CN107286238B

CN107286238B - Preparation, detection and the application of HCV-Ab IgG wide spectrum neutralizing antibody

Info

Publication number: CN107286238B
Application number: CN201710668070.8A
Authority: CN
Inventors: 廖化新; 张远旭; 袁晓辉; 王月明; 刘彤; 昝利鹏; 吴昌文
Original assignee: Zhuhai Microlab Biotechnology Co Ltd; GUANGZHOU TAINUODI BIOTECHNOLOGY CO Ltd; Jinan University
Current assignee: Guangzhou Tainuodi Biotechnology Co ltd; Zhuhai Tainuo Maibo Pharmaceutical Co ltd; Jinan University
Priority date: 2017-08-07
Filing date: 2017-08-07
Publication date: 2019-11-26
Anticipated expiration: 2037-08-07
Also published as: CN107286238A

Abstract

The invention discloses preparation, detection and the applications of a kind of HCV-Ab IgG wide spectrum neutralizing antibody.Antibody number of the invention is TRN1009, has heavy chain CDR region shown in NO.1~3 SEQ ID, light chain CDR region shown in NO.5~7 SEQ ID.Antibody combination activity with higher prepared by the present invention and affine activity, can be used as diagnosticum or therapeutic agent is applied in disease caused by HCV.

Description

Preparation, detection and the application of HCV-Ab IgG wide spectrum neutralizing antibody

Technical field

Invention belongs to cellular immunology, genetic engineering field, is related to a kind of system of HCV-Ab IgG wide spectrum neutralizing antibody Standby, detection and application.

Background technique

Hepatitis C Virus (Hepatitis C virus, HCV) is single-stranded positive RNA enveloped virus, belongs to flavivirus Hepatovirus is bitten by section, mainly through blood born, can cause acute chronic viral hepatitis, cirrhosis, then develop hepatoblast Cancer.It is reported that medical history 20 years or more patients with chronic hepatitis C, the Annual occurence rate of cirrhosis is 10%~15%, and every year about The liver cirrhosis patient development for having 1-7% is liver cancer.HCV infection is in global distribution, and China belongs to HCV grade and moderate infection area.China The number of the infected of most newly reported hepatitis C in 2016 is about 200,000, disease incidence 15%.Therefore, it grinds on the basis for reinforcing HCV Study carefully, exploitation treatment HCV drug and diagnostic reagent, to ensure that the health of our people has great importance.

Self component can be synthesized and replicated after infection with hepatitis C virus in body, constantly forms new progeny virus, Specific immune response is generated for HCV infection so as to cause body.The laboratory diagnosis of infection with hepatitis C virus is mainly wrapped Include serodiagnosis, HCV diagnostic nucleic acid.Wherein, HCV serodiagnosis includes HCV antibody diagnosis and HCV diagnostic antigen；HCV core Acid diagnosis is mainly the qualitative and quantitative detection of HCV RNA.Clinically, mainly according to clinical manifestation and laboratory check into Row HCV makes a definite diagnosis

The purpose of HCV antiviral therapy is to remove or persistently inhibit intracorporal HCV, to reduce hepatic injury, restores liver function, It prevents it from progressing to liver fibrosis, cirrhosis, liver failure or liver cancer, and improves the quality of life of patient.The world is more at present The widely used therapeutic scheme of state is the combination therapy of ethylene glycol interferon-' alpha ' and Ribavirin, but it is to 2 type of gene and 3 types SVR rate is 75%~90%, but very low to the SVR rate of 1 type of gene and 4 types, only 45% and 52%.Both medicine combination therapies There are many serious side effects and contraindication, recurrence rate is higher, and expensive.Therefore, to the improvement of existing therapeutic scheme With the exploitation to new safely and effectively drug for clinical treatment with regard to particularly necessary and important.

In recent years, effect of the antibody in HCV infection starts to be taken seriously, studies have shown that antibody block HCV particle with The combination of acceptor molecule inhibits cell entry cell aspect to show great potentiality by neutralizing.Although there is HCV antibody class Drug is carrying out clinical or preclinical laboratory, but also seldom into the antibody levels of preclinical study at present.It is necessary to open The more researchs of exhibition, excavate the treatment potentiality of HCV antigen/antibody combination as far as possible, provide more for the antiviral therapy of hepatitis patient Selection.

Summary of the invention

In order to make up for the deficiencies of the prior art, the object of the present invention is to provide in a kind of wide spectrum of the HCV-Ab IgG of full source of people And the application in the drug of antibody and the antibody disease caused by detection HCV and preparation treatment HCV infection.

To achieve the goals above, the present invention adopts the following technical scheme:

The present invention provides a kind of HCV-Ab IgG wide spectrum neutralizing antibody, the antibody heavy chain variable region contains comprising at least one The light chain variable region of the heavy chain variable region of three CDR and at least one CDR containing there are three；Wherein the heavy chain variable region includes:

(1) VH CDR1, amino acid sequence are SEQ ID NO.1；

(2) VH CDR2, amino acid sequence are SEQ ID NO.2；

(3) VH CDR2, amino acid sequence are SEQ ID NO.3；

Wherein the light chain variable region includes:

(4) VL CDR1, amino acid sequence are SEQ ID NO.5；

(5) VL CDR2, amino acid sequence are SEQ ID NO.6；

(6) VLCDR2, amino acid sequence are SEQ ID NO.7.

