CN101312988A

CN101312988A - Antibody polypeptide library screening and selected antibody polypeptides

Info

Publication number: CN101312988A
Application number: CNA2006800379007A
Authority: CN
Inventors: L·耶斯佩斯; J·克拉布
Original assignee: Domantis Ltd
Current assignee: Domantis Ltd
Priority date: 2005-10-11
Filing date: 2006-10-11
Publication date: 2008-11-26
Also published as: GB0520644D0

Abstract

The present invention provides further developments in the screening of antibody polypeptide libraries. The invention also provides novel isolated antibody polypeptides obtainable by the methods of the invention.

Description

The antibody polypeptides class of antibody polypeptides library screening and selection

The present invention further provides in the screening antibody polypeptides library dynamically.The new isolated antibody polypeptide class that can provide the inventive method to obtain also is provided in the present invention.

Be appointed as to have described among the applicant's the WO99/20749 and use general part, for example antibody or antibody fragment or comprise method from the arbitrary substance screening antibody library (for example heavy chain of antibody variable domain library) of one or more specific binding sites of antibody." antibody " is defined as the structure in combination (variable) district that uses this antibody-like.The content that WO99/20749 is disclosed is incorporated herein by reference, particularly the library generates (" structure in library of the present invention "), select polypeptide class (" selection of polypeptide class of the present invention ") from the library, part is selected the content of disclosure in (" antibody of the part in selecting as polypeptide ") and the industrial applications (" application of the polypeptide class of selecting according to the present invention ").Clearly all these parts are introduced the application so that the disclosure that can be used for feature of the present invention is provided, and those skilled in the art be easy to recognize-in the context of the application's claim-those can be used for feature of the present invention.

Unconventional antibody type-heavy chain antibody has been described in the document.Have the patient of heavy chain disease, the serum of the B-cell that EBV transforms, and the more important thing is and in the serum of Camelidae (Camelidae) (camel, Llama), found these high titer antibody.These heavy chain antibodies do not contain heavy chain, but only contain single right identical heavy chain.These heavy chains are that with those differences among the commonly used IgGs their lack the constant domain 1 (CH1) that works in the light chain pairing on the mediation heavy chain.Therefore, derived heavy chain antibody does not contain the light chain variable territory, and only contains two unpaired heavy chain variable domains.The research of relevant immunization Camelidae serum is verified, although there is not light chain, the certain specificity of these heavy chain antibodies is in conjunction with having the antigen of moderate to high affinity.Unpaired heavy chain variable domain (being called VHH) has carried out genetic adaptation from start to finish what evolve: many aminoacid replacement are taking place to take place in the interactional part with the VL structural domain by (even be level in kind) usually: the L45 that guards in VHs is replaced by Arg.Undergo mutation usually in other position: V37 is mutated into Phe, and G44 is mutated into Glu and W47 is mutated into Gly.Finally, on average than those (being respectively 9 and 12 amino acid) long (16-17 amino acid) of mouse and people VHS, compensated non-existent VL structural domain thus from the CDR3 of the VHHs of camel (but being not limited to Llama) so that conjugated antigen.With reference to WO05044858A1, WO04062551A2, WO04041867A2, WO04041865A2, WO04041863A2, the description of relevant Camelid VHH structural domain among the WO04041862A2, WO03050531A2 and EP0656946.

Since 1989, had realized that the single variable domain of antibody has treatment potential.Because its small size, so they rest on the antigen position of inaccessible conventional antibody (splitting valley, avtive spot).These structural domains can also form the product of the certain limit of making as required: for example can make their multimerizations (by chemistry or mode of inheritance) so that increase avidity, keep relatively little overall dimension simultaneously.Can pass through Pegylation (so that increase fluid size) or by with serum protein, such as the serum albumin that shows prolong half-life (in human body, reaching 19 days) covalently or non-covalently in conjunction with the persistence of regulating in the serum.Finally, can make single variable domain form IgG again so that have benefited from the Fc-effector function.In all situations, the genetic information of the antigen-specific antibodies variable domain of having the right to use is for for the absolute prerequisite condition of all above-mentioned strategies of mentioning.

Researched and developed the gene that several method separates the antibody variable domains of coding for antigens-specific antibody.One of these methods are based on early stage observations, and promptly in its R﹠D process, the B-lymphocyte is expressed the antibody (by combining with the hereditary syzygy mediation of film anchoring peptide) of single type on its cell surface separately.Conjugated antigen (participating in t helper cell) mediates the lymphocytic antigen-proliferated specifically of B-, and reaching maturity at subsequent stage is plasma cell.These cells are not expressed surface-bonded antibody, but secrete them in a large number.Therefore, in its growth course, the B-lymphocyte can be considered as hereditary demonstration package, wherein phenotype (antibody) is connected (antibody gene) with genotype.Therefore, in order to separate the gene of coding for antigens-specific antibody, researched and developed method, wherein (i) makes the B-lymphocyte gather thing (separating the animal from immunization) contact antigen (can on cell or solid phase immunization maybe can use dye marker); (ii) antigen-specific b-lymphocyte conjugated antigen; (iii) antigen-specific b-the lymphocyte of separation and combination from unconjugated B-lymphocyte; (iv) bonded antigen-specific b-lymphocyte is recovered into storage.Test tube, hole or plate; (v) from isolating B-lymphocyte, reclaim the gene (can be used as mono-clonal or polyclonal population stores) of coding for antigens-specific antibody variable domain.The example that uses the method for this scheme is those that describe in the following document:

(i) (1996) Proc.Natl.Acad.Sci.USA 93 such as Babcook, 7843-7848.

SLAM has overcome the limitation of the antibody library of hybridoma technology and bacterial expression by can separate the high-affinity antibody that generates in the immunne response process in vivo from any kind.SLAM can make single lymphocyte generation have the antibody of required specificity or function so that identified from this lymphocyte in the genetic information of the antibodies specific that lymphoidocyte large group and coding recover, for example described genetic information can be cloned into expression vector, so that can express a large amount of antibody.In one embodiment, the cell that produces antibody detects (Jerne and Nordin (1963) Science140,405) in conjunction with antigen of selecting and the suitable hemolytic bacteria patch test method of use.In this test, the antigen coated red corpuscle of use selecting and it is hatched with cell mass and complement source of producing antibody.The cell that produces monospecific antibody is identified in formation according to the hemolytic bacteria plaque.Use inverted microscope to identify cracked red corpuscle plaque, and use micromanipulative technique to take out the cell of the generation monospecific antibody of on the center of plaque, paying close attention to, be used to inoculate bag then by the single hole of EL4-B5T cell (great-hearted cell-cell interaction is provided and is used for the soluble factor of B-cell expressing).On average, distribute for each hole inoculation 0.3B-cell to guarantee the clone.Clone from the gene in these clone's equatorial B cell by reverse transcription antibody PCR then.Other method that is used for detecting the cell with required function that produces monospecific antibody has been described in international patent specification WO 92/02551.

(ii) (1997) J.Immunol.Methods 2073 such as de Wildt, 61-67.

Described method comprises the following steps: that (i) gathers the B-lymphocyte from people's donor; (ii) the flow cytometer (FACS instrument) with the fluorescent activation that the automated cell distribution member has been installed detects the CD19+/CD20+ cell; (iii) the CD19+/CD20+B cell has been allocated separately at the bag quilt in the single hole of EL4-B5T cell, described EL4-B5T cell provides cell-cell interaction (CD40-CD40L) and soluble growth factor; Single B-cell is expanded separately so that increase the amount of mRNA; (v) reclaim antibody gene by RT-PCR.Should notice that step 4 (expansion of B-cell) is optional, can carry out pcr amplification to the antibody gene from single cell effectively as long as PCR method can be designed to.

(iii) WO 2004106377 such as Lawson.

In this patent application, the author is by flow cytometry ((1997) J.Immunol.Methods such as de Wildt, 2073,61-67) or biological elutriation (Lagerkvist etc. (1995) Biotechniques 18 862-869) has separated antigen-specific b-cell.Opposite with these two kinds of means, the method for Lawson etc. need not to separate each B cell.The quantity of B cell is at 100-20 in every hole, 000 scope-remain after the B-cell expands, and the antibody variable Gene RT-PCR discloses the monoclonal antibody sequence in most of hole.

The B-cell subsets that all these methods have all been used antigen to separate to present antibody.This means reckon without, and in antigen-specific b-cell mass, the antibody of displaying can be different in directly not relating to antigen bonded district.For example, antibody commonly used is made of κ or lambda light chain.Only thus can not the different B-cell subsets of sorting based on antigenic selection.Must test the B-lymphocyte of each antigen-selection then, so that be used for the light chain isotype subsequently.Specified prior example according to observations to the Camelidae of immunization: in camel, think～20% B-lymphocyte expresses in its surface and/or secretes heavy chain antibody.Four-chain antibody that remaining B-lymphocyte displaying/expression is conventional.Therefore, can't distinguish between those express those B-cells of heavy chain antibody based on antigenic selection, described heavy chain antibody is expressed the B-cell of conventional antibodies from those.Use the present invention to address this problem.

Also with reference to following open source literature with the Celltech name:

WO04051268A1:ASSAY FOR IDENTIFYING ANTIBODYPRODUCING CELL, the document has disclosed and has been used to identify the homology mensuration that produces in conjunction with the cell of the antigenic antibody of selecting, and this method comprises hatches the cell that produces antibody with the anti-antibody antibody of antigen and mark.

WO04106377A1:METHODS FOR PRODUCING ANTIBODY, the document has disclosed the antibody that obtains to have required function through the following steps: make B cells contacting capture reagent, the B cell that B cell that separation is captured and cultivation and screening are captured is so that identify that the cell that produces antibody is so that obtain required antibody.

WO05019823A1:METHODS FOR OBTAINING ANTOBODY, the document has disclosed enrichment B cell mass, this B cell mass is used to produce the antigenic antibody of being paid close attention to by exposing cell identification, the antigen of being paid close attention to and the cell of antibody-particle composites, the wherein antigen paid close attention to of antibody recognition.

WO05019824A1:METHODS FOR OBTAINING ANTIBODY, the document has disclosed enrichment B cell mass in producing the cell of discerning the antigenic antibody of being paid close attention to, and this method comprises that use has the cell of specific antibody labeling cell and separation of double mark to B cell marking and antigen.

The antigen binding domains of antibody comprises two independent districts: can be V κ or V λ) heavy chain variable domain (VH) and light chain variable territory (VL).Antigen-binding site self is made of 6 polypeptide rings: 3 from (1 rearrangement of gene fragment of VH structural domain.The VH gene is by reorganization H1, and H2 and H3 produce) and 3 from VL structural domain (L1, L2 and L3).The different elementary repertoire (repertoire) of the V gene of coding VH and VL structural domain is by merging three kinds of gene fragment VH, and D and JH produce.In the people, (Cook and Tomlinson (1995) Immunol Today, 16:237), (Corbett etc. (1997) J.Mol.Biol. is 268:69) with 6 functional J for 25 functional D fragments about 51 functional VH fragments _H(Ravetch etc. (1981) cell, 27:583), this depends on haplotype to fragment.The VH fragment coding constitutes the polypeptide sequence of the first or second antigen coupling collar of VH structural domain (H1 and H2), and VH, D and J _HFragment is merged into the antigen iii coupling collar of VH structural domain (H3).Only two kinds of gene fragment VL and JL produce the VL gene by reorganization.In the people, exist about 40 functional V κ fragments (

And Zachau (1993) Biol.Chem.Hoppe-Seyler, 374:1001), 31 functional V λ fragments (Williams etc. (1996) J.Mol.Biol., 264:220; Kawasaki etc. (1997) Genome Res., 7:250), (Hieter etc. (1982) J.Biol.Chem. is 257:1516) with 4 functional J λ fragment (Vasicek and Leder (1990) J.Exp.Med. for 5 functional J κ fragments, 172:609), this depends on haplotype.The VL fragment coding constitutes the polypeptide sequence of the first or second antigen coupling collar of VL structural domain (L1 and L2), and VL and JL fragment are merged into the antigen iii coupling collar of VL structural domain (L3).Think that the antibody of selecting is enough to differently to have the antigen of moderate avidity at least in conjunction with nearly all from this elementary repertoire.Produce high-affinity antibody by " affinity maturation " of resetting gene, wherein generate by immunity system and the selected element sudden change based on the combination that improves.

The analysis of antagonist structure and sequence has confirmed that 5 in 6 antigen coupling collars (H1, H2, L1, L2 and L3) have main chain conformation or norm structure (Chothia and Lesk (1987) J.Mol.Biol., the 196:901 of limited quantity; Chothia etc. (1989) Nature, 342:877).Determine the main chain conformation according to following condition: (i) length of antigen coupling collar; (ii) specific residue or the residue type on some key position in antigen coupling collar and antibody framework.Analysis to ring length and Key residues can make us predict the H1 that is encoded by most people's antibody sequence, H2, L1, the main chain conformation of L2 and L3 (Chothia etc. (1992) J.Mol.Biol., 227:799; Tomlinson etc. (1995) EMBO J., 14:4628; Williams etc. (1996) J.Mol.Biol., 264:220).Although the H3 district is in sequence, length and configuration aspects different (because of using the D fragment) far away, but make it also constitute the main chain conformation of limited amount becate length, this depends on specific residue or residue type (Martin etc. (1996) J.Mol.Biol., the 263:800 that exists on the key position on length and ring and the antibody framework; Shirai etc. (1996) FEBS Letters, 399:1).

Can from the pattern that generates by somatic hypermutation, separate sequence polymorphism pattern in the elementary repertoire to the multifarious similar analysis of side chain in the human antibody sequence.Find two kinds of pattern complementations: the diversity in elementary repertoire concentrates on antigen in conjunction with the center, and somatic hypermutation passes to diversity at elementary repertoire camber conservative surrounding zone (Tomlinson etc. (1996) J.Mol.Biol., 256:813; Ignatovich etc. (1997) J.Mol.Biol, 268:69).This complementarity seems to develop as search sequence spatial available strategy, thereby has obtained the B cell of the limited quantity that can in officely mean fixes time can be used in selection.Therefore, at first from elementary repertoire, select antibody based on the supercentral diversity of binding site.Keep somatic hypermutation then so that optimize residue on the periphery under the favourable interactional situation of in not destroying primary response's process, setting up.

The appearance of display technique of bacteriophage (Smith (1985) Science, 228:1315; Scott and Smith (1990) Science, 249:386; McCafferty etc. (1990) Nature 348:552) makes the antibody of selecting at extensive target antigen external from " single jar " library.These phage-antibody libraries can be divided into two classes: use and gather from the natural library of the rearrangement V of human B cell gene (Marks etc. (1991) J.Mol.Biol., 222:581; Vaughan etc. (1996) NatureBiotech., 14:309) or to plant be that the V gene fragment is external ' rearrangement ' (Hoogenboom ﹠amp; Winter (1992) J.Mol.Biol., 227:381; Nissim etc. (1994) EMBO J., 13:692; Griffiths etc. (1994) EMBO J., 13:3245; (1995) J.Mol.Biol. such as De Kruif, 248:97) or synthetic CDRs mix single rearrangement V gene (Barbas etc. (1992) Proc.Natl.Acad.Sci.USA, synthetic library 89:4457).Although synthetic library helps to overcome the inherent bias that may limit by the natural repertoire of the gene constructed effective size of phage library of rearrangement V, they need use common long degenerate pcr primer with base-V gene that importing is assembled to disappearance.This height randomization also may cause can not correctly folding and also being thus the antibody generation of non-functional.In addition, the antibody of selecting from these libraries is beyond expression of words, and in many cases, these antibody comprise framework mutations, and these sudden changes can influence antibody mediated immunity originality when being used for people's therapy.

In the expansion of synthetic library means, prompting (WO97/08320, Morphosys) can be by synthetic one group ' key-gene ' optimize in advance people's antibody framework, this group ' key-gene ' have total frame sequence and mix to demonstrate and can improve aminoacid replacement folding and expression.Use oligonucleotide to mix the diversity of CDRs then.Owing to need to produce and can not be identified as ectogenic people workman's antibody by human immune system, thus in most of situation be not the application that is equivalent to the total framework of any natural framework be the shortcoming of these means.In addition, because possible situation is, CDR diversity also doubling is folded and/or express and have effect, optimizes folding and/or expresses (and removing any frameshit or terminator codon) so preferably assemble the back fully at the V gene.For this purpose, need selective system, it can be eliminated non-functional in the library or be difficult to the member that folds/express, after this uses target antigen to screen.

Use the additional problem in the library of prior art to be, because the main chain conformation is a heterology, so 3 d structure modelization is difficult, this is because suitable high resolving power crystallographic data can't utilize.This is the particular problem in H3 district, and is wherein most of; The antibody that derives from natural or synthetic antibody libraries has moderate-length or long ring and thus can not modeling.

Summary of the invention

First aspect of the present invention provides from the repertoire of polypeptide class the method for selecting in conjunction with the functional polypeptide types of populations of the general part on the target ligands on first binding site and second binding site, described general part can be in conjunction with the combined function member of repertoire, but irrelevant with the specificity of target ligands, this method comprises the following steps:

A) make repertoire contact general part and select bonded functional polypeptide class with it; With

B) make the functional polypeptide class contact target ligands of selection and select polypeptide types of populations in conjunction with target ligands.

The present invention provides method thus, by this method, according to as the repertoire in conjunction with the preselected polypeptide class of the ability mensuration of general part, and is used for extra selection cycle according to the ability in conjunction with target ligands then.In a preferred embodiment, although at first use general part to select repertoire, those skilled in the art are apparent, can make repertoire with opposite order contact part, promptly contact target ligands before the general part of contact.

The present invention allows those skilled in the art to remove those from the repertoire of the polypeptide class selected to be the polypeptide class of non-functional, for example, as importing phase shift mutation, terminator codon, folding mutant or express the result of mutant, they may be basically can not or can not be in conjunction with target ligands arbitrarily.This class is non--and functional mutants produces by normal randomization and the variable operation step that is used to make up the polypeptide repertoire.Simultaneously, it is that those belong to functional that the present invention allows those skilled in the art, the abundant polypeptide class repertoire of selecting with the enrichment of height polypeptide expressed class that fold.

Two or more subgroups of preferred polypeptide class by method of the present invention available from repertoire, for example by using two or more general part prescreen repertoires or by making repertoire under different condition, contact general part.Advantageously, thus obtained polypeptide class subgroup is merged into extra polypeptide class repertoire, can be by contacting target and/or general part to its further screening.

Preferred library of the present invention comprises the polypeptide class of immunoglobulin superfamily, such as antibody polypeptides class or T-cell receptors polypeptide class.Advantageously, this library can comprise each immunoglobulin domains, such as the VH of antibody or the V of VL structural domain or T-cell receptors _βOr V _αStructural domain.Therefore, in a preferred embodiment, for example, can use the general part repertoire of prescreen VH and VL polypeptide class separately, and merge then and produce the functional repertoire that comprises VH and VL polypeptide class.Use target ligands to screen this class repertoire then, comprise VH and VL structural domain and and to have a polypeptide class of required binding specificity so that separate.

In an advantageous embodiment, for the general part that is used for the selection of immunoglobulin (Ig) repertoire is a superantigen.Superantigen can the combined function immunoglobulin molecules or its comprise the subgroup of specific main chain conformation, and irrelevant with the target ligands specificity.Perhaps general part can be selected from can be in conjunction with any part of the general structure of polypeptide class, and described polypeptide class constitutes any specified repertoire, such as antibody self, and metal ion matrix, organic compound comprises protein or peptide class etc.

The present invention provides the library in aspect second, and wherein functional member has the binding site of general and target ligands.For this purpose designs the library by following manner: for example, make up the antibody library that has by the main chain conformation of specifying superantigen identification, or make up the library, wherein all possible basically functional member has can be by the structure of antibody ligand identification.

The present invention provides detection in aspect the 3rd, and is fixing, and the method for the one or more members in the polypeptide class repertoire that purifying or immunoprecipitation are selected according to the present invention in advance comprises and makes this member in conjunction with general part.

The present invention provides the library of the polypeptide class repertoire that comprises immunoglobulin superfamily in aspect the 4th, and wherein the member in the repertoire has known main chain conformation.

The present invention provides the method for the polypeptide of selecting to have required general and/or target ligands binding site from polypeptide class repertoire in aspect the 5th, comprises the following step:

A) library of the above-mentioned aspect of expression the present invention;

B) make the general and/or target ligands of polypeptide class contact and selecting in conjunction with those of general and/or target ligands; With

C) optional amplification is in conjunction with the polypeptide of the selection of general and/or target ligands;

D) optional repeating step a)-c).

Advantageously generate and keep the repertoire of the polypeptide class of nucleic acid library form.Therefore, the present invention provides the nucleic acid library of coded polypeptide class repertoire in aspect the 6th.

The present invention provides the method for selecting in conjunction with the functional variable domain colony of target ligands and general part in aspect the 7th from the repertoire of antibody polypeptides class, this general part can be in conjunction with the combined function member in the repertoire, and irrelevant with the specificity of target ligands, this method comprises the following steps:

A) make repertoire contact described general part and select the functional variable domain of bonded with it; With

B) make the functional variable domain contact target ligands of selection and select variable domain in conjunction with target ligands;

Wherein, (i) variable domain is that heavy chain variable domain and general part are the light chain of antibody variable domain; Or (ii) variable domain is that light chain variable territory and general part are the heavy chain of antibody variable domain; And

Wherein choose wantonly in (i), heavy chain variable domain is Camelid variable domain (VHH) or derives from Camelid heavy chain antibody (H2 antibody); Or choose wantonly at (i) and (ii), variable domain is separately for people's variable domain or derive from the people.

The preferred repertoire of antibody polypeptides class that at first makes contacts target ligands and contacts general part then.

Preferred general part is in conjunction with the subgroup of variable domain repertoire.

Preferred two or more subgroup is selected from the repertoire of polypeptide class.

With regard to preferred two or more general parts that use, optional two or more light chain variable territories (with regard to option i)) or two or more heavy chain variable domains (with regard to option (ii) with regard to) select.

Preferably after selection, merge two or more subgroups so that produce extra polypeptide class repertoire.

Preferably make two or more repertoires of polypeptide class contact general part and merge thus obtained polypeptide class subgroup then.

In the embodiment aspect the 7th, select heavy chain of antibody variable domain colony and select light chain of antibody variable domain colony, and merge thus obtained colony then.

The present invention also provides the method for selecting in conjunction with the functional T-cell receptors structural domain colony of target ligands and general part in aspect the 8th from the repertoire of polypeptide class, this general part can be in conjunction with the functional member in the repertoire, and irrelevant with the specificity of target ligands, this method comprises the following steps:

A) make repertoire contact described general part and select the functional T-cell receptors structural domain of bonded with it; With

B) make the functional T-cell receptors structural domain contact target ligands of selection and select T-cell receptors structural domain colony in conjunction with target ligands;

Wherein, (i) the T-cell receptors structural domain is V _αStructural domain and general part are T-cell receptors V _βStructural domain; Or (ii) T-cellularstructure territory is T-cell receptors V _βStructural domain and general part are T-cell receptors V _αStructural domain; And

Wherein choose in (i) T-cell receptors V wantonly _αStructural domain is the Camelid structural domain that derives from Camelid; Or choose wantonly at (i) and (ii), the T-cell receptors structural domain is separately for people's structural domain or derive from the people.

In the embodiment aspect the 8th, select T-cell receptors V _αStructural domain colony and selection T-cell receptors V _βStructural domain colony, and merge thus obtained colony then.

The present invention also provides the method for selecting at least a heavy chain of antibody variable domain from the antibody polypeptides types of populations in aspect the 9th, and this method comprises:

A) make described population exposed light chain of antibody variable domain; With

B) select at least a heavy chain of antibody variable domain in conjunction with the light chain variable territory.

In one embodiment, the initial colony that target antigen is used for antagonist polypeptide class carries out SLAM (the lymphocyte antibody method of selection), so that select the antibody polypeptides types of populations in conjunction with target antigen; And with the colony selected as the antibody polypeptides types of populations in the step a) in contact light chain variable territory.

Preferably before step a), exist to make antibody polypeptides class contact target ligands and selection, the described antibody polypeptides types of populations of using in the step a) is provided thus in conjunction with the step of the antibody polypeptides class of target ligands.

Preferably after step b), exist to make the heavy chain of antibody variable domain contact target ligands of in step b), selecting and select step in conjunction with the heavy chain variable domain of target ligands.

The heavy chain structural domain of preferably selecting in step b) is separately from organizing down: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.

The heavy chain structural domain of preferably selecting in step b) is humanization Camelid or mouse heavy chain variable domain or humanization Nanobody separately ^TM

The heavy chain structural domain of preferably selecting in step b) is people's heavy chain variable domain separately.

Preferred light chain variable territory behaviour light chain variable territory or the light chain variable territory that derives from the people or have the FW2 sequence, described FW2 sequence is identical with the FW2 that by kind is gene order DPK9 coding.

Preferred light chain variable territory is Camelid light chain variable territory or derives from Camelid.

The colony of preferred steps in a) provided by the B-cell mass.

Preferred B-cell is a peripheral blood lymphocyte.

Preferred B-cellular segregation is from the animal of immunization target antigen.

Preferred B-cellular segregation is from the animal of immunization target antigen not.

The colony that preferred steps is used in a) is provided by the repertoire of the antibody polypeptides class of the antibody gene coding of resetting with synthesis mode.

The colony that preferred steps is used in a) is by the phage display library body that comprises the phage of showing described antibody polypeptides class.

The colony that preferred steps is used in a) comprises: (i) each self-contained at least a not with the antibody polypeptides class of light chain variable territory paired heavy chain variable domain; (ii) each self-contained and antibody polypeptides class light chain variable territory paired heavy chain variable domain.

Preferred steps a) the middle colony that uses comprises single variable domain of Camelid heavy chain (VHH) or Nanobodies ^TM

The colony that preferred steps is used in a) is by comprising the single variable domain of people's heavy chain (VH).

Preferably in step b), at least a and protein portion in the heavy chain of antibody variable domain of selection merges or puts together.Preferred protein partly is selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

Preferably in step b), at least a in the heavy chain of antibody variable domain of selection is antibody moiety or antibody fragment, and it is selected from IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv and disulfide bonding Fv.

The particularly preferred embodiment of the 9th aspect of the present invention:

In this embodiment, the colony that uses in the step a) is by the B-cell mass, and the peripheral blood lymphocyte that has preferably separated the Camelid (for example Llama or camel) from the immunization target antigen provides.In this embodiment, preferred target antigen is selected from any one in the target listed in the target ligands of TNF α, serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP12, PDK1 and amyloid beta (A-β) or the appendix 1.The light chain variable territory of using in the step b) is: people's light chain variable territory; Derive from the people; Light chain variable territory with FW2 sequence, described FW2 sequence is identical with the FW2 that by kind is gene order DPK9 coding; Or Camelid, rabbit or mouse light chain variable territory.Therefore, this embodiment is paid close attention to from the application of peripheral blood lymphocyte group in step a), its from end user's light chain variable territory (or have the people interface region light chain structural domain of (comprising the 44-47 amino acids) at least according to this district of Kabat, promptly should the district usually with people VH/VL pairing in VH structural domain separation surface) Camelid of immunization of selection.

One aspect of the present invention provides the method for selecting at least a Camelid antibody VHH structural domain from Camelid antibody polypeptides types of populations, the B-cell of the Camelid of immunization target antigen provides described Camelid antibody polypeptides types of populations by separating certainly, and this method comprises:

A) make described colony basis light chain of antibody variable domain; With

B) select at least a VHH structural domain in conjunction with the light chain variable territory.

Preferred light chain variable territory behaviour light chain variable territory.

Preferably the B-cell is provided in a plurality of holes or the reservoir, wherein each hole or reservoir on average comprise a kind of B-cell type.

The present invention provides the isolated antibody polypeptide in the tenth each side, it comprises the heavy chain of antibody variable domain or is made up of the heavy chain of antibody variable domain, wherein polypeptide can obtain by the method for the 9th aspect of the present invention, and wherein light chain variable territory behaviour light chain variable territory in the method and heavy chain variable domain are from non--people's Mammals.The heavy chain variable domain of preferred antibody polypeptide is from organizing down: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.Preferably the heavy chain variable domain of antibody polypeptides is provided as the part of CamelidIgG or the IgG that derives from Camelid.Preferably with the heavy chain variable domain of antibody polypeptides as the part of human IgG or derive from people's IgG body, and wherein heavy chain variable domain in IgG and be different from the light chain variable territory pairing that is used for the method aspect the 9th of the present invention.The present invention also provides the derivative of antibody polypeptides, and wherein this derivative is compared with the CDR3 of the antibody polypeptides variable domain of the tenth aspect and had the CDR3 sudden change.The present invention also provides the derivative by the affinity maturation generation of the antibody polypeptides of the tenth aspect.

Antibody polypeptides of the present invention in one embodiment (for example the 10th or the 12nd aspect) is as drug use or be used for the human disease or the treating and/or preventing of illness.

In method of the present invention, use or an embodiment of antibody polypeptides in, heavy chain variable domain is in conjunction with being selected from down the target ligands of organizing: any one in the target ligands of TNF α, serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP12, PDK1 and amyloid beta (A-β) or the appendix 1 in the listed target.

The present invention provides the method for selecting at least a light chain of antibody variable domain from the antibody polypeptides types of populations in aspect the 11, and this method comprises:

A) make described population exposed heavy chain of antibody variable domain; With

B) select at least a light chain of antibody variable domain in conjunction with heavy chain variable domain.

Preferably after step b), exist to make the light chain of antibody variable domain contact target ligands of in step b), selecting and select step in conjunction with the light chain variable territory of target ligands.

The light chain structural domain of preferably selecting in step b) derives from Camelid separately.

The light chain structural domain behaviour light chain variable territory of preferably in step b), selecting.

