 6, or the conjugate any one of claim 7-11 are required, be enough to be formed
Under conditions of immune complex, the presence or absence of immune complex is detected, the presence of wherein immune complex represents raw
There is H10N8 subtype avian influenza virus hemagglutinins in thing sample.
18. methods as described in claim 17, wherein sample source are in the mankind or derive from birdss.
19. a kind of detection experimenters whether method with H10N8 subtype avian influenzas, it is characterised in which includes：
Biological sample containing the experimenter from suspected infection H10N8 subtype avian influenzas, and the right of effective dose will
The monoclonal antibody or Fab any one of 1-5, the bispecific described in claim 6 is asked to resist
Body, or the conjugate any one of claim 7-11, and monoclonal antibody is detected, Fab,
Bi-specific antibody, or the combination of conjugate, wherein monoclonal antibody, Fab, bi-specific antibody,
Or the combination of conjugate is compared negative control and increases expression experimenter with H10N8 subtype avian influenzas.
20. methods as described in claim 19, it is characterised in that wherein negative control is a reference standard, or monoclonal
The combination of antibody, Fab, bi-specific antibody, or conjugate and the sample from health volunteer.
21. methods as described in claim 19 or claim 20, it is characterised in that wherein sample is blood, serum,
Blood plasma, sputum, or biopsy samples.
22. a kind of Pharmaceutical compositions, it is characterised in that any one of claim 1-5 containing effective preventive dose
Monoclonal antibody or Fab, the bi-specific antibody described in claim 6 are appointed in claim 7-11
Conjugate described in one, the nucleic acid any one of claim 12-13, or in claim 14-15 it is arbitrary
Plasmid described in, and a kind of pharmaceutical acceptable carrier.
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