CN1968712A - Novel antigen-binding polypeptides and their uses - Google Patents

Abstract

Novel antigen-binding polypeptides (ABP) and uses thereof are provided.

Description

Novel antigen-binding polypeptides and its purposes

The cross case

The application's case is advocated the U.S. Provisional Patent Application case the 60/581st of filing an application on June 18th, 2004, the U.S. Provisional Patent Application case the 60/648th that on January 28th, No. 334 1 filed an application, No. 222 and the U.S. Provisional Patent Application case the 60/654th of filing an application on February 17th, 2005, No. 018 priority, the description of described patent application case are incorporated herein by reference in full.

Technical field

The present invention relates to comprise the novel antigen-binding polypeptides of at least a non-naturally encoded amino acids.The invention particularly relates to and use chemistry and recombinant DNA to produce by molecular biology method and the selection antigen-binding polypeptides, belong to biomedicine field.

Background technology

Four poly structures that the antibody of natural generation (Ab) is made up of two identical immunoglobulins (Ig) heavy chain and two identical light chains.The heavy chain of Ab and light chain are made up of same area not.Each light chain has a variable domain (VL) and a constant domain (CL), and each heavy chain has a variable domain (VH) and three or four constant domains (CH).Each functional domain of being made up of about 110 amino acid residues is folded into by two β lamellas packs formed characteristic β sandwich structure mutually, is called immunoglobulin folding.The VL territory respectively has three complementary determining regions (CDR1-3), and the VH territory respectively has nearly four complementary determining regions (CDR1-4), and complementary determining region is ring or the corner that connects the β chain at an end of functional domain.The variable region of light chain and heavy chain helps antigenic specificity usually, although and respective chains equates specific contribution is also nonessential.By the ring of CDR at random, antibody molecule has developed into and has been incorporated into many molecules.

Can be by Proteolytic enzyme and the function minor structure by recombinant methods Ab.These function minor structures comprise Fab fragment, Fv fragment and Fc part, wherein the Fab fragment comprises heavy chain VH-CH1 territory and the light chain VL-CL1 territory that connects by an intrachain disulfide bond, the Fv fragment only comprises VH and VL territory, and Fc partly comprises the non-antigen binding domain of molecule.In some cases, single VH territory keep with antigenic remarkable affinity (people such as Ward, 1989, Nature 341,554-546).Shown that also some single poly-κ light chain should be incorporated into its antigen specifically.(people such as L.Masat, 1994, PNAS 91:893-896).Have been found that isolating light chain or heavy chain also keep sometimes some antigen-binding activities (people such as Ward, 1989, Nature 341,554-546).

Another function minor structure is strand Fv (scFv), its comprise by the variable region of covalently bound heavy chain immunoglobulin of peptide connexon and light chain (people such as S-z Hu, 1996, Cancer Research, 56,3055-3061).These little (Mr 25,000) albumen keep specificity and the affinity with single polypeptide endoantigen usually, and can provide convenient means unit (building block) big, the antigenic specificity molecule.The short-half-life of scFV limits its treatment effectiveness in many cases in the circulation.

With part Ig VH territory as stencil design be called " miniantibody (minibody) " the small protein skeleton (people such as Pessi, 1993, Nature 362,367-369).Also then use the phage display method to select mutant to differentiate with interleukin-6 to have high affinity (dissociation constant (K by making corresponding to the CDR1 of VH and the ring randomization of CDR2 _d) be about 10 ^-7Miniantibody M) (people such as Martin, 1994, EMBO is J.13.5303-5309).

When analyzing the IgG sample material of camel serum, find that camel lacks variable light chain territory usually, show that enough antibody specificities and affinity may only derive from VH territory (three or four CDR rings).Made " camelization " VH territory with high-affinity, and can be only by making the CDR3 randomization produce high specific.

" miniantibody " one substitutes is " miniature bifunctional antibody (diabody) ".Miniature bifunctional antibody is little, bivalence, the bispecific antibody fragment with two antigen binding sites.Described fragment is included in same polypeptide chain (V _H-V _L) go up and light chain variable territory (V _L) heavy chain variable domain (V that is connected _H).Miniature bifunctional antibody size is similar to the Fab fragment.Can not make paired connexon between two functional domains on the same chain by using too short, make on functional domain and another chain the pairing of complementary function territory and produce two antigen binding sites.These dimerization antibody fragments or " miniature bifunctional antibody " be bivalence and have a bispecific.Referring to, people such as P.Holliger, PNAS 90:6444-6448 (1993).

Made CDR peptide and organic C DR analogies (people such as Dougall, 1994, Trends Biotechnol.12,372-379).The CDR peptide is the small peptide (being generally ring-type) of the aminoacid sequence of corresponding antibody CDR ring.The CDR ring is responsible for antibody-antigen and is done mutually.CDR peptide and organic C DR analogies have shown and have kept some binding affinities (Smyth ﹠amp; Von Itzstein, 1994, J.Am.Chem.Soc.116,2725-2733).Mice CDR has been transplanted to human Ig framework and do not lose affinity (people such as Jones, 1986, Nature 321,522-525; People such as Riechmann, 1988).

In health, select specificity Ab and from big amplified library (affinity maturation).Can use the described process of reorganization library technical profession replication in vitro.On the bacteriophage surface, successfully show the Ab fragment made produce and screen many CDR mutants become possible (people such as McCafferty, 1990, Nature 348,552-554; People such as Barbas, 1991, Proc.Natl.Acad.Sci.USA 88,7978-7982; People such as Winter, 1994, Annu.Rev.Immunol.12,433-455).Produce increasing Fab and Fv (and derivant) by this technology.Recombinant technique can combine with the Ab analogies.

The many albumen territory that may serve as the albumen skeleton has shown as the fusant with the phage capsid protein.Referring to Clackson ﹠amp; Wells, Trends Biotechnol.12:173-184 (1994).With in these albumen territories several as skeleton with the display random peptide sequence, comprise ox pancreas insulin inhibitor (people such as Roberts, PNAS 89:2429-2433 (1992)), human growth hormone (people such as Lowman, Biochemistry30:10832-10838 (1991), Venturini et al., Protein Peptide Letters 1:70-75 (1994)) and streptococcus (Streptococcus) IgG in conjunction with territory (people such as C ' Neil, Techniques in Protein ChemistryV (Crabb, L, editor) 517-524 page or leaf, Academic Press, San Diego (1994)).These folding single random loops or districts of having shown.With tendamistat (tendamistat) as on the filobactivirus M13 present skeleton (presentation scaffold) (McConnell and Hoess, 1995, J.Mol.Biol.250:460-470).

The receptor tyrosine kinase of ErbB family plays crucial effects in cell growth and differentiation.The abnormal activation of these receptors is relevant with human cancer.Dimerization (receptor pairing) is that all ErbB receptor signal transductions are active necessary.Show the active ability that directly suppresses ErbB2 and other ErbB2 receptor protein dimerization of blocking-up ErbB2 dimerization.Suppress the activation of receptor dimerization prevention ErbB signal transduction pathway.The antagonistic molecule of downward modulation ErbB signal transduction can be served as antitumor agent.At present, ErbB signal transduction network is the main target of developing anti-tumor medicaments.ErbB-1 is the specific receptor of EGF, and ErbB-2 has unknown native ligand.ErbB2 can form heterodimer with ErbB-1 when adding EGF.ErbB2 also serves as the first-selected dimerization companion of kinase dead (kinase-dead) ErbB-3 and ErbB-4, and described both are the receptor of neuroregulation element (neuregulin).Also can during signal transduction, activate ErbB signal transduction network with indirect mode by the part by cytokine and G coupling protein receptor, show that it plays an important role in the Growth Control of many different cell types.

The proto-oncogene c-erbB-1 EGF-R ELISA of encoding.Its name originates from from birds erythroblastosis virus (avian erythroblastosis virus, AEV) the viral homology v-erbB that separates, its fragment as the chicken c-ErbB-1 gene that lacks the amino terminal ligand binding domain is contained in this virus.The overexpression of erbB-1 gene takes place in comprising many tumors of various sites squamous cell carcinoma and adenocarcinoma.Human c-erbB-1 gene is arranged in chromosomal region 7p14 and 7p12.

ErbB-2 proto-oncogene (being called Neu, EGFR-2 or HER-2 again) is the member of transmembrane receptor family tyrosine kinase, and it also comprises EGF receptor and EGFR-3 (HER-3 or ErbB-3).The ErbB-2 coding has the 185kDa transmembrane receptor sample glycoprotein of inherent tyrosine kinase activity.Although ErbB-2 does not have any known high-affinity part, can under the situation of no part, activate its kinase activity by overexpression or with heterogeneous combination of other member of ErbB family receptors.Finding the amplification of ErbB-2 gene and the overexpression of its product in the human breast carcinoma of 40% constitutional nearly.The ErbB-2 overexpression also is found in ovarian cancer, gastric cancer and nonsmall-cell lung cancer.Usually activate ErbB-2 by neuroregulation with plain receptor ErbB-3 of neuroregulation and ErbB-4 formation heterodimer.The anti-ErbB-2 monoclonal antibody of humanization He Saiting (Herceptin) (from monoclonal 4D5) has obtained FDA approval and has been used for the treatment of the cancer of overexpression ErbB-2.The another kind of anti-ErbB-2 antibody of developing is handkerchief trastuzumab (Pertuzumab) (from monoclonal 2C4).The specific inhibitor of the tyrosine kinase activity of ErbB-1 (EGF receptor) is also carrying out clinical trial.

Anti-ErbB antibody is known in affiliated technical field, and includes, but is not limited to United States Patent (USP) the 4th, 753, No. 894, the 5th, 169, No. 774, the 5th, 677, No. 171, the 5th, 720, No. 937, the 5th, 720, No. 954, the 5th, 725, No. 856, the 5th, 770, No. 195, the 5th, 772, No. 997, the 5th, 783, No. 186, the 6th, 054, No. 561, the 6th, 165, No. 464, the 6th, 333, No. 169, the 6th, 015, No. 567, the 6th, 387, No. 371, the 6th, 399, No. 063, the 6th, 441, No. 143, the 6th, 458, No. 356, the 6th, 627, No. 196, each patent documentation is all incorporated this paper by reference into.

Poly-(ethylene glycol) (the abbreviating PEG as) of covalently bound hydrophilic polymer is a kind of increase water solublity, biological usability, increase serum half-life, increase the treatment half-life, regulate immunogenicity, the method for regulating biological activity or prolonging many bioactive molecules (comprising albumen, peptide and specific hydrophobic molecule) circulation time.PEG has been widely used in pharmaceuticals, artificial graft's body and other application, and biocompatibility, shortage toxicity and shortage immunogenicity are important in these are used.For desirable characteristics maximization with PEG, be connected in the total molecular weight of PEG polymer of bioactive molecule and hydration status must be enough high can giving usually the favorable characteristics (for example increasing water solublity and increase circulating half-life) with PEG polymer join dependency, and can influence the biological activity of parent molecule sharply.

The PEG derivant is connected with bioactive molecule through for example lysine, cysteine and histidine residues, N end and the reactive chemical functional group of carbohydrate part usually.Albumen has a limited number of reactive site that polymer is connected that can be used for usually with other molecule.Be suitable for most connecting and the site modified plays an important role in receptors bind usually, and be that will to keep the biological activity of molecule necessary through polymer.Therefore, these reactive site indiscriminate is connected the biological activity that causes usually through polymer-modified molecule and significantly reduces or even completely lose on polymer chain and the bioactive molecule.People such as R.Clark, (1996), J.Biol.Chem., 271:21969-21977.Have the conjugate that is enough to target molecule is given the polymer molecular weight of required advantage in order to form, prior art approach is usually directed to being connected at random of many polymeric arms and molecule, and then increases that the parent molecule biological activity reduces or even the risk that completely loses.

The reactive site that is formed for the locus that the PEG derivant is connected with albumen is determined by protein structure.The albumen that comprises enzyme is made up of various alpha amino acid sequences, and it has general structure H ₂N--CHR--COOH.The amino part of an amino acid whose α (H ₂N--) in conjunction with contiguous amino acid whose carboxy moiety (--COOH) forming amido link, can by--(NH--CHR--CO) _n--expression, wherein subscript " n " can equal hundreds of or thousands of.Can contain by the fragment shown in the R and to be useful on protein biological activity and to be used for the reactive site that the PEG derivant is connected.

For instance, under the situation of amino acid lysine, in the ε position and the α position exist--NH ₂Part.ε position--NH ₂Part is free for the reaction under the alkaline pH condition.Major part has been directed to ε position--the NH that exploitation PEG derivant is used for connecting the lysine residue that is present in albumen with the technology that PEG carries out the field of protein derivedization ₂Part." Polyethylene Glycol and Derivatives for Advanced PEGylation ", NektarMolecular Engineering Catalog, 2003, the 1-17 pages or leaves.All these PEG derivants all have general restriction, yet they can not optionally be placed on the protein surface in existing many lysine residues.Under lysine residue for protein active is important situation, for example be present in the enzyme active sites, perhaps under the situation that lysine residue is done to work to mediation albumen and other biomolecule mutually, as under the situation of receptor binding site, this can be one and significantly limit.

Have second of albumen PEGization method now and be equal to important complicated factor and be that the PEG derivant also bad side reaction can take place with the residue outside the required residue.The reactive imino group part shown in--N (H)--that histidine contains by structure, but many and ε position--NH ₂The chemical reactivity material of reaction also can react with--N (H)--.Equally, the side chain of amino acid cysteine has by the free sulfhydryl groups shown in structure-SH.In some cases, at lysine ε--NH ₂The PEG derivant of group also can be reacted with cysteine, histidine or other residue.This can produce the deutero-bioactive molecule of PEG complexity, heterogeneous mixture, and destroy the active risk of target biomolecule.Need exploitation to allow the chemical functional group introduce the PEG derivant in the single site in the albumen, clearly define on one or more PEG polymer and the protein surface and the bioactive molecule selectivity coupling of predictable specificity site thereby can make.

Except lysine residue, the extensive work in the affiliated field is aimed at other amino acid side chain of exploitation targeting active PEG reagent of (comprising cysteine, histidine and N end).Referring to, for example United States Patent (USP) the 6th, 610, No. 281 (it incorporates this paper by reference into) and " Polyethylene Glycol and Derivatives for AdvancedPEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages or leaves.Can use in rite-directed mutagenesis and the affiliated field other known technology that cysteine residues site selectivity ground is introduced in the protein structure, and gained free sulfhydryl groups part can with the PEG derivatives reaction with thiol-reactive functional group.Yet the complicated part of described method is to introduce free sulfhydryl groups can make the proteic expression of gained, folding and stability become complicated.Therefore, need have the chemical functional group is introduced method in the bioactive molecule, so that can make one or more PEG polymer and the coupling of albumen selectivity, and simultaneously be typically found at chemical functional group in the albumen compatible (that is, not with the bad side reaction of its generation) with sulfydryl and other.

As the sampling result in the affiliated field as seen, verified, being used for of being developed is connected with proteic side chain, especially with lysine side-chain on--NH ₂On part and the cysteine side chain-in these derivants that SH partly is connected many its synthetic with use in have problems.Some form labile bond with the albumen that suffers hydrolysis, and thereby decompose, degraded or unstable in water environment, for example in blood flow.Some form more stable key, but suffer hydrolysis before forming key, mean reactive group on the PEG derivant may be before albumen be connected with regard to inactivation.Some have a little toxicity, and therefore are not suitable in vivo using.Some reactions are unavailable too slowly and in fact.Some cause the protein active forfeiture because being connected in the site of being responsible for protein active.Some do not have specificity to the site that it connected, this also may cause the loss of activity of wanting and result's repeatability lack.In order to overcome and to use the poly-relevant difficult problem of (ethylene glycol) part modified protein, (for example United States Patent (USP) 6 to have developed more stable PEG derivant, 602,498, it incorporates this paper by reference into) or with the PEG derivant of molecule and lip-deep thiol moiety selective reaction (for example United States Patent (USP) 6,610,281, it incorporates this paper by reference into).Affiliated field need be chemical inertness clearly until specifying selective reaction to form the PEG derivant of stablizing chemical bond in physiological environment.

Recently, reported in a kind of albumen science new technique fully, its promise overcomes many and the relevant restriction of protein loci specificity modification.Clear and definite, new component has been made an addition to prokaryote escherichia coli (Escherichia coli) (E.coli) (people such as L.Wang for example, (2001), Science292:498-500) and eukaryote saccharomyces cerevisiae (Sacchromyces cerevisiae) (S.cerevisiae) (people such as J.Chin for example, Science301:964-7 (2003)) in the protein biology combination mechanism, makes it possible to non-genetic coding aminoacid is in vivo incorporated in the albumen.Used the method effectively and with high fidelity many amino acids (comprising light affinity labelling and photoisomerization aminoacid, keto amino acid and glycosylation aminoacid) with novel chemistry, physics or biological property to be incorporated in the escherichia coli and yeast protein in response to amber codon TAG.Referring to, people such as J.W.Chin for example, (2002), Journal of the American Chemical Society124:9026-9027; J.W.Chin ， ﹠amp; P.G.Schultz, (2002), ChemBioChem11:1135-1137; People such as J.W.Chin, (2002), PNAS United States of America99:11020-11024; With L.Wang ， ﹠amp; P.G.Schultz, (2002), Chem.Comm.,1-10.These researchs are verified, can be optionally and be introduced in routinely in the albumen undiscovered, all functional groups in the aminoacid of 20 common genetic codings are chemically inert and can be used for effectively and optionally reacting the chemical functional group who stablizes covalent bond to form, for example ketone group, alkynyl and nitrine part.

Incorporate non-genetic coding aminoacid in the albumen ability and allow to introduce the chemical functional group that can replace natural generation functional group valuably, described natural generation functional group is the ε-NH of lysine for example ₂, sulfydryl-SH of cysteine, the imino group of histidine or the like.Known some chemical functional group is inertia to the functional group that is found in 20 common, genetic coding aminoacid, but can be clearly and react effectively to form stable keys.For instance, known azido in affiliated field and acetenyl suffer Huisgen[3+2 in water condition under the situation that has catalytic amount copper] cycloaddition reaction.Referring to, people such as Tornoe for example, (2002) Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002) Angew.Chem.Int.Ed.41:2596-2599.For instance, by nitrine is partly introduced in the protein structure, can introduce being found in that amine, sulfydryl, carboxylic acid, hydroxyl in the albumen is chemical inertness but can steadily and effectively react to form the functional group of cycloaddition product with acetylene moiety.Importantly, under the situation that does not have acetylene moiety, nitrine keeps chemical inertness and non-reacted under other albumen side chain situation of existence and under physiological condition.

The present invention especially is devoted to solve with antigen-binding polypeptides and segmental activity thereof and produces relevant problem, also is devoted to have the generation of the antigen-binding polypeptides that improves biological property or pharmacological property (for example improving the treatment half-life) simultaneously.

Summary of the invention

The invention provides the antigen-binding polypeptides (ABP) that comprises one or more non-naturally encoded amino acids.In certain embodiments, ABP comprises complete antibody heavy.In certain embodiments, ABP comprises the complete antibody light chain.

In certain embodiments, ABP comprises the variable region of light chain of antibody.In certain embodiments, ABP comprises the variable region of heavy chain of antibody.In certain embodiments, ABP comprises at least one CDR of light chain of antibody.In certain embodiments, ABP comprises at least one CDR of heavy chain of antibody.In certain embodiments, ABP comprises at least one CDR of light chain and at least one CDR of heavy chain.In certain embodiments, ABP comprises Fab.In certain embodiments, ABP comprises two or more Fab.In certain embodiments, ABP comprises scFv.In certain embodiments, ABP comprises two or more scFv.In certain embodiments, ABP comprises miniantibody.In certain embodiments, ABP comprises two or more miniantibodies.In certain embodiments, ABP comprises miniature bifunctional antibody.In certain embodiments, ABP comprises two or more miniature bifunctional antibodies.In certain embodiments, ABP comprises variable region of light chain and variable region of heavy chain.In certain embodiments, ABP comprises complete light chain and complete heavy chain.In certain embodiments, ABP comprises one or more Fc territories or its part.In certain embodiments, ABP comprises the combination of any the foregoing description.In certain embodiments, ABP comprises homodimer, heterodimer, homology polymer or the heteromultimers of any the foregoing description.In certain embodiments, ABP comprises and the bonded polypeptide of binding partners, and wherein binding partners comprises antigen, polypeptide, nucleic acid molecules, polymer or other molecule or material.In certain embodiments, ABP is relevant with non-antibody molecule of the skeleton or material.

In certain embodiments, ABP comprises one or more post translational modifications.In certain embodiments, ABP is connected with connexon, polymer or bioactive molecule.In certain embodiments, ABP is connected with double functional copolymer, difunctionality connexon or at least one other ABP.In certain embodiments, to be connected in not be the polypeptide of ABP to ABP.In certain embodiments, the antigen-binding polypeptides that comprises non-naturally encoded amino acids is connected in other antigen-binding polypeptides that one or more also can comprise non-naturally encoded amino acids.

In certain embodiments, non-naturally encoded amino acids is connected in water-soluble polymer.In certain embodiments, water-soluble polymer comprises poly-(ethylene glycol) part.In certain embodiments, poly-(ethylene glycol) molecule is the double functional copolymer.In certain embodiments, the double functional copolymer is connected in second polypeptide.In certain embodiments, second polypeptide is an antigen-binding polypeptides.

In certain embodiments, antigen-binding polypeptides comprises at least two aminoacid that are connected in the water-soluble polymer that comprises poly-(ethylene glycol) part.In certain embodiments, at least one aminoacid is non-naturally encoded amino acids.

In certain embodiments, antigen-binding polypeptides comprises when comparing with the affinity of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and regulates replacement, interpolation or the disappearance of antigen-binding polypeptides to antigenic affinity.In certain embodiments, antigen-binding polypeptides comprises when comparing with the stability of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and increases replacement, interpolation or the disappearance of antigen-binding polypeptides stability.In certain embodiments, antigen-binding polypeptides comprises when comparing with the immunogenicity of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and regulates the immunogenic replacement of antigen-binding polypeptides, interpolation or disappearance.In certain embodiments, antigen-binding polypeptides comprise when with correspondingly do not have replacement, add or the serum half-life of the antigen-binding polypeptides of disappearance or circulation time when comparing replacement, interpolation or the disappearance of regulating antigen-binding polypeptides serum half-life or circulation time.

In certain embodiments, antigen-binding polypeptides comprises when comparing with the water solublity of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and increases the water miscible replacement of antigen-binding polypeptides, interpolation or disappearance.In certain embodiments, antigen-binding polypeptides comprises when comparing with the dissolubility of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and increases the deliquescent replacement of antigen-binding polypeptides, interpolation or the disappearance that are produced in the host cell.In certain embodiments, antigen-binding polypeptides comprise when with correspondingly do not have replacement, add or the expression of the antigen-binding polypeptides of disappearance when comparing, increase in the host cell or replacement, interpolation or the disappearance of the in vitro synthetic antigen-binding polypeptides expression of institute.In certain embodiments, antigen-binding polypeptides comprises when comparing with the protease resistant of the corresponding antigen-binding polypeptides that does not have replacement, adds or lack, and increases replacement, interpolation or the disappearance of antigen-binding polypeptides protease resistant.

In certain embodiments, can replace with the aminoacid that natural generation or non-natural generation aminoacid carry out among the ABP, its restrictive condition is that at least one replacement is to carry out with non-naturally encoded amino acids.

In certain embodiments, non-naturally encoded amino acids comprises carbonyl, acetyl group, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.

In certain embodiments, non-naturally encoded amino acids comprises carbonyl.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R ₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R ₃For H, aminoacid, polypeptide or amino terminal are modified base, and R ₄For H, aminoacid, polypeptide or carboxyl terminal are modified base.

In certain embodiments, non-naturally encoded amino acids comprises aminooxy group.In certain embodiments, non-naturally encoded amino acids comprises hydrazide group.In certain embodiments, non-naturally encoded amino acids comprises diazanyl.In certain embodiments, the non-naturally encoded amino acids residue comprises amino urea groups.

In certain embodiments, the non-naturally encoded amino acids residue comprises azido.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.

In certain embodiments, non-naturally encoded amino acids comprises alkynyl.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.

In certain embodiments, polypeptide is at least a active agonist of antigen, partial agonist, antagonist, partial antagonist or inverse agonist.In certain embodiments, agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprise the non-naturally encoded amino acids that is connected in water-soluble polymer.In certain embodiments, water-soluble polymer comprises poly-(ethylene glycol) part.In certain embodiments, agonist, partial agonist, antagonist, partial antagonist or inverse agonist comprise non-naturally encoded amino acids and one or more post translational modifications, connexon, polymer or bioactive molecule.

The present invention also provide comprise coding for antigens in conjunction with the polynucleotide of polypeptide through isolating nucleic acid, wherein polynucleotide comprises at least one and selects codon, it includes, but is not limited to SEQ ID NO:18,20,22,25,27,29.In certain embodiments, select codon to be selected from by amber codon, ochre codon, opal codon, unique codon, rare codon and the molecular group of four base passwords.

The present invention also provides the method for making the antigen-binding polypeptides that is connected in water-soluble polymer.In certain embodiments, described method comprises to make and comprises contacting with the water-soluble polymer that comprises the part of reacting with non-naturally encoded amino acids through separating antigen-binding polypeptides of non-naturally encoded amino acids.In certain embodiments, incorporate non-naturally encoded amino acids in the antigen-binding polypeptides into, and described water-soluble polymer is not had a reactivity to any 20 common amino acids to water-soluble polymer tool reactivity.In certain embodiments, incorporate non-naturally encoded amino acids in the antigen-binding polypeptides into, and described connexon, polymer or bioactive molecule are not had a reactivity to any 20 common amino acids to connexon, polymer or bioactive molecule tool reactivity.

In certain embodiments, comprise the antigen-binding polypeptides that the antigen-binding polypeptides that contains carbonylamino acid and poly-(ethylene glycol) the molecular reaction manufacturing that comprises aminooxy group, diazanyl, hydrazide group or amino urea groups are connected in water-soluble polymer by making.In certain embodiments, aminooxy group, diazanyl, hydrazide group or amino urea groups are connected in poly-(ethylene glycol) molecule via amido link.

In certain embodiments, by being reacted with the polypeptide that comprises the non-naturally encoded amino acids that contains aminooxy group, diazanyl, hydrazide group or amino urea groups, poly-(ethylene glycol) molecule that comprises carbonyl makes the antigen-binding polypeptides that is connected in water-soluble polymer.

In certain embodiments, comprise the antigen-binding polypeptides that the antigen-binding polypeptides that contains alkynyl amino acid and poly-(ethylene glycol) the molecular reaction manufacturing that comprises the nitrine part are connected in water-soluble polymer by making.In certain embodiments, azido or alkynyl are connected in poly-(ethylene glycol) molecule via amido link.

In certain embodiments, contain amino acid whose antigen-binding polypeptides of nitrine and the antigen-binding polypeptides that (ethylene glycol) molecular reaction manufacturing is connected in water-soluble polymer that gathers that comprises alkynyl moiety by making to comprise.In certain embodiments, azido or alkynyl are connected in poly-(ethylene glycol) molecule via amido link.

In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between about 0.1kDa and the about 100kDa.In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between about 0.1kDa and the about 50kDa.

In certain embodiments, poly-(ethylene glycol) molecule is a branch polymer.In certain embodiments, each branch of poly-(ethylene glycol) branch polymer has between 1kD and the 100kDa or the molecular weight between 1kDa and the 50kDa.

In certain embodiments, the water-soluble polymer that is connected in antigen-binding polypeptides comprises the ployalkylene glycol part.In certain embodiments, the non-naturally encoded amino acids residue of incorporating in the antigen-binding polypeptides comprises carbonyl, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.In certain embodiments, the non-naturally encoded amino acids residue of incorporating among the ABP comprises carbonyl moiety, and water-soluble polymer comprises aminooxy group, diazanyl, hydrazide group or amino urea groups.In certain embodiments, the non-naturally encoded amino acids residue of incorporating in the antigen-binding polypeptides comprises alkynyl moiety, and water-soluble polymer comprises the nitrine part.In certain embodiments, the non-naturally encoded amino acids residue of incorporating in the antigen-binding polypeptides comprises the nitrine part, and water-soluble polymer comprises alkynyl moiety.

The present invention also provides the compositions that comprises the antigen-binding polypeptides that contains non-naturally encoded amino acids and pharmaceutically acceptable supporting agent.In certain embodiments, non-naturally encoded amino acids is connected in water-soluble polymer.

The present invention also provides and comprises coding for antigens in conjunction with polypeptide and cell that contain the polynucleotide of selecting codon.In certain embodiments, described cell comprises in order to non-naturally encoded amino acids is replaced quadrature RNA synzyme and/or the quadrature tRNA in the antigen-binding polypeptides into.

The present invention also provides the method for making the antigen-binding polypeptides that comprises non-naturally encoded amino acids.In certain embodiments, described method is included in to cultivate under the condition that allows antigen-binding polypeptides to express and comprises the cell of coding for antigens in conjunction with polynucleotide, quadrature RNA synzyme and/or the quadrature tRNA of polypeptide; With from cell and/or culture medium purifying antigen in conjunction with polypeptide.

The present invention also provides the method for the treatment half-life, serum half-life or the circulation time that increase antigen-binding polypeptides.The present invention also provides the immunogenic method of antigen-binding polypeptides of regulating.In certain embodiments, described method comprises with non-naturally encoded amino acids replaces any or more than one aminoacid in the natural generation antigen-binding polypeptides, and/or antigen-binding polypeptides is connected in connexon, polymer, water-soluble polymer or bioactive molecule.

The present invention also provides the method that needs the patient of this treatment with the antigen-binding polypeptides treatment of the present invention of effective dose.In certain embodiments, described method comprises the medical composition of the patient being thrown the antigen-binding polypeptides that contains non-naturally encoded amino acids with comprising of treatment effective dose and pharmaceutically acceptable supporting agent.In certain embodiments, non-naturally encoded amino acids is connected in water-soluble polymer.

The present invention also provides and comprises SEQ ID NO:19, and 21,23,24,26,28,30, the antigen-binding polypeptides of sequence shown in 31 and fragment thereof or any other antigen-binding polypeptides sequence is replaced by non-naturally encoded amino acids but condition is at least one aminoacid.In certain embodiments, non-naturally encoded amino acids is connected in water-soluble polymer.In certain embodiments, water-soluble polymer comprises poly-(ethylene glycol) part.In certain embodiments, non-naturally encoded amino acids comprises carbonyl, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.

The present invention also provides and comprises the pharmaceutically acceptable supporting agent and the medical composition of antigen-binding polypeptides, described antigen-binding polypeptides comprises SEQ ID NO:19,21,23,24,26,28, sequence shown in 30,31 and fragment thereof or any other antigen-binding polypeptides sequence, wherein at least one aminoacid is replaced by non-naturally encoded amino acids.In certain embodiments, non-naturally encoded amino acids comprises sugar moieties.In certain embodiments, water-soluble polymer is connected in polypeptide via sugar moieties.In certain embodiments, connexon, polymer or bioactive molecule are connected in antigen-binding polypeptides via sugar moieties.

The present invention also provides and comprises the antigen-binding polypeptides of water-soluble polymer that is connected in the single amino acid place of antigen-binding polypeptides through covalent bond.In certain embodiments, water-soluble polymer comprises poly-(ethylene glycol) part.In certain embodiments, the aminoacid that is covalently attached to water-soluble polymer is the non-naturally encoded amino acids that is present in the polypeptide.

The invention provides the antigen-binding polypeptides that comprises at least one connexon, polymer or bioactive molecule, wherein said connexon, polymer or bioactive molecule are connected in polypeptide by the functional group that incorporates the non-naturally encoded amino acids in the polypeptide through ribosome into.In certain embodiments, polypeptide coverlet PEGization.The present invention also provides the ABP polypeptide that comprises the connexon, polymer or the bioactive molecule that are connected in one or more non-naturally encoded amino acids, and wherein said non-naturally encoded amino acids is incorporated preliminary election site in the polypeptide into through ribosome.

In another embodiment, owing to be used for and the bonded unique chemical reaction of alpha-non-natural amino acid, therefore comprise the antigen-binding polypeptides that provides pure in fact that combines that amino acid whose antigen-binding polypeptides and another molecule (including, but not limited to PEG) take place one or more non-naturals.Can carry out combining of the antigen-binding polypeptides that comprises one or more non-naturally encoded amino acids and another molecule (for example PEG), and before or after integrating step, implement other purification technique simultaneously, so that pure in fact antigen-binding polypeptides to be provided.

Description of drawings

Fig. 1 is the general structure chart of antibody molecule (IgG) and antigen-binding portion thereof thereof.Antigen recognition site includes CDR.

Fig. 2 show be used for the scFv-108 pericentral siphon (Fig. 2, A) and Cytoplasm (Fig. 2, B) construct of expression/inhibition.The position of indication succinum termination codon.Show and be used for Fab-108 fragment (Fig. 2, C) the bicistronic mRNA cascade of expression/inhibition.Show the construct (Fig. 2, D and E) that is used for scFv-4D5 fragment periplasmic expression/inhibition.Show be used for Fab-4D5 fragment expression/inhibition cistron (Fig. 2, F).

Fig. 3 show the inhibition of amber mutation in second serine in the GlySer connexon (S131Am) (Fig. 3, A) and the corresponding IMAC purity analysis that contains the scFv of pAcF (Fig. 3, B).

Fig. 4 shows that the amber mutation in the VL chain (L156) suppresses during the scFv cytoplasmic expression.

Fig. 5 A shows segmental PEGization of pAcF-scFv-108 and dimerization.Indicate single PEGization scFv and dimeric position with single arrow and double-head arrow respectively.Fig. 5, B show the PEGization of pAcF-scFv-108 fragment-(S136).Fig. 5, the segmental PEGization of WT scFv is not observed in the C demonstration.

Fig. 6 is the gel that is presented at elution fraction collected during the scFv-108 homodimer purification.

Fig. 7 A-C shows combining of scFv albumen that contains pAcF or pAcF-PEG and the A431 cell of expressing the EGF receptor.

Fig. 8 A is the segmental gel of the Fab that contains pAcF or pAcF-PEG that shows mAb 108.Fig. 8, B-D show the Fab fragment of mAb 108 and combining of the A431 cell of expressing the EGF receptor.

Fig. 9 shows the example of allos difunctionality ABP of the present invention.

Figure 10 be show the C end (Figure 10, A) or N end scFv-4D5 (Figure 10, B) gel that amber mutation suppresses in second serine of segmental GlySer connexon.

Figure 11 is presented at pAcF-Fab-4D5-(K139) and Fab-4D5-cys (Figure 11, SDS-PAGE analysis A) under reduction and the non-reduced condition.Figure 11, B shows the Figure 11 that uses anti-His antibody, the Western trace of sample shown in the A.

Figure 12 shows among the HIV-1 that is connected in peptide T20 and human Fab 4E10.

Figure 13 is the diagram that shows the dimerization process.

That Figure 14 shows that the scFv dimer forms is non-reduced (Figure 14, A) and reduction (Figure 14, B) SDS-PAGE analysis.

Figure 15 shows that the dimeric SDS-PAGE of purified scFv analyzes.

The specific embodiment

Definition

Should be appreciated that the present invention is not limited to ad hoc approach as herein described, experimental program, cell line, construct and reagent, and therefore can change.Should be appreciated that also term used herein only is the purpose that is used to describe specific embodiment, but not be intended to limit protection scope of the present invention, protection scope of the present invention is limited only by the accompanying claims.

Unless this paper spells out in addition, otherwise as used in this paper and the appended claims, singulative " " and " described " comprise plural form.Therefore, for example mention that " antigen-binding polypeptides " or " ABP " is meant one or more described albumen, and its equipollent that comprises that one of ordinary skill in the art are known, or the like.

Unless otherwise defined, otherwise all scientific and technical terminologies used herein all have with this aspect one of ordinary skill in the art the general identical meanings of understanding.Although in enforcement of the present invention or check, can use similar or the method, device and the material that are equal to any and described herein, present description method for optimizing, device and material.

All open cases and patent that this paper is mentioned all are incorporated herein with for referencial use, to be used for the construct and the method for the possible use related to the present invention described in the open case of description and open (for example).The open disclosure of case before the application's case applying date discussed in this article only is provided.This paper never should be interpreted as permitting the present inventor and have no right to shift to an earlier date the present invention by previous invention or for any other reason.

Term " pure in fact " be meant in fact or be substantially free of follow usually the albumen in natural generation environment, found or with the ABP of the component of described interactions between protein (that is, producing under the situation of ABP in reorganization is n cell or host cell).The ABP that can not contain cellular material in fact comprise have be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 10%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than the protein formulation of about 1% (in dry weight) contaminating protein.When producing ABP or its variant by host cell reorganization, this contaminating protein can about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% about 1% or dry cell weight still less exist.When producing ABP or its variant, can there be the contaminating protein of about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or about 1mg/L or dry cell weight still less in the culture medium by the host cell reorganization.Therefore, measure as proper method by for example SDS/PAGE analysis, RP-HPLC, SEC and capillary electrophoresis, by " pure in fact " ABP that method of the present invention produced can have at least about 30%, purity at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, especially at least about 75%, 80%, 85% purity, and more particularly at least about 90% purity, purity at least about 95% is at least about 99% or higher purity.

" recombinant host cell " or " host cell " is meant and no matter uses which kind of insertion method (for example, directly picked-up, transduction, f cooperate (f-mating) or affiliated field to become known for setting up other technology of recombinant host cell) and comprise the cell of external source polynucleotide.The external source polynucleotide can be maintained nonconformity carrier (for example plasmid), perhaps can be integrated in the host genome.

Term as used herein " medium " comprises any culture medium, solution, solid, the semi-solid rigid support thing that maybe can support or contain any host cell, and described any host cell comprises bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryote host cell, mammalian host cell, Chinese hamster ovary celI or escherichia coli and cellular content.Therefore, the medium that host cell is grown can be contained in described term, and for example ABP is secreted in medium wherein, comprises the medium of propagation before or after the step.Buffer or the reagent that contains the host cell lysate also contained in described term, for example produces in cell and host cell is dissolved or destroy and discharge under the situation of ABP at ABP.

Be defined as in the sulfydryl of keeping reducing condition and the original molecule or any chemical compound or the material of intermolecular disulfide bond as this paper about folding again (protein refolding) used " Reducing agent " of albumen.Appropriate reductant include, but is not limited to dithiothreitol, DTT (dithiothreitol, DTT), 2 mercapto ethanol, dithioerythritol, cysteine, cysteamine (2-aminoothyl mercaptan) and reduced glutathion.One of ordinary skill in the art understand many Reducing agents easily and are applicable in the method and composition of the present invention.

Be defined as any chemical compound or the material that can remove electronics as this paper about folding again used " oxidant " of albumen from oxidized chemical compound.Suitable oxidant comprises (but being not limited to) oxidized form of glutathione, cystine, cystamine, oxidized form dithiothreitol, DTT, the red threitol of oxidized form (erythreitol) and oxygen.One of ordinary skill in the art understand many oxidants easily and are applicable in the method for the present invention.

" denaturant (denaturing agent or denaturant) " as used herein is defined as and can causes reversible any chemical compound or the material of opening of albumen.The intensity that can determine denaturant by the character and the concentration of specific denaturant.Suitable denaturant can be chaotropic agent, detergent, organic solvent, with miscible solvent, phospholipid or two or more these combination of agents of water.Suitable chaotropic agent includes, but is not limited to carbamide, guanidine and sodium rhodanate.Available detergent can include, but is not limited to strong detergent, for example sodium lauryl sulphate and polyethenoxy ether class (for example Tween or Triton detergent), N-sarcosyl (Sarkosyl); Gentle nonionic detergent (for example digitonin (digitonin)); Gentle cationic detergent, for example N-〉2,3-(two oily acyloxy)-propyl group-N, N, N-trimethyl ammonium; Gentle ion detergent (for example sodium cholate or NaTDC); Or zwitterionic detergent, include, but is not limited to contain sulfobetaines class (sulfobetaine) (Zwittergent), 3-(3-gallbladder amido propyl propyl group) dimethyl amine-1-rosickyite hydrochlorate (3-(3-chlolamidopropyl) dimethylammonio-1-propane sulfate, CHAPS) and 3-(3-gallbladder amido propyl propyl group) dimethyl amine-2-hydroxyl-1-propane sulfonic acid salt (3-(3-chlolamidopropyl) dimethylammonio-2-hydroxy-1-propane sulfonate, CHAPSO).Can be with the organic solvent miscible with water, for example acetonitrile, than low carbon number alkanol (C especially ₂-C ₄Alkanol, for example ethanol or isopropyl alcohol) or than low carbon number alkane glycol (C especially ₂-C ₄Alkane glycol, for example ethylene glycol) as denaturant.Can be used for phospholipid of the present invention can be natural generation phospholipid, for example PHOSPHATIDYL ETHANOLAMINE, phosphatidylcholine, Phosphatidylserine and phosphatidylinositols or synthetic phospholipid derivant or variant, for example two caproyl phosphatidylcholines or two heptanoyl group phosphatidylcholines.

" again folding (refolding) " as used herein described the polypeptide that will contain disulfide bond and changed into any process, reaction or method about the natural or appropriate folded conformation of disulfide bond from incorrect folded state or open mode.

" altogether folding " as used herein specifically is meant at least two interactional polypeptide of employing and causes opening or incorrect folding polypeptide changes into folding process again, reaction or method natural, appropriately folding polypeptide.

Antibody is meant the albumen of specific antigen being showed binding specificity.Natural antibody is generally about 150,000 daltonian allos four polysaccharide albumen, and it is made up of two identical light (L) chain identical with two heavy (H) chains.Each light chain is connected in heavy chain by a covalent disulfide bonds, and the interchain disulfide bond number of the weight of different immunoglobulin isotypes difference.Each heavy chain and light chain also have disulphide bridges in the rule chain at interval.Each heavy chain at one end has variable domain (V _H), inoculation is many constant domains.Each light chain has variable domain (V at one section _L) and have constant domain at the other end; The constant domain of light chain aligns with first constant domain of heavy chain, and aligns with heavy chain variable domain in the light chain variable territory.It is believed that particular amino acid residue forms the interface between light chain and heavy chain variable domain.

Term " variable " is meant that in fact the sequence of some part of variable domain between the different antibodies is extensively different, and is responsible for the binding specificity of each specific antibodies and its specific antigen.Yet transmutability is not a uniform distribution in the variable domain of antibody.It concentrates in light chain and the weight chain variable zone in three sections that are called complementary determining region (CDR).More the variable domain of high conservative partly is called framework region (FR).The variable domain of natural heavy chain and light chain respectively comprise four the most of β of employing lamella configurations by three or four FR districts that CDR is connected, it forms articulating, and forms part β lamellar structure in some cases.CDR in each chain closely is retained on together by the FR district, CDR with other chain impels the antigen binding site that forms antibody (referring to people such as Kabat, Sequences ofProteins of Immunological Interest, the 5th edition Public Health Service, National Institutes ofHealth, Bethesda, MD. (1991)).

Constant domain does not directly relate to antibody and combines with antigenic, but shows various effector functions.The aminoacid sequence of apparent weight chain constant region and deciding can be assigned as antibody or immunoglobulin different classes of.There are five class main immunoglobulin: IgA, IgD, IgE, IgG and IgM, wherein have several classes can further be divided into subclass (isotype), for example IgG1, IgG2, IgG3 and IgG4; IgA1 and IgA2.The CH of corresponding different classes of immunoglobulin is called α, δ, ε, γ and μ.In various human immunoglobulin classifications, only known person IgG1, IgG2, IgG3 and IgM activating complement.

Antibody in vivo affinity maturation is driven by the selection of antigen of higher affinity antibody variants, and described higher affinity antibody variants mainly is to make by somatic hypermutation (somatic hypermutagenesis)." ingredient change (repertoire shift) " also takes place usually, wherein find to reply once more or the main germline gene (germline gene) of tertiary response be different from for the first time or reply once more those.

Can duplicate immune affinity maturation process by in vitro in antibody gene, introducing sudden change and use affine selection method to separate mutant with improvement affinity.Described mutant antibody can be illustrated on the surface of filamentous bacteria phage or microorganism (for example yeast), and can select antibody by antibody and antigenic affinity or by antibody and the dissociated kinetics of antigen (dissociation rate (off-rate)).People such as Hawkins, J.Mol.Biol.226:889-896 (1992).Adopted CDR step to move sudden change (walking mutagenesis) and made people's antibody in conjunction with people's envelope glycoprotein gp120 of human immunodeficiency virus type 1 (HIV-1) (people such as Barbas III, PNAS (USA) 91:3809-3813 (1994); With people such as Yang, J.Mol.Biol.254:392-403 (1995)) and anti-c-erbB-2 strand Fv fragment (people such as Schier, J.Mol.Biol.263:551567 (1996)) affinity maturation.Adopted antibody chain reorganization (clain shuffling) and CDR to suddenly change to make high affinity human affinity matured antibody at HIV the 3rd hypermutation ring people J.Mol.Biol.256:77-88 (1996) such as () Thompson.Balint and Larrick Gene 137:109-118 (1993) describe computer assisted oligodeoxynucleotide scanning sudden change, and then simultaneously and search for the improvement variant of all CDR of variable region gene up hill and dale.Use initial limited sudden change to make α v β 3 Humanized antibody specific affinity maturations, wherein each position of all six CDR all suddenlys change, and then expresses and screening comprises reorganization library people PNAS (USA) 95:6037-6-42 (1998) such as () Wu of high-affinity mutant.At Chiswell and McCafferty TIBTECH 10:80-84 (1992); With commented phage displaying antibody among Rader and the Barbas IIICurrent Opinion in Biotech.8:503-508 (1997).Compare with parental generation antibody under each situation with the mutant antibody that improves affinity at above-mentioned list of references report, mutant antibody has aminoacid and replaces in CDR.

This paper " affinity maturation " is meant the process that strengthens antibody and its antigen affinity.The affinity maturation method includes, but is not limited to calculate screening method and laboratory method.

This paper " antibody " is meant the albumen of being made up of all or part antibody gene encoded polypeptides in fact one or more.Immunoglobulin gene includes, but is not limited to κ, λ, α, γ (IgG1, IgG2, IgG3 and IgG4), δ, ε and μ constant region gene, and a large amount of immune globulin variable region gene.This paper antibody is meant and comprises full length antibody and antibody fragment, and comprises natural be present in any organism or through the antibody of genetic engineering procedure (for example variant).

