CN101039959A - Compositions and methods for treating inflammatory disorders - Google Patents

Compositions and methods for treating inflammatory disorders Download PDF

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CN101039959A
CN101039959A CN200580028518.5A CN200580028518A CN101039959A CN 101039959 A CN101039959 A CN 101039959A CN 200580028518 A CN200580028518 A CN 200580028518A CN 101039959 A CN101039959 A CN 101039959A
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single domain
domain antibody
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O·伊纳托维奇
R·德维尔德特
B·伍尔文
S·格兰特
P·琼斯
A·巴斯兰
N·布鲁伊斯
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Teva Pharmaceuticals Australia Pty Ltd
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Domantis Ltd
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Priority claimed from PCT/GB2004/002829 external-priority patent/WO2004081026A2/en
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Abstract

The invention relates to compositions and methods for treating inflammatory disorders. More specifically, the invention relates to antibody compositions and their use in the treatment of inflammatory disorders.

Description

The composition and the method that are used for the treatment of inflammatory diseases
The present invention relates to use method, the composition of these parts and the method for preparation and these parts of use of the antibody polypeptides construction treatment disease (comprising rheumatoid arthritis) that contains single domain antibody part and dual specific part.Specifically, the invention provides preparation method in conjunction with the single domain antibody of inflammatory cytokine (comprising TNF-α and VEGF).The invention also discloses and contain in conjunction with first single immunoglobulin variable structural domain of first antigen or epi-position with in conjunction with the dual specific part of second single immunoglobulin variable structural domain of second antigen or epi-position.More particularly, the present invention relates to the dual specific part, wherein with first and second antigen or epi-position at least one combined the effect that increases half life in the part body.The invention describes the opening and the sealing conformation part that contain more than one binding specificities.The invention discloses and use the single domain antibody construction and in conjunction with the method for first and second antigenic dual specific ligands for treating rheumatoid arthritis, wherein said dual specific part can comprise for example arbitrary combination of TNF-α, VEGF and HSA.
TNF-α：
As its name suggests, the molecule that tumor necrosis factor-alpha (TNF-α) is described to have antitumor characteristic at first, but find that subsequently this molecule plays a crucial role in other process, be included in the remarkable effect in transmitting inflammation and the autoimmune disease.TNF-α is crucial pro-inflammatory cytokine in inflammatory diseases, and these inflammatory diseasess comprise for example rheumatoid arthritis (RA), Crohn disease, ulcerative colitis and other enteropathy, psoriatic, toxic shock, graft versus host disease and multiple sclerosis.
The short scorching effect of TNF-α causes tissue injury, for example induce procoagulant activity (Pober etc. to vascular endothelial cell, J.Immunol.136:1680 (1986)), increase neutrophilic granulocyte and lymphocytic adhesion (Pober etc., J.Immunol.138:3319 (1987)) and stimulate scavenger cell, neutrophilic granulocyte and vascular endothelial cell to discharge platelet activation factor (Camussi etc., J.Exp.Med.166:1390 (1987)).
Synthesize and have the TNF-α that the 26kD of tail in the born of the same parents strides the film precursor protein, it is by TNF-α-conversion metalloprotease cutting, secretes then to be the soluble proteins of 17kD.Activity form is made up of the monomeric homotrimer of 17kD, and cell surface receptor p55TNFR1 that they are different with two kinds and p75 TNFR2 interact.Show on evidence that also the cell surface of TNF-α can mediate some biological action of this factor in conjunction with precursor forms.Most of cell is expressed the p55 and the p75 acceptor of the different biological function of mediation part simultaneously.The p75 acceptor participate in to trigger lymphopoiesis, and the p55 acceptor participates in cytotoxicity, apoptosis, antiviral activity, fibroblast proliferation and the NF-kB activation (referring to Locksley etc., 2001, Cell 104:487-501) of TNF mediation.
The TNF acceptor is the member of membranin family, and this family comprises the acceptor of NGF acceptor, Fas antigen, CD27, CD30, CD40, Ox40 and lymphotoxin α/β heterodimer.The zygotic induction receptor clustering of homotrimer and acceptor is the tuftlet of 2 or 3 p55 or p75 molecule.TNF-α is mainly produced by activatory scavenger cell and T lymphocyte, but is also produced by neutrophilic granulocyte, endotheliocyte, keratinocyte and inoblast in the middle of acute inflammatory reaction.
TNF-α be in the pro-inflammatory cytokine cascade the summit (summarize in Feldmann and Maini, 2001, Ann.Rev.Immunol.19:163).The expression of the pro-inflammatory cytokine (specifically being IL-1 and IL-6) that this cytokine induction is other or release (referring to for example Rutgeerts etc., 2004, Gastroenterology 126:1593-1610).Suppress the releasing and activity of inflammatory cytokines that TNF-α can suppress to comprise IL-1, IL-6, IL-8 and GM-CSF (Brennan etc., 1989, Lancet2:244).
Because the effect of TNF-α in inflammation is so it has become important inhibition target in the work that alleviates the inflammatory diseases symptom.Carry out the various TNF-α inhibition methods that are used for the disease clinical treatment, specifically comprised use soluble TNF-α acceptor and TNF-alpha specific antibody.The go through commodity of clinical use comprise for example antibody product Remicade TM(English monoclonal antibody of sharp former times; Centocor, Malvern, PA; The chimeric monoclonal IgG antibody of a kind of carrier IgG4 constant region and mouse variable region), Humira TM(adalimumab or D2E7; AbbottLaboratories is described in United States Patent (USP) the 6th, 090, No. 382) and soluble receptors product Enbrel TM(etanercept, a kind of p75 TNFR2 Fc fusion rotein of solubility; Immunex).
The effect of TNF-α in inflammatory arthritis summarized in for example Li and Schwartz, and 2003, Sringer Semin.Immunopathol.25:19-33.In RA, TNF-α (specifically at cartilage-pannus joint) in the synovial membrane of inflammation highly expresses (DiGiovine etc., 1988, Ann.Rheum.Dis.47:768; Firestein etc., 1990, J.Immunol.144:3347; With Saxne etc., 1988, Atrhritis Rheum.31:1041).Except the level that confirms TNF-α increase inflammatory cytokine IL-1, IL-6, IL-8 and GM-CSF, TNF-α also can cause arthritis and inoblast sample synovial cell propagation (Gitter etc., 1989, Immunology 66:196) alone; Induce collagenase, cause cartilage destruction (Dayer etc., 1985, J.Exp.Med.162:2163 thus; Dayer etc., 1986, J.Clin.Invest.77:645); By articular chondrocytes arrestin glycan synthetic (Saklatvala, 1986, Nature 322:547; Saklatvala etc., 1985, J.Exp.Med.162:1208); And can stimulate osteoclast to generate and bone resorption (Abu-Amer etc., 2000, J.Biol.Chem.275:27307; Bertolini etc., 1986, Nature 319:516).TNF-α induces the CD14+ monocyte of marrow to discharge to be increased.This monocyte can soak into the joint, and through RANK (receptor activation thing or NF-κ B)-RANKL signal transduction pathway amplification inflammatory reaction, in arthritis, cause osteoclast form (summarize in Anandarajah and Richlin, 2004, Curr.Opin.Rheumatol.16:338-343).
TNF-α is an acute phase protein, by its to IL-8 induce the increase vascular permeability, thus scavenger cell and neutrophilic granulocyte are raised to infection site.In case this situation occurs, the activatory scavenger cell just continues to produce TNF-α, keeps thus and amplifies inflammatory reaction.
Effective with soluble receptors construction etanercept titration TNF-α to the RA treatment, but the Crohn disease is failed to respond to any medical treatment.On the contrary, antibody TNF-alpha-2 antagonists English monoclonal antibody of sharp former times effectively treat RA and Crohn sick these two.Therefore, in only be not and relevant unique mechanism is renderd a service in the treatment of anti-TNF type with soluble TNF-α.On the contrary, sealing is by other short scorching signal of TNF-α inductive or molecule also work (Rutgeerts etc., ibid).For example, give the expression that English monoclonal antibody of sharp former times obviously reduces adhesion molecule, cause neutrophilic granulocyte that the infiltration of inflammation part is descended.In addition, English monoclonal antibody of sharp former times is treated the inflammatory cell disappearance on the intestinal mucosa that causes previous inflammation in the Crohn disease.In the lamina propria disappearance of activating T cell by activate in Fas dependency mode that Caspase 8,9 activates then that Caspase 3 causes have film in conjunction with the apoptosis mediation of the cell of TNF-α (referring to Lugering etc., 2001, Gastroenterology 121:1145-1157).Therefore, TNF-α membrane-bound or receptors bind the important target that is anti-TNF-α methods of treatment.Other people show, English monoclonal antibody of sharp former times is in conjunction with activatory peripheral blood cells and lamina propria cell, and by activation Caspase 3 apoptosis-induced (referring to Van den Brande etc., 2003, Gastroenterology 124:1774-1785).
In cell, trimerization TNF-α and its acceptor combine triggering signal transduction cascade of events, comprise and substitute to suppress molecule such as SODD (death domain silencer) and in conjunction with fit factor FADD, TRADD, TRAF2, c-IAP, RAIDD and TRIP, add that kinases RIP1 and some Caspase (summarize in Chen and Goeddel, 2002, Science 296:1634-1635; And Muzio and Saccani, be stated from: Methods in Molecular Medicine:Tumor Necrosis Factor, Methods and Protocols; Corti and Ghezzi edit, (Humana Press, New Jersey), 81-99 page or leaf).The signal transduction mixture of assembling can activate active cells survival path by NF-kB activation and downstream gene subsequently, or activates apoptosis pathway by Caspase.
In other disease, but outer downstream cytokine cascade of born of the same parents and intracellular signal transduction path like the TNF-α induction phase.Therefore, promote other disease or the obstacle of pathological condition for TNF-alpha molecule wherein, suppressing TNF-α provides a kind of methods of treatment.
VEGF：
Vasculogenesis plays an important role in the activity propagation of inflammatory synovial tissue.The RA synovial tissue invasion and attack periphery joint cartilage and the osseous tissue of height vascularization cause joint injury.
Vascular endothelial growth factor (VEGF) is the most effective known vasculogenesis cytokine.VEGF is that heparin-bounding secretion is with biglycan albumen, because the alternative splicing of its primary transcript and have (Leung etc., 1989, Science 246:1306) with several variable forms.Also known VEGF is vascular permeability factor (VPF), and this induces the ability of the significant process vascular leakage in the inflammation owing to it.In RA patient synovial tissue, identify VEGF and highlighted latent effect (Fava etc., 1994, the J.Exp.Med.180:341:346 of VEGF in the RA pathology; Nagashima etc., 1995, J.Rheumatol.22:1624-1630).According to the research that wherein gives VEGF antibody in mouse collagen protein induction type sacroiliitis (CIA) model, the effect of VEGF in the RA pathology consolidated.In these researchs, when inducing an illness, the vegf expression in the joint increases, and gives the development that the anti-VEGF antiserum(antisera) has been blocked joint disease, and improves existing disease (Sone etc., 2001, Biochem.Biophys.Res.Comm.281:562-568; Lu etc., 2000, J.Immunol.164:5922-5927).
Antibody polypeptides:
Antibody is high degree of specificity to it in conjunction with target, although antibody is derived from natural host defence mechanism, antibody is faced with several challenges when being used for the treatment of the human patients disease.Conventional antibody is the large protein molecule of many subunits, contains at least 4 polypeptide chains.For example, human IgG has 2 heavy chains and 2 light chains, and its disulfide bonding is to form functional antibodies.The size of conventional IgG is about 150kD.Because the size that it is relatively large, so complete antibody (for example IgG, IgA, IgM etc.) is limited aspect treatment validity, this is attributable to for example problem of tissue penetration aspect.Considerable research work concentrates on differentiates and produces reservation antigen combined function and deliquescent less antibody fragment.
The heavy chain of antibody and light chain polypeptide chain contain variable (V) that participate in AI directly and distinguish and provide structural support to distinguish with the non-antigen-specific of immune effector interactional constant (C) with rising.The antigen binding domains of conventional antibody is made up of two separated structures territories: weight chain variable structural domain (V H) and light chain variable structural domain (V L: it can be V κ or V λ).Antigen binding site self is made up of 6 polypeptide rings: 3 from V HStructural domain (H1, H2 and H3), 3 from V LStructural domain (L1, L2 and L3).In vivo, reset gene segment by combination and produce coding V HAnd V LThe diversity master library of the V gene of structural domain.The C district comprises that light chain C district (is called C LThe district) and heavy chain C district (be called C H1, C H2 and C H3 districts).
Identified the numerous less Fab of natural antibody according to protease digestion.These fragments for example comprise " Fab fragment " (V L-C L-C H1-V H), " Fab ' fragment " (Fab) and " F (ab ') with heavy chain hinge area 2Fragment " (by the segmental dimer of Fab ' of heavy chain hinge area joint).Used recombination method to produce littler Fab, be called " strand Fv " (variable fragment) or " scFv ", it is by the V that engages by synthetic peptide linker LAnd V HForm.
Single domain antibody:
Although the antigen bonding unit of common known natural antibody (for example in people and most of other Mammals) is by a pair of V district (V L/ V H) form, but the antibody of the global function of Camelidae (camelid) species expression vast scale, high specific, no sequence of light chain.Found that the Camelidae heavy chain antibody is the homodimer of single heavy chain through its constant region dimerization.The variable region of these Camelidae heavy chain antibodies is called as V HThe H structural domain is retained in and is separated into V HDuring the chain fragment with ability ((Hamers-Casterman etc., 1993, the Nature 363:446-448 of high specific conjugated antigen; Gahroudi etc., 1997, FEBS Lett.414:521-526).Also by for example by the amplification of the genomic dna of immune mouse spleen and at the mouse V of expression in escherichia coli HIdentify antigen bonded list V in the gene library HStructural domain (Ward etc., 1989, Nature 341:544-546).Ward etc. are with isolating single V HStructural domain called after " dAb ", representative " domain antibodies ".Term " dAb " refers to single immunoglobulin variable structural domain (V of specificity conjugated antigen at this paper H, V HHOr V L) polypeptide." dAb " is independent of other V structural domain conjugated antigen; But in situation used herein, " dAb " can be with other V as this term HOr V LSame or the heteromultimeric form of structural domain exists, and the dAb conjugated antigen does not need other structural domain in this case, and promptly dAb is independent of other V in this case H, V HHOr V LThe structural domain conjugated antigen.
Single immunoglobulin variable structural domain, for example V HH is the antigen binding antibody unit of known minimum.For being used for the treatment of, preferred people's antibody, this mainly is because its unlikely immunne response that excites when giving the patient.As mentioned above, isolating non-Camelidae V HStructural domain is often insoluble relatively, expresses relatively poor usually.Camelidae V HH and people's antibody V HThe contrast of structural domain discloses, corresponding to people V HThe V of structural domain H/ V LThe Camelidae V of joint HIn the H structural domain framework region several key difference are arranged.To people V HThese residues of 3 have carried out closer similar V HThe sudden change of H sequence (specifically being Gly 44 Glu, Leu 45 Arg and Trp 47Gly), keep antigen-binding activity (Davies and Riechmann to produce, 1994, FEBS Lett.339:285-290) and also have expression and deliquescent " camelization " people V that improves HStructural domain.(varied texture domain amino acid numbering used herein and Kabat numbering convention (Kabat etc., 1991, Sequences of Immunological Interest, the 5th edition, the healthy and human service department of the U.S., Washington, D.C.) unanimity).WO 03/035694 (Muyldermans) report, the non-Camelidae V of Trp 103Arg sudden change the improvement HThe solvability of structural domain.Davies and Riechmann (1995, Biotechnology N.Y.13:475-479) have also reported camelization people V HThe generation in structural domain phage display storehouse and with the selection of the avidity in the 100-400nM scope in conjunction with haptenic clone, but selected conjugated protein antigenic clone has more weak avidity.
The antigen binding domains of antibody comprises 2 separate areas: weight chain variable structural domain (V H) and light chain variable structural domain (V L: it can be V κPerhaps V λ).Antigen binding site self is made up of 6 polypeptide rings: 3 from V HStructural domain (H1, H2 and H3), 3 from V LStructural domain (L1, L2 and L3).Reset gene segment production coding V by combination HAnd V LThe diversity master library of the V gene of structural domain.V HGene is by 3 gene segment V H, D and J HReorganization produce.In the people, according to haplotype, 51 kinds of functional V have an appointment HSections (Cook and Tomlinson (1995) Immunol Today, 16:237), 25 kinds of functional D sections (Corbett etc., (1997) J.Mol.Biol., 268:69) and 6 kinds of functional J HSections (Ravetch etc., (1981) Cell, 27:583).V HThe sections coding forms V HThe polypeptide sequence of first of structural domain and second antigen coupling collar (H1 and H2), and V H, D and J HSections is combined to form V HThe 3rd the antigen coupling collar (H3) of structural domain.V LGene is by only 2 the gene segment V that recombinate LAnd J LProduce.In the people, according to haplotype, 40 kinds of functional V have an appointment κSections (Schable and Zachau (1993) Biol.Chem.Hoppe-Seyler, 374:1001), 31 kinds of functional V λSections (Williams etc., (1996) J.Mol.so Biol., 264:220; Kawasaki etc., (1997) Genome Res., 7:250), 5 kinds of functional J κ(Hieter etc., (1982) J.Biol.Chef. is 257:1516) with 4 kinds of functional J for sections λSections (Vasicek and Leder (1990) J.Exp.Med., 172:609).V LThe sections coding forms V LThe polypeptide sequence of first of structural domain and second antigen coupling collar (L1 and L2), and V LAnd J LSections is combined to form V LThe 3rd the antigen coupling collar (L3) of structural domain.It is generally acknowledged that the antibody variation that is selected from this master library must be enough to medium at least avidity in conjunction with nearly all antigen.Produce high affinity antibody by " the avidity sudden change " of resetting gene, wherein immunity system with improve be combined into benchmark production and selected element suddenlys change.
The structure and the sequential analysis of antibody show have 5 (H1, H2, L1, L2, L3) to have a small amount of main chain conformation or norm structure (Chothia and Lesk (1987) d:Mol.Biol., 196:901 in 6 antigen coupling collars; Chothia etc., (1989) Nature, 342:877).The main chain conformation is by the length of (i) antigen coupling collar and the (ii) specific residue or the residue type decided of some key position in antigen coupling collar and the antibody framework.Ring analysis long and Key residues can make us predict main chain conformation (Chothia etc., (1992) J.Mol.Biol., the 227:799 of H1, the H2, L1, L2 and the L3 that are encoded by most people's antibody sequence; Tomlinson etc., (1995) EMBOJ., 14:4628; Williams etc., (1996) J.Mol.Biol., 264:220).Although the H3 district is variation more (owing to using the D sections) with regard to sequence, length and structure, but it also forms the long main chain conformation of a small amount of becate, it depends on the length of the concrete residue of key position in ring and the antibody framework and has situation or residue type (Martin etc., (1996) J.Mol.Biol, 263:800; Shirai etc., (1996) FEBS Letters, 399:1).
Bi-specific antibody:
Contain V HAnd V LThe bi-specific antibody of district's complementary pair is known in the art.These bi-specific antibodies must comprise 2 couples of V HAnd V L, each V H/ V LTo in conjunction with single antigen or epi-position.Described method comprises heterozygosis hybridoma (Milstein and Cuello, Nature 305:537-40), miniantibody (Hu etc., (1996) bifunctional antibody (Holliger etc. Cancer Res 3056:3055-3061),, (1993) Proc.Natl.Acad.Sci.USA 90,6,444 6448; WO 94/13804), chelating recombinant antibodies (CRAb; (Neri etc., (1995) J.Mol.Biol.246,367-373), two scFv (Atwell etc. for example, (1996) Mol.Immunol.33,13011312), " knot (knobs in holes) in the hole " stabilization antibody (Carter etc., (1997) ProteinSci.6,781 788).In each case, each antibody type all comprises two antigen binding sites, and each antigen binding site is V HAnd V LThe form of structural domain complementary pair.Each antibody thus can be simultaneously in conjunction with 2 different antigens or epi-position, with combining by V of each antigen or epi-position HAnd complementary V LThe structural domain mediation.In these technology each all has its specific shortcoming; For example concerning the heterozygosis hybridoma, the V of inactivation H/ V LTo can greatly reducing the mark of dual specific IgG.
And most of dual specific method depends on different V H/ V LRight combination or V HAnd V LThe combination of chain is to rebuild two different V H/ V LBinding site.Therefore, can not control assembling molecule at the ratio of the binding site of each antigen or epi-position, therefore, many assembling molecules are in conjunction with antigen or epi-position, but debond another.In some cases, might be at engineered heavy chain of subunit's joint and light chain (Carter etc., 1997), so that promote two antigens or epi-position are all had the molecule number of binding site, but this causes never all molecules all in conjunction with two antigens or epi-position.
There are some evidences to show, two kinds of different antibody binding specificities can be joined in the same binding site, but these binding sites provide two or more specificitys corresponding to structurally associated antigen or epi-position usually, perhaps corresponding to the antibody with extensive cross reaction.For example, cross-reacting antibody has so been described, usually wherein two antigens are relevant on sequence or structure, for example hen's egg-white lysozyme and turkey N,O-Diacetylmuramidase (McCafferty etc., WO 92/01047), perhaps free haptens and the haptens (Griffiths AD etc., EMBO J 199413:14 3245-60) that is conjugated to carrier.In another example, WO 02/02773 (Abbott Laboratories) has described the antibody molecule with " dual specificity ".The antibody of the antibody molecule of being mentioned for producing or select at multiple antigen makes its specificity cover more than one antigen.The complementary V of in the antibody of WO 02/02773 each H/ V LTo having specified to the antigenic single binding specificity of two or more structurally associateds; V in this complementary pair HAnd V LStructural domain does not all have independent specificity.
Therefore, antibody has single specificity widely, and it comprises two kinds of structurally associated antigens.And, described for the natural autoantibodies of multiple reactionness (Casali and Notkins, Ann.Rev.Immunol.7,515-531), itself and at least two kinds of incoherent not synantigens of (more usually) structures or epi-position reaction.Also show, use display technique of bacteriophage to select random peptide library will identify the peptide sequence of a series of coupling antigen binding sites monoclonal antibody.Some are arranged in these sequences is height correlations, has consensus sequence, and other sequence is very different, be called as mimic epitopes (Lane and Stephen, Current Opinion in Immunology, 1993,5,268-271).Therefore, clearly, contain combination and complementary V HAnd V LNatural 4 chain antibodies of structural domain have in conjunction with the antigenic potentiality of multiple difference in the known antigens on a large scale.The unclear binding site of how in same antibody, setting up at two given antigens (antigen that may not be correlated with on the structure specifically).
Show that the protein engineering method has meaning to this respect.For example, also proposed to set up metal ion has been had in conjunction with active catalytic antibody by a variable domains, or haptens (substrate) is had in conjunction with active catalytic antibody (Barbae etc. by contacting with complementary variable domains to set up with metal ion, 5 1993 Proc.Natl.Acad.Sci USA 90,6385-6389).But, in the case, the combination of substrate (first antigen) and the combination that catalysis needs metal ion (second antigen) have been proposed.Therefore, with V H/ V LRight combination relates to single component rather than multi-component antigen.
Described the method for setting up bi-specific antibody by camel heavy chain of antibody single structure territory already, wherein in a variable domains, set up, in second variable domains, set up second antigenic in conjunction with contact to an antigenic combination contact.But variable domains is a complementary not.Therefore, select anti-first antigenic first weight chain variable structural domain and anti-second antigenic second weight chain variable structural domain, then two structural domains are connected on same the chain together, to produce bispecific antibody fragment (Conrath etc., J.Biol.Chem.270,27589-27594).But the unusual part in camel heavy chain single structure territory is that it is derived from the natural camel antibody with light chain, and in fact heavy chain single structure territory can not combine with the camel light chain to form complementary V HAnd V LRight.
Also described substance chain variable domains, it is derived from general and light chain bonded natural antibody (is derived from monoclonal antibody or structural domain storehouse; Referring to EP-A-0368684).Show that these weight chain variable structural domains interact with one or more related antigens specifically, but not with other heavy chain or the combination of light chain variable structural domain with set up to two or more not synantigen have specific part.And, show that these single structure territories have half life in the very short body.Therefore, the therapeutic value of this structural domain is limited.
Proposed by (as mentioned above) preparation bispecific antibody fragment that not homospecific weight chain variable structural domain is linked together.The shortcoming of this method is that the isolated antibody variable domains can have hydrophobic interfaces, and its general and light chain interaction is exposed to solvent, can be " viscosity ", makes the single structure territory in conjunction with hydrophobic surface.And when not having the mating partner light chain, two or more different heavy chains variable domains can be through combination of its hydrophobic interfaces and combination, and this can stop them can one or both parts of bonded when separating in conjunction with it.In addition, in the case, the weight chain variable structural domain can not combine with complementary light chain variable structural domain, thus potentially unstable and be opened easily (Worn and Pluckthun, 1998 Biochemistry 37,13120-7).
Summary of the invention
The present inventor has described the double specificity immunoglobulin part that contains immunoglobulin (Ig) list variable domains in the International Patent Application WO 03/002609 of its common pending trial and in the unexposed UK Patent Application 0230203.2 of common pending trial, wherein each variable domains can have different specificitys.Structural domain can play the competition effect mutually, or antigen or the epi-position on the binding target molecule independently.
The invention describes the method for this disease of treatment in the individuality of suffering from TNF-α dependency inflammatory diseases.This method comprises the single domain antibody polypeptide construction that gives this individual treatment significant quantity, preferred people's single domain antibody construction, and wherein single domain antibody polypeptide construction is in conjunction with the humanTNF-, and it is diseases related to treat TNF-α thus.
In one aspect, inflammatory diseases is a rheumatoid arthritis, and this method comprises uses one or more single domain antibody polypeptide constructions, and wherein one or more construction antagonisms humanTNF-and acceptor combines.The invention describes the composition of the single domain antibody polypeptide construction that contains one or more antagonisms humanTNF-and receptors bind, and described the dual specific part, wherein a specific specificity of part is at TNF α, and second specific specificity is at VEGF or HSA.The present invention has further described bi-specific antibody, and wherein a specific specificity of part is at VEGF, and second specific specificity is at HSA.
This paper also comprises polypeptide construction as described herein purposes in the preparation disease therapeuticing medicine, specifically the purposes in preparation rheumatoid arthritis treatment medicine.
In one aspect, the present invention includes the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, and it comprises the single domain antibody polypeptide construction of antagonism humanTNF-and receptors bind, treats rheumatoid arthritis thus.
In one embodiment, composition stops the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model.
In another embodiment, giving Tg197 transgenic mice composition may further comprise the steps: a) weekly peritoneal injection gives heterozygosis Tg197 transgenic mice composition, b) claim the weight of step a) mouse once in a week, and c) write down the sacroiliitis macroscopic view phenotype symptom of mouse once in a week according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), (the swelling of 2=moderate sacroiliitis, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In another embodiment, obviously give the mouse composition before the outbreak at arthritic symptom.In another embodiment, give composition when big for the first time in mouse 3 week.In another embodiment, give composition when big for the first time in mouse 6 week.
In another embodiment, the effectiveness that in Tg197 transgenic mice sacroiliitis experiment, has of composition in the statistical significance scope more than or equal to the effectiveness of equal dose (is benchmark with mg/kg) medicine (being selected from etanercept, English monoclonal antibody of sharp former times and D2E7).
In another embodiment, composition has effectiveness in the experiment of Tg197 transgenic mice sacroiliitis, and the sacroiliitis that makes treatment produce 0-0.5 is kept the score.In another embodiment, composition has effectiveness in the experiment of Tg197 transgenic mice sacroiliitis, and the sacroiliitis that makes treatment produce 0-1.0 is kept the score.In another embodiment, composition has effectiveness in the experiment of Tg197 transgenic mice sacroiliitis, and the sacroiliitis that makes treatment produce 0-1.5 is kept the score.In another embodiment, composition has effectiveness in the experiment of Tg197 transgenic mice sacroiliitis, and the sacroiliitis that makes treatment produce 0-2.0 is kept the score.
In another embodiment, treatment comprises that suppressing rheumatoid arthritis makes progress.In another embodiment, treatment comprises the outbreak of prevention or deferred class rheumatic arthritis.
In another embodiment, cause the statistics noticeable change of one or more RA indications.In another embodiment, one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
In another embodiment, one or more RA indications comprise that the sacroiliitis macroscopic view phenotype symptom in the Tg197 transgenic mice goes down, wherein give Tg197 transgenic mice composition, wherein write down the sacroiliitis macroscopic view phenotype symptom of Tg197 transgenic mice, wherein according to following system log (SYSLOG) sacroiliitis macroscopic view phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), (the swelling of 2=moderate sacroiliitis, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In another embodiment, one or more RA indications comprise that the sacroiliitis histopathology symptom in the Tg197 transgenic mice goes down, wherein give Tg197 transgenic mice composition, wherein write down the sacroiliitis histopathology symptom of Tg197 transgenic mice, wherein sacroiliitis histopathology symptom shows on the joint, and there is not detectable pathological condition according to following system log (SYSLOG): 0=, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
In another embodiment, single domain antibody polypeptide construction comprises people's single domain antibody polypeptide.In another embodiment, people's single domain antibody polypeptide is in conjunction with TNF α.In another embodiment, single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.In another embodiment, single domain antibody polypeptide construction is with the K of 100nM to 50pM scope dIn conjunction with human TNF alpha.In another embodiment, single domain antibody polypeptide construction is with the K of 30nM to 50pM dIn conjunction with human TNF alpha.In another embodiment, single domain antibody polypeptide construction is with the K of 10nM to 50pM dIn conjunction with human TNF alpha.In another embodiment, single domain antibody polypeptide construction is with the K of 1nM to 50pM scope dIn conjunction with human TNF alpha.
In another embodiment, according to the detection in the experiment of standard L929 cytotoxic cell, single domain antibody polypeptide construction antagonism human TNF alpha.
The present invention further comprises the method for the treatment of rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it comprises the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind, wherein single domain antibody polypeptide construction suppresses combining of human TNF alpha and TNF α acceptor, treats rheumatoid arthritis thus.
In one embodiment, combination of single domain antibody polypeptide construction specificity and cell surface receptor bonded human TNF alpha.
In another embodiment, single domain antibody polypeptide construction has t α half life in the body in 15 minutes to 12 hours scopes.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 1-6 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 2-5 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 3-4 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-60 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-48 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-26 hour scope.
In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 150mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 100mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 75mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 50mg. minute/ml.
In another embodiment, single domain antibody polypeptide construction is connected to the PEG molecule.In another embodiment, the single domain antibody polypeptide construction that PEG connects has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.In another embodiment, the single domain antibody polypeptide construction that connects of PEG has the hydrokinetics size of 200kDa and total PEG size of 20-60kDa at least.In another embodiment, PEGization albumen of the present invention on average can be connected to 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or more a plurality of peg molecule.
In another embodiment, the antibody construction thing contains two or more single immunoglobulin variable structural domain polypeptide in conjunction with human TNF alpha.In another embodiment, the antibody construction thing contains the homodimer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.In another embodiment, the antibody construction thing contains the homotrimer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.In another embodiment, the antibody construction thing contains the same tetramer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.
In another embodiment, construction also comprises has specific antibody polypeptides to non-TNF α antigen.In another embodiment, there is specific antibody polypeptides to comprise the single domain antibody polypeptide to non-TNF α antigen.In another embodiment, non-TNF α antigen is had specific antibody polypeptides and non-TNF α are antigenic to combine half life in the body that has increased the antibody polypeptides construction.In another embodiment, non-TNF α antigen comprises serum protein.In another embodiment, serum protein is selected from scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin (heptaglobin), albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.In another embodiment, non-TNF α antigen comprises HSA.
In another embodiment, treatment also comprises and gives at least a other therapeutical agent.
In another embodiment, single domain antibody polypeptide construction comprises the aminoacid sequence of the antibody polypeptides CDR3 that is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In another embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
The present invention further comprises the method for the treatment of rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it comprises the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind, wherein composition stops the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha, wherein according to the detection in standard L929 cell experiment, in the single domain antibody polypeptide construction and human TNF alpha, wherein treat rheumatoid arthritis.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein single domain antibody polypeptide construction antagonism human TNF alpha and acceptor combines, when giving the mouse of Tg197 transgenic mice arthritis model, prevent the sacroiliitis increase of keeping the score, wherein according to detection in standard L929 cell experiment, in the single domain antibody polypeptide construction and human TNF alpha.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein single domain antibody polypeptide construction antagonism human TNF alpha and acceptor combines, stop sacroiliitis increases of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein single domain antibody polypeptide construction suppresses rheumatoid arthritis and makes progress.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein single domain antibody polypeptide construction antagonism human TNF alpha and acceptor combines, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score, wherein single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein single domain antibody polypeptide construction antagonism human TNF alpha and acceptor combines, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score, wherein according to the detection in standard L929 cell experiment, in the single domain antibody polypeptide construction and human TNF alpha, wherein single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress, wherein single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
In the further embodiment of aforementioned 3 embodiments, single domain antibody polypeptide construction contains the aminoacid sequence of the antibody polypeptides CDR3 that is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
In further embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
The present invention further comprises the method for the treatment of rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it comprises the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, treats rheumatoid arthritis thus.
In one embodiment, composition stops the sacroiliitis increase of keeping the score when giving the mouse of collagen protein induction type sacroiliitis (CIA) mouse model.Bring out chronic recurrence type polyarthritis with mouse II collagen type immunity DBA/1 mouse, it provides human autoimmune arthritic powerful model.This model description is in for example Courtenay etc., and 1980, Nature 282:666-668, Kato etc., 1996, Ann.Rheum.Dis.55:535-539 and Myers etc., 1997, Life Sci.61:1861-1878, it is attached to herein separately by reference.
In one embodiment, giving the mouse composition may further comprise the steps: a) weekly peritoneal injection gives CIA mouse composition, b) claim the weight of step a) mouse once in a week, and c) write down the sacroiliitis macroscopic view phenotype symptom of mouse once in a week according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In one embodiment, treatment comprises the progress that suppresses rheumatoid arthritis.
In one embodiment, treatment comprises prevention and the outbreak of deferred class rheumatic arthritis.
In one embodiment, produce the statistics noticeable change of one or more RA indications.Change preferably reach at least 10% or more than.
In one embodiment, one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint index (are described in Ritchie etc., 1968, Q.J.Med.37:393-406) analyze and analyze the histopathology that obtains by one or more arthrodesis sections and indicate with the x ray contrast in refuse to budge morning continuous time, joint mobilization, arthroncus, one or more joints.Also can use state of an illness activity index (DAS) and/or chronic arthritis system index (CASI) to estimate the state of an illness activity and the variation of treatment influence, referring to Carotti etc., 2002, Ann.Rheum.Dis.61:877-882, with Salaffi etc., 2000, Rheumatology 39:90-96.
In one embodiment, one or more RA indications are included in that sacroiliitis macroscopic view phenotype symptom goes down in the mouse of collagen protein induction type sacroiliitis mouse model, wherein give the mouse composition, wherein write down the sacroiliitis macroscopic view phenotype symptom of mouse, wherein according to following system log (SYSLOG) sacroiliitis macroscopic view phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), (the swelling of 2=moderate sacroiliitis, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In one embodiment, the sacroiliitis histopathology symptom that one or more RA indications are included in the mouse of collagen protein induction type sacroiliitis mouse model goes down, wherein give the mouse composition, wherein write down the sacroiliitis histopathology symptom of mouse, wherein sacroiliitis histopathology symptom shows on the joint, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
In one embodiment, single domain antibody polypeptide construction comprises people's single domain antibody polypeptide.
In one embodiment, people's single domain antibody polypeptide is in conjunction with VEGF.
In one embodiment, single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with people VEGF.
In one embodiment, single domain antibody polypeptide construction with the Kd in 100nM to the 50pM scope in conjunction with people VEGF.
In one embodiment, single domain antibody polypeptide construction with the Kd of 30nM to 50pM in conjunction with people VEGF.
In one embodiment, single domain antibody polypeptide construction with the Kd of 10nM to 50pM in conjunction with people VEGF.
In one embodiment, single domain antibody polypeptide construction with the Kd in 1nM to the 50pM scope in conjunction with people VEGF.
In one embodiment, according to the detection in vegf receptor 1 experiment or vegf receptor 2 experiments, in the single domain antibody polypeptide construction and people VEGF.
The present invention further comprises the method for the treatment of rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it comprises the single domain antibody construction of antagonism people VEGF and receptors bind, wherein the single domain antibody construction suppresses combining of people VEGF and vegf receptor, treats rheumatoid arthritis thus.
In one embodiment, combination of single domain antibody polypeptide construction specificity and cell surface receptor bonded people VEGF.
In one embodiment, single domain antibody polypeptide construction is connected to the PEG molecule.
In one embodiment, the single domain antibody polypeptide construction that PEG connects has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.
In one embodiment, the single domain antibody polypeptide construction that connects of PEG has the hydrokinetics size of 200kDa and total PEG size of 20-60kDa at least.
In one embodiment, PEGization albumen of the present invention on average can be connected to 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or more a plurality of peg molecule.
In one embodiment, the antibody construction thing contains two or more single immunoglobulin variable structural domain polypeptide in conjunction with people VEGF.
In one embodiment, the antibody construction thing contains the homodimer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
In one embodiment, the antibody construction thing contains the homotrimer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
In one embodiment, the antibody construction thing contains the same tetramer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
In one embodiment, the antibody construction thing further contains non-VEGF antigen is had specific antibody polypeptides.
In one embodiment, non-VEGF antigen is had specific antibody polypeptides and comprise the single domain antibody polypeptide.
In one embodiment, non-VEGF antigen is had specific antibody polypeptides and non-VEGF are antigenic to combine half life in the body that increases the antibody polypeptides construction.
In one embodiment, non-VEGF antigen comprises serum protein.
In one embodiment, serum protein is selected from scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.
In one embodiment, non-VEGF antigen comprises HSA.
In one embodiment, single domain antibody polypeptide construction has t α half life in the body in 15 minutes to 12 hours scopes.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 1-6 hour scope.
In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 2-5 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 3-4 hour scope.
In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-60 hour scope.In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-48 hour scope.
In another embodiment, single domain antibody polypeptide construction has t β half life in the body in 12-26 hour scope.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 150mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 100mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 75mg. minute/ml.In another embodiment, single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 50mg. minute/ml.
In one embodiment, treatment further comprises and gives at least a other therapeutical agent.
In one embodiment, therapeutical agent is selected from etanercept, English monoclonal antibody of sharp former times and D2E7.
In one embodiment, therapeutical agent is selected from reflunomide, proteolytic ferment, non-steroidal anti-inflammatory drug (NSAID), acetylsalicylic acid, pyrazolone, fragrant that acid (fenamate), diflunisal, acetogenin, propanoic derivatives, former times health (oxicams), mefenamic acid, Ponstel, meclofenamic acid, Meclomen, Phenylbutazone, Butazolidin, diflunisal, Dolobid, diclofenac, diclofenac, indomethacin (indomethacin), INDOMETHACIN (Indocin), sulindac, Sulindac, R-ETODOLAC, Luo Ding, ketorolac, Toradol, nabumetone, Relafen, tolmetin, Tolectin, Ibuprofen BP/EP, Merrill Lynch, fenoprofen, Nalfon, flurbiprofen, Anthe, carprofen, Rimadyl, Ketoprofen, Orudis, Naproxen Base, Anaprox, Naprosyn, piroxicam and Feldene.
In one embodiment, single domain antibody polypeptide construction comprises the aminoacid sequence of the CDR3 of the antibody polypeptides that is selected from following clone: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
In one embodiment, single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, and wherein single domain antibody polypeptide construction contains and is selected from following CDR3 sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 85% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 90% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 92% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 94% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 96% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 98% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the composition of the single domain antibody polypeptide construction that contains antagonism people VEGF and receptors bind, wherein single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 99% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
The present invention further comprises the method for the treatment of rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, wherein composition comprises the bonded single domain antibody construction of combining of antagonism humanTNF-and acceptor and antagonism people VEGF and acceptor, treats rheumatoid arthritis thus.
In one embodiment, composition stops the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model.
In another embodiment, giving Tg197 transgenic mice composition may further comprise the steps: a) weekly peritoneal injection gives heterozygosis Tg197 transgenic mice composition, b) claim the weight of step a) mouse once in a week, and c) write down the sacroiliitis macroscopic view phenotype symptom of mouse once in a week according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), (the swelling of 2=moderate sacroiliitis, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In another embodiment, the effectiveness that in Tg197 transgenic mice sacroiliitis experiment, has of composition in the statistical significance scope more than or equal to the effectiveness of the medicine that is selected from etanercept, English monoclonal antibody of sharp former times and D2E7.
In another embodiment, treatment comprises the progress that suppresses rheumatoid arthritis.
In another embodiment, treatment comprises the outbreak of prevention or deferred class rheumatic arthritis.
In another embodiment, the statistics that produces one or more RA indications changes significantly.
In another embodiment, one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
In another embodiment, one or more RA indications comprise that the sacroiliitis macroscopic view phenotype symptom in the Tg197 transgenic mice goes down, wherein give Tg197 transgenic mice composition, wherein write down the sacroiliitis macroscopic view phenotype symptom of Tg197 transgenic mice, wherein according to following system log (SYSLOG) sacroiliitis macroscopic view phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), (the swelling of 2=moderate sacroiliitis, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
In another embodiment, one or more RA indications comprise that the sacroiliitis histopathology symptom in the Tg197 transgenic mice goes down, wherein give Tg197 transgenic mice composition, wherein write down the sacroiliitis histopathology symptom of Tg197 transgenic mice, wherein sacroiliitis histopathology symptom shows on the joint, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
In another embodiment, single domain antibody polypeptide construction comprises people's single domain antibody polypeptide.
In another embodiment, people's single domain antibody polypeptide is in conjunction with TNF α and VEGF.
In another embodiment, according to the detection in standard L929 cell experiment, in the single domain antibody polypeptide construction and human TNF alpha.
In another embodiment, single domain antibody polypeptide construction is connected to the PEG molecule.
In another embodiment, the single domain antibody polypeptide construction that PEG connects has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.
In another embodiment, the single domain antibody polypeptide construction that connects of PEG has the hydrokinetics size of 200kDa and total PEG size of 20-60kDa at least.
In another embodiment, the antibody polypeptides construction on average is connected to 1,2,3,4,5,6,7,8,9,10,12,15,17,20 or more a plurality of peg molecule.
In another embodiment, the antibody construction thing contains two or more single immunoglobulin variable structural domain polypeptide and/or two or more single immunoglobulin variable structural domain polypeptide in conjunction with people VEGF in conjunction with human TNF alpha.
In another embodiment, the antibody construction thing contains in conjunction with the homodimer of single immunoglobulin variable structural domain polypeptide of human TNF alpha and/or in conjunction with the homodimer of single immunoglobulin variable structural domain polypeptide of people VEGF.
In another embodiment, the antibody construction thing contains in conjunction with the homotrimer of single immunoglobulin variable structural domain polypeptide of human TNF alpha and/or in conjunction with the homotrimer of single immunoglobulin variable structural domain polypeptide of people VEGF.
In another embodiment, the antibody construction thing contains in conjunction with the same tetramer of single immunoglobulin variable structural domain polypeptide of human TNF alpha and/or in conjunction with the same tetramer of single immunoglobulin variable structural domain polypeptide of people VEGF.
In another embodiment, the antibody construction thing further contains non-TNF α or VEGF antigen is had specific antibody polypeptides.
In another embodiment, non-TNF α or VEGF antigen are had specific antibody polypeptides and comprise the single domain antibody polypeptide.
In another embodiment, non-TNF α or VEGF antigen are had specific antibody polypeptides and non-TNF α or VEGF are antigenic to combine half life in the body that increases the antibody polypeptides construction.
In another embodiment, non-TNF α or VEGF antigen comprise serum protein.
In another embodiment, serum protein is selected from scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.
In another embodiment, non-TNF α antigen comprises HSA.
In another embodiment, treatment also comprises and gives at least a other therapeutical agent.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein combining of single domain antibody polypeptide construction antagonism human TNF alpha and acceptor and combining of antagonism people VEGF and acceptor, stop sacroiliitis increases of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein single domain antibody polypeptide construction suppresses rheumatoid arthritis and makes progress.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein combining of single domain antibody polypeptide construction antagonism human TNF alpha and acceptor and combining of antagonism people VEGF and acceptor, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score, wherein single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
The present invention further comprises the composition that contains single domain antibody polypeptide construction, wherein combining of single domain antibody polypeptide construction antagonism human TNF alpha and acceptor and combining of antagonism people VEGF and acceptor, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score, wherein according to the detection in standard L929 cell experiment, in the single domain antibody polypeptide construction and human TNF alpha, wherein single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress, wherein single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
Another aspect is to select the method for single domain antibody polypeptide construction, wherein single domain antibody polypeptide construction antagonism human TNF alpha and acceptor combines, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score, wherein according to the detection in standard L929 cell experiment, in the described single domain antibody polypeptide construction and human TNF alpha, wherein said single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha, this method may further comprise the steps: the nucleic acid in the site, several hypervariable region of the described single domain antibody polypeptide construction of (1) sudden change coding, so that each site all produces all possible amino displacement, (2) nucleic acid in the site, encoding mutant hypervariable region that will produce in step (1) imports in the phagemid display carrier, to form a large amount of display carriers, its each all can express a kind of in the site, described sudden change hypervariable region, be illustrated on the phagemid surface display albumen; (3) on the filobactivirus particle surface, express the site, hypervariable region that suddenlys change, so that site, consequent sudden change hypervariable region as with each particle in the syzygy of gene III product of the M13 that packs, show with the unit price form by the filobactivirus particle, (4) phage particle of screening surface expression is in conjunction with the ability of TNF α, (5) separating can be in conjunction with the phage particle of the surface expression of TNF α, (6) selecting can be in conjunction with the surface expression phage particle of the step (5) of TNF α, it also stops the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, according to the detection in standard L929 cell experiment, in it and human TNF alpha, suppress described rheumatoid arthritis progress, with less than the Kd of 100nM in conjunction with human TNF alpha, select one or more phagemids that contains display protein thus, wherein display protein contains the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind.
Another aspect is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, treat described rheumatoid arthritis thus, wherein said single domain antibody polypeptide construction has t α half life in the body in 15 minutes to 12 hours, 1-6 hour, 2-5 hour or the 3-4 hour scope.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, and described thus single domain antibody polypeptide construction has t β half life in the body in 12-60 hour, 12-48 hour or 12-26 hour scope.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, and described thus single domain antibody polypeptide construction has AUC half life value in the body of 15mg. minute/ml to 150mg. minute/ml, 15mg. minute/ml to 100mg. minute/ml, 15mg. minute/ml to 75mg. minute/ml or 15mg. minute/ml to 50mg. minute/ml.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, wherein said composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind and antagonism people VEGF and receptors bind, treat described rheumatoid arthritis thus, wherein said composition stops the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein said single domain antibody polypeptide construction each with less than the Kd of 100nM in conjunction with human TNF alpha and VEGF, wherein said single domain antibody polypeptide construction each with the Kd in 100nM to the 50pm scope in conjunction with human TNF alpha and VEGF, wherein said single domain antibody polypeptide construction each with K of 30nM to 50pm dIn conjunction with human TNF alpha and VEGF, wherein said single domain antibody polypeptide construction each with the Kd of 10nM to 50pm in conjunction with human TNF alpha and VEGF, or wherein said single domain antibody polypeptide construction each with the K in 1nM to the 50pm scope dIn conjunction with human TNF alpha and VEGF.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it contains the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind and antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction suppresses combining of combining of human TNF alpha and TNF α acceptor and people VEGF and vegf receptor, treats described rheumatoid arthritis thus.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, it contains the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind and antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction suppresses combining of combining of human TNF alpha and TNF α acceptor and people VEGF and vegf receptor, treat described rheumatoid arthritis thus, wherein said single domain antibody polypeptide construction specificity combination and cell surface receptor bonded human TNF alpha.
Another embodiment is the method for treatment rheumatoid arthritis, this method comprises the composition that the individual treatment of needs significant quantity is arranged, wherein said composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha and receptors bind and antagonism people VEGF and receptors bind, treat described rheumatoid arthritis thus, wherein said single domain antibody polypeptide construction specificity combination and cell surface receptor bonded human TNF alpha.
Another embodiment of the invention is the method for treatment rheumatoid arthritis, it comprises the antibody construction thing that gives the TNF alpha specific, and wherein the antibody construction thing sequence identity percentage that comprises any anti-TNF alpha cloned sequence of mentioning with this paper is formed more than or equal to 85,90,95,96,97,98,99 or 100% sequence or by it.
Another embodiment of the invention is the composition that contains TNF alpha specific antibody construction, and wherein the antibody construction thing sequence identity percentage that comprises any anti-TNF alpha cloned sequence of mentioning with this paper is formed more than or equal to 85,90,95,96,97,98,99 or 100% sequence or by it.
Another embodiment of the invention is the method for treatment rheumatoid arthritis, it comprises and gives VEGF specific antibody construction thing, and wherein the antibody construction thing sequence identity percentage that comprises any anti-VEGF cloned sequence of mentioning with this paper is formed more than or equal to 85,90,95,96,97,98,99 or 100% sequence or by it.
Another embodiment of the invention is the composition that contains the specific antibody construction thing of VEGF, and wherein the antibody construction thing sequence identity percentage that comprises any anti-VEGF cloned sequence of mentioning with this paper is formed more than or equal to 85,90,95,96,97,98,99 or 100% sequence or by it.
In another embodiment, provide tetravalence dual specific antigen-binding polypeptides construction, its contain 2 copies, in conjunction with the V of first antigen or epi-position HOr V LSingle domain antibody; With 2 copies, in conjunction with the V of second antigen or epi-position HOr V LSingle domain antibody.First and second epi-position can be present on the same antigen, or are present in not on the synantigen.2 copies, all merge separately to IgG heavy chain constant domain separately in conjunction with the single domain antibody of first antigen or epi-position, 2 copies, all merge separately in conjunction with the single domain antibody of second antigen or epi-position to light chain constant domain separately.These tetravalence dual specific polypeptide constructions are IgG samples, because it has two antigen brachium conjunctivums that engage by heavy chain and light chain constant domain.They are with the difference of natural IgG: by means of two kinds that exist on each arm different antigen-specific single domain antibody polypeptide, each arm can be in conjunction with two kinds of different antigens or epi-position, makes construction become tetravalence and dual specific.In one embodiment, first is identical with second epi-position, and making has 4 specific binding sites at this epi-position to be present on the polypeptide construction.In another embodiment, first are different with second epi-position, and it is present on the identical or different antigen.
Dual specific tetravalence polypeptide construction as described herein can comprise that any two antigens or epi-position are had specific single domain antibody sequence, but specifically humanTNF-and VEGF there is specific single domain antibody sequence, any single domain antibody sequence described herein of more specifically saying so.In other embodiments, can use C κOr C λThe light chain constant domain also can be used the IgG heavy chain constant domain of non-IgG1.
The present invention also comprises this class construction that contains anti-TNF-Alpha antibodies clone in single structure territory and single structure territory VEGF antibody clone, the anti-TNF-Alpha antibodies in single structure territory stops sacroiliitis increases of keeping the score when being cloned in and giving the mouse of Tg197 transgenic mice arthritis model as monomer, the prevention sacroiliitis increase of keeping the score when single structure territory VEGF antibody is cloned in and gives the mouse of collagen protein induction type sacroiliitis mouse model as monomer.In further embodiment, employed single structure territory anti-TNF-Alpha antibodies clone when as monomer in L929 cytotoxicity experiment described herein in and TNF-α, VEGF antibody clone in employed single structure territory is antagonism vegf receptor combination in vegf receptor 2 combinations described herein are tested when as monomer.In further embodiment, employed single domain antibody clone is with the K less than 100nM dIn conjunction with its antigen or epi-position separately.In further embodiment, dual specific divalence construction is with the K less than 100nM dIn conjunction with its antigen or epi-position separately, and stop the sacroiliitis increase of keeping the score in any of Tg197 described herein and CIA arthritis model or in the two.
With regard to give, with regard to the monitoring of dosage and effectiveness, the mode that this tetravalence dual specific construction can be similar to other construction described herein is used for the treatment of rheumatoid arthritis.Can be as indicated above, for example by adding peg moiety, or by with the further fusion that increases the circulation albumen of half life (for example serum protein, as HSA) specificity bound fraction (for example other single domain antibody), change the half life of construction.
So, in one aspect, a kind of dual specific antigen-binding polypeptides has been described, it contains in conjunction with first antibody single structure domain polypeptide of TNF-α with in conjunction with second antibody single structure domain polypeptide of VEGF.This peptide species can be used for for example treating rheumatoid arthritis.
So, on the other hand, a kind of tetravalence dual specific antigen-binding polypeptides construction has been described, it comprises: a) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain; B) second of described first fusion rotein copy; C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain; D) second of described second fusion rotein copy; Described first and described second copy of wherein said first fusion rotein through its IgG heavy chain constant domain separately each other disulfide linkage close, the described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and wherein said polypeptide construction is in conjunction with described first and described second epi-position.
In an embodiment of this tetravalence dual specific antigen-binding polypeptides construction, in conjunction with the single domain antibody of first epi-position for being selected from V HAnd V LThe V structural domain.
In another embodiment, in conjunction with the single domain antibody of second epi-position for being selected from V HAnd V LThe V structural domain.
In another embodiment, IgG heavy chain constant domain is an IgG1 heavy chain constant domain.
In another embodiment, the light chain constant domain is C κOr C λThe light chain constant domain.In another embodiment, the light chain constant domain is C κThe light chain constant domain.
In another embodiment, first is present on the same antigen with second epi-position.
In another embodiment, first is present on different first and second antigen with second epi-position.
In another embodiment, described single domain antibody in conjunction with first epi-position and described in conjunction with one or two behaviour single domain antibody in the single domain antibody of second epi-position.
In another embodiment, in conjunction with the single domain antibody polypeptide of described first epi-position and/or contain following one or more in conjunction with the single domain antibody polypeptide of described second epi-position: by ethnic group be VH gene order coding FW1, by ethnic group be VH gene order coding FW2, be the FW3 of VH gene order coding and be the FW4 of VH gene order coding by ethnic group by ethnic group.
In another embodiment, all be people's single domain antibody in conjunction with the single domain antibody polypeptide of first epi-position with in conjunction with the single domain antibody of second epi-position.
In another embodiment, IgG heavy chain constant domain is a human IgG heavy chain constant domain.
In another embodiment, IgG heavy chain constant domain comprises C H1 structural domain.
In another embodiment, light chain constant domain behaviour C κThe light chain constant domain.
In another embodiment, construction is in conjunction with TNF-α and VEGF.In another embodiment, merge to IgG1 heavy chain constant domain, merge to C in conjunction with the single domain antibody of TNF-α in conjunction with the single domain antibody of VEGF κThe light chain constant domain.
In another embodiment, when giving the mouse of collagen protein induction type sacroiliitis mouse model, stop the sacroiliitis increase of keeping the score in conjunction with the single domain antibody of VEGF.
In another embodiment, the active single domain antibody polypeptide portion of antagonism people VEGF is with the K less than 100nM dIn conjunction with people VEGF.
In another embodiment, according to the detection in vegf receptor 1 experiment or vegf receptor 2 experiments, in the active single domain antibody polypeptide portion of antagonism people VEGF and people VEGF.
In another embodiment, combination of the active single domain antibody polypeptide portion of antagonism people VEGF specificity and cell surface receptor bonded people VEGF.
In another embodiment aspect this, when giving the mouse of Tg197 transgenic mice arthritis model, stop the sacroiliitis increase of keeping the score in conjunction with TNF-α or the active single domain antibody of antagonism humanTNF-.In another embodiment, according to the detection in standard L929 cytotoxicity experiment, single domain antibody antagonism humanTNF-activity.In another embodiment, single domain antibody is with the K less than 100nM dIn conjunction with the humanTNF-.
In any embodiment aspect this, the active single domain antibody polypeptide portion of antagonism humanTNF-can comprise the CDR3 aminoacid sequence of the antibody polypeptides that for example is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19, or comprise and described sequence for example at least 85%, alternatively at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identical aminoacid sequence.
In another embodiment aspect this, the active single domain antibody polypeptide portion of antagonism humanTNF-can comprise the aminoacid sequence of the antibody polypeptides that for example is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19, or comprise and described sequence for example at least 85%, alternatively at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identical aminoacid sequence.
In any embodiment aspect this, antagonism people VEGF and vegf receptor bonded single domain antibody polypeptide portion can comprise the CDR3 aminoacid sequence of the antibody polypeptides that for example is selected from following clone: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30, or comprise and described sequence for example at least 85%, alternatively at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identical aminoacid sequence.
In another embodiment aspect this, antagonism people VEGF and vegf receptor bonded single domain antibody polypeptide portion can comprise the aminoacid sequence of the antibody polypeptides that is selected from following clone: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30, or comprise and described sequence for example at least 85%, alternatively at least 90%, 92%, 94%, 96%, 98%, 99% or 100% identical aminoacid sequence.
Definition
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have the identical implication of common sense with this area (for example at cell cultures, molecular genetic, nucleic acid chemistry, hybridization technique and biochemical field) those skilled in the art.Use molecule, heredity and biochemical method (generally referring to Sambrook etc., Molecular Cloning:A LaboratoryManual, the 2nd edition, (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. with Ausubel etc., Short Protocols in Molecular Biology (1999) the 4th edition, John Wiley ﹠amp; Sons, Inc, it is attached to herein by reference) and the standard technique of chemical process.
Term used herein " structural domain " refers to be independent of proteic remainder and the folded protein structure that keeps its tertiary structure.In general, structural domain is responsible for proteic discrete function characteristic, can be added into, removes or be transferred to other albumen in many cases, and not lose the function of albumen remainder and/or structural domain.
Described " single immunoglobulin variable structural domain " or " single domain antibody polypeptide " refer to contain the folding polypeptide structure territory of the sequence signature and the specificity conjugated antigen (being 500nM or lower dissociation constant) of immunoglobulin variable structural domain.Therefore, " single domain antibody polypeptide " comprises the variable domains of complete antibody variable domains and modification, for example wherein one or more rings are by the variable domains of the sequence of no antibody variable domains feature replacement, or brachymemma or contain the N-end or the terminal antibody variable territory of extending of C-, and keep 500nM or lower (450nM or lower for example, 400nM or lower, 350nM or lower, 300nM or lower, 250nM or lower, 200nM or lower, 150nM or lower, the specific variable domains fold segments of the target antigen of dissociation constant 100nM or lower) and total length structural domain.Preferred antibody list variable domains is selected from V HAnd V L, comprise V κAnd V λ
Statement " single domain antibody polypeptide construction " not only comprises isolating single domain antibody polypeptide, and comprises and contain the monomeric mostly peptide construction of one or more single immunoglobulin variable structural domain peptide sequences.Be stressed that, for the single domain antibody polypeptide of big construction part by oneself specificity in conjunction with target antigen.Therefore, the single domain antibody polypeptide construction that contains an above single domain antibody polypeptide does not for example comprise wherein needs V for forming specificity in conjunction with the necessary binding site of monoclonal antibody original molecule jointly HAnd V LThe construction of structural domain.Connection between the single domain antibody polypeptide in the single domain antibody polypeptide construction can be peptide or peptide linker, perhaps can be other chemistry and connects, for example being connected by polypeptide monomer and multivalence PEG.The single domain antibody polypeptide that connects can be identical or different, and the target-specific of forming polypeptide also can be identical or different.
Complementary: two kinds of immunoglobulin domains belong to form connection to or the structure family of group or be derived from this family and keep under the situation of this feature, these two kinds of immunoglobulin domains are complementary.For example, the VH structural domain of antibody and VL structural domain are complementary; Two VH structural domains are not complementary, and two V structural domains are not complementary.The complementary structure territory can be present among other member of immunoglobulin superfamily, for example the V of TXi Baoshouti αAnd V β(or γ and δ) structural domain.Under second kind of configuration background of the present invention, the not collaborative binding target molecule of incomplementarity structural domain, but act on the different target epi-positions that may be positioned on the identical or different molecule independently.The artificial structure territory for example based on the structural domain of albumen support (except that engineered back in conjunction with debond epi-position the epi-position), is non-complementary.Equally, two structural domains based on (for example) immunoglobulin domains and fibronectin structural domain are not complementary.
Immunoglobulin (Ig): it refers to keep the immunoglobulin folding feature of antibody molecule, the peptide family that contains two βZhe Dies and contain conservative disulfide linkage usually.The immunoglobulin superfamily member relates to cells in vivo and the interactional many aspects of acellular, be included in extensive effect (for example antibody, TXi Baoshouti molecule etc.) in the immunity system, participate in cell adhesion (for example ICAM molecule) and intracellular signal transduction (for example acceptor molecule, as pdgf receptor).
The present invention is applicable to that all have the immunoglobulin superfamily molecule of binding domains.Preferably, the present invention relates to antibody.
Combination: variable domains of the present invention is combined to form one group of structural domain; For example, the complementary structure territory is capable of being combined, for example V LStructural domain combination VH structural domain.The incomplementarity structural domain is also capable of being combined.Structural domain can make up in many ways, comprises by mode syndeton territory covalently or non-covalently.
Sealing conformation polyspecific part: a kind of polyspecific part has as defined herein been described in this statement, and it contains the epi-position binding domains that at least two this paper thinks.The epi-position binding domains of term ' sealing conformation ' (polyspecific part) assignment body is arranged to such an extent that make the epi-position of an epi-position binding domains in conjunction with combining competition with the epi-position of another epi-position binding domains.That is, each epi-position binding domains can be independently but not side by side in conjunction with the connection epi-position.Can use method described herein to obtain the part of sealing conformation.
Antibody: antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (for example Fab, F (ab ') 2, Fv, FV, the scFv that disulfide linkage connects, the multi-specificity antibody of sealing conformation, scFv, the bifunctional antibody that disulfide linkage connects), no matter be any species that are derived from natural generation antibody, still produce by recombinant DNA technology; Separate from serum, B cell, hybridoma, transfectoma, yeast or bacterium no matter be.
The dual specific part: contain as defined herein first immunoglobulin (Ig) list variable domains and the part of second immunoglobulin (Ig) list variable domains, wherein the variable region can be in conjunction with general not synantigen or two kinds of epi-positions of two kinds of coverlet specific immunoglobulin bonded not on the same antigen.For example, two epi-positions can be positioned on the same haptens, but are not identical epi-positions, or in abutting connection with being not enough to the combination of coverlet ligands specific.Dual specific part of the present invention is formed by having not homospecific variable domains, does not conform to that to have mutually homospecific mutual complementary variable domains right.
Antigen: by part bonded molecule of the present invention.Usually, antigen can be produced antibody response in vivo by the antibody ligand combination.It can be polypeptide, albumen, nucleic acid or other molecule.In general, according to the target-specific of specific antigen being selected dual specific part of the present invention.With regard to conventional antibody and fragment thereof, can conjugated antigen by the definite antibody combining site of variable loop (L1, L2, L3 and H1, H2, H3).
Epi-position: usually by the VH/VL of immunoglobulin (Ig) to the bonded structural unit.Epi-position defines the minimum binding site of antibody, therefore represents the specific target of antibody.With regard to single domain antibody, epi-position representative is by variable domains bonded structural unit independently.
Metal ligand: in conjunction with the part of all members in the storehouse.In general, not by antigen binding site combination as defined above.Limiting examples comprises A albumen, L albumen and G albumen.
Select: obtain or obtain by screening, wherein producing binding interactions between structural domain and antigen or the epi-position or between antibody and antigen or epi-position by the Darwinian system of selection.Therefore, can under the situation that has or do not exist complementary variable domains, select first variable domains of conjugated antigen or epi-position.
General framework: corresponding to as Kabat (" Sequences of Proteins ofImmunological Interest ", U.S. HHS) the conservative antibody district in the defined sequence or corresponding to as Chothia and Lesk, the defined ethnic group of (1987) J.Mol.Biol.196:910-917 is the single antibody frame sequence of immunoglobulin (Ig) storehouse or structure.The invention provides the purposes of single framework and one group of this framework, found that this purposes allows the derivation of binding specificity in fact arbitrarily by independent hypervariable region variation.
Homogeneous phase immunization experiment: check and analysis thing and do not need combination and the immunization experiment of binding reagents separating step not wherein.
Essentially identical (or " basic homologous "): first amino acid or nucleotide sequence contain the identical with second amino acid or nucleotide sequence of enough numbers or are equal to and (for example have similar side chain, Bao Shou aminoacid replacement for example) amino-acid residue or Nucleotide make first have similar activity with second amino acid or nucleotide sequence.With regard to antibody, second antibody has identical binding specificity, has at least 50% of first antibody affinity.
" domain antibodies " or " dAb " is equal to term used herein " single immunoglobulin variable structural domain polypeptide " or " single domain antibody polypeptide ".
Statement used herein " specificity combination " refers to according to using for example BIAcore TMSurface plasma body resonant vibration system and BIAcore TMThe detection that the surface plasma body resonant vibration of kinetics evaluation software (for example 2.1 editions) is analyzed, the immunoglobulin variable structural domain is with 1 μ M or following dissociation constant (K d) conjugated antigen.The affinity of specificity binding interactions or K dBe preferably about 500nM or lower, more preferably from about 300nM or lower.
Term used herein " high affinity combination " refers to be less than or equal to the K of 100nM dIn conjunction with.
Statement used herein " people's single domain antibody polypeptide " refers to have the polypeptide of sequence that the ethnic group of being derived from is immunoglobulin (Ig) V district.When sequence is separated from individual human, when separating the human immunoglobulin gene sequence library (or library of people's antibody V district gene order) of cloning certainly, maybe when clone's ethnic group be the V region sequence be used to produce one or more select subsequently itself and the antigenic bonded diversity of target sequence (by at random or targeted mutagenesis) time, this sequence " being derived from ethnic group is the V district ".The human normal immunoglobulin variable domains is minimum to have at least 85% amino acid similarity (for example comprise 87%, 90%, 93%, 95%, 97%, 99% or higher similarity) with natural human immunoglobulin variable structural domain sequence.
Perhaps or in addition, " human normal immunoglobulin variable domains " is the variable domains that contains 4 people's immunoglobulin variable structural domain framework regions (W1-FW4), described framework region such as Kabat etc. (1991, ibid supra) set forth." human normal immunoglobulin variable domains framework region " comprises a) aminoacid sequence of people's framework region, and b) contain the framework region of at least 8 continuous amino acids of people's framework region aminoacid sequence.The human normal immunoglobulin variable domains can comprise the aminoacid sequence of FW1-FW4, it is identical with the aminoacid sequence of corresponding framework region that by ethnic group is antibody gene sections coding, perhaps it also can contain variable domains, wherein the FW1-FW4 sequence contains nearly 10 aminoacid sequence differences altogether with respect to the aminoacid sequence that by ethnic group is the corresponding framework region of antibody gene sections coding, reach 9 aminoacid sequence differences, reach 8 aminoacid sequence differences, reach 7 aminoacid sequence differences, reach 6 aminoacid sequence differences, reach 5 aminoacid sequence differences, reach 4 aminoacid sequence differences, reach 3 aminoacid sequence differences, reach 2 aminoacid sequence differences or reach 1 aminoacid sequence difference.
" the human normal immunoglobulin variable domains " of this paper definition self has the ability of specificity conjugated antigen, no matter variable domains is to exist with independent single immunoglobulin variable structural domain, still exists with the single immunoglobulin variable structural domain with one or more other peptide sequences.Term used herein " human normal immunoglobulin variable domains " does not comprise " humanization " immunoglobulin polypeptides, has promptly modified in constant region and makes less inhuman (for example mouse, the camel etc.) immunoglobulin (Ig) of its immunogenicity in the people.
Statement used herein " sequence signature of immunoglobulin variable structural domain " refers to the sequence formed with immunoglobulin variable structural domain homologous aminoacid sequence in 20 or more a plurality of, 25 or more a plurality of, 30 or more a plurality of, 35 or more a plurality of, 40 or more a plurality of, 45 or more a plurality of and even 50 or more a plurality of continuous amino acid.
Term used herein " divalence " refers to that antigen binding antibody polypeptide has two antigen-specific binding sites.The epi-position that antigen binding site is discerned can be identical or different.When antibody polypeptides when (be present on the different antigen, or be present on the same antigen), is called antibody polypeptides " dual specific " in conjunction with two kinds of different epi-positions through separately two antigen-specific binding sites.
Term used herein " tetravalence " refers to that antigen-binding polypeptides has 4 antigen-specific binding sites.The epi-position that antigen binding site is discerned can be identical or different." dual specific " tetravalent antibody polypeptide has two binding sites to an epi-position or antigen, and another different epi-positions or antigen are had two binding sites.
" tetravalence dual specific antigen-binding polypeptides construction " used herein has the structure that is similar to natural IgG, and promptly it has two antigen brachium conjunctivums that are connected with the light chain constant domain by heavy chain.But different with natural IgG, each arm all has two antigen binding domainss, and one is first antigen-specific, and one is second antigen-specific.In tetravalence dual specific antigen-binding polypeptides construction described herein, each antigen binding domains is single domain antibody, and promptly the antigen binding domains is unpaired forms single binding site together, as the single binding site in scFv.
Term used herein " IgG type " refers to artificial antigen in conjunction with polypeptide, and its similar that has is in natural IgG, and promptly construction has two antigen brachium conjunctivums that are connected with the light chain constant domain by the heavy chain that is bonded to each other.As described herein, the antigen-binding polypeptides of IgG type is made up of 4 polypeptide chains: contain in conjunction with first antigen or epi-position, merge to IgG heavy chain constant domain and (for example contain C H1-C H2-C HTwo copies of first fusion rotein of single domain antibody polypeptide 3 structural domain); Contain in conjunction with second antigen or epi-position, merge to light chain constant domain (C for example λOr C κ) two copies of second fusion rotein of single domain antibody polypeptide.In this type, when coexpression in cell, heavy chain constant domain disulfide linkage is each other closed, and the also equal separately disulfide linkage of these heavy chain constant domain is bonded to the light chain constant domain.When this term used in this article, the antigen-binding polypeptides of IgG type was a quaternary; Can select to merge single domain antibody to constant domain, (for example to merge to the heavy chain constant domain in conjunction with synantigen not, in conjunction with a kind of antigenic dAb1, merge to the light chain constant domain, in conjunction with another kind of antigenic dAb2), different epi-positions on the same antigen (for example merge to the heavy chain constant domain, the dAb1 of a kind of epi-position on the conjugated antigen, merge to the light chain constant domain, dAb2 in conjunction with the another kind of epi-position on the same antigen), perhaps whole 4 copies all can be in conjunction with the identical epi-position on the same antigen (dAb1 with dAb2 in conjunction with the identical epi-position on the same antigen).
