CN102702359A

CN102702359A - Identification of novel igE epitopes

Info

Publication number: CN102702359A
Application number: CN2011103434066A
Authority: CN
Inventors: 桑贾亚·辛格; 黄丹阳; 锡·钟·迈克尔·冯
Original assignee: Tanox Inc
Current assignee: Tanox Inc
Priority date: 2004-02-02
Filing date: 2004-07-29
Publication date: 2012-10-03
Also published as: KR20130088198A; CA2552999A1; EP1718669A1; KR101581659B1; SG149892A1; KR20120056297A; KR101562114B1; KR101365375B1; AU2009202408A1; JP2008507474A; EP1718669A4; KR20070008578A; KR20140021058A; WO2005075504A1; SG183683A1; AU2004315197A1; AU2004315197B2

Abstract

The present invention relates to novel peptide epitopes derived from the CH3 domain of IgE which are recognized by high affinity antibodies that specifically bind IgE. These novel peptides may be used for both active immunization of a subject by administering these peptides to generate high affinity antibodies in a subject, as well as for generating high affinity anti-IgE antibodies in non-human hosts that specifically bind to these regions of IgE for passive immunization of a subject.

Description

The discriminating of new IgE epi-position

The application for the applying date be that July 29, application number in 2004 are 200480041292.8, denomination of invention divides an application for the application for a patent for invention of " discriminating of new IgE epi-position ".

The crosscorrelation application

It is the PCT application PCT/US04/02892 on February 2nd, 2004 and the right of priority of PCT/US04/02894 that the application requires the applying date, and it is for referencial use that this paper is incorporated in these PCT applications into.

Background of invention

Transformation reactions (allergy) is by to extrinsic factor such as the excessive immunne response institute inductive allergic state of allergen (allergen).Be characterised in that with allergen contact after that allergic immediate hypersensitivity (I type) takes place immediately is cell-mediated by B, and based on antigen-antibody reaction.Delayed type hypersensitivity is cell-mediated by T, and based on cellular immunity mechanism.In recent years, term " transformation reactions " more and more becomes the synonym of I type allergy.

Immediate hypersensitivity is based on a kind of the replying that the B cell produces E type Tegeline (IgE antibody), and said B cellular exposure is divided into the plasmocyte of secretory antibody in allergen.The reaction of IgE inductive is the position that gets into body at allergen, promptly at mucomembranous surface and/or in regional nodes, and the local event of generation.The local IgE that produces at first makes local mastocyte responsive; Be that IgE antibody combines the lip-deep Fc epsilon receptor of its constant region and mastocyte, the IgE that " overflows (spill-over) " then get into circulation and with the circulation of whole machine body in the mastocyte of basophilic leukocyte and fixation of tissue on receptors bind.As bonded IgE during subsequently with allergen contact, the Fc epsilon receptor is crosslinked through allergenic combination, causes the cell threshing and disengages many transformation reactions media, like histamine, prostaglandin(PG), leukotrienes or the like.The release of these materials causes the typical clinical symptom of immediate hypersensitivity, said clinical symptom be respiratory tract or enteron aisle smooth muscle contraction, little vasodilation and to the permeability of water and plasma proteins increase, mucus secretion (causing for example allergic rhinitis, atopic eczema and asthma) and the stimulation of cutaneous nerve tip caused itch and pain.In addition, strengthen based on the reaction that contacts with the allergen secondary because first with allergen contact after, through on cell surface, expressing IgE, some B cells form the positive B cell of surperficial IgE (sIgE ⁺The B cell) " data base (memory pool) ".

IgE has two kinds of principal recipient: high-affinity receptor Fc ε RI and low-affinity receptor Fc ε RII.Fc ε RI is mainly at mastocyte and basophilic leukocyte surface expression, but on people's Langerhans cell, dendritic cell and monocyte, also finds low-level Fc ε RI, presents effect at the allergen of this its performance IgE-mediation.In addition, be reported on people's eosinophilic granulocyte and the thrombocyte find Fc ε RI (Hasegawa, S.et al., Hematopoiesis, 1999,93:2543-2551).On B cell, T cell or neutrophilic granulocyte, do not find Fc ε RI.The expression of Fc ε RI on Langerhans cell and skin dendritic cell for the allergy individuality in the antigen presentation of IgE bonded on function and biologically all be important (Klubal R.et al.; J.Invest.Dermatol.1997,108 (3): 336-42).

Low-affinity receptor Fc ε RII (CD23) is a kind of agglutinin molecule; Comprise three identical subunits; It has the long alpha-helix that stretches out from cytoplasmic membrane header structure (head the structure) (Dierks that stem structure (stalk) extends that curls; A.E.etal., J.Immunol.1993,150:2372-2382).With IgE bonded basis on, FceRII combines with CD21 on the B cell, participates in the IgE synthetic and regulates (Sanon, A.et al.; J.Allergy Clin.Immunol.1990,86:333-344, Bonnefoy; J.et al., Eur.Resp.J.1996,9:63s-66s).Fc ε RII be considered to for a long time be used for allergen present (Sutton and Gould, 1993, Nature, 366:421-428).With Fc ε RII bonded IgE on the epithelial cell cause the quick allergen of specificity present (Yang, P.P., J.Clin.Invest., 2000,106:879-886).Fc ε RII is present on some cell types, comprises B cell, eosinophilic granulocyte, thrombocyte, natural killer cell, T cell, follicular dendritic cell and Langerhans cell.

Differentiated with Fc ε RI and the interactional IgE molecule of Fc ε RII on structural solid.Mutagenesis research shows CH3 structural domain mediation IgE and Fc ε RI (Presta et al., J.Biol.Chem.1994,269:26368-26373; Henry A.J.et al., Biochemistry, 1997,36:15568-15578) with Fc ε RII (Sutton and Gould, Nature, 1993,366:421-428; Shi, J.et al., Biochemistry, 1997, interaction 36:2112-2122).The binding site of high-affinity and low-affinity receptor is all along the symmetry location via the centre rotational axis of two CH3 structural domains.Fc ε RI binding site is arranged in the CH3 structural domain near the outside of CH2 structural domain junction, and Fc ε RII binding site is positioned at the C-terminal of CH3.

Treat an allergic notion likely and comprise using monoclonal antibody, said antibody is that the IgE isotype is specific, and therefore can combine IgE.This scheme is based on regulating the IgE immunne response and transformation reactions is suppressed through decrement, said immunne response is to induce the incident that transformation reactions occurs the earliest and make the transformation reactions state be maintained.Therefore because other antibody classes other reply unaffectedly, is achieved to the instant and long term of allergy symptoms.The early stage research of people's basophilic leukocyte density is illustrated relation between the Fc ε RI acceptor number of IgE level and each basophilic leukocyte in patient's blood plasma (Malveaux et al., J.Clin.Invest., 1978,62:176).They notice that the Fc ε RI density of transformation reactions and non-transformation reactions philtrum is each basophilic leukocyte 10 ⁴-10 ⁶Individual acceptor.Illustrated afterwards with anti-IgE treatment allergic disease make the quantity of IgE in the circulation be reduced to level before the treatment 1% (MacGlashan et al., J.Immunol., 1997,158:1438-1445).MacGlashan analyzes the patient's of terrible personal complete anti-IgE antibodies (it is combined in the free IgE of round-robin among the patients serum) treatment serum.They have reported that the acceptor number that the circulation IgE level that reduces the patient causes existing on the basophilic leukocyte surface reduces.Therefore, their density of imagining basophilic leukocyte and the lip-deep Fc ε of mastocyte RI receives the direct or indirect adjusting of circulation IgE antibody horizontal.

Recently, WO 99/62550 discloses IgE molecule and segmental application, and the IgE binding site that these IgE molecules and fragment combine Fc ε RI and Fc ε RII is to block combining of IgE and acceptor.Yet, also very limited to the effective treat-ment that does not have harmful side effect of these allergic diseases.A kind of treat-ment of treatment allergic disease comprises uses humanization anti-IgE antibodies treatment rhinallergosis and asthma (Corne, J.et al., J.Clin.Invest.1997,99:879-887; Racine-Poon, A.et al., Clin.Pharmcol.Ther.1997,62:675-690; Fahy, J.V.et al., Am.J.Resp.Crit.Care Med.1997,155:1824-1834; Boulet, L.P.et al., Am.J.Resp.Crit.Care Med., 1997,155:1835-1840; Milgrom, E.et al., N.Engl.J.Med., 1999,341:1966-1973).These clinical datas show suppress IgE and its acceptor to combine be a kind of effective ways of treating allergic disease.

Be suitable as antiabnormal reaction agent antibody should be divided into the positive B cell response of the plasmacytic surperficial IgE that produces IgE, can be used for functionally eliminating those B cells thus.Yet the antibody of IgE also can be induced release medium from the mastocyte of IgE sensitization through crosslinked Fc epsilon receptor in principle, and antagonism is to SERUM IgE and sIgE thus ⁺The beneficial effect of b cell level performance.A kind of potentially dangerous problem of developing anti-IgE treatment be therapeutic antibodies with high-affinity receptor bonded IgE combine cause that IgE is crosslinked and excite histamine release to cause the anaphylactoid possibility of potential.

Therefore, can be used for treating allergic antibody can not with bonded IgE reaction on the mastocyte of sensitization and the basophilic leukocyte, discern sIgE but should keep ⁺The ability of B cell.This IgE isotype specific antibody is for example described in the United States Patent(USP) No. 5,449,760 at European patent No.EP0407392 and some USPs by (Biotechnology 8,122-126 (1990)) such as Chang.

The peptide that is used to produce anti-IgE antibodies also has the danger of inducing sensitizing antibody.If the IgE of antibodies that between duration of immunity, produces and high-affinity IgE receptors bind, or through other mechanism, the generation of anti-IgE antibodies perhaps can be to excite histamine release with the passive same mode of anti-IgE antibodies that gives during active immunity.

Therefore, need specificity to combine IgE but debond with the non-sensitizing antibody of the higher affinity of its high-affinity receptor bonded IgE, and the peptide that is used for active immunity of not inducing sensitizing antibody.The inventor has differentiated the specificity epitope of IgE, and it is with the high-affinity binding antibody but the IgE on debond mastocyte or the basophilic leukocyte.Next these specificity epitopes can be used for producing specific peptide; To be used for active immunity only to produce the regional bonded IgE antibody with the bind receptor of IgE; Therefore thereby guarantee said antibody with not crosslinked, and be non-sensitization with the IgE of receptors bind.

Summary of the invention

The present invention relates to new peptide epitopes derived from the CH3 structural domain of IgE.These peptide epitopes are combined the high-affinity antibody identification of IgE by specificity.Can the peptide that these are new be used for mammiferous active immunity avidity to produce high-affinity antibody in vivo through giving these new peptides of Mammals.Said peptide epitopes also is used in and produces the high-affinity anti-IgE antibodies that specificity combines these IgE zones in the inhuman host, and uses gained antibody passive immunization Mammals.

A kind of immunogen of the present invention (epi-position A Figure 11) comprises following aminoacid sequence:

Asn?Pro?Arg?Gly?Val?Ser?Xaa?Tyr?Xaa?XaaArg?Xaa(SEQ?ID?NO.72)

The instance of epi-position A is:

Asn?Pro?Arg?Gly?Val?Ser?Ala?Tyr?Leu?Ser?Arg?Pro(SEQ?ID?NO.73)

Another kind of immunogen (epi-position B Figure 11) comprises following aminoacid sequence:

Leu?Pro?Arg?Ala?Leu?Xaa?Arg?Ser?Xaa(SEQ?ID?NO.74)。

The example of epi-position B comprises:

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr(SEQ?ID?NO.75)

His?Pro?His?Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr(SEQ?ID?NO?76)

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr?Thr?Lys?Thr(SEQ?ID?NO?77)。

In SEQ ID NO:72 or SEQ ID NO:74, Xaa can be any amino acid.

These peptides can be included in the compsn, and said compsn comprises at least a said peptide and a kind of physiology acceptable carrier, thinner, stablizer or vehicle, and a kind of immunogenic carrier.Said immunogenic carrier can be for example BSA, KLH, Toxoid,tetanus and DT.The invention still further relates to coding SEQ ID NO.72-77 polynucleotide, comprise the carrier of said polynucleotide and contain the cell of said carrier.

The invention still further relates to the antibody of specificity combination epi-position A and/or epi-position B.The invention still further relates to a kind of method that specificity combines the antibody of epi-position A and/or epi-position B that produces.

The object that the present invention relates to suffer from disease or the pathology of IgE mediation comprises the peptide of SEQ ID NO:72 and/or SEQ ID NO:74.

The present invention relates to suffer from the high-affinity antibody that the Mammals of disease or the pathology of IgE mediation produces with the peptide that comprises SEQ ID NO:72 and/or SEQ ID NO.74.Said high-affinity antibody can be people's antibody, humanized antibody or chimeric antibody.Said antibody can be polyclonal antibody or monoclonal antibody.The disease or the pathology of this IgE mediation comprise for example asthma, atopic dermatitis, rubella, rhinallergosis and eczema.

The accompanying drawing summary

Fig. 1 phage vector is an antibody cloning with screening in the synoptic diagram of used phage vector.

Fig. 2 is the synoptic diagram that is used to produce the oligonucleotide of antibody variants.

Fig. 3 A TES-C21 and template comparison illustrate light chain and people's template L16 of combination and the contrast of JK4 of mouse-anti IgE antibody TES-C21.

Fig. 3 B TES-C21 and template comparison illustrate heavy chain and people's template DP88 of combination and the contrast of JH4b of TES-C21.

The frame sequence of Fig. 4 high-affinity material standed for illustrates the tabulation of comparing the framework residue variant with high-affinity with parental generation TES-C21.

Fig. 5 ELISA titration curve, A illustrate with the Fab of parental generation TES-C21 with B and compare with negative control group (5D12),

clone

4,49,72,78 and 136 ELISA titration curve.

Fig. 6 suppresses to measure, and illustrates with parental generation TES-C21 and compares with negative control antibody, and the inhibition of

clone

2C, 5A and 5I is measured.

Fig. 7 derives from the high-affinity material standed for tabulation in library, illustrates to have to cause that IgE is had the more clone's of the useful sudden change combination of high-affinity sequence.

Fig. 8 A and 8B illustrate the frame sequence of complete variable region of light chain of the

clone

136,1,2,4,8,13,15,21,30,31,35,43,44,53,81,90 and 113.

Fig. 9 A and 9B illustrate the frame sequence of 35 clones' complete variable region of heavy chain.

Figure 10 A-F illustrates complete heavy chain and the sequence of light chain of

clone

136,2C, 5I, 5A, 2B and 1136-2C.

The CH3 of Figure 11 people IgE zone aminoacid sequence illustrates into the CH3 zone aminoacid sequence of IgE and marks epi-position " A " and epi-position " B ".

The pepscan of Figure 12 epi-position B illustrates the overlapping peptide that is used to differentiate epi-position B.