" antibody fragment " is meant any form antibody except that the total length form.This paper antibody fragment comprise be present in the full length antibody than the antibody of small component with through the antibody of genetic engineering procedure.Antibody fragment includes, but is not limited to Fv, Fc, Fab and (Fab ¹) ₂, strand Fv (scFv), miniature bifunctional antibody, miniature three function antibodies, miniature four function antibodies, difunctional hybrid antibody, CDR1, CDR2, CDR3, CDR combination, variable region, framework region, constant region or the like (Maynard ﹠amp; Georgiou, 2000, Annu.Rev.Biomed.Eng.2:339-76; Hudson, 1998, Curr.Opin.Biotechnol.9:395-402).

This paper " calculating screening method " is meant any method that relates to one or more sudden changes in albumen, wherein said method adopt computer estimate potential amino acid side chain replace it interphase interaction and/or with the interactional energy of albumen remainder.

This paper " Fc " is meant the antibody moiety that comprises immunoglobulin territory C γ 2 and C γ 3 (C γ 2 and C γ 3).Fc also can comprise and is present between C γ 2 and the C γ 1 (C γ 1) the intrachain any residue of N end hinge.Fc can refer to isolating described district, perhaps the described district in antibody or antibody fragment content.Fc also can comprise the Fc of any modified form, and it includes, but is not limited to natural monomer, natural dimer (connecting through disulfide bond), modified dimer (connecting through disulfide bond and/or non-covalent bond) and modified monomer (that is derivant).

This paper " full length antibody " is meant the structure of the natural biological form that constitutes antibody H and/or L chain.In comprising most of mammal of people and mice, described form is the tetramer, and is made up of two pairs of two identical immunoglobulin chains, and each is to having a light chain and a heavy chain, and each light chain comprises immunoglobulin territory V _LAnd C _L, and each heavy chain comprises immunoglobulin territory V _H, C γ 1, C γ 2 and C γ 3.Each centering, variable region of light chain and variable region of heavy chain (V _LAnd V _H) be responsible for conjugated antigen together, and constant region (C _L, C γ 1, C γ 2 and C γ 3, especially C γ 2 and C γ 3) be responsible for the antibody mediated effect subfunction.In some mammals of for example camel (camel) and yamma (llama), full length antibody can only be made up of two heavy chains, and each heavy chain comprises immunoglobulin territory V _H, C γ 2 and C γ 3.

This paper " immunoglobulin (Ig) " is meant the albumen of being made up of the immunoglobulin gene encoded polypeptide in fact one or more.Immunoglobulin includes, but is not limited to antibody.Immunoglobulin can have many versions, and it includes, but is not limited to full length antibody, antibody fragment and indivedual immunoglobulin territory, includes, but is not limited to V _H, C γ 1, C γ 2, C γ 3, V _LAnd C _L

This paper " immunoglobulin (Ig) territory " is meant the albumen territory of being made up of the immunoglobulin gene encoded polypeptide by in fact.As shown in Figure 1, the Ig territory includes, but is not limited to V _H, C γ 1, C γ 2, C γ 3, V _LAnd C _L

" misfolded proteins sequence " used herein is meant to have one or more and another similar protein sequence different protein sequence on the aminoacid concordance.Described similar protein sequence can be natural wild-type protein sequence, or another variant of wild-type sequence.In general, homing sequence is called " parental generation " sequence, and can be wild type or variant sequence.For instance, the preferred embodiments of the present invention adopt humanization parental generation sequence, described humanization parental generation sequence are carried out computational analysis to produce variant.

" variable region " of this paper antibody is meant and comprises V _HImmunoglobulin territory, V _LImmunoglobulin territory or V _HAnd V _LThe polypeptide in immunoglobulin territory (comprising variant), as shown in Figure 1.The variable region can refer to the described polypeptide of unpack format, as the Fv fragment, as the scFv fragment, as this district in the big antibody fragment content, or as this district in the content of full length antibody or alternate, non-antibody molecule of the skeleton.

The present invention also can be used for from the antibody of many sources acquisition.Antibody can be coded by the antibody gene from any organism in fact, described organism includes, but is not limited to people, mice, rat, rabbit, camel, yamma, dromedary camel, monkey, especially mammal, especially people, and especially mice and rat.In one embodiment, described antibody can be human antibody, for example by using transgenic mice or other animal (Bruggemann ﹠amp; Taussig, 1997, Curr.Opin.Biotechnol.8:455-458) or people's antibody library in conjunction with selection method (Griffiths ﹠amp; Duncan, 1998, Curr.Opin.Biotechnol.9:102-108) from patient or person under inspection, obtained.Antibody can comprise artificial or natural generation from any source.For instance, the present invention adopts and to include, but is not limited to chimeric antibody and humanized antibody (Clark, 2000, the antibody through genetic engineering procedure Immunol.Today21:397-402), or derive from the reorganization library.In addition, can be in fact by the variant through genetic engineering procedure of the antibody of one or more natural antibody coded by said gene through optimized antibody.For instance, in one embodiment, can be the antibody of differentiating by affinity maturation through optimized antibody.

With regard to ABP of the present invention, term " antigenic specificity " or " specificity in conjunction with " are meant one or more epitopes of the interested binding partners of conjugated antigen or institute, but nonrecognition and combination contain the ABP of other molecule in the sample of hybrid antigen colony in fact.

Term used herein " bispecific ABP " or " polyspecific ABP " are meant the ABP that comprises two or more antigen binding sites or binding partners binding site, first binding site has the affinity with first antigen or epitope, and second binding site has different with first and affinity second antigen or epitope.

Term as used herein " epitope " is meant on antigen or the binding partners site by ABP discerned.If antigen comprises polypeptide, then epitope can be the aminoacid that linearity or conformation form sequence or profile.Epitope also can be ABP any position on the bonded any kind antigen.

" antigen-binding polypeptides " as used herein or " ABP " should comprise having at least specificity in conjunction with particular combination companion's (as antigen) bioactive polypeptide and albumen, and ABP analog, the ABP isomeric form, the ABP analogies, the ABP fragment, hybridization ABP albumen, fusion rotein, oligomer and polymer, congener, glycosylation type variant and mutein, and regardless of its biological activity, and further synthesize or manufacture method regardless of it, described method includes, but is not limited to reorganization and (no matter whether originates from cDNA, genomic DNA, synthetic DNA or other form nucleic acid), in vitro, in vivo, by the microinjection nucleic acid molecules, synthetic, transgenic and gene activation method.The instantiation of ABP includes, but is not limited to any aminoacid sequence of antibody molecule, heavy chain, light chain, variable region, CDR, Fab, scFv, substituting skeleton non-antibody molecule, part, receptor, peptide and conjugated antigen.

Term " ABP " or " antigen-binding polypeptides " are meant above-mentioned ABP, and keep natural generation antibody at least a biological activity (include, but is not limited to except that antigen in conjunction with activity) polypeptide.Except that antigen in conjunction with activity include, but is not limited to any or more than one activity relevant with Fc.

Antigen-binding polypeptides comprises pharmaceutically acceptable salt and prodrug, polymorph, hydrate, solvate, bioactive fragment, biological activity variant and the stereoisomer of prodrug and salt and agonist, analogies and antagonist variant and the polypeptide fusant thereof of the human ABP of natural generation of natural generation people ABP.Term " antigen-binding polypeptides " is encompassed in aminoterminal, c-terminus or both places and all comprises other amino acid whose fusant.Exemplary fusant for example includes, but is not limited to; methionyl ABP (wherein methionine is connected in the end by the N of the recombinant expressed ABP that obtains), be used for the purification purpose fusant (including, but is not limited to poly histidine or affinity epitope), be used for ABP be connected in the bioactive molecule purpose fusant, with the fusant of serum albumin binding peptide and with the fusant of serum albumin (serum albumin).

Term " antigen " or " binding partners " are meant the material in conjunction with active target that is showed by ABP.In fact antigen or binding partners that any material can be ABP.The example of antigen or binding partners includes, but is not limited to α-1 antitrypsin, angiostatin (Angiostatin), AHF (Antihemolytic factor), antibody, apolipoprotein (Apolipoprotein), apoprotein (Apoprotein), atrionatriuretic factor (Atrialnatriuretic factor), atrial natriuretic polypeptins (Atrial natriuretic polypeptide), atrial natriuretic peptide (Atrialpeptide), C-X-C chemotactic factor (C-X-C chemokine) (T39765 for example, NAP-2, ENA-78, Gro-a, Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), calcitonin (Calcitonin), CC chemotactic factor (monocyte chemoattractant protein-1 for example, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-1 β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), the CD40 part, the C-kit part, collagen, colony stimulating factor (Colony stimulating factor, CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine (epithelium neutrophil activation peptide-78 for example, GRO/MGSA, GRO, GRO, MIP-1, MIP-1, MCP-1), epidermal growth factor (EGF), erythropoietin (Erythropoietin, " EPO "), toxin (Exfoliating toxin) A and B come off, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (Fibroblast Growth Factor, FGF), fibrinogen (Fibrinogen), Fibronectin (Fibronectin), G-CSF, GM-CSF, glucocerebrosidase (Glucocerebrosidase), gonadotropin (Gonadotropin), somatomedin, Hedgelog protein (Hedgehog protein) (Sonic for example, Indian, Desert), hemoglobin (Hemoglobin), C-MET HGFr (Hepatocyte Growth Factor, HGF), hirudin (Hirudin), the human serum albumin, insulin, insulin like growth factor (Insulin-likeGrowth Factor, IGF), interferon (IFN-α for example, IFN-β, IFN-γ), interleukin (IL-1 for example, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 or the like), keratinocyte growth factor (Keratinocyte Growth Factor, KGF), lactoferrin (Lactoferrin), leukaemia inhibitory factor, luciferase (Luciferase), neurotrophic factor (Neurturin), neutrophil cell inhibitive factor (Neutrophil inhibitory factor, NIF), tumour inhibitor M (oncostatin M), BMP (Osteogenic protein), parathyroid hormone, PD-ECSF, PDGF, peptide hormone (for example human growth hormone), opsonin (Pleiotropin), a-protein, protein G, the pyrogen exotoxin A, B and C, relaxin (Relaxin), feritin (Renin), SCF, soluble complement receptor I, solubility I-CAM 1, soluble interleukin receptor (IL-1,2,3,4,5,6,7,9,10,11,12,13,14,15), soluble TNF acceptor, somatomedin (Somatomedin), somatostatin (Somatostatin), growth hormone (Somatotropin), streptokinase (Streptokinase), superantigen (Superantigen), promptly, staphyloentero-toxin (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), superoxide dismutase, toxic shock syndrome, TSS toxin (Toxic shock syndrome toxin, TSST-1), thymosin, tissue fibrin's zymoexcitator, tumor necrosis factor (TNF-β), Tumor Necrosis Factor Receptors (TNFR), tumor necrosis factor (TNF-α), VEGF (Vascular Endothelial Growth Factor, VEGEF), urokinase and other.

Many in these albumen is commercially available (referring to for example Sigma BioSciences 2002 catalogues and price lists), and corresponding protein sequence and gene and its common many variants are well-known (referring to for example Genbank).

Other antigen or binding partners include, but is not limited to the transcript and expression activator.Transcript and expression activator example comprises gene and the albumen of regulating cell growth, differentiation, regulation and control or the like.Expression and transcriptional activation agent are found in prokaryote, virus and the eukaryote that extensive treatment target is provided, and comprise fungus, plant and animal, comprise mammal.Should be appreciated that and express and the transcriptional activation agent is regulated by many mechanism and transcribed, for example expression by bind receptor, stimulus signal transductory cascade, regulative transcription factor, in conjunction with promoter and enhancer, in conjunction with promoter and the bonded albumen of enhancer, separate chain DNA, montage pre-mRNA, gather adenosine RNA and degradation of rna.Antigen or binding partners include, but is not limited to express activator, for example cytokine, inflammatory molecule, somatomedin, its receptor and oncogene product, for example interleukin (for example IL-1, EL-2, IL-8 or the like), interferon, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF, SCF/c-Kit, CD40L/CD40, VLA-4/VCAM-1, ICAM-1/LFA-1 and hyalurin/CD44; Signal transducers and corresponding oncogene product, for example Mos, Ras, Raf and Met; With transcriptional activation agent and inhibitor, for example p53, Tat, Fos, Myc, Jun, Myb, Rel and steroid hormone receptor, for example steroid hormone receptor of estrogen, Alfasone, testosterone, aldosterone (aldosterone), ldl receptor part and corticosterone.

Vaccine protein can be antigen or binding partners, and it includes, but is not limited to the albumen from the infectiousness fungus, and for example aspergillosis (Aspergillus), candidiasis (Candida) belong to; Albumen from antibacterial, especially serve as the escherichia coli of pathogenetic bacteria model and pharmaceutically important antibacterial, for example staphylococcus (Staphylococci) (for example staphylococcus aureus (Staphylococci aureus)) or streptococcus (Streptococci) (for example pneumonia Chain coccus (Streptococci pneumoniae)); From protozoacide albumen, for example sporozoon (sporozoa) (for example plasmodium (Plasmodia)), rhizopod (rhizopod) (for example entamoeba (Entamoeba)) and flagellate (flagellate) (trypanosomicide (Trypanosoma), Leishmania (Leishmania), trichomonacide (Trichomonas), giardia lamblia stiles (Giardia) or the like); From the albumen of virus, for example (example comprises poxvirus (Poxviruses) to (+) RNA viruses, for example cowpox (vaccinia); MiRNA sick plain (Picoruaviruses), for example poliomyelitis (polio); Coating virus (Togaviruses), for example rubella (rubella); Banzi virus (Flaviviruses), for example HCV; With hat virus (Coronaviruses), (-) RNA viruses (for example rhabdovirus (Rhabdoviruses), for example VSV; Paramyxovirus (Paramyxovimses), for example RSV; Influenza virus (Orthomyxovimses), for example influenza virus (influenza); This poplar virus (Bunyavirus); With sand grains virus (Arenaviruses)), dsDNA virus (for example reovirus (Reovirus)), RNA is to DNA viruses, be retrovirus (Retro virus), for example HIV and HTLV and some DNA be to RNA viruses, for example hepatitis B virus.

Antigen or binding partners can be enzymes, and it includes, but is not limited to amidase, amino acid racemase, acyltransferase, dehalogenase, dioxygenase, diaryl propane peroxidase, epimerase, Epoxide hydrolase, esterase, isomerase, kinases, glucose isomerase, glycosidase, glycosyl transferase, haloperoxidase, monooxygenase (for example p450s), lipase, lignin peroxidase, nitrile hydratase, nitrilase, protease, phosphate, subtilisin (subtilisin), transaminase and nuclease.

Agricultural dependency albumen, enzyme, plant and insect toxins, antitoxin albumen, the mycete rhzomorph (Mycotoxin) of for example anti-insect protein (for example Cry albumen), generation starch and lipid separated toxalbumin, (for example ribulose 1 for the plant growing enzyme, 5-diphosphonic acid carboxylase/oxygenase, Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, " RUBISCO "), lipoxygenase (lipoxygenase, LOX) and phosphoenolpyruvic acid (Phosphoenolpyruvate, PEP) carboxylase also can be antigen or binding partners.

For instance, antigen or binding partners also can be the disease association molecules, for example TSA, for example CD20 on B cell individual genotype, the Malignant B cell, the CD33 on the leukemia blastocyte and the HER2/neu on the breast carcinoma.Perhaps, antigen or binding partners also can be growth factor receptorses.The example of somatomedin includes, but is not limited to EGF-R ELISA (EGF), transferrins (transferrin), insulin like growth factor, transforming growth factor (TGF), interleukin-1 and interleukin II.For instance, in multiple human epithelium's primary tumor, find the high expressed of EGF receptor.Have been found that autocrine stimulation approach in the alpha mediated cancerous cell of TGF-.More verified murine monoclonal antibodies can be in conjunction with the EGF receptor, the propagation that combines and suppress various human cancerous cell line in culture and the xenotransplantation phantom type of block ligand and EGF receptor.Mendelsohn and Baselga (1995) Antibodies to growth factors and receptors, BiologicTherapy of Cancer, the 2nd edition, JB Lippincott, Philadelphia, 607-623 page or leaf.Therefore, ABP of the present invention can be used for the treatment of multiple cancer.

Antigen or binding partners also can be the cell surface protein relevant with coronary artery disease or receptor (for example platelet glycoprotein Iib/IIIa receptor), cell surface protein or the receptor (for example lipid A district of CD4, CAMPATH-1 and Glan (gram) negative bacteria lipopolysaccharide) relevant with autoimmune disease.The humanized antibody of having suffered from the anti-CD4 of test in patient's the clinical trial of cutaneous T cell lymphoma (Mycosis fungoides), generalized pustular psoriasis (generalized postular psoriasis), serious psoriasis and rheumatoid arthritis in treatment.In the treatment of septic shock (septic shock) clinical trial the antibody in lipid A district of anti-Glan negative bacteria lipopolysaccharide.Also in the treatment of healing property of difficulty rheumatoid arthritis clinical trial the antibody of anti-CAMPATH-1.Therefore, ABP of the present invention can be used for the treatment of multiple autoimmune disease.People such as Vaswani (1998) " Humanized antibodies as potential therapeutic drugs " Annals of Allergy, Asthma andImmunology 81:105-115.

Antigen or binding partners also can be albumen or the peptides relevant with human anaphylactic disease, for example inflammatory mediates albumen, for example interleukin-1 (IL-1), tumor necrosis factor (TNF), leukotriene receptor and 5-lipoxygenase, and adhesion molecule, for example V-CAM/VLA-4.In addition, because IgE plays crucial effects in the super immediately quick allergy of the I of for example asthma type, so IgE also can serve as antigen or binding partners.Studies show that it is relevant with the order of severity of disease (especially in asthma) that the content of total serum IgE trends towards.People such as Burrows (1989) " Association of asthma with serum IgE levels and skin-test reactivity toallergens " New Engl.L.Med.320:271-277.Therefore, in the treatment anaphylactic disease, can be used to reduce combining of IgE content or blocking-up IgE and mastocyte and basophilic leukocyte, and not influence normal immunologic function substantially at the selected ABP of IgE.

Antigen or binding partners also can be to serve as antigenic virus surface or the core protein that causes host immune response.The example of these virus proteins includes, but is not limited to glycoprotein (or surface antigen, for example GP120 and GP41) and capsid protein (or structural protein, for example P24 albumen); The core protein of surface antigen or first, second, third, fourth or hepatitis E virus (the little hbs antigen of hepatitis B virus (smallhepatitis B surface antigen for example, SHBsAg) and the core protein of hepatitis C virus, NS3, NS4 and NS5 antigen); Respiratory syncytial virus (respiratory syncytial virus, glycoprotein RSV) (G albumen) or fusion rotein (F albumen); The surface of herpes simplex virus HSV-1 and HSV-2 and core protein (for example from HSV-2 glycoprotein D).

Antigen or binding partners also can be its tumor suppression functions of forfeiture and can make cell to the cancer sudden change tumor inhibitor gene outcome of susceptible more.The tumor inhibitor gene is cell growth inhibiting and division cycle and the gene that therefore stops newborn tumor growth.Sudden change in the tumor inhibitor gene causes that the cell ignorance suppresses one or more components in the signal network, thereby overcomes the cell cycle checkpoint and cause higher controlled cell growth (cancer) speed.The example of tumor inhibitor gene includes, but is not limited to DPC-4, NF-1, NF-2, RB, p53, WT1, BRCA1 and BRCA2.

DPC-4 relates to cancer of pancreas and participates in suppressing fissional Cytoplasm approach.The NF-1 coding suppresses albumen-a kind of Cytoplasm Profilin of Ras.NF-1 relates to the neurofibroma (neurofibroma) and the pheochromocytoma (pheochromocytomas) of nervous system and myeloid leukemia.The NF-2 coding relates to the nucleoprotein in neural meningioma (meningioma), snow Wang Shi tumor (schwanoma) and the ependymoma (ependymoma).RB coding pRB albumen, pRB albumen is the nucleoprotein of the main inhibitor of cell cycle.RB relates to retinoblastoma (retinoblastoma) and osteocarcinoma, bladder cancer, small cell lung cancer and breast carcinoma.Division of p53 coding regulating cell and p53 albumen that can cell death inducing.In many cancers, find sudden change and/or the inertia of p53.WT1 relates to the nephroblastoma (Wilms tumor) of kidney.BRCA1 relates to breast carcinoma and ovarian cancer, and BRCA2 relates to breast carcinoma.Therefore, ABP can be used for the mutual work of blocking gene product and tumor generation and other albumen of evolutionary path and biochemical.

Antigen or binding partners can be the CD molecules, and it includes, but is not limited to CD1a, CD1b, CD1c, CD1d, CD2, CD3 γ, CD3 δ, CD3 ε, CD4, CD5, CD6, CD7, CD8 α, CD8 β, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45R, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CDw70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CD79 α, CD79 β, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD110-113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CDw124, CD125, CD126, CDw127, CDw128a, CDw128b, CD129, CDw130, CD131, CD132, CD133, CD134, CD135, CD136, CDw137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw145, CD146, CD147, CD148, CDw149, CD150, CD151, CD152, CD153, CD154, CD155, CD156, CD157, CD158a, CD158b, CD161, CD162, CD163, CD164, CD165, CD166 and TCR ζ.Antigen or binding partners can be VEGF, vegf receptor, EGFR, Her2, TNFa, the TNFRI receptor, GPIIb/IIIa, IL-2R α chain, IL-2R-β chain, RSV F albumen, α 4 integrates plain, IgE, the IgE receptor, digoxin (digoxin), phoorsa snake venom (carpet viper venom), complement C5, OPGL, the CA-125 tumor antigen, protein staphylococcus, staphylococcus epidermidis (Staphylococcus epidermidis) albumen, s. aureus protein, staphy lococcus infection associated protein (including, but is not limited to staphylococcus aureus and staphylococcus epidermidis), the IL-6 receptor, CTLA-4, RSV, IL-2 receptor Tac subunit, IL-5 and EpCam.Antigen or binding partners can be molecule fragments.

The example of bispecific ABP include, but is not limited to one of them ABP at tumor-cell antigen and another ABP at the bispecific ABP of cytotoxicity trigger molecule, the anti-CD 15 of for example anti-Fc γ RI/, anti-p185 ^HER2/ Fc γ RIII (CD 16), the anti-Malignant B cell of anti-CD3/ (1D10), the anti-p185 of anti-CD3/ ^HER2, the anti-p97 of anti-CD3/, the anti-renal cell carcinoma of anti-CD3/, the anti-OVCAR-3 of anti-CD3/, anti-CD3/L-D1 (resistive connection intestinal cancer), the anti-melanocyte stimulating hormone analogue of anti-CD3/, anti-EGF receptor/anti-CD3, the anti-CAMA1 of anti-CD3/, the anti-CD19 of anti-CD3/, anti-CD3/MoV18, anti-nerve cell adhesion molecule (neural cell adhesionmolecule, NCAM)/anti-CD3, anti-folic acid-binding protein (folate binding protein, FBP)/and anti-CD3, anti-general cancer associated antigen (pan carcinoma associated antigen, AMOC-31)/anti-CD3; ABP specificity in conjunction with tumor antigen another ABP in conjunction with the bispecific ABP of toxin, for example anti-Saponaria officinalis toxin (saporin)/anti-Id-1, the anti-Saponaria officinalis toxin of anti-CD22/, the anti-Saponaria officinalis toxin of anti-CD7/, the anti-Saponaria officinalis toxin of anti-CD38/, anti-CEA/ antiricin A chain, anti-interferon-α (IFN-α)/anti-hybridoma idiotype, the anti-catharanthus alkaloid of anti-CEA/ (vinca alkaloid); The bispecific ABP that is used for invertase activation prodrug, for example anti-CD30/ alkali resistance phosphatase (catalysis phosphoric acid mitomycin (mitomycin phosphate) prodrug changes into mitomycin alcohol); Can be used as the bispecific ABP of cellosolve (fibrinolytic agent), for example antifibrin/anti-tissue fibrin zymoexcitator (tPA), antifibrin/antiurokinase type brinase activator (uPA); Be used for bispecific ABP, for example anti-low density lipoprotein, LDL (LDL)/anti-Fc receptor (for example Fc γ RI, Fc γ RII or Fc γ RIII) with immune complex targeted cells surface receptor; The bispecific ABP that is used for the treatment of communicate illness, for example anti-CD3/ anti-herpes simplex virus (HSV), anti-T-cell receptors: CD3 complex/influenza emits virus, the anti-HrV of anti-Fc γ R/; Be used in vitro or the bispecific ABP of lesion detection in vivo the anti-EOTUBE of for example anti-CEA/, the anti-DPTA of anti-CEA/, anti-p185 ^HER2/ antihapten; As the bispecific ABP of vaccine adjuvant (referring to Fanger, people such as MW, Crit Rev Immunol.1992; 12 (3-4): 101-24, it incorporates this paper by reference into); With bispecific ABP as diagnostic tool, for example anti-rabbit igg/anti-ferritin, anti-horseradish peroxidase (HRP)/hormone antagonist, the long chalone of antibiosis/anti-Substance P, the anti-FITC of anti-HRP/, the anti-beta galactosidase of anti-CEA/ are (referring to Nolan, O et R.O ' Kennedy, BiochimBiophys Acta.1990 August 1; 1040 (1): 1-11, it incorporates this paper by reference into).The example of tri-specific ABP comprises the anti-CD37 of the anti-CD4/ of anti-CD3/, the anti-CD37 of the anti-CD5/ of anti-CD3/ and the anti-CD37 of the anti-CD8/ of anti-CD3/.

Various lists of references disclose and have been undertaken peptide modified by polymer combination or glycosylation.Term " ABP " or " antigen-binding polypeptides " include, but is not limited to the polypeptide that combines with polymer such as for example PEG, and can comprise one or more other derivatizations of cysteine, lysine, N or C terminal amino acid or other residue.In addition, ABP can comprise connexon, polymer or bioactive molecule, wherein connexon, polymer or the bonded aminoacid of bioactive molecule can be according to non-natural generation aminoacid of the present invention, for example perhaps can adopt with lysine or cysteine coupling etc. under known technology and combining in the field with natural coded amino acid.United States Patent (USP) the 4th, 904 discloses the polypeptide that exhausts PEGization lysine in No. 584, and wherein at least one lysine residue has lacked or replaced with any other amino acid residue.WO 99/67291 discloses a kind of with albumen and the bonded method of PEG, wherein at least one aminoacid deletion on the albumen, and described albumen be enough to reach with protein bound condition under contact PEG.WO 99/03887 discloses the PEGization variant of the polypeptide that belongs to the growth hormone superfamily, and wherein cysteine residues is replaced by the non essential amino acid residue that is positioned at the polypeptide appointed area.WO00/26354 discloses a kind of manufacturing and compares with the corresponding parent polypeptide that comprises at least one other glycosylation site, has the method for the glycosylated polypeptides variant of low irritability.

Term " antigen-binding polypeptides " also comprises glycosylation ABP, and for example (but being not limited to) is at the glycosylated polypeptide in any amino acid position place, connect or O connects the polypeptide of glycosylation form through N.The variant that contains single nucleotide alteration also is considered to the biological activity variant of ABP.In addition, also comprise splice variant.Term " antigen-binding polypeptides " also comprises ABP heterodimer, homodimer, heteromultimers or the homology polymer of any one or an above ABP or the bioactive molecule of any other polypeptide, albumen, carbohydrate, polymer, micromolecule, connexon, part or other any kind (connect or be expressed as fusion rotein by chemical method), and contains (for example) particular hole or other modification but still the polypeptide analog of retains biological activity.

In certain embodiments, antigen-binding polypeptides further comprises the bioactive interpolation of adjusting ABP, replacement or disappearance.For instance, interpolation, replacement or disappearance can be regulated one or more characteristics or the activity (including, but is not limited to regulate and antigenic affinity) of ABP; Regulate (including, but is not limited to increases or reduce) antigen conformation or other secondary, three grades or quarternary structure change; Stable antigen conformation or other secondary, three grades or quarternary structure change; Induce or cause that antigen conformation or other secondary, three grades or quarternary structure change; Regulate circulating half-life; The adjustment of treatment half-life; Adjusting polypeptide stability; Regulate dosage; Adjustment release or biological usability; Promote purification; Or improve or change specific dosing way.Equally, antigen-binding polypeptides can comprise the link molecule (including, but is not limited to biotin) of disappearance (including, but is not limited to GFP), purification or other characteristic of protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequence based on affinity (including, but is not limited to FLAG, poly-His, GST or the like) or improvement polypeptide.

ABP homodimer, heterodimer, homology polymer or the heteromultimers through connecting also contained in term " antigen-binding polypeptides ", include, but is not limited to directly through the non-naturally encoded amino acids side chain be connected in identical or different non-naturally encoded amino acids side chain or be connected in natural coded amino acid side chain as fusant or connect through connexon indirectly those.Exemplary connexon includes, but is not limited to the water-soluble polymer of little organic compound, multiple length, for example Polyethylene Glycol or polydextrose (polydextran), or the polypeptide of all lengths.

One of ordinary skill in the art should be appreciated that can identify easily in the fragment in conjunction with polypeptide etc. at antigen-binding polypeptides or related antigen corresponding specific antigen in conjunction with peptide sequence in the amino acid position of position.For instance, can use the sequence alignment program comparison of BLAST for example and differentiate the ad-hoc location that meets position in the correlated series in the albumen.

Term " antigen-binding polypeptides " is contained and is comprised the antigen-binding polypeptides that one or more aminoacid are replaced, added or lack.Antigen-binding polypeptides of the present invention can comprise with the modification of one or more natural amino acids and one or more alpha-non-natural amino acids to be modified.Exemplary replacement in the several amino acids position in the natural generation ABP polypeptide has been described, it includes, but is not limited to regulate one or more bioactive replacements in the antigen-binding polypeptides, for example (but being not limited to) increases agonist activity, increases the polypeptide dissolubility, polypeptide changed into antagonist or the like, and contained by term " ABP ".

" non-naturally encoded amino acids " is meant not to be a kind of aminoacid in 20 kinds of common amino acids or burnt lysine (pyrolysine) or the selenocystein (selenocysteine).To be that " alpha-non-natural amino acid (non-natural amino acid) ", " alpha-non-natural amino acid (unnatural amino acid) ", " non-natural generation aminoacid " and its are various have hyphen and not with the form of hyphen to other term that can use with the free burial ground for the destitute with term " non-naturally encoded amino acids ".Term " non-naturally encoded amino acids " also includes, but is not limited to by modifying (for example, post translational modification) natural coded amino acid (including, but is not limited to 20 kinds of common amino acids or burnt lysine and selenocystein) but self is not by the natural aminoacid that is taken place in the polypeptide chain in the growth of incorporating into of translation complex.Amino acid whose example takes place and includes, but is not limited to N-acetylglucosamine base-L-serine (N-acetylglucosaminyl-L-serine), N-acetylglucosamine base-L-threonine and O-phosphotyrosine in described non-natural.

" aminoterminal is modified base " is meant and can be connected in the N-terminal any molecule of polypeptide." c-terminus is modified base " similarly is meant any molecule that can be connected in the polypeptide c-terminus.End modified base includes, but is not limited to various water-soluble polymers, peptide or albumen (for example serum albumin) or other increases the part of the serum half-life of peptide.

Technical field and herein under term " functional group ", " active part ", " active group ", " leaving group ", " reaction site ", " chemical reaction base " and " chemical reaction part " are used for, be meant the molecule uniqueness, definable part or unit.Described term is a synonym at chemical field sometimes, and is used for representing carrying out some functions or active and have reactive molecular moiety with other molecule in this article.

Term used herein " key (linkage) " or " connexon (linker) " are meant because chemical reaction and usually formed and be generally the group or the key (bond) of covalent bond.The hydrolysis-stable key is that digital is stable in fact in water, and does not react with water under available pH value, and it includes, but is not limited to the long response time under physiological condition, possibility or even unlimited.Hydrolytically unstable or degradable linkage are meant described key (for example blood) degradable in water or aqueous solution.Enzyme instability or degradable key are meant that described key can be by one or more enzymatic degradation.Understand as affiliated technical field, PEG and dependency polymer can comprise degradable linkage between one or more functional end-groups of polymer backbone and polymer molecule in polymer backbone or connexon group.For instance, by on PEG carboxylic acid or active PEG carboxylic acid and the bioactivator between the alcohol groups reaction formed ester bond usually under physiological condition hydrolysis to discharge described reagent.Other hydrolysis degradable linkage includes, but is not limited to the carboxylic acid ester bond; By the imine linkage that amine and aldehyde reaction generated; The formed phosphoric acid ester bond of pure and phosphate-based reaction; Hydrazone key for hydrazides and aldehyde reaction product; Acetal bonds for aldehyde and pure product; Original acid ester key for formic acid esters and pure product; By the carboxyl formed peptide bond of amido (including, but is not limited to end) with peptide at the polymer of for example PEG; With by phosphoramidite base (including, but is not limited to end) and the formed oligonucleotide key of oligonucleotide 5 ' hydroxyl at polymer.The side chain connexon can be used in the antigen-binding polypeptides of the present invention.

Term used herein " bioactive molecule ", " biologically-active moiety " or " bioactivator " are meant can be influenced biosystem, approach, molecule or relate to any physics of mutual work of organism (including, but is not limited to virus, antibacterial, bacteriophage, transposon, Protein virus (prion), insecticide, fungus, plant, animal and human's class) or any material of biochemical property.Specifically, bioactive molecule as used herein includes, but is not limited to be used to diagnose, cure, alleviate, treat or prevent the mankind or the interior disease of other animal body or strengthen the mankind in addition or any material of animal body health or spiritual kilter.The example of bioactive molecule includes, but is not limited to peptide, albumen, enzyme, small-molecule drug, hard medicine (hard drug), soft medicine (soft drug), dyestuff, lipid, nucleoside, oligonucleotide, toxin, cell, virus, liposome, microgranule (microparticle) and micella (micell).Be suitable for including, but is not limited to medicine, prodrug, radionuclide, photographic developer, polymer, antibiotic, antifungal, antiviral agent, anti-inflammatory agent, antitumor agent, cardiovascular agents, antianxiety drug, hormone, somatomedin, steroid dose, the microorganism toxin or the like of deriving in conjunction with the bioactivator kind that the present invention uses.

In certain embodiments, ABP molecule of the present invention can be used for bioactive molecule or detectable label at tumor sites.This helps tumor to kill, detect and/or locate or other effect.Diagnostic probe or image probe also can be connected in ABP molecule of the present invention.In some particularly preferred embodiment, the bioactive molecule component of ABP is " radiopaque (rediopaque) " labelling, the labelling that for example can use (for example) x light to manifest easily.The radiopaque material is known by one of ordinary skill in the art.The most frequently used radiopaque material comprises iodide, bromide or barium salt.Other radiopaque material also is known, include, but is not limited to the organo-bismuth derivant (referring to, for example United States Patent (USP) the 5th, 939, No. 045), radiopaque polyuria alkane (multiurethane) (referring to, for example United States Patent (USP) the 5th, 346, No. 981), the organo-bismuth complex (referring to, for example United States Patent (USP) the 5th, 256, No. 334), radiopaque barium polymer complex (referring to, for example United States Patent (USP) the 4th, 866, No. 132) or the like.

ABP of the present invention can be directly and the coupling of radiopaque part, perhaps its " packing (package) " (for example chelating agen, liposome, polymer microballon or the like) that can be connected in carrying or contain the radiopaque material.

Except that the radiopaque labelling, other labelling also is applicable to the present invention.The detectable label that is suitable as the bioactive molecule component of ABP of the present invention comprises any composition that can be detected by Electronic Speculum, photochemistry, biochemistry, immunochemistry, electronics, optics or chemical method.Can be used for labelling among the present invention and comprise magnetic bead (Dynabeads for example ^TM), fluorescent dye (for example Fluorescein isothiocyanate, Dallas Pink (texas red), rhodamine (rhodamine), green fluorescent protein or the like), radioactive label (for example ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P), enzyme (for example horseradish peroxidase, alkali phosphatase and other are generally used for the enzyme among the ELISA) and colorimetric labelling, for example gluey gold or coloured glass or plastics (for example polystyrene (multistyrene), polypropylene (multipropylene), latex or the like) pearl.

Various preferred radioactive labels include, but is not limited to ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³MIn, ⁹⁷Ru, ⁶²Cu, ^64ICu, ⁵²Fe, ⁵²MMn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er, ¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ^Dy, ¹⁴⁹Pm, ¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh and ¹¹¹Ag.

Detecting the method for described labelling is known by one of ordinary skill in the art.Therefore, for example, can use photographic film, scintillation detector or the like detection of radioactive labels.Can detect the luminous fluorescent labeling that detects with photodetector.Usually by substrate being provided enzyme and detect the product that acts on the substrate to be produced by enzyme and detect enzyme labelling, and detect the colorimetric labelling by simple visual observation coloured marking.

In some specific embodiments, the present invention expects that immune conjugate (telescoping part) is used to detect the purposes of tumor and/or other cancerous cell.Therefore, for instance, bi-specific antibody of the present invention can with the luminous radiosiotope of γ (for example Na-22, Cr-51, Co-60, Tc-99, I-125, I-131, Cs-137, Ga-67, Mo-99) in conjunction with γ machine testing mutually; Combine on Positron Emission Tomography (PET) instrument, to detect with the luminous isotope of positron (for example C-11, N-13, O-15, F-18 or the like); And combine to be used for NMR (Nuclear Magnetic Resonance) imaging (MRI) with metal contrast medium (for example contain Gd reagent, contain Eu reagent or the like).In addition, bi-specific antibody of the present invention can be used for traditional immunohistochemistry (for example fluorescent labeling, nanocrystal labelling, enzyme labelling and colorimetric labelling or the like).

In another embodiment, bioactive molecule can be strengthen ionizing radiation on the cell (for example may by ⁶⁰Co or x light source produce) the radiosensitizer (radiosensitizer) of cytotoxic effect.Many radiosensitizers are known, include, but is not limited to benzoporphyrin (benzoporphyrin) derivative compound (referring to, for example United States Patent (USP) the 5th, 945, No. 439), 1,2, the 4-benzotriazine oxides (referring to, for example United States Patent (USP) the 5th, 849, No. 738), contain some diamidogen chemical compound (referring to, for example United States Patent (USP) the 5th, 700, No. 825), BCNT (referring to, for example United States Patent (USP) the 5th, 872, No. 107), Radiosensitizing nitrobenzoyl acid amide derivatives (referring to, for example United States Patent (USP) the 4th, 474, No. 814), various Hete rocyclic derivatives (referring to, for example United States Patent (USP) the 5th, 064, No. 849), platinum complex (referring to, for example United States Patent (USP) the 4th, 921, No. 963) or the like.

Bioactive molecule also can be a part, epitope label, peptide, albumen or another ABP.Part and antibody can be incorporated into the surface markers on the immunocyte.Serve as the difunctional connexon of getting in touch as the chimeric molecule of bioactive molecule with described antibody in foundation between the immunocyte of the binding partners of carrying part or ABP and the tumor cell of expressing the EGFR family member.

Especially under the situation that adopts pre-targeting (pre-targeting) strategy, the form that many pharmaceuticals as herein described and/or radioactive label can be used as chelating agen provides.Chelating molecule usually with the molecule that combines the epitope that is connected in bispecific and/or polyspecific ABP specifically (biological example element (biotin), antibiotin (avidin), streptavidin (streptavidin) or the like) coupling.

Chelate group is known by one of ordinary skill in the art.In certain embodiments, chelate group derives from ethylenediaminetetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), cyclohexyl 1,2-ethylenediamine tetraacetic acid (EDTA) (CDTA), ethylene glycol-O, O '-two (2-amino-ethyl)-N, N, N ', N '-tetraacethyl (EGTA), N, two (hydroxybenzyl)-ethylenediamine-N of N-, N '-oxalic acid (HBED), teiethylene tetramine-hexacetic acid (TTHA), 1,4,7,10-tetraazacyclododecanand-N, N ', N ", N_-tetraacethyl (DOTA); hydroxyethyl ethylenediamine triacetic acid (HEDTA); 1; 4; 8,11-tetraazacyclododecane tetradecane-N, N '; N ", N_-tetraacethyl (TETA), be substituted DTPA, be substituted EDTA or the like.

The example of some preferred sequestrants comprise be unsubstituted or be substituted 2-imino group sulfane (2-iminothiolane) and 2-imino group thia cyclohexane extraction, especially 2-imino group-4-mercapto methyl sulfane (2-imino-4-mercaptomethylthiolane) and SAPS (N-(4-[211At] Ai Shi phenethyl) succinate (N-(4-[211At] astatophenethyl) succinimate)).

A kind of chelating agen 1,4,7,10-tetraazacyclododecanand-N, N, N ", N_-tetraacethyl (DOTA) is especially interesting, this be because in the many diagnosis of its chelating and treatment go up the ability of important metal (for example radioactive nucleus thuja acid and radioactive label).

The proteic conjugate of DOTA and for example antibody has been described.For instance, United States Patent (USP) the 5th, 428, No. 156 teachings are with DOTA and antibody and the bonded method of ABP fragment.In order to prepare these conjugates, the carboxylic acid group of DOTA is changed into active ester, active ester can with amine or the sulfydryl reaction on ABP or the ABP fragment.People such as Lewis (1994) Bioconjugate Chem.5:565-576 describes a kind of similar methods, wherein a carboxyl with DOTA changes into active ester, and active DOTA mixed with ABP, ABP is connected through the ε of ABP lysine residue amino with DOTA, and then the carboxyl of DOTA is changed into amide moieties.

Perhaps, can directly or through connexon chelating agen be coupled to the epitope label or be coupled to the part that conjugated antigen decision disjunction mark is signed.The conjugate of DOTA and biotin has been described, referring to, Su (1995) J.Nucl.Med. for example, 36 (supplementary issues 5): the 154th page, the key that its open DOTA and biotin are connected via available amino end side chain biotin derivative (for example DOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin).People such as Yau, WO 95/15335 disclose a kind of manufacturing can with the method for the bonded nitro-benzyl of biotin-DOTA.Described method comprises: through the cyclization of hydroxyl temporary protection; The tosylation effect of amine; Temporary transient protected hydroxyl go the protection; Go to protect the tosylation effect of hydroxyl; With the cyclisation of intramolecularly tosylate.People such as Wu, (1992) Nucl.Med.BioL, 19 (2): disclose a kind of synthetic using that be used among the 239-244 ¹¹¹IN and ⁹⁰The method of Y radioactive marker protein's huge ring chelating agen.People such as Wu make stability and the bio distribution that has antibiotin (model protein that is used to study) through labelling DOTA-biotin conjugate with research.Use contains free amine group to make this conjugate with the biotin hydrazides of generated in-situ active DOTA derivatives reaction.

ABP of the present invention can merge with other bioactive molecule, and described other bioactive molecule includes, but is not limited to cell toxicity medicament, toxin, peptide, albumen, enzyme and virus (Chester, (2000) Dis.Markers 16:53-62; People Biochem J. (2000) Biochem such as Rippmann are (part 3): 805-812 J.349, Kreitman, RJ. (2001) Curr.Pharm.Biotechnol.2:313-325; Rybak, S.M. (2001) Expert Opin.Biol.Ther.1:995-1003; Van Beusechem, people J.Virol. (2002) 76:2753-2762 such as V.W.).

Effective cell toxic agents or payload (payload) can be incorporated into ABP, described ABP targeting and be combined on the target cell (including, but is not limited to cancerous cell) the main antigen of finding.Payload agent stable connexon in blood flow is connected in ABP, perhaps is easy to cracking at (for example) tumor sites under existing condition.Therefore the load agent, for example toxin is sent to target cell, and can initiator cell kills by the mechanism that depends on toxin.

The example of these toxin includes, but is not limited to micromolecule, derive calicheamycin (calicheamicin) (people (1993) Cancer Res.53:3336-3342 such as Hinman) and maytansinoid (people (1996) PNAS USA 93:8618-8623 such as Liu of fungus for example, Smith, S. (2001) Curr.Opin.Mol.Ther.3 (2): 198-203), trichothene and CC 1065; Or albumen, for example ricin A chain (people (2000) Clin.Cancer Res.6 (4) such as Messman: 1302-1313), Pseudomonas exotoxin (Pseudomonas exotoxin) (people (2001) Intl.J.Mol.Med.8 (5) such as Tur: 579-584), diphtheria toxin, diphtherotoxin (diphtheria toxin) (people (1998) Blood 91 (2) such as LeMaistre: 399-405) and ribosome inactivating protein (people (2001) such as Tazzari, J.Immunol.167:4222-4229).In specific embodiment, can use one or more calicheamycin molecule.The antibiotic member of calicheamycin family can produce the double-stranded DNA fracture under inferior picomole concentration.The also analog of known calicheamycin.Referring to people such as Hinman, Cancer Research 53:3336-42 (1993); People such as Lode (1998) Cancer Research 58:2925-28.The example that obtains the immunotoxin of FDA approval is Mylotarg_ (Wyeth the Ayerst)-a kind of calicheamycin that is used for acute myelogenous leukemia in conjunction with anti-CD 33 (people such as Sievers, (1999) Blood 93 (11): 3678-3684; Bemstein (2000) Leukemia14:474-475).ABP of the present invention can merge with toxin in a similar manner.Perhaps, ABP of the present invention can merge with bacillus botulinus A neurotoxin, and bacillus botulinus A neurotoxin is the albumen composition that is produced by antibacterial meat poison shuttle shape (Clostridiumbotulinum).