Term used herein " Fab type " refers to bivalent antibody polypeptide construction, and one of them single domain antibody merges to light chain constant domain C L(C for example λOr C κ), another single domain antibody merges to heavy chain C H1 constant domain, C separately H1 and C LConstant domain disulfide linkage is each other closed.Can select single domain antibody, with in conjunction with different epi-positions (also being dual specific) on different antigen (produce dual specific Fab type), the same antigen or the identical epi-position on the same antigen.The example of Fab type bi-specific antibody polypeptide comprises that fusion for example described herein is to for example C λThe anti-TNF-α single domain antibody of light chain, and merge as described herein to people's heavy chain C HThe anti-VEGF single domain antibody of 1 constant domain, wherein two kinds of fusion roteins through its constant domain separately each other disulfide linkage close.In the antibody polypeptides construction of this type, unpaired single binding site, for example single binding site in scFv of together forming of antigen binding domains; On the contrary, but each single domain antibody self equal conjugated antigen makes construction become divalence.
Described " rheumatoid arthritis " be the disease of inflammation in reference and joint and/or other internal's the lining (RA).RA is the many different joints of influence usually.It typically is chronicly, and can be the rapid property disease of sending out.RA is the systemic disease of invasion and attack whole machine body, is one of modal sacroiliitis form.The inflammation that it is characterized in that the joint lining form causes pain, stiff, warm sense, hyperemia and swelling.Inflamed joints lining synovial membrane can invasion and attack and damaged bone and cartilage.Inflammatory cell discharges the enzyme that can digest bone and cartilage.Affected joint can lose its shape and location, causes pain and can't move.Symptom comprises arthritis, swelling, is difficult to move and pain.Other symptom comprises forfeiture appetite, heating, energy loss, anaemia.Further feature comprises the lump (rheumatoid nodule) under the skin in the pressurized zone (for example behind the elbow).Rheumatoid arthritis based on the keeping the score according to grade of several clinical approvals, for example is described in United States Patent (USP) 5,698 clinically, 195 grade, and this patent is attached to herein by reference.In brief, clinical response research can be estimated following parameter:
1. the evaluation of the number in tenderness joint and pain/tenderness
Use following scoring:
0=does not have pain/tenderness
The 1=mild pain.The patient tells its tenderness when inquiry.
The 2=moderate pain.The patient tells its tenderness and shrinks back.
The serious pain of 3=.The patient tells its tenderness and shrinks back and withdraw.
2. the number of swollen joint
Estimate the tenderness and the swelling in each joint independently.
Morning deadlock time length (in minute)
4. grip
5. visual simulation pain scores (0-10cm)
6. inquire patient and blind valuator, to estimate clinical response to medicine.Use following subjective scoring system evaluation clinical response:
The reaction (best possible anticipation reaction) that 5=is splendid
4=sound response (being lower than best possible anticipation reaction)
3=common response (clearly improve but can better)
2=reactionless (no effect)
1=worsens (disease progression)
The cause of disease of unknown rheumatoid arthritis so far.But known RA is an autoimmune disease, causes immune system attack healthy joint tissue, causes inflammation and joint injury subsequently.Many RA patients have certain genetic marker that is called HLA-DR4.
Statement used herein " TNF-α is diseases related " refers to wherein independent or unites one or more other medicine or treatments, neutralize or the medicine of antagonism TNF-α function to treating effective disease of this disease or obstacle, term " treatment " is as defined herein.
Term used herein " treatment " refers to the outbreak of preventing disease or disease symptoms, the progress that suppresses disease disease or disease symptoms or reverse disease or disease symptoms.
Statement used herein " preventing disease outbreak " refer to tend to suffer from specified disease (for example rheumatoid arthritis) but individuality in one or more symptoms or the detect parameters of this disease do not appear.
The statement used herein progress of disease " suppress " refers to the progress when not having this treatment, adopts the treatment of medicine to stop or the seriousness that having delayed the disease symptoms that the treatment intrasubject showed increases.
Statement used herein " reverse disease " refers to that with respect to symptom or parameter before this giving after giving medicine, but one or more symptoms of disease or detect parameters are improved.But symptom or detect parameters " improvement " but evidence be the remarkable favourable difference of (but preferably at least 10%) of statistics of this detect parameters.
But but detect parameters can comprise the parameter that for example can directly detect and the parameter of indirect detection.Directly the limiting examples of detect parameters comprises that joint size, joint mobilization, sacroiliitis and histopathology are kept the score or the serum level of indication and indicator (for example cytokine).But the example of indirect detection parameter comprises the grade of the evaluation disease seriousness of patient's for example uncomfortable sensation or reactivity shortcoming or clinical approval.
Parameter used herein (but for example sacroiliitis is kept the score or other detect parameters) " increase " refers to that the significance,statistical of this parameter increases.Perhaps, " increase " refer at least 10% increase.Equally, " decline " of parameter refers to that the significance,statistical of parameter descends, or at least 10% decline.
Term used herein " antagonism " refers to the medicine interferon activity.When activity is for example TNF-α, VEGF or another kind of bioactive molecules or cytokine active, this term comprises the activity (reaching at least 10%) that suppresses this molecule or cytokine, comprise as limiting examples with receptors bind or interaction (external or on the cultured cells surface or in the body), intracellular signal transduction, cytotoxicity, mitotic division takes place or other is by this molecule or cytokine mediated downstream effects or process (for example gene activation).Antagonistic action comprises the receptors bind of interference factor (for example TNF, VEGF etc.) and the activity of interference factor when the factor is bonded to cell surface receptor.
Term used herein " more than or equal to " refer to that a value equals another value, or in the remarkable mode of statistics (P＜0.1, preferred p＜0.05, more preferably p＜0.01) greater than this value.When the effectiveness of the effectiveness of Comparative composition in for example disease treatment or receptors bind antagonism and another kind of composition, contrast should be carried out on equimolar basis.
" connection " used herein refers to that polymer moieties (for example PEG) is connected to the amino-acid residue of antibody polypeptides (single domain antibody for example as herein described).The PEG polymkeric substance is called as " PEGization " with being connected of amino-acid residue of antibody polypeptides (for example single domain antibody), can use several PEG connection portion to realize that these PEG connection portions include but not limited to N-hydroxy-succinamide (NHS) Acibenzolar, succinimide propionic ester (SPA), maleimide (MAL), vinyl sulfone(Remzaol (VS) or mercaptan.PEG polymkeric substance or other polymkeric substance can be connected to antibody polypeptides in the predetermined position, perhaps can be connected to antibody molecule at random.But preferred PEG polymkeric substance is connected to antibody polypeptides in the predetermined position.The PEG polymkeric substance can be connected to any residue in the antibody polypeptides, but, preferred polymers is connected to Methionin or halfcystine, natural in itself or the antibody polypeptides, perhaps by for example being halfcystine or Methionin and engineeredly go in the antibody polypeptides with the natural residue mutagenesis in the antibody polypeptides." connection " used herein also can refer to the monomeric combination of two or more antibody list variable domains, to form dimer, tripolymer, the tetramer or other polymer.The dAb monomer can be connected to form polymer by several method known in the art, described method includes but not limited to the connection of dAb monomer as expressing fusion protein, two or more monomers peptide linker between monomer, or connect or utilize the chemistry of the joint of disulfide linkage to connect, or by being connected to two, three or multivalence connection portion (for example multi-arm PEG) by translating the monomeric direct chemical each other in back.
With regard to polymkeric substance " directly connect " to the antibody polypeptides (for example single varied texture domain polypeptide), statement used herein " directly connects " and refers to that polymkeric substance is connected to the situation of residue, wherein said residue is the natural integral part of variable domains, for example is not included in constant region, hinge area or the joint peptide.On the contrary, statement used herein " indirectly connect " to antibody polypeptides refers to being connected of polymer molecule and antibody list variable domains, and wherein polymkeric substance is not attached to the amino-acid residue (for example can be connected to hinge area) for the integral part of natural variable region.If polymkeric substance is connected to antibody polypeptides through connection peptides, then polymkeric substance is " connecting indirectly ", and promptly polymkeric substance is not attached to the amino-acid residue into antibody self integral part.Perhaps, if polymkeric substance is connected to the terminal hinge area of the C-of polypeptide, or be connected to any residue of constant region of the integral part existence that can be used as antibody polypeptides, then polymkeric substance " connects " indirectly to antibody polypeptides.
Term used herein " homology " or " similarity " refer to the degree that two Nucleotide or aminoacid sequence are structurally similar each other.Term used herein " similarity " is the yardstick that aminoacid sequence has the degree of similar amino-acid residue at correspondence position when sequence alignment.Amino acid is similar each other when its side chain is similar.Specifically, " similarity " comprises and is the amino acid of conservative substitution each other." guarding " displacement is to have any displacement that is just dividing in blosum62 permutation matrix (Hentikoff and Hentikoff, 1992, Proc.Natl.Acad.Sci.USA 89:10915-10919).Described " sequence A is similar to sequence B n% " refers to that the n% of optimum overall comparison position between sequence A and the B is made up of same amino acid or conservative substitution.Can in the Needleman-Wunsch alignment algorithm, use following parameter to carry out optimum overall comparison:
For polypeptide:
Permutation matrix: blosum62.
Room scoring function :-A-B*LG, A=11 (gap penalty) wherein, B=1 (room length point penalty), LG is a room length.
For nucleotide sequence:
Permutation matrix: coupling is 10, and not matching is 0.
Room scoring function :-A-B*LG, A=50 (gap penalty) wherein, B=3 (room length point penalty), LG is a room length.
Typical conservative substitution is one of Met, Val, Leu and Ile; One of Ser and Thr; One of residue A sp, Glu and Ash; One of residue Gln, Lys and Arg; Or aromatic residue Phe and Tyr.
Just as used herein, when using Needleman-Wunsch algorithm or Tatusova and Madden, 1999, when " BLAST 2 sequences " algorithm that FEMS Microbiol Lett.174:247-250 describes is compared, two sequences have at least 85% sequence similarity each other, and these two sequences are " homologous " or " similar ".When using " BLAST 2 sequence algorithms " comparison aminoacid sequence, the Blosum62 matrix is a default matrix.
As known in the art, " identity " is the relation between two or more peptide sequences or two or more nucleotide sequences, and it is by the contrast sequencing.In this area, " identity " also refers to the serial correlation degree between polypeptide or the polynucleotide sequence, and it is measured by the coupling between these sequence chains according to circumstances.Can easily calculate the identity percentage by currently known methods, these methods include but not limited to be described in Computational MolecularBiology, Lesk, and A.M. edits, Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D.W. edits, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G. edits, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; With Sequence Analysis Primer, Gribskov, M. and Devereux, J. edits, M Stockton Press, New York, 1991; And Carillo, H. and Lipman, D., SIAM J.Applied Math., the method for 48:1073 (1988).The preferred method of measuring identity is designed for the maximum match that obtains between institute's cycle tests.Measuring the method for identity programmes in the computer program that can openly obtain.The preferred computer programmed method of measuring the sequence identity between two sequences includes but not limited to GCG routine package (Devereux, J. etc., 387 (1984)), BLASTP, BLASTN and FASTA (Atschul Nucleic Acids Research 12 (1):, S.F. etc., J.Molec.Biol.215:403-410 (1990)).BLAST X program can be by NCBI and open (BLAST Manual, Altschul, S. etc., NCBI NLM NIH Bethesda, the Md.20894 of obtaining in other source; Altschul, S. etc., J.Mol.Biol.215:403-410 (1990)).As an illustration, nucleotide sequence has at least for example polynucleotide of 95% " identity " with the reference nucleotide sequence of " SEQ ID NO:A ", implication is that the nucleotide sequence of these polynucleotide is identical with reference sequence except this polynucleotide sequence can comprise nearly 5 point mutation in per 100 Nucleotide of " SEQ ID NO:A " reference nucleotide sequence.In other words, for obtaining the identical polynucleotide of the nucleotide sequence had and reference nucleotide sequence at least 95%, maximum 5% Nucleotide can lack or by another kind of nucleotide subsitution in the reference sequence, maybe some Nucleotide of maximum 5% in the reference sequence total nucleotide can be inserted in the reference sequence.These sudden changes of reference sequence can appear at 5 or 3 terminal positions of reference nucleotide sequence or any position between these terminal positions, it is dispersed in the Nucleotide in the reference sequence independently, or is dispersed in one or more successive clusters (contiguous group) in the reference sequence.Similarly, with regard to the polypeptide that the reference aminoacid sequence of aminoacid sequence and " SEQ IDNO:B " has 95% identity at least for example, implication is that this amino acid sequence of polypeptide is identical with reference sequence except this peptide sequence can comprise maximum 5 amino acid variations in amino acid whose per 100 amino acid of " SEQ ID NO:B " reference.In other words, for obtaining the identical polypeptide of the aminoacid sequence had and reference aminoacid sequence at least 95%, maximum 5% amino-acid residue can lack or by another kind of amino-acid substitution in the reference sequence, maybe can be with some aminoacid insertion of total amino acid residue 5% in maximum reference sequence in reference sequence.These variations of reference sequence can appear at the amino of reference aminoacid sequence or any position between C-terminal position or these terminal positions, it is dispersed in the residue in the reference sequence independently, or is dispersed in one or more successive clusters in the reference sequence.
Term used herein " low severity ", " medium severity ", " high severity " or " very high stringent condition " have been described the condition of nucleic acid hybridization and rinsing.Be found in Current Protocols in Molecular Biology about the guidance of implementing hybridization, JohnWiley and Sons, N.Y. (1989), 6.3.1-6.3.6, its by reference integral body be attached to herein.In this reference, describe moisture and water-free method, can use any.Specific hybrid condition as referred to herein is following condition: (1) hangs down stringent hybridization condition: 6X sodium chloride/sodium citrate (SSC), about 45 ℃, follow with 0.2X SSC, 0.1%SDS at least 50 ℃ of (for low stringent hybridization condition, the rinsing temperature can increase to 55 ℃) rinsings 2 times; (2) medium stringent hybridization condition: 6X SSC, about 45 ℃, then in 60 ℃ of rinsings 1 time or repeatedly with 0.2X SSC, 0.1%SDS; (3) high stringent hybridization condition: 6X SSC, about 45 ℃, then in 65 ℃ of rinsings 1 time or repeatedly with 0.2X SSC, 0.1%SDS; And preferred (4) very high stringent hybridization condition: 0.5M sodium phosphate, 7%SDS, 65 ℃, then in 65 ℃ of rinsings 1 time or repeatedly with 0.2XSSC, 1%SDS.
Statement used herein " in ... concentration " is showed and is decided polypeptide and be dissolved in the solution (preferred aqueous solution) with mentioned per unit volume quality or molar weight.Therefore, " in X concentration " or " in the concentration of X at least " polypeptide that exists has been got rid of the drying and the crystallization goods of polypeptide.
Term used herein " storehouse " refers to the set of different variants, for example the different polypeptide variants of its primary sequence.Be used for library of the present invention and comprise the peptide library that contains at least 1000 members.
Term used herein " library " refers to the mixture of heterologous polypeptide or nucleic acid.The library all has the member composition of single polypeptide or nucleotide sequence by each.Thus, library and storehouse synonym.Sequence difference between the member of library is to produce the multifarious reason that exists in the library.The form of the simple mixtures of polypeptide or nucleic acid can be taked in the library, or can be the form of organism or cell, for example the bacterium that transforms with nucleic acid library, virus, animal or plant cell etc.Preferably, each independent organism or cell all only contain one or a spot of library member.Advantageously, nucleic acid is incorporated in the expression vector, so that allow the polypeptide of expression by nucleic acid encoding.Therefore, one preferred aspect, host living beings group's form can be taked in the library, each biology all contains one or more copies of expression vector, this carrier contains the single member in library of nucleic acid form, can express it to produce its corresponding polypeptide member.Therefore, the host living beings group has the potentiality in the big storehouse of genetic coding diversity polypeptide variants.
" polymkeric substance " used herein refers to the macromole be made up of repeated monomer unit, and can refer to nonsynthetic or naturally occurring polymkeric substance, for example optional straight or branched polyalkylene that replaces, poly-alkenylene or polyoxy alkylidene, or branch or ramose polysaccharide not.Specifically, " polymkeric substance " used herein refers to optional that replace or ramose chain poly-(ethylene glycol), poly-(propylene glycol) or poly-(vinyl alcohol) and derivative thereof.
" PEG " used herein or " PEG polymkeric substance " refers to polyoxyethylene glycol, the PEG that more particularly can refer to the derivatize form, N-hydroxy-succinamide (NHS) active ester that includes but not limited to PEG for example succinimide propionic ester, benzotriazole active ester, with the PEG of maleimide, vinyl sulfone(Remzaol or mercapto groups derivation.Concrete PEG goods can comprise PEG-O-CH 2CH 2CH 2-CO 2-NHS, PEG-O-CH 2-NHS, PEG-O-CH 2CH 2-CO 2-NHS, PEG-S-CH 2CH 2-CO-NHS, PEG-O 2CNH-CH (R)-CO 2-NHS, PEG-NHCO-CH 2CH 2-CO-NHS and PEG-O-CH 2-CO 2-NHS, wherein R is (CH 2) 4) NHCO 2(mPEG).Useful in the present invention PEG polymkeric substance can be linear molecule, perhaps can be ramose, and wherein a plurality of peg moieties are present in the single polymkeric substance.The particularly preferred PEG conformation of useful in the present invention some includes but not limited to following form:
Figure A20058002851801061
" sulfydryl selective reagent " used herein is to be used for the PEG polymkeric substance is connected to the reagent that contains mercaptoamino-acid.Mercapto groups on the amino-acid residue halfcystine is pair particularly useful with the interaction of sulfydryl selective reagent.Be used for sulfydryl selective reagent of the present invention and include but not limited to maleimide, vinyl sulfone(Remzaol or mercaptan.The purposes of sulfydryl selective reagent coupling cysteine residues is known in the art, can needs according to the present invention adopt (referring to Eg., Zalipsky, 1995, Biocojug.Chem.6:150; Greenwald etc., 2000, Crit. Rev.Ther.Drug Carrier Syst.17:101; Herman etc., 1994, Macromol Chem.Phys.195:203).
Term used herein " antigen " refers to by the land of antibody or antibody (for example variable domains) bonded molecule.Usually, antigen can produce antibody response in vivo.Antigen can be peptide, polypeptide, albumen, nucleic acid, lipid, sugar or other molecule.In general, select the immunoglobulin variable structural domain according to target-specific to specific antigen.
Term used herein " epi-position " refers to usually by immunoglobulin (Ig) V H/ V LTo the bonded structural unit.Epi-position defines the minimum binding site of antibody, has therefore represented the specific target of antibody.With regard to single domain antibody, epi-position is represented by isolating variable domains bonded structural unit.
Term used herein " neutralization " is when being used in reference to generation during single immunoglobulin variable structural domain polypeptide as herein described, but means detection of active or the function that this polypeptide disturbs target antigen.If but polypeptide is with the detection of active or the function reduction at least 50% of target antigen, preferably at least 60%, 70%, 80%, 90%, 95% or higher, be up to and comprise that 100% suppresses (promptly not having detectable target antigen effect or function), then this polypeptide is " neutralization " polypeptide.But this reduction of the detection of active of target antigen or function can use the standard method of one or more indexs that detect this activity or function to estimate by those skilled in the art.As an example, when target is TNF-α, can use the experiment of standard L929 cell killing or go up the ability that TNF-α inducibility ELAM-1 expresses (it detects TNF-α inductive cell activation) by the single immunoglobulin variable structural domain inhibition of detection HUVEC, evaluation neutralizes active.Similar with " neutralization " used herein, " cytotoxicity that suppresses cell " used herein refers to according to the detection of for example using the experiment of standard L929 cell killing, necrocytosis descends, wherein repressed cytotoxicity be necrocytosis descend at least 10% or more than.
" but the detection of active of target antigen or function " used herein includes but not limited to for example cell signalling, enzymic activity, combination activity, ligand dependent internalization, cell killing, cell activation, lifting cell survival rate and genetic expression.Those skilled in the art can implement to detect these active experiments of given target antigen.Preferably, following definite " activity " used herein: the ND50 in the experiment of (1) cellular type; (2) to the affinity of target ligands; (3) ELISA combination; Or (4) receptors bind experiment.The method of implementing these tests is well known by persons skilled in the art, is described in further detail hereinafter.
" retentive activity " used herein refers to the activity level of the antibody polypeptides (for example single variable domains) that PEG connects, it is at least 10% of the antibody polypeptides activity level that connects of non-PEG, be preferably antibody polypeptides active at least 20%, 30%, 40%, 50%, 60%, 70%, 80% that the non-PEG of identical sequence connects up to 90%, preferably reach 95%, 98% up to 100%, wherein active mensuration as described herein.More particularly, should be that benchmark is measured the activity of the antibody polypeptides of PEG connection than the antibody polypeptides of non-PEG connection with the antibody mole number; Promptly each PEG that should use mole number to equate in each experiment connects antibody polypeptides and is connected antibody polypeptides with non-PEG.Connect antibody polypeptides whether when " retentive activity " measuring specific PEG, the activity of the same antibody polypeptide when preferably the activity of the antibody polypeptides that PEG is connected is with no PEG contrasts.
Term used herein " homodimer ", " homotrimer ", " the same tetramer " and " same polymer " refer to respectively contain 2,3 or more a plurality of (for example 4,5 etc.) give the monomeric molecule of order immunoglobulin variable structural domain peptide sequence.For example, homodimer should comprise the identical V of two copies HSequence." monomer " of single immunoglobulin variable structural domain polypeptide is the single V of specificity conjugated antigen HOr V LSequence.Homodimer, homotrimer, can connect in the following manner: express (for example using the peptide linker between the monomer) as fusion polypeptide with the tetramer or with the monomer in the polymer, or by the monomer after will translating each other direct chemical connect or connect by the joint chemistry that utilizes disulfide linkage, or by being connected to two, three or the multivalence connection portion.In one embodiment, homodimer, homotrimer, can connect by multi-arm PEG polymkeric substance with the tetramer or with the monomer in the polymer, wherein each monomer in dimer, tripolymer, the tetramer or the polymer all is connected to the peg moiety of multi-arm PEG as mentioned above.
Term used herein " heterodimer ", " heterotrimer ", " the different tetramer " and " heteromultimeric " refer to contain 2,3 or more a plurality of (for example 4,5,6,7 up to 8 or more a plurality of) monomeric molecule of two or more different single immunoglobulin variable structural domain peptide sequences respectively.For example, heterodimer should comprise two V HSequence, for example V H1And V H2, perhaps alternately comprise V HAnd V LCombination.With homodimer, homotrimer or similar with the tetramer, monomer in heterodimer, heterotrimer, the different tetramer or the heteromultimeric can connect in the following manner: express (for example using the peptide linker between the monomer) as fusion polypeptide, or by the monomer after will translating each other direct chemical connect or connect by the joint chemistry that utilizes disulfide linkage, or by being connected to two, three or the multivalence connection portion.In one embodiment, monomer in heterodimer, heterotrimer, the different tetramer or the heteromultimeric can connect by multi-arm PEG polymkeric substance, and wherein each monomer in dimer, tripolymer, the tetramer or the polymer all is connected to the peg moiety of multi-arm PEG as mentioned above.
The serum-concentration of term used herein " half life " assignment body (for example antibody polypeptides, as single immunoglobulin variable structural domain) for example since ligand degradation and/or natural mechanism the removing of part and chelating were reduced for 50% spent time in vivo.Stabilization antibody polypeptides in vivo, its half life, increase by the molecule (for example PEG) that is bonded to opposing degraded and/or removing or chelating.If the cycle that antibody polypeptides (for example dAb) functionally active in vivo continues is longer than the similar dAb that is not connected to the PEG polymkeric substance, then its half life, increase.Usually, the half life of PEGization dAb, increase by 10%, 20%, 30%, 40%, 50% or higher with respect to non-PEGization dAb.The increase of half life in 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or higher scope is possible.Perhaps or in addition, the increase of half life in reaching 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x scope is possible.According to the present invention, the antibody list variable domains that PEG connects has 0.25-170 hour half life, and preferred 1-100 hour, more preferably 30-100 hour, more preferably 50-100 hour again, up to 170,180,190 and 200 hours or higher.
" to the resistance of degraded " used herein or " anti-degraded " are when the dAb monomer that connects at PEG or other polymkeric substance or polymer use, mean dAb monomer that PEG or other polymkeric substance connect or polymer and degrade in the time of 30 minutes at the stomach en-of contact pH 2.0 and be no more than 10%, preferably degrade.When specifically mentioning the dAb polymer (for example XOR homodimer, tripolymer, the tetramer etc.) that connects PEG or the connection of other polymkeric substance, the degraded in 30 minutes in the presence of the stomach en-of pH 2.0 of degradation-resistant molecule is lower than 5%, preferably not degraded.
" hydrokinetics size " used herein refers to the molecule apparent size based on the diffusion of molecule (for example protein molecular) in aqueous solution.Can handle diffusion or the motion of albumen in solution, to obtain proteic apparent size, wherein size obtains by " the Stokes radius " or " hydrodynamic radius " of protein grain.Proteic " hydrokinetics size " depend on quality and shape (conformation) these two, make two kinds of albumen to have different hydrokinetics sizes based on the whole conformation of albumen with same molecular amount.The big I of hydrokinetics of the antibody polypeptides (for example single variable domains comprises antibody variable described herein territory polymer) that PEG connects is in the scope of 24kDa to 500kDa, 30-500kDa, 40-500kDa, 50-500kDa, 100-500kDa, 150-500kDa, 200-500kDa, 250-500kDa, 300-500kDa, 350-500kDa, 400-500kDa and 450-500kDa.The hydrokinetics size of preferred PEGization dAb of the present invention is 30-40kDa, 70-80kDa or 200-300kDa.When needs were used for the radiography application with antibody variable territory polymer, polymer should have the hydrokinetics size of 50-100kDa.Perhaps, when needs were used for the treatment of application with antibody single structure territory polymer, polymer should have the above hydrokinetics size of 200kDa.
The accompanying drawing summary
Fig. 1 has shown the diversity that VH/HSA locates at position H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98 (respectively by DVT or NNK coding), and these positions are positioned at the antigen binding site of VH HSA.The VK sequence is diversified at position L50, L53.Aminoacid sequence, SEQ ID NO:219; Nucleotide sequence, cochain, SEQID NO:220.
Fig. 2 has shown library 1: kind is VK/DVT VH; Library 2: kind is VK/NNK VH; Library 3: kind is VH/DVT VK; Library 4: kind is VH/NNK VK.Be phage display/ScFv form.Select in advance in conjunction with metal ligand A albumen and proteic these libraries of L, so that there is function in the library of most of clone and selection.Select the library at HSA (first round) and β-gal (second takes turns) or HSA β-gal, or select the library at β-gal (first round) and HSA (second takes turns) β-gal HSA.These clones' of pcr amplification solubility scFv in order.Select the clone K8 of a coding bi-specific antibody further to study.Nucleotide sequence: SEQ ID NO:221; Aminoacid sequence: SEQ ID NO:222.
Fig. 3 has shown the comparison of VH chain and VK chain.V HSimulation, SEQ ID NO:1; K8, SEQ ID No 223 (VH) and 232 (VK); VH2, SEQ ID NO:224; VH4, SEQ ID NO:225; VHC11, SEQ ID NO:226; VHA10sd, SEQ ID NO:227; VHA1sd, SEQ ID NO:228; VHA5sd, SEQ ID NO:229; VHC5sd, SEQ ID NO:230; VHC11sd, SEQ ID NO:231; V κSimulation, SEQ ID NO:3; E5sd, SEQ ID NO:233; C3, SEQ ID NO:234.
Fig. 4 has shown the feature of K8 antibodies characteristic, and K8 antibodies characteristic is characterised in that mono-clonal phage E LISA, finds dual specific K8 antibodies HSA and β-gal, and be illustrated on the phage surface that absorption signal is greater than 1.0.Do not detect and other proteic cross reactivity.
Fig. 5 has shown the scFv ELISA of the K8 antibody fragment enforcement of using concentration known.With the BSA of 100 μ g HSA, 10 μ g/ml and β-gal and 100 μ g/ml A albumen with 1 μ g/ml concentration bag by the 96-orifice plate.Apply the serial dilution of the K8 scFv of 50 μ g, detect the bonded antibody fragment with L albumen-HR.ELISA result has confirmed the dual specific character of K8 antibody.
Fig. 6 shown the clone K8VK/ simulation VH that uses solubility scFv elisa assay in conjunction with feature.As Harrison etc., Methods Enzymol.1996; 267:83-109 is described to induce the segmental generation of solubility scFv with IPTG, directly detects the supernatant liquor that contains scFv.As described in embodiment 1, carry out solubility scFv ELISA, detect bonded scFv with L albumen-HRP.ELISA result discloses, and this clone still can be in conjunction with β-gal, and is eliminated in conjunction with BSA.
Fig. 7 has shown the sequence of varied texture domain vector 1 and 2.Nucleotide sequence: SEQ IDNO:221; Aminoacid sequence: SEQ ID NO:222.
Fig. 8 is the CH carrier figure that is used to make up VH1/VH2 polyspecific part.
Fig. 9 is the VK carrier figure that is used to make up VK1/VK2 polyspecific part.
Figure 10 has contrasted the monomeric TNF acceptor experiment of TAR1-5 dimer 4, TAR1-5-19 dimer 4 and TAR1-5-19.
Figure 11 has contrasted the TNF acceptor experiment of TAR1-5 dimer 1-6.All dimers are FPLC purifying all, has shown the result of the dimerization type of optimizing.
The TNF acceptor experiment of the veriform TAR1-519 homodimer of Figure 12: dAb joint-dAb type, Fab type and halfcystine knuckle joint type with 3U, 5U or 7U joint.
The simulation VH sequence in Figure 13 library 1.Based on kind is the VH frame sequence of sequence D P47-JH4b.Wherein NNK randomization (N=A or T or C or G Nucleotide; K=G or T Nucleotide) position that has been incorporated in the library 1 represents with the runic underline text.Aminoacid sequence: SEQ ID NO:1; Nucleotide sequence, cochain, SEQ ID NO:2.
The simulation VH sequence in Figure 14 library 2.Based on kind is the VH frame sequence of sequence D P47-JH4b.Wherein NNK randomization (N=A or T or C or G Nucleotide; K=G or T Nucleotide) position that has been incorporated in the library 2 represents with the runic underline text.Aminoacid sequence: SEQ ID NO:235; Nucleotide sequence, cochain, SEQ ID NO:236.
The simulation VK sequence in Figure 15 library 3.Based on kind is VK framework 5 sequences of sequence D PK9-J K1.Wherein NNK randomization (N=A or T or C or G Nucleotide; K=G or T Nucleotide) position that has been incorporated in the library 3 represents with the runic underline text.Aminoacid sequence: SEQ ID NO:3; Nucleotide sequence, cochain, SEQ ID NO:4.
Nucleotide and the aminoacid sequence of the anti-MSA dAb of Figure 16 MSA 16 and MSA 26.MSA 16: aminoacid sequence, SEQ ID NO:237; Nucleotide sequence, SEQ ID NO:238.MSA 26: aminoacid sequence, SEQ ID NO:239; Nucleotide sequence, SEQ IDNO:240.
Figure 17 MSA 16 and 26 inhibition Biacore.By measuring K dInhibition Biacore analyze the dAb MSA16 and the MSA26 of purifying.In brief, test dAb is to determine that obtaining 200RU on the Biacore CM5 chip of high-density MSA bag quilt replys required dAb concentration.In case determined required dAb concentration, just will be at expection K dThe MSA antigen and the dAb pre-mixing of near concentration range, and the incubation that spends the night.Detect dAb and MSA bag combining in each premixture with 30 μ l/ minutes high flow rate then by the Biacore chip.
MSA16 serum level after Figure 18 injection.In mouse, measure the serum half life of dAb MSA16.MSA16 goes into administration in the CD1 mouse with the single agent intravenous injection of about 1.5mg/kg.Adopt the modeling of 2 compartment models to show that MSA16 has 0.98 hour t1/2 β, 36.5 hours t1/2 β and the AUC of 913 hours .mg/ml.And have 0.06 hour t1/2 β and the HEL4 (anti-hen's egg-white lysozyme dAb) of 0.34 hour t1/2 β and compare, MSA16 have be extended quite long half life.
Figure 19 shows that suppressing TNF tests (c) with Fab print section bonded ELISA (a) that contains MSA26Ck and TAR1-5-19CH and TNF acceptor.Add MSA and Fab print section and reduced the inhibition level.Survey the elisa plate that wraps quilt with 1 μ g/ml TNF-α with dual specific VK CH and VK CK Fab print section, survey this elisa plate with the contrast TNF-α that on ELISA, produces the concentration of similarity signal as calculated in conjunction with dAb in addition.Having and do not having under the situation of 2mg/ml MSA, dual specific and contrast dAb all are used to survey elisa plate.Signal in the dual specific hole reduces above 50%, but the signal in the dAb hole does not have reduction (referring to Figure 19 a) at all.Having and do not having under the situation of MSA in addition, identical dual specificity protein is being placed the acceptor experiment, also showing MSA competition (referring to Figure 19 c).This shows, MSA and dual specific combine the competition that combines with itself and TNF-α.
The experiment of Figure 20 TNF acceptor has shown that the disulfide linkage of TNF and TAR1-5-19 dAb and MSA16 dAb closes heterodimer bonded inhibitor.MSA and dimeric adding reduce the inhibitor level in the dose-dependently mode.In the presence of the MSA of the heterodimer of constant density (18nM) and serial dilution and HSA, carry out the experiment of TNF acceptor.The HSA that exists with finite concentration scope (until 2mg/ml) does not cause that dimer suppresses the reduction of TNF-α ability.But, add MSA and make dimer suppress the ability dose-dependently reduction of TNF-α.This shows that MSA and TNF-α competition are in conjunction with the TAR1-5-19 of MSA16 dimer-cys bonding.Independent MSA and HSA do not influence TNF in the experiment in conjunction with level.
Figure 21 has shown that ethnic group is polynucleotide and the aminoacid sequence (in addition referring to Fig. 1) of framework sections DP47.Aminoacid sequence is SEQ ID NO:1; The polynucleotide sequence of cochain is SEQID NO:2.
Figure 22 has shown that ethnic group is polynucleotide and the aminoacid sequence of framework sections DPK9.Aminoacid sequence is SEQ ID NO:3; The polynucleotide sequence of cochain is SEQ ID NO:4.
Figure 23 has shown TAR1 clone's described herein aminoacid sequence (referring to for example embodiment 13).TAR1-5，SEQ?ID?NO：241；TAR1-27，SEQ?ID?NO：242；TAR1-261，SEQ?ID?NO：243；TAR1-398，SEQ?ID?NO：244；TAR1-701，SEQID?NO：245；TAR1-5-2，SEQ?ID?NO：246；TAR1-5-3，SEQ?ID?NO：247；TAR1-5-4，SEQ?ID?NO：248；TAR1-5-7，SEQ?ID?NO：249；TAR1-5-8，SEQ?ID?NO：250；TAR1-5-10，SEQ?ID?NO：251；TAR1-5-11，SEQ?IDNO：252；TAR1-5-12，SEQ?ID?NO：253；TAR1-5-13，SEQ?ID?NO：254；TAR1-5-19，SEQ?ID?NO：191；TAR1-5-20，SEQ?ID?NO：255；TAR1-5-21，SEQ?ID?NO：256；TAR1-5-22，SEQ?ID?NO：257；TAR1-5-23，SEQ?IDNO：258；TAR1-5-24，SEQ?ID?NO：259；TAR1-5-25，SEQ?ID?NO：260；TAR1-5-26，SEQ?ID?NO：261；TAR1-5-27，SEQ?ID?NO：262；TAR1-5-28，SEQ?ID?NO：263；TAR1-5-29，SEQ?ID?NO：264；TAR1-5-34，SEQID?NO：265；TAR1-5-35，SEQ?ID?NO：266；TAR1-5-36，SEQ?ID?NO：267；TAR1-5-464，SEQ?ID?NO：268；TAR1-5-463，SEQ?ID?NO：269；TAR1-5-460，SEQ?ID?NO：270；TAR1-5-461，SEQ?ID?NO：271；TAR1-5-479，SEQ?ID?NO：272；TAR1-5-477，SEQ?ID?NO：273；TAR1-5-478，SEQ?ID?NO：274；TAR1-5-476，SEQ?ID?NO：275；TAR1-5-490，SEQ?ID?NO：276；TAR1h-1，SEQ?ID?NO：277；TAR1h-2，SEQ?ID?NO：278；TAR1h-3，SEQ?ID?NO：279。
Figure 24 has shown the contrast of serum half life of the TAR1-5-19 of dAb haplotype after single agent intravenous injection or Fc pattern of fusion.
Figure 25 has summarized the dosage of the dAb construction that gives and has given the time in a series of Tg197 model experiments of using TAR1-5-19.
Figure 26 has summarized the weekly dosage of the veriform TAR1-5-19 dAb (Fc fusion, PEGization, anti-TNF/anti--SA dual specific) that uses in Tg197 mouse RA model research.
Figure 27 has summarized type (Fc fusion, PEG dimer, the PEG tetramer, anti-TNF/anti--SA dual specific), transfer mode and the dosage of the anti-TNF dAb that gives in Tg197 mouse RA model research, this research has contrasted the effectiveness of anti-TNF dAb construction and the effectiveness of existing anti-TNF product.
Figure 28 has shown the dosage regimen and the marking scheme of research, the effectiveness that this research checks anti-TNF dAb to resist existing disease symptoms in Tg197 mouse RA model.
Figure 29 has shown the SDS PAGE gel analysis of IgG sample bi-specific antibody, and described IgG sample bi-specific antibody contains the people VEGF specificity V that merges to human IgG1's constant domain κVariable domains and merge C to the people κHumanTNF-'s specificity V of constant domain κVariable domains.Swimming lane 1:InVitrogen Multimark molecular weight marker.Swimming lane 2: the anti-VEGF bi-specific antibody of anti-TNF x in 1X irreducibility load sample damping fluid.Swimming lane 3: the anti-VEGF bi-specific antibody of anti-TNF x in 1X load sample damping fluid+10mM beta-mercaptoethanol.
Figure 30 A and B shown TNF-alpha active and vegf receptor in conjunction with experiment in the inhibiting experimental result of the anti-VEGF bi-specific antibody of check anti-TNF x.In the A.L929TNF-α cytotoxicity and result of experiment.With the curve of square display data point, contrast anti-TNF-Alpha antibodies.With the curve at point triangle table registration strong point up, the anti-VEGF bi-specific antibody of anti-TNF x.With the curve at point triangle table registration strong point down, anti-TNF-α monomer.B. people's vegf receptor 2 is in conjunction with result of experiment.With the curve of square display data point, the anti-VEGF bi-specific antibody of anti-TNF x.With the curve at point triangle table registration strong point up, anti-VEGF contrast.With the curve at point triangle table registration strong point down, negative control.
Detailed Description Of The Invention
The bispecific antibody polypeptide:
The present inventor has described the further improvement of bispecific part in its International Patent Application WO 2004/003019, wherein a species specificity of part is for the albumen that exists in the organism body or polypeptide, can by with its in conjunction with playing increase part half life. WO 2004/003019 has described to contain to have first immunoglobulin (Ig) list variable domains of binding specificity and second antigen or epi-position is had bispecific part in conjunction with second active complementary immunoglobulin (Ig) list variable domains first kind of antigen or epi-position, in wherein said antigen or the epi-position one or both work to increase half life in the part body, wherein said first with the total mutually homospecific mutual complementary structure territory of second domain, prerequisite is that described bispecific part be can't help anti-HSA VH domain and anti-p galactosidase VK domain and formed.
Advantageously, the antigen of increase part as described herein half life or epi-position are present in the organism body on visible albumen or the polypeptide.
Example comprises extracellular matrix protein, blood protein and is present in albumen in the various tissues of organism. Albumen is for example by serving as filler or by part being anchored into the action site of needs, working to reduce part clearance rate in the blood. Annex 1 has hereinafter provided the example that increases antigen/epi-position of half life in the body.
The half life that increases, is useful to using in the body of immunoglobulin (Ig), and especially antagonist is useful, and is especially the most useful to undersized antibody fragment. This fragment (Fv that Fv, disulfide bond close, Fab, scFv, dAb) is subjected to the quick removing of body; Therefore, although it can arrive most of parts of body rapidly, and produce fast and easy operating, use in its body and be subject to its only retention time in the of short duration body. The present invention is by providing the part that half life increases in the body and therefore being provided at active this problem that solved of the ligand function that keeps the long period in the body.
Pharmacokinetic analysis and the assay method of part half life are well known to those skilled in the art. Detailed description is found in Kenneth, A etc.: Chemical Stability of Pharmaceuticals:A Handbook for Pharmacists and Peters etc., Pharmacokinetc analysis:A Practical Approach (1996). Also can be with reference to " Pharmacokinetics ", M Gibaldi and D Perron, Marcel Dekker publishes, the 2nd revised edition (1982), and it has described the pharmacokinetic parameter such as t α and t β half life and TG-AUC (AUC).
Can be by curve determination half life (T1/2 α and T1/2 β) and the AUC of part serum-concentration to the time. For example, can use WinNonlin analysis software package (can be by Pharsight Corp., Mountain View, CA, USA acquisition) to set up curve model. In the phase I (α phase), part main experience in the patient distributes, and some is removed. Second stage (β phase) is latter stage, and this moment, part distributed, and serum-concentration is along with part reduces by being eliminated among the patient. T α half life is the half life of phase I, and t β half life is the half life of second stage. Therefore, advantageously, the invention provides had 15 minutes or higher scope in t α half life part of the present invention or contain the composition of part. In one embodiment, the lower limit of scope is 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition or or, part of the present invention or composition have up to and comprise 12 hours t α half lifes in the scope. In one embodiment, the upper limit of scope is 11,10,9,8,7,6 or 5 hours. The example of OK range is 1-6 hours, 2-5 hour or 3-4 hour. Advantageously, the invention provides the part of the present invention of the t β half life that has in 2.5 hours or higher scope or contain the composition of part.
In one embodiment, the lower limit of scope is 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 10 hours, 11 hours or 12 hours. In addition or or, part of the present invention or composition have up to and comprise 21 days t β half lifes in the scope. In one embodiment, the upper limit of scope is 12 hours, 24 hours, 2 days, 3 days, 5 days, 10 days, 15 days or 20 days. Advantageously, part of the present invention or composition have the t β half life in 12-60 hour scope.
In further embodiment, it is in 12-48 hour scope. In embodiment further, it is in 12-26 hour scope.
Beyond the above standard or as an alternative, the invention provides the part of the present invention of the AUC value (TG-AUC) that has in 1mg. minute/ml or above scope or contain the composition of part. In one embodiment, the lower limit of scope is 5,10,15,20,30,100,200 or 300mg. minute/ml. In addition or or, part of the present invention or composition have up to the AUC in the 600mg. minute/ml scope.
In one embodiment, the upper limit of scope is 500,400,300,200,150,100,75 or 50mg. minute/ml. Advantageously, part of the present invention have AUC:15-150mg. minute/ml, 15-100mg. minute of being selected from the following scope/ml, 15-75mg. minute/ml and 15-50mg. minute/ml.
In first embodiment, the bispecific part contains 2 complementary variable domains, that is, even if two variable domains correlate epi-position in conjunction with it individually under background of the present invention, but in its natural surroundings, also can operate as correlating to or organizing effectively together. For example, complementary variable domains can be heavy chain immunoglobulin and light chain variable domain (VH and VL). VH and VL domain are advantageously provided by scFv or Fab antibody fragment. Can variable domains be joined together to form multivalent ligand by for example following mode: provide that the disulfide bond between the cysteine closes in the hinge area of each V domain C end and the hinge area; Or providing each all to have the dAb of cysteine at the C of domain end, the cysteine disulfide bond is combined; Or produce V-CH and V-CL, to produce the Fab type; Or use peptide linker (the Gly4Ser joint of for example hereinafter discussing), to produce dimer, tripolymer and other polymer. The present inventor finds, uses complementary variable domains to allow two domain surfactant packages together, isolates with solvent. And the complementary structure territory is stabilisation each other. In addition, it allows to set up bispecific IgG antibody, and does not have the shortcoming of the heterozygosis hybridoma that uses in the prior art or at the needs of the engineered heavy chain of subunit's joint or light chain.
The bispecific part of first aspect present invention has at least 1 VH/VL pair. Therefore, bispecific IgG of the present invention comprise two this right, have 1 pair on each arm of Y type molecule. Therefore, determined it whether to be successfully prepared with employed chain ratio and cause conventional bispecific antibody or the bifunctional antibody of operating difficulties different that bispecific part of the present invention does not have the problem of chain balance. Chain imbalance in the conventional bispecific antibody results from the combination of two different VL chains VH chain different with two, wherein VL chain 1 and VH chain 1 together can conjugated antigen or epi-positions 1, VL chain 2 and VH chain 2 together can conjugated antigen or 2, two correct pairings of epi-position interconnect in some way. Just produce bispecific when therefore, only VL chain 1 matches with VH chain 2 with 1 pairing of VH chain and VL chain 2 in individual molecule. This bispecific molecule can be set up by two kinds of different modes. At first, it can be by existingly setting up in conjunction with the VH/VL pairing (for example in bispecific IgG) of synantigen not or epi-position in conjunction with two separately. The VH/VL pairing must all flock together with 1: 1 ratio in the case, all is the molecular group of bispecific in order to set up. This occurs never (even if by " knot (knobs into holes) in the hole " when engineering method strengthens complementary CH domain), and the result produces bispecific molecule and only can be in conjunction with a kind of antigen or epi-position and can not be in conjunction with the mixture of alternative molecule. The mode that second is set up bispecific antibody be by means of two kinds of different VH chains from two kinds of different VL chains the time in conjunction with (for example in the bispecific bifunctional antibody). In the case, although VL chain 1 has preferential tendency with 1 pairing of VL chain and VL chain 2 with VH chain 2 pairings (it can strengthen by " knot (knobs into holes) in the hole " engineered VL and VH domain), but this pairing all realizes in all molecules never, the result produces mixed product, and occurring thus can not be in conjunction with the incorrect pairing of any antigen or epi-position.
The bispecific antibody that makes up according to the bispecific part method of first aspect present invention has overcome all these problems, because lay respectively in VH or the VL domain with the combination of antigen or epi-position 1, lay respectively in complementary VL or the VH domain with the combination of antigen or epi-position 2. Because VH and VL domain are take 1: 1 as the basis pairing, so all the VH/VL pairing all is bispecific, all models that therefore use these VH/VL pairings (Fv, scFv, Fab, miniantibody, IgG etc.) to make up all have 100% bispecific activity.
Under background of the present invention, first kind of epi-position that is understood that not identical with the second epi-position and can't help single monospecific ligand binding. In first kind of configuration of the present invention, it advantageously is in not on the synantigen, and one of them plays increases half life ground effect in the part body. Equally, first is advantageously not identical with second antigen.
Bispecific part of the present invention does not comprise the 02/02773 described part such as WO. Therefore, part of the present invention does not comprise collaborative complementary VH/VL pair in conjunction with any one or more antigen or epi-position. On the contrary, the part of first aspect present invention contains the VH/VL complementary pair, and wherein the V domain has different specificitys.
And the part of first aspect present invention contains the epi-position of non-structurally associated or antigen is had not homospecific VH/VL complementary pair. The epi-position of structurally associated or antigen are epi-position or the antigen that has enough structural similarities, played the conventional VH/VL complementary pair combination of conjugated antigen or epi-position effect with cooperative mode, with regard to the epi-position of structurally associated, epi-position is structurally similar as to be enough to make it " to meet " the identical combination bag that forms at the dimeric antigen binding site of VH/VL place.
In second aspect, the invention provides the part that contains first immunoglobulin variable domain (having first kind of antigen or epi-position binding specificity) and second immunoglobulin variable domain (having second antigen or epi-position binding specificity), in wherein said first and second variable domains one or two is in conjunction with the antigen that increases half life in the part body, and variable domains is each other not complementary.
In one embodiment, regulate the combination of part and second variable domains with the combination of a variable domains.
In this embodiment, variable domains for example can be the VH domain to or VL domain pair. Antigen can be regulated (for example enhancer or inhibitor) antigen in the combination in second site in the combination in first site. For example, the combination in first site suppresses antigen at least in part in the combination in second site. In this embodiment, part for example can be by remaining in the target organism body in conjunction with the albumen that increases the part half life, until for example its in conjunction with second target protein and moment of dissociating with the albumen that increases half life.
Combination is hereinbefore regulated owing to antigen binding site structure propinquity respect to one another is achieved. This structure propinquity can utilize the character of the structural constituent that connects two or more antigen binding sites and obtain, for example have the relative stiffness structure by the providing part of (it keeps the antigen binding site next-door neighbour). Advantageously, two or more antigen binding sites are physics next-door neighbour each other, so that the antigen combination in another site of procedure regulation by relating to steric hindrance in the immunoglobulin molecules and/or conformation change, site.
First and second covalently or non-covalently combination of antigen binding structural domain. In the covalently bound situation of domain, in conjunction with for example can be by disulfide bond or peptide linker ((Gly4Ser) n for example, n=1-8 wherein, for example 2,3,4,5 or 7) mediation.
According to the part of this aspect of the present invention one-tenth NIg capable of being combined multiple ligand structure, to form the multivalence compound, it provides good affinity thus in conjunction with the target molecule with same antigen, and at least one variable domains is in conjunction with increasing the polymer antigen of half life. For example, natural bacteria reporter molecules such as SpA are as the CDR stent graft, to produce the part of the one or more epi-positions of specific binding. The method is specified in United States Patent (USP) 5, S31,012. Other suitable support comprises the support based on fibronectin and affinity body (affibodies). The details of proper method is described in WO 98/58965. Other suitable support comprises such as van den Beuken etc., J.Mol.Biol. (2001) 310, the described lipocalin protein of 591-601 (lipocallin) and CTLA4, and such as the described support of WO0069907 (Medical Research Council), it is based on the circulus of for example bacterium GroEL or other companion's polypeptide.
The albumen support is capable of being combined, for example the CDR portable to the CTLA4 support, and and immunoglobulin (Ig) VHOr VLDomain is used to form part together.
Equally, fibronectin capable of being combined, lipocalin protein and other support.
Be selected from the situation of for example using the selected V gene pool of display technique of bacteriophage as herein described in variable domains, these variable domains can comprise the general framework district, so that it can be by the specificity metal ligand identification of this paper definition. The use of general framework, metal ligand etc. is described in WO 99/20749. In the present invention, the phage display of mentioning comprises the use of bacteriophage and/or phasmid.
When using the V gene pool, the variation of peptide sequence is preferably placed in the structure ring of variable domains. The peptide sequence of any variable domains all can be reset or the sudden change change by DNA, in order to strengthen the interaction of each variable domains and its complementary pair.
In a preferred embodiment of the invention, " bispecific part " is Single-Chain Fv Fragment of Murine. In alternate embodiment of the present invention, ' bispecific part ' is made up of the Fab district of antibody. Term " Fab district " comprises the Fab-sample district that wherein uses two VH or two VL domains.
The variable region can derive from the antibody of anti-target antigen or epi-position. Perhaps, it can derive from the storehouse in the monoclonal antibody body structure territory of for example expressing on the filobactivirus surface. Can as hereinafter with embodiment as described in select.
The preparation of dAb:
One aspect of the present invention not only relates to general bispecific part, and relate to the various constructions of bispecific form part that prolong the polypeptide of half life in conjunction with separately TNF-α, TNF-α and HSA or other, and in conjunction with the various constructions of the bispecific form part of TNF-α and VEGF. Also can prepare in conjunction with VEGF and HSA or other and prolong the part of the polypeptide of half life. Bispecific TNF-α/VEGF construction can comprise HSA or the another kind of combination that prolongs the molecule of half life in addition. In each of these embodiments, single part namely in conjunction with the part of TNF-α, HSA or VEGF, can be and be preferably dAb. The production theory of this dAb is set forth in hereinafter and embodiment.
In all fields, dAb disclosed herein can single aggressiveness form, dimeric forms, trimeric form, tetramer form so more the high polymer form exist. Except heterodimer form such as bispecific construction, the poly construction can be same polymer, namely homodimer, homotrimer, with tetramer etc. Heterotrimer, the different tetramer and more senior heteromultimeric have also specifically been imagined. In the various dAb conformations each all can be in addition and remaining part compound, polyethylene glycol (PEG) for example is in order to further prolong the serum half life of polypeptide construction. PEGization is known in the art, and at this paper discussion is arranged.
Prepare in many ways single immunoglobulin variable domain or dAb. One preferred aspect, dAb is the single immunoglobulin variable domain of people. For in these methods each, can use well-known nucleotide sequence preparation (such as amplification, sudden change etc.) and method of operating.
The method of a kind of dAb of preparation is the heavy chain of amplification and the clonal antibody of expressing known combining target antigen or the V of light chain geneHOr VLThe district. VHAnd VLThe border of domain is set forth in Kabat etc., (1991, ibid). Use the V of relevant heavy chain and light chain geneHAnd VLThe information design PCR primer on domain border is by clone's heavy chain or the light chain coded sequence amplification V domain of the known antibody in conjunction with given antigen of coding. The V domain of amplification is inserted in the suitable expression vector, such as pHEN-1 (Hoogenboom etc., 1991, Nucleic Acids Res.19:4133-4137), and individually or as expressing with the fusion of another peptide sequence. Screen then the V of the expression of high affinity combining target antigen when the remainder with heavy chain or light chain polypeptide separatesHOr VLDomain. For all aspects of the present invention, as known in the art dawn or as mentioned belowly carry out combination and screen.
By phage display for example, with respect to the elutriation of target antigen, screening VHOr VLThe domain storehouse. The method that makes up phage display library and bacteriophage lambda expression library is well-known in this area, says to be set forth in such as McCafferty etc., 1990, Nature 348:552; Kang etc., 1991, Proc.Natl.Acad.Sci.U.S.A., 88:4363; Clackson etc., 1991, Nature 352:624; Lowman etc., 1991, Biochemistry 30:10832; Burton etc., 1991, Proc.Natl.Acad.Sci U.S.A.88:10134; Hoogenboom etc., 1991, Nucleic Acids Res.19:4133; Chang etc., 1991, J.Immunol.147:3610; Breitling etc., 1991, Gene 104:147; Marks etc., 1991, J.Mol.Biol.222; 581; Barbas etc., 1992, Proc.Natl.Acad.Sci.U.S.A.89:4457; Hawkins and Winter (1992) J.Immunol., 22:867; Marks etc., (1992) J.Biol.Chem, 267:16007; With Lerner etc., (1992) Science, 258:1313. The scFv phage library is said and is set forth in such as Huston etc., 1988, Proc.Natl.Acad.Sci U.S.A.85:5879-5883; Chaudhary etc., 1990, Proc.Natl.Acad.Sci U.S.A.87:1066-1070; McCafferty etc., 1990, ibid; Clackson etc., 1991, ibid; Marks etc., 1991, ibid; Chiswell etc., 1992, Trends Biotech.10:80; With Marks etc., 1992, ibid. The various embodiments in the scFv library that is illustrated on the bacteriophage coat protein had been described already. The essence of phage display method will also be known, such as being described in WO 96/06213 and WO 92/01047 (Medical Research Council etc.) and WO 97/08320 (Morphosys, ibid).
V HOr VLThe domain storehouse can be natural storehouse or the synthetic storehouse of immunoglobulin sequences. Natural storehouse is for example by the storehouse from one or more individual immunoglobulin expression cell preparations of collecting. This storehouse can be " originally " storehouse, namely for example by human fetal or the preparation of neonatal immunity globulin express cell, or resets the storehouse, namely by for example becoming the human B cell preparation. Natural storehouse is described in such as Marks etc., 1991, J.Mol.Biol.222:581 and Vaughan etc., 1996, Nature Biotech.14:309. If needs are arranged, thus, then can be to carrying out mutagenesis and further screening by natural storehouse or in conjunction with the clone that any storehouse of target antigen is differentiated, in order to produce and select to have improvement in conjunction with the variant of feature.
By manually diversity being imported in clone's the V domain, prepare the synthetic storehouse of single immunoglobulin variable domain. Synthetic storehouse is described in for example Hoogenboom and Winter, 1992, J.Mol.Biol.227:381; Barbas etc., 1992, Proc.Natl.Acad.Sci. U.S.A.89:4457; Nissim etc., 1994, EMBO are J.13:692; Griffiths etc., 1994, EMBO are J.13:3245; DeKriuf etc., 1995, J.Mol.Biol.248:97; With WO 99/20749.
The antigen binding structural domain of conventional antibody contains two independent districts: weight chain variable domain (VH) and light chain variable domain (VL: it can be VκOr Vλ). The antigen binding site of this antibody is made of 6 polypeptide rings: 3 from VHDomain (H1, H2 and H3), 3 from VLDomain (L1, L2 and L3). The contour description of these rings is in such as (1991, ibid) such as Kabat. Reset production coding V in the gene segment body by combinationHAnd VLThe diversity master library of the V gene of domain. VHGene is by 3 gene segment VH, D and JHRestructuring produce. In the people, according to haplotype, 51 kinds of functional V have an appointmentHSections (Cook and Tomlinson (1995) Immunol Today, 16:237), 25 kinds of functional D sections (Corbett etc., (1997) J.Mol.Biol., 268:69) and 6 kinds of functional JHSections (Ravetch etc., (1981) Cell, 27:583). VHThe sections coding forms VHThe polypeptide sequence of first of domain and second antigen coupling collar (H1 and H2), and VH, D and JHSections is combined to form VHThe 3rd the antigen coupling collar (H3) of domain.
V LGene is by the only 2 kinds of gene segment V that recombinateLAnd JLProduce. In the people, according to haplotype, 40 kinds of functional V have an appointmentκSections (Schable and Zachau (1993) Biol.Chem. Hoppe-Seyler, 374:1001), 31 kinds of functional VλSections (Williams etc., (1996) J. Mol.so Biol., 264:220; Kawasaki etc., (1997) Genome Res., 7:250), 5 kinds of functional JκSections (Hieter etc., (1982) J.Biol.Chef, 257:1516) and 4 kinds of functional JλSections sections (Vasicek and Leder (1990) J.Exp.Med., 172:609). VLThe sections coding forms VLThe polypeptide sequence of first of domain and second antigen coupling collar (L1 and L2), and VLAnd JLSections is combined to form VLThe 3rd the antigen coupling collar (L3) of domain. It is generally acknowledged that the antibody variation that is selected from this original storehouse must be enough to at least medium affinity in conjunction with nearly all antigen. By producing high affinity antibody in " affinity sudden change " body of resetting gene, wherein immune system with improve be combined into benchmark production and selected element suddenlys change.
Structure and the sequence analysis of antibody show have 5 (H1, H2, L1, L2, L3) to have limited amount Conformation of the main chain or norm structure (Chothia and Lesk (1987) d:Mol.Biol., 196:901 in 6 antigen coupling collars; Chothia etc., (1989) Nature, 342:877). Conformation of the main chain is by the length of (i) antigen coupling collar and (ii) specific residue or the residue type decided of some key position in antigen coupling collar and the antibody framework. Ring analysis long and Key residues can make us predict Conformation of the main chain (Chothia etc., (1992) J.Mol.Biol., the 227:799 of H1, the H2, L1, L2 and the L3 that are encoded by most people's antibody sequence; Tomlinson etc., (1995) EMBO J., 14:4628; Williams etc., (1996) J.Mol.Biol., 264:220). H3 district although more diversified with regard to sequence, length and structure (reason is to use the D sections), but it also forms the long Conformation of the main chain of limited amount becate, it depends on the length of the concrete residue of key position in ring and the antibody framework and has situation or residue type (Martin etc., (1996) J.Mol. Biol, 263:800; Shirai etc., (1996) FEBS Letters, 399:1).
Yet, according to one embodiment of the invention, diversity can be joined any site of various antigen coupling collar CDR in the synthetic storehouse, the method produces the correct folding V domain of vast scale more and therefore makes the ratio of molecule of potential conjugated antigen littler. Understood the residue that helps antigen coupling collar Conformation of the main chain so that can differentiate VHOr VLMultifarious specificity residue in the synthetic storehouse of domain. That is, be preferably in the necessary residue of non-maintenance Conformation of the main chain and introduce diversity. As an example, for the diversity of ring L2, conventional method should be to make such as all variations of all residues (about 7 residues) among the corresponding CDR (CDR2) of the definition such as Kabat (1991, ibid). But for L2, known 50 and 53 is diversified in natural antibody, and observes it and contact with antigen. Method for optimizing should be these two residue variations that only make in this ring. This shows that the remarkable lifting of functional diversity need to be established the scope of antigen-binding specificity.
In one aspect, at VHOr VκIn the background, be V based on artificial diversified kindHOr VκSequence, the variable domains storehouse that preparation is synthetic. For example, VHThe domain storehouse is V based on clone's kindHGene segment V3-23/DP47 (Tomlinson etc., 1992, J.Mol.Biol.227:7768) and JH4b (referring to Fig. 1 and 2). VκThe domain storehouse is V based on for example plantingκGene segment O2/O12/DPK9 (Cox etc., 1994, Eur.J.Immunol.24:827) and Jκ1 (referring to Fig. 3). By PCR mutagenesis for example diversity is imported in these or other gene segment. Diversity can by such as error-prone PCR (Hawkins etc., 1992, J.Mol.Biol.226:889) or mutagenesis import at random. But as mentioned above, the target that preferred diversity imports is specific residue. Further preferred by using mutagenic primer to import codon NNK (using the IUPAC nomenclature, wherein N=G, A, T or C, K=G or T) target target residue, NNK encode whole amino acid and TAG terminator codon. Other codon that obtains similar end also is useful, and ((A/G/T) be (C/T) (A/G/C) to comprise NNN codon (it causes producing other terminator codon TGA and TAA), DVT codon ((A/G/T) be T (A/G/C)), DVC codon ((A/G/T) be C (A/G/C)) and DVY codon. DVT codon encode 22% serine and 11% tyrosine, asparagine, glycine, alanine, aspartic acid, threonine and cysteine, this amino acid residue of the most closely having simulated the antigen binding site of natural human antibody distributes. Use is treating that the PCR primer that diversified each position all has one or more selected degenerate codons prepares the storehouse. PCR mutagenesis is well-known in this area; But, be entitled as hereinafter to have discussed in the chapters and sections of " PCR mutagenesis " and be used for the design of primers of the inventive method and the Consideration of PCR mutagenesis.
In one aspect, as shown in Figure 1, multifarious site H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97 and H98 in corresponding to CDR1,2 and 3, using the NNK codon that diversity is imported to ethnic group is VHGene segment V3-23/DP47 (Tomlinson etc., 1992, J.Mol.Biol.227:7768) and in the sequence of JH4b.
On the other hand, as shown in Figure 2, multifarious site H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a and H100b in corresponding to CDR1,2 and 3, for example using the NNK codon that diversity is imported to ethnic group is VHIn the sequence of gene segment V3-23/DP47 and JH4b.
On the other hand, as shown in Figure 3, multifarious site L30, L31, L32, L34, L50, L53, L91, L92, L93, L94 and L96 in corresponding to CDR1,2 and 3, for example using the NNK codon that diversity is imported to ethnic group is VκGene segment O2/O12/DPK9 and JκIn 1 the sequence.
As known in the art and as described in WO 99/20749 for example diversified storehouse is cloned in the Vector for Phage Display. In general, implementing nucleic acid molecules required for the present invention and vector construct can be obtained by this area, and such as Sambrook etc., (1989) .Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, the described structure of standard laboratory method and the operation of USA.
Usually in recombinant vector, carry out the operation of nucleic acid of the present invention. " carrier " used herein refers to for the resolution element that allogeneic dna sequence DNA is imported to for its expression and/or the cell that copies. Selecting or making up and use subsequently the method for this carrier is that those skilled in the art are well-known. Many carriers can openly obtain, and comprise bacterial plasmid, bacteriophage, artificial chromosome and episomal vector. This carrier can be used for simple clone and mutagenesis; Perhaps, as the representative of the carrier that carries storehouse of the present invention (or front storehouse) member, use expression vector. Selected used according to the present invention carrier is generally 0.25 kilobase to (kb) polypeptid coding sequence to the target sizes of 40kb to hold length. After the body outer clone operation, transform suitable host cell with carrier. Each carrier all contains various function element, and it generally comprises clone's (or " polylinker ") site, origin of replication and at least one selectable marker gene. If given carrier is expression vector, then it has following one or more in addition: enhancer element, promoter, transcription terminator and burst, its each all be positioned at cloning site near so that it effectively is connected to code book invention peptide library member's gene.
In general, clone and expression vector these two all contain the nucleotide sequence that carrier is copied in one or more selected host cells. Usually, in cloning vector, this sequence is to make carrier be independent of the sequence that host chromosome DNA copies, and comprises origin of replication or autonomously replicating sequence. This type of sequence of various bacteriums, yeast and virus is well-known. The origin of replication of plasmid pBR322 is suitable for most of Gram-negative bacteria, and 2 μ plasmid starting points are suitable for yeast, and various viral starting point (for example SV 40, adenovirus) is used for the cloning vector of mammalian cell. In general, mammalian expression vector does not need origin of replication, unless these starting points are used in the mammalian cell that can copy high-level DNA COS cell for example.
Advantageously, clone or expression vector also contain the Select gene of the selected marker that is otherwise known as. The albumen of this gene code is the transformed host cell survival of cultivating in selective medium or grows necessary. Therefore, the host cell that does not transform with the carrier that contains Select gene can not be survived in this culture medium. Typical Select gene encoding proteins is given resistance, the defective that supplements the nutrients of antibiotic and other toxin (for example ampicillin, neomycin, amethopterin or tetracycline) or unavailable crucial nutrients in the growth medium is provided.
Because copying of carrier of the present invention carried out the most usually, so use the Escherichia coli selected marker, for example give the beta-lactamase gene to the antibiotic amicillin resistance in Escherichia coli. These carriers can derive from escherichia coli plasmid (for example pBR322) or pUC plasmid (for example pUC18 or pUC19).
Expression vector contains the promoter that also effectively is connected to the purpose coded sequence by host living beings identification usually. This promoter can be induction type or constitutive promoter. Term " effective connection " refers to this arranged side by side: the residing relation of wherein said component allows it to work in the expectation mode. " effectively connect " to the control sequence of coded sequence to realize under the suitable condition that the mode that coded sequence is expressed connects being connected with control sequence.
The promoter that is applicable to the prokaryotes host comprises for example beta-lactamase and lactase promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems and hybrid promoter, for example tac promoter. The promoter that is used for bacterial system generally also comprises the Shine-Dalgarno sequence that effectively is connected to coded sequence.
In library as herein described or storehouse, preferred vector is the expression vector that can express corresponding to polypeptide libraries member's nucleotide sequence. Therefore, by the single clone who expresses the polypeptide libraries member being bred separately and expresses, or by using any selection display systems, select. As mentioned above, preferably select display systems to use phage display. Therefore, can use bacteriophage or phagemid vector. Preferred carrier is phagemid vector, and it has Escherichia coli origin of replication (being used for two strands copies), also has phage replication starting point (for the production of single stranded DNA). The operation of this carrier and be expressed in this area well-known (Hoogenboom and Winter (1992), ibid; Nissim etc., (1994), ibid). Briefly, carrier contains gives the optionally lac promoter of beta lactamase or other selectable marker gene and expression cassette upstream of phasmid, this expression cassette by pelB targeting sequencing (it imports periplasmic space with the Peptide T of expressing) (N to C end), MCS (the library member who is used for clone's nucleotides form), optional one or more peptide-labeled (for detection of), optional one or more TAG terminator codons and bacteriophage albumen pIII form. Using colibacillary various suppressors and non-suppressor bacterial strain and adding in the situation of glucose, isopropylthio-β-D-galactoside (IPTG) or helper phage such as VCS M13, carrier can be as the plasmid replication of not expressing, produce a large amount of unique polypeptide libraries members, or producing bacteriophage, some of them contain at least one copy of polypeptide-pIII fusion in its surface.
The example of preferred vector is pHEN1 phagemid vector (Hoogenboom etc., 1991, Nucl.Acids Res.19:4133-4137; Can obtain for example is the sequence of the SEQ ID NO:7 among the WO 03/031611), wherein the production of pIII fusion is under the control of LacZ promoter, and this promoter is suppressed in the presence of glucose, induces with IPTG. When in Escherichia coli suppressor bacterial strain such as TG1, growing, produce gene III fusion, and be packed in the bacteriophage, and when in non-suppressor bacterial strain such as HB2151, growing, allow soluble fusion protein to be secreted into the bacterium pericentral siphon and be secreted in the culture medium. Because the infection of helper phage after the expression of gene III prevents so breed the bacterium with phagemid vector in the presence of glucose, uses VCSM 13 helper phages that are used for the bacteriophage rescue to infect then.
Vector construction of the present invention uses conventional interconnection technique. Carrier or dna fragmentation that cutting, finishing separate, and connect into again the desired form of carrier that generation needs. If needs are arranged, the Application standard method is carried out sequence analysis, to confirm there is correct sequence in the carrier that makes up. Be suitable for construction of expression vector, prepare the in-vitro transcription thing, import DNA in the host cell and express and the method for function evaluating analysis is well known by persons skilled in the art. Gene order exists situation in the test sample, or by quantitatively its amplification and/or expression, for example sequence analysis of some trace, in situ hybridization, immunocytochemistry or nucleic acid or the protein molecular of DNA or RNA analysis, Western blotting, DNA, RNA or albumen of conventional method. Those skilled in the art are easy to be susceptible to can how to revise these methods whenever necessary.
PCR mutagenesis:
The part of primer and target molecule is complementary, and it is present in the nucleic acid molecules pond for the preparation of coded polypeptide library member's nucleic acid library member group. The most frequent synthetic method preparation by chemistry or zymetology of primer. The mutagenic oligonucleotide primer is a long 15-100 nucleotides generally, and long 20-40 nucleotides ideally is although can use the oligonucleotides of different length.
Usually, when two nucleotide sequences substantially complementary (in the section at 14-25 nucleotides at least at least about 65% complementation, preferably at least about 75% complementation, more preferably at least about 85% or 90% complementation), the generation selective cross. Referring to Kanehisa, 1984, Nucleic Acids Res.12:203, it is incorporated herein by reference. Therefore, expection is allowed starting the site to a certain degree mispairing. This mispairing can be seldom, for example one, two or three nucleotides. Perhaps, it can comprise the nucleotides ring, and the nucleotides ring is defined as wherein mispairing at this paper and comprises the incessantly zone of continuous 4 or more nucleotides.
Generally speaking, the effectiveness of 5 factor affecting primers and second making nucleic acid molecular hybridization and selective. These factors are (i) primer length; (ii) nucleotide sequence and/or composition; (iii) hybridization temperature; (iv) buffer solution chemical property and (v) the steric hindrance potentiality in the district that need to hybridize of primer, these factors are important Considerations when the nonrandom initiating sequence of design.
