CN1926149A - Identification of novel IgE epitopes - Google Patents

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 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 immunoglobulin (Ig) (IgE antibody), and described B cellular exposure is divided into the plasmocyte of secretory antibody in allergen.The reaction of IgE inductive is the position that enters 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 sensitivity, be that IgE antibody combines the lip-deep Fc epsilon receptor of its constant region and mastocyte, the IgE that " overflows (spill-over) " then enter circulation and with the circulation of whole machine body in basophilic leukocyte and the receptors bind on the mastocyte of fixation of tissue.As bonded IgE during subsequently with allergen contact, the Fc epsilon receptor is crosslinked by allergenic combination, causes the cell threshing and disengages many transformation reactions media, as histamine, prostaglandin(PG), leukotrienes or the like.The release of these materials causes the typical clinical symptom of immediate hypersensitivity, described 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, by expressing IgE on cell surface, some B cells form the positive B cell of surperficial IgE (sIgE ⁺The B cell) " data base (memorypool) ".

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 also finds low-level Fc ε RI on people's Langerhans cell, dendritic cell and monocyte, 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 studies show that 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, described antibody is that the IgE isotype is specific, and therefore can be in conjunction with IgE.This scheme is based on regulating the IgE immunne response and transformation reactions is suppressed by decrement, described 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 the instant and long term to 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 the mastocyte RI direct or indirect adjusting of IgE antibody horizontal that is subjected to circulating.

Recently, WO 99/62550 discloses IgE molecule and segmental application, and these IgE molecules and fragment are in conjunction with the IgE binding site of Fc ε RI and Fc ε RII combining with blocking-up IgE and acceptor.Yet, also very limited to the effective methods of treatment that does not have harmful side effect of these allergic diseases.A kind of methods of treatment 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 that suppressing IgE is a kind of effective ways of treatment allergic disease with combining of its acceptor.

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 by 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 is a therapeutic antibodies and cause that IgE is crosslinked and excite histamine release to cause the anaphylactoid possibility of potential with combining of high-affinity receptor bonded IgE.

Therefore, can be used for treating allergic antibody can not with bonded IgE reaction on the mastocyte of sensitization and the basophilic leukocyte, but should keep identification sIgE ⁺The ability of B cell.This IgE isotype specific antibody is for example described in the U.S. Patent No. 5,449,760 at European patent No.EP0407392 and some United States Patent (USP)s 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 produces between duration of immunity and high-affinity IgE receptors bind, or by 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 in conjunction with 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 described 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 by the high-affinity antibody identification of specificity in conjunction with IgE.Can the peptide that these are new be used for mammiferous active immunity avidity to produce high-affinity antibody in vivo by giving these new peptides of Mammals.Described peptide epitopes also is used in and produces the high-affinity anti-IgE antibodies of specificity in conjunction with 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 Xaa Arg Xaa(SEQ ID NO.72)

The example 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 composition, and described composition comprises at least a described peptide and a kind of physiology acceptable carrier, thinner, stablizer or vehicle, and a kind of immunogenic carrier.Described immunogenic carrier can be for example BSA, KLH, Toxoid,tetanus and diphtheria toxoid.The invention still further relates to coding SEQ ID NO.72-77 polynucleotide, comprise the carrier of described polynucleotide and contain the cell of described carrier.

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

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

The present invention relates to suffer from the high-affinity antibody that the Mammals of the disease of IgE mediation or pathology produces with the peptide that comprises SEQ ID NO:72 and/or SEQ ID NO.74.Described high-affinity antibody can be people's antibody, humanized antibody or chimeric antibody.Described 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 is the synoptic diagram of used phage vector in antibody cloning and the screening.

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

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

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

Fig. 4 illustrates the tabulation of comparing the framework residue variant with high-affinity with parental generation TES-C21.

Fig. 5 A illustrates with the Fab of parental generation TES-C21 with B and compares with negative control group (5D12), clone 4,49,72,78 and 136 ELISA titration curve.

Fig. 6 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 A illustrates to have and causes 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.

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

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

Figure 13 illustrates the discriminating of important residue in the land of epi-position A.

Figure 14 illustrates the discriminating of important residue in the land of epi-position B.

Figure 15 illustrates the western engram analysis in conjunction with the MAb of mutant peptide.

Figure 16 is illustrated in the generation of anti-IgE antibodies in the transgenic animal of expressing human IgE.

Detailed Description Of The Invention

Definition

The implication that the term that uses among the application has those skilled in the art usually and the typical case understands. Yet the applicant wishes that following term has hereinafter specifically explanation.

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. Sequence and the reference nucleic acid sequence that can be interpreted as nucleotides about this term of nucleotide sequence present about at least 85% or 90% or 95% or 97% sequence homogeny.

Term " homogeny " or " homology " should be interpreted as after sequence being arranged contrast and importing breach (if need to reach the largest percentage homogeny of complete sequence) and do not think that any conservative replacement is the part of sequence homogeny, the percentage of the amino acid residue identical with the residue of the corresponding sequence that compares with it in the candidate sequence. N or C be terminal to be extended or inserts and all do not think and reduce homogeny or homology. The well known method and computer program that carries out the series arrangement contrast. The sequence homogeny can use sequence analysis software to measure.

Term " antibody " is used with implication the most widely, and contains especially monoclonal antibody (comprising full length monoclonal antibodies), polyclonal antibody and multiple specific antibody (for example bispecific antibody). Antibody (Ab) and immunoglobulin (Ig) (Ig) are the glycoprotein with same structure feature. Although antibody presents the binding specificity to specific target, immunoglobulin (Ig) comprises antibody and lacks other antibody sample molecule of target-specific. Natural antibody and immunoglobulin (Ig) normally about 150,000 daltonian allos tetramer glycoprotein are comprised 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 refers to have at least 10^-10, preferred 10^-12Those antibody of binding affinity.

As used herein, " anti human IgE antibody " refers to suppress or greatly reduce mode that IgE is combined with high-affinity receptor Fc ε RI in conjunction with the antibody of people IgE.

Some part that refers to the variable region about the term in the variable region of antibody " variable " is extensively different in the sequence of antibody, and is combination and the specificity institute foundation of each specific antibodies and its particular target. Yet changeability 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 β-folding configuration, are linked to each other by three CDR, form ring and link to each other with the beta sheet structure, and these rings consist of the part of beta sheet structure in some cases. CDR in every chain all closely links together by the FR district, and forms the target binding site (seeing that Kabat etc. is described) 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 " refers to 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 analog " 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 analog be stop or greatly reduce the IgE immunoglobulin molecules in conjunction with the mode of the ability of high-affinity receptor Fc ε RI in conjunction with the IgE immunoglobulin (Ig). As used herein, refer to Fv, F (ab) and F (ab ') about " function fragment " of antibody₂Fragment. " Fv " fragment is the minimum antibody fragment that contains complete target identification and binding site. This zone is by a heavy chain and the dimer (V that variable region of light chain forms of next-door neighbour's non-covalent combination_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 (half of Fv that perhaps only comprises three CDR that are specific to target) also has identification and in conjunction with the ability of target, but affinity is lower than complete binding site. " scFv " or " sFv " antibody fragment comprises the V of antibody_HAnd V_LDomain, wherein these domains are present in the single polypeptide chain. Normally, the Fv polypeptide further is included in V_HAnd V_LA peptide linker between the domain, it is so that sFv is formed for the structure of the hope of target combination.