In another embodiment, ABP of the present invention can comprise one or more enzyme activity toxin and/or its fragment.The example of these toxin comprises the non-binding active fragment of diphtheria toxin, diphtherotoxin, diphtheria toxin, diphtherotoxin A chain (from bacillus pyocyaneus (Pseudomonas aeruginosa)), ricin A chain, abrin (abrin) A chain, Mo Disu (modeccin) A chain, α-sarcina (alpha-sarcin), Dianthus carryophyllus (dianthin) albumen, Radix Phytolaccae (Phytolaca americana) albumen (PAPI, PAP AII and PAP-S), Fructus Momordicae charantiae (momordica charantia) inhibitor, curcin (curcin), crotin (crotin), sapaonaria officinalis inhibitor, spend more Rhizoma Melaleuca Viridiflora element (gelonin), mitogellin, restrictoein, phenomycin (phenomycin), enomycin (enomycin) and single-ended pityrosporion ovale mycotoxin (tricothecene).Referring to, for example WO 93/21232.Especially preferred cytotoxin comprises Pseudomonas exotoxin (PE), diphtheria toxin, diphtherotoxin, Ricin and abrin.Pseudomonas exotoxin and diphtheria toxin, diphtherotoxin are all known.The same with PE, diphtheria toxin, diphtherotoxin (DT) is by ADP ribosylation elongation factor 2 cell killing, and then Profilin is synthetic.Other citing document about immunotoxin comprises Brinkmann, U.In Vivo 14:21-28; People such as Niv (2001) Curr.Pharm.Biotechnol.2:19-46; People Adv.Cancer Res.81:93-124 such as Reiter; Kreitman, R.J. (1999) Curr.Opin.Immunol., 11:570-578; Hall (2001) Meth.Mol.Biol.166:139-154; Kreitman (2001) Curr.Opin.Investig.Drugs 2 (9): 1282-1293.One of ordinary skill in the art know PE that coding and various parts merge or DT gene clone method (referring to, people (1989) FASEB J. such as Siegall for example, 3:2647-2652; With people (1987) Proc.Natl.Acad.Sci.USA such as Chaudliary, 84:4538-4542).The all references document is all incorporated this paper by reference into.

Other suitable bioactive molecule comprises pharmacological agents or contains the package system of various pharmacological agents (encapsulation system).Therefore, the targeted molecular of chimeric molecule can be directly connected in the medicine for the treatment of directly to be passed to tumor.Described medicine is known by one of ordinary skill in the art, and includes, but is not limited to doxorubicin (doxirubicin), vinblastine (vinblastine), genistein (genistein), antisense molecule or the like.

Perhaps, bioactive molecule can be a package system, for example viral capsid, liposome or contain therapeutic combination (for example medicine, nucleic acid (for example antisensenucleic acids) or another kind of preferably through shielding in order to avoid directly be exposed to the treatment part of blood circulation) micella.Preparation is connected in the method for the liposome of antibody and knows for one of ordinary skill in the art.Referring to, for example United States Patent (USP) the 4th, 957, and No. 735, people such as Connor (1985) Pharm.Ther., 28:341-365.Owing to have an antigenic specificity, therefore ABP of the present invention can be used for the liposome of drug loading is sent to their target.Referring to Park, people such as J.W. (2002) Clin.Cancer Res.8,1172-1181 and Shi, people such as N. (2001) Pharm.Res.18,1091-1095.

ABP of the present invention can combine with the molecule of for example PEG to improve and in vivo transmit and the pharmacokinetics curve.People such as Leong describe has the low clearance rate that is better than PEGization form not and minimum or do not have the Fab ' segmental locus specificity PEGization (Leong, people such as S.R. (2001) Cytoltine16:106-119) of antigen in conjunction with the anti-IL-8 antibody of loss of activity.

ABP of the present invention can be connected in prodrug.Term as used herein " prodrug " is meant pharmacology's non-activity of active medicine or SA derivant.Thereby can arrive desirable action site by the characteristic (for example physical chemistry, biologic pharmacological science or pharmacokinetic properties) of handling medicine to the amount that prodrug designs to regulate medicine or bioactive molecule.Prodrug is in vivo through enzyme reaction or non-enzyme reaction and change into active medicine.Prodrug can provide improved physicochemical characteristics, and for example preferable dissolubility, enhanced transfer characteristic are for example selectively targeted in specific cells, tissue, organ or part and improved Drug therapy value.ABP of the present invention can merge with enzyme and be used for the prodrug activation (Kousparou, people such as C.A. (2002) Int.J.Cancer 99,138-148).Recombinant molecule can comprise ABP and act on the prodrug to discharge the enzyme of cytotoxin (for example cyanide).

The therapeutic agent that can prodrug forms comes into operation, and activate by the prodrug kinase that will change into the active anticancer medicine as the prodrug of peptidyl chemotherapeutant subsequently.Referring to, for example No. the 4th, 975,278, WO 88/07378, WO 81/01145, United States Patent (USP).In general, the enzyme component comprise any can with as convert it into the enzyme that the mode that has more active cytotoxicity form acts on prodrug.

Available enzyme include, but is not limited to can be used for the phosphoric acid ester prodrugs change into free drug alkali phosphatase, can be used for the sulfur acid ester prodrugs is changed into the aryl sulfatase of free drug; Can be used for nontoxic 5-flurocytosine is changed into the cytosine deaminase of cancer therapy drug 5-fluorouracil; Can be used for to contain the protease that the peptide prodrug changes into free drug, for example Serratieae (serratia) protease, thermolysin (thermolysin), subtilisin (subtilisin), carboxypeptidase and cathepsin (cathepsin) (for example cathepsin B and L); Can be used for transforming the D-alanyl carboxypeptidase of the prodrug that contains D-aminoacid replacement base; Can be used for the glycosylation prodrug is changed into the carbohydrate lyases of free drug, for example beta galactosidase and neuraminidase; Can be used for to change into the beta-lactamase of free drug with the deutero-medicine of beta-lactam; The penicillin amidase of free drug, for example penicillin V amidase or benzylpenicillin amidase will be changed into can be used at its amino nitrogen place respectively with phenoxy group acetyl group or the deutero-medicine of phenyl acetyl.

Perhaps, can use in affiliated technical field the antibody that is called " abzyme (abzyme) " again that prodrug of the present invention is changed into free active medicine with enzymatic activity.Referring to, Massey for example, (1987) 328:457-48.

One of ordinary skill in the art should be appreciated that bispecific of the present invention and/or polyspecific ABP and bioactive molecule part can link together with any order usually.Therefore, for example when targeted molecular was single chain protein, bioactive molecule can be connected in the aminoterminal or the c-terminus of targeted molecular.Bioactive molecule also can be connected in the inner area of bispecific and/or polyspecific ABP, and is perhaps opposite.Equally, bispecific and/or polyspecific ABP also can be connected in the interior location or the end of bioactive molecule.Under any circumstance, alternative point of contact does not consequently disturb indivedual activity of bispecific and/or polyspecific ABP or bioactive molecule.

Can connect bispecific and/or polyspecific ABP or bioactive molecule by the known any many modes of one of ordinary skill in the art.Bioactive molecule directly or through connexon (introns) combines with bispecific ABP usually.Yet, when bioactive molecule and bispecific ABP are polypeptide, may need to be the strand fusion rotein with chimeric molecule is recombinant expressed.

In one embodiment, bispecific and/or polyspecific ABP chemical bond are in bioactive molecule (for example cytotoxin, labelling, part, medicine, ABP, liposome or the like).The method of chemical bond molecule is known by one of ordinary skill in the art.

The connection procedure of reagent and ABP or other polypeptide targeted molecular can be different with the chemical constitution of reagent.Polypeptide contains multiple functional group usually, for example carboxylic acid (COOH) or unhindered amina (NH ₂) group, these functional groups can react to combine with bioactive molecule in this place with the appropriate functional group on the bioactive molecule.

Perhaps, bispecific ABP and/or bioactive molecule can be through deriving with exposure or connecting other reactive functionality.Described derivatization can relate to and connect any many link molecule, and for example those are available from PierceChemical Company, Rockford, 111 link molecule.

In some cases, when telescoping part has arrived its target site, may need to make bioactive molecule free from bispecific and/or polyspecific ABP, or the activation prodrug.Therefore, when dividing the period of the day from 11 p.m. to 1 a.m, can use the chimeric conjugate of the key that is included in contiguous target site place cleavable at the target site delivery of biologically active.Can by enzymatic activity or immune conjugate in target cell or the condition that in contiguous target site, the is stood cracking that promotes key with from the ABP release reagent.When target site is tumor, can use the connexon of (for example when being exposed to cancer-related enzyme or acid pH) cleavable under the condition that exists in the tumor sites place.

Many different cleavable connexons are known by one of ordinary skill in the art.Referring to, United States Patent (USP) the 4th, 618, No. 492, the 4th, 542, No. 225 and the 4th, 625, No. 014.Comprise from the mechanism of these connexon group release reagents, for example irradiates light unstability key and acid-catalyzed hydrolysis.For instance, United States Patent (USP) the 4th, 671 comprises the description of the immune conjugate that comprises connexon No. 958, described connexon at the target site place by the in vivo cracking of proteolytic enzyme of patient's complement system.The length of connexon can be scheduled to, and perhaps can be selected according to the relation of the requisite space between ABP and the connected molecule.In view of many report be used for method that multiple radiodiagnosis chemical compound, radiotherapy chemical compound, medicine, toxin and other reagent are connected with antibody, one of ordinary skill in the art should be able to determine appropriate method that giving reagent is connected with ABP or other polypeptide.

In certain embodiments, bioactive molecule comprises the chelating agen that is connected in ABP or is connected in the epitope label.Bispecific and/or polyspecific ABP carry corresponding antigen decision disjunction mark and sign or ABP, so bispecific and/or polyspecific ABP and the simple contact of chelating agen cause being connected of ABP and bioactive molecule.Can perhaps before transmitting chelating agen, target tissue be combined with bispecific and/or polyspecific ABP using described part (pre-targeting strategy) to carry out described integrating step afterwards.Be fit to the manufacture method of the link coupled chelate of various targeting moieties by one of ordinary skill in the art known (referring to, for example United States Patent (USP) the 6th, 190, No. 923, the 6th, 187, No. 285, the 6th, 183, No. 721, the 6th, 177, No. 562, the 6th, 159, No. 445, the 6th, 153, No. 775, the 6th, 149, No. 890, the 6th, 143, No. 276, the 6th, 143, No. 274, the 6th, 139, No. 819, the 6th, 132, No. 764, the 6th, 123, No. 923, the 6th, 123, No. 921, the 6th, 120, No. 768, the 6th, 120, No. 751, the 6th, 117, No. 412, the 6th, 106, No. 866, the 6th, 096, No. 290, the 6th, 093, No. 382, the 6th, 090, No. 800, the 6th, 090, No. 408, the 6th, 088, No. 613, the 6th, 077, No. 499, the 6th, 075, No. 010, the 6th, 071, No. 494, the 6th, 071, No. 490, the 6th, 060, No. 040, the 6th, 056, No. 939, the 6th, 051, No. 207, the 6th, 048, No. 979, the 6th, 045, No. 821, the 6th, 045, No. 775, the 6th, 030, No. 840, the 6th, 028, No. 066, the 6th, 022, No. 966, the 6th, 022, No. 523, the 6th, 022, No. 522, the 6th, 017, No. 522, the 6th, 015, No. 897, the 6th, 010, No. 682, the 6th, 010, No. 681, the 6th, 004, No. 533 and the 6th, 001, No. 329).

When bispecific and/or polyspecific ABP and/or bioactive molecule were single chain protein and relatively lack (promptly less than about 50 aminoacid), they can use standard chemical peptide synthetic technology to synthesize.When two kinds of components all relatively more in short-term, telescoping part can synthesize single contiguous polypeptide.Perhaps, can synthesize bispecific and/or polyspecific ABP and bioactive molecule separately, and merge by the aminoterminal of a molecule of condensation and the c-terminus of another molecule subsequently, thereby form peptide bond.Perhaps, bispecific and/or polyspecific ABP and bioactive molecule can be separately and a terminal condensation of peptide spacer molecule, and then form contiguous fusion rotein.

Solid phase synthesis is the method for optimizing of chemosynthesis polypeptide of the present invention, and wherein the C-terminal aminoacid of sequence is connected in insoluble supporter, then adds all the other aminoacid in the sequence in succession.Solid phase synthesis technique is by The Peptides:Analysis, Synthesis, Biology. the 2nd roll up: Special Methods in Peptide Synthesis, part A., people J.Am.Chem.Soc such as Merrifield, Barany and Merrifield among the 85:2149-2156 (1963), Solid-Phase Peptide Synthesis; People such as 3-284 page or leaf and Stewart, Solid Phase PeptideSynthesis, the 2nd edition .Pierce Chem.Co., Rockford, 111 (1984) describe.

" double functional copolymer " is meant the polymer that comprises two discontinuous functional groups, and these two discontinuous functional groups can react to form covalently or non-covalently key with other parts (including, but is not limited to amino acid side group) specifically.Can use one of them functional group can be with radical reaction on the particular organisms active component and another functional group can with the difunctionality connexon of radical reaction on second biological components, form the conjugate that comprises first biological active component, difunctionality connexon and second biological active component.The many processes and the link molecule that are used for all cpds is connected in peptide are known.Referring to, for example No. the 4th, 671,958, No. the 188th, 256, European patent application, United States Patent (USP), the 4th, 659, No. 839, the 4th, 414, No. 148, the 4th, 699, No. 784, the 4th, 680, No. 338, the 4th, 569, No. 789 and the 4th, 589, No. 071, it all incorporates this paper by reference into." polyfunctional poly compound " is meant the polymer that comprises two or more discontinuous functional groups, and affiliated two or more discontinuous functional groups can react to form covalently or non-covalently key with other parts (including, but is not limited to amino acid side group) specifically.Double functional copolymer or polyfunctional poly compound can have any desired molecule length or molecular weight, and can be through selecting so that intermolecular desired spacing or the conformation that is connected in ABP to be provided.

From left to right write by its conventional chemical formula and when specifying when substituent group, contain equally can from from right to left the identical substituent group of the resulting chemical property of the structure of writing, structure-CH for example ₂0-is equal to structure-OCH ₂-.

Term " substituent group " includes, but is not limited to " non-interfering substituent "." non-interfering substituent group " is meant that those produce the substituent group of stable compound.Suitable non-interfering substituent or group include, but is not limited to halogen, C ₁-C ₁₀Alkyl, C ₂-C ₁₀Thiazolinyl, C ₂-C ₁₀Alkynyl, C ₁-C ₁₀Alkoxyl, C ₁-C ₁₂Aralkyl, C ₁-C ₁₂Alkaryl, C ₃-C ₁₂Cycloalkyl, C ₃-C ₁₂Cycloalkenyl group, phenyl, be substituted phenyl, toluyl, xylyl, xenyl, C ₂-C ₁₂Alkoxyalkyl, C ₂-C ₁₂Alkoxy aryl, C ₇-C ₁₂Aryloxy alkyl, C ₇-C ₁₂Oxygen Ji Fangji, C ₁-C ₆Alkyl sulfinyl, C ₁-C ₁₀Alkyl sulphonyl,--(CH ₂) _m--O--(C ₁-C ₁₀Alkyl) (wherein m is 1 to 8), aryl, be substituted aryl, be substituted alkoxyl, fluoroalkyl, heterocyclic radical, be substituted heterocyclic radical, 4-nitro alkyl,--NO ₂,--CN,--NRC (O)--(C ₁-C ₁₀Alkyl),--C (O)--(C ₁-C ₁₀Alkyl), C ₂-C ₁₀Alkyl alkylthio base,--C (O) O--(C ₁-C ₁₀Alkyl),--OH,--SO ₂,=S,--COOH,--NR ₂, carbonyl,--C (O)-(C ₁-C ₁₀Alkyl)-CF ₃,--C (O)-CF ₃,--C (O) NR ₂,--(C ₁-C ₁₀Aryl)-S--(C ₆-C ₁₀Aryl),--C (O)--(C ₁-C ₁₀Aryl),--(CH ₂) _m--O--(--(CH ₂) _m--O--(C ₁-C ₁₀Alkyl) (wherein each m is 1 to 8),--C (O) NR ₂,--C (S) NR ₂,--SO ₂NR ₂,--NRC (O) NR ₂,--NRC (S) NR ₂, its salt or the like.Each R as used herein is H, alkyl or is substituted alkyl, aryl or is substituted aryl, aralkyl or alkaryl.

Term " halogen " comprises fluorine, chlorine, iodine and bromine.

Unless otherwise indicated, otherwise self or as term " alkyl " the expression straight chain of another substituent group part or be with branched chain or cyclic hydrocarbon group, or its combination, these groups can be fully saturated, single unsaturated or polyunsaturated, and can comprise that having the indication carbon number (is C ₁-C ₁₀Represent one to ten carbon) bivalence and multivalence group.The example of saturated hydrocarbyl includes, but is not limited to following group, for example the homologue and the isomer of methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, (for example) n-pentyl, n-hexyl, n-heptyl, n-octyl etc.Unsaturated alkyl is meant to have one or more pairs key or triple-linked alkyl.The example of unsaturated alkyl includes, but is not limited to vinyl, 2-acrylic, crotyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), acetenyl, 1-and 3-propinyl, 3-butynyl and higher carbon number homologue and isomer.Unless otherwise indicated, otherwise term " alkyl " also comprises hereinafter the alkyl derivative of specific definition more, for example " assorted alkyl ".The alkyl called after " high alkyl (homoalkyl) " that is limited to alkyl.

Self or derive from the divalent group of alkane, for example (but being not limited to) structure-CH as term " alkylidene " expression of another substituent group part ₂CH ₂-and-CH ₂CH ₂CH ₂CH ₂-, and comprise the group of " assorted alkylidene " as mentioned below in addition.Alkyl (or alkylidene) has 1 to 24 carbon atom usually, those have 10 or still less the group of carbon atom be first-selected in the present invention." than the low carbon number alkyl " or " than the low carbon number alkylidene " be have usually eight or still less carbon atom than short-chain alkyl or alkylidene.

Term " alkoxyl ", " alkyl amino " and " alkyl sulfenyl " (or thio alkoxy) use with its conventional meaning, and are meant the alkyl that is connected in the molecule remainder through oxygen atom, amino or sulphur atom respectively.

Unless otherwise indicated, otherwise separately or term " assorted alkyl " expression that is used in combination with other term by described number carbon atom and at least one be selected from the hetero atom of forming group by O, N, Si and S form (wherein nitrogen and sulphur atom can be according to circumstances through oxidation and nitrogen heteroatom can be according to circumstances through quaternized) stable straight chain or have branched chain or cyclic hydrocarbon group.Hetero atom O, N, S and Si can be positioned at any interior location place of assorted alkyl, perhaps are positioned at alkyl and are connected in molecule remainder office.Example includes, but is not limited to-CH ₂-CH ₂-O-CH ₃,-CH ₂-CH ₂-NH-CH ₃,-CH ₂-CH ₂-N (CH ₃)-CH ₃,-CH ₂-S-CH ₂-CH ₃,-CH ₂-CH ₂-S (O)-CH ₃,-CH ₂-CH ₂-S (O) ₂-CH ₃,-CH=CH-O-CH ₃,-Si (CH ₃) ₃,-CH ₂-CH=N-OCH ₃With-CH=CH-N (CH ₃)-CH ₃Maximum two hetero atoms can be successive, for example-and CH ₂-NH-OCH ₃With-CH ₂-O-Si (CH ₃) ₃Similarly, term " assorted alkylidene " expression partly derives from the divalent group of assorted alkyl, for example (but being not limited to)-CH separately or as another substituent group ₂-CH ₂-S-CH ₂-CH ₂-and-CH ₂-S-CH ₂-CH ₂-NH-CH ₂-.For assorted alkylidene, identical or different hetero atom also can occupy arbitrary chain end or two ends (including, but is not limited to alkylene oxide group, alkylene dioxo base, alkylidene amino, alkylidene diaminourea, aminooxy group alkylidene or the like).In addition, be connected base with assorted alkylidene, connect the orientation that direction that basic chemical formula writes does not represent to connect base for alkylidene.For instance, formula-C (O) ₂R '-expression-C (O) ₂R '-and-R ' C (O) ₂-.

Unless otherwise indicated, otherwise self or with the combination of other term in term " cycloalkyl " and " Heterocyclylalkyl " " alkyl " and " alkyl of mixing " of representing annular form respectively.Therefore, cycloalkyl or Heterocyclylalkyl comprise saturated and unsaturated ring key.In addition, for Heterocyclylalkyl, hetero atom can occupy the position that heterocycle is connected in the molecule remainder.The example of cycloalkyl includes, but is not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl or the like.The example of Heterocyclylalkyl includes, but is not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, Tetramethylene sulfide-2-base, Tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl or the like.In addition, dicyclo and tricyclic structure contained in described term.Similarly, self or derive from the divalent group of Heterocyclylalkyl as term " inferior Heterocyclylalkyl " expression of another substituent group part, and self or derive from the divalent group of cycloalkyl as term " cycloalkylidene " expression of another substituent group part.

Term as used herein " water-soluble polymer " be meant any in aqueous solution polymer soluble.The bonding of water-soluble polymer and ABP can produce following change, include, but is not limited to respect to for modified forms, increase or the adjusting serum half-life, perhaps increase or the adjustment of treatment half-life, regulate immunogenicity, regulate physics association feature (for example assembling and polymer formation), change receptors bind and change table receptor dimerizationization or multimerization effect.Water-soluble polymer can have or not have self biological activity, and can include, but is not limited to one or more ABP, one or more bioactive molecules as connexon to connect ABP and other material.Suitable polymers includes, but is not limited to Polyethylene Glycol, Polyethylene Glycol propionic aldehyde, its single C ₁-C ₁₀Alkoxyl or aryloxy group derivant (are described in United States Patent (USP) the 5th, 252, No. 714, it incorporates this paper by reference into), mono methoxy-Polyethylene Glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, divinyl ether-maleic anhydride, N-(2-hydroxypropyl)-Methacrylamide, glucosan, glucan derivative (comprising dextran sulfate), polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, the polyethoxylated polyhydric alcohol, heparin, the heparin fragment, polysaccharide, oligosaccharide, polysaccharide, cellulose and cellulose derivative (including, but is not limited to methylcellulose and carboxymethyl cellulose), starch and starch derivatives, polypeptide, ployalkylene glycol and derivant thereof, the copolymer of ployalkylene glycol and its derivant, polyvinyl ethylether and alpha-beta-poly-[(2-hydroxyethyl)-DL-agedoite or the like or its mixture.The example of these water-soluble polymers includes, but is not limited to Polyethylene Glycol and serum albumin.

Term as used herein " ployalkylene glycol " " poly-(alkylene glycol) " is meant Polyethylene Glycol, polypropylene glycol, polytetramethylene glycol and derivant thereof.Term " ployalkylene glycol " is contained straight chain and branch polymer and mean molecule quantity between 0.1kDa and 100kDa.Other one exemplary embodiment is listed in the commercial suppliers catalogue, for example the catalogue of ShearwaterCorporation " Polyethylene Glycol and Derivatives for Biomedical Applications " (2001).

Unless otherwise indicated, otherwise term " aryl " expression can be monocycle or condense in together or the how unsaturated aromatic hydrocarbon substituent group of covalently bound multi-ring (being preferably 1 to 3 ring).Term " heteroaryl " is meant and contains one to four heteroatomic aryl (aromatic ring) of selecting N, O and S, and wherein nitrogen-atoms and sulphur atom be according to circumstances through oxidation, and nitrogen-atoms is according to circumstances through quaternized.Heteroaryl can be connected in the remainder of molecule through hetero atom.The limiting examples of aryl and heteroaryl comprises phenyl, the 1-naphthyl, the 2-naphthyl, the 4-xenyl, the 1-pyrrole radicals, the 2-pyrrole radicals, the 3-pyrrole radicals, the 3-pyrazolyl, the 2-imidazole radicals, the 4-imidazole radicals, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridine radicals, the 3-pyridine radicals, the 4-pyridine radicals, the 2-pyrimidine radicals, the 4-pyrimidine radicals, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.The substituent group of each above-mentioned aryl and heteroaryl ring system is selected from the following substituent group that accepts.

Briefly, when being used in combination (including, but is not limited to aryloxy group, aryl sulfur oxygen base, aryl alkyl) with other term, term " aryl " comprises above defined aryl and heteroaryl ring.Therefore, term " aryl alkyl " comprises that aryl wherein is connected in those groups (including, but is not limited to benzyl, phenethyl, pyridylmethyl or the like) of alkyl, comprise that wherein carbon atom includes, but is not limited to phenoxymethyl, 2-pyridyloxy methyl, 3-(1-naphthoxy) propyl group or the like through the metathetical alkyl of (for example) oxygen atom (including, but is not limited to methylene).

Each above-mentioned term (comprising (but being not limited to " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ")) comprises the described group that is substituted and is unsubstituted form.The exemplary replacement of all kinds of groups is based on hereinafter providing.

The substituent group of alkyl and assorted alkyl (comprising the group that is commonly referred to alkylidene, thiazolinyl, assorted alkylidene, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) can be to be selected from one or more in the following multiple group: 0 to (2m '+1) number ^{-OR '},=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R_ ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ⁵,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) N " R_ ,-NR " C (O) ₂R ' ,-NR-C (NR ' R " R_)=NR_ ' ,-NR-C (NR ' R ")=NR_ ,-S (O) R ' ,-S (O) ₂R ' ,-S (O) ₂NR ' R " ,-NRSO ₂R ' ,-CN and-NO ₂, wherein m ' is the sum of carbon atom in the described group.R ', R ", R_ and R " " independently are meant hydrogen separately, are substituted or are unsubstituted assorted alkyl, are substituted or are unsubstituted aryl, its aryl that includes, but is not limited to replace through 1-3 halogen, be substituted or be unsubstituted alkyl, alkoxyl or thio alkoxy or aryl alkyl.For instance, when chemical compound of the present invention comprised an above R group, each R group independently was chosen as when having more than one these groups " the group as each R ', R ", R_ and R "." when being connected in identical nitrogen-atoms, it can combine with nitrogen-atoms to form 5 yuan, 6 yuan or 7 yuan of rings as R ' and R.For instance ,-NR ' R " includes, but is not limited to 1-pyrrolidinyl and 4-morpholinyl.Based on above-mentioned substituent description, one of ordinary skill in the art should be appreciated that term " alkyl " is meant and comprise and the group of the bonded carbon atom of group except that hydrogen atom that for example haloalkyl (includes, but is not limited to-CF ₃With-CH ₂CF ₃) and acyl group (include, but is not limited to-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂OCH ₃Or the like).

Similar to the described substituent group of alkyl, the substituent group of aryl and heteroaryl can change, and is selected from (but being not limited to): 0 to the aromatic ring system open valency (open valence) sum halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R_ ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R_ ,-NR " C (O) ₂R ' ,-NR-C (NR ' R " R_)=NR " " ,-NR-C (NR ' R ")=NR_ ,-S (O) R ' ,-S (O) ₂R ' ,-S (O) ₂NR ' R " ,-NRSO ₂R ' ,-CN and-NO ₂,-R ' ,-N ₃,-CH (Ph) ₂, fluorine (C ₁-C ₄) alkoxyl and fluorine (C ₁-C ₄) alkyl; And wherein R ', R ", R_ and R " " independent be hydrogen, alkyl, mix alkyl, aryl and heteroaryl.For instance, when chemical compound of the present invention comprised an above R group, each R group independently was chosen as when having more than one these groups " the group as each R ', R ", R_ and R ".

Term as used herein " through regulating serum half-life " is meant that the plus or minus with respect to the circulating half-life of modified ABP for modified forms changes.Get blood sample during by each time point behind throwing and ABP and measure serum half-life with the concentration of the described molecule of mensuration in each sample.The dependency of serum-concentration and time makes can calculate serum half-life.Serum half-life advantageously has the increase at least about twice, but less increase also is available, for example when making it possible to obtain satisfied dosage regimen or avoiding toxic action.In certain embodiments, described increase at least about three times, at least about five times or at least about ten times.

Term as used herein " through the adjustment of treatment half-life " is meant with respect to treatment effective dose ABP for modified forms or the plus or minus of half-life that comprises the ABP of modified bioactive molecule and changes.Measure the pharmacokinetics and/or the drug effect characteristic of molecule during by each time point after dispensing and measure the treatment half-life.The treatment half-life that increases advantageously makes it possible to obtain especially useful dosage regimen, especially useful accumulated dose, or when avoiding ill effect.In certain embodiments, the treatment half-life of increase comes from the raising of usefulness, modified molecule and the bonded increase of its target or minimizing or without the increase or the minimizing of another parameter of decorating molecule or mechanism of action.

When being used for nucleic acid or albumen, term " isolating " expression nucleic acid or albumen do not contain other relevant under native state cellular component in fact.It can be a homogeneous state.Separate substance can be dry state or half-dried attitude, perhaps in solution, includes, but is not limited to aqueous solution.Usually for example use analysis such as polyacrylamide gel electrophoresis or high performance liquid chromatography chemical technology to determine purity and homogeneity.Come down to purification as the albumen that is present in the main species in the preparation.Specifically, isolated genes is to obtain from being arranged in the proteic open reading frame separation except that gene of interest of gene flank and coding.Term " purification " expression nucleic acid or albumen produce a band in fact in running gel.Specifically, " purification " expression nucleic acid or albumen has at least 85%, at least 90%, at least 95%, at least 99% or high-purity more.

Term " nucleic acid " is meant deoxyribonucleotide, dezyribonucleoside, ribonucleotide or ribonucleotide and the polymer thereof of strand or double chain form.Unless in addition special restriction, otherwise described term is contained the known analog that contains natural nucleotide, is had the binding characteristic similar to reference nucleic acid and with the mode metabolic nucleic acid similar to natural generation nucleotide.Unless special in addition restriction, otherwise described term also refers to comprise the oligonucleotide analogs of PNA (peptide nucleic acid(PNA)), is used for the DNA analog (thiophosphate, phosphoramidate or the like) of antisense technology.Unless otherwise indicated, otherwise specific nucleic acid sequence also impliedly comprises its conservative sequence of modifying variant (including, but is not limited to degenerate codon replaces) and complementary series and clear and definite indication.Specifically, can finish degenerate codon and replace (people such as Batzer, Nucleic Acid Res.19:5081 (1991) through mixing sequence that base and/or deoxidation hypoxanthine residue replace by producing one of them or selected more than one (or all) codons the 3rd; People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); People (1992) such as and Cassol; People such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)).

Term " polypeptide ", " peptide " and " albumen " are used alternatingly and refer to the polymer of amino acid residue in this article.That is to say that be equally applicable to the description and the proteic description of peptide at the description of polypeptide, vice versa.Described term is applicable to natural generation amino acid polymer and one of them or the amino acid polymer that above amino acid residue is a non-naturally encoded amino acids.As used herein, the amino acid chain of any length contained in described term, comprises full-length proteins (being antigen), and wherein said amino acid residue connects by the covalency peptide bond.

Term " aminoacid " is meant the aminoacid that natural generation and non-natural take place, and with the amino acid analogue and the amino acid analog thing of natural generation amino acid similarity mode functionating.Natural coded amino acid is 20 kinds of common amino acids (alanine, arginine, agedoite, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) and burnt lysine and selenocystein.Amino acid analogue is meant the chemical compound that has with the identical basic chemical structure of natural generation aminoacid (promptly being incorporated into α carbon, carboxyl, amino and the R group of hydrogen), for example homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium (methionine methyl sulfonium).Described analog has modified R group (for example nor-leucine) or modified peptide backbone, but keeps and the identical basic chemical structure of natural generation aminoacid.

This paper according to IUPAC-IUB biochemistry noun validation board (IUPAC-IUB BiochemicalNomenclature Commission) recommended general known to a three-character doctrine number or a word symbol represent aminoacid.Equally also can represent nucleotide by the general individual character code of accepting.

" the conservative variant of modifying " is applicable to aminoacid and nucleotide sequence.With regard to specific nucleic acid sequence, " conservative modify variant " is meant the nucleic acid of the identical or substantially the same aminoacid sequence of coding, perhaps is not meant substantially the same sequence during encoding amino acid sequence when nucleic acid.Because the degeneracy of genetic code, the therefore identical any given albumen of nucleic acid coding of many functions.For instance, the equal coded amino acid alanine of codon GCA, GCC, GCG and GCU.Therefore,, codon can be changed over any above-mentioned corresponding codon and do not change encoded polypeptide by each specified position of codon at alanine.Described nucleic acid variation is " silent variant (silentvariation) ", is a kind of conservative modification variation.Each nucleotide sequence of this paper coded polypeptide is also described every kind of possible nucleic acid silent variant.One of ordinary skill in the art it should be understood that can modification of nucleic acids in each codon (except that AUG (generally only being codon methionine) and TGG (generally only being the tryptophan codon)) with generation function same molecular.Therefore, every kind of nucleic acid encoding silent variant is implicitly included in each described sequence.

About aminoacid sequence, one of ordinary skill in the art will be appreciated that, single amino acids or the amino acid whose indivedual replacements of little percentage ratio, disappearance or interpolation are " the conservative variants of modifying " in the change that nucleic acid, peptide, polypeptide or protein sequence are carried out, interpolation or the disappearance coded sequence, wherein change to cause aminoacid to be replaced through the similar aminoacid of chemistry.Provide the amino acid whose conservative substitution table of functional similarity to be known by affiliated technical field.Described conservative modification variant except that polymorphie variant of the present invention, plant between congener and the allele, but do not get rid of these.

Below eight groups respectively contain the promising conservative each other aminoacid of replacing:

1) alanine (A), glycine (G);

2) aspartic acid (D), glutamic acid (E);

3) agedoite (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V);

6) phenylalanine (F), tyrosine (Y), tryptophan (W);

7) serine (S), threonine (T); With

8) cysteine (C), methionine (M).

Referring to, Creighton for example, Proteins:Structures and Molecular Properties (W H Freeman﹠amp; Co.; The 2nd edition (in December, 1993).

In the content of two or more nucleic acid or peptide sequence, term " unanimity " or percentage ratio " concordance " are meant sequence or the subsequence that two or more are identical.When on comparison window or appointed area when using a kind of following sequence comparison algorithm or coming comparison and comparison maximum to response by manual comparison and visual observation, if its have same amino acid residue or nucleotide percentage ratio (promptly on the appointed area about 60% concordance, according to circumstances about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% concordance), then sequence is " consistent in fact ".Described definition also refers to the complementary series of cycle tests.Described concordance may reside on the zone at least about 50 aminoacid or length of nucleotides, on the zone of 75-100 aminoacid or length of nucleotides, perhaps is on whole sequence or polynucleotide or the polypeptide when not specifying perhaps.

For sequence relatively, a common sequence is served as the reference sequences that cycle tests compares.When using sequence comparison algorithm,, specify subsequence coordinate (if desired) and specified sequence algorithm routine parameter with cycle tests and reference sequences input computer.Can use the default program parameter, perhaps can designated parameter.Sequence comparison algorithm calculates the percentage ratio sequence identity of cycle tests with respect to reference sequences based on program parameter subsequently.

" comparison window " as used herein comprise be selected from by 20 to 600, about 50 to about 200, more generally about 100 to about 150 sections of forming any number continuous position of group usually, wherein can be after a sequence and the best comparison of reference sequences with the reference sequences comparison of this sequence and similar number continuous position.Being used to compare the comparative sequences method is known by affiliated field.Can carry out the best comparison of comparative sequences, include, but is not limited to local homology's algorithm of Smith and Waterman (1970) Adv.Appl.Math.2:482c, the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol.Biol.48:443, the search similarity method of Pearson and Lipman (1988) Proc.Nat ' l.Acad.Sci.USA 85:2444, the computer realization of these algorithms (WisconsinGenetics Software Package, Genetics Computer Group, 575Science Dr., Madison, GAP among the WI, BESTFIT, FASTA and TFASTA) or manually comparison and visual observation be (for example, referring to people such as Ausubel, Current Protocols in Molecular Biology (1995 supplementary issue)).

A kind ofly be applicable to that the algorithm examples of determining percentage ratio concordance and sequence similarity is BLAST and BLAST2.0 algorithm, it is described in respectively among people (1990) J.Mol.Biol.215:403-410 such as people such as Altschul (1977) Nuc.Acids Res.25:3389-3402 and Altschul.The software of carrying out the BLAST analysis is to disclose available through American National biotechnology information centre (National Center for Biotechnology Information).BLAST algorithm parameter W, T and X determine the sensitivity and the speed of comparison.BLASTN program (being used for nucleotide sequence) is used acquiescence word length (W) 11, expected value (E) or 10, M=5, N=-4 and relatively double-stranded.For aminoacid sequence, BLASTP uses acquiescence word length 3 and expected value (E) 10 and BLOSUM62 score matrix (referring to Henikoff and Henikoff (1989) Proc.Natl.Acad.Sci.USA 89:10915) comparison (B) 50, expected value (E) 10, M=5, N=-4 and two strands relatively.Usually carry out the BLAST algorithm closing under " low complex degree " filter.

The BLAST algorithm also carries out the statistical analysis (referring to, Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787 for example) of similarity between two sequences.The similarity measurement standard that the BLAST algorithm is provided is minimum and probability (smallest sum probability) (P (N)), the probability that mates between its indication occurrent two nucleotide of possibility or the aminoacid sequence.For instance, if the minimum of test nucleic acid and reference nucleic acid comparison and probability less than about 0.2, be more preferably less than about 0.01 and most preferably less than about 0.001, think that then nucleic acid is similar to reference sequences.

Phrase " selectivity (or specificity) hybridization " is meant that described molecule only combines with specific nucleotide sequence when sequence is present in the complex mixture (including, but is not limited to total cell or library DNA or RNA) under rigorous hybridization conditions, duplex or hybridization.

Phrase " rigorous hybridization conditions " is meant as known low ionic strength in affiliated field and hot conditions.Target sequence hybridization in the complex mixture (include, but is not limited to total cell or library DNA or RNA) of the common Ying Yuqi of probe under the rigorous condition at nucleic acid, and not with complex mixture in other sequence hybridization.Rigorous condition has serial correlation, and can be different under different situations.Longer sequence is specific hybrid under higher temperature.The extensive guidance of nucleic acid hybridization sees Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes is in " Overview of principles of hybridization andthe strategy ofnucleic acid assays " (1993).Usually rigorous condition is chosen as the heat fusion joint (T that is lower than specific sequence under defined ionic strength pH _m) about 5-10 ℃.T _mBe to hybridize balance (as the excessive existence of target sequence, T with 50% and target sequence of the complementary probe of target _mThe time, 50% probe occupies balance) time temperature (under defined ionic strength, pH and nucleic acid concentration).Rigorous condition can be that 7.0 to 8.3 times salinity of pH are lower than about 1.0M sodium ion (usually about 0.01 to 1.0M Na ion concentration), and for short probe temperature at least about 30 ℃ (including, but is not limited to 10 to 50 nucleotide) for long probe at least about 60 ℃ (including, but is not limited to) greater than 50 nucleotide.Also can the destabilizing agent of Methanamide reaches rigorous condition by for example adding.For selectivity or specific hybrid, positive signal can be twice background at least, is 10 times of background hybridizations according to circumstances.Exemplary rigorous hybridization conditions can be as follows: 50% Methanamide, 5 * SSC and 1%SDS, at 42 ℃ of following incubations; Perhaps 5 * SSC, 1%SDS are at 65 ℃ of following incubations; Wash down at 65 ℃ with 0.2 * SSC and 0.1%SDS.Described washing can be carried out 5,15,30,60,120 minutes or the longer time.

Term as used herein " eukaryote " is meant and belongs to kind of the organism that is generation eukaryote territory (Eucarya), for example animal (including, but is not limited to mammal, insecticide, reptile, birds or the like), ciliate, plant (including, but is not limited to monocotyledon, dicotyledon, algae or the like), fungus, yeast, flagellate, microparticle insect, a protista or the like.

Term as used herein " non-eukaryote " is meant non-most eukaryotes.For instance, non-most eukaryotes can belong to eubacteria (Eubacteria) (including, but is not limited to escherichia coli, thermus thermophilus (Thermitsthermophilus), bacillus stearothermophilus (Bacillus stearothermophilus), pseudomonas fluorescens (Pseudomonas fluorescens), bacillus pyocyaneus (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida) or the like) kind system generation territory; Or Archimycetes (Archaea) (includes, but is not limited to methane coccus (Methanococcus jannaschii), methanogen (Methanobacteriumthermoautotrophicum), Halobacterium (Halobacterium) (for example Wo Shi has a liking for richly endowed bacterium of salt (Haloferaxvolcanii) and method clone halophilic bacteria (Halobacterium species) NRC-1), the ancient green-ball bacterium (Archaeoglobus fulgidus) of glimmering, fierce hot-bulb bacterium (Pyrococcus furiosus), super hyperthermophilic archaeon strain (Pyrococcus horikoshii), quick hot bacterium of gas (Aeuropyrum pernix) or the like) plants system the territory takes place.

Term as used herein " person under inspection " is meant the animal as treatment, observation or experimental subject, preferred mammal, and optimum is chosen.

Term as used herein " effective dose " is meant that (modified) non-natural amino acid polypeptides can alleviate the dosage of one or more symptoms of disease, the state of an illness or the disease of being treated to a certain extent.The compositions of (modified) non-natural amino acid polypeptides of described herein containing of can coming into operation is preventative to be used for, enhancing property and/or therapeutic treatment.

Term " enhancing " expression increases or prolongs the effect or the persistent period of required effect.Therefore, just strengthen the therapeutic agent effect, term " enhancing " is meant increase or prolongs other therapeutic agent to the effect of systemic effect or the ability of persistent period." enhancing effective dose " as used herein is meant and is enough to strengthen the amount that another therapeutic agent acts in required system.When being used for the patient, the effective dose of this purposes will depend on the order of severity of disease, disease or the state of an illness and process, previous therapy, patient health situation and to the reaction of medicine and treatment doctor's judgement.

Term as used herein " modified " is meant the post translational modification of existence to polypeptide.Form " (modified) " term is meant that the polypeptide of being discussed is modified according to circumstances, that is to say that the polypeptide of being discussed can be modified or not modified.

Term " through post translational modification " and " modified " are meant described amino acid whose any modification that natural or alpha-non-natural amino acid is taken place after incorporating polypeptide chain into.Only for instance, in vivo translation modification altogether, in vivo post translational modification and in vitro post translational modification contained in this term.

In prophylactic use, the compositions that contains (modified) non-natural amino acid polypeptides throw with susceptible in specified disease, disease or the state of an illness or patient with these risks.Described amount is defined as " prevention effective dose ".In this purposes, accurately amount also depends on health status, body weight of patient or the like.In affiliated art scope, can consider fully to determine described prevention effective dose by normal experiment (for example dosage escalation clinical trial).

Term " protected " is meant and has " protecting group " or the part that prevents chemical reaction functional group reactions under some reaction condition.Protecting group should be looked the type of the chemical reaction base of being protected and be changed.For instance, if the chemical reaction base is amine or hydrazides, protecting group can be selected from the group of tert-butoxycarbonyl (t-Boc) and 9-fluorenyl methoxy carbonyl (Fmoc) so.If the chemical reaction base is a mercaptan, protecting group can be adjacent pyridyl disulfide (orthopyridyldisulfide) so.If the chemical reaction base is carboxylic acid (for example butanoic acid or propanoic acid) or hydroxyl, protecting group can be benzyl or alkyl so, for example methyl, ethyl or the tert-butyl group.Known other protecting group in affiliated field also can be used for method and composition as herein described, perhaps therewith uses.

Only for instance, sealing base (blocking group)/protecting group can be selected from:

Pi-allyl an Cbz allyloxycarbonyl Me

Et tert-butyl group TBDMS Tooc

Boc pMBn trityl acetyl group Fmoc

Other protecting group is described in Greene and Wuts, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, it incorporates this paper by reference into.

In treatment was used, the compositions that will contain (modified) non-natural amino acid polypeptides was to be enough to cure or stop to small part the amount throwing and the patient who has suffered described disease, the state of an illness or disease of the symptom of disease, disease or the state of an illness.This amount is defined as " treatment effective dose ", it will depend on the order of severity of disease, disease or the state of an illness and process, previous treatment, patient health situation and to the reaction of medicine and treatment doctor's judgement.In affiliated art scope, can consider fully to determine described treatment effective dose by normal experiment (for example dosage escalation clinical trial).

Term " treatment " is meant preventative and/or therapeutic treatment.

Unless otherwise indicated, conventional mass spectrography, NMR, HPLC, albumen chemical method, biochemical process, recombinant DNA technology and the pharmacology's method in the art scope otherwise under adopting.

Embodiment

I. foreword

The invention provides the ABP molecule that comprises at least a alpha-non-natural amino acid.In certain embodiments of the present invention, the ABP with at least a alpha-non-natural amino acid comprises at least a post translational modification.In one embodiment, described at least a post translational modification comprises molecule and connects, it includes, but is not limited to labelling, dyestuff, polymer, water-soluble polymer, polyethyleneglycol derivative, photocrosslinking agent, cytotoxic compound, radionuclide, medicine, affinity labeling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelator, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, the water solublity dendrimer, cyclodextrin, inhibition ribonucleic acid, biomaterial, nanoparticle, spin labeling, fluorogen, the containing metal part, the radioactivity part, novel functional group, the group of covalently or non-covalently doing mutually with other molecule, light is caught part (photocaged moiety), the photoisomerization part, biotin, biotin derivative, the biotin analog, incorporate the part that heavy atom is arranged into, but chemical cracking group, but photodestruciton group, long side chain, carbon connects sugar, redox active agent, amino thio-acid, the toxicity part, through the isotopic labeling part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the close group of electronics, the magnetic group, insert group (intercalating group), chromophore, energy transfer agent, bioactivator, detectable label, micromolecule or combinations thereof or any other required compound or material, it comprises the known chemical method of specific reactivity group and at least a alpha-non-natural amino acid that comprises first reactive group of being applicable to of employing one of ordinary skill in the art and has reactive second reactive group.For instance, first reactive group is that the alkynyl part (includes, but is not limited to alpha-non-natural amino acid in the alkynes propoxyl group phenylalanine, wherein propargyl is also referred to as acetylene moiety sometimes), and second reactive group is the azido part, and adopt [3+2] cycloaddition method.In another example, first reactive group is an azido part (including, but is not limited to alpha-non-natural amino acid to azido-L-phenylalanine), and second reactive group is the alkynyl part.In some embodiment of the modified ABP polypeptide of the present invention, use at least a alpha-non-natural amino acid (including, but is not limited to contain the alpha-non-natural amino acid of ketone group functional group) that comprises at least a post translational modification, wherein said at least a post translational modification comprises sugar moieties.In certain embodiments, in eukaryotic cell or in non-eukaryotic cell, in vivo carry out post translational modification.