Further, the antibody has VL shown in VH structural domain shown in SEQ ID NO.4 and/or SEQ ID NO.8 Structural domain.

Further, the antibody includes all or part of of heavy chain constant region and/or antibody light chain constant region.

Further, the antibody is the antigen-binding fragment of antibody.

Further, the antibody is attached at least second opinion agent or therapeutic agent.

The present invention provides a kind of composition, the composition includes exempting from for antibody recited above or antibody described above Epidemic disease conjugates.

The immune conjugate includes antibody recited above, the immune conjugate be attached at least second therapeutic agent or Diagnosticum.Wherein, the antibody is attached at least radiotherapy medicament, chemotherapeutic agents, anti-angiogenesis medicament, Apoptosis-and lures Lead medicament, anti-tubulin medicament, anti-cell or cytotoxic drugs, steroids, cytokine antagonist, cell factor Expression inhibiting agent, chemokine antagonists, chemokine expression inhibitor, atriphos enzyme inhibitor, anti-inflammatory agents, signal Pathway inhibitor, anti-cancer agents, other antibody, coagulant or antivirotic.The antivirotic be selected from by nucleosides, Nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitor and protease inhibitors.

Further, the composition is pharmaceutically acceptable composition.

The present invention provides a kind of clone or expression vector, the clone or expression vector include one or more DNA sequences Column, the heavy chain and/or light chain of the DNA sequence encoding antibody recited above.DNA sequence dna of the invention may include for example passing through It is chemically treated the synthetic DNA, cDNA, genomic DNA or any combination thereof generated.

The DNA sequence dna for encoding antibody molecule of the invention can be obtained by method well known to those skilled in the art.For example, The DNA sequence dna of coded portion or whole heavy chain of antibody and light chain from determining DNA sequence dna or can be based on corresponding amino as needed Acid sequence synthesizes.

The DNA for encoding acceptor framework sequence is widely available for those skilled in the art and can be based on known to it Amino acid sequence is easily synthesized.The standard technique of molecular biology, which can be used for preparing, encodes antibody molecule of the invention DNA sequence dna.Required DNA sequence dna can be synthesized completely or partially using oligonucleotide synthesis technology.It can suitably be lured using fixed point Become and polymerase chain reaction (PCR) technology.

The present invention provides a kind of host cell, the host cell includes the heavy chain of antibody recited above and/or light The DNA sequence dna of chain or clone recited above or expression vector.

In the present invention, any host cell/carrier system appropriate can be used for encoding the DNA of antibody molecule of the invention The expression of sequence.Bacterium (such as Escherichia coli) and other microfloras can be used, or also eucaryote can be used (such as to feed Newborn animal) host cell expression system.Mammalian host cell appropriate include CHO, 293 cells, myeloma or hybridoma Cell.

The pharmaceutical composition or detection of disease caused by treating HCV in preparation the present invention provides antibody recited above Application in the tool of HCV.The disease includes but is not limited to hepatitis, cirrhosis, liver failure or liver cancer.

Further, the tool includes kit, test paper, chip etc..Wherein, the chip includes protein-chip；Institute Stating protein-chip includes solid phase carrier and the said monoclonal antibody for being fixed on solid phase carrier；The protein immunization detection examination Agent box；The protein immunization detection kit includes said monoclonal antibody.

As skilled in the art should understand, the immune binding reagents that term " antibody " includes expand to all anti- Body and its antigen-binding fragment, including entire antibody, dimerization, trimerization and multimeric antibody；Bispecific antibody；Chimeric antibody；Weight Group and engineered antibody and its segment.

Term " antibody " is accordingly used in referring to any antibody-based molecules with antigen binding domain, and the term includes composition The antibody fragment of antigen binding domain, such as Fab ', Fab, F (ab ') 2, single domain antibody (DAB), TandAbs dimer, Fv, scFv (scFv), dsFv, ds-scFv, Fd, linear antibodies, miniantibody (minibodies), bivalent antibody (diabodies) are double Specific Ab fragments, two antibody (bibody), three antibody (tribody) (scFv-Fab fusion, respectively bispecific or three Specificity)；Sc- bivalent antibody (sc-diabody)；κ (λ) antibody (kappa (lamda) bodies) (scFv-CL fusion)；It is double Specific T-cells adapter (Engager (BiTE)) (scFv connects to attract T cell)；Double variable regions (DVD)-Ig is (double special Personality formula)；Small immune protein (SIP) (a kind of miniantibody)；SMIP (" little module immune drug ") scFV-Fc dimerization Body；DART (double-strand stablizes bivalent antibody " double affinity target again ")；Small antibody comprising one or more CDR is quasi- like object etc. Deng.

Routine techniques fragmentation can be used in antibody.For example, can be 2 by pepsin antibody tormation F (ab ') Section.Resulting 2 segment of F (ab ') can be processed to reduce fragment of the disulfide bond to produce Fab '.Papain disappears Change the formation that can lead to fab segment.Fab, Fab ' and F (ab '), scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, Dimer, miniantibody, bivalent antibody, bispecific antibody fragment and other segments can also be synthesized by recombinant technique or can be with Chemical synthesis.The technology of production antibody fragment is well known and describes in the field.