The Fab fragment contains the constant region of light chain and first constant region (CH1) of heavy chain. The difference of Fab ' fragment and Fab fragment is to add several residues, comprise one or more cysteine from the antibody hinge region at the carboxyl terminal of heavy chain CH1 domain. F (ab ') fragment is by cracking F (ab ')₂The disulfide bond of the hinge cysteine of pepsin digestion product produces. Other chemical coupling mode of those skilled in the art's known antibodies fragment.

As used herein, term " monoclonal antibody " refers to derive from the antibody of the antibody of the basic homology of a group, namely comprises except may being each identical antibody the sudden change with the natural generation that exists very in a small amount. Monoclonal antibody is the antibody for the high degree of specificity of single target site. In addition, opposite from (polyclone) antibody preparations (its typical case comprises the different antibodies for different determinants (epi-position)) of routine, each monoclonal antibody is all for a single determinant on the target. Except its specificity, the advantage of monoclonal antibody is can cultivate by hybridoma to synthesize, and is not polluted by other immunoglobulin (Ig). Modifier " monoclonal " expression antibody derives from basically this feature of the antibody population of homogeneity, rather than refers to and need to produce this antibody by any specific process. For example, the technical point that can know by use of the monoclonal antibody used of the present invention is from from the phage antibody library. 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 by recombination method.

" humanization " form of inhuman (for example mouse) antibody is to contain chimeric immunoglobulin (Ig), immunoglobulin chain or its fragment derived from the minmal sequence of non-human immunoglobulin (for example Fv of antibody, Fab, Fab ', F (ab ')₂Fragment or other subsequence in conjunction with target). Generally speaking, humanized antibody comprises basically whole at least one, two variable regions of typical case, wherein all or basically all CDR zone all corresponding to those zones of non-human immunoglobulin, and all or basically all FR zones are those zones of human immunoglobulin(HIg) 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 immunoglobulin(HIg) 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 function 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 " refers to DNA is imported in the organism, and this DNA can be used as extra-chromosomal element or copies by 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 coded sequence in fact is expressed. Term " host cell of transfection " and " host cell of conversion " refer to wherein import the cell of DNA. Described cell is called " host cell ", and it can be prokaryotic or eukaryotic. Typical prokaryotic host cell comprises colibacillary various bacterial strain. Typical eukaryotic host cell is mammal 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 from host cell or from the species different with host cell, contains some external sources and some homologous dnas.

Term " carrier " refers to a kind of DNA construct, and it contains with energy so that the dna sequence dna that the suitable control sequence that described DNA expresses in suitable host operably is connected. These control sequences comprise the sequence of the promoter 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. Described carrier can be plasmid, phage particle, perhaps only is that potential genome inserts body. In case transform suitable host, described carrier can copy and irrespectively bring into play function with host genome, perhaps is integrated in the genome in some cases. In this manual, " plasmid " and " carrier " sometimes can Alternate, 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 as known in the art.

" control sequence " refers to express the necessary dna sequence dna of coded sequence that operably connects in specific host organisms. Be suitable for procaryotic control sequence and for example comprise promoter, randomly operon sequence, and ribosome bind site. The known genuine nucleus utilizes promoter, polyadenylation signal and enhancer. If it is expressed as the front albumen that participates in described polypeptide secretion, then the DNA of presequence (presequence) or secretion targeting sequencing can operably be connected with the DNA of polypeptide; If it affects transcribing of this sequence, then promoter or enhancer are operably to be connected with coded sequence; If perhaps it affects transcribing of this sequence, then ribosome bind site is operably to be connected with coded sequence; If perhaps it is positioned in and can promotes the position of translating, then ribosome bind site is operably to be connected with coded sequence. Normally, " operably connect " refers to that the dna sequence dna that connects is continuous, and is continuous in the situation of secretion targeting sequencing and is to separate read states (in reading phase). Yet it is continuous that enhancer needs not to be.

" mammal " treated refers to classify as mammiferous any animal, comprises people, domestic animal and farm-animals, non-human primate, reaches zoo animal, motion animal, perhaps pet such as dog, horse, cat, ox etc.

Be meant a peptide species that merges with " epi-position mark " at this paper used term " the epi-position mark " with regard to polypeptide.Described epi-position labeling polypeptide has enough residues can produce the epi-position of antibody at it to provide, yet is the enough short activity of not disturbing polypeptide thus.Described epi-position mark preferably also is fairly individual, thus antibody substantially 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.Example 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)) at 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 in the Fc zone that described epi-position mark can be the IgG molecule (for example IgG1, IgG2, IgG3 or IgG4), 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 composition, its can with molecule or protein for example antibody directly or indirectly put together.Described mark can be self detectable (for example labelled with radioisotope or fluorescent mark), perhaps can catalysis in the situation of enzyme labelling can be detected substrate compounds or the chemically changed of composition.

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), polyacrylamide, polystyrene, polyvinyl alcohol and silicone.In certain embodiments, according to the context of article, described solid phase can comprise the hole of assay plate; In other embodiments, described 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 comprising the pathology relevant with atopic allergy with the supersensitivity of sensitization, for example comprise 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, snakebite, food or drug-induced by for example honeybee also is encompassed in the scope of this term.

Production of antibodies

Initial or " parental generation " antibody can use the existing technology preparation that is used to produce these antibody in this area.These technology are known.Produce illustrative methods more detailed description in following chapters and sections of initial antibody.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 determined before producing high-affinity antibody of the present invention.In addition, antagonist can carry out other biologic activity 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 described antibody.

In order to screen antibody (for example blocking IgE and its those antibody of high-affinity receptor bonded) in conjunction with 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, EdHarlow and David Lane (1988)).Perhaps, can carry out epitope mapping to determine the position of the corresponding epi-position of antibodies.Randomly, antibody can use technology known in the art to determine for the binding affinity of the analogue (described 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 described antibody in preclinical study.Therefore, described species can be inhuman primates, as rhesus monkey, macaque, baboon, chimpanzee and macaque.In other embodiments, described species for example can be rodent, cat or dog.