Primer length is with having positive correlation between the effectiveness of primer and target sequence annealing and the accuracy; Longer sequence has the melting temperature (T that is higher than shorter sequenceM), can not in given target sequence, repeat, make thus the hybridization minimum that mixes. The primer sequence that has high G-C content or contain palindromic sequence tends to from hybridization, and the target site of its expection is owing to for unimolecule rather than bimolecular also are so, generally be conducive to hybridization kinetics in solution; Simultaneously, importantly design contains the right primer of G-C oligonucleotide ligand of sufficient amount, with the target sequence of combining closely because these to each by 3 Hydrogenbonds, rather than 2 Hydrogenbonds that exist when A and T base pairing. Hybridization temperature and primer annealing are renderd a service inverse change, with the concentration of the organic solvent that may comprise in the hybridization mixture (for example formamide) also be inverse change, and the increase of salinity is beneficial to combination. Under stringent hybridization condition, more effectively hybridize than the shorter probe that under advantage more, is enough to hybridize than long probe. The stringent hybridization condition of primer generally includes the salinity that is lower than about 1M, more generally is lower than the salinity of about 500mM, preferably is lower than the salinity of about 200mM. The scope of hybridization temperature is higher than about 30 ℃ by being low to moderate 0 ℃ to being higher than 22 ℃, and (the most frequent) is above about 37 ℃. For specific hybrid, long fragment may need higher hybridization temperature. Because several factor affecting hybridization stringency, so parameters combination is than any absolute measure is more important separately.
Design reasoningly primer with these Considerations. Although those skilled in the art can estimate with brains the relative merit of numerous sequences, designed computer program and helped estimate these several parameters and optimize primer sequence. The example of these programs is DNAStarTMSoftware kit (DNAStar, Inc.; Madison, WI) " PrimerSelect " and OLIGO 4.0 (National Biosciences, Inc.). In case design is finished, can be by suitable method, for example Beaucage and Carruthers, 1981, phosphoramidite method or Matteucci and Caruthers that Tetrahedron Lett.22:1859 describes, the triester method of 1981, J.Am.Chem.Soc.103:3185 (these two documents are all incorporated herein by reference), or use commercially available automated oligonucleotide synthesizer or VLSIPS for exampleTMOther chemical method of technology prepares suitable oligonucleotides.
Use template DNA (1fg at least; 1-1000ng more generally) and at least the 25pmol Oligonucleolide primers carries out PCR; When the severe allos of primer pond, use relatively large primer possibility favourable, because each sequence by the representative of the only sub-fraction molecule in the pond, becomes restrictive in the amount amplification cycles afterwards. Typical reactant mixture comprises: the 1.25 μ M dNTP of 2 μ l DNA, 25pmol Oligonucleolide primers, 2.5 μ l 10X PCR buffer solutions, 1 (Perkin-Elmer), 0.4 μ l, 0.15 μ l (or 2.5 units) Taq archaeal dna polymerase (Perkin Elmer) and to the deionized water of cumulative volume 25 μ l. Cover mineral oil, and use the programmable heat circulating instrument to carry out PCR.
The length in each step of PCR circulation and temperature and period are regulated according to the needed stringency of reality. By the expection primer annealing of template is renderd a service and the mispairing degree of allowing these two determine annealing temperature and time; Obviously, when nucleic acid molecules increases with mutagenesis simultaneously, need mispairing in synthesizing in the first round at least. For attempting using the mutagenic primer pond amplifier molecule group who mixes, with respect to the mix annealing of primer to sequence beyond the target spot, weigh the loss of potential sudden change product under strict (high temperature) annealing conditions (it should only be obtained by low melting temperature). The ability of optimizing primer annealing condition stringency belongs to those skilled in the art's ken fully. Use the annealing temperature between 30 ℃-72 ℃. With 92 ℃ of initial sex change that template molecule took place in-99 ℃, 4 minutes, then be by sex change (94-99 ℃, 15 seconds to 1 minute), the annealing (temperature of determining as mentioned above generally; 1-2 minute) and extend (72 ℃ 1-5 minute, depend on the length of amplified production) 20-40 circulation forming. Last extension is generally 72 ℃, 4 minutes, can after connect nothing fixed number (0-24 hour) step of 4 ℃.
The dAb of screening antigen combination:
Be to express after the dAb storehouse on the phage surface, phage library is contacted with the immobilization target antigen, rinsing is selected thus to remove unconjugated bacteriophage and to breed the bacteriophage of combination, and whole process often is called as " elutriation ". Perhaps, by with respect to only by immobilization metal ligand (for example A albumen or the L albumen) elutriation of folding member's combination, the bacteriophage of correct folding member's variant is expressed in preliminary election. This have reduce nonfunctional member ratio, increasing thus may be in conjunction with the advantage of member's ratio of target antigen. Instruct in WO 99/20749 with the metal ligand preliminary election. The screening general description of phage antibody library is in such as Harrison etc., 1996, Meth.Enzymol.267:83-109.
Usually, use and to be fixed on solid support (for example plastic tube or hole) or chromatography substrate (Sepharose for exampleTM(Pharmacia)) purifying antigen on screens. Screening or selection also can be carried out at complex antigen, such as cell surface (Marks etc., 1993, BioTechnology 11:1145; De Kruif etc., 1995, Proc.Natl.Acad.Sci.U.S.A.92:3938). Another alternative method comprises by the biotinylated antigen in the binding soln to be selected, and then is captured on the coated pearl of streptavidin.
One preferred aspect, by at pipe or plate mesopore (Nunc MAXISORP for exampleTMImmunity pipe 8 hole bars) upper immobilized antigen (genus or specific) carries out elutriation. With the coated hole of 150 μ l antigens (the PBS solution of 100 μ g/ml), and the incubation that spends the night. Use then PBS rinsing hole 3 times, and sealed 2 hours in 37 ℃ with 400 μ l PBS-2% skimmed milks (2%MPBS). With PBS drip washing hole 3 times, in 2%MPBS, add bacteriophage. Mixture is in room temperature incubation 90 minutes, removes the liquid that contains not in conjunction with bacteriophage. With PBS-0.1%Tween 20 drip washing holes 10 times, use then PBS drip washing 10 times, to remove denaturant. By adding the 100mM triethylamine of the fresh preparation of 200 μ l, mix aperture and in room temperature incubation 10 minutes, the bacteriophage of elution of bound. The bacteriophage of wash-out is transferred in the pipe of the 1M Tris-HCl, the pH 7.4 that contain 100 μ l, vortex, with in and triethylamine. By in 37 ℃ of incubations 30 minutes, with for example e. coli host cell of the phage-infect exponential growth of 150ml wash-out (for example TG1). The lower cell that infects of rotation be resuspended in the fresh culture, and plating is in top agar. The plaque elution entered or pick up in the fresh culture of host cell and breed, be used for analyzing or the next round screening. If need to guarantee the equal a group of the bacteriophage selected, then plaque be carried out taking turns or taking turns more purifying. Other screening technique is described in Harrison etc., and 1996, ibid.
After the bacteriophage that identifies single immunoglobulin variable domain of expressing the combining target target, if used phagemid vector such as pHEN1, then by infecting non-suppressor bacterial isolates, for example allow the HB2151 of secretion solubility gene III fusion, can easily produce the varistructure domain fusion protein of soluble form. Perhaps, V domain sequence subclone can be entered in the suitable expression vector, to produce soluble protein according to methods known in the art.
The purifying of dAb and concentrated:
Collect and purifying (Harrison etc., 1996, ibid) is secreted into the dAb polypeptide of periplasmic space or bacteria culture media according to known method. Skerra and Pluckthun (1988, Science 240:1038) and Breitling etc. (1991, Gene 104:147) described by pericentral siphon collection antibody polypeptides, Better etc. (1988, Science 240:1041) have described by culture supernatant and have collected. Also can pass through in conjunction with metal ligand, for example A albumen or L albumen are realized purifying. Perhaps, can express and have the variable domains that is beneficial to by peptide-labeled (for example for Myc, HA or 6X-His mark) of affinity chromatography purification.
Concentrate polypeptide by several method well-known in the art, these methods comprise for example ultrafiltration, diafiltration and tangential flow filtration. Ultra-filtration process uses pellicle and pressure with based on size and dimension isolated molecule classification. Pressure is by air pressure or centrifugal providing. Can widely utilize and for example derive from Millipore (Bedford, MA; Example comprises CentriconTMAnd MicroconTMInspissator) and Vivascience (Hannover, Germany; Example comprises VivaspinTMInspissator) commercially available ultrafiltered product. By selecting the molecular weight that dams (being generally 1/3 to 1/6 of target polypeptides molecular weight, although can successfully use the difference that is low to moderate 10kD) less than target polypeptides, when solvent and littler solute passed film, polypeptide was kept. Therefore, the molecular cut off of about 5kD is concentrated to dAb polypeptide described herein is useful.
When the salt in needs removal or replacing polypeptide product or buffer solution, use diafiltration, diafiltration is used milipore filter in " cleaning " process. Concentrate polypeptide by making solvent and little solute pass film, when continuing ultrafiltration, remove remaining salt or buffer solution with the polypeptide of new buffer solution or salting liquid or water dilution reservation as required. In continuous diffusion, to add new buffer solution with the identical speed of speed that filtered solution passes film. The diafiltration volume is the diafiltration volume that begins front polypeptide solution-continuous diafiltration of use, can see through more than 99.5% of solute by removing with the new buffer solution for cleaning of 6 times of diafiltration volumes all. Perhaps, can discontinuous mode implement this process, wherein dilute sample is repeatedly then filtered back its original volume, to remove or to change salt or buffer solution, finally concentrates polypeptide. The method detailed of diafiltration equipment and its use can derive from for example Pall Life Sciences (Ann Arbor, MI) and Sartorius AG/Vivascience (Hannover, Germany).
Tangential flow filtration (TFF) is also referred to as " cross-flow filtration ", and it also uses milipore filter. The fluid that contains target polypeptides is tangentially pumped into along the film surface. Pressure makes a part of fluid pass film, and target polypeptides is retained on the film simultaneously. But opposite with the standard ultrafiltration, the molecule of reservation is not accumulated on the film surface, but is taken away by slipstream. Can be repeatedly with the solution (containing target polypeptides) that do not pass filter membrane in the film cocycle, to obtain aimed concn. The method detailed of TFF equipment and use thereof for example can derive from Millipore, and (for example ProFlux M 12TMBenchtop TFF system and PelliconTMSystem), Pall Life Sciences (Minim for exampleTMThe tangential flow filtration system).
Detect protein concentration in numerous modes well-known in the art. These methods comprise for example absorption, " Bradford " and " Lowry " method and the SDS-PAGE of amino acid analysis, 280nm. The most accurate method is complete hydrolysis, succeeded by the HPLC analysis of amino acid, then by with known dAb peptide sequence comparative measurements concentration. Although the method is the most accurate, its costliness is consuming time. Absorb by the UV that detects 280nm that to carry out protein determination very fast, spend much lower, also relatively accurate, preferably as the compromise algorithm of amino acid analysis. The protein concentration that the absorption measurement of use 280nm is reported in embodiment as herein described.
The experiment of " Bradford " and " Lowry " albumen (Bradford, 1976, Anal.Biochem. 72:248-254; Lowry etc., 1951, J.Biol.Chem.193:265-275) comparative sample protein concentration and the most common calibration curve based on bovine serum albumin(BSA) (BSA). These methods are not enough accurate, often underestimated the concentration of single immunoglobulin domains. But, by using VHOr VκThe single domain polypeptide can promote its accuracy as standard items.
Remaining method of protein detection is to be described in United States Patent (USP) the 4th, 839, the dicinchonine acid experiment of No. 295 (incorporated herein by reference), by Pierce Biotechnology (Rockford, IL) with " BCA Protein Assay " (for example the Pierce catalog number (Cat.No.) 23227) list marketing.
The SDS-PAGE method is used gel electrophoresis and Coomassie blue stain, with the standard items contrast of concentration known, for example single immunoglobulin variable domain polypeptide of known quantity. Quantitatively can be undertaken by naked eyes or photo densitometry.
In the third aspect, the invention provides the method for producing part, described part contains first immunoglobulin (Ig) list variable domains with first kind of binding specificity and second immunoglobulin (Ig) list variable domains with second (different) binding specificity, in the binding specificity one or both are specific to the antigen that increases half life in the part body, and the method may further comprise the steps: (a) select first variable domains according to its ability in conjunction with first epi-position; (b) select second variable domains according to its ability in conjunction with second epi-position; (c) combination variable domains; (d) select part according to its ability in conjunction with described first epi-position and described second epi-position.
Part can be side by side in conjunction with first and second epi-position, or exist between the binding structural domain of epi-position combination in the situation of competition being used for, the combination of a domain can be got rid of the combination of another domain and its connection epi-position. Therefore, in one embodiment, above step (d) needs simultaneously the epi-position in conjunction with first and second (and may exist other); In another embodiment, the combination with first and second epi-position is not simultaneously.
Epi-position is preferably on the antigen that separates.
Advantageously, part contains VH/VL combination or VH/VH or the VL/VL combination of aforesaid immunoglobulin variable domain. In addition, part can contain Camelidae VHH domain, and prerequisite is that the antigen that increases half life in the part body is had specific VHH domain not in conjunction with HEL (HEL), Porcine pancreatic α-amylase or NmC-A; Hog, RR6 ado 5 dyestuffs or Streptococcus mutans (S.mutates) HG982 cell that BSA connects, such as Conrath etc., (2001) JBC 276:7346-7350 and WO 99/23221 are described, and wherein any does not all describe the specificity of using for the antigen that increases half life increases half life in the part body.
In one embodiment, do not having in the situation of complementary variable domains according to selecting described first variable domains (namely selecting it as dAb as indicated above) with the combination of described first epi-position. In further embodiment, basis is selected described first variable domains with the combination of described first epi-position/antigen in the situation that has the 3rd variable domains, wherein said the 3rd variable domains is different from described second variable domains, and with first domain complementation. Equally, can be not or have and select second domain in the situation of complementary variable domains.
Except the half life Enhancin, the antigen of part target of the present invention or epi-position can be any antigen or epi-position, but advantageously for having target antigen or the epi-position for the treatment of benefit. The invention provides part, comprise open conformation, sealing conformation and the dAb monomer part that separates, it all is specific to any this target, especially the further target of differentiating of this paper. This target can be natural or synthetic polypeptide, albumen or nucleic acid or its part. In this regard, part of the present invention can be in conjunction with epi-position or antigen, and play antagonist or activator (for example EPO receptor stimulating agent). One skilled in the art will recognize that selection is on a large scale with variable.
For example, it can be enzyme cofactor or the DBP of human or animal's albumen, cell factor, cytokine receptor, enzyme. Can be included but not limited to by suitable cell factor and the growth factor of list as herein described or bispecific Binding peptide target: ApoE, Apo-SAA, BDNF, BLyS, heart nutrient-1, EGF, the EGF acceptor, ENA-78, ECF, ECF-2, Exodus-2, EpoR, aFGF, basic FGF, FGF-10, FLT3L, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-01, insulin, IFN-y, IGF-I, IGF-II, IL-, IL-1p, 20IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 amino acid), IL-8 (77 amino acid), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin B IP-10, keratinocyte growth factor 2 (KGF-2), KGF, leptin, LIF, lymphocyte chemotactic factor (LCF), the Mullerian mortifier, the monocyte colony inhibition factor, the monocyte bait protein, M-CSF, MDC (67 amino acid), MDC (69 amino acid), MCP-I (MCAF), MCP-2, MCP-3, MCP-4, MIG, MIP1 α, MIP1 β, MIP3 α, MEP3 β, MIP-4, bone marrow precursor inhibiting factor-1 (MPIF-1), NAP-2, Neurturin, nerve growth factor, β-NGF, NT-3, NT-4, the plain M of tumorigenesis, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β 2, TGF-β 3, TNF (TNF), TNF-α, TNF-β, TNF acceptor I, the TNF receptor II, TNIL-1, TPO, VEGF, vegf receptor 1, vegf receptor 2, VEGFR3, GCP-2, GRO/MGSA, GRO-β, GRO-8, HCC1,1-309, HER1, HER2, HER3, HER4, the TACE recognition site, TNF BP-I and TNF BP-II, and in the annex 2 that invests this paper or annex 3 disclosed any target, no matter be with the combination described in the annex, different combination or independent.
Be noted that preferred part comprises independent, together and/or has anti-HSA in conjunction with TNF-α and the VEGF of activity.
Cytokine receptor comprises the acceptor of aforementioned cell factor. Should recognize that this enumerates anything but detailed.
In one embodiment of the invention, variable domains derives from the separately antibody of antigen or epi-position. In a preferred embodiment, variable domains derives from the storehouse in single variable antibody structure territory.
In yet another aspect, the invention provides one or more nucleic acid molecules of at least bispecific part of coding this paper definition.
The bispecific part can be encoded at the single core acid molecule; Perhaps, each domain can be by the nucleic acid molecule encoding that separates. When part during by single nucleic acid molecule encoding, the mode that domain can the scFv molecule is expressed as fused polypeptide, perhaps can express individually, for example uses subsequently chemical bridging agent to link together. Utilize suitable method to be linked together by the part of independent expression of nucleic acid.
Nucleic acid can further encode for when expressing by the burst of host cell output polypeptide, and can be when expressing and the surface component fusion of filobactivirus particle (or select other component of the display systems).
On the other hand, the invention provides the carrier of the code nucleic acid that contains bispecific part of the present invention.
More on the other hand, the invention provides the host cell with the carrier transfection of code book invention bispecific part.
Can design the expression of this carrier, for example to produce the variable domains that is used for selection on the phage particle surface. This uses method of the present invention to select ' bispecific part ' so that can select the variable region showed thus.
The present invention further provides the kit that contains bispecific part at least of the present invention.
Bispecific part of the present invention preferably comprises the combination of heavy chain and light chain domain. For example, the bispecific part can comprise VH domain and VL domain, and the form that it can scFv links together. In addition, part can comprise one or more CH or CL domain. For example, part can comprise CH1 domain, CH2 or CH3 domain and/or CLDomain, C μ, C μ 2, C μ 3 or C μ 4 domains, or its any combination. Also can comprise the hinge area domain. This domain combination for example can be simulated natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2 molecules. Also imagine other construction, for example contained VH, VL, CH1 and CLThe IgG molecule single armed of domain.
In a preferred embodiment of the invention, select the variable region by single domain V gene pool. In general, storehouse, monoclonal antibody body structure territory is illustrated on the filobactivirus surface. In a preferred embodiment, select each monoclonal antibody body structure territory according to the combination of phage library and antigen.
In a preferred embodiment of the invention, can not have in the situation of complementary variable region according to selecting each single variable domains with the combination of its target antigen or epi-position. In an alternate embodiment, can exist in the situation of complementary variable region according to selecting single variable domains with the combination of its target antigen or epi-position. Therefore, first variable domains can be in the situation that has the 3rd complementary variable domains, selected, second variable domains can be in the situation that has the 4th complementary variable domains, selected. Complementary the 3rd or the 4th variable domains can be with the survey single domain has mutually homospecific natural connection variable domains, or is non-connection complementary structure territory, for example " simulation " variable domains.
Preferably, bispecific part of the present invention only contains two variable domains, although several this parts can be incorporated in the same albumen together, for example two this parts can be incorporated among IgG or poly-ig such as the IgM. Perhaps, in another embodiment, make up a plurality of bispecific parts, to form polymer. For example, make up two different bispecific parts, to set up four specific moleculars.
One skilled in the art will recognize that light chain and the variable region of heavy chain of the bispecific part of producing according to the inventive method can be positioned on the same polypeptide chain, perhaps are positioned on the different polypeptide chains. Be positioned at situation on the different polypeptide chains for the variable region, it can connect through joint (being generally elastomeric joint (for example polypeptide chain)), cytotoxic compounds or arbitrarily other method known in the art.
On the other hand, the invention provides a kind of composition, it contains can be by bispecific part and medicine acceptable carrier, diluent or the excipient of the inventive method acquisition.
And, the invention provides ' bispecific part ' of the present invention or combination treatment and/or the prophylactic method used.
In the second configuration, the invention provides the polyspecific part that contains at least two noncomplementation variable domains. For example, part can comprise a pair of VH domain or a pair of VL domain. Advantageously, domain is non-Camelidae source; Preferably be people's domain or contain people's framework region (FW) and one or more allos CDR. CDR and framework region are to have the district that is considered as the immunoglobulin variable domain in the protein sequence Kabat database of Immunological Significance.
Preferred people's framework region is to be people's framework region of gene segment DP47 and DPK9 coding by kind. Advantageously, FW1, the FW2 of VH or VL domain and FW3 have FW1, FW2 or the FW3 sequence of DP47 or DPK9. People's framework region optionally comprises sudden change, and is for example always total up to about 5 amino acid variations or up to about 10 amino acid variations at the people's framework region that is used for part of the present invention.
Variable domains in the polyspecific part of second configuration of the present invention can be opened or seal conformation and be arranged; That is to say that it can arrange to such an extent that make the variable domains can be independently or simultaneously in conjunction with its connection part, or make that only wherein a kind of variable domains can be at any one time in conjunction with its connection part.
The present inventor has realized that, under some structural condition, in part, can there be incomplementarity variable domains (for example two light chain variable domains or two weight chain variable domains), so that the combination in conjunction with second epi-position of inhibition and second variable domains of first epi-position and first variable domains, even if this incomplementarity domain is as correlating effectively linking together.
Advantageously, part contains two pairs or many to variable domains; Be that it contains at least 4 variable domains. Advantageously, 4 variable domains contain the framework that the people originates.
In a preferred embodiment, people's framework and ethnic group are that the framework of sequence is identical.
The present inventor thinks, this antibody is particularly useful in the ligand binding experiment that is used for the treatment of with other purposes.
Therefore, aspect first of second configuration, the invention provides the method for producing the polyspecific part, it comprises following steps: (a) select first epi-position binding structural domain according to its ability in conjunction with first epi-position; (b) select second epi-position binding structural domain according to its ability in conjunction with second epi-position; (c) combination epi-position binding structural domain; (d) according to its polyspecific part in conjunction with the ability selection sealing conformation of described first epi-position and described second epi-position.
Aspect another of second configuration, the invention provides the method for the polyspecific part of preparation sealing conformation, this part contains first epi-position binding structural domain with first kind of epi-position binding specificity and has second epi-position binding structural domain of noncomplementation of second epi-position binding specificity, wherein first kind and the combination of second binding specificity competition epi-position, so that the polyspecific part of sealing conformation can not simultaneously in conjunction with two epi-positions, said method comprising the steps of: (a) select first epi-position binding structural domain according to its ability in conjunction with first epi-position; (b) select second epi-position binding structural domain according to its ability in conjunction with second epi-position; (c) combination epi-position binding structural domain makes domain become the sealing conformation; (d) in conjunction with described first epi-position and described second epi-position but do not select simultaneously the polyspecific part of sealing conformation in conjunction with the ability of described first and second epi-position according to it.
And, the invention provides the polyspecific part of sealing conformation, second epi-position binding structural domain of noncomplementation that it contains first epi-position binding structural domain with first kind of epi-position binding specificity and has second epi-position binding specificity, wherein first kind and the combination of second binding specificity competition epi-position be not so that the polyspecific part of sealing conformation can be simultaneously in conjunction with two epi-positions.
The alternate embodiment of the above aspect of second configuration of the present invention randomly comprises further step (b1), and it comprises selects the three or more epi-position binding structural domain. Produce in this way or polyspecific part open or the sealing conformation, it contains two or more epi-position binding specificities. Aspect second configuration of the present invention preferred, when the polyspecific part contained plural epi-position binding structural domain, at least two in the described domain were in the sealing conformation, and the competition combination; Other domain can be competed combination, perhaps can be independently and the free combination of its connection epi-position.
According to the present invention, term " polyspecific part " refers to have the part of the epi-position binding specificity of more than one this paper definition.
The epi-position binding structural domain of term " sealing conformation " (polyspecific part) the assignment body of this paper definition randomly is connected to each other or combination by the albumen skeleton, so that the epi-position of an epi-position binding structural domain is in conjunction be combined competition with the epi-position of another epi-position binding structural domain. That is, each epi-position binding structural domain can be independently but not simultaneously in conjunction with the connection epi-position. Use method described herein can obtain the sealing conformation of part.
The epi-position binding structural domain of " open conformation " assignment body randomly is connected to each other or combination by the albumen skeleton, so that the epi-position of an epi-position binding structural domain is in conjunction with or not competition with the epi-position of another epi-position binding structural domain.
Term mentioned in this article " competition " refer to when second epi-position when its connection epi-position binding structural domain is combined, the combination of first epi-position and its connection epi-position binding structural domain is suppressed. For example, can by for example physical containment binding structural domain or by changing structure or the environment of binding structural domain, so that its affinity or affinity to epi-position descends, spatially suppress combination.
In another embodiment of second configuration of the present invention, epi-position in conjunction with the time can substitute each other. For example, first epi-position can be present on the antigen, and it causes the steric hindrance of second binding structural domain or conformation change wherein in conjunction with first binding structural domain of its connection the time, and this first epi-position has substituted the epi-position in conjunction with second binding structural domain.
Advantageously, in conjunction with reduce by 25% or more than, advantageously 40%, 50%, 60%, 70%, 80%, 90% or more than, preferably up to 100% or near 100%, so that in conjunction with fully suppressed. Can by conventional antigen in conjunction with experiment (such as ELISA), fluorescent type technology (comprising FRET) or the technology of the surface plasma body resonant vibration of detection molecules quality for example, detect the epi-position combination.
Advantageously, according to method of the present invention, each epi-position binding structural domain has different epi-position binding specificities.
Under background of the present invention, first is understood that not identical with second " epi-position " and not by the epi-position of single monospecific ligand binding. It can be in not on the synantigen, or is on the same antigen, but separate enough distances so that its do not form can be by the single monospecific VH/VL of conventional antibody in conjunction with the single entity to combination. On experience, if by the monospecific VH/VL Ligand Competition of anti-two epi-positions, then these two epi-positions are away from being not enough to be considered to separation epi-position of the present invention respectively for the single variable domains of two single-chain antibody forms (domain antibodies or dAb).
Sealing conformation polyspecific part of the present invention does not comprise the 02/02773 described part such as WO. Therefore, part of the present invention does not comprise collaborative complementary VH/VL pair in conjunction with any one or more antigen or epi-position. On the contrary, part of the present invention preferably comprises noncomplementation VH or VL pair. Advantageously, each VH of each VH or VL centering or VL domain have different epi-position binding specificities, the epi-position binding site arrange so that the epi-position in a site in conjunction be combined competition with the epi-position in another site.
Advantageously, according to the present invention, each epi-position binding structural domain comprises the immunoglobulin variable domain. More advantageously, each immunoglobulin variable domain is variable light chain domain (VL) or variable heavy chain domain VH. In second configuration of the present invention, immunoglobulin domains is non-complementary on being present in part of the present invention the time, and namely it is in conjunction with forming the VH/VL antigen binding site. Therefore, the polyspecific part that second configuration of the present invention is approved contains the immunoglobulin domains of identical hypotype, and it is variable light chain domain (VL), perhaps is variable heavy chain domain (VH). And when part of the present invention was the sealing conformation, immunoglobulin (Ig) can be Camelidae VHH type.
In alternate embodiment, part of the present invention does not comprise Camelidae VHH domain. More particularly, part of the present invention does not comprise and compares Camelidae VHH domain with people VH domain specific one or more amino acid residue is arranged.
Advantageously, single variable domains derive from according to not synantigen or epi-position in conjunction with the active antibody of selecting. For example, can partly separate variable domains by human body effect immunity at least. The method that substitutes is known in this area, comprises by the separation of people's antibody library and artificial antibody's gene synthetic.
Variable domains is advantageously in conjunction with super antigen, for example A albumen or L albumen. Being combined with super antigen is the characteristic in the correct antibody variable territory that folds, and allows to separate this domain by for example restructuring or library, mutation structure territory. Epi-position binding structural domain of the present invention comprises the albumen support and epi-position is done site (it is advantageously located on the albumen rack surface) mutually. The epi-position binding structural domain also can be based on albumen support or the skeleton beyond the immunoglobulin domains. For example, natural bacteria acceptor such as SpA are as the CDR stent graft, to produce the part of the one or more epi-positions of specific binding. The method is specified in United States Patent (USP) 5,831,012. Other suitable support comprises the support based on fibronectin and affinity body (affibodies). Suitable method is specified in WO 98/58965. Other suitable support comprises such as van den Beuken etc., J.Mol.Biol. (2001) 310, the described lipocalin protein of 591-601 and CTLA4, and such as the described support of WO 0069907 (Medical Research Council), it is based on the circulus of for example bacterium GroEL or other companion's polypeptide. The albumen support is capable of being combined; For example the CDR portable is to the CTLA4 support, and and immunoglobulin (Ig) VH or VL domain be used to form together multivalent ligand. Equally, fibronectin capable of being combined, lipocalin protein and other support.
The epi-position binding structural domain that those of skill in the art will recognize that the polyspecific part of the sealing conformation of producing according to the inventive method can be on the identical polypeptide chain, or is on the different polypeptide chains. Be positioned at situation on the different polypeptide chains for the variable region, it can connect through joint (advantageously being elastomeric joint (for example polypeptide chain)), cytotoxic compounds or arbitrarily other method known in the art.
First be connected an epi-position binding structural domain and can covalently or non-covalently connect. For the covalently bound situation of domain, for example can connect by the disulfide bond mediation.
In second configuration of the present invention, first are preferably different with second epi-position. It can be natural or synthetic polypeptide, albumen or nucleic acid or its part. In this regard, part of the present invention can be in conjunction with epi-position or antigen, and play antagonist or activator (for example EPO receptor stimulating agent). In one embodiment, the epi-position binding structural domain of part has identical epitope specificity, and for example can be simultaneously in conjunction with its epi-position when a plurality of copies of epi-position are present on the same antigen. In another embodiment, be provided at these epi-positions on the synantigen not, so that part can be in conjunction with epi-position and bridge joint antigen. One skilled in the art will recognize that the selection of epi-position and antigen is on a large scale with variable. It can for example be enzyme cofactor or the DBP of people or animal protein, cell factor, cytokine receptor, enzyme.
Can be included but not limited to by suitable cell factor and the growth factor of list as herein described or bispecific Binding peptide target: ApoE, Apo-SAA, BDNF, BLyS, heart nutrient-1, EGF, the EGF acceptor, ENA-78, ECF, ECF-2, Exodus-2, EpoR, aFGF, basic FGF, FGF-10, FLT3L, Fractalkine (CX3C), GDNF, G-CSF, GM-CSF, GF-01, insulin, IFN-y, IGF-I, IGF-II, IL-, IL-1p, 20IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 amino acid), IL-8 (77 amino acid), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin B IP-10, keratinocyte growth factor 2 (KGF-2), KGF, leptin, LIF, lymphocyte chemotactic factor (LCF), the Mullerian mortifier, the monocyte colony inhibition factor, the monocyte bait protein, M-CSF, MDC (67 amino acid), MDC (69 amino acid), MCP-I (MCAF), MCP-2, MCP-3, MCP-4, MIG, MIP1 α, MIP1 β, MIP3 α, MEP3 β, MIP-4, bone marrow precursor inhibiting factor-1 (MPIF-1), NAP-2, Neurturin, nerve growth factor, β-NGF, NT-3, NT-4, the plain M of tumorigenesis, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF12, SDF1 β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF-β, TGF-β 2, TGF-β 3, TNF (TNF), TNF-α, TNF-β, TNF acceptor I, the TNF receptor II, TNIL-1, TPO, VEGF, vegf receptor 1, vegf receptor 2, VEGFR3, GCP-2, GRO/MGSA, GRO-β, GRO-8, HCC1,1-309, HER1, HER2, HER3, HER4, the TACE recognition site, TNF BP-I and TNF BP-II, and in the annex 2 that invests this paper or annex 3 disclosed any target, no matter it is described combination, different combination or independent.
Cytokine receptor comprises the acceptor of aforementioned cell factor, and for example IL-1R1, IL-GR, IL-10R, IL-18R, and the acceptor of the cell factor of statement in annex 2 or the annex 3 are together with disclosed acceptor in annex 2 and 3.
Will be appreciated that this inventory is anything but detailed. When two epi-positions of polyspecific ligand binding (on the identical or different antigen), can select antigen by this inventory.
Advantageously, the bispecific part can be used for cell factor and other molecule of target Synergistic in the treatment situation of living organism. Therefore the present invention provides the collaborative method that strengthens two or more cytokine activities, and it comprises that give can be in conjunction with the bispecific part of described two or more cell factors. Of the present invention aspect this in, the bispecific part can be any bispecific part, comprises the part of the part that is made up of complementation and/or incomplementarity domain, open conformation and the part of sealing conformation. For example, this aspect of the present invention relates to the combination of VH domain and VL domain, only VH domain and VL domain only.
May be implemented in a variety of ways the Synergistic under the treatment background. For example, only two targets during simultaneously by the part target target combination just therapeutic activity may be arranged, and the independent target of target is invalid in treatment. In another embodiment, an independent target can provide a little slow or smaller curative effect, but provides the collaborative curative effect that increases with second target combination together.
Preferably, selected the cell factor of the bispecific ligand binding of this aspect of the present invention by the inventory shown in the annex 2.
And the bispecific part can be used for the oncology purposes, and during for the CD89 that expressed by cytotoxic cell, another kind of specificity is tumour-specific in a species specificity. But provided the example of the tumour antigen of target in the annex 3.
In an embodiment of second configuration of the present invention, variable domains derives from the antibody of anti-first and/or second antigen or epi-position. In a preferred embodiment, variable domains derives from the storehouse in single variable antibody structure territory. In an example, this storehouse is storehouse or the synthetic storehouse of not setting up in animal. In another embodiment, (at least in part) be not used for separating single variable domains by animal immune. Therefore, can separate single domain by neural library.
On the other hand, second configuration of the present invention provides the polyspecific part, second epi-position binding structural domain of noncomplementation that it contains first epi-position binding structural domain with first kind of epi-position binding specificity and has second epi-position binding specificity. First kind can be identical or different with the second binding specificity.
More on the one hand, the invention provides the polyspecific part of sealing conformation, second epi-position binding structural domain of noncomplementation that it contains first epi-position binding structural domain with first kind of epi-position binding specificity and has second epi-position binding specificity, wherein first kind and second binding specificity can be competed the epi-position combination, so that the polyspecific part of sealing conformation can not be simultaneously in conjunction with two epi-positions.
More on the other hand, the invention provides the part of open conformation, it contains the noncomplementation binding structural domain, and wherein domain is specific to the different epi-positions on the same target. This antibody with the affinity that increases in conjunction with target.
Equally, the invention provides multivalent ligand, it contains has specific incomplementarity binding structural domain to identical epi-position, and guiding contains the target of a plurality of copies of described epi-position, for example IL-5, PDGF-AA, PDGF-BB, TGF β, TGF β 2, TGF β 3 and TNF α, for example people TNF acceptor 1 and human TNF alpha.
Aspect similar, ligand design of the present invention can be become with low affinity in conjunction with each epi-position, so that meaning is not treated in the combination of single epi-position; But provide the treatment interests by be combined the increase affinity that produces with two epi-positions. In a specific embodiments, but this epi-position of target: and it is individualism on the normal cell type, but only exists together on unusual or diseased cells such as tumour cell. In this case, bispecific the part of the present invention unusual or tumour sufferer cell of efficient targeting only. Have specific part (being called chelating dAb) also to can be trimerization or poly (four poly-or more than) part to the identical epi-position of a plurality of copies on the same target or contiguous epi-position, it contains 3,4 or more noncomplementation binding structural domain. For example, can make up the part that contains 3 or 4 VH domains or VL domain.
And, the part in conjunction with many subunits target is provided, wherein each binding structural domain is specific to the subunit of described target. Part can be dimerization, trimerization or polymer. Preferably, can utilize the polyspecific part of the above aspect of method acquisition the present invention of first aspect present invention.
According to the above aspect of second configuration of the present invention, advantageously, first epi-position binding structural domain and second epi-position binding structural domain are noncomplementation immunoglobulin variable domains as defined herein. It is VH-VH variable domains or VL-VL variable domains.
Specifically, can prepare chelating dAb according to preferred aspect of the present invention, namely use grappling dAb, wherein use vector construction dimerization, trimerization or the poly dAb library contain joint sequence upstream or the constant dAb in downstream, the storehouse of second, the 3rd and all the other dAb is inserted into the opposite side of joint. For example, grappling or guiding dAb can be TAR1-5 (VK), TAR1-27 (V), TAR2h-5 (VH) or TAR2h-6 (VK).
In the methodology that substitutes, can avoid using joint, for example by using non-covalent bonding or the natural affinity between the binding structural domain (for example VH and VL). Therefore the present invention provides the method for preparing chelating poly part, and it may further comprise the steps:
(a) provide the carrier that contains nucleotide sequence, this sequential coding has specific unijunction to close domain to first epi-position on the target;
(b) provide the carrier of code database, this storehouse contains has specific second binding structural domain to second epi-position on the described target, and this epi-position can be identical or different with first epi-position, contiguous described first epi-position of described second epi-position; With
(c) express described first and second binding structural domain; With
(d) separate the combination of first and second binding structural domain, these two domain combinations produce the dimer in conjunction with target together.
First and second epi-position are contiguous, so that the poly part can be simultaneously in conjunction with two epi-positions. This provides the part of the binding affinity advantage with increase. When epi-position is identical, obtain the affinity of increase by the epi-position of a plurality of copies that exist at target, so that can be simultaneously in conjunction with at least two copies, so that the affinity effect that acquisition increases.
Can and use joint to connect binding structural domain by several method.
For example, binding structural domain can comprise cys residue, avidin and streptavidin group and is connected other means for the synthesis of rear non-covalent connection; Separate effective combination in conjunction with target. Perhaps, can have joint between first and second joint binding structural domain, these domains are expressed as single polypeptide by single carrier, and this polypeptide contains for example storehouse of aforesaid first binding structural domain, joint and second binding structural domain.
One preferred aspect, first and second binding structural domain natural combination when being bonded to antigen; For example, VH and VK domain are when being bonded to contiguous epi-position, with the natural combination of the interaction of three-dimensional, to form stable dimer. Thisly can separate in conjunction with experiment at target in conjunction with albumen. An advantage of the method is in correct conformation to only have the just combination of binding structural domain in conjunction with closely adjacent epi-position, so this binding structural domain will be separated owing to its affinity to target increases.
In the alternate embodiment aspect second configuration of the present invention above, at least one epi-position binding structural domain comprises NIg ' albumen support ' or ' the albumen skeleton ' of this paper definition. Suitable NIg albumen support includes but not limited to any following albumen support that is selected from: SpA, fibronectin, GroEL and other chaperone, lipocalin protein, CCTLA4 and the affinity body (affibodies) of as above stating.
According to the above aspect of second configuration of the present invention, advantageously, the epi-position binding structural domain is connected to ' albumen skeleton '.
Advantageously, albumen skeleton of the present invention is the immunoglobulin (Ig) skeleton. According to the present invention, the albumen that term ' immunoglobulin (Ig) skeleton ' refers to contain at least a immunoglobulin folding and is used as the core of one or more epi-position binding structural domains as defined herein.
Preferred " the immunoglobulin (Ig) skeleton " of this paper definition comprises and is selected from following any one or more: immunoglobulin molecules, and it contains CL (κ or the λ hypotype) domain of (i) antibody at least; Or (ii) the CH1 domain of heavy chain of antibody; Contain the CH1 of heavy chain of antibody and the immunoglobulin molecules of CH2 domain; Contain CH1, the CH2 of heavy chain of antibody and the immunoglobulin molecules of CH3 domain; Or with (ii) group that CL (κ or the λ hypotype) domain of antibody engages in any. Also can comprise the hinge area domain. This domain combination for example can be simulated natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2 molecules.
Those skilled in the art should know, and this inventory does not have detailed implication.
Can realize the skeleton of this paper definition and being connected of epi-position binding structural domain in the polypeptide level, namely after the nucleic acid of expressing coding skeleton and/or epi-position binding structural domain. Perhaps, can implement Connection Step in nucleic acid level. The method that albumen skeleton of the present invention is connected to one or more epi-position binding structural domains comprises use protein chemistry well known to those skilled in the art and disclosed herein and/or Protocols in Molecular Biology.
Advantageously, the polyspecific part of sealing conformation can comprise can binding target molecule first domain and can be in conjunction with the molecule that prolongs the part half life or second domain of group. For example, described molecule or group can be big material (bulky agent), for example HSA or cellular matrix albumen. Statement used herein " prolongs part molecule or the group of half life " and refers to following molecule or chemical group, with respect to not in conjunction with the part of this molecule or group, it increases half life in the body of this bispecific part when giving animal in conjunction with the situation of bispecific part described herein. The molecule of prolongation part half life or the example of group have hereinafter been described. In a preferred embodiment, the polyspecific part of sealing conformation possible binding target molecule just when the molecule that replace to strengthen half life or group only. Therefore, for example, the polyspecific part of sealing conformation remains in experimenter's the blood circulation by means of big molecule (for example HSA). When running into target molecule, the competition between the binding structural domain of polyspecific part of sealing conformation causes HSA replaced and be combined with target.
Advantageously, the part of any aspect of the present invention and be used for to make up the dAb monomer of this part can 300nM to 5pM (namely 3 * 10-7To 5 * 10-12M) KdAnd/or 5 * 10-1To 1 * 10-7S -1KoffSpeed constant (fixed according to surface plasmon resonance measurement) and 20 targets of its connection dissociate KdPreferred 50nM-20pM, or 5nM-200pM or 1nM-100pM, 1 * 10-7M or following, 1 * 10-8M or following, 1 * 10-9M or following, 1 * 10-10M or following, 1 * 10-11M or following; KoffSpeed constant preferred 1 * 10-2To 1 * 10-6S -1, or 5 * 10-3To 1 * 10-5S -1, or 5 * 10-1S -1Or below, or 1 * 10-2S -1Or below, or 1 * 10-3S -1Or below, or 1 * 10-4S -1Or below, or 1 * 10-5S -1Or below, or 1 * 10-6S -1Or below. KdSpeed constant is defined as Koff/K on。
Specifically, the invention provides TNF alpha antibody dAb monomer (or contain this dAb bispecific part), homodimer, heterodimer or homotrimer part, wherein each dAb is all in conjunction with TNF-α. Part is with 300nM to 5pM (namely 3 * 10-7To 5 * 10-12M) KdIn conjunction with TNF-α, preferred 50nM-20pM, more preferably 5nM-200pM, most preferably 1 nM-100pM; Or represent K in the mode that substitutesdBe 1 * 10-7M or following, preferred 1 * 10-8M or following, more preferably 1 * 10-9M or following, advantageously 1 * 10-10M or following, most preferably 1 * 10-11M or following; And/or fixed according to surface plasmon resonance measurement, KoffSpeed constant is 5 * 10-1To 1 * 10-7S -1, preferred 1 * 10-2To 1 * 10-6S -1, more preferably 5 * 10-3To 1 * 10-5S -1, for example 5 * 10-1S -1Or below, preferred 1 * 10-2S -1Or below, more preferably 1 * 10-3S -1Or below, advantageously 1 * 10-4S -1Or below, further advantageously 1 * 10-5S -1Or below, most preferably 1 * 10-6S -1Or below.
Preferably, part in standard L929 experiment with the ND50 of 500nM-50pM in and TNF-α, preferred 100nM-50pM, advantageously 10nM-100pM, more preferably 1 nM-100pM; 50nM or following for example, preferred 5nM or following, advantageously 500pM or following, more preferably 200pM or following, most preferably 100pM or following.
Preferably, part is with the IC50 inhibition TNF-α of 500nM-50pM and the combination of TNF-α acceptor I (p55 acceptor), preferred 100nM-50pM, more preferably 10nM-100pM, advantageously 1nM-100pM; 50nM or following for example, preferred 5nM or following, more preferably 500pM or following, advantageously 200pM or following, most preferably 100pM or following. Preferably, TNF-α is the humanTNF-α.
In addition, the invention provides anti-TNF acceptor I dAb monomer or contain the bispecific part of this dAb, it is with 300nM to 5pM (namely 3 * 10-7To 5 * 10-12M) KdAnd/or 5 * 10-1To 1 * 10-7S -1KoffSpeed constant (fixed according to surface plasmon resonance measurement) is in conjunction with TNF acceptor I, KdPreferred 50nM-20pM, more preferably 5nM-200pM, most preferably 1 nM-100pM; For example 1 * 10-7M or following, preferred 1 * 10-8M or following, more preferably 1 * 10-9M or following, advantageously 1 * 10-10M or following, most preferably 1 * 10-11M or following; Koff speed constant preferred 1 * 10-2To 1 * 10-6S -1, more preferably 5 * 10-3To 1 * 10-5S -1, for example 5 * 10-1S -1Or below, preferred 1 * 10-2S -1Or below, more preferably 1 * 10-3S -1Or below, advantageously 1 * 10-4S -1Or below, further advantageously 1 * 10-5S -1Or below, most preferably 1 * 10-6S -1Or below.
Preferably, dAb monomer or part in standard test (for example L929 described herein or Hela experiment) with the ND50 of 500nM-50pM in and TNF-α, preferred 100nM-50 pM, more preferably 10nM-100pM, advantageously 1nM-100pM; 50nM or following for example, preferred 5nM or following, more preferably 500pM or following, advantageously 200pM or following, most preferably 100pM or following.
Preferably, dAb monomer or part are with the IC50 inhibition TNF-α of 500nM-50pM and the combination of TNF-α acceptor I (p55 acceptor), preferred 100nM-50pM, more preferably 10 nM-100pM, advantageously 1nM-100pM; 50nM or following for example, preferred 5nM or following, more preferably 500pM or following, advantageously 200pM or following, most preferably 100 pM or following. Preferably, the target of TNF acceptor I is the humanTNF-α.
In addition, the invention provides with 1nM to 500 μ M (namely 1 * 10-9To 5 * 10-4), the K of preferred 100nM-10 μ MdIn conjunction with the dAb monomer of seralbumin (SA) (or contain this dAb bispecific part). Preferably, for containing first anti-SA dAb and for the bispecific part of second dAb of another target, second dAb to the affinity of its target (Kd that for example detects by the surface plasma body resonant vibration that uses BiaCore and/or Koff be first dAb to the 1-100000 of the affinity of SA doubly (preferred 100-100000 doubly, more preferably 1000-100000 doubly or 10000-100000 doubly). For example, first dAb with the affinity of about 10 μ M in conjunction with SA, and second dAb with the affinity of 100pM in conjunction with its target. Preferably, seralbumin is human serum albumins (HSA).
In one embodiment, first dAb (or dAb monomer) is with the K of about 50nMdIn conjunction with SA (for example HSA), preferred 70nM, more preferably 100,150 or 200nM.
And, the invention provides dimer, tripolymer and the polymer of the aforementioned dAb monomer that meets the aforementioned aspect of the present invention.
Part of the present invention comprises dAb monomer, dimer and tripolymer, can be connected to the antibody Fc district, and it contains in CH2 and the CH3 domain one or two, and randomly contains hinge area. For example, can use the carrier of the part that is connected with the Fc district as the mononucleotide sequential coding to prepare this peptide species.
Aspect another of second configuration of the present invention, the invention provides the nucleic acid molecules of the polyspecific part of one or more this paper definition of encoding at least. In one embodiment, part is sealing conformation part. In another embodiment, it is open conformation part. Can be at single core acid molecule coding polyspecific part; Perhaps each epi-position binding structural domain can be by independent nucleic acid molecule encoding. When part during by single nucleic acid molecule encoding, domain can be used as fused polypeptide and expresses, but perhaps single expression, and for example use subsequently chemical bridging agent to link together. To be linked together by the part of independent expression of nucleic acid by suitable method.
Nucleic acid can further encode for when expressing by the burst of host cell output polypeptide, and can be when expressing and the surface component fusion of filobactivirus particle (or select other component of the display systems). The targeting sequencing that can be used for the displaying of bacterial expression and/or bacteriophage or phasmid comprises pelB, stII, ompA, phoA, bla and pelA.
Aspect another of second configuration of the present invention, the invention provides the carrier that contains nucleic acid of the present invention.
More on the other hand, the invention provides the host cell with carrier transfection of the present invention.
Can design the expression of this carrier, for example to produce the epi-position binding structural domain that is used for selection on the phage particle surface. This can use method of the present invention to select ' polyspecific part ' so that can select the domain showed thus.
In a preferred embodiment of second configuration of the present invention, the epi-position binding structural domain is immune globulin variable region, and is selected by single domain V gene pool. In general, storehouse, monoclonal antibody body structure territory is illustrated on the filobactivirus surface. In a preferred embodiment, select each monoclonal antibody body structure territory according to the combination of phage library and antigen.
The present invention further provides the kit that contains polyspecific part at least of the present invention, this part can be open conformation or sealing conformation part. Kit of the present invention can be such as diagnostic kit, treats the detection kit of kit, chemistry or biological substance etc.
Again one side at second configuration of the present invention the invention provides the homogeneous phase immunization experiment that uses part of the present invention.
Again one side at second configuration of the present invention the invention provides a kind of composition, and it contains by the polyspecific part of the obtainable sealing conformation of the inventive method and medicine acceptable carrier, diluent or excipient. And, the invention provides the polyspecific part of use sealing conformation of the present invention or the method for composition therapeuticing disease. In a preferred embodiment of the invention, described disease is cancer or inflammatory disease, for example rheumatoid arthritis, asthma or Crohn disease.
Again one side at second configuration of the present invention the invention provides a kind of diagnostic method, comprises and uses polyspecific part or the composition of sealing conformation of the present invention to diagnose the illness.
Therefore, in general, can utilize analyte and the combination of the polyspecific part of sealing conformation to replace a kind of material, this causes producing signal when replacing. For example, the enzyme in conjunction with replaceable binding antibody of analyte (second antigen) (first kind of antigen), this provides the basis of immunization experiment, especially keeps in the situation of antibody by its avtive spot at enzyme.
Therefore, aspect last of second configuration, the invention provides the method that has situation that detects target molecule, it comprises:
(a) provide the polyspecific part of the sealing conformation of being combined with material, described part is specific to target molecule and described material, is wherein replaced in by part by the described material of ligand binding to cause producing detectable signal when coming out; (b) make the polyspecific part of sealing conformation contact target molecule; (c) detect the signal that produces owing to replacing this material.
According to the above aspect of second configuration of the present invention, advantageously, described material is enzyme, and it is inactivation when being closed the polyspecific ligand binding of conformation. Perhaps, described material can be and is selected from following any one or more: the substrate of enzyme and inactivation or by the fluorescence of quencher, luminous or chromonic molecule by ligand binding the time.
Sequence similar with sequence disclosed herein or homology (for example at least about 70% sequence homogeneity) also is a part of the present invention. In certain embodiments, the sequence homogeneity on amino acid levels can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. On nucleic acid level, sequence homogeneity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Perhaps, when lower in selective cross condition (for example very high stringency hybridization condition) complement with chain when the nucleic acid sections is hybridized, there is remarkable homogeneity. Nucleic acid can be present in the intact cell, in the cell lysate, or be partial purification or basic purified form.
The following calculating of carrying out " homology " or " sequence homogeneity " between two sequences or " similitude " (these terms are used interchangeably at this paper). Be the best contrast purpose aligned sequences (for example, for the best contrast, introduce the room in one or two that can be in first and second amino acid or nucleotide sequence, for purpose relatively, can ignore non-homogeneous sequence).
In a preferred embodiment, the reference sequence length that is used for purpose relatively and compares is at least 30% of reference sequence length, preferably at least 40%, more preferably at least 50%, again more preferably at least 60%, more more preferably at least 70%, 80%, 90%, 100%. Compare then amino acid residue or nucleotides at corresponding amino acid position or nucleotide position. When the position of first sequence is occupied by the amino acid residue identical with correspondence position in second sequence or nucleotides, molecule identical (amino acid used herein or nucleic acid " homology " are equal to amino acid or nucleic acid " homogeneity ") when this position then. Homogeneity percentage between two sequences is the function of the total same position number of sequence, and is thought of as two sequences of optimum comparison and the room number that needs to introduce and the length in each room.
Advantageously, use BLAST algorithm (2.0 editions) to carry out sequence alignment, parameter uses as default. " in " blast_help.html " file under the catalogue. Search argument advantageously is set at the default parameters of restriction as giving a definition.
BLAST (basic Local Alignment gopher) is the heuristic searching algorithm that program blastp, blastn, blastx, tblastn and tblastx use; The meaning of these programs ascribes to uses Karlin and the Altschul that some enhancings are arranged, the result that the statistical method of 1990,20 Proc.Natl.Acad.Sci.USA 87 (6): 2264-8 (referring to aforesaid " blast_help.html " file) obtains. Blast program is suitable for the sequence similarity retrieval, for example differentiates the homology to search sequence. This program generally is not used in the retrieval of motif type. About the discussion of the basic problem of sequence library similarity retrieval, referring to Altschul etc., (1994).
Can carry out following task at 5 blast programs that American National biotechnology information centre website obtains: " blastp " is with respect to protein sequence database comparing amino acid search sequence; " blastn " compares the nucleotide query sequence with respect to nucleotide sequence database; " blastx " compares 6 frame conceptual translation products of nucleotide query sequence (two chain) with respect to the protein sequence database; " tblastn " compares the albumen search sequence with respect to the nucleotide sequence database with whole 6 frames (two chain) dynamic translation. " tblastx " compares 6 frame translation products of nucleotide query sequence with respect to 6 frame translation products of nucleotide sequence database.
BLAST uses following search argument:
Histogram (HISTOGRAM): the histogram that shows each retrieval scoring; Be defaulted as yes. (seeing the Parameter H in the BLAST guide).
Describe (DESCRIPTIONS): the Short Description number that will report matching sequence is limited to designation number; The acquiescence limit value is 100 descriptions. (seeing the V parameter in the guide page or leaf). Other sees EXPECT and CUTOFF.
Aim at (ALIGNMENTS): will report that the high score sections is defined in the appointment number to the database sequence of (HSP); The acquiescence limit value is 50. If there is the report statistics that satisfies learn the database sequence of conspicuousness threshold value and exceed this acquiescence limit value (seeing following EXPECT and CUTOFF), then only report the coupling of tool significance,statistical. (seeing the B parameter in the BLAST guide).
Desired value (EXPECT): be used for the significance,statistical threshold value of report and database sequence coupling, default value is 10, and this is so that according to the stochastic model of Karlin and Altschul (1990), expection only chances on 10 couplings. If the significance,statistical that coupling has is not then reported coupling greater than the EXPECT threshold value. The EXPECT threshold value is more low more strict, causes reporting that the chance of coupling is more few. Can accept fractional value. (seeing the parameter E in the BLAST guide).
Cutoff value (CUTOFF): the right cutoff value of report high score sections. Default value calculates (seeing above) by the EXPECT value. The significance,statistical that only has at its significance,statistical and the independent HSP that equals the CUTOFF value of scoring is at least the same when high, just the HSP of report database sequence. The CUTOFF value is more high more strict, and the MM that causes reporting can be more few. (seeing the parameter S of BLAST guide). Usually, use EXPECT can process more intuitively the conspicuousness threshold value.
Matrix (MATRIX): for BLASTP, BLASTX, TBLASTN and TBLASTX specify the matrix of alternately scoring. Default matrix is that (Henikoff and Henikoff, 1992, Proc.Natl.30Acad.Sci.USA 89 (22): 10915-9) for BLOSUM62. Effectively alternative comprises PAM40, PAM120, PAM:250 and IDENTITY. The matrix of alternately scoring that is not used for BLASTN; In the BLASTN request, specify the MATRIX order to return wrong the response.
Chain (STRAND): the TBLASTN retrieval is limited to only cochain or the lower chain of database sequence; Or BLASTN, BLASTX or TBLASTX retrieval is limited to the frame of search sequence cochain only or lower chain.
Filter (FILTER): according to the mensuration of the SEG program of Wootton and Federhen (1993) Computers and Chemisty 17:149-163, shielding has the low search sequence sections that forms complexity, or according to Claverie and States, 1993, the XNU program determination of Computers and Chemistry 17:191-201, perhaps measure the sections that shielding is made up of short period property internal repeat according to the DUST program (referring to the Web site of NCBI) of Tatusov and Lipman for BLASTN. Filtration can eliminate statistically significant from the blast Output rusults but the report of the abiology meaning hit results of common acidity, alkalescence or proline-rich region territory (for example with respect to), and staying obtainablely in the search sequence more has a biological significance zone to database sequence-specific coupling. In nucleotide sequence, use letter " N " (for example " N " repeats 13 times), in protein sequence, use letter " X " (for example " X " repeats 9 times), the low-complexity sequence that the displacement filter program is found.
Filtration is only applicable to search sequence (or its translation product), is not suitable for database sequence. The acquiescence filter of BLASTN is DUST, and the acquiescence filter of other program is SEG. During sequence in being applied to SWISS-PROT, usually at all useless with SEG, XNU or these two shielding, so the expectability filtration does not always tell on. And, in some cases, the whole conductively-closed of sequence, this shows the significance,statistical that should suspect with respect to any coupling of not filtering the search sequence report.
NCBI-gi: in Output rusults, except showing accession number and/or locus title, also show NCBI gi identification number.
Most preferably use the simplification BLAST searching algorithm that under "/the BLAST " of above-mentioned NCBI Web site catalogue, provides to carry out sequence relatively.
Preparation based on the immunoglobulin (Ig) of polyspecific part
Bispecific part of the present invention, no matter be open in the conformation of target configuration of the present invention or sealing, all can be according to technology preparation previous foundation, be used for scFv, " bacteriophage " antibody or the preparation of other engineered antibody molecule in the antibody engineering field. Antibody production techniques, bispecific antibody technology of preparing specifically, summary and the list of references mentioned below for example being described in: Winter and Milstein, (1991) Nature 349:293-299; Pluckthun (1992) Immunological Reviews 5 130:151-188; Wright etc., (1992) Crit. Rev.Immunol.12:125-168; Holliger, P. and Winter, G., (1993) Curr.Op. Biotechn.4,446-449; Carter etc., (1995) J.Hematother.4,463-470; Chester, K.A. and Hawkins, R.E., (1995) Trends Biotechn.13,294-300; Hoogenboom, H.R. (1997) Nature Biotechnol.15,125-126; Fearon, D., (1997) Nature Biotechnol.15,618-619; Pluckthun, A. and Pack, P., (1997) Irnmunotechnology 3,83-105; Carter, P. and Merchant, A.M., (1997) Curr. Opin.Biotechnol.8,449-454; Holliger, P. and Winter, G., (1997) Cancer Immunol.Immunother.45,128-130.
The invention provides with respect to two not the variable domains of synantigen or epi-position select and the subsequently combination of variable domains. Be used for selecting utilization library known in the art and the system of selection of variable domains. Use is by natural library (Marks etc., (1991) J.Mol.Biol., the 222:581 of the rearrangement V gene of human B cell collection; Vaughan etc., (1996) Nature Biotech., 14:309) be that those skilled in the art are well-known. Usually use PCR, by clone's immunoglobulin (Ig) V gene preparation synthetic library (Hoogenboom and Winter (1992) J.Mol.Biol., 227:381; Barbas etc., (1992) Proc.Nafl.Acad.Sci.USA, 89:4457; Nissim etc., (1994) EMBO J., 13:692; Griffiths etc., (1994) EMBO], 13:3245; De Kruif etc., (1995) J.MoL Biol., 248:97). Mistake in the PCR process can cause height random. Can (directly select in this case the single domain combination) individually or select together VH and/or VL library with respect to target antigen or epi-position.
The method for optimizing for preparing bispecific part of the present invention comprises the use selective system, wherein according to itself and first antigen or epi-position in conjunction with selecting a variable domains storehouse, and according to its with second antigen or epi-position in conjunction with variable domains storehouse of selection. Then selected first of combination and second variable domains, and according to its and first and second antigen or epi-position in conjunction with selection bispecific part. According to itself and first and second antigen or epi-position these two individually but be combined simultaneously the part of selecting to seal conformation.
A. carrier library system
Be applicable to that various selective system of the present invention is known in the art. The example of this system has hereinafter been described.
Can directly screen the phageλ expression system into plaque or lysogen colony, these two had before all had description (Muse etc., (1989) 20 Science, 246:1275; Caton and Koprowski, (1990) Proc.Natl.Acad.Sci.U.S.A., 87; Mullinax etc., (1990) Proc.Natl.Acad.Sci.U.S.A., 87:8095; Persson etc., (1991) Proc.Natl. Acad.Sci.U.S.A., 88:2432), useful to the present invention. Although this expression system can be used for screening the library up to 106Individual different members, but it is unsuitable for screening greater number really (above 106Individual member).
Useful especially in library construction is to select display systems, and it can make nucleic acid be connected with the polypeptide of its expression. Selection display systems used herein is to allow to utilize suitable methods of exhibiting by select the single member's in library system in conjunction with metal ligand and/or target ligands.
The system of selection that separates the target member in big library is known in the art, take display technique of bacteriophage as representative. In this system, the variation peptide sequence is expressed in (Scott and Smith (1990) Science on the filobactivirus surface, 249:386), proved that this system can be used for setting up the antibody fragment library of (with its nucleotide sequence of coding), it is used for external selection and increases in conjunction with the specific antibody fragment (McCafferty etc., WO 92/01047) of target antigen. The nucleotide sequence in coding VH and VL district is connected to the s genetic fragment of the targeting signal of encoding, and this fragment is with this nucleotide sequence colibacillary periplasmic space that leads; Resultant antibody fragment is illustrated on the phage surface, is generally the fusion with bacteriophage coat protein (for example pIII or pVIII).
Perhaps, the antibody fragment outside is illustrated on the bacteriophage lambda capsid (phagebodies). The advantage of phage type display systems is: because it be biosystem, so only just can simply increase and select the library member by cultivate the bacteriophage that contains selected library member in bacterial cell. And, because coded polypeptide library member's nucleotide sequence is included on bacteriophage or the phagemid vector, so check order, express relative simple and easy with genetic manipulation subsequently.
The method of structure phage antibody display libraries and bacteriophage lambda expression library is well-known (McCafferty etc., (1990) Nature, 348:552 in this area; Kang etc., (1991) Proc. Natl.Acad.Sci.U.S.A., 88:4363; Clackson etc., (1991) Nature, 352:624; Lowman etc., (1991) Biochemistry, 30:10832; Burton etc., 20 (1991) Proc. Natl.Acad.Sci U.S.A., 88:10134; Hoogenboom etc., (1991) Nucleic Acids Res., 19:4133; Chang etc., (1991) J.Immunol., 147:3610; Breitling etc., (1991) Gene, 104:147; Marks etc., (1991), ibid; Barbas etc., (1992), ibid; Hawkins and Winter (1992) J.Immunol., 22:867; Marks etc., 1992, J.Biol.Chem., 267:16007; Lerner etc., (1992) Science, 258:1313, it is incorporated herein by reference).
A particularly advantageous method is to use scFv phage library (Huston etc., 1988, Proc.Natl.Acad.Sci U.S.A., 85:5879-5883; Chaudhary etc., (1990) Proc. Natl.Acad.Sci U.S.A., 87:1066-1070; McCafferty etc., (1990), ibid; Clackson etc., (1991) Nature, 352:624; Marks etc., (1991) J.Mol.Biol., 222:581; Chiswell etc., 30 (1992) Trends Biotech., 10:80; Marks etc., (1992) J.Biol.Chem., 267). The various embodiments of showing the scFv library at bacteriophage coat protein had been described already. The essence of phage display method will also be known, and such as being described in WO 96/06213 and WO 92/01047 (Medical Research Council etc.) and WO 97/08320 (Morphosys), it is incorporated herein by reference.
Other system that produces peptide library comprises the acellular enzyme machine that uses for external synthetic library member. In a method, by alternately selection and the pcr amplification with respect to target ligand of round are selected RNA molecule (Tuerk and Gold (1990) Science, 249:505; Ellington and Szostak (1990) Nature, 346:818). Can use similar technology discriminating in conjunction with dna sequence dna (Thiesen and Bach (1990) Nucleic Acids Res., the 18:3203 of set human transcription factor; Beaudry and Joyce (1992) Science, 257:635; WO 92/05258 and WO 92/14843). Can use in a similar manner In Vitro Translation as the synthetic polypeptide of the method for producing big library. In general these methods comprise the stabilisation polyribosome camplex, and these methods are further described in WO 88/08453, WO 90/05785, WO 90/07003, WO 91/02076, WO 91/05058 and WO 92/02536. The alternative display systems of non-phage type, for example disclosed system in WO 95/22625 and WO 95/11922 (Affymax) uses polysome to show the polypeptide that is used for selection.
The technology of other type comprises the storehouse of selecting in the artificial compartment again, and it allows gene to be connected with its gene outcome. For example, at WO 99/02671, WO 00/40712 and Tawfik and Griffiths (1998) Nature Biotechnol 16 (7), describe selective system among the 652-6, wherein can in the microcapsule that forms by water in oil emulsion, select the nucleic acid of coding genes of interest product. The genetic elements compartmentation that coding is had the gene outcome of targeted activity enters in the microcapsule, transcribes then and/or translates, to produce its gene outcome (RNA or albumen) separately in microcapsule. Subsequently sorting produces the genetic elements of the gene outcome with targeted activity. The method detects targeted activity by ining all sorts of ways and selects the genes of interest product.
B. library construction
Can use technology known in the art (technology of for example above stating) to make up or can be used for by the commercial source purchase plan library of selection. Be used for library of the present invention and be described in for example WO 99/20749. In case selected carrier system and the nucleotide sequence of one or more coding desired polypeptides is cloned in the carrier library, people just can produce diversity by carry out mutagenesis before expression in clone's molecule; Perhaps, encoding proteins be can express as mentioned above and select, mutagenesis and the in addition selection of round implemented afterwards. By the standard molecule method nucleotide sequence of the polypeptide of coding structure optimization is carried out mutagenesis. Useful especially is polymerase chain reaction or PCR (it is incorporated herein by reference for Mullis and Faloona (1987) Methods E ' nzymol., 155:335). PCR is well-known in this area, and it uses the dna replication dna by a plurality of circulations of heat-staple DNA dependent dna-polymerases catalysis, amplification target sequence. The structure of various antibody libraries has been discussed in Winter etc., (1994) Ann.Rev.Immunology 12,433-55 and the list of references of wherein mentioning.
Use template DNA (1fg at least; The most common 1-1000ng) and at least the 25pmol Oligonucleolide primers carries out PCR; It can advantageously use relatively large primer during heavy different degree source, primer pond, because each sequence only accounts for the sub-fraction of pond molecule, amount becomes restrictive in follow-up amplification cycles. Typical reactant mixture comprises: 2 μ l DNA, 25pmol Oligonucleolide primers, 2.5 μ l 10X PCR buffer solutions, 1 (Perkin-Elmer, Foster City, CA), the 1.25 μ M dNTP of 0.4 μ l, 0.15 μ l (or 2.5 units) Taq archaeal dna polymerase (Perkin Elmer, Foster City, CA) and make the deionized water of cumulative volume to 25 μ l. Cover mineral oil, and use the programmable heat circulating instrument to carry out PCR. The length in each step of PCR circulation and temperature and period are according to the needed stringency adjustment of reality. These two determines annealing temperature and time to the effectiveness of template annealing and the mispairing degree of allowing by the expection primer; Obviously, when nucleic acid molecules increases with mutagenesis simultaneously, need mispairing in synthesizing in the first round at least. The ability of optimizing primer annealing condition stringency belongs to those of ordinary skills' ken fully. The annealing temperature of use between 30 ℃-72 ℃. With 92 ℃ of initial sex change that template molecule took place in-99 ℃, 4 minutes, then be that 20-40 is individual by sex change (94-99 ℃, 15 seconds to 1 minute), the annealing (temperature of determining as mentioned above generally; 1-2 minute) and extend (72 ℃ 1-5 minute, depend on the length of the amplified production) circulation that forms. Last extension is generally 72 ℃, 4 minutes, can after connect nothing fixed number (0-24 hour) step of 4 ℃.
C. make up single variable domains
Be used for domain of the present invention in case selected, it just can by the whole bag of tricks combination known in the art, comprise covalency and non-covalent method.
Method for optimizing comprise use as described such as with the peptide linker (Bird etc., (1988) Science 242:423-426) of scFv branch sub-connection. Bird etc., Science 242,423-426; Hudson etc., Journal Immunol.Methods 231 (1999) 177-189; Hudson etc., Proc Nat Acad Sci USA 85,5,879 5883 provides the discussion of suitable joint. Joint is preferably flexible, and two single domains can be interacted. A joint example is (Gly4Ser) n joint, n=1-8 wherein, for example 2,3,4,5 or 7. Also can use the less flexible joint (Holliger etc., (1993) PNAS (USA) 90:6444-6448) that in bifunctional antibody, uses.
In one embodiment, the joint of use is not immunoglobulin hinge region.
Can use joint Combination of Methods variable domains in addition. For example, the disulfide bond that can utilize the cysteine residues by natural or engineered mistake to provide is stablized VH-V H、V L-V LOr VH-V LDimer (Reiter etc., (1994) Protein Eng.7:697-704), or by the interaction between the reconstruct variable domains, to improve " phase adaptive ", therefore and improve interactional stability (Ridgeway etc., (1996) Protein Eng.7:617-621; Zhu etc., (1997) Protein Science 6:781-788).
When suitable, can use other technology of connection or stabilizing immunoglobulin variable domains (specifically being antibody VH domain).
According to the present invention, the bispecific part can be " sealing " conformation in solution. " sealing " configuration be wherein two domains (for example VH and VL) with the configuration that combining form exists, for example form antibody combining site in conjunction with the right configuration of VL. For example, scFv can be the sealing conformation, and this depends on the arrangement for the joint that connects VH and VL domain. If joint elastic is enough to allow the domain combination, or rigidly domain is remained on binding site, then domain might take to seal conformation.
Similarly, the VH domain is to existing sealing conformation with the VL domain. Generally speaking, combine closely (for example by means of the rigid joint) of domain has function in the ligand molecular. Part in the sealing conformation can not be simultaneously in conjunction with increasing part molecule and second target molecule of half life. Therefore, part usually only with the molecular dissociation that increases the part half life time just in conjunction with second target molecule.
And the competition that non junction ground VH/VH, VL/VL or VH/VL dimer are configured between the domain is prepared.
In addition, part of the present invention can be open conformation. In this conformation, part can be simultaneously in conjunction with increasing part molecule and second target molecule of half life. Usually, with regard to VH VL to regard to, the variable domains in the open configuration keeps enough far, domain is not interacted and forms antibody combining site, does not compete in conjunction with its epi-position separately. With regard to VH/VH or VI/VL dimer, by rigid joint domain is not forced together. This domain is not to competing natively the antigen combination or forming antibody combining site.
Fab fragment and complete antibody mainly exist with the sealing conformation, but will recognize, under varying environment, bispecific part open and sealing might exist with various poised states. The combination of part and target may be shifted to open configuration with balance. Therefore, some part of the present invention can two kinds in solution conformations exist, wherein a kind of (opening mode) can be independently in conjunction with two antigens or epi-position, and another kind of conformation (closing form) only can be in conjunction with a kind of antigen or epi-position; Therefore in this conformation, antigen or epi-position are competed binding partner.
Although therefore the bispecific part of opening mode can exist with the poised state with closing form in solution, the sensation poised state is conducive to closing form; And opening mode can be by target in conjunction with being chelated into the sealing conformation. Therefore, preferably, some bispecific part of the present invention exists with the poised state between two kinds of (open and sealing) conformations.
Can modify bispecific part of the present invention, in order to be conducive to open or the sealing conformation. For example, stablize V with disulfide bondH-V LThe sealing conformation has been stablized in interaction. And, can make up for the syndeton territory and (comprise VH domain and VLDomain to) joint so that be conducive to opening mode; For example, joint can spatially hinder the combination of domain, and for example by adding big amino acid residue in appropriate position, or design keeps the suitable rigid structure of domain physical space spacing.