It will also be appreciated by those of skill in the art that the present invention covers the amino acid sequence modifications of the HCV antibody.Citing For, it may be desired to improve the binding affinity and/or other biological characteristics of antibody.The amino acid sequence variation of HCV antigen/antibody combination It is from introducing appropriate nucleotide variation into HCV antigen/antibody combination nucleic acid or being prepared by peptide synthesis.The equal modifications include (for example) HCV-Ab IgG Residue deletions and/or insertion and/or substitution in antibody amino acids sequence.Any combination for being lacked, being inserted into and being replaced with Reach final construct, restrictive condition is that the final construct has desired characteristics.It is anti-that HCV-Ab IgG also can be changed in amino acid variation Process after the translation of body, such as number of change glycosylation site or position.

Amino acid sequence insertions include that length is amino of the residue to the polypeptide for containing 100 or more And/or insert in carboxyl terminal fusion, and the sequence with single or multiple amino acid residues.End insert is real Example includes the antibody for having the HCV antigen/antibody combination of N-terminal methionyl residue or merging with cytotoxic polypeptide.HCV antigen/antibody combination Other insertion variants of molecule include that the N-terminal of HCV antigen/antibody combination or C-terminal resist with enzyme (for example, for ADEPT) or increase The fusion of the polypeptide of the serum half-life of body.

Another type of variant is amino acid substitution variant.These variants have at least one in HCV antigen/antibody combination molecule Amino acid residue through different residue displacements.Most noticeable substitution Mutagenesis Site includes hypervariable region, but also covers FR or perseverance Determine the variation in area.The amino acid substitution of FR or constant region, preferably 1~5 amino acid, more preferably 1 or 2 amino acid Replace.Card Buddhist nun with such amino acid substitution is preferably functionally same with unsubstituted antibody.Therefore, amino acid substitution is excellent It is selected as conservative amino acid substitution, this is the substitution between the kin amino acid of charge, side chain, polarity, armaticity etc..Property The similar amino acid of matter can for example classify are as follows: basic amino acid (arginine, lysine, histidine), acidic amino acid (asparagus fern ammonia Acid, glutamic acid), uncharged polar amino acid (glycine, asparagine, glutamine, serine, threonine, cysteine, Tyrosine), apolar amino acid (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, first Methyllanthionine), branched-chain amino acid (threonine, valine, isoleucine), aromatic amino acid (phenylalanine, tyrosine, color ammonia Acid, histidine) etc..Two sulphur are imported and the amino acid residue in framework region to be replaced to the amino acid substitution of cysteine Key, it is possible thereby to make stabilized antibody and antigen-binding antibody segment (dsFv etc.), such antibody and antigen binding Property antibody fragment is also contained in the scope of the present invention.Here, " functionally same " refers to, the antibody with amino acid substitution With same biology or biochemical activity, particularly there is HCV infection inhibitory activity with the antibody before substitution.

Generally also can with serine replace with maintenance HCV antigen/antibody combination be suitably configured unrelated any cysteine residues with It improves the oxidation stability of molecule and prevents abnormal crosslinking.On the contrary, cysteine key can be added in antibody steady to improve it It is qualitative (especially wherein antibody be such as Fv segment antibody fragment).

Present invention also contemplates that other modifications of antibody.It for example, can be by it in antibody and a variety of non-protein polymer One (for example, copolymer of polyethylene glycol, polypropylene glycol, polyalkylene oxide or polyethylene glycol and polypropylene glycol) is connect.Antibody is also It can be wrapped in for example by condensation technique or the microcapsules of interfacial polymerization preparation (for example, respectively hydroxymethyl cellulose or gelatin is micro- Capsule and poly- (methyl methacrylate) microcapsules) in, colloidal drug delivery systems are wrapped in (for example, liposome, albumin Microsphere, microemulsion, nanometer particle and nanometer capsule) or huge lotion in.

The present invention, which covers, has one with the amino acid sequence of the antibody or any nucleotide sequence for encoding the antibody Determine the sequence identity of degree or the sequence of sequence homology, term " homologue " implies that and individual amino acids sequence and individual core Nucleotide sequence has the entity of certain homologys.Herein, term " homology " can be equal to " consistency ".

In the present invention, homologous sequence be used for include can with individual amino acids at least 75,85 or 90% consistency, preferably extremely The amino acid sequence of few 95% or 98% consistency.In general, homologue will include the active sites identical as individual amino acids sequence Point etc..Although also can about similitude (also that is, have the function of similar chemical characteristic/amino acid residue) consider homology, In the content of present invention, homology preferably is expressed about sequence identity.

In the present invention, homologous sequence be used for include can be with the nucleotide sequence (individual sequence) of coding as many as present invention peptide The consistency of at least 75%, 85% or 90%, the nucleotide sequence of preferably at least 95% or 98% consistency.In general, homologue will Sequence comprising active site identical as individual sequential coding etc..Although also can be about similitude (also that is, having similar chemistry special Property/function amino acid residue) consider homology, but in the content of present invention, preferably express homology about sequence identity.