The change according to the present invention of described 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, described surface (Jones, the S.﹠amp of coincideing that produces uniqueness complimentary to one another; Thornton, J.M. (1996) Proc.Natl.Acad.Sci.USA 93:13-20).By 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 by importing or improveing various noncovalent forces, and it finally reduces interactional energy and produces higher avidity.

Van der Waals force is noncovalent interaction (Voet, the D.﹠amp that takes place between two electric neutrality molecules; Voet, J.G. (1990) Biochemistry JohnWiley and Sons, NY, NY).By from electrostatic interaction permanent or that the inductive dipole produces, can associate between two surfaces.These dipoles can exist along the terminal of α spiral or near polare Aminosaeren.By 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 that participate 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.Therefore, a kind of mode that 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 exists in advantageous environment.By 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 by improvement.Therefore, by importing favourable contact and improveing the interface by 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 wish 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. acquisition or selection are in conjunction with the parental generation antibody that comprises heavy chain and variable region of light chain of interested target.This can be by 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 by producing random amino acid in each and each possible CDR position.Also can replace amino acid in people's architecture template at random with all possible amino acid, for example contiguous CDR or influence in conjunction with or folding amino acid, produce the library that framework replaces.These frameworks replace and can estimate target combination and the folding latent effect of antibody at it.Amino acid whose replacement can or be carried out in succession with the aminoacid replacement while among the CDR in the framework.A kind of method that produces the variant library is synthetic by 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.Contain these light chains and sequence of heavy chain carrier an example as shown in Figure 1.

5. screening is at the clone library of specific target, and screening in conjunction with those clones of target 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, as glycosylation site or the potential immunogenicity site that has 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 to stay people's framework integral body and produce high-affinity antibody by only replacing the CDR zone at random from people's parental generation antibody completely.

Because the high throughput screening technology and the carrier for example shown in Figure 1 of improvement, the technician can be rapidly and the comprehensive replacement library of screening all sites in given CDR and/or framework region effectively.By replacing all amino acid at random simultaneously in all positions, the technician can screen may making up of remarkable increase avidity, and this class combination is because synergy and can not be predicted or differentiate by single replacement for example.

The parental generation antibody preparation

The target preparation

Soluble target or its fragment can be as the immunogens that produces antibody.Described antibody is to produce at interested target.Preferably, described target is the important polypeptide of biology, and the Mammals that described antibody suffers from disease or functional disorder can be produced the treatment benefit in this Mammals.Yet, can produce antibody at non-polypeptide target.When described target was polypeptide, it can be a for example somatomedin 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.Described target can be that reorganization produces or use synthetic method to produce.Described 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), thyroglobulin, 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 be promoted inducing of strong immune response.

Polyclonal antibody

The relevant target that polyclonal antibody normally makes up by 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.

By 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 at target, immunogenic conjugate or derivative.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, described antibody can be in conjunction with the anti-IgE target of people, and binding affinity (Kd) numerical value is about 1 * 10 ^-8M.Affinity of antibody can be determined by 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 HarborLaboratory, the described mensuration of Ed Harlow and David Lane (1988).Perhaps, can carry out epitope mapping and determine combination, for example Champe et al.J.Biol.Chem.270:1388-1394 (1995) is described.

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 by recombinant DNA method.

In hybridoma method, with mouse or other appropriate host animal such as rodent such as above-mentionedly carry out immunity, produce or can produce the lymphocyte of specificity in conjunction with the proteinic antibody that is used for 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 described substratum.For example, if the parental generation myeloma cell lacks hypoxanthine-guaninephosphoribosyl transferase (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.Preferred myeloma cell is effective fusion, support the cytotostatic of the generation antibody selected to produce antibody and to those myeloma cells of substratum such as HAT substratum sensitivity high-levelly.Human myeloma and mouse-people's allos myeloma cell line is at the generation of human monoclonal antibodies and be 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 and by standard method growth (Goding by restriction dilution program, Monoclonal Antibodies:Principals and Practice, pp.59-103, Academic Press, 1986)).For this purpose, suitable medium comprises.By routine immunization sphaeroprotein purification process appropriate separation from substratum, described method is albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography for example by subclone excretory monoclonal antibody.

The DNA of coding monoclonal antibody is easy to use ordinary method to separate and order-checking (for example use can specificity in conjunction with the oligonucleotide probe of the gene of the heavy chain of coding monoclonal antibody and light chain).Described hybridoma is as the source of this DNA.After the separation, DNA can 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 described DNA also can for example pass through personnel selection heavy chain and constant region of light chain replaces homology mouse sequence (U.S. Patent No. 4,816,567; Morrison et al., Proc.Natl Acad.Sci.USA 81:6851 (1984)) or by 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; Riechmanet al., Nature332:323-327 (1988); Verhoeyens et al., Science 239:1534-1536 (1988)) method is carried out (seeing for example U.S. Patent No. 4,816,567) by replace in people's antibody corresponding sequence with inhuman CDR or CDR sequence.Put into practice as the present invention, humanized antibody can have number of C DR residue and some the FR residues by the residue replacement in similar site in the murine antibody.

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.The human sequence of approaching inhuman parental generation antibody is acknowledged as people's framework (Sims etal., the J.Immunol.151:2296 (1993) of humanized antibody then; 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 subgroup 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. by what the complete antibody of proteolysis produced, Journal ofBiochemical and Biophysical Methods 24:107-117 (1992) and Brennanetal., Science 229:81 (1985)).Yet these fragments can directly produce by 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 ') ₂Fragment (Carter etal., Bio/Technology 10:163-167 (1992)).According to another kind of method, F (ab ') ₂Fragment can directly be separated 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 patent application 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, can replace one or more framework residue in parental generation antibody, this antibody is for example improved 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 239 400 B1) 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 by 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 by 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 synthetic method, 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.By 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 by synthesis cycle isolating or subsequently.The method of synthetic oligonucleotide is known in the art, also can be purchased.The method that produces antibody variants from these oligonucleotide 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, described 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, determine the biologic activity of variant with respect to parental generation antibody.As mentioned above, this comprises the binding affinity of definite variant for target.The method that many high yields are arranged is with the ability of rapid screening antibody variants in conjunction with interested target.

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, described 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).From measuring, calculates SPR bonded equilibrium dissociation constant K _D, be expressed as k _Off/ k _OnBecause equilibrium dissociation constant K _DWith k _OffBe inversely proportional to, therefore by 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 to have enhanced still keeps hope in conjunction with active antibody variants therapeutic property.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 with the binding affinity that the is significantly higher than parental generation antibody ability in conjunction with target.