D. the feature of bispecific part
Can utilize method test bispecific part well known to those skilled in the art, as to comprise ELSIA and the combination of its specific antigen or epi-position. In a preferred embodiment of the invention, use monoclonal Phage-ELISA test combination.
Can implement Phage-ELISA according to the method for any appropriate: exemplary process is set forth in hereinafter.
Can by each wheel of ELISA screening select phage-infest of producing to selected antigen or epi-position in conjunction with situation, to differentiate " polyclone " phage antibody. Can screen by ELISA then the bacteriophage of the Simple infection bacterial clump of these groups, to differentiate " monoclonal " phage antibody. Also need to screen the soluble antibody fragment of conjugated antigen or epi-position, this also can be undertaken (referring to such as Winter etc. by using such as the ELISA for C-or the end-labelled reagent of N-, (1994) Ann.Rev.Immunology 12,433-55 and the list of references of wherein mentioning).
Gel 5 electrophoresis that also can be by the PCR product (Marks etc., 1991, ibid; Nissim etc., 1994, ibid), survey (Tomlinson etc., 1992) J.Mol.Biol.227,776) or the carrier DNA order-checking estimate the diversity of selected bacteriophage monoclonal antibody.
E. the structure of ' bispecific part '
As mentioned above, antibody is defined as following antibody (for example IgG, IgM, IgA, IgA, IgE) or fragment (Fv that Fab, Fv, disulfide bond close, scFv, bifunctional antibody) at this paper: it comprises at least one heavy chain and light chain variable domain, at least two weight chain variable domains or at least two light chain variable domains. (term antibody also comprises dAb). It derives from any species of natural generation antibody at least in part, or sets up by recombinant DNA technology; No matter it separates from serum, B cell, hybridoma, transfectoma, yeast or bacterium.
In a preferred embodiment of the invention, the bispecific part contains at least one 20 substance chain variable domains of antibody and single light chain variable domain of an antibody, or two substance chains or light chain variable domain. For example, part can comprise VH/VL to, a pair of VH domain or a pair of VLDomain.
First of this part can be positioned on the identical polypeptide chain with second variable domains. Perhaps, it can be positioned on the independent polypeptide chain. Under it was positioned at situation on the identical polypeptide chain, it can connect by joint, and joint is preferably aforesaid peptide sequence.
First be connected a variable domains and can covalently or non-covalently connect. In its covalently bound situation, covalent bond can be disulfide bond.
Be selected from variable domains in the situation of the V gene pool that for example uses display technique of bacteriophage selection described herein, these variable domains comprise the general framework district, so that it can be by the specificity metal ligand identification of this paper definition. The use of general framework, metal ligand etc. is described in WO 99/20749.
When using the V gene pool, the variation of peptide sequence is preferably placed in the structure ring of variable domains. The peptide sequence of any variable domains all can be reset or the sudden change change by DNA, in order to strengthen the interaction of each variable domains and its complementary pair. It is known that DNA is rearranged in this area, instruct in for example Stemmer, and No. the 6th, 297,053,1994, Nature 370:389-391 and United States Patent (USP), these two is all incorporated herein by reference. Other method of mutagenesis is that those skilled in the art are well-known.
In a preferred embodiment of the invention, ' bispecific part ' is Single-Chain Fv Fragment of Murine. In alternate embodiment of the present invention, ' bispecific part ' is made up of the Fab type.
On the other hand, the invention provides the nucleic acid of coding ' bispecific part ' at least as defined herein.
One skilled in the art will recognize that, according to this aspect of the present invention, antigen or epi-position these two can be simultaneously in conjunction with identical same antibody molecule. Perhaps, it can be competed in conjunction with same antibody molecule. For example, when two epi-positions by simultaneously in conjunction with the time, two variable domains of bispecific part can be independently in conjunction with its target epi-position. In the situation of domain competition, variable domains can be in conjunction with its target, but is combined its connection target not at one time with other variable domains; Perhaps first variable domains can be in conjunction with its target, but is combined its connection target not at one time with second variable domains.
The variable region can derive from the antibody of anti-target antigen or epi-position. Perhaps, it can derive from storehouse, monoclonal antibody body structure territory, the storehouse, antibody structure territory of for example expressing on the filobactivirus surface. Can as described belowly select.
In general, can be such as the standard laboratory handbook (such as Sambrook etc., (1989) Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, USA) statement make up and nucleic acid molecules and vector construct required for the present invention are implemented in operation.
The operation that is used for nucleic acid of the present invention is carried out at recombinant vector usually.
Therefore, on the other hand, the invention provides carrier, it contains the nucleic acid of coding ' bispecific part ' at least as defined herein.
Carrier used herein refers to for the resolution element that allogeneic dna sequence DNA is imported to for its expression and/or the cell that copies. Selecting or making up and use subsequently the method for this carrier is that persons skilled in the art are well-known. Countless carriers can openly obtain, and comprise bacterial plasmid, bacteriophage, artificial chromosome and episomal vector. This carrier can be used for simple clone and mutagenesis; Perhaps, use expression vector. Can select used according to the present invention carrier, be generally 0.25 kilobase to (kb) polypeptid coding sequence to 40kb or longer target sizes to hold length. After the body outer clone operation, transform suitable host cell with carrier. Each carrier all contains various function element, and it generally comprises clone's (or " polylinker ") site, origin of replication and at least one selectable marker gene. If given carrier is expression vector, then it has following one or more in addition: enhancer element, promoter, transcription terminator and burst, each all be positioned at cloning site near so that it effectively is connected to the gene of code book invention part.
In general, clone and expression vector these two all contain the nucleotide sequence that 30 carriers are copied in one or more selected host cells. Usually, in cloning vector, this sequence is to make carrier be independent of the sequence that host chromosome DNA copies, and comprises origin of replication or autonomously replicating sequence. This type of sequence of various bacteriums, yeast and virus is well-known. The origin of replication of plasmid pBR322 is suitable for most of gramnegative bacterium, and 2 μ plasmid starting points are suitable for yeast, and various viral starting point (for example SV 40, adenovirus) is used for the cloning vector of mammalian cell. In general, mammalian expression vector does not need origin of replication, unless these starting points are used in the mammalian cell that can copy high-level DNA COS cell for example.
Advantageously, clone or expression vector also can contain the Select gene of the selected marker that is otherwise known as. The survival of the transformed host cell that this gene code is cultivated in selective medium or the essential albumen of growing. Therefore, the host cell that does not transform with the carrier that contains Select gene can not be survived in this culture medium. Typical Select gene encoding proteins is given resistance, the defective that supplements the nutrients of antibiotic and other toxin (for example ampicillin, neomycin, amethopterin or tetracycline) or unavailable crucial nutrients in the growth medium is provided. Because copying of the carrier of code book invention part carried out the most usually, so use the Escherichia coli selected marker, for example give the beta-lactamase gene to the antibiotic amicillin resistance in Escherichia coli. These selected markers can derive from escherichia coli plasmid (for example pBR322) or pUC plasmid (for example pUC18 or pUC19).
Expression vector contains the promoter of host living beings identification usually, and effectively is connected to the purpose coded sequence. This promoter can be induction type or constitutive promoter. Term " effective connection " refers to this arranged side by side: the residing relation of wherein said component allows it to work in the expectation mode. " effectively connecting " mode of expressing with realization coded sequence under the condition compatible with control sequence to the control sequence of coded sequence is connected.
The promoter that is applicable to the prokaryotes host comprises for example beta-lactamase and lactase promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems and hybrid promoter, for example tac promoter. In general, the promoter for bacterial system also comprises the Shine-Dalgarno sequence that effectively is connected to coded sequence. Preferred vector is the expression vector that can express corresponding to polypeptide libraries member's nucleotide sequence. Therefore, can be by the single clone who expresses the polypeptide libraries member being bred separately and express, or by using any selection display systems, select with first kind and/or second antigen or epi-position. As mentioned above, preferably selecting display systems is phage display. Therefore, can use bacteriophage or phagemid vector, for example pIT1 or pIT2. Be used for targeting sequencing of the present invention and comprise pelB, stII, ompA, phoA, bla and pelA. An example is phagemid vector, and it has Escherichia coli origin of replication (being used for two strands copies), also has phage replication starting point (for the production of single stranded DNA). The operation of this carrier and be expressed in this area well-known (Hoogenboom and Winter (1992), ibid; Nissim etc., (1994), ibid). Briefly, carrier contains gives the optionally lac promoter of beta lactamase gene and expression cassette upstream of phasmid, this expression cassette by the pelB targeting sequencing (it imports periplasmic space with the Peptide T of expressing) of (N to C end), a plurality of cloning site (the library member who is used for clone's nucleotides form), optional one or more peptide-labeled (for detection of), optional one or more TAG terminator codons and bacteriophage albumen pIII form. Therefore, using colibacillary various suppressors and non-suppressor bacterial strain and adding in the situation of glucose, isopropylthio-β-D-galactoside (IPTG) or helper phage such as VCS M13, carrier can be as the plasmid replication of not expressing, produce a large amount of unique polypeptide libraries members, or producing bacteriophage, some of them contain at least one copy of polypeptide-pIII fusion in its surface.
The Vector construction of code book invention part uses conventional interconnection technique. Carrier or dna fragmentation that cutting, finishing separate, and connect into again desired form, produce the carrier that needs. If needs are arranged, analyze in a known way, to confirm in the carrier that makes up, having correct sequence. Be suitable for construction of expression vector, prepare the in-vitro transcription thing, import DNA in the host cell and express and the method for function evaluating analysis is well known by persons skilled in the art. Gene order exists situation in the test sample, or by quantitatively its amplification and/or expression, for example sequence analysis of some trace, in situ hybridization, immunocytochemistry or nucleic acid or the protein molecular of DNA or RNA analysis, Western blotting, DNA, RNA or albumen of conventional method. How those skilled in the art can revise these methods whenever necessary if being easy to imagination. The structure of the polyspecific part of sealing conformation
According to an aspect of second configuration of the present invention, connect two or more noncomplementation epi-position binding structural domains, make it become as defined herein sealing conformation. Advantageously, it can further be connected to skeleton, and this skeleton as an alternative or can be conducive to form and/or keep epi-position binding site sealing conformation each other when adding joint described herein.
(I) skeleton
Skeleton can be based on immunoglobulin molecules, or can be the NIg of above statement origin. The preferred immunoglobulin skeleton of this paper definition comprises and is selected from following any or a plurality of: immunoglobulin molecules, and it contains CL (κ or the λ hypotype) domain of (i) antibody at least; Or (ii) the CH1 domain of heavy chain of antibody; Contain the CH1 of heavy chain of antibody and the immunoglobulin molecules of CH2 domain; Contain CH1, the CH2 of heavy chain of antibody and the immunoglobulin molecules of CH3 domain; Or with (ii) group that CL (κ or the λ hypotype) domain of antibody engages in any. Also can comprise the hinge area domain. This domain combination for example can be simulated natural antibody, for example IgG or IgM or its fragment, for example Fv, scFv, Fab or F (ab ') 2 molecules. Those skilled in the art should know, and this inventory does not have detailed implication.
(II) albumen support
Each epi-position binding structural domain all contains albumen support and one or more interactional CDR of specificity that relates to domain and one or more epi-positions. Advantageously, epi-position binding structural domain of the present invention contains 3 CDR. Suitable albumen support comprises any following support that is selected from: based on the albumen support of immunoglobulin domains, based on the albumen support of fibronectin, based on the albumen support of affinity body (affibodies), based on the albumen support of CTLA4, based on the albumen support of molecular chaperones such as GroEL, based on the albumen support of lipocalin protein with based on the albumen support of bacterium Fc acceptor SpA and SpD. Those skilled in the art should know, and this inventory does not have detailed implication.
F: the support that is used to make up the dual specific part
The selection of main chain conformation
Immunoglobulin superfamily member's polypeptide chain all has similar folding.For example, although antibody is highly diversified with regard to its primary sequence, but sequence and crystallography contrast disclose, opposite with expection, 5 (H1, H2, L1, L2, L3) in 6 antigen coupling collars of antibody adopts limited amount main chain conformation or norm structure (Chothia and Lesk (1987) J.Mol.Biol., 196:901; Chothia etc., (1989) Nature, 342:877).Therefore, the analysis of ring length and Key residues can indicate H1, H2, L1, L2 and L3 main chain conformation (Chothia etc., (1992) J.Mol.Biol, the 227:799 that exists in most people's antibody; Tomlinson etc., (1995) EMBO J., 14:204628; Williams etc., (1996) J.Mol.Biol., 264:220).Although the H3 district is much diversified (reason is to use the D sections) with regard to sequence, length and structure, but it also forms the long main chain conformation of limited amount becate, it depends on the length of the concrete residue of key position in ring and the antibody framework and has situation or residue type (Martin etc., (1996) J.Mol.Biol, 263:800; Shirai etc., (1996) FEBS Letters, 399:1).
Dual specific part of the present invention is advantageously by domain libraries assembling, for example VH domain libraries and/or V LDomain libraries.And self can the library form provide dual specific part of the present invention.In one aspect of the invention, the long and Key residues of certain ring is wherein selected in the library of design dual specific part and/or structural domain, and is known with the main chain conformation of guaranteeing the member.As mentioned above, advantageously, these libraries are real conformations of naturally occurring immunoglobulin superfamily molecule, so that the chance minimum of its NOT-functionization.Plant is that the V gene segment is as a kind of basic boom that is suitable for making up antibody or TXi Baoshouti library; Other sequence also is useful.Can low frequency morph, make a spot of main chain conformation that has the function member can have change, this conformation does not influence its function.
The norm structure theory also can be used for estimating a large amount of different main chain conformations by the part coding, with the main chain conformation and the diversified residue of selecting do not influence norm structure of prediction based on ligand sequence.Known in people VK structural domain, L1 ring can adopt a kind of in 4 kinds of norm structures, and the L2 ring has single norm structure, and 90% people VK structural domain adopts a kind of (ibid for Tomlinson etc., (1995)) in 4 kinds of the L3 ring or the 5 kinds of norm structures; Therefore, in independent VK structural domain, different norm structure capable of being combined is to set up a series of different main chain conformation.The norm structure of the different series of known V αJie Gouyu coding L1, L2 and L3 ring, VK and V αJie Gouyu can with any VH structural domain pairing of several norm structures of codified H1 and H2 ring, very big to these 5 the viewed norm structure number of combinations of ring.This hint, multifarious generation may be to produce that binding specificity is necessary widely in the main chain conformation.But opposite with expection, by making up antibody library based on single known main chain conformation, the diversity of having found the main chain conformation is not to produce to be enough to target all antigenic diversity are necessary substantially.Even be that single main chain conformation needs not to be apokoinou construction-single native conformation and can be used as the basis in whole library more astoundingly.Therefore, one preferred aspect, dual specific part of the present invention has single known main chain conformation.
The single main chain conformation of selecting is preferably common in described immunoglobulin superfamily types of molecules.When the natural molecule of finding remarkable quantity adopted this conformation, this conformation was common.Therefore, of the present invention one preferred aspect, the natural appearance of the different main chain conformations of each coupling collar of immunoglobulin domains is considered to independently, so select different rings to have the natural variable domains of required main chain conformation combination.If do not obtain any conformation, then can choose the immediate isomorphic map of Denging.Be gene segment by the kind of selecting coding target main chain conformation preferably, different rings is set up the target combination of main chain conformation.More preferably the kind of Xuan Zeing is the usually natural expression of gene segment, and most preferably it is the most frequent expression in the gene segment for all natural kinds.
When design dual specific part or its library, can consider the occurrence rate of the different main chain conformations of each in 6 antigen coupling collars separately.For H1, H2, L1, L2 and L3, the known conformation of selecting 20%-100% natural molecule antigen coupling collar to adopt.
Usually, observe occurrence rate (being between the 35%-100%) more than 35%, ideally more than 50% and even more than 65%.Because most of H3 rings do not have norm structure, so preferably be chosen in main chain conformation common in the ring that shows norm structure really.Therefore, for each ring, be chosen in the most frequent observed conformation in the natural storehouse.In people's antibody, the most general norm structure (CS) of each ring is as follows: the L1-CS 2 (39%) of H1-CS 1 (expression library 79%), H2-CS 3 (46%), VK, (calculation assumption α k: the λ ratio is 70: 30 to the L3-CS 1 (36%) of L2-CS 1 (100%), VK, Hood etc., (1967) Cold Spring Harbor Symp.Quant.Biol, 48:133).For H3 ring with norm structure, have the 7 residue CDR3 length (Kabats etc. of residue 94 to the salt bridge of residue 101, (1991) Sequences ofproteins of immunological interest, the healthy and human service department of the U.S.) as if the most common.In the EMBL database, there are at least 16 human antibody sequences to have the desired H3 length of this conformation of formation and Key residues, at least 2 crystallography structures that can be used as antibody modeling (2cgr and 1tet) basis are arranged in albumen database.The kind of this norm structure combination of the most normal expression is that gene segment is VH sections 3-23 (DP-47), JH sections JH4b, VK sections 02/012 (DPK9) and JK sections JK1.VH sections DP45 and DP38 are also suitable.Therefore, these sections are capable of being combined as the basis that makes up the library with required single main chain conformation.
Perhaps, the natural occurrence rate based on different main chain conformations is not that each coupling collar is selected single main chain conformation separately, and is to use the basis of the natural occurrence rate of main chain conformation combination as the single main chain conformation of selection.For example, for antibody, can measure any 2,3,4,5 or the natural occurrence rate of the norm structure of whole 6 antigen coupling collars combination.At this, preferred selected conformation is modal in natural antibody, and most preferably it the most often is observed in natural storehouse.Therefore, for example in people's antibody, when considering the natural combination of 5 antigen coupling collar H1, H2, L1, L2 and L3, determine modal norm structure combination, then with the most general H3 ring conformation combination, as the basis of selecting single main chain conformation.
Ii. the variation of canonical sequence
If selected several known main chain conformations or preferred single known main chain conformation, then can make up dual specific part of the present invention or be used for library of the present invention, so that produce storehouse with structure and/or functional diversity by the binding site that changes molecule.This means the generation variant, make it on its structure and/or its function, have enough diversity, so that it can provide a series of activity.
Produce the diversity that needs by change the molecule of selecting in one or more positions.Position to be changed can be selected at random, or preferably optionally.Can realize changing by randomization then, in randomisation process, original amino acid is by natural or synthetic amino acid or the replacement of its analogue arbitrarily, produce very a large amount of variations, or by realizing changing with the original amino acid of the aminoacid replacement of one or more qualification hypotypes, producing more has a spot of variant.
Reported already and imported this multifarious the whole bag of tricks.Can use error-prone PCR (Hawkins etc., (1992) J.Mol.Biol., 226:889), chemomorphosis (Deng etc., (1994) J.Biol.Chem., 269:9533) or bacterium mutator (Low etc., (1996) J.Mol.Biol 260:359) imports to random mutation in the gene of coding molecule.The method of sudden change select location also is well-known in this area, is included in to be with or without oligonucleotide or the degenerate oligonucleotide that uses mispairing under the situation of using PCR.For example, several synthetic antibody libraries have been set up by target to the antigen coupling collar that will suddenly change.The H3 district of randomization people Toxoid,tetanus associativity Fab, set up a series of new binding specificities (Barbas etc., (1992) Proc.Natl.Acad.Sci.USA, 89:4457).Will be at random or partly to add to kind be the V gene segment H3 and L3 district at random, produce big library (Hoogenboom and Winter, (1992) J:Mol.Biol, 227:381 with the framework region that does not suddenly change; Barbas etc., (1992) Proc.Natl.Acad.Sci.USA, 89:4457; Nissim etc., (1994) EMBO J., 13:692; Griffiths etc., (1994) EMBO J., 13:3245; De Kruif etc., (1995) J.Mol.Biol., 248:97).This species diversity has extended to part or all of (Crameri etc., (1996) NatureMed., 5 2:100 that comprise other antigen coupling collar; Riechmann etc., (1995) Bio/Techrology, 13:475; Morphosys, WO 97/08320, ibid).Because the potential foundation of ring randomization approximately surpasses 10 15The variant of individual independent H3 structure and similar a large amount of other 5 rings, thus use existing transformation technology so that use cell free system production represent the library that might make up infeasible.For example, in one of maximum library that makes up so far, produce 6 * 10 10Plant different antibodies (ibid for Griffiths etc., (1994)), it only is the potential multifarious part in the design library.
The just quilt variation of residue of the objective function of foundation participated in directly or decorating molecule is only arranged in a preferred embodiment.For many molecules, function is in conjunction with target, so diversity should concentrate on the target binding site, and avoids changing to the entire package of molecule or keep the residue of selected main chain conformation key.
The diversity of canonical sequence when using the antibody structure territory
For antibody dual specific part, the binding site of target is the most common to be antigen binding site.Therefore, aspect highly preferred, the invention provides the antibody dual specific ligand library that wherein only changes the residue in the antigen binding site or be used for the assembly of this antibody dual specific part.These residues diversity very in people's antibody library knownly forms contact in high resolving power antibody/antigen mixture.For example, in L2, known location 50 and 53 is diversified in natural antibody, observes this position and contacts with antigen.On the contrary, ordinary method can make as all the residue variations in the corresponding complementary determining area (CDR1) of definition such as Kabat (1991, ibid), 7 residues of having an appointment, and compare used according to the present invention two residue variations in the library with it.This expression is set up the needed functional diversity of a series of antigen-binding specificities and is significantly promoted.
In fact, antibody diversity is the result of two processes: kind is the somatocyte reorganization of V, D and J gene segment, sets up master library (so-called kind system and junctional diversity) originally, and the somatic hypermutation of the rearrangement V gene that is obtained.The human antibody sequence analysis shows, diversity in the master library concentrates on the center of antigen binding site, and somatic hypermutation extends to diversity in the zone of antigen binding site periphery, these zones are that high conservative is (referring to Tomlinson etc. in master library, (1996) J.Mol.Biol., 256:813).This complementarity perhaps becomes retrieve sequence spatial available strategy gradually, although obviously be that antibody is exclusive, it can easily be applied to other peptide libraries.The residue that changes is a part that forms the residue of target binding site.If needs are arranged, the residue of different (comprising overlapping) part in the different steps variation target binding site in chosen process.
For antibody library, some in antigen binding site but be not under the diversified situation of whole residues set up initial ' originally ' storehouse.The antibody molecule that the term that this paper uses under this background " originally " refers to not have pre-determined target.These molecules are similar to by the molecule that does not experience the individual immunoglobulin gene coding of immunity variation, and fetus and newborn infant's individuality that immunity system also is not subjected to large-scale antigenicity stimulator attack are exactly this situation.A series of relatively then antigens or epi-position are selected this storehouse.If needs are arranged, then more diversity can be imported outside the variation district in the initial storehouse.Can select this ripe storehouse according to the function, specificity or the affinity that change.
Two kinds of differences that the invention provides binding domains are the storehouse originally, is used to make up the storehouse originally of dual specific part or dual specific part, and wherein the part or all of residue in the antigen binding site changes.Natural master library is simulated in " master " library, and it is the residue at antigen binding site center of variation (kind is a diversity) or variation (junctional diversity) in regrouping process in the V gene segment that diversity is confined in kind.Diversified residue includes but not limited to H50, H52, H52a, H53, H55, H56, HS8, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96.In " somatocyte " library, diversity is confined to the residue of variation (junctional diversity) in the regrouping process or the residue of height somatic mutation.Diversified residue includes but not limited to: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96.Knownly list in above being suitable for diversified all residues all contact one or more antigen-antibody complex in these libraries.Because be not that all residues in the antigen binding site all change in two libraries, thus in chosen process, adds other diversity by changing remaining residue, if needs are like this.It will be apparent for a person skilled in the art that the arbitrary portion of these residues (or other residue of composition antigen binding site) all can be used for initially and/or antigen binding site variation subsequently arbitrarily.
Be used for library construction of the present invention, usually on nucleic acid level, realize the variation of select location by changing encoding sequence, described encoding sequence has been determined peptide sequence, makes to add numerous possible amino acid (whole 20 kinds or its part) in this position.Use the IUPAC nomenclature, most of multi-usage codon is NNK, its encode whole amino acid and TAG terminator codon.The preferred NNK codon that uses is so that import the diversity that needs.Other codon that obtains same end also is useful, comprises the NNN codon, and it causes producing other terminator codon TGA and TAA.
The multifarious obvious bias of tending to some amino-acid residue that is characterised in that of side chain in the antigen binding site of people's antibody.If with VH, V κAnd V λThe amino acid of each 10 most diverse positions is formed addition in the district, then side chain multifarious more than 76% from 7 different residues only, these residues are Serine (24%), tyrosine (14%), l-asparagine (11%), glycine (9%), L-Ala (7%), aspartic acid (6%) and Threonine (6%).This bias tendency with elastic wetting ability residue of main chain and little residue can be provided, main chain elasticity may reflect to be tended in conjunction with the extensively evolution on the surface of antigen or epi-position, and has and help explain the mixing property of antibody that needs in the master library.
Distribute because preferably simulate this amino acid, preferably simulate observed distribution in the antigen binding site of antibody so wait the amino acid distribution that changes the position.This bias in amino-acid substitution allows to select some polypeptide (being not only antibody polypeptides) with respect to a series of target antigens, is applied to any peptide library easily.The method that the amino acid that has multiple bias to wait to change the position distributes (comprise and use trinucleotide mutagenesis) referring to WO 97/08320, preferred method wherein uses conventional degenerate codon, and reason is to be easy to synthesize.By contrast by the amino acid of whole degenerate codon assembly codings distribute (have in each position equal ratio single, double, three and the quadruple degeneracy) and natural amino acid selection, might calculate most representative codon.Codon (AGT) (AGC) T, (AGT) (AGC) C and (AGT) (AGC) (CT)-promptly use the IUPAC nomenclature to be respectively DVT, DVC and DVY, be the codon that approaches target amino acid spectrum most: its encode 22% Serine and 11% tyrosine, l-asparagine, glycine, L-Ala, aspartic acid, Threonine and halfcystine.Therefore, preferably, any in each diversified position use DVT, DVC or 30 DVY codons makes up library.
G: can increase the part antigen of half life
Dual specific part of the present invention is enough in one or more molecules that increase half life in the part body with its a kind of configurational energy.Usually, this molecule is natural existence and the anti-polypeptide of degrading or removing in the body, and this degraded or removal do not need the endogenous mechanism of material to carry out by removing body.For example, increasing the molecule of organism half life can be selected from:
Extracellular matrix protein; For example collagen protein, layer fibronectin, integrin and fibronectin.Collagen protein is the major protein of extracellular matrix.Present known about 15 types collagen molecules, be present in the different piece of body, for example be present in type i collagen albumen in bone, skin, tendon, ligament, cornea, the internal organs (account for body collagen protein 90%) or be present in II collagen type in the vitreous humour of cartilage, invertebrates intervertebral disk, notochord, eye.
Be present in the albumen in the blood, comprise: plasma proteins, for example scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen B, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin; Enzyme and inhibitor, for example proplasmin, N,O-Diacetylmuramidase, cysteine proteinase inhibitor C, α-1-antitrypsin and pancreas kypsin inhibitor.Proplasmin is the inactive precursor of trypsin-like serine protease Tryptase.It circulates general discovery in whole blood flow.When become activity and when changing Tryptase into of proplasmin, it launches the fibriilar effective enzymatic structure of solution fibrin territory, and the scleroproein protofibril is with the hemocyte tangle up in the clot.This is called fibrinolysis.
Immune system protein, for example IgE, IgG, IgM.
Translocator, for example retinol conjugated protein, α-1 microglobulin.
Defensin, for example beta-defensin 1, neutrophilic granulocyte defensin 1,2 and 3.
Be present in the albumen in hemato encephalic barrier or the nervous tissue, for example melanotropin acceptor, myelin, xitix transporter.
TfR ligands specific-neurologic agent fusion rotein (referring to US5977307); Cartilago cranialis endothelial cell receptor, Transferrins,iron complexes, TfR, Regular Insulin, type-1 insulin like growth factor (IGF 1) acceptor, rhIGF-1 2 (IGF 2) acceptor, insulin receptor.
Be positioned the albumen of kidney, for example many capsules albumen, IV collagen type, organic anion transporter K1, Heymann antigen.
Be positioned the albumen of liver, for example alcoholdehydrogenase, G250.
Blood coagulation factor X
α-1 antitrypsin
HNF1α
Be positioned the albumen of lung, for example secretory component (in conjunction with IgA).
Be positioned the albumen of heart, for example HSP 27.It is relevant with dilated cardiomyopathy.
Be positioned the albumen of skin, for example Keratin sulfate.
The bone specific proteins, Delicious peptide (BMP) for example, it is a group of transforming growth factor, shows osteogenic activity.
Example comprises BMP-2 ,-4 ,-5 ,-6 ,-7 (being also referred to as bone morphogenic protein (OP-1)) and-8 (OP-2).
Tumour-specific albumen comprises people's TA, Herceptin acceptor, estrogen receptor, kethepsin, for example cathepsin B's (being present in liver and the spleen).
Disease specific albumen, for example antigen of only on the activated T cell, expressing: comprise LAG-3 (lymphocyte activation gene); Osteoprotegerin part (OPGL) is referring to Nature 402,304-309,1999; OX40 (a member of TNF receptor family is expressed on activating T cell, is the known unique T cellular elements that stimulates altogether that is clearly raised in the cell that produces human T-cell leukemia virus's I type (HTLV-I))-referring to J.Immunol.2000 January 1; 16561): 263-70; Metalloprotease (relevant with sacroiliitis/cancer) comprises CG6512 Drosophila, people Paraplegin, people FtsH, people AFG3L2, mouse ftsH; Angiogenesis growth factor comprises acid fibroblast growth factor (FGF-1), Prostatropin (FGF-2), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), transforminggrowthfactor-(TGF-α), tumor necrosis factor-alpha (TNF-α), angiogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), Platelet-derived Endothelial Cell Growth Factor (PD-ECGF), placenta growth factor (PlGF), factor thrombocyte derivation growth factor-B B in mid-term (PDGF), Fractalkine.
Stress protein (heat shock protein(HSP))
HSP generally is present in the born of the same parents.When it existed outside born of the same parents, it was that cell is dead and overflow the indicator of its content.This non-procedural necrocytosis (necrosis) is only owing to just take place during the influencing of wound, disease, damage, and therefore triggers immunity system to anti-infective and reaction disease by the outer HSP of born of the same parents in vivo.Dual specific in conjunction with the outer HSP of born of the same parents can be positioned disease sites.
Participate in the albumen of Fc transhipment
Brambell acceptor (being also referred to as FcRB)
This Fc acceptor has two functions, and these two functions all can be used for transmitting potentially.These two functions are: (1) passes placenta, and IgG is transported to fetus by mother; (2) protection IgG avoids degraded, prolongs the serum half life of IgG thus.It is generally acknowledged that acceptor reclaims IgG by endosome.Referring to Holliger etc., Nat Biotechnol in July, 1997; 15 (7): 632-6.
Can design part of the present invention, and not need any increase of half life in the body above target-specific.For example, part of the present invention can be specific to being selected from aforesaid tissue specificity target, allow the dual specific part or the monomeric tissue specificity target of dAb of the relevant target of conjunctive tissue specific treatment thus, irrelevant with any increase of half life, although this increase may take place.And under the situation of part or dAb monomer target kidney or liver, this may be removing approach in the another kind of body (for example, part may break away from hepatic clearance and turn to kidney to remove) with part or the break-in of dAb monomer.
Increase other method of half life in the body:
Except design wherein the dual specific part of a specific specificity at the target protein that increases antibody polypeptides construction serum half life, can further stablize antibody polypeptides as described herein by being connected to the chemical part that increases the serum half life.For the improvement of antibody molecule pharmacokinetics is provided, the invention provides variable region, single structure territory polypeptide, this polypeptide chain is connected to stability that increase is provided and the polymkeric substance of half life.Polymer molecule (polyoxyethylene glycol for example; PEG) continue to use for a long time with proteic the connection, shown the pharmacokinetic properties of its adjusting institute modified protein.For example, having shown that proteic PEG modifies changes proteic body-internal-circulation half life, antigenicity, solvability and to proteoclastic resistance (Abuchowski etc., J.Biol.Chem.1977,252:3578; Nucci etc., Adv.Drug Delivery Reviews 1991,6:133; Francis etc., Pharmaceutical Biotechnology, the 3rd volume, (Borchardt, R.T. edits); With Stability of Protein Pharmaceuticals:in vivo Pathways of Degradation andStrategies for Ptotein Stabilization 1991 235-263 pages or leaves, Plenum, NY).
The locus specificity of protein molecular and at random PEGization in this area be known (referring to for example Zalipsky and Lee, Poly (ethylene glycol) Chemistry:Biotechnical and Biomedical Applications1992,347-370 page or leaf, Plenum, NY; Goodson and Katre, 1990, Bio/Technology, 8:343; Hershfield etc., 1991, PNAS88:7185).More particularly, at random PEGization and mercaptan derivative (Ling and Mattiasson, 1983, the Immunol.Methods 59:327 of antibody molecule at the lysine residue place described already; Wilkinson etc., 1987, Immunol.Letters, 15:17; Kitamura etc., 1991, Cancer Res.51:4310; Delgado etc., 1996 Br.J.Cancer, 73:175; Pedley etc., 1994, Br.J. Cancer, 70:1126).
The PEGization method has hereinafter been described.U.S. Provisional Application the 60/535th in common co-pending application PCT/GB2004/002829 of submission on June 30th, 2004 (specifying U.S) and submission on January 8th, 2004, the PEGization example of antibody polypeptides (being particularly for dAb) also is provided in No. 076, and each of these patents all integral body is attached to herein by reference.
Affinity/determination of activity:
Isolating single domain antibody as herein described (for example dAb) polypeptide has 300nM or following affinity (dissociation constant, Kd=K at least Off/ K On), preferred 300nM-50pM at least, 200nM-50pM, more preferably 100nM-50pM at least, 75nM-50pM, 50nM-50pM, 25nM-50pM, 10nM-50pM, 5nM-50pM, 1nM-50pM, 950pM-50pM, 900pM-50pM, 850pM-50pM, 800pM-50pM, 750pM-50pM, 700pM-50pM, 650pM-50pM, 600pM-50pM, 550pM-50pM, 500pM-50pM, 450pM-50pM, 400pM-50pM, 350pM-50pM, 300pM-50pM, 250pM-50pM, 200pM-50pM, 150pM-50pM, 100pM-50pM, 90pM-50pM, 80pM-50pM, 70pM-50pM, 60pM-50pM and even be low to moderate 50pM.
(Pharmacia Biosensor, Piscataway NJ.) pass through the antigen of surface plasma body resonant vibration (SPR) conventional sense varied texture domain polypeptide in conjunction with affinity can to use the BIAcore system.In the method, antigen is coupled to the BIAcore chip with concentration known, imports the varied texture domain polypeptide.The varied texture domain polypeptide combines with specificity between the immobilized antigen that the protein concentration that causes on the chip matrix increases and the spr signal change.Spr signal changes and to be recorded as resonance units (RU), and along the Y-axis of Sensorgram with respect to time display.Background signal adopts the independent solvent (for example PBS) that feeds chip.The associated value of the given sample of clean poor representative between the signal after background signal and the injection of varied texture domain polypeptide.For mensuration is left speed (K Off), association rate (K On) and the speed (K that dissociates d) constant, use BIAcore kinetics evaluation software (for example 2.1 editions).
High affinity depends on the complementarity between the CDR of antigenic surface and antibody or antibody fragment.Complementary type and intensity decision by the interaction of molecules that may exist between part target and the CDR, for example, potential ionic interaction, van der Waals gravitation, hydrogen bonding or contingent other interaction.CDR3 often more promotes the antigen binding interactions than CDR 1 and 2, and reason may be its general bigger size, this provide the more multimachine meeting that helps surface interaction (referring to for example Padlan etc., 1994, Mol.Immunol.31:169-217; Chothia and Lesk, 1987, J.Mol.Biol.196:904-917; With Chothia etc., 1985, J.Mol.Biol.186:651-663).High affinity shows that single immunoglobulin variable structural domain/psma ligand is highly complementary to having, and this complementarity is directly related with the structure of variable domains and target.
Can use permission to dock antigenic molecule modeling software, highlight and give the structure of single immunoglobulin variable structural domain polypeptide given antigenic high affinity with polypeptide structure.In general, the computer model of the target antigen of available polypeptide or other known structure docks the computer model of single immunoglobulin variable structural domain structure of known affinity, to determine interactive surfaces.The structure of known this known interactional interactive surfaces, people just can predict that conservative or not too cautious displacement in the variable domains sequence to the front or the negative impact of interaction strength, allows the improved binding molecule of appropriate design thus.
The antibody list variable domains of poly form:
In one aspect, antibody polypeptides construction as herein described (for example dAb) poly is turned to for example XOR homodimer, XOR homotrimer, XOR with the tetramer or the same polymer of high-grade XOR (for example XOR with pentamer to eight aggressiveness) more.Multimerization can increase the antigen bonding strength by the avidity effect, and wherein bonding strength is relevant with the summation in conjunction with affinity of a plurality of binding sites.
Prepare different and same polymer by expressing the single domain antibody that for example merges, produce configuration dAb-joint-dAb or this more multiple arrangement by peptide linker.Polymer also can be connected to other part, for example increases serum peptide sequence or another effector part, for example toxin or the targeting moiety of half life; PEG for example.The joint peptide sequence all can be used for producing the same polymer of XOR arbitrarily, and for example this area is used to produce the joint sequence of scFv.A common useful joint comprises multiple peptide sequence (Gly 4Ser) n(SEQ ID NO:7), wherein n=1 is to about 10 (for example n=1,2,3,4,5,6,7,8,9 or 10).For example, joint can be (Gly 4Ser) 3(SEQ ID NO:8), (Gly 4Ser) 5(SEQ ID NO:9), (Gly 4Ser) 7(SEQ ID NO:10) or another multiple (Gly 4Ser) (SEQ ID NO:7) sequence.
Expressing polymeric alternative method as the monomer that connects by peptide sequence is to close or other chemistry connection by for example disulfide linkage, and the translation back connects the monomeric immunoglobulin variable domains.For example, engineered for example at the free cysteine of monomer peptide C end, make that disulfide linkage closes between the monomer.Aspect this or need the others of free cysteine, by halfcystine codon (TGT, TGC) is included in abutting connection with last codon of dAb sequence (for the C-terminal cysteine, sequence in the primer is actually reverse complemental, be ACA or GCA, because it will be incorporated in the downstream PCR primer) and just in the PCR primer before one or more terminator codons, import halfcystine.If needs are arranged, can be with the joint peptide sequence as (Gly 4Ser) n(SEQ ID NO:7) places between dAb sequence and the free cysteine.Monomeric expression with free cysteine residues produces the mixture of monomer and dimerization form with about 1: 1 mixture.Use gel chromatography (for example using the ion exchange chromatography of salt gradient wash-out) to separate dimerization and monomer.
Perhaps, use the free cysteine of engineered mistake, by the mercaptan key monomer is coupled to multivalence chemistry joint, for example three poly maleimide molecules (for example Tris[2-maleimide ethyl] amine, TMEA) or bismaleimides PEG molecule (deriving from for example Nektar (Shearwater)).
In one embodiment, homodimer of the present invention or heterodimer comprise covalently bound to immunoglobulin (Ig) C with the C-end amino acid respectively H1 structural domain or C KThe V of structural domain HOr V LStructural domain.Therefore, the XOR homodimer can be Fab sample molecule, and wherein the antigen binding domains contains covalently bound to C with its C-terminal respectively H1 and C KStructural domain in conjunction with V HAnd/or V LStructural domain.In addition or or, dAb polymer of the present invention can be based on the Camelidae species modeling of the antibody of expressing most of global function, high specific, no sequence of light chain.The Camelidae heavy chain antibody exists with the homodimer of single heavy chain by its constant region dimerization.The variable domains of these Camelidae heavy chain antibodies is called as V HThe H structural domain, when its as V HWhen the fragment of chain is separated, keep ability (Hamers-Casterman etc., 1993, Nature 363:446-448 with the high specific conjugated antigen; Gahroudi etc., 1997, FEBS Lett.414:521-526).Therefore, can use methods known in the art and aforesaid method to make up antibody list variable domains polymer of the present invention, so that it has the V of Camelidae species heavy chain antibody HThe H conformation.
The PEGization of antibody polypeptides
The invention provides antibody polypeptides (for example dAb) monomer and the polymer of PEGization, with respect to the antibody polypeptides of PEGization not, it provides the half life of increase, anti-degraded, and do not lose activity (for example in conjunction with affinity).
Can use methods known in the art, antibody polypeptides molecule as herein described is coupled to the polymer molecule (preferred PEG) that is used to the half life that obtains to increase and degradation-resistant characteristic.Can be used for polymer moieties of the present invention and can be synthetic or natural, include but not limited to straight or branched polyalkylene, poly-alkenylene or polyoxyalkylene polymers, or branch or not ramose polysaccharide, for example homopolysaccharide or mixed polysaccharide.The preferred embodiment that can be used for synthetic polymer of the present invention comprises the form of straight or branched polyoxyethylene glycol (PEG), polypropylene glycol or polyvinyl alcohol and derivative or replacement.The particularly preferred substituted polymer that is used to be connected to antibody polypeptides described herein comprises and comprises methoxyl group (polyoxyethylene glycol) by the PEG of replacement.Natural polymer spendable or that replace PEG to use partly comprises lactose, amylose starch, dextran or glycogen except that PEG, and those skilled in the art's its derivative of generally acknowledging.The polymer molecule of derivation form comprises the other part that for example wherein exists or reactive group allows and the interactional derivative of amino-acid residue of antibody polypeptides described herein.This derivative comprises N-hydroxy-succinamide (NHS) active ester, succinimide propionic ester polymkeric substance and sulfydryl selective reaction thing, for example maleimide, vinyl sulfone(Remzaol and mercaptan.Particularly preferred derivation polymkeric substance includes but not limited to have the PEG polymkeric substance of following formula: PEG-O-CH 2CH 2CH 2-CO 2-NHS, PEG-O-CH 2-NHS, PEG-O-CH 2CH 2-CO 2-NHS, PEG-S-CH 2CH 2-CO-NHS, PEG-O 2CNH-CH (R)-CO 2-NHS, PEG-NHCO-CH 2CH 2-CO-NHS and PEG-O-CH 2-CO 2-NHS; Wherein R is (CH 2) 4) NHCO 2(mPEG).The PEG polymkeric substance can be linear molecule, perhaps can be ramose, and wherein a plurality of peg moieties exist with single polymkeric substance.Can be used for particularly preferred PEG derivatives more of the present invention and include but not limited to following material:
Figure A20058002851801831
Figure A20058002851801841
Reactive group (for example MAL, NHS, SPA, VS or mercaptan) can be connected directly to the PEG polymkeric substance, perhaps can be connected to PEG through linkers.
The size that is used for polymkeric substance of the present invention can be between 500Da-60kDa, and for example 1000Da-60kDa, 10kDa-60kDa, 20kDa-60kDa, 30kDa-60kDa, 40kDa-60kDa are between 50kDa-60kDa.Being used for polymkeric substance of the present invention, is PEG specifically, can be straight-chain polymer, perhaps can have a chain conformation.According to the combination of molecular weight and conformation, polymer molecule produces the molecule of average flow body dynamics size between 24-500kDa when being connected to antibody construction thing (for example dAb) monomer or polymer.The apparent size of the big little finger of toe molecule of the hydrokinetics of polymer molecule used herein (for example protein molecular), it is based on the diffusion of molecule in whole aqueous solution.Can handle diffusion or the motion of albumen in whole solution, to obtain proteic apparent size, wherein size obtains by the Stokes radius or the hydrodynamic radius of protein grain.Proteic " hydrokinetics size " depend on quality and shape (conformation) these two, make two kinds of albumen can have different fluid kinetics size based on the whole conformation of albumen with same molecular quality.The antibody list variable domains (comprising single domain antibody polymer described herein) that PEG connects) the big I of hydrokinetics is in the scope of 24kDa to 500kDa, 30-500kDa, 40-500kDa, 50-500kDa, 100-500kDa, 150-500kDa, 200-500kDa, 250-500kDa, 300-500kDa, 350-500kDa, 400-500kDa and 450-500kDa.The hydrokinetics size of preferred PEGization dAb is 30-40kDa, 70-80kDa or 200-300kDa.Therefore, the big I that is connected to the polymer molecule of antibody polypeptides (for example dAb or dAb polymer) changes to some extent, and this depends on the purpose purposes.For example, when will making PEGization dAb leave circulation and when entering peripheral tissues, need with connection polymkeric substance size remain on low-levelly, be beneficial to by oozing out in the blood flow.Perhaps, when PEGization dAb need be kept longer time cycle in circulation the time, can use the polymkeric substance (for example polymkeric substance of 30-60kDa) of higher molecular weight.
Can use method well-known in the art, will be used for polymkeric substance of the present invention (PEG) molecule and be connected to the antibody polypeptides construction.PEG or other polymer moieties are connected to antibody polypeptides monomer of the present invention or the polymeric the first step is to contain the hydroxyl terminal groups that close electric functional group replaces the PEG polymkeric substance.Specifically, the PEG polymkeric substance is connected to halfcystine or the lysine residue that exists in antibody polypeptides monomer or the polymer.Halfcystine and lysine residue can be natural, perhaps can be engineered go in the antibody polypeptides molecule.For example, the cysteine residues recombined engineering can be gone into the C-terminal of dAb polypeptide, but perhaps available halfcystine or Methionin are substituted in the residue at place, specific solvent approximated position in dAb or other antibody polypeptides.In a preferred embodiment, peg moiety is connected to the cysteine residues that is present in dAb monomer as herein described or the polymer C-terminal hinge area.
In one embodiment, the PEG polymkeric substance is connected to the framework region (FW) that is present in dAb and one or more halfcystines or the lysine residue among one or more allos CDR.CDR and framework region are to have the zone (Kabat etc. that are defined as the immunoglobulin variable structural domain in the protein sequence Kabat database of immunology importance, (1991) Sequences of proteins ofimmunological interest, U.S. HHS).In a preferred embodiment, the PEG polymkeric substance is connected to V HFramework sections DP47 or V κHalfcystine or lysine residue among the framework sections DPK9.The halfcystine and/or the lysine residue that can be connected to the DP47 of PEG comprise the halfcystine of the middle position 22 of SEQ ID NO:1 (Figure 21) or 96 and the Methionin of position 43,65,76 or 98.Can be connected to the halfcystine of DPK9 of PEG and/or lysine residue according to the present invention and comprise the cysteine residues of position 23 among the SEQ ID NO:2 (Figure 22) or 88 and the lysine residue of position 39,42,45,103 or 107.In addition, V HSpecific halfcystine or lysine residue among standard framework region DP38 or the DP45 can be connected to PEG.
In addition, the specificity solvent of non-natural halfcystine or lysine residue can sport halfcystine or the Methionin that is used to connect the PEG polymkeric substance near the site in the dAb molecule.Can use methods known in the art, for example analyze the crystalline structure of given dAb, determine that the solvent in any given dAb monomer or the polymer can be near residue.For example, use V H(it is in conjunction with hen's egg-white lysozyme for dAb HEL4; Vide infra) the crystalline structure of having illustrated,
The one-level aminoacid sequence of HEL4 (SEQ ID NO:5).
1 EVQLLESGGG?LVQPGGSLRL?SCAASGFRIS?DEDMGWVRQA?PGKGLEWVSS
51 IYGPSGSTYY?ADSVKGRFTI?SRDNSKNTLY?LQMNSLRAED?TAVYYCASAL
101 EPLSEPLGFW?GQGTLVTVSS
V κThe one-level aminoacid sequence of stand-in (SEQ ID NO:6).
1 DIQMTQSPSS?LSASVGDRVT?ITCRASQSIS?SYLNWYQQKP?GKAPKLLIYA
51 ASSLQSGVPS?RFSGSGSGTD?FTLTISSLQP?EDFATYYCQQ?SYSTPNTFGQ
101 GTKVEIKR
Having identified residue Gln-12, Pro-41, Asp-62, Glu-89, Gln-112, Leu-115, Thr-117, Ser-119 and Ser-120 is that solvent is come-at-able, should be to sport to be used to connect the halfcystine of PEG polymkeric substance or the attractive candidate of lysine residue.In addition, use V κThe crystalline structure of having illustrated of stand-in dAb (referring to above), having identified residue Val-15, Pro-40, Gly-41, Ser-56, Gly-57, Ser-60, Pro-80, Gly-71, Gln-100, Lys-107 and Arg-108 is that solvent is come-at-able, should be to sport to be used to connect the halfcystine of PEG polymkeric substance or the attractive candidate of lysine residue.In one embodiment, the PEG polymkeric substance is connected to come-at-able halfcystine of a plurality of solvents or lysine residue, or be connected to the solvent that sports halfcystine or lysine residue can be near residue.Perhaps, only have at specific antibody polypeptides construction under the situation of a come-at-able halfcystine of solvent or the Methionin residue of halfcystine or Methionin (or be revised as), or be selected under the situation of several this PEGization residues at the come-at-able residue of specific solvent, only a solvent can be connected to PEG near residue.
(SanCarlos CA) provides several connectivity scenarios of the present invention that are used in Nektar company.For example, when needs are connected to lysine residue with PEG or other polymkeric substance, can use the active ester of the PEG polymkeric substance of having used N-hydroxy-succinamide (for example succinimide propionic ester) derivation.In the time that cysteine residues will be connected to, can use the PEG polymkeric substance of having used sulfydryl selective reagent (for example maleimide, vinyl sulfone(Remzaol or mercaptan) derivation.Can be used for being found in Nektar catalogue (can on the World Wide Web nektar.com place obtain) according to other example of particular that the present invention produces the PEG derivative of PEGization dAb.In addition, the PEG of several derivation forms be can use, PEG polymkeric substance and dAb monomer of the present invention or polymeric the connection are beneficial to according to the present invention.Be used for PEG, PEG benzene carbonic ether, PEG succinimdyl carbonate, PEG-carboxymethyl trinitride, dimethyl maleic anhydride PEG, PEG dithiocarbonic acid ester derivative, PEG-trifluoro esilate (2 that PEG derivative of the present invention includes but not limited to that PEG-succinimide succinate, urethane connect, 2,2-trifluoro esilate), mPEG polyurethane and as Zalipsky and Lee, (1992) (" Use of functionalizedpoly (ethylene glycol) s for modification of peptides " is stated from Poly (Ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, J.MiltonHarris edits, Plenum Press, NY) described other PEG derivative.
In each of above embodiment, the PEG polymkeric substance all can be connected to the arbitrarily suitable residue that is present in the antibody polypeptides construction peptide, or preferably, the one or more residues of construction can be modified or sport halfcystine or lysine residue, can use its tie point then as the PEG polymkeric substance.Preferably, the residue of modifying in this way is that solvent can be near residue; The i.e. residue that aqueous environments and derivation PEG polymkeric substance can be approaching when the antibody polypeptides construction is its natural folding configuration.In case one or more in these residues sport cysteine residues according to the present invention, it just can be used for using the PEG of linearity or ramose MAL derivation PEG (MAL-PEG) to connect.
In one embodiment, provide the antibody construction thing that contains antibody list variable domains and PEG polymkeric substance, wherein the ratio of PEG polymkeric substance and antibody list variable domains is at least 0.25: 1 molar ratio.In another embodiment, the molar ratio of PEG polymkeric substance and antibody list variable domains is 0.33: 1 or higher.In another embodiment again, the molar ratio of PEG polymkeric substance and antibody list variable domains is 0.5: 1 or higher.
H: the purposes of part as herein described
1) purposes of the polyspecific part of second kind of configuration of the present invention
The polyspecific part of second kind of configuration method of the present invention can be used for interior therapeutic and preventive use, external and in-vivo diagnostic purposes, experiment in vitro and reagent purposes etc.For example, antibody molecule can be used for the anitibody type experimental technique according to method known to those skilled in the art, for example the μ elisa technique.
As implied above, ligands specific of the present invention can be used for diagnosis, prevention and methods of treatment.Ligands specific of the present invention has diagnostic uses in protein analysis that utilizes standard immunoassay group method and original position Protein Detection; For being used for these purposes, can be according to technical mark part known in the art.In addition, this antibody polypeptides during to chromatographic support such as resin, is used for to preparation property the affinity chromatography method when compound.All these technology all are that those skilled in the art are well-known.
The diagnostic uses of the polyspecific part of sealing conformation of the present invention comprises the homogeneous phase experiment that is used to analyze, the polyspecific part competition that this experiment has utilized the sealing conformation in conjunction with two targets make two targets can not be simultaneously in conjunction with the ability of (sealing conformation), or it is simultaneously in conjunction with the ability (open conformation) of two targets.
The diagnosis and the manufacturer of research experiment system that are used for drug development and development thirst for seeking real homogeneous phase immunization experiment form always.Main diagnosis market comprises the human test in hospital, consulting room and clinic, commercial reference laboratory, blood bank and the family, inhuman diagnosis (for example food test, water check, environmental test, biophylaxis and veterinary inspector) and last research (comprising drug development, fundamental research and academic research).
At present, all these markets all utilize the immunization experiment system that sets up round chemoluminescence, ELSIA, fluorescence or the radioimmunoassay experiment technology that is of little use.Each of these assay format all needs separating step (separation and combination with unconjugated reagent).In some cases, need several separating steps.Increasing these extra steps increases reagent and automated operation, expends time in and influences the net result of experiment.In people's diagnosis, but the separating step automatization, and it has covered problem, but can not remove problem.Robot technology, extra reagent, extra incubation time etc. have increased considerable cost and complicacy.In drug development, for example wherein use the high flux screening of the similar millions of samples of very low-level test molecule one-time detection, add extra separating step and can eliminate the ability of screening.But cancellation is separated in the too many noise of generation among the reading result.Therefore, need real homogeneous phase form, it provides sensitivity in by the obtainable scope of this assay format.Advantageously, experiment has complete quantitative reading result, has highly sensitive and big dynamics range.Sensitivity is an important requirement, and it has reduced the sample size that needs.These two features are features that homogeneous system provides.This is for careful check and wherein the drug development of sample preciousness is extremely important.The present available multiphase system in this area needs a large amount of samples and expensive reagent.
The application of homogeneous phase experiment comprises the cancer test, and wherein Zui Da experiment is the experiment that is used for prostate specific antigen, and this experiment is used to screen the prostate cancer male sex.Other application comprises the fertility check, and it provides a series of checks for attempting conceived women, comprises pregnant β-hcg.The infectious diseases check comprises hepatitis, HIV, rubella and other virus and microorganism and sexually transmitted disease (STD).Blood bank's service test, especially HIV check, first type, B-mode, third type, non-A non-B hepatitis detect.Therapeutic type drug surveillance check comprise the monitoring prescription drug in the patient effectively and avoid toxic level, described toxicity for example be digoxin to ARR toxicity, and the phenylethyl barbituric acid level in the psychosis case; Theophylline is for the toxicity of asthma.And diagnostic test can be used for the Drug abuse check, for example is used for the check of Cocaine, hemp etc.The metabolism check is used to detect thyroid function, anaemia and other physiology obstacle and function.
And homogeneous phase immunization experiment form can be used for the production of standard clinical chemical experiment.Including immunization experiment and chemical experiment on same instrument in is highly favourable in diagnostic test.Suitable chemical experiment comprises test glucose, cholesterol, potassium etc.
The other main application of homogeneous phase immunization experiment is drug discovery and exploitation: high flux screening is included in the superelevation volume with respect to target check combinatorial chemistry library.Detection signal becomes positive component less group then, checks in cell at last, checks in animal then.The homogeneous phase experiment can be used for the check of all these types.In drug development, especially in zooscopy and clinical experiment, use immunization experiment in a large number.Homogeneous phase experiment is greatly quickened and has been simplified these methods.Other application comprises the food and drink check: the intestinal bacteria of check meat and other food, Salmonellas etc.; The water check comprises all types of pollutents in the check waterplant, comprises intestinal bacteria; And veterinary inspector.
In the generalized embodiment, the invention provides in conjunction with experiment, it comprises the detected thing of the polyspecific part that is bonded to sealing conformation of the present invention, changes the detected characteristic that can detect thing by the polyspecific part that analyte is bonded to described sealing conformation.This experiment can be disposed by several different modes, and every kind of mode is all utilized the above-mentioned characteristic of the polyspecific part of sealing conformation.
Experiment depends on analyte to detecting the direct or indirect replacement of thing, and the detected characteristic that can detect thing is changed.For example, in the time can detecting thing for the enzyme of the reaction that can catalysis has end point detectable, described enzyme can be reached the degree that hinders its avtive spot by its part combination, makes enzyme deactivation thus.Also be closed the polyspecific part bonded analyte substituted enzyme of conformation, make enzyme activation by discharging avtive spot.Endonuclease capable and substrate reactions then, generation can detect incident.In an alternate embodiment, part can influence the conformation of enzyme in conjunction with the enzyme of avtive spot outside, changes its activity thus.For example, but part perhaps can prevent active necessary cofactor combination in conjunction with restricted activity site structure.
The physical apparatus of experiment can adopt any form known in the art.Polyspecific part/the enzyme complex of sealing conformation for example, can be provided on test strip; Can provide substrate in the different zones of test strip, part/enzyme complex is passed through in the solvent migration that allows to contain analyte, substituted enzyme, and carry it into substrate zone, to produce signal.Perhaps, can on test rod or other solid phase, provide part/enzyme complex, and be immersed in analyte/substrate solution, under the analyte effect that exists, enzyme is released in the solution.
Because each molecule of analyte discharges an enzyme molecule potentially, experiment is quantitatively, and the strength of signal that produces in preset time depends on the analyte concentration in the solution.
It is possible using other configuration of the analyte of sealing conformation.For example, can design the polyspecific part of sealing conformation, with at allosteric site desmoenzyme, activating enzymes thus.In this embodiment, enzyme has activity when not having analyte.Add the analyte substituted enzyme, remove the allosteric activation, thus fermentoid.
Under the embodiment background of above use enzymic activity as the analyte concentration detection method, the activation of enzyme or inactivation refer to the increase or the reduction of enzymic activity, detect to be the aitiogenic ability of enzyme catalysis signal.For example, but but the undetectable substrate of enzyme catalysis to the transformation of its test format.For example, horseradish peroxidase is widely used in this area with commercially available product look or chemical luminous substrate.The level that enzymic activity increases or reduces can be between 10%-100%, and for example 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%; Under the situation that activity increases, increase can surpass 100%, and promptly 200%, 300%, 500% or higher, if perhaps be subjected to the active undetectable words of the baseline of inhibitory enzyme, then may detect by percentage.
In other configuration, but the right substrate of polyspecific part desmoenzyme/substrate of sealing conformation, rather than enzyme.Therefore substrate is not useable for enzyme, until discharging in by the polyspecific part that seals conformation by analyte.The realization of this configuration is as the realization of the configuration of desmoenzyme.
And, configurable experiment, with the combined with fluorescent molecule, for example fluorescein or another kind of fluorophore, its conformation make fluorescence when being bonded to part by quencher.In the case, analyte will replace fluorescence molecule with combining of part, produce signal thus.The substitute that is used for fluorescence molecule of the present invention comprises luminous agent, and for example luciferin/luciferase, and chromogenic reagent for example is generally used for the material of immunization experiment, for example HRP.
Comprise according to the treatment of the polyspecific part of the present invention preparation and preventive use part of the present invention is accepted Mammals, for example the people.Polyspecific can allow antibody with high affinity in conjunction with poly antigen.The polyspecific part can allow two antigen cross-linkings, for example when raising cytotoxic T cell, with killing and wounding of mediation tumor cell line.
At least the pure substantially part of 90-95% homogeneity or its are conjugated protein, and for example the dAb monomer preferably gives Mammals, and 98-99% or higher homogeneity most preferably are used for pharmaceutical usage, are man-hour Mammals especially.In case purifying or be purified to the homogeneity that needs partly, diagnosticability or therapeutic ground (comprising the ground that exsomatizes) use part, or use part (Lefkovite and Pernis in exploitation with when implementing experimental technique, immunofluorescence dyeing etc., (1979 and 1981) Immunological Methods, I and II volume, Academic Press, NY).
Part of the present invention or its are conjugated protein, for example the dAb monomer can be used for prevention usually, suppresses or treatment inflammatory states, abnormal allergy, cancer, bacterium or virus infection and autoimmune disease (it includes but not limited to type i diabetes, asthma, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous, Crohn disease and myasthenia gravis).
Except rheumatoid arthritis, anti-TNF described herein-α polypeptide can be used for treating autoimmune disease, for example (in the bracket expression infected organ) but be not limited to: Addison disease (suprarenal gland), the autoimmune disease of ear (ear), the autoimmune disease (eye) of eye, autoimmune hepatitis (liver), autoimmunity parotitis (parotid gland), sick and the inflammatory bowel (small intestine) of Crohn, type i diabetes (pancreas), epididymitis (epididymis), glomerulonephritis (kidney), hyperthyroidism (Tiroidina), Guillain-Barre syndromes (neurocyte), Hashimoto disease (Tiroidina), hemolytic anemia (red corpuscle), systemic lupus erythematous (a plurality of tissue), male sterility (sperm), multiple sclerosis (neurocyte), myasthenia gravis (myoneural junction), pemphigus (mainly being skin), psoriatic (skin), rheumatic fever (heart and joint), sarcoidosis (a plurality of tissues and organ), scleroderma (skin and reticular tissue), Sjogren syndromes (exocrine gland and other tissue), SpA (axial skeleton and other tissue), thyroiditis (Tiroidina), ulcerative colitis (small intestine) and vasculitis (blood vessel).Except rheumatoid arthritis and other chronic inflammation disease (for example Crohn disease, psoriatic etc.), anti-VEGF polypeptide as herein described can be used for treating diabetes, acute myelogenous leukemia, leukemia and ophthalmic diseases, comprises macular degeneration and diabetic retinopathy.
In this application, granting asylum property composition before term ' prevention ' is included in and induces an illness." inhibition " refers to inducing after the incident but gave composition before clinical manifestation goes out disease.
Can obtain to can be used for screening antibody or its conjugated protein protection in order to avoid the animal experimental model of the validity aspect ill or the treatment disease.The method of checking system lupus erythematosus (SLE) is (Knight etc., (1978) J.Exp.Med., 147:1653 known in the art in the susceptible mouse; Reinersten etc., (1978) New Eng.J.Med., 299:515).By the solubility AchR albumen with another species induce an illness in the SJL/J female mice, check myasthenia gravis (MG) (Lindstrom etc., (1988) Adv.Immurzol., 42:233).By injection II collagen type in the susceptible mouse species, bring out sacroiliitis (Stuart etc., (1984) Ann.Rev.Immunol., 42:233).Described already by injection mycobacterium heat shock protein(HSP) in the susceptible rat, bring out adjuvant arthritis model (Van Eden etc., (1988) Nature, 331:171).By give described thyroglobulin in mouse, bring out thyroiditis (Maron etc., (1980) J.Exp.Med., 152:1115).The natural generation of insulin-dependent diabetes (IDDM) perhaps can be as Kanasawa etc., (1984) Diabetologia, and 27:113 is described to be brought out in some mouse species.EAE in mouse and the rat is as the MS model of philtrum.In this model, bring out demyelinating disease (referring to Paterson (1986) Textbookof Immuopathology, editors such as Mischer, Grune and Stratton, New York, 179-213 page or leaf by giving myelin basic protein; McFarlin etc., (1973) Science, 179:478; With Satoh etc., (1987) J.Immunol., 138:179).
In general, part of the present invention will use with pharmaceutically suitable carrier with the form of purifying.Usually, these carriers comprise water-based or contain the solution of alcohol/water, emulsion or suspension that wherein any all comprises salt solution and/or buffer medium.The outer carrier of enteron aisle comprises sodium chloride solution, Ringer glucose, glucose and sodium-chlor and lactic acid Ringer.In the time of need in suspension, keeping polypeptide complex, can from thickening material such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and alginates, select suitable physiology can accept adjuvant.
Intravenous vehicles comprises fluid and nutritional supplement and electrolyte supplements, for example based on the carrier of Ringer glucose.Also can there be sanitas and other compound, for example biocide, antioxidant, sequestrant and rare gas element (Mack, (1982) Remington ' sPharmaceutical Sciences, the 16th edition).
Part of the present invention can be used as the composition that gives separately and uses, or with other material coupling.These materials can comprise various immunotherapy medicaments, for example S-Neoral, methotrexate, Zorubicin or cis-platinum, and immunotoxin.Pharmaceutical composition can comprise various cytotoxic agents or other material together with part of the present invention and even have " mixture " of not homospecific ligand combination of the present invention (part that for example uses different target antigens or epi-position to select), and no matter whether they mix before giving.
The approach that gives of medicinal compositions of the present invention can be that persons skilled in the art are usually known to give in the approach any.With regard to treatment (including but not limited to immunotherapy), can give any patient with its selected part of the present invention according to standard technique.Can be any suitable mode, comprise parenteral, intravenously, intramuscular, intraperitoneal, transdermal, through pulmonary route, or this external when suitable by directly inculcating with conduit.Dosage that gives and frequency give, avoid when depending on patient's age, sex and physical appearance, other medicines and other the clinical parameter that will consider.
As what the technician recognized, give the result that approach and/or mode depend on needs.In certain embodiments, available protection compound exempts from the preparing carriers active compound of snap-out release, and for example controlled release preparation comprises implant, percutaneous plaster and microencapsulation transfer system.The single domain antibody construction is suitable for fully being mixed with and prolongs the preparation that discharges, and partly cause is that the mole number of its small size one every dosage can be significantly higher than for example dosage of complete big or small antibody.Can use biodegradable, biocompatible polymer, for example ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen protein, poe and poly(lactic acid).Include in the composition by the material (for example monostearate and gelatin) that will postpone to absorb, can produce the injectable composition that postpones absorption.The many methods that prepare this preparation have all obtained patent, perhaps in general are well known by persons skilled in the art.Referring to for example Sustained and ControlledRelease Drug Delivery Systems, J.R.Robinson edits, Marcel Dekker, Inc., New York, 1978.Be applicable to sustained release or prolong other method that discharges peptide material (single immunoglobulin variable structural domain polypeptide for example disclosed herein) and be described in for example United States Patent (USP) the 6th, 306,406 and 6,346, No. 274, and for example being described in U.S. Patent application US20020182254 and US20020051808, these documents all are attached to herein by reference.
But part freeze-drying described herein stores, and rebuilds with suitable carriers before use.Shown that this technology is effective to the routine immunization sphaeroprotein, can use freeze drying technology known in the art and reconstruction technique.One skilled in the art will recognize that freeze-drying and reconstruction can cause the antibody activity loss (for example with regard to the routine immunization sphaeroprotein, IgM antibody often has the loss of activity bigger than IgG antibody) of indefinite degree, in order to compensate, have to raise usage level.
The composition that can contain part of the present invention or its mixture is used for preventative and/or therapeutic treatment.In some therapeutic is used, but be enough to realize suppressing, checking, regulate, kill and wound or the amount of some other detect parameters is defined as " treatment effective dose " to small part to selected cell mass.Obtain the general state that the required amount of this dosage depends on severity of disease and patient's autoimmunization system, but in general between 0.005-5.0mg part (for example antibody, acceptor (for example TXi Baoshouti) or its conjugated protein)/kg body weight, more generally use the dosage of 0.05-2.0mg/kg/ agent.For prophylactic application, can also contain the composition of part of the present invention or its mixture with dosage similar or that reduce a little.
If the treatment of using composition described herein to implement make one or more symptoms with respect to this symptom that exists before the treatment or with respect to this sx in the individuality of not using this combination treatment (for example reach at least 10% or the clinical evaluation grade at least one point), think that then this treatment is " effectively ".Symptom will obviously change with target disease or obstacle, but can be detected by common clinicist or technician.The level of one or more biochemical index that for example can be by monitoring of diseases or obstacle is (for example with the level of the enzyme or the metabolite of disease-related, infected cell count etc.), monitoring physical manifestations (inflammation for example, tumour size etc.) or clinical evaluation standard by generally acknowledging, for example expand disabled state scoring (being used for multiple sclerosis), Irvine inflammatory bowel questionnaire (is estimated and intestinal function, physical symptom, social function, 32 evaluation and tests one of the quality of life that emotional status is relevant are kept the score by 32 to 224, quality of life is preferably represented in higher keeping the score), rheumatoid arthritis quality of life standard or other generally acknowledged clinical evaluation standard known in the art detect this symptom.Disease or obstacle symptom continue (for example 1 day or more than, preferably longer) alleviate and reach at least 10% or reach one or more points in the given clinical grading, be indicated as " effectively " and treat.Equally, if the prevention of using composition as herein described to implement makes the outbreak of one or more symptoms or seriousness postpone, alleviate or eliminate with respect to the symptom in the similar individuality of not using combination treatment (human or animal's model), then this prevention is " effectively ".
Can under prevention and treatment background, utilize the composition that contains part of the present invention or its mixture, to help change, deactivation, to kill and wound or remove the selected target cell group in the Mammals.In addition, selected peptide library described herein can exsomatize or external use, is used for selective killing, minimizing or effectively takes out the target cell group by heterogencity cell harvesting thing on the contrary.Mammalian can exsomatize and part (for example antibody, cell surface receptor or its conjugated protein) combination, kills and wounds unwanted cells thus, or opposite, and it by taking out in the blood, is used for turning back to Mammals according to standard technique.
2: the purposes that strengthens the dual specific part of the present invention of half life
Dual specific part according to the inventive method can be used for interior therapeutic and preventive use, in-vivo diagnostic purposes etc.
Comprise according to the treatment of the dual specific part of the present invention preparation and preventive use part of the present invention is accepted Mammals, for example the people.Bi-specific antibody of the present invention comprises at least a specificity that is strengthened molecule half life; One or more other specificitys can be at target molecule.For example, dual specific IgG can be 4 kinds of epitope specificities, and wherein a specific specificity strengthens molecule at half life.Dual specific can allow antibody with big avidity in conjunction with poly antigen.Bi-specific antibody can allow two kinds of antigen cross-linkings, for example when raising cytotoxic T cell, with killing and wounding of mediation tumor cell line.
The pure substantially part of 90-95% homogeneity or its are conjugated protein at least preferably to give Mammals, dAb monomer for example, and 98-99% or higher homogeneity most preferably are used for pharmaceutical usage, are man-hour Mammals especially.In case purifying or be purified to the homogeneity that needs partly, diagnosticability or therapeutic ground (comprising the ground that exsomatizes) use part, or use part (Lefkovite and Pernis in exploitation with when implementing experimental technique, immunofluorescence dyeing etc., (1979 and 1981) Immunological Methods, I and II volume, Academic Press, NY).
Part of the present invention can be used for prevention usually, suppresses or treatment inflammatory states, abnormal allergy, cancer, bacterium or virus infection and autoimmune disease (it includes but not limited to type i diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous, Crohn disease and myasthenia gravis).