In the present invention, immunohistochemistry technique known in the art can be used for the combination to antibody and cell or sample into Row scoring.It is this to test the specificity that can be used for testing antibody specific, or expression of the detection HCV in tissue sample.In short It, such as antibody can test in the tissue of a high density arrays, which includes that positive control (is known to be HCV- Positive cell) and negative control (cell for being known to be HCV- feminine gender).Film staining power can be logical with 4 grade standards (0,1,2,3) It crosses and visually observes assessment.HCV+ is organized, preferred antibody is scored at faint or strong (such as 0 or more score), preferably It is strong immunization group score.

Drug of the invention can be the pharmaceutical composition containing pharmaceutically acceptable carrier.It is " pharmaceutically acceptable Carrier " refer to can in the technical field of preparation conventional use of solvent and/or additive.Pharmaceutically acceptable solvent includes (such as ethyl alcohol, propylene glycol, ethoxylation second stearyl alcohol, polyoxygenated are different hard for (but being not limited to) water, pharmaceutically acceptable organic solvent Lipidol, polyoxyethylene sorbitan fatty esters of gallic acid).These solvents preferably sterilize, and as needed, are preferably regulated as and blood It is isotonic.

In addition, pharmaceutically acceptable additive includes but is not limited to collagen, polyvinyl alcohol, polyvinylpyrrolidone Ketone, carboxyl vinyl polymer, sodium carboxy methyl cellulose, Sodium Polyacrylate, sodium alginate, water-soluble dextrin, carboxymethyl form sediment Powder sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum arabic, agar, polyethylene glycol, two glycerol, glycerol, Propylene glycol, vaseline, paraffin, hard ester alcohol, stearic acid, human albumin, mannitol, D-sorbite, lactose, as drug add Acceptable surfactant of object etc..

It also may include excipient, adhesive, disintegration as needed in addition to this about pharmaceutically acceptable additive It is agent, filler, emulsifier, flowing addition regulator, lubricant, drug flavoring, cosolvent (solvable agent), suspending agent, dilute Release agent, surfactant, stabilizer, sorbefacient, increment drug, assign humectant, moisturizer (such as glycerol, starch), adsorbent, Disintegration inhibitor, coating agent, colorant, preservative agent, antioxidant, fragrance, flavouring agent, sweetener, buffer etc..

It, can independent or appropriately combined use according to dosage form about solvent and additive.For example, making as injection preparation Purified antibody can be dissolved in solvent (such as physiological saline, buffer, glucose solution), add thereto by the used time Add preparation obtained by adsorption inhibitor (for example, Tween 80, polysorbas20, gelatin, human albumin).Alternatively, in order to before use It is dissolved and the dosage form reconstructed is made, can be freeze-dried products.For example, excipient (example can be used in order to carry out freeze-drying The sugar alcohol or carbohydrate of such as mannitol, glucose)

Antibody or antigen-binding antibody segment of the invention or drug can by it is oral give, organize in give (example As subcutaneous administration, muscle are given, intravenous administration), administer locally to (for example, for percutaneous administration of) or per rectum give carry out to It gives.Dosage form is preferably suitable for the form in administration way.Such as when being given in tissue, preferably via the injection of blood flow, at this time Dosage form, representative is liquid.

When being injected, injection position is not particularly limited.In including but not limited to intravenous, intra-arterial, liver, In intramuscular, intra-articular, marrow, in pulp cavity, in intra-ventricle, percutaneous, subcutaneous, intradermal, intraperitoneal, nasal cavity, enteral or sublingual etc.. The preferably intravascular injections such as intravenous injection or intra-arterial injection.Drug of the invention can be made directly to reach entirely via blood flow Body, and invasion is relatively low, bears to the objects bring such as patient small.Alternatively, also may be injected into liver or vena portae hepatica, Drug of the invention is set to directly act on position where HCV.

It is preferably each when giving antibody of the invention or antigen-binding antibody segment or drug to object (patient etc.) It gives in unit containing a effective amount of antibody antigen binding antibodies segment of the invention that can play HCV infection inhibitory activity. " effective quantity ", which refers to, plays amount necessary to its function with regard to effective component, i.e., refers in the present invention necessary to inhibiting HCV infection Amount, and refer to the amount for not generating harmful side effect nearly or completely to object to be administered.The effective quantity can be according to object The information of (patient etc.), dosage form and the various conditions of giving approach etc. and change." information of object (patient etc.) " packet Contain: progress extent or severe degree, the health status of whole body, age, weight, gender, the dietetic life, medicaments insensitive of disease Property, whether there is or not concomitant medicament and to patience for the treatment of etc..The final administered dose and effective quantity of drug of the present invention, based on each right As the information etc. of person, it is determined by the judgement of doctor.Obtaining HCV infection inhibitory effect Fang Min, it is necessary to largely give When drug of the present invention, in order to mitigate the burden for object, it also can be divided into and given for several times.

In order to supplement or enhance the treatment or prevention effect of drug of the present invention or reduce administered dose, this can also be sent out Bright drug and other medicines are used in mixed way in right amount or and with uses.As drug, can enumerate: existing antivirotic, Such as interferon, virazole etc..