Preferred heavy chain variable domain is: people's heavy chain variable domain; Derive from the people; Heavy chain variable domain with FW2 sequence, described FW2 sequence is that the FW2 of gene order DP47 coding is identical with planting; Or to have and plant be 44,45 44,45 and 47 heavy chain variable domains identical with 47 of gene order DP47 coding.

Preferred heavy chain variable domain is Camelid heavy chain variable domain (VHH or VH) or derives from Camelid.

Preferably the colony in step a) is provided by the B-cell mass.

Preferred B-cell is a peripheral blood lymphocyte.

Preferred B-cellular segregation from immunization the animal of target antigen.

Preferred steps a) the middle colony that uses is provided by the phage display library that comprises the phage of showing described antibody polypeptides class.

The colony that preferred steps is used in a) comprises: (i) each self-contained not with the antibody polypeptides class at least a light chain variable of heavy chain variable domain paired territory; (ii) each self-contained and antibody polypeptides class heavy chain variable domain paired light chain variable territory.

Preferred steps a) the middle colony that uses comprises the single variable domain of people's light chain (VL).

Preferably in step b), at least a and protein portion in the light chain of antibody variable domain of selection merges or puts together.Preferred protein partly is selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

In the embodiment aspect the 11, target antigen is used for initial antibody polypeptides class is carried out SLAM (the lymphocyte antibody method of selection), so that select the antibody polypeptides types of populations in conjunction with target antigen; And with the antibody polypeptides types of populations in the step a) of selecting that colony is used as with heavy chain variable domain contacts.

The present invention provides the isolated antibody polypeptide in aspect the 12, it comprises the light chain of antibody variable domain or is made up of the light chain of antibody variable domain, wherein polypeptide can obtain by the method for the 11 aspect of the present invention, and wherein heavy chain variable domain behaviour heavy chain variable domain in the method and light chain variable territory are from non--people's Mammals.The light chain variable territory of preferred antibody polypeptide is from Camelid.Preferably the light chain variable territory of antibody polypeptides is provided as the part of Camelid IgG or the IgG that derives from Camelid.Preferably the light chain variable territory of antibody polypeptides is provided as the part of human IgG or the IgG that derives from the people, and wherein make the light chain variable territory in IgG and be different from the heavy chain variable domain pairing that is used for the heavy chain variable domain aspect the 11 of the present invention.The present invention also provides the derivative of the antibody polypeptides of the 12 aspect, and wherein this derivative is compared with the CDR3 of the polypeptide variable domain of the 12 aspect and had the CDR3 sudden change.The present invention also provides the derivative by the affinity maturation generation of the antibody polypeptides of the 12 aspect.

Polypeptide is provided among the present invention in one aspect, and it comprises the part or the derivative of the transformation period prolongation that is connected with the antibody polypeptides of the 10th or the 12nd aspect, and wherein this part is selected from: PEG; The antibody constant domain; The antibody Fc district; Albumen or its fragment; In conjunction with albuminised peptide or antibody fragment, white protein fragment; Neonatal Fc receptor; Transhipment; Or transhipment acceptor.Preferred polypeptide is medicine or is used for the human disease or the treating and/or preventing of illness.The variable domain of preferred antibody polypeptide is in conjunction with being selected from down the target ligands of organizing: any one in the target ligands of TNF α, serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP12, PDK1 and amyloid beta (A-β) or the appendix 1 in the listed target.

In one embodiment of the invention, this method further comprises the mutant of the variable domain that produces selection or the step of derivative.

In one embodiment of the invention, wherein use the B-cell mass, the B-cell mass is provided in a plurality of holes or the reservoir, wherein each hole or reservoir comprise single B-cell type.

In one embodiment of the invention, wherein use the B-cell mass, the B-cell mass is provided in a plurality of holes or the reservoir, wherein each hole or reservoir on average comprise a kind of B-cell type.

In one aspect of the invention, provide the method for separating IgG in the single variable domain of antibody from the colony of the antibody polypeptides class that comprises single variable domain and IgG, this method comprises:

A) make the general part of described population exposed; With

B) select the generally subgroup of part of combination, from single variable domain, separate IgG thus;

Wherein general ligand gene antagonist CH1 structural domain, light chain constant domain (CL), the binding specificity of IgG hinge or light chain of antibody variable domain.

Preferred general part is selected from: protein L, protein L structural domain or in conjunction with the protein L derivative in light chain variable territory; Protein G; Protein G structural domain or in conjunction with the protein G derivative of CH1; Antibody and antibody fragment; Affibody; The ldl receptor structural domain; With the EGF structural domain.

Preferred general part is selected from dAb, Nanobody ^TM, scFv, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv or disulfide bonding Fv.

Behave single variable domain and IgG of preferred variable domain is human IgG.

Preferred general part is with 1mM or the avidity binding antibody CH1 structural domain below the 1mM, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain.

Preferred general part is with 1 micromole or avidity binding antibody CH1 structural domain, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain below 1 micromole.

Preferred general part is with 100nM or avidity binding antibody CH1 structural domain, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain below the 100nM.

The present invention provides the method for the single variable domain of separation of C amelid VHH among the IgG from the colony of the antibody polypeptides class that comprises Camelid VHH structural domain and IgG in the 13 each side, this method comprises:

A) make the general part of described population exposed; With

B) select the generally subgroup of part of combination, from IgG, separate single variable domain thus.

Preferred general part is selected from the light chain of antibody variable domain, antibody and antibody fragment.

Preferred general part with 1mM or the avidity below the 1mM in conjunction with VHH or heavy chain antibody (H2) hinge.

Preferred general part with 1 micromole or below 1 micromole in conjunction with VHH or heavy chain antibody (H2) hinge.

Preferred general part with 100nM or the avidity below the 100nM in conjunction with VHH or heavy chain antibody (H2) hinge.

Preferred antibody polypeptide colony is provided by the B cell.

Preferred variable domain is that Camelid VHH and IgG are Camelid IgG.

Preferred variable domain is provided by Camelid heavy chain (H2) antibody.

The general part of preferred mark (labelled) or mark (tagged).

In method of the present invention, antibody polypeptides, in the embodiment of derivative or application, general part is for being selected from appendix 2a), c), d) or e) antibody variable domains.

The present invention provides the method for selecting in conjunction with the single variable domain of target ligands and general part in aspect the 14 from the repertoire of antibody polypeptides class, this method comprises the following steps:

A) make repertoire contact target ligands and select the single variable domain of bonded with it; With

B) make the variable domain of selection contact general part and select the generally variable domain of part of combination;

Wherein general part is for being selected from appendix 2 (c) or antibody variable domains (e); And

Wherein (i), when the variable domain of selecting was heavy chain variable domain, general part was the light chain variable territory; Or (ii) when the variable domain of selecting is the light chain variable territory, general part is a heavy chain variable domain.

The repertoire of preferred antibody polypeptide class is the repertoire of heavy chain variable domain, and general part is the light chain variable territory.

The repertoire of preferred antibody polypeptide class is the repertoire in light chain variable territory, and general part is a heavy chain variable domain.

Preferred this method comprises the mutant of the variable domain that produces selection or the step of derivative.

Preferred general part combination and the identical target ligands kind of selecting of variable domain.

Preferred general part combination and the different target ligands kind of selecting of variable domain.

The variable domain that preferred this method comprises the following steps: to merge selection with and the general identical antibody variable domains or derivatives thereof of part so that the product of generation gene target ligands binding specificity.

The present invention provide in aspect the 15 production appendix 2 (c) (i)-(iv) in conjunction with the method for the antibody or the antibody fragment derivative of target ligands, this method comprises:

A) use described antibody or segmental heavy chain variable domain or identical variable domain as the general part in the method for the 14th aspect, and the target ligands that wherein is used for step a) is antibody or fragment bonded target ligands, selects the single variable domains of light chain in conjunction with target ligands and heavy chain variable domain thus; With

B) with the light chain variable territory of selecting; Identical light chain variable territory or derivatives thereof replaces at least a in antibody or the fragment light chain variable territory.

The present invention provide in aspect the 16 production appendix 2 (c) (i)-(iv) in the arbitrarily multi-specificity antibody in mutually or the method for antibody fragment derivative, this method comprises:

A) use described antibody or segmental heavy chain variable domain (or identical variable domain) as the general part in the 14th the aspect method, and the target ligands that wherein is used for step a) is the target ligands that is different from antibody or fragment bonded target ligands, selects the single variable domain of light chain in conjunction with different target ligands and heavy chain variable domain thus; With

B) with the light chain variable territory of selecting; Identical light chain variable territory or derivatives thereof replaces at least a in antibody or the fragment light chain variable territory, produces the polyspecific product thus.

In preferably aspect the 15th and the 16th, the light chain variable territory is selected from: people's light chain variable territory; Derive from people's light chain variable territory; Light chain variable territory with FW2 sequence, described FW2 sequence is identical with the FW2 that by kind is gene order DPK9 coding; Camelid light chain variable territory; Derive from the light chain variable territory of Camelid; With humanization Camelid or mouse light chain variable territory.

The present invention provides among production appendix 2 (c) any in (i)-(iv) the method in conjunction with the antibody or the antibody fragment derivative of target ligands in aspect the 17, and this method comprises:

A) use described antibody or segmental light chain variable territory (or identical variable domain) as the general part in the method for the 14th aspect, and the target ligands that wherein is used for step a) is antibody or fragment bonded target ligands, selects the single variable domain of heavy chain in conjunction with target ligands and light chain variable territory thus; With

B) with the heavy chain variable domain of selecting; Identical heavy chain variable domain or derivatives thereof replaces at least a in antibody or the fragment heavy chain variable domain.

The present invention provides the antibody among production appendix 2 (c) any in (i)-(iv) or the method for antibody fragment degree specificity derivative in aspect the 18, and this method comprises:

A) use described antibody or segmental light chain variable territory (or identical variable domain) as the general part in the method for claim 94, and the target ligands that wherein is used for step a) is the target ligands that is different from antibody or fragment bonded target ligands, selects the single variable domain of heavy chain in conjunction with different target ligands and light chain variable territory thus; With

B) with the heavy chain variable domain of selecting; Identical heavy chain variable domain or derivatives thereof replaces at least a in antibody or the fragment heavy chain variable domain, produces the polyspecific product thus.

In preferably aspect the 17th and the 18th, heavy chain variable domain is selected from: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH; Humanization Camelid or mouse heavy chain variable domain; Humanization Nanobody ^TMPeople's heavy chain variable domain; Derive from people's heavy chain variable domain; Heavy chain variable domain with FW2 sequence, described FW2 sequence is that the FW2 of gene order DP47 coding is identical with planting; Or to have with planting be 44,45 and 47 44,45 and 47 identical heavy chain variable domains of gene order DP47 coding.

In preferably aspect 15-18, a) variable domain of Xuan Zeing is heavy chain variable domain and antibody or the selecteed separately heavy chain variable domain of segmental heavy chain variable domain; Identical heavy chain variable domain or derivatives thereof replaces; Or b) variable domain of Xuan Zeing is light chain variable territory and antibody or selecteed separately light chain variable territory, segmental light chain variable territory; Identical light chain variable territory or derivatives thereof replaces.

The derivative that the method that the present invention also provides can provide any in the 18th aspect of 15-obtains.

The present invention provides the derivative of antibody or antibody fragment in aspect nineteen, and it is selected from Panorex ^TMRituxin ^TMZevalin ^TMMylotarg ^TMCampath ^TMHerceptin ^TMReoPro ^TMSynagis ^TMXolair ^TMRemicade ^TMSimulect ^TMOKT3 ^TMOrthoclone ^TMZenapax ^TMHumira ^TMBexxar ^TMRaptiva ^TMAntegren ^TMErbitux ^TMAnd Avastin ^TM, it can provide any acquisition in the 18th aspect of the present invention 15-.

The present invention provides the application in the treating and/or preventing of human disease or illness of described derivative or product in aspect the 20.

The present invention provides the application in the treating and/or preventing of human disease or illness of described derivative or product in aspect the 21.

The present invention provides the application in the treating and/or preventing of human disease or illness of described derivative or product in aspect the 22, wherein said illness be to appendix 2 (c) (i) in antibody or the listed illness of antibody fragment.

Selecting the VL structural domain or application during vice versa if the present invention relates to the general part of VH or VHH, can help selecting through the pairing of inherence, interface region by this class formation territory so, for example, the related interfaces district can comprise VH and VHH structural domain (with the equivalence district of VL structural domain) 44,45 and 47, and this class numbering is followed Kabat.Therefore, application with the VL (for example 44G, 45L and 47W) at people interface can be used for the structural domain from library selection Camelid VHH, because this can select to have the VHH structural domain with paired interface region, people VL interface region, therefore, this has selected the people-category feature in the VHH structural domain, provides the useful VHH product of philtrum (for example being used for physics) for us thus and/or has been used for further research and development useful leading of people's consistency product.

The accompanying drawing summary

Fig. 1: show extensive natural diversity and can contact antigenic people's antibody repertoire VH and V _κThe stick plot of the position in the district is (referring to (1996) J.Mol.Biol. such as Tomlinson, 256:813).Shown H3 and L3 end in this synoptic diagram, but, they also are highly different and can contact antigen.Although fully being characterized, the sequence polymorphism in the people λ gene (, 268:69), with regard to the three-dimensional lambda structure, almost do not have the data of relevant antigen contact to exist at present referring to (1997) J.Mol.Biol such as Ignatovich.

Fig. 2: the scFv sequence that constitutes basis, library of the present invention.The library that has two kinds of forms at present: " elementary " library, wherein 18 positions change; " somatocyte " library, wherein 12 positions change.6 ring districts H1, H2, H3, L1, L2 and L3 have been shown.Underline for CDR district (Kabat etc. (1991) .Sequences of proteinsofimmunological interest, U.S.Department of Health and Human Services) according to the Kabat definition.

Fig. 3: use the functional selection in the library of the present invention, general ligandin matter A and protein selection front and back.Protein L herein is positioned on the ELISA flat board, and the scFv supernatant liquor is combination and use a-protein-HRP detection scFv combination with it.Therefore, only those can conjugated protein A and the scFv of protein L produce the ELISA signal.

Fig. 4: using the ox ubiquitin, rat BIP, the ox histone, NIP-BSA, FITC-BSA, people's Leptin, human thyroglobulin, BSA, hen-egg lysozyme is selected from the clone's in library of the present invention sequence after mouse IgG and the human IgG elutriation.The underscore that adds on sequence is illustrated in the position that changes in the corresponding library.

Fig. 5: 5a: the comparison of the scFv concentration that produces by non-selected and preselected " elementary " DVT library in the host cell.5b: as the ELISA typical curve of measuring according to the known standard product.

Fig. 6:, use anti--phage pIII antibody to survey described library so that measure phage per-cent with scFv from the western blotting of the phage in preselected and unselected " elementary " DVT library.

Detailed Description Of The Invention

Definition

Repertoire (repertoire)

Repertoire is the colony of different variants, for example lists different nucleic acid variants or at polypeptide variants different aspect the aminoacid sequence at nucleotides sequence.Library of the present invention comprises the repertoire of polypeptide class or nucleic acid.Design the repertoire of polypeptide class according to the present invention, so that have the binding site of general part and the binding site of target ligands.Binding site is can be in the same zone of molecule overlapping or be positioned at wherein, but its specificity difference.

Organism

The cell life form that term used herein " organism " intention is all is such as prokaryotic cell prokaryocyte and eukaryotic cell; And the basis that contains nucleic acid of non--cell, such as phage and virus.

Functional

Term used herein " functional " intention has proteinic natural bioactive or any required active polypeptide of specificity of its type of natural generation, for example according to the hereinafter ability judgement of its binding partner molecule of definition.The example of " those " polypeptide class comprises the antibody by its antigen-binding site specificity conjugated antigen, in conjunction with the acceptor molecule (for example T-cell receptors) of its feature part with in conjunction with the enzyme of its substrate.For of the present invention functional according to polypeptide is categorized as, follow at first and must carry out suitable processing treatment and folding it so that keep its overall structure integrity, as according to definition also as hereinafter its in conjunction with the ability judgement of part.

For avoiding query, functionally be not and the ability equivalence that combines target ligands.For example, functional anti--the CEA monoclonal antibody can not specificity in conjunction with target ligands, such as bacterium LPS.Yet, because it can be in conjunction with target ligands (being that it can be in conjunction with CEA, as long as CEA is a target ligands), so it is categorized as " functional " antibody molecule and can be as described below according to selecting in conjunction with general part.Generally speaking, non--functional antibodies molecule can not be in conjunction with any target ligands.

General part

General part is in conjunction with the part of specifying the most of functional member in the repertoire.Therefore, identical general part can be in conjunction with the many members in the repertoire, and with its target ligands specificity irrelevant (vide infra).Generally speaking, exist functional general ligand-binding site point to show that the repertoire member is correctly expressed and folds.Therefore, general part combines the method that preselected functional polypeptide class from the repertoire of polypeptide class is provided with its binding site.

Target ligands

Target ligands is the part that specific binding members in the repertoire or a plurality of member are identified.If the member in the repertoire is an antibody molecule, target ligands can be antigen so, and if the member in the repertoire be enzyme, target ligands can be substrate so.Depend on as mentioned described in the general part in the repertoire member for functional in conjunction with target ligands, and depend on the accurate specificity of the binding site of target ligands.

Subgroup

Subgroup is the integral part of repertoire.For the present invention, common situation is that only the subgroup of repertoire is functional and has functional general ligand-binding site point thus.In addition, also possible is that only the functional member's of repertoire part (obviously surpassing in conjunction with specified target ligands) is in conjunction with general part.Can select these subgroups according to the present invention.

Can merge or gather the library subgroup so that produce new according to the preselected repertoire of required standard.The repertoire that merges or gather can maybe can be operated so that merge two polypeptide subgroups for by the single mixture of general part in conjunction with preselected polypeptide member.For example, prescreen VH and VL polypeptide class and merging on single carrier on the hereditary level subsequently make them as the VH-VL dimer that merges separately, are expressed such as scFv.

The library

The mixture of term library intention heterologous polypeptide class or nucleic acid.This library is by the member composition with single polypeptide or nucleotide sequence.With regard to this degree, library and repertoire are synonym.Sequence difference causes the diversity that exists in the library between the member of library.But this library adopts the single mixture of polypeptide class or nucleic acid maybe can be organism or identical form, for example bacterium, virus, the animal or plant cell etc. that use nucleic acid library to transform.Preferred each organism or an identical member who only comprises separately in this library.Advantageously, nucleic acid is mixed expression vector, so that can express polypeptide class by described nucleic acid encoding.Therefore, one preferred aspect in, the form of host organisms colony can be adopted in the library, and each organism comprises one or more copies of expression vector, and described expression vector comprises can express so that produce single member in its corresponding polypeptide member's the library of nucleic acid form.Therefore, host organisms colony has the potential of a large amount of repertoires of polypeptide variants different on the genetic coding.

Immunoglobulin superfamily

Its intention keeps the polypeptide class family of the immunoglobulin folding characteristic of immunoglobulin (Ig) (antibody) molecule, and it comprises two βZhe Dies and conservative usually disulfide linkage.Member in the immunoglobulin superfamily relates to many aspects of cells in vivo and non--cell interaction, be included in extensive effect in the immunity system (antibody for example, T-cell receptors molecule etc.), relate to cell adhesion (for example ICAM molecule) and intracellular signal conduction (for example acceptor molecule, such as pdgf receptor).The present invention is suitable for all immunoglobulin superfamily molecules, because variation wherein realizes in a similar fashion.Preferably the present invention relates to immunoglobulin (Ig) (antibody).

The main chain conformation

The C skeleton vestige of the structure of main chain conformation intention three dimensional form.When considering each hypermutation ring of antibody or TCR molecule, main chain conformation and norm structure are synonym.Such as Chothia and Lesk (1987) J.Mol.Biol., 196:901 and Chothia etc. (1989) Nature, 342:877 is described, antibody shows the norm structure of limited quantity, in its 6 each hypermutation ring 5 (H1, H2 is arranged, L1, L2 and L3), but, in ring self, there is suitable side chain diversity.The accurate norm structure that demonstrates depends on the length of ring and relates to the individual character of some Key residues of its packing.Say nothing of different that the 6th ring (H3) has aspect length and sequence, and only show norm structure (Martin etc. (1996) J.Mol.Biol., the 263:800 of some becate length thus; Shirai etc. (1996) FEBS Letters, 399:1).In the present invention, all 6 rings preferably have norm structure and known thus complete antibody molecular backbone conformation.

Antibody polypeptides

Antibody is for being produced and constituted the immunoglobulin (Ig) of the center integral part of vertebrate host immune defense system by the B cell.Antibody polypeptides used herein is a polypeptide, and it is the antibody or the antibody moiety of modification or unmodified.Therefore, the term antibody polypeptides comprises the heavy chain light chain, heavy chain-light chain dimer, and the Fab fragment, F (ab ') 2 fragments, Dab fragment or Fv fragment comprise strand Fv (scFv).The method that makes up this antibody-like molecule is well-known in the art.

Superantigen

Superantigen is most of antigen for the toxin form expressed in bacterium, the member in the outside and immunoglobulin superfamily interacts at the ligand-binding site point commonly used of these molecules for they.Staphylococcus (Staphylococcal) enterotoxin class and T-cell receptors take place to interact and have the effect that stimulates the CD4+T-cell.The superantigen of antibody comprises molecule protein G (Bjorck and Kronvall (1984) J.Immunol, the 133:969 in conjunction with the IgG constant region; Reis etc. (1984) J.Immunol., 132:3091), the a-protein of IgG constant region and VH structural domain (Forsgren and Sjoquist (1966) J.Immunol., 97:822) and in conjunction with the protein L of VL structural domain (Bjorck (1988) J.Immunol., 140:1994).

The preferred embodiments of the invention

The invention provides eliminate in the polypeptide libraries non--functional or be difficult to the member (or significantly reducing its ratio) that folds/express, enriched with functional the specificity of " target ligands " is selected before simultaneously, folding and member's that give full expression to selective system.Make the repertoire contact " general part " of peptide molecule, promptly have protein, the protein of correlation type for example complete and/or that correctly fold the avidity of the common constitutional features of all functions.Notice that term " part " is used for the of the present invention minute extensive term of the period of the day from 11 p.m. to 1 a.m relating to.Term used herein " part " intention is in conjunction with the member in the polypeptide libraries or by any body of they institute's bonded.

Being present in a large amount of members in the deficient protein matter in the initial repertoire is difficult in conjunction with general part and is eliminated thus.This from the library selectivity remove non--functional polypeptide class and cause its actual size obviously to reduce, its functional size is maintained the wherein corresponding raising of its quality simultaneously.By the polypeptide class that keeps in conjunction with general part constituted original repertoire ' the at first set of Xuan Zeing ' or ' subgroup '.Therefore, functional in the initial repertoire of enrichment this ' subgroup ', fully folding and give full expression to the member.

Make set or the subgroup polypeptide class contact at least a " target ligands " at first selected subsequently, this target ligands is in conjunction with having the specific polypeptide class of appointed function.This class target ligands comprises, but be not limited to the half receptor/ligand to (for example hormone or other cell-signal transduction molecule, such as neurotransmitter and the connection acceptor) in separately, cell adhesion molecule in conjunction with centering separately, by enzyme, protein, peptide or little organic compound and even antibody self at the specific antibodies that is oriented to.Therefore, the library labour intensity that is applied in time and materials aspect ratio routine in this class library is lower and more economical.In addition, compare with the repertoire that does not use general part to select, because at first the set of Xuan Zeing comprise far above those can not be in conjunction with the ratio of target ligands can be in conjunction with the molecule of target ligands, so the background in using " target ligands " chosen process significantly reduces.

The present invention also pays close attention to the combination selection scheme.Can use unusual and/or target ligands is parallel or the multiple selection of the identical initial polypeptide repertoire of contacting.Therefore, can at first use single general part to select repertoire, and use different target ligands to carry out parallel selection then immediately.Be used alone or in combination the gained subgroup then, wherein the subgroup of this combination has the target ligands specificity of certain limit, and has single general ligand specificity.Perhaps, can at first use single target ligands to select repertoire, and use unusual part to carry out parallel selection then immediately.Be used alone or in combination the gained subgroup then, wherein the subgroup of this combination has the general ligand specificity of certain limit, and has single target ligands specificity.Also pay close attention to the more application of elaborate schemes.For example, can use two kinds of different general parts that initial repertoire is carried out two round-robin and select, use target ligands to select subsequently.This process produces a subgroup, and wherein all members are both in conjunction with general part, again in conjunction with target ligands.Perhaps, if use two kinds of general parts that initial repertoire is carried out parallel selection and merge the gained subgroup and use target ligands to select then, at least a in conjunction with in two kinds of general parts and the target ligands of gained subgroup so.The repertoire that merges or gather can maybe can be operated so that connect subgroup with physics mode for the single mixture of described subgroup.For example, can be by selecting VH and VL polypeptide class abreast separately in conjunction with two kinds of different general parts, and be incorporated on the single carrier with hereditary level subsequently, make them be expressed as the VH-VL that merges.Can select this repertoire to target ligands then, the member who make to select can either be in conjunction with general part, again can be in conjunction with target ligands.

The present invention includes the library of the functional polypeptide class of selecting by broadly described method above that maybe can select by aforesaid method, and coding can be used for the nucleic acid library of the peptide molecule (molecule that preferably comprises the first target ligands binding site and the second general ligand-binding site point) of the selection carried out according to these methods.In addition, the invention provides detection, fixing, purifying or immunoprecipitation use the method for the one or more members in general or the functional polypeptide class repertoire that target ligands is selected of the present invention.

The present invention is particularly suitable for the molecular library in the enrichment immunoglobulin superfamily.This is being definite especially aspect generation antibody and T-cell receptors colony, and described antibody and T-cell receptors are functional and have required specificity that because this is to be used for diagnosis, treatment or prevention operation are required.In order to reach this purpose, the invention provides antibody and T-cell receptors library, wherein all members all have the ring of natural framework and known main chain conformation, and the invention provides the strategy of selecting subsequently of the functional variant of the useful mutagenesis of homing sequence and generation thus.This class polypeptide libraries can comprise VH or V _βStructural domain, or alternatively, it can comprise VL or V _αStructural domain and even two kinds of VH or V _βWith VL or V _αStructural domain.

Exist in the art the antibody of improvement or the demand of T-cell receptors molecular library.For example, although in generating " single jar " phage-antibody library, there is progress, still there are several problems.Use and gather from the natural library of the rearrangement V of human B cell gene (Marks etc. (1991) J.Mol.Biol., 222:581; Vaughan etc. (1996) Nature Biotech. 14:309) selects offsets in height because of the positive and negative of B cell in the body.This result can limit by effective size of resetting the gene constructed phage library of V.In addition, the clone who derives from natural library comprises framework mutations unchangeably, and when being used for people's therapy, these sudden changes can influence antibody mediated immunity originality.Synthetic library (Hoogenboom ﹠amp; Winter (1992) J.Mol.Biol., 227:381; Barbas etc. (1992) Proc.Natl.Acad.Sci.USA, 89:4457; Nissim etc. (1994) EMBO J., 13:692; Griffiths etc. (1994) EMBO J., 13:3245; (1995) J.Mol.Biol. such as De Kruif 248:97) can overcome the bias problem, but they need use long degenerate pcr primer, and these primers are usually with base-to importing the V gene of assembling.The randomization of this height can also cause can not correctly folding and also being thus the antibody generation of non-functional.In many cases, possible situation is that these non--functional members surpass the functional member in the library on number.Although can be by using the synthetic one group ' key-gene of total frame sequence ' and improve folding and express and optimize the folding of framework in advance and/or express (WO97/08320 by the aminoacid replacement shown in mixing, Morphosys), but still there is immunogenic problem, because in most of situation, consensus sequence is not equivalent to any natural framework.In addition and since the CDR diversity also folding the and/or expressional function of gene be possible, so after assembling the V gene fully, can preferentially optimize folding and/or express (and removing any frameshit or terminator codon).

Use another problem in existing library to be, because the main chain conformation is allogenic, so the modeling of three-dimensional structure is difficult, because can't obtain suitable high resolving power crystallographic data.This is the particular problem in H3 district, and wherein the major part antibody that derives from natural or synthetic antibody libraries has moderate-length or long ring and thus can not modeling.

Use another problem in existing library to be dependence to the epitope tag (such as myc, FLAG or HIS mark) of the antibody fragment that is used to detect expression.Because they are usually located on the N or C-terminal of antibody fragment, so their trends are easy to take place proteolytic cleavage.Superantigen, can be used for by self detect the antibody fragment of expression in conjunction with the pleated sheet structure territory such as a-protein and protein, so but because they be VH and VL family specificity in any existing antibody library, only the relatively small amount member can in conjunction with one of these reagent and even less ratio in conjunction with they both.

For this purpose, need have selective system, that this system can eliminate is non-in the library-and functional or be difficult to the member (or reducing its ratio) that folds/express, after this can select target, enrichment simultaneously wherein can be in conjunction with general part, functional such as superantigen a-protein and protein L, folding and give full expression to AllThe member.In addition, advantageously make up antibody library, wherein all members have natural framework and have the ring that possesses known main chain conformation.

The present invention provides method thus, can select the polypeptide repertoire so that remove non--functional member by this method.This causes actual library size significantly to reduce (and the corresponding increase of library quality), but can not reduce functional library size.The present invention also provides the method that generates new polypeptide repertoire, and wherein all functions member can be in conjunction with specified general part.Identical general part can be used for any one or a plurality of members' of repertoire subsequently detection, fixing, purifying or immunoprecipitation.