The scanning of the L-Ala of Figure 13 epi-position A illustrates the discriminating of important residue in the land of epi-position A.

The scanning of the L-Ala of Figure 14 epi-position B illustrates the discriminating of important residue in the land of epi-position B.

1. Figure 15 western engram analysis illustrates the western engram analysis of the MAb that combines mutant peptide.

Anti-IgE in Figure 16 transgenic mice replys, and is illustrated in the generation of anti-IgE antibodies in the transgenic animal of expressing human IgE.

Detailed Description Of The Invention

Definition

The term that uses among the application has the common implication of understanding with the typical case of those skilled in the art.Yet the applicant hopes that following term has the specific explanation of hereinafter.

Can be interpreted as antibody chain and the reference polypeptide sequence presents at least 70% or 80% or 90% or 95% sequence homogeny about the phrase " substantially the same " of antibody chain peptide sequence.The sequence and the reference nucleic acid sequence that can be interpreted as Nucleotide about this term of nucleotide sequence present about at least 85% or 90% or 95% or 97% sequence homogeny.

Term " homogeny " perhaps " homology " should be interpreted as after sequence being arranged contrast and importing breach (reaching the largest percentage homogeny of complete sequence if desired) and do not think that any conservative replacement is the part of sequence homogeny the per-cent of the amino-acid residue identical with the residue of the corresponding sequence that compares with it in the candidate sequence.N or C-terminal extend or insert all not to be thought and reduces homogeny or homology.The well known correlated method and computer program of series arrangement that carries out.The sequence homogeny can use sequence analysis software to measure.

Term " antibody " is used with implication the most widely, and contains monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody and multiple specific antibody (for example bi-specific antibody) especially.Antibody (Ab) and Tegeline (Ig) are the gp with same structure characteristic.Although antibody presents the binding specificity to specific target, Tegeline comprises antibody and lacks other antibody molecule of target-specific.Natural antibody and Tegeline normally about 150,000 daltonian allos tetramer gp are made up of with two identical weights (H) chain two identical light (L) chains.Each heavy chain at one end has a variable region (V _H), be some constant regions subsequently.Each light chain at one end has a variable region (V _L), have a constant region at the other end." high-affinity " antibody is meant to have at least 10 ^-10, preferred 10 ^-12Those antibody of binding affinity.

As used herein, " anti human IgE antibody " is meant to suppress perhaps to reduce greatly IgE combines people IgE with high-affinity receptor Fc ε RI bonded mode antibody.

Some part that is meant the variable region about the term in the variable region of antibody " variable " is extensively different in the sequence of antibody, and is each antibodies specific and the combining and specificity institute foundation of its particular target.Yet mutability is not equally distributed in the variable region of antibody.It concentrates in three sections that are called complementary determining region (CDR), and CDR is also referred to as hypervariable region, and they all exist in light chain and variable region of heavy chain.The conservative part of the height of variable region is called framework (FR).Each self-contained four FR district, the variable region of natural heavy chain and light chain mainly are to take the beta sheet configuration, are linked to each other by three CDR, form ring and link to each other with the beta sheet structure, and these rings constitute the part of beta sheet structure in some cases.CDR in every chain all closely links together through the FR district, and forms the target binding site (seeing that Kabat etc. is said) of antibody with the CDR of other chain.As used herein; Unless otherwise indicated; The numbering of immunoglobulin amino acid residue is carried out (Sequences of Proteins of Immunological Interest according to the immunoglobulin amino acid residue numbering system of Kabat etc.; National Institute of Health, Bethesda, Md.1987).

Term " antibody fragment " is meant the part of full length antibody, and normally target is decided land or variable region.Antibody fragment for example comprises Fab, Fab ', F (ab ') ₂With the Fv fragment." function fragment or the analogue " of phrase antibody is the compound that has with the ejusdem generis BA of full length antibody.For example, the function fragment of anti-IgE antibodies or analogue are to combine the mode of the ability of high-affinity receptor Fc ε RI to combine the IgE Tegeline to stop or to reduce the IgE immunoglobulin molecules greatly.As used herein, be meant Fv, F (ab) and F (ab ') about " function fragment " of antibody ₂Fragment." Fv " fragment is the minimum antibody fragment that contains complete target identification and binding site.This zone is by heavy chain of non-covalent bonded and the dimer (V that variable region of light chain is formed of next-door neighbour _H-V _LDimer) forms.In this configuration, three CDR of each variable region interact and define V _H-V _LThe lip-deep target binding site of dimer.In a word, six CDR make antibody have the target binding specificity.Yet even single variable region (Fv half that perhaps only comprises three CDR that are specific to target) also has identification and combine the ability of target, but avidity is lower than complete binding site." strand Fv " perhaps " sFv " antibody fragment comprises the V of antibody _HAnd V _LStructural domain, wherein these structural domains are present in the single polypeptide chain.Normally, the Fv polypeptide further is included in V _HAnd V _LA peptide linker between the structural domain, it makes sFv be formed for the structure that the target bonded is hoped.

The Fab fragment contains the constant region of light chain and first constant region (CH1) of heavy chain.The segmental difference of Fab ' fragment and Fab is to add several residues, comprise one or more halfcystine from the antibody hinge region at the C-terminal of heavy chain CH1 structural domain.F (ab ') fragment is through cracking F (ab ') ₂The disulfide linkage of the hinge halfcystine of gastric pepsin digestion product produces.Segmental other chemical coupling mode of those skilled in the art's known antibodies.

As used herein, term " monoclonal antibody " is meant the antibody that derives from the basic homologous antibody of a group, promptly comprises except possibly being each identical antibody the sudden change with the natural generation that exists very in a small amount.Monoclonal antibody is the antibody to the high degree of specificity of single target site.In addition, opposite with (polyclone) antibody preparations (its typical case comprises the different antibodies to different determinants (epi-position)) of routine, each monoclonal antibody is all to a single determinant on the target.Except its specificity, the advantage of monoclonal antibody is can cultivate through hybridoma to synthesize, and is not polluted by other Tegeline.Modifier " mono-clonal " expression antibody derives from this characteristic of the antibody population of homogeneity basically, rather than refers to that needs produce this antibody through any special methods.For example, the monoclonal antibody of the present invention's use can leave from the phage antibody library through the technical point that use is known.Parental generation monoclonal antibody used according to the invention can be passed through by Kohler and Milstein, and the hybridoma method that Nature 256,495 (1975) at first describes produces, and perhaps can produce through recombination method.

" humanization " form of inhuman (for example mouse) antibody is to contain chimeric Tegeline, immunoglobulin chain or its fragment derived from the minmal sequence of non-human immunoglobulin (the for example Fv of antibody, Fab, Fab ', F (ab ') ₂Fragment or other combine the subsequence of target).Generally speaking; Humanized antibody comprises whole basically at least one, typical two variable regions; Wherein all or basically all CDR zone all corresponding to those zones of non-human immunoglobulin, and all perhaps basically all FR zones are those zones of human normal immunoglobulin consensus sequence.Humanized antibody also can comprise at least a portion constant region for immunoglobulin (Fc), and the typical case is at least a portion constant region for immunoglobulin of selected human normal immunoglobulin template.

Term " cell ", " clone " and " cell culture " comprise the offspring.Also should understand since have a mind to or unintentionally sudden change cause all offsprings' the DNA content needn't be identical.The present invention includes and have and the identical functions of in initial transformant, screening or the variant offspring of biological property." host cell " that the present invention uses be prokaryotic hosts or eucaryon host normally.

" with DNA transformant organism " is meant DNA imported in the organism, and this DNA can be used as extra-chromosomal element or duplicates through chromosomal integration thus." with DNA transfectional cell organism " is that phalangeal cell or organism absorb for example expression vector of DNA, and no matter whether any encoding sequence is in fact expressed.Term " host cell of transfection " and " transformed host cells " are meant the cell that has wherein imported DNA.Said cell is called " host cell ", and it can be prokaryotic cell prokaryocyte or eukaryotic cell.Typical prokaryotic host cell comprises colibacillary various bacterial strain.Typical eukaryotic host cell is Mammals such as Chinese hamster ovary cell or human archeocyte.The dna sequence dna that imports can be the crossbred dna sequence dna perhaps from the species identical with host cell or from the species different with host cell, contains some external sources and some homologous dnas.

Term " carrier " is meant a kind of DNA construct, and it contains and the dna sequence dna that can make that appropriate control sequence that said DNA expresses operably is connected in suitable host.These control sequences comprise the sequence of the promotor of transcribing, this operon of transcribing of optional control, the suitable mRNA ribosome bind site of encoding and control the sequence of transcribing with translation termination.Said carrier can be plasmid, phage particle, perhaps only is that the potential genome inserts body.In case transform appropriate host, said carrier can duplicate and irrespectively bring into play function with host genome, perhaps is integrated in the genome in some cases.In this manual, " plasmid " and " carrier " can exchange use sometimes, because the plasmid the most frequently used form that is carrier.Yet, the present invention includes other form carrier of bringing into play said function, these forms are known in the art or become to known in the art.

" control sequence " is meant and in specific host organisms, expresses the necessary dna sequence dna of encoding sequence that operably connects.Be suitable for procaryotic control sequence and for example comprise promotor, randomly operon sequence, and ribosome bind site.The known genuine karyocyte utilizes promotor, polyadenylation signal and enhanser.If it is expressed as participating in albumen before the said polypeptide excretory, then presequence (presequence) or the DNA that secretes leader sequence can operably be connected with the DNA of polypeptide; If it influences transcribing of this sequence, then promotor or enhanser are operably to be connected with encoding sequence; If perhaps it influences transcribing of this sequence, then ribosome bind site is operably to be connected with encoding sequence; If perhaps it is positioned in and can promotes the position of translating, then ribosome bind site is operably to be connected with encoding sequence.Normally, " operably connect " is meant that the dna sequence dna of connection is a successive, and in the situation of secretion leader sequence, is successive and is to separate read states (in reading phase).Yet enhanser needs not to be successive.

" Mammals " treated be meant and classify as mammiferous any animal, comprises people, domestic animal and farm-animals, non-human primate, and zoo animal, motion animal, perhaps pet such as dog, horse, cat, ox or the like.

Be meant a peptide species that merges with " epi-position mark " at this paper used term " the epi-position mark " with regard to polypeptide.Said epi-position labeling polypeptide has enough residues can produce the epi-position of antibody to it to provide, yet is the enough short activity of not disturbing polypeptide thus.Said epi-position mark preferably also is fairly individual, thus antibody basically not with other epi-position cross reaction.Suitable labeling polypeptide has at least 6 amino-acid residues usually, has about 8-50 amino-acid residue (a preferably approximately 9-30 residue) usually.Instance comprises flu HA labeling polypeptide and antibody 12CA5 (Field etal, Mol Cell.Biol.8:2159-2165 (1988)) thereof; The c-myc mark reaches 8F9,3C7,6E10, G4, B7 and the 9E10 antibody (Evan et al., Mol Cell.Biol.5 (12): 3610-3616 (1985)) to it; (Paborsky etal., Protein Engineering 3 (6): 547-553 (1990)) to reach herpes simplex virus glycoprotein D (gD) mark and antibody thereof.In certain embodiments, the epi-position (for example IgG1, IgG2, IgG3 or IgG4) in the Fc zone that said epi-position mark can be the IgG molecule, it causes serum half-life in the body that increases the IgG molecule.

Term used herein " mark " is meant a kind of detectable compound or compsn, and it can for example antibody be directly or put together indirectly with molecule or protein.Said mark can be self detectable (for example labelled with radioisotope or fluorescent mark), perhaps can catalysis in the situation of enzyme labelling can be to be detected substrate compounds or the chemically changed of compsn.

As used herein, " solid phase " is meant the nonaqueous phase matrix that antibody of the present invention can adhere to.The solid phase that the present invention is contained for example comprises some or all of those solid phases that formed by glass (for example controlled pore glass), polysaccharide (for example agarose), SEPIGEL 305, PS, Z 150PH and silicone.In certain embodiments, according to the context of article, said solid phase can comprise the hole of assay plate; In other embodiments, said solid phase can be purification column (a for example affinity column).

As used herein, term " IgE mediation illness " is meant the excessive generation that is characterised in that Immunoglobulin IgE and/or the pathology or the disease of supersensitivity.Especially, this term can be interpreted as and comprise the pathology relevant with atopic allergy with the supersensitivity of sensitization, for example comprises asthma, rhinallergosis and conjunctivitis (spring fever), eczema, rubella, atopic dermatitis and food anaphylaxis.The serious physiology pathology of being stung the anaphylactic shock of thorn, venom, food or drug-induced by for example honeybee also is encompassed in the scope of this term.

Production of antibodies

Initial perhaps " parental generation " antibody can use the existing technology preparation that is used to produce these antibody in this area.These technology are known.The illustrative methods that produces initial antibody is more described in detail in following chapters and sections.These descriptions are the other alternative methods that produce or select parental generation antibody, rather than in order to limit the method that can produce this molecule.

The binding affinity of antibody was confirmed before producing high-affinity antibody of the present invention.In addition, antagonist can carry out other BA analysis, for example estimates the effectiveness as therapeutical agent.These analyses are known in the art and depend on the target position and the appointed purposes of said antibody.

In order to screen the antibody (for example blocking IgE and its those antibody of high-affinity receptor bonded) that combines defined epitope; Can carry out conventional intersection blocking-up analyzes; As carry out in the analysis described in the Antibodies:A Laboratory Manual (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)).Perhaps, can carry out epitope mapping to confirm the position of the corresponding epi-position of antibodies.Randomly, antibody can use technology known in the art to confirm for the binding affinity of the analogue (said analogue is from different plant species) of the target that is used to produce this antibody.In one embodiment, other species are the non-human mammals that give said antibody in preclinical study.Therefore, said species can be inhuman primates, like rhesus monkey, macaque, baboon, chimpanzee and macaque.In other embodiments, said species for example can be rodent, cat or dog.

The change according to the present invention of said parental generation antibody has antibody higher or strong binding affinity to produce to compare for target with parental generation antibody.Antibodies specific gets the unique interface that forms between comfortable antibody and its target, said surface coincide (Jones, S.& Thornton, J.M. (1996) the Proc.Natl.Acad.Sci.USA 93:13-20) that produces uniqueness complimentary to one another.Through the contact surface of further improvement along this interface, whole avidity can be owing to required lower the increasing of energy of association that promotes binding partner.

The mating surface of antibody is made up of 6 complementary determining regions (CDR) usually, and they are the annulars of stretching out from core.CDR is made up of the amino acid of the unique sequences with binding specificity target.In order to increase antibody avidity antigenic with it, the environment around these amino acid must become more favourable through importing or improveing various noncovalent forces, and it finally reduces interactional energy and produces higher avidity.