The object for giving object as antibody of the invention or antigen-binding antibody segment or drug is not limited, It is preferred that for the mammal of primate, domestic animal, pet, experiment (test) animal of people etc. etc., it is particularly preferably long for the spirit of people etc. Class.Object can be to suffer from hepatitis C or can be and not suffer from hepatitis C but have pair for suffering from hepatitis C risk As person.Object can be the chronic hepatitis C patient for receiving liver transplant or can be the operation for receiving to need to transfuse blood Patient.

Antibody or antigen-binding antibody segment of the invention can be used as the HCV detection reagent in sample.Sample refers to, It may include the various samples of HCV (HCV particle or its film albumen).Such as are as follows: culture cell, culture clasmatosis liquid, culture Liquid supernatant or sample, such as people's sample from object.People's sample refers to, the tissue for picking up from people (is taken for example, postoperative Tissue) or blood, serum, serum, urine, bone marrow fluid, saliva, lymph, sperm etc. all living bodies from people such as body fluid Sample, preferably blood, serum, blood plasma or urine.In addition, not only can be liquor sample, it is also possible to solid sample.Such as It can be the donor organ or tissue section preparation etc. of organ transplant.

The detection of HCV can by ELISA method, EIA method, fluorescence immunoassay, radioimmunoassay or shine exempt from The well known immunological detection method, surface plasmon resonance (SPR method) or stone using labelled antibody of epidemic disease measuring method etc. English crystal microbalance measuring method is implemented, but is preferably implemented by using the immunological detection method of labelled antibody.

In addition, above-mentioned immunological assay method can also be implemented as follows: by immunoturbidimetry, latex agglutination reaction, glue The generation of the immune complex agglutinator of newborn turbidimetry, red cell agglutination or particle agglutination reaction etc., optically Its transmitted light or scattering light, or the measuring method that measures by visual observation are measured to implement.At this point it is possible to using phosphate buffer, Glycine buffer, Tris buffer or Good buffer etc. are used as solvent, and may include the reaction promotion of polyethylene glycol etc. Agent or non-specific reaction suppressor.

HCV-Ab IgG neutralizing antibody number disclosed in this invention is TRN1009.

The advantages of the present invention:

The present invention provides a kind of HCV-Ab IgG neutralizing antibody, which has antibody titer height, high specificity, affinity, surely The characteristics of qualitative good, non-immunogenicity, complete people endogenous binding protein structure.

The present invention provides a kind of pharmaceutical composition, which can be used for (such as the third type liver of disease caused by HCV Inflammation, cirrhosis, liver cancer etc.) treatment.

Antibody provided by the invention can be used for preparing the tool of detection HCV virus, finds effective Neutralization and crystallization and opens Send out hepatitis C recombinant protein and subunit vaccine.

Detailed description of the invention

Fig. 1 is antibody TRN1009 expression and purification figure, wherein figure A shows the SDS-PAGE detection figure of TRN1009 antibody, Figure B shows the Western-blot detection figure of TRN1009 antibody.

Fig. 2 is that antibody TRN1009 schemes from the detection of the neutralization activity of different HCV virus strains；Wherein, figure A shows antibody The neutralization activity figure of TRN1009 and Strain h77；Figure B shows the neutralization activity figure of antibody TRN1009 Yu Strain Con1； Figure C shows the neutralization activity of antibody TRN1009 Yu Strain JFH1；Figure D shows antibody TRN1009's and Strain s52 Neutralization activity；Figure E shows the neutralization activity of antibody TRN1009 Yu Strain ED43；Figure F shows antibody TRN1009 and disease The neutralization activity of strain SA13；Figure G shows the neutralization activity figure of antibody TRN1009 Yu Strain HK6a.

Fig. 3 is the combination Activity determination figure of the antigen of antibody TRN1009 and different genotype.

Fig. 4 is the affine Activity determination figure of antibody TRN1009 and different HCV virus antigen E2-core albumen, wherein figure A For antibody TRN1009 and the affine Activity determination figure of E2-core-2a genotype albumen；Scheming B is antibody TRN1009 and E2-core- 4a genotype albumen is affine Activity determination figure；Figure C is the affine Activity determination of antibody TRN1009E2-core-6a genotype albumen Figure；Scheming D is antibody TRN1009 and the affine Activity determination figure of E2-core-7a genotype albumen.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:ColdSpring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

1 separation of lymphocytes of embodiment and single memory B cells sort

One, material:

1. reagent: Ficoll lymphocyte separation medium is Cedarlane Products.CD3-APC,CD14-APC,CD16- PE, CD20-APC, CD27-PE, IgD-FITC etc. are Invirogen Products etc..

Two, methods and results

1, sample acquisition: HCV infection person's anticoagulation cirumferential blood picks up from Yunnan Province Kunming, which confirms that infection time is 2010.

2, separation of lymphocytes and single memory B cells sort

HCV-I002 blood sample 100mL is acquired, with separation of lymphocytes pipe separating peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC), utilizes BD FACSria flow cytometer (BDBiosciences, San Jose, CA) carries out cell sorting, removes cell fragment first, is adhered cell and dead cell, leads to It crosses fluorescent antibody staining and obtains the cell of CD3-/CD14-/CD16-/IgM-, the B of selection expression CD235a-/IgD-/CD20+ is thin Born of the same parents iris out CD27ALL memory B cell, obtain the target cell of E2 double fluorescence labeling with the antigen of specific marker fluorescein.

