CN102702348B - Single source antibody of antagonism enteric virus71 type and uses thereof - Google Patents

Abstract

The present invention relates to the single source antibody be combined with enterovirus (EV) 71 type, or its binding fragment.The present invention discloses and possesses the antibody of enteric virus71 type-binding ability, still retains the fragment of enteric virus71 type-binding ability therewith, complete human antibodies or humanized antibody in antibody-like, and comprise these disclose the medical component of antibody.The present invention also discloses the isolated nucleic acid of code book invention antibody, and through the host cell of these nucleic acid transfections.In addition, the present invention disclose again utilize antibody of the present invention and nucleic acid prevention, treatment and diagnostic method.

Description

Single source antibody of antagonism enteric virus71 type and uses thereof

Technical field

The present invention relates to the purposes of field for neurocyte protection of immunology, particularly relate to the single source antibody being combined with enteric virus71 type and/or neutralizing.

Background technology

Enteric virus71 type is the underlying stock RNA viruses that one ranges small nut candy nucleic acid virus section (Picornaviridae).Since in 1969 since California, USA occurs that first EV71 type infects case, all parts of the world regularly there will be the case of this virus infects human.Be subject to enteric virus71 type (the corresponding body that some heredity is above close with it, infect as Coxsackie virus 16 (CA16) and CA10), notify and caused slight dermexanthesis symptom, stomatopod hand disease (HFMD)/herpangina may be shown as with it baby and child.In the past in 10-12, the regularly outburst enteric virus71 type infection of Asian-Pacific area is very popular, and has caused the pulmonary edema/bleeding that can cause many infected death of child.In enteric virus71 type gene group, the virus of gene group B and C has occurred in being very popular of TaiWan, China and has been separated between 1986 to 2008.Popular activity between 1999 to 2002, mainly relevant with gene group B virus.In 2006 and 2007, identify a kind of enteric virus71 type with genotype B5, and during the TaiWan, China coming across 2008 is once again very popular.

The enteric virus71 type of constantly scurrying out infects, and is discussed the problem that global public health threatens by as a kind of.In the past, not yet have and effective antiviral drug infected to treatment enteric virus71 type, and be used for preventing and treating effective preventative vaccine of virus pandemic, and expection in recent future still without can user.The enteric virus71 type being occurred in China the past over 10 years is very popular, and has utilized the compound mankind's character used in proper names and in rendering some foreign names agate sphaeroprotein of dispensing, has treated to suffer from and infect relevant Patients With Encephalitis to enteric virus71 type.But this test does not reach gratifying result.The existing SP70 that one to be made is exposed to VP1 capsid protein surface, Immunodominant Antigenic determines base and key wellhole worm relative hemocyanin (KLH) conjugation, produces in energy and the muroid list source antibody of the enteric virus71 type strain isolated (it is touring popular in China) of genotype C.

For the selection being used for being delivered to immune suitable immune composite, may decisive influence produce the specificity of antibody.There is no available single source antibody at present, the single-minded antagonism of energy is in recent years in the touring popular genotype B enteric virus71 C-type virus C strain of TaiWan, China.

Summary of the invention

In one side, the present invention relates to a kind of single source antibody or its binding fragment, it is exclusively be combined with the polypeptide listed by SEQIDNO:55.

In another aspect, system of the present invention is about single source antibody of being combined with enterovirus (EV) 71 type of one or its binding fragment, it comprises: heavy chain complementarity determining region 1 (CDR1) polypeptide a) with the Amino acid sequence listed by SEQIDNO:70, with heavy chain complementarity determining region 2 (CDR2) polypeptide of the Amino acid sequence had listed by SEQIDNO:71; And b) there is the light chain CDR1 polypeptide of the Amino acid sequence listed by SEQIDNO:72 or 73, there is the light chain CDR2 polypeptide of the Amino acid sequence listed by SEQIDNO:74 or 75, with the light chain CDR3 polypeptide of the Amino acid sequence had listed by SEQIDNO:76 or 77.

In another aspect, the present invention relates to a kind of isolated nucleic acid, it comprises the nucleotide sequence that coding one comprises the heavy chain polypeptide of the Amino acid sequence listed by SEQIDNO:82.

In another aspect, the present invention relates to a kind of isolated nucleic acid, it comprises the nucleotide sequence of an encoded packets containing the light chain polypeptide of the Amino acid sequence be selected from listed by SEQIDNO:78,79,80 and 81.

In another aspect, the present invention relates to a kind of host cell, it comprises foregoing isolated nucleic acid.

In another aspect, the present invention relates to a kind of composition for neutralizing or protect the enteric virus71 type of antagonism genotype B to infect, it comprises one or more foregoing single source antibody or its binding fragment; And a kind of pharmaceutically acceptable carrier.

In on the other hand, the present invention relates to a kind of composition, it comprises a kind of foregoing single source antibody or its binding fragment; And a kind of pharmaceutically acceptable carrier.

Again on the other hand, the present invention relates to a kind of enteric virus71 type of in vitro detecting and whether there is method in a biological specimen, it comprises: a) contacted with aforesaid composition by this sample; And combination b) analyzing this antibody is to measure the existence of enteric virus71 type.

Again on the other hand, the present invention relates to a kind of single source antibody of being combined with enteric virus71 type or its binding fragment, it comprises: heavy chain polypeptide a) comprising the Amino acid sequence listed by SEQIDNO:82; And b) comprise be selected from SEQIDNO:78,79, the light chain polypeptide of Amino acid sequence listed by 80 and 81.

These and the other side of the present invention, will describing from following preferred embodiment, and combine listed graphic and show out, but under the spirit not departing from the present invention's novelty creation concept and scope, can carry out various variation and amendment.

Subsidiary graphic be implement example to exemplify one or more that the present invention is described, and the embodiment one contained with this specification sheets is used from the principle explaining the present invention.Respectively graphicly middle use the symbolic module with same No. number, in each enforcement example, all represent same or analogous meaning.