Van der Waals force be the noncovalent interaction that between two electroneutral molecules, takes place (Voet, D.& Voet, J.G. (1990) Biochemistry John Wiley and Sons, NY, NY).Through from electrostatic interaction permanent or that the inductive dipole produces, can associate between two surfaces.These dipoles can or exist near polare Aminosaeren along the terminal of α spiral.Through increasing, can produce more favourable association along the quantity of the Van der Waals force of bonding interface.

Import hydrogen bond and also will increase interactional specificity between antibody and its antigen.The common donor and the acceptor of participating in hydrogen bond are nitrogen, oxygen and sulphur atom, amino acid mainly by they form (see Voet, et al., as previously mentioned).Hydrogen bond only intersects very short distance (being generally 2.7-3.1

) often, so binding partner must be closely approaching so that these interactions to take place.A kind of mode that therefore, can improve avidity is that potential donor is closely contacted to set up hydrogen bond with acceptor molecule.

At last, the improvement hydrophobic interaction also can increase the favourable energetics between two binding partners.The non-polar residue that approaches mating surface should be centered on by other non-polar residue, and therefore in advantageous environment, exists.Through fully burying of non-polar sidechain, interactional energetics is favourable for strong bonding interface.

The interaction of stabilizing protein-protein interface reduces the energy expenditure of keeping these contacts, and has therefore increased whole avidity.Near the environment around each amino acid of bonding interface, can produce the more advantageous environment that causes higher binding affinity through improvement.Therefore, through importing favourable contact and improveing the interface through further complementary, then the whole binding interactions between antibody and the antigen can improve greatly.

The gained high-affinity antibody is compared for the binding affinity of target with parental generation antibody, preferably exceeds about at least 10 times, perhaps exceeds about at least 20 times, perhaps exceeds about at least 500 times, perhaps can be to exceed 1000-5000 doubly.The enhancing degree of binding affinity needs or that hope depends on the initial binding affinity of parental generation antibody.

Usually, the method for generation high-affinity antibody comprises the steps: from parental generation antibody

1. obtain or select to combine the parental generation antibody that comprises heavy chain and variable region of light chain of interested target.This can be through traditional hybridoma technology, display technique of bacteriophage or any other method realization that produces target-specific antibody.

2. select and the approaching frame sequence of parental generation frame sequence preferred people's template sequence.This template can be based on the size of for example its suitable total length, CDR, the amino-acid residue, whole homology etc. of joint selected between framework and CDR.The template of selecting can be the mixture of an above sequence, perhaps can be total template.

3. replace the generation clone library through producing random amino acid in each and each possible CDR position.Also can use all possible amino acid to replace the amino acid in people's architecture template at random, for example contiguous CDR or influence combine perhaps folding amino acid, produce the substituted library of framework.These frameworks replace and can estimate the latent effect that target combines and antibody is folding to it.Amino acid whose replacement can or be carried out with the aminoacid replacement while among the CDR in succession in the framework.A kind of method that produces the variant library is synthetic through oligonucleotide.

4. structure comprises the heavy chain of generation in the step (3) and/or the expression vector of light chain variant, and these variants can comprise following chemical formula:

FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 (I) and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 (II); Wherein FRL1, FRL2, FRL3, FRL4, FRH1, FRH2, FRH3 and FRH4 represent the architecture template light chain of selection in step 3 and the variant of sequence of heavy chain, and CDR represents the variant CDR of parental generation antibody CDR.An instance of carrier that contains these light chains and sequence of heavy chain is as shown in Figure 1.

5. screening is directed against the clone library of specific target, and those clones that screening combines target are with the improvement binding affinity.Can select to compare with higher those clones of avidity bonded with the parental generation molecule.Best high-affinity material standed for is compared with parental generation antibody has maximum binding affinity, preferably exceeds 20,100,1000 or 5000 times.If the variant of selecting contains some undesirable amino acid, like the glycosylation site or the potential immunogenicity site that have imported, then those amino acid can be used more useful radical amino acid replacement, and reappraise binding affinity.

The technician also can use this method from people's parental generation antibody completely, to stay people's framework integral body and produce high-affinity antibody through only replacing the CDR zone at random.

Since the high throughput screening of improvement technology and carrier for example shown in Figure 1, the comprehensive replacement library that the technician can also screen all sites in given CDR and/or framework region rapidly effectively.Through replacing all amino acid at random simultaneously in all positions, the technician can screen possibly making up of remarkable increase avidity, and this type combination can not be through single replacement by prediction or discriminating owing to for example synergy.

The parental generation antibody preparation

The target preparation

Soluble target or its fragment can be as the immunogens that produces antibody.Said antibody is to produce to interested target.Preferably, said target is the important polypeptide of biology, and the Mammals that said antibody suffers from disease or functional disorder can be produced the treatment benefit in this Mammals.Yet, can produce antibody to non-polypeptide target.When said target was polypeptide, it can be a for example growth factor of a kind of transmembrane molecule (for example acceptor) or part.A kind of target of the present invention is IgE.Intact cell can be used as the immunogen that produces antibody.Said target can be that reorganization produces or use compound method to produce.Said target also can separate from natural origin.

The antigen that is used to produce antibody of the present invention can comprise polypeptide of the present invention and fragment thereof, comprises epi-position A and/or B.The polypeptide that is used for immune animal can pass through standard weight group of methods, chemical synthesis process or purification process and obtain.As known in the art, in order to increase immunogenicity, antigen can be puted together with a kind of carrier proteins.Normally used carrier comprises but non-keyhole chirp hemocyanin (KLH), Thyroprotein, bovine serum albumin (BSA) and the Toxoid,tetanus of being limited to.Then the link coupled peptide is used for immune animal (for example mouse, rat or rabbit).Except these carriers, the adjuvant of knowing can be given with antigen and promote inducing of strong immune response.

Polyclonal antibody

The relevant target that polyclonal antibody normally makes up through injection of repeatedly subcutaneous (sc) or intraperitoneal (ip) and adjuvant in non-human mammal produces.The well known multiple medicament that can excite immunological response.

Through with protein or conjugate (respectively for rabbit or mouse) and Freund's complete adjuvant combination, and this solution of intradermal injection, and animal is carried out immunity to target, immunogenic conjugate or verivate.After one month, with animal at the peptide of the 1/5-1/10 original vol of a plurality of positions subcutaneous injection in Freund's incomplete adjuvant or conjugate and booster immunization.After 7-14 days, from this animal drainage blood, serum analysis antibody titer.Animal is continued booster immunization be in steady state until tiring.

The Mammals antibody of selecting usually and target have enough strong binding affinity.For example, said antibody can combine the anti-IgE target of people, and binding affinity (Kd) numerical value is about 1 * 10 ^-8M.Affinity of antibody can be confirmed through saturated combination, enzyme-linked immunosorbent assay (ELISA) and competition assay (for example radioimmunoassay).

In order to screen and interested target bonded antibody; Can carry out conventional crosslinked mensuration, as carrying out Antibodies, A Laboratory Manual; Cold Spring Harbor Laboratory, the described mensuration of Ed Harlow and David Lane (1988).Perhaps, can carry out epitope mapping and confirm to combine, for example Champe et al.J.Biol.Chem.270:1388-1394 (1995) is said.

Monoclonal antibody

Monoclonal antibody is the antibody of the single antigenic site of identification.Its consistent specificity makes monoclonal antibody more useful than polyclonal antibody, and polyclonal antibody contains the antibody of discerning multiple different antigenic sites usually.Monoclonal antibody can be used at first by Kohler et al., Nature, and the hybridoma method that 256:495 (1975) describes produces, and perhaps can produce through recombinant DNA method.

In hybridoma method, mouse or other appropriate host animal such as rodent such as above-mentioned carried out immunity, produce or can produce the lymphocyte that specificity combines to be used for the proteinic antibody of immunity to excite.Perhaps, lymphocyte can carry out immunity external.Use suitable fusogen such as polyoxyethylene glycol to merge in lymphocyte and myeloma cell then; Form hybridoma (Goding; Monoclonal Antibodies:Principals and Practice, pp.590-103 (Academic Press, 1986)).

The hybridoma of preparation thus is seeded in the suitable medium and growth therein, preferably contains parental generation myeloma cell's growth that inhibition do not merge or one or more material of survival in the said substratum.For example, if the parental generation myeloma cell lacks HGPRT (HGPRT or HPRT), then the substratum typical case of hybridoma comprises xanthoglobulin, aminopterin-induced syndrome and the thymidine (HAT substratum) of the cell growth that stops the HGPRT defective.The cytotostatic of the generation antibody that preferred myeloma cell is effective fusion, support to select produces antibody and to substratum such as those responsive myeloma cells of HAT substratum high-levelly.Human myeloma and mouse-people's allos myeloma cell line is directed against the generation of human monoclonal antibodies and is described (Kozbar, J.Immunol.133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

After the hybridoma of having differentiated the specificity, avidity and/or the active antibody that produce hope; Can the clone be carried out subclone also through standard method growth (Goding through restriction dilution program; Monoclonal Antibodies:Principals and Practice; Pp.59-103, Academic Press, 1986)).For this purpose, suitable medium comprises.Through routine immunization sphaeroprotein purification process appropriate separation from substratum, said method is albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography for example by subclone excretory monoclonal antibody.

The DNA of the monoclonal antibody of encoding is easy to use ordinary method to separate and order-checking (oligonucleotide probe of the heavy chain of for example use ability specificity combination coding monoclonal antibody and the gene of light chain).Said hybridoma is as the source of this DNA.After the separation, can DNA be placed expression vector, be transferred to host cell then for example among Bacillus coli cells, NS0 cell, Chinese hamster ovary cell (CHO) or the myeloma cell, with synthetic monoclonal antibody in the host cell of reorganization.The encoding sequence that said DNA also can for example pass through personnel selection heavy chain and constant region of light chain replaces homology mouse sequence (United States Patent(USP) No. 4,816,567; Morrison et al., Proc.Natl Acad.Sci.USA 81:6851 (1984)) or through being modified with the immunoglobulin polypeptides covalent attachment.

Humanized antibody

Humanization is a kind of technology that produces chimeric antibody, and wherein abundant part less than complete people variable region is replaced by the corresponding sequence of inhuman species.Humanized antibody has importing one or more amino-acid residue from inhuman species wherein.These inhuman amino-acid residues so-called " introducing " residue, its typical case takes from " introducing " variable region.Humanization can pass through Winter and colleague (Jones et al, Nature 321:522-525 (1986) basically; Riechman et al., Nature332:323-327 (1988); Verhoeyens et al., Science 239:1534-1536 (1988)) method is carried out (seeing for example United States Patent(USP) No. 4,816,567) through replace in people's antibody corresponding sequence with inhuman CDR or CDR sequence.Such as the present invention practice, humanized antibody can have by the substituted number of C DR residue of the residue in similar site in the murine antibody and some FR residues.

The selection of people variable region (light chain and heavy chain) that is used to produce humanized antibody is extremely important for reducing antigenicity.According to so-called " best coincideing " method, non-human antibody's the variable region sequences and the library of known people's variable region sequences are compared.Be acknowledged as people's framework (Sims etal., the J.Immunol.151:2296 (1993) of humanized antibody then near the human sequence of inhuman parental generation antibody; Chothia etal., J.Mol.Biol.196:901 (1987)).Another kind method is used the specific framework derived from the consensus sequence of everyone antibody of a special inferior group light chain or heavy chain.Identical framework can be used for some different humanized antibodies (Carter et al., Proc.Natl.Acad.Sci.USA, 89:4285 (1992); Presta et al., J.Immunol.151:2623 (1993)).

Antibody fragment

The various technology that produce antibody fragment have been developed.Traditionally; These fragments are (to see for example Morimoto et al. through what the complete antibody of proteolysis produced; Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan etal., Science 229:81 (1985)).Yet these fragments can directly produce through recombinant host cell now.For example, antibody fragment can separate from the antibody phage library.Perhaps, F (ab ') 2-SH fragment can directly reclaim from intestinal bacteria and by chemical coupling to form F (ab ') 2 fragments (Carter etal., Bio/Technology 10:163-167 (1992)).According to another kind of method, F (ab ') ₂Fragment can direct separation from recombinating the host cell culture.Other method of the known generation antibody fragment of those skilled in the art.In other embodiments, the antibody of selection is strand Fv fragment (scFv) (PCT patented claim WO 93/16185).

The preparation of high-affinity antibody

In case discriminated union has separated parental generation antibody, one or more amino-acid residue in one or more variable region of parental generation antibody can be changed.Perhaps in addition, in parental generation antibody, can replace one or more framework residue, this antibody for example is able to improvement for the binding affinity of people IgE like this.Framework region residue to be finished for example comprises and the non-covalent direct bonded residue of target (Amit et al.Science 233:747-753 (1986)); Interact/influence the conformation of CDR those (Chothia et al.J.Mol.Biol.196:901-917 (1987)) with the conformation of CDR; And/or the residue (EP 239400B1) at participation VL-VH interface.In certain embodiments, the modification of one or more this framework region residue causes the binding affinity of antibody and interested target to strengthen.

The modification of the biological property of antibody can realize the remarkable different replacement of the effect of keeping following aspect through selecting it, for example keep the structure that (a) replaces polypeptide main chain in the zone, for example folding or helical conformation; (b) molecule is in the electric charge or the hydrophobicity of target site, perhaps the volume of (c) side chain.Non-conservative replacement need be replaced a member of one of these classifications with another kind of other member.

The nucleic acid molecule of encoding amino acid sequence variant prepares through several different methods known in the art.These methods comprise but the non-cassette mutagenesis that is limited to the species dependency antibody of oligonucleotide mediated (perhaps directed) mutagenesis, PCR mutagenesis, the variant that reaches previous preparation or non-variant form.The preferred method that produces variant is oligonucleotide mediated synthetic.In certain embodiments, for example in approximately 2-15 kind hypervariable region replaced, antibody variants only had a single hypervariable region residue and is substituted.

A kind of method that produces the variant library is oligonucleotide mediated compound method, and is as shown in Figure 2.Each of three kinds of oligonucleotide of about 100 Nucleotide all can synthesize crosses over whole light chains or variable region of heavy chain.Every kind of oligonucleotide can comprise: (1) is by triplet (NNK) ₂₀One section 60 the amino acid whose sequence that produces, wherein N is any Nucleotide, K is G or T; (2) each terminal and ensuing oligonucleotide or with the about 15-30 of a carrier sequence eclipsed Nucleotide.The annealing of these three kinds of oligonucleotide in the PCR-based reaction, the chain that polysaccharase is opposite with filling produces complete double-stranded heavy chain or light chain variable region sequence.The number of triplet can be adjusted to the repetition of any length, and can select its position in oligonucleotide so that only be substituted in given CDR or the amino acid in the framework region.Through using (NNK), all 20 amino acid each position in the variant of coding all is possible.The overlap of 5-10 amino acid (15-30 Nucleotide) is not substituted, but this can select to be positioned at the accumulation area of framework, perhaps can replace through synthesis cycle isolating or subsequently.The method of synthetic oligonucleotide is known in the art, also can be purchased.The method that from these oligonucleotide, produces antibody variants also is known in the art, for example PCR.