2 single-cell RT-PCR separation antibody variable region gene of embodiment

One, material

1. reagent: primer (Invitrogen synthesis), Superscript III reverse transcriptase, HotStarTaq Plus enzyme (Invitrogen, Carlsbad, CA), agar etc..

Two, methods and results

1. single-cell RT-PCR

The PCR of 96 orifice plates containing single bone-marrow-derived lymphocyte is predicted to each subtype heavy chain and light chain of 0.5 μM of addition in mixed liquid Constant region primers and Superscript IV reverse transcriptase (Invitrogen, Carlsbad, CA), while positive and yin is set Property control；Reverse transcription PCR condition: 55 DEG C of 60min are down to 4 DEG C.First chain of cDNA is synthesized, it is cDNA-20 DEG C of product long-term to protect It deposits.

2. the amplification of antibody variable region target gene

Using nested PCR method, with reverse transcription product (cDNA) for template, AmpliTaq Gold 360Master is added The specific primer of Mix (Invitrogen, Carlsbad, CA) and 0.5 μM of each subtype heavy chain and light chain antibody.React item Part: 95 DEG C of 5min of initial denaturation, then carry out 35 PCR cycles: 95 DEG C × 30s, 55 DEG C × 60s, 72 DEG C × 90s, finally with 72 DEG C extend 7min.PCR product is identified with 1.2% agarose gel electrophoresis.

3.DNA gel electrophoresis identification

Electrophoresis experiment is carried out using 2% Ago-Gel to be identified.Take 2 μ l PCR reaction products and 18 μ l0.1%'s Loading after Loading Buffer is mixed carries out electrophoresis 12min under the EG mode of E-base electrophoresis system.Gel imaging system Detect PCR product size.

The building of the expression vector of 3 recombinant antibodies of embodiment

One, material

1. reagent: agarose, Tris, LB, ampicillin, PolyFect (Qiagen, Valencia, CA), FCS, DMEM, PBS etc..

2. carrier: pcDNA3.3 carrier

3. bacterial strain: bacillus coli DH 5 alpha

Two, methods and results

1.PCR product purification clone identification

2 μ l amplified productions are taken to detect through 2% agarose gel electrophoresis, the purpose of light chain about 800bp and heavy chain about 1500bp Segment.Gel electrophoresis is accredited as the positive, and heavy chain can match pairs of antibody variable gene PCR product with light chain and utilize TA The method of clone is connected on pcDNA3.3 carrier (engineered and contain antibody leader and constant region), and connection product is turned Change in DH5 α competent bacteria, 37 DEG C of overnight incubations on the plate containing ampicillin, 10 single colonies of picking are used immediately Specific primer carries out PCR, reaction condition: 94 DEG C of initial denaturation 3min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 1min40s, 28 circulations, last 72 DEG C re-extend 5min.

2. result

It takes 2 μ lPCR products to carry out electrophoresis detection on 1% Ago-Gel, chooses PCR and be accredited as positive bacterium colony progress Gene sequencing, comparison result are correctly the recombinant expression plasmid of heavy chain of antibody and light chain.

The expression of 4 target gene of embodiment and the selective mechanisms of antibody

One, material

1. reagent: PolyFect (Qiagen, Valencia, CA), FBS, DMEM, PBS, Luciferase, IgG antibody, BSA, PBST, sodium sulphate, acetone etc..

2. cell: 293T, CHO, Huh7.

3. virus: HCV pseudovirus (HCVpp).

Two, method

1. the expression of target gene

The plasmid of massive amplification expression positive antibody heavy chain and light chain gene, endotoxin-free extracting in bacillus coli DH 5 alpha Kit extracting.With transfection reagent Polyetherimide corotation transfected cho cell, 4-6h adds fresh serum free culture after transfection Base is placed in 37 DEG C, 8%CO₂96h is cultivated in constant incubator, is collected cell conditioned medium and is detected.

2. the selective mechanisms of antibody

Screening: cell conditioned medium uses the antibody of direct competive ELISA testing goal gene expression after culture 72h.ELISA sieve Choosing: using different HCV envelope glycoproteins as antigen, and it is 100ng/ml by antigen diluent concentration with coating buffer, is coated in ELISA 96 orifice plates, every 100 μ l of hole, 4 DEG C overnight.37 DEG C of closing 2h of confining liquid.After closing plus primary antibody, antibody initial concentration are 25 μ g/ml, 3 times of gradient dilutions, every pore volume is 100 μ l, 37 DEG C of incubation 1h, while using HCV positive patients serum as positive control, mad dog Antibody is negative control.Use goat anti-human igg's (1:2000 dilution) of HRP label as 37 DEG C of incubation 1h of secondary antibody.It is aobvious that substrate is added Color liquid (TMB) after 37 DEG C of avoid light place 5min, with 2M sulfuric acid stopped reaction, carries out colorimetric with 450nm wavelength.