Can obtain animal model system, it can be used for screening the dual specific part and avoids validity aspect ill or the treatment disease in protection.The method of checking system lupus erythematosus (SLE) is (Knight etc., (1978) J.Exp.Med., 147:1653 known in the art in the susceptible mouse; Reinersten etc., (1978) New Eng.J.Med., 299:515).By the solubility AchR albumen with another species induce an illness in the SJL/J female mice, check myasthenia gravis (MG) (Lindstrom etc., (1988) Adv.Immurzol., 42:233).By injection II collagen type in the susceptible mouse species, bring out sacroiliitis (Stuart etc., (1984) Ann.Rev.Immunol., 42:233).Described already by injection mycobacterium heat shock protein(HSP) in the susceptible rat, bring out adjuvant arthritis model (Van Eden etc., (1988) Nature, 331:171).By give described thyroglobulin in mouse, bring out thyroiditis (Maron etc., (1980) J.Exp.Med., 152:1115).The natural generation of insulin-dependent diabetes (IDDM) perhaps can be at for example Kanasawa etc., and (1984) Diabetologia brings out in described some mouse species of 27:113.EAR in mouse and the rat is as the MS model of philtrum.In this model, bring out demyelinating disease (referring to Paterson (1986) Textbook of Immuopathology, editors such as Mischer, Grune and Stratton, NewYork, 179-213 page or leaf by giving myelin basic protein; McFarlin etc., (1973) Science, 179:478; With Satoh etc., (1987) J.Immunol., 138:179).
Dual specific part of the present invention and dAb monomer can be in conjunction with the born of the same parents' external targets (for example clathrin) that participates in endocytosis, and these targets can make the dual specific part by endocytosis, can allow to be transmitted into environment in the born of the same parents in conjunction with the another kind of specificity of target in the born of the same parents.This strategy need have the dual specific part of the physical property that can make its reservation function in cell.Perhaps, if final objective intracellular region chamber just in oxidation, then good folding part may need not to be no disulfide linkage.
In general, dual specific part of the present invention will use with pharmaceutically suitable carrier with the form of purifying.Usually, these carriers comprise water-based or contain the solution of alcohol/water, emulsion or suspension that wherein any includes salt solution and/or buffer medium.The parenteral carrier comprises sodium chloride solution, Ringer glucose, glucose and sodium-chlor and lactic acid Ringer.Keep polypeptide complex if desired in suspension, can be by selecting suitable physiology can accept adjuvant in thickening material such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and the alginates.Intravenous vehicles comprises fluid and nutritional supplement and electrolyte supplements, for example based on the carrier of Ringer glucose.Also can there be sanitas and other compound, for example biocide, antioxidant, sequestrant and rare gas element (Mack, (1982) Remington ' sPharmaceutical Sciences, the 16th edition).
Part of the present invention can be used as the composition that gives separately and uses, or with other material coupling.These materials can comprise various immunotherapy medicaments, for example S-Neoral, methotrexate, Zorubicin or cis-platinum, and immunotoxin.Pharmaceutical composition can comprise various cytotoxic agents or other material " mixture " together with part of the present invention.
The approach that gives of medicinal compositions of the present invention can be that persons skilled in the art are usually known to give in the approach any.With regard to treatment (including but not limited to immunotherapy), can give any patient with part of the present invention according to standard technique.Can be any suitable mode, comprise parenteral, intravenously, intramuscular, intraperitoneal, transdermal, through pulmonary route or when suitable by directly inculcating with conduit.Dosage that gives and frequency give when depending on patient's age, sex and physical appearance, other medicines, taboo and clinical other parameter that will consider.
But part freeze-drying of the present invention stores, and rebuilds with suitable carriers before use.Show that this technology is effective to the routine immunization sphaeroprotein, can use freeze drying technology known in the art and reconstruction technique.One skilled in the art will recognize that freeze-drying and reconstruction can cause the antibody activity loss (for example with regard to the routine immunization sphaeroprotein, IgM antibody often has the loss of activity bigger than IgG antibody) of indefinite degree, in order to compensate, have to raise usage level.
The composition that can contain part of the present invention or its mixture is used for preventative and/or therapeutic treatment.In some therapeutic is used, but be enough to realize suppressing, checking, regulate, kill and wound or the amount of some other detect parameters is defined as " treatment effective dose " to small part to selected cell mass.Obtain the general state that the required amount of this dosage depends on severity of disease and patient's autoimmunization system, but in general between 0.005-5.0mg part/kg body weight, more generally use the dosage of 0.05-2.0mg/kg/ agent.For prophylactic application, can also contain the composition of part of the present invention or its mixture with dosage similar or that reduce a little.
Can under prevention and treatment background, utilize the composition that contains part of the present invention, to help change, deactivation, to kill and wound or remove the selected target cell group in the Mammals.
In addition, selected peptide library described herein can exsomatize or external selective killing, minimizing or on the contrary by effectively taking out the target cell group in the heterogencity cell harvesting thing.Mammalian can exsomatize and part (for example antibody, cell surface receptor or its conjugated protein) combination, kills and wounds unwanted cells thus, or opposite, and it by taking out in the blood, is used for turning back to Mammals according to standard technique.Only, in following examples, further described the present invention for illustration purpose.For the purpose to the dAb name, humanTNF-used herein is called as TAR1, and human TNF alpha acceptor 1 (p55 acceptor) is called as TAR2.
3. the treatment of rheumatoid arthritis
In a preferred embodiment, part described herein can be used for treating rheumatoid arthritis.
In one aspect, the invention provides the method for treatment rheumatoid arthritis, it comprises and uses one or more single domain antibody polypeptide constructions, and wherein one or more construction antagonism human TNF alphas and acceptor combines.The present invention includes the single domain antibody polypeptide construction that contains one or more antagonism human TNF alphas and receptors bind and the composition of dual specific part, in the dual specific part, one specific specificity of part is the single domain antibody at TNF α, and second specific specificity is the single domain antibody at VEGF or HSA.The present invention further comprises the dual specific part, and wherein a specific specificity of part is at VEGF, and second specific specificity is at HSA.
In one embodiment, the invention provides the method for treatment rheumatoid arthritis, it comprises the composition that contains one or more single domain antibody polypeptide constructions, wherein one or more construction antagonism human TNF alphas and acceptor combines, and/or when giving the mouse of Tg197 transgenic mice arthritis model, prevent the sacroiliitis increase of keeping the score, and/or in the L929 cytotoxicity experiment and TNF-α.Specifically, the method of treatment of arthritis comprises the composition that contains one or more single domain antibody polypeptide constructions, combining of one or more construction antagonism human TNF alphas and acceptor wherein wherein gives Tg197 transgenic mice composition and prevents the sacroiliitis increase of keeping the score.
A) receptors bind experiment
The part of treatment rheumatoid arthritis can disturb combining of TNF-α and TNF-α acceptor.Acceptor can be isolating (being generally membrane-bound) acceptor, perhaps it can be the acceptor that is present on the cell in external or body.
Detection TNF-α receptors bind and part have been described in the following Example 6 to this bonded interferential experiment.These experiments comprise ELISA, and (embodiment 6, chapters and sections 1.3.1), (embodiment 6 in the BIAcore analysis, chapters and sections 1.3.2) and use isolating (or membrane-bound) acceptor (embodiment 6, chapters and sections 1.3.3) and be expressed in the biological chemistry receptors bind experiment of the lip-deep acceptor of culturing cell (embodiment 6, chapters and sections 1.3.3).
Term used herein " antagonism (acceptor) combination " is showed and is decided ability and the effect that the antibody polypeptides construction disturbs TNF-α (or VEGF or other factor) and connection receptors bind.Use that experiment detects antagonistic action in one or more external, cellular types as described herein or the body.Therefore, acceptor can be isolating, membrane-bound or is present on the cell surface.If detected combination has statistics to descend significantly when not having construction when construction exists, then construction interference or antagonism and connection acceptor (for example TNFR1, TNFR2, VEGFR1, VEGFR2's) combines.Perhaps, if detected combination has at least 10% decline when it does not exist when construction exists, then construction disturbs combination.
B) L929 cytotoxicity experiment
The part that is used for rheumatoid arthritis treatment can disturb the cytotoxic effect of TNF-α at the L929 cytotoxicity experiment.This experiment is described in the chapters and sections 1.3.3 of embodiment 6, and it is based on Evans etc., and 2000, the experiment that Molecular Biotechnology 15:243-248 describes.Be used for the treatment of the anti-TNF-alpha ligands of rheumatoid arthritis can be in this cell experiment in and the activity of TNF-α.
Term used herein " neutralization " when being used in reference to antibody as herein described or dAb polypeptide, but refer to that polypeptide disturbs the detection of active or the function of target antigen., polypeptide reaches at least 50% if but reducing the detection of active of target antigen or function, preferably at least 60%, 70%, 80%, 90%, 95% or more than, until and comprise that 100% suppresses (promptly not having detectable target antigen effect or function), then polypeptide " neutralization " polypeptide.Therefore, when target is TNF-α, can uses standard L929 cell killing as herein described experiment or suppress HUVEC and go up ability (this ability detects TNF-α inductive cell activation) that TNF-α inducibility ELAM-1 expresses to estimate neutralization active by detecting anti-TNF-α polypeptide construction.
Antibody polypeptides disturbs other experiment of the receptor-binding activity of TNF-α to comprise HeLa IL-8 experiment, and it also is described in the chapters and sections 1.3.3 of embodiment 6.
C) experiment in the body
Can use Tg197 transgenic mice arthritis model to estimate the effectiveness of anti-TNF-alpha ligands described herein.The Tg197 mouse is the transgenic animal of people TNF-sphaeroprotein heterozygous genes, heterozygote develops into age such as chronic, the carrying out property polyarthritis (Keffer etc. with histologic characteristics identical with rheumatoid arthritis in 4-7 week, 1991, EMBO J.10:4025-4031).Can keep the score to the sacroiliitis phenotype by estimating joint mobilization and arthroncus.X-ray imaging that can be by the joint and by the keep the score sacroiliitis phenotype in joint of the histopathological analysis of knee and ankle/pawl arthrodesis section.
Following enforcement experimental therapy is to estimate the effectiveness of given antibody polypeptides construction.
1) be test antibody polypeptide construction to arthritic prevention, following treatment animal:
A) heterozygosis Tg197 mouse is divided into 10 male and female animal groups with equal number.Treatment is when 3 ages in week, and intraperitoneal gives the PBS solution of antibody polypeptides weekly, or gives independent PBS in control animal;
B) claim mouse weight weekly;
C) according to the keep the score sacroiliitis macroscopic view phenotype symptom of mouse of following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
Should implement research best, so that individual score is blind to test group.The preferred antibody pass through mechanism that is used for this experiment is the IP injection.But experiment can be adopted subcutaneous injection, IV injection (for example through the tail vein), intramuscularly or oral, suction or topical administration.
Be lower than (according to the statistics significant quantity) only the keeping the score of control group of carrier if the average sacroiliitis of treatment group is kept the score, it is effective then treating in the Tg197 model system.If average sacroiliitis is kept the score than only low 0.5 unit of control animal, 1.0 units, 1.5 units or 2 units of carrier at least at least at least at least, then treatment also is considered to effective.Perhaps, if average sacroiliitis is kept the score and remained on or be low to moderate 0-0.25 in whole treatment plan process, then treatment is effective.
If the average sacroiliitis in the treatment group is kept the score in experimentation, increase, but when the start delay of this increase when only the contrast of carrier is compared, it is effective then treating in the Tg197 model.If the average sacroiliitis of treatment group is kept the score and increased when only the contrast of carrier is compared 0.5 week of start delay, at least 1 week, at least 1.5 weeks, at least 2 week or 3 weeks of surpassing, then treatment also is counted as effectively.
As substituting that macroscopical phenotype is kept the score, the different interval in can therapeutic process is ankle/pawl and knee joint fixedly, and uses following system that it is carried out histopathological analysis: 0=and do not have detectable pathology; 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs; 2=pannus and fibrous tissue form and the focus subchondral bone corrodes; 4=joint cartilage widely destroys and bone erosion.Be lower than (according to the statistics significant quantity) only control group of carrier if the average tissue pathology are kept the score, then treatment is considered to effective.If the average tissue pathology are kept the score than low 0.5 unit of control group, 1.0 units, 1.5 units, 2.0 units, 2.5 units, 3.0 units or 3.5 units of carrier only at least at least at least at least at least at least at least, then treatment also is considered to effective.Perhaps, if the average tissue pathology are kept the score and remained on or be low to moderate 0-0.5 in whole treatment plan process, then treatment is effective.
2) be test antibody polypeptide construction (anti-TNF-α, anti-VEGF etc.) to existing arthritic effect, can be as mentioned above the Tg197 animal be implemented experiment, just 6 age in week begin treatment, this time is the time that animal has remarkable sacroiliitis phenotype.Keep the score and Validity Analysis also as mentioned above.Anti-TNF-α dAb construction as herein described can stop or reverse existing arthritic progress in one or more described model systems.
The methods of treatment of any form includes the anti-TNF-α (anti-TNF-α dAb for example as herein described) of monomer, dimer or other polymer form, the anti-VEGF of monomer, dimer or other polymer form (anti-VEGF dAb for example as herein described, also comprise the anti-VEGF dAb of Camelidae), the anti-VEGF of anti-TNF/ of dual specific form carries anti-HSA, PEG or other half life and revises list or dual specific construction partly.In addition, anti-VEGF composition described herein can give with other anti-TNF composition (for example sharp former times monoclonal antibody (Remicade) of etanercept (Enbrel), D2E7 (Humira) and English) combination.Can use for example validity of cell culture and this combination therapy of body inner model system evaluation described herein.
Other sacroiliitis animal model of generally acknowledging comprises the sacroiliitis (CIA) that collagen protein brings out, and it is by for example Horsfall etc., 1997, the sacroiliitis that J.of Immunol.159:5687 describes and pristane brings out, it is by for example Stasluk etc., 1997, Immunol.90:81 describes.The experiment of anti-VEGF polypeptide construction validity:
A) vegf receptor 2 is in conjunction with experiment
This method has been described vegf receptor in conjunction with experiment, and it is used to detect solubility domain antibodies (dAb) and stops VEGF 165Ability in conjunction with vegf receptor 2.
VEGF is specificity mitogen and intravital effective angiogenesis factor of external endotheliocyte, and this albumen is high level expression in various types of tumours.It is the glycoprotein of 45kDa, as homodimer activity is arranged.Up to now, 5 kinds of different isotypes that produce by variable mRNA montage had been described already.Isotype VEGF wherein 121And VEGF 165Be the abundantest.
VEGF mainly is subjected to two types receptor tyrosine kinase (RTK)-VEGF R1 (FIt-1) and the adjusting of VEGF R2 (KDR/Flk-1) to the specific effect of endotheliocyte.But, VEGF R2 mediation VEGF activity as if, even if for example mitogenesis, chemotaxis and induce morphological change are two kinds of acceptors experience phosphorylation in conjunction with VEGF time all.
Use recombinant human VEGF R2/Fc mosaic in this experiment, it comprises the people VEGF R2 ectodomain that merges to human IgG1's Fc district.Say that simply on elisa plate, closure plate then is to prevent non-specific binding with receptor capture.Add VEGF then 165With the proteic mixture of dAb, rinsing plate, use biotinylation VEGF antibody and the anti-biotin antibodies of puting together HRP to detect VEGF 165The bonded acceptor.Use the colorimetric substrates color plate, and read the OD of 450nm.If dAb sealing VEGF combines with acceptor, then do not have color and can detect.
Following enforcement experiment.With every hole 100 μ l recombinant human VEGF R2/Fc (R﹠amp; D Systems, catalog number (Cat.No.): 0.5 μ g/ml carbonate buffer solution 357-KD-050) spends the night in 4 ℃ and wraps by 96 hole Nunc Maxisorp brassboards.With 0.05%Tween/PBS rinsing hole 3 times, and with PBS rinsing 3 times.The PBS solution that adds the 2%BSA in 200 μ l/ holes is with closure plate, with plate in the room temperature incubation minimum 1 hour.
Rinsing hole (as mentioned above), the purifying dAb albumen with 50 μ l/ holes joins each hole then, and the 6ng/ml diluent (final concentration 3ng/ml) with 50 μ l VEGF joins each hole then, and plate (was used for the supernatant liquor experiment in 2 hours in the room temperature incubation; 80 μ l supernatant liquors are added to each hole, add the 15ng/ml VEGF of 20 μ l then).
Should comprise following contrast: 0ng/ml VEGF (only diluent); 3ng/ml VEGF (R﹠amp; D Systems, catalog number (Cat.No.): 293-VE-050); 3ng/ml VEGF contains the anti-VEGF neutralizing antibody of 0.1 μ g/ml (R﹠amp; D Systems, catalog number (Cat.No.): MAB293).
Rinsing plate (as mentioned above) adds 100 μ l biotinylation VEGF antibody (R﹠amp then; DSystems, catalog number (Cat.No.): 0.5 μ g/ml diluent BAF293), and in room temperature incubation 2 hours.
Rinsing hole (as mentioned above) adds the anti-biotin antibodies (diluent of dilution in 1: 5000 that 100 μ l put together HRP then; Stratech, catalog number (Cat.No.): 200-032-096), then in room temperature incubation 1 hour.
Any residual Tween-20 is guaranteed to have removed in rinsing hole (as mentioned above), with the background in the restriction superoxide experiment subsequently, helps to prevent to test the bubble in the plate hole, and bubble will produce inaccurate OD reading.
100 μ l SureBlue 1-Compent TMB MicroWell superoxide enzyme solution are added to each hole, place room temperature to reach 20 minutes plate.Because mark conjugate and substrate reactions in conjunction with HRP show navy blue solvability product.By adding 100 μ l 1M hydrochloric acid termination reactions (blueness will become yellow).Should add in sour 30 minutes, read the plate OD of 450nm with 96 orifice plate readers.The amount of OD450nm and bonded streptavidin-HRP conjugate is proportional.
As follows by the expected results that contrast obtains: 0ng/ml VEGF should obtain＜low signal of 0.15OD; 3ng/ml VEGF should obtain＞signal of 0.5OD; Should produce with the 3ng/ml VEGF of 0.1 μ g/ml neutralizing antibody preincubation＜signal of 0.2OD.
B) vegf receptor 1 is in conjunction with experiment
This experiment detects VEGF 165With combining and this interactional ability of dAb blocking-up of VEGF R1.
This paper uses recombinant human VEGF R1/Fc mosaic, and it comprises and merges to human IgG 1The people VEGF R1 ectodomain in Fc district.On elisa plate, closure plate then is to prevent non-specific binding with receptor capture.Add VEGF then 165With the proteic mixture of dAb, rinsing plate, use biotinylation VEGF antibody and the anti-biotin antibodies of puting together HRP to detect VEGF 165The bonded acceptor.Use the colorimetric substrates color plate, and read the OD of 450nm.If the dAb blocking VEGF combines with acceptor, then there is not color to manifest.
Following enforcement experiment.With every hole 100 μ l recombinant human VEGF Rl/Fc (R﹠amp; D Systems, catalog number (Cat.No.): 0.1 μ g/ml carbonate buffer solution 321-FL-050) spends the night in 4 ℃ and wraps by 96 hole Nunc Maxisorp brassboards.With 0.05%Tween/PBS rinsing hole 3 times, and with PBS rinsing 3 times.
The PBS solution that adds the 2%BSA in 200 μ l/ holes is with closure plate, with plate in the room temperature incubation minimum 1 hour.
Rinsing hole (as mentioned above), the purifying dAb albumen with 50 μ l/ holes joins each hole then.1ng/ml diluent (final concentration 500pg/ml) with 50 μ l VEGF joins each hole then, and (supernatant liquor was tested in 1 hour in the room temperature incubation with plate; 80 μ l supernatant liquors are added to each hole, add the VEGF of 20 μ l 2.5ng/ml then).
Should comprise following contrast: 0ng/ml VEGF (only diluent); 500pg/ml VEGF; With 500pg/ml VEGF, contain the anti-VEGF neutralizing antibody of 1 μ g/ml (R﹠amp; D Systems, catalog number (Cat.No.): MAB293).
Rinsing plate (as mentioned above) adds the 50ng/ml diluent of 100 μ l biotinylation VEGF antibody then, and in room temperature incubation 1 hour.
Rinsing hole (as mentioned above) adds the anti-biotin antibodies (1: 5000 dilution diluent) that 100 μ l put together HRP then, then in room temperature incubation 1 hour.
Rinsing plate (as mentioned above) guarantees to have removed any residual Tween-20, with the background in the restriction peroxidase experiment subsequently, helps to prevent to test the bubble in the plate hole, and bubble will produce inaccurate OD reading.
100 μ l SureBlue 1-Compent TMB Micro Well superoxide enzyme solution are added to each hole, place room temperature to reach 20 minutes plate.Because mark conjugate and substrate reactions in conjunction with HRP show navy blue solvability product.By adding 100 μ l 1M hydrochloric acid termination reactions (blueness will become yellow).Should add in sour 30 minutes, read the plate OD of 450nm with 96 orifice plate readers.The amount of OD450nm and bonded streptavidin-HRP conjugate is proportional.
The expected results that obtains by contrast: 0ng/ml VEGF should obtain＜low signal of 0.15OD; 500pg/ml VEGF should obtain＞signal of 0.8OD; Should produce with the 500pg/ml VEGF of 1 μ g/ml neutralizing antibody preincubation＜signal of 0.3OD.
C) be used for the active cellular type experiment of VEGF:
This Bioexperiment detects the ability of antibody polypeptides (for example dAb) and other inhibitor neutralize VEGF inductive HUVE cell proliferation.The VEGF of HUVE cell in 96 orifice plates and pre-equilibration and dAb albumen incubation 72 hours will be seeded in.Use the cell survival dyestuff to detect cell count then.
Following enforcement experiment.By the 175cm that does not converge 2Flask trypsinized HUVE cell.The sucking-off substratum cleans cell with 5ml trypsinase, then with cell and 2ml trypsinase in room temperature incubation 5 minutes.Generally, gently cell is removed by drag by knocking to setting about.Then the 8ml inducing culture is joined in the flask, suction of cells is to separate any cell of trooping.Use trypan blue staining counting survivaling cell.
Rotation sedimentation cell cleans 2 times with inducing culture, and rotation sedimentation cell is at each back sucking-off substratum that cleans.The last time after the sucking-off, with cell dilution to 10 5Cell/ml (using inducing culture), and go in 96 orifice plates (10,000 cells/well) with 100 μ l/ hole platings.With plate in 37 ℃ of incubations more than 2 hours, to allow cell adhesion.
With 60 μ l dAb albumen with contain 40ng/ml VEGF 165The 60 μ l inducing cultures of (final concentration 10ng/ml) join in 96 orifice plates at the bottom of the V-type, use diaphragm seal.Then with the dAb/VEGF mixture in 37 ℃ of incubation 0.5-1 hours.
The dAb/VEGF plate by taking out in the incubator, is added 100 μ l solution (final volume 200 μ l) to each hole of the plate that contains HUVEC.This plate is played back in 37 ℃ of incubators then, places for some time of at least 72 hours.
Control wells comprises: contain cell but do not contain the hole of VEGF; Contain the positive control of cell, neutrality VEGF antibody and the hole of VEGF; The control wells that only contains cell and VEGF.
Estimate cell viabilities by adding 20 μ l/ hole Celltiter96 reagent, with plate in 37 ℃ of incubation 2-4 hours, until brown occurring.By adding 10% (w/v) SDS termination reaction in 20 μ l/ holes.Use the Wallac microplate to read the absorbancy of 490nm then.
With the absorbancy of the control wells of no VEGF by all deduction in other value.Absorbancy and cell count are proportional.Contain the control wells that contrasts VEGF antibody and also should show minimum cell proliferation.The hole that only contains VEGF should have maximum cell proliferation.
D) be used for experiment in the active body of VEGF:
Also can in the Tg197 of arthritis disease transgene mouse model, check the effectiveness of anti-VEGF polypeptide construction (monomer, polymer or dual specific or polyspecific).Dosage regimen and keep the score basic identical with the description of anti-TNF-α polypeptide construction.
4.Crohn sick treatment
Anti-TNF-α polypeptide described herein is used in philtrum treatment Crohn disease.In one embodiment, the invention provides treatment and wherein relate to the Crohn disease of TNF-α or the method for other inflammatory bowel (IBD).This method comprises the composition that contains one or more single domain antibody polypeptide constructions, combining of one or more construction antagonism human TNF alphas and acceptor wherein, and/or when giving the Tnf of IBD Δ AREPrevent increase acute or that chronic inflammatory bowel disease is kept the score during the mouse of transgene mouse model, and/or in the L929 cytotoxicity experiment and TNF-α.Specifically, treatment Crohn method sick or other inflammatory bowel comprises the composition that contains one or more single domain antibody polypeptide constructions, and wherein combining of one or more construction antagonism human TNF alphas and acceptor wherein gives Tnf Δ AREThe transgenic mice composition prevents acute or increase that chronic inflammatory bowel disease is kept the score or make the minimizing of keeping the score.
The Tnf of Crohn disease Δ ARETransgene mouse model is at first by Kontoyiannis etc., and 1999, Immunity 10:387-398 describes; In addition referring to Kontoyiannis etc., 2002, J.Exp.Med.196:1563-1574.These mouse have by the deletion mutantion of target in 3 ' AU enrichment element (ARE) of TNF-α mRNA.AU-enrichment element participates in keeping low mRNA stability, and its destruction causes mouse TNF-α to cross expression in these animals.Animal occurs beginning to occur the sick obviously similar IBD phenotype to Crohn between age in week to 4-8.Standard weave's pathological characteristics comprises inflammation under fine hair rust and the mucous membrane, have general PMN/ scavenger cell and lymphocyte transudate, continue to develop into the inflammation of the whole layer of mottling, polymerization of lymph sample and immature granuloma (Kontoyiannis etc. occur, 2002, ibid).The sacroiliitis phenotype also appears in these animals, therefore also can be used for estimating individually the effectiveness of anti-TNF-α treatment to RA.
In the time will estimating treatment, with initially IP dosage begin treatment when 3 ages in week for example weekly of given antibody polypeptides construction in the effectiveness of prevention among the IBD.Those skilled in the art can select the dosing interval of higher or lower frequency according to the result of preliminary research.Then can be according to Kontoyiannis etc., 2002, the inflammatory bowel of ibid described standard class monitoring animal.According to of the paraffin embedding small intestine section of following grade: followingly estimate acute and chronic inflammation-acute inflammation 0=0-1 polymorphonuclear cell (PMN)/hpf (PMN/hpf) that keeps the score individually with minimum 8 high power fields (hpf) with blind mode Histological evaluation ileum; 2-10PMN/hpf in the 1=mucous membrane; 11-20PMN/hpf in the 2=mucous membrane; 21-30PMN/hpf or extend to 11-20PMN/hpf under the sheath muscularis mucosae in the 3=mucous membrane; And 30PMN/hpf or extend under the sheath muscularis mucosae＞20PMN/hpf in 4=＞mucous membrane.Chronic inflammatory diseases 0-10 monokaryon lymphocyte (ML) in the 0=mucous membrane/hpf (ML/hpf) that keeps the score; 11-20ML/hpf in the 1=mucous membrane; 21-30ML/hpf or extend to 11-20ML/hpf under the sheath muscularis mucosae in the 2=mucous membrane; 31-40ML/hpf or extend to 21-30ML/hpf or FH under the sheath muscularis mucosae in the 3=mucous membrane; And 40ML/hpf or extend under the sheath muscularis mucosae＞30ML/hpf or FH in 4=＞mucous membrane.Acute inflammation by each mouse of addition or the chronic inflammatory diseases total disease of calculating every mouse of keeping the score is kept the score.
For estimating the effect of treatment to existing disease, treatment can during age in week, be kept the score in an identical manner at 6-8.
Be lower than (according to the statistics significant quantity) only control group of carrier if the average tissue pathology disease in the treatment animal is kept the score, then treatment is considered to effective.If the average tissue pathology are kept the score than low 0.5 unit of control group, 1.0 units, 1.5 units, 2.0 units, 2.5 units, 3.0 units or 3.5 units of carrier only at least at least at least at least at least at least at least, then treatment also is considered to effective.Perhaps, if the average tissue pathology are kept the score and remained on or be low to moderate 0-0.5 in whole treatment plan process, then treatment is effective.
Other IBD model comprises chronic colitis DSS (glucosamine sulphate sodium) model in the BALB/c mouse for example.The DSS model is at first by Okayasu etc., and 1990, Gastroenterology 98:694-702 describes, by Kojouharoff etc., 1997, Clin Exp.Immunol.107:353-358 improvement (referring to the WO 2004/041862 that specifies the U.S., it is attached to herein by reference in addition).In the recovery circulation at interval of treatment in 7 days and 12 days no DSS, by in its drinking-water, giving the BALB/c mouse that DSS handles heavy 21-22g, to bring out chronic colitis with 5%w/v.The 4th decubation can extend to 12-21 days, showing chronic inflammatory state, rather than with short recovery mimic acute state.After last decubation, give the antibody polypeptides treatment, anti-TNF-α polypeptide for example as herein described.Recommend during beginning to give once weekly, but those skilled in the art can adjust (when especially for example estimating the formulation with different half lifes modification parts) as required.In the middle of treatment, put to death animal at set intervals, dissect small intestine, as described herein or as Kojouharoff etc., 1997, ibid, and described evaluation of tissue pathology are kept the score.
Other animal model of inflammatory bowel comprises that rectum instils 2,4,6-trinitro-benzene-sulfonic acid (TNBS; Neurath etc., 1995, the method that J.Exp.Med.182:1281 describes; In addition referring to United States Patent (USP) 6,764,838, it is attached to herein by reference) chronic intestinal inflammations of bringing out.Can use above-mentioned identical standard to carry out histopathology keeps the score.
Contrast with other anti-TNF-alpha substance:
Herein disclosed is the anti-TNF-α dAb construction of effective treatment RA, Crohn disease and the alpha mediated disease of other TNF-.In one aspect, the validity of anti-TNF-α dAb construction is more than or equal to the validity that is selected from following material: etanercept (ENBREL), the sharp former times monoclonal antibody (REMICADE) of English and D2E7 (HUMIRA; Referring to United States Patent (USP) the 6th, 090, No. 382, it is attached to herein by reference).
Be connected to recombinant forms soluble TNF R (the p75) (sTBFR (p75): Fc of human IgG1's Fc part, ENBREL, Immunex) clinical experiment shows, it produces significantly the RA disease activity and the (Moreland etc. that descend fast, 1997, N.Eng.J.Med., 337:141-147).In addition, the preliminary secure data of the paediatrics clinical experiment of sTNFR (p75): Fc shows, general well tolerable this medicine (Garrison etc. of juvenile rheumatoid arthritis (JRA) patient, 1998, Am.College of Rheumatology meeting, on November 9th, 1998, summary 584).
Dimerization fusion rotein as implied above, that ENBREL is made up of the outer ligand binding moiety of born of the same parents of people 75kDa (p75) TNFR (GenBank accession number P20333) of the Fc part that is connected to the human IgG1.The Fc component of ENBREL contains CH2 structural domain, CH3 structural domain and hinge area, but does not contain the CH1 structural domain of IgG1.ENBREL produces in Chinese hamster ovary (CHO) mammalian cell expression system.It is made up of 934 amino acid, has apparent molecular weight (Smith etc., 1990, the Science 248:1019-1023 of about 150kDa; Mohler etc., 1993, J.Immunol.151:1548-1561; United States Patent (USP) the 5th, 395, No. 760 (ImmunexCorporation, Seattle, Wash.; It is attached to herein by reference); United States Patent (USP) the 5th, 605, No. 690 (Immunex Corporation, Seattle, Wash.; It is attached to herein by reference).
(English monoclonal antibody of sharp former times, REMICADE Centocor) have shown clinical efficacy (Elliott etc., 1993, the Arthritis Rheum.36:1681-1690 of treatment RA and give when not having methotrexate the monoclonal antibody of anti-TNF-α; Elliott etc., 1994, Lancet 344:1105-1110).These data show that in the 4th, 12 and 26 weeks, Paulus 20% and 50% standard significantly reduce.Intravenously gives this treatment, and the anti-TNF monoclonal antibody disappears from circulation in the bimestrial time.As if the effectiveness time length of repeated doses descend.The patient can produce the antibody at anti-TNF antibodies, and it has limited the validity and the time length (Kavanaugh etc., 1998, Rheum.Dis.Clin.North Am.24:593-614) of this treatment.The combination that gives methotrexate and Ying Li former times monoclonal antibody helps to prevent the appearance (Maini etc., 1998, Arthritis Rheum.41:1552-1563) of anti-English antibody mab of sharp former times.English monoclonal antibody of sharp former times has also shown the clinical efficacy (Baert etc., 1999, Gastroenterology 116:22-28) of treatment inflammatory bowel Crohn disease.
As the argumentation in background section, English monoclonal antibody of sharp former times is the chimeric monoclonal IgG antibody of carrier IgG4 constant region and mouse variable region.English monoclonal antibody polypeptide of sharp former times is described in United States Patent (USP) the 5th, 698, and 195 and 5,656, No. 272, it is attached to herein by reference.
Be the effectiveness of the anti-TNF-α composition of comparison these or other, people only need to use anti-TNF-α dAb construction together with prior composition, carry out experiment in one or more receptors bind as indicated above, cellular type or the body.Therefore, this method is differentiated anti-TNF-α dAb construction, and the inhibiting validity of TNF-α that it shows in one or more experiments is equal to or greater than the validity of (with the statistics significant quantity) Comparative composition.The example of this construction is provided among the embodiment and has shown and equated or the analytical procedure of high-efficiency more.
Embodiment
(the selection of the dual specific scFv antibody (K8) of β-gal) of embodiment 1. AHS's albumin (HSA) and beta-galactosidase enzymes
This embodiment has illustrated the method for the bi-specific antibody for preparing anti-β-gal and HAS, wherein select to be connected to kind of a VK variable domains storehouse that is (simulation) VH structural domain, select to be connected to kind of a VH variable domains storehouse that is (simulation) VK structural domain according to combination to HSA according to combination to β-gal.Make up selected variable VH HSA and V then κThe B-gal structural domain is selected antibody according to the combination to β-gal and HSA.HSA is the albumen of the increase half life that exists in the human blood.
In this experiment, use 4 kinds of people's phage antibody libraries.
The library is V for a kind κ/ DVT V H8.46 * 10 7
The library is V for 2 kinds κ/ NNK V H9.64 * 10 7
The library is VH/DVT V for 3 kinds κ1.47 * 10 8
The library is V for 4 kinds H/ NNK V κ1.45 * 10 8
All libraries are all based on V H(V3-23/DP47 and J H4b) and V κ(O12/O2/DPK9 and J κ1) single people's framework, side chain diversity mix in the complementary determining region (CDR2 and CDR3).
Library 1 and library 2 contain simulation V κSequence, and V HSequence in site H50, H52, H52a, H53, H55, H56, H58, H95, H97 and H98 variation (respectively by DVT or NNK coding) (Fig. 1).Library 3 and library 4 contain simulation V HSequence, and V κSequence in site L50, L53, L91, L92, L93, L94 and L96 variation (respectively by DVT or NNK coding) (Fig. 1).The library is a phagemid pIT2/ScFv form (Fig. 2), and with proteic in conjunction with preselected to metal ligand A albumen and L, makes that the major part clone in the non-selected library has function.More than shown in the size in the library size after corresponding to preliminary election.Mix library 1 and library 2, then to antigen selection, to produce single V H/ simulation V κLibrary 3 and library 4 are mixed, to form single V in the library κ/ simulation V HThe library.
Use V κ/ simulation V HThe library is carried out 3 to β-gal and is taken turns selection, uses V H/ simulation V κThe library is carried out 3 to HSA and is taken turns selection.For β-gal, phage titre is by 1.1 * 10 of the first round 6Rise to 2.0 * 10 of third round 8For HSA, phage titre is by 2 * 10 of the first round 4Rise to 1.4 * 10 of third round 9As Griffith etc., (1993) are described to be selected, and the exception part is to use KM13 helper phage (it contains the pIII albumen that has proteolytic enzyme cutting site between D2 and D3 structural domain), and with the tryptic PBS eluant solution of 1mg/ml phage.The trypsinase cutting that adds is derived from the pIII albumen (but do not cut from phagemid albumen) of helper phage, and by scFv-phage syzygy (Fig. 2) in c-myc mark cutting elution of bound, the further enrichment of functional scFv of phage expression and corresponding decline (Kristensen and Winter, the Folding ﹠amp of background are provided thus; Design 3:321-328, on July 9th, 1998).Use the HSA of 100 μ g/ml concentration or the immune pipe of β-gal bag quilt to select.
For the check combination, screen 24 bacterium colonies that each selects third round by mono-clonal phage E LISA.As Harrison etc., Methods Enzymol.1996; The described production phage particle of 267:83-109.With the PBS solution of 100 μ l HSA of 10 μ g/ml concentration or β-gal in 4 ℃ of bags that spend the night by 96 hole elisa plates.According to standard ELISA method (Hoogenboom etc., 1991), detect the bonded phage with anti-M13-HRP conjugate.The clone who selects produces ELISA signal greater than 1.0 with 50 μ l supernatant liquors.
Then, use QIAprep Spin Miniprep test kit (Qiagen), by the V that selects at HSA H/ simulation V κLibrary and the V that selects at β-gal κ/ simulation V HThe library prepares the DNA goods.
For obtaining most of diversity, select preparation DNA goods by each third round, then each antigenic DNA goods are lumped together.Then use SalI/NotI in 37 ℃ of digested overnight DNA goods.After the gel-purified fragment, connect the V that selects at β-gal κ/ simulation V HThe V in library κChain replaces the V that selects at HSA H/ simulation V κThe simulation V in library κChain sets up 3.3 * 10 9Clone's library.
Select this library (HSA/ β-gal selects) at HSA (first round) and β-gal (second takes turns) then, perhaps select this library (β-gal/HSA selects) at β-gal (first round) and HSA (second takes turns).Select as mentioned above.In each case, second take turns selection after, by combining of 48 clones of mono-clonal phage E LISA (as mentioned above) and solubility scFv fragment ELISA check and HSA and β-gal.As Harrison etc., (1996) described production soluble antibody fragment, and implementation criteria ELISA method (Hoogenboom etc., (1991) NucleicAcids Res., 19:4133), the exception part is to use 2%Tween/PBS as the sealing damping fluid, and detects bonded scFv with L albumen-HRP.2 clones (K8 and K10) that 3 clones (E4, E5 and E8) that HSA/ β-gal selects and β-gal/HSA selects can be in conjunction with two kinds of antigens.Use primer LMB3 and pHENseq, these clones' of pcr amplification scFv, as Ignatovich etc., (1999) J.Mol.Biol.1999Nov.26; 294 (2): the described order-checking of 457-65.Sequential analysis discloses, and all clones are identical.Therefore, only select the clone (K8) of a coding bi-specific antibody further to study (Fig. 3).
The feature of embodiment 2.K8 antibodies characteristic
At first, utilize mono-clonal phage E LISA to characterize the binding characteristic of K8 antibody.Arise from 4 ℃ of bags that spend the night by 96 orifice plates with 100 μ l HSA and β-gal together with 10 μ g/ml concentration PBS solution one of alkaline phosphatase (APS), bovine serum albumin (BSA), peanut agglutinin, N,O-Diacetylmuramidase and cytochrome c (to detect cross reactivity).As Harrison etc., (1996) described phagemid with KM13 rescue K8 clone directly detects the supernatant liquor (50 μ l) that contains phage.According to standard ELISA method (Hoogenboom etc., 1991), detect the bonded phage with anti-M13-HRP conjugate.In conjunction with HSA and β-gal, absorbance signal is greater than 1.0 (Fig. 4) when finding that dual specific K8 antibody is on being illustrated in phage surface.Also observe strong combination the (Fig. 4) with BSA.
Because HSA and BSA be 76% homology on amino acid levels, so the albumen of these two kinds of structurally associateds of K8 antibody recognition is not unexpected.Do not detect and other proteic cross reactivity (Fig. 4).
Secondly, check the binding characteristic of K8 antibody with solubility scFvELISA.As Harrison etc., (1996) described IPTG of utilization induces the segmental production of solubility scFv.For measuring the expression level of K8 scFV, as Harlow and Lane, Antibodies:a Laboratory Manual, (1988) Cold Spring Harbor is described, uses the supernatant liquor purification of soluble antibody fragment of A Protein S epharose post by the 50ml inductor.Detect OD then 280, as Sambrook etc., (1989) described calculating protein concentration.In supernatant liquor, produce K8 scFv with 19mg/l.
Use the K8 antibody fragment of concentration known to carry out solubility scFv ELISA then.With the A albumen bag of the 1 μ g/ml concentration of HSA, the BSA of the 10 μ g/ml concentration of 100 μ l and β-gal and 100 μ l by 96 orifice plates.Apply the serial dilution of 50 μ l K8 scFv, detect the bonded antibody fragment with L albumen-HRP.ELISA result has confirmed the dual specific character (Fig. 5) of K8 antibody.
For confirming the V of combining with β-gal by K8 scFv antibody κStructural domain decision and with the V of combining of HSA/BSA by K8 scFv antibody HThe structural domain decision digests by downcutting V on the K8 scFv DNA by SalI/NotI κStructural domain, and be connected to contain the simulation V HIn the pIT2 carrier of the SalI/NotI digestion of structural domain (Fig. 1 and 2).The clone K8V that is obtained by solubility scFv elisa assay κ/ simulation V HIn conjunction with feature.As Harrison etc., (1996) are described induces the segmental production of solubility scFv with IPTG, directly detects the supernatant liquor (50 μ l) that contains scFv.As enforcement solubility scFv ELISA as described in the embodiment 1, detect bonded scFv with L albumen-HRP.ELISA result discloses, and this clone still can be in conjunction with β-gal, and is eliminated (Fig. 6) with combining of BSA.
Single V of embodiment 3. antigen As and B HSingle V of domain antibodies and antigens c and D κThe selection of domain antibodies
This embodiment has described a kind of method, and this method prepares single V of antigen A and B by in the initial monoclonal antibody body variable domains storehouse that does not have to select under the situation of complementary variable domains conjugated antigen A, B, C and D HSingle V of domain antibodies and antigens c and D κDomain antibodies.
(referring to embodiment 5, PCT/GB 02/003014) as discussed previously carries out selection and the sign in conjunction with the clone.Select 4 clones further to study:
The anti-AV of VH1- H
The anti-BV of VH2- H
The anti-CV of VK1- κ
The anti-DV of VK2- κ
Can be similar to described mode and use aforesaid method among the embodiment 1-3, contain V with production HStructural domain (is V HPart) combination and V LStructural domain (V L-V LPart) Zu He dimer molecule.
Embodiment 4. sets up and characterizes dual specific ScFv antibody (VK1/VK2 of the VH1/VH2 of antigen A and B and antigens c and D)
This embodiment shows, can pass through V κ and the V that combination is selected each antigen in anti-ScFv carrier HDual specific ScFv antibody (VK1/VK2 of the VH1/VH2 of antigen A and B and antigens c and D) is set up in the single structure territory.For setting up bi-specific antibody VH1/VH2, downcut VH1 single structure territory by NcoI/XhoI digestion by varied texture domain vector 1 (Fig. 7) is middle, and connect in the varied texture domain vector 2 (Fig. 7) of NcoI/XhoI digestion, to set up VH1/ varied texture domain vector 2.Use primer by varied texture domain vector 1 pcr amplification VH2 single structure territory, to introduce the NotI restriction site at 5 ' terminal introducing SalI restriction site with at 3 ' end.Digest the PCR product with SalI/NotI then, and connect into the VH1/ varied texture domain vector 2 of SalI/NotI digestion, to set up VH1/VH2/ varied texture domain vector 2.
Set up VK1/VK2/ varied texture domain vector 2 in a similar manner.As previously mentioned (referring to embodiment 6, PCT/GB02/003014) VH1/VH2 ScFv that is produced with solubility ScFv ELISA check and the dual specific character of VK1/VK2 ScFv.Implement competitive ELISA as previously mentioned (referring to embodiment 8, PCT/GB02/003014).
Possible result:
-VH1/VH2 ScFv is conjugated antigen A and B simultaneously.
-VK1/VK2 ScFv is conjugated antigen C and D simultaneously.
-VH1/VH2 ScFv is in conjunction with being emulative (when conjugated antigen A, VH1/VH2ScFv can not conjugated antigen B).
-VK1/VK2 ScFv is in conjunction with being emulative (when conjugated antigen C, VK1/VK2ScFv can not conjugated antigen D).
Embodiment 5. dual specific VH1/VH2 Fab and the structure of VK1/VK2 Fab and the analysis of binding characteristic thereof
For setting up VH1/VH2 Fab, VH1 single structure territory is connected in the CH carrier (Fig. 8) of NcoI/XhoI digestion, to set up VH1/CH, VH2 single structure territory is connected in the CK carrier (Fig. 9) of SalI/NotI digestion, to set up VH2/CK.It is as discussed previously that (referring to embodiment 8, PCT/GB02/003014), use derives from the plasmid DNA cotransformation competence Bacillus coli cells of VH1/CH and VH2/CK.
Then, (referring to embodiment 8, PCT/GB02/003014), utilize IPTG to induce to contain the clone of VH1/CH and VH2/CK plasmid, as previously mentioned to produce solubility VH1/VH2Fab.
Produce VK1/VK2 Fab in a similar manner.
As previously mentioned (referring to embodiment 8, the PCT/GB02/003014) binding characteristic of the Fab that is produced by competitive ELISA check.
Possible result:
-VH1/VH2 Fab is conjugated antigen A and B simultaneously.
-VK1/VK2 Fab is conjugated antigen C and D simultaneously.
-VH1/VH2 Fab is in conjunction with being emulative (when conjugated antigen A, VH1/VH2 Fab can not conjugated antigen B).
-VK1/VK2 Fab is in conjunction with being emulative (when conjugated antigen C, VK1/VK2 Fab can not conjugated antigen D).
Embodiment 6. chelating dAb dimers
General introduction
Use the elasticity peptide linker to set up the VH and the VK homodimer of dAb-joint-dAb type.The carrier of dAb-joint-dAb type of setting up contains glycine-Serine joint: the 3U:(Gly of different lengths 4Ser) 3, 5U:(Gly 4Ser) 5, 7U:(Gly 4Ser) 7Use the guiding dAb of joint: TAR1-5 (VK), 5TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK) upstream and set up the dimer library corresponding to the library of second dAb behind the joint.Use this method to select new dimerization dAb.By ELISA and BIAcore research and with cell neutralization and the definite dimerization of receptors bind experiment to the effect of antigen bonded.These two dimerization of TAR1-5 and TAR1-27 all promotes producing significantly in conjunction with affinity and neutralization levels.
1.0 method
1.1 the library produces
1.1.1 carrier
Design pEDA3U, pEDA5U and pEDA7U carrier are to import the different joint length compatible with dAb-joint-dAb type.For pEDA3U, use slow cycle of annealing (95 ℃-5 minutes, 80 ℃-10 minutes, 70 ℃-15 minutes, 56 ℃-15 minutes, 42 ℃ until using), annealing has the oligonucleotide joint of 73 base pairs of justice and antisense in the damping fluid that contains 0.1M NaCl, 10mM Tris-HCl pH7.4, and uses XhoI and NotI restriction site clone.
Joint comprises 3 (Gly 4Ser) (SEQ ID NO:7) unit and the stuffer district (flow process 1) between SalI and NotI cloning site.Select the possibility of monomer dAb in order to reduce phage display, design stuffer district is to comprise 3 terminator codons, SacI restriction site and frame phase shift mutation, when not having second dAb this district is placed outside the frame.For pEDA5U and 7U, owing to the joint length that needs, design the overlapping oligomerization joint of each carrier, annealing uses Klenow to extend.Use suitable enzyme purification and digestion fragment then, use XhoI and NotI restriction site clone afterwards.
1.1.2 library preparation
Use NcoI and XhoI restriction site clone joint upstream corresponding to the terminal V gene of the N-of guiding dAb.The VH gene has existing compatible site, but cloning the VK gene need import suitable restriction site.These can be by 2: 1 mixtures of use SuperTaq (HTBiotechnology Ltd) and pfu turbo (Stratagene), the PCR primer that use is revised in the pcr amplification in 30 cycles (VK-DLIBF:5 ' cggccatggcgtcaacggacat-3 '; VKXhoIR:5 ' atgtgcgctcgagcgtttgattt-3 ') realizes.This has destroyed the SalI site of adjacency simultaneously in the 5 ' terminal NcoI site that keeps, and in the 3 ' terminal XhoI site that imports.5 guiding dAb:TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH), TAR2-6 (VK) and TAR2-7 (VK) are cloned in each of 3 dimer carriers.All constructions are all verified by sequential analysis.
Guiding dAb:TAR1-5 (VK), TAR1-27 (VK), TAR2-5 (VH) or TAR2-6 (VK) have been cloned afterwards in joint upstream in each carrier (pEDA3U, 5U and 7U), in the library of joint rear clone corresponding to second dAb.For realizing this target, the anti-humanTNF-'s who is selected by the first round VK library (is about 1 * 10 the 1st after taking turns 6Density) VH of (at TAR1-5 or TAR1-27 during for guiding dAb) or anti-people p55 TNF acceptor or VK library (all are about 1 * 10 the 1st after taking turns 5Density) (when TAR2-5 or TAR2-6 are respectively guiding dAb) the complementary dAb of phage pcr amplification library of reclaiming.For the VK library, use 2: 1 mixtures of SuperTaq and pfu turbo, in the pcr amplification in 30 cycles, use primer to carry out pcr amplification.Use primer PCR amplification VH library, so that introduce the SalI restriction site at 5 of gene ' end.With suitable restriction enzyme digestion dAb library PCR, and use the SalI/NotI restriction site to connect into the joint downstream of corresponding carrier, electroporation is gone in the competence TG1 cell of prepared fresh.
The titre that each library obtains is as follows:
TAR1-5：pEDA3U＝4×10 8，pEDA5U＝8×10 7，pEDA7U＝1×10 8
TAR1-27：pEDA3U＝6.2×10 8，pEDA5U＝1×10 8，pEDA7U＝1×10 9
TAR2h-5：pEDA3U＝4×10 7，pEDA5U＝2×10 8，pEDA7U＝8×10 7
TAR2h-6：pEDA3U＝7.4×10 8，pEDA5U＝1.2×10 8，pEDA7U＝2.2×10 8
1.2 select
1.2.1TNF-α
Use the passive human TNF alpha that is coated on the immune pipe to select.Say simply, spend the night bag by the immunity pipe with the required antigen of 1-4ml.Then with PBS rinsing immunity pipe 3 times, sealed 1-2 hour with the PBS solution of 2% milk powder, use the PBS rinsing again 3 times.With the PBS solution dilution phage solution of 2% milk powder, and in room temperature incubation 2 hours.Use the PBS rinsing tube then, with 1mg/ml trypsinase-PBS wash-out bacteriophage.For TAR1-5 dimer library, 3 kinds of selection strategies have been studied.The 1st take turns be chosen in the human TNF alpha bag that uses 1 μ g/ml or 20 μ g/ml by and immune pipe with PBS 0.1%Tween rinsing 20 times in carry out.With the phage-infect TG1 cell of wash-out, and measure titre (Marks etc. for example, J Mol Biol.1991 December 5; 222 (3): 581-97, Richmann etc., Biochemistry.1993 August 31; 32 (34): 8848-55).
The titre that reclaims is:
pEDA3U＝2.8×10 7(1μg/ml?TNF)，1.5×10 8(20μg/ml?TNF)，
pEDA5U＝1.8×10 7(1μg/ml?TNF)，1.6×10 8(20μg/ml?TNF)，
pEDA7U＝8×10 6(1μg/ml?TNF)，7×10 7(20μg/ml?TNF)。
Use 3 kinds of different methods to carry out the 2nd and take turns selection.
1. in immune pipe, the rinsing 20 times and the incubation that spends the night, post rinse is 10 times afterwards.
2. in immune pipe, rinsing 20 times, afterwards in the rinsing damping fluid that contains 1 μ g/ml TNF-α in room temperature incubation 1 hour, post rinse 10 times.
3. use 33pmol biotinylation humanTNF-on the streptavidin pearl, to select (Henderikx etc., 2002, Selection of antibodies against biotinylatedantigens.Antibody Phage Display:Methods and protocols, O ' Brien and Atkin edit, Humana Press).The 2nd single clone who takes turns selection is chosen in 96 orifice plates, prepare thick supernatant liquor goods with 2ml 96 well plate format.
The 1st takes turns human TNF alpha immunity pipe bag by concentration The 2nd takes turns system of selection 1 The 2nd takes turns system of selection 2 The 2nd takes turns system of selection 3
pEDA3U 1μg/ml 1×10 9 1.8×10 9 2.4×10 10
pEDA3U 20μg/ml 6×10 9 1.8×10 10 8.5×10 10
pEDA5U 1μg/ml 9×10 8 1.4×10 9 2.8×10 10
pEDA5U 20μg/ml 9.5×10 9 8.5×10 9 2.8×10 10
pEDA7U 1μg/ml 7.8×10 8 1.6×10 8 4×10 10
pEDA7U 20μg/ml 1×10 10 8×10 9 1.5×10 10
For TAR1-27, select with following modification as previously mentioned.The 1st take turns be chosen in the human TNF alpha bag that uses 1 μ g/ml or 20 μ g/ml by and immune pipe with PBS 0.1%Tween rinsing 20 times in carry out.The 2nd takes turns to be chosen in and uses 20 rinsings and the incubation that spends the night carries out in the immune pipe of post rinse 20 times afterwards.The 2nd single clone who takes turns selection is chosen in 96 orifice plates, prepare thick supernatant liquor goods with 2ml 96 well plate format.
The TAR1-27 titre is as follows:
Human TNF alpha immunity pipe bag is by concentration The 1st takes turns The 2nd takes turns
pEDA3U 1μg/ml 4×10 9 6×10 9
pEDA3U 20μg/ml 5×10 9 4.4×10 10
pEDA5U 1μg/m1 1.5×10 10 1.9×10 10
pEDA5U 20μg/ml 3.4×10 9 3.5×10 10
pEDA7U 1μg/ml 2.6×10 9 5×10 9
pEDA7U 20μg/ml 7×10 9 1.4×10 10
1.2.2TNF acceptor 1 (p55 acceptor; TAR2)
Only the TAR2h-5 library is selected as previously mentioned.Carry out 3 and take turns selection in immune pipe, described immunity pipe uses 1 μ g/ml people p55 TNF acceptor or 10 μ g/ml people p55 TNF acceptors, and with PBS 0.1%Tween rinsing 20 times, the incubation that spends the night, post rinse is 20 times afterwards.Take turns with the 3rd single clone who takes turns selection the 2nd and to choose in 96 orifice plates, prepare thick supernatant liquor goods with 2ml 96 well plate format.
The TAR2h-5 titre is as follows:
The 1st takes turns people p55 TNF acceptor immunity pipe bag by concentration The 1st takes turns The 2nd takes turns The 3rd takes turns
pEDA3U 1μg/ml 2.4×10 6 1.2×10 7 1.9×10 9
pEDA3U 10μg/ml 3.1×10 7 7×10 7 1×10 9
pEDA5U 1μg/ml 2.5×10 6 1.1×10 7 5.7×10 8
pEDA5U 10μg/ml 3.7×10 7 2.3×10 8 2.9×10 9
pEDA7U 1μg/ml 1.3×10 6 1.3×10 7 1.4×10 9
pEDA7U 10μg/ml 1.6×10 7 1.9×10 7 3×10 10
1.3 screening
Under suitable situation, take turns or the 3rd single clone who takes turns selection by selection the 2nd in each 3U, the 5U that obtain with the different choice method and the 7U library.To be cloned among the 2xTY that contains 100 μ g/ml penbritins and 1% glucose in 37 ℃ of incubated overnight.This culture of 1/100 dilution is inoculated among the 2xTY that 2ml contains 100 μ g/ml penbritins and 1% glucose with 2ml, 96 well plate format, and cultivated, be about 0.9 until OD600 in 37 ℃ of joltings.Then with 1mM IPTG in 30 ℃ of inducing culture things that spend the night.
By in the dull and stereotyped whizzer of Sorval in centrifugal 15 minutes clarified supernatant of 4000rpm.The supernatant liquor goods are used for initial screening.
1.3.1?ELISA
By the A/L protein ELISA or by antigen ELISA contrast dimerization recombinant protein and monomeric in conjunction with active.In brief, with antigen or A/L albumen in 4 ℃ of bags that spend the night by 96 orifice plates.With 0.05%Tween-PBS rinsing plate, and with 2%Tween-PBS sealing 2 hours.Sample is added in the entering plate, in room temperature incubation 1 hour.The rinsing plate, and with plate and second reagent in room temperature incubation 1 hour.The rinsing plate develops the color with tmb substrate.Use A/L albumen HRP or India-HRP as second reagent.For antigen ELISA, the antigen concentration of use is the PBS solution of 1 μ g/ml human TNF alpha and people TNF acceptor 1.Owing in most of the cases there is guiding dAb, so dimer produces positive ELISA signal-therefore leave the rate determination value with the BIAcore check.
1.3.2?BIAcore
TAR1-5 and TAR2h-5 clone are carried out the BIAcore analysis.For screening, with high-density (about 10,000RU) humanTNF-is coupled to the CM5 chip.
With the chip of 50 μ l human TNF alphas (50 μ g/ml) to be coupled in acetate buffer pH5.5 in 5 μ l minutes.Because humanTNF-'s unstable, so after analysis, can not use standard method regeneration chip.Therefore, after analyzing each sample, use damping fluid rinsing chip 10 minutes.
For TAR1-5, screen the 2nd clone's supernatant liquor of taking turns selection by BIAcore.By filtering out 48 clones in each of the 3U, the 5U that use following system of selection acquisition and 7U library:
R1:1 μ g/ml humanTNF-immunity pipe, R2 1 μ g/ml humanTNF-immunity pipe, the rinsing of spending the night.
R1:20 μ g/ml humanTNF-immunity pipe, R2 20 μ g/ml humanTNF-s immunity pipe, the rinsing of spending the night.
R1:1 μ g/ml humanTNF-immunity pipe, R2 33pmol biotinylation humanTNF-pearl.
R1:20 μ g/ml humanTNF-immunity pipe, R2 33pmol biotinylation humanTNF-pearl.
For screening, with high-density (about 4,000RU) people p55 TNF acceptor is coupled to the CM5 chip.With the chip of 100 μ l people p55 TNF acceptors (10 μ g/ml) to be coupled in acetate buffer pH5.5 in 5 μ l/ minutes.Inspecting standard regeneration condition (glycine pH2 or pH3), but in each case antigen by be removed on the chip surface-therefore, the same with the situation of TNF-α, after analyzing each sample, with damping fluid rinsing chip 10 minutes.
For TAR2-5, screen clone's supernatant liquor that this takes turns selection.
Use following system of selection, by all filtering out 48 clones in each of 3U, 5U and 7U library:
R1:1 μ g/ml people p55 TNF acceptor immunity pipe, R2 1 μ g/ml people p55 TNF acceptor immunity pipe, the rinsing of spending the night.
R1:10 μ g/ml people p55 TNF acceptor immunity pipe, R2 10 μ g/ml people p55 TNF acceptors immunity pipe, the rinsing of spending the night.
1.3.3 acceptor and cell experiment
The neutralising capacity of following enforcement dimer in the acceptor experiment:
Receptors bind
Check anti-TNF dAb to suppress TNF and reorganization TNF acceptor 1 (p55) bonded ability.In brief, with Maxisorp plate and the anti-people Fc of 30mgml mouse monoclonal antibody (Zymed, San Francisco, USA) incubation that spends the night.With the phosphate-buffered saline that contains 0.05%Tween-20 (PBS) rinsing hole, the PBS solution with 1%BSA seals then, afterwards with 100ng/ml TNF acceptor 1Fc fusion rotein (R﹠amp; D Systems, Minneapolis, USA) incubation.Anti-TNFdAb is mixed with the TNF that is added to the rinsing hole with the 10ng/ml final concentration.With 0.2mg/ml biotinylation anti-TNF antibodies (HyCuIt biotechnology, Uben, Netherlands), after meet the streptavidin (AmershamBiosciences of horseradish peroxidase-labeled of dilution in 1: 500, UK), (USA) incubation detects the TNF combination for KPL, GaitherSburg with tmb substrate then.By adding the HCl termination reaction, read absorbancy in 450nm.Anti-TNFdAb activity causes TNF in conjunction with decline, therefore causes absorbancy than the only contrast increase of TNF.
The L929 cytotoxicity experiment
Also tested among the anti-TNF dAb and TNF to the ability of the fibroblastic cytotoxic activity of mouse L929 (Evans, T. (2000) Molecular Biotechnology 15,243-248).In brief, will be at the L929 cell of microtiter plate middle plateform inoculation and anti-TNFdAb, 100pg/ml TNF and 1mg/ml dactinomycin (Sigma, Poole, UK) incubation that spends the night.By with [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl p-methoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium _ (Promega, Madison USA) read the absorbance detection cell viability of 490nm behind the incubation.Anti-TNF dAb activity causes the TNF cytotoxicity to descend, and therefore causes absorbancy than the only contrast increase of TNF.
In initial screening, also in the acceptor experiment, use as mentioned above and analyze the supernatant liquor for preparing as BIAcore.Also use purifying protein in acceptor and cell experiment, to select dimeric further analysis.
HeLa IL-8 experiment
(method of employing derives from Akeson with TNF inductive IL-8 excretory ability in the HeLa cell to test anti-TNFR1 or anti-TNF alpha dAb, L. etc., (1996) Journal ofBiological Chemistry 271,30517-30523, it has described IL-1 inducing IL-8 in HUVEC; We investigate inducing of human TNF alpha at this, and we use the Hela cell to replace HUVEC clone).In brief, will be at the HeLa cell of microtiter plate middle plateform inoculation and dAb and the 300pg/ml TNF incubation that spends the night.By sucking-off supernatant liquor in the cell, utilize sandwich ELISA (R﹠amp behind the incubation; D Systems) detects IL-8 concentration.Anti-TNFR1 dAb activity causes being secreted into the only contrast decline of TNF of IL-8 ratio in the supernatant liquor.
In following experimentation, use the L929 experiment all the time; But, preferably use HeLa IL-8 experiment to detect anti-TNF acceptor 1 (p55) part; The mouse p55 that exists in the L929 experiment uses it and produces certain limitation.
1.4 sequential analysis
Verify the dimer order-checking that has target property in tangible BIAcore and the acceptor experiment screening.Sequence is specified in hereinafter sequence table, the drawings and Examples.
1.5 typing
1.5.1 TAR1-5-19 dimer
Reset shown have good in and the TAR1-5 dimer of characteristic, and in cell and acceptor experiment, analyze.Replace TAR1-5 guiding dAb with the sophisticated clone of avidity TAR1-5-19.For realizing this target, with TAR1-5 clone single dimer to outside, and with the TAR1-5-19 displacement by pcr amplification.In addition, also in 3U, 5U and 7U carrier, make up the TAR1-5-19 homodimer.By the N-terminal copy of pcr amplification gene, and clone as mentioned above, use existing SalI and NotI restriction site clone C-terminal gene fragment.
1.5.2 mutagenesis
By site-directed mutagenesis the amber terminator codon that exists among one of the terminal dAb of the C-of the TAR1-5 dimer centering dAb2 is sported glutamine.
1.5.3?Fab
The dimer that will contain TAR1-5 or TAR1-5-19 is reset in the Fab expression vector.Use SfiI and NotI restriction site that dAb is cloned into and contain in CK or the CH expression carrier, and check by sequential analysis.The CK support source is from pUC type amicillin resistance carrier, and the CH support source is from pACYC chlorampenicol resistant carrier.Express for Fab, dAb-CH and dAb-CK construction corotation are dissolved in the HB2151 cell, and in the 2xTY that contains 0.1% glucose, 100 μ g/ml penbritins and 10 μ g/ml paraxin, cultivate.
1.5.3 hinge area dimerization
The dAb dimerization that check forms through the halfcystine key.The engineered C-terminal district of the short sequence EPKSGDKTHTCPPCP (SEQ ID NO:11) of human IgG C1 hinge-amino acid with improved form at dAb.The oligomerization joint of synthetic as previously mentioned and this sequence of annealing coding.Use XhoI and NotI restriction site that joint is cloned in the pEDA carrier that contains TAR1-5-19.Dimerization original position in pericentral siphon takes place.
1.6 express and purifying
1.6.1 express
Prepare supernatant liquor with 2ml, 96 well plate format that are used for initial screening as previously mentioned.After initial screening process, further analyze selected dimer.In TOP10F ' or HB2151 cell, express the dimer construction as supernatant liquor.In brief, with the single bacterium colony that derives from fresh streak plate in the 2xTY that contains 100 μ g/ml penbritins and 1% glucose in 37 ℃ of incubated overnight.This culture of 1/100 dilution is inoculated among the 2xTY that contains 100 μ g/ml penbritins and 0.1% glucose, cultivated, be about 0.9 until OD600 in 37 ℃ of joltings.Then with 1mM IPTG in 30 ℃ of inducing culture things that spend the night.By centrifugal removal cell, with A or L albumen agarose purifying supernatant liquor.
In the HB2152 cell, express Fab and halfcystine hinge dimer as periplasm protein.
The overnight culture of 1/100 dilution is inoculated and contained among 0.1% glucose and the suitable antibiotic 2xTY, and cultivate, be about 0.9 until OD600 in 37 ℃ of joltings.Use 1mMIPTG in 30 ℃ of inducing culture things 3-4 hour then.By centrifugal collecting cell, pellet resuspended is prepared in the damping fluid (30mM Tris-HCl pH8.0,1mM EDTA, 20% sucrose) at pericentral siphon.Keep supernatant liquor in centrifugal back, with pellet resuspended at 5mM MgSO 4In.By centrifugal collection supernatant liquor, mix and purifying again.
1.6.2A/L protein purification
Check by L albumen agarose (Affitech, Norway) or A albumen agarose (Sigma, UK) top condition of the dimer protein of purifying.Use peristaltic pump to pass through wash-out or post wash-out eluted protein in batches.Check 3 kinds of damping fluids: 0.1M phosphate-citrate salts pH of buffer 2.6; 0.2M glycine pH 2.5; With 0.1M glycine pH 2.5.The optimal conditions of determining is to use 0.1M glycine pH 2.5 under the peristaltic pump condition, by 10 column volumes.Use 0.1M glycine pH 2.5 under the peristaltic pump condition, to carry out purifying by A albumen.
1.6.3 FPLC purifying
By the further purifying of the analysis carrying out of the FPLC in AKTA Explorer 100 systems (Amersham Biosciences Ltd).By the cation-exchange chromatography (1ml Resource S-AmershamBiosciences Ltd) with the 50mM acetate buffer pH4 wash-out of 0-1M NaCl gradient, fractional separation TAR1-5 and TAR1-5-19 dimer.By ion exchange chromatography (1mlResource Q Amersham Biosciences Ltd) purifying hinge dimer with 25mM Tris HCl pH 8.0 wash-outs of 0-1M NaCl gradient.By using the molecular exclusion chromatography of Superose 12 (Amersham Biosciences Ltd) post, move purifying Fab with 0.5ml/ minute flow velocity with the PBS that contains 0.05%Tween.Behind purifying, use Vivaspin 5K molecular weight thickener (Vivascience Ltd) concentrating sample that dams.
2.0 result
2.1 TAR1-5 dimer
Take turns 6 * 96 clones of sort out in the selection by the 2nd, it comprises all libraries and selection condition.Preparation supernatant liquor goods detect by antigen and L protein ELISA, BIAcore and acceptor experiment.In EUSA, the positive of differentiating each system of selection is in conjunction with the clone, and it is distributed between 3U, 5U and the 7U library.But, owing to always there is guiding dAb, thus utilize this method can not distinguish high and low affinity combination-therefore carry out the BIAcore analysis.
Using the 2ml supernatant liquor to carry out BIAcore analyzes.BIAcore analyzes announcement, and dimer Koff speed is compared monomer TAR1-5 very big lifting.Monomer Koff speed is 10 -1In the M scope, dimer Koff speed is 10 by contrast -3-10 -4In the M scope.Select 16 and seem to have the clone who leaves speed very slowly, these are cloned from 3U, 5U and 7U library, to its order-checking.In addition, analytically clear liquid in acceptor experiment in and humanTNF-'s ability.
6 guiding clones (following d1-d6) neutralization in these experiments is to its order-checking.The result shows that 3 the 2nd different dAb (dAb1, dAb2 and dAb3) are only arranged outside 6 clones that obtained, still, when observing the 2nd dAb more than once, it is connected with the joint of different lengths.
TAR1-5d1:3U joint the 2nd dAb=dAb1-1 μ g/ml Ag immunity pipe, the rinsing of spending the night.
TAR1-5d2:3U joint the 2nd dAb=dAb2-1 μ g/ml Ag immunity pipe, the rinsing of spending the night.
TAR1-5d3:5U joint the 2nd dAb=dAb2-1 μ g/ml Ag immunity pipe, the rinsing of spending the night.
TAR1-5d4:5U joint the 2nd dAb=dAb3-20 μ g/ml Ag immunity pipe, the rinsing of spending the night.
TAR1-5d5:5U joint the 2nd dAb=dAb1-20 μ g/ml Ag immunity pipe, the rinsing of spending the night.
TAR1-5d6:7U joint the 2nd dAb=dAb1-R1:1 μ g/ml Ag immunity pipe, the rinsing of spending the night, R2: pearl
Further 6 guiding of check are cloned.Produce albumen by pericentral siphon and supernatant liquor, with L albumen agarose purifying, and check in cell and acceptor experiment.Neutralization levels is variable (table 1).Determine the optimal conditions of protein Preparation.The albumen of being produced as supernatant liquor by the HB2151 cell obtains production peak (about 10mg/L culture).With supernatant liquor and L albumen agarose in room temperature incubation 2 hours or in 4 ℃ of incubations that spend the night.With PBS/NaCl rinsing pearl, and use peristaltic pump to be loaded on the FPLC post.With the PBS/NaCl rinsing pearl of 10 column volumes, with 0.1M glycine pH 2.5 wash-outs.In general, dimer protein is eluted after monomer.
By FPLC purifying TAR1-5d1-6 dimer.Obtain 3 kinds of materials by the FPLC purifying, differentiate by SDS PAGE.1 kind of material is corresponding to monomer, and 2 kinds of materials are corresponding to different big or small dimers in addition.Bigger one may come from and has the C-terminal mark in two kinds of materials.These albumen of check in the acceptor experiment.The data represented optimal result (Figure 11) that obtains by two dimer classifications that provides in the table 1.3 kind of the 2nd dAb (being dAb1, dAb2 and dAb3) clone that dimer is right is monomer, and by ELISA and cell and acceptor examination.Whole 3 kinds of dAb all by the antigen ELISA specificity in conjunction with TNF, not with plastics or BSA cross reaction.As for monomer, none dAb neutralization in cell or acceptor experiment.