So the antibody variants of selecting can further be modified according to earmarking of antibody usually.These modifications can comprise further change aminoacid sequence, merge and/or those covalent modifications as described below with heterologous polypeptide.For example, any cysteine residues that does not participate 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 described nucleic acid, and produces the recombinant technology of described 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 (for example by use can specificity in conjunction with the oligonucleotide probe of the gene of the heavy chain of 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, do not show on the bacterial cell surface, 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.Described variant can also be expressed as the fusion polypeptide that merges with heterologous polypeptide, and described 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 and processing natural antibody signal sequence, signal sequence can the prokaryotic signal sequence of alkaline phosphatase, 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 kluyveromyces (Kluyveromyces) α-factor leader sequence) or acid phosphatase leader sequence, Candida albicans (C.albicans) glucoamylase leader sequence, and perhaps for example WO 90/13646 described signal replaces.In mammalian cell expression, can utilize for example herpes simplex gD signal of obtainable mammalian signal sequence and viral secretory leader sequence.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 described 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, described 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 example of selection scheme is the growth that utilizes the drug block host cell.Authorize the protein of drug resistance with those cells generations that heterologous gene successfully transforms, and therefore in selection scheme, survive.The example that this dominance is selected is to use medicine Xin Meisu, mycophenolic acid and Totomycin.

The example 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, as DHFR, thymidine kinase, metallothionein(MT) 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 by 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 by 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 (U.S. Patent 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 the selective marker that is used for lacking 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 described.The existence of trp1 damage provides by 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 by carrying the known plasmid complementary of Leu2 gene.

D: promotor composition

Express and cloning vector contains the promotor of being discerned 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, alkaline phosphatase, 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 upstream base place.Another sequence at many gene transcription section start upstream 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 may be the signal that adds poly-A afterbody at 3 ' end of encoding sequence.All these sequences all are fit to insert in the carrier for expression of eukaryon.

The example 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 as Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, 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 by growth conditions control) is alcoholdehydrogenase 2, cytochrome C (isocytochrome C), acid phosphatase, degrading enzyme, metallothionein(MT), the glyceraldehyde-3-phosphate dehydrogenase relevant with nitrogen metabolism and is used for maltose 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 (described virus for example is polyomavirus, avipoxvirus, adenovirus (as 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 in U.S. Patent No. 4,419, describe in 446.To this system be modified in U.S. Patent 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 by insert enhancer sequence in carrier of higher eucaryotic cells.Known many enhancer sequence (sphaeroprotein, elastoser, albumin, alpha-fetoprotein and Regular Insulin) from mammalian genes at present.Yet, typically, can use enhanser from eukaryotic cell virus.Described 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.Described 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 (karyocyte 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 transcribing as the polyadenylation fragment is contained in these zones in the untranslated part of the mRNA of encoding said antibody.A kind of useful Transcription Termination composition is Trobest polyadenylation zone.See that for example WO94/11026 is described.

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 microorganism such as eucaryon host such as grade.Yet many other Pseudomonas, bacterial classification and bacterial strain also can obtain and utilize in this article, as schizosaccharomyces pombe (Schizosaccharomycespombe); Kluyveromyces (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 as Aspergillus nidulans (A.nidulans) and aspergillus niger (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 example of invertebral zooblast comprises plant and insect cell, Luckow et al., and Bio/Technology 6,47-55 (1988); Miller et al., GeneticEngineering, 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 (Drosophilamelanogaster) (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 (Autographacalifornica NPV) and Bombyx mori nuclear polyhydrosis virus (Bombyx moriNPV) 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, potato, 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 described.The example of useful mammalian host cell line be monkey-kidney cells system, human embryonic kidney cell system, baby hamster kidney cell, Chinese hamster ovary cell/-DHFR (CHO Urlaubet al., Proc.Natl.Acad.Sci.USA 77:4216 (1980)), mouse support (sertoli) cell, human cervical carcinoma cell (HELA), Madin-Darby canine kidney(cell line), 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 ' sMedium.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 or 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 somatomedins (as Regular Insulin, transferrin or Urogastron) as required, (as the X-muriate, wherein X is sodium, calcium, magnesium to salt; And phosphoric acid salt), damping fluid (as HEPES), Nucleotide (as adenosine and thymidine), microbiotic (as 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 to produce,, can for example remove the particulate fragment of host cell or crack fragment by centrifugal or ultrafiltration as the first step in cell.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 phenylmethylsulfonyl fluoride (PMSF).Cell debris can be removed by 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 for preparing from cell 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 accompanying matrix of affinity ligand is agarose normally, but also can utilize other matrix.The matrix of mechanically stable such as controlled pore glass or poly-(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, as 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 (as the poly aspartic acid 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 described polypeptide or antibody, storing as freeze dried preparation or aqueous solution form, described 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 by having the polypeptide of wishing purity.For example, and buffer reagent, stablizer, sanitas, isotonic agent, nonionic detergent, antioxidant and other various additives (see Remington ' s Pharmaceutical Sciences, 16thedition, A.Osol, Ed. (1980) is described).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, as the citrate buffer (mixture of sodium dihydrogen citrate and disodium citrate for example, the mixture of citric acid and trisodium citrate, mixture of citric acid and sodium dihydrogen citrate or the like), succinate damping fluid (succsinic acid-succsinic acid one sodium mixture for example, succsinic acid-sodium hydroxide mixture, succsinic acid-disodium succinate mixture or the like), tartrate buffer (tartrate-sodium tartrate mixture for example, 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 (fumaric acid-fumaric acid one sodium mixture for example, fumaric acid-fumaric acid disodium mixture, fumaric acid one sodium-fumaric acid disodium mixture or the like), gluconate damping fluid (glyconic acid-gluconic acid sodium salt mixture for example, glyconic acid-sodium hydroxide mixture, glyconic acid-potassium gluconate mixture or the like), oxalate damping fluid (oxalic acid-sodium oxalate mixture for example, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture or the like), lactate buffer (lactic acid-Sodium.alpha.-hydroxypropionate mixture for example, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate 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 isotonicity of liquid composition of the present invention, isotonic agent comprises the poly-hydroxy sugar alcohol, preferably trihydroxy-or more poly-hydroxy sugar alcohol are as glycerine, erythritol, arabitol, Xylitol, Sorbitol Powder and mannitol.