' pure ' or ' immunity ' antibody repertoire can be used for the present invention arbitrarily, so that enriched with functional member and/or enrichment are in conjunction with the member of specified general part or multiple part.In fact and since only all ethnic groups of less per-cent be the VH fragment with high-affinity conjugated protein A, and only all ethnic groups of less per-cent be the VL fragment with high-affinity conjugated protein L, these superantigens are highly favourable.Perhaps, epitope tag is preselected can remove non--functional variant from synthetic library by using.Be easy to preselected library and include, but are not limited to, the library of the V genomic constitution of resetting in (1996) Nature Biotech. such as 222:581 and Vaughan, the body of the described type of 14:309 by (1991) J.Mol.Biol. such as Marks; Synthetic library, wherein planting is that the V gene fragment is at external ' rearrangement ' (Hoogenboom ﹠amp; Winter (1992) J.Mol.Biol., 227:381; Nissim etc. (1994) EMBO J., 13:692; Griffiths etc. (1994) EMBO J., 13:3245; (1995) J.Mol.Biol. such as De Kruif, 248:97) or will wherein synthesize V gene (Barbas etc. (1992) Proc.Natl.Acad.Sci.USA that CDRs mixes single rearrangement, 89:4457) or mix a plurality of main truss (WO97/08320, Morphosys).

The selection of polypeptide class of the present invention

In case generated the polypeptide class set of variant, just used selection of the present invention.Two kinds of orders of using based on general and target ligands of selection operation step widely; The combination version of relevant these schemes is included in to specify selects to use in the step a plurality of general and/or target ligands.When using assembled scheme, for example, can make peptide molecule gather several target ligands of contact at once or separately separately polyphone; In the later case, can preserve the polypeptide class set of the selection of gained separately, or collect they self.These selection schemes can be summarized as follows:

A. selection operation 1:

Use the elementary polypeptide of general part to select

In order to remove the non--functional member in the library, select general part, make general part only by the functional molecular combination.For example, general part can be metal ion, antibody (monoclonal antibody or antibody polyclone form of mixtures), half enzyme/ligand complex or organism; The part of noting any kind in these types is used as target ligands of the present invention in addition or selectively.Having gone through antibody hereinafter produces and metal affinity chromatography.In fact, these ligand-binding site points (for example peptide-labeled or superantigen binding site) on the member of library have constant structure or sequence, and this structure is easy to not exist or change in non--functional member.With regard to antibody library, this method has from only those have the application of selecting functional member's the library of the binding site of specifying superantigen or monoclonal antibody; These class means are used for from natural and synthetic set selection function antibody polypeptides class.

Superantigen a-protein and/or protein L have the application as the general part of selecting the antibody repertoire in the present invention, because their are respectively correct in folding VH and VL structural domain (belonging to some VH and VL family), and with the sequence and the structure-irrelevant of target ligands binding site.In addition, a-protein or another kind of superantigen protein G have the application as general part, and described general part can be selected to fold and/or express by the heavy chain constant domain of binding antibody.Anti--κ and anti--λ antibody also have the application in selecting the light chain constant domain.Antibody or other are conjugated protein, and (Li etc. (1998) Nature Biotech. also is useful 16:190) such as the little organic molecule stand-in of a-protein.

When using this selection operation, general part can be in conjunction with all functions member in the preselected repertoire because of its definite character; Therefore, this general part (or its some conjugate) can be used for detecting, and is fixing, and purifying or immunoprecipitation are from any member or the member colony (no matter whether by selecting in conjunction with the specified target part, as described below) of repertoire.Can by the relevant test antibody of hereinafter discussing select the technology of part application in the present invention carry out by immunoassay protein detection and to the immunoprecipitation (referring to " Antibody for use as ligands in polypeptideselection ") of member's polypeptide class of repertoire of the present invention.Specificity general with the present invention by repertoire polypeptide member or target ligands combines fixes, general or the target ligands self of described the present invention is connected with solid phase or semi-solid phase support, (comprise such as filter membrane (for example nitrocotton or nylon leaching film) or chromatogram support, but be not limited to Mierocrystalline cellulose, polymkeric substance, resin or silicon-dioxide support); Can use any member who well known to a person skilled in the art in the chemical cross-linking agent to make member's polypeptide and general or target ligands carry out covalent attachment.(referring to " Metallicligands as use for the selection of polypeptides ") fixing as described below on technology affinity chromatography support.The chromatogram that purifying can comprise any kind in these technology or its combination, particularly immunoprecipitation and be undertaken by the well-known method of those skilled in the art.

Use this means, can use the selection that hockets of multiple general part, so that generation repertoire, wherein all members are separately abreast in conjunction with two or more general parts, so that can merge subgroup (thus subsequently, member in the preselected repertoire is at least a in well matched in conjunction with one) or mixed identical polypeptide chain subsequently respectively, in big functional library, all members can be in conjunction with all general parts that use in the preselected process thus.For example, can use the heavy chain of binding antibody molecule and the unusual part (vide infra) of light chain from one or more libraries, to select subgroup, and any heavy chain/light chain library that is merged into, wherein, for example by use VH in the strand Fv background and VL structural domain make heavy chain and light chain non--covalent attachment or covalent attachment.

Use the secondary polypeptide of target ligands to select

After using the selection step of general part, the screening library is so that identify member in conjunction with target ligands.Because using general part to select back enriched with functional polypeptide class, so advantageously in using the target ligands chosen process, reduce non-specific (" background ").In addition, significantly reduce (improving) owing to use the selection of general part that actual library size is produced with corresponding library quality, and can not reduce functional library size, so less repertoire should cause and identical target ligands specificity and the avidity diversity of bigger initial repertoire (comprise many non--functional and be difficult to the member that folds/express).

One or more target ligands can be used for selecting the polypeptide class from the polypeptide of selecting the first set of using general part to generate.Be used for generating the situation of many different subgroups at two or more target ligands, in these Asias two or more can be merged into single more complicated subgroup.Single general part can merge each member in the subgroup in conjunction with gained; Yet specified target ligands is only in conjunction with the library member of subgroup.

B. selection operation 2:

Use the repertoire member's of target ligands initial selection

Using general part to select herein, BeforeUse the selection of target ligands.Obviously, polypeptide class on the same group can be produced by arbitrary scheme mutually, as long as this class result is an ideal.Use this means, can be parallel or use the selection of multiple target ligands by the target ligands that is mixed for selecting.If parallel carrying out so, if desired, can merge the gained subgroup.

Use the secondary polypeptide of general part to select

The target ligands that can use one or more general parts to carry out is subsequently then selected in conjunction with subgroup.Although the selection of this and NOT-function is definite functional owing to having in conjunction with the repertoire member of target ligands, so it can become the subgroup in conjunction with isolating unusual part.Therefore, can be with a kind of general part or the colony of selecting target ligands to select with two or more general parts.In this case, can be used alternatingly general part so that generate repertoire, so wherein the member in conjunction with or separately abreast in conjunction with target ligands and two or more general parts, so that generate different (list may be overlapping) subgroup in conjunction with target ligands and unusual part.Can merge their (member in conjunction with in the general part at least a) thus, then.

Immunoglobulin (Ig)-family polypeptides library member's selection

Repertoire of selecting among the present invention or the member in the library be contactin molecule, particularly antibody polypeptides class or T-cell receptors polypeptide class advantageously.With regard to antibody, estimate that method of the present invention can be suitable for any kind (no matter be natural or synthetic) in the existing antibody library as known in the art or be suitable for using the antibody library (vide infra) of general part specific design.

The structure in library of the present invention

A. the selection of main chain conformation

Member in the immunoglobulin superfamily all the similar of total its polypeptide chain folds.For example, although antibody is very different aspect its basic sequence, but sequence contrast and crystalline structure disclose, with estimate opposite, 5 (H1, H2, L1 in 6 antigen coupling collars of antibody, L2 and L3) (Chothia and Lesk (1987) are referring to above to have adopted the main chain conformation of limited quantity or norm structure; Chothia etc. (1989) are referring to above).Analysis to ring length and Key residues can be predicted the H1 that finds thus in most people's antibody, H2, and L1, (Chothia etc. (1992) are referring to above for the main chain conformation of L2 and L3; Tomlinson etc. (1995) are referring to above; Williams etc. (1996) are referring to above).Although the H3 district is in sequence, length and configuration aspects different (due to the segmental application of D) far away, but it also is formed for the main chain conformation of the limited quantity of becate length, this depends on the length and the existence of specific residue or residue type, (Martin etc. (1996) are referring to above for key position on ring and antibody framework; Shirai etc. (1996) are referring to above).

According to designerantibodies polypeptide class of the present invention library, wherein select certain ring length and Key residues to guarantee known member's main chain conformation.Advantageously, they are the definite conformation of the immunoglobulin superfamily molecule of actual discovery, thereby they are reduced to bottom line for the chance of non-functional as mentioned above.Plant is that the V gene fragment is as a kind of suitable base frame that makes up antibody or T-cell receptors library; Other sequence also is useful.Variation can take place with low frequency, make a small amount of functional member can have the main chain conformation of change, but this can not influence its function.

The norm structure principle also is applied to the present invention, so that estimate by the member in the different main chain conformations of antibody coding, thus the diversified residue that prediction is used for not influencing norm structure based on the main chain conformation and the selection of antibody sequence.Known at present, at people V _κIn the structural domain, the L1 ring can adopt one of 4 kinds of norm structures, and the L2 ring has the people V of single norm structure and 90% _κStructural domain adopts 4 or one of 5 kind of norm structure that is used for the L3 ring (Tomlinson etc. (1995), referring to above); Therefore, at independent V _κIn the structural domain, the different specification structure can be merged into the different main chain conformations of certain limit.Because V _λThe L1 of structural domain coding different range, the standard conformation and the V of L2 and L3 ring _κAnd V _λStructural domain can with any VH structural domain pairing of several norm structures of codified H1 and H2 ring, so it is very big to spend these the 5 kinds observed norm structure number of combinations of ring.This means in the main chain conformation and to produce diversity binding specificity may be absolutely necessary for producing widely.Yet, opposite with prediction, by making up the antibody library based on single known main chain conformation, the diversity of discovery in the main chain conformation is not that mainly all antigenic enough diversity of target are required in generation.Even more surprisingly, single main chain conformation needn't can be used as the basis in complete library for apokoinou construction-single naturally occurring conformation.Therefore, the present invention one preferred aspect in provide the library, the single known main chain conformation of encoding of member wherein.Yet, should understand accidental variation can take place, making a small amount of functional member can have selectable may be unknown main chain conformation.

The single main chain conformation of selecting is conventional in described immunoglobulin superfamily types of molecules preferably.Conformation when observing a large amount of naturally occurring molecules and adopt it for conventional.Therefore, of the present invention one preferred aspect in, consider the different main chain conformations of each coupling collar of naturally occurring immunoglobulin superfamily molecule separately, and select to have the required main chain conformation that is used for different rings then CombinationNaturally occurring immunoglobulin superfamily.If none can utilize, can select immediate equivalent so.Have the non-natural framework or comprise framework mutations (referring to above) because the defective in the immunoglobulin (Ig) of prior art-family polypeptides library is many members, so with regard to antibody or T-cell receptors, be the required combination that gene fragment generates the main chain conformation that is used for different rings preferably by the kind of selecting the required main chain conformation of coding.More preferably frequent express the kind of selecting be gene fragment and most preferably they for the most frequent expression.

Therefore.In the designerantibodies library, can consider the incidence of 6 antigen coupling collars different main chain conformations separately separately.With regard to H1, H2, L1, L2 and L3, select the appointment conformation that adopts by the natural antigen coupling collar that has a 20%-100% of molecule.Generally speaking, its observed incidence is higher than 35% (being 35%-100%), and in fact, is higher than 50% and even be higher than 65%.Because most of H3 ring does not have norm structure, be conventional main chain conformation so preferentially be chosen in those rings that show norm structure really.With regard to each ring, select major part observed conformation in natural repertoire usually thus.In people's antibody, the modal norm structure (CS) of each ring is as follows: H1-CS 1 (repertoire of 79% expression); H2-CS 3 (46%); V _κL1-CS 2 (39%); L2-CS 1 (100%); V _κL3-CS 1 (36%), the ratio of calculation κ: λ is 70: 30, Hood etc. (1967) Cold SpringHarbor Symp.Quant.Biol ., 48:133).With regard to H3 ring with norm structure, it is obviously common to the CDR3 length (Kabat etc. (1991) Sequences of proteins of immunological interest, U.S.Department ofHealth and Human Services) of 7 residues of the salt-bridge of residue 101 to have residue 94.In EMBL data library, there are at least 16 kinds and have the human antibody sequence of required H3 length and Key residues, and in Protein Data Bank, have at least two kinds of crystalline structure (2cgr and 1tet) that can be used as antibody modeling basis so that constitute this conformation.The kind of frequent expression is a gene fragment, and promptly this norm structure is combined as VH fragment 3-23 (DP-47), J _HFragment JH4b, V _κFragment O2/O12 (DPK9) and J _κFragment J _κ1.These fragments can be used as the basis that makes up the library with required single main chain conformation thus and are used in combination.

Perhaps, naturally occurring main chain conformation is made up the basis of using the single main chain conformation that elects, rather than select single main chain conformation based on coupling collar naturally occurring different main chain conformations separately in separating.With regard to antibody, for example, can determine in the antigen coupling collar any two kinds, three kinds, four kinds, five kinds or all naturally occurring norm structures combinations of 6 kinds.Herein, the preferred conformation of selecting is common and most preferably the most frequent observed in natural repertoire in the naturally occurring antibody.Therefore, in people's antibody, for example, and when considering five kinds of antigen coupling collar H1, H2, L1 during the natural combination of L2 and L3, determines the combination of the most frequent norm structure and merges with the most common conformation of H3 ring then, as the basis of selecting single main chain conformation.

B. the variation of canonical sequence

Because selected several known main chain conformations, or preferred single known main chain conformation, so make up library of the present invention so that generate repertoire with structure and/or functional diversity by the binding site that changes molecule.This means the generation variant, make them have enough diversity, make them that the activity of certain limit can be provided in its structure and/or its function aspects.For example, if described polypeptide class cell-surface receptor, they can have various target ligands binding specificity so.

General by on one or more positions, changing the required diversity of selecting of molecule generation.Can select or preferentially select the position of change at random.Can change by randomization then, in this process, with natural or synthetic amino acid or its analogue replacement inherent amino acid, use the aminoacid replacement inherent amino acid of determining subgroup thereby produce a large amount of variants or pass through, thereby produce the more variant of limited quantity.

Reported and introduced the multifarious the whole bag of tricks of this class.Fallibility PCR (Hawkins etc. (1992) J.Mol.Biol., 226:889), chemomorphosis (Deng etc. (1994) J.Biol.Chem., 269:9533) or the bacterium mutator (Low etc. (1996) J.Mol.Biol. 260:359) can be used for random mutation is imported the gene of coding molecule.The method that makes the position sudden change of selection also is well-known in the art and comprises oligonucleotide or the degenerate oligonucleotide that uses mispairing, wherein uses or do not use PCR.For example, by making sudden change targeting antigen coupling collar generate several synthetic antibody libraries.Made the new binding specificity (Barbas etc. (1992), referring to above) of people's Toxoid,tetanus-produced certain limit in conjunction with the H3 district randomization of Fab.Made at random or to be additional to kind be to produce big library with the framework region that does not suddenly change on the V gene fragment (Hoogenboom and Winter (1992) are referring to above half-at random H3 and L3 district; Nissim etc. (1994) are referring to above; Griffiths etc. (1994) are referring to above; (1995) such as De Kruif are referring to above).Made this class variation expansion so that comprise some or all other antigen coupling collar (Crameri etc. (1996) Nature Med., 2:100; Riechmann etc. (1995) Bio/Technology, 13:475; Morphosys, WO97/08320 is referring to above).

Because having, the ring randomization generates about 10 ¹⁵The potential of the variant of individual above independent H3 structure and similar a large amount of other five kinds of rings, thus use present transformation technology so by use not celliferous identical generation represent the library that might make up be not practical.For example, in one of maximum library that makes up up to now, produced 6 * 10 ¹⁰Individual different antibody, this only is the possible multifarious part (Griffiths etc. (1994), referring to above) in the library of this design.

-functional member non-and using the single known main chain conformation, the present invention except removing also by only make those directly design produce or the residue variation that changes the required functions of molecule has solved these restrictions.With regard to many molecules, function in conjunction with target ligands and thus diversity should concentrate on the target ligands binding site, avoid changing for the molecule overall package simultaneously or keep the conclusive residue of main chain conformation of selection; Therefore, the invention provides the library, wherein the selected location of Gai Bianing can constitute the position of target ligands binding site for those.

The variation of canonical sequence when being applied to antibody

With regard to antibody library, the binding site of target ligands is modal to be antigen-binding site.Therefore, aspect very preferably in, the invention provides antibody library, wherein only the residue on those antigen-binding sites changes.These residues are extremely different in people's antibody repertoire, and known permission contact high resolving power antibody/antigen mixture.For example, known 50 different and observe and allow to contact antigen in naturally occurring antibody in L2 with 53.On the contrary, conventional means make all residue variations in the complementary determining region (CDR1) of definition such as corresponding Kabat (1991, referring to above), and the seven kinds of residues of having an appointment are compared in two kinds of variations in the library of the present invention.This has represented the remarkable improvement of the required functional diversity aspect of the antigen-binding specificity that produces certain limit.

In fact, antibody diversity is the result of two kinds of processes: kind is V, and the somatocyte of D and J gene fragment is recombinated and generated pure elementary repertoire (so-called kind system and somatocyte reorganization); Somatic hypermutation with gained rearrangement v gene.Diversity in the verified elementary repertoire of analyst's antibody sequence concentrate on antigen-binding site in the heart, and somatic hypermutation is passed to district on the periphery of the conservative antigen-binding site of elementary repertoire camber (referring to (1996) such as Tomlinson, referring to above) with diversity.This complementarity may be as search sequence spatial available strategy development, although and antagonist obviously be unique, it is easy to be suitable for other polypeptide repertoire of the present invention.According to the present invention, the residue of change is those subgroup of the binding site that constitutes target ligands.If desired, the different steps variation of the residue of different (comprising overlapping) subgroups in selecting formation in the target ligands binding site.

With regard to the antibody repertoire, two-footwork of the present invention antibody maturation in human immune system is similar.Produce the repertoire of initial ' pure ', and some in the residue in the antigen-binding site, but be not whole variations.The term that uses in this paper context " pure " intention does not have the antibody molecule of pre-determined target part.These molecules are similar to by not carrying out those of immune diversified individual immunity globulin gene coding, thus, use fetus and newborn infant's individuality, and its immunity system is not subjected to the attack of various antigenic stimulation things as yet.Then to this repertoire of antigen selection of certain limit.If desired, diversified district is outside in initial repertoire subsequently imports extra diversity.Can select to change function, this sophisticated repertoire of specificity or avidity.

The invention provides two kinds of different pure repertoires of antibody, wherein some or all in the residue in the antigen-binding site changes.Natural elementary repertoire is simulated in " elementary " library, and wherein diversity is limited at the supercentral residue of antigen-binding site, and they are different (kind is a diversity) or variation (junctional diversity) in regrouping process in the V gene fragment in kind.Those diversified residues include, but are not limited to H50, H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96.In " somatocyte " library, diversity is limited in regrouping process variation (junctional diversity) or the residue of height somatic mutation takes place).Those diversified residues include, but are not limited to: H31, H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96.Known above-mentioned conduct is suitable for that diversified all residues can contact one or more antibody-antigenic compounds in these libraries.Because in two kinds of libraries, be not that residue in all antigen-binding site changes, thus in chosen process, mix extra diversity by changing remaining residue, as long as need so.It will be apparent to those skilled in the art that any any subgroup of these residues extra residue of antigen-binding site (or comprise) can be used at first and/or antigen-binding site variation subsequently.

In making up library of the present invention, general encoding sequence by change appointment peptide sequence is realized the variation of selected location on nucleic acid level, make and possible amino acid quantity (all 20 kinds or its subgroup) can be introduced this position.Use the IUPAC nomenclature, the most general codon is coding all amino acid whose NNK and TAG terminator codon.The preferred NNK codon that uses is so that introduce required diversity.Other codon of realizing same purpose also is useful, comprises the NNN codon, and it produces extra terminator codon TGA and TAA.

Side chain diversity in people's antibody antigen combining site is characterized as the remarkable bias that helps some amino-acid residue.If amount to VH, V _κAnd V _λThe amino acid of the position that the tool in district in separately 10 changes is formed, side chain diversity more than 76% only derives from 7 kinds of different residues so, they are Serine (24%), tyrosine (14%), l-asparagine (11%), glycine (9%), L-Ala (7%), aspartic acid (6%) and Threonine (6%).This to the wetting ability residue and the evolution that can provide the bias of main chain handiness may reflect the surface on a small quantity, described surface is easy in conjunction with antigen widely and helps to explain required the mixing property of antibody in the elementary repertoire.

Because preferred this seed amino acid of simulation distributes,, wherein distribute at the locational amino acid distribution simulation of change observed amino acid in the antibody antigen combining site so the invention provides the library.The bias of this class in allowing the certain limit target ligands is selected the aminoacid replacement of some polypeptide class (not being only the antibody polypeptides class) is easy to be applied to the present invention's polypeptide repertoire arbitrarily.Have the various methods that change locational amino acid distributions shift (comprise and use trinucleotide mutagenesis, WO97/08320, Morphosys is referring to above) that are used to make, wherein because of synthetic convenience preferable methods for using member's degenerate codon.So by relatively by the amino acid profile of degenerate codon assembly coding (use list in each first-class ratio in position, two, three and the quadruple degeneracy) with the application of natural amino acid, can calculate the most representative codon.Codon (AGT) is T (AGC), (AGT) (AGC) C and (AGT) (AGC) (CT)-promptly use the DVT of IUPAC nomenclature respectively, DVC and DVY-are the most approaching amino acid needed profile: Serine of their codings 22% and 11% tyrosine, l-asparagine, glycine, L-Ala, aspartic acid, Threonine and halfcystine.Therefore, the DVT that preferably uses diversified position to go up separately, DVC or DVY codon make up the library.

As mentioned above, the polypeptide class that constitutes antibody library of the present invention can be complete antibody or its fragment, such as Fab, F (ab ') ₂, Fv or scFv fragment or independent VH or VL structural domain, any kind wherein is for modifying or unmodified.Wherein strand Fv fragment or scFvs have specific application.ScFv fragment and other antibody polypeptides class generate by antibody remodeling method well-known in the art reliably.By using the suitably connection peptides of design of coding, such as (Gly-Gly-Gly-Gly-Ser) ₃Or the oligonucleotide of the connection peptides of equivalence connects VH and the VL gene constitutes scFv.Joint is according to the C-end in first V district of order bridging of VH-joint-VL or VL-joint-VH and the N-end in second V district.The binding site of scFv mainly can reproduce the specificity of corresponding complete antibody reliably, and vice versa.

Make up Fv, Fab and F (ab ') ₂The similar techniques of fragment and chimeric antibody molecule is not well-known in the art.When expressing the Fv fragment, answer careful consideration to guarantee correct chain folding and to be connected.With regard to Fab and F (ab ') ₂Fragment, with VH and VL polypeptide class with can separate from resetting gene, kind is the C gene or is merged by relevant V district segmental antibody sequence data synthetic constant region fragment.Library of the present invention is VH or VL library not.Therefore, can make up the independent library that comprises single VH and VL structural domain, and their optional C that comprises respectively _HOr C _LStructural domain, thus the Dab molecule generated.

C. carrier library of the present invention system

Library of the present invention can be used to make general and/or target ligands directly to screen or be used to comprise the selection scheme of hereditary demonstration package.

Can directly bacteriophage λ expression system directly be screened (1989) Science such as Huse as mentioned above as plaque screening or as the lysogen bacterium colony, 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) and they be useful in the present invention.Although can being used for screening, this class expression system reaches 10 ⁶The library of individual different members, but in fact and be not suitable for screening bigger quantity (greater than 10 they ⁶Individual member).It is synthetic that other screening system depends on library member's for example direct chemical.A kind of early stage method comprises one group of pin or rod peptide class, described in WO84/03564.The peptide synthetic similar approach that relates on pearl is described in U.S. Pat 4,631, and in 211, this method forms peptide library, pearl each library member that respectively does for oneself wherein, and relevant method is described among the WO92/00091.Comprise based on the remarkable improvement of the method for pearl and to use unique identifying mark, such as each pearl of oligonucleotide mark, so that help the evaluation of each library member's aminoacid sequence.These are improved based among the WO93/06121 under methods description of pearl.

Another kind of chemical synthesis process comprises according to the array that different library members (for example unique peptide sequence) is placed the synthetic from the teeth outwards peptide of mode (or the plain peptide of intending) on the array dispersive predetermined position.Determine each library member's identity according to the locus in the array.Measure the position in the array of generation binding interactions between predetermined molecules (for example acceptor) and the reactive library member, thus based on identification reaction library, locus member's sequence.These methods are described in U.S. Pat 5,143,854; WO90/15070 and WO92/10092; Fodor etc. (1991) Science, 251:767; Dower and Fodor (1991) Ann.Rep.Med.Chem. is among the 26:271.

Useful especially in library construction of the present invention is to select display systems, and they can make the expressed polypeptide in nucleic acid and it be connected.Selection display systems used herein is for allowing by suitable exhibition method according to the system of selecting each member in the library in conjunction with general and/or target ligands.

Select arbitrarily display systems can with library of the present invention coupling.The selection scheme that is used for separating the big required member in library is as known in the art, being the typical case by display technique of bacteriophage.Verified this type systematic, wherein different peptide sequences are illustrated in (Scott and Smith (1990) on the filobactivirus surface, referring to above), can be used for generating the antibody fragment library of (with their nucleotide sequence of coding), so that in conjunction with segmental external selection of the specific antibody of target antigen and amplification.The nucleotide sequence in coding VH and VL district is connected with the gene fragment of coding targeting signal, described targeting signal makes them be oriented to intestinal bacteria (E.coli) periplasmic space, and as a result of, the gained antibody fragment is illustrated on the phage surface, general as with the syzygy of phage coat protein (for example pIII or pVIII).Perhaps, antibody fragment is showed on lambda particles phage capsid (phage) outside.Advantage based on the display systems of phage is, because they are biosystem, so the library member that can select by the phage growth amplification that makes the library member who comprises selection in bacterial cell merely.In addition, because surperficial polypeptide libraries member's nucleotide sequence is included on phage or the phagemid carrier,, express relative direct with genetic manipulation subsequently so check order.

The method that makes up phage antibody display libraries and lambda particles phage expression library is that well-known in the art (McCafferty etc. (1990) are referring to above; 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.SciU.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) are referring to above; Barbas etc. (1992) are referring to above; Hawkins and Winter (1992) J.Immunol., 22:867; Marks etc., 1992, J.Biol.Chem., 267:16007; Lerner etc. (1992) Science, 258:1313 is incorporated herein by reference these documents).

A kind of particularly advantageous means for 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) are referring to above; Clackson etc. (1991) are referring to above; Marks etc. (1991) are referring to above; Chiswell etc. (1992) Trends Biotech., 10:80; Marks etc. (1992) are referring to above).The various embodiments in the scFv library of showing on the phage coat protein have been described.For example, described in the elite of phage display means such as WO96/06213 and WO92/01047 (MedicalResearch Council etc.) and the WO97/08320 (Morphosys, referring to above), these documents are incorporated herein by reference.

Other system that is used to generate polypeptide class or Nucleotide library comprises the not celliferous enzymatic device that is used for external synthetic library member of use.In one approach, select RNA molecule (Tuerk and Gold (1990) Science, 249:505 by alternate cycles and pcr amplification that target ligands is selected; Ellington and Szostak (1990) Nature, 346:818).Similar techniques can be used to identify dna sequence dna (Thiesen and Bach (1990) Nucleic Acids Res., the 18:3203 in conjunction with predetermined human transcription factor; Beaudry and Joyce (1992) Science, 257:635; WO92/05258 and WO92/14843).According to similar originating party formula, external translation can be used for synthetic polypeptide class as the method that generates big library.These methods that generally comprise stable polysome mixture further describe at WO88/08453, WO90/05785, and WO90/07003, WO91/02076 is among WO91/05058 and the WO92/02536.Be not alternately display systems, such as those polypeptide classes of using the polysome displaying to be used to select that are disclosed among WO95/22625 and the WO95/11922 (Affymax) based on phage.In addition the above-mentioned documents of these and all is incorporated herein by reference.

The present invention provides from the repertoire of polypeptide class the method for the polypeptide of selecting to have required general and/or target ligands binding site thus, and this method comprises the following steps:

A) library of the above-mentioned aspect of expression the present invention;

B) described polypeptide class contact with general and/or target ligands and selects combination generally and/or those of target ligands; With

C) optional amplification is in conjunction with the polypeptide (class) of the selection of general and/or target ligands;

D) optional repeating step a)-c).

The preferred phage display system that uses carries out step a)-d).

Owing to the invention provides the library that general and target ligands are all had the polypeptide class of binding site, so above-mentioned system of selection can be suitable for using the selection of general part or target ligands.Therefore, can use general part, and use target ligands then or use target ligands, and use general part to select initial library then.The present invention also provides before using target ligands to select or multiple selection parallel afterwards or the unusual part of polyphone use.

Preferred method of the present invention further comprises the polypeptide (class) that makes selection and carries out extra variation (as described herein) and repeating step step a)-d).

Because general part can be used in combination all library members that general part is selected according to its definite character, so further comprising, method of the present invention use general part (or its some conjugate) to detect, fixing, purifying or immunoprecipitation are from any functional member or the member colony (no matter whether passing through to select in conjunction with target ligands) in described library.

Owing to the invention provides the library, wherein the member has known main chain conformation, so method of the present invention further comprises the 3 d structure model (no matter whether passing through to select in conjunction with target ligands) that produces any functional member in the library.The structure of preferred this class model comprises homology modeling and/or molecular replacement.Can be by with secondary structure prediction or generate the rudimentary model of main chain conformation by the sequence of the screening many peptide sequences in structure library and known three-dimensional structure.Can also be with software for calculation because the secondary structure of prediction polypeptide.In order to predict the side chain conformation on the variable position, can use side chain rotational isomer library.

Generally speaking, to be used to implement nucleic acid molecule required for the present invention and vector construction body be available in the art and can be as the standard laboratory handbook, such as (1989) Molecular Cloning:A Laboratory Manual such as Sambrook, Cold Spring Harbor makes up described in the USA and operation.