In certain embodiments, albumen comprises at least a in the post translational modification of in vivo being carried out by a kind of host cell, and wherein said post translational modification be can't help another host cell type usually and carried out.In certain embodiments, albumen comprises at least a in the post translational modification of in vivo being carried out by a kind of eukaryotic cell, and wherein said post translational modification be can't help non-eukaryotic cell usually and carried out.The example of post translational modification includes, but is not limited to acetylation, acidylate, lipid-modified, palmitoylation, cetylate addition, phosphorylation, glycolipid key (glycolipid-linkage) is modified or the like.In one embodiment, post translational modification comprises oligosaccharide and agedoite (including, but is not limited to wherein, oligosaccharide comprises (GlcNAc-Man) by GlcNAc-agedoite key ₂-Man-GlcNAc-GlcNAc or the like) connects.In another embodiment, post translational modification comprises oligosaccharide (including, but is not limited to Gal-GalNAc, Gal-GlcNAc or the like) and is connected with serine or threonine by GalNAc-serine, GalNAc-threonine, GlcNAc-serine or GlcNAc-threonine key.In certain embodiments, albumen of the present invention or polypeptide can comprise that secretion or positioning sequence, epitope label, FLAG label, poly are histidine-tagged, GST fusant and/or similar substance.The example of secretory signal sequence include, but is not limited to prokaryote secretory signal sequence, eukaryote secretory signal sequence, 5 '-be used for the eukaryote secretory signal sequence of bacterial expression, novel secretory signal sequence, pectate lyases secretory signal sequence, Omp A secretory signal sequence and phage secretory signal sequence through optimization.The example of secretory signal sequence includes, but is not limited to STII (prokaryote), Fd GIII and M13 (phage), Bgl2 (yeast) and derives from the signal sequence bla of transposon.

Can use the antigen-binding polypeptides that comprises alpha-non-natural amino acid to regulate treatment half-life, serum half-life or the circulation time of bioactive molecule, described bioactive molecule includes, but is not limited to micromolecule, peptide and oligonucleotide.Described micromolecule, peptide and oligonucleotide can have the combination of including, but is not limited to and/or identification target molecule or the active biological activity of cell type, anti-tumor activity, anti-angiogenic activity, antiviral activity and apoptosis.In addition, the antigen-binding polypeptides that comprises alpha-non-natural amino acid can provide required activity, include, but is not limited to effector function, for example ADCC, phagocytosis or CDC, prodrug activation, enzymatic activity, catalytic activity, blocks protein-interactions between protein, in conjunction with required antigen with the required site of micromolecule targeting.ABP blocks protein-interactions between protein can regulate connect one or more activity of bioactive molecule.Can micromolecule is active with the combination of disturbing other albumen or molecule as antagonist.

Antigen-binding polypeptides can be connected by connexon, polymer or covalent bond with micromolecule.Connexon, polymer or micromolecule self can comprise and 20 kinds of common amino acids reactive functional group of tool not.Connexon or polymer can be dual functional.At desired conditions, one or more to relate to antigen-binding polypeptides can be irreversible, reversible or unsettled through connexon, polymer or covalent bonds in the key of bioactive molecule.One or more relate to antigen-binding polypeptides can allow antigen-binding polypeptides or other molecule in the key of molecule through connexon, polymer or covalent bonds the adjustment release that is subjected to.One of ordinary skill in the art can be by chemical method, produce multiple micromolecule as Separation of Natural Products or other method.

People Proc Natl Acad Sci USA.2003 such as Rader April 29; 100 (9): describe a kind of method that described molecule is provided effector function and long serum half-life via little synthetic molecules and general antibody molecule are reacted among the 5396-400, this incorporates this paper into by reference with reference to case.By mAb 38C2 (simulating the catalytic antibody of natural aldolase) through reactive lysine residue on the antibody and and the reversible covalent bonds between the plain derovatives of the integration of targeting Arg-Gly-Asp peptide mimics produce described complex.Except the half-life that increases peptide mimics, complex is also showed antibody selectivity targeted expression integrin alpha again _Vβ ₃, α _Vβ ₅Cell surface.

Institute's protein of interest or polypeptide can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten or ten above alpha-non-natural amino acids.Alpha-non-natural amino acid can be identical or different, for example comprise 1,2,3,4,5,6,7,8,9,10 or the albumen of how different alpha-non-natural amino acids in can have 1,2,3,4,5,6,7,8,9,10 or more different loci.In certain embodiments, at least one (but be less than all) replaced by alpha-non-natural amino acid with the specific amino acids that proteic natural generation form exists.

The invention provides method and composition based on antigen-binding polypeptides that comprises at least one non-naturally encoded amino acids or ABP.Do not use with the chemistry that combines of common generation 20 seed amino acids reaction allowing to relate to including, but is not limited to one or more non-naturally encoded amino acids generation specificity chemical reactions among at least one non-naturally encoded amino acids introducing ABP.In certain embodiments, the ABP that comprises non-naturally encoded amino acids is connected with water-soluble polymer (for example Polyethylene Glycol (PEG)) via the side chain of non-naturally encoded amino acids.The invention provides high efficiency method with PEG derivatives selectively modified protein, described method relates to be incorporated into non-genetic coding aminoacid selectivity in response to the albumen of selecting codon and with suitable reactive PEG derivant these aminoacid is modified subsequently, it not is to be found in 20 kinds of natural functional group or substituent aminoacid of incorporating in the aminoacid that described non-genetic coding aminoacid includes, but is not limited to contain, and described functional group or substituent group include, but is not limited to ketone, nitrine or acetylene moiety.In case after incorporating into, promptly can come amino acid side chain is modified by using the known particular functional group or the substituent chemical method that are present in natural coded amino acid of being applicable to of one of ordinary skill in the art.Known number of chemical method is applicable among the present invention to be incorporated water-soluble polymer in the albumen into.Described method includes, but is not limited to respectively and includes, but is not limited to acetylene or azido derivant and carry out Huisgen[3+2] cycloaddition reaction (referring to, Padwa for example, A.'s Comprehensive Organic Synthesis, the 4th volume, (1991) editor Trost, B.M., Pergamon, Oxford, 1069-1109 page or leaf; And Huisgen, R.'s 1,3-Dipolar Cvcloaddition Chemistry, (1984) editor Padwa, A., Wiley, New York, 1-176 page or leaf).

Because Huisgen[3+2] the cycloaddition method relates to cycloaddition rather than nucleophilic substitution, so albumen can be through the very modification of high selectivity.Can come at room temperature to carry out described reaction with good area selectivity (1,4＞1,5) in the aqueous conditions by Cu (I) salt that in reactant mixture, adds catalytic amount.Referring to, people such as Tornoe for example, (2002) Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002) Angew.Chem.Int.Ed.41:2596-2599; With WO 03/101972.The molecule that can add in [3+2] cycloaddition in the albumen of the present invention comprises any have appropriate functional group or substituent molecule in fact, includes, but is not limited to azido or acetylene-derivative.These molecules can be made an addition in the alpha-non-natural amino acid (including, but is not limited to) with acetenyl respectively or have in the alpha-non-natural amino acid (including, but is not limited to) of azido azido-phenylalanine to alkynes propoxyl group phenylalanine.

By Huisgen[3+2] five-membered ring that cycloaddition produced is irreversible in reducing environment usually, and in aqueous environments for a long time to hydrolysis-stable.Therefore, the physics of multiple material and chemical feature can be modified with active PEG derivant of the present invention under required aqueous conditions.What is more important is because nitrine and acetylene moiety have specificity (for example, with any 20 kinds of common genetic coding aminoacid reaction) each other, so can be in one or more specificity site with high selectivity modified protein very.

The present invention also provides the water solublity and the hydrolysis-stable derivant of PEG derivant, with the relevant hydrophilic polymer with one or more acetylene or nitrine part.The PEG polymer derivant that contains acetylene moiety has high selectivity to selectivity introducing in response to the coupling partly of the nitrine in the albumen of selecting codon.Similarly, the PEG polymer derivant that contains nitrine part coupling that selectivity is introduced in response to the acetylene moiety in the albumen of selecting codon has high selectivity.

More particularly, the nitrine part is including (but not limited to) the derivant of alkyl azide, aryl azide and these nitrine.The derivant of alkyl azide and aryl azide can comprise other substituent group, as long as keep the acetylene specific reaction.Acetylene moiety comprises alkyl acetylene and aryl ethane and derivant separately.The derivant of alkyl acetylene and aryl ethane can comprise other substituent group, as long as keep the nitrine specific reaction.

The ABP that comprises non-naturally encoded amino acids can be used for adopting the test of antibody specificity.For instance, ABP molecule of the present invention can be used to screen potential antigen colony.

The invention provides and have multiple functional group, the material of substituent group or part and the conjugate of other material, described other material includes, but is not limited to labelling, dyestuff, polymer, water-soluble polymer, polyethyleneglycol derivative, photocrosslinking agent, cytotoxic compound, radionuclide, medicine, affinity labeling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelator, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, the water solublity dendrimer, cyclodextrin, inhibition ribonucleic acid, biomaterial, nanoparticle, spin labeling, fluorogen, the containing metal part, the radioactivity part, novel functional group, the group of covalently or non-covalently doing mutually with other molecule, light is caught part, the photoisomerization part, biotin, biotin derivative, the biotin analog, incorporate the part that heavy atom is arranged into, but chemical cracking group, but photodestruciton group, long side chain, carbon connects sugar, redox active agent, amino thio-acid, the toxicity part, through the isotopic labeling part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the close group of electronics, the magnetic group, insert group, chromophore, energy transfer agent, bioactivator, detectable label, micromolecule or combinations thereof or any other required compound or material.The present invention also provides the conjugate of material with nitrine or acetylene moiety and the PEG polymer derivant with corresponding acetylene or nitrine part.For instance, the PEG polymer that contains nitrine part can contain the amino acid whose position of non-genetic coding with acetylene functional group and is coupled to bioactive molecule in albumen.The link coupled bonding that is used for PEG and bioactive molecule includes, but is not limited to Huisgen[3+2] the cycloaddition product.

Under technical field fully definite, PEG can be used for the modified biological material the surface (referring to, for example United States Patent (USP) 6,610,281; Mehvar, R., J.Pharmaceut.Sci., 3 (1): 125-136 (2000), describedly incorporate this paper by reference into) with reference to case.The present invention also provides and comprises surface with one or more reactive nitrine or acetylene site and one or more through Huisgen[3+2] the cycloaddition key is coupled to the biomaterial that contains nitrine or acetylene polymer of the present invention on described surface.Biomaterial and other material also can be coupled to nitrine or the activatory polymer derivant of acetylene by the key except that nitrine or acetylene union (for example by comprising the key of carboxylic acid, amine, alcohol or thiol moiety), to stay nitrine or acetylene moiety can be used for afterreaction.

The present invention includes and synthesize the method that the present invention contains the polymer of nitrine and acetylene.Under the situation of the PEG derivant that contains nitrine, nitrine can directly be incorporated into the carbon atom of polymer.Perhaps, can be connected in conventional living polymer by the bridging agent that an end is had the nitrine part and prepare the PEG derivant that contains nitrine, so that resulting polymers has the nitrine part at its end.Under the situation of the PEG derivant that contains acetylene, acetylene can directly be incorporated into the carbon atom of polymer.Perhaps, can be connected in conventional living polymer by the bridging agent that an end is had acetylene moiety and prepare the PEG derivant that contains acetylene, so that resulting polymers has acetylene moiety at its end.

More particularly, under the situation of the PEG derivant that contains nitrine, water-soluble polymer with at least one activity hydroxy part has the polymer that is substituted that has more reactive part through reaction to generate on it, described reactive part for example methanesulfonates, three fluoro ethane sulfonic acid esters (tresylate), tosylate or the halogen leaving group of having more.One of ordinary skill in the art know the preparation and the purposes of the PEG derivant that contains sulfonylamino acid halogenide, halogen atom and other leaving group.Gained be substituted polymer with after the reaction partly replace described reactive part that has more with described end with nitrine at polymer.Perhaps, water-soluble polymer experience and the reaction that has the bridging agent of nitrine at an end with at least one active nucleophilic or electrophilic part, so that form covalent bond between PEG polymer and bridging agent, and nitrine partly is positioned at the described end of polymer.One of ordinary skill in the art know nucleophilic and electrophilic part, comprise amine, mercaptan, hydrazides, hydrazine, alcohol, carboxylate, aldehyde, ketone, thioesters or the like.

More particularly, under the situation of the PEG derivant that contains acetylene, have halogen or other activity leaving group of water-soluble polymer in reacting the predecessor that contains acetylene moiety with displacement of at least one activity hydroxy part.Perhaps, water-soluble polymer experience and the reaction that has the bridging agent of acetylene at an end with at least one active nucleophilic or electrophilic part, so that form covalent bond between PEG polymer and bridging agent, and acetylene moiety is positioned at the described end of polymer.One of ordinary skill in the art improve and have established halogen part, activity leaving group, nucleophilic and electrophilic part in the preparation of organic synthesis content and PEG derivant and the purposes in the application.

The present invention also provides selective modification proteic method, to add other material to modified albumen, includes, but is not limited to water-soluble polymer, for example contains the PEG and the PEG derivant of nitrine or acetylene moiety.Can optionally connect PEG derivant and proteic method and provide simultaneously with the characteristic (wherein mainly being biocompatibility, stability, dissolubility and shortage immunogenicity) of PEG derivant modification of surfaces that contains nitrine and acetylene and molecule than having more of affiliated technical field previously known.

II. antigen-binding polypeptides

There are many kinds of ABP.ABP self has specificity to many kinds of antigens.The antigenic specificity ABP fragment that also has a large amount of multiple classes.Therefore, ABP is intended to comprise any polypeptide of showing specificity binding target molecule or antigenic ability.Any known antibody or antibody fragment are ABP.

ABP of the present invention can comprise Fc district or Fc sample district.The Fc territory provides and being connected of effector function, for example complement or phagocyte.The Fc of immunoglobulin partly has long plasma half-life, and Fab have short life (people (1989) such as Capon, Nature, 337:525-531).When making up together with treatment albumen, the long half-lift that the Fc territory can providing more or incorporate into Fc receptors bind for example, protein A in conjunction with, complement fixation and may or even the function that shifts of Placenta Hominis.For instance, the Fc district of IgG1 antibody has been blended in the N end of CD30-L, CD30-L is in conjunction with the CD30 receptor (United States Patent (USP) the 5th that is expressed on Hokdkin disease (Hodgkin ' s Disease) tumor cell, anaplastic lymphoma cell, T chronic myeloid leukemia cell and other malignant cell type, 480, No. 981).Anti-inflammatory and anti-repellents IL-10 have been blended in murine Fc γ 2a to increase the short circulating half-life of cytokine.Zheng, people such as X. (1995), The Journal of Immunology, 154:5590-5600.Also studied to estimate the Tumor Necrosis Factor Receptors that is connected with the Fc albumen of IgG 1 treatment has been suffered from septic shock (Fisher, people such as C., N.Engl.J.Med., 334:1697-1702 (1996); Van Zee, people such as K., TheJournal of Immunology, 156:2221-2230 (1996)) and rheumatoid arthritis (Moreland waits people (1997), N.Engl.J.Med., 337 (3): patient's 141-147) purposes.Fc also with the CD4 receptor merge with generation be used for the treatment of AIDS treatment albumen (people (1989) such as Capon, Nature, 337:525-531).In addition, the Fc part that the N of interleukin II end also has been blended in IgGl or IgG3 with the short-half-life that overcomes interleukin II and general toxicity thereof (people (1995) such as Harvill, Immunotechnology, 1:95-105).

As everyone knows, the Fc district of antibody is by forming by disulfide bond or by the poly-polypeptide sections of list that non-covalent association connects into dimerization or poly form.The number of the intermolecular disulfide bond between the poly-subunit of the list of natural Fc molecule depends on the kind (for example, IgG, IgA, IgE) of related antibody or subclass (for example, IgG1, IgG2, IgG3, IgA1, IgGA2) and is 1 to 4.Term as used herein " Fc " is the general name of the Fc molecule of single poly-, dimerization and poly form.It should be noted that when having suitable Cys residue, unless there is the specific condition that stops the dimerization effect that forms through disulfide bond, the Fc monomer is with spontaneous dimerization.Even usually form in the Fc dimer that the Cys residue of disulfide bond is removed or by other residue displacement, Dan Julian usually will be via non-covalent mutual work dimerization.These forms that term used herein " Fc " expression is any: natural monomer, natural dimer (connecting), modified dimer (connecting) and modified monomer (being derivant) through disulfide bond and/or non-covalent bond through disulfide bond.

For instance, can make up Fc variant, analog or derivant partly by on residue that comprises non-naturally encoded amino acids or sequence, carrying out various replacements.Variant (or analog) polypeptide comprises the insertion variant, and one of them or an above amino acid residue replenish the Fc aminoacid sequence.Insertion can be positioned at proteic arbitrary end or two ends, perhaps can be arranged in the inner area of Fc aminoacid sequence.The insertion variant that has other residue at arbitrary end or two ends can comprise (for example) fusion rotein and comprise the albumen of amino acid label or labelling.For instance, the Fc molecule can contain N end Met according to circumstances, especially when described molecule is recombinant expressed in bacterial cell (for example escherichia coli).In Fc disappearance variant, remove one or more amino acid residues in the Fc polypeptide.Can realize disappearance in the one or both ends of Fc polypeptide, perhaps can realize disappearance with removing in the Fc aminoacid sequence one or more residues.Therefore, the disappearance variant comprises all fragments of Fc peptide sequence.In Fc replaces variant, remove one or more amino acid residues of Fc polypeptide and replace with other residue.On the one hand, replace with natural conservative, yet the present invention is also contained and is nonconservative replacement.For instance, cysteine residues can lack, perhaps can be with other amino acid replacement to prevent some or all two sulfur-crosslinked formation of Fc sequence.Albumen can contain one or more cysteine residues, and can remove each cysteine residues or replace one or more described cysteine residues with other aminoacid (for example Ala or Ser or non-naturally encoded amino acids).As another example, also can modify to introduce aminoacid and replace consequently (1) excision Fc receptor binding site; (2) excision complement (Clq) binding site; And/or (3) excision antibody dependent cellular mediated cell toxin (ADCC) site.Described site is that affiliated technical field is known, and any known replacement all is in the scope of Fc used herein.For instance, with regard to the ADCC site among the IgG1, please referring to Molecular Immunology, Vol.29, No.5,633-639 (1992).Equally, one or more tyrosine residues also can be replaced by phenylalanine residue.In addition, also contain other variant aminoacid insertion, disappearance (for example 1-25 aminoacid) and/or replacement, and these are in scope of the present invention.Conserved amino acid is replaced normally first-selected.In addition, change can be for through changing amino acid form, for example peptide mimics or D-aminoacid.

The Fc sequence also can be through derivatization, promptly bears insertion except that amino acid residue, the modification lacking or replacing it.Modify natural covalency preferably, and comprise the chemical bonding of (for example) and polymer, lipid, other organic moiety and inorganic part.Can prepare derivant of the present invention to increase circulating half-life, perhaps can design derivant of the present invention to improve the ability of polypeptide target to required cell, tissue or organ.Also can be with the Fc part that receptor binding domains (salvage receptor binding domain) is used as The compounds of this invention of remedying of complete Fc molecule, for example be described in WO 96/32478, title is in " Altered Polypeptides with Increased Half-Life ".Other member that this paper is appointed as the molecule of Fc is described in WO 97/34631, and title is in " Immunoglobulin-Like Domains with Increased Half-Lives ".This section open PCT application case of two of being quoted is incorporated this paper by reference into.

May find other ABP afterwards.Area of computer aided secondary that can be by predetermined protein sequence and tertiary structure analysis and be used to differentiate that by appointment the selection technology of the molecule that is incorporated into particular target differentiates novel ABP.The described ABP that was found afterwards also is included among the present invention.

Therefore, the description that ABP is provided to be being used for illustration purpose, and only as an example, and not as the restriction to the scope of methods described herein, compositions, strategy and technology.In addition, mention in the application's case that ABP is intended to use the example of this general name as any ABP.Therefore, should be appreciated that this paper is equally applicable to any antigen-binding polypeptides about specific antigen in conjunction with polypeptide or the described modification of albumen and chemical method, comprises those that this paper specifically lists.

III. the general recombinant nucleic acid method used of the present invention

In many embodiment of the present invention, separate, the clone and use usually recombination method change coding the nucleic acid of interested ABP.Described embodiment is used for including, but is not limited to protein expression or is used for variant, derivant, expression cascade or other derives from the generation of the sequence of antigen-binding polypeptides.In certain embodiments, encode the sequence of polypeptide of the present invention through being operatively connected in allogeneic promoter.

Can and then change nucleotide sequence with the introducing that realizes the related amino acid residue (promptly incorporate into or replaces) or remove (i.e. disappearance or replacement) based on the aminoacid sequence of parent polypeptide, come composite coding to comprise the nucleotide sequence of the antigen-binding polypeptides of non-naturally encoded amino acids.Can come modified nucleotide sequence expediently by rite-directed mutagenesis according to conventional methods.Perhaps, can prepare nucleotide sequence by chemosynthesis, include, but is not limited to by using the oligonucleotide synthetics,, and preferably be chosen in recombinant polypeptide and will result from the codon of being liked in wherein the host cell wherein according to required amino acid sequence of polypeptide design oligonucleotides.For instance, can pass through several small oligonucleotides that the required polypeptide portion of coding is synthesized and assembles in PCR, connection (ligation) or connection chain reaction.Referring to, people such as Barany for example, Proc.Natl.Acad.Sci.88:189-193 (1991); United States Patent (USP) 6,521,427, it incorporates this paper by reference into.

The present invention adopts genetic recombination to learn the routine techniques in field.The substance of the conventional method that open the present invention is used comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With CurrentProtocols in Molecular Biology (people such as Ausubel edits 1994).

The general content of describing Protocols in Molecular Biology comprises Berger and Kimmel. Guide to Molecular Cloning Techniques, Methods in Enzvmology volume 152Academic Press, Inc., SanDiego, CA (Berger); People such as Sambrook, Molecular Cloninfi-A Laboratory Manual (the 2nd edition), The 1-3 volume,Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and Current Protocols in Molecular Biology, people such as F.M.Ausubel edit, Current Protocols, a joint venture between Greene Publishing Associates, Inc.and JohnWiley ﹠amp; Sons, Inc., (replenishing 1999) (" Ausubel ").These content description mutation effects, support applications, promoter and many other related subjects, include, but is not limited to produce and comprise that the gene that is used to produce proteic selection codon, described albumen comprise alpha-non-natural amino acid, quadrature tRNA, quadrature synzyme and pairing thereof.

Various types of mutation effects are used for multiple purpose of the present invention, and the selection codon that includes, but is not limited to produce the library of tRNA, the library that produces synzyme, generation selection codon, the alpha-non-natural amino acid of will encoding inserts in institute's protein of interest or the polypeptide.Described mutation effect include, but is not limited to the to fix a point sudden change that random point mutation, homologous recombination, DNA reorganization or other cycle mutant (recursive mutagenesis) method, chimeric construct, use contain sudden change, oligonucleotide orthomutation, the sudden change of thiophosphate modifying DNA that the template of uracil carries out, use breach double-stranded DNA etc. to carry out or its any combination.Other appropriate method comprises a mispairing reparation, use sudden change, restricted selection and restricted purification that the repair-deficiency host strain carries out, deletion mutation, break by synthetic sudden change, the two strands of carrying out of total gene and to repair etc.Including, but is not limited to relate to the chimeric construct body is also included among the present invention at interior mutation effect.In one embodiment, can instruct sudden change by the Given information of natural generation molecule or natural generation molecule, include, but is not limited to sequence, sequence comparison, physical property, secondary structure, tertiary structure or quarternary structure, crystal structure or the like through changing or making a variation.

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Acids Res.16:803-814; People such as Kramer, Thegapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucl. Acids Res.12:9441-9456 (1984); Kramer ﹠amp; Fritz Oligonucleotide-directedconstruction of mutations via gapped duplex DNA, Methods in Enzymol.154:350-367 (1987); People such as Kramer, Improved enzymatic in vitro reactions in the gapped duplex DNAapproach to oligonucleotide-directed construction of mutations, Nucl. Acids Res.16:7207 (1988); People such as Fritz, Oligonucleotide-directed construction of mutations:agappedduplex DNA procedure without enzymatic reactions in vitro, Nucl. Acids Res.16:6987-6999 (1988); People such as Kramer, Different base/base mismatches are corrected withdifferent efficiencies by the methyl-directed DNA mismatch-repair system of E.coli, Cell38:879-887 (1984); People such as Carter, Improved oligonucleotide site-directed mutagenesisusing M13 vectors, Nucl. Acids Res.13:4431-4443 (1985); Carter, Improvedoligonucleotide-directed mutagenesis using Ml 3 vectors, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh﹠amp; Henikoff, Use of oligonucleotides to generate largedeletions, NucL Acids Res.14:5115 (1986); People such as Wells, Importance of hydrogen-bondformation in stabilizing the transition state of subtilisin, Phil.Trans.R.Soc.Lond.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning of a gene coding for theribonuclease S protein, Science223:1299-1301 (1984); Sakmar and Khorana, Totalsynthesis and expression of a gene for the alpha-subunit of bovine rod outer segmentguanine nucleotide-bindingprotein (transducin), Nucl.Acids Res.14:6361-6372 (1988); People such as Wells, Cassette mutagenesis:an efficient method for generation of multiplemutations at defined sites, Gene34:315-323 (1985); People such as Grundstrom, Oligonucleotide-directed mutagenesis by microscale ' shot-gun ' gene synthesis, Nucl. Acids Res.13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strandbreak repair in plasmids of Escherichia coli:a method for site-specific mutagenesis, Proc. Natl.Acad.Sci.USA.83:7177-7181 (1986); Arnold, Protein engineering for unusualenvironments, Current Opinion in BiotechnoloRV4:450-455 (1993); People such as Sieber, NatureBiotechnology, 19:456-460 (2001); W.P.C.Stemmer, Nature370,389-91 (1994); And, I.A.Lorimer, I.Pastan, Nucleic Acids Res.23,3067-8 (1995).Other details about many said methods is seen Methods in EnzymologyThe 154th volume, this also describes with various mutation methods with reference to case and carries out effective control to fault detection problem (trouble-shooting problem).

The present invention also relates to be used for through quadrature tRNA/RS to the eukaryotic host cell of in vivo incorporating alpha-non-natural amino acid into, non-eukaryotic host cell and organism.With polynucleotide of the present invention or comprise that the construct (including, but is not limited to carrier of the present invention, for example can be cloning vehicle or expression vector) of polynucleotide of the present invention comes genetic engineering procedure (including, but is not limited to conversion, transduction or transfection) host cell.For instance, carrier can be for plasmid, antibacterial, virus, exposed polynucleotide or in conjunction with the form of polynucleotide.By standard method carrier is introduced in cell and/or the microorganism, described standard method comprise electroporation (people such as From, Proc.Natl. Acad.Sci.USA82,5824 (1985)); Viral vector infects; By small-particle with nucleic acid in globule or particle substrate or carry out from the teeth outwards the high speed ballistic penetration (people such as Klein, Nature327,70-73 (1987)).

Can obtain to be applicable to the host cell of cultivating in the active conventional Nutrient medium that screens step, activation promoter or selection transformant etc. through the engineering operation in the process improvement.These cells can be cultivated in transgenic organism according to circumstances.Other the useful list of references that includes, but is not limited to be used for cell separation and cultivation (for example, being used for separate nucleic acid subsequently) comprises Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and the list of references of wherein being quoted; People such as Payne (1992) Plant Cell and Tissue Culture in Liquid SystemsJohn Wiley ﹠amp; Sons, Inc.New York, NY; Gamborg and Phillips (editor) (1995) Plant Cell Tissue and Organ CultureFundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (editor) The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, FL.

Some well-known process that target nucleic acid is introduced in the cell can be used, and wherein any all can be used for the present invention.These methods comprise: recipient cell and the antibacterial protoplast fusion that contains DNA, electroporation, emission bombarded (projectile bombardment) and infected (further discussing hereinafter) with viral vector, or the like.The plasmid number that bacterial cell can be used for increasing and contains DNA construct of the present invention.Make bacterial growth to exponential phase, and can be by the plasmid in technical field known method under multiple (referring to, the Sambrook for example) separation of bacterial.In addition, many commercial reagent box can be used for from the bacteria purification plasmid (referring to, EasyPrep for example ^TM, FlexiPrep ^TM, all available from Pharmacia Biotech; StrataClean available from Stratagene ^TMWith QIAprep available from Qiagen ^TM).Further handle through separate and purified plasmid to produce other plasmid, be used for transfectional cell or incorporate relevant carriers into to infect organism.Typical carriers contains transcribes with translation termination, transcribes and translation initiation sequence and the promoter that can be used for regulating and control the particular target expression of nucleic acid.Carrier comprises general expression cascade according to circumstances, and described expression cascade contains at least one independent terminator sequence, the permission level is associated in sequence of duplicating in eukaryote or prokaryote or both (the including, but is not limited to shuttle vector) and the selected marker that is used for protokaryon and eukaryotic system.Carrier is applicable to and duplicates and be integrated into eukaryote, prokaryote or be preferably both.Referring to, Giliman﹠amp; Smith, Gene8:81 (1979); People such as Roberts, Nature, 328:731 (1987); Schneider, people such as B., Protein Expr.Purif.6435:10 (1995); Ausubel, Sambrook, Berger (all with above).For instance, provide the antibacterial that can be used for cloning and the catalogue of bacteriophage, for example by ATCC delivered by ATCC The ATCC CataloRue of Bacteria and Bacteriophage(1992) people (editor) such as Gherna.Other is used to check order, the basic process of clone and molecular biology others and rationale consider that item asks for an interview people such as Watson (1992) Recombinant DNA Second EditionScientific American Books, NY.In addition, basically any nucleic acid is (with any through labeling nucleic acid basically, the standard criteria of right and wrong also no matter) all can be article made to order or the standard substance of ordering from any multiple commercial source, described commercial source is Midland Certified ReagentCompany (Midland.TX available on the World Wide Web at mcrc.com) for example, The GreatAmerican Gene Company (Ramona.CA available on the World Wide Web atgenco.com), ExpressGen Inc. (Chicago.IL available on the World Wide Web atexpressgen.com), Operon Technologies Inc. (Alameda, CA), etc.

Select codon

Selection codon of the present invention enlarges the genetic code subframe of protein biology synthesis machine.For instance, select codon to include, but is not limited to unique three base codons, nonsense codon, for example termination codon (amber codon (UAG) or opal codon (UGA)), non-natural codon, four or more base codon, rare codon or the like.One of ordinary skill in the art understand easily, the selection codon in the required gene can introduced that has many numbers, one or more in the single polynucleotide of at least a portion ABP that includes, but is not limited to encode, two or more, more than three, 4,5,6,7,8,9,10 or more a plurality of.

In one embodiment, described method relates to the purposes of selecting codon, and described selection codon is a termination codon of in vivo incorporating alpha-non-natural amino acid in eukaryotic cell into.For instance, produce the O-tRNA of identification termination codon, include, but is not limited to UAG, and carry out the aminoacylation effect with required alpha-non-natural amino acid by O-RS.Described O-tRNA can't help natural generation host's aminoacyl-tRNA synthetase and discerns.Conventional rite-directed mutagenesis is used in institute's interested site introducing termination codon of the interested polypeptide of institute, includes, but is not limited to TAG.Referring to, Sayers for example, people such as J.R. (1988), 5 ', 3 ' Exonuclease in phosphorothioate-basedoligonucleotide-directed mutagenesis. Nucleic Acids Res,791-802.When coding O-RS, the O-tRNA of interested polypeptide and nucleic acid in vivo in conjunction with the time, thereby alpha-non-natural amino acid is incorporated in response to the UAG codon and is created in the polypeptide that ad-hoc location contains alpha-non-natural amino acid.

Can significantly not carry out in vivo incorporating into of alpha-non-natural amino acid under the disturbance eukaryotic host cell.For instance, because the inhibition efficient of UAG codon depends on the competition between O-tRNA (including, but is not limited to amber suppressor tRNA) and the eucaryon releasing factor (including, but is not limited to eRF) (its combination termination codon and cause the growth peptide discharge from ribosome), can regulate and control by the expression that includes, but is not limited to increase O-tRNA and/or suppressor gene tRNA so suppress efficient.

Select codon also to comprise long codon, include, but is not limited to four or more base codon, for example four, five, six or polybase base codon more.The example of four base codons comprises: include, but is not limited to AGGA, CUAG, UAGA, CCCU or the like.The example of five base codons comprises: include, but is not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC or the like.Feature of the present invention comprises the long codon that use suppresses based on frameshit.Four or more base codon can insert in the same albumen including, but is not limited to one or more alpha-non-natural amino acids.For instance, existing under the sudden change O-tRNA frame shift suppressor tRNA of (include, but is not limited to have anticodon loop (for example having 8-10 nucleotide anticodon loop)), four or more base codon is read to be monamino acid.In other embodiments, anticodon loop can be decoded and be included, but is not limited at least four base codons, at least five base codons or hexabasic at least basic codon or more.Owing to there are 256 kinds of four possible base codons, therefore can use four or more base codon a plurality of alpha-non-natural amino acids of in same cell, encoding.Referring to, people such as Anderson, (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selection of EfficientSuppressors of Four-base Codons and Identification of " Shifty " Four-base Codons with aLibrary Approach in Escherichia coli J. Mol.Biol.307:755-769.

For instance, having used in vitro, biological synthesis method is used for four base codons to incorporate alpha-non-natural amino acid into albumen.Referring to, people such as Ma for example, (1993) Biochemistry, 32:7939; With people such as Hohsaka, (1999) J.Am, Chem.Soc.121:34.Turn the frame shift suppressor tRNA of usefulness into two chemical acyl groups of process, use CGGG and AGGU simultaneously the NBD derivant of 2-naphthyl alanine and lysine in vitro to be incorporated in the streptavidin.Referring to, people such as Hohsaka for example, (1999) J.Am.Chem.Soc.121:12194.In vivo in the research, people detection such as Moore have a NCUA anticodon the tRNALeu derivant suppress the ability of UAGN codon (N can be U, A, G or C), and find that tetrad UAGA can be decoded by the tRNALeu with UCUA anticodon with 13 to 26% efficient, and few decoding wherein takes place in 0 or-1 framework.Referring to, people such as Moore, (2000) J.Mol.Biol..298:195.In one embodiment, can be used for the present invention based on the long codon of rare codon or nonsense codon, it can reduce in other non-missense of wishing the site reads over (missense readthrough) and frameshit suppresses.

For giving fixed system, select codon also can comprise a kind of natural three base codons, wherein the Nei Sheng system does not use (or few use) described natural base codon.For instance, it comprise the system that lacks the tRNA that discerns natural three base codons and/or wherein three base codons be the system of rare codon.

Select codon to comprise the non-natural base pair according to circumstances.These non-natural base pairs further enlarge existing genetic alphabet.A kind of extra base pair is increased to 125 with the number of triplet codon from 64.The characteristic of the 3rd base pair comprises stable and the selectivity base pairing, incorporate the primer extension that effectively continues after DNA and the synthetic nascent non-natural base pair into by the enzyme process of the effective high-fidelity of polymerase.Description applicable to the non-natural base pair of described method and composition comprises people such as (for example) Hirao, (2002) An unnatural base pair for incorporatingamino acid analogues into protein, Nature Biotechnology, 20:177-182.Other relevant publication is listed in hereinafter.

For in vivo using, non-natural nucleoside can be worn film, and forms corresponding triguaiacyl phosphate through phosphorylation.In addition, the hereditary information of increase is stable and is not destroyed by cellular enzymes.Benner and other people previous work have utilized and have been different from the right hydrogen bond type of typical Watson-Crick, it should be noted that wherein iso-C:iso-G is right most.Referring to, people such as Switzer for example, (1989) J.Am.Chem.Soc.111:8322; With people such as Piccirilli, (1990) Nature, 343:33; Kool, (2000) Curr.Opin.Chem.BioL,4:602.Common and the natural base mispairing to a certain degree of these bases, and can not duplicate by enzyme process.Kool and partner prove that the mutual work of hydrophobic packing between the base can replace hydrogen bond to form to impel base pair.Referring to Kool, (2000) Curr. Opin.Chem.BioL,4:602; With Guckian and Kool, (1998) Angew.Chem.Int.Ed.Engl.36,2825.Satisfy in the work of non-natural base pair of all above-mentioned requirements in exploitation, Schultz, Romesberg and partner are systematically synthetic and studied a series of non-natural hydrophobicity bases.Find that PICS:PICS self is more more stable than natural base pair to (self-pair), and can incorporate among the DNA effectively by the Klenow fragment (KF) of e. coli dna polymerase I.Referring to, people such as McMinn for example, (1999) J.Am.Chem.Soc,121:11586; With people such as Ogawa, (2000) J.Am.Chem.Soc, 122:3274.Can be by KF in that to have under the efficient that enough satisfies biological function and the selectivity a synthetic 3MN:3MN self right.Referring to, people such as Ogawa for example, (2000) L Am.Chem.Soc,122:8803.Yet two kinds of bases are all served as the chain terminating agent that further duplicates.Develop recently and can be used for duplicating the right mutation DNA polymerase of PICS self.In addition, it is right to duplicate 7AI self.Referring to, people such as Tae for example, (2001) J.Am.Chem.Soc, 123:7439.Developed recently and stablized right novel metal base pair Dipi:Py in conjunction with forming behind the Cu (II).Referring to people such as Meggers, (2000) J.Am.Chem.Soc,122:10714.Because long codon and non-natural codon itself is orthogonal thereto with natural codon, so method of the present invention can utilize described characteristic to come it is produced quadrature tRNA.

Translation is walked around system (translational bypassing system) and also can be used for incorporating alpha-non-natural amino acid into required polypeptide.Walk around in the system in translation, big sequence is incorporated in the gene, but be not translated as albumen.Described sequence contains the structure of the insert (cue) that serves as the downstream translation of inducing ribosome to jump and continuing to insert again on sequence.

In certain embodiments, in method of the present invention and/or the compositions institute's protein of interest or polypeptide (or its part) by nucleic acid coding.Nucleic acid comprises at least one usually and selects codon, at least two selection codons, at least three selection codons, at least four selection codons, at least five selection codons, at least six selection codons, at least seven selection codons, at least eight selection codons, at least nine selection codons, selects codon more than ten or ten.

Can the gene of coding institute's protein of interest or polypeptide be undergone mutation with one of ordinary skill in the art's method known and described herein, be used to incorporate into one or more selection codons of alpha-non-natural amino acid to comprise (for example).For instance, the nucleic acid of institute's protein of interest is undergone mutation to comprise that one or more that be used to incorporate into one or more alpha-non-natural amino acids select codons.The present invention includes any described variant, it includes, but is not limited to mutant, any albumen form, for example comprises at least one alpha-non-natural amino acid.The present invention comprises corresponding nucleic acid too, promptly has any nucleic acid of one or more selection codons of one or more alpha-non-natural amino acids of coding.

Can make the nucleic acid molecules sudden change of coding institute's protein of interest (for example ABP) introduce cysteine easily with any desirable position at polypeptide.Cysteine is widely used in and introduces reactive molecule, water-soluble polymer, albumen or multiple other molecule on institute's protein of interest.Being suitable for method that cysteine is introduced the desired part of antigen-binding polypeptides is that affiliated technical field is known (for example United States Patent (USP) the 6th, 608, and described in No. 183, this patent is incorporated this paper by reference into) and standard mutating technology.

IV. non-naturally encoded amino acids

Multiple non-naturally encoded amino acids is applicable to the present invention.The non-naturally encoded amino acids of any number can be introduced among the ABP.In general, the non-naturally encoded amino acids of being introduced becomes chemical inertness in fact to 20 kinds of common genetic coding aminoacid (being alanine, arginine, agedoite, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine).In certain embodiments, non-naturally encoded amino acids comprises the side chain functionalities that forms the stable bond thing with not being found in that 20 kinds of functional groups's (including, but is not limited to azido, ketone group, aldehyde radical and aminooxy group) in the common amino acid effectively and optionally react.For instance, the antigen-binding polypeptides that comprises the non-naturally encoded amino acids that contains azido functional group can react with polymer (including, but is not limited to gather (ethylene glycol)), perhaps react to form the stable bond thing with second polypeptide that contains alkynyl moiety, the selective response that produces nitrine and alkynes functional group is to form Huisgen[3+2] the cycloaddition product.

The general structure of a-amino acid (formula I) as follows:

Non-naturally encoded amino acids is generally has the above any structure of listed formula, and wherein the R group is any substituent group except that being used for 20 kinds of natural amino acids, and is applicable to the present invention.Because non-naturally encoded amino acids of the present invention is usually only different in side-chain structure with described natural amino acid, thus described non-naturally encoded amino acids and other aminoacid (including, but is not limited to natural or non-naturally encoded) with natural generation polypeptide in the identical mode of amido link generation type form amido link.Yet non-naturally encoded amino acids has the side chain different with natural amino acid.For instance, R comprise according to circumstances alkyl-, aryl-, acyl group-, ketone group-, azido-, hydroxyl-, hydrazine, cyano group-, halogen-, hydrazides, thiazolinyl, alkynyl, ether, mercaptan, selenium-, sulfonyl-, boron (III) acid group (borate), boron (V) acid group (boronate), phosphate, phosphono, phosphine, heterocyclic radical, ketenes (enone), imines, aldehyde, ester, thio-acid, azanol, amino or the like or its combination.Other goes for interested non-natural generation aminoacid of the present invention and includes, but is not limited to comprise the aminoacid that light can activate cross-linking agent, through spin labeling aminoacid, fluorescence aminoacid, melts combine aminoacid, containing metal aminoacid, radioactivity aminoacid, aminoacid with novel functional group, the aminoacid of covalently or non-covalently doing mutually with other molecule, light is caught and/or photoisomerization aminoacid, the aminoacid that comprises biotin or biotin analog, the glycosylation aminoacid that for example replaces serine through sugar, other carbohydrate modification aminoacid, ketone group containing aminoacid, the aminoacid that comprises Polyethylene Glycol or polyethers, aminoacid through the heavy atom replacement, chemistry cleavable and/or light cleavable aminoacid, compare the have long side chain aminoacid of (include, but is not limited to polyethers or long chain hydrocarbon, include, but is not limited to) greater than about 5 or greater than about 10 carbon with natural amino acid, contain glycoprotein amino acid through the carbon connection, redox active amino acids, contain the aminoacid of amino thio-acid and comprise one or more toxicity aminoacid partly.

Applicable to the present invention and can be used for that exemplary non-naturally encoded amino acids with water-soluble polymer reaction includes, but is not limited to have carbonyl, aminooxy group, hydrazine, hydrazides, semicarbazides, the aminoacid of nitrine and alkyne reaction group.In certain embodiments, non-naturally encoded amino acids comprises sugar moieties.These amino acid whose examples comprise M acetyl group-L-glucosamine base-L-serine, N-acetyl group-L-galactosaminyl-L-serine, M acetyl group-L-glucosamine base-L-threonine, N-acetyl group-L-glucosamine base-altheine and O-epichitosamine base-L-serine.These amino acid whose examples comprise following example, and wherein natural generation N-between aminoacid and the sugar or O-key are by covalent bond (including, but is not limited to alkene, oxime, thioether and amide or the like) displacement that usually is not found in natural.These amino acid whose examples also comprise the sugar that is not typically found in the naturally occur protein, for example 2-deoxy-glucose, 2-deoxy galactose or the like.

Many non-naturally encoded amino acids that this paper provided are commercially available, for example can be available from Sigma-Aldrich (St.Louis, MO, USA), Novabiochem (branch of EMD Biosciences, Darmstadt, Germany) or Peptech (Burlington, MA, USA).Described commercially available aminoacid can provide or use the known standard method of one of ordinary skill in the art to synthesize according to circumstances and obtain according to this paper.About organic synthesis technology, please referring to, for example Fessendon and Fessendon Organic Chemistry, (1982, the 2 editions, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and Sundberg Advanced Organic ChemistryThe 3rd edition, part A and B, 1990, Plenum Press, New York).Also referring to the open case 2003/0082575 and 2003/0108885 of U. S. application, it incorporates this paper by reference into.Except the alpha-non-natural amino acid that contains novel side chain, go for alpha-non-natural amino acid of the present invention and also comprise modified framing structure according to circumstances, include, but is not limited to by the structure shown in formula II and the III:

Wherein Z comprises OH, NH usually ₂, SH, NH-R ' or S-R '; X and Y can be identical or different, comprise S or O usually, and R and R ' according to circumstances can be identical or different, are selected from above same composition inventory and hydrogen to the described R group of the alpha-non-natural amino acid with formula I usually.For instance, alpha-non-natural amino acid of the present invention comprises according to circumstances by amino shown in formula II and the III or the replacement in the carboxyl.The alpha-non-natural amino acid of this type includes, but is not limited to alpha-hydroxy acid, α-thio-acid, alpha-amido carbothioic acid ester, includes, but is not limited to have the side chain or the non-natural side chain person of corresponding common 20 kinds of natural amino acids.In addition, the replacement at the alpha-carbon place includes, but is not limited to L, D or α-α-disubstituted amino acid according to circumstances, for example D-glutamic acid, D-alanine, D-methyl-O-tyrosine, aminobutyric acid or the like.Other optional structure comprises cyclic amino acid, and for example auxilliary amino analog and 3,4,6,7,8 and 9 yuan of rings auxilliary propylhomoserin analog, β and alpha amino acids for example are substituted Beta-alanine and butyrine.

Many alpha-non-natural amino acids are based on natural amino acid, for example tyrosine, glutamine, phenylalanine or the like, and be applicable to the present invention.The tyrosine analog includes, but is not limited to para-orientation tyrosine, the ortho position replaces tyrosine and a position replaces tyrosine, wherein is substituted tyrosine and comprises: include, but is not limited to ketone group (including, but is not limited to acetyl group), benzoyl, amino, hydrazine, azanol, mercapto, carboxyl, isopropyl, methyl, C ₆-C ₂₀Straight or branched hydrocarbon, saturated or unsaturated hydrocarbons, O-methyl, polyether group, nitro, alkynyl or the like.In addition, also contain polysubstituted aryl rings.Include, but is not limited to Alpha-hydroxy derivant, γ replacement-substitutive derivative, cyclic derivatives and amide applicable to glutamine of the present invention and replace the glutamine derivant.Example applicable to phenylalanine of the present invention includes, but is not limited to para-orientation phenylalanine, ortho position substituted benzene alanine and a position substituted benzene alanine, and wherein substituent group comprises: include, but is not limited to hydroxyl, methoxyl group, methyl, pi-allyl, aldehyde radical, azido, iodine, bromine, ketone group (including, but is not limited to acetyl group), benzoyl, alkynyl or the like.Instantiation applicable to alpha-non-natural amino acid of the present invention includes, but is not limited to acetyl group-L-phenylalanine; O-methyl-L-tyrosine; L-3-(2-naphthyl) alanine; the 3-methylphenylalanine; O-4-pi-allyl-L-tyrosine; 4-propyl group-L-tyrosine; three-O-acetyl group-GlcNAc β-serine; L-Dopa; fluoridize phenylalanine; isopropyl-L-phenylalanine; to azido-L-phenylalanine; to acyl group L-phenylalanine; to benzoyl-L-phenylalanine; the L-phosphoserine; the phosphono serine; phosphono tyrosine; to the iodo-phenylalanine; to bromophenyl alanine; to amino-L-phenylalanine; isopropyl-L-phenylalanine and to alkynes propoxyl group-phenylalanine or the like.Structure example applicable to multiple alpha-non-natural amino acid of the present invention is provided in (for example) WO 2002/085923, and title is in " In vivo incorporation of unnatural amino acids. ".About other methionine analog, also referring to people such as Kiick, (2002) Incorporation ofazides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS99:19-24.