Stablizer is meant a wide class 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 mentioned above); Amino acid such as arginine, Methionin, glycine, glutamine, l-asparagine, Histidine, L-Ala, ornithine, L-leucine, 2-phenylalanine, L-glutamic acid, Threonine etc., organic sugar or sugar alcohol, as 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 as 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 immunoglobulin (Ig); Hydrophilic polymer is as polyvinylpyrrolidone; Monose is as wood sugar, seminose, fructose, glucose; Disaccharides such as lactose, maltose, sucrose and trisaccharide such as raffinose; Polysaccharide such as dextran.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 ") and treat the gathering that the anti-stirring of protein causes to help dissolution treatment agent and protection, 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.), Pluronic  polyvalent alcohol, polyoxyethylene sorbitol acid anhydride monoether (TWEEN -20, TWEEN -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), antioxidant (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 may be desirable further providing a kind of immunosuppressor.These molecules are planned purpose suitably to be effective to amount combination exists.Activeconstituents also can be trapped in the micro-capsule, described micro-capsule for example is respectively Walocel MT 20.000PV or gelatin microcapsule and poly-(methyl methacrylate) micro-capsule () by for example preparing by condensation technique or by interfacial polymerization in colloidal state delivery system (for example liposome, albumin microsphere, microemulsion, nano particle and nanometer packing) or coarse emulsion (macroemulsion).These technology are in Remington ' sPharmaceutical 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 by 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 described matrix is the shaped object form, for example film or micro-capsule.The example that continues release matrix comprises polyester, hydrogel (for example poly-(2-hydroxyethyl meth acrylate) or poly-(vinyl alcohol)), polylactide (United States Patent (USP) 3,773,919), the multipolymer of L-L-glutamic acid and ethyl-L-glutamate, nondegradation ethane-acetic acid ethyenyl ester, degradation property lactic acid-ethanol copolymer such as LUPRON DEPOT ^TM(Injectable microspheres of forming by lactic acid-ethanol copolymer and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyric acid.Although the polymkeric substance as 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 biologic activity 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 by sulfo--disulfide exchange, then can be by modifying sulfydryl, freeze-drying from acidic solution, control moisture content, using suitable additive and exploitation specificity polymer matrix composition to realize stablizing.

Effective therapeutical peptide, antibody or its segmental amount determine according to the character of described illness or pathology in treatment particular disorder or pathology, and can determine by standard clinical techniques.If possible, hope at first at external definite pharmaceutical composition dose response curve of the present invention, was used for useful animal model system then before human experimentation.

In a preferred embodiment, therapeutical peptide, antibody or its segmental aqueous solution give by 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, described 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, described target is combined on the fixed antibody variants.At last, upholder is washed with other suitable solvent such as glycine buffer, this will disengage described target from antibody variants.

Described 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, described antibody variants is typically used detectable component mark.Many marks can utilize.The technology of quantification change in fluorescence as mentioned above.Chemical luminous substrate becomes electric excited state by chemical reaction, can launch the light that can measure (for example use chemoluminescence instrumentation fixed) then or provide energy as fluorescent receptor.The example of enzyme labelling comprises luciferase (for example Lampyridea luciferase and bacteriofluorescein enzyme; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydro phthalazine diketone (dihydrophthalazinediones), malate dehydrogenase (malic acid dehydrogenase), urase, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase enzymes, glucoamylase, N,O-Diacetylmuramidase, carbohydrate oxidase (for example glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase (G6PD)), heterocycle oxydase (as uriKoxidase and XOD), lactoperoxidase, microperoxisome or the like.The technology that enzyme and antibody are puted together is at O ' Sullivan etal., Methodsfor the Preparation of Enzyme-Antibody Conjugates for Use in EnzymeImmunoassay, in Methods in Enzym. (Ed.J.Langone ﹠amp; H.Van Vunakis), Academic press describes among the New York, 73:147-166 (1981).

Sometimes, described 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, in order to reach puting together indirectly of mark and antibody variants, described antibody variants and little haptens (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.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, as competitive binding assay, and direct and indirect sandwich assay, and immune precipitation determination.Zola，MonoclonalAntibodies：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 determining and the amount of the standard substance of antibodies that described 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 be in conjunction with 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 U.S. Patent No. 4,376,110 is described.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.

Described antibody also can be used for in-vivo diagnostic and measure.Normally, with described antibody variants with a kind of radionuclide (as ¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or 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 described test kit promptly has the agent combination of the packaged predetermined amount of the specification sheets that carries out diagnostic assay.With in the situation of enzyme labelling, described test kit can comprise needed substrate of described enzyme and cofactor (substrate that can detect chromophore or fluorophore precursor for example is provided) at antibody variants.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 perhaps can be used to study the toxicity of antibody interested with the animal model of having set up of the disease of Antybody therapy.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 by 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, by drug administration by injection, most preferably by 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 suitable dose of antibody or polypeptide depend on the disease that will treat type, severity of disease and the course of disease, antibody variants be used to prevent replying and doctor in charge's judgement of purpose or therapeutic purpose, previous treatment, patient's clinical history and antagonist variant.The anti human IgE antibody of very high-affinity of the present invention can suitably once be given the patient or given the patient in a series of treatments.

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 by 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 by 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 composition can be to prepare, to determine dosage and administration with the corresponding to mode of good medical practice.Comprise the particular disorder 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 by the item of these considerations, and is to prevent, alleviate or treat disease or the required minimum of illness.Described 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 as 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 snakebite, 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 forms from continuous amino acid still typically keeps when being exposed to the sex change solvent, and typically no longer exists after with the sex change solvent treatment by 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 described 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 determine the specific epitopes of blocking-up IgE and its high-affinity receptor bonded IgE.Described 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 be about 80 amino acid in interval in linear order, 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 determines by ELISA.

Determine in these epi-positions for amino-acid residue (Cunningham et al., " High-Resolution Epitope Mappingof hGH-Receptor Interactions by Alanine-Scanning Mutagenesis " Science244:1081-1085) by alanine scanning mutagenesis in conjunction with the high-affinity antibody key.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 described 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 at IgE mediation disease or pathology such as transformation reactions and atopic dermatitis to the method that Mammals carries out immunity, comprise the above-mentioned immunogenic peptide that needs the patient treatment of this treatment significant quantity.

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

The initial dose of peptide (for example about 0.2mg-5mg) can for example give by intramuscularly, repeats to give same dosage (reinforcement) subsequently after 14-28 days.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 " immunogenic carrier " comprises having those materials that independently excite immunne response character in host animal herein, its can and free carboxy, amino or the hydroxyl of polypeptide by 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 by the bonding of conventional difunctional linking group.

The example of these immunogenic carriers comprises: albumin, as BSA; Sphaeroprotein; Thyroglobulin; 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, Am.J.Physio.199[1960] described those roundworm extracts of 575-578 or its purified product; Polylysine; Polyglutamic acid; The LYS-GLU multipolymer; The multipolymer that contains Methionin or ornithine; Or the like.Vaccine used diphtheria toxoid or Toxoid,tetanus produce as the immunogenic carrier material (Lepow M.L.etal., J.of Infectious Diseases150[1984] 402-406; Coen Beuvery, E.etal., Infection and Immunity 40[1983] 39-45), these toxin materials also can be used for the present invention.The protein derivatives of the purifying of tuberculin (PPD) is particularly preferred in the active immunity scheme, because: (1) himself not inducing T cell reply (being that it is effective T cell haptens), but work and therefore by the T cell recognition as the antigen of complete processing; (2) known its in chain recognition mode, be one of the most powerful hapten-carrier; (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 described polynucleotide and carry the cell of described carrier.In addition, active immunity can realize by 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 by the disease of suffering from the IgE mediation or patient's anti-IgE antibodies of the present invention of pathology.