Detection: there is the active antibody of combination to be named as TRN1009 for what ELISA was screened, and carry out neutralization experiment: will not Same HCV envelope glycoprotein is packaged into plasmid, and fluorescein Luciferase gene transfection 293T cell is added for packing HCV Pseudovirus (HCVpp) collects supernatant for infecting.Huh7 cell is spread in 96 orifice plates, every hole cell about 1 × 10⁴It is a, every hole body Product is 100 μ l, is incubated overnight, and cell about 30% is paved with when infection.HCVpp is mixed with antibody, is placed at room temperature for 30min.It is added HCVpp and antibody mixed liquor infect Huh7 cell in 96 orifice plates.HCVpp is only added in control group, and infection changes liquid afterwards for 24 hours, continues to train It supports 1-2 days.Luciferase activity is measured after infection after 2-3 days, measurement method is to remove cell conditioned medium, and every hole is cracked with 30 μ l Liquid sufficiently takes 20 μ l cell pyrolysis liquids to add 30 μ l substrates, reads fluorescent value after cracking.Compare antibody and control group, calculates and neutralize Efficiency.

3. result

ELISA experiment screening to have in conjunction with active antibody have extraordinary neutralization efficiency.

5 antibody great expression of embodiment and purifying

One, material

1. reagent: PolyFect (Qiagen, Valencia, CA), PBS, FBS, DMEM (Gibco), Pro293A-CDM training It supports base (LONZA), Protein-A affinity column (Amersham), SDS-PAGE electrophoresis kit (Invitrogen), low point Son amount protein standard, bromophenol blue, coomassie brilliant blue R250.

2. cell: CHO.

Two, method

1.TRN1009 antibody great expression and purifying

The expression vector of heavy chain of antibody and light chain that the number for having neutralization activity of experimental identification out is TRN1009 will be neutralized (wherein, the amino acid sequence of heavy chain variable region is as shown in SEQ ID NO.4；The amino acid sequence of antibody's light chain variable region such as SEQ Shown in ID NO.8) corotation transfected cho cell (cell fusion degree reaches 90% or more), 6-8h benefit culture medium after transfection, 37 DEG C, 8% CO₂96h is cultivated in incubator.Then transfection supernatant is collected, 4000rp 4 DEG C, is centrifuged 1h, abandons precipitating, utilize albumen (Protein) A affinity chromatography carries out purifying and obtains TRN1009 antibody.

2.SDS-PAGE and Western-blot is examined

Using utilization lauryl sodium sulfate-polyacrylamide gels (polyacrylamide Gelelectrophoresis, SDS-PAGE) and immunoblotting (western blot test, i.e. Western Blot) inspection The result of the expression and purification of TRN1009 antibody: prepare sample (200-800ng/w), loading (10% pre-prepared colloid), electrophoresis, Fixed, dyeing, decoloration；Or carry out Western blot analysis.

3. result

As a result as shown in Figure 1, obtaining the very high protein ingredient of purity after purification, the molecular weight of antibody TRN1009 is more than 120kD, and heavy chain of antibody, light chain after unwinding can be obviously observed, heavy chain is about 70kD, and light chain is about 30kD.

The binding specificity of 6 TRN1009 antibody of embodiment detects

One, material

1. reagent: PBS, Luciferase, IgG antibody, BSA, PBST, sodium sulphate, acetone etc..

2. virus: HCV pseudovirus (HCVpp).

Two, methods and results

1. the ELISA method mentioned with embodiment 4 detects the combination binding specificity of the antibody of expression and purification: will Different HCV envelope glycoproteins is packaged into plasmid, and fluorescein Luciferase gene transfection 293T cell is added for packing HCV pseudovirus (HCVpp) collects supernatant for infecting.Huh7 cell is spread in 96 orifice plates, every hole cell about 1 × 10⁴It is a, mistake Night culture.HCVpp is mixed with the antibody TRN1009 of various concentration, and 75 μ g/ml of initial concentration, 3 times of dilutions are placed at room temperature for 30min.HCVpp and antibody TRN1009 mixed liquor is added in 96 orifice plates, infects Huh7 cell.HCVpp is only added in control group, sense Dye changes liquid afterwards for 24 hours, continues culture 1-2 days.Luciferase activity is measured after infection after 2-3 days, measurement method is to remove cell Supernatant, 30 μ l lysates of every hole sufficiently take 20 μ l cell pyrolysis liquids to add 30 μ l substrates, read fluorescent value after cracking.

2. result: fluorescent value reduces, and shows that TRN1009 antibody and HCV envelope glycoprotein 100% are specifically bound.

The neutralization activity of 7 TRN1009 antibody of embodiment detects

One, material

1. reagent: DMEM, BSA, PBST, sodium sulphate etc..

2. virus: HCV virus strain (h77, Con1, JFH1, S52, ED43, SA13, HK6a).

Two, methods and results

1. spreading Huh7 cell in 96 orifice plates, it is incubated overnight, cell about 30% is paved with when infection.The strain of HCV euvirus (h77, Con1, JFH1, S52, ED43, SA13, HK6a) it is mixed with the antibody of various concentration, 25 μ g/ml of initial concentration, 3 times of dilutions, room Temperature places 30min.HCV virus strain (h77, Con1, JFH1, S52, ED43, SA13, HK6a) and antibody mixed liquor is added in 96 holes Plate infects Huh7 cell.HCV virus strain 2G9 is only added in control group, and liquid is changed in infection afterwards for 24 hours, continues culture 1-2 days.2- after infection Activity is measured after 3 days, reads fluorescent value.Compare antibody and control group, calculates and neutralize efficiency.