In one embodiment, provide the ABP compositions that comprises alpha-non-natural amino acid (for example to (alkynes propoxyl group)-phenylalanine).Also provide and comprise the various compositionss of (alkynes propoxyl group)-phenylalanine and include, but is not limited to albumen and/or cell.On the one hand, comprise that the compositions to (alkynes propoxyl group)-phenylalanine alpha-non-natural amino acid further comprises quadrature tRNA.Alpha-non-natural amino acid can include, but is not limited to be covalently bonded in quadrature tRNA through amino-acyl bond in conjunction with (including, but is not limited to covalency) in quadrature tRNA, is covalently bonded in the 3 ' OH or the 2 ' OH of the terminal ribose of quadrature tRNA, or the like.

The chemical part that can incorporate in the albumen through alpha-non-natural amino acid provides proteic multiple advantage and operation.For instance, ketone group functional group unique reactive allows with any multiple reagent that contains hydrazine or azanol selective modification albumen in vitro and in vivo.For instance, the heavy atom alpha-non-natural amino acid can be used for phasing (phasing) X-ray structured data.Use alpha-non-natural amino acid to carry out the introducing of heavy atom locus specificity and also providing selectivity and motility aspect the position of selecting heavy atom.For instance, effectively in vivo and in vitro opalescence is crosslinked in photoreactivity alpha-non-natural amino acid (including, but is not limited to have the aminoacid of benzophenone and aryl azide (including, but is not limited to aziminobenzene) side chain) permission.The amino acid whose example of photoreactivity includes, but is not limited to azido-phenylalanine with to benzoyl-phenylalanine.That albumen with photoreactivity alpha-non-natural amino acid can be able to by the exciting light reactive group subsequently is crosslinked-temporary transient control is provided.In an example, the methyl of alpha-non-natural amino acid can replace through isotopic labeling, and the methyl that it includes, but is not limited to as partial structurtes and kinetics probe includes, but is not limited to by using nuclear magnetic resonance, NMR and vibrational spectroscopy.For instance, alkynyl or azido functional group allow with molecule through [3+2] cycloaddition reaction selective modification albumen.

The alpha-non-natural amino acid of incorporating in the polypeptide at aminoterminal can (be different from the NH that is present in usually in the a-amino acid by the R group (the R group is any substituent group except that being used for 20 kinds of natural amino acids) and second reactive group ₂Group) (seeing formula I) forms.Can introduce similar alpha-non-natural amino acid at carboxyl terminal with second reactive group that is different from the COOH group (seeing formula I) that is present in usually in the a-amino acid.

The chemosynthesis of alpha-non-natural amino acid

Manyly be applicable to that alpha-non-natural amino acid of the present invention is commercially available, for example can be available from Sigma (USA) or Aldrich (Milwaukee, WL USA).Described commercially available aminoacid can provide as this paper according to circumstances or obtain as providing in the various publications or using the known standard method of one of ordinary skill in the art to synthesize.About organic synthesis technology, referring to, for example Fessendon and Fessendon Organic Chemistry, (1982, the 2 editions, Willard Grant Press, Boston Mass.); March's Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and Sundberg Advanced Organic ChemistryThe 3rd edition, part A and B, 1990, Plenum Press, New York).Describe synthetic other publication of alpha-non-natural amino acid and comprise that (for example) WO 2002/085923 title is " In vivo incorporation ofUnnatural Amino Acids; " people such as Matsoukas, (1995) J.Med.Chem.,38,4660-4669; King, F.E. ﹠amp; Kidd, D.A.A. (1949) A New Synthesis of Glutamine and of y-Dipeptides ofGlutamic Acid from Phthylated Intermediates. J.Chem.Soc,3315-3319; Friedman, O.M.﹠amp; Chatterrji, R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates forAnti-Tumor Agents. J.Am.Chem.Soc.81,3750-3752; Craig, people such as J.C. (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl] amino] quinoline (Chloroquine). J.Org.Chem. 53,1167-1170; Azoulay, M., Vilmont, M.﹠amp; Frappier, F. (1991) Glutamine analogues asPotential Antimalarials. Eur.J.Med.Chem.26,201-5; Koskinen, A.M.P. ﹠amp; Rapoport, H. (1989) Synthesis of 4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J.Org.Chem.54,1859-1866; Christie, B.D. ﹠amp; Rapoport, H. (1985) Synthesisof Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization. J. Org.Chem.1989:1859-1866; People such as Barton, (1987) Synthesis of Novel a-Amino-Acids andDerivatives Using Radical Chemistry:Synthesis of L-and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; With people such as Subasinghe, (1992) Quisqualic acid analogues:synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J.Med.Chem.35:4602-7.Also the title referring to December in 2003 application on the 22nd is No. the 10/744th, 899, the patent application case of " Protein Arrays " and the 60/435th, No. 821 of December in 2002 application on the 22nd.

A. carbonyl reaction group

The aminoacid that wherein has the carbonyl reaction group allows various reactions to come link molecule (including, but is not limited to PEG or other water soluble molecules) via nucleophilic addition or aldol reaction.

The exemplary carbonylamino acid that contains can be as follows:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R ₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R ₃For H, aminoacid, polypeptide or amino terminal are modified base, and R ₄For H, aminoacid, polypeptide or carboxyl terminal are modified base.In certain embodiments, n is 1, R ₁Be phenyl and R ₂For simple alkyl (being methyl, ethyl or propyl group) and ketone group partly are positioned at para-position with respect to alkyl side chain.In certain embodiments, n is 1, R ₁Be phenyl and R ₂For simply alkyl (being methyl, ethyl or propyl group) and ketone group partly are positioned at respect to position between alkyl side chain.

Acetyl group-(+/-)-phenylalanine and an acetyl group-(+/-)-phenylalanine synthetic is described in people such as Zhang, Z.., among the Biochemistry 42:6735-6746 (2003), it incorporates this paper by reference into.One of ordinary skill in the art can similarly prepare other and contain carbonylamino acid.

In certain embodiments, the polypeptide that comprises non-naturally encoded amino acids through chemical modification to produce reactive carbonyl functional group.For instance, can produce the aldehyde functional group that can be used for association reaction from functional group with adjacent amino and hydroxyl.When bioactive molecule was polypeptide, for example N terminal filament propylhomoserin or threonine (it can exist usually or expose through chemistry or enzymic digestion) can be used for producing the aldehyde functionality with periodate under the mild oxidation cracking condition.Referring to, people such as Gaertner for example, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K.﹠amp; Stroh, I, Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J. Biol Chem.269:7224-7230 (1994).Yet the known method of affiliated technical field only limits to the aminoacid of peptide or albumen n end.

In the present invention, carrying adjacent hydroxyl and amino non-naturally encoded amino acids can incorporate in the polypeptide as " hidden (masked) " aldehyde functional group.For instance, the 5-oxylysine has the hydroxyl adjacent to ε amine.The reaction condition that is used to produce aldehyde be usually included in add molar excess under the temperate condition sodium metaperiodate (sodiummetaperiodate) to avoid the oxidation of other site in polypeptide.The pH value of oxidation reaction is generally about 7.0.Type reaction comprises in the buffer solution of polypeptide the sodium metaperiodate that adds about 1.5 molar excess, then about 10 minutes of incubation in the dark.Referring to, for example United States Patent (USP) the 6th, 423, and No. 685, it incorporates this paper by reference into.

The carbonyl functional group can with contain hydrazine, hydrazides, azanol or semicarbazides reagent under the temperate condition in aqueous solution selective reaction to be respectively formed at corresponding hydrazone stable under the physiological condition, oxime or semicarbazones key.Referring to, Jencks for example, W.P., J.Am.Chem.Soc.81,475-481 (1959); Shao, J. and Tam, J.P., J.Am.Chem.Soc.117:3893-3899 (1995).In addition, unique reactive permission of carbonyl carried out selective modification in the presence of other amino acid side chain.Referring to, Cornish for example, people such as V.W., J Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F. and Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L.K., Science 276:1125-1128 (1997).

B. hydrazine, hydrazides or semicarbazides reactive group

The non-naturally encoded amino acids that contains nucleophilic group (for example hydrazine, hydrazides or semicarbazides) allows to react to form conjugate (including, but is not limited to and PEG or other water-soluble polymer) with multiple electrophilic group.

Exemplary contain hydrazine, hydrazides or semicarbazides aminoacid as follows:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl or do not exist; X is O, N, S or does not exist; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.

In certain embodiments, n is 4, R ₁Do not exist, and X is N.In certain embodiments, n is 2, R ₁Do not exist, and X does not exist.In certain embodiments, n is 1, R ₁Be phenyl, X is O, and oxygen atom is positioned at the para-position of aliphatic group on the aryl rings.

Contain hydrazine, hydrazides and semicarbazides aminoacid can purchase white commercial source.For instance, L-glutamic acid-y-hydrazides can available from Sigma Chemical (St.Louis, MO).Other non-commercially available aminoacid can be prepared by one of ordinary skill in the art.Referring to, for example United States Patent (USP) the 6th, 281, and No. 211, it incorporates this paper by reference into.

The polypeptide that contains the non-naturally encoded amino acids that carries hydrazides, hydrazine or semicarbazides functional group can react with similar chemical reactivity to the multiple molecule that contains aldehyde radical or other functional group effectively with optionally.Referring to, Shao for example, J. and Tarn, J., J.Am.Chern.Soc.117:3893-3899 (1995).With compare being present in 20 kinds of nucleophilic groups (including, but is not limited to the hydroxyl of serine or threonine or lysine amino and N end) on the common amino acid, unique reactivity of hydrazides, hydrazine and semicarbazides functional group makes it significantly have more reactivity to aldehyde, ketone and other electrophilic group.

C. contain aminooxy group aminoacid

The non-naturally encoded amino acids that contains aminooxy group (being also referred to as azanol) allows to react to form conjugate (including, but is not limited to and PEG or the reaction of other water-soluble polymer) with multiple electrophilic group.The same as hydrazine, hydrazides and semicarbazides, the enhanced nucleophilicity of aminooxy group allow its to the multiple molecule that contains aldehyde or other functional group with similar chemical reactivity effectively and selective reaction.Referring to, Shao for example, J. and Tarn, J., J.Am.Chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, Ace.Chem.Res.34:727-736 (2001).Although the result who reacts with diazanyl group is corresponding hydrazone, oxime derives from the reaction of aminooxy group and carbonyl group-containing groups (for example ketone group) usually.

The exemplary aminooxy group aminoacid that contains can be as follows:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; Y=C (O) or do not exist; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.In certain embodiments, n is 1, R ₁Be phenyl, X is O, and m is 1, and Y exists.In certain embodiments, n is 2, R ₁Do not exist with X, m is 0, and Y does not exist.

The aminoacid that can contain aminooxy group by the aminoacid predecessor that can get easily (homoserine, serine and threonine) preparation.Referring to, for example M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858 (2003).Isolate some and contain aminooxy group aminoacid, for example L-2-amino-4-(aminooxy group) butanoic acid (Rosenthal, people such as G., Life Sci.60:1635-1641 (1997)) from natural origin.One of ordinary skill in the art can prepare other and contain aminooxy group aminoacid.

D. nitrine and alkyne reaction group

Unique reactivity of nitrine and alkynes functional group makes it extremely can be used for the selective modification of polypeptide and other biomolecule.Organic nitrine (especially α nitrine) and alkynes are stable to the common response electrochemical conditions usually.Specifically, nitrine and alkynes functional group are inertia to the side chain (being the R group) that is found in 20 kinds of common amino acids in the natural generation polypeptide.Yet, when placing closely (close proximity), demonstrate " load on spring (spring-loaded) " character of nitrine and ethynylene group, and they are through Huisgen[3+2] and cycloaddition reaction selectivity and reacting effectively to generate corresponding triazole.Referring to, people such as Chin J. for example, Science 301:964-7 (2003); Wang, people such as Q., J.Am.Chem.Soc.125,3192-3193 (2003); Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction relate to the selectivity cycloaddition reaction (referring to, Padwa for example, the Comprehensive Organic Synthesis of A., the 4th volume, (Trost, B.M., 1991 editors), 1069-1109 page or leaf; Huisgen, R. 1,3-Depolar Cycloaddition Chemistry, (Padwa, A., 1984 editors) rather than nucleophilic displacement of fluorine, 1-176 page or leaf), thus incorporate into carry the non-naturally encoded amino acids that contains nitrine and alkynes side chain allow the gained polypeptide in the position of non-naturally encoded amino acids through selective modification.Can at room temperature under aqueous conditions, (include, but is not limited to catalytic amount CuSO by the Cu (II) that adds catalytic amount ₄Form), be used for Cu (II) is reduced in existence in the presence of the Reducing agent of Cu (I) and relate to the cycloaddition reaction that contains nitrine or alkynes ABP.Referring to, Wang for example, people such as Q., J.Am.Chem.Soc.125,3192-3193 (2003); Tornoe, people such as C.W., J.Org.Chem.67:3057-3064 (2002); People such as Rostovtsev, Angew.Chem.Int.Ed.41:2596-2599 (2002).Exemplary Reducing agent includes, but is not limited to ascorbic acid, metallic copper, quinine, hydroquinone, vitamin K, glutathion, cysteine, Fe ²⁺, Co ²⁺With apply electromotive force.

In some cases, as the Huisgen[3+2 that needs between nitrine and the alkynes] during cycloaddition reaction, antigen-binding polypeptides comprises the non-naturally encoded amino acids that contains alkynyl moiety, and be interconnected in amino acid whose water-soluble polymer and comprise the nitrine part.Perhaps, also can carry out back reaction (being the nitrine part and the alkynyl moiety that is present on the water-soluble polymer on the aminoacid).

Nitrine functional group also can with the water-soluble polymer selective reaction that contains aryl ester, and suitably functionalized to produce amido link with aryl phosphine part.Aryl phosphine group in-situ reducing nitrine, and gained amine reacts to produce corresponding amide effectively with immediate ester bond subsequently.Referring to, for example E.Saxon and C.Bertozzi, Science 287,2007-2010 (2000).The aminoacid that contains nitrine can be alkyl azide (including, but is not limited to 2-amino-6-azido-1-caproic acid) or aryl azide (to the triazobenzene alanine).

The exemplary water-soluble polymer that contains aryl ester and phosphine part can be as follows:

Wherein X can or not exist for O, N, S, and Ph is a phenyl, W be water-soluble polymer and R can be for H, alkyl, aryl, be substituted alkyl and be substituted aryl.Exemplary R group includes, but is not limited to-CH ₂,-C (CH ₃) ₃,-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O) ₂R ' ,-S (O) ₂NR ' R " ,-CN and-NO ₂R ', R ", R_ and R " " independently are meant hydrogen separately, are substituted or are unsubstituted assorted alkyl, are substituted or are unsubstituted aryl, the aryl that includes, but is not limited to replace through 1-3 halogen, be substituted or be unsubstituted alkyl, alkoxyl or thio alkoxy or aryl alkyl.For instance, when chemical compound of the present invention comprised an above R group, each R group independently was chosen as when having more than one these groups " the group as each R ', R ", R_ and R "." when being connected in same nitrogen-atoms, they can combine with nitrogen-atoms, form 5 yuan, 6 yuan or 7 yuan of rings as R ' and R.For instance ,-NR ' R " includes, but is not limited to 1-pyrrolidinyl and 4-morpholinyl.For above-mentioned substituent group, one of ordinary skill in the art should be appreciated that term " alkyl " is meant and comprise and the group of the bonded carbon atom of group except that hydrogen atom that for example haloalkyl (includes, but is not limited to-CF ₃With-CH ₂CF ₃) and acyl group (include, but is not limited to-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂OCH ₃Or the like).

Nitrine functional group also can with the water-soluble polymer selective reaction that contains thioesters, and suitably functionalized and produce amido link with aryl phosphine part.Aryl phosphine group in-situ reducing nitrine, and gained amine reacts to produce corresponding amide effectively with thioester bond subsequently.The exemplary water-soluble polymer that contains thioesters and phosphine part can be as follows:

Wherein n is 1-10; X can or not exist for O, N, S, and Ph is a phenyl, and W is a water-soluble polymer.

The exemplary alkynyl amino acid that contains can be as follows:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.In certain embodiments, n is 1, R ₁Be phenyl, X does not exist, and m is 0, and acetylene moiety is positioned at the para-position with respect to alkyl side chain.In certain embodiments, n is 1, R ₁Be phenyl, X is O, and m is 1, and the alkynes propoxyl group is positioned at the para-position (being O-propargyl-tyrosine) with respect to alkyl side chain.In certain embodiments, n is 1, R ₁Do not exist with X, m is 0 (being PGIY).

It is commercially available containing alkynyl amino acid.For instance, PGIY can available from Peptech (Burlington, MA).Perhaps, can contain alkynyl amino acid according to the standard method preparation.For instance, can (for example) as Deiters, A. wait the people, synthetic described in the J.Am.Chem.Soc.125:11782-11783 (2003) to alkynes propoxyl group phenylalanine, and as Kayser, B. wait the people, Tetrahedron 53 (7): synthetic 4-alkynyl-L-phenylalanine described in the 2475-2484 (1997).One of ordinary skill in the art can prepare other and contain alkynyl amino acid.

The exemplary nitrine aminoacid that contains can be as follows:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.In certain embodiments, n is 1, R ₁Be phenyl, X does not exist, and m is 0, and nitrine partly is positioned at the para-position with respect to alkyl side chain.In certain embodiments, n is 0-4, and R ₁Do not exist with X, and m is 0.In certain embodiments, n be 1, R ₁Be phenyl, X is O, m be 2 and β-azido ethyoxyl partly be positioned at para-position with respect to alkyl side chain.

The aminoacid that contains nitrine can be buied from commercial source.For instance, 4-triazobenzene alanine can derive from Chem-Impex International, and Inc. (Wood Dale, IL).About the non-commercially available nitrine aminoacid that contains; can use the known standard method of one of ordinary skill in the art to prepare azido relatively simply; include, but is not limited to via suitable leaving group (including, but is not limited to halogen, methanesulfonates, tosylate) displacement, perhaps via the lactone of opening through due care.Referring to, March for example Advanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York).

E. amineothiot reactive group

Make it extremely can be used for polypeptide and other bioactive molecule that contains aldehyde radical carries out selective modification via forming tetrahydro-thiazoles through unique reactivity of the amineothiot functional group of beta substitution.Referring to, for example J.Shao and J.Tam, J.Am.Chem.Soc.1995,117 (14) 3893-3899.In certain embodiments, can incorporate in the ABP polypeptide, and react with the water-soluble polymer that comprises aldehyde functional group subsequently through the amineothiot aminoacid of beta substitution.In certain embodiments, water-soluble polymer, drug conjugates or other payload (payload) can be coupled to via forming tetrahydro-thiazoles and comprise through the amino acid whose ABP polypeptide of the amineothiot of beta substitution.

The cellular uptake of alpha-non-natural amino acid

When specifying and selecting alpha-non-natural amino acid when being used to include, but is not limited to incorporate albumen into, must consider problem usually by eukaryotic cell picked-up alpha-non-natural amino acid.For instance, the high charge density of a-amino acid hints that these chemical compounds unlikely have cell permeable.Natural amino acid is absorbed by eukaryotic cell based on proteic movement system through one group.Can carry out rapid screening to assess (if existence) which alpha-non-natural amino acid by cellular uptake.Referring to, for example the title of December in 2003 application on the 22nd be the toxicity test in No. the 10/744th, 899, the application case of " Protein Arrays " and the December in 2002 applied in 22nd the 60/435th, No. 821; And Liu, D.R. ﹠amp; Schultz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code. PNAS United States96:4780-4785.Analyze picked-up although in all sorts of ways easily, to the alternative biosynthesis pathway that provides of the anticipation alpha-non-natural amino acid of obedience cellular uptake approach in vivo to produce aminoacid.

The biosynthesis of alpha-non-natural amino acid

Existed many biosynthesis pathwaies to be used to produce aminoacid and other chemical compound in the cell.Although may not have the biological synthesis method of specific alpha-non-natural amino acid in natural (including, but is not limited to eukaryotic cell), the invention provides described method.For instance, by adding new enzyme or modifying existing host cell approach and in host cell, produce the biosynthesis pathway of alpha-non-natural amino acid according to circumstances.Other new enzyme is the natural generation enzyme or the enzyme that manually develops according to circumstances.For instance, the biosynthesis of p-Aminophenylalanine (for example WO 2002/085923 title is for shown in " In vivo incorporation of unnatural amino acids ") depends on the combination of adding the known enzyme that derives from other organism.By these genes being incorporated in the eukaryotic cell with the plasmid transformant of the gene that comprises described enzyme.When expressing in cell, described gene provides enzymatic pathway with synthetic required compound.The example of the enzyme type of Tian Jiaing provides in the example hereinafter according to circumstances.Other enzyme sequence is seen (for example) Genbank.The enzyme that also will manually develop in the same manner according to circumstances is added in the cell.Manipulating cells machine and cell source is to produce alpha-non-natural amino acid in this way.

Several different methods can be used for producing and is used for biosynthesis pathway or is used for the novel enzymes that existing approach develops.For instance, include, but is not limited to Maxygen, the recurrence reorganization (recursive recombination) that Inc. (www.maxygen.com is last available) is developed is used to develop novel enzymes and approach according to circumstances.Referring to, Stemmer (1994) for example, Rapid evolution of a protein in vitro by DNA shuffling, Nature370 (4): 389-391; And Stemmer, (1994), DNA shuffling by random fragmentation and reassembly:In vitrorecombination for molecular evolution, Proc.Natl.Acad.Sci.USA., 91:10747-10751.The DesignPath that is developed by Genencor (www.genencor.com is last available) ^TMBe used for metabolic pathway engineering according to circumstances, include, but is not limited to engineered approach in cell, to produce O-methyl-L-tyrosine.Described technology adopts the combination of new gene (including, but is not limited to differentiate through functional genomics, molecular evolution and design) to rebuild existing approach in host organisms.Diversa Corporation (www.diversa.com is last available) also is provided for the technology in rapid screening gene and gene approach library, includes, but is not limited to set up new way.

Usually produce alpha-non-natural amino acid with the present invention through the biosynthesis pathway of engineering operation to be enough to the carrying out synthetic concentration of effective protein biology, described concentration includes, but is not limited to the n cell amount, but does not reach the degree that influences other aminoacid or exhaust cell source concentration.The typical concentration that in vivo produces is that about 10mM is to about 0.05mM in this way.In case with the plasmid transformant that comprises the gene that is used to produce the required enzyme of specificity approach and produce alpha-non-natural amino acid, just use the generation of in vivo selecting to be used for the alpha-non-natural amino acid of the synthetic and cell growth of ribosomal protein according to circumstances with further optimization.

Polypeptide with alpha-non-natural amino acid

Can carry out incorporating into of alpha-non-natural amino acid for multiple purpose, include, but is not limited to adjust the change of protein structure and/or function; Change size, acidity, nucleophilicity, hydrogen bond, hydrophobicity, protease target site accessibility; Targeting moiety (including, but is not limited to be used for protein arrays); Add bioactive molecule; Connect polymer; Connect radionuclide; Regulate serum half-life; Regulate tissue infiltration (for example tumor); Regulate active transport; Regulate tissue, cell or organ specificity; Regulate immunogenicity; Regulate protease resistant or the like.The albumen that comprises alpha-non-natural amino acid can have enhanced or or even complete new catalysis or bio-physical property.For instance, according to circumstances by alpha-non-natural amino acid being comprised into albumen changes following character: toxicity, bio distribution, structural property, spectroscopic property, chemistry and/or spectrochemical property, catalytic capability, half-life (including, but is not limited to serum half-life), with the ability of other molecular reaction, include, but is not limited to covalently or non-covalently, or the like.Comprise that the proteic compositions that contains at least a alpha-non-natural amino acid can be used for (including, but is not limited to) novel treatment, diagnosis, catalyzing enzyme, industrial enzyme, conjugated protein (including, but is not limited to antibody) and includes, but is not limited to study protein structure and function.Referring to, Dougheny for example, (2000) Unnatural Amino Acids as Probesof Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.

In one aspect of the present invention, compositions comprises at least a albumen with at least one (including, but is not limited to more than at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or ten) alpha-non-natural amino acid.Alpha-non-natural amino acid can be identical or different, include, but is not limited to comprise 1,2,3,4,5,6,7,8,9,10 or the albumen of how different alpha-non-natural amino acids in can have 1,2,3,4,5,6,7,8,9,10 or more different loci.On the other hand, compositions comprises a kind of albumen, in this albumen at least one but be less than in whole albumen existing specific amino acids and replace by alpha-non-natural amino acid.For given albumen with an above alpha-non-natural amino acid, alpha-non-natural amino acid can identical or different (include, but is not limited to the alpha-non-natural amino acid that described albumen can comprise two or more type, perhaps can comprise two kinds of identical alpha-non-natural amino acids).For given albumen with two above alpha-non-natural amino acids, alpha-non-natural amino acid can be identical, the combination of different or a plurality of same type alpha-non-natural amino acids and at least one different alpha-non-natural amino acid.

Interesting ABP with at least one alpha-non-natural amino acid is a feature of the present invention.The present invention also comprises having at least one polypeptide or albumen with the alpha-non-natural amino acid of the compositions and methods of the invention generation.Also can provide excipient (including, but is not limited to pharmaceutically acceptable excipient) for albumen.

By produce interesting albumen or the polypeptide with at least one alpha-non-natural amino acid in eukaryotic cell, albumen or polypeptide generally include the eukaryotic cell post translational modification.In certain embodiments, albumen comprises at least one alpha-non-natural amino acid and at least a post translational modification of in vivo being carried out by eukaryotic cell, and wherein said post translational modification also be can't help prokaryotic cell and carried out.For instance, post translational modification include, but is not limited to that acetylation, acidylate, lipid-modified, palmitoylation, cetylate addition, phosphorylation, glycolipid key are modified, glycosylation or the like.On the one hand, post translational modification comprises that oligosaccharide (includes, but is not limited to (GlcNAc-Man) ₂-Man-GlcNAc-GlcNAc) be connected in agedoite by GlcNAc-agedoite key.Referring to table 1, some N that list eukaryotic protein connect the example (also may have other not shown residue) of oligosaccharide.On the other hand, post translational modification comprises that oligosaccharide (including, but is not limited to Gal-GalNAc, Gal-GlcNAc or the like) is connected with serine or threonine by GalNAc-serine or GalNAc-threonine key or GlcNAc-serine or GlcNAc-threonine key.

Table 1: through the oligosaccharide example of GlcNAc-key connection

On the other hand, post translational modification comprises the Proteolytic enzyme processing of predecessor (including, but is not limited to calcitonin predecessor, calcitonin-gene-related peptide predecessor, Pre Pro PTH, preproinsulin, proinsulin, pre-pro-opiomelanocortin (prepro-opiomelanocortin), proopiomelanocortin or the like); Be assembled into multi-subunit protein or macromolecular assemblies; Be sent to another site in the cell (include, but is not limited to organelle, for example endoplasmic reticulum, Gorky (Golgi) body, nuclear, lysosome, peroxisome, mitochondrion, chloroplast, vacuole or the like or through secretory pathway).In certain embodiments, albumen comprise that secretion or positioning sequence, epitope label, FLAG label, poly are histidine-tagged, GST fusant or the like.

An advantage of alpha-non-natural amino acid is that its existence can be used in other chemical part that adds other molecule.Can in eucaryon or non-eukaryotic cell, in vivo or in vitro carry out these modifications.Therefore, in certain embodiments, post translational modification is to carry out via alpha-non-natural amino acid.For instance, post translational modification can be carried out via nucleophilic-electrophilic reaction.The major part that is used for the albumen selective modification is at present reacted the covalent bond that relates between nucleophilic and the electrophilic reaction companion, includes, but is not limited to the reaction of α-Lu Daitong and histidine or cysteine side chain.Number by nucleophilic residues in the albumen and accessibility are determined the selectivity under these situations.In albumen of the present invention, can use other to have more optionally reaction, for example non-natural ketone group-aminoacid and hydrazides or the reaction in vitro and in vivo of aminooxy group chemical compound.Referring to, people such as Cornish for example, (1996) Am.Chem.Soc, 118:8150-8151; People such as Mahal, (1997) Science, 276:1125-1128; People such as Wang, (2001) Science292:498-500; People such as Chin, (2002) Am.Chem.Soc.124:9026-9027; People such as Chin, (2002) Proc.Natl.Acad. ScL99:11020-11024; People such as Wang, (2003) Proc.Natl.Acad.ScL100:56-61; People such as Zhang, (2003) Biochemistry, 42:6735-6746; With people such as Chin, (2003) Science(in press).This allows with any in fact albumen of many reagent (including, but is not limited to fluorogen, cross-linking agent, sugar derivatives and cytotoxicity molecule) selected marker.Also referring to, U.S. Provisional Patent Application case the 60/419th based on application on October 16th, 2002, the U.S. Provisional Patent Application case the 60/420th of application on October 23rd, No. 265 1, the U.S. Provisional Patent Application case the 60/441st of No. 990 and on January 16th, 2003 application, the title of No. 450 application in 15 days October in 2003 is the United States Patent (USP) the 10/686th of " Glycoprotein synthesis ", No. 944, these all incorporate this paper into by reference with reference to case.Including, but is not limited to also can to connect (including, but is not limited to use triaryl phosphine reagent) via azido aminoacid by Staudinger in interior post translational modification carries out.Referring to, people such as Kiick for example, (2002) Incorporation of azides into recombinant proteins forchemoselective modification by the Staudinger ligation, PNAS99:19-24.

The invention provides and be used for proteic another high efficiency method of selective modification, described method relates to heredity and incorporates alpha-non-natural amino acid into, includes, but is not limited to nitrine or alkynyl are partly comprised in response in the albumen of selecting codon.Can modify these amino acid side chains by the following method subsequently, described method includes, but is not limited to respectively and includes, but is not limited to acetylene or azido derivant and carry out Huisgen[3+2] cycloaddition reaction (referring to, Padwa for example, A.'s Comprehensive Organic Synthesis, the 4th volume, (1991) editor Trost, B.M., Pergamon, Oxford, 1069-1109 page or leaf; And Huisgen, R.'s 1,3-Dipolar Cvcloaddition Chemistry, (1984) editor Padwa, A., Wiley, New York, 1-176 page or leaf).Because described method relates to cycloaddition rather than nucleophilic displacement of fluorine, so albumen can be modified with high selectivity.Can come at room temperature in aqueous conditions, to react by Cu (I) salt that in reactant mixture, adds catalytic amount with good area selectivity (1,4＞1,5).Referring to, people such as Tornoe for example, (2002) Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002) Angew.Chem.Int.Ed.41:2596-2599.Another kind of methods availalbe is to carry out ligand exchange with four cysteine motifs on two arsenic compounds, referring to, people such as Griffin for example, (1998) Science281:269-272.

Can comprise any molecule in fact by the molecule that [3+2] cycloaddition reaction is added in the albumen of the present invention with nitrine or alkynyl derivatives.Molecule includes, but is not limited to dyestuff, fluorogen, cross-linking agent, sugar derivatives, polymer (including, but is not limited to polyethyleneglycol derivative), photocrosslinking agent, cytotoxic compound, affinity labeling, biotin derivative, resin, globule, second albumen and polypeptide (or more), polynucleotide (including, but is not limited to DNA, RNA or the like), metal-chelator, cofactor, fatty acid, carbohydrate or the like.These molecules can be made an addition in the alpha-non-natural amino acid (including, but is not limited to) with acetenyl respectively or have in the alpha-non-natural amino acid (including, but is not limited to) of azido azido-phenylalanine to alkynes propoxyl group phenylalanine.

V. in vivo produce and comprise the amino acid whose ABP of non-genetic coding

Can with modified tRNA and tRNA synzyme add to or replace and the non-natural generation systems in coded aminoacid in vivo produce antigen-binding polypeptides of the present invention.

Use and the non-natural generation systems in coded amino acid whose tRNA and the production method of tRNA synzyme be described in (for example) U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, No. 931) in, it incorporates this paper by reference into.These methods relate to generation and to translation system are the machine translator (therefore being sometimes referred to as " quadrature ") that endogenous synzyme and tRNA irrespectively work.Translation system comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) usually.O-RS is usually preferred with at least one the non-natural generation aminoacid aminoacylation O-tRNA in the translation system, and O-tRNA discerns at least one and can't help the selection codon that other tRNA discerned in the system.Therefore translation system is in the albumen in response to coded selection codon that is produced in the non-naturally encoded amino acids insertion system, and then aminoacid " replacement " is advanced in the position of coded polypeptide.

Affiliated technical field has been described quadrature tRNA and the aminoacyl tRNA synthetase that many kinds are used for specific synthesizing amino acid is inserted polypeptide, and is applicable to the present invention usually.For instance, ketone group specificity O-tRNA/ aminoacyl-tRNA synthetase is described in Wang, people such as L., and Proc.Nail.Acad.Sci.USA 100:56-61 (2003) and Zhang, people such as Z. are among Biochem.42 (22): the 6735-6746 (2003).Exemplary O-RS or its part are coded by the polymerized nucleoside acid sequence, and comprise the aminoacid sequence that is disclosed in U.S. Patent Application Publication case 2003/0082575 and 2003/0108885, and it incorporates this paper by reference into.Be used for also being described in U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927) and 2003/0108885 (the 10/126th, No. 931) with the corresponding O-tRNA molecule of a use of O-RS, it incorporates this paper by reference into.

The case description of nitrine specificity O-tRNA/ aminoacyl-tRNA synthetase system is in Chin, and people such as J.W. are among the J.Am.Chem.Soc.124:9026-9027 (2002).Exemplary O-RS sequence to azido-L-Phe includes, but is not limited to as U.S. Patent Application Publication case 2003/0108885 the (the 10/126th, No. 931) in disclosed nucleotide sequence SEQ ID NO:14-16 and 29-32 and aminoacid sequence SEQ ID NO:46-48 and 61-64, described open case is incorporated this paper by reference into.Be applicable to that exemplary O-tRNA sequence of the present invention includes, but is not limited to as U.S. Patent Application Publication case 2003/0108885 the (the 10/126th, No. 931) in disclosed nucleotide sequence SEQ ID NO:1-3, described open case is incorporated this paper by reference into.Specific non-naturally encoded amino acids is had specific O-tRNA/ aminoacyl-tRNA synthetase to being described in the U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927), and described open case is incorporated this paper by reference into.O-RS and O-tRNA that the aminoacid of ketone group containing and nitrine is incorporated in the saccharomyces cerevisiae are described in Chin, and people such as J.W. are among the Science 301:964-967 (2003).

The purposes of O-tRNA/ aminoacyl-tRNA synthetase comprises the specificity codon of selecting the coding non-naturally encoded amino acids.Although can use any codon, usually need to select in the expressed cell of O-tRNA/ aminoacyl-tRNA synthetase rare or from original codon.For instance, exemplary codon comprises nonsense codon, for example termination codon (succinum, Haematitum and milky white); Four or more base codon and other are rare or from original natural three base codons.

Field known mutation method under can using (including, but is not limited to locus specificity sudden change, cascade sudden change, restricted selection sudden change or the like) selects codon to introduce in the appropriate location in the ABP polypeptid coding sequence specificity.

The method that is used for producing the component (for example O-RS, O-tRNA and the can be used for quadrature O-tRNA/O-RS that incorporates non-naturally encoded amino acids into to) of protein biology synthesis machine is described in Wang, people such as L., Science 292:498-500 (2001); Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002); Zhang, people such as Z., Biochemistry 42:6735-6746 (2003).The method and composition that is used in vivo incorporating into non-naturally encoded amino acids is described in the U.S. Patent Application Publication case 2003/0082575 (the 10/126th, No. 927), and described open case is incorporated this paper by reference into.Be used to select to be used for organism in vivo the right method of quadrature tRNA-tRNA synzyme of translation system also be described in U.S. Patent Application Publication case 2003/0082575 the (the 10/126th, No. 927) and 2003/0108885 the (the 10/126th, No. 931) in, it incorporates this paper by reference into.

The method that is used to produce at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) comprises: (a) produce the RS library (being mutant according to circumstances) that derives from least a aminoacyl-tRNA synthetase (RS) from first organism, described first organism includes, but is not limited to the prokaryote body, the for example ancient green-ball bacterium of methane coccus, methanogen, Halobacterium, escherichia coli, flicker, fierce hot-bulb bacterium, super hyperthermophilic archaeon strain, the hot bacterium of quick gas, thermus thermophilus or the like, or most eukaryotes; (b) select in (and/or screening) RS library (be mutant according to circumstances) member of aminoacylation quadrature tRNA (O-tRNA) in the presence of non-naturally encoded amino acids and natural amino acid, and then activity (being mutant RS according to circumstances) RS pond is provided; And/or (c) select in (selecting through feminine gender according to circumstances) described pond in the presence of no non-natural coded amino acid the preferably active RS (including, but is not limited to mutant RS) of aminoacylation O-tRNA, and then at least a reorganization O-RS is provided; Wherein said at least a reorganization O-RS preferably uses non-naturally encoded amino acids aminoacylation O-tRNA.

In one embodiment, RS is nonactive RS.Can produce nonactive RS by active RS is suddenlyd change.For instance, can by will at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or become different aminoacids (including, but is not limited to alanine) to produce nonactive RS at least about 10 or 10 above amino acid mutations.

Can produce mutant RS library with the various known technologies of affiliated technical field, described technology includes, but is not limited to the appropriate design based on the three-dimensional RS structure of protein, the perhaps sudden change of RS nucleotide at random or in the appropriate design technology.For instance, can be by locus specificity sudden change, random mutation, multiformity recombination mutation (diversity generating recombination mutation), chimeric construct, appropriate design, and produce mutant RS by known other method of described herein or affiliated technical field.

In one embodiment, select that active (including, but is not limited to aminoacylation quadrature tRNA (O-tRNA) in the presence of non-naturally encoded amino acids and natural coded amino acid) member comprises in (and/or screening) RS library (being mutant RS according to circumstances): the positive is selected or introduce in a plurality of cells in selection markers (including, but is not limited to antibiotics resistance gene or the like) and (mutant according to circumstances) RS library, wherein positive selection and/or selection markers comprise at least one selection codon (including, but is not limited to succinum, Haematitum or opal codon); Described a plurality of cell is grown in the presence of selective agent; In the presence of selection and/or selective agent, select codon to differentiate survival (or showing specific reaction) cell, and then provide a subgroup to contain the cell through positive selection in activity (being mutant according to circumstances) RS pond by at least one that suppresses in positive selection and/or the selection markers.Selection and/or selective agent concentration can change according to circumstances.

On the one hand, positive selectable marker is chloramphenicol acetyltransferase (CAT) gene, and the selection codon is the succinum termination codon in the CAT gene.Positive selectable marker is the beta-lactamase gene according to circumstances, and the selection codon is the succinum termination codon in the beta-lactamase gene.On the other hand, positive selectable marker comprises fluorescence or luminous selection markers or based on the selection markers (including, but is not limited to cell surface marker) of affinity.

In one embodiment, the negative selection/or the screening pond in preferred in the presence of no non-natural coded amino acid the active RS library (being mutant according to circumstances) of aminoacylation O-tRNA comprising: select or screen activity (being mutant according to circumstances) the RS pond that obtains and introduce in a plurality of cells of second organism with feminine gender selection or selection markers with by the positive, wherein said negative selection or selection markers comprise at least one and select codon (include, but is not limited to antibiotics resistance gene, include, but is not limited to chloramphenicol acetyltransferase (CAT) gene); With differentiate in being supplemented with first culture medium of non-naturally encoded amino acids and screening or selective agent survival or show the specificity screening reaction but can not in being supplemented with second culture medium of non-naturally encoded amino acids and screening or selective agent, survive or show the cell of specificity screening reaction, and then provide survivaling cell or through screening cell with at least a O-RS of reorganization.For instance, CAT identification experiment scheme is served as positive-selecting and/or the negative screening in the determining of suitable O-RS recombinant according to circumstances.For instance, duplicate the clone pond containing CAT (comprise at least one select codon) and contain or do not contain on the growth plate of one or more non-naturally encoded amino acids according to circumstances.Therefore will be only be considered as containing reorganization O-RS containing the colony of growing on the plate of non-naturally encoded amino acids.On the one hand, select the concentration of (and/or screening) agent to change.In some respects, the first and second organism differences.Therefore, first and/or second organism comprises according to circumstances: prokaryote, eukaryote, mammal, escherichia coli, fungus, yeast, archeobacteria (archaebacteria), eubacteria, plant, insecticide, protista or the like.In other embodiments, selection markers comprises fluorescence or luminous selection markers or based on the selection markers of affinity.

In another embodiment, active (being mutant according to circumstances) RS comprises in screening or selection (including, but is not limited to negative the selection) pond: isolate active mutant RS pond from positive selection step (b); Feminine gender selected or introduce in a plurality of cells of second organism in selection markers (wherein negative select or selection markers comprises at least one and selects codon (include, but is not limited to the toxicity marker gene, include, but is not limited to comprise the ribonuclease barnase gene that at least one selects codon)) and active (being mutant according to circumstances) RS pond; Discriminating is survived in first culture medium of not replenishing non-naturally encoded amino acids or is showed that specificity screening reacts, still can not survive or show the cell of specificity screening reaction in being supplemented with second culture medium of non-naturally encoded amino acids, and then provide survivaling cell or through the screening cell, wherein said at least a reorganization O-RS is specific to described non-naturally encoded amino acids with at least a reorganization O-RS.On the one hand, described at least one selection codon comprises two or more selection codons approximately.Described embodiment can comprise according to circumstances: wherein said at least one select codon to comprise two or more to select codon and the situation of first and second organism difference (each organism includes, but is not limited to prokaryote, eukaryote, mammal, escherichia coli, fungus, yeast, archeobacteria, eubacteria, plant, insecticide, protista or the like according to circumstances) wherein.Some aspects comprise that also wherein negative selection marker comprises the ribonuclease barnase gene situation of (it comprises at least one and selects codon).Others comprise that selection markers wherein comprises fluorescence or luminous selection markers according to circumstances or based on the situation of the selection markers of affinity.In the embodiment of this paper, screening and/or the variation of selecting to comprise screening according to circumstances and/or selecting preciseness.

In one embodiment, the method that is used to produce at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) can further comprise: (d) separating out at least one reorganization O-RS; (e) produce the second group of O-RS (according to circumstances through sudden change) that derives from least a reorganization O-RS; (f) repeating step (b) and (c) until the O-RS that obtains to comprise preferred aminoacylation O-tRNA ability.Repeating step (d)-(f) includes, but is not limited at least about twice according to circumstances.On the one hand, can produce the second group of sudden change O-RS that derives from least a reorganization O-RS by suddenling change, described sudden change includes, but is not limited to random mutation, locus specificity sudden change, reorganization or its combination.

In the said method include, but is not limited to positive selections/screening step (b), negative selections/screening step (c) or the positive and feminine gender selection/screening step (b) and (c) both comprise change selection/screening step preciseness according to circumstances at the preciseness of interior selection/screening step.In another embodiment, positive selection/screening step (b), negative selection/screening step (c) or positive and negative selection/screening step (b) are with (c) both comprise use report, wherein detect report, perhaps wherein by luminous detection report by fluorescence-activated cell sorting (FACS).Report is illustrated on the cell surface according to circumstances, on the phage display surface or the like, and select based on affinity that relates to non-naturally encoded amino acids or analog or catalytic activity.In one embodiment, the sudden change synzyme be illustrated on the cell surface, on the phage display surface or the like.

The method that is used for generation reorganization quadrature tRNA (O-tRNA) comprises: (a) produce the mutant tRNA library that derives from least a tRNA (including, but is not limited to suppressor gene tRNA) from first organism; (b) select (including, but is not limited to negative the selection) or screening to derive from nothing that origin comes from (mutant according to circumstances) tRNA library of the RS aminoacylation of second organism in the presence of the aminoacyl-tRNA synthetase (RS) of first organism, and then tRNA (mutant according to circumstances) is provided the pond; (c) select or screening tRNA (mutant according to circumstances) pond in by the member of introducing quadrature RS (O-RS) aminoacylation, and then provide at least a reorganization O-tRNA; Wherein said at least a reorganization O-tRNA identification selection codon, and can't help to derive from the effectively identification of RS institute of second organism, and preferably by the O-RS aminoacylation.In certain embodiments, at least a tRNA is suppressor gene tRNA and/or comprise natural and/or non-natural unique three base codons, perhaps is nonsense codon, rare codon, non-natural codon, the codon that comprises at least 4 bases, amber codon, ochre codon or opal temination codon.In one embodiment, reorganization O-tRNA has improved orthogonality.Should be appreciated that, in certain embodiments, need not to modify and O-tRNA is introduced first organism from second organism according to circumstances.In various embodiments, first and second organism is identical or different, and is selected from (including, but is not limited to) prokaryote (including, but is not limited to methane coccus, methanogen, escherichia coli, Halobacterium or the like), eukaryote, mammal, fungus, yeast, archeobacteria, eubacteria, plant, insecticide, protista or the like according to circumstances.In addition, tRNA is according to circumstances by the non-naturally encoded amino acids aminoacylation in reorganization, and wherein non-naturally encoded amino acids is natural or through genetic manipulation biosynthesis in vivo.According to circumstances non-naturally encoded amino acids is made an addition to the growth medium that is used at least the first or second organism.