The different heavy chain of random site and the library of light chain variant can make up in any expression vector in its sequence, said carrier such as phage, and carrier particularly shown in Figure 1, it all contains the DNA of special heavy chain of coding and light chain variant.

After producing antibody variants, confirm the BA of variant with respect to parental generation antibody.As stated, this comprises the binding affinity of definite variant for target.There is the method for many high yields to combine the ability of interested target with the rapid screening antibody variants.

Then to one or more antibody screening of being selected from this initial screening its with respect to parental generation antibody enhanced binding affinity.A kind of method of definite binding affinity commonly used is to use BIAcore ^TM(BIAcore Inc.) estimates the combination and the velocity constant of dissociating in the surface plasma resonance system.Instruct according to manufacturer (BIAcore), activate biologic sensor chip with the target covalent coupling.With target dilution and be expelled on the chip to obtain a signal, said signal is with the unit of replying (RU) expression of fixed substance then.Because signal and fixed substance quality among the RU are proportional, so this has represented the density range of fixed target on the matrix.The single site of the data fit of dissociating model obtains k _Off± s.d. (standard deviation of mensuration).Calculate false one-level velocity constant (k for each binding curve _s), and, obtain k as the function plotting of protein concn _On± s.e. (identical standard error)., calculates SPR bonded equilibrium dissociation constant K from measuring _D, be expressed as k _Off/ k _OnBecause equilibrium dissociation constant K _DWith k _OffBe inversely proportional to, therefore through supposition association rate (k _On) all be constant for all variants, can estimate the improvement of avidity.

The material standed for that gained has a high-affinity can be chosen wantonly and carry out one or more further Determination of biological activity, to confirm that having enhanced combines active antibody variants still to keep the therapeutic property of hope.For example, in the situation of anti-IgE antibodies, can screen those antibody of blocking-up IgE and its receptors bind and inhibition histamine release.Best antibody variants keeps the ability that combines target with the binding affinity that is significantly higher than parental generation antibody.

The antibody variants of so selecting can further be modified according to earmarking of antibody usually.These modifications can comprise further change aminoacid sequence, merge with heterologous polypeptide and/or as those covalent modifications of hereinafter description.For example, any cysteine residues of not participating in keeping the suitable conformation of antibody variants all can be substituted (being replaced by Serine usually), with the oxidative stability of improvement molecule and stop unusual crosslinked.On the contrary, the halfcystine key can add in the antibody, to improve its stability (being in a kind of antibody fragment such as the segmental situation of Fv at antibody particularly).

Carrier

The present invention also provides the isolating nucleic acid of the antibody variants of code book invention announcement, comprises the carrier and the host cell of said nucleic acid, and produces the recombinant technology of said antibody variants.For the generation antibody variants of recombinating, separate coding its nucleic acid and be inserted in the reproducible carrier with further clone (amplification of DNA) or express.The DNA of encoding antibody variant is easy to use ordinary method to separate and order-checking (the for example oligonucleotide probe of the gene through using the heavy chain that can specificity combines the encoding antibody variant and light chain).

Many carriers are obtainable.Carrier components generally includes but non-ly is limited to following one or more composition: signal sequence, replication orgin, one or more marker gene, enhancer element, promotor, and transcription termination sequence.

Phage expression vector shown in Figure 1 is made up of M13 carrier commonly used and the autogene III viral secretory signal of M13, has the variant Fab of suitable binding specificity and minimum avidity standard with quick secretion and screening.Therefore this carrier does not use complete genome III sequence, on the bacterial cell surface, do not show, but Fab still secretes in the periplasmic space.Perhaps, Fab can express in tenuigenin and be separated.Heavy chain and light chain all have its oneself viral secretory signal, but dependency is expressed from a single strong inducible promoter.

Carrier shown in Figure 1 also provides a His mark and a myc mark to be easy to purifying and detection.Those skilled in the art recognize that Fab can express independently or secretion signal needs not be the virus sequence of selection from independent promotor, and can be to be suitable for antibody fragment excretory protokaryon or eucaryon signal sequence from the host cell of selecting.Should recognize that also heavy chain can be positioned at different carriers with light chain.

A: signal sequence composition

The antibody variants of the present invention generation of can recombinating.Said variant can also be expressed as the fusion polypeptide that merges with heterologous polypeptide, and said heterologous polypeptide is signal sequence or have other polypeptide of specificity cleavage site at the N-terminal of mature protein or polypeptide preferably.Preferred selected allos signal sequence is by host cell identification and processing (promptly by the signal peptidase cracking).For the prokaryotic host cell of nonrecognition with processing natural antibody signal sequence, signal sequence can the prokaryotic signal sequence of SEAP, penicillinase, Ipp or heat-staple enterotoxin 1 I leader sequence replaces by for example being selected from.Perhaps in the situation of the carrier of Fig. 1, the signal sequence of selection is the viral signal sequence from gene III.For yeast secretary; The natural signals sequence can be by for example yeast invertase leader sequence, α-factor leader sequence (comprising yeast (Saccharomyces) and yeast kluyveromyces fragilis (Kluyveromyces) α-factor leader sequence) or acid phosphatase leader sequence, Candida albicans (C.albicans) glucoamylase leader sequence, and perhaps for example WO 90/13646 said signal replaces.In mammalian cell expression, obtainable mammalian signal sequence capable of using and viral secretory leader sequence be herpes simplex gD signal for example.The DNA in this precursor zone meets frame ground and is connected with the DNA of encoding antibody variant.

B: the composition of replication orgin

Carrier contains the nucleotide sequence that makes that it duplicates in the host cell of one or more selection usually.Normally, this sequence is to make said carrier not rely on host chromosome DNA and the sequence of duplicating, and comprises replication orgin or autonomously replicating sequence.These sequences of various bacteriums, yeast and virus are known.Replication orgin from plasmid pBR322 is suitable for most of gram negative bacteriums, and 2 μ plasmid starting points are suitable for yeast, and various viral starting points (SV40, polyomavirus, adenovirus, VSV or BPV) are used for the carrier of mammalian cell.Normally, the composition of replication orgin is for mammalian expression vector no requirement (NR) (can typically use the SV40 starting point, for no other reason than that it contains early promoter).

C: select gene element

Carrier can contain one selects gene, is also referred to as selective marker.The typical such protein of genes encoding of selecting; Said protein (a) is authorized microbiotic or other toxin for example penbritin, Xin Meisu, methotrexate or tetracyclin resistance; (b) complementary auxotrophy; Perhaps (c) provides critical nutrients unavailable from complex medium, the gene of the D-alanine racemase of the genus bacillus of for example encoding.

A kind of instance of selection scheme is the growth that utilizes the drug block host cell.The protein of drug resistance is authorized in those cells generations with heterologous gene successfully transforms, and therefore in selection scheme, survives.The instance that this dominance is selected is to use medicine Xin Meisu, mycophenolic acid and Totomycin.

The instance that another kind is suitable for the selective marker of mammalian cell is to make those marks of ability that can identification of cell picked-up antibody nucleic acid, like DHFR, thymidine kinase, rhMT I and II (preferred primate metallothionein gene), adenosine deaminase, ornithine decarboxylase or the like.

For example, select the cell of gene transformation at first to differentiate with DHFR through in the substratum that contains methotrexate (Mtx, the competitive antagonist of DHFR), cultivating all transformant.When using wild-type DHFR, proper host cell is to lack the active Chinese hamster ovary of DHFR (CHO) clone.

Perhaps; With encoding antibody, wild-type dhfr protein and other selective marker such as aminoglycoside 3 '-dna sequence dna of phosphotransferase (APH) transforms or the host cell (the wild-type host of particularly containing endogenous DHFR) of cotransformation; Can through contain for the selective agent of selective marker such as aminoglycoside antibiotics for example the cell growth in the substratum of kantlex, Xin Meisu or G418 select (United States Patent(USP) No. 4; 965,199).

The suitable selection gene that is used for yeast is that yeast plasmid Yrp7 goes up the trp1 gene (Stinchcomb et al., Nature 282:39 (1979)) that exists.The trp1 gene provides and has been used for lacking the selective marker in the yeast variant strain of tryptophane energy for growth, and such yeast variant strain is ATCC No.44076 or PEP4-1.Jones for example, and Genetics 85:12 (1977) is said.The existence of trp1 damage provides through growth in not having the situation of tryptophane and has detected the effective environment that transforms in the yeast host cell genome.Similarly, the yeast strains of Leu2-defective (ATCC20,622 or 38,626) is through carrying the known plasmid complementary of Leu2 gene.

D: promotor composition

Express with cloning vector and contain the promotor of discerning by host organisms and operably be connected with antibody nucleic acid usually.The promotor that is applicable to prokaryotic hosts comprises phoA promotor, β-Nei Xiananmei and lactose promoter systems, SEAP, tryptophane (trp) promoter systems and crossbred promotor such as tac promotor.Yet other known bacterium promotor also is fit to.The promotor that is used for bacterial system also can contain Shine-Dalgarno (S.D.) sequence that operably is connected with the DNA of encoding antibody.

Known eukaryotic promoter sequence.In fact all eukaryotic genes all have a zone of being rich in AT, are positioned at the about 25-30 in a transcription initiation site upper reaches base place.Another sequence at many gene transcription section start upper reaches 70-80 base finds is the CNCAAT zone, and wherein N can be any Nucleotide.3 ' end at most of eukaryotic genes is the AATAAA sequence, and it possibly be that 3 ' end at encoding sequence adds the signal that gathers the A afterbody.All these sequences all are fit to insert in the carrier for expression of eukaryon.

The instance of the suitable promoter sequence that uses with yeast host comprises the promotor of following enzyme: glycerol 3-phosphate acid kinase or other glycolytic ferment, and like Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, HK, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, triose-phosphate isomerase and glucokinase.

Other Yeast promoter (inducible promoters with the additional advantage of transcribing through growth conditions control) is alcoholdehydrogenase 2, Lrax (isocytochrome C), acid phosphatase, degrading enzyme, rhMT, the glyceraldehyde-3-phosphate dehydrogenase relevant with nitrogen metabolism and is used for SANMALT-S and the promoter region of the enzyme of galactose utilization.The suitable carrier and the promotor that are used for yeast expression further describe in EP 73,657.It also is favourable that the yeast enhanser uses with Yeast promoter.

Antibody in mammalian host cell from carrier transcribe by the genomic promotor control that for example derives from virus (said virus for example is polyomavirus, avipoxvirus, adenovirus (like adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40) most preferably), by derive from for example actin promoter or the immunoglobulin promoter control of allos mammalian promoter, by the control of heat-shocked promotor, these promotors and the host cell systems that provide are compatible.

Early stage and the late promoter of SV40 virus obtains as also containing the SV40 restricted fragment of SV40 virus replication starting point expediently.The immediate early promoter of human cytomegalic inclusion disease virus obtains as HindIII E restricted fragment easily.In mammalian hosts, use bovine papilloma virus as the system of vector expression DNA at United States Patent(USP) No. 4,419, describe in 446.To this system be modified in United States Patent(USP) No. 4,601, describe in 978.Perhaps, human cDNA expresses in mouse cell under the control from the thymidine kinase promoter of hsv.Perhaps, rous sarcoma virus length is terminal repetition can be used as promotor.

E: enhancer element composition

The DNA of antibody of the present invention of encoding is increased by transcribing usually through in carrier, inserting enhancer sequence of higher eucaryotic cells.Known many enhancer sequence (sphaeroprotein, Pancreatopeptidase E, BSA, ALPHA-FP and Regular Insulin) from mammalian genes at present.Yet, typically, can use enhanser from eukaryotic cell virus.Said enhanser for example is included in the sub-enhanser of SV40 enhanser, cytomegalovirus early promoter of replication orgin side in late period (bp 100-270), at the polyomavirus enhanser and the adenovirus enhanser of replication orgin side in late period.See Yaniv, Nature 297:17-18 (1982) is for the description that strengthens the element that activates the eukaryotic cell promotor.Said enhanser can be at carrier 5 ' or 3 ' engagement position antibody coding sequence, but be preferably placed at 5 ' position of promotor.

F: Transcription Termination composition

The expression vector that is used for eukaryotic host cell (nucleated cell of yeast, fungi, insect, plant, animal, people or other multinuclear organism) also can contain the essential sequence essential with stable mRNA of Transcription Termination.These sequences can obtain the untranslated district from 5 ' (once in a while from 3 ') of eukaryotic cell or viral DNA or cDNA usually.The Nucleotide sections of in the untranslated part of the mRNA of encoding said antibody, transcribing as the polyadenylation fragment is contained in these zones.A kind of useful Transcription Termination composition is Trobest polyadenylation zone.See that for example W094/11026 is said.

The selection of host cell and conversion

The suitable host cell of clone or expressible dna is prokaryotic cell prokaryocyte, yeast or higher eucaryotic cells in carrier of the present invention.For this purpose; Suitable prokaryotic cell prokaryocyte comprises Gram-negative and Gram-positive biology; For example enterobacteria section such as intestinal bacteria (E.coli), enterobacter (Enterobacter), erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), Salmonellas (Salmonella), Serratia (Serratia) and Shigellae (Shigella), and genus bacillus (Bacilli), pseudomonas (Pseudomonas) and streptomycete (Streptomyces).A kind of preferred escherichia coli cloning host is intestinal bacteria 294 (ATCC 31,446), and other bacterial strain such as intestinal bacteria B, intestinal bacteria X1776 (ATCC31,537) and intestinal bacteria W3110 (ATCC 27,325) also are suitable.These just illustrate and unrestricted meaning.

Except prokaryotic cell prokaryocyte, eukaryotic microorganisms such as filamentous fungus or yeast also are the suitable clone or the expressive hosts of antibody coding carrier.Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) is the most frequently used low mikrobe such as eucaryon host such as grade.Yet many other Pseudomonas, bacterial classification and bacterial strain also can obtain and utilize in this article, like schizosaccharomyces pombe (Schizosaccharomyces pombe); Yeast kluyveromyces fragilis (Kluyveromyces); Candiyeast (Candida); Wood mould (Trichoderma); Coarse arteries and veins spore mould (Neurospora crassa); With filamentous fungus Neurospora (Neurospora) for example, mould (Penicillium), curved neck mould (Tolypocladium), and aspergillus (Aspergillus) host are like Aspergillus nidulans (A.nidulans) and black mold (A.niger).