Accompanying drawing explanation

Fig. 1 is the light absorption value figure of display homotype N1, N3, N4 and N6 antibody typing;

Fig. 2 is the photo of the gel electrophoresis analysis result of the ascites fluid sample of display respectively containing indication order source antibody N1, N3, N4 and N6;

Fig. 3 is display E71/E59 viral protein is turned stain analytical results respectively photo by the west of indication order source antibody N1, N3, N4 and N6 combination existed in ascites fluid sample;

Fig. 4 lists the single-letter Amino acid sequence of the variable region of light chain (VL) of N1, N3, N4 and N6 antibody.FW: skeleton; CDR: complementarity determining region;

Fig. 5 lists the total Amino acid sequence of the variable region of heavy chain (VH) of N1, N3, N4 and N6 mono-source antibody;

Fig. 6 A-6D shows the result of epitope positioning analysis;

Fig. 7 shows distinctly and the analytical results figure of the Neutralization effect of antibody cocktail.

Embodiment

The term used in this specification sheets usually in technical field that the present invention belongs to, in the present invention in literary composition and single term by the specific interior literary composition that uses, be there is its general significance.Below (or specification sheets other local) discuss some for describing the term of the present invention, implement this tactful person about the extra guide of content of the present invention to provide.For convenience's sake, some specific terms will use italics and/or quotation marks to emphasize out, but use the sign emphasized can't affect scope and the meaning of a certain term itself; No matter in same interior literary composition, whether it is labeled a certain term, its scope and meaning are identical; Same thing has more than one saying to be intelligible.Therefore, herein one or more term of discussing can use substituting language and synonym, whether it is described in detail in this article or not particularly important is discussed.The quoting not get rid of of one or more synonym of the synonym of some specific terms can use other synonym.Used herein include arbitrary herein the enforcement example of term discussed only as aid illustration, scope and definition not for limiting the present invention or any exemplified term.Equally, the present invention is not limited to various enforcement example contained in this manual.

Unless otherwise prescribed, technology used herein and scientific terminology, have with technical field that the present invention belongs in have and usually know the knowledgeable, the identical meaning of common understanding.If when conflicting to some extent, at presents (comprising definition), control will be proposed.

" about " used herein, " approximately " or " probably " should broadly mean, within 20% of given numerical value or scope, preferably within 10%, and more preferably within 5%.Herein the quantity that gives be approximation, represent without under clear and definite instruction, term " about ", " approximately " or " probably " can be quoted.

It " preparation " used herein generally should mean something and be produced, manufacture, and a certain material is through special preparation.

Term used herein " antibody " means a kind of immunoglobulin molecules, or has the immunoglobulin molecules fragment of the ability that can be combined with specific antigen specificity.Antibody is known known by the knowledgeable for having in field of immunology usually.Term used herein " antibody " not only refers to full length antibody molecule, also comprises the Antibody molecule fragments possessing antigen binding capacity.This type of fragment also, known by this skill, is usually used in vitro and in vivo.Especially, term used herein " antibody " not only refers to the immunoglobulin molecules of total length, also comprises the fragment of tool antigen-binding activity, the active fragments F (ab ') 2 such as known, Fab, Fv and Fd.

The Variable Area system of one antibody is called FV district, and it is the most important region be combined with antigen.

Ig monomer is a kind of " Y "-font molecule, and it is be made up of four polypeptide chains; Article two, the light chain that identical heavy chain is identical with two, is connected with cystine linkage to each other.The position that arm (such as) contains and antigen combines of Y-font, and therefore can the special exotic of identification.This region of antibody is called Fab (fragment, antigen combines) region.It is by from each heavy chain of antibody and light chain, and a constant region and a variable region formed.

The present invention relates to four kinds of enterovirus EV71 type-specificity neutralizing antibodies, called after N1, N3, N4 and N6.These antibody systems use original fusion knurl technology separation to obtain, and comprise and BALB/c (H-2d) mouse spleen of isolated for the EV71/E59 through making a living strain immunity and NS-1 myeloma cell being merged.Neutralizing antibody can be used for preparing the medical composition infected for treatment, disseminates especially for blocking virus.

By the non-CDR regional replacement of a mammalian antibody with the zone similarity of allogenic animal or heterogenous animal antibody, and the specificity of the epitope of original antibodies can be still kept in this skill is known.This most easily represents when research and development and use " humanization " antibody, be wherein that non-human CDRs and the mankind FR and/or Fc/pFc ' region are carried out covalency joint, and generation has functional antibody.Therefore, such as, PCT International Publication case WO92/04381 teaching, wherein will muroid FR region at least partially about the manufacture of humanization muroid RSV antibody and use, replaces the FR region with human origin.This antibody-like (comprising the full length antibody fragment with antigen binding capacity), is referred to as " mosaic type " antibody usually.Some or all FR region of this type of wherein anti-EV71 antibody can be produced, be replaced as the Chimeric antibodies in other homology mankind FR region.

The humanization pattern of non-human (such as muroid) antibody is mosaic type immunoglobulin (Ig), it contains derived from the immunoglobulin chain of the minmal sequence of non-human immunoglobulin or fragment (such as, Fv, Fab, Fab ', other antigen-binding subsequence of F (ab ') 2 or antibody).Humanized antibody comprises human immunoglobulin (recipient antibody), wherein carry out the residue of autoreceptor complementarity determining region (CDR), replaced with the residue of the CDR from non-human species (donor antibody) such as such as mouse, rat or rabbits, it has desired specificity, affinity and ability.In some case, be with corresponding non-human residues by the displacement of the Fv framework residue of human immunoglobulin.Humanized antibody also can comprise and not appear in recipient antibody, or be not present in introduce or residue in frame sequence.Generally, humanized antibody can comprise in fact all at least one (typically two kinds) Variable Areas, wherein all or in fact whole CDR region, system is equivalent to the CDR region of non-human immunoglobulin, and all or in fact whole FR regions, be the FR region of human immunoglobulin consensus sequence.Humanized antibody the best also comprises constant region for immunoglobulin (Fc) at least partially, and representational system is from human immunoglobulin [people such as Jones, Nature, 321:522-525 (1986); The people such as Riechmann, Nature, 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol., 2:593-596 (1992)].