The general nucleic acid operation of in recombinant vectors, carrying out among the present invention.Carrier intention used herein is used for the allogeneic dna sequence DNA transfered cell so that its expression and/or the dispersive element that duplicates.Select or make up and use the method for this class carrier well-known subsequently as those skilled in the art.A large amount of carriers are that the public is available, comprise bacterial plasmid, phage, artificial chromosome and episomal vector.This class carrier can be used for simple clone and mutagenesis; Perhaps,, wherein carry repertoire of the present invention (or preceding-repertoire) member, use genetic expression to carry as the typical case of carrier.Can select to adapt to the carrier that the present invention of the polypeptid coding sequence of required size uses, required size is generally 0.25 kilobase (kb)-40kb length.Use described carrier to transform appropriate host in body outer clone operation back.Each carrier comprises different functional ingredient, and they generally comprise clone's (or " polylinker ") site, replication orgin and at least a selectable marker gene.If specified carrier is an expression vector, it also has one or more in following so: enhancer element; Promotor; Transcription Termination and signal sequence, they are positioned near the cloning site separately, make them operationally be connected with code book invention polypeptide repertoire member's gene.

Clone and expression vector generally all comprise the nucleotide sequence that this carrier is duplicated in the host cell of one or more selections.Generally in cloning vector, this sequence is independent of the sequence that host chromosome DNA duplicates and comprises replication orgin or autonomously replicating sequence for making carrier.Well-known this class sequence is used for various bacteriums, yeast and virus.Replication orgin from plasmid pBR322 is suitable for most of Gram-negative bacteria, and 2 microns 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.Generally speaking, replication orgin is not that mammalian expression vector is required, unless they are used for duplicating the mammalian cell of high-level DNA, such as the COS cell.

Advantageously, clone or expression vector can comprise the selection gene that is also referred to as selectable marker.The transformed host cells survival that this genes encoding is grown in selecting substratum or the necessary protein of growing.The typical protein of selecting genes encoding to give microbiotic and other toxin resistance, described microbiotic and other toxin for example are Ampicillin Trihydrate, Xin Meisu, methotrexate or tsiklomitsin; The extra-nutrition defective type lacks or the crucial nutrition that can't obtain in the growth medium is provided.

Because duplicating the most expediently of carrier of the present invention carried out in intestinal bacteria, so intestinal bacteria-selectable marker, for example, the β-Nei Xiananmei gene of giving microbiotic Ampicillin Trihydrate resistance is useful.They can be available from escherichia coli plasmid, such as pBR322 or pUC plasmid, such as pUC18 or pUC19.

Expression vector comprises the promotor of being discerned and operationally being connected with the encoding sequence of being paid close attention to by host organisms usually.This class promotor can be for induction type or composing type.The term intention that " is operably connected " is arranged side by side, and wherein said composition is a dependency, allows them to work according to its specific mode.Express the control sequence that this class mode is connected " being operably connected " encoding sequence according under the condition compatible, carrying out encoding sequence with control sequence.

The promotor that is applicable to eucaryon host comprises, for example, and β-Nei Xiananmei and lactose promoter systems; Alkaline phosphatase; Tryptophane (trp) promoter systems; And hybrid promoter, such as the tac promotor.The promotor that is used for bacterial system generally also comprises the SD sequence that operationally is connected with encoding sequence.

In library of the present invention, preferred carrier is for expressing the expression vector of the nucleotide sequence that is equivalent to the polypeptide libraries member.The independent propagation of monospecific polyclonal that therefore, can be by express polypeptide library member and expression or by using any selection display systems to use the selection of general and/or target ligands.As mentioned above, preferably selecting display systems is phage display.Therefore, can use phage or phagemid carrier.Preferred carrier is for having the phagemid carrier of intestinal bacteria replication orgin (being used for two strands duplicates) and other phage replication starting point (being used to produce single stranded DNA).The operation of this class carrier and be expressed as that well-known in the art (Hoogenboom and Winter (1992) are referring to above; Nissim etc. (1994) are referring to above).In brief, this carrier is included in the β-Nei Xiananmei gene of that imparts selective on the phagemid and expresses the lac promotor downstream of cartridge clip, this expression cartridge clip is by pelB leader sequence (making polypeptide expressed be oriented to periplasmic space), multiple clone site (the Nucleotide form that is used for the clone library member), choose any one kind of them or multiple peptide-labeled (being used for detecting), choose any one kind of them or multiple TAG terminator codon and phage protein pIII composition (N-C end).Therefore, use colibacillary different the inhibition and non--inhibition bacterial strain and interpolation glucose, isopropylthio-(IPTG) or helper phage, such as VCS M13, described carrier can be as plasmid replication under situation about not expressing, only produce a large amount of polypeptide libraries members or produce phage, wherein some is included at least a copy of its lip-deep polypeptide-pIII syzygy.

The structure of carrier of the present invention has used conventional interconnection technique.Cracking isolated vectors or dna fragmentation constitute and connect into the required form of required carrier that produces.If desired, can confirm that correct sequence is present in the analysis in the carrier of structure according to known way.Be used for construction of expression vector, preparation in-vitro transcription thing, it is known in those skilled in the art with the suitable method of carrying out the analysis of evaluation expression and function that DNA is imported host cell.The gene order of operating in the test sample or by quantitative its amplification of ordinary method and/or expression, described ordinary method such as DNA or RNA analyze, western blotting, DNA, RNA or proteinic Dot blot, in situ hybridization, the sequential analysis of immunocytochemistry or nucleic acid or protein molecule.If desired, those skilled in the art are easy to estimate how to improve these methods.

Use the mutagenesis of polymerase chain reaction (PCR)

In case select carrier system and will encode that paid close attention to be cloned into carrier library with one or more nucleotide sequences polypeptide class, just can be by the intramolecularly generation diversity of before expression, taking mutagenesis cloning; Perhaps, as mentioned above, can before mutagenesis, express and select encoded protein matter and carry out extra selection circulation.As mentioned above, carry out the mutagenesis of the nucleotide sequence of the polypeptide class that coding structure optimizes by the standard molecule method.Useful especially is polymeric enzyme reaction or PCR (Mullis and Faloona (1987) Methods Enzymol., 155:335 is incorporated herein by reference the document).The PCR of the target sequence that the catalytic a plurality of dna replication dna cyclic amplifications of use thermostability DNA-dependent dna-polymerases are paid close attention to is well-known in the art.

Being used for Oligonucleolide primers of the present invention is single stranded DNA or RNA molecule, and they and nucleic acid-templated hybridization are so that cause that the enzymatic of the second chain nucleic acid is synthetic.The part complementation of this primer and the target molecule that is present in the nucleic acid molecule storehouse that is used for preparing array group of the present invention.Concern prepares this quasi-molecule by synthetic method, no matter is chemical process or enzymatic means.Perhaps, this quasi-molecule or the natural existence of its fragment and separation are from its natural origin or available from goods providers.Although the oligonucleotide of different lengths is useful, the mutagenic oligonucleotide primer is a 15-100 length of nucleotides, it is desirable to 20-40 Nucleotide.

Generally speaking, when two kinds of nucleotide sequences complementary basically (in one section sequence at 14-25 Nucleotide at least be complementary at least about 65%,, be complementary more preferably) at least about 90% preferably at least about 75%, the generation selective cross.Referring to Kanehisa (1984) Nucleic Acids Res.12:203, the document is incorporated herein by reference.As a result of, estimate that the mispairing to a certain degree on priming site is acceptable.This class mispairing can be for little, such as one-, two-or three-Nucleotide.Perhaps, it can comprise the Nucleotide ring, and we are defined as the district, and wherein mispairing comprises the continuous string of 4 or 4 above Nucleotide.

In a word, the validity and the selectivity of 5 factor affecting primers and second kind of making nucleic acid molecular hybridization.These factors are: (i) primer length; (ii) nucleotide sequence and/or composition; (iii) hybridization temperature; (iv) buffer reagent chemistry; (v) need sterically hindered possibility in the district that primer hybridizes, these factors are considerable when the nonrandom initiation sequence of design.

Between the efficient of target sequence and tolerance range, there is positive correlation at primer length and primer annealing; Long sequence has the melting temperature(Tm) (T that is higher than shorter sequence _M), and the multiple possibility is lower in the specified target sequence, thus mixed and disorderly hybridization is reduced to bottom line.The primer sequence that has high G-C content or comprise palindromic sequence trends towards oneself's hybridization, trends towards specified target site as it, because unit molecule but not the bimolecular hybridization kinetics generally is favourable in solution; Simultaneously, importantly design primer, its G-C oligonucleotide ligand that comprises sufficient amount is to so that combine closely target sequence because this class to separately by three hydrogen bonds, but not two hydrogen bonded of when A and T base pairing, finding.The change that is inversely proportional to of hybridization temperature and primer degeneration efficient, as the organic solvent that can be included in the hybridization mixture, for example the concentration of methane amide is the same, and the hybridization of salt concn helps combination.Under stringent hybridization condition, probe sufficient hybridization efficiency height under the condition of more permission that long probe is relatively shorter.Strict hybridization conditions generally comprises and is lower than about 1M, more generally be the salt concn that is lower than about 500mM and preferably is lower than about 200mM.Hybridization temperature be low to moderate 0 ℃-be higher than 22 ℃, be higher than about 30 ℃ and (the most common is) above about 37 ℃.Long fragment may need higher hybridization temperature so that carry out specific hybrid.As the Several Factors of influence hybridization severity, the combination of parameter is more even more important than any one independent absolute measured value.

Use the consideration design primer in these thinkings.Although those skilled in the art can use one's brains the relative merits of a large amount of sequences is assessed, designed this Several Parameters of computer program auxiliary evaluation and optimized primer sequence.The example of this class method is DNAStar ^TM" PrimerSelect " (DNAStar, Inc. of software package; Madison, WI) and OLIGO4.0 (National Biosciences, Inc.).In case design, just prepare oligonucleotide by suitable method, Beaucage and Carruthers (1981) Tetrahedron Lett. for example, 22:1859) described phosphoramidite method or Matteucci and Caruthers (1981) J.Am.Chem.Soc., the triester method of 103:3185 is incorporated herein by reference these two pieces of documents; Or be purchased other chemical process or the VLSIPS of automated oligonucleotide synthesizer by use ^TMTechnology.

Use template DNA (1fg at least; 1-1000ng more usefully) and at least the 25pmol Oligonucleolide primers carries out PCR; Advantageously be evident as in the primer storehouse and use a large amount of primers when allogenic, because sequence is only divided subrepresentation by the small part in the storehouse separately, and amount becomes limited in amplification cycles subsequently.Typical reaction mixture comprises: 2 μ l DNA, the 25pmol Oligonucleolide primers, 2.5 μ l of 10X PCR damping fluid 1 (Perkin-Elmer, Foster City, CA), 0.4 μ l, 1.25 μ M dNTP, 0.15 μ l (or 2.5 units) Taq archaeal dna polymerase (PerkinElmer, Foster City is CA) with deionized water to 25 μ l cumulative volume.Cover mineral oil and the controlled thermal cycler of service routine and carry out PCR.

In fact adjust PCR circulate length and temperature and cycle index in each step according to the requirement of severity.Primer annealing to the efficient and the acceptable mispairing degree of template determined annealing temperature and time limit on the estimation; Obviously, when simultaneously the amplification and mutagenized nucleic acid divide the period of the day from 11 p.m. to 1 a.m, in first synthesis cycle, need mispairing at least.In the process of the mixing storehouse amplifier molecule colony that attempt to use mutagenic primer, to primer to the sequence of non-target site mix that annealing weighs only may be under severity (high temperature) annealing conditions because of the product loss that may suddenly change of low melting temperature(Tm) generation.The ability of optimizing the severity of primer annealing condition fully belongs to those skilled in the art's ken.Use 30C-72 ℃ annealing temperature.The initial sex change of template molecule took place 4 minutes down at 92 ℃-99 ℃ usually, was that (temperature of determining as mentioned above in annealing by sex change (94-99 ℃ following 15 seconds-1 minute) subsequently; 1-2 minute) and stretch 20-40 the circulation that (72 ℃ following 1-5 minute, this depends on the length of amplified production) are formed.Final stretching, extension be generally 72 ℃ following 4 minutes and can be uncertain (0-24 hour) step under 4 ℃ subsequently.

Repertoire member's structural analysis

Owing to the invention provides the repertoire of known main chain conformation, so be easy to generate the 3 d structure model of any member in the repertoire.Generally speaking, the structure of this class model comprises homology modeling and/or molecular replacement.By the similar sequence of many peptide sequences, by secondary structure prediction or by screening the preparing model that the structure library generates the main chain conformation in known three-dimensional structure.The molecular model software package be purchased and be used to predict the polypeptide secondary structure.In order to predict the conformation of side chain on the variable position, can use side chain rotational isomer library.

Antibody as the part in the polypeptide selection

General or the target ligands self that is used for polypeptide selection of the present invention can be antibody.This is definite especially for general part, and they are combined in to being essentially conservative constitutional features in the functional polypeptide class that comprises repertoire selection of the present invention.If suitable antibody is not for the public can obtain, can pass through so phage display method (referring to above) or following production it:

The albumen that recombinant protein or those derive from natural origin can be used for using those well-known standard techniques of this area to generate antibody.For example, give protein (or " immunogen ") so that attack Mammals, such as monkey, goat, rabbit or mouse.Gained antibody can be collected as polyclonal serum, maybe can make the cell immortalization (for example by merging so that produce hybridoma with the immortalization fusion partner) from the generation antibody of the animal of attacking, this cell produces monoclonal antibody subsequently.

A. polyclonal antibody

Antigen protein can use separately or put together so that increase its immunogenicity with common carrier, and the antiserum(antisera) to this peptide-carrier conjugate is increased in animal.Can carry out as described peptide and carrier protein couplet and immunization (Dymecki etc. (1992) J.Biol.Chem., 267:4815).By ELISA, or selectable by spot or the some mark to proteantigen titration serum (Boersma and Van Leeuwen (1994) J.Neurosci.Methods, 51:317).By ELISA, for example, according to operation steps (1982) Cell of Green etc., 28:477 confirms serum and suitable peptide class generation kickback.

B. monoclonal antibody

The technology of preparation monoclonal antibody is well-known, and can use any candidate antigens, preferably with carrier-bound antigen, and as (1981) Nature such as Arnheiter, 294,278 described preparation monoclonal antibodies.Monoclonal antibody is generally available from the hybridoma tissue culture or from the animal ascites fluid available from the introductive crossing tumor tissue.Yet, monoclonal antibody can be described as " protein is increased " or " by protein induce ".

After increase, by the function and the specificity of any kind test monoclonal antibody in many modes.The similar operation step also can be used to test the recombinant antibodies that produces by phage display or other external selection technology.Can screen the hybridoma (or polyclonal serum) that produces monoclonal antibody equally with immunogen bonded antibody.Particularly preferred immunity test comprises enzyme-linked immunoassay (ELISA), and immunoblotting and immunoprecipitation (referring to Voller, (1978) DiagnosticHorizons, 2:1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller etc. (1978) J.Clin.Pathol., 31:507; U.S.ReissuePat.No.31,006; UK Patent 2,019,408; Butler (1981) Methods Enzymol., 73:482; Maggio, E. (ed.), (1980) Enzyme Immunoassay, CRC Press, Boca Raton, FL) or radioimmunoassays (RIA) (Weintraub, B., Principles ofradioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March 1986, pp.1-5,46-49 and 68-78), all these methods have only detected antibody and have combined with making the immunogenic of its increase.Those skilled in the art are apparent, must the traget antibody molecule or immunogen detect so that help this class.The technology that is used for the traget antibody molecule well-known to those skilled in the art (referring to Harlour and Lane (1989) Antibodies, Cold Spring Harbor Laboratory, pp.1-726).

Perhaps, other technology can be used for detection and combine with immunogenic, confirms the integrity as the antibody of general antigen of the present invention or target antigen thus.They comprise chromatography, such as SDSPAGE, and isoelectrofocusing, western blotting, HPLC and capillary electrophoresis.

" antibody " is defined as in this article construct and other antibody modification thing in combination (variable) district that uses this antibody-like.Therefore, be used for antibody of the present invention and can comprise complete antibody, antibody fragment, multi-functional antibody aggregation thing or generally speaking for comprising is from the arbitrary substance of one or more specific binding sites of antibody.Antibody fragment can be fragment, such as Fv, and Fab and F (ab ') ₂Fragment or its be derivative arbitrarily, such as strand Fv fragment.Antibody or antibody fragment can be non--reorganization, reorganization or humanized.This antibody can have immunoglobulin (Ig) isotype arbitrarily, IgG for example, IgM etc.In addition, if suitable, can use the aggregation of immunoglobulin (Ig), polymkeric substance, derivative and conjugate or its fragment.

Further describe the present invention in the following example, purpose only is to explain.

Metal ion as the part that is used for the selection of polypeptide class

As mentioned above, the part of non-antibody is useful in polypeptide class of the present invention is selected.A kind of this class part is a metal ion.For example, wish to use functional Histidine (HIS) the mark preselected repertoire of Ni-NTA matrix to existing.Immobilized metal affinity chromatography (IMAC; Hubert and Porath (1980) J.Chromatography 98:247) has utilized on a large amount of protein surfaces that expose, the melts combine characteristic of the amino-acid residue that Histidine and cysteine amino and other can bond.It uses resin, is generally agarose, and it comprises the bidentate metal chelator (iminodiethanoic acid for example of coordination of metal ion, IDA, the dicarboxylic acid group), so that generate the metal-ionic resin that has of the present invention, with agarose/IDA and metal-salt (for example, CuCl ₂2H ₂O) mix, the IDA chelating is from wherein divalent cation.A kind of agarose that is purchased/the IDA goods are " CHELATING SEPHAROSE 6B " (PharmaciaFine Chemicals; Piscataway, NJ).Useful metal ion includes, but are not limited to divalent cation Ni ²⁺, Cu ²⁺, Zn ²⁺And Co ²⁺Prepare the peptide molecule storehouse with the binding buffer liquid of mainly forming by the salt (being generally NaCl or KCl) and the weak part (such as Tris or ammonia) of 0.1-1.0M concentration, the latter in binding buffer liquid has avidity to the metal ion of resin, but degree is lower than the polypeptide that the present invention selects.The weak useful concentration of part in binding buffer liquid is in the scope of 0.01-0.1M.

Make peptide library contact pressure resin under allow the to have metal binding domain polypeptide class bonded condition of (vide infra); After washing away impurity, the polypeptide class of using buffer solution elution to select is in damping fluid, weak part exists with the concentration that is higher than in binding buffer liquid, especially, although lower to the binding affinity of metal ion, its concentration is enough to replace the polypeptide class of selection for weak part.The weak useful concentration of part in elution buffer is higher than that 10--50-doubly is generally 0.1-0.3M in binding buffer liquid; Notice that the concentration of salt in elution buffer equals the concentration in binding buffer liquid.According to method of the present invention, the metal ion of resin is generally as general part; Yet, pay close attention to them and also can be used as target ligands.

Use standard colour chart device (post aspirates damping fluid by it by gravity, by vacuum attraction or pass through pressure-driven) to carry out IMAC; Perhaps, use operation in enormous quantities, the resin territory that wherein will have metal is mixed into slurry form from the peptide library of selecting repertoire member of the present invention.

Described by IMAC partial purification serum T april protein (Staples etc., U.S. Pat 5,169,936); Yet, the wide spectrum protein that comprises the metal binding domain that the surface exposes also comprises other soluble T 4 albumen, human serum protein (IgG for example, haptoglobin, hemopexin, the Gc-sphaeroprotein, Clq, C3, C4), people desmoplasmin, Dolichos biflorus lectin, the Tyr of zinc-inhibition (P) Phosphoric acid esterase, phenolase, the carboxypeptidase isozyme, Cu, Zn superoxide-dismutase (comprise people and all other Eukaryotic those), nucleoside diphosphatase, LeIF, lactoferrin, human plasma α ₂-SH glycoprotein, β ₂-macroglobulin, α ₁-antitrypsin, plasminogen activator, gi tract polypeptide class, stomach en-, people and bovine serum albumin(BSA) are from granulocytic granule protein, N,O-Diacetylmuramidase, nonhistone protein, human fibrinogen, human serum transferrin, human lymphotoxin, calmodulin, a-protein, avidin, myosin, the somatomedin class, human growth hormone, transforming growth factor, Thr6 PDGF BB, α-human atrial natriuretic polypeptide, cardiodilatin etc.In addition, can use the protein-bonded ectodomain sequence of IMAC purification membrane.Attention can be according to nonproductive comprising in the above-mentioned protein of the present invention ArbitrarilyThe repertoire of kind or its melts combine variant.

Behind wash-out, from the melts combine damping fluid, take out the polypeptide class of selecting and put into the damping fluid that is suitable for its next step application.If metal ion is used to generate the peptide library that the present invention at first selects, so the molecule in this storehouse is put into the damping fluid that is used for second kind of part of selection function polypeptide repertoire member for combination.If select step and metal is used for second, the polypeptide class with repertoire changes damping fluid (for example 0.5% glycine buffer) that is suitable for storing or the application of specifying them over to so.This class damping fluid includes, but are not limited to: water, organic solvent, water and water are easy to the mixture of molten mixed organic solvent, normal saline buffer solution and protein/nucleic acid or protein/protein bound damping fluid.Perhaps, can make peptide molecule dehydration (promptly by freeze-drying) or be fixed on solid or semi-solid upholder, go up or crosslinked with chromatography resin such as nitrocotton or nylon leaching film or gel matrix (being agarose or polyacrylamide matrix).

Can from elution buffer, take out peptide molecule by many known method in this area.Can be to water or the another kind of solution dialysis polypeptide elutriant of selecting; If freeze-drying polypeptide class is used the water added proteinase inhibitor (for example pepstatin, Trypsin inhibitor,Trasylol, leupeptin etc.) so.Perhaps, can make sample carry out ammonium sulfate precipitation well-known in the art, after this medium in suspend again.

The application of the polypeptide class that the present invention selects

The polypeptide class of selecting according to the inventive method can be used for arbitrary method basically, and it comprises part-polypeptide combination, comprises interior therapeutic and prophylactic applications, and external and in-vivo diagnostic is used external test and reagent application etc.For example, with regard to antibody, according to well known to a person skilled in the art method, antibody molecule can be used for the determination techniques based on antibody, such as elisa technique.

As mentioned above, the molecule that the present invention selects has diagnosis, the application in prevention and the treatment operation.For example, can use technology well-known in the art in external or body, to measure the activity of the enzyme variants that generates and select by these methods,, the conversion of substrate to product hatched and analyzed in they and candidate's substrate molecule by these methods.Can in cultured cells, express cell-surface receptor or the adhesion molecule selected, and test the avidity of the ability of its reaction that biochemistry is stimulated or they and other cell type then, described other cell type expression can estimate that diversified adhesion molecule is not distinguished bonded cell-surface molecular.The antibody polypeptides class that the present invention selects has diagnostic use in western blot analysis of operating by standard immunoassay histological chemistry and original position protein detection; Use the antibody of the repertoire that can select according to technical mark well known in the art in order to be used for these.In addition, when with the chromatogram upholder, during such as resin compounded, can use this antibody-like polypeptide class with preparation method according to avidity chromatographic run step.It is well-known that all these class technology are those skilled in the art.

The proteinic treatment and the prophylactic applications of the present invention's preparation comprise the Mammals to the recipient, give the polypeptide class that the present invention selects such as the people.Concrete in this respect use antibody arranged; Other acceptor (including, but are not limited to T-cell receptors), and thus, wherein antibody or acceptor are as general or target ligands; In conjunction with their protein.

Preferably have at least the homogeneous pure basically antibody of 90-95% or conjugated proteinly give Mammals, and most preferably 98-99% or 99% above homogeneity are used for medicinal application, especially when Mammals be man-hour.In case partial purification or to homogeneity as required, just can be in diagnosis or treatment (comprising external) or in research and development with carry out measurement operation, use polypeptide class (Lefkovite and the Pernis that selects in the immunofluorescence dyeing etc., (1979 and 1981) ImmunologicalMethods, Volumes I and II, Academic Press, NY).

The antibody that the present invention selects or its conjugated protein generally being applied to are prevented, suppress or the treatment inflammatory conditions, anaphylactic hypersensitivity, cancer, bacterium or virus infection and autoimmune disease (include, but are not limited to type i diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematous, Crohn's disease and myasthenia gravis).

In this application, term " prevention " is included in Before inducing an illnessGive prophylactic compositions." inhibition " intention is after the situation of bringing out, but in disease Before the clinical manifestationGive composition." treatment " is included in and gives prophylactic compositions after disease symptoms becomes obviously.

Can utilize and to be used to screen antibody or its conjugated protein animal model system in prevention or treatment disease.The method that is used for testing susceptible mouse systemic lupus erythematous (SLE) is (Knight etc. (1978) J.Exp.Med., 147:1653 well known in the art; Reinersten etc. (1978) NewEng.J.Med., 299:515).By use solubility AchR protein from another kind induce an illness test SJL/J female mice myasthenia gravis (MG) (Lindstrom etc. (1988) Adv.Immunol., 42:233).By injection II collagen type in the susceptible mouse species, bring out sacroiliitis (Stuart etc. (1984) Ann.Rev.Immunol., 42:233).Described by injection mycobacterium heat shock protein in the susceptible rat, bring out the adjuvant-induced arthritis model (VanEden etc. (1988) Nature, 331:171).By give as described thyroglobulin in mouse, bring out thyroiditis (Maron etc. (1980) J.Exp.Med., 152:1115).Insulin-dependent diabetes (IDDM) spontaneous generation or can in the mouse of some strain, bring out, such as described those (1984) Diabetologia such as Kanasawa, 27:113.EAE in mouse and the rat is as the model of people MS.In this model, by give myelin basic protein bring out demyelinating disease (referring to Paterson (1986) Textbook of Immunopathology, Mischer etc., eds., Grune and Stratton, New York, pp.179-213; McFarlin etc. (1973) Science, 179:478: and (1987) J.Immunol. such as Satoh, 138:179).

The antibody that the present invention selects, acceptor (including, but are not limited to T-cell receptors) or its conjugated protein can also with other antibody coupling, particularly with the people's cell that causes disease on the monoclonal antibody (MAbs) of other labeled reactant.For example, suitable T-cell marking can comprise those that are categorized into so-called " differentiation bunch ", according to First International LeukocyteDifferentiation Workshop name (Bernhard etc. (1984) Leukocyte Typing, Springer Verlag, NY).

Generally speaking, the antibody that the present invention selects, acceptor or conjugated proteinly use with the pharmacology suitable carriers with purified form.Generally speaking, these carriers comprise water or alcohol/aqueous solution, and emulsion or suspension comprise salt solution and/or buffer medium arbitrarily.Non-enteron aisle vehicle comprises sodium chloride solution, woods lattice Glucose Liquid, glucose and sodium-chlor and lactated Ringer's.If by polypeptide complex is remained in the suspension necessity, the acceptable adjuvant of so suitable physiology can be selected from thickening material, such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and alginate.

Intravenously vehicle comprises fluid and nutritional supplement and electrolyte supplements, such as based on those of woods lattice Glucose Liquid.Can also there be sanitas and other additive, such as antiseptic-germicide, antioxidant, sequestrant and rare gas element (Mack (1982) Remington ' s PharmaceuticalSciences, 16 ^ThEdition).

The polypeptide class that the present invention can be selected use as independent administration composition or with other promoting agent coupling.They can comprise various immunotherapy medicines, such as S-Neoral, methotrexate, Zorubicin or cis-platinum and immunotoxin class.Pharmaceutical composition can comprise the antibody that various cytotoxic agents or other promoting agent and the present invention are selected, acceptor or its are conjugated protein, and even the present invention has the polypeptide class of not homospecific selection, such as " mixture " of the combination of the polypeptide class of using different target ligands to select, no matter whether before administration, collect them.

The route of administration of pharmaceutical composition of the present invention can for those skilled in the art generally well-known those.With regard to therapy, include, but are not limited to immunotherapy, can give the antibody that the present invention selects to patient arbitrarily according to standard technique, acceptor or its are conjugated protein.Administration can comprise non-enteron aisle by the mode of any appropriate, intravenously, muscle, intraperitoneal, transdermal, by lung by way of, or also can be by using the direct infusion of conduit.Dosage and frequency depend on patient's age, sex and situation, the other medicines that give simultaneously, other parameter that contraindication and clinicist are considered.

Can be for storing freeze-drying of polypeptide class and the dissolving more before use that the present invention is selected.Verified this technology is effectively when the lyophilization of using conventional immunoglobulin (Ig) and generally acknowledging and can uses dissolving technology again.It will be appreciated by those skilled in the art that freeze-drying and dissolve the antibody activity that can cause in various degree again and lack (activity of for example using immunoglobulin (Ig) commonly used, IgM antibody to trend towards having greater than IgG antibody lacks) and must adjust upward application level so that compensate.

Can comprise the polypeptide class that the present invention selects or the composition of its mixture for prevention and/or prophylactic treatment.In some treatment of the cell mass of selecting is used, be not inhibition at least, retardance, adjusting kills and wounds or the q.s of some other parameter that can measure is defined as " treatment effective dose ".Reach the general situation that the required amount of this dosage depends on severity of disease and patient's autoimmunization system, but the general antibody of selecting at 0.005-5.0mg, the scope of acceptor (for example T-cell receptors) or its conjugated protein/kg body weight, dosage more commonly used is 0.05-2.0mg/kg/ dosage.In order to carry out prophylactic application, can also comprise the polypeptide class of the present invention's selection or the composition of its mixture with the dosage of similar or appropriate reduction.

The composition that comprises the polypeptide that the present invention selects can be used for prevention and treatment environment,, make its inactivation, kill and wound or remove them so that auxiliaryly change the target cell group who selects in the Mammals.In addition, the repertoire that can select in external use polypeptide class described herein or be chosen in external killing and wounding exhausts and even effectively remove the target cell group from the allos cell mass.Can external will be from the antibody of mammiferous blood and selection, cell-surface receptor or its conjugated protein merging kill and wound unwanted cells according to standard technique thus and even remove them so that Mammals recovers from blood.