2.1.2 TAR1-5-19 dimer
With the TAR1-5 among 6 guiding clones of TAR1-5-19 displacement.(table 2) except as otherwise noted, otherwise use the whole TAR1-5-19 dimers in total protein (only L protein purification) pair cell and the acceptor experiment to analyze.TAR1-5-19d4 and TAR1-5-19d3 have optimum N D in cell experiment 50(about 5nM), this is consistent with the acceptor experimental result, than TAR1-5-19 monomer (ND 50About 30nM) improvement is arranged.Although the TAR1-5 dimer of purifying obtains variable result in acceptor and cell experiment, the TAR1-5-19 dimer is more consistent.When in the protein purification process, using different elution buffers, demonstrate viability.Use 0.1M phosphate-citrate salts pH of buffer 2.6 or 0.2M glycine pH 2.5 wash-outs, although, make the idle substantially energy of albumen in most of the cases by having taken off all albumen in the L albumen agarose.
In fermentor tank, express TAR1-5-19d4, and on cationic exchange FPLC purifying, produce pure dimer fully.The same with TAR1-5d4, obtain 3 kinds of materials by the FPLC purifying, corresponding to monomer and two kinds of dimers.This dimer is carried out amino acid sequencing.In the acceptor experiment, check TAR1-5-19 monomer and TAR1-5-19d4, the monomer I C of acquisition then 50Be 30nM, dimeric IC 50Be 5nM.
The acceptor result of experiment of contrast TAR1-5-19 monomer, TAR1-5-19d4 and TAR1-5d4 is shown in Figure 10.
Preparation TAR1-5-19 homodimer is expressed, and is used the L protein purification in 3U, 5U and 7U carrier.In cell and acceptor experiment, check albumen, measure the IC that is produced 50(for the acceptor experiment) and ND 50(for cell experiment) (table 3, Figure 12).
2.2?Fab
In addition TAR1-5 is cloned into the Fab type with the TAR1-5-19 dimer, express, and with L albumen agarose purifying.With acceptor experimental evaluation Fab (table 4).The result shows, for TAR1-5-19 and TAR1-5 dimer these two, neutralization levels is similar to the original Gly that produces it 4Ser joint dimer.Express the TAR1-5-19Fab that TAR1-5-19 wherein shows simultaneously on CH and CK, the L protein purification, and use the acceptor experimental evaluation.The IC that obtains 50Be about 1nM.
2.3 TAR1-27 dimer
Take turns 3 * 96 clones of sort out in the selection by the 2nd, it comprises all libraries and selection condition.Preparation 2ml supernatant liquor goods are used for ELISA and Bioexperiment analysis.Antigen ELISA produces 71 positive colonies.The acceptor experiment of thick supernatant liquor produces 42 clones (TNF is in conjunction with 0-60%) with rejection characteristic.Under most of situation, rejection characteristic and strong ELISA signal correction.42 clones, wherein 39 the 2nd dAb sequences with uniqueness check order.Further analyze 12 dimers that produce the optimal inhibition characteristic.
With 12 neutrality clonal expressions is 200ml supernatant liquor goods, and on L albumen purifying.By L albumen and antigen ELISA, BIAcore with these clones of acceptor experimental evaluation.All obtain strong positive ELISA signal in all cases.BIAcore analyzes announcement, and all clones have combination fast and leave speed.Leaving speed has improvement than monomer TAR1-27, leaves speed (Koff is about 10 but TAR1-27 is dimeric -1-10 -2In the scope of M) (Koff is about 10 than the previous TAR1-5 dimer of checking -3-10 -4In the scope of M) faster.The dimeric stability of purifying is a problem, therefore in order to promote stability, comprises adding 5% glycerine, 0.5%Triton X100 or 0.5%NP40 (Sigma) when 2 kinds of TAR1-27 dimers of purifying (d2 and d16).Adding NP40 or Triton X100 promote about 2 times with the output of purified product.In the acceptor experiment, estimate two kinds of dimers.Under all purification conditions, the IC that TAR1-27d2 produces 50Be about 30nM.When not using the stablizer purifying, TAR1-27d16 does not demonstrate neutralizing effect, but produces the IC of about 50nM when purifying under stable condition 50Further analyze.
2.4 TAR1-5 dimer
Take turns 3 * 96 clones of sort out in the selection by the 2nd, it comprises all libraries and selection condition.Preparation 2m1 supernatant liquor goods are used for analyzing.Every block of plate is carried out A albumen and antigen ELISA.Identifying 30 by BIAcore has and well leaves speed (Koff is 10 -2-10 -3In the scope) the target clone.Order-checking is cloned, and identifies the dimer of 13 uniquenesses by sequential analysis.
Table 1:TAR1-5 dimer
Dimer Cell type Purifying Albumen sepn Elution requirement Acceptor/cell experiment
TAR1-5d1 HB2151 L albumen+FPLC Young waiter in a wineshop or an inn's aggressiveness type 0.1M glycine pH 2.5 The about 30nM of RA
TAR1-5d2 HB2151 L albumen+FPLC Young waiter in a wineshop or an inn's aggressiveness type 0.1M glycine pH 2.5 The about 50nM of RA
FPLC Type pH?2.5 M
TAR1-5d3 HB2151 L albumen+FPLC Big dimer type 0.1M glycine pH 2.5 The about 300nM of RA
TAR1-5d4 HB2151 L albumen+FPLC Young waiter in a wineshop or an inn's aggressiveness type 0.1M glycine pH 2.5 The about 3nM of RA
TAR1-5d5 HB2151 L albumen+FPLC Big dimer type 0.1M glycine pH 2.5 The about 200nM of RA
TAR1-5d6 HB2151 L albumen+FPLC Big dimer type 0.1M glycine pH 2.5 The about 100nM of RA
*Note: dimer 2 has identical the 2nd dAb (being called dAb2) with dimer 3, but has different joint length (d2=(Gly 4Ser) 3, d3=(Gly 4Ser) 3).DAb1 is the mating partner dAb of dimer 1,5 and 6.DAb3 is the mating partner dAb of dimer 4.None mating partner dAb neutralizes alone.Except as otherwise noted, otherwise all carry out the FPLC purifying by cationic exchange.In these experiments, determine each dimeric optimum dimer kind by the FPLC acquisition.
Table 2:TAR1-5-19 dimer
Dimer Cell type Purifying Albumen sepn Elution requirement Acceptor/cell experiment
TAR1-5-19d1 top10F′ L albumen Total protein 0.1M glycine pH 2.0 The about 15nM of RA
TAR1-5-19d2 (not having the codon of termination) top10F′ L albumen Total protein 0.1M glycine pH 2.0+0.05% NP40 The about 2nM of RA
TAR1-5-19d3 (not having the codon of termination) top10F′ L albumen Total protein 0.1M glycine pH 2.5+0.05% NP40 The about 8nM of RA
TAR1-5-19d4 top10F′ L albumen+FPLC FPLC purifying fraction 0.1M glycine pH 2.0 The about 12nM of the about 2nM 5 nM CA of RA
TAR1-5-19d5 top10F′ L albumen Total protein 0.1M glycine pH 2.0+NP40 The about 10nM of the about 8nM CA of RA
TAR1-5-19d6 top10F′ L albumen Total protein 0.1M glycine pH 2.0 The about 10nM of RA
Table 3:TAR1-5-19 homodimer
Dimer Cell type Purifying Albumen sepn Elution requirement Acceptor/cell experiment
Homodimer The about 30nM of nM CA
TAR1-5-19 5U homodimer HB2151 L albumen Total protein 0.1M glycine pH 2.5 The about 3nM of the about 2nM CA of RA
TAR1-5-19 7U homodimer HB2151 L albumen+FPLC The dimer fraction of FPLC purifying 0.1M glycine pH 2.5 The about 15nM of the about 10nM CA of RA
TAR1-5-19 cys hinge HB2151 Albumen Total protein 0.1M glycine pH 2.5 The about 2nM of RA
TAR1-5-19 CH/TAR1-5- 19CK HB2151 Albumen Total protein 0.1M glycine pH 2.5 The about 1nM of RA
Table 4:TAR1-5/TAR1-5-19Fab
Dimer Cell type Purifying Albumen sepn Elution requirement Acceptor/cell experiment
TAR1-5CH/ dAb1?CK HB2151 L albumen Total protein 0.1M Citrate trianion pH 2.5 The about 90nM of RA
TAR1-5CH/ dAb2?CK HB2151 L albumen Total protein 0.1M Citrate trianion pH 2.5 The about 60nM of the about 30nM CA of RA
dAb3CH/ TAR1-5CK HB2151 L albumen Total protein 0.1M Citrate trianion pH 2.6 The about 10nM of RA
TAR1-5-19CH/ dAb1?CK HB2151 L albumen Total protein 0.1M Citrate trianion pH 2.0 The about 6nM of RA
dAb1CH/ TAR1-5-19CK HB2151 L albumen 0.1M glycine pH 2.0 Myc/flag The about 6nM of RA
TAR1-5-19CH/ dAb2?CK HB2151 L albumen Total protein 0.1M glycinate pH 2.0 The about 12nM of the about 8nM CA of RA
TAR1-5-19CH/ dAb3?CK HB2151 L albumen Total protein 0.1M glycinate pH 2.0 The about 3nM of RA
The dimeric PCR construction of TAR1-5-19CYS
Referring to describing the trimerical embodiment 8 of dAb.The tripolymer method produces monomer, dimer and trimerical mixture.
Dimeric expression of TAR1-5-19CYS and purifying
Such as embodiment 8 general introduction, by being captured on the L albumen agarose, by culture supernatants purifying dimer.
The TAR1-5-19CYS monomer separates with TAR1-5-19CYS is dimeric
Before cationic exchange is separated, according to manufacturer's guide, use PD-10 post (Amersham Pharmacia), blended monomer/dimer sample buffering exchange is gone among the 50mM sodium citrate buffer solution pH 4.0.Then with sample on the sample to 1mL Resource S cationic exchange coloum (Amersham Pharmacia), this post has been used 50mM Trisodium Citrate pH 4.0 pre-equilibrations.Following salt gradient separating monomer and the dimer of use in 50mM Trisodium Citrate pH 4.0:
150-200mM sodium-chlor is by 15 column volumes
200-450mM sodium-chlor is by 10 column volumes
450-1000mM sodium-chlor is by 15 column volumes.
Use SDS-PAGE to differentiate and only contain dimeric fraction, merge then, 1M Tris pH by adding 1/5 volume 8.0 increases to 8 with pH.
External function is in conjunction with experiment: experiment of TNF acceptor and cell experiment
Use TNF acceptor and cell experiment to measure the affinity of dimer to people TNFcz.IC in the acceptor experiment 50Be about 0.3-0.8nM; ND in the cell experiment 50Be about 3-8nM.
The TAR1-5-19CYS dimeric forms that other is possible
PEG dimer and customization synthetic maleimide dimer
Nektar (Shearwater) provides a series of bismaleimides PEG[mPEG2-(MAL) 2 or mPEG-(MAL) 2], it allows to make monomer form dimer with the little joint of separating dAb, and the two all is connected to the PEG of 5-40kDa size.Show that 5kDamPEG-(MAL) 2 (i.e. [TAR1-5-19]-Cys-maleimide-PEG * 2, wherein maleimide links together) has the avidity that about 1-3nM leaves speed in the experiment of TNF acceptor in dimer.In addition, also can use TMEA (Tris[2 5-maleimide ethyl] amine) (Pierce Biotechnology) or other bifunctional linker to produce dimer.
Also might use to adopt 2,2 '-dithio two pyridines (Sigma Aldrich) and the monomeric chemical coupling method production of reduction disulfide linkage dimer.
C-terminal to dAb adds peptide linker or hinge
Can be between dAb and terminal cysteine residue an engineered little joint, it is (Gly 4Ser) n, n=1-10 wherein, for example 1,2,3,4,5,6 or 7, perhaps be immunoglobulin (Ig) (for example IgG hinge area), perhaps be peptide sequence (for example being selected from peptide sequence library at random) at random.Can use its preparation dimer as above general introduction then.
Embodiment 8.dAb trimerizing
General introduction
For the dAb trimerizing, need free cysteine at proteic C-terminal.In a single day cysteine residues is reduced the generation free sulfhydryl groups, just can be used for protein-specific is coupled to trimerizing maleimide amine molecule, for example TMEA (Tris[2-maleimide ethyl] amine).
The PCR of TAR1-5-19CYS makes up
Following oligonucleotide is used for specific PCR and has the SalI that is used to clone and the TAR1-5-19 in BamHI site, also is used to import C-terminal cysteine residue:
Sal?I
Trp?Ser?Ala?Ser?Thr?Asp*Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val
TGG?AGC?GCG?TCG?ACG?GAC?ATC?CAG?ATG?ACC?CAG?TCT?CCA?TCC?TCT?CTG?TCT?GCA?TCT?GTA
ACC?TCG?CGC?AGC?TGC?CTG?TAG?GTC?TAC?TGG?GTC?AGA?GGT?AGG?AGA?GAC?AGA?CGT?AGA?CAT
Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Asp?Ser?Tyr?Leu?His?Trp
GGA?GAC?CGT?GTC?ACC?ATC?ACT?TGC?CGG?GCA?AGT?CAG?AGC?ATT?GAT?AGT?TAT?TTA?CAT?TGG
CCT?CTG?GCA?CAG?TGG?TAG?TGA?ACG?GCC?CGT?TCA?GTC?TCG?TAA?CTA?TCA?ATA?AAT?GTA?ACC
Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Glu?Leu?Gln
TAC?CAG?CAG?CCA?CCA?GGG?AAA?GCC?CCT?AAG?CTC?CTG?ATC?TAT?AGT?GCA?TCC?GAG?TTG?CAA
ATG?GTC?GTC?TTT?GGT?CCC?TTT?CGG?GGA?TTC?GAG?GAC?TAG?ATA?TCA?CGT?AGG?CTC?AAC?GTT
Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile
AGT?GGG?GTC?CCA?TCA?CGT?TTC?AGT?GGC?AGT?GGA?TCT?GGG?ACA?GAT?TTC?ACT?CTC?ACC?ATC
TCA?CCC?CAG?GGT?AGT?GCA?AAG?TCA?CCG?TCA?CCT?AGA?CCC?TGT?CTA?AAG?TGA?GAG?TGG?TAG
Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Val?Trp?Arg?Pro
AGC?AGT?CTG?CAA?CCT?GAA?GAT?TTT?GCT?ACG?TAC?TAC?TGT?CAA?CAG?GTT?GTG?TGG?CGT?CCT
TCG?TCA?GAC?GTT?GGA?CTT?CTA?AAA?CGA?TGC?ATG?ATG?ACA?GTT?GTC?CAA?CAC?ACC?GCA?GGA
BamHI
Phe?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Cys?***?***?Gly?Ser?Gly
TTT?ACG?TTC?GGC?CAA?GGG?ACC?AAG?GTG?GAA?ATC?AAA?CGG?TGC?TAA?TAA?GGA?TCC?GGC
AAA?TGC?AAG?CCG?GTT?CCC?TGG?TTC?CAC?CTT?TAG?TTT?GCC?ACG?ATT?ATT?CCT?AGG?CCG
( *The starting point of TAR1-5-19CYS sequence; Aminoacid sequence=SEQ ID NO:12; Nucleotide sequence=SEQ ID NO:13)
Forward primer 5 '-TGGAGCGCGTCGACGGACATCCAGATGACCCAGTCTCCA-3 ' (SEQ ID NO:14)
Reverse primer 5 '-TTAGCAGCCGGATCCTTATTAGCACCGTTTGATTTCCAC-3 ' (SEQ ID NO:15)
Following preparation PCR reactant (50 μ L volume): 200 μ M dNTP, 0.4 the various primers of μ M, 5 μ L 10x PfuTurbo damping fluids (Stratagene), 100ng template plasmid (coding TAR1-5-19), 1 μ L PfuTurbo enzyme (Stratagene), and use sterilized water that volume is adjusted to 50 μ L.Use following PCR condition: initial denaturing step 94 ℃, 2 minutes, 94 ℃ then, 30 seconds; The circulation of 64 ℃, 30 seconds and 72 ℃, 30 seconds.Last extension step also comprises 72 ℃, 5 minutes.Purified pcr product with SalI and BamHI digestion, and connects in the carrier of also using the cutting of Serine restriction enzyme.By the correct clone of dna sequencing checking.
The expression of TAR1-5-19CYS and purifying
According to manufacturer's method, the TAR1-5-19CYS carrier is transformed in BL21 (DE3) the pLysS chemoreception attitude cell (Novagen).Use 100 μ g/mL Pyocianils and 37 μ g/mL paraxin to select to carry the cell of dAb plasmid.Culture contained in the band baffle plate flask (baffled flask) of 500mLTerrific Broth (Sigma-Aldrich), 100 μ g/mL Pyocianils and 37 μ g/mL paraxin at 2L cultivate.With 200rpm culture is cultured to O.D.6001-1.5 in 30 ℃, uses 1mM IPTG (sec.-propyl-β-D-sulfo-galactopyranoside derives from Melford Laboratories) to induce then.DAb was expressed in 30 ℃ of lasting 12-16 hours.Find that most of dAb is present in the substratum.Therefore, by centrifugal (8,000 * g, 30 minutes) isolated cell and substratum, use supernatant liquor purifying dAb.Whenever go up clear liquid and add 30mL L albumen agarose (Afftech), under agitation make dAb in batches in conjunction with 2 hours.Make resin sedimentation 1 hour more under gravity then, siphon off afterwards supernatant liquor.XK 50 posts (Amersham Phamacia) of then agarose being packed into are washed with 10 column volume PBS streams.With the dAb of 100mM glycine pH 2.0 elution of bound, then by among the 1M Tris pH 8.0 that adds 1/5 volume and protein-contg fraction.Every liter of culture supernatant is isolated the 20mg pure protein, and it contains the monomer of 50: 50 ratios: dimer.
The trimerizing of TAR1-5-19CYS
With the 100M TAR1-5-19CYS of 5mM dithiothreitol (DTT) reductase 12 .5ml, be placed on room temperature 20 minutes.Use PD-10 post (Amersham Pharmacia) buffering exchange sample then.With 5mM EDTA, 50mM sodium phosphate pH 6.5 pre-equilibration columns, according to manufacturer's guide with sample on the sample and wash-out.Sample is placed on ice, when needs.TMEA (Tris[2-maleimide ethyl] amine) available from Pierce Biotechnology.20mM TMEA mother liquor among the preparation 100%DMSO (dimethyl sulfoxide (DMSO)).Discovery is higher than 3: 1, and the TMEA concentration of (molar ratio of dAb: TMEA) causes albumen rapid precipitation and crosslinked.In addition, precipitation and cross-linked speed increase with pH.Therefore, use 100 μ M reductive TAR1-5-19CYS, add 25 μ M TMEA,, make to react on room temperature and carried out 2 hours with trimerizing albumen.Discovery adds additive such as glycerine or ethylene glycol to 20% (v/v) and has significantly reduced the tripolymer precipitation along with linked reaction is carried out.After coupling, the SDS-PAGE analysis revealed exists monomer, dimer and tripolymer in solution.
The purifying of trimerizing TAR1-5-19CYS
Every mL TMEA-TAR1-5-19cys reactant adds 40 μ L, 40% Glacial acetic acid, so that pH is reduced to 4.Then with sample on the sample to 1mL Resource S cationic exchange coloum (Amersham Pharmacia), this post is with 50mM sodium acetate pH 4.0 pre-equilibrations.Use 340-450nM sodium chloride salt gradient, 50mM sodium acetate pH 4.0 partly to separate dimer and tripolymer by 30 column volumes.Use SDS-PAGE to differentiate and only contain trimerical fraction, merge then, 1M Tris pH by adding 1/5 volume 8.0 increases to 8 with pH.For preventing that enrichment step from (using the dam Viva rotary concentrator of molecular weight of 5K; Vivascience) the tripolymer precipitation in adds 10% glycerine in sample.
Vitro functional is in conjunction with experiment: experiment of TNF acceptor and cell experiment
Use TNF acceptor and cell experiment to measure the avidity of tripolymer to the humanTNF-.IC in the acceptor experiment 50Be 0.3nM; ND in the cell experiment 50In the 3-10nM scope (for example 3nM).
The TAR1-5-19CYS trimeric form that other is possible
Also use following reagent that TAR1-5-19CYS is made tripolymer:
PEG tripolymer and customization synthetic maleimide tripolymer
Nektar (Shearwater) provides a series of multi-arm PEG, can be at the PEG end to its chemically modified.Therefore use the PEG tripolymer that has the maleimide amine functional group at each arm end, can with the similar mode trimerizing of the use THEA trimerizing dAb of above general introduction.PEG also can have increases the tripolymer solvability, prevent the advantage of aggegation problem thus.Therefore, people can produce the dAb tripolymer, and wherein each dAb all has the C-terminal cysteine that is connected to the maleimide amine functional group, and the maleimide amine functional group is connected to the PEG tripolymer.
C-terminal to dAb adds peptide linker or hinge
Can be between dAb and terminal cysteine residue an engineered little joint, it is (Gly 4Ser) n, n=1-10 wherein, for example 1,2,3,4,5,6 or 7, perhaps be immunoglobulin (Ig) (for example IgG hinge area), perhaps be peptide sequence (for example being selected from peptide sequence library at random) at random.When being used to prepare polymer (for example dimer or tripolymer), this can import the elasticity and the distance of higher degree again between single monomer, the elasticity of this higher degree and distance can be improved the binding characteristic to target, and described target is many subunits target for example, as the humanTNF-.
The selection of single domain antibody (dAb) gleanings of embodiment 9. AHS's albumin (HSA) and mice serum albumin (MSA)
This embodiment has set forth the method for preparing the albuminous single domain antibody of antiserum(antisera) (dAb).The selection of antagonism mice serum albumin (MSA) and both dAb of human serum albumin (HSA) has been described.Use 3 kinds of people's phage displaying antibody libraries in this experiment, each is all based on single people VH (referring to Figure 13: based on the simulation VH sequence of V3-23/DP47 and JH4b) or V κ(referring to Figure 15: based on the simulation V of 012/02/DPK9 and Jk1 κSequence) framework has the side chain diversity by the NNK codon coding that mixes in complementary determining region (CDR1, CDR2 and CDR3).
Library 1 (V H):
At the diversity with upper/lower positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98
Library size: 6.2 * 10 9
Library 2 (V H):
At the diversity with upper/lower positions: H30, H31, H33, H35, H50, H52, H52a, H53, H55, H56, H58, H95, H97, H98, H99, H100, H100a, H100b
Library size: 4.3 * 10 9
Library 3 (V κ):
At the diversity with upper/lower positions: L30, L31, L32, L34, L50, L53, L91, L92, L93, L94, L96
Library size: 2 * 10 9
Respectively according to proteic in conjunction with preselected V to metal ligand A albumen and L HAnd V κThe library makes that the major part clone in non-selected library has function.More than shown in the size in library corresponding to the size after preselected.Using each library that serum albumin is carried out two-wheeled respectively selects.Take turns selection for every, with 100 μ g/ml concentration in 4ml PBS with antigen coated on immunity pipe (nunc).In selecting in the first round, respectively in HSA (Sigma) and 3 libraries of MSA (Sigma) elutriation each.Take turns in the selection second, (i) once more at same antigen (for example the 1st take turns MSA, the 2nd takes turns MSA), (ii) (for example the 1st take turns MSA at reciprocity antigen, the 2nd takes turns HSA), the phage of each produces and takes turns selection for 12 the 2nd altogether in 6 first round selections of elutriation.In each case, after the 2nd takes turns selection, test combining of 48 clones and HSA and MSA.As Harrison etc., MethodsEnzymol.1996; 267:83-109 is to the segmental description production of scFv solubility dAb fragment, operative norm ELISA method (Hoogenboom etc., (1991) Nucleic Acids Res., 19:4133), the exception part is that 2%Tween PBS is used as the sealing damping fluid, uses L albumen-HRP (Sigma) (for V κ) and A albumen-HRP (Amersham Pharmacia Biotech) (for V H) detection bonded dAb.
Produce the combination of the dAb of the above signal of background (representative in conjunction with MSA, HSA or these two) with the insoluble formal testing of ELISA, but it is that serum albumin is specific all to independent plastics.(seeing table) cloned in order-checking then, and the result discloses and identifies 21 unique dAb sequences.Minimum similarity (on amino acid levels) between the selected VK dAb clone is 86.25% ((69/80) * 100; The asynchronous result of whole diversified residues for example clones 24 and 34).Selected V HMinimum similarity between the dAb clone is 94% ((127/136) * 100).
Then, the ability of the biotinylated antigen in the solution is caught in check in conjunction with sero-abluminous dAb.Implement ELISA method (as mentioned above), the exception part is (for V with 1 μ g/ml L albumen κClone) and 1 μ g/ml A albumen (for V HThe clone) bag is by elisa plate.As at the solubility dAb that catches described in the method in the solution, and detect with biotinylation MSA or HSA and strepto-HRP.Explanation according to the manufacturer prepares biotinylation MSA and HSA, and purpose is that each serum albumin molecule on average obtains 2 vitamin Hs.Identify 24 clones that in ELISA, catch the biotinylation MSA in the solution.Wherein 2 (following clones 2 and 38) also catch biotinylation HSA.Then, test dAb is in conjunction with the ability that is coated on the MSA on the CM5 Biacore chip.Find that 8 clones are in conjunction with the MSA on the Biacore.
DAb (all catching biotinylation MSA) H or κ ? ? ? CDR1 ? ? ? CDR2 ? ? ? CDR3 In biacore in conjunction with MSA? Do you catch biotinylation HSA?
V κ library 3 templates (simulation) 2,4,7,41,38,54 46,47,52,56 13,15 30,35 19,22,23,24,31,33,34,53,11 ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? XXXLX； SEQ?ID NO：16 SSYLN； SEQ?ID NO：19 SSYLN； SEQ?ID NO：22 FKSLK； SEQ?ID NO：25 YYHLK； SEQ?ID NO：28 RRYLK； SEQ?ID NO：31 YNWLK ；SEQ?ID NO：34 LWHLR； SEQ?ID NO：37 FRYLA； SEQ?ID NO：40 FYHLA； SEQ?ID NO：43 IWHLN； SEQ?ID NO：46 YRYLR； SEQ?ID NO：49 LKYLK； SEQ?ID NO：52 LRYLR； SEQ?ID NO：55 LRSLK； ? ? XASXLQS； SEQ?ID?NO： 17 RASPLQS； SEQ?ID?NO： 20 RASPLQS； SEQ?ID?NO： 23 NASYLQS； SEQ?ID?NO： 26 KASTLQS； SEQ?ID?NO： 29 QASVLQS； SEQ?ID?NO： 32 RASSLQS； SEQ?ID?NO： 35 HASLLQS； SEQ?ID?NO： 38 HASHLQS； SEQ?ID?NO： 41 PASKLQS； SEQ?ID?NO： 44 RASRLQS； SEQ?ID?NO： 47 KASSLQS SEQ?ID?NO： 50 NASHLQS； SEQ?ID?NO： 53 KASWLQS ；SEQ?ID?NO： 56 AASRLQS； ? ? QQXXXXPXT； SEQ?ID?NO：18 ? QQTYSVPPT； SEQ?ID?NO：21 ? QQTYRIPPT； SEQ?ID?NO：24 ? QQVVYWPVT； SEQ?ID?NO：27 ? QQVRKVPRT； SEQ?ID?NO：30 ? QQGLYPPTT； SEQ?ID?NO：33 ? QQNVVIPRT； SEQ?ID?NO：36 ? QQSAVYPKT； SEQ?ID?NO：39 ? QQRLLYPKT； SEQ?ID?NO：42 ? QQRARWPRT； SEQ?ID?NO：45 ? QQVARVPRT； SEQ?ID?NO：48 ? QQYVGYPRT SEQ?ID?NO：51 ? QQTTYYPIT； SEQ?ID?NO：54 ? QQVLYYPQT； SEQ?ID?NO：57 ? QQVVYWPAT； ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? √ √ whole 4 in conjunction with two combinations of √
? ? 12， ? ? 17， ? ? 18， ? ? 16，21 ? ? 25，26 ? ? 27， ? ? 55， ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ ? ? κ SEQ?ID NO：58 FRHLK； SEQ?ID NO：61 RKYLR； SEQ?ID NO：64 RRYLN； SEQ?ID NO：67 IKHLK； SEQ?ID NO：70 YYHLK； SEQ?ID NO：73 YKHLK； SEQ?ID NO：76 FKSLX； SEQ?ID NO：79 SEQ?ID?NO： 59 AASRLQS； SEQ?ID?NO： 62 TASSLQS； SSQ?ID?NO： 65 AASSLQS； SEQ?ID?NO： 68 GASRLQS； SEQ?ID?NO： 71 KASTLQS； SEQ?ID?NO： 74 NASHLQS； SEQ?ID?NO： 77 NASYLQS； SEQ?ID?NO： 80 ?SEQ?ID?NO：60 ? ?QQVALYPKT； ?SEQ?ID?NO：63 ? ?QQNLFWPRT； ?SEQ?ID?NO：66 ? ?QQMLFYPKT； ?SEQ?ID?NO：69 ? ?QQGARWPQT； ?SEQ?ID?NO：72 ? ?QQVRKVPRT； ?SEQ?ID?NO：75 ? ?QQVGRYPKT； ?SEQ?ID?NO：78 ? ?QQVVYWPVT； ?SEQ?ID?NO：81 ? ? √ ? ? √ ? ? √ ? ? √ ? ? √ ? ? √ ? ? √
V HLibrary 1 (with 2) template (simulation) 8; 10 36 ? ? ? H ? ? H ? ? H ? ? ? XXYXXX； SEQ?ID?NO： 82 WVYQMD； SEQ?ID?NO： 85 WSYQMT； SEQ?ID?NO： 88 ? ? ? XIXXXGXXTXYADSVKG； SEQ?ID?NO；83 ? SISAFGAKTLYADSVKG； SEQ?ID?NO：86 ? SISSFGSSTLYADSVKG； SEQ?ID?NO：89 ? ? ? XXXX(XXXX)FDY； SEQ?ID?NO：84 ? LSGKFDY；SEQ?ID?NO： 87 ? GRDHNYSLFDY；SEQ ID?NO：90
Framework is all identical with framework in the corresponding simulated series in all cases, shown in the diversity among the CDR is as above shown.
In 8 clones, there are 2 to be cloned in intestinal bacteria camber expression (clone MSA16 and MSA26), select it further to study (referring to embodiment 10) in conjunction with the MSA on the BIAcore.
The complete Nucleotide and the aminoacid sequence of MSA16 and 26 in Figure 16, have been provided.Embodiment 10. measures avidity and the serum half life in mouse in conjunction with the dAb MSA16 of MSA and MSA26
In colibacillary pericentral siphon, express dab MSA16 and MSA26, and use is adsorbed to L albumen-agarose affinity resin in batches, and (Affitech Norway) then carries out purifying with glycine pH 2.2 wash-outs.Analyze the dAb of purifying then by inhibition BIAcore, to measure K dSay that simply the MSA16 and the MSA26 of test purifying obtain the required dAb concentration of 200RU reaction to be determined on the BIAcore CM5 chip with high-density MSA bag quilt.In case determined required dAb concentration, just will be at expection K dThe MSA antigen and the dAb pre-mixing of near concentration range, and the incubation that spends the night.Detect dAb and MSA bag combining in each premixture with 30 μ l/ minutes high flow rate then by the BIAcore chip.Use the curve that is obtained to set up Klotz figure, the estimation K that this figure provides MSA16 dBe 200nM, the estimation K of MSA26 dBe 70nM (Figure 17 A and B).
Then, to clone MSA16 and MSA26 is cloned in have the HA mark expression vector of (nucleotide sequence: TATCCTTATGATGTTCCTGATTATGCA (SEQ ID NO:91) and aminoacid sequence: YPYDVPDYA (SEQ ID NO:92)), amount at expression in escherichia coli 2-10mg, with L albumen-agarose affinity resin (Affitech, Norway) by the supernatant liquor purifying, and with glycine pH 2.2 wash-outs.Measure the dAb serum half life in the mouse.MSA26 and MSA16 go in the CD1 mouse with the single agent intravenous injection of about 1.5mg/kg.
(Abeam UK) catches and uses 4%Marvel sealed L albumen-HRP (Invitrogen) detection elisa assay serum level by the anti-HA of goat.0.05%TweenPBS is adopted in rinsing.In the presence of the 1x mice serum, set up the typical curve of concentration known dAb, to guarantee the comparability with specimen.Show with 2 kinds of compartment modelings, MSA-26 has 0.16 hour t1/2 α, 14.5 hour t1/2 β, the area under curve of 465 hours mg/ml (AUC) (data are unlisted), MSA-16 has 0.98 hour t1/2 α, 36.5 hour t1/2 β, the AUC of 913 hours .mg/ml (Figure 18).And have 0.06 hour t1/2 α and the HEL4 (anti-hen's egg-white lysozyme dAb) of 0.34 hour t1/2 β and compare, two kinds of anti-MSA clones have prolong quite long half life.
Embodiment 11.V H-V HAnd V κ-V κThe segmental foundation of dual specific Fab sample
This embodiment has described preparation as the segmental V of Fab sample HAnd V κThe method of dual specific.Before making up described each Fab print section, at first select in conjunction with the dAb that selectes target by the dAb library in the library that is similar to embodiment 9 descriptions.The V of separation and combination hen's egg-white lysozyme (Sigma) HDAb-HEL4, separation and combination TNF-α acceptor (R ﹠amp again; D Systems) second V HDAb (TAR2h-5).These sequences have been provided in the sequence table.V by selection and the ripe separation and combination TNF-α of avidity (TAR1-5-19) κDAb, this sequence also is shown in the sequence table.In these experiments, also use second V that is described in embodiment 9 κDAb (MSA 26), its sequence is in Figure 17 B.
Contain the DNA of the expression vector of above-mentioned 4 dAb with enzyme SalI and NotI digestion, with the DNA of excision coding dAb.By electrophoresis digest on sepharose and excise band, then use Qiagen gel-purified test kit (Qiagen, UK) gel-filtration, the band of purifying expection size (300-400bp).The DNA of dAb of will encoding then is inserted in CH as shown in the table or the CK carrier (Fig. 8 and 9).
dAb Target antigen dAb?V HOr dAb V κ Insert in the carrier Mark (C end) Antibiotics resistance
HEL4 Hen's egg-white lysozyme V H C H myc Paraxin
TAR2-5 The TNF acceptor V H C κ flag Penbritin
TAR1-5-19 TNFα V κ C H myc Paraxin
MSA26 The mice serum albumin V κ C κ flag Penbritin
With VH CH and VH C κThe construction corotation dissolves in the HB2151 cell.Independently with V κCH and V κC κThe construction corotation dissolves in the HB2151 cell.The culture of each cotransformation clone of incubated overnight (is cultivated in the 2x TY that contains 5% glucose, 10 μ g/ml paraxin and 100 μ g/ml penbritins, to keep CH and C κThe microbiotic selectivity of plasmid).Use overnight culture inoculation fresh culture (2x TY, 10 μ g/ml paraxin and 100 μ g/ml penbritins), and be cultured to OD 0.7-0.9, induce by adding IPTG then, to express its CH and C κConstruction.
Then by A protein purification (the VH CH and the VH C that are used for cotransformation κ) and the MSA affinity resin purifying (V that is used for cotransformation κCH and V κC κ), by the Fab print section of pericentral siphon purifying expression.
V H-V HDual specific
By electrophoresis test for protein VH CH and VH C on gel κThe expression of dual specific.Blot gel, can be by myc mark and flag mark these two on western blotting, detects to expecting the segmental band of big or small Fab, this shows and has segmental VH CH of Fab sample and VH C simultaneously κPart.Then, in order to measure in the same Fab print section whether have two half dual specifics, with the 3mg/ml sodium bicarbonate buffer liquid of the hen's egg-white lysozyme (HEL) in 100 μ l/ holes in 4 ℃ of bags that spend the night by elisa plate.Then with 2%Tween PBS sealing (as described in embodiment 1) this plate, then with VH CH/VH C κDual specific Fab print section incubation.Use 9e10 (, Roche) and anti-mouse IgG-HRP (Amersham Pharmacia Biotech), to detect combining of bi-specific antibody and HEL by non-connection chain in conjunction with the monoclonal antibody of myc mark.VH CH/VH C κThe segmental signal of dual specific Fab sample is 0.154, by contrast the VH C of single expression κThe background signal of chain is 0.069.This shows that Fab print section has binding specificity to target antigen.
V κ-V κDual specific
The V of purifying cotransformation on the MSA affinity resin κCH and V κC κAfter the dual specific Fab print section, the albumen detection that use is obtained is wrapped the elisa plate of quilt with 1 μ g/ml TNF-α and is wrapped the elisa plate of quilt with 10 μ g/ml MSA.As expected, signal (data are unlisted) more than the background appears when detecting on two kinds of elisa plates with L albumen-HRP.This shows, protein part can confirm the dual specific of antibody fragment in conjunction with TNF-α in ELISA subsequently again in conjunction with MSA (and therefore purifying on MSA affinity post).Then this protein part is used for two experiments subsequently.At first, use dual specific V κCH and V κC κFab print section is surveyed the elisa plate with 1 μ g/ml TNF-α bag quilt, also uses the contrast dAb in conjunction with TNF-α to survey this plate with the concentration that produces similarity signal as calculated on ELISA.Having and do not having under the situation of 2mg/ml MSA, using dual specific and contrast dAb to survey elisa plate.Signal in the dual specific hole reduces above 50%, but the signal in the dAb hole does not descend (referring to Figure 19 a) at all.Also same protein is placed and do not have the acceptor experiment of MSA, shown the competition (referring to Figure 19 c) of MSA again.This shows the competition that combines with itself and TNF-α of combining of MSA and dual specific.
Embodiment 12. sets up has specific V to mice serum albumin and TNF α κ-V κThe dual specific of dual specific cys bonding
This embodiment has described through disulfide linkage all has the method for specific bispecific antibody fragment by the chemical coupling preparation to mice serum albumin and TNF-α.These two is cloned into and has in C-terminal halfcystine and the unmarked pET type carrier with MSA16 (deriving from embodiment 1) and TAR1-5-19 dAb.Two kinds of dAb are with the 4-10mg horizontal expression, and albumen-(Affitiech is Norway) by the supernatant liquor purifying for the agarose affinity resin to use L.Then with the dAb of dithiothreitol (DTT) reduction halfcystine mark.Then, form again, cause forming PEP 1-5-19 homodimer with the blocking-up disulfide linkage with TAR1-5-19 dAb and dithio two pyridine couplings.Mix two kinds of different dAb in pH 6.5 then, form and produce the heterodimer of TAR1-5-19, MSA16 cys bonding to promote disulfide linkage.This method of producing two kinds of proteic conjugates of dissmilarity is at first by (King TP, Li Kochoumian L Biochemistry.1978 17 volumes: 1499-506 Preparation of protein conjugates via intermoleculardisulfide bond formation) describe such as King.Separate heterodimer and monomeric substance by cationic exchange.Confirm to separate by the band that on sds gel, has the expection size.With the different dimerization material that TNF acceptor examination produces, find its have about 18nM in the IC of TNF 50Then, repeat the acceptor experiment with the heterodimer (18nM) of constant density and the MSA and the HSA of serial dilution.There is the ability drop that can not cause dimer to suppress TNF-α the HSA of certain limit concentration (being up to 2mg/ml) in it.
But, add MSA and make dimer suppress the ability dose-dependently decline of TNF-α: (Figure 20).This shows that MSA and TNF-α competition are in conjunction with TAR1-19, the MSA16 dimer of cys bonding.
Data gather
Chen Shu the data that experiment obtained gather and are shown in annex 4 in the aforementioned embodiment.
Embodiment 13: the gathering of the nucleic acid of anti-TNF-α dAb and peptide sequence
In the whole process of the research of relevant anti-TNF-α dAb described herein, identify many different dAb in conjunction with people and/or mouse TNF-α.Sequence and more information hereinafter are provided.
Clone in conjunction with mouse TNF-α:
Nucleotide and the aminoacid sequence of 4 kinds of anti-mouse TNF-α dAb below are provided.Wherein two kinds (TAR1-2m-9 and TAR1-2m-30) suppresses mouse TNF-alpha active, two kinds of combinations but do not suppress (TAR1-2m-1 and TAR1-2m-2).
TAR1-2m-9：
TAR-2m-9 is V κThe clone, IC50 is 6 μ M, ND50 is 5 μ M.IC50 and ND50 do not improve when the L protein-crosslinking.This clone does not have effect (having estimated the kind cross reactivity with two concentration in cell experiment) to the humanTNF-, but rat TNF-α is had similar neutralization activity.
Aminoacid sequence (CDR3 is a runic) (SEQ ID NO:93):
DIQMTQSPSSLSASVGDRVTITCRASQPIGSFLWWYQQKPGKAPKLLIYYSSYLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYRWHPNTFGQGTKVEIKR
Nucleotide?sequence(SEQ?ID?NO：94)：
Nucleotide sequence (SEQ ID NO:94):
1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc
61
atcacttgccgggcaagtcagcctattgggagttttttatggtggtaccagcagaaacca
121
gggaaagcccctaaactcctgatctattatagttcctatttgcaaagtggggtcccatca
181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct
241
gaagattttgctacgtactactgtcaacagtatcgttggcatcctaataccttcggccaa
301?gggaccaaggtggaaatcaaacgg
TAR1-2m-30：
TAR1-2m-30 is V κThe clone, ND50 is 10 μ M.ND50 does not improve when the L protein-crosslinking.This clone does not have effect (having estimated the kind cross reactivity with two concentration in cell experiment) to the humanTNF-, but and the mouse validity that compares rat TNF reduce a little.
Aminoacid sequence (CDR3 is a runic) (SEQ ID NO:95):
DIQMTQSPSSLSASVGDRVTITCRASQSIYSWLNWYQQKPGKAPKLLIYRASHLQSGV
PSRFSGSGSGTDFTLTISSLQPEDFAlYYC FGQGTKVEIKR
Nucleotide sequence (SEQ ID NO:96):
1
gacatceagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc
61
atcacttgccgggcaagtcagtcgatttatagttggttaaattggtaccagcagaaaCca
121
gggaaagcccctaagctcctgatctatagggcgtcccatttgcaaagtggggtcccatca
181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct
241
gaagattttgctacgtactactgtcaacagatttggaatatgccttttacgttcggccaa
301?gggaecaaggtggaaatcaaacgg
TAR1-2m-1：
This is cloned in conjunction with mouse TNF-α, but does not suppress receptor-binding activity.
Aminoacid sequence (SEQ ID NO:97):
DIQMTQSPSSLSASVGDRVTITCRASQPIGYDLFWYQQKPGKAPKLLIYRGSVLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQRWRWPFTFGQGTKVEIKR
Nucleotide sequence (SEQ ID NO:98):
1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc
61
atcacttgccgggcaagtcagcctattggttatgatttattttggtaccagcagaaacca
121
gggaaagcccctaagctcctgatctatcggggttccgtgttgcaaagtggggtcccatca
181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct
241
gaagattttgctacgtactactgtcaacagcggtggcgttggccttttacgttcggccaa
301
ggcaccaaggtggaaatcaaacgg
TAR1-2m-2：
This is cloned in conjunction with mouse TNF-α, but does not suppress receptor-binding activity.
Aminoacid sequence (SEQ ID NO:99):
DIQMTQSPSSLSASVGDRVTTTCRASLPIGRDLWWYQQKPGKAPKLLIYRGSFLQSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQRWYYPHTFGQGTKVEIKR
Nucleotide sequence (SEQ ID NO:100):
1
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc
61
atcacttgccgggcaagtctgcctattggtcgtgatttatggtggtatcagcagaaacca
121
gggaaagcccctaagctcctgatctatcgggggtcctttttgcaaagtggggtcccatca
181
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct
241
gaagattttgctacgtactactgtcaacagaggtggtattatcctcatacgttcggccaa
301?gggaccaaggtggaaatcaaacgg
DAb clone in conjunction with the humanTNF-
Below listed the nucleotide sequence that identifies in conjunction with humanTNF-'s dAb.Corresponding amino acid sequence is provided among Figure 23.
TAR1-5(SEQ?ID?NO：101)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTATGAATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGC
TAR1-27(SEQ?ID?NO：102)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTGGACGAAGTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATATG
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTGGTTTAGTAATCCTAGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACG
TAR1-261(SEQ?ID?NO：103)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGAGCATTATTTAT
GGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCTATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGAGTTTGGCGTGTCCTCCTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-398(SEQ?ID?NO：104)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTATGGTCATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGCCTTTGGTGCGGCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-701(SEQ?ID?NO：105)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGCTAAGTTGTTAT
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGAT
GCATCCTCTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTGGTGGGGGTATCCTGGTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-2(SEQ?ID?NO：106)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTCCTGCTTTAC
TTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATATTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-3(SEQ?ID?NO：107)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATGCGTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAG
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-4(SEQ?ID?NO：108)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTTATGAATTTAT
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGGTTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-7(SEQ?ID?NO：109)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTGAATTCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCACTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-8(SEQ?ID?NO：110)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTTTGAATTCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAT
GCATCCACTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-10(SEQ?ID?NO：111)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-11(SEQ?ID?NO：112)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-12(SEQ?ID?NO：113)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAATTATGCTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATCAG
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-13(SEQ?ID?NO：114)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-19(SEQ?ID?NO：115)
GACATCCAGATGACCCAGTCTCCATCCTCTCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-20(SEQ?ID?NO：116)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GCATCCAATTTGCAAAGTGAGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-21(SEQ?ID?NO：117)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-22(SEQ?ID?NO：118)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATTCTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCCTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-23(SEQ?ID?NO：119)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCTTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACATACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-24(SEQ?ID?NO：120)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAAAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTGT
GCATCCCAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTACATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-25(SEQ?ID?NO：121)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGCGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCCTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-26(SEQ?ID?NO：122)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGTGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACCCTCACCATCAGCAGTCTGCAGCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-27(SEQ?ID?NO：123)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCTCGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-28(SEQ?ID?NO：124)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGTTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CAACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-29(SEQ?ID?NO：125)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-34(SEQ?ID?NO：126)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTCAGACTGCGTTAC
TGTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACATACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-35(SEQ?ID?NO：127)
GACATCCAGAGGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GCATCCAATTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-36(SEQ?ID?NO：128)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCCAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-464(SEQ?ID?NO：129)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-463(SEQ?ID?NO：130)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAACCCCCTAAGCTCCTGATCTATTCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-460(SEQ?ID?NO：131)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATCATTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCCGAGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-461(SEQ?ID?NO：132)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCG
GCATCCATGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-479(SEQ?ID?NO：133)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCCAAGCTCCTGATCTATTCT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGCATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-477(SEQ?ID?NO：134)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTTTTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCG
GCATCCGCTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-478(SEQ?ID?NO：135)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATGAGTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCT
GCATCCATTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCACCCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-476(SEQ?ID?NO：136)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAATTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCT
GCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGATGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTGCGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1-5-490(SEQ?ID?NO：137)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGATAGTTATTTAC
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCATCAAATTTAGAAACAGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGGTTGTGTGGCGTCCTTTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1h-1(SEQ?ID?NO：138)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGGTGATTTGGGATGCGTTAG
ATTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAGT
GCGTCCCGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCATCCTGAAGATTTTG
CTACGTACTACTGTCAACAGTATGCTGTGTTTCCTGTGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1h-2(SEQ?ID?NO：139)
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGACTATTTATGATGCGTTAA
GTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GGTTCCAGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCGGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG
CTACGTACTACTGTCAACAGTATAAGACTAAGCCTTTGACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
TAR1h-3(SEQ?ID?NO：140)
GACATCCAGATGACCCAGTCCCCATCCTCCCTGTCTGCATCTGTAGGAGA
CCGTGTCACCATCACTTGCCGGGCAAGTCAGACTATTTATGATGCGTTAA
GTTGGTACCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGT
GGTTCCAGGTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGTAGTGGATC
TGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCCGAAGATTTTG
CTACGTACTACTGTCAACAGTATGCTCGTTATCCTCTTACGTTCGGCCAA
GGGACCAAGGTGGAAATCAAACGG
Anti-humanTNF-dAb clone in addition comprises following clone:
Several clones have experienced the avidity maturation.Clone TAR1-100-47 is the sophisticated clone of avidity, and the ND50 in the L929 cell experiment is 30-50nM, and ND50 is 3-5nM when with the L protein-crosslinking.TAR1-100-47 and rhesus monkey TNF cross reaction.Its aminoacid sequence and many other clones' aminoacid sequence below is provided.TAR1-2-100 and TAR1-2-109 are the parental generation clones who is used to make up the library.TAR1 clone good in this group has following consensus sequence:
D/E30, W32, R94 and F96 show with runic in TAR1-100-47.
TAP1-100-29(SEQ?ID?NO：141)，
DIQMTQSPSSLSASVGDRVTITCRASQDIEEWLMWYQQKPGKAPKLLIYNSSTLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQPLSRRFTFGQGTKVEIKR
TAR1-100-35(SEQ?ID?NO：142)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYRASFLQ
SGVPSKFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAP1-100-43(SEQ?ID?NO：143)
DIQMTQSPSSLSASVGDRVTITCRASQFIEDWLFWYQQKPGKAPKLLIYQASKLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-47(SEQ?ID?NO：144)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQASRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ
Figure A20058002851802561
TGQGTKVEIKR
TAR1-100-52(SEQ?ID?NO：145)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYRASFLQ
SGVPPRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-109(SEQ?ID?NO：146)
DIQMTQSPSSLSASVGDRVTITCRASQNIDDHLMWYQQKPGKAPKLLIYSSSILQS
GVPPRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100(SEQ?ID?NO：147)
DIQMTQSPSSLSASVGDRVTITCRASQDIDHALLWYQQKPGKAPRLLIYNGSMLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLRRPFTFGQGTKVEIKR
TAR1-100-34(SEQ?ID?NO：148)
DIQMTQSPSSLSASVGDRVTITCRASQHIGDWLLWYQQKPGKAPMLLIYQSSRLQ
SGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-36(SEQ?ID?NO：149)
DIQMTQSPSSLSASVGDRVTITCRASQHIDSYLMWYQQKPGKAPKLLIYNTSVLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-38(SEQ?ID?NO：150)
DIQMTQSPSSLSASVGDRVTITCRASQWIDDHLFWYQQKPGKAPKLLIYNTSTLQ
SGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-39(SEQ?ID?NO：151)
DIQMTQSPSSLSASVGDRVTITCRASQFIDEHLMWYQQKPGKAPKLLIYRSSELQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-40(SEQ?ID?NO：152)
DIQMTQSPSSLSASVGDRVTITCRASQWINNWLLWYQQKPGKAPKLLIYESSNLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-41(SEQ?ID?NO：153)
DIQMTQSPSSLSASVGDRVTITCRASQLIDDHFWYQQKPGKAPTLLIYNSSVLQSG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-45(SEQ?ID?NO：154)
DIQMTQSPSSLSASVGDRVTITCRASQDIDQWLMWYQQKPGKAPKLLIYQSSML
QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-60(SEQ?ID?NO：155)
DIQMTQSPSSLSASVGDRVTITCQASQDIDNWLLWYQQKPGKAPKLLIYQASNLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-62(SEQ?ID?NO：156)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQASRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSGPFTFGQGTKVEIKR
TAR1-100-64(SEQ?ID?NO：157)
DIQMTQSPSSLSASVGDRVTTTCRASQYIDYGLMWYQQKPGKAPKLLIYRTSELQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-65(SEQ?ID?NO：158)
DIQMTQSPSSLSASVGDRVTITCRASQWIDSFLMWYQQKPGKAPKLLIYNGSVLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-75(SEQ?ID?NO：159)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQGSRLQ
SGVPLRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIRR
TAR1-100-76(SEQ?ID?NO：160)
DIQMTQSPSSLSASVGDRVTITCRASQHIDSWLLWYQQKPGKAPKLLIYNGSVLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSGPFTFGQGTKVEIKR
TAR1-100-77(SEQ?ID?NO：161)
DIQMTQSPSSLSASVGDRVTITCRASQHIDTHLFWYQQKPGKAPKLLIYNTSTLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-78(SEQ?ID?NO：162)
DIQMTQSPSSLSASVGDRVTITCRASQFIDTHLMWYQQKPGKAPRLLIYNTSTLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-80(SEQ?ID?NO：163)
DIQMTQSPSSLSASVGDRVTITCRASQDIDDWLLWYQQKPGKAPKLLIYQGSRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDRATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-82(SEQ?ID?NO：164)
DIQMTQSPSSLSASVGDRVTITCRASQWIDDTLMWYQQKPGKAPKLLIYRSSMLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFIFGQGTKVEIKR
TAR1-100-83(SEQ?ID?NO：165)
DIQMTQSPSSLSASVGDRVTITCRASQYIDSHLMWYQQKPGKAPKLLIYDTSRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-84(SEQ?ID?NO：166)
DIQMTQSPSSLSASVGDRVTITCRASQHIDQHLFWYQQKPGKAPKLLIYNSSSLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-89(SEQ?ID?NO：167)
DIQMTQSPSSLSASVGDRVTITCRASQHIERWLLWYQQKPGKAPKLLIYNSSKLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-90(SEQ?ID?NO：168)
DIQMTQSPSSLSASVGDRVTISCRASQHIERWLLWYQQKPGKAPKLLIYNSSKLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-91(SEQ?ID?NO：169)
DIQMTQSPSSLSASVGDRVTITCRASQDIGSWLMWYQQKSGKAPKLLIYNGSALQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-92(SEQ?ID?NO：170)
DIQMTQSPSSLSASVGDRVTITCRASQHIDKWLMWYQQKPGKAPKLLIYQASKL
QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-93(SEQ?ID?NO：171)
DIQMTQSPSSLSASVGDRVTITCRASQDIEEWLMWYQQKPGKAPKLIYNSSTLQ
SGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-94(SEQ?ID?NO：172)
DIQMTQSPSSLSASVGDRVTITCRRASQYIDYGLMWYQQKPGKAPKLLIYRTSELQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQ
TAR1-100-95(SEQ?ID?NO：173)
DIQMTQSPSSLSASVGDRVTITCRASQNIDIHLMWYQQKPGKAPKLLIYQSSNLQS
GVPSPFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-96(SEQ?ID?NO：174)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLLWYQQKPGKAPKLLIYQSSELQS
GVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-97(SEQ?ID?NO：175)
DIQMTQSPSSLSASVGDRVTITCRASQEIGVWLMWYQQKPGKAPKLLIYEGSRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFVFGQGTKVEIKR
TAR1-100-98(SEQ?ID?NO：176)
DIQMTQSPSSLSASVGDRVTITCRASQSIGKWLMWYQQKPGKAPKLLIYQSSLLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-99(SEQ?ID?NO：177)
DIQMTQSPSSLSASVGDRVTIICRASQDIDTWLFWYQQKPGKAPKLLIYNGSRLQ
SGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-100(SEQ?ID?NO：178)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLMWYQQKPGKAPKLLIYQASRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-101(SEQ?ID?NO：179)
DIQMTQSPSSLSASVGDRVTITCRASQDIEGWLLWYQQKPGKAPKLLIYNSSTLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-102(SEQ?ID?NO：180)
DIQMTQSPSSLSASVGDRVTITCRASQHIDDWLFWYQQKPGKAPKLLIYRASFLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-103(SEQ?ID?NO：181)
DIQMTQSPSSLSASVGDRYTITCRASQDIDTWLFWYQQKPGKAPKLLIYNGSRLQ
SGVPSRFSGSGSGTDFTLTISGLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-105(SEQ?IDNO：182)
DIQMTQSPSSLSASVGDRVTITCRASQPIEEWLLWYQQKPGKAPKLLIYNGSHLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-106(SEQ?ID?NO：183)
DIQMTQSPSSLSASVGDRVTITCRASQHIDKWLMWYQQKPGKAPKLLIYQASKL
QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-107(SEQ?ID?NO：184)
DIQMTQSPSSLSASVGDRVTITCRASQDUEEWLMWYQQKPGKAPKLLIYNSSTLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDYATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-108(SEQ?ID?NO：185)
DIQMTQSPSSLSASVGDRVTITCRASQPIDYGLMWYQQKPGKAPKLLIYRSSQLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-109(SEQ?ID?NO：186)
DIQMTQSPSSLSASVGDRVTTTCRASQEIGSWLMWYQQKPGKAPKLLIYQSSKLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-110(SEQ?ID?NO：187)
DIQMTQSPSSLSASVGDRVTITCRASQPIDSWLLWYQQKPGKAPKLLIYNASSLQS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-111(SEQ?ID?NO：188)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQASALQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-112(SEQ?ID?NO：189)
DIQMTQSPSSLSASVGDRVTITCRASQNIHEWLMWYQQKPGKAPKLLIYQGSRLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQPLSRPFTFGQGTKVEIKR
TAR1-100-113(SEQ?ID?NO：190)
DIQMTQSPSSLSASVGDRVTITCRASQDIGPWLMWYQQKPGKAPKLLIYQASALQ
SGVPSRFSGSGSGTDFTLTISSLQPEDSATYYCQQPLSRPFTFGQGTKVEIKR
The sequence that is suitable for the anti-humanTNF-dAb of various forms of TAR1-5-19 in these embodiments is as follows:
TAR1-5-19
Amino acid (SEQ ID NO:191)
DIQMTQSPSSLSASVGDRVTITCRASQSVKEFLWWYQQKPGKAPKLLIYMASNLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQKFKLPRTFGQGTKVEIKR
Nucleotide (SEQ ID NO:115)
gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagaccgtgtcacc
atcacttgccgggcaagtcagagcgttaaggagtttttatggtggtaccagcagaaacca
gggaaagcccctaagctcctgatctatatggcatccaatttgcaaagtggggtcccatca
cgtttcagtggcagtggatctgggacagatttcactctcaccatcagcagtctgcaacct
gaagattttgctacgtactactgtcaacagaagtttaagctgcctcgtacgttcggccaa
gggaccaaggtggaaatcaaacgg
The effect research of embodiment 14:PEGization TAR1-5-19 in the sacroiliitis prophylaxis model
The Tg197 mouse is that people TNF-sphaeroprotein heterozygous genes is genetically modified, chronic progressive external polyarthritis [the Keffer of the histologic characteristics the same with rheumatoid arthritis appears having in heterozygote when age in 4-7 week, J., Probert, L, Cazlaris, H., Georgopoulos, S., Kaslaris, E., Kioussis, D., Kollias, G. (1991) .Transgenic mice expressing humantumor necrosis factor:a predictive genetic model of arthritis.EMBO J., the 10th volume, the 4025-4031 page or leaf].
DAb (is the 2 * 20k ramose PEG form with two dAb connection site for test PEGization, [being 40K mPEG2 MAL2], dAb is TAR1-5-19cys) the arthritic effectiveness of prevention in the Tg197 model, the heterozygote transgenic mice is divided into every group of group that 10 male and female animals of number such as has.Treatment is in 3 weeks during ages, weekly the peritoneal injection test substances.Monomeric expression of TAR1-5-19cys and PEGization are summarized in the 1.3.3 joint of embodiment 1.All protein products all in phosphate-buffered saline, are tested the acceptable level of endotoxin of all protein products.
Carry out blind research.Weigh weekly animal and according to following system log (SYSLOG) sacroiliitis macroscopic view phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
Result of study is clear to be shown, 10mg/kg PEGization TAR1-5-19 suppresses the sacroiliitis development, and the sacroiliitis between saline control and the treatment group is kept the score, and there were significant differences.The PEGization TAR1-5-19 of 1mg/kg dosage also produces the intermediate value sacroiliitis that significantly is lower than the saline control group on the statistics keep the score (P＜0.05% uses the normal approximation of Wilcoxon check).
The effect research of embodiment 15:PEGization TAR1-5-19 in the arthritis treatment model
Be test PEGization dAb effectiveness to the arthritis treatment model in the Tg197 model, the heterozygote transgenic mice is divided into every group of group that 10 male and female mouse of number such as has.Treatment is when 6 ages in week, and this moment, mouse had remarkable sacroiliitis phenotype.Treat with 4.6mg/kg peritoneal injection test substances for twice weekly.Specimen preparation and disease are kept the score and are carried out as described in above embodiment 14.
Sacroiliitis is kept the score clear showing, the sacroiliitis development in the PEGization TAR1-5-19 suppression therapy model.4.6mg/kg the PEGization of dosage TAR1-5-19 is producing the intermediate value sacroiliitis that significantly is lower than the saline control group on the statistics keep the score (P＜0.01% uses the normal approximation of Wilcoxon check) the 9th week.
Embodiment 16: the dAb of slowly-releasing form renders a service
Be the effectiveness of the dAb of test slowly-releasing form, the dAb (wherein PEG is 4 * 5k, and 4 dAb connection site have the terminal cys residue of the C-of containing, and dAb is that TAR1-5-19[is 20K PEG4 arm MAL]) that will have little PEG molecule is loaded in the 0.2ml osmotic pump.This pump has the rate of release of 0.2ml in 4 cycles, the 6th week with the aforesaid therapeutic Tg197 model of the subcutaneous implantation of this pump in.Compare with the animal of implanting the salt solution load pump, the sacroiliitis of these animals is kept the score to be increased with obviously slower speed.This shows that dAb is effective when being transmitted by the slowly-releasing form.
Embodiment 17: half life,, stable anti-humanTNF-dAb prevented that RA from showing effect in the Tg197 mouse model
DAb monomer TAR1-5-19 described herein is the sophisticated dAb monomer of avidity, derives from the dAb of the TNF-α initial selected of using passive bag quilt.Initially be cloned in the L929TNF-cytotoxicity and experiment in have ND50 greater than 5 μ M.TAR1-5-19 has the ND50 less than 30nM.When making Fc syzygy described herein, TAR1-5-19 is cloned in the ND50 that has in the L929 experiment less than 5nM.
TAR1-5-19 dAb Fc-syzygy is being injected in the mouse its serum of back check half life.The results are shown in Figure 24.When TAR1-5-19 dAb monomer had about 20 minutes t1/2 β, the Fc-syzygy configuration form of this dAb had the t1/2 β above 24 hours, showed that serum half life increase is above 70 times.
Test TAR1-5-19 dAb Fc syzygy construction in above-described Tg197 mouse RA model.Mouse is divided into 5 groups, 10 every group, the male and female mice that quantity such as has.Twice IP injection TAR1-5-19 dAb Fc syzygy, ENBREL or salt solution are treated weekly, and treatment is when 3 ages in week, and be the time that the RA symptom does not also show 3 ages in week.Research was carried out for 7 weeks.As shown in figure 25, give two doses of TAR1-5-19 dAb Fc syzygys of 1mg/kg and 10mg/kg.Anti--β-gal Fc syzygy that the negative control animal is accepted the negative control of twice 10mg/kg weekly, 1 group weekly twice usefulness saline injection treat.For comparing, accept 10mg/kg ENBREL weekly twice for 1 group.
The sacroiliitis of estimating animal in blind mode as described herein is kept the score.When 7 weeks treatment time-histories finishes, its sacroiliitis that has of animal of accepting the TAR1-5-19 dAb Fc syzygy of twice 10mg/kg dosage is weekly kept the score and is lower than the animal of accepting 10mg/kg ENBREL, and, compare experience arthritis disease prevention substantially completely with the not treatment animal of accepting negative control dAb Fc syzygy.
TNF-α is relevant with emaciation.In the whole process of anti-TNF-α dAb treatment, animal is weighed.The body weight of accepting the animal of TAR1-5-19 dAb Fc syzygy is significantly higher than the body weight of accepting negative control dAb Fc syzygy and untreated animal, and is similar to the weight of animals of accepting the ENBREL injection.
In a word, 10mg/kg TAR1-5-19 prevents the sacroiliitis outbreak fully in the Tg197 model.This reaction is a dose-dependently, and 1mg/kg dosage produces partial action, and reaction is better than the reaction that the existing anti-TNF-α medicine ENBREL with similar dosage observes.This studies show that dAb is as the effectiveness of therapeutical agent in clinical generally acknowledged human disease model.
The histopathological analysis of joint of animal immobilization section is consistent with these data (unlisted).
Embodiment 18: study in the body to different prolongation half life forms
In the research of a series, the effect of checking the anti-TNF-α dAb forms of 3 kinds of different prolongation half lifes that sacroiliitis is kept the score.These forms are that anti-TNF-α dAb Fc syzygy is (by merging to human IgG C H2/C H3 districts are with two anti-humanTNF-dAb of dimerization), the anti-TNF-α dAb construction that connects of two different PEG (homodimer that 2 identical dAb and the PEG of 2 * 20K branch form by cys-maleimide key and 4 identical dAb and the PEG of 4 * 10K branch pass through the same tetramer of cys-maleimide key formation) and the anti-TNF-α of dual specific/anti-SA dAb, it comprises 2 anti-TNF-α dAb that meet anti-mice serum albumin dAb after identical.
In independently studying, give pharmaceutical composition weekly 1 time with 10mg/kg or 1mg/kg as shown in figure 26, or give pharmaceutical composition weekly twice with the dosage that changes, give when 3 ages in week, and continued for 7 weeks.
Keep the score according to sacroiliitis, in weekly infusion protocol, the anti-TNF dAb of the PEGization of 10mg/kg homodimer is effective to thorough prevention sacroiliitis.Be used for correlated existing anti-TNF-α medicine than not treating declines of keeping the score of joint of animal inflammation, be higher than keeping the score of PEGization dAb construction acquisition in the remarkable mode of statistics but keep the score.Anti-TNF-α/anti-SA dual specific and Fc syzygy are than the treatment demonstration is ineffective.
In the weekly infusion protocol of 1mg/kg, 100% effective although none treatment shows effect to preventing disease, than not treating and have anti-TNF-α medicine, the anti-TNF-α of PEGization dAb construction still makes progress highly effective to the preventing disease symptom.In this dosage regimen, anti-TNF-α dAb Fc syzygy and dual specific construction are also more effective than existing medicine.
In a word, prolong weekly dosage regimen research that dAb carry out with 3 kinds of multi-form half lifes and further verified the effectiveness for the treatment of in clinical generally acknowledged human disease's model.
Embodiment 19: compare anti-humanTNF-dAb effectiveness to existing disease in Tg197 mouse RA model with existing anti-TNF-α therapeutical agent
In this research, with the effectiveness of the anti-existing disease of anti-TNF-α dAb construction of Tg197 model contrast various forms and dosage regimen with etc. existing anti-TNF-α therapeutical agent ENBREL, the HUMIRA of molar dose and the effectiveness of REMICADE.When the 6th week rather than the 3rd week, begin to give the treatment of animals agent, make arthritic symptom show.With blind mode by histology (the 9th week) and sacroiliitis keep the score (1 time weekly) monitoring symptom.
Figure 27 has shown and has given twice various formulations and dosage weekly.Formulation comprises that the Fc syzygy is (by merging the C to the human IgG1 H2/C H3 districts are with the TAR1-5-19 dAb of two copies of dimerization), TAR1-5-19 dAb PEG dimer (homodimer that 2 identical dAb and the PEG of 2 * 20K branch form by cys-maleimide key), the TAR1-5-19 dAb PEG tetramer (the same tetramer that 4 identical dAb and the PEG of 4 * 10K branch form by cys-maleimide key) and the anti-mouse SA of TAR1-5-19 dAb/ dual specific (afterwards connecing the linear syzygy of two identical anti-THF-α dAb of anti-mice serum albumin dAb).Dosage regimen is illustrated in Figure 28.Also estimated the 4 * 5k PEGization TAR1-5-19 construction that continues to give by the osmotic pump of implanting.
Result of study shows, do not have a kind of existing biotechnological formulation to reverse sacroiliitis considerablely and keeps the score and reached for 9 weeks.Compare with saline control, the TAR form is higher all or stablize sacroiliitis than low degree ground and keep the score, and this is that statistics is significant.And, when comparing with the 6th journal branch, the sign that has disease to reverse.
When checking the histopathology morbid state, to compare with the joint in the 6th week, the arthritis joint in the 9th week also demonstrates disease seriousness decline after using the TAR form of therapy.This has confirmed that the TAR form can excite the reverse of existing disease sacroiliitis phenotype.
These studies confirm that the anti-TNF-α dAb construction the tested validity to existing disease, comprise that TNF-α dAb reverses the ability of the course of disease at least in part.
Embodiment 20: anti-TNF dAb as with the effect research of effectiveness A TAR1-5-19/ antiserum(antisera) albumin dAb syzygy in preventative arthritis model of the syzygy of antiserum(antisera) albumin dAb
The Tg197 mouse is that people TNF-sphaeroprotein heterozygous genes is genetically modified, chronic progressive external polyarthritis [the Keffer of the histologic characteristics the same with rheumatoid arthritis appears having in heterozygote when age in 4-7 week, J., Probert, L, Cazlaris, H., Georgopoulos, S., Kaslaris, E., Kioussis, D., Kollias, G. (1991) .Transgenic mice expressing humantumor necrosis factor:a predictive genetic model of arthritis.EMBO J., the 10th volume, the 4025-4031 page or leaf].
Be test TAR1-5-19/ antiserum(antisera) albumin dAb syzygy (the straight-line type tripolymer of 3 dAb, 3 dAb are TAR1-5-19, TAR1-5-19 and anti-mice serum albumin dAb) the arthritic effectiveness of prevention in the Tg197 model, the heterozygote transgenic mice is divided into the group that every group of male and female 10 animals of number such as has.Treatment is in 3 weeks during ages, weekly peritoneal injection test substances.Have the TAR1-5-19/ antiserum(antisera) albumin dAb syzygy of terminal 6 histidine marks of C-at expression in escherichia coli, and by Ni affinity chromatography, IEX and gel-filtration purifying.All protein products all in phosphate-buffered saline, are tested the acceptable level of endotoxin of all protein products.
Carry out blind research.Weigh weekly animal and according to following system log (SYSLOG) sacroiliitis macroscopic view phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
Result of study clearlys show that 10mg/kg TAR1-5-19/ antiserum(antisera) albumin dAb syzygy suppresses the sacroiliitis development, and the sacroiliitis between saline control and the treatment group is kept the score, and there were significant differences.The TAR1-5-19/ antiserum(antisera) albumin dAb syzygy of 1mg/kg dosage also produces the intermediate value sacroiliitis that significantly is lower than the saline control group on the statistics keep the score (P＜2% uses the normal approximation of Wilcoxon check).
The effect research of B TAR1-5-19/ antiserum(antisera) albumin dAb syzygy in the therapeutic arthritis model
Be test TAR1-5-19/ antiserum(antisera) albumin dAb syzygy effectiveness to the arthritis treatment model in the Tg197 model, the heterozygote transgenic mice be divided into the group that every group of male and female 10 mouse of number such as has.Treatment is when 6 ages in week, and this moment, mouse had remarkable sacroiliitis phenotype.Treat with 2.7mg/kg peritoneal injection test substances for twice weekly.Specimen preparation and disease are kept the score and are carried out as mentioned above.
Sacroiliitis is kept the score and is clearly show, the sacroiliitis development in the TAR1-5-19/ antiserum(antisera) albumin dAb syzygy suppression therapy model.2.7mg/kg the TAR1-5-19/ antiserum(antisera) albumin dAb syzygy of dosage is producing the intermediate value sacroiliitis that significantly is lower than the saline control group on the statistics keep the score (P＜0.05% uses the normal approximation of Wilcoxon check) the 9th week.
This clearlys show, and is effective with the anti-TNF dAb of anti-SA dAb moulding, and the expection half life of the serum half life that anti--SAdAb will resist TNF dAb by independent anti-TNF dAb prolongs.