By humanized for non-human antibody method, known by this skill.Generally, humanized antibody has one or more amino acid residue imported from nonhuman origin, and these non-human amino acid residue are commonly referred to " introduction " residue, take from one " introduction " Variable Area typically.Humanization effect substantially can according to method [people such as Jones, Nature, the 321:522-525 (1986) described in Winter and colleague; The people such as Riechmann, Nature, 332:323-329 (1988); The people such as Verhoeyen, Science, 239:1534-1536 (1988)], by corresponding sequence rodents CDRs or CDR sequence being replaced human antibodies.So this type of " humanization " antibody is Chimeric antibodies (U.S.Pat.No.4,816,567), wherein will be less than in fact a complete human variable domain, replaced by the corresponding sequence from non-human species.In practical application, humanized antibody is wherein some CDR residue typically, and may be replaced by from the residue of similar position in rodent antibodies by some FR residues.

Also can use the technology that this skill various is known, comprise phage displaying antibody storehouse (phagedisplaylibraries) and carry out manufacturer's antibody-like.The technology of the people such as people and Boerner such as Cole also can be used for the preparation mankind single source antibody (people such as Cole, single source antibody and cancer therapy, AlanR.Liss, p.77 (1985); And the people such as Boerner, J.Immunol., 147 (1): 86-95 (1991)).Equally, animal can be grown by being turned by human immunoglobulin gene seat quiding gene, such as, in the mouse that wherein endogenous immunoglobulin genes is partially or completely deactivated.During attacking poison, observed human antibodies manufacture, it is very similar in mankind's each side viewed, comprises gene rearrangement, assembling and antibody repertoire (antibodyrepertoire).This processing procedure is through being described in (such as) United States Patent (USP) case 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661, No. 016.These type of complete mankind or humanization list source antibody, be particularly useful for the immune response person not wishing to cause antagonism this antibody itself.See United States Patent (USP) case 7,622, No. 113, it is complete is incorporated herein by reference.

Antibody can carry out demarcating and being fixed on solid support.Words and phrases " mark " directly or indirectly engage with antibody for meaning herein, thus produce " by demarcating " antibody can detect compound or composition.Mark this as (such as, labelled with radioisotope or fluorescent mark) that can detect, or the situation of ferment mark, can catalysis carry out being can detecting by the chemical transformation of matter compound or composition.

For cause produce can cross neutralization in recent years at single source antibody of the touring popular enteric virus71 type genotype B virus of TaiWan, China, by the isolated strain of TaiWan, China (having the EV71/59 of genotype B4) that mouse inoculation is lived, and animal capable initiation antagonism wider range is made to there is the bone-marrow-derived lymphocyte of the interior epitope of natural viral antigen.Then, via selecting culturing method to isolate, the fusion knurl with the antibody of desired characteristic can be manufactured.The present invention describes merges by four kinds the feature that knurl clones manufactured single source antibody.

Be used in cells produce, and through sucrose gradient centrifugation purifying it, the enteric virus71 type (called after EV71/E59) with genotype B4 of living, for being used for producing the immunogen of EV71-specificity list source antibody.Filter out four kinds from causing BALB/c (H-2d) mouse spleen of exempting from advance through EV71/E59-, with the NS-1 myeloma cell presenting stable growth carry out merging the fusion knurl that derives clone, for further signature analysis.Showing different complementarity determining regions (CDRs) based on observing they in its κ light chain gene, proving that the fusion knurl produced is independently clone really.The purified ascites manufactured by distinctly clone, turns during stain is analyzed and can react for viral capsid proteins (VP1), and identification is positioned at the different positions of the common epitope of C-end in west.Each single source antibody all presents effectively to resist to cause exempts from virus strain, and other two strain belongs to the Neutralization effect of time gene group B4 and B5, called after N0781-TW-01 and N2838 (often appearing at TaiWan, China to be very popular closely several times) respectively.Also observe in and EV71/E59 virus time, single source antibody can be cooperated with each other action.

In one side, the present invention relates to a kind of single source antibody or its binding fragment, it is exclusively be combined with the polypeptide listed by SEQIDNO:55.

In one of the present invention item specific embodiments, this single source antibody is humanized antibody.

In another specific embodiments of the present invention, this single source antibody is antibody fragment.

In another aspect, the present invention relates to a kind of single source antibody of being combined with enterovirus (EV) 71 type or its binding fragment, it comprises: heavy chain complementarity determining region 1 (CDR1) polypeptide a) with the listed Amino acid sequence of SEQIDNO:70, with heavy chain complementarity determining region 2 (CDR2) polypeptide of the Amino acid sequence had listed by SEQIDNO:71; And b) there is the light chain CDR1 polypeptide of SEQIDNO:72 or 73 listed Amino acid sequence, there is the light chain CDR2 polypeptide of SEQIDNO:74 or 75 listed Amino acid sequence, with the light chain CDR3 polypeptide with SEQIDNO:76 or 77 listed Amino acid sequence.

In one of the present invention item specific embodiments, this binding fragment comprises a Fv fragment.

In another specific embodiments of the present invention, this binding fragment comprises a Fab fragment.

In another specific embodiments of the present invention, this antibody is the complete mankind single source antibody.

In another specific embodiments of the present invention, this antibody is humanization list source antibody.

In another specific embodiment party case of the present invention, this antibody or the neutralization of binding fragment system have the enteric virus71 type of genotype B.

In another specific embodiments of the present invention, this antibody or binding fragment comprise the heavy chain polypeptide with the listed Amino acid sequence of SEQIDNO:82.

In another specific embodiments of the present invention, this antibody or binding fragment comprise have be selected from SEQIDNO:78,79, the light chain polypeptide of 80 and 81 listed Amino acid sequence.