Embodiment 1

The antibody library design

A. main chain conformation

With regard to 5 kinds (L1, L2, L3, H1 and H2) in 6 kinds of antigen coupling collars of people's antibody, there be main chain conformation or norm structure ((Chothia etc. (1992) J.Mol.Biol., the 227:799 of limited quantity; Tomlinson etc. (1995) EMBO J., 14:4628; Williams etc. (1996) J.Mol.Biol., 264:220).These rings modal main chain conformation separately is used to provide the present invention single known main chain conformation.They are: H1-CS 1 (79% repertoire of expressing), H2-CS 3 (46%), V _κL1-CS 2 (39%), L2-CS 1 (100%), V _κL3-CS 1 (36%).The H3 ring constitutes main chain conformation (Martin etc. (1996) J.Mol.Biol., the 263:800 of the becate length of limited quantity; Shirai etc. (1996) FEBS Letters, 399:1).Therefore, (defined if H3 has the CDR3 length of 7 residues as (1991) such as Kabat.Sequences of proteins of immunological interest, U.S.Department of Health and Human Services) and have Methionin or arginine residues on the H94 position and on the H101 position, having asparagicacid residue, between these two kinds of residues, form salt bridge so, and in most of situation, can produce single main chain conformation.In EMBL data library, there are 16 kinds of human antibody sequences at least, and in Protein Data Bank, have two kinds of crystalline structure (2cgr and 1tet) that can be used as antibody modeling basis at least with this conformation of formation of required H3 length and Key residues.

In this case, the required ring length of coding of frequent expression and Key residues be so that producing the kind of required norm structure combination is that gene fragment is VH fragment 3-23 (DP-47), J _HFragment JH4b, V _κFragment O2/O12 (DPK9) and J _κFragment J _κ1.These fragments can be used in combination thus so that have the basis in the library of required single main chain conformation as structure.V _κFragment O2/O12 (DPK9) is V _κMember in 1 family and thus in conjunction with superantigen protein L.VH fragment 3-23 (DP-47) is the member in the VH3 family and should can be used as general part then in conjunction with the superantigen a-protein thus.

B. the selection of variant position

People VH and V _κSequential analysis be presented in the ripe repertoire most of different position for allow to contact the most widely antigenic those (referring to Tomlinson etc., (1996) J.Mol.Biol., 256:813; Fig. 1).These positions have constituted the functional antigen binding site and have selected (Fig. 2) for the side chain variation thus.H54 is for away from the residue of the key of antigen-binding site in the H2 norm structure of selecting 3 and point (observed diversity is because of due to norm structure 1,2 and 4 on this position, wherein H54 point sensing binding site).In this case, and H55 (sensing binding site) variation.These locational diversity because of the kind system in elementary repertoire or junctional diversity or somatic hypermutation produce (Tomlinson etc., (1996) J.Mol.Biol., 256:813; Fig. 1).The different residue subgroups of in the antigen-binding site two change over two kinds of different library forms thus.In " elementary " library, for residue that change to select from H2, H3, L2 and L3 (diversity in these rings mainly is kind to be or the result of junctional diversity).The position that changes in this library is: H50, and H52, H52a, H53, H55, H56, H58, H95, H96, H97, H98, L50, L53, L91, L92, L93, L94 and L96 (amount to 18 residues, Fig. 2).In " somatocyte " library, for residue that change to select from H1, H3, the end of L1 and L3 (diversity herein mainly is the result of somatic hypermutation or junctional diversity).The position that changes in this library is: H31, and H33, H35, H95, H96, H97, H98, L30, L31, L32, L34 and L96 (amount to 12 residues, Fig. 2).

C. change the selection that locational amino acid is used

By mixing codon NNK (the 20 all seed amino acids of encoding, comprise the TAG terminator codon, but non-TGA and TAA terminator codon) or codon DVT (Serine of coding 22% and 11% tyrosine, l-asparagine, glycine, L-Ala, aspartic acid, Threonine and halfcystine and on each position, use the list of equal proportion, dual, triple and quadruple degeneracy, the amino-acid residue of the most closely simulating in the natural human antibody antigen combining site distributes) the side chain diversity is introduced " elementary ' ' and " somatocyte " library.

Embodiment 2

The selection of library construction and the general part of use

By listed oligonucleotide in the table 1 and plant be the PCR assembling " elementary " of V gene fragment DPK9 and " somatocyte " library (Cox etc. (1994) Eur.J.Immunol., 24:827) and DP-47 (Tomlinson etc. (1992) J.Mol.Biol., 227:7768).In brief, to use 5 ' (oppositely) primers and NNK or DVT 3 ' (forward) primer pair and corresponding kind be the V gene fragment carries out first round-robin increase (referring to table 1) as template.This process produces 8 independent dna fragmentations that are used for NNK and DVT library separately.Use 5 shown in the table 1 ' (oppositely) primer then and carry out second round-robin amplification with 3 ' (forward) primers and from two in the purifying fragment of first amplification cycles.This process produces 4 the independent dna fragmentations that are used for NNK and DVT library separately (" elementary " VH fragment, 5A; " elementary " V _κFragment, 6A; " cell " VH fragment, 5B; With " somatocyte " V _κFragment, 6B).

Cut each in these fragments and be connected into pCLEANVH (with regard to the VH fragment) then or pCLEANVK (with regard to V _κFragment), it comprises virtual VH and the V that does not contain accordingly any TAG codon or peptide-labeled pHEN1 form _κStructural domain (Hoogenboom ﹠amp; Winter (1992) J.Mol.Biol., 227:381).Then this connection electroporation is gone into the coli strain HB2151 of non-inhibitor.Generation from these libraries each phage and use separately immunity pipe (immunotubes) to select, described immunity pipe bag is respectively applied for VH and V _κThe 10 μ g/ml general ligandin matter A and the protein L in library.Prepare DNA and cutting by the intestinal bacteria that infected the phage of selecting then, so that use corresponding V _κThe library replaces virtual V _κInsert fragment.The telex machine in these libraries produces following insertion fragment library size: 9.21 * 10 ⁸(" elementary " NNK), 5.57 * 10 ⁸(" elementary " DVT), 1.00 * 10 ⁹(" somatocyte " NNK) and 2.38 * 10 ⁸(" somatocyte " DVT).As preselected control group, generate 4 kinds of extra libraries, but do not use general ligandin matter A and protein L to select: the insertion fragment library size in these libraries is 1.29 * 10 ⁹(" elementary " NNK), 2.40 * 10 ⁸(" elementary " DVT), 1.16 * 10 ⁹(" somatocyte " NNK) and 2.17 * 10 ⁸(" somatocyte " DVT).

In order to verify the success of preselected step, will go into based on expression vector and the electroporation of pUC to go into HB2151 from the dna clone in selection and unselected " elementary " NNK library.From the library that each is cloned again, select 96 clones and induce them with random fashion so that express solubility scFv fragment.The generation of ELISA measurement function scFv by using protein L so as to capture scFv and a-protein-HRP conjugate subsequently so that the detection combination.Only expressive function VH and V _κThe scFv of structural domain (no frameshit, terminator codon, folding or expression sudden change) produces the signal that uses this assay method.The member of functional antibodies in each library (the ELISA signal that is higher than background) is 5% when using unselected " elementary " NNK library and is 75% (Fig. 3) when using identical selection form.Negative clone's order-checking alleged occurrence frameshit in this assay method, terminator codon must prevent PCR sudden change folding and/or that express on critical framework residues and the amino acid in antigen-binding site.

Embodiment 3

Library to target ligands is selected

Use the 5 kinds of antigens (ox ubiquitin, rat BIP, ox histone, NIP-BSA and hen-egg lysozyme) that are coated on the immune pipe of different concns to select " elementary " and " somatocyte " NNK library (no preselected) separately.After 2-4 selection circulation, obtain antibody at all the antigenic high degree of specificity except that hen-egg lysozyme.Select the clone at random so that check order, thereby confirm each antigenic antibody scope (Fig. 4).

Two interim, will mix so that generate single " elementary " library and single " somatocyte " library according to 1: 1 from the phage in preselected NNK and DVT library.Use the 7 kinds of antigens (FITC-BSA, people's Leptin, human thyroglobulin, BSA, hen-egg lysozyme, mouse IgG and human IgG) on the immune pipe of being coated on of different concns to select these libraries separately then.After 2-4 selection circulation, obtain the antibody of all antigenic high degree of specificity, described all antigens are included in and are difficult to produce the male hen-egg lysozyme in the early stage selection, and it is preselected that the library that wherein early stage selection is used does not use general part to carry out.Select the clone at random so that check order, thereby confirm each antigenic different antibodies scope (Fig. 4).

Embodiment 4

Preselected effect of scFv being expressed and having the phage generation of scFv

For the further preselected result of checking, will go into based on expression vector and the electroporation of pUC to go into HB2151 from the dna clone in " elementary " DVT library of unselected and pre--selection, in two kinds of situations, all produce 10 ⁵Individual clone.From the library that each is cloned again, select 96 clones and induce them with random fashion so that express solubility scFv fragment.Use protein L so that capture scFv, uses a-protein-HRP to detect bonded scFv in the generation of surveying measurement function scFv subsequently.The per-cent of functional antibodies in each library is 35.4% (unselected) and 84.4% (pre--as to select), show as use a-protein and protein L pre--result that selects, functional member's quantity has increased by 2.4 times, and (this increase is starkly lower than the NNK library of equivalence, the TAG terminator codon because the DVT codon is not encoded.In unselected NNK library, at the bacterial strain of non-inhibition, such as existing the TAG terminator codon to cause stopping among the HB2151 and preventing that thus functional scFv from expressing.NNK library preselected removed the clone who comprises the TAG terminator codon and produced the library, and wherein a high proportion of member expresses solubility scFv).

In order to estimate the preselected effect that overall scFv is expressed in " elementary " DVT library, induce again unselected and pre--selection of clone the library (in based on the expression vector of pUC each self-contained 10 ⁵Individual clone) expresses so that carry out the segmental polyclone of scFv.Then by on the ELISA flat board of protein L bag quilt, hatching 2 times of diluents (the 1-12 hurdle among Fig. 5 a) of supernatant liquor, the concentration of scFv in supernatant liquor of using a-protein-HRP detection assay to express subsequently, the ScFvs of parallel detection concentration known is so that quantitative to the expression level of scFv in the DVT library of unselected and pre--selection.They are used for drawing standard curve (Fig. 5 b) and from wherein the expression level in unselected and pre--" elementary " DVT library of selecting being calculated as 12.9 μ g/ml and 67.1 μ g/ml respectively, promptly because of use a-protein and protein L preselected cause expressing increase by 5.2 times.

In order to estimate the amount of the phage with scFv, make " elementary " DVT library growth of unselected and pre--selection and produce the polyclone phage.In the isopyknic phage that moves on the 4-12%Bis-Tris NuPAGE gel that uses the MES electrophoretic buffer and under the sex change condition from two libraries.The gained gel is carried out western blotting, use anti--pIII antibody to survey and in X-ray sheet exposure (Fig. 6).Xia Mian band is equivalent to independent pIII protein in each case, and the band of higher position comprises the pIII-scFv fusion rotein.Use software package NIH image to the quantificational expression of band strength pre--amount of selecting to cause being present in the fusion rotein in the phage increases by 11.8 times.In fact, in advance-phage selected in 43% among total pIII exist as the pIII-scFv syzygy, thereby point out most of phage particle to have at least a scFv that shows from the teeth outwards.

Therefore, not only use general part pre--select can be from repertoire the enriched with functional member, and cause on phage surface, to cause high-caliber displaying, but not by the bacterioprotein enzymatic lysis to the preferential selection of those members who gives full expression to and (if desired).

Advantageously can select not match or loose paired from the single variable domain of antibody of antibody polypeptides types of populations.Can also select not match or loose paired from the T-cell receptors structural domain (V of T-cell receptors polypeptide types of populations _αOr V _β).The present invention has satisfied these demands.

All embodiments of the present invention described herein and aspect in, wherein mentioned target ligands, this target ligands is selected from any one in the target listed in TNF α serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP12, PDK1 and amyloid beta (A-β) or the appendix 1.

With reference to WO05044858A1, WO04062551A2, WO04041867A2, WO04041865A2, WO04041863A2, relevant Camelid VHH structural domain and Nanobodies among the WO04041862A2, WO03050531A2 and EP0656946 ^TMDescription.Especially with VHH and Nanobodies ^TMThese disclosure contents and the sequence and the example of definition and disclosure be incorporated herein by reference.

With regard to method of the present invention, in one embodiment, polypeptide class (for example antibody polypeptides class) colony is provided by B cell (for example peripheral blood lymphocyte), wherein the B-cell mass is provided in a plurality of holes or the reservoir, each hole or reservoir comprise single B-cell type or on average comprise a kind of B-cell type.With reference to (1997) j.Immunol.Methods 2073 such as de Wildt, (1996) Proc.Natl.Acad.Sci.USA 93 such as 61-67 and Babcook, relevant disclosure content how to control B cell mean value among the 7843-7848.These reference intactly are incorporated herein by reference.

The present invention for example selects the concrete application from the B cell mass of Camelids

Selective system is provided among the present invention in one aspect, it can eliminate antigen-specific b-cell (as subgroup) (or significantly reducing their ratio), described antigen-specific b-cell (as subgroup) can not show preferred antibody type, and those show the antigen-specific b-cell (as subgroup) of preferred antibody type really enrichment simultaneously.Use general part to carry out this enriching method, described general part is protein or little chemical molecular, and they are to being common in all the antigen-specific b that shows preferred antibody type really-cells and being not to be common in all antigen-specific bs-cells that do not show the preferred antibody type to have avidity.For example, general part can the antagonist structural domain or antibody surface spot have avidity, described antibody structure territory or antibody surface spot are common in all the antigen-specific b that shows the preferred antibody type really-cells and are not to be common in all the antigen-specific b that does not show the preferred antibody type-cells.Obviously, before selecting antigen-binding activity, in the process or use the selection of general part afterwards, final result is to express the antigen-specific b-cell subsets of the antibody with required type.

In the context of the relevant Camelidae B-cell that shows antibody commonly used or heavy chain antibody, can estimate two kinds of methods of one of two kinds of subgroups of enrichment:

In first kind of means, the superantigen protein G is in the present invention as general part.Protein G is in conjunction with the CH1 constant domain in the heavy chain.Verified (Hamers-Casterman etc. (1993) Nature, 363,446-448) heavy chain antibody of larger proportion can conjugated protein G (not can conjugated protein A) yet, and this species diversity is used for separating camel antibody commonly used by the chromatogram mode from heavy chain camel antibody when analyzing animal serum.Obviously, the disappearance in the CH1 structural domain in the heavy chain antibody causes not existing in conjunction with immobilized protein G.In the present invention, protein G (preferably being fixed on the solid support) can be used for the B-cell (these cell conjugated proteins G) of enrichment expression antibody commonly used or the B-cell (these cell debond protein Gs) that heavy chain antibody is expressed in enrichment.Perhaps, can use fluorescence dye (such as fluorescein, Cy3, Cy5, Texas is red etc.) labelled protein G and hatch with the B-cell mass.Can from the B-cell of expressing heavy chain antibody, separate the B-cell of expressing antibody commonly used by the different subgroups of cell sorter (FACS) sorting of using fluorescent activation then.Obviously, can use monoclonal antibody that Camelidae antibody CH1 structural domain is increased or polyclonal antiserum but not protein G.By expanding this means, monoclonal antibody or polyclonal antiserum that antagonist light chain (light chain variable territory, light chain constant domain or they both) increases also can separate the B-cell subsets of expressing the conventional structure territory from the B-cell subsets of expressing heavy chain antibody.

In second kind of means, the light chain variable territory is useful in the present invention.Described in preface part of the present invention, in antibody commonly used, find conduct and the variable light chain structural domain of heavy chain variable domain paired all the time.On the contrary, heavy chain antibody does not have the light chain variable territory.Therefore, on the one hand-VL binding site on antibody commonly used-VH structural domain is occupied by the VL structural domain, and in heavy chain antibody, the VL binding site on the VHH structural domain is not still occupied.The VHH variable domain of Camelidae heavy chain prevents to obtain on the VL bonded VL-binding site thinking.Yet, it is worthy of note at the VHH structural domain, these sudden changes (37,44,45 with 47 on-Kabat numbering) be not identical all the time and the more important thing is, be not to be present in simultaneously on all positions all the time.Although this mark of this variability degree prompting is to the biophysics characteristic, such as solubleness in solution and free state is useful, but it can advantageously be substituted by further feature, such as long CDR3, the sudden change on W103 position (Kabat numbering) and also have amino acid composition in the CDRs.The example of this class camel VHH sequence can find (table 1 of WO2004041862,4,5,6 in patent; WO 2004041865-table 4; WO 2004041863-table 4,5,7,8).Therefore, the camelid heavy chain variable domain of larger proportion provides and similar above-mentioned VL-interface, heavy chain of antibody variable domain VL-interface commonly used.This has just produced as drawing a conclusion: can separate this class B-cell subsets from the B-cell of expressing antibody commonly used by the VL structural domain of contact isolating VL structural domain of immobilization (from Camelidae or from other Mammals) or dye marker.VL and VH structural domain are miscellaneous according to its combinatorial property, can by chain reorganization make affinity matured antibody commonly used (Marks etc. (1992) Biotechnology (NY) 10,779-783).Therefore, this class is selected to find that the possibility in miscellaneous light chain variable territory is good relatively.Be lower than the interaction of VL structural domain and VH structural domain commonly used although it shall yet further be noted that the monomer interaction of VL structural domain and monomer heavy chain VHH, the selection scheme that proposes can be alleviated this problem.In fact, by fixing VL structural domain with by making it contact the B-cell, bigger avidity effect meeting occurs because of show many identical antibody copies on the B-cell surface.

A significant embodiment of the present invention is to use the VL structural domain also can distinguish between antibody commonly used dissimilar on the B-cell surface.The VL structural domain can change to the avidity of VH structural domain that (dissociation constant is 10 ^-9M-10 ^-6M-is by Pl ü ckthun (1992) Immunol.Rev.130, and 151-188 summarizes).Therefore, can estimate, even in antibody commonly used, also have VL and the VH structural domain paired subgroup that has in various degree.By select to show the B-cell of antibody commonly used with immobilization VL structural domain, can separate the B-cell that those express antibody commonly used, wherein a little less than the pairing between interior VL of antibody and the VH structural domain, and immobilization VL structural domain is excessive in a large number, VH structural domain and the pairing of immobilization VL structural domain can be promoted, cause the B-cell fixation thus.This means are meaningful especially, because it helps to select to express to have the very B-cell subsets of the antibody commonly used of miscellaneous VH structural domain, this has importance when thinking single variable domain format conversion.

I. use the selection of light chain of antibody variable domain

This provides and is used for selecting method in conjunction with the functional variable domain colony of target ligands and general part from the repertoire of antibody polypeptides class an embodiment in the present invention, described general part can be in conjunction with the functional member in the repertoire, and irrelevant with the target ligands specificity, this method comprises the following steps:

Wherein variable domain is that heavy chain variable domain and general part are the light chain of antibody variable domain.

The heavy chain variable domain that the present invention pays close attention to selection may reside in IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv and disulfide bonding promptly has at least a heavy chain and light chain variable territory paired antibody or the antibody fragment.Do not wish to be subjected to any theory constraint, think in some example of these paired, variable domain is paired into loose complementary, and promptly structural domain can be only be connected antigen or mainly be connected one of variable domain in conjunction with main by antigen, but is not to be connected in the pairing another to any outstanding degree.For example, in some VH/VL pairing, for example in some people or Camelid 4-chain IgG, that heavy chain variable domain can provide is main/unique with antigenic in conjunction with contacting.Therefore, the application has from antibody polypeptides colony the antibody selected in this specification sheets or the application of antibody fragment, because the light chain variable territory that is used for step a) is in conjunction with at least a antibody or fragment (IgG, Fab with loose VH/VL complementarity, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv or disulfide bonding) and the VH that provides.This provides the useful mode of selecting VH, and described VH can be used as single variable domain (dAb or Nanobody ^TM) diagnosis, treat and/or prevent product or research and develop their starting point.Antibody or the antibody fragment self selected can have the purposes of this series products, for example find antigen, and wherein " survival ability or loose VH/VL pairing may be favourable.Therefore, for example, the present invention is used for carrying out this class and selects as this class from the ideal IgG of the subgroup of IgG colony, and described IgG colony is camel or human IgG or humanization or chimeric IgG colony for example.As the extension of this notion, this paper can be used to select antibody or antibody fragment (IgG for example, Fab, Fab ', F (ab) in the present invention described in I and the II part ₂, F (ab ') ₂Bispecific antibody, scFv matches dimer), it comprises as WO03002609A2, the dual specific part that discloses in any one piece among WO04003019A2 and the WO04058821A2, wherein this dual specific part has at least a VH/VL or VH/VH or VL/VL pairing, and wherein the variable domain in the pairing is separately in conjunction with corresponding antigen.Antigen type can be identical (for example all in conjunction with TNF α, vWF, any other target antigen that the VH of serum albumin and VL or this paper disclose) or variable domain can be in conjunction with different antigen types, for example in conjunction with the antigen of serum albumin and other TNF α or other target antigen arbitrarily.

In one embodiment, the antibody polypeptides types of populations is the single variable domain of an antibody colony.Therefore, this colony can be Camelid or human antibody heavy chain's variable domain colony.In one embodiment, it is VHH structural domain or Nanobodies ^TMColony.

Optional step b) heavy chain variable domain in is Camelid variable domain (VHH), Nanobodies ^TMOr derive from Camelid heavy chain antibody (H2 antibody); Or optional heavy chain variable domain is people's variable domain separately or derives from the people.

The preferred repertoire of antibody polypeptides class that at first makes contacts target ligands and contacts general part then.Perhaps, make the antibody polypeptides class contact general part and contact target ligands then.

Preferred general part is in conjunction with the variable domain subgroup in the repertoire.In an example, two or more subgroups of heavy chain variable domain are selected from the repertoire of polypeptide class.Selection in this case can be used two or more general parts, and promptly carry out in two or more different light chain variable territories.Preferably after selection, merge two or more subgroups, according to the present invention the light chain variable territory is selected then so that produce the extra repertoire of polypeptide class.

In one embodiment, make two or more repertoires in the polypeptide class contact general part (identical or different general part) and merge thus obtained polypeptide class subgroup then.

The method of selecting at least a heavy chain of antibody variable domain from the antibody polypeptides types of populations is provided in another aspect of the present invention, and this method comprises:

The same with above-mentioned embodiment, pay close attention to the heavy chain variable domain of selecting and may reside in IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv and disulfide bonding promptly has at least a heavy chain and light chain variable territory paired antibody or the antibody fragment.Above-mentioned about from loose VH/VL pairing, selecting the discussion of VH also to be fit to herein.

Preferably before step a), exist to make antibody polypeptides class contact target ligands and selection, the described antibody polypeptides types of populations that is used for step a) is provided thus in conjunction with the step of the antibody polypeptides class of target ligands.

Preferably after step b), exist to make heavy chain of antibody variable domain contact target ligands of selecting in the step b) and the step of selecting in conjunction with the heavy chain variable domain of target ligands.

Preferred steps b) the heavy chain structural domain of selecting in is separately from organizing down: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.Numbering according to the Kabat coding rule carry out (Kabat etc., 1991, Sequences of Immunological Interest, 5 ^ThEd.U.S.Dept.Health ﹠amp; People Services, Washington, D.C.).

Preferred steps b) the heavy chain structural domain of selecting in is humanization Camelid or mouse heavy chain variable domain or humanization Nanobody separately ^TM

Preferred steps b) the heavy chain structural domain of selecting in is people's heavy chain variable domain separately; Derive from people's heavy chain variable domain; With humanization heavy chain variable domain (for example humanization Camelid or mouse variable domain).

Preferred light chain variable territory behaviour light chain variable territory; Derive from the people; Light chain variable territory with FW2 sequence, described FW2 sequence is that the FW2 of gene order DPK9 coding is identical with planting; Or has a light chain structural domain (for example Camelid-deutero-VL) of people interface region (promptly usually and the interfacial district of people VH/VL paired VH structural domain).In another example, the light chain variable territory is Camelid light chain variable territory or derives from Camelid.

The colony of preferred steps in a) is by the B-cell mass, and for example peripheral blood lymphocyte provides.In an example, the B-cellular segregation is from the Mammals of immunization target antigen (for example mouse or Camelid, for example Llama or camel).In another example, the B-cellular segregation is from the Mammals of immunization target antigen (for example mouse or Camelid, for example Llama or camel) not as yet.

In a kind of selection, the colony that uses in the step a) is provided by the repertoire of the antibody polypeptides class of the antibody gene coding of resetting by synthesis mode.

In one embodiment, the colony that uses in the step a) is provided by the phage display library that comprises the phage of showing described antibody polypeptides class.The example in this class library is disclosed among the WO99/20749.Also with reference to WO04003019A2, WO05044858A1, WO04062551A2, WO04041867A2, the example of relevant phage display library among the WO04041865A2, WO04041863A2 and WO04041862A2.

In one embodiment, the colony that is to use in the step a) comprises: (i) each self-contained not with the antibody polypeptides class of at least a heavy chain variable domain of light chain variable territory paired; (ii) each self-contained and light chain variable territory paired antibody polypeptides class.Therefore, for example, method of the present invention is used for selecting the single variable domain of antibody (being unpaired V structural domain) from for example also comprising the population mixture of the pairing V structural domain of IgG form.Therefore, the present invention is applied to select the single variable domain of VHH from the population mixture (for example by the B-cell, providing such as peripheral blood lymphocyte) that also comprises Camelid 4-chain IgG (having paired VH/VL structural domain).Similarly, there is the application of from the population mixture that also comprises people 4-chain IgG, selecting people VH.If select single variable domain and IgG, wherein VH/VL is paired into as mentioned above " great-hearted ", after step b), can there be extra step so, wherein from the IgG that selects, separates single variable domain (for example based on size or by any other routine techniques).

Can separate the nucleotide sequence of the single variable domain that the coding that uses any embodiment of the present invention selects and insert the expression vector of expressing variable domain from B-cell or phage (or yeast or other is used for connecting the system in phenotype and genotypic library arbitrarily).Optional can make nucleotide sequence sudden change (for example in CDR3 and/or FW, introducing one or more sudden changes) and/or operationally with one or more antibody structure territories (for example another kind of single variable domain), the antibody Fc structural domain, mark or effect group connect, and after this express from expression vector.

The colony that is preferred for step a) comprises single variable domain of camelid heavy chain (VHH) or Nanobodies ^TM

The colony that is preferred for step a) comprises the single variable domain of people's heavy chain (VH).

In an especially preferred embodiment, the method of selecting at least a Camelid antibody VHH structural domain from Camelid antibody polypeptides types of populations is provided, the B-cell of the Camelid of immunization target antigen provides described Camelid antibody polypeptides class by separating certainly, and this method comprises:

B) select at least a VHH structural domain in conjunction with the light chain variable structural domain.

In this embodiment preferred, preferred light chain variable territory behaviour light chain variable territory.

In this embodiment preferred, preferably the B-cell is provided in a plurality of holes or the reservoir, wherein on average comprise a kind of B-cell type in each hole or the reservoir.

The present invention also provides method, comprises: the initial colony that (i) uses target antigen antagonist polypeptide class carries out SLAM (the lymphocyte antibody method of selection) so that select antibody polypeptides types of populations in conjunction with this target antigen; (ii) use the colony of selecting as the antibody polypeptides types of populations that is used for the inventive method step a).

In the method for the invention, in step b), at least a and protein portion in the heavy chain of antibody variable domain of selection is merged or put together with it.Preferred protein partly is selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

The present invention also provides and has comprised the heavy chain of antibody variable domain or by its isolated antibody polypeptide of forming, wherein polypeptide can be by comprising step a) and b) method obtain, wherein light chain variable territory behaviour light chain variable territory in this method and heavy chain variable domain are from inhuman Mammals, for example Camelid.Preferred heavy chain variable domain is from organizing down: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.

Preferably heavy chain variable domain is provided as Camelid IgG or the integral part that derives from the IgG of Camelid.

Preferably heavy chain variable domain is provided as human IgG or the integral part that derives from people's IgG, and wherein make heavy chain variable domain in IgG with the light chain variable territory pairing that is different from the light chain variable territory that this method steps uses in a).

The present invention also provides the application of this antibody-like polypeptide as medicine.

The present invention also provides the application in human disease or illness treat and/or prevent of this antibody-like polypeptide.Accommodable illness and disease are disclosed in WO04003019A2, WO05044858A1, and WO04062551A2, WO04041867A2, WO04041865A2 among WO04041863A2 and the WO04041862A2, as sends, administration and preparation approach.The disclosure content that all these are concrete is introduced this specification sheets clearly as being applied to suitable example reference of the present invention.

II. use the selection of heavy chain of antibody variable domain

In one embodiment, the invention provides from the repertoire of antibody polypeptides class the method for selecting in conjunction with the functional variable domain colony of target ligands and general part, described general part can be in conjunction with the functional member in the repertoire, and irrelevant with the target ligands specificity, this method comprises the following steps:

B) make the functional variable domain contact target ligands of selection and select variable domain colony in conjunction with target ligands;

Wherein variable domain is that light chain variable territory and general part are the heavy chain of antibody variable domain.