On the one hand, select in (including, but is not limited to negative the selection) or the screening library to comprise: toxicity marker gene (its toxic marker gene comprises at least one described selection codon) (or cause the gene of toxigenicity agent or electrostatic agent (static agent) or be the necessary gene of organism, wherein said marker gene comprises at least one and selects codon) and (being mutant according to circumstances) tRNA library introducing are derived from a plurality of cells of second organism by (being mutant according to circumstances) tRNA of aminoacyl-tRNA synthetase aminoacylation (step (b)); With the selection survivaling cell, wherein said survivaling cell contains (being mutant according to circumstances) the tRNA pond that comprises at least a quadrature tRNA or non-functional tRNA.For instance, can select survivaling cell by using compa-ratios cell density test (comparison ratio cell density assay).

On the other hand, the toxicity marker gene can comprise two or more selection codons.In another embodiment of described method, the toxicity marker gene is a ribonuclease barnase gene, and wherein ribonuclease barnase gene comprises at least one amber codon.Ribonuclease barnase gene can comprise two or more amber codons according to circumstances.

In one embodiment, select or screening (being mutant according to circumstances) tRNA pond in comprise by the member of quadrature RS (O-RS) aminoacylation of being introduced: the positive is selected or selection markers gene (wherein positive mark's gene comprises drug resistance gene (including, but is not limited to comprise the beta-lactamase gene of at least one described selection codon (for example at least one succinum termination codon))) or for the necessary gene of organism or cause toxic agents antidotal gene, is introduced and derive from a plurality of cells of second organism together with O-RS and (being mutant according to circumstances) tRNA pond; With the survival of differentiating growth in the presence of selection or selective agent (including, but is not limited to antibiotic) or through the screening cell; and then provide cell pool with at least a reorganization tRNA; wherein said at least a reorganization tRNA is by the O-RS aminoacylation, and aminoacid insertion selected codon in response at least one and in the translation product by positive mark's gene code.In another embodiment, select the concentration of (and/or screening) agent to change.

The right method of specificity O-tRNA/O-RS that produces is provided.Method comprises: (a) produce the mutant tRNA library that derives from least a tRNA from first organism; (b) negative select or the screening library in the presence of not having from the aminoacyl-tRNA synthetase (RS) of first organism by (being mutant according to circumstances) tRNA from the RS aminoacylation of second organism, and then provide (being mutant according to circumstances) tRNA pond; (c) select or screening (being mutant according to circumstances) tRNA pond in by the member of quadrature RS (O-RS) aminoacylation of being introduced, and then provide at least a reorganization O-tRNA.Described at least a reorganization O-tRNA identification selection codon, and can't help effectively to discern from the RS of second organism, and preferably by the O-RS aminoacylation.Described method comprises that also (d) produces (being mutant according to circumstances) the RS library that derives from least a aminoacyl-tRNA synthetase (RS) from the 3rd organism; (e) select or screening mutant RS library in preferred in the presence of non-naturally encoded amino acids and natural amino acid the member of at least a reorganization of aminoacylation O-tRNA, and then provide active (being mutant according to circumstances) RS pond; (f) negative select or the screening pond in preferred in the presence of no non-natural coded amino acid activity (the being mutant according to circumstances) RS of at least a O-tRNA of reorganization of aminoacylation; and then providing at least a specificity O-tRNA/O-RS right, wherein said at least a specificity O-tRNA/O-RS is to comprising at least a reorganization O-RS and at least a reorganization O-tRNA that is specific to described non-naturally encoded amino acids.Comprise that the specificity O-tRNA/O-RS that produces by described method is right.For instance, specificity O-tRNA/O-RS is right to comprising (including, but is not limited to) mutRNATyr-mutTyrRS, for example mutRNATyr-SS12TyrRS is right to, mutRNAGlu-mutGluRS to, mutRNAThr-mutThrRS to, mutRNALeu-mutLeuRS, or the like.In addition, described method comprises the wherein situation of first identical with the 3rd organism (including, but is not limited to the methane coccus).

The present invention comprises that also selection is used for the in vivo right method of quadrature tRNA-tRNA synzyme of translation system of second organism.Described method comprises: with marker gene, tRNA and separate from or the aminoacyl-tRNA synthetase (RS) that derives from first organism introduce in first group of cell from second organism; Marker gene and tRNA are introduced in the repetitive cell group from second organism; With first group that selects in the repetitive cell group, to survive in survivaling cell, or screening is showed the specificity screening reaction and can not produce the cell of this reaction in the repetitive cell group, wherein first group and repetitive cell group select or selective agent in the presence of growth, wherein survival or comprise and be used for second organism in vivo the quadrature tRNA-tRNA synzyme of translation system is right through screening cell.In one embodiment, comparison and selection or screening comprise in vivo complementary assay.Selection or selective agent concentration can change.

Organism of the present invention comprises multiple organism and multiple combination.For instance, first and second organisms in the method for the present invention can be identical or different.In one embodiment, organism is a prokaryote according to circumstances, includes, but is not limited to methane coccus, methanogen, Halobacterium, escherichia coli, the ancient green-ball bacterium of flicker, fierce hot-bulb bacterium, super hyperthermophilic archaeon strain, the hot bacterium of quick gas, thermus thermophilus or the like.Perhaps, organism comprises eukaryote according to circumstances, include, but is not limited to plant (including, but is not limited to complicated plant, for example monocotyledon or dicotyledon), algae, protista, fungus (including, but is not limited to yeast or the like), animal (including, but is not limited to mammal, insecticide, arthropod or the like) or the like.In another embodiment, second organism is a prokaryote, includes, but is not limited to methane coccus, methanogen, Halobacterium, escherichia coli, the ancient green-ball bacterium of flicker, Halobacterium, fierce hot-bulb bacterium, super hyperthermophilic archaeon strain, the hot bacterium of quick gas, thermus thermophilus or the like.Perhaps, second organism can be eukaryote, includes, but is not limited to yeast, zooblast, plant cell, fungus, mammalian cell or the like.In various embodiments, the first and second organism differences.

VI. the position of non-natural generation aminoacid in ABP

The present invention is contained one or more non-natural generation aminoacid is incorporated among the ABP.One or more non-natural generation aminoacid can be incorporated in the specific location of not destroying polypeptide active.This can be achieved by carrying out " conservative " replacement, include, but is not limited to replace that hydrophobic amino acid, big aminoacid are replaced big aminoacid, hydrophilic amino acid is replaced hydrophilic amino acid, and/or be not to be that insert the necessary position of activity non-natural generation aminoacid with hydrophobic amino acid.

Can adopt multiple biochemistry and structural approach to select to be used in the antigen-binding polypeptides the required site of replacing with non-naturally encoded amino acids.One of ordinary skill in the art understand easily, and any position in the polypeptide chain is suitable for selecting incorporating non-naturally encoded amino acids into, and select can be based on appropriate design or by selecting to be used for any or non-special required purpose at random.The selection in required site can be used for generation and have any required character or active ABP molecule, includes, but is not limited to agonist, super-agonists, inverse agonist, antagonist, receptors bind regulator, receptor activity modulators, dimer or polymer and forms, do not change activity or character than natural molecule; Perhaps handle any physics or the chemical property of polypeptide, for example dissolubility, aggregation or stability.For instance, can use known alanine scanning of affiliated technical field or homologue scanning method to differentiate the necessary position of ABP biological activity in the polypeptide.The required activity that depends on polypeptide is except that the residue of being differentiated by alanine or homologue scanning sudden change for the residue of biological activity key can be the good candidate of replacing with non-naturally encoded amino acids.Perhaps, depend on the required activity of polypeptide once more, differentiated for the site of biological activity key also can be the good candidate of replacing with non-naturally encoded amino acids.The another kind of selection is to replace continuously with non-naturally encoded amino acids in the position of each on polypeptide chain simply, and observes the influence to polypeptide active.One of ordinary skill in the art understand easily, anyly be used for selecting to replace into alpha-non-natural amino acid means, technology or the method for the position of any polypeptide are applicable to the present invention.

In case eliminate and to stand the residue of replacing with non-naturally encoded amino acids, promptly can check the influence of the replacement that propose in each rest position place from the secondary of antigen-binding polypeptides and binding partners thereof, three grades or quarternary structure or three-dimensional crystalline structure.Therefore, one of ordinary skill in the art can differentiate the amino acid position that can be replaced by non-naturally encoded amino acids easily.

The exemplary residue of incorporating non-naturally encoded amino acids into includes, but is not limited to following description person: do not contain potential antigen binding domain; Solvent exposes wholly or in part; Have minimum with contiguous residue or do not have hydrogen bond and do mutually; Can be exposed to minimum degree contiguous reactive residue; Can be on one or more exposures of ABP; Can be and the 2nd ABP or other molecule or the juxtaposed ABP of its fragment site; As by in conjunction with or the three-dimensional, secondary, three grades or the quarternary structure that are not incorporated into its antigen or coupling or are not coupled to the ABP of another ABP or other bioactive molecule predicted, can be in the zone of highly flexible or structural rigidity; Perhaps optionally ABP self be can regulate or dimer or the polymeric conformation of one or more ABP comprised by the flexibility or rigidity that changes complete structure.The residue that is used to incorporate into alpha-non-natural amino acid can be cleavage sequence, a part that connects connexon sequence, antibody binding domain (including, but is not limited to myc label, FLAG or poly-His) or other sequence based on affinity (including, but is not limited to FLAG, poly-His, GST or the like) of antibody fragment or ABP.The residue that is used to incorporate into alpha-non-natural amino acid can be N end or the C end residue of ABP, and perhaps the non-antigen of ABP is in conjunction with residue.

Can replace or incorporate into the given position of ABP with many kinds of non-naturally encoded amino acids.In general, according to the specific non-naturally encoded amino acids of selecting to get off to be used to incorporate into: have the three-dimensional crystalline structure of its antigenic ABP, the perhaps secondary of ABP, three grades or quarternary structure by any other means inspection; The preferred property that conservative is replaced (promptly, for example acetyl phenyl alanine or O-propargyl tyrosine being replaced Phe, Tyr or Trp) based on the non-naturally encoded amino acids of aryl; With hope introduce in the antigen-binding polypeptides specificity in conjunction with chemistry (for example, if wish to realize with the water-soluble polymer with alkynyl moiety between Huisgen[3+2] cycloaddition reaction or and have aryl ester and incorporate amido link formation between the water-soluble polymer of phosphine part into, so just introduce 4-triazobenzene alanine).

In one embodiment, described method further comprises to be incorporated alpha-non-natural amino acid in the albumen into, and wherein alpha-non-natural amino acid comprises first reactive group; And the molecule that makes the albumen contact comprise second reactive group (includes, but is not limited to labelling, dyestuff, polymer, water-soluble polymer, polyethyleneglycol derivative, photocrosslinking agent, radionuclide, cytotoxic compound, medicine, affinity labeling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelator, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, the water solublity dendrimer, cyclodextrin, inhibition ribonucleic acid, biomaterial, nanoparticle, spin labeling, fluorogen, the containing metal part, the radioactivity part, novel functional group, the group of covalently or non-covalently doing mutually with other molecule, light is caught part, the photoisomerization part, biotin, biotin derivative, biotin derivative, the biotin analog, incorporate the part that heavy atom is arranged into, but chemical cracking group, but photodestruciton group, long side chain, carbon connects sugar, redox active agent, amino thio-acid, the toxicity part, through the isotopic labeling part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the close group of electronics, the magnetic group, insert group, chromophore, energy transfer agent, bioactivator, detectable label, micromolecule or any combinations thereof or any other required compound or material).First reactive group and second reaction-ity group reaction are to be connected in alpha-non-natural amino acid through [3+2] cycloaddition reaction with molecule.In one embodiment, first reactive group is alkynyl or azido part, and second reactive group is azido or alkynyl part.For instance, first reactive group is an alkynyl part (including, but is not limited to alpha-non-natural amino acid to alkynes propoxyl group phenylalanine), and second reactive group is the azido part.In another example, first reactive group is an azido part (including, but is not limited to alpha-non-natural amino acid to azido-L-phenylalanine), and second reactive group is the alkynyl part.

In some cases, non-naturally encoded amino acids is replaced and can be combined to influence other biological characteristics of ABP with other interpolation, replacement or the disappearance in the antigen-binding polypeptides.In some cases, described other adds, replaces or disappearance can increase the stability (including, but is not limited to the resistance of Degradation that Proteolytic enzyme is caused) of ABP, perhaps increases ABP and ABP receptor or antigenic affinity.In some cases, other interpolation, replacement or disappearance can increase the stability (including, but is not limited to when expressing) of antigen-binding polypeptides in escherichia coli or other host cell.In certain embodiments, behind escherichia coli or other recombinant host cell invading the exterior soothing the liver, interpolation, replacement or disappearance can increase the polypeptide dissolubility.In certain embodiments, except another is used for incorporating alpha-non-natural amino acid into and makes and causing the site that the polypeptide dissolubility increases, select to be used for the site of replacing after escherichia coli or other recombinant host cell are expressed through natural coding or alpha-non-natural amino acid.In certain embodiments, antigen-binding polypeptides comprise that affinity, adjusting (including, but is not limited to increases or the reduce) receptor dimerization regulated with the ABP receptor turn usefulness into, stablize receptor dimer, adjusting circulating half-life, adjustment release or biological usability, promotion purification or improvement or change another increase, replacement or the disappearance of specific dosing way.Similarly, antigen-binding polypeptides also can comprise chemistry or enzymatic lysis sequence, protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequence (including, but is not limited to FLAG, poly-His, GST or the like) based on affinity or improve disappearance (including, but is not limited to GFP), purification, through tissue or cell membrane transporter, prodrug release or activation, the ABP size reduces or the link molecule of other polypeptide characteristic (including, but is not limited to biotin).

In some cases, 1,2,3,4,5,6,7,8,9,10 or more aminoacid replace through one or more non-naturally encoded amino acids.In some cases, ABP comprises that further one or more non-naturally encoded amino acids are to natural generation amino acid whose 1,2,3,4,5,6,7,8,9,10 or more a plurality of replacement.In certain embodiments, ABP is replaced by one or more non-naturally encoded amino acids with two residues in the inferior segment at least.In some cases, two or more non-naturally encoded residues are connected in the straight or branched PEG (on the whole approximately～5-20kDa or littler) of one or more lower molecular weights, and then strengthen binding affinity and comparable serum half-life for the material of, larger molecular weight PEG single with respect to being connected in.

In certain embodiments, nearly two residues of antigen-binding polypeptides are replaced by one or more non-naturally encoded amino acids.

VII. the expression in non-eukaryote and eukaryote

Clone the high level expression of ABP polypeptide in order to obtain, usually the polynucleotide sub-clone of code book invention antigen-binding polypeptides is advanced to contain guide transcribes, transcribes/expression vector of the strong promoter of translation termination in, and if, then contain the ribosome binding site that is useful on translation initiation for the nucleic acid of encoding proteins.Suitable antibacterial promoter is known by affiliated technical field, and is described in philtrums such as people such as (for example) Sambrook and Ausubel.

Be used for expressing the bacterial expression system of ABP polypeptide of the present invention at (including, but is not limited to) escherichia coli, bacillus stearothermophilus, pseudomonas fluorescens, bacillus pyocyaneus, pseudomonas putida and Salmonella (Salmonella) available (people such as Palva, Gene 22:229-235 (1983); People such as Mosbach, Nature302:543-545 (1983)).The test kit that is used for these expression systems is commercially available.The eukaryotic expression system that is used for mammalian cell, yeast and insect cell is known by affiliated technical field, and also is commercially available.Using quadrature tRNA and aminoacyl-tRNA synthetase (above-mentioned) to express under the situation of antigen-binding polypeptides of the present invention, using the ability of quadrature component to select the host cell that is used to express according to it.Exemplary host cell comprises Glan positive bacteria (including, but is not limited to bacillus subtilis or streptomycete (Streptomyces)) and Glan negative bacteria (escherichia coli, pseudomonas fluorescens, bacillus pyocyaneus, pseudomonas putida) and yeast and other eukaryotic cell.Can use as described herein comprise the right cell of O-tRNA/O-RS.

Eukaryotic host cell of the present invention or non-eukaryotic host cell provide the synthetic proteic ability that comprises a large amount of available alpha-non-natural amino acids.On the one hand, compositions comprises (including, but is not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram according to circumstances or more comprises the albumen of alpha-non-natural amino acid, perhaps the amount that can reach with process for generation of protein in vivo (this paper provides about recombiant protein and produces and the details of purification).On the other hand, in (including, but is not limited to) cytolysate, buffer, medical buffer or other liquid suspension (including, but is not limited to about 1nl), exist according to circumstances in the compositions to the volume of about 100L (including, but is not limited to) whenever rise to few 10 microgram albumen, whenever rise to few 50 microgram albumen, whenever rise to few 75 microgram albumen, whenever rise to few 100 microgram albumen, whenever rise to few 200 microgram albumen, whenever rise to few 250 microgram albumen, whenever rise to few 500 microgram albumen, whenever rise to and lack 1 milligram of albumen, whenever rise to the albumen that lacks 10 milligrams of albumen or bigger concentration.Produce in the eukaryotic cell a large amount of (include, but is not limited to more than usually with include, but is not limited in vitro to translate other interior method can obtainable amount) the albumen that comprises at least a alpha-non-natural amino acid be a feature of the present invention.

Eukaryotic host cell of the present invention or non-eukaryotic host cell provide biosynthesis to comprise the proteic ability of a large amount of available alpha-non-natural amino acids.For instance, can produce the albumen that comprises alpha-non-natural amino acid that includes, but is not limited to following concentration: cell extract, cytolysate, culture medium, at least 10 micrograms per litre in buffer or the like, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre, at least 1 mg/litre, at least 2 mg/litre, at least 3 mg/litre, at least 4 mg/litre, at least 5 mg/litre, at least 6 mg/litre, at least 7 mg/litre, at least 8 mg/litre, at least 9 mg/litre, at least 10 mg/litre, at least 20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900 mg/litre, 1 grams per liter, 5 grams per liters, 10 grams per liters or bigger.

I. expression system, cultivation and separate

Can express ABP in the suitable expression system of any number, suitable expression system includes, but is not limited to yeast, insect cell, mammalian cell and antibacterial.The description of exemplary expression system hereinafter is provided.

Yeast term as used herein " yeast " comprises any various yeast that can express coding ABP gene.Described yeast includes, but is not limited to belong to ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), the basidiosporogenous yeast of Fungi Imperfecti (Fungi imperfecti) (spore guiding principle (Blastomycetes)).Ascosporogenous yeast is divided into two sections: Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises four subfamilies, Schizosaccharomycoideae (for example schizosaccharomyces pombe belongs to (genus Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), Lipomycoideae and Saccharomycoideae (for example Pichia sp. (Pichia) belongs to, kluyveromyces (Kluyveromyces) belongs to and saccharomyces cerevisiae (Saccharomyces) belongs to).The Basidiosporogenous yeast comprises that white winter spore yeast (Leucosporidium) belongs to, red winter spore yeast (Rhodosporidium) belongs to, lock is thrown yeast (Sporidiobolus) genus, Filobasidium and cross-arm bacterium (Filobasidiella) and belonged to.The yeast that belongs to Fungi Imperfecti (spore guiding principle) is divided into two sections: Sporobolomycetaceae (Sporobolomycetaceae) (for example Sporobolomyces (Sporobolomyces) and cloth are reined in and played spore Saccharomyces (Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example Candida (Candida)).

Especially interesting kind used in the present invention is with subordinate's kind: Pichia sp., kluyveromyces, saccharomyces cerevisiae, schizosaccharomyces pombe belongs to, Hansenula yeast (Hansenula), torulopsis (Torulopsis) and candidiasis include, but is not limited to pichia pastoris (P.pastoris), P.guillerimondii, saccharomyces cerevisiae, Ka Ersibai yeast (S.carlsbergensis), streptomyces diastaticus (S.diastaticus), S.douglasii, Ke Shi yeast (S.kluyveri), S.norbensis, ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), candida albicans (C.albicans), maltose candida mycoderma (C.maltosa) and multiple-shaped nuohan inferior yeast (H.polymorph).Selection is used for the suitable yeast of ABP expression within one of ordinary skill in the art's technical ability.At the yeast host of selecting to be used for expressing, suitable host can comprise the host with (for example) good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein generation and overall robustness.Yeast can obtain from multiple source usually, include, but is not limited to YeastGenetic Stock Center, Department of Biophysics and Medical Physics, University ofCalifornia (Berkeley, CA), with American Type Culture Collection (" ATCC ") (Manassas, VA).

Term " yeast host " or " yeast host cell " comprise the yeast of the receptor that can be used as or be used as recombinant vector or other transhipment DNA.Described term comprises the offspring of the initial yeast host cell that receives recombinant vector or other transhipment DNA.Should be appreciated that, because accidental or sudden change intentionally, so the offspring of single parent's cell is may not must identical on form or genome or total DNA complement with initial parental generation.Described definition indication offspring comprises the offspring who is the parental cell of correlation properties (nucleotide sequence that for example has coding ABP) with the enough similarities of parental generation.

Developed the expression and the conversion carrier that comprise extrachromosomal replication or integration vector and advanced many yeast hosts to be used for conversion.For instance, developed expression vector (people such as Sikorski, Genetics (1998) 112:19 that is used for saccharomyces cerevisiae; People such as Ito, J.Bacteriol. (1983) 153:163; People such as Hinnen, PROC Natl.Acad.Sci.USA (1978) 75:1929); The expression vector of candida albicans (people such as Kurtz, Mol.Cell.Biol. (1986) 6:142); The expression vector of maltose candida mycoderma (people such as Kunze, J.BASICMICROBlOL. (1985) 25:141); The expression vector of multiple-shaped nuohan inferior yeast (people such as Gleeson, J.Gen.Microbiol. (1986) 132:3459; People such as Roggenkamp, Mol.Gen.Genet. (1986) 202:302); The expression vector of Kluyveromyces fragilis (people such as Das, J.BACTERIOL. (1984) 158:1165); The expression vector of Kluyveromyces lactis (people such as De Louvencourt, J.Bacteriol. (1983) 154:737; People such as Van den Berg, Bio/Technology (1990) 8:135); The expression vector of P.guillerimondii (people such as Kunze, J.BASICMlCROBlOL. (1985) 25:141); The expression vector of pichia pastoris (United States Patent (USP) the 5th, 324, No. 639, the 4th, 929, No. 555 and the 4th, 837, No. 148; With people such as Gregg, Mol.Cell.Biol. (1985) 5:3376); The expression vector of schizosaccharomyces pombe (Schizosaccharomyces pombe) (Beach and Nurse, Nature (1981) 300:706); Conciliate expression vector (people such as Davidow, Curr.GENET. (1985) 10:380 (1985) of fat Ye Shi yeast (Y.lipolytica); People such as Gaillardin, CURR.GENET. (1985) 10:49); The expression vector of aspergillus nidulans (A.nidulans) (people such as Ballance, BIOCHEM.Biophys.Res.Common. (1983) 112:284-89; People such as Tilburn, Gene (1983) 26:205-221; With people such as Yelton, Proc.Natl.Acad.Sci.USA (1984) 81:1470-74); The expression vector of aspergillus niger (A.niger) (Kelly and Hynes, EMBOJ. (1985) 4:475479); The expression vector of trichoderma reesei (T.reesei) (EP 0 244234); And filamentous fungi, the expression vector (WO 91/00357) of Neurospora (Neurospora), Penicillium (Penicillium), the curved mould genus of neck (Tolypocladium) for example, each document is incorporated this paper by reference into.

The control sequence that is used for yeast vector is known by one of ordinary skill in the art, and include, but is not limited to derive from the promoter region of following gene: alcoholdehydrogenase (alcohol dehydrogenase, ADH) (EP 0,284 044), enolase, glucokinase, G-6-P isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase and pyruvate kinases (PyK) (EP 0 329 203) for example.The yeast PHO5 gene of coding acid phosphoric acid esterase also provides promoter sequence people such as (, Proc.Natl.Acad.Sci.USA (1983) 80:1) Myanohara of usefulness.Other the suitable promoter sequence that uses for yeast host can comprise and is used for the kinase whose promoter of 3-phosphoglycerate people such as (, J.BIOL.CHEM. (1980) 255:2073) Hitzeman; With other glycolytic ferment, pyruvic carboxylase, phosphotriose isomerase and phosphogvlucoisomerase (people such as Holland, BIOCHEMISTRY (1978) 17:4900 for example; People such as Hess, J.Adv.Enzyme Reg. (1968) 7:149).Have other the induced Yeast promoter that is subjected to the advantage of transcribing of growth conditions control and can comprise the promoter region of following material: alcoholdehydrogenase 2, different cell pigment C, acid phosphoric acid esterase, metallothionein (metallothionein), glyceraldehyde-3-phosphate dehydrogenase, the digestive enzyme relevant and the enzyme of responsible maltose and galactose utilization with nitrogen metabolism.The carrier and the promoter that are applicable to yeast expression are further described among the EP 0 073 657.

The yeast enhancer also can be used for Yeast promoter.In addition, synthetic promoter also can serve as Yeast promoter.For instance, (upstream activating sequences's upstream activating sequence of Yeast promoter UAS) can link to each other with the transcriptional activation domain of another Yeast promoter, to produce synthetic heterozygote promoter.The example of affiliated heterozygote promoter comprises the ADH regulating and controlling sequence that is connected in the GAP transcriptional activation domain.Referring to, United States Patent (USP) the 4th, 880, No. 734 and the 4th, 876, No. 197, it incorporates this paper by reference into.Other example of heterozygote promoter comprises the promoter of being made up of in conjunction with the glycolytic ferment gene transcription active region of for example GAP or PyK the regulating and controlling sequence of ADH2, GAL4, GAL10 or PHO5 gene.Referring to EP 0 164 556.In addition, Yeast promoter can comprise the natural generation promoter in the non-yeast source with combining yeast RNA polymerase and initial ability of transcribing.

Other control element can comprise and comprise (for example) part (people such as Holland, J.BiOL.Chem. (1981) 256:1385) from the Yeast expression carrier of the terminator of GAPDH or enolase gene.In addition, the origin of replication from 2 μ plasmid starting points is suitable for yeast.Be applicable to that zymic selection gene is the trp1 gene that is present in the plasmid.Referring to people such as Tschemper, GENE (1980) 10:157; People such as Kingsman, GENE (1979) 7:141.Trp1 gene pairs shortage zymic mutant strain of energy for growth in tryptophan provides selected marker.Similarly, the known plasmid with Leu2 gene is contained Leu2 disappearance yeast strain (ATCC 20,622 or 38,626).

The method that is used for foreign DNA is introduced yeast host is known by one of ordinary skill in the art, and generally includes (but being not limited to) spheroplast (spheroplast) or the conversion of the complete yeast host cell handled through alkaline kation.For instance, can be according to people such as Hsiao, people such as Proc.Natl.Acad.Sci.USA (1979) 76:3829 and Van Solingen, the method described in J.Bact. (1977) 130:946 is carried out yeast conversion.Yet, also can be as people such as Sambrook, summarize among the Molecular Cloning:A Lab.Manual (2001) and use other that DNA is introduced method in the cell, for example nuclear is injected (nuclear injection), electroporation or protoplast fusion.Can use the known standard technique culture yeasts host cell of one of ordinary skill in the art subsequently.

Other is used for being known by one of ordinary skill in the art in the method for yeast host cell expression heterologous protein.Usually referring to, No. the 20020055169th, U.S. Patent Publication case; United States Patent (USP) the 6th, 361, No. 969, the 6th, 312, No. 923, the 6th, 183, No. 985, the 6th, 083, No. 723, the 6th, 017, No. 731, the 5th, 674, No. 706, the 5th, 629, No. 203, the 5th, 602, No. 034 and the 5th, 089, No. 398; Not reexamination patent of U.S. RE37, No. 343 and RE35, No. 749; PCT publication application case WO 99/078621, WO 98/37208 and WO 98/26080; European patent application EP 0 946 736, EP 0 732 403, EP 0 480 480, EP 0 460 071, EP 0 340 986, EP 0 329 203, EP 0 324 274 and EP 0 164 556.Also referring to people such as Gellissen, Antonie VanLeeuwenhoek (1992) 62 (1-2): 79-93; People such as Romanos, Yeast (1992) 8 (6): 423-488; Goeddel, METHODS IN ENZYMOLOGY (1990) 185:3-7, these all incorporate this paper into by reference with reference to case.

Can use the known standard fed-batch fermentation of one of ordinary skill in the art method at the amplification stage yeast host bacterial strain of in fermentation tank, growing.Fermentation process is suitable for also explaining that specific yeast host carbon utilizes the difference of approach or expression control model.For instance, the fermentation of saccharomyces cerevisiae yeast host may need single glucose feed supplement, complicated nitrogenous source (for example caseic hydrolysate) and multivitamin fill-in.On the contrary, the methanol yeast pichia pastoris may need glycerol, methanol and trace minerals feed supplement, but only simple ammonium (nitrogen) salt is used for optimum growh and expression.Referring to, for example United States Patent (USP) the 5th, 324, No. 639; People such as Elliott, J.PROTEIN Chem. (1990) 9:95; With people BIOTECH.BlOENG. (1987) 29:1113 such as Fieschko, its mode of quoting is incorporated this paper into.

Yet described fermentation process can have the irrelevant general feature of some and used yeast host strain.For instance, can growth limitation nutrient (being generally carbon) be added in the fermentation tank so that reach at utmost growth in the amplification phase.In addition, fermentation process adopts through design usually to contain the fermentation medium of capacity carbon, nitrogen, basic salt (basal salt), phosphorus and other trace nutrient (vitamin, trace minerals and salt or the like).The case description of fermentation medium that is applicable to Pichia sp. is in United States Patent (USP) the 5th, 324, and No. 639 and the 5th, 231, in No. 178, it incorporates this paper by reference into.

The insect cell term " insect host " that infects through baculovirus or " insect host cell " be meant can as or as the insecticide of the receptor of recombinant vector or other transhipment DNA.Described term comprises the offspring of the initial insect host cell of transfection.Should be appreciated that owing to accidental or sudden change intentionally, so the offspring of single parent's cell is may not must identical on form or genome or total DNA complement with initial parental generation.The offspring of described definition indication comprises the offspring who is the parental cell of correlation properties (nucleotide sequence that for example has coding ABP) with the enough similarities of parental generation.

The one of ordinary skill in the art that are chosen as that are applicable to the insect cell that ABP expresses know.Some castes have fully been described in affiliated field and have been had commercially availablely, and it comprises greedy noctuid (Spodopterafrugiperda) of Aedes aegypti (Aedes aegypti), Bombyxmori Linnaeus (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), meadow and cabbage looper (Trichoplusia ni).At the insect host of selecting to be used for expressing, suitable host can comprise especially have good secretion capacity, the host of the active and overall robustness of low proteolytic.Insecticide can obtain from multiple source usually, includes, but is not limited to Insect Genetic Stock Center, Departmentof Biophysics and Medical Physics, University of California (Berkeley, CA); With American Type Culture Collection (" ATCC ") (Manassas, VA).

Generally comprise the transport vehicle that is generally bacterial plasmid (contain the baculovirus genomic fragment and be used for inserting the convenient restriction site of heterologous gene to be expressed), have and the wild-type baculovirus (this allows heterologous gene homologous recombination to advance in the baculovirus genome) of the homologous sequence of the shaft-like virus-specific fragment of transport vehicle and suitably insect host cell and growth medium through the component of the insect expression system that baculovirus is infected.Be used for carrier construction, transfectional cell, select bacterial plaque, the material at culture medium auxocyte or the like, method and technology be that affiliated field is known, and can obtain to describe the handbook of these technology.

After inserting heterologous gene in the transport vehicle, carrier and the transfection of wild-type virus genome are advanced in the insect host cell carrier and viral genome reorganization therein.Expression is through the packing recombinant virus, and discriminating and purification of Recombinant bacterial plaque.(Carlsbad, kit form CA) is commercially available with (for example) Invitrogen Corp. to be used for the material of baculovirus/insect cell expression system and method.It is known that these technology are generally one of ordinary skill in the art, and the complete Summers and Smith that is described in, and among the Texas Agricultural Experiment Station Bulletin the 1555th phase (1987), it incorporates this paper by reference into.Also referring to Richardson, 39 Methods inMolecular Biology:Baculovirus Expression Protocols (1995); People such as Ausubel, CurrentProtocols in Molecular Biology 16.9-16.11 (1994); King and Possee, The Baculo virusSystem:A Laboratory Guide (1992); And people such as O ' Reilly, Baculovirus Expression Vectors:A Laboratory Manual (1992).

In fact using baculovirus/insect cell expression system to produce various heterologous proteins is that affiliated technical field is known.Referring to, for example United States Patent (USP) the 6th, 368, No. 825, the 6th, 342, No. 216, the 6th, 338, No. 846, the 6th, 261, No. 805, the 6th, 245, No. 528, the 6th, 225, No. 060, the 6th, 183, No. 987, the 6th, 168, No. 932, the 6th, 126, No. 944, the 6th, 096, No. 304, the 6th, 013, No. 433, the 5th, 965, No. 393, the 5th, 939, No. 285, the 5th, 891, No. 676, the 5th, 871, No. 986, the 5th, 861, No. 279, the 5th, 858, No. 368, the 5th, 843, No. 733, the 5th, 762, No. 939, the 5th, 753, No. 220, the 5th, 605, No. 827, the 5th, 583, No. 023, the 5th, 571, No. 709, the 5th, 516, No. 657, the 5th, 290, No. 686, WO 02/06305, WO 01/90390, WO 01/27301, WO 01/05956, WO 00/55345, WO 00/20032, WO 99/51721, WO 99/45130, WO 99/31257, WO99/10515, WO 99/09193, WO 97/26332, WO 96/29400, WO 96/25496, WO 96/06161, WO 95/20672, WO 93/03173, WO 92/16619, WO 92/03628, WO 92/01801, WO90/14428, WO 90/10078, WO 90/02566, WO 90/02186, WO 90/01556, WO 89/01038, WO 89/01037, WO 88/07082, and it incorporates this paper by reference into.

The carrier that can be used for baculovirus/insect cell expression system is that affiliated field is known, and comprise that (for example) derives from baculovirus autographa california nuclear polyhedrosis virus (Autographacalifornica nuclearpolyhedrosis virus, AcNPV) insecticide is expressed and transport vehicle, and it is a kind of virus expression carrier that does not rely on helper virus.The virus expression carrier that derives from this system uses the strong virus polyhedrin gene promoter to drive expression of heterologous genes usually.Usually referring to, people such as Reilly, BACULOVIRUS EXPRESSIONVECTORS:A LABORATORY MANUAL (1992).

Before exogenous gene is inserted shaft-like viral genome, will comprise usually promoter, conductor (if desired), the said components of interested coded sequence and transcription terminator be assembled in the middle of in the dislocation construct (transport vehicle).Middle dislocation construct is maintained in the replicon usually, for example can be in the host of for example antibacterial the extra-chromosomal element (for example plasmid) of stable maintenance.Therefore replicon can have dubbing system, allows it to maintain among the host who is suitable for cloning and increases.More particularly, plasmid can contain (amp) gene and be used for the origin of replication selecting and breed escherichia coli of the poly-adenosine signal of polyhedron albumen people such as (, Ann.Rev.Microbiol. (1988) 42:177) Miller and protokaryon amicillin resistance (ampicillin-resistance).

A transport vehicle that is generally used for exogenous gene is introduced AcNPV is pAc373.Field known many other carriers under also having designed comprise (for example) pVL985, and it changes over the polyhedron start codon ATT and introduce BamHI cloning site 32 base pairs in the ATT downstream from ATG.Referring to Luckow and Summers, 17 VIROLOGY 31 (1989).Other commercially available carrier comprise (for example) PBlueBac4.5/V5-His, pBlueBacHis2, pMelBac, pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).

Insert after the heterologous gene, with transport vehicle and wild-type baculovirus genome cotransfection to the insect cell host.The method that is used for allogeneic dna sequence DNA is introduced the required site of baculovirus is that affiliated technical field is known.Referring to Summers and Smith, Texas Agricultural Experiment Station Bulletin the 1555th phase (1987); People such as Smith, Mol.Cell.Biol. (1983) 3:2156; Luckow and Summers, Virology (1989) 17:31.For instance, can be inserted in the gene of polyhedron gene for example by homology dual crossing reorganization; Also can be inserted into and be operable in the restriction enzyme site of required baculovirus gene through through engineering approaches.Referring to people such as Miller, B1OESSAYS (1989) 4:91.

Can finish transfection by electroporation.Referring to TROTTER and Wood, 39Methods in MolecularBiology (1995); Mann and King, J.Gen.Virol. (1989) 70:3501.Perhaps, can infect insect cell with recombinant expression carrier and baculovirus with liposome.Referring to, people such as Liebman for example, Biotechniques (1999) 26 (1): 36; People such as Graves, Biochemistry (1998) 37:6050; People such as Nomura, J.Biol.Chem. (1998) 273 (22): 13570; People such as Schmidt, Protein Expression andPurification (1998) 12:323; People such as Siffert, Nature Genetics (1998) 18:45; People such as Tilkins, CellBiology:A Laboratory Handbook 145-154 (1998); People such as Cai, Protein Expression andPurification (1997) 10:263; People such as Dolphin, Nature Genetics (1997) 17:491; People such as Kost, Gene (1997) 190:139; People such as Jakobsson, J.Biol.Chem. (1996) 271:22203; People such as Rowles, J.Biol.Chem. (1996) 271 (37): 22376; People such as Reversey, J. Biol.Chem. (1996) 271 (39): 23607-10; People such as Stanley, J.Biol.Chem. (1995) 270:4121; People such as Sisk, J.Virol. (1994) 68 (2): 766; With people such as Peng, BioTechniques (1993) 14.2:274.Commercially available liposome comprise (for example) Cellfectin_ and Lipofectin_ (Invitrogen, Corp., Carlsbad, CA).In addition, can use calcium phosphate transfection.Referring to Trotter and Wood, 39Methods in Molecular Biology (1995); Kitts, NAR (1990) 18 (19): 5667; With Mann and King, J.Gen.Virol. (1989) 70:3501.

Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter is any DNA sequence that can be transcribed into mRNA in conjunction with shaft-like viral rna polymerase and start code sequence (3 ') downstream (for example structural gene).Promoter has the transcription initiation region that is usually located near coded sequence 5 ' end.This transcription initiation region generally includes RNA polymerase binding site and transcriptional start site.Bacilliform virus promoter also can have second territory that is called enhancer, if exist, it is usually away from structural gene so.In addition, expression can be regulated and control or is composing type.

Provide especially available promoter sequence infecting the structural gene that later stage in cycle abundance transcribes.Example comprises the sequence that derives from coding viral polyhedron proteic gene (people such as Friesen, The Regulation of BaculovirusGene Expression in The MOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP0 127 839 and 0 155 476) and the sequence of coding p10 proteic gene people such as (, J.Gen.Virol. (1988) 69:765) Vlak.

Recently the rhabdovirus expression vector of Xing Chenging is packaged in the infectiousness recombinant baculovirus, and the bacterial plaque of growth subsequently can be passed through the known technology purification of one of ordinary skill in the art.Referring to people such as Miller, BiOESSAYS (1989) 4:91; Summers and Smith, Texas Agricultural Experiment Station Bulletin N the 1555th phase (1987).

Having developed recombination rhabdovirus expression vector is used to infect to some insect cells.For instance, wherein develop recombinant baculovirus and be used for Aedes aegypti (ATCC CCL-125 number), Bombyxmori Linnaeus (ATCC CRL-8910 number), Drosophila melanogaster (No. the 1963rd, ATCC), the greedy noctuid in meadow and cabbage looper.Referring to WO 89/046,699; Wright, Nature (1986) 321:718; People such as Carbonell, J.VIROL. (1985) 56:153; People such as Smith, MOL.CELL.Biol. (1983) 3:2156.Usually referring to people such as Fraser, In VitroCell.Dev.Biol. (1989) 25:225.More particularly, the cell line that is used for the rhabdovirus expression vector system generally includes (but being not limited to) Sf9 (noctuid is coveted on the meadow) (ATCC CRL-1711 number), Sf21 (noctuid is coveted on the meadow) (Invitrogen Corp., catalog number (Cat.No.) 11497-013 (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five ^TMBTI-TN-5B1-4 (cabbage looper).

The cell and the culture medium that are used for and amalgamation and expression heterologous polypeptide direct in baculovirus/expression are commercially available, and cell culture technology is substantially known to the one of ordinary skill in the art.

Escherichia coli, pseudomonas kind and other prokaryote bacterial expression technology are that affiliated technical field is known.

Variety carrier can be used in the bacterial host.Carrier can be single copy or low or high multi-copy vector.Carrier can be used for the clone and/or expresses.In view of the handbook that can commercial buy and describe carrier and its restriction map and feature in a large number about the document of carrier, many carriers, this paper need not to remake extensive argumentation.As everyone knows, carrier is usually directed to allow the labelling selected, and described labelling can provide cytotoxic agent resistance, former nutrition or immunity.Usually there is the multiple labelling that different characteristic is provided.

The antibacterial promoter is any DNA sequence that can be transcribed into mRNA in conjunction with bacteria RNA polymerase and start code sequence (3 ') downstream (for example structural gene).Promoter has the transcription initiation region that is usually located near coded sequence 5 ' end.This transcription initiation region generally includes RNA polymerase binding site and transcriptional start site.The antibacterial promoter also can have second territory that is called operator, and it can be overlapping with contiguous RNA polymerase binding site at the synthetic place that begins of RNA.Because gene repression albumen can be in conjunction with operator, and and then suppress transcribing of specific gene, so operator allows negative regulation (can induce) to transcribe.Not existing under the situation of the negative regulatory element of operator for example, constitutive expression may take place.In addition, can just reach, if this sequence exists, then usually near (5 ') RNA polymerase binding sequence by the gene activation protein binding sequence.The proteic example of gene activation is catastate activator protein (catabolite activator protein, CAP), it helps the transcribing of lac operon in the initial escherichia coli (E.coli) people such as [, Annu.Rev.Genet. (1984) 18:173] Raibaud.Therefore, modulated expression can be plus or minus, and then strengthens or reduce and transcribe.

The sequence of encoding metabolic pathway enzyme provides especially available promoter sequence.Example comprises the promoter sequence that derives from the carbohydrate metabolism enzyme, for example galactose, lactose (lac) [people such as Chang, NATURE (1977) 198:1056] and maltose.Other example comprises the promoter sequence that derives from biosynthetic enzyme, for example tryptophan (trp) [people such as Goeddel, Nuc.ACIDS RES. (1980) 8:4057; People such as Yelverton, NUCL.Acids Res. (1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; The open case of European patent No. 036776 and the 121 No. 775, it incorporates this paper by reference into].People such as beta galactosidase (bla) promoter systems [Weissmann (1981) " The cloning ofinterferon and other mistakes. " In Interferon 3 (editor I.Gresser)], bacteriophage λ PL[Shimatake, NATURE (1981) 292:128] and T5[United States Patent (USP) the 4th, 689, No. 406, it incorporates this paper by reference into] promoter systems also provides available promoter sequence.The preferred method of the present invention adopts strong promoter to induce high-level ABP, for example the T7 promoter.The example of described carrier is that affiliated technical field is known, and comprises the pET29 series that derives from Novagen and be described in pPOP carrier among the WO99/05297, and it incorporates this paper by reference into.Described expression system produces high-level ABP and does not influence host cell vigor or growth parameter(s) in the host.PET19 (Novagen) is the known another kind of carrier in affiliated field.

In addition, the synthetic promoter that does not take place in natural also serves as the antibacterial promoter.For instance, the transcriptional activating sequence of a kind of antibacterial or bacteriophage promoter can be connected with the operon sequence of another kind of antibacterial or bacteriophage promoter, to produce synthetic heterozygote promoter [United States Patent (USP) the 4th, 551, No. 433, it incorporates this paper by reference into].For instance, the tac promoter is the heterozygote trp-lac promoter that comprises trp promoter and lac operon sequence, and it is subjected to lac repressor regulation and control [people such as Amann, Gene (1983) 25:167; People such as de Boer, Proc.Natl.Acad.Sci. (1983) 80:21].In addition, the antibacterial promoter can comprise the natural generation promoter that has in conjunction with the non-bacterial origin of bacteria RNA polymerase and initial ability of transcribing.The natural generation promoter of non-bacterial origin also can with compatible RNA polymerase coupling in prokaryote, to produce the high expression level of some genes.Bacteriophage t7 rna polymerase/promoter systems is example [people such as Studier, J.Mol.BlOL. (1986) 189:113 of coupling promoter systems; People such as Tabor, Proc Natl.Acad.Sci. (1985) 82:1074].In addition, the heterozygote promoter also can comprise bacteriophage promoter and escherichia coli operator region (No. the 267 851, the open case of European patent).

Except serving as promoter sequence, effectively ribosome binding site also is used in expression alien gene in the prokaryote.In escherichia coli, ribosome binding site is called Shine-Dalgarno (SD) sequence, and comprise start codon (ATG) and be positioned at the 3-9 length of nucleotides sequence [people such as Shine, NATURE (1975) 254:34] at upstream from start codon 3-11 nucleotide place.Think that the SD sequence promotes mRNA to combine [people " Genetic signals andnucleotide sequences in messenger RNA " such as Steitz with ribosome by the base pairing between SD sequence and 3 of the escherichia coli 16S rRNA ' end, In Biological Regulation and Development:Gene Expression (editor R.F.Goldberger, 1979)].In order to express eukaryotic gene and prokaryotic gene [people " Expression of cloned genes in Escherichia coli " such as Sambrook, Molecular Cloning:A Laboratory Manual, 1989] with weak ribosome binding site.

Term " bacterial host " or " bacterial host cell " are meant the antibacterial of the receptor that can be used as or be used as recombinant vector or other transhipment DNA.Described term comprises the offspring of the initial bacterial host cell of transfection.Should be appreciated that owing to accidental or sudden change intentionally, so the offspring of single parent's cell is may not must identical on form or genome or total DNA complement with initial parental generation.The offspring of described definition indication comprises the offspring who is the parental cell of correlation properties (nucleotide sequence that for example has coding ABP) with the enough similarities of parental generation.