In another specific embodiments of the present invention, this as previously mentioned single source antibody or its binding fragment system through demarcating.

In another specific embodiments of the present invention, single source antibody or its binding fragment system are combined with enterovirus capsid protein VP1 as previously mentioned for these.

In another aspect, the present invention relates to a kind of isolated nucleic acid, it comprises the nucleotide sequence that coding one comprises the heavy chain polypeptide of the listed Amino acid sequence of SEQIDNO:82.

In on the other hand, the present invention relates to a kind of isolated nucleic acid, its comprise an encoded packets containing be selected from SEQIDNO:78,79, the nucleotide sequence of the light chain polypeptide of 80 and 81 listed Amino acid sequence.

In another aspect, the present invention relates to a kind of host cell, it comprises isolated nucleic acid as previously mentioned.

In another aspect, the present invention relates to a kind of composition for neutralizing or protect the enteric virus71 type of antagonism genotype B to infect, it comprises one or more single source antibody or its binding fragment as previously mentioned; And a kind of pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a kind of composition, it comprises one single source antibody or its binding fragment as previously mentioned; And a kind of pharmaceutically acceptable carrier.

Again on the other hand, the present invention relates to one and in vitro detect enteric virus71 type and whether there is method in a biological specimen, it comprises: a) contacted with aforementioned composition by this sample; And combination b) analyzing this antibody is to measure the existence of enteric virus71 type.

Again on the other hand, the present invention relates to a kind of single source antibody of being combined with enteric virus71 type or its binding fragment, it comprises: heavy chain polypeptide a) comprising the listed Amino acid sequence of SEQIDNO:82; And b) comprise be selected from SEQIDNO:78,79, the light chain polypeptide of 80 and 81 listed Amino acid sequence.

Embodiment

Be not intended to limit the scope of the invention, the instrument, device, method and its correlated results that exemplify according to the various embodiments of the present invention are below provided.To it should be noted that in embodiment the title that conveniently readers ' reading uses or subtitle, be not limited within the scope of the present invention.In addition, also propose in this manual and disclose some theory; But no matter they are right or mistake, as long as this invention implemented according to the present invention, all should be limited within the scope of the present invention, and do not need to consider any specific theory or embodiment.

Materials and methods

Mouse and immunity

Will purchased from Experimental Animal Center (TaiWan, China), and be maintained at the animal care facility being looked after and use council's assay approval by this central animals, six to seven week age female Balb/c (H-2d) mouse distinctly carry out immunization three times, each via intraperitoneal routes, immunity 106 bacteriolyze spots formed unit (PFUs) it, through sucrose gradient purified EV71/E59 virus.After 10 days, animal is drawn blood in last supplementary immunization, and use direct ferment connection immuning adsorpting analysis (ELISA) of EV71/E59 (HI-EV71/E59) the virus coating through heat deactivation, measure serum antibody.To present the mouse of the antibody response resisting the most by force this virus, bestow last supplementary immunization with the live virus of same dose, after 7 days, its spleen is taken out and is used for merging.

The production of virus, purifying and signature analysis

American Type Culture Collecti (ATCC, Rockville, MD, USA) will be derived from be grown in VP-SFM serum free medium (Gibco/Invitrogen, Carlsbad, CA, USA).The isolated strain of EV71/E59 derives from TaiWan, China disease Control Centre (CDC).(medical technologies system of National Cheng Kung University, platform south, TaiWan, China) is taught by Jen-RenWang by the N0781-TW-01 of tool genotype B4 and the N2838-TW-03 system of tool genotype B5 to be provided.By by various virus with the infection multiplicity of 10-5 (MOI) inoculate into, be incubated in 1.5-2.0 × 108VERO cell culture of 850cm2 rotating and culturing bottle (Corning, NY, USA), complete a large amount of of virus and manufacture.Be fixed on by culturing bottle on swivel mount (Wheaton, Millville, NJ, USA), being placed in 37 DEG C of culturing room's setting speeds is that 0.33rpm is to promote viral growth.Culture supernatants is collected after 5 days, by 0.65 μm of filter membrane (Sartorius, Hayward, CA, USA) filter to remove cell debris, finally by being passed through Minimate100K tangential stress flow filtration (TFF) sleeve pipe (Pall life science, AnnArbor, MI, USA), and be concentrated into about 50.0mL.Concentrated sample is placed on the continuous saccharose gradient of 10-50% (Merck, WestPoint, PA, USA), and under 32,000rpm centrifugal 3 hours.The strip portion that will be positioned at 35-37% gradient is collected, and exchanges three times to 1.0L1 × PBS, pH7.2 (Gibco/Invitrogen, Carlsbad, CA, USA) and dialyse.Use immune bacteriolysis spot to be formed to analyze, measure the power valency of the purified viral preps obtained.

Produce EV71/E59-specificity list source antibody

By 1.0 × 107 from selected experiment mice obtain not containing erythrocytic spleen cell, with 2 × 107NS-1 cell ( rockville, MD, USA), method (Kohler and Milstein (1975) " the continuous culture secretion of fused cell the has predetermined narrow spectrum antibody " Nature256 set up according to Prior Art, 495-497), use purchased from Sigma/Aldrich (StLouis, MO, USA) HybriMax [p7306, PEG3000-3700Da are dissolved in methyl-sulphoxide (DMSO)] merges.Culture supernatants is collected from the fusion knurl of well-grown in HT (xanthoglobulin and thymidine) substratum (Sigma/Aldrich, StLouis, MO, USA).With every wellhole containing 0.3 cell, in substratum [CM: supplement with 10.0% foetal calf serum (BiologicalIndustries, KibbutzBeitHaemek, Israel) with penicillin/streptomycin mixture (Gibco/Invitrogen, Carlsbad, CA, USA) Eagle ' s substratum (the DMEM) (Gibco/Invitrogen that Dubecco ' s revises, Carlsbad, CA, USA)], under having 5.0 × 105 BALB/c (H-2d) spleen cells (as feeder cell) through irradiating (2000rads) to exist, carry out producing the fusion knurl pure lines that can neutralize and cause the EV71/E59-specific antibodies of exempting from virus.The fusion knurl hyperplasia of growth will be had, and determine its Clonal (clonality) through signature analysis, and the characteristic of single source antibody that produces.