The light chain variable territory that the present invention pays close attention to selection may reside in IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv and disulfide bonding promptly has at least a heavy chain and light chain variable territory paired antibody or the antibody fragment.Do not wish to be subjected to any theory constraint, think in some example of these paired, variable domain is paired into loose complementary, and promptly structural domain can be only be connected antigen or mainly be connected one of variable domain in conjunction with main by antigen, but is not to be connected in the pairing another to any outstanding degree.For example, in some VH/VL pairing, for example in some people or Camelid 4-chain IgG, that the light chain variable territory can provide is main/unique with antigenic in conjunction with contacting.Therefore, the application has from antibody polypeptides colony the antibody selected in this specification sheets or the application of antibody fragment, because the heavy chain variable domain that is used for step a) is in conjunction with at least a antibody or fragment (IgG, Fab with loose VH/VL complementarity, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv or disulfide bonding) and the VL that provides.This provides the useful mode of selecting VL, and described VL can be used as single variable domain (dAb or Nanobody ^TM) diagnosis, treat and/or prevent product or research and develop their starting point.Antibody or the antibody fragment self selected can have the purposes of this series products, for example find antigen, and wherein " vigor or loose VH/VL pairing may be favourable.Therefore, for example, the present invention is used for carrying out this class and selects as this class from the ideal IgG of the subgroup of IgG colony, and described IgG colony is camel or human IgG or humanization or chimeric IgG colony for example.As the extension of this notion, this paper can be used to select antibody or antibody fragment (IgG for example, Fab, Fab ', F (ab) in the present invention described in I and the II part ₂, F (ab ') ₂Bispecific antibody, scFv matches dimer), it comprises as WO03002609A2, the dual specific part that discloses in any one piece among WO04003019A2 and the WO04058821A2, wherein this dual specific part has at least a VH/VL or VH/VH or VL/VL pairing, and wherein the variable domain in the pairing is separately in conjunction with corresponding antigen.Antigen type can be identical (for example all in conjunction with TNF α, vWF, any other target antigen that the VH of serum albumin and VL or this paper disclose) or variable domain can be in conjunction with different antigen types, for example in conjunction with the antigen of serum albumin and other TNF α or other target antigen arbitrarily.

In one embodiment, the antibody polypeptides types of populations is the single variable domain of an antibody colony.Therefore, this colony can be Camelid or human antibody light chain variable domain colony.

Optional step b) the light chain variable territory in is Camelid light chain variable territory (VHH) or derives from Camelid; Or optional light chain variable territory is people's variable domain separately or derives from the people.

Preferred general part is in conjunction with the variable domain subgroup in the repertoire.In an example, two or more subgroups of heavy chain variable domain are selected from the repertoire of polypeptide class.Selection in this case can be used two or more general parts, and promptly two or more different heavy chain variable domains carry out.Preferably after selection, merge two or more subgroups, according to the present invention heavy chain variable domain is selected then so that produce the extra repertoire of polypeptide class.

The method of selecting at least a light chain of antibody variable domain from the antibody polypeptides types of populations is provided in another aspect of the present invention, and this method comprises:

The same with above-mentioned embodiment, pay close attention to the light chain variable territory of selecting and may reside in IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, the Fv of Fv and disulfide bonding promptly has at least a heavy chain and light chain variable territory paired antibody or the antibody fragment.Above-mentioned about from loose VH/VL pairing, selecting the discussion of VL also to be fit to herein.

Preferably after step b), exist to make light chain of antibody variable domain contact target ligands of selecting in the step b) and the step of selecting in conjunction with the light chain variable territory of target ligands.

Preferred steps b) the light chain structural domain of selecting in is humanization Camelid or mouse light chain variable territory.

Preferred steps b) the light chain structural domain of selecting in is people's light chain variable territory separately; Derive from people's light chain variable territory; Or humanization light chain variable territory (for example humanization Camelid or mouse variable domain).

Preferred heavy chain variable domain behaviour heavy chain variable domain; Derive from the people; Light chain variable territory with FW2 sequence, described FW2 sequence is that the FW2 of gene order DP47 coding is identical with planting; Or to have and plant be 44,45 44,45 and 47 heavy chain variable domains identical with 47 of gene order DP47 coding; Or has a heavy chain structural domain (for example Camelid-deutero-VH) of people interface region (promptly usually with the interfacial district of VL structural domain of people VH/VL pairing weight).Heavy at another example, heavy chain variable domain is the Camelid heavy chain variable domain or derives from Camelid.

In one embodiment, the colony that is to use in the step a) comprises: (i) each self-contained not with the antibody polypeptides class at least a light chain variable of heavy chain variable domain paired territory; (ii) each self-contained and antibody polypeptides class light chain variable territory paired heavy chain variable domain.Therefore, for example, method of the present invention is used for selecting the single variable domain of antibody (being unpaired V structural domain) from for example also comprising the population mixture of the pairing V structural domain of IgG form.Therefore, the present invention is applied to select the single variable domain of VL from the population mixture (for example by the B-cell, providing such as peripheral blood lymphocyte) that also comprises Camelid 4-chain IgG (having paired VH/VL structural domain).If select single variable domain and IgG, wherein VH/VL is paired into as mentioned above " great-hearted ", after step b), can there be extra step so, wherein from the IgG that selects, separates single variable domain (for example based on size or by any other routine techniques).

Preferred steps a) the middle colony that uses comprises the single variable domain of camelid light chain.

In the method for the invention, in step b), at least a and protein portion in the light chain of antibody variable domain of selection is merged or put together with it.Preferred protein partly is selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

Preferably in step b), at least a in the light chain of antibody variable domain of selection is antibody moiety or antibody fragment, and it is selected from IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv and disulfide bonding Fv.

The present invention also provides and has comprised the light chain of antibody variable domain or by its isolated antibody polypeptide of forming, wherein polypeptide can be by comprising step a) and b) method obtain, wherein heavy chain variable domain behaviour heavy chain variable domain in this method and heavy chain variable domain are from inhuman Mammals, for example Camelid.Preferred light chain variable territory derives from Camelid.Preferably the light chain variable territory is provided as Camelid IgG or the IgG integral part that derives from Camelid.

Preferably the light chain variable territory is provided as human IgG or the integral part that derives from people's IgG, and wherein make the light chain variable territory in IgG with the heavy chain variable domain pairing that is different from the heavy chain variable domain that this method steps uses in a).

The present invention also provides the application in the treating and/or preventing of human disease or illness of this antibody-like polypeptide.The present invention also provides the application in human disease or illness treat and/or prevent of this antibody-like polypeptide.Accommodable illness and disease are disclosed in WO04003019A2, WO05044858A1, and WO04062551A2, WO04041867A2, WO04041865A2 among WO04041863A2 and the WO04041862A2, as sends, administration and preparation approach.The disclosure content that all these are concrete is introduced this specification sheets clearly as being applied to suitable example reference of the present invention.

In one embodiment, select heavy chain of antibody variable domain colony, and select the light chain of antibody variable domain, and merge thus obtained colony then according to method listed in the II part according to method listed in the I part.

III. use the selection of T-cell receptors structural domain

The invention provides from the repertoire of polypeptide class the method for selecting in conjunction with the functional T-cell receptors structural domain colony of target ligands and general part, described general part can be in conjunction with the functional member in the repertoire, and irrelevant with the specificity of target ligands, this method comprises the following steps:

Wherein a) the T-cell receptors structural domain is V _αStructural domain and general part are T-cell receptors V _βStructural domain; Or b) T-cellularstructure territory is T-cell receptors V _βStructural domain and general part are T-cell receptors V _αStructural domain.

Choose in a) T-cell receptors V wantonly _αStructural domain is the Camelid structural domain that derives from Camelid; Or choose wantonly a) and b) in, the T-cell receptors structural domain is people's structural domain or derive from the people separately.

Preferably select T-cell receptors V according to this method _αStructural domain colony and select T-cell receptors V according to this method _βStructural domain, and merge thus obtained colony then.

Other embodiment

Embodiment A 1: the variable domain of in two biological elutriation circulations, from the Llama of immunization, separating antigen-specificity heavy chain antibody

The 0th, 7,14,21,28,35, give 1 adult Llama injection 1mg people's Toxoid,tetanus (TT) in the time of 42,49 and 54 days.Before per injection, gather serum, take place subsequently immunogenic immunne response.From the immunization animal, gather anticoagulation (150ml) and use Unisep (WAK Chemie, Germany) preparation peripheral blood lymphocyte (PBLs).

Use the protein G bag by aseptic ELISA flat board (4 ℃ down 10 μ g/ml spend the night), then with aseptic PBS washing, the FCS (foetal calf serum) that exhausts with aseptic PBS-10% IgG-seals and and then washs with PBS.

Use the TT bag by aseptic ELISA flat board (4 ℃ down 10 μ g/ml spend the night), then with aseptic PBS washing, the FCS (foetal calf serum) that exhausts with aseptic PBS-10%IgG-seals and and then washs with PBS.

At first the PBLs (in PBS) of purifying is joined in the hole of protein G-Bao quilt and make its at 37 ℃ down in conjunction with at least 1 hour.Unconjugated cell (mainly show heavy chain antibody or do not have antibody) and merging in the careful taking-up supernatant liquor from each hole of protein G-Bao quilt.

The cell mass of expressing enrichment in the B-cell of heavy chain is joined in the hole (with the density of 300 cells/well and 1,500 cells/well) of TT-bag quilt and make its at 37 ℃ down in conjunction with at least 1 hour.By removing unconjugated cell in the supernatant liquor 10 times with the substratum washing.Envelope antigen is being arranged, T cell conditioned medium (3%) and EL-4 cell (5 * 10 ⁴/ hole) exists down the cell cultures of antigen expressed-specificity heavy chain antibody 7 days.

The positive hole that antibodies in the hole of the fresh TT-bag of the elisa assay quilt by using a-protein-horseradish peroxidase conjugate and tmb substrate in the culture supernatants of small volume is identified secretion antigen-specific antibody.

Select positive B-cell hole to be used for further processing: to make the B-cell precipitation, remove supernatant liquor and cell precipitation is suspended in 10 μ l fresh cultures (DMEM or the RPMI that contain the 1-6%T-cell conditioned medium) again.Get the PCR that aliquot (each 2 μ l) is used to use MJ ResearchRobus RT-PCR test kit (Catalogue No.F-580L), wherein use following mixture/pipe to separate heavy chain variable domain:

DEPC water 35.5 μ l

10x damping fluid 5 μ l

dNTPS 1μl

10％NP-40 2.5μl

RNAsin 0.5μl

RT 1μl

Polysaccharase 2 μ l

Primer mixture ( ^*) 1 μ l (each 10 μ M)

MgCl2 1.5

The PCR program: 50 ℃ following 30 minutes, subsequently 94 ℃ following 2 minutes and be 40 circulations of [94 ℃/1 minute, 55 ℃/1 minute, 72 ℃/1 minute] then.Tissue stretches: 72 ℃/5 minutes.

^*: this mixture comprises following two kinds of primers: the FR1 primer of widow-dT primer and single sex change: 5 '-GAGGTBCARCTGCAGGASTCYGG-3 ', its coding PstI restriction site.

With PstI and BstEII cutting 1,300bp fragment.A kind of enzyme in back cuts in the dna fragmentation of the framework 4 of encoding heavy chain antibody usually.

In electrophoresis and UV irradiation back from sepharose separating digesting product and be connected into the corresponding site of cloning vector, described cloning vector can be bred intestinal bacteria under the selectivity microbiotic condition that is used for growing.

Connection carrier is used for the transformed into escherichia coli cell and PstI/BstEII is inserted sequencing fragment so that disclose the dna sequence dna of heavy chain variable domain.

Embodiment B: the variable domain of from the Llama of immunization, separating antigen-specificity heavy chain antibody by biological elutriation and flow cytometry

Use VL structural domain VL structural domain ((2005) J.Mol Biol.350 such as Conrath for example, the described VL structural domain of 112-25) bag is by aseptic ELISA flat board (10 μ g/ml spend the night under 4 ℃), then with aseptic PBS washing, the FCS (foetal calf serum) that exhausts with aseptic PBS-10%IgG-seals and and then washs with PBS.

Use and use texas Red or fluorescein isothiocyanate (FITC) the mark Toxoid,tetanus that the lysine residue that exposes is had specific N-hydroxy-succinamide base (NHS).Remove excessive fluorescent mark by the TT that makes mark by PD10 post (Amersham Biosciences).Use the degree of texas Red or FITC mark by mass spectrum (MALDI-TOF) evaluation.

At first at room temperature the PBLs of purifying is hatched 30 minutes (with regard to 10 with the TT of mark ⁶Individual cell uses the TT of 1ug mark).Optional step: by with centrifugal 5 minutes sedimentation cells of 200g and be suspended in again among the 0.5ml PBS.Then with the TT-positive cell in flow cytometer (for example from Coulter, Florida, the Coulter EpicsElite flow cytometer of the USA) sorting cells that the automated cell storage means has been installed.All TT-bonded positive cells are collected in the single reservoir.

In this stage, conventional antibody or heavy chain antibody on antigen-specific b-cell expressing cell surface.The following further separation of carrying out the B-cell subsets of antigen expressed-specificity heavy chain antibody.

The cell mass of enrichment in the B-cell that merges is joined in the hole of protein L-bag quilt and make its at 37 ℃ down in conjunction with at least 1 hour.By remove unconjugated cell in the supernatant liquor (the main displaying used antibody always or do not have antibody) for 10 times with the substratum washing.Take out bonded cell (antigen expressed-specificity heavy chain antibody), count and be used for the hole (5 * 10 of inoculating fresh EL4-B5 T-cell envelope in the presence of the T cell conditioned medium (3%) with the inoculative proportion of 0.3 cells/well having ⁴/ hole).With cell cultures 7 days, the positive hole that the antibodies in the culture supernatants of small volume is identified secretion antigen-specificity heavy chain antibody in the hole of the fresh TT-bag of the elisa assay quilt by using a-protein-horseradish peroxidase conjugate and tmb substrate after this.

DEPC water 35.5 μ l

10x damping fluid 5 μ l

dNTPS 1μl

10％NP-40 2.5μl

RNAsin 0.5μl

RT 1μl

Polysaccharase 2 μ l

Primer mixture ( ^*) 1 μ l (each 10 μ M)

MgCl2 1.5

Appendix 1

A) cytokine, cytokine receptor, enzymes etc. comprise cytokine, cytokine receptor, enzyme, enzyme cofactor or DNA are conjugated protein.Suitable cytokine and somatomedin include, but are not limited to: ApoE, Apo-SAA, BDNF, heart nutrient substance-1, EGF, EGF acceptor, ENA-78, the eotaxin, eotaxin-2, Exodus-2, FGF-acidity, FGF-alkalescence, fibroblast growth factor-10, FLT3 part, chemotactic bone molecule (CX3C), GDNF, G-CSF, GM-CSF, GF-β 1, Regular Insulin, IFN-γ, IGF-I, IGF-II, IL-1 α, IL-1 β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), statin α, statin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF, Leptin, LIF, the lymphocyte chemotactic protein, mullerian inhibitory substance, monocyte colony inhibition factor, the monocyte bait protein, M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1 α, MIP-1 β, MIP-3 α, MIP-3 β, MIP-4, marrow progenitor cell supressor-1 (MPIF-1), NAP-2, Neurturin, nerve growth factor, β-NGF, NT-3, NT-4, oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1 α, SDF1 β, SCF, SCGF, STEM CELL FACTOR (SCF), TARC, TACE recognition site, TGF-α, TGF-β, TGF-β 2, TGF-β 3, tumour necrosis factor (TNF), TNF-α, TNF-β, TNF acceptor I (p55), TNF receptor II, TNIL-1, TPO, VEGF, vegf receptor 1, vegf receptor 2, vegf receptor 3, GCP-2, GRO/MGSA, GRO-β, GRO-γ, HCC1,1-309, HER 1, and HER 2, HER 3 and HER 4.Cytokine receptor comprises above-mentioned cytokine acceptor separately, IL-1R for example, IL-6R, IL-10R, IL-18R etc.Be appreciated that the limit absolutely not of this inventory.The target of the single variable domain polypeptide of the preferred antigen of the present invention class be disclosed among the WO04/041867 (with the contents intact of the document be incorporated herein by reference), and include, but are not limited to TNF α, IgE, IFN γ, MMP-12, EGFR, CEA, helicobacter pylori (H.pylori), TB, influenza, PDK-1, GSK1, Bad, Caspase, Forkhead and Feng's von willebrand's factor (vWF).Target can also be the fragment of above-mentioned target.Therefore, target is also for causing the fragment of the above-mentioned target that immunity is answered.Target also is can be in conjunction with the fragment of the above-mentioned target of the single variable domain polypeptide of antibody that the total length target is increased.

B) can increase the antigen (part of back is, for example treatment or imaging protein portion, for example antibody or antibody fragment, antibody variable domains for example is such as people VH or VL structural domain or Camelid VHH) of partial half-life.

α-1 glycoprotein (seromucoid) (AAG)

α-1 chymotrypsin inhibitor (Antichyromotrypsin) (ACT)

α-1 antitrypsin (AAT)

α-1 microglobulin (protein HC) (AIM)

α-2 macroglobulin (A2M)

Antithrombin III (AT III)

APoA-I (Apo A-1)

Apolipoprotein B (Apo B)

Beta-2-microglobulin (B2M)

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) [Lpa]]

Seminose-conjugated protein (MBP)

Myosin (Myo)

Profibrinolysin (PSM)

Prealbumin (transthyretin) (PAL)

Vogan-Neu-conjugated protein (RBP)

The Rheomatoid factor (RF)

Serum amyloid A protein (SAA)

Solubility transferrin (Tranferrin) acceptor (sTfR)

Transferrin (Tf)

G: the antigen that can increase the part transformation period

From the protein of extracellular matrix, for example collagen protein, ln, integrin and fibronectin.Collagen protein is the main protein in the extracellular matrix.The present collagen molecules of known about 15 types of in the different sites of health, finding, the type i collagen albumen of for example in bone, skin, tendon, ligament, cornea, internal organs, finding (account for health collagen protein 90%) or the II collagen type of in the vitreous humor of cartilage, intervertebral disk, notochord, eye, finding.

The protein of finding in blood comprises:

Plasma proteins, such as fibrin, α-2 macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein a-protein, heptaglobulin, protein, ubiquitin, Clara cell 10kDa protein and p-2-microglobulin;

Enzyme and inhibitor, such as Profibrinolysin, N,O-Diacetylmuramidase, Guang proteinase inhibitor C, α-1-antitrypsin and pancreas kypsin inhibitor.Profibrinolysin is the inactive precursor of trypsinase-sample 2s serine protease Tryptase.It circulates general discovery by blood flow.When Profibrinolysin becomes activation and changes into Tryptase, its not folding effective enzymatic structure territory, the fibrin protofibril of hemocyte in the clot is mixed in this structural domain dissolving.This is called fibrinolysis;

Immune system protein, such as IgE, IgG, IgM;

Translocator, such as retinol conjugated protein, the o-1 microglobulin;

Alexin, such as beta-alexin 1,

neutrophilic granulocyte alexin

1,2 and 3;

The protein of on hemato encephalic barrier or nervous tissue, finding, such as melanocortin receptor, myelin, ascorbate salt translocator;

Transferrin receptor ligands specific-neurologic agent promoting agent fusion rotein (referring to US5977307);

The brain capillary endothelial cell acceptor, transferrin receptor, Regular Insulin, Regular Insulin-like growth factor 1 (IGF 1) acceptor, Regular Insulin-like growth factor 2 (IGF 2) acceptor, insulin receptor;

Be positioned at the protein of kidney, such as many capsules albumen, IV class collagen protein, organic anion transferrin K1, Heymann antigen.

Be positioned at the protein of liver, alcoholdehydrogenase for example, G250;

Factor X;

Alpha1 Anti-trypsin;

HNF 1α；

Be positioned at the protein of lung, such as secretory component (in conjunction with IgA);

Be positioned at the protein of heart, for example HSP 27, and it is relevant with dilated cardiomyopathy;

Be positioned at the protein of skin, for example Keratin sulfate;

The bone specific proteins, such as Delicious peptide (BMPs), it is the transforming growth factor subgroup that is shown as bone active, example comprises that BMP-2 ,-4 ,-5 ,-6 ,-7 (is also referred to as bone morphogenic protein (OP-1) and-8 (OP-2);

Tumour-specific albumen comprises people's TA, Trastuzumab acceptor, estradiol receptor, kethepsin, for example cathepsin B's (finding in liver and spleen);

Disease-specific proteins, such as the antigen of only expressing on activated T-cell: comprise LAG-3 (lymphocyte activation gene), osteoprotegerin part (OPGL) is referring to Nature 402,304-309; 1999, OX40 (TNF receptor family member is expressing on the activated T cell and the known T cellular elements specificity incremental adjustments in the cell of generation human T-cell leukemia virus's I type (HTLV-I) that only stimulates altogether) is referring to J Immunol.2000 Jul 1; 165 (1): 263-70; Metalloprotease (relevant with sacroiliitis/cancer) comprises CG6512 Drosophila (Drosophila), people's paraplegia albumen, people FtsH, people AFG3L2, mouse ftsH; Generate the vessel growth factor, comprise acid fibroblast growth factor (FGF-1), Prostatropin (FGF-2), vascular endothelial growth factor/blood vessel permeability factor (VEGF/VPF), (TGF a) for transforming growth factor-a, tumor necrosis factor-alpha (TNF-α), angiogenin, interleukin 3 (IL-3), interleukin 8 (IL-8), platelet-derived endothelial cell growth factor (ECGF) (PD-ECGF), placenta growth factor (P1GF), factor Thr6 PDGF BB-BB in mid-term (PDGF), the CXXXX chemotactic molecule;

Stress protein (heat shock protein);

HSPs is generally at born of the same parents' inner analysis.When finding that they are outside born of the same parents, it is necrocytosis and the indicator that exhales its inclusion.This non-procedural necrocytosis (necrosis) is only as wound, and the i or I result takes place, and thus in vivo the outer HSPs of born of the same parents cause from anti-infective and immune reaction disease.Dual specificity in conjunction with the outer HSP of born of the same parents can be confined to disease location;

The protein that relates to the Fc transhipment;

Brambell acceptor (being also referred to as FcRB);

This Fc acceptor has two kinds of functions, and the two all can be used in and sends;

These functions are:

1) IgG is transitted to children by placenta from parent;

2) prevent the IgG degraded, prolong the serum half-life of its IgG thus.Think that acceptor makes the IgG recirculation from endosome.

Referring to Holliger etc., Nat Biotechnol 1997 Jul; 15 (7): 632-6.

C) target antigen can also be in the following table any one, and as shown in Table whether pairing is irrelevant with this antigen :-

Pairing	The treatment coherent reference
Pairing	The treatment coherent reference	TNF α/TGF-β	TGF-b and TNF have significantly strengthened arthritis when injecting the ankle joint of the arthritis model that collagen protein brings out.No effect in the mouse that non--collagen protein is attacked.
TNF α/IL-1	TNF and IL-1 play synergy in uveitic pathology situation.TNF and IL-1 in the pathology situation of malaria, rise synergy (hypoglycemia, NO).TNF and IL-1 play synergy so that induce polymorphonuclear leukocyte in the inflammation (PMN) cell migration.IL-1 and TNF play synergy inducing PMN to be impregnated in the peritonaeum.IL-1 and TNF play synergy so that at inducing endothelial cell secretion IL-1.Important in inflammation.IL-1 or TNF induce some cellular infiltration to go into the knee synovial membrane separately.IL-1 induces PMNs, the TNF-monocyte.They induce the more infiltration of seriousness jointly because of PMNs increases.The myocardium inhibitory substance that circulates (being present in the Sepsis) is for low-level IL-1 and with synergy mode TNFacting.	TNF α/TGF-β
TNF α/IL-1		TNF α/IL-2	Most of relevant with the collaborative activation of killer T-cell.

TNF α/IL-3	Interleukin and the tumor necrosis factor alpha synergy in stimulating the paotoblastic clonal growth of acute myelogenous leukemia is the result that tumor necrosis factor alpha is induced secondary hematopoietic cytokine.·Cancer Res.1992 Apr 15；52(8)：2197-201。
TNF α/IL-3		TNF α/IL-4	IL-4 and TNF play synergy so that VCAM expresses on the inducing endothelial cell.Intention has effect in asthma.For synovial membrane identical-relate to RA.TNF and IL-4 play synergy, and IL-6 expresses in the keratinocyte so that induce.TNF-α and IL-4 or IL-13 combination realize the lasting elevated levels of VCAM-1 in the inoblast sample synovial cell who cultivates by the mRNA stability that increases.Am J Pathol.1999 Apr; 154 (4): 1149-58
TNF α/IL-5	System of tumor necrosis factor in grownup and children's thereof the bronchial hyperreactivity and serum interleukin 4, interleukin 5, interleukin 8, the dependency between eosinophile cationic protein and the immunoglobulin E level.Allergy Asthma Proc.2003Mar-Apr；24(2)：111-8。	TNF α/IL-4
TNF α/IL-5		TNF α/IL-6	TNF and IL-6 are effective OH-2, the somatomedin that promptly a kind of new human lymphoma cell is.Eur J Haematol.1994 Jul；53(1)：31-7。
TNF α/IL-8	TNF and IL-8 and PMNs synergy are so that activated blood platelet.Relate to adult respiratory distress syndrome.Referring to IL-5/TNF (asthma).Be used for the interleukin 8 of platelet activation of neutrophilic granulocyte-mediation and the synergy between the tumor necrosis factor-alpha.Eur cytokine Netw.1994 Sep-Oct; 5 (5): 455-60 (adult respiratory distress syndrome (ARDS))	TNF α/IL-6
TNF α/IL-8		TNF α/IL-9
TNF α/IL-10	IL-10 induces and acts synergistically with TNF among the HIV in inducing chronically infected T-cell.	TNF α/IL-9

TNF α/IL-11	Cytokine is induced the osteoclast differentiation in the synergy mode: supported by immortalization or normal braincap cell.Am J Physiol Cell Physiol.2002 Sep；283(3)： C679-87。(bone loss)
TNF α/IL-11		TNF α/IL-12
TNF α/IL-13	TNF-α and IL-4 or IL-13 combination realize the lasting elevated levels of VCAM-1 in the inoblast sample synovial cell who cultivates by the mRNA stability that increases.Am J Pathol.1999 Apr；154(4)：1149-58。Interleukin-13 and tumor necrosis factor-alpha induce the eotaxin in people's nose inoblast to produce in the synergy mode.Clin Exp Allergy.2000 Mar；30(3)： 348-55。Interleukin-13 and tumor necrosis factor-alpha are induced eotaxin's generation in people's nose inoblast in the synergy mode.Clin Exp Allergy.2000 Mar; 30 (3): nephrotic syndrome reaction Childhood that 348-55 (allergic inflammation) serum TNF-β and IL-13 relating to treatment.Cytokine.2003 Feb 7；21(3)：155-9。	TNF α/IL-12
TNF α/IL-13		TNF α/IL-14	The effect of tumor necrosis factor alpha in having the experimenter of mild asthma that sucks.Thorax.2002Sep；57(9)：774-8。
TNF α/IL-15	The effect of tumor necrosis factor alpha in having the experimenter of mild asthma that sucks.Thorax.2002Sep；57(9)：774-8。	TNF α/IL-14
TNF α/IL-15		TNF α/IL-16	Tumor necrosis factor-alpha-inductive interleukin 16 is synthetic the airway epithelia cell: causing serotonin stimulates.Am J Respir Cell Mol Biol.2003 Mar; 28 (3): the circulating leukocyte that 354-62. (airway inflammation) suffers from the patient of rheumatoid arthritis is situated between plain 16 relevant with pro-inflammatory cytokine.Rheumatology(Oxford).2001Apr；40(4)：474-5。There is not available summary.Interleukins 16 is incremental adjustments and ginseng in Crohn's disease

	TNBS colitis with mouse.Gastroenterology.2000 Oct；119(4)：972-82。
		TNF α/IL-17	Sacroiliitis takes place in the inoculation mouse that the inhibition Interleukin-17 prevents to use B. burgdorferi (Borrelia burgdorferi) to attack.Infect Immun.2003 Jun；71(6)：3437-42。Interleukin 17 acts synergistically with tumor necrosis factor alpha so that the outer cartilage destruction of inductor.Ann Rheum Dis.2002 Oct； 61(10)：870-6。The effect of the GM-CSF that IL-17 and TNF-α cause during neutrophilic leukocyte is accumulated in air flue.Eur Respir J. 2003 Mar； 21(3)：387-93。(airway inflammation) summary, interleukin 1, tumor necrosis factor alpha and Interleukin-17 produce with nitrogen protoxide and prostaglandin E2 in the synergy mode incremental adjustments people osteoarthritic knee joint dish explant.Arthritis Rheum.2001Sep；44(9)： 2078-83。
TNF α/IL-18	The level of interleukin-18 expression rising in suffering from patient's knee synovial tissue of rheumatoid arthritis with interleukin-1 ' beta ' and tumor necrosis factor alpha is relevant.Arthritis Rheum. 2003Feb；48(2)：339-47。Summary suffers from the elevated levels of interleukin-18 and tumor necrosis factor-alpha among the patients serum of diabetes B: with the dependency of diabetic nephropathy.Metabolism.2003 May； 52(5)：605-8。	TNF α/IL-17
TNF α/IL-18		TNF α/IL-19	Summary, IL-19 are induced IL-6 and TNF-α to produce and are caused the cell apoptosis by TNF-α.J Immunol. 2002 Oct 15；169(8)：4288-97。
TNF α/IL-20	Summary, the effector in cytokine: IL-20-new skin inflammation.Curr Biol.2001 Jul 10; 11 (13): R531-4	TNF α/IL-19
TNF α/IL-20		TNF α/complement	Inflammation and solidifying: with septic patient's dependency.Clin Infect Dis.2003 May 15；36(10)：1259-65.Epub

	2003 May 08。Summary.
	2003 May 08。Summary.	TNF α/IFN-γ	MHC induces in the brain.Synergy during anti--virus reaction/IFN induces.Neutrophil activation/respiratory burst.Activated endothelial cell.The toxicity of when using TNF/IFN-to treat the patient, noticing as anti--virus therapy.The Astrocytic CXXXC chemotactic molecule of people is expressed.Relevant inflammatory reaction-be LPS, also be many papers of macrophage activation.Anti-TNF and anti--IFN-γ play synergy and so that prevent mouse the mortality endotoxemia take place.