The one of ordinary skill in the art that are chosen as that are applicable to the host bacteria that ABP expresses know.The bacterial host of selecting to be used for expressing, suitable host can comprise especially having the host that good inclusion body forms ability, the active and overall robustness of low proteolytic.Bacterial host can obtain from multiple source usually, include, but is not limited to Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, and University of California (Berkeley, CA); With American Type Culture Collection (" ATCC ") (Manassas, VA).The antibacterial (for example BL21) that derives from the antibacterial (for example W3110) of K bacterial strain or derive from the B bacterial strain is used in the fermentation of industry/medicine usually.Owing to very be familiar with the growth parameter(s) of these bacterial strains and its for stablizing, so these bacterial strains are particularly useful.In addition, these bacterial strain right and wrong are pathogenic, consider that for safety and environment reason it has commercial significance.In an embodiment of the inventive method, escherichia coli host is the BL21 bacterial strain.In another embodiment of the inventive method, escherichia coli host is the negative bacterial strain of protease, includes, but is not limited to OMP-and LON-.Among another embodiment in the methods of the invention, the host cell bacterial strain is the pseudomonas kind, includes, but is not limited to pseudomonas fluorescens, bacillus pyocyaneus and pseudomonas putida.Known pseudomonas fluorescens biotype 1 (specified bacterial strains MB101) can be used for reorganization and produces, and can be used for the human cytokines production process.The example of pseudomonas expression system comprises as the system (Midland, MI www.dow.com can use) of host strain available from Dow Chemical Company.Incorporate the United States Patent (USP) of this paper with way of reference into and describe the purposes that pseudomonad strain is used as the host cell of human growth hormone's generation the 4th, 755, No. 465 and the 4th, 859, No. 600.

In case set up recombinant host cell bacterial strain (promptly expression construct is introduced in the host cell, and separated host cell), promptly cultivate the recombinant host cell bacterial strain being suitable for producing under the condition of ABP with appropriate expression construct.One of ordinary skill in the art should be appreciated that the cultural method of recombinant host cell bacterial strain will depend on the character of used expression construct and the identity of host cell.Usually the known method of technical field is cultivated the recombinant host bacterial strain under using.Usually also contain vitamin, aminoacid, somatomedin according to circumstances and cultivate recombinant host cell in the known fluid medium of technical field under other at the similar source of containing carbon, nitrogen and inorganic salt (assimilatable source) like albumen cultivation fill-in.The fluid medium that is used to cultivate host cell can contain antibiotic and antifungal according to circumstances, and preventing the growth of non-required microorganism and/or chemical compound, it includes, but is not limited to be used to select contain the antibiotic of the host cell of expression vector.

Can be in batches or cultivate recombinant host cell in a continuous manner, wherein with collecting cell (under the situation about in the ABP cell, accumulating) in batches or in a continuous manner or collect culture supernatants.For producing in prokaryotic host cell, batch culture and cell harvesting are first-selected.

Usually purification antigen-binding polypeptides of the present invention behind recombination system invading the exterior soothing the liver.Can be by the known several different methods of affiliated technical field purification ABP from host cell.The common dissolubility of the ABP that in bacterial host cell, is produced relatively poor or insoluble (being the inclusion body form).In one embodiment of the invention, can in antigen-binding polypeptides, carry out aminoacid easily and replace, wherein use method disclosed herein and the known method of affiliated technical field that aminoacid is replaced and select to reach the proteic deliquescent purpose that increase is produced by reorganization.Under the situation of insoluble protein, can from the host cell lysate, collect albumen by centrifugal, and then further make cell homogenization.Under the proteic situation of relatively poor dissolubility, (polyethylene imine, chemical compound PEI) is to induce part soluble protein precipitation can to add (including, but is not limited to) polymine.Can pass through centrifugal collecting precipitation protein expediently subsequently.The known several different methods of technical field is destroyed or the recombinant host cell that homogenizes under can using, so that discharge inclusion body in cell.Can use and know technology and carry out host cell and destroy or homogenize, it includes, but is not limited to enzyme cytoclasis, ultrasonic Treatment, Du Ensi (dounce) homogenizes or high pressure discharges and destroys.In an embodiment of the inventive method, use the high pressure release tech to destroy e. coli host cell to discharge the inclusion body of ABP.When the inclusion body of operation A BP, advantageously reduce to minimum with repeating the time of homogenizing, can not incur loss so that the output of inclusion body is maximum because of factors such as for example dissolving, mechanical shearing or Proteolytic enzyme.

The insoluble or precipitate A BP of any dissolving under can using subsequently in the known many suitable lytic agents of technical field.Preferably with carbamide or guanidine hydrochloride dissolution ABP.The volume of dissolving ABP should be reduced to minimum, so that can produce in enormous quantities with the batch size of being convenient to manage.When growing recombinant host with thousands of batches that rise volume, this factor is extremely important in the large-scale commercial applications device.In addition, when in the large-scale commercial applications device, producing ABP, especially when for human medical usage, if possible then should avoid to damage the harsh chemical product of machine and container or protein product self.Show that in the method for the invention comparatively Wen He denaturant carbamide can be used to dissolve the ABP inclusion body to replace comparatively harsh denaturant guanidine hydrochloride.Use carbamide to reduce the risk of damage used stainless steel equipment in the manufacturing of ABP and purifying process significantly, and effective dissolving ABP inclusion body simultaneously.

Under the situation of solubility ABP, ABP can secrete to periplasmic space (periplasmic space) or secrete to culture medium.In addition, solubility ABP may reside in the Cytoplasm of host cell.May before carrying out purification step, concentrate solubility ABP.The affiliated known standard technique of technical field can be used for concentrating solubility ABP from (for example) cytolysate or culture medium.In addition, the known standard technique of affiliated technical field can be used to destroy host cell, and discharges solubility ABP from Cytoplasm or all marginal plasmas space of host cell.

When producing the ABP of fusion rotein form, preferably remove fusion sequence.Can finish removing of fusion sequence by enzymatic lysis or chemical cracking (being preferably enzymatic lysis).Can remove with the enzyme process that the method that affiliated technical field is known is finished fusion sequence.Can be identified for removing the selection of the enzyme of fusion sequence by the identity of fusant, and reaction condition is specified in the selection of the enzyme that can understand by one of ordinary skill in the art.Preferably by well-known process from through cracked fusion sequence purification through cracked ABP.Understand as one of ordinary skill in the art, can determine described method by identity and the character of fusion sequence and ABP.Purification process can include, but is not limited to the size exclusion chromatography, hydrophobicly do chromatography, ion exchange chromatography or dialysis or its any combination mutually.

Also preferred purification ABP is to remove DNA from protein solution.Can remove DNA by the known any appropriate method of affiliated technical field, for example precipitation or ion exchange chromatography remove but preferably use the nucleic acid precipitant to precipitate, for example (but being not limited to) protamine sulfate.Can use (including, but is not limited to) centrifugal or filtering standard well-known process to separate ABP from deposit D NA.When and of the present invention method human with ABP treatment reduced to pharmaceutically acceptable level with host cell DNA, removing of host's nucleic acid molecules was a key factor in the device.

Small-scale or large scale fermentation method also can be used for protein expression, and it includes, but is not limited to fermentation tank, vibration flask, fluidized bed bioreactor, hollow-fiber bioreactor, roller flask culture system and stirring type bioreactor system.Can add formula (fed-batch) or described each method of continuous mode technology execution with batch-type, stream.

Usually can reclaim people ABP of the present invention with the method standard of affiliated technical field.For instance, can be centrifugal or filter culture medium or cytolysate to remove cell debris.Can or be diluted to volume requiredly with supernatant concentration, perhaps saturating filter be advanced to regulate in the suitable buffer preparation condition and is further purified being used to.Be further purified ABP of the present invention and comprise the ABP variant of isolating demidizate form and clipped form from complete form.

Can adopt any following example procedure purification antigen-binding polypeptides of the present invention: affinity chromatography, anion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE), the silicon dioxide chromatography, the reverse hplc method, gel filtration (use includes, but is not limited to SEPHADEXG-75), the hydrophobic chromatography of doing mutually, the size exclusion chromatography, metal chelate chromatography, ultrafiltration/diafiltration, ethanol precipitation, ammonium sulfate precipitation method, the chromatofocusing method, displacement chromatography, electrophoresis process (including, but is not limited to the isoelectrofocusing of preparation type), difference dissolubility (including, but is not limited to ammonium sulfate precipitation), SDS-PAGE or extraction method.

Can be according to one of ordinary skill in the art the known and standard procedure used with albumen of the present invention (include, but is not limited to comprise alpha-non-natural amino acid albumen, comprise alpha-non-natural amino acid proteic antibody, comprise proteic binding partners of alpha-non-natural amino acid or the like) purification becomes part or homogenizing in fact.Therefore, in the many methods that can know by affiliated field any reclaims and purification polypeptide of the present invention, and it includes, but is not limited to ammonium sulfate or ethanol precipitation, acid or alkali extraction method, column chromatography, affinity column chromatography method, anion or cation-exchange chromatography, phosphocellulose chromatography method, hydrophobic chromatography, hydroxyapatite chromatography, agglutinin chromatography, gel electrophoresis or the like done mutually.In producing correct folding maturation protein, optionally can carry out albumen folding step again.When the needs high-purity, in final purification step, can adopt high performance liquid chromatography (HPLC), affinity chromatography or other appropriate method.In one embodiment, the antibody of the prepared anti-alpha-non-natural amino acid albumen of alpha-non-natural amino acid (or comprise) can be used as purified reagent, includes, but is not limited to be used to comprise the proteic purification based on affinity of one or more alpha-non-natural amino acids.Dawn partial purification or be purified to (if desired) after the homogenizing promptly is used for polypeptide multiple function according to circumstances, includes, but is not limited to as test component, treatment reagent, prevention reagent, diagnostic reagent, research reagent and/or the immunogen that produces as antibody.

Except other mentioned list of references of this paper, multiple purification/protein folding method is known by affiliated technical field, includes, but is not limited to described in the following document: R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol.182:Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins, Academic Press, Inc.; People such as Bollag (1996) Protein Methods. the 2nd edition Wiley-Liss, NY; Walker, (1996) The Protein Protocols HandbookHumana Press, NJ; Harris and Angal, (1990) Protein Purification Applications:A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods:A Practical ApproachIRL Press at Oxford, Oxford, England; Scopes, (1993) Protein Purification:Principles And Practice the 3rd editionSpringer Verlag, NY; Janson and Ryden, (1998) Protein Purification: Principles, High Resolution Methods and Applications,The 2nd edition Wiley-VCH, NY; And Walker (1998), Protein Protocols on CD-ROMHumana Press, NJ; The list of references of being quoted wherein.

An advantage that produces interesting albumen with alpha-non-natural amino acid or polypeptide in eukaryotic host cell or non-eukaryotic host cell is that common albumen or polypeptide fold with its native conformation.Yet in certain embodiments of the present invention, one of ordinary skill in the art it should be understood that albumen can have the conformation that is different from the required conformation of related polypeptide after synthetic, expression and/or purification.In one aspect of the invention, through expressed proteins degeneration and renaturation subsequently according to circumstances.The known method of technical field was reached under this can adopt, and included, but is not limited to by add chaperonin (chaperonin) in institute's protein of interest or polypeptide; By soluble protein in chaotropic agent (for example guanidine hydrochloride); Adopt protein disulfide isomerase or the like.

In general, need degeneration and reduction through polypeptide expressed once in a while, and cause that subsequently polypeptide is folded into preferred conformation again.For instance, guanidine, carbamide, DTT, DTE and/or chaperonin can make an addition in the interested translation product of institute.The method of reduction, degeneration and recombinant protein is known (referring to, people (1993) such as above-mentioned list of references and Debinski by one of ordinary skill in the art J.Biol.Chem..268:14065-14070; Kreitman and Pastan (1993) Bioconiug.Chem., 4:581-585; With people such as Buchner, (1992) Anal.Biochem..205:263-270).For instance, people such as Debinski describes the degeneration and the reduction of inclusion body protein among guanidine-DTE.Albumen can be folding again in containing the arginic potential buffer solution of (including, but is not limited to) oxidized glutathione and L-.Again folding reagent can flow or move to one or more polypeptide or other expression product and contact, and perhaps vice versa.

Produce at protokaryon under the situation of ABP, the ABP that is produced thus may misfolding and thereby is lacked or have a low biological activity.Can be by the biological activity of " folding again " recoverin.In general, by use (for example) one or more chaotropic agents (for example carbamide and/or guanidine) and can reduce disulfide bond Reducing agent (for example dithiothreitol, DTT DTT or 2 mercapto ethanol 2-ME) dissolving (wherein ABP is also for insoluble), open with the reducing polypeptide chain and come to fold again misfolding ABP.Under the debita spissitudo chaotropic agent, add oxidant (for example oxygen, cystine or cystamine) subsequently, to allow to form again disulfide bond.The folding again ABP of the known standard method in field for example is described in United States Patent (USP) the 4th, 511 under can using, and No. 502, the 4th, 511, No. 503 and the 4th, 512, in No. 922, it incorporates this paper by reference into.Also ABP and other albumen can be folded to form heterodimer or heteromultimers altogether.Again after folding or folding altogether, preferably be further purified ABP.

General purification process can carry out any in the multiple separating step on the cytolysate that comprises ABP or any ABP mixture that any separating step produced, it includes, but is not limited to affinity chromatography, ion exchange chromatography, hydrophobicly does chromatography, gel filtration chromatography, high performance liquid chromatography (" HPLC "), reverse hplc method (" RP-HPLC "), expanded bed adsorption method or any combination and/or its repetition mutually and with any suitable order.

Equipment and other essential material of being used to carry out technology described herein are commercially available.Pump, fraction collector, watch-dog, monitor and whole system can be available from (for example) Applied Biosystems (Foster City, CA), Bio-RadLaboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway, NJ).Including, but is not limited to exchange base material, medium and buffer also can be available from these company at interior chromatographic material.

Can reach balance in the column chromatography process described herein and other step more apace with the Special Equipment of for example pump, for example washing and eluting.Commercially available pump include, but is not limited to HILOAD_Pump P-50, PeristalticPump P-1, Pump P-901 and Pump P-903 (Amersham Biosciences, Piscataway, NJ).

The example of fraction collector comprise RediFrac Fraction Collector, FRAC-100 and FRAC-200Fraction Collectors and SUPERFRAC_Fraction Collector (Amersham Biosciences, Piscataway, NJ).Also can use blender to form pH value and linear concentration gradient.Commercially available blender comprise Gradient Mixer GM-1 and In-line Mixer (Amersham Biosciences, Piscataway, NJ).

Can monitor chromatographic process with any commercially available watch-dog.Described watch-dog can be used to collect for example information such as UV value, pH value and conductivity.The example of detector comprises Monitor UV-1, UVICORD_S II, MonitorUV-M II, Monitor UV-900, Monitor UPC-900, Monitor pH/C-900 and ConductivityMonitor (Amersham Biosciences, Piscataway, NJ).In fact whole system is commercially available, comprises various available from Amersham Biosciences (Piscataway, AKTA_ system NJ).

For instance, in one embodiment of the invention, can reduce ABP and by in carbamide first degeneration gained purification ABP come degeneration, then under suitable pH value, it is diluted in the TRIS buffer that contains Reducing agent (for example DTT).In another embodiment, about 2M to the concentration range of about 9M in carbamide degeneration ABP, then in about 5.0 to about 8.0 pH value scope, be diluted in the TRIS buffer.Folding again mixture that subsequently can the incubation present embodiment.In one embodiment, at room temperature incubation folds mixture 4 to 24 hours again.Subsequently further isolated or purified through the reduction and degeneration ABP mixture.

As described herein, can before carrying out any separating step subsequently, regulate the pH value of an ABP mixture.In addition, can concentrate an ABP mixture or its any subsequent mixtures with affiliated technical field known technology.In addition, the elution buffer that can use the known technology of one of ordinary skill in the art will comprise an ABP mixture or its any subsequent mixtures replaces with the buffer that is applicable to separating step then.

Ion exchange chromatography can carry out ion exchange chromatography as optional additional step to an ABP mixture in one embodiment.Usually referring to ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion exchange column comprises HITRAP ^_, HIPREP ^_And HILOAD ^_Columns (AmershamBiosciences, Piscataway, NJ).Described post adopts strong anion exchanger, for example Q SEPHAROSE ^_Fast Flow, Q SEPHAROSE ^_High Performance and Q SEPHAROSE ^_XL; Strong cation exchanger, for example SP SEPHAROSE ^_High Performance, SP SEPHAROSE ^_Fast Flow and SPSEPHAROSE ^_XL; Weak anion exchanger, for example DEAE SEPHAROSE ^_Fast Flow; And weak cation exchanger, for example CM SEPHAROSE ^_Fast Flow (Amersham Biosciences, Piscataway, NJ).Can carry out anion or cation exchange column chromatography chromatography to separate purified in fact ABP to ABP in any stage of purge process.Can use any suitable cation exchange substrate to carry out the cation-exchange chromatography step.That available cation exchange substrate includes, but is not limited to is fibrous, porous, atresia, microgranular, pearl or cross-linked cationic exchange base material.Described cation exchange substrate includes, but is not limited to the complex of cellulose, agarose, glucosan, polyacrylate, polyethylene, polystyrene, silicon dioxide, polyethers or any aforementioned substances.

Cation exchange substrate can be any suitable cation exchanger that comprises strong and weak cation exchanger.Strong cation exchanger can keep ionizing in wide pH value scope, and therefore can be in conjunction with ABP in wide pH value scope.Yet weak cation exchanger can be used as pH function forfeiture ionizing.For instance, when being reduced to about pH 4 or pH 5 under the pH value, weak cation exchanger may lose electric charge.Suitable strong cation exchanger includes, but is not limited to charged functional groups, for example sulfopropyl (sulfopropyl, SP), methylmesylate (methylsulfonate, S) or sulfoethyl (sulfoethyl, SE).Cation exchange substrate can be preferably to have about 2.5 to about 6.0 the ABP strong cation exchanger in conjunction with the pH value scope.Perhaps, strong cation exchanger can have about 2.5 to about 5.5 ABP in conjunction with the pH value scope.Cation exchange substrate can be to have about 3.0 the ABP strong cation exchanger in conjunction with pH value.Perhaps, cation exchange substrate can be preferably to have about 6.0 to about 8.0 the ABP strong cation exchanger in conjunction with the pH value scope.Cation exchange substrate can be preferably to have about 8.0 to about 12.5 the ABP strong cation exchanger in conjunction with the pH value scope.Perhaps, strong cation exchanger can have about 8.0 to about 12.0 ABP in conjunction with the pH value scope.

Before load ABP, for example can use rare weak acid (for example the 20mM acetic acid of four column volumes, pH 3) of some column volumes to come balance cation exchange substrate.After the balance, before the purified in fact ABP of eluting, can add ABP, and (weak acetic acid or the phosphoric acid solution) column scrubber that also can use weak acid solution is once to for several times.For instance, can use the 20mM acetic acid (pH 3) of about 2-4 column volume to come column scrubber.Also can use the 0.05M sodium acetate (pH 5.5) of (for example) 2-4 column volume or the mixture (pH 5.5) of 0.05M sodium acetate and 0.1M sodium chloride to add washing.Perhaps, under using, during the known method of technical field, also can use rare weak base balance cation exchange substrate of some column volumes.

Perhaps, can contact the purified in fact ABP of eluting with cationite substrate with buffer, thereby ABP is cemented out from substrate with enough low pH value or ion concentration.The pH value of elution buffer can be at about pH 2.5 to about pH 6.0 scopes.More particularly, the pH value of elution buffer can be at about pH2.5 to about pH 5.5 scopes, and about pH 2.5 is to about pH 5.0 scopes.Elution buffer can have about 3.0 pH value.In addition, the amount of elution buffer can extensively change, and usually about 2 to about 10 column volume scopes.In addition, this paper can use the known suitable buffer of one of ordinary skill in the art, includes, but is not limited at least about 5mM extremely at least about the citrate in the 100mM concentration range, phosphate, formates, HEPES and MES buffer.

After the ABP polypeptide is adsorbed in cationite substrate, can contact the purified in fact ABP polypeptide of eluting with buffer with enough high pH value or ion concentration and substrate, from substrate so that ABP is cemented out.The suitable buffer that is used for the purified in fact ABP of high pH value eluting can include, but is not limited at least about 5mM extremely at least about the citrate in the 100mM concentration range, phosphate, formates, acetate, HEPES and MES buffer.

Reverse-phase chromatography can be carried out RP-HPLC according to the known suitable experimental program of one of ordinary skill in the art and come purifying protein.Referring to, people such as Pearson for example, Anal Biochem. (1982) 124:217-230 (1982); People such as Rivier, J.Chrom. (1983) 268:112-119; People such as Kunitani, J.Chrom. (1986) 359:391-402.Can carry out RP-HPLC to separate purified in fact ABP to ABP.Thus, can use to have for multiple length and (include, but is not limited at least about C ₃Extremely at least about C ₃₀, at least about C ₃Extremely at least about C ₂₀Or at least about C ₃Extremely at least about C ₁₈) the silicon dioxide resins derived therefrom of alkyl functional group.Perhaps can use polymer resin.For instance, can use TosoHaas Amberchrome CG1000sd resin, it is a kind of styrenic polymer resins.Also can use cyano group or polymer resin with multiple alkyl chain length.In addition, can use for example alcoholic acid solvent wash RP-HPLC post.Source RP post is another example of RP-HPLC post.

Can use the suitable elution buffer eluting ABP from the RP-HPLC post that contains ion-pairing agent and organic modifier, described organic modifier is methanol, isopropyl alcohol, oxolane, propionitrile or ethanol for example.The most frequently used ion-pairing agent includes, but is not limited to acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, acetic acid tetramethyl-ammonium, tetrabutylphosphoniuacetate acetate ammonium and acetic acid triethyl ammonium.Can use one or more gradients or etc. degree (isocratic) condition carry out eluting, wherein the preferred gradient condition reduces disengaging time and reduces peak width.Other method relates to uses the gradient with different solvents concentration range.The example that is used for the suitable elution buffer of this paper includes, but is not limited to ammonium acetate and acetonitrile solution.

The hydrophobic chromatography purification technique of doing mutually can be carried out the hydrophobic chromatography (HIC) of doing mutually to ABP.Usually referring to HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND Methods (catalog number (Cat.No.) 18-1020-90, Amersham Biosciences (Piscataway, NJ)), it incorporates this paper by reference into.Appropriate H IC substrate can include, but is not limited to alkyl or aryl and replace substrate, for example butyl, hexyl, octyl group or phenyl substituent matter, comprise agarose, Sepharose, agarose gel, cellulose, silicon dioxide, glucosan, polystyrene, poly-(methacrylate) substrate and mixed model resin, include, but is not limited to polyethyene diamine resin or butyl or phenyl and replace poly-(methacrylate) substrate.Be used for the hydrophobic commercially available source of making column chromatography mutually and include, but is not limited to HITRAP ^_, HIPREP ^_And HILOAD ^_Post (Amersham Biosciences, Piscataway, NJ).

Briefly, before loading, can use the known standard buffer solution balance HIC post of one of ordinary skill in the art, described standard buffer solution is acetic acid/sodium chloride solution or contain the HEPES of ammonium sulfate for example.Can be with ammonium sulfate as the buffer that loads the HIC post.Load after the ABP, then can be with standard buffer solution and standard conditions column scrubber removing non-required material, but on the HIC post, stay ABP.Can wherein for example contain EDTA and the HEPES that is lower than the ammonium sulfate concentrations of level pad with about 3 standard buffer solution eluting ABP to about 10 column volumes, or acetic acid/sodium chloride buffer.Also the linearity of using (for example) potassium phosphate gradient can be reduced salt gradient and be used for eluting ABP molecule.For instance, can come concentrate eluant by filtering (filter or ultrafiltration thoroughly) subsequently.Can adopt filter to remove the salt that is used for eluting ABP.

Other purification technique for instance, can use gel filtration (Gel Filtration:Principles and Methods (catalog number (Cat.No.) 18-1022-18 to an ABP mixture or its any subsequent mixtures, AmershamBiosciences, Piscataway, NJ), it incorporates this paper by reference into), (suitable matrix includes, but is not limited to HA-Ultrogel to hydroxyapatite chromatography, High Resolution (Calbiochem), CHT CeramicHydroxyapatite (BioRad), Bio-Gel HTP Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, saturating filter, another separating step of lyophilizing or the like, with remove any excess salt and be applicable to separating step subsequently or even the buffer that forms of final pharmaceutical product replace described buffer.

The non-naturally encoded amino acids that is present among the ABP also can be used to provide other cell protein that never contains non-naturally encoded amino acids to separate.Because non-naturally encoded amino acids can comprise unique chemical functional group, therefore the coupling of unique functional group and another molecule can provide substantive purification step.For instance, non-naturally encoded amino acids can be coupled to another and helps molecule from other Protein Separation.The described molecule that is used for the coupling alpha-non-natural amino acid includes, but is not limited to PEG, other polymer, globule and other solid matter.

Can use the known technology of one of ordinary skill in the art to comprise the ABP productive rate of purified in fact ABP in each step place monitoring described herein.Described technology can be used to assess the last separating step productive rate of purified in fact ABP afterwards.For instance, can use any some anti-phase high pressure liquid chromatography post with multiple alkyl length (for example cyano group RP-HPLC, C ₁₈RP-HPLC and cation exchange HPLC and gel filtration HPLC) monitoring ABP productive rate.

In specific embodiments of the invention, the ABP productive rate after each purification step can for ABP in each purification step initial substance at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99%.

Can use for example standard technique of SDS-PAGE, perhaps by using Western trace and ELISA test determination ABP to determine purity.For instance, can create antagonism from negative control culture propagation and the isolated proteic polyclonal antibody of cation exchange recovery.Described antibody also can be used to survey the existence of polluting host cell proteins.

RP-HPLC material Vydac C ₄(Vydac) be made up of silica gel particle, its surface has C ₄Alkyl chain.Separate ABP from quasiprotein impurity and be based on the hydrophobic difference of making intensity mutually.The acetonitrile gradient that is used in rare trifluoroacetic acid carries out eluting.Use stainless steel column (being full of 2.8 to 3.2 liters Vydac C4 silica gel) to carry out preparation HPLC.By interpolation trifluoroacetic acid acidify Hydroxyapatite Ultroge eluent, and be loaded on the Vydac C4 post.For washing and eluting, use the acetonitrile gradient in rare trifluoroacetic acid.Collect elution fraction and neutralize with phosphate buffer immediately.Compile IPC and limit interior ABP elution fraction.

DEAE Sepharose (Pharmacia) material is made up of diethylamino ethyl (DEAE) group that is covalently bonded in the little bead surface of Sepharose.Mediate combining of ABP and DEAE group mutually by ion.Acetonitrile and trifluoroacetic acid are not kept here by post.After these materials of flush away, by under low pH value, removing trace impurity with the acetate buffer column scrubber.Use neutral phosphor phthalate buffer column scrubber subsequently, and with having the buffer solution elution ABP that increases ion concentration.Chromatographic column is full of DEAE Sepharose high velocity stream.The adjustable column volume is to guarantee that the ABP useful load is in 3-10mg ABP polypeptide/milliliter gel range.Water and level pad (sodium phosphate/potassium phosphate) column scrubber.Load the HPLC eluent through compiling elution fraction, and use the level pad column scrubber.Use lavation buffer solution (sodium acetate buffer) column scrubber subsequently, then wash with level pad.Subsequently, use elution buffer (sodium chloride, sodium phosphate/potassium phosphate) eluting ABP from post, and be collected as single elution fraction according to main elution curve.The eluent of DEAE Sepharose post is adjusted to specified conductivity.The aseptic filtration of gained drug substance to teflon (Teflon) bottle, and is stored under-70 ℃.

Adoptable other method includes, but is not limited to remove the step of endotoxin (endotoxin).Endotoxin be loaded into lipid polysaccharide on negative host cell (for example escherichia coli) adventitia of Glan (lipopoly-saccharide, LPS).The method that is used to reduce endotoxin content is known for one of ordinary skill in the art, and includes, but is not limited to use the purification technique of silicon dioxide supporter, glass dust or hydroxyapatite; Reverse-phase chromatography, affinity chromatography, size exclusion chromatography, anion-exchange chromatography, the hydrophobic chromatography of doing mutually; Combination of these methods or the like.Can require improvement or addition method to come to remove polluter, for example move albumen altogether from interested polypeptide.

Several different methods and process can be used to assess proteic productive rate of the ABP that contains one or more non-naturally encoded amino acids and purity, include, but is not limited to the method that Bradford algoscopy, SDS-PAGE, silver are dyed SDS-PAGE, coomassie (coomassie) dyeing SDS-PAGE, mass spectrography (including, but is not limited to MALDI-TOF) and the known profiling protein of other one of ordinary skill in the art.

VIII. the expression in the alternate system

Some strategies be used for alpha-non-natural amino acid introduce the non-recombinant hosts cell, in the albumen of sudden change host cell or cell free system.These systems also are applicable to and make antigen-binding polypeptides of the present invention.(for example Lys, Cys and Tyr) makes amino acid derivedization cause that lysine changes into N with reactive side chain ²-acetyl group-lysine.Chemosynthesis also provides the straightforward procedure of incorporating alpha-non-natural amino acid into.Use the enzyme connection of the fragments of peptides of developing recently to be connected, may make than large protein with native chemical.Referring to, for example P.E.Dawson and S.B.H.Kent, Annu. Rev.Biochem, 69:923 (2000).A kind of common in vitro biological synthesis method has been used for incorporating the albumen of multiple any in fact size into surpassing 100 alpha-non-natural amino acid locus specificities, will make an addition to the suppressor gene tRNA of required alpha-non-natural amino acid chemical acylation in the described method and can support in the synthetic in vitro extract of protein biology.Referring to, V.W.Cornish for example, D.Mendel and P. G. Schultz, Angew.Chem.Int.Ed. EngL1995,34:621 (1995); CJ.Noren, S J.Anthony-Cahill, M.C.Griffith, P.G. Schultz, Ageneral method for site-specific incorporation of unnatural amino acids into proteins, Science244:182-188 (1989); And J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specific incorporation of a non-natural amino acid into apolypeptide, J.Am.Chem.Soc.111:8013-8014 (1989).The functional group of broad range has introduced and has been used to study protein stability, protein folding, enzyme mechanism and signal transduction in the albumen.

Develop and be called selection pressure and incorporate mix (promiscuity) that the in vivo method of (selective pressure incorporation) is developed the wild type synzyme into.Referring to, N.Budisa for example, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber, FASEB J., 13:41 (1999).Growing nutrient deficient strain in containing the amino acid whose minimal medium of limited concentration natural, and suppress transcribing of target gene simultaneously closes wherein that pair cell provides specific natural amino acid whose associated metabolic approach in the described auxotrophic strain.In the fixed growth phase, the disappearance natural amino acid is also alternative with the alpha-non-natural amino acid analog.Induce Recombinant Protein Expression to cause to contain the proteic accumulation of non-natural analog.For instance, use this strategy, incorporate in the albumen with adjacent fluorophenylalanine, a fluorophenylalanine with to phenylalanine, and in UV spectrum, show two the characteristic shoulders (shoulder) that to be differentiated easily, referring to, C.Minks for example, R.Huber, L.Moroder and N.Budisa Anal.Biochem., 284:29 (2000); TFM S-Trifluoromethyl-L-homocysteine has been used for replacing the methionine of bacteriophage T4 lysozyme to pass through ¹⁹The mutual work of F NMR research and oligochitosan (chitooligosaccharide), referring to, H.Duewel for example, E.Daub, V.Robinson and J.F.Honek, Biochemistry, 36:3404 (1997); And the trifluoro leucine has been incorporated into to replace leucine, produces leucine heat stability and the chemical stability that (leucine-zipper) albumen increases of marching, referring to, Y.Tang for example, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew. Chem.Int.Ed.EngL, 40:1494 (2001).In addition, Selenomethionine and tellurium methionine have been incorporated in the various recombiant proteins to promote the phased soln in the x light crystallography.Referring to, W.A.Hendrickson for example, J.R.Horton and D.M.Lemaster, EMBO J., 9:1665 (1990); J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and M.Hatada, Nat.Struct.Biol., 1:283 (1994); N.Budisa, B.Steipe, P.Demange, C.Eckerskorn, J.Kellermann and R.Huber, Eur.J.Biochem..230:788 (1995); And N.Budisa, W.Karnbrock, S.Steinbacher, A.Hum, L.Prade, T.Neuefeind, L.Moroder and R.Huber, J.Mol.Biol..270:616 (1997).Effectively incorporated methionine analog into, made to allow by the other modified protein of chemical means with alkene or alkynes functional group.Referring to, for example J.C.M.vanHest and D.A.Tirrell, FEBS Lett., 428:68 (1998); J.C.M.van Hest, K.L.Kiick and D.A.Tirrell, J.Am.Chem.Soc, 122:1282 (2000); With K.L.Kiick and D.A.Tirrell, Tetrahedron, 56:9487 (2000); United States Patent (USP) the 6th, 586, No. 207; U.S. Patent Publication case 2002/0042097 is describedly incorporated this paper into by reference with reference to case.

The identification of aminoacyl-tRNA synthetase to the alpha-non-natural amino acid analog is depended in the success of the method, and this needs high selectivity to guarantee the fidelity of protein translation usually.A kind of approach that enlarges the method scope is to loosen the substrate specificity of aminoacyl-tRNA synthetase, and this reaches in limited kind of situation.For instance, replace Ala by escherichia coli phenylalanyl-tRNA synzyme (PheRS) with Gly ²⁹⁴Can increase the size of substrate binding pocket, and cause by p-Cl-phenylalanine (p-Cl-Phe) acidylate tRNAPhe.Referring to M.Ibba, P.Kast and H.Hennecke, Biochemistry, 33:7107 (1994).Coli strain with this mutant PheRS allows to incorporate into p-Cl-phenylalanine or p-Br-phenylalanine to replace phenylalanine.Referring to, for example M.Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); And N.Sharma, R.Furter, P.Kast and D.A.Tirrell, FEBS Lett., 467:37 (2000).Similarly, the point mutation Phe130Ser demonstration of the aminoacid binding site of close escherichia coli tyrosyl--tRNA synzyme allows azepine tyrosine to incorporate into more effectively than tyrosine.Referring to, F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura, J.Biol.Chem.275:40324 (2000).

Be used in vivo incorporating alpha-non-natural amino acid into proteic another strategy and be to modify synzyme with check and correction mechanism.These synzyme can not distinguish and therefore can not activation structure on the aminoacid similar to the homology natural amino acid.In independent this mistake of site correction, by the wrong electric charge aminoacid (mischarged aminoacid) of tRNA deacylation, to keep the fidelity of protein translation.If the check and correction activity of synzyme can not act on, can escape editting function and be merged in through the activated analog of mistake so.Recently proved the method with valyl-tRNA synzyme (ValRS).Referring to V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere, Science, 292:501 (2001).ValRS can use Cys, Thr or the wrong aminoacylation tRNAVal of aminobutyric acid ester (Abu); Subsequently by the non-homogeneous aminoacid of edit field hydrolysis.After the random mutation escherichia coli chromosome, be chosen in ValRS and edit the mutant Escherichia coli bacterial strain that has sudden change in the site.Editor's deficiency ValRS makes tRNAVal charged with Cys mistakenly.Because Abu space assembling Cys (among the Abu Cys-the SH group is by-CH3 displacement), therefore when this mutant Escherichia coli bacterial strain was grown in the presence of Abu, mutant ValRS also incorporated Abu in the albumen into.Mass spectral analysis shows at about 24% valine in each valine position of native protein is replaced by Abu.

Solid phase synthesis and semisynthesis also allow synthetic many albumen that contain new amino acid.For instance, referring to following discloses case and the list of references wherein quoted: Crick, FJ.C, Barrett, L.Brenner, S.Watts-Tobin, R.General nature of the genetic code for proteins. Nature, 192:1227-1232 (1961); Hofmann, K., Bonn, H.Studies on polypeptides.XXXVI.The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptidefragment, J.Am Chem.88 (24): 5914-5919 (1966); Kaiser, E.T.Synthetic approaches tobiologically active peptides and proteins including enyzmes, Ace Chem Res, 47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T.Peptide segment coupling catatyzed by thesemisynthetic enzyme thiosubtilisin, J Am Chem Soc, 3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256 (5054): 221-225 (1992); Chaiken, I.M.Semisynthetic peptides and proteins, CRC Grit Rev Biochem.11 (3): 255-301 (1981); Ofrord, R.E.Protein engineering by chemical means? Protein Eng., 1 (3): 151-157 (1987); And, Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A DesignedPeptide Ligasefor Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science, 266 (5183): 243 (1994).

Chemical modification has been used for multiple non-natural side chain is in vitro introduced albumen, and described non-natural side chain comprises cofactor, spin labeling and oligonucleotide.Referring to, Corey for example, D.R., Schultz, P.G.Generation of ahybrid sequence-specific single-stranded deoxyribonuclease, Science, 238 (4832): 1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemicalmodification of enzymatic specificity, Annu Rev Biochem, 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S.Chemical mutation ofenyzme active sites, Science, 226 (4674): 505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin, J Biol.Chem, 243 (24): 6392-6401 (1968); Polgar, L.B., M.L.Anew enzymecontaining a synthetically formed active site.Thiol-subtilisin. J.Am Chem Soc, 3153-3154 (1966); And Pollack, S.J., Nakayama, G. Schultz, P.G. Introduction ofnucleophiles and spectroscopicprobes into antibody combining sites, Science, 242 (4881): 1038-1040 (1988).

Perhaps, adopt the biological synthesis method of chemical modification aminoacyl-tRNA to be used for incorporating some biophysics probes in vitro synthetic albumen.Referring to following discloses case and the list of references wherein quoted: Brunner, J.New Photolabeling and crosslinking methods, Annu.Rev Biochem, 62:483-514 (1993); And Krieg, U.C., Walter, P., Hohnson, A.E.Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle Proc.Natl.Acad.Set83 (22): 8604-8608 (1986).

Before showed, can be by adding in the protein synthesis reaction that carry out and alpha-non-natural amino acid in vitro incorporated in the albumen on locus specificity ground through chemical aminoacylation suppressor gene tRNA with the gene that contains required succinum non-sense mutation.By using these methods, can use for the auxotrophic strain of specific amino acids and replace many 20 common seed amino acids with the close congener of structure, for example replace phenylalanine with fluorophenylalanine.Referring to, Noren for example, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G.A general method forsite-specific incorporation of unnatural amino acids into proteins, Science, 244:182-188 (1989); People such as M.W.Nowak, Science268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specific Incorporation of anon-natural amino acid into a polypeptide, J.Am Chem Soc, 111:8013-8014 (1989); People such as N.Budisa, FASEB J.13:41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins. Methods in Enz., 301-336 (1992); And Mendel, D., Cornish, V.W. ﹠amp; Schultz, P.G.Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys.Biomol Struct.24,435-62 (1995).

For instance, the suppressor gene tRNA of preparation identification termination codon UAG, and with the chemical aminoacylation of alpha-non-natural amino acid.Conventional rite-directed mutagenesis is used for introducing termination codon TAG in the interesting site of protein gene.Referring to, Sayers for example, J.R., Schmidt, W.Eckstein, F.5 ', 3 ' Exonuclease inphosphorothioate-based olignoucleotide-directed mutagensis, Nucleic Acids Res, 16 (3): 791-802 (1988).When in vitro transcribe/translation system in when combination acidylate suppressor gene tRNA and mutant gene, incorporate alpha-non-natural amino acid in response to the UAG codon, described UAG codon is created in specified location and contains described amino acid whose albumen.Use [ ³H]-experiment of Phe and use that experimental results show that of alpha-hydroxy acid is only amino acid needed incorporates in the specified position of UAG codon, and this aminoacid is not incorporated in proteic any other site.Referring to, people such as Noren for example, supra; People such as Kobayashi, (2003) Nature StructuralBiology 10 (6): 425-432; And Ellman, J.A., Mendel, D., Schultz, P.G.Site-specificincorporation of novel backbone structures into proteins, Science, 255 (5041): 197-200 (1992).

The microinjection technology also has been used for incorporating alpha-non-natural amino acid into albumen.Referring to, M.W.Nowak for example, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester, Science, 268:439 (1995); And D.A.Dougherty, Curr.Opin.Chem.Biol., 4:645 (2000).Two RNA species of in vitro preparation are injected in the xenopus leavis oocytes (Xenopus oocyte) altogether: interested amino acid position place have UAG termination codon the coding target protein mRNA and with the amber suppressor tRNA of required alpha-non-natural amino acid aminoacylation.The machine translator of oocyte inserts alpha-non-natural amino acid in the specified position of UAG subsequently.The method allows the research of the proteic in vivo structure function of integral membrane, and this is common and disobey in vitro expression system.Example comprises incorporates in tachykinin neurokinin-2 receptor fluorescence aminoacid with by the FRET (fluorescence resonance energy transfer) measuring distance into, referring to, G.Turcatti for example, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet J.Biol.Chem., 271:19991 (1996); Incorporate biotin labeling aminoacid into differentiating surperficial exposed residue in the ion channel, referring to, J.P. Gallivan for example, H.A.Lester and D.A.Dougherty, Chem.Biol., 4:739 (1997); Use is caught the tyrosine analog with the conformational change in the real-time monitoring ion passage, referring to, J.C.Miller for example, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619 (1998); With use the α hydroxy-amino-acid to change the ion channel skeleton being used to survey its door control mechanism, referring to, P.M.England for example, Y.Zhang, D.A.Dougherty and H.A.Lester, Cell, 96:89 (1999); And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang, Nat.Neurosci., 4:239 (2001).

Directly in vivo incorporate alpha-non-natural amino acid into proteic ability following advantage is provided: in high yield mutant protein, technology facility, the research cell or the probability of the mutant protein in may live organism and in therapeutic treatment, use these mutant proteins.The alpha-non-natural amino acid that will have all size, acidity, nucleophilicity, hydrophobicity and other characteristic comprises that into proteic ability can greatly enlarge the ability of rationally and systematically handling protein structure, to survey new albumen or the organism that protein function and generation have novel characteristics.Yet, because requirement reaches the complicated character that the tRNA synzyme of the high fidelity (Hi-Fi) degree of protein translation is done mutually, so this process is difficult.

Incorporate in the trial of para-F-Phe at locus specificity, phenylalanyl-the tRNA synzyme is right to use yeast amber suppressor tRNAPheCUA/ in para-F-Phe resistance, Phe auxotroph coli strain.Referring to, R.Furter for example, Protein Sci,7:419 (1998).

Use acellular (in vitro) translation system also may obtain the expression of ABP polynucleotide of the present invention.In these comprise that mRNA is as template (in vitro translation) or the system of DNA as template (combination is in vitro transcribed and translated), instruct in vitro synthetic by ribosome.Carry out extensive work and developed the acellular albumen expression system.Referring to, for example, Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering, 74:309-316 (2001); Kim, D.-M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology Progress, 16,385-390, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering, 66,180-188, (1999); And Patnaik, R. and J.R.Swartz, Biotechniques 24,862-868, (1998); United States Patent (USP) the 6th, 337, No. 191; No. the 2002/0081660th, U.S. Patent Publication case; WO 00/55353; WO 90/05785, describedly incorporates this paper by reference into reference to case.The another kind of method that can be used to express the antigen-binding polypeptides that comprises non-naturally encoded amino acids comprises mRNA-peptide integration technology.Referring to, for example R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94:12297-12302 (1997); People such as A.Frankel, Chemistyy ﹠amp; Biology 10:1043-1050 (2003).In the method, the mRNA template that is connected in puromycin (puromycin) is translated as peptide on ribosome.If modified one or more tRNA molecules then also can be incorporated alpha-non-natural amino acid in the peptide into.After reading last mRNA codon, puromycin is caught the C end of peptide.Have interesting characteristic if find gained mRNA-peptide conjugate in vitro in measuring, can manifest its identity so easily from the mRNA sequence.The library that can screen the antigen-binding polypeptides that comprises one or more non-naturally encoded amino acids in this way has the polypeptide of desirable characteristics with discriminating.Recently, reported with the translation of the in vitro ribosome of purified component and allowed the synthetic of the peptide replaced through non-naturally encoded amino acids.Referring to, people such as A.Forster for example, Proc.Natl Acad.Sci. (USA) 100:6353 (2003).

IX. be coupled to the macromolecule polyalcohol of ABP

Can use compositions described herein, method, technology and strategy realization various modifications to non-natural amino acid polypeptides described herein.These modifications comprise other functional group are incorporated on the alpha-non-natural amino acid component of polypeptide that described other functional group includes, but is not limited to labelling, dyestuff, polymer, water-soluble polymer, polyethyleneglycol derivative, photocrosslinking agent, radionuclide, cytotoxic compound, medicine, affinity labeling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelator, cofactor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, the antisense polynucleotide, the water solublity dendrimer, cyclodextrin, inhibition ribonucleic acid, biomaterial, nanoparticle, spin labeling, fluorogen, the containing metal part, the radioactivity part, novel functional group, the group of covalently or non-covalently doing mutually with other molecule, light is caught part, the photoisomerization part, biotin, biotin derivative, the biotin analog, incorporate the part that heavy atom is arranged into, but chemical cracking group, but photodestruciton group, long side chain, carbon connects sugar, redox active agent, amino thio-acid, the toxicity part, the isotopic labeling part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the close group of electronics, the magnetic group, insert group, chromophore, energy transfer agent, bioactivator, detectable label, micromolecule or above-mentioned any combination or any other required compound or material.Illustrative, limiting examples as compositions described herein, method, technology and strategy, below describe and macromolecule polyalcohol is added in the non-natural amino acid polypeptides concentrating on, should be appreciated that simultaneously, wherein said compositions, method, technology and strategy are applicable to also that (then suitably revise if desired, and one of ordinary skill in the art can utilize this paper disclosure to make suitable modification) adds and include, but is not limited to above be listed in other interior functional group).

Multiple macromolecule polyalcohol and other molecule can be connected in antigen-binding polypeptides of the present invention and provide neoplasm character with the biological property of adjusting ABP and/or to the ABP molecule.These macromolecule polyalcohols can be through natural coded amino acid, through non-naturally encoded amino acids, any sense substituent of perhaps natural or alpha-non-natural amino acid, or any substituent group or the functional group that make an addition in natural or the alpha-non-natural amino acid are connected in ABP.The molecular weight of polymer can have broad range, includes, but is not limited to about 100Da and about 100, between the 000Da or bigger.