The suitable host of expressing glycosylated antibody is cell-derived from multicellular organisms.In principle, any higher eucaryotic cells culture all can use, and no matter it derives from vertebrates still is the invertebrates culture.The instance of invertebral zooblast comprises plant and insect cell, Luckow et al., and Bio/Technology 6,47-55 (1988); Miller et al., Genetic Engineering, Setlow et al.eds.Vol.8, pp.277-279 (Plenam publishing1986); Mseda et al., Nature 315,592-594 (1985).From the greedy noctuid (Spodoptera frugiperda) (caterpillar) of host such as meadow, yellow-fever mosquito Aedes (mosquito), drosophila melanogaster (Drosophila melanogaster) (fruit bat) and silkworm (Bombyx mori), differentiated the insect host cell of many baculovirus strains and variant and corresponding approval.Can openly obtain to be used for many virus strain of transfection; The Bm-5 strain of L-1 variant of autographa california nuclear polyhedrosis virus (Autographa californica NPV) and Bombyx mori nuclear polyhydrosis virus (Bombyx mori NPV) for example; These viruses can be used as virus of the present invention at this, are used in particular for the transfection of the greedy frugiperda cell in meadow.In addition, plant cell cultures such as cotton, corn, yam, soybean, petunia, tomato and tobacco are also as the host.

Vertebrate cells known in the art and the vertebrate cells propagation in cultivating (tissue culture).See Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973) is said.The instance of useful mammalian host cell line be MK cells system, human embryonic kidney cell system, baby hamster kidney cell, Chinese hamster ovary cell/-DHFR (CHO Urlaub et al., Proc.Natl.Acad.Sci.USA 77:4216 (1980)), mouse support (sertoli) cell, human cervical carcinoma cell (HELA), MDCK, human pneumonocyte, human liver cell, mouse mammary tumor cell and NS0 cell.

Host cell is transformed with generation antibody with above-mentioned carrier, and in the conventional nutritional medium of revising for the gene that is suitable for evoked promoter, selection transformant or the desirable sequence of amplification coding, cultivate.

The host cell that is used for producing antibody variants of the present invention can be cultivated at various substratum.Commercially available substratum such as Ham ' s F10 (Sigma), Minimal Essential Medium (MEM, Sigma), (DMEM Sigma) is suitable for cultivating host cell for RPMI-1640 (Sigma) and Dulbecco ' s Modified Eagle ' s Medium.In addition, Ham et al., Meth.Enzymol.58:44 (1979), Barnes etal., Anal.Biochem.102:255 (1980), United States Patent(USP) Nos. 4; 767,704,4,657,866,4; 560,655,5,122,469,5; 712,163, perhaps 6,048,728 described any substratum all can be as the substratum of host cell.Any of these substratum all can be added hormone and/or other growth factors (like Regular Insulin, transferrin or Urogastron) as required, (like the X-muriate, wherein X is sodium, calcium, magnesium to salt; And phosphoric acid salt), damping fluid (like HEPES), Nucleotide (like adenosine and thymidine), microbiotic (like the GENTAMYCIN.TM. medicine), trace element (mineral compound that exists with the final concentration of micro-molar range usually), and glucose or the energy source that is equal to.Any other essential fill-in that also can comprise the known proper concn of those skilled in the art.Culture condition such as temperature, pH etc. are previous used for to expressing those conditions of selecting host cell, and these are well known to those skilled in the art.

Antibody purification

When using recombinant technology, antibody variants can be in cell, in the periplasmic space generation, and perhaps direct secretion is advanced in the substratum.If antibody variants is in cell, to produce,, can for example remove the particulate fragment of host cell or crack fragment through centrifugal or ultrafiltration as the first step.Carter et al., Bio/Technology 10:163-167 (1992) have described a kind of method that the antibody in the colibacillus periplasm gap is advanced in secretion of separating.In brief, the cell paste was thawed about 30 minutes under the situation that has sodium acetate (pH 3.5), EDTA and PMSF (PMSF).Cell debris can be removed through centrifugal.Secrete at antibody variants under the situation of substratum into, at first use commercially available protein to concentrate the supernatant that strainer concentrates this expression system usually, for example use Amicon or Millipore Pellicon ultra-filtration equipment to filter.In aforementioned any step, all can comprise proteinase inhibitor such as PMSF,, and can comprise that microbiotic is to prevent the growth of external contaminant with the arrestin hydrolysis.

The antibody compositions that from cell, prepares for example can use hydroxyapatite, gel electrophoresis, dialysis and affinity chromatography to carry out purifying, and wherein affinity chromatography is as preferred purification technique.Albumin A depends on the kind and the isotype of any immunoglobulin Fc domain that exists in the antibody variants as the suitability of affinity ligand.Albumin A can be used for the antibody (Lindmark et al., J.Immunol Meth.62:1-13 (1983)) of purifying based on human IgG1, IgG2 or IgG4 heavy chain.Recommended all mouse isotypes and the human IgG 3 (Guss et al., EMBO is (1986) J.5:1567-1575) of being used for of Protein G.The matrix that affinity ligand adhered to is agarose normally, but also can utilize other matrix.The matrix of mechanically stable such as controlled pore glass or gather (vinylbenzene divinyl) benzene can reach than agarose the flow velocity faster that can reach and shorter process period.When antibody variants comprised the CH3 structural domain, (J.T.Baker, Phillipsburg N.J.) can be used for purifying to Bakerbond ABXTM resin.Based on the antibody variants that will reclaim; Also can use other purified technology of protein, like the SEPHAROSE on the chromatography on the fractional separation on the ion exchange column, ethanol sedimentation, reversed-phase HPLC, the silicon-dioxide, chromatography, negatively charged ion or the Zeo-karb (like the SAP 73 post) on the heparin ^TMChromatography, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any preliminary purification step, can use the elution buffer of pH between about 2.5-4.5 that the mixture that comprises interested antibody variants and pollutent is hanged down the pH hydrophobic interaction chromatography, preferably under low salt concn (for example about 0-0.25M salt), carry out.

Medicament preparation

The therapeutic preparation that can prepare said polypeptide or antibody; Storing as freeze dried preparation or aqueous solution form, said preparation randomly mixes with " pharmacology is acceptable " carrier, vehicle or the stablizer (all these all are called vehicle) of this area typically used and carries out through having the polypeptide of hoping purity.For example, and buffer reagent, stablizer, sanitas, isotonic agent, nonionic detergent, inhibitor and other various additives (see Remington ' s Pharmaceutical Sciences, 16thedition, A.Osol, Ed. (1980) is said).These additives must be avirulent in dosage and the concentration range used to acceptor.

Buffer reagent helps to keep pH in the scope near physiological conditions.They preferably exist with the concentration range of about 2mM-50mM.Comprise organic and mineral acid and salt thereof for application suitable reducing of the present invention, like citrate buffer (for example mixture of the mixture of the mixture of sodium dihydrogen citrate and disodium citrate, Hydrocerol A and trisodium citrate, Hydrocerol A and sodium dihydrogen citrate or the like), SUMATRIPTAN SUCCINATE damping fluid (for example succsinic acid-succsinic acid one sodium mixture, succsinic acid-sodium hydroxide mixture, succsinic acid-disodium succinate mixture or the like), tartrate buffer (for example tartrate-sodium tartrate mixture, tartrate-soluble tartrate mixture, tartrate-sodium hydroxide mixture or the like), fumaric acid salt buffer (for example fumaric acid-fumaric acid one sodium mixture or the like), fumaric acid salt buffer (for example fumaric acid-fumaric acid one sodium mixture, fumaric acid-fumaric acid disodium mixture, fumaric acid one sodium-fumaric acid disodium mixture or the like), glyconate damping fluid (for example glyconic acid-gluconic acid sodium salt mixture, glyconic acid-sodium hydroxide mixture, glyconic acid-potassium gluconate mixture or the like), oxalate damping fluid (for example oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture or the like), lactate buffer (for example lactic acid-Sodium.alpha.-hydroxypropionate mixture, lactic acid-sodium hydroxide mixture, lactic acid-Potassium.alpha.-hydroxypropionate mixture or the like) and acetate buffer (for example, acetate-sodium acetate mixture, acetate-sodium hydroxide mixture or the like).That can also mention in addition, has phosphate buffered saline buffer, histidine buffering liquid and trismethylamine salt such as a Tris.Can add sanitas to stop microorganism growth, sanitas can add with the amount of 0.2%-1% (w/v) scope.Be used for suitable sanitas of the present invention and comprise phenol, phenylcarbinol, m-cresol, para methyl paraben, propylparaben, stearyl dimethyl benzyl ammonium chloride, benzene diformazan hydrocarbon ammonium (benzalconium) halogenide (for example, muriate, bromide, iodide), chlorination hexane diamine, oxybenzene alkyl formate such as para methyl paraben or propylparaben, catechol, Resorcinol, hexalin and 3-amylalcohol.

Can add sometimes be also referred to as " stablizer " isotonic agent (isotonicifier) to guarantee the isotope of liquid compsn of the present invention; Isotonic agent comprises the poly-hydroxy sugar alcohol; Preferably trihydroxy-or more poly-hydroxy sugar alcohol are like glycerine, erythritol, arabitol, Xylitol, Sorbitol Powder and mannitol.

Stablizer is meant one wide type of vehicle, and their scope is from weighting agent to the dissolution treatment agent or help the additive that prevents sex change or adhere to wall of container on function, and typical stablizer can be poly-hydroxy sugar alcohol (as stated); Amino acid such as l-arginine, Methionin, glycocoll, Stimulina, l-asparagine, Histidine, L-Ala, ornithine, L-leucine, 2-phenylalanine(Phe), L-glutamic acid, Threonine etc.; Organic sugar or sugar alcohol; Like lactose, trehalose, stachyose, mannitol, Sorbitol Powder, Xylitol, ribitol, myoinisitol, melampyrum, glycerine etc., comprise cyclic alcohol such as inositol; Polyoxyethylene glycol; Aminoacid polymers; The sulfur-bearing reductive agent is like urea, gsh, Thioctic Acid, Thioglycolic acid sodium salt, mercapto glycerol, α-single mercapto glycerol and Sulfothiorine; Low molecular weight polypeptide (residue promptly＜10); Protein such as human serum albumin, bovine serum albumin, gelatin or Tegeline; Hydrophilic polymer is like Vinylpyrrolidone polymer; Monose is like wood sugar, seminose, fructose, glucose; Disaccharides such as lactose, SANMALT-S, sucrose and trisaccharide such as raffinose; Polysaccharide such as VISOSE.Stablizer can exist with the amount of every weight part active protein 0.1-10000 weight part.

Can add nonionogenic tenside or stain remover (being also referred to as " wetting agent ") to help the dissolution treatment agent gathering that anti-stirring causes with protection treatment protein, it also makes preparation can be exposed to the shear surface of pressurization and does not cause protein denaturation.Suitable ionic surfactant pack is drawn together polysorbate (20; 80 etc.); Polyoxamer (184; 188 etc.);

polyvalent alcohol; Polyoxyethylene sorbitol acid anhydride monoether (

-20;

-80 etc.).Nonionogenic tenside can with about 0.05mg/ml to about 1.0mg/ml, preferably about 0.07mg/ml extremely the scope of about 0.2mg/ml exist.

Complementary vehicle in addition comprises weighting agent (for example starch), sequestrant (for example EDTA), inhibitor (for example xitix, methionine(Met), vitamin E) and cosolvent.If the specific adaptations disease needs of being treated, preparation of the present invention can also contain more than one active compound, preferably has the active compound of the not negative impact each other of complementary activity.For example, it possibly be desirable a kind of immunosuppressor further being provided.These molecules are planned purpose suitably to be effective to amount combination exists.Activeconstituents also can be trapped in the micro-capsule; Said micro-capsule for example is respectively Walocel MT 20.000PV or gelatin microcapsule and gathers (TEB 3K) micro-capsule () through in colloidal state delivery system (for example liposome, albumin microsphere, microemulsion, nano particle and nanometer packing) or coarse emulsion (macroemulsion), for example preparing through condensation technique or through interfacial polymerization.These technology are in Remington ' s Pharmaceutical Sciences, 16 ^ThEdition, A.Osal, open among the Ed. (1980).The preparation that is used for vivo medicine-feeding must be aseptic.This can easily for example realize through aseptic membrane filtration.Can prepare extended release preparation.The suitable example of extended release preparation comprises half penetrating matrix of the solid hydrophobic polymkeric substance that contains antibody variants, and said matrix is the shaped object form, for example film or micro-capsule.The example that continues release matrix comprises polyester, hydrogel (for example gather (2-hydroxyethyl meth acrylate) or gather (vinyl alcohol)), polylactide (USP 3; 773,919), the multipolymer of L-L-glutamic acid and ethyl-L-glutamate, nondegradation ethene-vinyl-acetic ester, degradation property lactic acid-ethanol copolymer such as LUPRON DEPOT ^TM(Injectable microspheres of forming by lactic acid-ethanol copolymer and TAP-144) and gather-D-(-)-3-hydroxybutyric acid.Although the polymkeric substance like ethane-acetic acid ethyenyl ester and lactic acid-ethanol can discharge molecule above 100 days, some hydrogel release protein time is shorter.When capsulation antibody keeps when long-time in vivo, they may be owing to being exposed to wet environment and sex change or gathering at 37 ℃, thereby cause BA forfeiture and immunogenicity to change.According to related mechanism can be reasonable in design strategy to carry out stabilization.For example; If find that aggregation of multiple forms intermolecular S-S key through sulfo--disulfide exchange, then can be through modifying sulfydryl, freeze-drying from acidic solution, control moisture content, using suitable additive and exploitation specificity polymer matrix composition to realize stablizing.

Efficacious therapy property polypeptide, antibody or its segmental amount determine according to the character of said illness or pathology in treatment particular condition or pathology, and can confirm through standard clinical techniques.If possible, hope was used for useful animal model system then at first at external definite pharmaceutical composition dose response curve of the present invention before human experimentation.

In a preferred embodiment, therapeutical peptide, antibody or its segmental aqueous solution give through subcutaneous injection.Dosage is about 0.5 μ g-50 μ g/kg body weight, more preferably about 3 μ g-30 μ g/kg body weight.

Subcutaneous injection time of administration table according to many clinical factors can be one month once to once a day, said clinical factor comprises disease type, the severity of disease, and object is for the susceptibility of therapeutical agent.

The application of antibody variants

Antibody variants of the present invention can be used as the affinity purification agent.In this process, use method well known in the art that antibody is fixed on solid phase such as SEPHADEX ^TMOn resin or the filter paper.The fixed antibody variants is contacted with the sample that contains the target of wanting purifying, use this upholder of suitable solvent wash afterwards, removed in the sample all material except the target of wanting purifying so basically, said target is combined on the fixed antibody variants.At last, upholder is washed with other suitable solvent such as glycine buffer, this will disengage said target from antibody variants.