TGF-β/IL-1	IL-6 and IL-8 in IL-6 generation (inflammatory model) the stimulation lung fibroblast of the synthetic intestinal epithelial cells of osteoblastic prostaglandin(PG) in IL-11 and IL-6 (inflammatory model) retina produce
TGF-β/IL-1		TGF-β/IL-6	Chrondrocarcinoma (Chondrocarcoma) propagation
IL-1/IL-2	The synergy of IL-1 and IL-2 during the killer cell of B-cell activation LAK cell activation T-cell activation TNF-α and β (lymphotoxin) mediation lymphokineactivation goes down to posterity.Cytokine.1992 Nov；4(6)：479-87。	TGF-β/IL-6	Chrondrocarcinoma (Chondrocarcoma) propagation
IL-1/IL-2		IL-1/IL-3
IL-1/IL-4	B-cell activation IL-4 induces the IL-1 in activated endothelial cell to express	IL-1/IL-3
IL-1/IL-4		IL-1/IL-5
IL-1/IL-6	B cell activation T cell activation (can replace helper) IL-1 induces IL-6 to express C3 and serum amyloid protein is expressed (acute phase reaction)	IL-1/IL-5

	HIV expresses the chondrogen proteolysis
	HIV expresses the chondrogen proteolysis	IL-1/IL-7	IL-7 is that IL-1-inductive thymocyte proliferation is necessary.The IL-7 that relates in the synergy of granulocyte-macrophage colony stimutaing factor or tumour necrosis factor and IL-1.J Immunol.1992 Jan 1；148(1)：99-105。
IL-1/IL-8		IL-1/IL-7
IL-1/IL-8		IL-1/IL-10
IL-1/IL-11	Cytokine is induced the osteoclast differentiation in the synergy mode: immortalization or normal braincap cell are supported.Am J Physiol Cell Physiol.2002 Sep；283(3)： C679-87。(bone loss)	IL-1/IL-10
IL-1/IL-11		IL-1/IL-16	It is plain 16 relevant with pro-inflammatory cytokine that the circulating leukocyte of suffering from the patient of rheumatoid arthritis is situated between.Rheumatology(Oxford).2001 Apr；40(4)： 474-5。There is not available summary.
IL-1/IL-17	Sacroiliitis takes place in the inoculation mouse that the inhibition Interleukin-17 prevents to use B. burgdorferi to attack.Infect Immun. 2003 Jun；71(6)：3437-42。Interleukin 17 impels the synovial membrane inflammation in human cartilage degraded and the osteoarthritis.Osteoarthritis Cartilage.2002 Oct；10(10)：799-807。Summary, interleukin 1, tumor necrosis factor alpha and Interleukin-17 are sternly living with nitrogen protoxide and prostaglandin E2 in the synergy mode incremental adjustments people osteoarthritic knee joint dish explant.Arthritis Rheum.2001 Sep；44(9)： 2078-83。	IL-1/IL-16
IL-1/IL-17		IL-1/IL-18	The level of interleukin-18 expression rising in suffering from patient's knee synovial tissue of rheumatoid arthritis with interleukin-1 ' beta ' and tumor necrosis factor alpha is relevant.Arthritis Rheum.2003 Feb；48(2)：339-47。
IL-1/IFN-g		IL-1/IL-18

IL-2/IL-3	T-cell proliferation B cell proliferation
IL-2/IL-3	T-cell proliferation B cell proliferation	IL-2/IL-4	B-cell proliferation T-cell proliferation (activation of CD8 and NK lymphocyte selective induction) IL-2R beta-agonists P1-30 and IL-2, IL-4, IL-9 and IL-15 play synergy: biology and molecularity.J Immunol.2000 Oct 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-4
IL-2/IL-5		IL-2/IL-6	Cytotoxic T-cell development
IL-2/IL-7		IL-2/IL-6	Cytotoxic T-cell development
IL-2/IL-7		IL-2/IL-9	Referring to IL-2/IL-4 (NK-cell)
IL-2/IL-10	The B-cell activation	IL-2/IL-9	Referring to IL-2/IL-4 (NK-cell)
IL-2/IL-10	The B-cell activation	IL-2/IL-12	IL-12 and IL-2 synergy is so that induce perforin and granzyme genetic expression in lymphokine-activatory cytotoxicity and the NK cells of human beings.Cell Immunol.1995 Oct 1； 165(1)：33-43。(T-cell activation)
IL-2/IL-15	Referring to IL-2/IL-4 (NK cell) (T cell activation and propagation) IL-15 and IL-2: the survival and the dead material of T cell in the body.Nat Med.2001 Jan； 7(1)：114-8。	IL-2/IL-12
IL-2/IL-15		IL-2/IL-16	IL-16 and IL-2 are to the collaborative activation of CD4+T cell.J Immunol.1998Mar 1；160(5)：2115-20。
IL-2/IL-17	The evidence of the early stage dependency of interleukin 17 in people and experiment allograft rejection.J Pathol.2002 Jul； 197(3)：322-32。	IL-2/IL-16
IL-2/IL-17		IL-2/IL-18	The synergy inducing mouse mortality injury of lung of interleukin-18 (IL-18) and IL-2: cytokine, chemokine and the natural killer cell latent effect in the interstitial pneumonia pathogenesis.Blood.2002Feb 15；99(4)：1289-98。
IL-2/TGF-β	The control of CD4 effector destiny: transforminggrowthfactor-and	IL-2/IL-18

	The interleukin II synergy is so that prevent apoptosis and the expansion of accelerating effect thing.J Exp Med.1995 Sep 1；182(3)：699-709。
		IL-2/IFN-γ	The Ig secretion IL-2 of B-cell induces the IFN-γ of T-cell to express
IL-2/IFN-α/β	Do not have	IL-2/IFN-γ
IL-2/IFN-α/β	Do not have	IL-3/IL-4	The synergy that synergy IL-4 in the mastocyte growth and GM-CSF or IL-3 induce CD23 to express to the person monocytic cell: the regulating effect of IFN-α and IFN-γ.Cytokine.1994 Jul；6(4)：407-13。
IL-3/IL-5		IL-3/IL-4
IL-3/IL-5		IL-3/IL-6
IL-3/IFN-γ	IL-4 and IFN-γ increase total polymerization IgA receptor level in human intestinal epithelial's cell in the synergy mode.The effect of protein tyrosine kinase.J Immunol.1996 Jun 15；156(12)： 4807-14。	IL-3/IL-6
IL-3/IFN-γ		IL-3/GM-CSF	Cytokine is to people's eosinophilic granulocyte IL-3, and IL-5 and GM-CSF acceptor α-chain expression difference is regulated: IL-3, and IL-5 and GM-CSF decrement are regulated IL-5 acceptor alpha expression, and wherein IL-5 is reactive lacks, and incremental adjustments IL-3 acceptor alpha expression.J Immunol.2003 Jun 1; 170 (11): 5359-66. (allergic inflammation)
IL-4/IL-2	IL-4 promotes IL-2-and IL-12-inductive IFN-{ γ in the mouse NK cell in the synergy mode } express.Blood. the electronic edition before 2003 Mar 13[print]	IL-3/GM-CSF
IL-4/IL-2		IL-4/IL-5	The increase of serotonins such as mastocyte histamine relates to bullous pemphigoid, i.e. IL-4 and the IL-5 effect in this disease pathogenesis as the reaction Th2-like cell factor reaction to IgE.Int J Immunopathol Pharmacol.1999 May-Aug； 12(2)：55-61。
IL-4/IL-6		IL-4/IL-5

IL-4/IL-10
IL-4/IL-10		IL-4/IL-11	Cooperative interaction in the original hematopoiesis progenitor cell propagation of support mouse between interleukin 11 and the interleukin 4.Blood.1991 Sep 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 generation of dendritic cell.J Immunol.2000 Jan 1； 164(1)：64-71。(increasing the Th1/Th2 differentiation) IL-4 strengthens IL-2-and IL-12-inductive IFN-{ γ in the mouse NK cell in the synergy mode } express.Electronic edition before the Blood.2003 Mar 13[printing]	IL-4/IL-11
IL-4/IL-12		IL-4/IL-13	Summary, interleukin 4 and Interleukin-13 signal conduction correlation diagram.Science.2003 Jun 6; 300 (5625): 1527-8. (anaphylaxis, asthma) suppresses the IL-4/IL-13 receptor system and prevents allergic sensitization, but does not influence the anaphylaxis of setting up in the allergic asthma mouse model.J Allergy Clin Immunol.2003 Jun； 111(6)：1361-1369。
IL-4/IL-16	(asthma) interleukin-(IL)-4/IL-9 and exogenous IL-16 induce the BEAS-2B cell, and promptly the IL-16 of bronchial epithelial cell system produces.Cell Immunol.2001 Feb 1; 207 (2): 75-80	IL-4/IL-13
IL-4/IL-16		IL-4/IL-17	Interleukin-(IL)-4 and IL-17 stimulate the IL-6 in people's colon myofibroblast to secrete in the synergy mode.Int J Mol Med.2002 Nov；10(5)：631-4。(enteritis)
IL-4/IL-24	IL-24 is expressed by rat and people's myofibroblast.Immunobiology.2002 Jul；205(3)：321-34。	IL-4/IL-17
IL-4/IL-24		IL-4/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.2002 Jul 1；169(1)：443-53。(allergic inflammation) summary, mastocyte produce interleukin II 5 when the activation of Fc-ε RI-mediation.Blood.2003 May 1；101(9)：

	3594-6.Epub 2003 Jan 02. (allergic inflammation)
	3594-6.Epub 2003 Jan 02. (allergic inflammation)	IL-4/IFN-γ	Summary, interleukin-4 inducing endothelial cell interleukin 6 produces: with the synergy of interferon-gamma.Eur J Immunol.1991 Jan；21(1)：97-101。
IL-4/SCF	STEM CELL FACTOR and IL-4 are to the adjusting of people's intestinalmast cell.Immunol Rev.2001 Feb；179：57-60。Summary.	IL-4/IFN-γ
IL-4/SCF		IL-5/IL-3	Cytokine is to people's eosinophilic granulocyte IL-3, and IL-5 and GM-CSF acceptor α-chain expression difference is regulated: IL-3, and IL-5 and GM-CSF decrement are regulated IL-5 acceptor alpha expression, and wherein IL-5 is reactive lacks, but incremental adjustments IL-3 acceptor alpha expression.J Immunol.2003 Jun 1; 170 (11): 5359-66. (allergic inflammation is referring to summary)
IL-5/IL-6		IL-5/IL-3
IL-5/IL-6		IL-5/IL-13	Dexamethasone suppresses supersensitivity airway inflammation and airway hyperreactivity in the mouse: eosinophilic granulocyte, IL-5, the effect of eotaxin and IL-13.J Allergy Clin Immunol.2003 May；111(5)：1049-61。
IL-5/IL-17	Compile (orchestrates) granulocyte after the allergen of Interleukin-17 in the allergic asthma mouse model sucks and flow into air flue.Am Respir Cell Biol.2003 Jan；28(1)： 42-50。	IL-5/IL-13
IL-5/IL-17		IL-5/IL-25	Summary, new IL-17 family member promotes the reaction of Th1 in the lung or Th2: function in the body of new cytokine IL-25.J Immunol.2002 Jul 1；169(1)：443-53。(allergic inflammation) summary, mastocyte produce interleukin II 5 when the activation of Fc-ε RI-mediation.Blood.2003 May 1；101(9)： 3594-6.Epub 2003 Jan 02。(allergic inflammation)
IL-5/IFN-γ		IL-5/IL-25
IL-5/IFN-γ		IL-5/GM-CSF	Cytokine is to people's eosinophilic granulocyte IL-3, and IL-5 and GM-CSF acceptor α-chain expression difference is regulated: IL-3, IL-5 and GM-CSF decrement are regulated IL-5 acceptor alpha expression, wherein

	The reactive disappearance of IL-5, and incremental adjustments IL-3 acceptor alpha expression.J Immunol.2003 Jun 1；170(11)：5359-66。(allergic inflammation)
		IL-6/IL-10
IL-6/IL-11		IL-6/IL-10
IL-6/IL-11		IL-6/IL-16	Interleukin 16 stimulates the person monocytic cell to express and produces pro-inflammatory cytokine.Immunology.2000 May；100(1)： 63-9。
IL-6/IL-17	Interleukin-(IL)-17 stimulates by IL-6 paracrine/autocrine loop.J Biol Chem.2003 May 9；278(19)： 17036-43.Epub 2003 Mar 06。(airway inflammation, asthma)	IL-6/IL-16
IL-6/IL-17		IL-6/IL-19	Summary, IL-19 are induced IL-6 and TNF-α to produce and are caused the cell apoptosis by TNF-α.J Immunol. 2002 Oct 15；169(8)：4288-97。
IL-6/IFN-g		IL-6/IL-19
IL-6/IFN-g		IL-7/IL-2	Interleukin-17 worsens graft versus host disease (GVH disease).Blood. 2002 Oct 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-2
IL-7/IL-12		IL-7/IL-15	Interleukin 7 and Interleukin-15 are regulated bcl-2 and the expression of c-myb gene in skin T-cell lymphoma cell.Blood.2001 Nov 1；98(9)：2778-83。(somatomedin)
IL-8/IL-11	Interleukin-(IL)-11 and the IL-8 unusual generation in true property erythrocytosis.Cytokine.2002 Nov 21； 20(4)：178-83。	IL-7/IL-15
IL-8/IL-11		IL-8/IL-17	The effect of IL-17 in the joint is damaged.Drug News Perspect.2002 Jan；15(1)：17-23。(sacroiliitis) summary, Interleukin-17 stimulate the popularity tract epithelial cell to express interleukin 8, relevant oncogene-α of growth and granulocyte colony-stimulating factor.Am J Respir Cell Mol Biol.

	2002 Jun；26(6)：748-53。(airway inflammation)
	2002 Jun；26(6)：748-53。(airway inflammation)	IL-8/GSF	Interleukin 8: promote the autocrine/paracrine somatomedin of the artificial blood progenitor cell of monocyte-scavenger cell growth and differentiation with colony-stimulating factor-1 synergy.Exp Hematol.1999 Jan；27(1)：28-36。
IL-8/VGEF	VEGF in the chamber in primary and the recurrent glioblastoma, bFGF, IL-8, IL-12 level.J Neurooncol. 2003 May；62(3)：297-303。	IL-8/GSF
IL-8/VGEF		IL-9/IL-4	Anti--interleukin 9 Antybody therapy suppresses airway inflammation and hyperergy in mouse asthmatic model.Am J Respir Crit Care Med.2002 Aug 1；166(3)：409-16。
IL-9/IL-5	The lung overexpression of IL-9 is induced the Th2 cytokine-expressing, causes the immunopathology situation.J Clin Invest.2002 Jan； 109(1)：29-39。Th2 cytokine and asthma.Interleukin 9 is as the treatment of asthma target.Respir Res.2001；2(2)：80-4.Epub 2001 Feb 15。Summary.Summary, interleukin 9 promotes the interleukin 5 expression of receptor of people's eosinophilic granulocyte, differentiation and survival.Blood. 2000 Sep 15; 96 (6): 2163-71 (asthma)	IL-9/IL-4
IL-9/IL-5		IL-9/IL-13	Anti--interleukin 9 Antybody therapy suppresses airway inflammation and hyperergy in mouse asthmatic model.Am J Respir Crit Care Med.2002 Aug 1；166(3)：409-16。Interleukin-13 causes mucus mucus in airway hyperreactivity and the asthma to epithelial direct effect.Nat Med. 2002 Aug；8(8)：885-9。
IL-9/IL-16	Referring to IL-4/IL-16	IL-9/IL-13
IL-9/IL-16	Referring to IL-4/IL-16	IL-10/IL-2	Interleukin 10 (IL-10) and interleukin II (IL-2) interaction in humoral immunoresponse(HI): IL-10 and IL-2 synergy are so that promote the human B lymphocyte reaction in the mechanism that is different from CD25 expression incremental adjustments.Cell Immunol.1994Sep；157(2)：478-88。

IL-10/IL-12
IL-10/IL-12		IL-10/TGF-β	IL-10 and TGF-β are concuring in the allergenic regulatory T cells reaction the mucous membrane in normal immunity and the specific immunotherapy.Eur J Immunol.2003 May； 33(5)：1205-14。
IL-10/IFN-γ		IL-10/TGF-β
IL-10/IFN-γ		IL-11/IL-6	Interleukin-6 and interleukin 11 form by RANKL-dependency mechanism backer osteoclast.Bone.2003 Jan；32(1)：1-7。(bone resorption in the inflammation)
IL-11/IL-17	The polarization expression in vivo of IL-11 and IL-17 between acute and chronic skin damages.J Allergy Clin Immunol.2003 Apr；111(4)：875-81。The disappearance of (allergic dermatitis) IL-17 by NF-κ B part/osteoprotegerin equilibrated receptor activator promotes the bone erosion in the sacroiliitis of mouse collagen protein-bring out.J Immunol.2003 Mar 1； 170(5)：2655-62。	IL-11/IL-6
IL-11/IL-17		IL-11/TGF-β	The polarization expression in vivo of IL-11 and IL-17 between acute and chronic skin damages.J Allergy Clin Immunol.2003 Apr；111(4)：875-81。(allergic dermatitis)
IL-12/IL-13	The unbalance dependency with classification-specificity Rheumatoid factors, polyclonal and allergic dermatitis antibody of interleukin 12 and Interleukin-13 in the systemic lupus erythematous.Clin Rheumatol.2003 May；22(2)：107-11。	IL-11/TGF-β
IL-12/IL-13		IL-12/IL-17	The incremental adjustments of interleukin 12 and-17 in the reactivity inflammatory bowel.Scand J Gastroenterol.2003 Feb； 38(2)：180-5。
IL-12/IL-18	Interleukin 12 and interleukin-18 are to the collaborative propagation and the activation of natural killer cell.Cytokine.1999 Nov； 11(11)：822-30。The struvite liver steatosis that IL-12 and IL-18 cause.J Interferon Cytokine Res.2003 Mar；23(3)： 155-62。	IL-12/IL-17

IL-12/IL-23	Interleukin-(nterleukin)-23 but not interleukin 12 is the key cytokines of brain autoimmunization inflammation.Nature.2003 Feb 13；421(6924)：744-8。Summary, the unique effect of IL-23 in promoting cellular immunity.J Leukoc Biol.2003 Jan；73(1)：49-56。Summary.
IL-12/IL-23		IL-12/IL-27	Summary, the 7 different dimerization cytokine IL-2 that are made up of EBI3 and p28 albumen induce pure CD4 (+) T cell proliferation.Immunity.2002 Jun；16(6)：779-90。
IL-12/IFN-γ	IL-12 induces the IFN-γ of B and T-cell to express as immunostimulating integral part.	IL-12/IL-27
IL-12/IFN-γ		IL-13/IL-5	Referring to 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.2002 Jul 1；169(1)：443-53。(allergic inflammation) summary, mastocyte produce interleukin II 5 when the activation of Fc-ε RI-mediation.Blood.2003 May 1；101(9)： 3594-6.Epub 2003 Jan 02。(allergic inflammation)	IL-13/IL-5	Referring to IL-5/IL-13
IL-13/IL-25		IL-15/IL-13	Interleukin-(IL)-13 and IL-15 are in the women who suffers from endometriosis and normally can educate women's dystopy and the differential expression in the normotopia uterine endometrium.Am J Reprod Immunol.2003 Feb；49(2)：75-83。
IL-15/IL-16	Express IL-15 and IL-16 overexpression in the skin T-cell lymphoma: in the mycosis fungoides progress the stage-dependency increases.Exp Dermatol.2000 Aug；9(4)：248-51。	IL-15/IL-13
IL-15/IL-16		IL-15/IL-17	Summary, the IL-17 that lymphocyte and neutrophilic leukocyte produce is that lipopolysaccharides-inductive air flue neutrocytophilia is requisite: IL-15 is as possible triggering thing.J Immunol. 2003 Feb 15；170(4)：2106-12。(airway inflammation)
IL-15/IL-21	IL-21 and IL-15 or IL-18 synergy promote that IFN-γ produces in people NK and the T cell.J Immunol.2003 Jun 1； 170(11)：5464-9。	IL-15/IL-17

IL-17/IL-23	Interleukin II 3 promotes to be characterised in that the different cd4 t cell active state of Interleukin-17 generation.J Biol Chem.2003 Jan 17；278(3)：1910-4.Epub 2002 Nov 03。
IL-17/IL-23		IL-17/TGF-β	The polarization expression in vivo of IL-11 and IL-17 between acute and chronic skin damages.J Allergy Clin Immunol.2003 Apr；111(4)：875-81。(allergic dermatitis)
IL-18/IL-12	Interleukin 12 and interleukin-18 are to the collaborative propagation and the activation of natural killer cell.Cytokine.1999 Nov; 11 (11): the external immunoglobulin (Ig) that 822-30. summary, IL-12 suppress in the mouse chronic graft versus host disease produces: act synergistically with IL-18.Eur J Immunol.1998 Jun；28(6)：2017-24。	IL-17/TGF-β
IL-18/IL-12		IL-18/IL-21	IL-21 and IL-15 or IL-18 synergy promote that IFN-γ produces in people NK and the T cell.J Immunol.2003 Jun 1； 170(11)：5464-9。
IL-18/TGF-β	Interleukin-18 and transforminggrowthfactor-in the patients serum who suffers from the graves' ophthalmopathy of using corticosteroid treatment.Int Immunopharmacol.2003 Apr；3(4)： 549-52。	IL-18/IL-21
IL-18/TGF-β		IL-18/IFN-γ
Anti-TNF alpha/anti--CD4	Synergistic therapeutic action in the DBA/1 arthritis type mouse.	IL-18/IFN-γ

Target	Disease	With following pairing
Target	Disease	With following pairing	CD89*	Application as the cytotoxic cell person of raising	All
			CD89*	Application as the cytotoxic cell person of raising	All
			CD19	B cell lymphoma	HLA-DR CD5
HLA-DR	B cell lymphoma	CD89 CD19 CD5	CD19	B cell lymphoma	HLA-DR CD5
HLA-DR	B cell lymphoma	CD89 CD19 CD5	CD38	Malignant lymphoma	CD138 CD56 HLA-DR
CD138	Malignant lymphoma	CD38 CD56 HLA-DR	CD38	Malignant lymphoma	CD138 CD56 HLA-DR
CD138	Malignant lymphoma	CD38 CD56 HLA-DR	CD138	Lung cancer	CD56 CEA
CD33	Acute myelogenous lymphoma	CD34 HLA-DR	CD138	Lung cancer	CD56 CEA
CD33	Acute myelogenous lymphoma	CD34 HLA-DR	CD56	Lung cancer	CD138 CEA
CEA	Carcinoma of the pancreas	The MET acceptor	CD56	Lung cancer	CD138 CEA
CEA	Carcinoma of the pancreas	The MET acceptor	VEGF	Carcinoma of the pancreas	The MET acceptor
Vegf receptor	Carcinoma of the pancreas	The MET acceptor	VEGF	Carcinoma of the pancreas	The MET acceptor
Vegf receptor	Carcinoma of the pancreas	The MET acceptor	IL-13	Asthma/pneumonia	IL-4 IL-5 eotaxin MDC TARC TNF α IL-9

		EGFR CD40L IL-25 MCP-1 TGFβ
		EGFR CD40L IL-25 MCP-1 TGFβ	IL-4	Asthma	IL-13 IL-5 eotaxin MDC TARC TNF α IL-9 EGFR CD40L IL-25 MCP-1 TGF β
The eotaxin	Asthma	IL-5 eotaxin-2 eotaxin-3	IL-4	Asthma
The eotaxin	Asthma	IL-5 eotaxin-2 eotaxin-3	EGFR	Cancer	HER2/neu HER3 HER4
HER2	Cancer	HER3 HER4	EGFR	Cancer	HER2/neu HER3 HER4
HER2	Cancer	HER3 HER4	TNFR1	The RA/ Crohn's disease	IL-1R IL-6R IL-18R
TNFα	The RA/ Crohn's disease	IL-1α/β IL-6 IL-18 ICAM-1	TNFR1	The RA/ Crohn's disease	IL-1R IL-6R IL-18R

		IL-15 IL-17
		IL-15 IL-17	IL-1R	The RA/ Crohn's disease	IL-6R IL-18R
IL-18R	The RA/ Crohn's disease	IL-6R	IL-1R	The RA/ Crohn's disease	IL-6R IL-18R

EpCAM

CD20

CD33

CD52

Her-2/neu

GPIIb/IIIa

RSV

CD25

CD3

a4B3

E) a) people's form of target ligands-d).

Appendix 2

The example of general part

A) the single variable domain of Gong Buing

Be disclosed in WO030020609, WO2004101790, WO2005035572, WO2004081026, variable domain arbitrarily among WO2004003019 and the WO2004058821, with these variable domains that disclose, its sequence and production and system of selection are incorporated herein by reference clearly, so that be provided for the example of general part of the present invention for those skilled in the art.

Be disclosed in WO9404678, WO9748905, WO9933221, WO9937681, WO0024884, WO0043507, WO0065057, WO0140310, WO03035694, WO03053531, WO03054015, WO0305527, WO2004015425, WO2004041862, WO2004041863, WO20040401865, WO2004062551, any VHH structural domain among WO2005044858 and the EP1134231 or other variable domain arbitrarily are with these variable domains that disclose, its sequence and production and system of selection are incorporated herein by reference clearly, so that be provided for the example of general part of the present invention for those skilled in the art.

B) (i) derive from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be the segmental people VH of VH.

The people VH that (ii) has the FW1 aminoacid sequence, described FW1 aminoacid sequence with from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be that the aminoacid sequence of the segmental corresponding FW of VH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

The people VH that (iii) has the FW2 aminoacid sequence, described FW2 aminoacid sequence with from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be that the aminoacid sequence of the segmental corresponding FW of VH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

The people VH that (iv) has the FW3 aminoacid sequence, described FW3 aminoacid sequence with from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be that the aminoacid sequence of the segmental corresponding FW of VH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(the people VH that v) has the FW4 aminoacid sequence, described FW4 aminoacid sequence with from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be that the aminoacid sequence of the segmental corresponding FW of VH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(vi) has FW1,2, the people VH of 3 and 4 aminoacid sequences, described FW1,2,3 with 4 aminoacid sequences with from Fig. 7 in a) 3-23 position locus or arbitrarily the kind of other locus be that the aminoacid sequence of the segmental corresponding FW of VH is identical or wherein

FW

1,2,3 and 4 aminoacid sequences have jointly and reach 10 kinds of amino acid differences from described corresponding FWs.

DPK9 locus in (vii) deriving from Fig. 7 b) or any genomic people V _k

(the people V that viii) has the FW1 aminoacid sequence _k, described FW1 aminoacid sequence with from Fig. 7 b) in the DPK9 locus or arbitrarily the kind of locus be V _kThe aminoacid sequence of segmental corresponding FW is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(ix) has the people V of FW2 aminoacid sequence _k, described FW2 aminoacid sequence with from Fig. 7 b) in the DPK9 locus or arbitrarily the kind of locus be V _kThe aminoacid sequence of segmental corresponding FW is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(x) has the people V of FW3 aminoacid sequence _k, described FW3 aminoacid sequence with from Fig. 7 b) in the DPK9 locus or arbitrarily the kind of locus be V _kThe aminoacid sequence of segmental corresponding FW is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xi) has the people V of FW4 aminoacid sequence _k, described FW1 aminoacid sequence with from Fig. 7 b) in the DPK9 locus or arbitrarily the kind of locus be V _kThe aminoacid sequence of segmental corresponding FW is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xii) has

FW

1,2, the people V of 3 and 4 aminoacid sequences _k, described FW1,2,3 and 4 aminoacid sequences with from Fig. 7 b) in the DPK9 locus or arbitrarily the kind of locus be V _kIdentical or the FW1 wherein of the aminoacid sequence of segmental corresponding FW, 2,3 and 4 aminoacid sequences have jointly and reach 10 kinds of amino acid differences from described corresponding FWs.

(xiii) derive from Fig. 7 c) in any kind be the segmental people V of V λ λ.

(xiv) have the people V λ of FW1 aminoacid sequence, described FW1 aminoacid sequence with from Fig. 7 c) in any kind be that the aminoacid sequence of corresponding FW of V λ is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xv) have the people V λ of FW2 aminoacid sequence, described FW2 aminoacid sequence with from Fig. 7 c) in any kind be that the aminoacid sequence of corresponding FW of V λ is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xvi) have the people V λ of FW3 aminoacid sequence, described FW3 aminoacid sequence with from Fig. 7 c) in any kind be that the aminoacid sequence of corresponding FW of V λ is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xvii) have the people V λ of FW4 aminoacid sequence, described FW4 aminoacid sequence with from Fig. 7 c) in any kind be that the aminoacid sequence of corresponding FW of V λ is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xviii) has FW1, the people V λ of 2,3 and 4 aminoacid sequences, described FW 1 aminoacid sequence with from Fig. 7 c) in any kind be the identical or FW 1 wherein of the aminoacid sequence of corresponding FWs of V λ, 2,3 and 4 aminoacid sequences have jointly and reach 10 kinds of amino acid differences from described corresponding FWs.