These antibody can prepare by giving non-human mammal immunogenic peptide of the present invention and collect the gained antiserum(antisera).By 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 by 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 vehicle 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 vehicle separately or with suitable adjuvant, and described adjuvant is as the adjuvant based on aluminium hydroxide.

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 some standards, comprise the 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 (VK subgroup 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 by making up the Fab library with the synthetic and overlapping PCR method of DNA shown in Figure 2 as mentioned above.Described 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 combination and carry out under Standard PC R condition to the biotinylated forward primer of the terminal outstanding sequence of leader sequence (GeneIII) with from the reverse primer of the constant region of guarding (C κ or CH1).The PCR product is by the agarose gel electrophoresis purifying, and the perhaps PCR purification kit purifying by 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 cumulative volume 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.

With the magnetic bead of 100 μ L streptavidin bag quilts with 200 μ L 2x B﹠amp; W damping fluid washed twice is resuspended in 200 μ L, 2 * B﹠amp; In the W damping fluid.The PCR product of phosphorylation is mixed with pearl, shook incubation gently 16 minutes in room temperature (RT).

With pearl deposition and with 200 μ L, 2 * B﹠amp; 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 gently in room temperature.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) by 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.By having on bromination second pyridine (EtBr) agarose plate of DNA standard substance point sample (spotting) or by measuring OD ₂₆₀SsDNA is quantized.

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

By hybridizing mutagenesis with V _HAnd V _LThe clone advances in the phage expression vector.Uridine acidifying template infects CJ236 coli strain (dut by 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] by 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 T4DNA polysaccharase (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 by electroporation transfection.The gained phage by 37 ℃ on XL-1 Blue bacterium lawn grow overnight determine to tire.The clone is checked order to determine 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 wishing.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 with 96 hole flat boards 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.To move in the hole with filtering the transfer pipet point from the single phage plaque of the library flat board that spends 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 hole flat boards (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 precipitation 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 by 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 (wobbleresidue) in framework.V _HIn the 73rd to keep in the humanization library be the mouse threonine residues because determine this position influence combination.Yet, notice that the Threonine at VH73 is to be total to somebody's residue in people's reproductive tract VH subgroup 1 and 2.

Framework residues different between TES-C21 sequence and people's template are replaced at random as above-mentioned, 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, described 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 is in conjunction with (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 described 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 to parental generation TES-C21.These clones combine people IgE with mouse TES-C21 competition, show during humanization is handled not change in conjunction with epi-position.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 residues (=94.3% people VH framework homology), and 100% people's light chain framework of selecting for affinity maturation.This paper has shown that humanization Fab is to the restraining effect (Fig. 6) of IgE in conjunction with Fc ε RI.

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

Flat board is used for the 2 μ g/mL sheep anti people Fd that the carbonate bag is cushioned liquid to be spent the night at 4 ℃ of bags.Remove bag by 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 of DMB sealing, 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 0.05%TWEEN  then.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%TWEEN , 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 the carbonate bag is cushioned liquid to be spent the night at 4 ℃ of bags.Remove bag by solution, flat board was sealed 1 hour at 37 ℃ with 200 μ L/ hole 3%BSA/PBS.

Flat board is washed 4 times with PBS/0.1%TWEEN  (PBST).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%TWEEN  20.With flat board room temperature incubation 2 hours.

Flat board with PBST washing 4 times, is added 50 μ L/ holes vitamin H-sheep anti people Fd that 1: 1000 (0.8 μ g/ml) dilutes in 0.5%BSA/PBS and 0.050%TWEEN  20.With flat board at room temperature incubation 2 hours once more.

After with PBST washing 4 times, be incorporated in 50 μ L/ hole Neutra-avidin-AP (0.9 μ g/mL) of dilution in 1: 2000 among 0.5%BSA/PBS and the 0.05%TWEEN  20, 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 precipitation 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 by 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 by 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 effluent and carry out the SDS-PAGE analysis.With post 20mL damping fluid (50mM NaH ₂PO ₄, 300mMNaCl, the 15mM imidazoles is 8.0 with NaOH 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 is 8.0 with NaOH with pH regulator) and wash-out 6 times, and analyze by SDS PAGE.With the post fraction 4 ℃ of storages.By SDS-PAGE analytical column fraction, select to have the fraction of maximum Fab and in PBS 4 ℃ of dialysis.

Embodiment 8: soluble receptor determination

The 96 hole assay plates that will be suitable for ELISA with 0.05mL 0.5 μ g/mL Fc ε RI α-chain acceptor bag be cushioned liquid (50mM carbonate, pH 9.6) at 4-8 ℃ of bag by 12 hours.Aspirate described 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 by with measuring damping fluid (0.5%BSA and 0.05%Tween 20; PBS; pH 7.2) carry out titration with dilution in 1: 4 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 of Fc ε R1 bag quilt 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 flat board is washed as described above 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 by 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 by determining 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 by SA-HRP.Mouse MAb E-10-10 detects by the Ab that mountain sheep anti mouse Ig Fc HRP-puts together.

Embodiment 10: the clone identifies

Measure the binding affinity of each material standed for, and positive colony is checked order.The antibody variants that has the useful sudden change that increases binding affinity in the CDR zone 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 of various CDR that has the avidity of improvement through demonstration is shown in table 1.Fig. 7 illustrates the high-affinity material standed for of the combination with replacement.

Table 1

CDRL1：		CDRH1：
				P RASQSIGTNIH	SEQ ID NO 5	P MYWLE	SEQ ID NO 15
#1 RASRSIGTNIH	SEQ ID NO 6	#1WYWLE	SEQ ID NO 16
				#2 RASQRIGTNIH	SEQ ID NO 7	#2YYWLE	SEQ ID NO 17
CDRL2：		CDRH2：
				P YASESIS	SEQ ID NO 8	P EISPGTFTTNYNEKFKA	SEQ ID NO 18
#1 YAYESIS	SEQ ID NO 9	#1 EIEPGTFTTNYNEKFKA	SEQ ID NO 19
				#2 YASESIY	SEQ ID NO 10	#2 EIDPGTFTTNYNEKFKA	SEQ ID NO 20
#3 YASESDS	SEQ ID NO 11	#3 EISPDTFTTNYNEKFKA	SEQ ID NO 21
				#4 YASESES	SEQ ID NO 12	#4 EISPETFTTNYNEKFKA	SEQ ID NO 22
CDRL3：		#5 EISPGTFETNYNEKFKA	SEQ ID NO 23
				P QQSDSWPTT	SEQ ID NO 13	#6 EIEPGTFETNYNEKFKA	SEQ ID NO 24
#1 AASWSW PTT	SEQ ID NO 14	#7 EIDPGTFETNYNEKFKA	SEQ ID NO 25
						CDRH3：
		P FSHFSGSNYDYFDY	SEQ ID NO 26
						#1 FSHFSGMNYDYFDY	SEQ ID NO 27
		#2 FSHFSGQNYDYFDY	SEQ ID NO 28
						#3 FSHFTGSNYDYFDY	SEQ ID NO 29

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 under the CMV promoter expression separately subclone advance in H-and the L-chain expression vector.Make up 6 complete antibody clones, shown in Figure 10 A-F.By 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.JImmunol.143 (12): 3967-75 (1989) is described.Use albumin A sepharose (Pharmacia) antibody purification from single stable clone supernatant.The concentration of antibody uses spectrophotometer to determine 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 determined through the flat board of monoclonal human IgE (SE44) bag quilt with ELISA.