The manufacture of ascites and purifying

Indivedual > 6-monthly age will be injected into by 5 × 105, in the BALB/c mouse of pristane process, fusion knurl out of the ordinary clones the ascites fluid produced, with 10% sulfuric acid glucosan solution (Sigma/Aldrich, the StLouis of 0.04mL, MO, and 1.0M calcium chloride (Sigma/Aldrich, StLouis, the MO of 1.0mL USA), USA), its degrease matter is made with the mixing of the ratio of 1: 0.04: 1 (v/v).After 5 minutes, by mixture in 10, under 000 × g centrifugal 10 minutes (whizzer model: LegendMicro17R, SorvallThermoScientific).Collect supernatant fraction, and be dissolved in dH2O 1 × Tris buffer saline (TBS) in 1.0L, pH7.2 (137mMNaCl and 10mMTris, Sigma/Aldrich, StLouis, MO, USA) dialysed overnight is carried out in, be loaded on afterwards on the protein G tubing string of use AKTA starting up pump (GEHealthcare, salt lake city, USA).The antibody that will be combined with protein G, with 10.0mM glycine-HCl (Sigma/Aldrich, StLouis, MO, USA), pH3.0 damping fluid dissolved goes out.Dissolved liquid is flowed part collection with every 1.0mL, and immediately 1.0L 1 × PBS.pH7.2 (Gibco/Invitrogen, Carlsbad, CA, USA) is dialysed.

Analyze the V of EV71/E59-specificity list source antibody _hwith V _lregion

The RNeasy cover group purchased from Qiagen (Valencia, CA, USA) will be used, the RNA extracted is cloned from each fusion knurl, synthesize with TranscriptorHighFidelitycDNA the reagent provided in cover group (Roche, IN, USA) and carry out reverse transcription.Will by the κ chain complementarity determining region (CDR) distinctly merging knurl performance, use forward direction introduction 5 '-GGATACAGTTGGTGCAGCATC-3 ' (SEQIDNO:1) and the reverse introduction 5 ' of degeneracy-GAYATTGTGMTSACMCARWCT-3 ' (SEQIDNO:2), with following cycling program: at 94 DEG C 1 minute, at 94 DEG C 1.5 minutes, at 50 DEG C 2.0 minutes, at 72 DEG C 3.0 minutes, at 72 DEG C 1.0 minutes, the polymerase chain reaction (PCR) carrying out 40 circulations completed amplification.The PCR primer obtained is processed with BciVI restriction enzyme (Fermentas, StLeonRot, Germany), the κ chain DNA of the variation derived from NSI fusion partners is digested.Electrophoresis will be carried out through the PCR primer of digestion on 2.0% sepharose (Sigma/Aldrich, StLouis, MO, USA), DNA fragmentation is separated.Use gel fragment extraction cover group (Geneaid, Xi Zhi city, TaiWan, China), the band that will be positioned at about 360bp extracts, and is addressed to MissionBioteck (TaiWan, China, Nan Gang, the Taibei, TaiWan, China) and carries out sequencing.

CDRs (hypervariable region of the heavy chain) linkwork of VH uses containing 9 kinds of degeneracy forward introductions, with the reverse introduction of single kind (5 '-AGGGGCCAGTGGATAGAC-3 '; SEQIDNO:3) increase.These forward introductions are: OVH1 (5 '-SAGGTCCAGCTGCAGCAGYYTGG-3 '; SEQIDNO:4), OVH2 (5 '-CAGGTRCAGCTGAAGSAGTCAGG-3 '; SEQIDNO:5), OVH3 (5 '-GAKGTGCAGCTTCAGCAGTCRGG-3 '; SEQIDNO:6), OVH5-1 (5 '-GAVGTGAWGCTGGTGGAGTCTGA-3 '; SEQIDNO:7), OVH5-2 (5 '-GAVGTGAWGCTGGTGGAGTCTGG-3 '; SEQIDNO:8), OVH11 (5 '-GAAGTGCAGCTGTTGGAGACTGG-3 '; SEQIDNO:9), OVH14-1 (5 '-GAGGTTCAGCTGCAGCAGTCTGG-3 '; SEQIDNO:10), OVH14-2 (5 '-GAGGTTCAGCTGCAGCAGTCTGT-3 '; SEQIDNO:11) and OVH15 (5 '-CAGGTTCACCTACAACAGTCTGG-3 '; SEQIDNO:12).PCR program setting for generation of the DNA fragmentation with about 300bp is as follows: at 92 DEG C 3 minutes, at 92 DEG C 1 minute, at 68 DEG C 30 seconds, at 72 DEG C 1 minute, at 72 DEG C 10 minutes, carries out 25 circulations.Extract desired PCR primer from 2.0% sepharose, and processed sequencing.