Embodiment 21: the effect of checking anti-TNF-α dAb disclosed herein in Tg197 mouse RA model sacroiliitis and histopathology to be kept the score
Carry out two other researchs, the effect of checking anti-TNF-α dAb in Tg197 mouse RA model, sacroiliitis and histopathology to be kept the score.
In first research, begin weekly twice during 3 all ages before the RA symptom occurs and give aforesaid TAR1-5-19 dAb Fc syzygy with 10mg/kg.Inject the judged result of comparing with salt solution, ENBREL and the contrast Fc syzygy of same approach.
Keep the score and Histological section's analysis and judgement according to sacroiliitis, TAR1-5-19 dAb Fc syzygy prevents the RA paresthesia epilepsy more effective than ENBREL in mouse.
In second research, began during ages in 3 weeks with 10 or 1mg/kg inject once anti-TNF-α dAb Fc syzygy, PEG dimer and the anti-SA of the anti-TNF/ of dual specific weekly, its effect is contrasted with ENBREL and HUMIRA.
When comparing with saline control, the sacroiliitis of all TAR forms that give with 1mg/kg or 10mg/kg dosage is kept the score and is all descended.And, evidence suggests that seizure of disease postpones.Compare with Enbrel with Humira, PEGization and anti-SA dual specific form are more effective aspect reduction sacroiliitis seriousness.In addition, the joint tissue credit in the 10th week is analysed also and is shown, compares with saline control, and the TAR form is effectively, reduces disease seriousness.
In a word, the anti-TNF-dAb of TAR1-5-19 of Fc syzygy, PEGization and anti-SA dual specific form is all effective anti-RA symptom in the Tg197 model, no matter be before the arthritic symptom outbreak or after give.In all researchs, the most effective anti-TNF dAb form and HUMIRA equivalence or more effective, the most effective anti-TNF dAb form is obviously more effective than ENBREL.
Embodiment 22: anti-people VEGF dAb
TAR15 (anti-people VEGF)
VK dAb in conjunction with people VEGF has below been described.RBA refers to that vegf receptor 2 described herein is in conjunction with experiment.
Guiding dAb RBA (R2) IC50-L albumen (nM) RBA (R2) IC50+L albumen (nM) With the cross reactivity of mouse VEGF in ELISA
TAR15-1 VK 171 7.4 ?+
TAR15-10 VK 12.2 0.3 ?+
TAR15-16 VK 31 1.7 ?+/-
TAR15-17 VK 38 0.5 ?+/-
TAR15-18 VK 174 0.4 ?+
TAR15-20 VK 28 0.3 ?-
When testing on low density BIAcore chip with various concentration, the TAR15-1 clone has the Kd of 50-80nM.Other VK clone is with a certain concentration (50nM) process low density chip.Different clones demonstrates different kinetics models.
Aminoacid sequence:
Consensus sequence: W28, G30, E32, S34, H50 and Y93.
The anti-people VEGF of other TAR15 dAb clone has consensus sequence:
W28, G30, E32, S34, H50 and Y93 are shown in following TAR15-10.
TAR15-1(SEQ?ID?NO：192)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLIYHGSILQS
GV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRMYRPATFGQGTKVEIKR
TAR15-3(SEQ?ID?NO：193)
DIQMTQSPSSLSASVGDRVTITCRASQWIGRELKWYQQKPGKAPRLLIYHGSVL
QSG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDFFVPDTFGQGTKVEIKR
TAR15-4(SEQ?ID?NO：194)
DIQMTQSPSSLSASVGDRVTITCRASQDIANDLMWYQQKPGKAPKLLIYRNSRLQ
GG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLVHRPYTIGQGTKVEIKR
TAP15-9(SEQ?ID?NO：195)
DIQMTQSPSSLSASVGDRVTITCRASQFIGPHLTWYQQKPGKAPKLLIYHSSLLQS
GV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMYYPSTFGQGTKVKIKP
TAR15-10(SEQ?ID?NO：196)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYHTSILQS
GVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYMFQPRTFGQGTKVEIRR
TAR15-11(SEQ?ID?NO：197)
DIQMIQSPSSLSASVGDRVTITCRASQFIGNELSWYQQKPGKAPKLLIYHASSLQS
GV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLGYPYTFGQGTKVEIKR
TAR15-12(SEQ?ID?NO：198)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYHGSILQ
SG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLYSPLTFGQGTKVEIKR
TAR15-13(SEQ?ID?NO：199)
DIQMTQSPSSLSASVGDRVTITCRASQWIGNELKWYQQKPGKAPKLLIYMSSLL
QSG
VPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQTLLLPFTFGQGTKVEIKR
TAR15-14(SEQ?ID?NO：200)
DIQMTQSPSSLSASVGDRVTITCRASQWIGPELSWYQQKPGKAPKLLIYHGSILQ
SG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQRLYYPGTFGQGTKVEIKR
TAR15-15(SEQ?ID?NO：201)
DIQMTQSPSSLSASVGDRVTITCRASQSIGRELSWYQQKPGKAPMLLIYHSSNLQ
SG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGMYWPYTFGQGTKVEIKR
TAR15-16(SEQ?ID?NO：202)
DIQMTQSPSSLSASVGDRVTITCRASQWIKPALHWYQQKPGKAPKLLIYHGSILQS
GV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTLFMPYTFGQGTKVEIKR
TAR15-17(SEQ?ID?NO：203)
DIQMTQSPSSLSASVGDRVTTTCRASQSISTALLWYQQKPGKAPKLLIYNGSMLPN
GVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQTWDTPMTFGQGTKVEIKR
TAR15-18(SEQ?ID?NO：204)
DIQMTQSPSSLSASVGDRVTITCRASQWIGHDLSWYQQKPGKAPKLLIYHSSSLQS
GV
PSRFSGSGSGTDFTLTISSLQPEDVATYYCQQLMGYPFTFGQGTKVEIKR
TAR15-19(SEQ?ID?NO：205)
DIQMTQSPSSLSASVGDRVTITCRASQDIGGLLVWYQQKPGKAPKLLIYRSSYLQ
SGV
PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTWGIPHTFGQGTKVEIkR
TAR15-20(SEQ?ID?NO：206)
DIQMTQSPSSLSASVGDRVTITCRASQKIFNGLSWYQQKPGKAPKLLIYHSSTLQS
GVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQVLLYPYTFGQGTKVEIKR
TAR?15-22(SEQ?ID?NO：207)
DIQMTQSPSSLSASVGDRVTITCRASQSIGTNLSWYQQKPGKAPRLLIYRTSMLQ
SG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQQFFWPHTFGQGTKVEIKR
VH dAb in conjunction with people VEGF has below been described.These are cloned in and produce among the supernatant liquor PBA (R2) descend (surpassing 50%):
Guiding ?dAb In supernatant liquor RBA (R2), descend and surpass 50% In ELISA with the cross reactivity of mouse VEGF
TAR15-5 ?VH ?+ +
TAR15-6 ?VH ?+ +/-
TAR15-7 ?VH ?+ +/-
TAR15-8 ?VH ?+ +
TAR15-23 VH + -
TAR15-24 VH + -
TAR15-25 VH + -
TAR15-26 VH + +/-
TAR15-27 VH + +/-
TAR15-29 VH + -
TAR15-30 VH + -
The VH clone is with the low density VEGF chip on a certain concentration (50nM) the process BIAcore.Different clones produces different kinetics models.
Aminoacid sequence:
TAR15-5(SEQ?ID?NO：208)
EVQLLESGGGLVQPGGSLRLSCAASGFTFRLYDMVWVRQAPGKGLEWVSYISSGGSGTYYADS
VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGGRASFDYWGQGTLVTVSS
TAR15-6_(SEQ?ID?NO：209)
EVQLLESGGGLVQPGGSLRLSCAASGFTFHLYDMMWVRQAPGKGLEWVSFIGGDGLNTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGTQFDYWGQGTLVTVSS
TAR15-7(SEQ?ID?NO：210)
EVQLLESGGGLVQPGGSLRLSCAASGFTFNKYPMMWVRQAPGKGLEWVSEISPSGQDTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAETAVYYCAKNPQILSNFDYWGQGTLVTVSS
TAR15-8(SEQ?ID?NO：211)
EVQLLESGGGLVQPGGSLRLSCAASGFTFQWYPMWWVRQAPGKGLEWVSLIEGQGDRTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAGDRTAGSRGNSFDYWGQGTLVTVSS
TAR15-23(SEQ?ID?NO：212)
EVQLLESGGGLVQPGGSLRLSCAASGFTFKAYEMGWVRQAPGKGLEWVSGISPNGGWTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKESISPTPLGFDYWGQGTLVTVSS
TAR15-24(SEQ?ID?NO：213)
EVQLLESGGGLVQPGGSLRLSCAASGFTFTGYEMGWVRQAPGKGLEWVSYISRGGRWTYYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSDTMFDYWGQGTLVTVSS
TAR15-2S(SEQ?ID?NO：214)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMGWVRQAPGKGLEWVSFISGGGRWTYYAD
SVKGRFTISRDNSKNTLYLQMNSLFRAEDTAVYYCAKYSEDFDYWGQGTLVTVSS
TAR15-26(SEQ?ID?NO：215)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGAYPMMWVRQAPGKGLEWVSEISPSGSYTYYADS
VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDPRKFDYWGQGTLVTVSS
TAR15-27(SEQ?ID?NO：216)
EVQLLESGGGLVQPGGSLRLSCAASGFTFQFYKMGWVRQAPGKGLEWVSSISSVGDATYYADS
VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKMGGGPPTYVVYFDYWGQGTLVTVSS
TAR15-29(SEQ?ID?NO：217)
EVQLLESGGGLVQPGGSLRLSCAASGFTFGEYGMYWVRQAPGKGLEWVSSISERGRLTYYADS
VKGRFTISRDNSKNTLYLQMNNLRAEDTAVYYCAKSALSSEGFSRSFDYWGQGTLVTVSS
TAR15-30(SEQ?ID?NO：218)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMYWVRQAPGKGLEWVSSITARGFITYYADS
VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSGFPHKSGSNYFDYWGQGTLVTVSS
Embodiment 23: other research of carrying out with anti-VEGF dAb
Can be as above described to various anti-TNF-α dAb, the effectiveness of testing the anti-VEGF dAb of various forms as herein described, these forms comprise for example Fc syzygy, Fab, PEGization form, dimer, the tetramer and anti-SA dual specific form.In addition, not only estimate anti-VEGF dAb, and estimate anti-VEGF dAb with other anti-TNF-α goods such as HUMIRA, ENBREL and/or REMICADE with anti-TNF-α dAb described herein.
Can carry out other research, with the effect of checking anti-VEGF dAb in Tg197 RA model, for example sacroiliitis and histopathology to be kept the score.
For example, in 3 ages (before the RA paresthesia epilepsy) in week or begin when (behind the paresthesia epilepsy) weekly 6 ages in week or be similar to the TAR15 dAb Fc syzygy of above-described TAR1-5-19Fc syzygy with 1mg/kg or 10mg/kg IP twice, and continue to reach 7 weeks or more than.Preferably with equimolar amount, than salt solution, contrast Fc syzygy (anti-β-gal), independent TAR1-5-19, ENBREL, REMICADE and/or HUMIRA judged result.
Writing down macroscopical phenotype indication (for example sacroiliitis is kept the score) and the histopathology of animal as mentioned above keeps the score.Render a service by each following confirmation:
I) when when 3 weeks begin to give animal ages, can not occur the disease symptom (with sacroiliitis or histopathology keep the score be according to),
Ii) than control animal, when the disease symptom seriousness that occurred when 3 weeks, began to give ages weakens,
Iii) than control animal, when can not developing into more serious disease when begin to give ages in 6 weeks or with than the low rate development,
Iv) when when 6 weeks begin to give animal ages, in 7,8,9,10,11,12 or 14 weeks, reversing symptom (utilize sacroiliitis to keep the score once more or histopathology is kept the score) arbitrary week.
Each of available above-mentioned multi-form (for example Fab, PEGization form, dimer, the tetramer and anti-SA dual specific form) is carried out similar research.
Anti-VEGF dAb (for example TAR15 dAb) also can unite with HUMIRA, ENBREL and/or REMICADE and gives Tg197 mouse model.To carry out this research, be used to test independent VEGF dAb, and measure in the same manner and render a service with above-mentioned identical mode.
Embodiment 24: estimate anti-TNF-α dAb with the Crohn disease model
For estimating the validity of anti-TNF-α dAb (and/or anti-VEGF dAb) in the Crom disease, use at first by Kontoyiannis etc., 1999, the TNF that Immunity 10:387-398 describes Δ ARETransgenic mice Crohn disease model (all right similar manner uses the DSS model).The IBD phenotype appears in animal, and the Crohn disease that begins between age in week at 4-8 has similarity.Therefore, when 3 ages in week (to test disease prevention) or 6 all ages (stable, prevention with the test disease symptoms develop or reverse), give anti-TNF-α dAb, for example various forms of TAR1-5-19 (Fc syzygy, Fab, PEGization (dimer, the tetramer etc.), with the dual specific of VEGF, with the dual specific of SA etc.) as described hereinly keep the score to animal by body weight and histology.Preliminary research is used the IP dosage of 1mg/kg and 10mg/kg, adjusts according to the result of these preliminary research.Give test composition once or twice weekly, perhaps can give continuously, for example use osmotic pump.Perhaps, also can use the oral delivery formulation, for example with Zantac per os tube feed or utilize the enteric coating preparation.Research continue at the very start to reach 7 the week or more than.
Following each all shown at TNF Δ AREEffectiveness in the Crohn disease model:
I) when when 3 weeks begin to give animal ages, disease symptoms can not occurring,
Ii) than control animal, when the disease symptoms seriousness that occurred when 3 weeks, began to give ages weakens,
Iii) than control animal, when can not developing into more serious disease when begin to give ages in 6 weeks or with than the low rate development,
Iv) when the arbitrary week reverse symptom in 7,8,9,10,11,12 or 14 weeks when 6 weeks, began to give animal age.
Specifically, be lower than keeping the score of (according to the statistics significant quantity) vehicle Control group if the average tissue pathology disease in the treatment animal is kept the score, then treatment is considered to effective.If the average tissue pathology are kept the score than low 0.5 unit of control group, 1.0 units, 1.5 units, 2.0 units, 2.5 units, 3.0 units or 3.5 units of carrier only at least at least at least at least at least at least at least, then treatment also is considered to effective.Perhaps, if the average tissue pathology are kept the score and remained on or be low to moderate 0-0.5 in whole treatment plan process, then treatment is effective.
The same with the RA model, also with this model evaluation with the effect of the combination therapy of VEGF specificity dAb or other anti-TNF alpha composition (for example ENBREL, REMICADE and/or HUMIRA).
Embodiment 25: the dual specific IgG of anti-humanTNF-and people VEGF
In engineered IgG sample dual specific form described below, two kinds of not homospecific dAb are merged respectively to heavy chain and light chain constant domain.When coexpression in cell, produce the IgG sample molecule of both arms, wherein can be present in conjunction with two kinds of variable domains of two kinds of treatment targets (for example a specific specificity is at TNF-α, and a specific specificity is at VEGF) on each arm of two target IgG.
The DNA construction: the mammalian expression vector of use is based on Invitrogen pcDNA3.1 skeleton, and it helps in the mammalian cell genetic expression through the CMV immediate early promoter.For heavy chain expression, to be inserted into by the expression cassette that people CD33 signal peptide and human IgG1's heavy chain constant domain are formed among the NheI and XbaI restriction site of carrier pcDNA3.1 (+), use HindIII and NotI restriction site, VEGF is specific and be cloned in this expression cassette between CD33 signal peptide and the IgG1 heavy chain constant domain as the variable domains that the part of heavy chain polypeptide is expressed.For light chain expression, to be inserted into by the expression cassette that people CD33 signal peptide and people C κ constant domain are formed among the NheI and XbaI restriction site of carrier pcDNA3.1zeo (+), use HindIII and NotI restriction site, with the TNF-alpha specific and be cloned in this expression cassette between CD33 signal peptide and the C κ constant domain as the variable domains that the part of light chain polypeptide is expressed.
Protein expression and purifying: use Qiagen EndoFree plasmid Mega test kit, the DNA for preparing heavy chain and light chain expression vector according to manufacturer's explanation, and with Roche transfection reagent Fugene ó, according to manufacturer's explanation, use this DNA transfection HEK293 (deriving from European cell culture preservation center) or Cos-7 cell (deriving from American type culture collection).After 5 days, cultivate culture supernatant by centrifugal collection, and use the purifying secreted bi-specific antibody of two step protein affinity purifications.At first, adding culture supernatant to final concentration with phosphate-buffered saline (PBS) is 1.5 * PBS, with antibody capture on Amersham Streamline A protein resin.Clean resin with 2 * PBS, then clean resin, use the antibody of 0.1M glycine pH 2 elution of bound with 10mM Tris pH 8.By among the 1M TrispH8 that adds 25% volume and elutriant, recombinant antibodies is captured on the Affitech L albumen agarose resin.Re-use 2 * PBS and clean resin, then clean resins, use the recombinant antibodies of 0.1M glycine pH 2 elution of bound, by among the 1M Tris pH8 that adds 25% volume and elutriant with 10mM Tris pH 8.
The analysis of recombinant antibodies.On spectrophotometer, be used in the recombinant antibodies of the quantitative purifying of absorbancy that 280nm reads, and use Invitrogen NuPAGE 4-12%Bis-Tris gel and SilverQuest to dye, according to manufacturer's explanation, analyze the recombinant antibodies of this purifying by SDS-PAGE.Figure 29 has shown that the SDS-PAGE of the bi-specific antibody that contains people VEGF specificity κ variable domains (it merges to human IgG1's heavy chain constant domain) and humanTNF-'s specificity κ variable domains (it merges the constant domain to people C κ) analyzes.Swimming lane 1 loads Invitrogen MultiMark molecular weight marker, swimming lane 2 is carried in the bi-specific antibody in 1 * InvitrogenNuPAGE LDS sample buffer, and swimming lane 3 is carried in the bi-specific antibody in 1 * Invitrogen NuPAGE LDS sample buffer of adding the 10mM beta-mercaptoethanol.In swimming lane 3, heavy chain is regarded as the 50kDa band, and light chain is regarded as the 25kDa band.
The check of dual specific.Antibody by detecting each purifying batch in conjunction with the effectiveness in the experiment, confirms the dual specific character of the antibody of expressing at people TNF cell experiment and people's vegf receptor.
(2000, MolecularBiotechnology 15,243-248) the L929 cytotoxicity experiment of Miao Shuing as Evans in the people TNF cellular type experiment of using.Say simply, will be at the L929 cell of microtiter plate upper flat plate inoculation and bi-specific antibody, 100pg/mlTNF and 1mg/ml dactinomycin (Sigma, Poole, UK) incubation that spends the night.By with [3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxyl p-methoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium _ (Promega, Madison USA) read the absorbance detection cell viability of 490nm behind the incubation.Anti-TNF activity causes the TNF cytotoxicity to descend, and therefore causes absorbancy than the only contrast increase of TNF.
The VEGFR2 that uses the chapters and sections description that is entitled as " preparation of immunoglobulin (Ig) type polyspecific part " substantially as mentioned detects the VEGF activity in conjunction with experiment.Say simply, with 0.5 μ g/ml recombinant human VEGF R2/Fc (R﹠amp; D Systems, catalog number (Cat.No.): carbonate buffer solution 357-KD-050) spends the night bag by 96 hole Nunc Maxisorp brassboards.With connecing PBS rinsing hole repeatedly behind the 0.05%Tween/PBS.The PBS solution that adds 2%BSA is with closure plate.Rinsing hole (as mentioned above), the bi-specific antibody with purifying is added in each hole then.The diluent (final concentration 3ng/ml) that in each hole, adds 6ng/ml VEGF then, with plate in room temperature incubation 2 hours.Wash-out hole adds 0.5 μ g/ml biotinylation VEGF antibody (R﹠amp then as mentioned above; D Systems, catalog number (Cat.No.): diluent BAF293), and in room temperature incubation 2 hours.The rinsing hole then adds the anti-biotin antibodies (diluent of dilution in 1: 5000 that HRP puts together as mentioned above; Stratech, catalog number (Cat.No.): 200-032-096).Then with plate in room temperature incubation 1 hour.The rinsing plate guarantees to have removed any residual Tween-20 as mentioned above.For detecting, add 100 μ l SureBlue 1-Component TMB MicroWell superoxide enzyme solution to each hole.By adding 1M hydrochloric acid termination reaction, then use plate reader to read OD 450
Figure 30 has shown the result of the bi-specific antibody that contains people VEGF specificity κ variable domains (it merges to human IgG1's heavy chain constant domain) and humanTNF-'s specificity κ variable domains (it merges the constant domain to people C κ).Bi-specific antibody (representing anti-TNF-α * anti-VEGF) is simultaneously in conjunction with humanTNF-and people VEGF.Antibody is two valencys to two kinds of targets: the ND50 (24nM) of TNF-α significantly is lower than in bispecific molecule merges to the anti-TNF-α monomer (200nM) that merges to C κ as variable domains.The EC50 of VEGF (75pM) is far below merge anti-VEGF monomer (12nM to the heavy chain constant domain as variable domains in bispecific molecule, do not show), also be lower than anti-VEGF monomer by L protein-crosslinking oligomerization (with the line of square display data point, 990pM).
The construction of this embodiment is the dual specific antigen-binding polypeptides construction of 4 valencys, and it comprises the V in conjunction with 2 copies of first epi-position HOr V LSingle domain antibody; With V in conjunction with 2 copies of second epi-position HOr V LSingle domain antibody.All merge to IgG heavy chain constant domain in conjunction with in the single domain antibody of 2 copies of first epi-position each, all merge to the light chain constant domain in conjunction with in the single domain antibody of 2 copies of second epi-position each.
Those skilled in the art for example can use other anti-TNF-α and VEGF antibody sequence (for example described herein any), produce other dual specific 4 valency polypeptide constructions that are similar to the construction that this embodiment describes.In other embodiments, can use C κOr C λThe light chain constant domain also can be used the IgG heavy chain constant domain of non-IgG1.The most interesting material that is used to develop such construction is that anti-TNF-Alpha antibodies clone in single structure territory (its prevent when giving the mouse of Tg197 transgenic mice arthritis model as the dAb monomer sacroiliitis from keeping the score increases) and single structure territory VEGF antibody are cloned (its prevent sacroiliitis from keeping the score increase) when giving the mouse of the sacroiliitis mouse model that collagen protein brings out as the dAb monomer.The anti-TNF-Alpha antibodies clone's in also preferred single structure territory monomer in L929 cytotoxicity experiment as herein described in and the humanTNF-, single structure territory VEGF antibody clone's monomer vegf receptor 2 as herein described in conjunction with experiment in the combination of antagonism vegf receptor.The preferred single domain antibody clone who uses is with the K less than 100nM dIn conjunction with epi-position separately.Also preferred these dual specific 4 valency constructions are with the K less than 100nM dIn conjunction with epi-position separately, and in any Tg197 described herein and CIA arthritis model or both, prevent the sacroiliitis increase of keeping the score.
This construction can give, aspect dosage and the effectiveness monitoring to be used for the treatment of rheumatoid arthritis with the similar mode of other construction described herein.Half life that can modification construction as indicated above, is for example by adding peg moiety and/or to increasing the circulation albumen of half life (for example serum protein, as HSA) specific bound fraction (for example another single domain antibody) being arranged by further merging.
The reference of mentioning in all publications of mentioning in this specification sheets, patent and publication application and the described publication all is attached to herein by reference.The various modifications and the change of the method for the invention and system it will be apparent to those skilled in the art that, and not depart from scope and spirit of the present invention.Although the present invention who describes in conjunction with concrete embodiment preferred should be understood that, the present invention who requires to protect should be confined to this specific embodiments excessively undeservedly.In fact, the various modifications of described embodiment of the present invention are conspicuous to molecular biology or those skilled in the relevant art, and these modifications are intended to belong to the scope of following claim.
Annex 1: the polypeptide that strengthens half life in the body
α-1 glycoprotein (seromucoid) (AAG)
α-1 chymotrypsin inhibitor (ACT)
α-1 antitrypsin (AAT)
α-1 microglobulin (albumen HC) (AIM)
α-2 macroglobulin (A2M)
Antithrombin III (AT III)
APoA-I (Apo A-1)
Apolipoprotein B (Apo B)
Beta-2-microglobulin (β 2M)
Ceruloplasmin (Cp)
Complement component (C3)
Complement component (C4)
C1 esterase inhibitor (C1 INH)
C-reactive protein (CRP)
Cysteine proteinase inhibitor C (Cys C)
Ferritin (FER)
Fibrinogen (FIB)
Fibronectin (FN)
Haptoglobin (Hp)
Hemopexin (HPX)
Immunoglobulin A (IgA)
Immunoglobulin D (IgD)
Immunoglobulin E (IgE)
Immunoglobulin G (IgG)
Immunoglobulin M (IgM)
Light chain immunoglobulin (κ/λ)
Lipoprotein (a) [Lp (a)]
Mannose-binding protein (MBP)
Myohaemoglobin (Myo)
Proplasmin (PSM)
Prealbumin (transthyretin) (PAL)
Retinol conjugated protein (RBP)
Rheumatoid factors, polyclonal (RF)
Serum amyloid A protein (SAA)
Solubility is changeed iron acceptor (sTfR)
Transferrins,iron complexes (Tf)
Annex 2
Pairing The bibliography relevant with therapeutical agent
TNF α/TGF-β ● TGF-β and TNF significantly strengthen arthritis in the ankle joint that is injected into the arthritis model that collagen protein brings out the time.Not effect in the mouse that noncollagen protein is attacked.
TNF α/IL-1 ● TNF and IL-1 be synergy in uveitis pathology.● TNF and IL-1 are at malaria pathology (hypoglycemia, NO) middle synergy.● synergy when TNF and IL-1 induce polymorphonuclear cell (PMN) migration in inflammation.● the PMN in IL-1 and the TNF co-induction peritonaeum soaks into.● IL-1 and TNF co-induction endotheliocyte secretion IL-1.This is very important in inflammation.● independent IL-1 or TNF induce some cellular infiltration in the knee synovial membrane.● IL-1 induces PMN, TNF-monocyte.Because the PMN that increases is so they induce more serious infiltration simultaneously.● the myocardium inhibition (being present in the septicemia) that circulates is the low-level IL-1 and the TNF of co-action.
TNF α/IL-2 ● major part relates to the collaborative activation of killer T cell.
TNF α/IL-3 ● the collaborative stimulation of interleukin-13 and tumor necrosis factor alpha acute myelogenous leukemia protoblast clonal growth is the result that tumor necrosis factor alpha is induced secondary hematopoietic cytokine.Cancer Res.1992 April 15; 52 (8): 2197-201.
TNF α/IL-4 ● IL-4 and TNF co-induction VCAM express on endotheliocyte.Hint has effect in asthma.There is identical result-hint that effect is arranged in RA to synovial membrane.● TNF and IL-4 co-induction IL-6 in keratinocyte expresses.
● can utilize the combination of TNF-α and IL-4 or IL-13, by increasing
MRNA stability, the VCAM-1 level that is implemented among the inoblast sample synovial cell of cultivation continues to promote.Am J Pathol. in April, 1999; 154 (4): 1149-58.
TNF α/IL-5 ● in adult and children's segmental bronchus overreaction thereof system of tumor necrosis factor and serum interleukin-4, interleukin-5, interleukin-8, have a liking for the relation between eosin cationic protein and the immunoglobulin (Ig) B level.The Allergy Asthma Proc.2003 3-4 month; 24 (2): 111-8.
TNF α/IL-6 ● TNF and IL-6 are effective somatomedins of new human myeloma cell line OH-2.EurJ Haematol.1994 July; 53 (1): 31-7.
TNF α/IL-8 ● TNF and IL-8 and PMN work in coordination with activated blood platelet.Participate in acute respiratory distress syndrome.● referring to IL-5/TNF (asthma).The synergy of the platelet activation that between IL-8 and the tumor necrosis factor alpha neutrophilic granulocyte is mediated.The Eur Cytokine Netw.1994 9-10 month; 5 (5): 455-60.(adult respiratory distress syndrome (ARDS))
TNF α/IL-9
TNF α/IL-10 ● IL-10 induces and expresses with HIV in the chronically infected T cell of TNF co-induction.
TNF α/IL-11 ● cytokine co-induction osteoclast differentiation: support by immortalization or normal braincap cell.Am J Physiol Cell Physiol.2002 September; 283 (3): C679-87.(bone loss)
TNF α/IL-12
TNF α/IL-13 ● can utilize the combination of TNF-α and IL-4 or IL-13, by increasing mRNA stability, the VCAM-1 level that is implemented among the inoblast sample synovial cell of cultivation continues to promote.Am J Pathol. in April, 1999; 154 (4): 1149-58.
● IL-13 and tumor necrosis factor alpha are collaborative in people's nose inoblast
Induce eotaxin (Eotaxin) to produce.Clin Exp Allergy.2000 March; 30 (3): 348-55.
● IL-13 and tumor necrosis factor alpha co-induction in people's nose inoblast is bitten acid granulocyte chemokine (Eotaxin) and is produced.Clin Exp Allergy.2000 March; 30 (3): 348-55.(alterative inflammation) ● serum TG F-β and IL-13 are related with child's nephrotic syndrome therapeutic response.Cytokine.2003 February 7; 21 (3): 155-9.
TNF α/IL-14 ● the effect of the tumor necrosis factor alpha of suction in slight asthmatic patient.Thorax.2002 September; 57 (9): 774-8.
TNF α/IL-15 ● the effect of the tumor necrosis factor alpha of suction in slight asthmatic patient.Thorax.2002 September; 57 (9): 774-8.
TNF α/IL-16 ● tumor necrosis factor alpha induces IL-16 synthetic in tracheal epithelial cell: thrombotonin stimulates triggering.Am J Respir Cell Mol Biol. in March, 2003; 28 (3): 354-62.(trachitis) ● interrelated in patient with rheumatoid arthritis of circulation IL-16 and pro-inflammatory cytokine.Rheumatology (Oxford) .2002 April; 40 (4): 474-5.Do not obtain summary.● IL-6 is raised in the Crohn disease, and participates in the TNBS colitis in the mouse.Gastroenterology.2000 October; 119 (4): 972-82.
TNF α/IL-17 ● being suppressed at of IL-17 prevents the sacroiliitis development in the inoculation mouse of attacking with Borrelia burgdoyferi.Infect Immun.2003 June; 71 (6): 3437-42.● IL-17 and the external cartilage destruction of tumor necrosis factor alpha co-induction.Ann Rheum Dis.2002 October; 61 (10): 870-6.● GM-CSF is in the tracheae neutrophilia that is caused by IL-17 and TNF-α
Effect in the granulocyte accumulation.Eur Respir is year March J.2003; 21 (3): 387-93.(trachitis) ● summary: collaborative nitrogen protoxide and the prostaglandin E2 production of going up in mediator's osteoarthritis knee meniscal implantation body of IL-1, tumor necrosis factor alpha and IL-17.Arthritis Rheum.2001 September; 44 (9): 2078-83.
TNP α/IL-18 ● the IL-18 expression is relevant with the two level increase of tumor necrosis factor alpha with the IL-1 β in the patient with rheumatoid arthritis knee synovial tissue.Arthritis Rheum.2003 February; 48 (2): 339-47.● summary: IL-18 and the tumor necrosis factor alpha level in the diabetes B patients serum promotes: with the relation of diabetic nephropathy.Metabolism.2003 May; 52 (5): 605-8.
TNF α/IL-19 ● summary: IL-19 induces IL-6 and TNF-α to produce, and causes apoptosis by TNF-α.J Immunol.2002 October 15; 169 (8): 4288-97.
TNF α/IL-20 ● new effect of summary: cytokine: IL-20-in skin inflammation.Curr Biol.2001 July 10; 11 (13): R531-4.
TNF α/complement ● inflammation and aggegation: relate to septic patient.Clin Infect Dis.2003 May 15; 36 (10): 1259-65.Electronic edition, on May 8th, 2003 summarized
TNF α/IFN-γ ● the MNC in brain induces ● collaborative in antiviral response/IFN-is beta induced ● neutrophil activation/respiratory burst ● activated endothelial cell ● be recorded to toxicity when treating the patient with TNF/IFN-γ as antiviral therapy
● the Astrocytic Fractalkine of people expresses
● many about inflammatory reaction-be the LIP paper of (also being called macrophage activation)
● anti-TNF and anti-IFN-γ coordinating protection mouse avoid the lethality endotoxemia
TGF-β/IL-1 ● osteoclast synthesis of prostaglandins ● intestinal epithelial cell (inflammatory model) produces IL-6 ● at lung fibroblast (inflammatory model) moderate stimulation IL-11 and IL-6 ● in retina, produce IL-6 and IL-8
TGF-β/IL-6 ● chrondrocarcinoma propagation
IL-1/IL-2 ● B cell activation ● LAK cell activation ● T cell activation ● IL-1 and IL-2 be co-action in the lymphokin activated killer cells of TNF-α and β (lymphotoxin) mediation is produced.Cytokine. in November, 1992; 4 (6): 479-87.
IL-1/IL-3
IL-1/IL-4 ● B cell activation ● IL-4 induces IL-1 to express in activated endothelial cell
IL-1/IL-5
IL-1/IL-6 ● B cell activation ● T cell activation, (alternative helper) ● IL-1 induces IL-6 to express ● C3 and serum amyloid protein are expressed, (acute phase reaction) ● HIV expresses ● the chondrogen proteolysis
IL-1/IL-7 ● IL-7 is that IL-1 inductive thymocyte proliferation is essential.IL-7 relates to the synergy of granulocyte-macrophage colony stimutaing factor or tumour necrosis factor and IL-1.J Immunol.1992 January;
148 (1): 99-105
IL-1/IL-8
IL-1/IL-10
IL-1/IL-11 ● cytokine co-induction osteoclast differentiation: support by immortalization or normal braincap cell.Am J Physiol Cell Physiol.2002 September; 283 (3): C679-87.(bone loss)
IL-1/IL-16 ● interrelated in patient with rheumatoid arthritis of circulation IL-16 and pro-inflammatory cytokine.Rheumatology (Oxford) .2002 April; 40 (4): 474-5.Do not obtain summary
IL-1/IL-17 ● being suppressed at of IL-17 prevents the sacroiliitis development in the inoculation mouse of attacking with Borrelia burgdoyferi.Infect Immun.2003 June; 71 (6): 3437-42.● IL-7 impels human cartilage degraded and synovial membrane inflammation in osteoarthritis.Osterarthritis Cartilage.2003 June; 71 (6): 3437-42 ● summary: collaborative nitrogen protoxide and the prostaglandin E2 production of going up in mediator's osteoarthritis knee meniscal implantation body of IL-1, tumor necrosis factor alpha and IL-17.Arthritis Rheum.2001 September; 44 (9): 2078-83.
IL-1/IL-18 ● the IL-18 expression is relevant with the two level enhancing of tumor necrosis factor alpha with the IL-1 β in the patient with rheumatoid arthritis knee synovial tissue.Arthritis Rheum.2003 February; 48 (2): 339-47.
IL-1/IFN-g ●
IL-2/IL-3 ● T cell proliferation ● B cell proliferation
IL-2/IL-4 ● B cell proliferation ● T cell proliferation ● (selective induction activation CD8 and NK lymphocyte) IL-2R beta-agonists P1-30 and IL-2, IL-4, IL-9 and IL-15 have worked in coordination with work
With: biology and molecularity.J.Immunol.2000 October 15; 165 (8): 4312-8.
IL-2/IL-5 ● B cell proliferation/Ig secretion.● IL-5 induces the IL-2 acceptor on the B cell.
IL-2/IL-6 ● cytotoxic T cell appears.
IL-2/IL-7 ●
IL-2/IL-9 ● see IL-2/IL-4 (NK cell).
IL-2/IL-10 ● the B cell activation.
IL-2/IL-12 ● the cytotoxicity of IL-12 and IL-2 co-induction lymphokineactivation in fresh NK cells of human beings and granzyme genetic expression.Cell Immunol.1995 October 1; 165 (1): 33-43. (T cell activation)
IL-2/IL-15 ● see IL-2/IL-4 (NK cell) ● (T cell activation and propagation) IL-15 and IL-12: the life of T cell and dead problem in the body.Nat Med.2001 January; 7 (1): 114-8.
IL-2/IL-16 ● the collaborative activation of IL-16 and IL-2 CD4+T cell.J Immunol. on March 1st, 1998; 160 (5): 2115-20
IL-2/IL-17 ● the early stage evidence that participates in of IL-17 in people and experimental rectum allograft rejection.J Pathol.2002 July; 197 (3): 322-32
IL-2/IL-18 ● il-1 8 (IL-18) and IL-2 co-induction lethality injury of lung in mouse: cytokine, chemokine and the natural killer cell latent effect in the interstitial pneumonia disease is sent out.Blood.2002 February 15; 99 (4): 1289-98.
IL-2/TGF-β ● the control of CD4 effector destiny: collaborative apoptosis and accelerating effect of preventing of transforminggrowthfactor-and IL-2 increases.J Exp Med on September 1st, 1995; 182 (3): 699-709.
IL-2/IFN-γ ● B emiocytosis Ig
● the IFN-γ of IL-2 inducing T cell expresses
IL-2/IFN-α/β ● do not have
IL-3/IL-4 ● the synergy in the mastocyte growth ● the synergy that IL-4 and GM-CSF or IL-3 express the CD23 that induces the person monocytic cell: the regulating effect of IFN-α and IFN-γ.Cytokine.1994 July; 6 (4): 407-13
IL-3/IL-5
IL-3/IL-6
IL-3/IFN-γ ● the collaborative total multimerization IgA receptor level that increases in people's intestinal epithelial cell of IL-4 and IFN-γ.The effect of protein tyrosine kinase.J Immunol.1996 June 15; 156 (12): 4807-14.
IL-3/GM-CSF ● cytokine is regulated people's eosinocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 acceptor alpha expression, lose the IL-5 reactivity, but raise IL-3 acceptor alpha expression.J Immunol.2003 June 1; 170 (11): 5359-66. (alterative inflammation)
IL-4/IL-2 ● collaborative IL-2 and the two the inductive IFN-γ of IL-12 that strengthens in the mouse NK cell of IL-4 expresses.Blood.2003 March 13.(electronic edition before publishing)
IL-4/IL-5 ● strengthen the secretions such as histamine of mastocyte under the IgE effect ● the reaction of the Th2-like cell factor participates in bullous pemphigoid.IL-4 and the IL-S effect in disease pathogenesis.The Int J Immunol Pharmacol.1999 5-8 month; 12 (2): 55-61.
IL-4/IL-6
IL-4/IL-10
IL-4/IL-11 ● between IL-11 and the IL-4 to supporting the cooperative interaction of mouse hematopoiesis group originally cell proliferation.Blood.1991 September 15;
78 (6): 1448-51
IL-4/IL-12 ● IL-4 and IL-18 are to the synergy of the IL-12 dependency IFN-γ production of dendritic cell.J Immunol.2000 January 1; 164 (1): 64-71. (increasing the Th1/Th2 differentiation) ● IL-2 and the two inductive IFN-γ of IL-12 express in the collaborative enhancing of the IL-4 mouse NK cell.Blood.2003 March 13.[electronic publication before publishing]
IL-4/IL-13 ● summary: IL-4 and IL-13 signal transduction contact figure.Science.2003 June 6; 300 (5625): 1527-8. (transformation reactions, asthma) ● suppress the IL-4/IL-13 receptor system and prevent allergic sensitization, and do not influence transformation reactions existing in the atopic asthma mouse model.J Allergy Clin Immunol.2003 June; 111 (6): 136-1369.
IL-4/IL-16 ● it is the IL-16 production of BEAS-2B cell that (asthma) interleukin (IL)-4/IL-9 and external source IL-16 induce bronchial epithelial cell.Cell Immunol.2001 February 1; 207 (2): 75-80.
IL-4/IL-17 ● the fibroblastic IL-6 of the collaborative people of stimulation of interleukin (IL)-4/IL-9 and IL-17 colonic muscle secretes.Int J Mol Med.2002 November; 10 (5): 631-4.(enteritis)
IL-4/IL-24 ● IL-24 is expressed by rat and human macrophage.Immunobiology. in July, 2002; 205 (5): 321-34.
IL-4/IL-25 ● summary: new IL-17 family member promotes Th1 or the Th2 reaction in the lung: function in the body of novel cytokine IL-25.J Immunol. on July 1st, 2002; 169 (1): 443-453.(alterative inflammation) ● summary: mastocyte is produced IL-25 when Fcepsilon RI mediation activation.Blood.2003 May 1; 101 (9): 3594-6.Electronic edition on January 2nd, 2003.(alterative inflammation)
IL-4/IFN-γ ● summary: the IL-4 inducing endothelial cell is produced IL-6: with IFN-γ co-action.Eur J Immunol.1991 January; 21 (1): 97-101
IL-4/SCF ● the little intestinalmast cell of STEM CELL FACTOR and IL-4 mediator.Immunol Rev.2001 February; 179:57-60. summary
IL-5/IL-3 ● cytokine is regulated people's eosinocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 acceptor alpha expression, lose the IL-5 reactivity, but raise IL-3 acceptor alpha expression.J Immunol.2003 June 1; 170 (11): 5359-66. (alterative inflammation is referring to summary)
IL-5/IL-6
IL-5/IL-13 ● utilize dexamethasone in mouse, to suppress allergy trachitis and tracheae hyperreactive: the effect of eosinocyte, IL-5, eotaxin (Eotaxin) and IL-13.J Allergy Clin Immunol.2003 May; 111 (5): 1049-61
IL-5/IL-17 ● IL-17 organizes granulocyte to flow in the tracheae after the atopic asthma mouse model sucks allergen.Am JRespir Cell Mol Biol.2003 January; 28 (1): 42-50
IL-5/IL-25 ● summary: new IL-17 family member promotes Th1 or the Th2 reaction in the lung: function in the body of novel cytokine IL-25.J Immunol. on July 1st, 2002; 169 (1): 443-453.(alterative inflammation) ● summary: mastocyte is produced IL-25 when Fcepsilon RI mediation activation.Blood.2003 May 1; 101 (9): 3594-6.Electronic edition on January 2nd, 2003.(alterative inflammation)
IL-5/IFN-γ
IL-5/GM-CSF ● cytokine is regulated people's eosinocyte IL-3, IL-5 and GM-CSF receptor alpha chain expression difference: IL-3, IL-5 and GM-CSF downward modulation IL-5 acceptor alpha expression, lose the IL-5 reactivity, but raise
IL-3 acceptor alpha expression.J Immunol.2007 June 1; 170 (11): 5359-66. (alterative inflammation)
IL-6/IL-10
IL-6/IL-11
IL-6/IL-16 ● IL-16 stimulates the expression and the production of person monocytic cell's pro-inflammatory cytokine.Immunology.2000 May; 100 (1): 63-9.
IL-6/IL-17 ● utilize interleukin (IL)-17 to stimulate the tracheae mucin gene to express by IL-6 paracrine/autocrine loop.J Biol Chem.2003 May 9; 278 (19): 17036-43.Electronic edition on May 6th, 2003.(trachitis, asthma)
IL-6/IL-19 ● summary: IL-19 induces IL-6 and TNF-α to produce, and causes apoptosis by TNF-α.J Immunol.2002 October 15; 169 (8): 4288-97
IL-6/IFN-g
IL-7/IL-2 ● IL-7 worsens graft versus host disease.Blood.2002 October 1; 100 (7): 2642-9.
IL-7/IL-12 ● IL-7 and IL-12 act synergistically to human T-cell's activatory.J Immunol.1995 May 15; 154 (10): 5093-102
IL-7/IL-15 ● IL-7 and IL-15 regulate bc1-2 and the c-myb expression of gene in the cutaneous T cell lymphoma cell.Blood.2001 November 1; 98 (9): 2778-83.(somatomedin)
IL-8/IL-11 ● interleukin (IL)-11 and the IL-8 unusual generation in polycythemia vera.Cytokme.2002 November 21; 20 (4): 178-83.
IL-8/IL-17 ● the effect of IL-17 in destruction of joint.Drug News Perspect. in January, 2002; 15 (1): 17-23.(sacroiliitis) ● summary: IL-17 stimulates people's tracheae epithelial cell to express IL-8, the relevant oncogene-α of growth and granulocyte-G CFS.Am?J
Respir Cell Mol Biol.2002 June; 26 (6): 748-53. (trachitis)
IL-8/GSF ● IL-8: play the collaborative autocrine/paracrine somatomedin that promotes the artificial blood progenitor cell of monocyte-scavenger cell growth and Differentiation with colony-stimulating factor 1.Exp Hematol.1999 January; 27 (1): 28-36
IL-8/VEGF ● VEOF, bFGF, IL-8, IL-12 level in the chamber in initial and recurrence glioblastoma.J Neurooncol.2003 May; 62 (3)-297-303.
IL-9/IL-4 ● anti-IL-9 Antybody therapy suppresses trachitis and hyperreactive in mouse asthmatic model.Am JRespir Crit Care Med.2002 August 1; 166 (3): 409-16.
IL-9/IL-5 ● induced expression Th2 cytokine-expressing is crossed by the lung of IL-9, causes immune symptom.J Clin Invest.2002 January; 109 (1): 29-39. ● Th2 cytokine and asthma.IL-9 is as the treatment of asthma target.Respir Res.2001; 2 (2): 80-4.2001 electronic edition on February 15 summary ● summary: IL-9 strengthens the eosinophilic IL-5 expression of receptor of people, differentiation and survival.Blood.2000 September 15; 96 (6): 2163-71.(asthma)
IL-9/IL-13 ● anti-IL-9 Antybody therapy suppresses trachitis and hyperreactive in mouse asthmatic model.Am J Respir Crit Care Med.2002 August 1; 166 (3): 409-16. ● IL-13 causes tracheae hyperreactive and the excessive production of mucus to epithelial acting directly in the asthma.NatMed.2002 August; 8 (8): 885-9
IL-9/IL-16 ● see IL-4/IL-16
IL-10/IL-2 ● interleukin-10 (IL-10) and interleukin-2 (IL-2) are anti-at hormone immunity
Interaction in answering: IL-b and IL-2 are with the collaborative reaction that strengthens human B lymphocyte of the mechanism different with raising the CD25 expression.Cell Immunol.1994 September; 157 (2): 478-88.
IL-10/IL-12
IL-10/TGF-β ● IL-10 and TGF-β work in coordination with metering needle to the allergenic t cell responses of mucous membrane in normal immunity and specific active immunotherapy.Eur J Immunol.2003 May; 33 (5): 1205-14.
IL-10/IFN-γ
IL-11/IL-6 ● IL-6 and IL-11 utilize non-RANKL dependency mechanism backer osteoclast to form.Bone.2003 January; 32 (1): 1-7. (bone resorption in the inflammation)
IL-11/IL-17 ● polarization IL-11 and IL-17 expression in vivo between the acute and chronic skin focus.J Allergy Clin Immunol.2003 April; 111 (4): 875-81. (allergic dermatitis) ● the receptor activator of IL-17 by losing NP-κ B part/protect ossein is equilibrated in the collagen protein inductive mouse sacroiliitis and promotes bone erosion.J Immunol.2003 March 1; 170 (5): 2655-62.
IL-11/TGF-β ● IL-11 between the acute and chronic skin focus and IL-17 polarization expression in vivo.J Allergy Clin Immunol.2003 April; 111 (4): 875-81. (allergic dermatitis)
IL-12/IL-13 ● IL-12 and IL-13 uneven and type specificity Rheumatoid factors, polyclonal and the relation of anticardiolipin antibody in systemic lupus erythematous.Clin Rheumatol.2003 May; 22 (2): 107-11
IL-12/IL-17 ● IL-12 in active inflammatory bowel and the rise of IL-17.Scand J Gastroenterol.2003 February; 38 (2): 180-5
IL-12/IL-18 ● collaborative propagation of IL-12 and IL-18 and active natural killer cell.Cyrokine.1999 November; 11 (11): 822-30 ● the inflammatory fatty degeneration of liver that IL-12 and IL-18 cause.JInterferon
Cytokine Res.2003 March; 23 (3): 155-62.
IL-12/IL-23 ● IL-23 rather than IL-12 are the key cytokines of brain autoimmunization inflammation.Nature.2003 February 13; 421 (6924): 744-8.● the unique effect of summary: IL-23 in promoting cellular immunity.J Leukoc Biol.2003 January; 73 (1): 49-56.Summary
IL-12/IL-27 ● summary: a kind of different dimerization cytokine IL-27 that is made up of EBI3 and p28 albumen induces CD4 (+) T cell proliferation originally.Immunity.2002 January; 16 (6): 779-90.
IL-12/IFN-γ ● IL-12 induces and is the B of an immunostimulation part and T cell expressing IFN-γ.
IL-13/IL-5 ● see IL-5/IL-13.
IL-13/IL-25 ● summary: new IL-17 family member promotes Th1 or the Th2 reaction in the lung: function in the body of new cytokine IL-25.J Immunol. on July 1st, 2002; 169 (1): 443-53.(alterative inflammation).● summary: mastocyte is produced IL-25 when Fcepsilon RI mediation activation.Blood.2003 May 1; 101 (9): 3594-6.Electronic edition on January 2nd, 2003.(alterative inflammation).
IL-15/IL-13 ● the differential expression of interleukin (IL)-13 and IL-15 in the women's of film dystopy women and normal reproductive performance dystopy and the normotopia uterine endometrium in uterus.AmJ Reprod Immunol.2003 February; 49 (2): 75-83.
IL-15/IL-16 ● IL-15 and IL-16 in cutaneous T cell lymphoma cross expression: the stage dependency in the mycosis fungoides progress increases.Exp Dermatol.2000 August; 9 (4): 248-51.
IL-15/IL-17 ● summary: the IL-17 of lymphocyte and neutrophilic granulocyte production is that lipopolysaccharide-induced tracheae neutrophilia is necessary: IL-15 is possible triggering agent.J Immunol.2003 February 15; 170 (4): 2106-12. (trachitis).
IL-15/IL-21 ● the IFN-γ in collaborative people NK of enhancing of IL-21 and IL-15 or IL-18 and the T cell produces.J Immunol.2003 January 1; 170 (11): 5464-9.
IL-17/IL-23 ● IL-23 promotes that to produce IL-17 be the different cd4 t cell active state of feature.J Biol Chem.2003 January 17; 278 (3): 1910-4. electronic edition on November 3rd, 2002.
IL-17/TGF-β ● IL-11 between the acute and chronic skin focus and IL-17 polarization expression in vivo.J Allergy Clin Immunol.2003 April; 111 (4): 875-81. (allergic dermatitis).
IL-18/IL-12 ● IL-12 and IL-18 are collaborative to be strengthened and the active natural killer cell.Cytokine.1999 November; 11 (11): 822-30.● summary: the inhibition that IL-12 produces external immunoglobulin (Ig) in mouse chronic graft versus host disease: with the synergy of IL-18.Eur J Immunol.1998 June; 28 (6): 2017-24.
IL-18/IL-21 ● the IFN-γ in collaborative people NK of enhancing of IL-21 and IL-15 or IL-18 and the T cell produces.J Immunol.2003 January 1; 170 (11): 5464-9.
IL-18/TGF-β ● IL-18 and transforminggrowthfactor-in the Grave persons suffering from ocular disorders serum of corticosteroid treatment.Int Immunopharmacol.2003 April; 3 (4): 549-52.
IL-18/IFN-γ ●
Anti-TNF alpha/anti-CD4 ● the synergistic therapeutic action in DAB/1 sacroiliitis mouse
Annex 3: oncology combination
Target Disease Pairing
CD89 * Raise thing as cytotoxic cell All
CD19 B cell lymphoma HLA-DR CD5
HLA-DR B cell lymphoma CD89 CD19 CD5
CD38 Multiple myeloma CD138 CD56 HLA-DR
CD138 Multiple myeloma CD38 CD56 HLA-DR
CD138 Lung cancer CD56 CEA
CD33 Acute marrow lymphoma CD34 HLA-DR
CD56 Lung cancer CD138 CEA
CEA General cancer The MET acceptor
VEGF General cancer The MET acceptor
Vegf receptor General cancer The MET acceptor
IL-13 Asthma/pneumonia IL-4 IL-5 Eotaxin MDC TARC TNFα IL-9 EGFR
CD40L IL-25 MCP-1 TGF-β
IL-4 Asthma IL-13 IL-5 Eotaxin MDC TARC TNFα IL-9 EGFR CD40L IL-25 MCP-1 TGF-β
Eotaxin Asthma IL-5 Eotaxin-2 Eotaxin-3
EGFR Cancer HER2/neu HER3 HER4
HER2 Cancer HER3 HER4
TNFR1 The RA/Crohn disease IL-1R IL-6R IL-18R
TNFα The RA/Crohn disease IL-1α/β IL-6 IL-18 ICAM-1
IL-15 IL-17
IL-1R The RA/Crohn disease IL-6R IL-18R
IL-18R The RA/Crohn disease IL-6R
Annex 4:
Data are summed up
Target dAb Equilibrium dissociation constant (Kd=Koff/Kon) Koff The IC50 of part experiment In the cellular type and the experiment ND50
TAR1 The TAR1 monomer 300nM-5pM (promptly 3 * 10 -7-5×10 -12), preferred 50nM-20pM 5×10 -1-1×10 -7 500nM-100pM 500nM-50pM
The TAR1 dimer With the TAR1 monomer With the TAR1 monomer With the TAR1 monomer With the TAR1 monomer
The TAR1 tripolymer With the TAR1 monomer With the TAR1 monomer With the TAR1 monomer With the TAR1 monomer
TAR1-5
TAR1-27
The TAR1-5-19 monomer 30nM
The TAR1-5-19 homodimer Has (Gly 4Ser) 3Joint=20nM has (Gly 4Ser) 5Joint=2nM has (Gly 4Ser) 5Joint=10nM Fab form=1nM 30nM ＝3nM ＝15nM
The TAR1-5-19 heterodimer Has (Gly 4Ser) nJoint TAR1-5-19 d2=2nM 8nM TAR1-5-19 d4=2-5nM TAR1-5-19 d5=8nM Fab form TAR1-5-19 CH d1 CK=6nM TAR1-5-19 CK d1 CH=6nM ? ＝12nM ? ? ? ＝12nM
TAR1-5-19?CH?d2?CK＝8nM
TAR1-5-19?CH?d3?CK＝3nM
The TAR1-5 heterodimer Has (Gly 4Ser) nJoint TAR1-5 d1=30nM TAR1-5 d2=50nM TAR1-5 d3=300nM TAR1-5 d4=3nM TAR1-5 d5=200nM TAR1-5 d6=100nM Fab form ? ? ? ? ? ? ＝60nM
TAR1-5?CH?d2?CK＝30nM TAR1-5?CK?d3?CH＝100nM
The TAR1-5 homotrimer 0.3nM 3-10nM (for example 3nM)
TAR2 The TAR2 monomer With the TAR1 monomer With the TAR1 monomer 500nM-100pM 500nM-50pM
TAR2-10
TAR2-5
Serum albumin Anti-SA monomer 1mM-500 μ M, preferred 100nM-10 μ M dual specific form, the target affinity is the 1-100 of SA dAb affinity, 000 * affinity, for example 100 pM (target) and 10 μ M SA affinitys 1mM-500 μ M, preferred 100 nM-10 μ M dual specific forms, the target affinity is the 1-100 of SA dAb affinity, 000 * affinity, for example 100pM (target) and 10 μ M SA affinitys
MSA-16 200nM
MSA-26 70nM
Sequence table
<110>Domantis?Ltd.
Ignatovich，Olga
DeWildt，Rudolph?M.T.