Following culture has been preserved in American type culture collection (AmericanTytpe 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 to keep this work culture 30 years from preservation.Under the scope of Budapest agreement, can obtain this organism by ATCC, this has guaranteed that the public can obtain the offspring of this culture for good and all, without restriction after relevant United States Patent (USP) mandate.

The application's transferee agreed if dead or lose or when being compromised by the preservation culture when what cultivate under conditions suitable, will be promptly after notified with the live body sample replacement of identical culture.The acquisition of preservation strain is as implementing permission of the present invention under the right of giving according to its patent law in any responsible departments of the government of violation.

The specification sheets of first above written it is believed that is enough to make those skilled in the art can implement the present invention.The present invention is not subjected to 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 all within the scope of the present invention on any function.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 presents yet.In fact, by top description, to of the present invention various except shown in this article and described modification 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 is in conjunction with the location of epi-position

A: the synthetic and anti-IgE of peptide is in conjunction with mensuration

Research illustrates IgE and passes through C _H3 structural domains are in conjunction with 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 by expressing in in-vitro transcription-translation, and are used for the Western trace and measure to detect the anti-IgE Mab of HA combination.CL-2C and CL-5I MAb all can be in conjunction with 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 before peptide.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 by chemoluminescence and is measured the IgE reactivity after 3 times 15 minutes in TBS-TWEEN .

Experimental result shows the C of the anti-IgE Mab of HA in conjunction with 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

Substituted amino acid along those peptides of finding in IgG1 carries out L-Ala scanning, to determine which amino acid is crucial for the anti-IgE MAb of HA with combining of these peptides.Through determine that anti-IgE MAb combination is that important amino acid uses the vitro mutagenesis method to replace for HA in the ε of IgE chain.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.By 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 mutant thereof use based on the in-vitro transcription of reticulocyte lysate and translation coupling system (Promega, Madison, WI) expression.

The lysate (10 μ l reaction mixture) that derives from this in-vitro transcription and translation coupling system is carried out SDS-PAGE (12%), move on the nitrocellulose membrane then.5% milk power solution that this film is used for Tris buffer saline (TBS) seals, subsequently with the anti-IgE MAb dyeing of primary antibody.The specific reaction band uses the anti-human IgG Fc of goat 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-IgEMAb 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 in conjunction with 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, MI) the 20 μ g immunogenic peptides in.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.As shown in figure 16, described peptide excites anti-IgE antibodies in these transgenic mices.

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

Sequence table

＜110〉Tyler Corporations

＜120〉discriminating of new IgE epi-position

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<151>2003-02-01

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Tyr Ala Tyr Glu Ser Ile Ser

1 5

<210>10

<211>7

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRL2 VARIANT #2(TABLE 1)

<400>10

Tyr Ala Ser Glu Ser Ile Tyr

1 5

<210>11

<211>7

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRL2 VARIANT #3(TABLE 1)

<400>11

Tyr Ala Ser Glu Ser Asp Ser

1 5

<210>12

<211>7

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRL2 VARIANT #4(TABLE 1)

<400>12

Tyr Ala Ser Glu Ser Glu Ser

1 5

<210>13

<211>9

<212>PRT

<213>ARTIFICIAL

<220>

<223>TES-C21 CDRL3(TABLE 1)

<400>13

Gln Gln Ser Asp Ser Trp Pro Thr Thr

1 5

<210>14

<211>9

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRL3 VARIANT(TABLE)

<400>14

Ala Ala Ser Trp Ser Trp Pro Thr Thr

1 5

<210>15

<211>5

<212>PRT

<213>ARTIFICIAL

<220>

<223>TES-C21 CDRH1

<400>15

Met Tyr Trp Leu Glu

1 5

<210>16

<211>5

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH1 VARIANT #1(TABLE 1)

<400>16

Trp Tyr Trp Leu Glu

1 5

<210>17

<211>5

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH1 #2(TABLE 1)

<400>17

Tyr Tyr Trp Leu Glu

1 5

<210>18

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>TES-C21 CDRH2(TABLE 1)

<400>18

Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>19

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #1(TABLE 1)

<400>19

Glu Ile Glu Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>20

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #2(TABLE 1)

<400>20

Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>21

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #3(TABLE 1)

<400>21

Glu Ile Ser Pro Asp Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>22

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #4(TABLE 1)

<400>22

Glu Ile Ser Pro Glu Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>23

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #5(TABLE 1)

<400>23

Glu Ile Ser Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>24

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #6(TABLE 1)

<400>24

Glu Ile Glu Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>25

<211>17

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH2 VARIANT #7(TABLE 1)

<400>25

Glu Ile Asp Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Ala

<210>26

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>TES-C21 CDRH3(TABLE 1)

<400>26

Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210>27

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH3 VARIANT #1(TABLE 1)

<400>27

Phe Ser His Phe Ser Gly Met Asn Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210>28

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH3 VARIANT #2(TABLE 1)

<400>28

Phe Ser His Phe Ser Gly Gln Asn Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210>29

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>CDRH3 VARIANT #3(TABLE 1)