Said variant antibody also can be used for dialysis to be measured, and for example detects interested target in specific cells, tissue or expression of serum.Use for diagnostic, said antibody variants is typically used detectable component mark.Many marks can utilize.The technology of quantification change in fluorescence as stated.Chemical luminous substrate becomes electric excited state through chemical reaction, can launch the light (for example using the chemoluminescence instrumentation to decide) that can measure then or as fluorescent receptor energy is provided.The instance of enzyme labelling comprises luciferase (for example Lampyridea luciferase and bacteriofluorescein enzyme; United States Patent(USP) No. 4; 737; 456), luciferin, 2,3-dihydro phthalazine diketone (dihydrophthalazinediones), MDH, urase, px such as horseradish peroxidase (HRPO), SEAP, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase (for example P-FAD, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), heterocycle oxydase (like uriKoxidase and XOD), POD, microperoxisome or the like.The technology that enzyme and antibody are puted together is at O ' Sullivan etal.; Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay; In Methods in Enzym. (Ed.J.Langone & H.Van Vunakis); Academic press describes among the New York, 73:147-166 (1981).

Sometimes, said mark and antibody variants are puted together indirectly.The technician understands has various technology can reach this purpose.For example, antibody variants can with biotin-conjugated, and the kind widely of above-mentioned three kinds of marks all can put together with avidin, vice versa.Vitamin H selective binding avidin, mark is puted together with this indirect mode and antibody variants thus.Perhaps,, said antibody variants and little haptin (for example digoxin) are puted together, and above-mentioned a kind of dissimilar marks and antihapten antibody variant (for example anti digoxin antibody) are puted together in order to reach puting together indirectly of mark and antibody variants.Therefore can reach puting together indirectly of mark and antibody variants.

In another embodiment of the invention, antibody variants does not need mark, and its existence can be used the antibody test that is labeled of binding antibody variant.

Antibody of the present invention can be used in any known measuring method, like competitive binding assay, and direct and indirect sandwich assay, and immune precipitation determination.Zola，Monoclonal?Antibodies：A?Manual?of?Techniques，pp.147-158(CRC?Press，Inc.1987)。

The standard substance that competitive binding assay depends on mark combines limited amount antibody variants with the specimen competition.The amount of the amount that specimen hits and the standard substance of binding antibody is inversely proportional to.For the ease of confirming and the amount of the standard substance of antibodies that said antibody is normally undissolved before or after competition.As a result, can be easily from unconjugated standard substance and specimen, separate in conjunction with the standard substance of this antibody and specimen.

Sandwich assay comprises uses two kinds of antibody, and every kind of antibody all can combine different immunogenicity part or epi-position or protein to be detected.In sandwich assay, specimen to be analyzed is by the first kind of antibodies that is fixed on the solid support, and second kind of antibody combines with specimen afterwards, forms insoluble three part mixtures thus.See for example United States Patent(USP) No. 4,376,110 is said.Second kind of available a kind of detectable component mark of antibody itself (directly sandwich assay) perhaps can use through the AIA of detectable component mark and measure (sandwich assay indirectly).For example, one type sandwich assay is that ELISA measures, and wherein detectable component is a kind of enzyme.

For immunohistochemical method, tumor sample can be fresh or refrigerated or can be embedded in the paraffin and with sanitas formalin fixed for example.

Said antibody also can be used for in-vivo diagnostic and measure.Normally, with said antibody variants with a kind of radionuclide (as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P perhaps ³⁵S) mark is so that can use immune imaging method (immunoscintiography) positioning tumor.For example, high-affinity anti-IgE antibodies of the present invention can be used for the amount of the IgE that test example such as asthmatic patient lung exist.

Antibody of the present invention can provide in test kit, and said test kit promptly has the agent combination of the packaged predetermined amount of the specification sheets that carries out diagnostic assay.In the situation of antibody variants with enzyme labelling, said test kit can comprise needed substrate of said enzyme and cofactor (substrate that can detect chromophore or fluorophore precursor for example is provided).Additive such as stablizer, the damping fluid (for example sealing damping fluid or lysis buffer) etc. that can comprise in addition, other.The relative quantity of all ingredients can extensively change so that the concentration of reagent in solution can fully be optimized the sensitivity of detection.

Especially, reagent can provide with the dry powder form that comprises vehicle, and is normally freeze dried, and it will provide the reagent solution with suitable concn after dissolving.

Use in the body of antibody

Be understood that antibody of the present invention can be used for treating Mammals.In one embodiment, antibody is given non-human mammal to be used for for example obtaining clinical preceding data.Comprised that by the illustrative non-human mammal of being treated non-human primates, dog, cat, rodents and other carry out the Mammals of preclinical study.These Mammalss can be the animal models of having set up that will use the disease of Antybody therapy, perhaps can be used to study the toxicity of antibody interested.In each of these embodiments, can on Mammals, carry out dosage and progressively increase research.Antibody or polypeptide by administration, comprise in parenteral, subcutaneous, intraperitoneal, the lung and in the nose through any suitable method, and are used for the local immunity suppression therapy if desired, can damage interior administration.The parenteral infusion comprises intramuscular, intravenously, intra-arterial, intraperitoneal or subcutaneous administration.In addition, with the administration of pulse infusion mode, particularly wherein antibody variants dosage descends antibody variants gradually suitably.Preferably, through drug administration by injection, most preferably through intravenously or subcutaneous injection administration, it is short-term or long-term that this point depends in part on administration.

In order to prevent or treat disease, the appropriate dose of antibody or polypeptide depends on the replying and physician in charge surgeon in charge attending doctor doctor in charge's judgement of clinical history and antagonist variant that is used to prevent purpose or therapeutic purpose, previous treatment, patient of type, severity of disease and the course of disease, the antibody variants of the disease that will treat.The anti human IgE antibody of very high-affinity of the present invention can suitably once be given the patient or in a series of treatments, given the patient.

According to the type and the seriousness of disease, about 0.1mg/kg to 150mg/kg (for example 0.1-20mg/kg) antibody is the initial candidate's dosage that gives the patient, for example can one or repeatedly administration or through the continuous infusion administration respectively.Typical per daily dose can be from about 1mg/kg to 100mg/kg or higher, and this depends on above-mentioned factor.For the repeat administration in several days or longer time, according to symptom, can continued treatment until the desirable inhibition of generation to disease symptoms.But other dosage also is an available.The process of this treatment can easily be monitored through routine techniques and detection.A kind of illustrative dosage of anti-LFA-1 or anti-ICAM-1 antibody is open in WO 94/04188.

The antibody variants compsn can be to prepare, to confirm dosage and administration with the corresponding to mode of good medical practice.Comprise the particular condition of being treated, the specific Mammals of being treated, each patient's clinical setting, the factor that causes illness, the position that gives medicament, medication, administration time table, reach the known other factors of medical practitioner in this factor that need consider." the treatment significant quantity " of administered antibodies variant determines through the item of these considerations, and is to prevent, alleviate or treat disease or the required minimum of illness.Said antibody variants needs not be, but randomly prepares with one or more medicament that is used at present to prevent or treat the illness of being considered.The significant quantity of these other medicaments depends on type, and the factor discussed of other front of the illness of the antibody amount that exists in the preparation, treatment.These medicaments are usually with same dose application, and with used to give approach consistent, or the 1-99% of application dose before using.

Identification IgE can be used for treatment " illness of IgE mediation " as the antibody of the present invention of its target.These illnesss comprise like asthma, rhinallergosis and conjunctivitis (spring fever), eczema, rubella, atopic dermatitis and food anaphylaxis.By for example honeybee bite, these serious physiology pathologies of anaphylactic shock of venom, food or drug-induced also contain within the scope of the invention.

Antibody epitope location (MAPPING)

Term " epi-position " is meant the site of B on the antigen and/or t cell response.The B cell epitope can form from successive amino acid, perhaps from causing forming the contiguous discontinuous amino acid owing to protein folds for three grades.The epi-position that from continuous amino acid, forms still typically keeps when being exposed to the sex change solvent, and typically no longer exists after with the sex change solvent treatment through three grades of epi-positions that are folded to form.Epi-position typical case in a unique space conformation comprises at least 3, more commonly comprises at least 5 or 8-10 amino acid.Discerning the antibody of identical epi-position can differentiate in easy immunoassay, and said immunoassay illustrate another kind of antibody of a kind of antibody blocking and target antigen bonded ability.

The epitope mapping of the binding site of high-affinity antibody IgE of the present invention comprises amino-acid substitution and the directed mutagenesis for the respective regions of the pepscan of the CH3 structural domain of bonded Western engram analysis, IgE, the L-Ala scanning that the bonded zone is shown, IgG1.

The pepscan of the complete CH3 structural domain of IgE needs 73 overlapping peptides.Each peptide all carries out the combination of the anti-IgE antibodies of mark of the present invention, to confirm the specific epitopes of blocking-up IgE and its high-affinity receptor bonded IgE.Said pepscan has differentiated it is two peptides in the anti-IgE MAb contact of potential site on IgE, be called epi-position A and epi-position B (seeing shown in Figure 11).Although epi-position A and epi-position B sequence are at about 80 amino acid of linear order interval, their positions in the three-dimensional structure of IgE are closely adjacent each other.These two epi-positions all are that the surface exposes, and the Fc ε RI binding site of they and IgE is overlapping, and in these two peptides, positively charged Arg residue and hydrophobic residue Pro is arranged all.Figure 12 illustration use the calmodulin binding domain CaM of the epi-position B that pepscan confirms through ELISA.

Confirm in these epi-positions for combining the crucial amino-acid residue (Cunningham et al., " High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis " Science244:1081-1085) of high-affinity antibody through alanine scanning mutagenesis.With each residue of L-Ala replacement epi-position A and epi-position B, and the combination of definite high-affinity monoclonal antibody (embodiment 12 and Figure 13 and 14 see below).

Active and passive immunization

The invention still further relates to pharmaceutical composition, vaccine for example, it comprises peptide based immunogens molecule of the present invention (comprising SEQ ID NO:72 and/or SEQ ID NO:74) and thinner, vehicle, adjuvant or carrier.The invention further relates to immunogenic preparation method of the present invention, comprise a kind of composition covalent coupling of at least a peptide of the present invention with the immunne response that can excite the said peptide of antagonism.

The invention still further relates to above-mentioned immunogenic peptide and be used as medicine in disease of for example treating the IgE mediation or the application in pathology such as transformation reactions and the atopic dermatitis.

The invention still further relates to a kind of disease or pathology such as transformation reactions to IgE mediation and Mammals is carried out immune method, comprise the above-mentioned immunogenic peptide that needs the patient treatment of this treatment significant quantity with atopic dermatitis.

Though immunogenic peptide of the present invention can not mediate acellular solvability histamine release basically, the antibody that it can excite the target amino acid sequence with epi-position A and/or epi-position B to have strong serological cross reaction property.

The initial dose of peptide (for example about 0.2mg-5mg) can for example give through intramuscularly, after 14-28 days, repeats to give same dosage (reinforcement) subsequently.The dosage that gives will be decided according to patient's age, body weight and general situation certainly.Immunity can be active immunity or passive immunization.In active immunity, object is accepted immunogenic peptide of the present invention, and anti-IgE replys by the immunity system of object and initiatively induces.

Active immunity is preferred for human body, but also can other mammalian species of similar processing, for example dog.Term among this paper " immunogenic carrier " comprises having those materials that in host animal, independently excite immunne response character; It can and free carboxy, amino or the hydroxyl of polypeptide through in polypeptide and immunogenic carrier material on form peptide or ester bond between the corresponding group and direct covalent coupling; Perhaps, perhaps form fusion rotein with polypeptide by covalent coupling through the bonding of conventional difunctional linking group.

The instance of these immunogenic carriers comprises: BSA, like BSA; Sphaeroprotein; Thyroprotein; Oxyphorase; Hemocyanin (particularly keyhole chirp hemocyanin [KLH]); The protein that from roundworm, extracts, for example J.Immunol.111 [1973] 260-268, J.Immunol.122 [1979] 302-308, J.Immun.98 [1967] 893-900, described those roundworm extracts of Am.J.Physio.199 [1960] 575-578 or its purified product; Polylysine; Polyglutamic acid; The LYS-GLU multipolymer; The multipolymer that contains Methionin or ornithine; Or the like.Vaccine has used DT or Toxoid,tetanus to produce (Lepow M.L.etal., J.of Infectious Diseases150 [1984] 402-406 as the immunogenic carrier material; Coen Beuvery, E.etal., Infection and Immunity 40 [1983] 39-45), these toxin materials also can be used for the present invention.The purified proteins matter verivate (PPD) of tuberculin is particularly preferred in the active immunity scheme; Because: (1) himself not inducing T cell reply (being that it is effective T cell haptin), but work and therefore by the T cell recognition as the antigen of complete processing; (2) known its is one of the most powerful hapten-carrier in chain recognition mode; (3) it can be used for human body and need not further test.

The invention still further relates to coding peptide of the present invention polynucleotide, comprise the carrier of said polynucleotide and carry the cell of said carrier.In addition, active immunity can realize through the polynucleotide of the peptide of the present invention of encoding.The carrier that is suitable for this treatment known in the art comprises for example adenovirus carrier.

Passive immunization is to realize through the disease of suffering from the IgE mediation or patient's anti-IgE antibodies of the present invention of pathology.

These antibody can prepare through giving non-human mammal immunogenic peptide of the present invention and collect the gained antiserum(antisera).Through duplicate injection for some time tiring of can obtaining to improve.Excite the mammalian species of antibody not have particular restriction for can be used for; Usually preferred use rabbit or cavy, but also can use horse, cat, dog, goat, pig, rat, ox, sheep or the like.Collects after week by the blood of immune animal through 1-2 after giving the last time, centrifugal this blood and from blood separation of serum, obtain antibody thus.Monoclonal antibody can for example be people or murine antibody.

When object was carried out immunity, antibody of the present invention can import in the Mammals through for example intramuscularly.Yet, can use any type of antibody to give mode.Can use and can be object that accept and any conventional liq or solid vector that do not have adverse side effect.The phosphate buffered saline (PBS) (PBS) that is in physiological pH value (for example about pH 6.8-7.2, preferably approximately pH 7.0) can be used as vector separately or with suitable adjuvant, and said adjuvant is like the adjuvant based on white lake.

Provide following embodiment the present invention to be described and unrestricted meaning.

Embodiment

Embodiment 1: the humanization of anti-IgE mouse MAb TES-C21

Variable region of heavy chain (V with mouse mAb TES-C21 _H) and variable region of light chain (V _L) sequence compare with the people's antibody reproductive tract sequence that derives from the public data storehouse.When decision as the described template of above-mentioned step 1, adopt several criteria, comprise size of similar CDR position, whole homology, CDR in total length, the framework or the like.Whole all these standards of consideration can be selected best people's template, shown in the contrast of the series arrangement between TES-C21MAb heavy chain and sequence of light chain and the representative people's template sequence, shown in Fig. 3 A and 3B.

In this case, use more than one people's architecture template to design this antibody.For V _HPeople's template that chain is selected is the combination (seeing Fig. 3 B) of DP88 (1-95 amino acids residue) and JH4b (103-113 amino acids residue).For V _LPeople's template that chain is selected is the combination (seeing Fig. 3 A) of L16 (the inferior group of VK III, 1-87 amino acids residue) and JK4 (98-107 amino acids residue).Framework homology between mouse sequence and the people's template is for V _HFor about 70%, for V _LBe about 74%.