(xix) derive from Fig. 7 d) or be disclosed in Nguyen etc., EMBO J, 2000, Vol19, No.5, any kind among Fig. 2 of pp921-930 (924 pages) (document that will disclose the VHH kind especially and be sequence is incorporated herein by reference) is segmental Camelid VHH or Nanobody ^TM

(xx) have the Camelid VHH or the Nanobody of FW1 aminoacid sequence ^TM, described FW1 aminoacid sequence be disclosed in from Nguyen etc., EMBO J, 2000, Vol19, No.5, any kind among Fig. 2 of pp921-930 (924 pages) is that the aminoacid sequence of the segmental corresponding FW of VHH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xxi) have the Camelid VHH or the Nanobody of FW2 aminoacid sequence ^TM, described FW2 aminoacid sequence be disclosed in from Nguyen etc., EMBO J, 2000, Vol19, No.5, any kind among Fig. 2 of pp921-930 (924 pages) is that the aminoacid sequence of the segmental corresponding FW of VHH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xxii) have the Camelid VHH or the Nanobody of FW3 aminoacid sequence ^TM, described FW3 aminoacid sequence be disclosed in from Nguyen etc., EMBO J, 2000, Vol19, No.5, any kind among Fig. 2 of pp921-930 (924 pages) is that the aminoacid sequence of the segmental corresponding FW of VHH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xxiii) have the Camelid VHH or the Nanobody of FW4 aminoacid sequence ^TM, described FW4 aminoacid sequence be disclosed in from Nguyen etc., EMBO J, 2000, Vol 19, No.5, and any kind among Fig. 2 of pp921-930 (924 pages) is that the aminoacid sequence of the segmental corresponding FW of VHH is identical or have and reach 5 kinds of amino acid differences from described corresponding FW.

(xxiv) has FW1, the Camelid VHH or the Nanobody of 2,3 and 4 aminoacid sequences ^TMDescribed FW1,2,3 and 4 aminoacid sequences be disclosed in from Nguyen etc., EMBO J, 2000, Vol 19, No.5, and any kind among Fig. 2 of pp921-930 (924 pages) is the identical or FW1 wherein of the aminoacid sequence of the segmental corresponding FW of VHH, 2,3 and 4 aminoacid sequences have jointly and reach 10 kinds of amino acid differences from described corresponding FW.

c)

(i) from the VH or the VL of any kind in the following antibody product:

Target product A b class illness

EpCAM Panorex mouse colon cancer

The chimeric IgG1 non-Hodgkin lymphoma of CD20 Rituxin (NHL)

CD20 Ze Waling mouse 90Y NHL

Humanization IgG4

CD33 Mai Luota toxin medicine is sewed AML (acute myeloid leukaemia)

CD52 Campath-1H humanization IgG1 B-CLL

Her-2/neu Trastuzumab humanization IgG1 mammary cancer

GPIIb/IIIa ReoPro chimeric Fab angina

RSV Synagis humanization IgG1 respiratory syncytial virus (RSV)

IgE Xolair humanization IgG1 asthma, allergic rhinitis

TNF-α class restrains chimeric IgG1 rheumatoid arthritis (RA),

Crohn's disease, psoriatic, tetanic

Spondylitis, the psoriasis joint

Inflammation, ulcerative colitis

The chimeric IgG1 transplant rejection of CD25 Shu Lai

OKT3，

CD3 Orthoclone mouse IgG2a transplant rejection

CD25 Zenapax humanization IgG1 renal transplantation

RA, Crohn's disease, psoriatic,

Ankylosing spondylitis, psoriasis

TNF-α Humira human IgG1 sacroiliitis, ulcerative colitis

CD20 Bexxar NHL

CD11a Raptiva psoriatic

A4B3 Antegren multiple sclerosis

EGFR Erbitux colorectal carcinoma

VEGF Zarator colorectal carcinoma, kidney

(ii) be disclosed in US6090382, US20030235585A1, US20040166111A1, EP1500329A2, US20040131613A1, US20030144484A1, US5698195, EP1159003A1, US20020119152A1, EP0871641A4, EP0710121B1, US5702705, anti-TNF antibody among the EP0487610B1 or the variable domain of antibody fragment (VH or VL), the content that will wherein disclose (particularly antibody and the segmental generation that wherein discloses, sequence and application) is incorporated herein by reference especially, so that be provided for this category information of the present invention for those skilled in the art.

(iii) with 1 * 10 of the mensuration that resonates by surperficial plasmon ^-8M or 1 * 10 ^-8The K that M is following _dHeavy or the light chain variable territory of dissociated antibody from target ligands (for example VH, VL or VHH).In a preferred embodiment, target ligands is a target ligands described in the appendix 1.In order to instruct surperficial plasmon resonance, those skilled in the art are with reference to US6090382 or WO04003019A2.

(iv) with 1 * 10 of the mensuration that resonates by surperficial plasmon ^-3s ^-1Or 1 * 10 ^-3s ^-1Following K _OffHeavy or the light chain variable territory of velocity constant dissociated antibody from target ligands (for example VH, VL or VHH).In a preferred embodiment, target ligands is a target ligands described in the appendix 1.In order to instruct surperficial plasmon resonance, those skilled in the art are with reference to US6090382 or WO04003019A2.

(v) with 1 * 10 of the mensuration that resonates by surperficial plasmon ^-8M or 1 * 10 ^-8The K that M is following _dWith 1 * 10 ^-3s ^-1Or 1 * 10 ^-3s ^-1Following K _OffHeavy or the light chain variable territory of velocity constant dissociated antibody from target ligands (for example VH, VL or VHH).In a preferred embodiment, target ligands is a target ligands described in the appendix 1.In order to instruct surperficial plasmon resonance, those skilled in the art are with reference to US6090382 or WO04003019A2.

(vi) with all by the resonance of surperficial plasmon measure 1 * 10 ^-8M or 1 * 10 ^-8The K that M is following _dWith 1 * 10 ^-3s ^-1Or 1 * 10 ^-3s ^-1Following K _OffHeavy or the light chain variable territory of velocity constant dissociated antibody from target ligands (for example VH, VL or VHH), and they in standard test with 1 * 10 ^-7M or 1 * 10 ^-7The IC that M is following ₅₀In and the target ligands cytotoxicity.In a preferred embodiment, target ligands is a target ligands described in the appendix 1.In order to instruct surperficial plasmon resonance, those skilled in the art are with reference to US6090382 or WO04003019A2.In one embodiment, target ligands is that TNF α and standard test are measured for L929 described in US6090382.

In the above-described embodiment, preferred general part is with 1 * 10 ^-9M or 1 * 10 ^-9Below the M, 1 * 10 ^-9M or 1 * 10 ^-9Below the M, 1 * 10 ^-11M or 1 * 10 ^-11Below the M, 1 * 10 ^-12M or 1 * 10 ^-12The K that M is following _dIn conjunction with.

Preferred K _OffVelocity constant is 1 * 10 ^-4s ^-1Or 1 * 10 ^-4s ^-1Below, 1 * 10 ^-5s ^-1Or 1 * 10 ^-5s ^-1Below, 1 * 10 ^-6s ^-1Or 1 * 10 ^-6s ^-1Below, 1 * 10 ^-7s ^-1Or 1 * 10 ^-7s ^-1Below, 1 * 10 ^-8s ^-1Or 1 * 10 ^-8s ^-1Below.

Preferred IC ₅₀Be 1 * 10 ^-8M or 1 * 10 ^-8Below the M, 1 * 10 ^-9M or 1 * 10 ^-9Below the M, 5 * 10 ^-10M or 5 * 10 ^-10Below the M, 1 * 10 ^-10M or 1 * 10 ^-10Below the M, 5 * 10 ^-11M or 5 * 10 ^-11Below the M.

D) have with appendix 2 (a) in the antibody variable domains of sequence of sequence at least 90% homology.

Have with appendix 2 (c) in the antibody variable domains of sequence of sequence at least 90% homology.

Claims

1. the method for selection function variable domain colony from the repertoire of antibody polypeptides class, described functional variable domain is in conjunction with target ligands and general part, this general part can be in conjunction with the functional member in the repertoire, but irrelevant with the specificity of target ligands, this method comprises the following steps:

A) make repertoire contact and select the functional variable domain of bonded with it with described general part; With

Wherein (i) variable domain is that heavy chain variable domain and general part are the light chain of antibody variable domain; Or (ii) variable domain is that light chain variable territory and general part are the heavy chain of antibody variable domain; And

2. the process of claim 1 wherein that the repertoire that at first makes the antibody polypeptides class contacts target ligands, and contact general part then.

3. claim 1 or 2 method, wherein general part is in conjunction with the subgroup of variable domain repertoire.

4. the method for claim 3, wherein two or more subgroups are selected from the repertoire of polypeptide class.

5. the method for claim 4 is wherein used two or more general parts, optional two or more light chain variable territories (just optional (i) with regard to) or two or more heavy chain variable domains (with regard to choose wantonly (ii) with regard to) select.

6. claim 4 or 5 method wherein merge two or more subgroups so that produce other repertoire of polypeptide class after selection.

7. the method that any aforesaid right requires wherein makes two or more repertoires of polypeptide class contact general part and merge thus obtained polypeptide class subgroup then.

8. the method for selection function T-cell receptors structural domain colony from the repertoire of polypeptide class, described functional T-cell receptors structural domain is in conjunction with target ligands and general part, this general part can be in conjunction with the functional member in the repertoire, but irrelevant with the specificity of target ligands, this method comprises the following steps:

A) repertoire is contacted with described general part and select the functional T-cell receptors structural domain of bonded with it; With

Wherein (i) T-cell receptors structural domain is V _αStructural domain and general part are T-cell receptors V _βStructural domain; Or (ii) T-cellularstructure territory is T-cell receptors V _βStructural domain and general part are T-cell receptors V _αStructural domain; And

Choose in (i) T-cell receptors V wantonly _αStructural domain is the Camelid structural domain that derives from Camelid; Or choose wantonly at (i) and (ii), the T-cell receptors structural domain is separately for people's structural domain or derive from the people.

9. a method is wherein selected heavy chain of antibody variable domain colony according to claim 1, and selects light chain of antibody variable domain colony according to claim 1, and merges thus obtained colony then.

10. a method is wherein selected T-cell receptors V according to claim 8 _αStructural domain colony, and according to claim 8 selection T-cell receptors V _βStructural domain colony, and merge thus obtained colony then.

11. select the method for at least a heavy chain of antibody variable domain from the antibody polypeptides types of populations, this method comprises:

12. the described method of claim 11 before described method is included in step a), makes antibody polypeptides class contact target ligands and the selection step in conjunction with the antibody polypeptides class of target ligands, is provided for the colony of the described antibody polypeptides class of step a) thus.

13. the described method of claim 11 after described method is included in step b), makes the heavy chain of antibody variable domain contact target ligands of selecting and selects step in conjunction with the heavy chain variable domain of target ligands in step b).

14. any described method among the claim 11-13, the heavy chain structural domain of wherein selecting in step b) are separately from organizing down: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.

15. any described method among the claim 11-13, the heavy chain structural domain of wherein selecting in step b) are humanization Camelid or mouse heavy chain variable domain or humanization Nanobody separately ^TM

16. any described method among the claim 11-13, the heavy chain structural domain of wherein selecting in step b) are people's heavy chain variable domain separately.

17. any described method among the claim 11-16, wherein behaviour light chain variable territory, light chain variable territory or the light chain variable territory that derives from the people or have the FW2 sequence, described FW2 sequence is that the FW2 of gene order DPK9 coding is identical with planting.

18. any described method among the claim 11-16, wherein the light chain variable territory is Camelid light chain variable territory or derives from Camelid.

19. any described method among the claim 11-18, wherein the colony in step a) is provided by the B-cell mass.

20. the described method of claim 19, wherein the B-cell is a peripheral blood lymphocyte.

21. claim 19 or 20 described methods are wherein separated the B-cell from the animal of using the target antigen immunization.

22. claim 19 or 20 described methods are wherein never used and are separated the B-cell in the animal of target antigen immunization.

23. any described method among the claim 11-16, the colony that wherein uses in step a) is provided by the repertoire of the antibody polypeptides class of the antibody gene coding of resetting with synthesis mode.

24. any described method among the claim 11-16, the colony that wherein uses in step a) is provided by the phage display library that comprises the phage of showing described antibody polypeptides class.

25. any described method among the claim 11-24, the colony that wherein uses in step a) comprises: (i) each self-contained at least a not with the antibody polypeptides class of light chain variable territory paired heavy chain variable domain; (ii) each self-contained and antibody polypeptides class light chain variable territory paired heavy chain variable domain.

26. any described method among the claim 11-25, the colony that wherein uses in step a) comprises single variable domain of camelid heavy chain (VHH) or Nanobodies ^TM

27. any described method among the claim 11-25, the colony that wherein uses in step a) comprises the single variable domain of people's heavy chain (VH).

28. select the method for at least a Camelid antibody VHH structural domain from Camelid antibody polypeptides types of populations, the B-cell of the Camelid of immunization target antigen provides described Camelid antibody polypeptides types of populations by separating certainly, this method comprises:

29. the described method of claim 28, wherein behaviour light chain variable territory, light chain variable territory.

30. claim 28 or 29 described methods wherein provide B-cell in a plurality of holes or consent, wherein each hole or consent comprise average a kind of B-cell type.

31. a method, described method comprises:

A) the initial colony of antagonist polypeptide class uses target antigen to carry out SLAM (the lymphocyte antibody method of selection) so that select antibody polypeptides types of populations in conjunction with target antigen; With

B) use the colony of selection as the antibody polypeptides types of populations that is used for any described method of claim 11-30.

32. any described method among the claim 11-31 wherein in step b), makes at least a in the heavy chain of antibody variable domain of selection merge with protein portion or put together with it.

33. the described method of claim 32, wherein protein portion is selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

34. any described method among the claim 11-31, wherein in step b), at least a in the heavy chain of antibody variable domain of selection for being selected from IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv and disulfide bonding antibody moiety or the antibody fragment of Fv.

35. the isolated antibody polypeptide, it comprises the heavy chain of antibody variable domain or is made up of it, and wherein this polypeptide can obtain by the described method of claim 11, and wherein light chain variable territory behaviour light chain variable territory in this method and heavy chain variable domain are from non--people's Mammals.

36. claim 35 described antibody polypeptides, wherein heavy chain variable domain is from the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH.

37. the described antibody polypeptides of claim 36, wherein heavy chain variable domain provides as Camelid IgG or the integral part that derives from the IgG of Camelid.

38. the described antibody polypeptides of claim 36, wherein heavy chain variable domain provides as human IgG or the integral part that derives from people's IgG, and wherein heavy chain variable domain in IgG with the light chain variable territory pairing that is different from the territory of light chain variable described in the claim 11.

39. any one antibody polypeptides is as the application of medicine among the claim 35-38.

40. any one antibody polypeptides application in the treating and/or preventing of human disease or illness among the claim 35-38.

41. use described in any described antibody or claim 39 or 40 among any described method or the claim 35-38 among the claim 11-34, wherein heavy chain variable domain is in conjunction with in the target listed in target ligands that is selected from TNF α, serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP 12, PDK1 and amyloid beta (A-β) or the appendix 1 any one.

42. select the method for at least a light chain of antibody variable domain from the antibody polypeptides types of populations, this method comprises:

43. the described method of claim 42 before described method is included in step a), makes antibody polypeptides class contact target ligands and the selection step in conjunction with the antibody polypeptides class of target ligands, is provided for the described colony of the antibody polypeptides class of step a) thus.

44. the described method of claim 42 after described method is included in step b), makes the light chain of antibody variable domain contact target ligands of selecting in the step b) and selects step in conjunction with the light chain variable territory of target ligands.

45. any described method among the claim 42-44, the light chain structural domain of wherein selecting in the step b) derives from Camelid separately.

46. any described method among the claim 42-44, the light chain structural domain of wherein selecting in the step b) are people's light chain variable territory separately.

47. any described method among the claim 42-44, wherein heavy chain variable domain is: people's heavy chain variable domain; Derive from the people; Heavy chain variable domain with FW2 sequence, described FW2 sequence is that the FW2 of gene order DP47 coding is identical with planting; Or to have with planting be 44,45 and 47 44,45 and 47 identical heavy chain variable domains of gene order DP47 coding.

48. any described method among the claim 42-44, wherein heavy chain variable domain is Camelid heavy chain variable domain (VHH or VH) or derives from Camelid.

49. any described method among the claim 42-48, wherein the colony in the step a) is provided by the B-cell mass.

50. the described method of claim 49, wherein the B-cell is a peripheral blood lymphocyte.

51. claim 49 or 50 described methods, wherein the B-cellular segregation is from the animal of immunization target antigen.

52. claim 49 or 50 described methods, wherein the B-cellular segregation is from the animal of immunization target antigen not.

53. any described method among the claim 42-48, wherein the colony that uses in the step a) is provided by the repertoire of the antibody polypeptides class of the antibody gene coding of resetting with synthesis mode.

54. any described method among the claim 42-48, wherein the colony in the step a) is provided by the phage display library that comprises the phage of showing described antibody polypeptides class.

55. any described method among the claim 42-54, wherein the colony that uses in the step a) comprises: (i) each self-contained at least a not with the antibody polypeptides class in heavy chain variable domain paired light chain variable territory; (ii) each self-contained and antibody polypeptides class heavy chain variable domain paired light chain variable territory.

56. any described method among the claim 42-54, wherein the colony that uses in the step a) comprises the single variable domain of people's light chain (VL).

57. a method, described method comprises:

B) use the colony of selection as the antibody polypeptides types of populations that is used for any described method of claim 42-56.

58. any described method among the claim 42-57, wherein in step b), at least a and protein portion in the light chain of antibody variable domain of selection merges or puts together with it.

59. the described method of claim 58, wherein said protein portion are selected from phage coat protein, one or more antibody structure territories, antibody Fc structural domain, enzyme, toxin, mark and effect group.

60. any described method among the claim 42-59, wherein in step b), at least a in the heavy chain of antibody variable domain of selection for being selected from IgG, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv and disulfide bonding antibody or the antibody fragment of Fv.

61. the isolated antibody polypeptide, it comprises the light chain of antibody variable domain or is made up of it, and wherein this polypeptide can obtain by the described method of claim 42, and wherein heavy chain variable domain behaviour heavy chain variable domain in this method and light chain variable territory are from non--people's Mammals.

62. the described antibody polypeptides of claim 61, wherein the light chain variable territory is from Camelid.

63. the described antibody polypeptides of claim 61, wherein the light chain variable territory provides as Camelid IgG or the integral part that derives from the IgG of Camelid.

64. the described antibody polypeptides of claim 61, wherein the light chain variable territory provides as human IgG or the integral part that derives from people's IgG, and wherein the light chain variable territory in IgG with the heavy chain variable domain pairing that is different from heavy chain variable domain described in the claim 42.

65. any one antibody polypeptides derivative among claim 35-38 and the 61-64, wherein this derivative has the CDR3 sudden change of comparing with the variable domain CDR3 of any described polypeptide among the 61-64 with claim 35-38.

66. pass through the antibody polypeptides derivative that the affinity maturation of antibody polypeptides any among claim 35-38 and the 61-64 produces.

67. polypeptide, it comprises the transformation period prolongation that is connected with the derivative of any one antibody polypeptides or claim 65 or 67 among claim 35-38 and the 61-64, and wherein this part is selected from: PEG; The antibody constant domain; The antibody Fc district; White protein or its fragment; In conjunction with albuminised peptide or antibody fragment; The white protein fragment; Neonatal Fc receptor; Shift; Or transfer receptor.

68. any one antibody polypeptides is as the application of medicine among the claim 61-67.

69. any one antibody polypeptides application in the treating and/or preventing of human disease or illness among the claim 61-67.

70. any described antibody or claim 68 or 69 described application among any described method or the claim 61-67 among the claim 42-66, wherein the variable domain of light chain variable territory or antibody polypeptides is in conjunction with being selected from the target listed in the target ligands of TNF α, serum albumin, Feng's von willebrand's factor (vWF), IgE, interferon-gamma, EGFR, IgE, MMP12, PDK1 and amyloid beta (A-β) or the appendix 1 any one.

71. claim 1,11 or 42 described methods, described method comprise the mutant of the variable domain that produces selection or the step of derivative.

72. any described method wherein is provided at the B-cell mass in a plurality of holes or the consent among claim 19-22 and the 49-52, wherein each hole or consent comprise single B-cell type.

73. any described method wherein is provided at the B-cell mass in a plurality of holes or the consent among claim 19-22 and the 49-52, wherein each hole or consent comprise average a kind of B-cell type.

74. the method for separating IgG in the single variable domain of antibody from the antibody polypeptides types of populations that comprises single variable domain and IgG, this method comprises:

A) make the general part of described population exposed; With

Wherein general part antagonist CH1 structural domain, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain have binding specificity.

75. the described method of claim 74, wherein general part are selected from protein L, protein L structural domain or in conjunction with the derivative of the protein L in light chain variable territory; The structural domain of protein G, protein G or in conjunction with the derivative of the protein G of CH 1; Antibody and antibody fragment, affibody, ldl receptor structural domain and EGF structural domain.

76. the described method of claim 75, wherein general part is selected from dAb, Nanobody ^TM, scFv, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv or disulfide bonding Fv.

The single variable domain 77. any described method among the claim 74-76, variable domain are behaved, and IgG is a human IgG.

78. any described method among the claim 74-77, wherein general part is with 1mM or avidity binding antibody CH1 structural domain, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain below the 1mM.

79. any described method among the claim 74-77, wherein general part is with 1 micromole or avidity binding antibody CH1 structural domain, light chain constant domain (CL), IgG hinge or light chain of antibody variable domain below 1 micromole.

80. any described method among the claim 74-77, wherein general part is with 100nM or be lower than avidity binding antibody CH1 structural domain, light chain constant domain (CL), IgG hinge or the light chain of antibody variable domain of 100nM.

81. the method for the single variable domain of separation of C amelid VHH among the IgG from the antibody polypeptides types of populations that comprises Camelid VHH structural domain and IgG, this method comprises:

A) make the general part of described population exposed; With

B) select the generally subgroup of part of combination, from IgG, separate single variable domain thus;

Wherein general part is to (i) VHH but not to VH; Or (ii) heavy chain antibody (H2) hinge has binding specificity.

82. the described method of claim 81, wherein general part is selected from light chain of antibody variable domain, antibody and antibody fragment.

83. the described method of claim 82, wherein general part is selected from dAb, Nanobody ^TM, scFv, Fab, Fab ', F (ab) ₂, F (ab ') ₂, scFv, Fv or disulfide bonding Fv.

84. any described method among the claim 81-83, wherein general part with 1mM or the avidity below the 1mM in conjunction with VHH or heavy chain antibody (H2) hinge.

85. any described method among the claim 81-33, wherein general part with 1 micromole or the avidity below 1 micromole in conjunction with VHH or heavy chain antibody (H2) hinge.

86. any described method among the claim 81-83, wherein general part with 100nM or the avidity below the 100nM in conjunction with VHH or heavy chain antibody (H2) hinge.

87. any described method among the claim 74-86, wherein antibody polypeptides colony is provided by the B cell.

88. any described method among the claim 74-87, wherein variable domain is Camelid VHH, and IgG is Camelid IgG.

89. any described method among the claim 74-88, wherein variable domain is provided by Camelid heavy chain (H2) antibody.

90. any described method among the claim 74-89, the wherein general part of mark or label to it.

91. any described method, antibody polypeptides, derivative or an application among claim 1-7 and the 11-90, wherein general part is for being selected from appendix 2a), c), d) or antibody variable domains e).

92. select the method in conjunction with the single variable domain of target ligands and general part from the repertoire of antibody polypeptides class, described method comprises the following steps:

Wherein general part is for being selected from appendix 2c) or antibody variable domains e); And

Wherein, (i) when the variable domain of selecting is heavy chain variable domain, general part is the light chain variable territory; Or (ii) when the variable domain of selecting is the light chain variable territory, general part is a heavy chain variable domain.

93. the described method of claim 92, wherein the repertoire of antibody polypeptides class is the repertoire of heavy chain variable domain, and general part is the light chain variable territory.

94. the described method of claim 92, wherein the repertoire of antibody polypeptides class is the repertoire in light chain variable territory, and general part is a heavy chain variable domain.

95. any described method among the claim 92-94, described method comprise the mutant of the variable domain that produces selection or the step of derivative.

96. any described method among the claim 92-94, wherein general part combination and the identical target ligands kind of selecting of variable domain.

97. any described method among the claim 92-94, wherein general part combination and the different target ligands kind of selecting of variable domain.

98. any described method among the claim 92-97, described method comprise the variable domain and the antibody variable domains merging identical with general part or derivatives thereof that will select, so that produce the product with target ligands binding specificity.

99. produce appendix 2c in conjunction with target ligands) (i)-(iv) in arbitrarily antibody or the method for the derivative of antibody fragment, this method comprises:

A) use described antibody or segmental heavy chain variable domain or with the described method of claim 94 in the identical variable domain of general part, and wherein the target ligands that uses in the step a) is described antibody or fragment bonded target ligands, selects the single variable domain of light chain in conjunction with target ligands and heavy chain variable domain thus; With

B) with the light chain variable territory of selecting; Identical light chain variable territory or derivatives thereof replaces at least a in described antibody or the segmental light chain variable territory.

100. produce appendix 2c) (i)-(iv) in the method for polyspecific derivative of antibody in arbitrarily or antibody fragment, this method comprises:

A) use described antibody or segmental heavy chain variable domain or with the described method of claim 94 in the identical variable domain of general part, and wherein the target ligands that uses in the step a) is the target ligands that is different from described antibody or fragment bonded target ligands, selects the single variable domain of light chain in conjunction with the variable domain of different target ligands and heavy chain thus; With

B) with the light chain variable territory of selecting; Identical light chain variable territory or derivatives thereof replaces at least a in described antibody or the fragment light chain variable territory, consequent polyspecific product.

101. produce appendix 2c) (i)-(iv) in the method for derivative of antibody in arbitrarily or antibody fragment, this method comprises:

A) use described antibody or segmental light chain variable territory or with the described method of claim 94 in the identical variable domain of general part, and wherein the target ligands that uses in the step a) is described antibody or fragment bonded target ligands, selects the single variable domain of heavy chain in conjunction with target ligands and light chain variable territory thus; With

B) with the heavy chain variable domain of selecting; Identical heavy chain variable domain or derivatives thereof replaces at least a in described antibody or the segmental heavy chain variable domain.

102. produce appendix 2c) (i)-(iv) in the method for polyspecific derivative of antibody in arbitrarily or antibody fragment, this method comprises:

A) use described antibody or segmental light chain variable territory or with the described method of claim 94 in the identical variable domain of general part, and wherein the target ligands that uses in the step a) is the target ligands that is different from described antibody or fragment bonded target ligands, selects the single variable domain of heavy chain in conjunction with different target ligands and light chain variable territory thus; With

B) with the heavy chain variable domain of selecting; Identical heavy chain variable domain or derivatives thereof replaces at least a in described antibody or the segmental heavy chain variable domain, produces the polyspecific product thus.

103. the described method of claim 100 to 102, wherein heavy chain variable domain is selected from: the heavy chain variable domain that derives from Camelid; The VHH structural domain; Nanobody ^TMThe VHH that on 44, has glycine; On 45, has leucic VHH; The VHH that on 47, has tryptophane; Have glycine on 44 and on 45, having leucic VHH; Have glycine on 44 and on 47, having the VHH of tryptophane; Have leucine on 45 and on 47, having the VHH of tryptophane; On 44, have glycine, have leucine on 45 and on 47, having the VH of tryptophane; On 103, have tryptophane or arginic VHH; Humanization Camelid or mouse heavy chain variable domain; Humanization Nanobody ^TMPeople's heavy chain variable domain; Derive from people's heavy chain variable domain; Heavy chain variable domain with FW2 sequence, described FW2 sequence is that the FW2 of gene order DP47 coding is identical with planting; Or to have with planting be 44,45 and 47 44,45 and 47 identical heavy chain variable domains of gene order DP47 coding.

104. claim 99 or 101 described methods, wherein the light chain variable territory is selected from people's light chain variable territory; Derive from people's light chain variable territory; Light chain variable territory with FW2 sequence, described FW2 sequence is that the FW2 of gene order DPK9 coding is identical with planting; Camelid light chain variable territory; Derive from the light chain variable territory of Camelid; With humanization Camelid or mouse light chain variable territory.

105. any described method among the claim 99-104, wherein the variable domain of a) selecting is a heavy chain variable domain, and antibody or the selecteed separately heavy chain variable domain of segmental heavy chain variable domain; Identical heavy chain variable domain or derivatives thereof replaces; Or b) variable domain of Xuan Zeing is the light chain variable territory, and antibody or selecteed separately light chain variable territory, segmental light chain variable territory; Identical light chain variable territory or derivatives thereof replaces.

106. can be by any derivative that described method obtains among the claim 99-105.

107. antibody or antibody fragment derivative, it is selected from Panorex ^TMRituxin ^TMZevalin ^TMMylotarg ^TMCampath ^TMHerceptin ^TMReoPro ^TMSynagis ^TMXolair ^TMRemicade ^TMSimulect ^TMOKT3 ^TMOrthoclone ^TMZenapax ^TMHumira ^TMBexxar ^TMRaptiva ^TMAntegren ^TMErbitux ^TMAnd Avastin ^TM, wherein this derivative can obtain by any described method among the claim 99-105.

108. the described method of claim 100, wherein general part is the antibody variable domains from antibody or antibody fragment, and described antibody or antibody fragment are selected from Panorex ^TM, Rituxin ^TM, Zevalin ^TM, Mylotarg ^TM, Campath ^TM, Herceptin ^TM, ReoPro ^TM, Synagis ^TM, Xolair ^TM, Remicade ^TM, Simulect ^TM, OKT3 ^TM, Orthoclone ^TM, Zenapax ^TM, Humira ^TM, Bexxar ^TM, Raptiva ^TM, Antegren ^TM, Erbitux ^TMAnd Avastin ^TMOr in conjunction with the identical variable domain or derivatives thereof of this antibody or antibody fragment institute bonded target ligands.

109. have the product of target ligands binding specificity, wherein this product can obtain by the described method of claim 108.

110. any described derivative or product are as the application of medicine in claim 106-107 and 109.

111. any described derivative or the product application in the treating and/or preventing of human disease or illness in claim 106-107 and 109.

112. any described derivative or the product application in the treating and/or preventing of human disease or illness in claim 106 or 109, wherein said illness is appendix 2c) (i) in antagonist or the listed illness of antibody fragment.