Benjamin，Woolven
Grant，Steven
Jones，Philip
Basran，Amrik
Brewis，Neil
＜120〉be used for the treatment of the composition and the method for inflammatory diseases
<130>P59456L-WO；8039/2108
＜140〉also do not specify
<141>2005-06-30
<150>PCT/GB2004/002829
<151>2004-06-30
<150>US?10/925,366
<151>2004-08-24
<150>US?11/098,758
<151>2005-04-04
<160>351
＜170〉PatentIn version 3 .1
<210>1
<211>116
＜212〉protein
＜213〉people
<400>1
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Tyr?Gly?Ala?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>2
<211>348
<212>DNA
＜213〉people
<400>2
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttagc?agctatgcca?tgagctgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgtgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaagttat 300
ggtgcttttg?actactgggg?ccagggaacc?ctggtcaccg?tctcgagc 348
<210>3
<211>108
＜212〉protein
＜213〉people
<400>3
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Thr?Pro?Asn
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>4
<211>324
<212>DNA
＜213〉people
<400>4
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattagc?agctatttaa?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?ggcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?agttacagta?cccctaatac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>5
<211>120
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial antibody's structural domain sequence
<400>5
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Arg?Ile?Ser?Asp?Glu
20 25 30
Asp?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Tyr?Gly?Pro?Ser?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Ser?Ala?Leu?Glu?Pro?Leu?Ser?Glu?Pro?Leu?Gly?Phe?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>6
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial antibody's structural domain sequence
<400>6
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr?Ser?Thr?Pro?Asn
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>7
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic joint sequence
<400>7
Gly?Gly?Gly?Gly?Ser
1 5
<210>8
<211>15
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic joint sequence
<400>8
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1 5 10 15
<210>9
<211>25
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic joint sequence
<400>9
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
1 5 10 15
Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
20 25
<210>10
<211>35
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic joint sequence
<400>10
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
1 5 10 15
Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
20 25 30
Gly?Gly?Ser
35
<210>11
<211>15
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic joint sequence
<400>11
Glu?Pro?Lys?Ser?Gly?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro
1 5 10 15
<210>12
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial antibody's structural domain sequence
<220>
＜221〉further feature
<222>(115)..(116)
＜223〉X=terminator codon
<400>12
Trp?Ser?Ala?Ser?Thr?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu
1 5 10 15
Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln
20 25 30
Ser?Ile?Asp?Ser?Tyr?Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala
35 40 45
Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro
50 55 60
Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile
65 70 75 80
Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val
85 90 95
Val?Trp?Arg?Pro?Phe?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105 110
Arg?Cys?Xaa?Xaa?Gly?Ser?Gly
115
<210>13
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉DNA of coding artificial antibody structural domain sequence
<400>13
tggagcgcgt?cgacggacat?ccagatgacc?cagtctccat?cctctctgtc?tgcatctgta 60
ggagaccgtg?tcaccatcac?ttgccgggca?agtcagagca?ttgatagtta?tttacattgg 120
taccagcaga?aaccagggaa?agcccctaag?ctcctgatct?atagtgcatc?cgagttgcaa 180
agtggggtcc?catcacgttt?cagtggcagt?ggatctggga?cagatttcac?tctcaccatc 240
agcagtctgc?aacctgaaga?ttttgctacg?tactactgtc?aacaggttgt?gtggcgtcct 300
tttacgttcg?gccaagggac?caaggtggaa?atcaaacggt?gctaataagg?atccggc 357
<210>14
<211>39
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic PCR primer
<400>14
tggagcgcgt?cgacggacat?ccagatgacc?cagtctcca 39
<210>15
<211>39
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic PCR primer
<400>15
ttagcagccg?gatccttatt?agcaccgttt?gatttccac 39
<210>16
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the CDR sequence of synthetic antibody structural domain
<220>
＜221〉further feature
<222>(1)..(3)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(1)..(3)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(5)..(5)
＜223〉X=arbitrary amino acid
<400>16
Xaa?Xaa?Xaa?Leu?Xaa
1 5
<210>17
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<220>
＜221〉further feature
<222>(1)..(1)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(4)..(4)
＜223〉X=arbitrary amino acid
<400>17
Xaa?Ala?Ser?Xaa?Leu?Gln?Ser
1 5
<210>18
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<220>
＜221〉further feature
<222>(3)..(6)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(8)..(8)
＜223〉X=arbitrary amino acid
<400>18
Gln?Gln?Xaa?Xaa?Xaa?Xaa?Pro?Xaa?Thr
1 5
<210>19
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>19
Ser?Ser?Tyr?Leu?Asn
1 5
<210>20
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>20
Arg?Ala?Ser?Pro?Leu?Gln?Ser
1 5
<210>21
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>21
Gln?Gln?Thr?Tyr?Ser?Val?Pro?Pro?Thr
1 5
<210>22
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>22
Ser?Ser?Tyr?Leu?Asn
1 5
<210>23
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>23
Arg?Ala?Ser?Pro?Leu?Gln?Ser
1 5
<210>24
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>24
Gln?Gln?Thr?Tyr?Arg?Ile?Pro?Pro?Thr
1 5
<210>25
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>25
Phe?Lys?Ser?Leu?Lys
1 5
<210>26
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>26
Asn?Ala?Ser?Tyr?Leu?Gln?Ser
1 5
<210>27
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>27
Gln?Gln?Val?Val?Tyr?Trp?Pro?Val?Thr
1 5
<210>28
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>28
Tyr?Tyr?His?Leu?Lys
1 5
<210>29
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>29
Lys?Ala?Ser?Thr?Leu?Gln?Ser
1 5
<210>30
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>30
Gln?Gln?Val?Arg?Lys?Val?Pro?Arg?Thr
1 5
<210>31
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>31
Arg?Arg?Tyr?Leu?Lys
1 5
<210>32
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>32
Gln?Ala?Ser?Val?Leu?Gln?Ser
1 5
<210>33
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>33
Gln?Gln?Gly?Leu?Tyr?Pro?Pro?Ile?Thr
1 5
<210>34
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>34
Tyr?Asn?Trp?Leu?Lys
1 5
<210>35
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>35
Arg?Ala?Ser?Ser?Leu?Gln?Ser
1 5
<210>36
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>36
Gln?Gln?Asn?Val?Val?Ile?Pro?Arg?Thr
1 5
<210>37
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>37
Leu?Trp?His?Leu?Arg
1 5
<210>38
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>38
His?Ala?Ser?Leu?Leu?Gln?Ser
1 5
<210>39
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>39
Gln?Gln?Ser?Ala?Val?Tyr?Pro?Lys?Thr
1 5
<210>40
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>40
Phe?Arg?Tyr?Leu?Ala
1 5
<210>41
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>41
His?Ala?Ser?His?Leu?Gln?Ser
1 5
<210>42
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>42
Gln?Gln?Arg?Leu?Leu?Tyr?Pro?Lys?Thr
1 5
<210>43
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>43
Phe?Tyr?His?Leu?Ala
1 5
<210>44
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>44
Pro?Ala?Ser?Lys?Leu?Gln?Ser
1 5
<210>45
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>45
Gln?Gln?Arg?Ala?Arg?Trp?Pro?Arg?Thr
1 5
<210>46
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>46
Ile?Trp?His?Leu?Asn
1 5
<210>47
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>47
Arg?Ala?Ser?Arg?Leu?Gln?Ser
1 5
<210>48
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>48
Gln?Gln?Val?Ala?Arg?Val?Pro?Arg?Thr
1 5
<210>49
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>49
Tyr?Arg?Tyr?Leu?Arg
1 5
<210>50
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>50
Lys?Ala?Ser?Ser?Leu?Gln?Ser
1 5
<210>51
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>51
Gln?Gln?Tyr?Val?Gly?Tyr?Pro?Arg?Thr
1 5
<210>52
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>52
Leu?Lys?Tyr?Leu?Lys
1 5
<210>53
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>53
Asn?Ala?Ser?His?Leu?Gln?Ser
1 5
<210>54
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>54
Gln?Gln?Thr?Thr?Tyr?Tyr?Pro?Ile?Thr
1 5
<210>55
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>55
Leu?Arg?Tyr?Leu?Arg
1 5
<210>56
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>56
Lys?Ala?Ser?Trp?Leu?Gln?Ser
1 5
<210>57
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>57
Gln?Gln?Val?Leu?Tyr?Tyr?Pro?Gln?Thr
1 5
<210>58
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>58
Leu?Arg?Ser?Leu?Lys
1 5
<210>59
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>59
Ala?Ala?Ser?Arg?Leu?Gln?Ser
1 5
<210>60
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>60
Gln?Gln?Val?Val?Tyr?Trp?Pro?Ala?Thr
1 5
<210>61
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>61
Phe?Arg?His?Leu?Lys
1 5
<210>62
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>62
Ala?Ala?Ser?Arg?Leu?Gln?Ser
1 5
<210>63
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>63
Gln?Gln?Val?Ala?Leu?Tyr?Pro?Lys?Thr
1 5
<210>64
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>64
Arg?Lys?Tyr?Leu?Arg
1 5
<210>65
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>65
Thr?Ala?Ser?Ser?Leu?Gln?Ser
1 5
<210>66
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>66
Gln?Gln?Asn?Leu?Phe?Trp?Pro?Arg?Thr
1 5
<210>67
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>67
Arg?Arg?Tyr?Leu?Asn
1 5
<210>68
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>68
Ala?Ala?Ser?Ser?Leu?Gln?Ser
1 5
<210>69
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>69
Gln?Gln?Met?Leu?Phe?Tyr?Pro?Lys?Thr
1 5
<210>70
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>70
Ile?Lys?His?Leu?Lys
1 5
<210>71
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>71
Gly?Ala?Ser?Arg?Leu?Gln?Ser
1 5
<210>72
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>72
Gln?Gln?Gly?Ala?Arg?Trp?Pro?Gln?Thr
1 5
<210>73
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>73
Tyr?Tyr?His?Leu?Lys
1 5
<210>74
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>74
Lys?Ala?Ser?Thr?Leu?Gln?Ser
1 5
<210>75
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>75
Gln?Gln?Val?Arg?Lys?Val?Pro?Arg?Thr
1 5
<210>76
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<400>76
Tyr?Lys?His?Leu?Lys
1 5
<210>77
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>77
Asn?Ala?Ser?His?Leu?Gln?Ser
1 5
<210>78
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>78
Gln?Gln?Val?Gly?Arg?Tyr?Pro?Lys?Thr
1 5
<210>79
<211>5
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR1 sequence
<220>
＜221〉further feature
<222>(5)..(5)
＜223〉X=arbitrary amino acid
<400>79
Phe?Lys?Ser?Leu?Xaa
1 5
<210>80
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR2 sequence
<400>80
Asn?Ala?Ser?Tyr?Leu?Gln?Ser
1 5
<210>81
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉artificial CDR3 sequence
<400>81
Gln?Gln?Val?Val?Tyr?Trp?Pro?Val?Thr
1 5
<210>82
<211>6
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR1 in the VH library 1
<220>
＜221〉further feature
<222>(1)..(2)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(4)..(6)
＜223〉X=arbitrary amino acid
<400>82
Xaa?Xaa?Tyr?Xaa?Xaa?Xaa
1 5
<210>83
<211>17
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR2 in the VH library 1
<220>
＜221〉further feature
<222>(1)..(1)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(3)..(5)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(7)..(8)
＜223〉X=arbitrary amino acid
<220>
＜221〉further feature
<222>(10)..(10)
＜223〉X=arbitrary amino acid
<400>83
Xaa?Ile?Xaa?Xaa?Xaa?Gly?Xaa?Xaa?Thr?Xaa?Tyr?Ala?Asp?Ser?Val?Lys
1 5 10 15
Gly
<210>84
<211>11
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR3 in the VH library 1
<220>
＜221〉further feature
<222>(1)..(8)
＜223〉X=arbitrary amino acid
<400>84
Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Xaa?Phe?Asp?Tyr
1 5 10
<210>85
<211>6
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR1 in the VH library 1
<400>85
Trp?Val?Tyr?Gln?Met?Asp
1 5
<210>86
<211>17
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR2 in the VH library 1
<400>86
Ser?Ile?Ser?Ala?Phe?Gly?Ala?Lys?Thr?Leu?Tyr?Ala?Asp?Ser?Val?Lys
1 5 10 15
Gly
<210>87
<211>7
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR3 in the VH library 1
<400>87
Leu?Ser?Gly?Lys?Phe?Asp?Tyr
1 5
<210>88
<211>6
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR1 in the VH library 1
<400>88
Trp?Ser?Tyr?Gln?Met?Thr
1 5
<210>89
<211>17
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR2 in the VH library 1
<400>89
Ser?Ile?Ser?Ser?Phe?Gly?Ser?Ser?Thr?Leu?Tyr?Ala?Asp?Ser?Val?Lys
1 5 10 15
Gly
<210>90
<211>11
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the total CDR3 in the VH library 1
<400>90
Gly?Arg?Asp?His?Asn?Tyr?Ser?Leu?Phe?Asp?Tyr
1 5 10
<210>91
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of HA mark
<400>91
tatccttatg?atgttcctga?ttatgca 27
<210>92
<211>9
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of HA mark
<400>92
Tyr?Pro?Tyr?Asp?Val?Pro?Asp?Tyr?Ala
1 5
<210>93
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>93
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Gly?Ser?Phe
20 25 30
Leu?Trp?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Tyr?Ser?Ser?Tyr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Arg?Trp?His?Pro?Asn
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>94
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>94
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gcctattggg?agttttttat?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaaactcct?gatctattat?agttcctatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tatcgttggc?atcctaatac?cttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>95
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>95
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Tyr?Ser?Trp
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?His?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ile?Trp?Asn?Met?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>96
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>96
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gtcgatttat?agttggttaa?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagg?gcgtcccatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?atttggaata?tgccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>97
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>97
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Gly?Tyr?Asp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Gly?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Arg?Trp?Arg?Trp?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>98
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>98
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gcctattggt?tatgatttat?tttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcgg?ggttccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cggtggcgtt?ggccttttac?gttcggccaa 300
ggcaccaagg?tggaaatcaa?acgg 324
<210>99
<211>107
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>99
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Leu?Pro?Ile?Gly?Arg?Asp
20 25 30
Leu?Trp?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Gly?Ser?Phe?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Arg?Trp?Tyr?Tyr?Pro?His
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105
<210>100
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>100
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtct?gcctattggt?cgtgatttat?ggtggtatca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcgg?gggtcctttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?aggtggtatt?atcctcatac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>101
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>101
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?atgaatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgc 324
<210>102
<211>323
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>102
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttgg?acgaagttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatatg?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tggtttagta?atcctagtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acg 323
<210>103
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>103
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgag?cattatttat?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatcctatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?agtttggcgt?gtcctcctac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>104
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of encoding synthetic antibody
<400>104
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttat?ggtcatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cctttggtgc?ggccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>105
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>105
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgct?aagttgttat?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgat?gcatcctctt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tggtgggggt?atcctggtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>106
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>106
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?cctgctttac?tttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagatattg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>107
<211>224
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>107
attggtacca?gcagaaacca?gggaaagccc?ctaagctcct?gatctatcag?gcatccattt 60
tgcaaagtgg?ggtcccatca?cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca 120
ccatcagcag?tctgcaacct?gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc 180
gtccttttac?gttcggccaa?gggaccaagg?tggaaatcaa?acgg 224
<210>108
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>108
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?atgaatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacaggt?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>109
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>109
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttg?aattctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccactt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>110
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>110
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttg?aattctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccactt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>111
<211>325
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleic acid in coding synthetic antibody structure territory
<400>111
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatcccatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acggv 325
<210>112
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>112
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattaat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>113
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>113
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattaat?tatgctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcag?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>114
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>114
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>115
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>115
gacatccaga?tgacccagtc?tccatcctct?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>116
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>116
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?gcatccaatt?tgcaaagtga?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>117
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>117
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>118
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>118
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?tcttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccctgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>119
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>119
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatcccttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacatacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>120
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>120
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?aagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattgt?gcatcccagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctacatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>121
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>121
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gcgtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccctgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>122
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>122
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aggtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcaccctca?ccatcagcag?tctgcagcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>123
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>123
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcctcgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>124
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>124
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?caacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>125
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>125
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>126
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>126
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattcag?actgcgttac?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacatacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>127
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>127
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?gcatccaatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>128
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>128
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcccagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>129
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>129
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>130
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>130
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaacccc?ctaagctcct?gatctattct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>131
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>131
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cattttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>132
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>132
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattcg?gcatccatgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>133
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>133
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ccaagctcct?gatctattct?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>134
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>134
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattcg?gcatccgctt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>135
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>135
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaccct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>136
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>136
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gatgattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttgc?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>137
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>137
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcaaatt?tagaaacagg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>138
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>138
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?ggtgatttgg?gatgcgttag?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcgtcccgtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?tatgctgtgt?ttcctgtgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>139
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>139
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gactatttat?gatgcgttaa?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?ggttccaggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcggtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tataagacta?agcctttgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>140
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence in coding synthetic antibody structure territory
<400>140
gacatccaga?tgacccagtc?cccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gactatttat?gatgcgttaa?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?ggttccaggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gtagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaaccc 240
gaagattttg?ctacgtacta?ctgtcaacag?tatgctcgtt?atcctcttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>141
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>141
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Glu?Glu?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ash?Ser?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Tyr?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>142
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>142
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Asp?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?Phe?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>143
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>143
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Phe?Ile?Glu?Asp?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>144
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>144
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Asp?Ser?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>145
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>145
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Asp?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?Phe?Leu?Gln?Ser?Gly?Val?Pro?Pro?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>146
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>146
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asn?Ile?Asp?Asp?His
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ser?Ser?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Pro?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>147
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>147
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Asp?His?Ala
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Arg?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Met?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Leu?Arg?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>148
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>148
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Gly?Asp?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Met?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ile?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>149
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>149
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Ser?Tyr
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ash?Thr?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>150
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>150
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Asp?Asp?His
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Thr?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Ile?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>151
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>151
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Phe?Ile?Asp?Glu?His
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ser?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>152
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>152
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Asn?Asn?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Glu?Ser?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>153
<211>107
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>153
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Leu?Ile?Asp?Asp?His
20 25 30
Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Thr?Leu?Leu?Ile?Tyr
35 40 45
Asn?Ser?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser
50 55 60
Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu
65 70 75 80
Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe?Thr
85 90 95
Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>154
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>154
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Asp?Gln?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Met?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>155
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>155
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Gln?Ala?Ser?Gln?Asp?Ile?Asp?Asn?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Ash?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>156
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>156
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Asp?Ser?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Gly?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>157
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>157
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Tyr?Ile?Asp?Tyr?Gly
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Thr?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>158
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>158
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Asp?Ser?Phe
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>159
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>159
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Pro?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Leu?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Arg?Arg
100 105
<210>160
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>160
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Ser?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Gly?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>161
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>161
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Thr?His
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ash?Thr?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>162
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>162
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Phe?Ile?Asp?Thr?His
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Arg?Leu?Leu?Ile
35 40 45
Tyr?Asn?Thr?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>163
<211>108
＜212〉protein
＜213〉artificial sequence
<400>163
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Asp?Asp?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>164
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>164
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Asp?Asp?Thr
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ser?Ser?Met?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>165
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>165
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Tyr?Ile?Asp?Ser?His
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asp?Thr?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>166
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>166
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Gln?His
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>167
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>167
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Glu?Arg?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>168
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>168
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Ser?Cys?Arg?Ala?Ser?Gln?His?Ile?Glu?Arg?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>169
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>169
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Ser?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Ser?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Ala?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>170
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>170
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Lys?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>171
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>171
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Glu?Glu?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>172
<211>100
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>172
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Tyr?Ile?Asp?Tyr?Gly
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Thr?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln
100
<210>173
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>173
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asn?Ile?Asp?Ile?His
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Pro?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>174
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>174
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Pro?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Leu?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>175
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>175
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Glu?Ile?Gly?Val?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Glu?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Val?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>176
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>176
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Lys?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>177
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>177
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Asp?Thr?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Gly?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>178
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>178
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Asp?Ser?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>179
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>179
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Glu?Gly?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>180
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>180
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Asp?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?Phe?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>181
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>181
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Asp?Thr?Trp
20 25 30
Leu?Phe?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Gly?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>182
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>182
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Glu?Glu?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?His?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>183
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>183
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?His?Ile?Asp?Lys?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>184
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>184
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Glu?Glu?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ser?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Tyr?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>185
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>185
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Asp?Tyr?Gly
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ser?Ser?Gln?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>186
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>186
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Glu?Ile?Gly?Ser?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ser?Ser?Lys?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>187
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>187
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Pro?Ile?Asp?Ser?Trp
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>188
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>188
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Pro?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Ala?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>189
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>189
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asn?Ile?His?Glu?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Gly?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>190
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>190
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Pro?Trp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gln?Ala?Ser?Ala?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Ser?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Leu?Ser?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>191
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>191
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Val?Lys?Glu?Phe
20 25 30
Leu?Trp?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Met?Ala?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Lys?Phe?Lys?Leu?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>192
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>192
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Pro?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Gly?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Arg?Met?Tyr?Arg?Pro?Ala
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>193
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>193
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Arg?Glu
20 25 30
Leu?Lys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Arg?Leu?Leu?Ile
35 40 45
Tyr?His?Gly?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asp?Phe?Phe?Val?Pro?Asp
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>194
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>194
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Ala?Asn?Asp
20 25 30
Leu?Met?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Asn?Ser?Arg?Leu?Gln?Gly?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Val?His?Arg?Pro?Tyr
85 90 95
Thr?Ile?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>195
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>195
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Phe?Ile?Gly?Pro?His
20 25 30
Leu?Thr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Ser?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Met?Tyr?Tyr?Pro?Ser
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Lys?Ile?Lys?Arg
100 105
<210>196
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>196
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Pro?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Thr?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Tyr?Met?Phe?Gln?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Arg?Arg
100 105
<210>197
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>197
Asp?Ile?Gln?Met?Ile?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Phe?Ile?Gly?Asn?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Leu?Gly?Tyr?Pro?Tyr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>198
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>198
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Pro?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Gly?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Leu?Tyr?Ser?Pro?Leu
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>199
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>199
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Asn?Glu
20 25 30
Leu?Lys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Met?Ser?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Leu?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Thr?Leu?Leu?Leu?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>200
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>200
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?Pro?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Gly?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Arg?Leu?Tyr?Tyr?Pro?Gly
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>201
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>201
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Arg?Glu
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Met?Leu?Leu?Ile
35 40 45
Tyr?His?Ser?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gly?Met?Tyr?Trp?Pro?Tyr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>202
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>202
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Lys?Pro?Ala
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Gly?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Thr?Leu?Phe?Met?Pro?Tyr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>203
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>203
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Thr?Ala
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Gly?Ser?Met?Leu?Pro?Asn?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Thr?Trp?Asp?Thr?Pro?Met
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>204
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>204
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Trp?Ile?Gly?His?Asp
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Ser?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Val?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Met?Gly?Tyr?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>205
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>205
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Ile?Gly?Gly?Leu
20 25 30
Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ser?Ser?Tyr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Thr?Trp?Gly?Ile?Pro?His
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>206
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>206
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Lys?Ile?Phe?Asn?Gly
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Ser?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Leu?Leu?Tyr?Pro?Tyr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>207
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>207
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Thr?Asn
20 25 30
Leu?Ser?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Arg?Leu?Leu?Ile
35 40 45
Tyr?Arg?Thr?Ser?Met?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gln?Phe?Phe?Trp?Pro?His
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>208
<211>118
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>208
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Leu?Tyr
20 25 30
Asp?Met?Val?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Tyr?Ile?Ser?Ser?Gly?Gly?Ser?Gly?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ala?Gly?Gly?Arg?Ala?Ser?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
100 105 110
Leu?Val?Thr?Val?Ser?Ser
115
<210>209
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>209
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?His?Leu?Tyr
20 25 30
Asp?Met?Met?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Phe?Ile?Gly?Gly?Asp?Gly?Leu?Asn?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ala?Gly?Thr?Gln?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>210
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>210
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asn?Lys?Tyr
20 25 30
Pro?Met?Met?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Glu?Ile?Ser?Pro?Ser?Gly?Gln?Asp?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Asn?Pro?Gln?Ile?Leu?Ser?Asn?Phe?Asp?Tyr?Trp?Gly?Gln?Gly
100 105 110
Thr?Leu?Val?Thr?Val?Ser?Ser
115
<210>211
<211>124
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>211
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gln?Trp?Tyr
20 25 30
Pro?Met?Trp?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Leu?Ile?Glu?Gly?Gln?Gly?Asp?Arg?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ala?Gly?Asp?Arg?Thr?Ala?Gly?Ser?Arg?Gly?Asn?Ser?Phe?Asp
100 105 110
Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>212
<211>121
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>212
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Lys?Ala?Tyr
20 25 30
Glu?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Gly?Ile?Ser?Pro?Asn?Gly?Gly?Trp?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Glu?Ser?Ile?Ser?Pro?Thr?Pro?Leu?Gly?Phe?Asp?Tyr?Trp?Gly
100 105 110
Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>213
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>213
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Thr?Gly?Tyr
20 25 30
Glu?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Tyr?Ile?Ser?Arg?Gly?Gly?Arg?Trp?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Asp?Thr?Met?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>214
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>214
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ala?Tyr
20 25 30
Glu?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Phe?Ile?Ser?Gly?Gly?Gly?Arg?Trp?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Tyr?Ser?Glu?Asp?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>215
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>215
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gly?Ala?Tyr
20 25 30
Pro?Met?Met?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Glu?Ile?Ser?Pro?Ser?Gly?Ser?Tyr?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Asp?Pro?Arg?Lys?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>216
<211>123
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>216
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gln?Phe?Tyr
20 25 30
Lys?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Ser?Ser?Val?Gly?Asp?Ala?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Met?Gly?Gly?Gly?Pro?Pro?Thr?Tyr?Val?Val?Tyr?Phe?Asp?Tyr
100 105 110
Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>217
<211>123
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>217
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gly?Glu?Tyr
20 25 30
Gly?Met?Tyr?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Ser?Glu?Arg?Gly?Arg?Leu?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Asn?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Ala?Leu?Ser?Ser?Glu?Gly?Phe?Ser?Arg?Ser?Phe?Asp?Tyr
100 105 110
Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>218
<211>123
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉synthetic antibody structure territory
<400>218
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Asp?Tyr
20 25 30
Ala?Met?Tyr?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Thr?Ala?Arg?Gly?Phe?Ile?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Gly?Phe?Pro?His?Lys?Ser?Gly?Ser?Asn?Tyr?Phe?Asp?Tyr
100 105 110
Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>219
<211>240
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the synthetic antibody sequence that is connected by the Gly4Ser joint of VH and VL,
<400>219
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Tyr?Gly?Ala?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
115 120 125
Gly?Gly?Ser?Thr?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser
130 135 140
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser
145 150 155 160
Ile?Ser?Ser?Tyr?Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro
165 170 175
Lys?Leu?Leu?Ile?Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser
180 185 190
Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser
195 200 205
Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Tyr
210 215 220
Ser?Thr?Pro?Asn?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
225 230 235 240
<210>220
<211>720
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the synthetic antibody sequence that is connected by the Gly4Ser joint of coding VH and VL,
<400>220
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttagc?agctatgcca?tgagctgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgtgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaagttat 300
ggtgcttttg?actactgggg?ccagggaacc?ctggtcaccg?tctcgagcgg?tggaggcggt 360
tcaggcggag?gtggcagcgg?cggtggcggg?tcgacggaca?tccagatgac?ccagtctcca 420
tcctccctgt?ctgcatctgt?aggagaccgt?gtcaccatca?cttgccgggc?aagtcagagc 480
attagcagct?atttaaattg?gtaccagcag?aaaccaggga?aagcccctaa?gctcctgatc 540
tatgctgcat?ccagttggca?aagtggggtc?ccatcacgtt?tcagtggcag?tggatctggg 600
acagatttca?ctctcaccat?cagcagtctg?caacctgaag?attttgctac?gtactactgt 660
caacagagtt?acagtacccc?taatacgttc?ggccaaggga?ccaaggtgga?aatcaaacgg 720
<210>221
<211>359
<212>DNA
＜213〉artificial sequence
<220>
＜223〉phage vector expression cassette nucleotide sequence
<400>221
caggaaacag?ctatgaccat?gattacgcca?agcttgcatg?caaattctat?ttcaaggaga 60
cagtcataat?gaaataccta?ttgcctacgg?cagccgctgg?attgttatta?ctcgcggccc 120
agccggccat?ggccgaggtg?tttgactact?ggggccaggg?aaccctggtc?accgtctcga 180
gcggtggagg?cggttcaggc?ggaggtggca?gcggcggtgg?cgggtcgacg?gacatccaga 240
tgacccaggc?ggccgcagaa?caaaaactcc?atcatcatca?ccatcacggg?gccgcaatct 300
cagaagagga?tctgaatggg?gccgcataga?ctgttgaaag?ttgtttagca?aaacctcat 359
<210>222
<211>96
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉expression cassette aminoacid sequence
<400>222
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Glu?Val?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
20 25 30
Leu?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
35 40 45
Gly?Gly?Gly?Gly?Ser?Thr?Asp?Ile?Gln?Met?Thr?Gln?Ala?Ala?Ala?Glu
50 55 60
Gln?Lys?Leu?His?His?His?His?His?His?Gly?Ala?Ala?Ile?Ser?Glu?Glu
65 70 75 80
Asp?Leu?Asn?Gly?Ala?Ala?Thr?Val?Glu?Ser?Cys?Leu?Ala?Lys?Pro?His
85 90 95
<210>223
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone K8
<400>223
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?His?Ile?Ser?Pro?Tyr?Gly?Ala?Asn?Thr?Arg?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Gly?Leu?Arg?Ala?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>224
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VH2
<400>224
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Asp?Ile?Gly?Ala?Thr?Gly?Ser?Lys?Thr?Gly?Tyr?Ala?Asp?Pro?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Lys?Val?Leu?Thr?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>225
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VH4
<400>225
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Arg?Ile?Asn?Gly?Pro?Gly?Ala?Thr?Gly?Tyr?Ala?Asp?Ser?Val?Lys
50 55 60
Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu
65 70 75 80
Gln?Ile?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Lys?His?Gly?Ala?Pro?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110
Val?Ser?Ser
115
<210>226
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VHC11
<400>226
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Asn?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Pro?Ala?Ser?Gly?Leu?His?Thr?Arg?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Pro?Gly?Leu?Gly?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>227
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VHA10sd
<400>227
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Asp?Ile?Glu?Arg?Thr?Gly?Tyr?Thr?Arg?Tyr?Ala?Asp?Ser?Val?Lys
50 55 60
Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu
65 70 75 80
Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Lys?Lys?Val?Leu?Val?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110
Val?Ser?Ser
115
<210>228
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VHA1sd
<400>228
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Glu?Ile?Ser?Ala?Asn?Gly?Ser?Lys?Thr?Gln?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Leu?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Lys?Val?Leu?Gln?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>229
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VHA5sd
<400>229
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Thr?Ile?Pro?Ala?Asn?Gly?Val?Thr?Arg?Tyr?Ala?Asp?Ser?Val?Lys
50 55 60
Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr?Leu
65 70 75 80
Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala
85 90 95
Lys?Ser?Leu?Leu?Gln?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110
Val?Ser?Ser
115
<210>230
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence of clone VHC5sd
<400>230
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Asp?Ile?Ala?Ala?Thr?Gly?Ser?Ala?Thr?Ser?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Lys?Ile?Leu?Lys?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>231
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the VH sequence sd of clone VHC11
<400>231
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Ser?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Thr?Ile?Ser?Ser?Val?Gly?Gln?Ser?Thr?Arg?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Asn?Leu?Met?Ser?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>232
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the Vk sequence of clone K8
<400>232
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?His?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Pro?Trp?Arg?Ser?Pro?Gly
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>233
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the Vk sequence of clone E5sd
<400>233
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Leu?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asn?Trp?Trp?Leu?Pro?Pro
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>234
<211>107
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the Vk sequence of clone C3
<400>234
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ser?Ser?Tyr
20 25 30
Leu?Asn?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser
50 55 60
Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu
65 70 75 80
Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Arg?Val?Tyr?Asp?Pro?Leu?Thr
85 90 95
Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>235
<211>120
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉the simulation VH in library
<220>
＜221〉further feature
<222>(103)..(106)
＜223〉Xaa of 103-106 position is by NNK codon amino acids coding, and wherein N is any among G, A, T or the C
<400>235
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Ser?Ser?Tyr
20 25 30
Ala?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ser?Tyr?Gly?Ala?Xaa?Xaa?Xaa?Xaa?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>236
<211>360
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the simulation VH in library
<220>
＜221〉further feature
<222>(307)..(317)
＜223〉N is G, A, T or C
<400>236
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttagc?agctatgcca?tgagctgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgtgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaagttat 300
ggtgctnnkn?nknnknnktt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcgagc 360
<210>237
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉sequence of anti-mouse serum albumin domain antibodies
<400>237
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Ile?Lys?His
20 25 30
Leu?Lys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Gly?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gly?Ala?Arg?Trp?Pro?Gln
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>238
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the anti-mouse serum albumin domain antibodies of coding
<400>238
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattatt?aagcatttaa?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?gcatcccggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?ggggctcggt?ggcctcagac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>239
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉sequence of anti-mouse serum albumin domain antibodies
<400>239
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Tyr?Tyr?His
20 25 30
Leu?Lys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Lys?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Arg?Lys?Val?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>240
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of the anti-mouse serum albumin domain antibodies of coding
<400>240
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttat?tatcatttaa?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataag?gcatccacgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttcggaagg?tgcctcggac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>241
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>241
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?atgaatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgc 324
<210>242
<211>323
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>242
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttgg?acgaagttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatatg?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tggtttagta?atcctagtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acg 323
<210>243
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>243
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgag?cattatttat?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatcctatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?agtttggcgt?gtcctcctac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>244
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>244
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttat?ggtcatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cctttggtgc?ggccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>245
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>245
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgct?aagttgttat?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgat?gcatcctctt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tggtgggggt?atcctggtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>246
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>246
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?cctgctttac?tttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagatattg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>247
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>247
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aatgcgttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcag?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>248
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>248
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?atgaatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacaggt?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>249
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>249
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttg?aattctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccactt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>250
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>250
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttg?aattctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccactt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>251
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>251
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatcccatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>252
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>252
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattaat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>253
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>253
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattaat?tatgctttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcag?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>254
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>254
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>255
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>255
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?gcatccaatt?tgcaaagtga?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>256
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>256
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>257
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>257
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?tcttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccctgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>258
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>258
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatcccttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacatacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>259
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>259
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?aagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattgt?gcatcccagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctacatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>260
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>260
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gcgtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccctgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>261
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>261
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aggtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcaccctca?ccatcagcag?tctgcagcct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>262
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>262
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcctcgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>263
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>263
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?caacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>264
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>264
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>265
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>265
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattcag?actgcgttac?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacatacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>266
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>266
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?gcatccaatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>267
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>267
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcccagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>268
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>268
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>269
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>269
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaacccc?ctaagctcct?gatctattct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>270
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>270
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?cattttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>271
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>271
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattcg?gcatccatgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>272
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>272
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ccaagctcct?gatctattct?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>273
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>273
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtttttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattcg?gcatccgctt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>274
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>274
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?gagtatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaccct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>275
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>275
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?aattatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gatgattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttgc?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>276
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>276
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatcaaatt?tagaaacagg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>277
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>277
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?ggtgatttgg?gatgcgttag?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcgtcccgtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcatcct 240
gaagattttg?ctacgtacta?ctgtcaacag?tatgctgtgt?ttcctgtgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>278
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>278
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gactatttat?gatgcgttaa?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?ggttccaggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcggtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tataagacta?agcctttgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>279
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of anti-mouse serum albumin domain antibodies
<400>279
gacatccaga?tgacccagtc?cccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gactatttat?gatgcgttaa?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatggt?ggttccaggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gtagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaaccc 240
gaagattttg?ctacgtacta?ctgtcaacag?tatgctcgtt?atcctcttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>280
<211>120
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>280
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Arg?Ile?Ser?Asp?Glu
20 25 30
Asp?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Tyr?Gly?Pro?Ser?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Ser?Ala?Leu?Glu?Pro?Leu?Ser?Glu?Pro?Leu?Gly?Phe?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>281
<211>360
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>281
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?taggattagc?gatgaggata?tgggctgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtatcaagc?atttatggcc?ctagcggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgtgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attattgcgc?gagtgctttg 300
gagccgcttt?cggagcccct?gggcttttgg?ggtcagggaa?ccctggtcac?cgtctcgagc 360
<210>282
<211>116
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>282
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asp?Leu?Tyr
20 25 30
Asn?Met?Phe?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Phe?Ile?Ser?Gln?Thr?Gly?Arg?Leu?Thr?Trp?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Thr?Leu?Glu?Asp?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser?Ser
115
<210>283
<211>348
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>283
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttgat?ctttataata?tgttttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcattt?attagtcaga?ctggtaggct?tacatggtac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaacgctg 300
gaggattttg?actactgggg?ccagggaacc?ctggtcaccg?tctcgagc 348
<210>284
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>284
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Val?Lys?Glu?Phe
20 25 30
Leu?Trp?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Met?Ala?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Lys?Phe?Lys?Leu?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>285
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>285
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcgttaag?gagtttttat?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatatg?gcatccaatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?aagtttaagc?tgcctcgtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>286
<211>120
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>286
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Glu?Trp?Tyr
20 25 30
Trp?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Val?Lys?Leu?Gly?Gly?Gly?Pro?Asn?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
115 120
<210>287
<211>362
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>287
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttgag?tggtattgga?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaagttaag 300
ttgggggggg?ggcctaattt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcgagc 360
gc 362
<210>288
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>288
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Asp?Ser?Tyr
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ser?Ala?Ser?Glu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Val?Trp?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>289
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>289
gacatccaga?tgacccagtc?tccatcctct?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgat?agttatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatagt?gcatccgagt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgc 324
<210>290
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>290
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Phe?Met?Asn
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asn?Ala?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Val?Trp?Arg?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>291
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>291
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattttt?atgaatttat?tgtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataat?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttgtgtggc?gtccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>292
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>292
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Tyr?Asp?Ala
20 25 30
Leu?Glu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Thr?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Val?Met?Gln?Arg?Pro?Val
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>293
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>293
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttat?gatgcgttag?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatact?gcatcccggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gttatgcagc?gtcctgttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>294
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(34)..(34)
＜223〉terminator codon
<400>294
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Tyr?Asp?Ala
20 25 30
Leu?Xaa?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Thr?Ala?Ser?Arg?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?His?Cys?Gln?Gln?Val?Met?Gln?Arg?Pro?Val
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>295
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>295
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttat?gatgctttac?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatact?gcatcccggt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacca?ctgtcaacag?gttatgcagc?gtcctgttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>296
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>296
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Val?Lys?Glu?Phe
20 25 30
Leu?Trp?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Met?Ala?Ser?Asn?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Lys?Phe?Lys?Leu?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>297
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>297
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcgttaag?gagtttttat?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatatg?gcatccaatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?aagtttaagc?tgcctcgtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>298
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>298
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Trp?Thr?Lys
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Met?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp?Phe?Ser?Asn?Pro?Ser
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>299
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>299
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttgg?acgaagttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatatg?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tggtttagta?atcctagtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgc 324
<210>300
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>300
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Xaa?Pro?Ile
20 25 30
Leu?Cys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ile?Gln?His?Ile?Pro?Val
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>301
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>301
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttag?ccgattttat?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?attcagcata?ttcctgtgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>302
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(31)..(31)
＜223〉terminator codon
<400>302
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Xaa?Asp
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Thr?Ala?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gln?Ser?Ala?Phe?Pro?Asn
85 90 95
Thr?Leu?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>303
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>303
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattggg?taggatttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatacg?gcatcccttt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cagagtgctt?ttcctaatac?gctcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>304
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(50)..(50)
＜223〉terminator codon
<400>304
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Thr?Lys?Asn
20 25 30
Leu?Leu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Xaa?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Arg?His?Lys?Pro?Pro
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>305
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>305
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ccgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcataacg?aagaatttac?tttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattag?gcatcctctt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cttcgtcata?agcctccgac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>306
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>306
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Xaa?Lys?Ser
20 25 30
Leu?Arg?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?His?Ala?Ser?Asp?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Met?Val?Asn?Ser?Pro?Val
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>307
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>307
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttag?aagtctttaa?ggtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcat?gcatccgatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?atggttaata?gtcctgttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>308
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>308
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Xaa?Thr?Ala
20 25 30
Leu?His?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ser?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Ser?Phe?Leu?Pro?Phe
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>309
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>309
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcatttag?acggcgttac?attggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tcgagttttt?tgccttttac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>310
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>310
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Pro?Ash
20 25 30
Leu?Glu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Ala?Ala?Ser?Ser?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Gln?Met?Gly?Arg?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>311
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>311
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattggg?ccgaatttag?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgct?gcatccagtt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cagatggggc?gtcctcggac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>312
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(32)..(32)
＜223〉terminator codon
<400>312
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Lys?His?Xaa
20 25 30
Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Lys?Ala?Ser?Val?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Leu?Arg?Arg?Arg?Pro?Thr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>313
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>313
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattaag?cattagttag?cttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataag?gcatccgtgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?cttaggcgtc?gtcctactac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>314
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(32)..(32)
＜223〉terminator codon
<400>314
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Val?Lys?Ala?Xaa
20 25 30
Leu?Thr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Lys?Ala?Ser?Thr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?His?Ser?Ser?Arg?Pro?Tyr
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>315
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>315
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcgttaag?gcttagttaa?cttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctataag?gcatccactt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?catagttcta?ggccttatac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>316
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(50)..(50)
＜223〉terminator codon
<400>316
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Glu?Asn?Arg
20 25 30
Leu?Gly?Glu?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Xaa?Ala?Ser?Leu?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asp?Ser?Tyr?Phe?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>317
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>317
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattgag?aatcggttag?gttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattag?gcgtccttgt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gattcgtatt?ttcctcgtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>318
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(50)..(50)
＜223〉terminator codon
<400>318
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Met?Asp?Lys
20 25 30
Leu?Lys?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Xaa?Ala?Ser?Ile?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Asp?Ser?Gly?Gly?Pro?Asn
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>319
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>319
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattatg?gataagttaa?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctattag?gcatccattt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gatagtgggg?gtcctaatac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>320
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>320
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Gly?Arg?Asn
20 25 30
Leu?Glu?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Asp?Ala?Ser?His?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ser?Arg?Glu?Leu?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>321
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>321
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattggg?aggaatttag?agtggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatgat?gcatcccatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?tcgcgttggc?ttcctcgtac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>322
<211>108
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>322
Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly
1 5 10 15
Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Ser?Ile?Arg?Lys?Met
20 25 30
Leu?Val?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile
35 40 45
Tyr?Arg?Ala?Ser?Tyr?Leu?Gln?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly
50 55 60
Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro
65 70 75 80
Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Ala?Phe?Arg?Arg?Pro?Arg
85 90 95
Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
100 105
<210>323
<211>324
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>323
gacatccaga?tgacccagtc?tccatcctcc?ctgtctgcat?ctgtaggaga?ccgtgtcacc 60
atcacttgcc?gggcaagtca?gagcattagg?aagatgttag?tttggtacca?gcagaaacca 120
gggaaagccc?ctaagctcct?gatctatcgg?gcatcctatt?tgcaaagtgg?ggtcccatca 180
cgtttcagtg?gcagtggatc?tgggacagat?ttcactctca?ccatcagcag?tctgcaacct 240
gaagattttg?ctacgtacta?ctgtcaacag?gcttttcggc?ggcctaggac?gttcggccaa 300
gggaccaagg?tggaaatcaa?acgg 324
<210>324
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>324
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asp?Leu?Tyr
20 25 30
Asn?Met?Phe?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Phe?Ile?Ser?Gln?Thr?Gly?Arg?Leu?Thr?Trp?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Thr?Leu?Glu?Asp?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser
115
<210>325
<211>345
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>325
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttgat?ctttataata?tgttttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcattt?attagtcaga?ctggtaggct?tacatggtac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaacgctg 300
gaggattttg?actactgggg?ccagggaacc?ctggtcaccg?tctcg 345
<210>326
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>326
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Pro?Val?Tyr
20 25 30
Met?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Asp?Ala?Leu?Gly?Gly?Arg?Thr?Gly?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Thr?Met?Ser?Asn?Lys?Thr?His?Thr?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>327
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>327
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttccg?gtttatatga?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcatcg?attgatgctc?ttggtgggcg?gacaggttac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaactatg 300
tcgaataaga?cgcatacgtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>328
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(57)..(57)
＜223〉terminator codon
<400>328
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Val?Ala?Tyr
20 25 30
Asn?Met?Thr?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Asn?Thr?Phe?Gly?Asn?Xaa?Thr?Arg?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Gly?Ser?Arg?Pro?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser
115
<210>329
<211>345
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>329
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttgtg?gcttataata?tgacttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcaagt?attaatactt?ttggtaatta?gacaaggtac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaggtagt 300
aggccttttg?actactgggg?ccagggaacc?ctggtcaccg?tctcg 345
<210>330
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>330
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Xaa?Gly?Tyr
20 25 30
Arg?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Trp?Ile?Thr?Arg?Thr?Gly?Gly?Thr?Thr?Gln?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Pro?Ala?Lys?Leu?Val?Gly?Val?Gly?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>331
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>331
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?caccttttag?gggtatcgta?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcatgg?attacgcgta?ctggtgggac?gacacagtac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaccggcg 300
aagcttgttg?gggttgggtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>332
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(33)..(33)
＜223〉terminator codon
<400>332
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Lys?Tyr
20 25 30
Xaa?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Gln?Ile?Gly?Ala?Lys?Gly?Gln?Ser?Thr?Asp?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Ash?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Lys?Lys?Arg?Gly?Glu?Asn?Tyr?Phe?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>333
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>333
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttcgg?aagtattaga?tggggtgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcacag?attggtgcga?agggtcagtc?tacagattac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaaagaag 300
aggggggaga?attatttttt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>334
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>334
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Arg?Tyr
20 25 30
Ser?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Asp?Ile?Ser?Arg?Ser?Gly?Arg?Tyr?Thr?His?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Arg?Ile?Asp?Ser?Ser?Gln?Asn?Gly?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>335
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>335
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttcgg?cggtatagta?tgtcgtgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagat?atttctcgtt?ctggtcggta?tacacattac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaacgtatt 300
gattcttctc?agaatgggtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>336
<211>115
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>336
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Xaa?Gly?Tyr
20 25 30
Lys?Met?Phe?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Gln?Lys?Glu?Asn?Phe?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val
100 105 110
Thr?Val?Ser
115
<210>337
<211>345
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>337
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?caccttttag?gggtataaga?tgttttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaacagaag 300
gagaattttg?actactgggg?ccagggaacc?ctggtcaccg?tctcg 345
<210>338
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>338
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Gly?Asp?Tyr
20 25 30
Ala?Met?Trp?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Val?Ile?Ser?Ser?Asn?Gly?Gly?Ser?Thr?Phe?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Ash?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Arg?Val?Arg?Lys?Arg?Thr?Pro?Glu?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>339
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>339
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttggg?gattatgcta?tgtggtgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagtg?attagttcga?atggtgggag?tacattttac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaacgtgtt 300
cgtaagagga?ctcctgagtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>340
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>340
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Arg?Tyr
20 25 30
Lys?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Gly?Arg?Ash?Gly?Thr?Lys?Thr?Asn?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Ash?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ile?Tyr?Thr?Gly?Lys?Pro?Ala?Ala?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>341
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>341
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttagg?aggtataaga?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagcg?attgggagga?atggtacgaa?gacaaattac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaatttat 300
acggggaagc?ctgctgcgtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>342
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(33)..(33)
＜223〉terminator codon
<400>342
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Lys?Lys?Tyr
20 25 30
Xaa?Met?Ser?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Met?Leu?Arg?Thr?Lys?Asn?Lys?Val?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>343
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>343
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttaag?aagtattaga?tgtcttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaatgctg 300
aggactaaga?ataaggtgtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>344
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>344
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Arg?Arg?Tyr
20 25 30
Lys?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Gly?Arg?Asn?Gly?Thr?Lys?Thr?Asn?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Ash?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Ile?Tyr?Thr?Gly?Lys?Pro?Ala?Ala?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>345
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>345
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cacctttagg?aggtataaga?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagcg?attgggagga?atggtacgaa?gacaaattac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaatttat 300
acggggaagc?ctgctgcgtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>346
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>346
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Xaa?Ser?Tyr
20 25 30
Arg?Met?Gly?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ser?Ile?Ser?Ser?Arg?Gly?Arg?His?Thr?Ser?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Arg?Val?Pro?Gly?Arg?Gly?Arg?Ser?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>347
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>347
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?caccttttag?agttatcgga?tgggttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcaagt?atttcgtcga?ggggtaggca?tacatcttac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaagggtt 300
ccgggtcggg?ggcgttcttt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>348
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<400>348
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Pro?Phe?Arg?Arg?Tyr
20 25 30
Arg?Met?Arg?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Gly?Ile?Ser?Pro?Gly?Gly?Lys?His?Thr?Thr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Gly?Glu?Gly?Gly?Ala?Ser?Ser?Ala?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>349
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>349
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?cccctttcgt?cggtatcgga?tgaggtgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcaggt?atttctccgg?gtggtaagca?tacaacgtac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaaggtgag 300
gggggggcga?gttctgcgtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357
<210>350
<211>119
＜212〉protein
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of domain antibodies
<220>
＜221〉further feature
<222>(30)..(30)
＜223〉terminator codon
<400>350
Glu?Val?Gln?Leu?Leu?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Xaa?Arg?Tyr
20 25 30
Gly?Met?Val?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ser?Ala?Ile?Ser?Gly?Ser?Gly?Gly?Ser?Thr?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Lys?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Lys?Arg?His?Ser?Ser?Glu?Ala?Arg?Gln?Phe?Asp?Tyr?Trp?Gly?Gln
100 105 110
Gly?Thr?Leu?Val?Thr?Val?Ser
115
<210>351
<211>357
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide sequence of domain antibodies
<400>351
gaggtgcagc?tgttggagtc?tgggggaggc?ttggtacagc?ctggggggtc?cctgcgtctc 60
tcctgtgcag?cctccggatt?caccttttag?cggtatggga?tggtttgggt?ccgccaggct 120
ccagggaagg?gtctagagtg?ggtctcagct?attagtggta?gtggtggtag?cacatactac 180
gcagactccg?tgaagggccg?gttcaccatc?tcccgcgaca?attccaagaa?cacgctgtat 240
ctgcaaatga?acagcctgcg?tgccgaggac?accgcggtat?attactgtgc?gaaacggcat 300
agttctgagg?ctaggcagtt?tgactactgg?ggccagggaa?ccctggtcac?cgtctcg 357

Claims (202)

1. the composition that contains single domain antibody polypeptide construction is used to prepare the purposes of medicine, and the external antagonism human TNF alpha of described construction bind receptor, described medicine are used for the treatment of, prevent rheumatoid arthritis, suppress its progress or postpone its outbreak.
2. the composition that contains single domain antibody polypeptide construction is used to prepare the purposes of medicine, the external antagonism human TNF alpha of described construction bind receptor, and described medicine is used to bring out the statistics noticeable change of one or more rheumatoid arthritis indications.
3. claim 1 or 2 purposes, wherein said composition prevent the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model.
4. the purposes of claim 3, the wherein said statistics noticeable change that causes one or more RA indications.
5. the purposes of claim 4, wherein said one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
6. the purposes of claim 4, wherein said one or more RA indications comprise that the sacroiliitis macroscopic view phenotype symptom in the Tg197 transgenic mice goes down, wherein give the Tg197 transgenic mice described composition, wherein write down the described sacroiliitis macroscopic view phenotype symptom of described Tg197 transgenic mice
Wherein according to the described sacroiliitis macroscopic view of following system log (SYSLOG) phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
7. the purposes of claim 4, wherein said one or more RA indications comprise that the sacroiliitis histopathology symptom in the Tg197 transgenic mice goes down, wherein give the Tg197 transgenic mice described composition, wherein write down the described sacroiliitis histopathology symptom of described Tg197 transgenic mice
Wherein on the joint, show described sacroiliitis histopathology symptom, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
8. the purposes of claim 3 wherein gives the described composition of Tg197 transgenic mice and may further comprise the steps:
A) weekly peritoneal injection gives heterozygosis Tg197 transgenic mice described composition,
B) weigh weekly a step a) mouse and
C) write down once the sacroiliitis macroscopic view phenotype symptom of described mouse weekly according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
9. the purposes of claim 3 wherein gave described mouse described composition before showing the arthritic symptom outbreak.
10. the purposes of claim 3 wherein gives described composition for the first time when described mouse was 3 ages in week.
11. the purposes of claim 3 wherein gives described composition for the first time when described mouse was 6 ages in week.
12. each purposes in the aforementioned claim, the effectiveness that wherein said composition is had in the experiment of Tg197 transgenic mice sacroiliitis is greater than or equal to the effectiveness of the material that is selected from etanercept, English monoclonal antibody of sharp former times and D2E7.
13. each purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-0.5 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
14. each purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-1.0 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
15. each purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-1.5 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
16. each purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-2.0 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
17. treatment rheumatoid arthritis method, described method comprises the composition that the individual treatment of needs significant quantity is arranged, described composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, treats described rheumatoid arthritis thus.
18. treatment rheumatoid arthritis method, described method comprises the composition that the individual treatment of needs significant quantity is arranged, described composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, wherein said single domain antibody polypeptide construction inhibition human TNF alpha combines with TNF α acceptor, treats described rheumatoid arthritis thus.
19. the method for claim 17 or 18, wherein said treatment comprises the progress that suppresses described rheumatoid arthritis.
20. comprising, the method for claim 17 or 18, wherein said treatment prevent or the outbreak of deferred class rheumatic arthritis.
21. the purposes of the method for claim 17 or 18 or claim 3 or 4, wherein said composition prevent the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model.
22. the method for claim 21, the wherein said statistics noticeable change that causes one or more RA indications.
23. the method for claim 22, wherein said one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
24. the method for claim 22, wherein said one or more RA indications comprise that the sacroiliitis macroscopic view phenotype symptom in the Tg197 transgenic mice goes down, wherein give the Tg197 transgenic mice described composition, wherein write down the described sacroiliitis macroscopic view phenotype symptom of described Tg197 transgenic mice
Wherein according to the described sacroiliitis macroscopic view of following system log (SYSLOG) phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
25. the method for claim 22, wherein said one or more RA indications comprise that the sacroiliitis histopathology symptom in the Tg197 transgenic mice goes down, wherein give the Tg197 transgenic mice described composition, wherein write down the described sacroiliitis histopathology symptom of described Tg197 transgenic mice
Wherein on the joint, show described sacroiliitis histopathology symptom, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
26. the method for claim 21 wherein gives the described composition of Tg197 transgenic mice and may further comprise the steps:
A) weekly peritoneal injection gives heterozygosis Tg197 transgenic mice described composition,
B) weigh weekly a step a) mouse and
C) write down once the sacroiliitis macroscopic view phenotype illness of described mouse weekly according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
27. the method for claim 21 wherein gave described mouse described composition before showing the arthritic symptom outbreak.
28. the method for claim 21 wherein gives described composition for the first time when described mouse was 3 ages in week.
29. the method for claim 21 wherein gives described composition for the first time when described mouse was 6 ages in week.
30. each method or purposes in the aforementioned claim, the effectiveness that wherein said composition is had in the experiment of Tg197 transgenic mice sacroiliitis is greater than or equal to the effectiveness of the material that is selected from etanercept, English monoclonal antibody of sharp former times and D2E7.
31. each method or purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-0.5 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
32. each method or purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-1.0 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
33. each method or purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-1.5 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
34. each method or purposes in the aforementioned claim, effectively, keep the score by the sacroiliitis that makes treatment produce 0-2.0 in the experiment of Tg197 transgenic mice sacroiliitis for wherein said composition.
35. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction contains people's single domain antibody polypeptide.
36. each method or purposes in the aforementioned claim, wherein said people's single domain antibody polypeptide is in conjunction with TNF α.
37. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is with the K less than 100nM dIn conjunction with human TNF alpha.
38. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is with the K of 100nM to 50pM scope dIn conjunction with human TNF alpha.
39. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is with the K of 30nM to 50pM dIn conjunction with human TNF alpha.
40. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is with the K of 10nM to 50pM dIn conjunction with human TNF alpha.
41. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is with the K of 1nM to 50pM scope dIn conjunction with human TNF alpha.
42. each method or purposes in the aforementioned claim, wherein according to the detection of standard L929 cell experiment, described single domain antibody polypeptide construction antagonism human TNF alpha.
43. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction specificity combination and cell surface receptor bonded human TNF alpha.
44. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t α half life in the body in 15 minutes to 12 hours scopes.
45. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t α half life in the body in 1 to 6 hour scope.
46. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t α half life in the body in 2 to 5 hours scopes.
47. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t α half life in the body in 3 to 4 hours scopes.
48. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t β half life in the body in 12 to 60 hours scopes.
49. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t β half life in the body in 12 to 48 hours scopes.
50. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have t β half life in the body in 12 to 26 hours scopes.
51. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have AUC half life value in the body of 15mg. minute/ml to 150mg. minute/ml.
52. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have AUC half life value in the body of 15mg. minute/ml to 100mg. minute/ml.
53. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have AUC half life value in the body of 15mg. minute/ml to 75mg. minute/ml.
54. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction have AUC half life value in the body of 15mg. minute/ml to 50mg. minute/ml.
55. each method or purposes in the aforementioned claim, wherein said single domain antibody polypeptide construction is connected to the PEG molecule.
56. the method for claim 55 or purposes, wherein the single domain antibody polypeptide construction of PEG connection has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.
57. the method for claim 55 or purposes, wherein the single domain antibody polypeptide construction of PEG connection has the hydrokinetics size of 200kDa at least, and total PEG size is 20-60kDa.
58. the method for claim 55 or purposes, wherein the albumen of PEG connection on average is connected to 1-20 peg molecule.
59. each method or purposes in the aforementioned claim, wherein said antibody construction thing contain two or more single immunoglobulin variable structural domain polypeptide in conjunction with human TNF alpha.
60. each method or purposes in the aforementioned claim, wherein said antibody construction thing contain the homodimer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.
61. each method or purposes in the aforementioned claim, wherein said antibody construction thing contain the homotrimer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.
62. each method or purposes in the aforementioned claim, wherein said antibody construction thing contain the same tetramer in conjunction with single immunoglobulin variable structural domain polypeptide of human TNF alpha.
63. each method or purposes in the aforementioned claim, wherein said antibody construction thing also comprise non-TNF α antigen there is specific antibody polypeptides.
64. the method for claim 63 or purposes, wherein said have specific antibody polypeptides to comprise the single domain antibody polypeptide to non-TNF α antigen.
65. the method for claim 63 or 64 or purposes, wherein said have to non-TNF α antigen that specific antibody polypeptides and described non-TNF α are antigenic to combine half life in the body that has increased described antibody polypeptides construction.
66. each method or purposes among the claim 63-65, wherein said non-TNF α antigen comprises serum protein.
67. the method for claim 66 or purposes, wherein said serum protein is selected from: scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.
68. the method for claim 63 or purposes, wherein said non-TNF α antigen comprises HSA.
69. also comprising, each method among the claim 17-68, wherein said treatment give at least a other therapeutical agent.
70. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprises the aminoacid sequence of the antibody polypeptides CDR3 that is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
71. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
72. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
73. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
74. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
75. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
76. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
77. each method or purposes among the claim 1-69, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
78. the composition that contains single domain antibody polypeptide construction is used to prepare the purposes of medicine, described construction antagonism human TNF alpha bind receptor, described pharmacological agent, the prevention rheumatoid arthritis, suppress its progress or postpone its outbreak,
Wherein said composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model,
Wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha,
Wherein detect, in the described single domain antibody polypeptide construction and human TNF alpha according to standard L929 cell experiment.
79. a method for the treatment of rheumatoid arthritis, described method comprises the composition that the individual treatment of needs significant quantity is arranged, and described composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor,
Wherein said composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model,
Wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha,
Wherein detect according to standard L929 cell experiment, in the described single domain antibody polypeptide construction and human TNF alpha, and
Wherein said rheumatoid arthritis obtains medical treatment.
80. each method or purposes among the claim 1-79, wherein said single domain antibody polypeptide construction comprises tetravalence bi-specific antibody polypeptide construction, and it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first and described second copy of wherein said first fusion rotein through its IgG heavy chain constant domain separately each other disulfide linkage close, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and second epi-position.
81. the method for claim 80 or purposes, wherein said first and/or second epi-position are TNF-α epi-position.
82. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, described composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein detect, in the described single domain antibody polypeptide construction and human TNF alpha according to standard L929 cell experiment.
83. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, described composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein said single domain antibody polypeptide construction suppresses the progress of rheumatoid arthritis.
84. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, described composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
85. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor, described composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein detect according to standard L929 cell experiment, in the described single domain antibody polypeptide construction and human TNF alpha, wherein said single domain antibody polypeptide construction suppresses the progress of rheumatoid arthritis, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha.
86. each composition among the claim 82-85, wherein said single domain antibody polypeptide construction comprises tetravalence bi-specific antibody polypeptide construction, and it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first of wherein said first fusion rotein closes through its IgG heavy chain constant domain disulfide linkage separately each other with described second copy, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and second epi-position.
87. the composition of claim 86, wherein said first and/or described second epi-position are TNF-α epi-position.
88. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprises the aminoacid sequence of the antibody polypeptides CDR3 that is selected from following clone: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
89. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
90. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
91. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
92. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
93. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
94. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
95. each composition among the claim 82-87, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR1-2m-9, TAR1-2m-30, TAR1-2m-1, TAR1-2m-2, TAR1-5, TAR1-27, TAR1-261, TAR1-398, TAR1-701, TAR1-5-2, TAR1-5-3, TAR1-5-4, TAR1-5-7, TAR1-5-8, TAR1-5-10, TAR1-5-11, TAR1-5-12, TAR1-5-13, TAR1-5-19, TAR1-5-20, TAR1-5-21, TAR1-5-22, TAR1-5-23, TAR1-5-24, TAR1-5-25, TAR1-5-26, TAR1-5-27, TAR1-5-28, TAR1-5-29, TAR1-5-34, TAR1-5-35, TAR1-5-36, TAR1-5-464, TAR1-5-463, TAR1-5-460, TAR1-5-461, TAR1-5-479, TAR1-5-477, TAR1-5-478, TAR1-5-476, TAR1-5-490, TAR1h-1, TAR1h-2, TAR1h-3, TAR1-100-29, TAR1-100-35, TAR1-100-43, TAR1-100-47, TAR1-100-52, TAR1-109, TAR1-100, TAR1-100-34, TAR1-100-36, TAR1-100-38, TAR1-100-39, TAR1-100-40, TAR1-100-41, TAR1-100-45, TAR1-100-60, TAR1-100-62, TAR1-100-64, TAR1-100-65, TAR1-100-75, TAR1-100-76, TAR1-100-77, TAR1-100-78, TAR1-100-80, TAR1-100-82, TAR1-100-83, TAR1-100-84, TAR1-100-89, TAR1-100-90, TAR1-100-91, TAR1-100-92, TAR1-100-93, TAR1-100-94, TAR1-100-95, TAR1-100-96, TAR1-100-97, TAR1-100-98, TAR1-100-99, TAR1-100-100, TAR1-100-101, TAR1-100-102, TAR1-100-103, TAR1-100-105, TAR1-100-106, TAR1-100-107, TAR1-100-108, TAR1-100-109, TAR1-100-110, TAR1-100-111, TAR1-100-112, TAR1-100-113 and TAR1-5-19.
96. the composition that contains single domain antibody polypeptide construction is used to prepare the purposes of medicine, described construction antagonism people VEGF is in conjunction with vegf receptor, described pharmacological agent, prevention rheumatoid arthritis, suppresses its progress or postpones its outbreak.
97. treatment rheumatoid arthritis method, described method comprises the composition that the individual treatment of needs significant quantity is arranged, described composition contains the single domain antibody polypeptide construction of antagonism people VEGF in conjunction with vegf receptor, treats described rheumatoid arthritis thus.
98. the purposes of claim 96 or the method for claim 97, wherein said composition prevent the sacroiliitis increase of keeping the score when giving the mouse of sacroiliitis (CIA) mouse model that collagen protein brings out.
99. the method for claim 98 or purposes wherein give described mouse with described composition and may further comprise the steps:
A) weekly peritoneal injection gives the CIA model mice described composition,
B) weigh weekly a step a) mouse and
C) write down once the sacroiliitis macroscopic view phenotype symptom of described mouse weekly according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
100. each method among the claim 97-99, wherein said treatment comprises the progress that suppresses described rheumatoid arthritis.
101. each method among the claim 97-99, wherein said treatment comprise the outbreak of prevention or deferred class rheumatic arthritis.
102. each method or purposes among the claim 97-99, the wherein said statistics noticeable change that causes one or more RA indications.
103. the method for claim 102 or purposes, wherein said one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
104. the method for claim 102 or purposes, wherein said one or more RA indications comprise that sacroiliitis macroscopic view phenotype symptom goes down in the mouse of the sacroiliitis mouse model that collagen protein brings out,
Wherein give described mouse described composition,
Wherein write down the described sacroiliitis macroscopic view phenotype symptom of described mouse,
Wherein according to the described sacroiliitis macroscopic view of following system log (SYSLOG) phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
105. the method for claim 102 or purposes, wherein said one or more RA indications comprise that sacroiliitis histopathology symptom goes down in the mouse of the sacroiliitis mouse model that collagen protein brings out,
Wherein give described mouse described composition,
Wherein write down the described sacroiliitis histopathology symptom of described mouse,
Wherein on the joint, show described sacroiliitis histopathology symptom, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
106. the purposes of claim 134 or method, wherein said bi-specific antibody construction comprise tetravalence bi-specific antibody polypeptide construction, it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first of wherein said first fusion rotein closes through its IgG heavy chain constant domain disulfide linkage separately each other with described second copy, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and described second epi-position.
107. the method for claim 106 or purposes, wherein said first and/or second epi-position are the VEGF epi-position.
108. each method or purposes among the claim 96-107, wherein said single domain antibody polypeptide construction comprises people's single domain antibody polypeptide.
109. the method for claim 108 or purposes, wherein said people's single domain antibody polypeptide is in conjunction with VEGF.
110. each method or purposes among the claim 96-109, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with people VEGF.
111. each method or purposes among the claim 96-109, wherein said single domain antibody polypeptide construction with the Kd in 100nM to the 50pM scope in conjunction with people VEGF.
112. each method or purposes among the claim 96-109, wherein said single domain antibody polypeptide construction with the Kd of 30nM to 50pM in conjunction with people VEGF.
113. each method or purposes among the claim 96-109, wherein said single domain antibody polypeptide construction with the Kd of 10nM to 50pM in conjunction with people VEGF.
114. each method or purposes among the claim 96-109, wherein said single domain antibody polypeptide construction with the Kd in 1nM to the 50pM scope in conjunction with people VEGF.
115. each method or purposes among the claim 96-114 wherein detect according to vegf receptor 1 experiment or vegf receptor 2 experiments, in the described single domain antibody polypeptide construction and people VEGF.
116. each method or purposes among the claim 96-115, wherein said single domain antibody polypeptide construction specificity combination and cell surface receptor bonded people VEGF.
117. each method or purposes among the claim 96-116, wherein said single domain antibody polypeptide construction is connected to the PEG molecule.
118. the method for claim 117 or purposes, wherein the single domain antibody polypeptide construction of PEG connection has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.
119. the method for claim 117 or purposes, wherein the single domain antibody polypeptide construction that connects of PEG has the hydrokinetics size of 200kDa and total PEG size of 20-60kDa at least.
120. each method or purposes among the claim 117-119, wherein the single domain antibody polypeptide construction of PEG connection on average is connected to two or more peg molecules.
121. each method or purposes among the claim 117-119, wherein the single domain antibody polypeptide construction of PEG connection on average is connected to 2-20 PEG molecule.
122. each method or purposes among the claim 96-121, wherein said antibody construction thing contain two or more single immunoglobulin variable structural domain polypeptide in conjunction with people VEGF.
123. each method or purposes among the claim 96-121, wherein said antibody construction thing contain the homodimer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
124. each method or purposes among the claim 96-121, wherein said antibody construction thing contain the homotrimer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
125. each method or purposes among the claim 96-121, wherein said antibody construction thing contain the same tetramer in conjunction with single immunoglobulin variable structural domain polypeptide of people VEGF.
126. each method or purposes among the claim 96-125, wherein said construction also contain non-VEGF antigen had specific antibody polypeptides.
127. the method for claim 126 or purposes, wherein said have specific antibody polypeptides to non-VEGF antigen and comprise the single domain antibody polypeptide.
128. the method for claim 126 or 127 or purposes, wherein said have to non-VEGF antigen that specific described antibody polypeptides and described non-VEGF are antigenic to combine half life in the body that has increased described antibody polypeptides construction.
129. each method or purposes among the claim 126-128, wherein said non-VEGF antigen comprises serum protein.
130. the method for claim 129 or purposes, wherein said serum protein is selected from: scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.
131. each method or purposes among the claim 126-129, wherein said non-VEGF antigen comprises HSA.
132. also comprising, each method among the claim 97-131, wherein said treatment give at least a other therapeutical agent.
133. the method for claim 132, wherein said therapeutical agent are selected from etanercept, English monoclonal antibody of sharp former times and D2E7.
134. the method for claim 132, wherein said therapeutical agent is selected from: reflunomide, non-steroidal anti-inflammatory drug (NSAID), acetylsalicylic acid, pyrazolone, fragrant that acid, diflunisal, acetogenin, propanoic derivatives, former times health, mefenamic acid, Ponstel, meclofenamic acid, Meclomen, Phenylbutazone, Butazolidin, diflunisal, Dolobid, diclofenac, diclofenac, indomethacin, INDOMETHACIN, sulindac, Sulindac, R-ETODOLAC, Luo Ding, ketorolac, Toradol, nabumetone, Relafen, tolmetin, Tolectin, Ibuprofen BP/EP, Merrill Lynch, fenoprofen, Nalfon, flurbiprofen, Anthe, carprofen, Rimadyl, Ketoprofen, Orudis, Naproxen Base, Anaprox, Naprosyn, piroxicam and Feldene.
135. each method or purposes in the claim 134, wherein said single domain antibody polypeptide construction comprises the aminoacid sequence of the antibody polypeptides CDR3 that is selected from following clone: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
136. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 85% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
137. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 90% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
138. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 92% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
139. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 94% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
140. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 96% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
141. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 98% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
142. each method or purposes among the claim 96-134, wherein said single domain antibody polypeptide construction comprise the antibody polypeptides that is selected from following clone aminoacid sequence or with its at least 99% identical aminoacid sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
143. containing, a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction be selected from following CDR3 sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
144. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 85% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
145. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 90% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
146. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 92% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
147. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 94% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
148. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 96% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
149. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 98% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
150. a composition that contains the single domain antibody polypeptide construction of antagonism people VEGF and receptors bind, wherein said single domain antibody polypeptide construction contain be selected from following aminoacid sequence or with its at least 99% identical sequence: TAR15-1, TAR15-3, TAR15-4, TAR15-9, TAR15-10, TAR15-11, TAR15-12, TAR15-13, TAR15-14, TAR15-15, TAR15-16, TAR15-17, TAR15-18, TAR15-19, TAR15-20, TAR15-22, TAR15-5, TAR15-6, TAR15-7, TAR15-8, TAR15-23, TAR15-24, TAR15-25, TAR15-26, TAR15-27, TAR15-29 and TAR15-30.
151. each composition among the claim 143-150, wherein said single domain antibody polypeptide construction comprises tetravalence bi-specific antibody polypeptide construction, and it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first and described second copy of wherein said first fusion rotein through its IgG heavy chain constant domain separately each other disulfide linkage close, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and described second epi-position.
152. the composition of claim 151, wherein said first and/or described second epi-position are the VEGF epi-position.
153. contain the purposes of composition of the single domain antibody polypeptide construction of the active and antagonism people VEGF bind receptor of antagonism human TNF alpha, this purposes is used to prepare treatment, prevention rheumatoid arthritis, suppresses the medicine that rheumatoid arthritis is made progress or deferred class rheumatic arthritis is shown effect.
154. contain the purposes of composition of the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, this purposes is used to the medicine for preparing treatment, prevention rheumatoid arthritis, suppress rheumatoid arthritis progress or the outbreak of deferred class rheumatic arthritis.
155. method for the treatment of rheumatoid arthritis, described method comprises the composition that the individual treatment of needs significant quantity is arranged, wherein said composition contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, treats described rheumatoid arthritis thus.
156. the purposes of claim 153 or 154 or the method for claim 155, wherein said polypeptide construction contains the bi-specific antibody construction.
157. the purposes of claim 134 or method, wherein said bi-specific antibody construction contain tetravalence bi-specific antibody polypeptide construction, it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first and described second copy of wherein said first fusion rotein through its IgG heavy chain constant domain separately each other disulfide linkage close, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and described second epi-position.
158. each method or purposes among the claim 153-157, wherein said composition prevent the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model.
159. the method for claim 158 or purposes wherein give the Tg197 transgenic mice described composition, it may further comprise the steps:
A) weekly peritoneal injection gives heterozygosis Tg197 transgenic mice described composition,
B) weigh weekly a step a) mouse and
C) write down once the sacroiliitis macroscopic view phenotype symptom of described mouse weekly according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
160. the method for claim 158 or purposes, the effectiveness that wherein said composition is had in the experiment of Tg197 transgenic mice sacroiliitis is greater than or equal to the effectiveness that is selected from following material: etanercept, English monoclonal antibody of sharp former times and D2E7.
161. each method or purposes among the claim 153-157, wherein said composition prevent the sacroiliitis increase of keeping the score when giving the mouse of sacroiliitis (CIA) mouse model that collagen protein brings out.
162. the method for claim 161 or purposes wherein give described mouse described composition, it may further comprise the steps:
A) weekly peritoneal injection gives the CIA model mice described composition,
B) weigh weekly a step a) mouse and
C) write down once the sacroiliitis macroscopic view phenotype symptom of described mouse weekly according to following system: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
163. the method for claim 158 or purposes, the wherein said statistics noticeable change that causes one or more RA indications.
164. each method among claim 131 or the 134-137, wherein said treatment comprises the progress that suppresses described rheumatoid arthritis.
165. each method among the claim 155-163, wherein said treatment comprise the outbreak of prevention or deferred class rheumatic arthritis.
166. the method for claim 163, wherein said one or more RA indications comprise following one or more: erythrocyte sedimentation rate (ESR), Ritchie joint exponential sum refuse to budge morning continuous time, joint mobilization, arthroncus, the x ray contrast in one or more joints and the histopathological analysis of one or more arthrodesis sections.
167. the method for claim 163, wherein said one or more RA indications comprise that the sacroiliitis macroscopic view phenotype symptom in the Tg197 transgenic mice goes down,
Wherein give the Tg197 transgenic mice described composition,
Wherein write down the described sacroiliitis macroscopic view phenotype symptom of described Tg197 transgenic mice,
Wherein according to the described sacroiliitis macroscopic view of following system log (SYSLOG) phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
168. the method for claim 140, wherein said one or more RA indications comprise that the sacroiliitis histopathology symptom in the Tg197 transgenic mice goes down,
Wherein give the Tg197 transgenic mice described composition,
Wherein write down the described sacroiliitis histopathology symptom of described Tg197 transgenic mice,
Wherein on the joint, show described sacroiliitis histopathology symptom, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
169. the method for claim 161 or purposes, the wherein said statistics noticeable change that causes one or more RA indications.
170. the method for claim 169 or purposes, wherein said one or more RA indications comprise that sacroiliitis macroscopic view phenotype symptom goes down in the mouse of the sacroiliitis mouse model that collagen protein brings out,
Wherein give described mouse described composition,
Wherein write down the described sacroiliitis macroscopic view phenotype symptom of described mouse,
Wherein according to the described sacroiliitis macroscopic view of following system log (SYSLOG) phenotype symptom: 0=does not have sacroiliitis (normal appearance and flexion), 1=mild osteoarthritis (joint distortion), 2=moderate sacroiliitis (swelling, joint deformity), 3=severe sacroiliitis (move and be badly damaged).
171. the method for claim 169, wherein said one or more RA indications comprise that sacroiliitis histopathology symptom goes down in the mouse of the sacroiliitis mouse model that collagen protein brings out,
Wherein give described mouse described composition,
Wherein write down the described sacroiliitis histopathology symptom of described mouse,
Wherein on the joint, show described sacroiliitis histopathology symptom, and use following system log (SYSLOG): 0=not have detectable pathological condition, 1=synovial hyperplasia and polymorphonuclear leukocyte infiltration occurs, 2=pannus and fibrous tissue form and the focus subchondral bone corrodes, the 3=joint cartilage is destroyed and bone erosion, and 4=joint cartilage widely destroys and bone erosion.
172. each method or purposes among the claim 153-171, wherein said single domain antibody polypeptide construction comprises people's single domain antibody polypeptide.
173. the method for claim 172 or purposes, wherein said single domain antibody polypeptide construction is in conjunction with TNF α and VEGF.
174. the method for claim 172 or purposes wherein detect according to vegf receptor 1 experiment or vegf receptor 2 experiments, in the active single domain antibody polypeptide portion of described antagonism people VEGF and people VEGF.
175. each method or purposes among the claim 153-174 wherein detect according to standard L929 cell experiment, in the described single domain antibody polypeptide construction and human TNF alpha.
176. each method or purposes among the claim 153-175, wherein said single domain antibody polypeptide construction is connected to the PEG molecule.
177. the method for claim 176 or purposes, wherein the single domain antibody polypeptide construction of PEG connection has the hydrokinetics size of 24kDa at least, and wherein total PEG size is 20-60kDa.
178. the method for claim 176 or purposes, wherein the single domain antibody polypeptide construction that connects of PEG has the hydrokinetics size of 200kDa and total PEG size of 20-60kDa at least.
179. each method or purposes among the claim 176-178, wherein said antibody polypeptides construction on average is connected to 2 or a plurality of peg molecule.
180. each method or purposes among the claim 176-178, wherein said antibody polypeptides construction on average is connected to 2-20 PEG molecule.
181. each method or purposes among the claim 153-180, wherein said antibody construction thing contain two or more single immunoglobulin variable structural domain polypeptide and/or two or more single immunoglobulin variable structural domain polypeptide in conjunction with people VEGF in conjunction with the humanTNF-.
182. each method or purposes among the claim 153-180, wherein said antibody construction thing contain in conjunction with the homodimer of single immunoglobulin variable structural domain polypeptide of humanTNF-and/or in conjunction with the homodimer of single immunoglobulin variable structural domain polypeptide of people VEGF.
183. each method or purposes among the claim 153-180, wherein said antibody construction thing contain in conjunction with the homotrimer of single immunoglobulin variable structural domain polypeptide of humanTNF-and/or in conjunction with the homotrimer of single immunoglobulin variable structural domain polypeptide of people VEGF.
184. each method or purposes among the claim 153-180, wherein said antibody construction thing contain in conjunction with the same tetramer of single immunoglobulin variable structural domain polypeptide of humanTNF-and/or in conjunction with the same tetramer of single immunoglobulin variable structural domain polypeptide of people VEGF.
185. each method or purposes among the claim 153-180, wherein said construction also contain non-TNF α or VEGF antigen had specific antibody polypeptides.
186. the method for claim 185 or purposes, wherein said have specific antibody polypeptides to non-TNF α or VEGF antigen and comprise the single domain antibody polypeptide.
187. the method for claim 185 or 186 or purposes, wherein said have to non-TNF α or VEGF antigen that specific antibody polypeptides and described non-TNF α or VEGF are antigenic to combine half life in the body that increases described antibody polypeptides construction.
188. each method or purposes among the claim 185-187, wherein said non-TNF α or VEGF antigen comprise serum protein.
189. the method for claim 188 or purposes, wherein said serum protein is selected from: scleroproein, α-2 macroglobulin, serum albumin, Fibrinogen A, Fibrinogen, serum amyloid A protein, heptoglobin, albumen, ubiquitin, Clara cell 10kDa protein and beta-2-microglobulin.
190. each method or purposes among the claim 185-189, wherein said non-TNF α antigen comprises HSA.
191. also comprising, each method among the claim 155-190, wherein said treatment give at least a other therapeutical agent.
192. the method for claim 191, wherein said other therapeutical agent is selected from: reflunomide, non-steroidal anti-inflammatory drug (NSAID), acetylsalicylic acid, pyrazolone, fragrant that acid, diflunisal, acetogenin, propanoic derivatives, former times health, mefenamic acid, Ponstel, meclofenamic acid, Meclomen, Phenylbutazone, Butazolidin, diflunisal, Dolobid, diclofenac, diclofenac, indomethacin, INDOMETHACIN, sulindac, Sulindac, R-ETODOLAC, Luo Ding, ketorolac, Toradol, nabumetone, Relafen, tolmetin, Tolectin, Ibuprofen BP/EP, Merrill Lynch, fenoprofen, Nalfon, flurbiprofen, Anthe, carprofen, Rimadyl, Ketoprofen, Orudis, Naproxen Base, Anaprox, Naprosyn, piroxicam and Feldene.
193. a dual specific antigen-binding polypeptides, it contains in conjunction with first antibody single structure domain polypeptide of TNF-α with in conjunction with second antibody single structure domain polypeptide of VEGF.
194. the dual specific antigen-binding polypeptides of claim 193, wherein said construction contain tetravalence bi-specific antibody polypeptide construction, it comprises:
A) contain in conjunction with first epi-position, merge to first copy of first fusion rotein of the single domain antibody polypeptide of IgG3 heavy chain constant domain;
B) second of described first fusion rotein copy;
C) contain in conjunction with second epi-position, merge to first copy of second fusion rotein of the single domain antibody polypeptide of light chain constant domain;
D) second of described second fusion rotein copy;
Described first and described second copy of wherein said first fusion rotein through its IgG heavy chain constant domain separately each other disulfide linkage close, and
The described light chain constant domain disulfide linkage of described first copy of wherein said second fusion rotein is bonded to the IgG heavy chain constant domain of described first copy of described first fusion rotein, and
Wherein the described light chain constant domain disulfide linkage of described second copy of second fusion rotein is bonded to the IgG heavy chain constant domain of described second copy of described first fusion rotein, and
Wherein said polypeptide construction is in conjunction with described first and described second epi-position.
195. the dual specific antigen-binding polypeptides of claim 165, wherein said first epi-position are TNF-α epi-position, described second epi-position is the VEGF epi-position.
196. the dual specific antigen-binding polypeptides of claim 165, wherein said first epi-position is the VEGF epi-position, and described second epi-position is TNF-α epi-position.
197. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, it prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein said single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress.
198. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, wherein said composition prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha, wherein said single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress.
199. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, it prevents the sacroiliitis increase of keeping the score when giving the mouse of Tg197 transgenic mice arthritis model, wherein detect according to standard L929 cell experiment, in the described single domain antibody polypeptide construction and human TNF alpha, wherein said single domain antibody polypeptide construction with less than the Kd of 100nM in conjunction with human TNF alpha, wherein said single domain antibody polypeptide construction suppresses the rheumatoid arthritis progress.
200. composition that contains the single domain antibody polypeptide construction of antagonism human TNF alpha bind receptor and antagonism people VEGF bind receptor, it prevents the sacroiliitis increase of keeping the score when giving the arthritis model mouse that collagen protein brings out, wherein said single domain antibody polypeptide construction suppresses the progress of rheumatoid arthritis.
201. each composition among the claim 193-200, wherein said composition gives with at least a other therapeutical agent.
202. the composition of claim 201, wherein said other therapeutical agent is selected from: reflunomide, non-steroidal anti-inflammatory drug (NSAID), acetylsalicylic acid, pyrazolone, fragrant that acid, diflunisal, acetogenin, propanoic derivatives, former times health, mefenamic acid, Ponstel, meclofenamic acid, Meclomen, Phenylbutazone, Butazolidin, diflunisal, Dolobid, diclofenac, diclofenac, indomethacin, INDOMETHACIN, sulindac, Sulindac, R-ETODOLAC, Luo Ding, ketorolac, Toradol, nabumetone, Relafen, tolmetin, Tolectin, Ibuprofen BP/EP, Merrill Lynch, fenoprofen, Nalfon, flurbiprofen, Anthe, carprofen, Rimadyl, Ketoprofen, Orudis, Naproxen Base, Anaprox, Naprosyn, piroxicam and Feldene.
CN200580028518.5A 2004-06-30 2005-06-29 Compositions and methods for treating inflammatory disorders Pending CN101039959A (en)

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CN101502490B (en) * 2009-02-26 2013-07-31 沈阳药科大学 Ketoprofen bioadhesive gel microsphere and preparation method thereof
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101502490B (en) * 2009-02-26 2013-07-31 沈阳药科大学 Ketoprofen bioadhesive gel microsphere and preparation method thereof
CN102639715A (en) * 2009-11-24 2012-08-15 凯蒂生化株式会社 Method and apparatus for prediction of pharmacological efficacy of humanized anti-TNF[alpha]antibody drug on rheumatoid arthritis
CN102639715B (en) * 2009-11-24 2014-09-10 凯蒂生化株式会社 Method and apparatus for prediction of pharmacological efficacy of humanized anti-TNF[alpha]antibody drug on rheumatoid arthritis
CN103842380A (en) * 2011-07-27 2014-06-04 葛兰素集团有限公司 Anti-VEGF single variable domains fused to Fc domains
CN107106672A (en) * 2015-01-12 2017-08-29 新加坡科技研究局 The monoclonal antibody for muramyl peptide for preventing and treating immune-mediated disease
CN107454905A (en) * 2015-03-31 2017-12-08 韦斯夸尔德有限公司 Polypeptide
CN107454905B (en) * 2015-03-31 2023-04-28 索里索制药公司 Polypeptides
WO2023025258A1 (en) * 2021-08-26 2023-03-02 广东暨安特博生物科技有限公司 Composition and method for controlling heat generation within organism, and use
CN114395574A (en) * 2022-01-18 2022-04-26 长沙爱科博生物科技有限公司 Porcine epidemic diarrhea virus fusion protein and coding gene and application thereof
CN114395574B (en) * 2022-01-18 2023-08-11 长沙爱科博生物科技有限公司 Porcine epidemic diarrhea virus fusion protein, and encoding gene and application thereof

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