In case selected template, then through making up the Fab library with the synthetic and overlapping PCR method of DNA shown in Figure 2 as stated.Said library is by forming with the people's template DP88/JH4b and the L16/JK4 synthetic TES-C21CDR that select respectively.The complicacy in library is 4096 (=2 ¹²).Encoding part V _HAnd V _LThe overlapping oligonucleotide of sequence is at about 63-76 Nucleotide scope synthetic, and it is overlapping to have 18-21 Nucleotide.

To V _LAnd V _HGene carries out pcr amplification, uses to contain the sequence that is specific to framework region FR1 and annealed and carry out under Standard PC R condition to the biotinylated forward primer of the outstanding sequence of leader sequence (GeneIII) end with from the reverse primer of the constant region of guarding (C κ or CH1).The PCR product is through the agarose gel electrophoresis purifying, and the perhaps PCR purification kit purifying through being purchased is to remove uncorporated biotinylated primer and non-specific PCR.

Using ddH ₂O is adjusted to and uses 2 μ g PCR products, 1 μ L T4 polynucleotide kinase (10 units/μ L), 2 μ L, 10 * PNK damping fluid, 1 μ L 10mM ATP that the PCR product is carried out 5 ' phosphorylation in the TV of 20 μ L.After 10 minutes, add ddH 37 ℃ of incubations 45 minutes and 65 ℃ of thermally denatures ₂O is adjusted to 200 μ L to carry out next step with reaction volume.

The magnetic bead that 100 μ L streptavidins are encapsulated is resuspended in 200 μ L, 2 * B&W damping fluid with 200 μ L 2x B&W damping fluid washed twice.The PCR product of phosphorylation is mixed with pearl, shook incubation gently 16 minutes in room temperature (RT).

With the pearl deposition and with 200 μ L, 2 * B&W damping fluid washed twice.Not biotinylated ssDNA (minus strand) shook wash-out 10 minutes with the 0.15M NaOH of 300 μ L prepared fresh in room temperature gently.The NaOH wash-out can slightly increase output (choosing wantonly) again.Eluate is centrifugal to remove the pearl of any trace.

The 3M NaOAc (pH 5.2) through adding 1 μ L glycogen (10mg/mL), 1/10 volume and the EtOH of 2.5 volumes precipitate ssDNA from supernatant.Then sedimentary ssDNA is washed with 70%EtOH, freeze-drying subsequently 3 minutes also is dissolved in 20 μ L ddH ₂Among the O.Through having on bromination second pyridine (EtBr) agarose plate of DNA standard substance point sample (spotting) or through measuring OD ₂₆₀SsDNA is quantized.

Embodiment 2:V _HAnd V _LThe clone advances phage expression vector

Through hybridizing mutagenesis with V _HAnd V _LThe clone advances in the phage expression vector.Uridine acidifying template infects CJ236 coli strain (dut through using the phage (phage expression vector TN003) based on M13 ^-Ung ^-) and prepare.

With following composition [200ng uridine acidifying phage vector (8.49kb); 92ng phosphorylation strand H chain (489 bases); 100ng phosphorylation strand L chain (525 bases); 1 μ L10 * annealing buffer is used ddH ₂O is 10 μ L with volume-adjustment] through with temperature maintenance 85 ℃ 5 minutes (sex change), gradient is reduced to 55 ℃ PCR and anneals (inserting body and carrier is about 8 times of molar ratios) among 1 hour then.With sample in cold shock on ice.

In the annealing product, add following composition: 1.4 μ L 10 * synthetic damping fluid, 0.5 μ L T4DNA ligase enzyme (1 unit/μ L), 1 μ L T4 archaeal dna polymerase (1 unit/μ L), subsequently incubation on ice 5 minutes, 37 ℃ of incubations 1.5 hours.Then with product through ethanol sedimentation, be dissolved in 10 μ L ddH ₂Among O or the TE.

With DNA with 1 μ L Xbal (10 units/μ L) digestion 2 hours, 65 ℃ of heated and inactivated 20 minutes.The DNA of digestion is advanced in the 50 μ L electroreception attitude DH10B cells through electroporation transfection.The gained phage through 37 ℃ on XL-1Blue bacterium lawn grow overnight confirm to tire.The clone is checked order to confirm its composition.

Embodiment 3: deep hole is cultivated to carry out library screening

A: bed board phage library

Phage library is diluted in the LB substratum to reach each dull and stereotyped phage number of hoping.The phage that height is tired mixes with 200 μ L XL-1B cell cultures.Mix the 3mLLB top-layer agar, pour on the LB flat board, left standstill 10 minutes in room temperature.Should be incubated overnight at 37 ℃ by flat board.

B: phage wash-out

In each hole that 96 holes at the bottom of the aseptic U-shaped of 100 μ L phage elution buffers (10mM Tris-CI, pH7.5,10mM EDTA, 100mM NaCl) adding are dull and stereotyped.To move in the hole with filtering the transfer pipet point from the dull and stereotyped single phage plaque in the library of spending the night.With this phage wash-out flat board 37 ℃ of incubations 1 hour.Behind the incubation with flat board 4 ℃ of storages.

C: deep hole is dull and stereotyped to be cultivated

To add in 2 * YT substratum with 1: 100 extent of dilution from the XL1B cell of 50mL culture.Cell is grown until A in shaking table at 37 ℃ ₆₀₀Reach 0.9-1.2.

C: in the deep hole flat board, use phage-infect

When cell reaches suitable OD, in the XL1B culture, add 1M IPTG (1: 2000).The final concentration of IPTG is 0.5mM.750 μ L cell cultures are moved in each hole of 96 hole depth holes dull and stereotyped (Fisher Scientific).The phage of 25 μ L wash-outs is inoculated in each hole.This deep hole flat board is placed shaking table (250rpm), be incubated overnight at 37 ℃.

D: the preparation supernatant is to carry out the ELISA screening

Behind incubation, use the dull and stereotyped rotor of Beckman JA-5.3 3 the deep hole flat board, centrifugal 20 minutes of 250rpm.From each hole, extract 50 μ L supernatants and carry out ELISA.

The inoculation of E:15mL XL-1 cell liquid culture

XL-1 is grown until A in the 2 * YT that contains 10 μ g/mL tsiklomitsins in shaking table (250rpm) at 37 ℃ ₆₀₀=0.9 to 1.2.Adding final concentration is the IPTG of 0.5mM, and the 15mL culture is moved in the 50mL tapered tube to identify each clone.Pair cell inoculate 10 μ L from height tire (tire=about 10 ¹¹Pfu/mL) phage of original seed and 37 ℃ of incubations 1 hour.Cell is shaken grow overnight in room temperature.

F: the separation of soluble Fab from pericentral siphon

With cell in the IEC whizzer with 4, centrifugal 20 minutes of 500rpm and precipitating.Remove substratum and deposition is resuspended in the resuspended damping fluid of 650 μ L (50mM Tris, pH 8.0, contain 1mM EDTA and 500mM sucrose), carry out vortex (vortex), place and shook gently on ice 1 hour.Cell debris through 4 ℃ with 9, centrifugal 10 minutes of 000rpm and removing.Collection contains the supernatant of soluble Fab and 4 ℃ of storages.

Embodiment 4: framework is modified

Above-mentioned potential key position has 12 mouse/people to become even residue (wobble residue) in framework.V _HIn the 73rd in the humanization library, to keep be the mouse threonine residues combine because confirm this position influence.Yet, notice that the Threonine at VH73 is to be total to somebody's residue in the

inferior group

1 and 2 of people's reproductive tract VH.

Framework residues different between TES-C21 sequence and people's template are replaced as above-mentioned at random, estimate it then to target combination and the folding potential impact of antibody.Discriminating can influence the potential framework residue of bonded.In this case, said residue is V _HIn the 12nd, 27,43,48,67,69 residue, V _LIn the 1st, 3,4,49,60,85 residue (Kabat numbering system) (see figure 4).Show subsequently and have only V _HThe the 27th and the 69th residue remarkably influenced combines (clone 1136-2C) in the zone.

The elementary screening of using is to use the single-point ELISA (SPE) (as following) of substratum.The clone of the target molecule of binding antibody has been selected in said elementary screening.Selection provides to compare with the parental generation molecule and has equated or the clone of better signal carries out the screening of next round.

Take turns in the screening second, each phage is grown in the 15ml bacterial cultures, and the pericentral siphon prepared product is used for SPE and ELISA titration determination.To keeping higher bonded clone further to identify in measuring at this.In case all selected elementary clones have all passed through this process, the highest 10-15% clone is checked order and according to sequence the clone arranged.The representative sequence of each sequence set is contrasted and selects best clone each other.Sequence to the clone of these selections makes up, and estimates the effect of various combinations.

The ELISA screening is carried out with improvement and recombinant human IgE, the combination of SE44 in the library that makes up.Separation and combination avidity is higher than clone and the order-checking of mouse TES-C21 Fab, and

clone ID#

4,49,72,76 and 136 is

further identified.Clone

4,49,72,78 and 136 ELISA titration curve is shown in Fig. 5 A and 5B, shows that its avidity is similar with parental generation TES-C21.These clones combine people IgE with mouse TES-C21 competition, are illustrated in to combine epi-position not change during humanization is handled.Humanized Fab debond is combined in the IgE on the FcsRI, and when prompting made up among the divalence IgG when it, the crosslinked histamine release that causes of humanized antibody and acceptor was unlikely

Humanization clone 136 keeps 5 mouse heavy chain framework residue (=94.3% people V _HAnd 100% people's light chain framework of selecting for affinity maturation the framework homology).This paper has shown that humanization Fab combines the restraining effect (Fig. 6) of Fc ε RI to IgE.

Embodiment 5: the single-point ELISA scheme that is used to screen anti-IgE

Flat board is used for 2 μ g/mL sheep anti people Fd that carbonate encapsulates damping fluid to encapsulate at 4 ℃ and spends the night.Remove and encapsulate solution, flat board is used in 37 ℃ with 200 μ L/ hole 3%BSA/PBS sealing 1 hour.After dull and stereotyped 4 times of PBS/0.1%TWEEN (PBST) washing, add 50 μ L/ hole Fab samples (promptly contain height tire the supernatant of phage and excretory Fab or the pericentral siphon prep that DMB seals, perhaps 15mL prep).With flat board room temperature incubation 1 hour, subsequently with PBST washing 4 times.Add the biotinylated SE44 in 50 μ L/ holes (the 0.015 μ g/mL that in 0.5%BSA/PBS, dilutes); Add then 0.05% with flat board room temperature incubation 2 hours, with PBST washing 4 times.Add the mould antibiotin HRP of 50 μ L/ pore chains (in 0.5%BSA/PBS 1: 2000 dilution) and add 0.05%

with flat board room temperature incubation 1 hour.Flat board is washed 6 times with PBST.Add the colour developing of 50 μ L/ hole tmb substrates (sigma), add 50 μ L/ hole 0.2M H then ₂SO ₄Stop.

Embodiment 6:ELISA titration: anti-IgE

Flat board is used for 0.25 μ g/mL (Fab for purifying the is 0.1 μ g/mL) SE44 that carbonate encapsulates damping fluid to encapsulate at 4 ℃ and spends the night.Remove and encapsulate solution, flat board was sealed 1 hour at 37 ℃ with 200 μ L/ hole 3%BSA/PBS.

Flat board (PBST) is washed 4 times with PBS/0.1%

.Add 50 μ L/ hole Fab (from 15mL pericentral siphon prep); The Fab that is added was since dilution in 1: 2, and serial dilution is 3 times in 0.5%BSA/PBS and 0.05%

.With flat board room temperature incubation 2 hours.

Flat board with PBST washing 4 times, is added the vitamin H-sheep anti people Fd of 50 μ L/ holes, 1: 1000 (0.8 μ g/ml) dilution in 0.5%BSA/PBS and 0.05%

.With flat board at room temperature incubation 2 hours once more.

After with PBST washing 4 times; Be incorporated among the 0.5%BSA/PBS and 0.05%

50 μ L/ hole Neutra-avidin-AP (0.9 μ g/mL) of dilution in 1: 2000, with flat board room temperature incubation 1 hour.

Flat board with PBST washing 4 times, is added 50 μ L/ hole pNPP substrate colour developings, add 50 μ L/ hole 3M NaOH color development stopping.Absorbancy in 405nm or the every hole of 410nm reading.

Embodiment 7: the scheme of the solvable Fab of affinity purification M13 phage expression

A: first day

With two 500mL cultures that contain the 10mg/mL tsiklomitsin (2 * YT) inoculation 5mL spend the night original seed XL1B and 37 ℃ of growths until A600=0.9-1.2.Add IPTG to concentration be 0.5mM.Then with each cell culture with 200 μ L phage-infects, shook incubation 1 hour at 37 ℃.After the infection, cell is shaken grow overnight at 25 ℃.

B: second day

With cell in the 250mL centrifuge tube 4 ℃ with 3500 * g centrifugal 30 minutes.The sucking-off substratum also is resuspended in deposition in the lysis buffer (buffer A+protease inhibitor cocktail) of common 12-15mL.

Buffer A (1 liter):

50mM NaH ₂PO ₄6.9g NaH ₂PO ₄H ₂O (perhaps 6g NaH ₂PO ₄)

300mM?NaCl 17.54g?NaCl

10mM imidazoles 0.68g imidazoles (MW 68.08)

Using NaOH is 8.0 with pH regulator.

Lysis buffer:

With 25mL buffer A and a slice adequate proteins enzyme inhibitors mixture (Roche, Basel, Switzerland) mixing.

Resuspended cell is moved in the 50mL tapered tube, use 100 μ l 100mg/mL N,O-Diacetylmuramidases to drip motion together (because due to cracking) as group and with lysis until mixture several times through managing upset.Cell is carried out supersound process on ice, add 10 μ L DNase I (about 1000 units) subsequently, shook gently 30 minutes at 4 ℃.Cell debris is through using the 50mL centrifuge tube to precipitate in centrifugal 30 minutes with 12000 * g at 4 ℃.Supernatant is moved in the new tapered tube, 4 ℃ of storages.

(Qiagen, Valencis CA) instruct the soluble Fab of purifying according to manufacturer to use the Ni-NT agarose.With lysate mix with Ni-NTA and application of sample in post.Collect elute and carry out the SDS-PAGE analysis.With post with 20mL damping fluid (50mM NaH ₂PO ₄, 300mM NaCl, the 15mM imidazoles, using NaOH is 8.0 with pH regulator) wash, use the 50mM NaH of 20mL subsequently ₂PO ₄, 300mM NaCl, the washing of 20mM imidazoles.Fab is with 500 μ L elution buffer (50mM NaH ₂PO ₄, 300mM NaCl, the 450mM imidazoles, using NaOH is 8.0 with pH regulator) and wash-out 6 times, and analyze through SDS PAGE.The post level is divided 4 ℃ of storages.Through SDS-PAGE analytical column level branch, the level of selecting to have maximum Fab divide and in PBS 4 ℃ of dialysis.