SDS-PAGE and west turn stain analysis

By 20 microlitres via BCA protein analysis (Pierce, Rockford, IL, USA) measure, the EV71/E59 virus of deactivate through sucrose gradient purified, heat (in 85 DEG C of hot water 10 minutes) containing 0.12 μ g overall protein matter, with 5.0 μ L 5 × concentrated Loading Dye (BionovasBiotechCo., Ltd., Toronto, Canada) mixing, and in 10%SDS-polyacrylamide gel, in 1 × Tris-glycine SDS-electrophoretic buffer (AmershamBiosciences, Piscataway, NJ, USA) in carry out electrophoretic analysis.Use MiniTrans-BlotCell (Biorad), will be transferred on pvdf membrane (Biorad, Hercules, CA, USA) through the virus protein of electrophoretic separation.Film containing protein is formulated in PBS in 5.0%, pH7.2 (Gibco/Invitrogen, Carlsbad, CA, USA) skimmed milk (pacify good, New Zealand) in blockade, cleaning, is then placed in Mini-ProteneIIMultiscreen device (Biorad, Hercules, CA, USA) in.The test single source antibody (N1, N3, N4 or N6) out of the ordinary 2.0 μ g being formulated in analysis buffer (1.0% skimmed milk is dissolved in PBS, pH7.2) adds in reaction chamber.By film with 0.5mL through 1: 10, the donkey anti-mouse antiserum(antisera) (JacksonImmuno-ResearchLab. of mountain horseradish peroxidase (the HRP)-conjugation of 000 dilution (in analysis buffer), WestGrove, PA, USA) process.After 0.5 hour cultivates, by Membrane cleaning, be placed in print on toilet paper dry, then it chemoluminescence flooded by matter (ThermoFisherScientific, Rockford, IL, USA) and be placed on this film.By film in the upper exposure of X-exographX (Kodak, Rochester, NY, USA) 0.5,1.5 and 10 minutes, to detect bonding state.

Peptide class is synthesized

By (triple city of Kelowna international scientific company, TaiWan, China) synthesis 57 kinds altogether, cross over the total length VP1 capsid protein of the isolated strain of enteric virus71 type (called after TW/2086/98) of tool genotype C2,15-person's peptide class (table 1) of mutual overlapping 10 Amino acids, in order to analyze the fine and closely woven specificity feature can resisting N1, N3, N4 and N6 mono-source antibody of the VP1 capsid protein of EV71/E59.By the high-effect liquid tomography (HPLC) of use, the peptide class out of the ordinary that will be used for this research has been purified to > 90% purity.Table 1 lists 15-person's peptide class of the fine and closely woven specificity feature for analyzing N1, N3, N4 and N6 mono-source antibody.

Table 1

Peptide class system out of the ordinary, based on the protein sequence of the isolated strain of genotype C2 enteric virus71 type (TW/2086/98) deriving from CDC (TaiWan, China), is synthesized by Kelowna international scientific company (triple city, TaiWan, China).Each peptide class in use before high-effect liquid tomography (HPLC) be purified to > 90% purity.By the peptide VC43 of each single source antibody identification, mark with boldface letter.

Epitope location ELISA

Use two kinds of different types to relate to MaxiSorp and the CovaLinkNHELISA square position used purchased from NUNC (Napperville, IL, USA), carry out the peptide class of set tested person.The tested person peptide class will rebuild in 5.0%DMSO (Sigma/Aldrich, StLouis, MO, USA) is diluted to 10.0 μ g/mL with carbonate-bicarbonate coating buffer (Sigma/Aldrich, StLouis, MO, USA).The mixture that five kinds of adjacent peptide classes are collected with 2.0mg/mL in each square position applies, or tested person peptide class is coated on severally in each wellhole of MaxiSorp square position.Described in the people such as Sondergard-Andersen (1990) (" being used for ferment connection immuning adsorpting analysis through the peptide class of covalent attachment " J.Immunol.Methods131,99-104), complete the coating program of peptide class on square position.In hold over night (for MaxiSorp square position) or after left at room temperature minute (for CovaLink square position), square position is cleaned, and be formulated in PBS in 3.0%, pH7.2 (Gibco/Invitrogen, Carlsbad, CA, USA) skimmed milk in blockade.Then the out of the ordinary purified single source antibody various different concns being formulated in analysis buffer (1.0% skimmed milk is dissolved in PBS, pH7.2) adds in each test wellhole.After 1 hour, by 100.0 μ L with 1: 10,000 dilution, the donkey anti-mouse antiserum(antisera) (JacksonImmuno-ResearchLab., WestGrove, PA, USA) being formulated in the HRP-conjugation of analysis buffer adds to detect.Clean each wellhole, and the TMB peroxidase of 50 μ L is added in each analysis well wellhole by matter (SureBlueTM, KPL, Gaithersburg, MD, USA).After 20 minutes, add each well wellhole to stop further colour generation by by the 2.0NH2SO4 (Sigma/Aldrich, StLouis, MO, USA) of 50.0 μ L.Read, in machine (SpectraMaxM2model, Sunnydale, CA, USA), under 450nm, to record light absorption value in ELISA meter.

Immunity-bacteriolyze spot forms inhibition analysis

By 2.5 × 105 settling flux in 1.0mLCM rhabdosarcoma (RD) cell ( rockville, MD, USA) be incubated in each wellhole of 24-wellhole tissue culture plates (Corning life science, Corning, NY, USA).Cell is placed in and spends the night formation individual layer with 37 of 5.0%CO2 balance DEG C of incubators.Take out culture supernatants, and 200.0 μ L are contained 100PFUs with the out of the ordinary single source antibody all belonging to IgG2a class of various different concns, or cultivate each Test Virus of (spending the night) in advance containing the mixture of four kinds of single source antibody, CM add to out of the ordinary containing in the cultivation wellhole of RD cell.Also analyze without adding single source antibody, or have the culture adding IgG2a mono-source antibody (clone #20102, R & DSystems, Minneapolis, MN, USA), respectively as positive and negative control group.Cultivate after 1 hour at 37 DEG C, the 1.1% methyl alcohol Mierocrystalline cellulose (Merck, WestPoint, PA, USA) of 0.5mL is elaborated on cell.To continue to cultivate two days in 37 of 5.0%CO2 balance DEG C of incubators.Take out culture supernatants, and 3.7% formalin (Merck, WestPoint, PA, USA) solution 0.5mL being formulated in PBS (pH7.2) adds to cell.Next day, formalin is removed, then by the anti-mouse EV71-specificity list source antibody MAB979 (Chemicon of 1 to 5000 of 100.0 μ L dilution (being formulated in analysis buffer), Temecula, CA, USA) add in test wellhole out of the ordinary.After 1 hour, square position is cleaned and the anti-mouse IgG-HRP conjugated antibodies (Serotec, Kidlington, Oxford, UK) of 1 to 50,000 of 100.0 μ L dilution (being formulated in analysis buffer) is added to detect.Cleaning square position, and the TMB peroxidase of 100.0 μ L is added in each analysis wellhole by matter (KPL, Gaithersburg, MD, USA).Order to react in dark colour generation 30 minutes.Under opticmicroscope, counting presents the bacteriolyze spot of black splotch.Virus power valency system with two groups every milliliter test gained PFUs mean value in the most high dilution of EV71/E59 preparation and represent (people (2011) such as the Chang manufacture of muroid list source antibody of the isolated strain of cross neutralization mankind enterovirus genotype B " can " JournalofVirologicalMethods173:189-195, it is incorporated herein by reference).