2. result

TRN1009 antibody and the neutralization activity testing result of 7 plants of HCV virus strains are as shown in Figure 2；TRN1009 antibody with not The neutralization efficiency of same HCV virus strain is as shown in table 1, and the TRN1009 antibody of low concentration is with the neutralization efficiency of HCV virus strain Reach 100%.

The neutralization efficiency of table 1 TRN1009 antibody and different HCV virus strains

The combination Activity determination of 8 TRN1009 antibody of embodiment

One, material

Reagent: IgG, PBST, TMB, sulfuric acid etc..

Two, methods and results

1. with the packet of different HCV subviral strains (HCV1a, HCV1b, HCV2, HCV3, HCV4, HCV5, HCV6, HCV7) Antigen diluent concentration is 100ng/ml as antigen, and with coating buffer by memebrane protein, is coated in 96 orifice plate of ELISA, every 100 μ of hole L, 4 DEG C overnight.37 DEG C of closing 2h of confining liquid.After closing plus primary antibody, TRN1009 initial concentration is 25 μ g/ml, 3 times of gradient dilutions, Every pore volume is 100 μ l, 37 DEG C of incubation 1h, while using HCV positive patients serum as positive control, and Rabies virus antibody is negative right According to.Use goat anti-human igg's (1:2000 dilution) of HRP label as 37 DEG C of incubation 1h of secondary antibody.Substrate developing solution (TMB) 100 μ is added The hole l/ after 37 DEG C of avoid light place 5min, with 2M sulfuric acid stopped reaction, carries out colorimetric with 450nm wavelength.

3. result

As a result as shown in figure 3, the antibody TRN1009 of expression and purification is carried out TRN1009 antibody after diluting greater than 2000 times Still there can be extremely strong combination activity with antigen binding.

The affine Activity determination of 9 TRN1009 antibody of embodiment

One, material

1. reagent: IgG antibody, Biacore coupling reagent kit etc..

2. chip: CM5.

Two, methods and results

1. the coupling prize law using BiaCore analyzer carries out affine Activity determination, BiaCore X- is utilized 100System software is analyzed.

2. result

Envelope protein E2-core (E2-core-2a, E2- of TRN1009 antibody and the HCV of 4 kinds of different genotypes Core-4a, E2-core-6a and E2-core-7a) it is affine activity as shown in Figure 4；Concrete outcome is as shown in Table 2 below, The affinity of the envelope glycoprotein of TRN1009 antibody and the HCV of 4 kinds of different genotypes is high, and equilibrium dissociation constant reaches 1*10^- ¹¹KD (M) or more.

The affinity kinetic results parameter list of 2 TRN1009 antibody of table and HCV virus antigen

Ligand	ka(1/Ms)	kd(1/s)	KD(M)
				E2-core-2a	4.88E+04	6.25E-07	1.28E-11
E2-core-4a	4.25E+04	8.24E-08	1.94E-12
				E2-core-6a	2.81E+04	4.97E-07	1.77E-11
E2-core-7a	9.85E+04	5.80E-09	5.88E-14

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

SEQUENCE LISTING

<110>Guangzhou Tylenol enlightening Biotechnology Co., Ltd Zhuhai Tylenol Mai Bo Bioisystech Co., Ltd and south are big It learns

<120>preparation, detection and application of HCV-Ab IgG wide spectrum neutralizing antibody

<160> 8

<170> PatentIn version 3.5

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Claims

1. a kind of HCV-Ab IgG wide spectrum neutralizing antibody, which is characterized in that the antibody variable region includes CDR there are three at least one contains Heavy chain variable region and at least one contain there are three CDR light chain variable region；Wherein the heavy chain variable region includes:

(1) VH CDR1, amino acid sequence are SEQ ID NO.1;

(2) VH CDR2, amino acid sequence are SEQ ID NO.2;

(3) VH CDR3, amino acid sequence are SEQ ID NO.3;

Wherein the light chain variable region includes:

(4) VL CDR1, amino acid sequence are SEQ ID NO.5;

(5) VL CDR2, amino acid sequence are SEQ ID NO.6;

(6) VLCDR3, amino acid sequence are SEQ ID NO.7.

2. antibody according to claim 1, which is characterized in that the antibody has VH structure shown in SEQ ID NO.4 VL structural domain shown in domain and/or SEQ ID NO.8.

3. a kind of composition, which is characterized in that the composition includes antibody of any of claims 1 or 2.

4. composition according to claim 3, which is characterized in that the composition is pharmaceutically acceptable composition.

5. a kind of clone or expression vector, which is characterized in that the clone or expression vector include DNA molecular, the DNA molecular Encode the heavy chain and light chain of antibody of any of claims 1 or 2.

6. a kind of host cell, which is characterized in that including encoding the heavy chain of antibody of any of claims 1 or 2 and the DNA of light chain Molecule or right want 5 described in clone or expression vector.

The pharmaceutical composition or preparation detection HCV of disease caused by 7. antibody of any of claims 1 or 2 treats HCV in preparation Tool in application.