Embodiment 8: soluble receptor determination

The 96 hole assay plates that will be suitable for ELISA encapsulate damping fluid (50mM carbonate, pH 9.6) and encapsulated 12 hours at 4-8 ℃ with 0.05mL 0.5 μ g/mL Fc ε RI α-chain acceptor.Aspirate said hole, and add 250 μ L sealing damping fluid (pH 7.2 for PBS, 1%BSA), 37 ℃ of incubations 1 hour.In an independent assay plate; Sample and with reference to TES-C21MAb through with measuring damping fluid (0.5%BSA and 0.05%Tween 20; PBS; PH 7.2) with 1: 4 the dilution and carry out titration from 200 μ g/mL to 0.001 μ g/mL, add the biotinylated IgE of isopyknic 100ng/mL, and with flat board at 25 ℃ of incubation 2-3 hours.The hole that Fc ε Rl encapsulates is washed 3 times with PBS and 0.05%TWEEN 20, shift out 50 μ L, stirred incubations 30 minutes at 25 ℃ from sample well.Will be in measuring damping fluid stir incubation 30 minutes, then with flat board such as aforementioned washing with the 1mg/mL streptavidin-HRP in 50 μ L/ holes of dilution in 1: 2000.The tmb substrate colour developing that adds 50 μ L/ holes.Reaction is through adding isopyknic 0.2M H ₂SO ₄And stop, measure absorbancy at 450nm.

Embodiment 9: the Fc ε RI of antibody and load IgE combines

In conjunction with the antibody of the associating people IgE of α subunit of Fc ε RI through confirming in 30 minutes 4 ℃ of preincubation with 10 μ g/mL people IgE.With flat board washing 3 times, subsequently with the mouse anti human IgE MAb E-10-10 of different concns or humanized Fab variant incubation 1 hour.Use biotin labeled anti-people Fd antibody, detect the combination of Fab subsequently through SA-HRP.Mouse MAb E-10-10 detects through the Ab that goat anti mouse Ig FcHRP-puts together.

Embodiment 10: the clone identifies

Measure the binding affinity of each material standed for, and positive colony is checked order.Antibody variants in the CDR zone, having the useful sudden change that increases binding affinity is further identified.Mensuration comprises that Biacore measures, the inhibition of IgE and its receptors bind, and the IgE's of receptors bind is crosslinked.

Produced a variant library.The aminoacid sequence that warp is proved the various CDR of the avidity with improvement is shown in table 1.Fig. 7 illustrates the high-affinity material standed for substituted combination.

Table 1

The P=parental generation

19 heavy chain variants are shown in Fig. 9, and 35 light chain variants are shown in Fig. 8.3 material standed fors are further identified binding affinity, and these material standed fors are shown in table 2.

Table 2 binding affinity

Mab	K _D	The increase multiple of binding affinity
			TES-C21	614±200pM	?
MAb?1(CL-5A)	0.158pM	3886
			MAb?2(CL-2C)	1.47±0.5pM	417
MAb?3(CL-5I)	3.2±2.2pM	191

Embodiment 11: expression that anti-IgE antibodies and HRP put together and purifying

Produced high-affinity MAb material standed for.In order to produce complete anti-IgE MAb, with heavy chain and variable region of light chain from the phage vector template through pcr amplification, and independent subclone advances in H-and the L-chain expression vector under the CMV promoter expression.Make up 6 complete antibody clones, shown in Figure 10 A-F.Through electroporation technology well known in the art suitable heavy chain and light chain plasmid co-transfection are advanced among the mouse myeloma cell line NS0.See that for example Liou et al.J Immunol.143 (12): 3967-75 (1989) is said.Use albumin A sepharose (Pharmacia) antibody purification from single stable clone supernatant.The concentration of antibody uses spectrophotometer to confirm at 280nm and FCA detection (IDEXX).

(Zymed Labs, San Francisco CA) put together antibody purified according to manufacturers protocol with horseradish peroxidase (HRP) with the peroxidase conjugated test kit.Tiring of every kind of anti-IgE MAb that puts together confirmed through the flat board that monoclonal human IgE (SE44) encapsulates with ELISA.

Following culture has been preserved in American type culture collection (American Type Culture Collection, 10801 University Boulevard, Manassas Va.20110-2209 USA (ATCC)):

Hybridoma	The ATCC numbering	Preservation date
			Anti-IgE CL-2C	PTA-5678	On December 3rd, 2003
Anti-IgE CL-5A	PTA-5679	On December 3rd, 2003
			Anti-IgE CL-5I	PTA-5680	On December 3rd, 2003

This preservation is to be used for the microbial preservation budapest treaty of patented procedure and the relevant regulations of detailed rules and regulations (budapest treaty) is carried out according to international recognition.This has guaranteed from preservation, to keep this work culture 30 years.Under the scope of Budapest agreement, can obtain this organism through ATCC, this has guaranteed that the public can obtain the offspring of this culture for good and all, without restriction after relevant USP mandate.

The application's transferee agreed if dead or lose or when being compromised by the preservation culture when what under conditions suitable, cultivate, will be promptly after by notice with the live body sample replacement of identical culture.The acquisition of preservation strain is the permission of conduct embodiment of the present invention under the right that any responsible departments of the government of violation give according to its patent law not.

The specification sheets of first above written it is believed that is enough to make that those skilled in the art can embodiment of the present invention.The present invention does not receive the qualification of scope of the culture of institute's preservation, because the embodiment of institute's preservation is just as illustrating one aspect of the present invention, culture of equal value on any function is all within scope of the present invention.The preserved material of this paper does not constitute that the contained description of being write is not enough to put into practice admitting of any aspect of the present invention (comprising optimal mode of the present invention) to this paper, and it is not used to the scope of these claims is limited to specific the illustrating that this paper appears yet.In fact, through top description, to of the present invention various except the modification shown in this paper with said will be conspicuous for those skilled in the art, and all within the scope of claims of appendix.

Embodiment 12: the high-affinity of people IgE combines the location of epi-position

A: the synthetic and anti-IgE of peptide combines to measure

Research illustrates IgE and passes through C _H3 structural domains combine its acceptor.Because HA of the present invention is anti--and IgE antibody blocks IgE and its receptors bind very effectively, and the peptide that whole CH3 structural domains are contained in use positions epi-position.At first, the peptide of two V5 marks of preparation, whole constant regions that comprise people IgE, a C who only comprises people IgE _H2-C _H3 zones.These two peptides are through expressing in in-vitro transcription-translation, and are used for the Western trace and measure to detect the anti-IgE Mab of HA and combine.CL-2C and CL-5I MAb all can combine complete people IgE and this two peptides.

In order to locate epi-position more specifically, the synthetic 141-368 amino acids residue that comprises people IgE (comprises whole C _H3 structural domains) 73 overlapping peptides.Each peptide is formed by 12 amino-acid residues, at 3 ' terminal overlapping 3 amino acid of peptide before.With fluorenylmethyloxycarbonyl (fluorenylmethoxycarboyl, Fmoc) amino acid synthetic SPOT film on cellulose membrane.With the rinsing in methyl alcohol of this film, in TBS (pH 7.5), washed 3 times 10 minutes then.After in lock solution (5% milk or the 3%BSA in TBS), being incubated overnight, the anti-IgE Mab of the HRP mark that will in lock solution, dilute and this film incubation 3 hours.Washing is used SuperSignal HRP substrate (Pierce) to be exposed to BioMax MS film (Kodak) desired time through chemoluminescence and is measured the IgE reactivity after 3 times 15 minutes in TBS-TWEEN

.

Experimental result shows that the anti-IgE Mab of HA combines the C of IgE _HTwo zones in 3 parts are represented with following two peptide sequences; NPRGVSAYLSRP (epi-position A) and HPHLPRALMRST (epi-position B) (seeing Figure 12).With a little less than the combining of the binding ratio of epi-position A and epi-position B several times.

B: alanine scanning mutagenesis

Carry out L-Ala scanning along the substituted amino acid of those peptides of in IgG1, finding, with confirm which amino acid for the anti-IgE MAb of HA and these peptides to combine be crucial.Through confirming that combining for the anti-IgE MAb of HA is that important amino acid uses the vitro mutagenesis method in the ε of IgE chain displace.Also use the another kind of peptide (seeing Figure 13 and 14) in foregoing covering C ε 2 and C ε 3 zones in this research.

Use is for the EU numbering plan of people IgE amino-acid residue.Use whole Fc zone of polymerase chain reaction (PCR) amplification IgE, and only contain the IgE Fc of the clipped form of CH2-CH3 structural domain.With the DNA product directly the clone advance the pcDNA3 expression vector (Invitrogene, Carlsbad, CA) in, (Invitrogene, Carlsbad CA) carry out to use TOPO cloning.

Use overlapping PCR (Ho et al., 1989) in IgE-Fc, to carry out mutagenesis.Through the agarose gel electrophoresis purifying, with suitable restriction enzyme digestion, and subclone advances in the pcDNA3 expression vector with the DNA product.For each variant construct, the order-checking fully from two chains of DNA of dideoxy nucleotide method is used in the zone of pcr amplification.Recombinant human IgE Fc and two mutants thereof use the in-vitro transcription and translation coupling system (Promega, Madison, WI) expression based on reticulocyte lysate.

Carry out SDS-PAGE (12%) with deriving from the lysate (10 μ l reaction mixture) of this in-vitro transcription, move on the nitrocellulose membrane then with the translation coupling system.5% milk power solution that this film is used for Tris BS (TBS) seals, subsequently with the anti-IgE MAb dyeing of primary antibody.The specific reaction band uses the goat anti human IgG Fc that puts together with horseradish peroxidase, and (Jackson Labs, Bar Harbor Maine) detect, and the immunoreactivity band uses SuperSignal Western trace detection kit (Pierce) observation.Anti-V5 antibody detects the V5 mark in the C-terminal importing of these peptides as positive control.The Western trace of anti-V5 antibody shows that all peptides are all at almost equal horizontal expression.Interesting ground, the anti-IgE MAb of HA can be combined in the peptide that epi-position A has sudden change, but their debonds have the peptide of sudden change at epi-position B, and this shows that this second site is for combining more important (seeing Figure 15).

Embodiment 13: use the active immunity of the immunogenic peptide of epi-position B to transgenic mice

The active of antibody of using the transgenic mice of constitutive expression people IgE to prove people's immunogenic peptide of epi-position B produces.Two fusogenic peptides of chemosynthesis, each peptide all comprises immunogenic peptide of the present invention, cysteine residues and KLH.The sequence of peptide 1 is:

(KLH-Cys)-Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr，

The sequence of peptide 2 is:

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr-(Cys-KLH)。

With transgenic mice in the PBS pH 7.4 of subcutaneous injection 200 μ L in complete Freund's adjuvant (Difco Laboratories, Detroit, 20 μ g immunogenic peptides in MI).At interval 2 weeks, be the peptide based immunogens of the 20 μ gs of mouse secondary subcutaneous injection in incomplete Freund's adjuvant.Then, in two week backs and in execution preceding 3 days, be the mouse 20 μ gs identical immunogen of peritoneal injection in PBS once more.Collect serum and also test the existence of the anti-IgE antibodies that is specific to epi-position B.Shown in figure 16, said peptide excites anti-IgE antibodies in these transgenic mices.

Those skilled in the art use and are no more than many Equivalents that particular embodiment of the present invention described herein will be recognized or can be confirmed to normal experiment.This Equivalent is encompassed in following claims.

Claims

1. isolated antibody, the epi-position in its specificity binding amino acid sequence, said epi-position is selected from down group:

Leu?Pro?ArgAla?Leu?XaaArg?Ser?Xaa；

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr；

His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr; Perhaps

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr?Thr?Lys?Thr。

2. the antibody of claim 1, it further comprises a kind of mark.

3. claim 1 or 2 antibody, wherein said antibody is:

A) chimeric antibody,

B) single-chain antibody,

C) Fab fragment,

D) F (ab ') fragment,

E) people's antibody, perhaps

F) humanized antibody.

4. each antibody of claim 1-3, wherein said antibody is polyclonal antibody.

5. each antibody of claim 1-3, wherein said antibody is monoclonal antibody.

6. compsn, it comprises each antibody and a kind of physiology acceptable carrier, thinner, stablizer and/or vehicle of claim 1-5.

7. method for preparing antibody, said method comprises:

A) exciting immune animal under the condition of antibody response with the polypeptide that comprises anti--IgE epi-position, wherein said epi-position is made up of following aminoacid sequence:

Leu?Pro?Arg?Ala?Leu?Xaa?Arg?Ser?Xaa；

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr；

His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr; Perhaps

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr?Thr?Lys?Thr，

B) from said animal separation antibody and

C) antibody of the polypeptide of discriminating specificity integrating step (a).

8. antibody that can the method through claim 7 obtains.

9. compsn, it comprises antibody and a kind of physiology acceptable carrier, thinner, stablizer and/or the vehicle of claim 8.

10. one kind prepares monoclonal antibody method, and said method comprises:

Leu?Pro?Arg?Ala?Leu?Xaa?Arg?Ser?Xaa；

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr；

His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr; Perhaps

Leu?Pro?Arg?Ala?Leu?Met?Arg?Ser?Thr?Thr?Lys?Thr，

B) separate the cell that produces antibody from said animal,

C) with the cytogamy of the cell and the immortalization of said generation antibody, form the hybridoma that produces monoclonal antibody,

D) cultivate said hybridoma, and

E) the polypeptid specificity bonded monoclonal antibody of separation and step (a) from culture.

11. monoclonal antibody that can obtain through the method for claim 10.

12. a compsn, it comprises monoclonal antibody and a kind of physiology acceptable carrier, thinner, stablizer and/or the vehicle of claim 11.

13. each antibody of claim 1-5, wherein said antibody are isolating through screening Fab expression library.

14. each antibody of claim 1-5, wherein said antibody are isolating through screening combination Tegeline library.

15. coding claim 1-5,8,11,13 or 14 each the isolating nucleic acid of antibody.

16. comprise the carrier of the isolating nucleic acid of claim 15.

17. comprise the isolating host cell of the carrier of claim 16.

18. one kind comprises claim 1-5,8,11,13 or 14 each the test kits of antibody.

19. claim 1-5,8,11,13 or 14 each antibody are used for treating disease or the pathology of suffering from IgE mediation or have the disease of suffering from the IgE mediation or the application of the mammiferous medicine of pathology risk in preparation.

20. the application of claim 19, the disease or the pathology of wherein said IgE mediation are transformation reactions, asthma, rhinallergosis, atopic dermatitis, rubella or eczema.

21. the application of claim 19 or 20, wherein said Mammals is the people.