Result

The qualification of IgG antibody hypotype (isotyping) and the purifying of EV71-specificity list source antibody

Utilize original fusion knurl technology, be separated to four kinds of EV71-specificity neutralizing antibodies, respectively called after N1, N3, N4 and N6.Use single source antibody subtype qualification cover group (ThermoScientific, USA), determine the antibody subtype (isotypes) of N1, N3, N4 and N6.The antibody subtype that Fig. 1 shows each test antibody is all IgG2a.

Use by by the purified mouse ascites fluid (be called N1, N3, N4 and N6) of 20.0 μ L through thermal treatment (lower 5 minutes of 95oC), with 5.0 μ L 5 × concentrated Loading Dye (Bionovas, TaiWan, China) mix and load on 10%SDS-polyacrylamide (AmershamBiosciences) gel, the SDS-PAGE carried out analyzes purity and the identity of immunoglobulin (Ig) (Ig).Then by gel under 100V, in carrying out electrophoresis 90 minutes in 1 × Tris-glycine SDS-electrophoretic buffer.By gel with 1x Kao Masi indigo plant (CB) solution-dyed purchased from Biorad, go dye with being placed in 7.0% acetic acid.By SDS-polyacrylamide gel (SDS-PAG) electrophoresis showed, know that protein band (being equivalent to heavy chain and the light chain of immunoglobulin G (IgG)) judges from what be positioned at 54KDa and 25KDa, obtain highly purified ascites fluid (Fig. 2).

The west of the ascites fluid of purifying turns stain analysis

As shown in Figure 3, single source out of the ordinary antibody meeting identification, from through sucrose gradient purified enterovirus EV71/E59 sample, is positioned at the virus protein of about 36kDa after carrying out electrophoresis parsing.According to reports, the restructuring finding expression in baculovirus has this molecular weight (people such as Chung, 2006).What is interesting is, EV71/E59-specificity list source out of the ordinary antibody also can with one comparatively VP1 slightly large (about 38kDa), also there is the protein part (Pr) separated through electrophoresis in EV71/E59 preparation and combine.The characteristic of this albumen still needs further research.Fig. 3 is shown in west and turns in stain analysis, resists the reactivity of purifying HI-EV71/E59 preparation through single source antibody (N1, N3, N4 and N6) that protein G is column purified.This analyzes system with 2.0mg/mL single source out of the ordinary antibody, enters passerby to the electrophoretic analysis result of 1.2 μ g/mL purifying EV71/E59 virus formulation.

CDR sequential analysis

Fig. 4 lists the Amino acid sequence of light chain (VL) variable region of N1, N3, N4 and N6 antibody.Term " FW " represents skeleton; Anomaly Amino acid system in region shows to add frame table.The sequence data display of gained, the fusion knurl of secretion N1 and N4 antibody has identical CDR1 and CDR2, but the CDR3 of they is different; And the fusion knurl producing N3 and N6 antibody shows different groups, there is identical CDR1 and CDR2, but the κ chain of different CDR3 combination (Fig. 4).These observationss support that N1, N3, N4 and N6 antibody system merges knurl by difference and clones manufacture.Light (L) chain of κ that the fusion knurl that Fig. 1 is shown in manufacture N1, N3, N4 and N6 antibody shows and the CDRs sequence weighed in (H) chain.

For merging the heavy chain showed in knurl, there is no and find that they contain different CDRs.The common Amino acid sequence of heavy (H) chain of N1, N3, N4 and N6 antibody is listed in Fig. 5.This represents, antibody N1, N3, N4 and N6 have identical VH Amino acid sequence.

Epitope Position Research

From the result display that two kinds of ELISA patterns obtain, collect mixture 9 containing can by the peptide class (Fig. 6 A-B) of various single source antibody identification.Distinctly be coated on MaxiSorp and CovaLinkNHELISA square position by peptide class contained in mixture 9 will be collected, the fine and closely woven anti-source carried out determines that base Analysis and Identification goes out the peptide class of called after VC43, comprise Amino acid sequence FGEHKQEKDLEYGAC (SEQIDNO:55), it is can by the target thing (Fig. 6 C-D) of various single source antibody identification.Fig. 6 A-D shows the result of epitope Position Research.Maxisorp square position and CovaLink square position is used to apply 15-person VP1 peptide class.10.0 μ g/mL single source out of the ordinary antibody are used in each test.Shownschematically result representative often pair respectively be coated on Maxisorp square position (A), CovaLink square position (B) collect peptide class, and the average absorbance value of two repeating groups that the peptide class (C and D) out of the ordinary being coated on these square positions is carried out.