<400>29

Phe Ser His Phe Thr Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210>30

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 136

<400>30

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>31

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 1

<400>31

Asp Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>32

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 2

<400>32

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>33

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 4

<400>33

Asp Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>34

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 13

<400>34

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>35

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 18

<400>35

Glu Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>36

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 25

<400>36

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>37

<211>23

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL1 VARIANT 27

<400>37

Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys

20

<210>38

<211>15

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL2 VARIANT 136

<400>38

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

1 5 10 15

<210>39

<211>15

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL2 VARIANT 1

<400>39

Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys

1 5 10 15

<210>40

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL3 VARIANT 136

<400>40

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210>41

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL3 VARIANT 1

<400>41

Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys

20 25 30

<210>42

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL3 VARIANT 13

<400>42

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Asp Tyr Tyr Cys

20 25 30

<210>43

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL3 VARIANT 18

<400>43

Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Asp Tyr Tyr Cys

20 25 30

<210>44

<211>10

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRL4 VARIANT

<400>44

Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

1 5 10

<210>45

<211>30

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH1 VARIANT 136

<400>45

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser

20 25 30

<210>46

<211>30

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH1 VARIANT 2

<400>46

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser

20 25 30

<210>47

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH2 VARIANT 136

<400>47

Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met Gly

1 5 10

<210>48

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH2 VARIANT 2

<400>48

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly

1 5 10

<210>49

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH2 VARIANT 8

<400>49

Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val Gly

1 5 10

<210>50

<211>14

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH2 VARIANT 21

<400>50

Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Val Gly

1 5 10

<210>51

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH3 VARIANT 136

<400>51

Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>52

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH3 VARIANT 1

<400>52

Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>53

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH3 VARIANT 43

<400>53

Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>54

<211>32

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH3 VARIANT 103

<400>54

Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu

1 5 10 15

Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210>55

<211>11

<212>PRT

<213>ARTIFICIAL

<220>

<223>FRH4 VARIANT 136

<400>55

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210>56

<211>19

<212>PRT

<213>Bacteriophage M13mp18

<220>

<221>misc_feature

<223>Gene III signal Sequence

<400>56

Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly

1 5 10 15

Val His Ser

<210>57

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE 136

<400>57

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>58

<211>106

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN CONSTANT REGION OF CLONE 136

<400>58

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

1 5 10 15

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

20 25 30

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

35 40 45

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

50 55 60

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

65 70 75 80

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

85 90 95

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210>59

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE 136

<400>59

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>60

<211>330

<212>PRT

<213>ARTIFICIAL

<220>

<223>CONSTANT REGION OF HUMAN IgG1

<400>60

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210>61

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE CL-2C

<400>61

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>62

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE CL-2C

<400>62

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Trp Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>63

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE CL-5I

<400>63

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>64

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE CL-5I

<400>64

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asp Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>65

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE CL-5A

<400>65

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>66

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE CL-5A

<400>66

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Trp Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Glu Pro Gly Thr Glu Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>67

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE CL-2B

<400>67

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>68

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE CL-2B

<400>68

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>69

<211>107

<212>PRT

<213>ARTIFICIAL

<220>

<223>LIGHT CHAIN VARIABLE REGION OF CLONE CL-1136-2C

<400>69

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210>70

<211>123

<212>PRT

<213>ARTIFICIAL

<220>

<223>HEAVY CHAIN VARIABLE REGION OF CLONE CL-1136-2C

<400>70

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr

20 25 30

Trp Leu Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210>71

<211>108

<212>PRT

<213>Homo sapiens

<400>71

Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro

1 5 10 15

Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val

20 25 30

Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala

35 40 45

Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg

50 55 60

Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp

65 70 75 80

Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu

85 90 95

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

100 105

<210>72

<211>12

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE

<220>

<221>misc_feature

<222>(7)..(7)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(9)..(10)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(12)..(12)

<223>Xaa can be any naturally occurring amino acid

<400>72

Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa

1 5 10

<210>73

<211>12

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE

<400>73

Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro

1 5 10

<210>74

<211>9

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE

<220>

<221>misc_feature

<222>(6)..(6)

<223>Xaa can be any naturally occurring amino acid

<220>

<221>misc_feature

<222>(9)..(9)

<223>Xaa can be any naturally occurring amino acid

<400>74

Leu Pro Arg Ala Leu Xaa Arg Ser Xaa

1 5

<210>75

<211>9

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE

<400>75

Leu Pro Arg Ala Leu Met Arg Ser Thr

1 5

<210>76

<211>12

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE

<400>76

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

1 5 10

<210>77

<211>12

<212>PRT

<213>ARTIFICIAL

<220>

<223>PEPTIDE DERIVED FROM CH3 DOMAIN OFI GE

<400>77

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

1 5 10

Claims (21)

1. isolating peptide, it is made up of following aminoacid sequence basically:

Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa(SEQ IDNO：72)。

2. the amino acid of claim 1, wherein said aminoacid sequence is:

Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro(SEQ ID NO：73)。

3. isolating peptide, it is made up of following aminoacid sequence basically:

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

4. the peptide of claim 3, wherein said aminoacid sequence is:

a)Leu Pro Arg Ala Leu Met Arg Ser Thr(SEQ ID NO：75)；

B) Hi Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr (SEQ IDNO:76); Perhaps

c)Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr(SEQ IDNO：77)。

5. composition, it comprises peptide and a kind of physiology acceptable carrier, thinner, stablizer or the vehicle of claim 1 or 3.

6. the composition of claim 5, it further comprises a kind of immunogenic carrier.

7. the composition of claim 6, wherein said immunogenic carrier is selected from BSA, KLH, Toxoid,tetanus and diphtheria toxoid.

8. isolated antibody, its specificity is in conjunction with each peptide of claim 1-4.

9. the antibody of claim 8, it further comprises a kind of mark.

10. the antibody of claim 8, 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.

11. a composition, it comprises antibody and a kind of acceptable carrier of claim 8.

12. a method for preparing polyclonal antibody, described method comprises:

A) use the polypeptide of forming by aminoacid sequence SEQ ID NO:72 or SEQ ID NO:74 exciting immune animal under the condition of antibody response,

B) in the described animal body separation antibody and

C), thereby differentiate and comprise the polypeptide of aminoacid sequence SEQ ID NO:72 or SEQ ID NO:74 with high-affinity specificity bonded polyclonal antibody with the antibody of described polypeptide screening and separating.

13. polyclonal antibody that produces by the method for claim 12.

14. a composition, it comprises polyclonal antibody and a kind of suitable carriers of claim 13.

15. one kind produces monoclonal antibody method, described method comprises:

A) use the polypeptide of forming by aminoacid sequence SEQ ID NO:72 or SEQ ID NO:74 exciting immune animal under the condition of antibody response,

B) in described animal body, separate the cell that produces antibody,

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

D) cultivate described hybridoma, and

E) from culture, separate and comprise aminoacid sequence SEQ ID NO:72 or SEQID NO:74 polypeptide of sequence with high-affinity specificity bonded monoclonal antibody.

16. monoclonal antibody that produces by the method for claim 15.

17. a composition, it comprises monoclonal antibody and a kind of suitable carriers of claim 16.

18. the antibody of claim 8, wherein said antibody produces by screening Fab expression library.

19. the antibody of claim 8, wherein said antibody produces by screening combination immunoglobulin (Ig) library.

20. one kind comprises each the test kit of antibody of claim 8-19.

21. a method of inducing the IgE immunological response in mammalian body comprises each peptide or the composition of claim 1-7 that gives to be enough to induce the amount of replying in described mammalian body.

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