The viral Neutralization effect of single source antibody

The result display of institute's acquisition such as thus, single source out of the ordinary antibody effectively resists the isolated strain of the EV71/E59 being used for causing the effect of exempting from, and has the N0781-TW-01 of genotype B4 and have the Neutralization effect of N2838-TW-03 (having found often to appear in TaiWan, China eruption and prevalence in recent years) of genotype B5.Under 3log10 μ g/mL, N3 and N6 mono-source antibody completely (100.0%) block the isolated strain of each virus tested to the infectivity (Fig. 7 B and 7D) of RD cell.At this concentration, N1 and N4 single source antibody also can completely in and EV71/E59 and N0781-TW-01 viral, but the N2838-TW-03 infectivity be neutralized reaches 81.0% and 88.0% (Fig. 7 A and 7C) respectively.Notice, single source antibody in and the isolated strain of EV71/E59 in can cooperation execution effect.Under significantly lower antibody collects mixture concentration of specimens (2.04log10 μ g/mL), can reach and suppress EV71/E59 viral infection (Fig. 7 E) completely.Fig. 4 shows the viral Neutralization effect measurement result of EV71/E59-specificity list source antibody that is out of the ordinary and that collect.The EV71 that the institute out of the ordinary of result display 100PFUs tests purifying is viral: EV71/E59 (▲), N0781-TW-01 (■) or N2838-TW-03 (●) are to out of the ordinary single source antibody (A-D) of various different concns, and the % neutralizing effect of single source antibody sample (E) gained collected.Also muroid IgG2a class list source antibody (clone #20102, R & DSystems, Minneapolis, MN, USA) is analyzed as control group (◆).

Conclusion

The object of this example is single source antibody that initiation generation can present the strong Neutralization effect of the isolated strain of EV71 (often appearing in TaiWan, China eruption and prevalence in recent years) of antagonism genotype B, as the target compound of research and development therapeutical agent.Use EV71/E59 alive to carry out immunity, be separated four strains fusion knurls can manufacture the single source antibody belonging to IgG2a class, energy identification is positioned at the different antigenic portion of common anti-source decision base (VC43) of the Amino acid 211-225 of VP1.These single source antibody present the Neutralization effect of the anti-genotype B virus of appropriate difference.Under the higher concentration of 3log10 μ g/mL, N3 and N6 mono-source antibody can suppress completely, 100PFUs for cause the effect of exempting from EV71/E59 virus, there is the N0781-TW-01 of homologous genes type and there is the infection of N2838-TW-03 of genotype B5.Although N1 and N4 single source antibody is at this concentration, also can completely in and genotype B virus, they in the isolated strain of N2838-TW-03 in, comparatively N3 and N6 mono-source antibody efficacy is slightly poor.About these single source antibody in and EV71/E59 virus time have and be added the discovery of effect, illustrates that they can different structure position in identification VC43 anti-source decision base.

The aforementioned illustrative example having disclosed the present invention, its just as an example with the object of explanation, but not for limiting the present invention to disclosed particular form.Many modifications and variations may be produced via above-mentioned teaching.

The specific embodiment of selected and description, the principle in order to explain the present invention and practical application, enable to have in technical field that the present invention belongs to and usually know that the knowledgeable utilizes the present invention and various enforcement example, various modification is carried out to meet desired special purposes to it.Enforcement example can be replaced it and usually know that the knowledgeable will be aobvious and easy to know to having in technical field that the present invention belongs to, spirit and the scope of the present invention can't be departed from.Therefore, scope system of the present invention defined by rear attached claim, is not limited to previous explanation and illustrative described herein enforcement example.

Quote in the present invention illustrates and discuss some reference, comprising Patent Case, patent application case and various public data.The explanation that the quoting and/or discuss of this type of reference is only and illustrates the present invention and providing, not approves that this type of reference any is all the present invention's it " prior art ".The all reference quoted and discuss in this case specification sheets are all intactly cited in full with it.

The above; be only the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; change can be expected easily or replace, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should described be as the criterion with the protection domain of claim.

Claims (14)

1. the single source antibody be combined with enterovirus (EV) 71 type, or its Fv fragment or Fab fragment, it comprises:

A) aminoacid sequence is heavy chain complementarity determining region 1 (CDR1) polypeptide of SEQIDNO:70, is heavy chain complementarity determining region 2 (CDR2) polypeptide of SEQIDNO:71 with aminoacid sequence; And

B) aminoacid sequence is the light chain CDR1 polypeptide of SEQIDNO:72 or 73, and aminoacid sequence is the light chain CDR2 polypeptide of SEQIDNO:74 or 75, is the light chain CDR3 polypeptide of SEQIDNO:76 or 77 with aminoacid sequence.

2. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein this single source antibody is the complete mankind single source antibody.

3. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein this single source antibody is a humanization list source antibody.

4. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein in this single source antibody or its Fv fragment or Fab fragment system and enteric virus71 type.

5. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein this single source antibody or its Fv fragment or Fab fragment comprise the heavy chain polypeptide that aminoacid sequence is SEQIDNO:82.

6. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein this single source antibody or its Fv fragment or Fab fragment comprise aminoacid sequence be selected from SEQIDNO:78,79, the light chain polypeptide of 80 and 81.

7. single source antibody according to claim 1 or its Fv fragment or Fab fragment, it is for detecting.

8. single source antibody according to claim 1 or its Fv fragment or Fab fragment, wherein this single source antibody or its Fv fragment or Fab fragment are combined with enterovirus capsid protein VP1.

9. an isolated nucleic acid, it comprises the nucleotide sequence that coding amino acid sequence is the heavy chain polypeptide of SEQIDNO:82.

10. an isolated nucleic acid, its comprise an encoding amino acid sequence be selected from SEQIDNO:78,79, the nucleotide sequence of the light chain polypeptide of 80 and 81.

11. 1 kinds of host cells, it comprises the isolated nucleic acid according to claim 9 or 10.

12. 1 kinds of compositions infected for the enteric virus71 type neutralizing or protect antagonism to have genotype B, it comprises one or more single source antibody according to claim 1 or its Fv fragment or Fab fragment; And a kind of pharmaceutically acceptable carrier.

13. 1 kinds of compositions, it comprises single source antibody according to claim 1 or its Fv fragment or Fab fragment; And a kind of pharmaceutically acceptable carrier.

14. single source antibody according to claim 1 or its Fv fragment or Fab fragment, it comprises:

A) amino acid sequence is the heavy chain polypeptide of SEQIDNO:82; And

B) amino acid sequence be selected from SEQIDNO:78,79, the light chain polypeptide of 80 and 81.