The invention provides polymer: the substantial homogeneous preparation of protein conjugates." homogenizing in fact " as used herein meaning is to observe polymer: the protein conjugates molecule is greater than half of total protein.Polymer: protein conjugates biologically active, and that this paper provided was described " homogenizing in fact ", and PEGization ABP preparation has the homogeneity that is enough to show the homogeneous preparation advantage, described advantage for example in the clinical practice batch between the simplification of (lot to lot) pharmacokinetics predictability.

Also can select to prepare polymer: the mixture of protein conjugates molecule, and the advantage that this paper provided is can to select to be included in the single polymer in the mixture: the ratio of protein conjugates.Therefore, if desired, then can prepare various albumen and various numbers through be connected (promptly two-, three-, four-or the like) mixture of polymer moieties, and make up described conjugate and use the prepared single polymer of the inventive method: protein conjugates, and make mixture have single polymer of predetermined ratio: protein conjugates.

Selected polymer can be water miscible, so that does not precipitate in aqueous environments (for example physiological environment) with its albumen that is connected.Polymer can have side chain or unbranched.For the therapeutic use of end-product preparation, polymer is preferably pharmaceutically acceptable.

The ratio of peg molecule and protein molecular can change, and their concentration in reactant mixture also can change.In general, can determine best ratio (with regard to reaction efficiency, being to exist minimum excessive unreacted albumen or polymer) by the molecular weight of selected Polyethylene Glycol and the useful number of available reactive group.As for molecular weight, usually polymer molecular weight is big more, and it is few more to be connected in proteic polymer molecule number.Equally, when these parameters of optimization, should be taken into account the polymer side chain.Common molecular weight high more (or side chain is many more), then polymer: Protein ratios is high more.

As used herein and when considering the PEGrABP conjugate, term " treatment effective dose " is meant the amount that the patient is produced required benefit.Described amount is different between Different Individual, and depends on many factors, comprises patient's the overall health and the basic cause of the state of an illness to be treated.The volume production life of the used ABP polypeptide of therapy can be accepted the change of ratio, and keeps required reaction in useful level.One of ordinary skill in the art use and disclose the treatment effective dose that available material and process can be found out the present composition easily.

Water-soluble polymer can be any structure form, includes, but is not limited to straight chain, forked or branch-like.Water-soluble polymer is generally poly-(alkylene glycol), and for example poly-(ethylene glycol) (PEG) still also can use other water-soluble polymer.For instance, use PEG to describe some embodiment of the present invention.

PEG is a kind of water-soluble polymer of knowing, it can be buied from the market or prepare (Sandier and Karo according to the method that affiliated technical field is known by ring-opening polymerisation ethylene glycol, Polymer Synthesis, Academic Press, New York, the 3rd volume, the 138-161 page or leaf).Be extensive use of term " PEG " and contain any peg molecule, and do not consider the modification of size or consideration PEG end, and be expressed from the next to being connected with ABP:

XO-(CH ₂CH ₂O) _n-CH ₂CH ₂-Y，

Wherein n be 2 to 10,000 and X be H or end modified, include, but is not limited to C _1-4Alkyl.

In some cases, with hydroxyl or methoxyl group end-blocking, promptly X is H or CH to PEG used herein at an end ₃(" methoxyl group PEG ").Perhaps, PEG can use the reactive group end-blocking, and then forms the double functional copolymer.The type reaction group can comprise group (including, but is not limited to the maleimide base), activated carbon acid esters (including, but is not limited to the p-nitrophenyl ester), active ester (including, but is not limited to N-hydroxy-succinamide, p-nitrophenyl ester) and the aldehyde that is generally used for and is found in functional group reactions in 20 kinds of common amino acids); And to 20 kinds of common amino acids be inertia but be present in the non-naturally encoded amino acids functional group that replenishes functional group's specific reaction and (include, but is not limited to azido, alkynyl.It should be noted that another end of the PEG shown in the Y can directly or through natural generation or non-naturally encoded amino acids be connected in antigen-binding polypeptides indirectly in the above-mentioned formula.For instance, Y can be amide, carbamate or the urea bond that is connected in polypeptide amido (including, but is not limited to the ε amine or the N end of lysine).Perhaps, Y can be the maleimide key that is connected in thiol group (including, but is not limited to the thiol group of cysteine).Perhaps, Y can be and the key that can not be connected via the residue that 20 kinds of common amino acids arrive usually.For instance, the azido on the PEG can be gone up the alkynyl reaction to form Huisgen[3+2 with ABP] the cycloaddition product.Perhaps, the alkynyl on the PEG can react to form similar product to the azido in being present in non-naturally encoded amino acids.In certain embodiments, strong nucleopilic reagent (including, but is not limited to hydrazine, hydrazides, azanol, semicarbazides) can react to form hydrazone, oxime or semicarbazones with the aldehyde radical or the ketone group that are present in the non-naturally encoded amino acids, in the time of suitably, can further handle and be reduced in some cases through suitable Reducing agent.Perhaps, strong nucleopilic reagent can be incorporated among the ABP through non-naturally encoded amino acids, and is used for preferably reacting with ketone group that is present in water-soluble polymer or aldehyde radical.

Can look the PEG that actual needs uses any molecular weight, include, but is not limited to about 100 dalton (Da), 000Da or optionally bigger (including, but is not limited to 0.1-50kDa or 10-40kDa sometimes) to 100.Also can use side chain PEG, include, but is not limited to the PEG molecule that each chain has MW in 1-100kDa (including, but is not limited to 1-50kDa or the 5-20kDa) scope.The PEG molecule of broad range is described in (including, but is not limited to) Shearwater Polymers, and in Inc. catalogue, the Nektar Therapeutics catalogue, each incorporates this paper into by reference with reference to case.

Usually at least one end of PEG molecule can be used for reacting with non-naturally encoded amino acids.For instance, as described herein, have the alkynyl moiety and the nitrine PEG derivant partly that are used for reacting and can be used for PEG is connected with non-naturally encoded amino acids with amino acid side chain.If non-naturally encoded amino acids comprises nitrine, then PEG contains alkynyl moiety usually to form [3+2] cycloaddition product, and the active PEG kind (being ester, carbonic ester) that perhaps contains phosphino-is to form amido link.Perhaps, if non-naturally encoded amino acids comprises alkynes, then PEG contains the nitrine part usually to form [3+2] Huisgen cycloaddition product.If non-naturally encoded amino acids comprises carbonyl, then PEG comprises effective nucleopilic reagent (including, but is not limited to hydrazides, hydrazine, azanol or semicarbazides functional group) usually to form corresponding hydrazone, oxime and semicarbazones key respectively.In other alternate embodiment, can use the reverse azimuth of above-mentioned reactive group, promptly the nitrine in non-naturally encoded amino acids part can with the PEG derivatives reaction that contains alkynes.

In certain embodiments, the ABP variant with PEG derivant contain with the side chain that is present in non-naturally encoded amino acids on the chemical functional group have reactive chemical functional group.

In certain embodiments, the invention provides to comprise and have about 800Da, the containing nitrine and contain the polymer derivant of acetylene of the water-soluble polymer skeleton of 000Da mean molecule quantity to about 100.The polymer backbone of water-soluble polymer can be a Polyethylene Glycol.Yet, should be appreciated that, the multiple water-soluble polymer that includes, but is not limited to gather (ethylene glycol) and other related polymer (comprising poly-(glucose) and poly-(propylene glycol)) also is applicable to enforcement the present invention, and uses term PEG or poly-(ethylene glycol) to be intended to the molecule of containing and comprise that all are such.Term PEG includes, but is not limited to any type of poly-(ethylene glycol), comprises difunctionality PEG, multi-arm PEG, derivatization PEG, forked PEG, side chain PEG, hanging type PEG (pendent PEG) (PEG or the related polymer that promptly have one or more functional groups that overhang polymer backbone) or wherein has the PEG of degradable linkage.

PEG normally transparent, colourless, tasteless, water soluble, to thermally-stabilised, many chemical reagent are inertia, not hydrolysis or rotten and nontoxic usually.Think that poly-(ethylene glycol) has biocompatibility, that is to say that PEG can coexist and be safe from harm with living tissue or organism.More particularly, PEG is non-immunogenicity in fact, and promptly PEG is not inclined to and produces immunne response in vivo.When in vivo with the molecule with some required functions (as bioactivator) when being connected, PEG tends to cover this reagent, and can reduce or eliminate any immunne response, causes organism can stand the existence of described reagent.The PEG conjugate is not inclined to produce the essence immunne response or cause and condenses or other ill effect.Have formula--CH ₂CH ₂O--(CH ₂CH ₂O) _n-CH ₂CH ₂--the PEG of (wherein n is about 3 to about 4000, common about 20 to about 2000) is applicable to the present invention.Have about 800Da to about 100, the PEG of 000Da molecular weight especially can be used as polymer backbone in some embodiments of the invention.

Polymer backbone can be straight chain or branch-like.Technical field was known under the branch polymer skeleton was generally.Branch polymer has branch core, center and a plurality of straight chain polymer chain that is connected in described center branch core usually.The PEG of common employed branch form can prepare by ethylene oxide is made an addition in the various polyhydric alcohol, and described polyhydric alcohol is glycerol, glycerol oligomer, tetramethylolmethane and sorbitol for example.Center branch part also can derive from some aminoacid, for example lysine.Side chain poly-(ethylene glycol) can be by general formula R (PEG-OH) _mExpression, wherein R derives from the core, for example glycerol, glycerol oligomer or tetramethylolmethane, and m represents the arm number.The multi-arm PEG molecule that for example is described in the following document also can be used as polymer backbone: United States Patent (USP) the 5th, 932, No. 462, the 5th, 643, No. 575, the 5th, 229, No. 490, the 4th, 289, No. 872; U.S. patent application case 2003/0143596; WO 96/21469; With WO 93/21259, each patent case is all incorporated this paper by reference into.

Side chain PEG also can be for by PEG (YCHZ ₂) _nShown forked PEG form, wherein Y is for connecting base, and Z by by the atomic link of definition length be connected in the reactive terminal group of CH.

Another kind of branch's form (hanging type PEG) has reactive group along the PEG skeleton rather than at the PEG chain end, for example carboxyl.

Except the PEG of these forms, also can prepare polymer with the weak or degradable linkage in the skeleton.For instance, can prepare PEG with the ester bond of experience hydrolysis in the polymer backbone.Shown in hereinafter, this hydrolysis causes polymer cracking is become the fragment of lower molecular weight:

-PEG-CO ₂-PEG-+H ₂O→PEG-CO ₂H+HO-PEG-。

One of ordinary skill in the art should be appreciated that term poly-(ethylene glycol) or PEG represent or comprise the known form of ownership in affiliated field, include, but is not limited to form disclosed herein.

Many other polymer also are applicable to the present invention.In certain embodiments, water solublity and have 2 polymer backbones and especially can be used for the present invention to about 300 ends.The example of suitable polymers includes, but is not limited to other poly-(alkylene glycol), for example poly-(propylene glycol) (" PPG "), its copolymer (including, but is not limited to the copolymer of ethylene glycol and propylene glycol), its trimer, its mixture or the like.Although the molecular weight of each chain of polymer backbone can be different, its usually at about 800Da to about 100, in the 000Da scope, usually about 6,000Da is extremely about 80, in the 000Da scope.

One of ordinary skill in the art it should be understood that the listed water solublity in fact of preamble skeleton is by no means detailed, and only are illustrative, and all polymeric materials with above-mentioned molecular weight all are applicable to the present invention.

In some embodiments of the invention, polymer derivant is " multi-functional ", and the meaning is that polymer backbone has at least two through the functionalized or activatory end of functional group, and may be up to about 300.Multifunctional polymer derivant includes, but is not limited to have the straight chain polymer of two ends, and wherein each end is incorporated into the identical or different functional group of possibility.

In one embodiment, polymer derivant has following structure:

X-A-POLY-B-N＝N＝N，

Wherein:

N=N=N is the nitrine part;

B is the coupling part, and it can exist or not exist;

POLY is the non-antigen polymer of water solublity;

A is the coupling part, and it can exist or not exist and can be identical or different with B; And

X is second functional group.

The example of the coupling part of A and B includes, but is not limited to contain maximum 18 and be preferably the multiple functionalized alkyl of 1-10 carbon atom.Alkyl chain can comprise for example hetero atom of nitrogen, oxygen or sulfur.Alkyl chain also can be at hetero atom punishment branch.Other example of the coupling part of A and B includes, but is not limited to contain maximum 10 and be preferably the multiple functionalized aryl of 5-6 carbon atom.Aryl can replace through another carbon atom, nitrogen, oxygen or sulphur atom.Suitable other example that connects base comprises the connection base that is described in the following patent case: United States Patent (USP) the 5th, 932, No. 462, the 5th, 643, No. 575; With case 2003/0143596 please be disclosed in the United States Patent (USP), each patent case is incorporated this paper by reference into.One of ordinary skill in the art it should be understood that the listed coupling part of preamble by no means for detailed and only for illustrative, and all coupling parts with above-mentioned amount all are applicable to the present invention.

The functional group's example that is suitable as X includes, but is not limited to hydroxyl; Protected hydroxyl; Alkoxyl; Active ester, for example N-hydroxy-succinamide base ester and 1-benzotriazole base ester; Activated carbon acid esters, for example N-hydroxy-succinamide base carbonic ester and 1-benzotriazole base carbonic ester; Acetal; Aldehyde; Aldehyde hydrate; Thiazolinyl; Acrylate; Methacrylate; Acrylamide; Active sulfone; Amine; Aminooxy group; Protected amine; Hydrazides; Protected hydrazides; Protected sulfur alcohol; Carboxylic acid; Protected carboxylic acid; Isocyanates; Isothiocyanate; Maleimide; Vinyl sulfone; Two thiopyridines; Vinylpyridine; Iodoacetamide; Epoxide; The Biformyl class; Diketone; Methanesulfonates; Tosylate; Three fluoro ethane sulfonic acid esters; Alkene; Ketone and nitrine.One of ordinary skill in the art should be appreciated that selected X should be compatible with azido, do not cause to react with azido.Contain the azide polymer derivant and can have same difunctionality, the meaning is that second functional group (being X) also is the nitrine part; Perhaps have Heterobifunctional, the meaning is that second functional group is a different functional groups.

Term " protected " is meant to exist and prevents protecting group or the part that chemical reaction functional group reacts under some reaction condition.The type that protecting group is looked the chemical reaction base of being protected is different and different.For instance, if the chemical reaction base is amine or hydrazides, then protecting group can be selected from the group of tert-butoxycarbonyl (t-Boc) and 9-fluorenyl methoxy carbonyl (Fmoc).If the chemical reaction base is a mercaptan, then protecting group can be adjacent pyridyl disulfide.If the chemical reaction base is carboxylic acid (for example butanoic acid or propanoic acid) or hydroxyl, then protecting group can be benzyl or alkyl, for example methyl, ethyl or the tert-butyl group.Affiliated known other blocking group of technical field also can be used for the present invention.

In the document instantiation of functional end-group include, but is not limited to the N-succinimidyl carbonate (referring to, for example United States Patent (USP) the 5th, 281, No. 698, the 5th, 468, No. 478); Amine (referring to, people Makromol.Chem.182:1379 (1981) such as Buckmann for example, people Eur.Polym.J.19:1177 (1983) such as Zaplipsky); Hydrazides (referring to, people Makromol.Chem.179:301 (1978) such as Andresz for example); Succinyl phosphorons amino propyl acid ester and succinimido butyrate (referring to, people's such as Olson Poly (ethylene glycol) Chemistry﹠amp for example; Biological Applications, 170-181 page or leaf, Harris ﹠amp; Zaplipsky edits, ACS, Washington, D.C., 1997; Also referring to United States Patent (USP) the 5th, 672, No. 662); The succinimido succinate (referring to, people Macrolol.Chem.180:138l (1979) such as people Cancer Biochem.Biophys.7:175 (1984) such as Abuchowski and Joppich for example); The succinimido ester (referring to, for example United States Patent (USP) the 4th, 670, No. 417); The benzotriazole carbonic ester (referring to, for example United States Patent (USP) the 5th, 650, No. 234); Glycidyl ether (referring to, people Eur.J Biochem.94:11 (1979) such as Pitha for example, people such as Elling, Biotech.Appl.Biochem.13:354 (1991)); Oxygen base carbonylic imidazole (referring to, people such as Beauchamp for example, Anal.Biochem.131:25 (1983), people J.Controlled Release 1:251 (1985) such as Tondelli); The p-nitrophenyl carbonic ester (referring to, people such as Veronese for example, Appl.Biochem.Biotech., 11:141 (1985); With people such as Sartore, Appl.Biochem.Biotech., 27:45 (1991)); Aldehyde (referring to, people J.Polym.Sci.Chem.Ed.22:341 (1984) such as Harris for example, United States Patent (USP) the 5th, 824, No. the 5th, 252,714, No. 784, United States Patent (USP)); Maleimide (referring to, people Bio/Technology 8:343 (1990) such as Goodson for example, people's such as Romani Chemistryof Peptides and Proteins 2:29 (1984)); And Kogan (Synthetic Comm.22:2417 (1992)); Adjacent pyridyl disulfide (referring to, people Bioconj.Chem.4:314 (1993) such as Woghiren for example); Acrylic alcohol (acrylol) (referring to, people such as Sawhney for example, Macromolecules, 26:581 (1993)); Vinyl sulfone (referring to, for example United States Patent (USP) the 5th, 900, No. 461).All above-mentioned lists of references and patent are all incorporated this paper by reference into.

In certain embodiments of the present invention, polymer derivant of the present invention comprises the polymer backbone with following structure:

X-CH ₂CH ₂O--(CH ₂CH ₂O) _n--CH ₂CH ₂-N＝N＝N，

Wherein:

X is a functional group as indicated above; And

N is about 20 to about 4000.

In another embodiment, polymer derivant of the present invention comprises the polymer backbone with following structure:

X-CH ₂CH ₂O--(CH ₂CH ₂O) _n--CH ₂CH ₂-O-(CH ₂) _m-W-N＝N＝N，

Wherein:

W is aliphatic or the aromatic series connexon part that comprises 1-10 carbon atom;

N is about 20 to about 4000; And

X is a functional group as indicated above.M is 1 to 10.

Can prepare the nitrine PEG derivant that contains of the present invention according to affiliated technical field several different methods known and/or disclosed herein.In a kind of method shown in hereinafter, to have about 800Da to about 100, (it can match with the suitable counter of any number for the water-soluble polymer skeleton of 000Da mean molecule quantity and nitrine anion, comprise sodium, potassium, uncle's fourth ammonium or the like) reaction, wherein polymer backbone has first end that is incorporated into first functional group and second end that is incorporated into suitable leaving group.Leaving group experiences nucleophilic displacement of fluorine, and is partly replaced by nitrine, obtains the required nitrine PEG polymer that contains.

X-PEG-L+N ₃ ^-→X-PEL-N ₃

As shown, be applicable to that suitable polymers skeleton of the present invention has formula X-PEG-L, wherein PEG is poly-(ethylene glycol), and X be the functional group of not reacting with azido, and L is suitable leaving group.The example of appropriate functional group includes, but is not limited to hydroxyl, protected hydroxyl, acetal, thiazolinyl, amine, aminooxy group, protected amine, protected hydrazides, protected mercaptan, carboxylic acid, protected carboxylic acid, maleimide, two thiopyridines and vinylpyridine and ketone.The example of suitable leaving group includes, but is not limited to chlorine, bromine, iodine, methanesulfonates, three fluoro ethane sulfonic acid ester and tosylates.

In the preparation another kind of method that contains the azide polymer derivant of the present invention, make bridging agent and have about 800Da to about 100 with nitrine functional group, the contact of the water-soluble polymer skeleton of 000Da mean molecule quantity (wherein bridging agent have can with the chemical functional group of chemical functional group's selective reaction on the PEG polymer), contain the azide polymer derivative products with formation, wherein nitrine is separated from polymer backbone by linking group.

Exemplary reaction process is as follows:

X-PEG-M+N-connexon-N=N=N → PG-X-PEG-connexon-N=N=N

Wherein:

PEG is END CAPPED GROUP (for example alkoxyl) or functional group as indicated above for gathering (ethylene glycol) and X; And

M is not with the nitrine functional group reactions but the functional group of and selective reaction effective with N functional group.

The example of appropriate functional group includes, but is not limited to: if N is an amine, then M is carboxylic acid, carbonic ester or active ester; If N is hydrazides or aminooxy group part, then M is a ketone; If N is a nucleopilic reagent, then M is a leaving group.

Can reach the purification of crude product by known method, include, but is not limited to make the product precipitation, then (if desired) carries out chromatography.

Under the situation of PEG diamidogen, hereinafter show example more specifically, the protected group part of one of them amine (for example the tert-butyl group-Boc) protection, and gained list protection PEG diamidogen reacts with the coupling part with nitrine functional group:

BocHN-PEG-NH ₂+HO ₂C-(CH ₂) ₃-N＝N＝N。

In the case, can use multiple activator (for example thionyl chloride or carbodiimides reagent and N-hydroxy-succinamide or N-hydroxybenzotriazole) with amine groups and hydroxy-acid group coupling, with in monoamine PEG derivant and contain between the nitrine connexon part and produce amido link.Success forms after the amido link, and gained contains azido derivant and can be directly used in the modified biological bioactive molecule through the N-tert-butyl group-Boc protection, perhaps its can be further through refining to settle other useful functional group.For instance, can come hydrolyzing N-t-Boc group to generate omega-amino--PEG-nitrine with strong acid treatment usually.Gained amine can be as synthetic handle (synthetic handle) so that for example other useful functional groups such as maleimide base, active disulfide bond, active ester to be installed, to be used to produce valuable Heterobifunctional reagent.

In the time different molecular need being connected in each end of polymer, the Heterobifunctional derivant is especially available.For instance, ω-N-amino-N-azido PEG can allow (for example will have active electrophilic group, aldehyde, ketone, active ester, activated carbon acid esters or the like) molecule be connected in the end of PEG, and the molecule that will have an acetenyl is connected in another end of PEG.

In another embodiment of the present invention, polymer derivant has following structure:

X-A-POLY-B-C≡C-R

Wherein:

R is H or alkyl, thiazolinyl, alkoxyl or aryl or is substituted aryl;

B is the coupling part, and it can exist or not exist;

POLY is the non-antigen polymer of water solublity;

A is the coupling part, and it can exist or not exist and can be identical or different with B; And

X is second functional group.

The example of the coupling part of A and B includes, but is not limited to contain the multiple functionalized alkyl that reaches 18 and be preferably the 1-10 carbon atom.Alkyl chain can comprise for example hetero atom of nitrogen, oxygen or sulfur.Alkyl chain also can be at hetero atom punishment branch.Other example of the coupling part of A and B includes, but is not limited to contain the multiple functionalized aryl that reaches 10 and be preferably the 5-6 carbon atom.Aryl can replace through another carbon atom, nitrogen, oxygen or sulphur atom.Suitable other example that connects base comprises the connection base that is described in the following patent case: United States Patent (USP) the 5th, 932, No. 462, the 5th, 643, No. 575; Disclose case 2003/0143596 clearly with the United States Patent (USP) Shen, each patent case is incorporated this paper by reference into.One of ordinary skill in the art it should be understood that the listed coupling part of preamble only is illustrative for detailed by no means, and multiple coupling part with above-mentioned amount all can be used for the present invention.

The functional group's example that is suitable as X comprises hydroxyl; Protected hydroxyl; Alkoxyl; Active ester, for example N-hydroxy-succinamide base ester and 1-benzotriazole base ester; Activated carbon acid esters, for example N-hydroxy-succinamide base carbonic ester and 1-benzotriazole base carbonic ester; Acetal; Aldehyde; Aldehyde hydrate; Thiazolinyl; Acrylate; Methacrylate; Acrylamide; Active sulfone; Amine; Aminooxy group; Protected amine; Hydrazides; Protected hydrazides; Protected sulfur alcohol; Carboxylic acid; Protected carboxylic acid; Isocyanates; Isothiocyanate; Maleimide; Vinyl sulfone; Two thiopyridines; Vinylpyridine; Iodoacetamide; Epoxide; The Biformyl class; Diketone; Methanesulfonates; Tosylate; With three fluoro ethane sulfonic acid esters; Alkene; Ketone and acetylene.Should be appreciated that selected X part should be compatible with acetenyl, thereby do not react with acetenyl.Contain the acetylene polymer derivant and can have same difunctionality, the meaning is that second functional group (being X) also is acetylene moiety; Perhaps have Heterobifunctional, the meaning is that second functional group is a different functional groups.

In another embodiment of the present invention, polymer derivant comprises the polymer backbone with following structure:

X-CH ₂CH ₂O--(CH ₂CH ₂O) _n--CH ₂CH ₂-O-(CH ₂) _m-C≡CH，

Wherein:

X is a functional group as indicated above;

N is about 20 to about 4000; And

M is 1 to 10.

The instantiation of each Heterobifunctional PEG polymer is as shown in hereinafter.

Technical field method known and/or disclosed herein prepares the acetylene PEG derivant that contains of the present invention under can using.In a method, make and have about 800Da to about 100, the water-soluble polymer skeleton of 000Da mean molecule quantity with have acetylene functional group and be suitable for going up the chemical compound reaction of the leaving group of nucleophilic group reaction with PEG, wherein polymer backbone has and is incorporated into the first terminal and be incorporated into second end of suitable nucleophilic group of first functional group.Have the PEG polymer and the branch period of the day from 11 p.m. to 1 a.m with leaving group of nucleophilic part when combination, leaving group experiences nucleophilic displacement of fluorine, and is partly replaced by nucleophilic, to generate the required polymer that contains acetylene.

As shown, the preferred polymers skeleton that is used for described reaction has formula X-PEG-Nu, and wherein PEG is poly-(ethylene glycol), Nu be nucleophilic part and X for not with the functional group of Nu, L or acetylene functional group reactions.

The example of Nu includes, but is not limited to mainly through machine-processed amine, alkoxyl, aryloxy group, sulfydryl, imido grpup, carboxylate, hydrazides, the aminooxy group that reacts of SN2 type.Other example of Nu group comprises the functional group of reacting through nucleophilic addition mainly.The L examples of groups comprises chlorine, bromine, iodine, methanesulfonates, three fluoro ethane sulfonic acid ester and tosylates, with the group of other expection through nucleophilic displacement of fluorine, and the electrophilic group of ketone, aldehyde, thioesters, alkene, alpha-beta unsaturated carbonyl, carbonic ester and other expection experience nucleopilic reagent additive reaction.

In another embodiment of the present invention, A is the aliphatic connexon of 1-10 carbon atom, or 6-14 carbon atom be substituted aryl rings.X be not with the functional group of azido reaction, and L is suitable leaving group.

In the preparation other method that contains the acetylene polymer derivant of the present invention; make the acetylene anion and have about 800 Da to about 100; the contact of the PEG polymer of 000Da mean molecule quantity, described PEG polymer has protected functional group or end-capping reagent and has suitable leaving group at another end at an end.

Exemplary reaction process is as follows:

Wherein:

PEG is an END CAPPED GROUP for gathering (ethylene glycol) and X, for example alkoxyl or functional group as indicated above; And

R ' is H, alkyl, alkoxyl, aryl or aryloxy group, or is substituted alkyl, alkoxyl, aryl or aryloxy group.

In above-mentioned example, when contacting with the acetylene anion of enough concentration, leaving group L should have enough reactivities and replace with experience SN2 type.Known by affiliated technical field through the required reaction condition of the anionic SN2 replacement of acetylene for reaching leaving group.

Can reach the purification of crude product by the known method of affiliated technical field, it includes, but is not limited to make the product precipitation, and then (if desired) carries out chromatography.

Water-soluble polymer can be connected with antigen-binding polypeptides of the present invention.Water-soluble polymer can be in incorporating antigen-binding polypeptides into non-naturally encoded amino acids, the any functional group or the substituent group of perhaps non-naturally encoded or natural coded amino acid, any functional group or the substituent group that perhaps make an addition to non-naturally encoded or natural coded amino acid connect.Perhaps, water-soluble polymer is connected in and incorporates the antigen-binding polypeptides that non-naturally encoded amino acids is arranged into through non-natural generation aminoacid (including, but is not limited to the amino of cysteine, lysine or N end residue).In some cases, ABP of the present invention comprises 1,2,3,4,5,6,7,8,9,10 alpha-non-natural amino acid, and one of them or more than one non-naturally encoded amino acids are connected in water-soluble polymer (including, but is not limited to PEG and/or oligosaccharide).In some cases, ABP of the present invention further comprises 1,2,3,4,5,6,7,8,9,10 or more a plurality of non-naturally encoded amino acids that is connected in water-soluble polymer.In some cases, ABP of the present invention comprises one or more non-naturally encoded amino acids that are connected in water-soluble polymer and one or more and is connected in the natural generation aminoacid of water-soluble polymer.In certain embodiments, be used for the serum half-life of water-soluble polymer wild phase of the present invention for the ABP of non-binding form.

Can regulate the water-soluble polymer number (being PEGization or degree of glycosylation) that is connected in antigen-binding polypeptides of the present invention to change (including, but is not limited to increases or reduce) pharmacology, pharmacokinetics or pharmacodynamic profile, for example half-life in vivo.In certain embodiments, the half-life of ABP is than increasing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2 times, 5 times, 10 times, 50 times or at least about 100 times without modified polypeptide.

The PEG derivant that contains strong nucleophilic group (being hydrazides, hydrazine, azanol or semicarbazides)

In one embodiment of the invention, comprise the antigen-binding polypeptides that contains the carbonyl non-naturally encoded amino acids with the PEG derivant modification that contains the terminal hydrazine, azanol, hydrazides or the semicarbazides part that are directly connected in the PEG skeleton.

In certain embodiments, the terminal PEG derivant of azanol has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-O-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 (being the mean molecule quantity of 5-40kDa).

In certain embodiments, contain hydrazine or contain hydrazides PEG derivant and have following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-X-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, contain semicarbazides PEG derivant and have following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-NH-C(O)-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, modify and comprise the antigen-binding polypeptides that contains carbonylamino acid with containing the PEG derivant that is connected in the terminal azanol, hydrazides, hydrazine of PEG skeleton or semicarbazides part by amido link.

In certain embodiments, the terminal PEG derivant of azanol has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)(CH ₂) _m-O-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 (being the mean molecule quantity of 5-40kDa).

In certain embodiments, contain hydrazine or contain hydrazides PEG derivant and have following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)(CH ₂) _m-X-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is 2-10, and n is 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, contain semicarbazides PEG derivant and have following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)(CH ₂) _m-NH-C(O)-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, comprise the ABP that contains carbonylamino acid with the side chain PEG derivant modification that contains terminal hydrazine, azanol, hydrazides or semicarbazides part, each chain of wherein said side chain PEG derivant has the MW in the 10-40kDa scope, more preferably 5-20kDa.

In another embodiment of the present invention, modify the ABP that comprises non-naturally encoded amino acids with PEG derivant with branched structure.For instance, in certain embodiments, contain hydrazine or contain hydrazides PEG derivant and have following structure:

[RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)] ₂CH(CH ₂) _m-X-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, the PEG derivant that contains the semicarbazides group has following structure:

[RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-C(O)-NH-CH ₂-CH ₂] ₂CH-X-(CH ₂) _m-NH-C(O)-NH-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and X is NH, O, S, C (O) or does not exist that m is that 2-10 and n are 100-1 according to circumstances, 000.

In certain embodiments, the PEG derivant that contains the azanol group has following structure:

[RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-C(O)-NH-CH ₂-CH ₂] ₂CH-X-(CH ₂) _m-O-NH ₂，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and X is NH, O, S, C (O) or does not exist that m is that 2-10 and n are 100-1 according to circumstances, 000.

Combining of ABP and antigen or receptor can be regulated in the degree that water-soluble polymer is connected in ABP and site.

Be used for activated polymer and be used for the method for binding peptide and chemistry is described in the literature to some extent and for affiliated technical field known.The method that is generally used for activated polymer includes, but is not limited to activate functional group with following material: Bromine cyanide., periodate, glutaraldehyde, diepoxide (biepoxide), chloropropylene oxide, divinylsulfone, carbodiimides, sulfonyl halogenide, three chlorotriazines or the like (referring to, R.F.Taylor, (1991), PROTEINIMMOBILISATION.FUNDAMENTAL AND APPLICATIONS, Marcel Dekker, N.Y.; S.S.Wong, (1992), CHEMISTRY OF PROTEIN CONJUGATION AND CROSSLINKING, CRC Press, BocaRaton; People such as G.T.Hermanson, (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES, Academic Press, N.Y.; Dunn, people such as R.L., editor POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series the 469th volume, American ChemicalSociety, Washington.D.C.1991).

Can obtain some about functionalized and bonded summary of PEG and monograph.Referring to, Harris for example, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, CriticalReviews in Tlierapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, BioconjugateChem.6:150-165 (1995).

The method that is used for activated polymer also can be referring to WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO 94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO 90/13540, United States Patent (USP) 5,281, No. 698 and WO 93/15189, and be used for combining between living polymer and the enzyme, described enzyme includes, but is not limited to blood coagulation factor VIII (WO 94/15625), hemoglobin (WO94/09027), oxygen carrier molecule (United States Patent (USP) the 4th, 412, No. 989), ribonuclease and superoxide dismutase (people such as Veronese, App.Biochem.Biotech.11:141-45 (1985)).Used list of references and patent are all incorporated this paper by reference into.

The PEGization (promptly adding any water-soluble polymer) that contains the antigen-binding polypeptides of non-naturally encoded amino acids (for example to azido-L-phenylalanine) by any conventional method.For instance, be terminal mPEG derivant PEGization ABP in order to alkynes.In simple terms, follow stirring in containing, to add excessive solid mPEG (5000)-O-CH under the room temperature to the ABP aqueous solution of azido-L-Phe ₂-G ≡ CH.Usually with having near the pK that carries out reaction pH value _aThe buffer of (about usually pH 4-10) cushions described aqueous solution.For instance, the example that is used for carrying out for 7.5 times at pH the suitable buffer of PEGization includes, but is not limited to HEPES, phosphate, borate, TRIS-HCl, EPPS and TES.Continue monitoring and (if desired) and regulate pH value.Usually make reaction continue about 1-48 hour.

Product with after the hydrophobic chromatography of doing mutually with from free mPEG (5000)-O-CH ₂Any high molecular weight component of-C ≡ CH and PEGization ABP is isolated PEGization ABP variant, and described high molecular weight component is all to be activated at two ends of molecule as non-block PEG, and then crosslinked ABP variant molecule and forming.The hydrophobic condition of doing mutually in the chromatography is feasible free mPEG (5000)-O-CH ₂-C ≡ CH the chromatographic column of flowing through, simultaneously any through the ABP of crosslinked PEGization variant complex after desired form molten from, described ABP variant complex contains an ABP variant molecule that is incorporated into one or more PEG groups.Appropraite condition is looked the relative size of cross-linked composite and required conjugate and is changed, and can be determined easily by one of ordinary skill in the art.Concentrate the eluat that contains required conjugate by ultrafiltration, and by saturating filter desalination.

If desired, then can be further purified PEGization ABP available from hydrophobic chromatography by one or more known processes of one of ordinary skill in the art, described process includes, but is not limited to affinity chromatography, anion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE), the silicon dioxide chromatography, reversed-phase HPLC, gel filtration (use includes, but is not limited to SEPHADEX G-75), the hydrophobic chromatography of doing mutually, the size exclusion chromatography, metal chelate chromatography, ultrafiltration/diafiltration, ethanol precipitation, ammonium sulfate precipitation method, the chromatofocusing method, displacement chromatography, electrophoresis process (including, but is not limited to the isoelectrofocusing of preparation type), difference dissolubility (including, but is not limited to ammonium sulfate precipitation) or extraction method.Can be by GPC by relatively assessing apparent molecular weight (PROTEIN PURIFICATION METHODS, APRACTICALAPPROACH (Harris with the globulin standard; Angal, Eds.) IRL Press 1989,293-306).The Degradation (including, but is not limited to the insulin cracking) that can cause by Proteolytic enzyme is then carried out the purity that the ABP-PEG conjugate is assessed in mass spectral analysis.People such as Pepinsky B., J.Pharmcol. ﹠amp; Exp.Ther.297 (3): 1059-66 (2001).

Can further carry out derivatization or replacement to the amino acid whose water-soluble polymer that is connected in ABP of the present invention down unrestricted.

Contain nitrine PEG derivant

In another embodiment of the present invention, combine polypeptide with the PEG derivant modified antigen that contains the nitrine part that to react with the alkynyl moiety on being present in the non-naturally encoded amino acids side chain.In general, the PEG derivant can have the mean molecule quantity in the 1-100kDa scope, and is 10-40kDa in certain embodiments.

In certain embodiments, the terminal PEG derivant of nitrine has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-N ₃，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 (being the mean molecule quantity of 5-40kDa).

In another embodiment, the terminal PEG derivant of nitrine has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-NH-C(O)-(CH ₂) _p-N ₃，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is 2-10, and p is that 2-10 and n are 100-1,000 (being the mean molecule quantity of 5-40kDa).

In another embodiment of the present invention, comprise the ABP that contains alkynyl amino acid with the side chain PEG derivant modification that contains terminal nitrine part, each chain of wherein said side chain PEG has the MW in the 10-40kDa scope, more preferably 5-20kDa.For instance, in certain embodiments, the terminal PEG derivant of nitrine has following structure:

[RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)] ₂CH(CH ₂) _m-X-(CH ₂) _pN ₃，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is 2-10, and p is that 2-10 and n are 100-1,000 and X be O, N, S or carbonyl (C=O) according to circumstances, it can exist or not exist under each situation.

Contain alkynes PEG derivant

In another embodiment of the present invention, can combine polypeptide with the PEG derivant modified antigen of the alkynyl moiety of nitrine partial reaction on being present in the non-naturally encoded amino acids side chain with containing.

In certain embodiments, the terminal PEG derivant of alkynes has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-C≡CH，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is that 2-10 and n are 100-1,000 (being the mean molecule quantity of 5-40kDa).

In another embodiment of the present invention, modify and comprise the antigen-binding polypeptides that contains the alkynes non-naturally encoded amino acids with containing the PEG derivant that is connected in the terminal nitrine part of PEG skeleton or terminal alkynyl moiety by amido link.

In certain embodiments, the terminal PEG derivant of alkynes has following structure:

RO-(CH ₂CH ₂O) _n-O-(CH ₂) _m-NH-C(O)-(CH ₂) _p-C≡CH，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is 2-10, and p is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, to use the side chain PEG derivant that contains terminal alkynyl moiety to modify to comprise and contain the amino acid whose antigen-binding polypeptides of nitrine, each chain of wherein said side chain PEG has the MW in the 10-40kDa scope, more preferably 5-20kDa.For instance, in certain embodiments, the terminal PEG derivant of alkynes has following structure:

[RO-(CH ₂CH ₂O) _n-O-(CH ₂) ₂-NH-C(O)] ₂CH(CH ₂) _m-X-(CH ₂) _pC≡CH，

Wherein R is simple alkyl (methyl, ethyl, propyl group or the like), and m is 2-10, and p is that 2-10 and n are 100-1,000, and X is O, N, S or carbonyl (C=O) according to circumstances, or do not exist.

Contain phosphine PEG derivant

In another embodiment of the present invention, with the PEG derivant modified antigen that contains active function groups (including, but is not limited to ester, carbonic ester) in conjunction with polypeptide, wherein said PEG derivant further comprise can with the aryl phosphine group that is present in nitrine partial reaction on the non-naturally encoded amino acids side chain.In general, the PEG derivant can have the mean molecule quantity in the 1-100kDa scope, and is 10-40kDa in certain embodiments.

In certain embodiments, the PEG derivant has following structure:

Wherein n is 1-10; X can or not exist for O, N, S, and Ph is a phenyl, and W is a water-soluble polymer.

In certain embodiments, the PEG derivant has following structure:

Wherein X can or not exist for O, N, S, and Ph is a phenyl, W be water-soluble polymer and R can be for H, alkyl, aryl, be substituted alkyl and be substituted aryl.Exemplary R group includes, but is not limited to-CH ₂,-C (CH ₃) ₃,-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O) ₂R ' ,-S (O) ₂NR ' R " ,-CN and-NO ₂R ', R ", R_ and R " " refer to hydrogen independently of one another, be substituted or be unsubstituted assorted alkyl, be substituted or be unsubstituted aryl (include, but is not limited to replace aryl), be substituted or be unsubstituted alkyl, alkoxyl or thio alkoxy through 1-3 halogen, or aryl alkyl.For instance, when chemical compound of the present invention comprised an above R group, each R group independently was chosen as when having more than one these groups " the group as each R ', R ", R_ and R "." when being connected in same nitrogen-atoms, both can combine with nitrogen-atoms and form 5 yuan, 6 yuan or 7 yuan of rings as R ' and R.For instance ,-NR ' R " is meant and includes, but is not limited to 1-pyrrolidinyl and 4-morpholinyl.For above-mentioned substituent group, one of ordinary skill in the art should be appreciated that, term " alkyl " is meant and comprises and the group of the bonded carbon atom of group except that hydrogen atom that for example haloalkyl (includes, but is not limited to-CF ₃With-CH ₂CF ₃) and acyl group (include, but is not limited to-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂OCH ₃Or the like).

Other PEG derivant and general PEGization technology

Other the exemplary PEG molecule and the PEGization method that can be connected in antigen-binding polypeptides comprise the method that is described in the following patent documentation, and for example the U.S. Patent Publication case 2004/0001838,2002/0052009,2003/0162949,2004/0013637,2003/0228274,2003/0220447,2003/0158333,2003/0143596,2003/0114647,2003/0105275,2003/0105224,2003/0023023,2002/0156047,2002/0099133,2002/0086939,2002/0082345,2002/0072573,2002/0052430,2002/0040076,2002/0037949,2002/0002250,2001/0056171,2001/0044526,2001/0027217,2001/0021763; United States Patent (USP) case 6,646,110,5,824,778,5,476,653,5,219,564,5,629,384,5,736,625,4,902,502,5,281,698,5,122,614,5,473,034,5,516,673,5,382,657,6,552,167,6,610,281,6,515,100,6,461,603,6,436,386,6,214,966,5,990,237,5,900,461,5,739,208,5,672,662,5,446,090,5,808,096,5,612,460,5,324,844,5,252,714,6,420,339,6,201,072,6,451,346,6,306,821,5,559,213,5,612,460,5,747,646,5,834,594,5,849,860,5,980,948,6,004,573,6,129,912; WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO95/13312, EP 921131, WO 98/05363, EP 809996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503 and EP 154 316 describedly incorporate this paper into by reference with reference to case.Any type of any PEG molecule as herein described be can use, strand, branch, multi-arm chain, simple function, difunctionality, multifunctional or its any combination included, but is not limited to.

Strengthen and sero-abluminous affinity

Also various molecules and antigen-binding polypeptides of the present invention can be merged to regulate the half-life of ABP in the serum.In certain embodiments, molecule is connected with antigen-binding polypeptides of the present invention or merge with strengthen with animal in endogenous sero-abluminous affinity.

For instance, in some cases, the reorganization of carrying out antigen-binding polypeptides and albumin bound sequence is merged.Exemplary albumin bound sequence include, but is not limited to from streptococcus protein G the albumin bound territory (referring to, people such as Makrides for example, J. people such as Pharmacol.Exp.Ther.277:534-542 (1996) and Sjolander, J, Immunol.Methods 201:115-123 (1997)), or people such as Dennis for example, the albumin binding peptide described in the J.Biol Chem.277:35035-35043 (2002).

In other embodiments, with fatty acid acidylate antigen-binding polypeptides of the present invention.In some cases, fatty acid promotes to combine with sero-abluminous.Referring to, people such as Kurtzhals for example, Biochem.J. 312:725-731 (1995).

In other embodiments, direct and serum albumin (the including, but is not limited to the human serum albumin) fusion of antigen-binding polypeptides of the present invention.One of ordinary skill in the art it should be understood that multiple other molecule also can be connected in ABP the combining with adjusting and serum albumin or other serum component among the present invention.

The glycosylation of X.ABP

The present invention includes to incorporate into has one or more to have the antigen-binding polypeptides of the non-naturally encoded amino acids of saccharide residue.Saccharide residue can be natural (including, but is not limited to the N-acetyl glucosamine) or non-natural (including, but is not limited to 3-fluorine galactose).Sugar can connect glycosidic bond (including, but is not limited to N-acetyl group galactose-L-serine) or non-natural key (include, but is not limited to oxime or corresponding C or S and connect glucosides) is connected with non-naturally encoded amino acids by N or O.

Can in vivo or in vitro sugar (including, but is not limited to glycosyl) part be added in the antigen-binding polypeptides.In some embodiments of the invention, use through aminooxy group derivatization sugar-modified comprise contain the carbonyl non-naturally encoded amino acids antigen-binding polypeptides to produce the corresponding glycosylated polypeptides that connects through the oxime key.In case be connected in non-naturally encoded amino acids, promptly by handling further refined sugar to be incorporated into the oligosaccharide of antigen-binding polypeptides with generation with glycosyl transferase and other enzyme.Referring to, people J. Am.Chem.Soc.125:1702-1703 (2003) such as H.Liu for example.

In some embodiments of the invention, directly apparatus comprises the antigen-binding polypeptides that contains the carbonyl non-naturally encoded amino acids just like the polysaccharide modification that the aminooxy group derivant prepares defined structure.One of ordinary skill in the art it should be understood that other functional group (comprising nitrine, alkynes, hydrazides, hydrazine and semicarbazides) also can be used to be connected sugar and non-naturally encoded amino acids.

In some embodiments of the invention, subsequently can by (including, but is not limited to) respectively with the Huisgen[3+2 of (including, but is not limited to) alkynyl or azido derivant] cycloaddition reaction modifies the antigen-binding polypeptides that comprises the non-naturally encoded amino acids that contains nitrine or contain alkynyl.Described method allows with high selective modification albumen.

XI.ABP dimer and polymer

The present invention also provides the combination that includes, but is not limited to ABP homodimer, heterodimer, homology polymer or heteromultimers (being trimer, tetramer or the like), the ABP polypeptide that wherein contains one or more non-naturally encoded amino acids is incorporated into another ABP or its variant or any other polypeptide for non-ABP polypeptide or its variant, and it is directly to be incorporated into polypeptide backbone or through the connexon combination.Because compare molecular weight increases with monomer, therefore ABP dimer or polymer conjugate can show new features or desirable characteristics, include, but is not limited to pharmacologys different for monomer A BP, pharmacokinetics, pharmacodynamic profiles, through the adjustment of treatment half-life or through regulating plasma half-life.In certain embodiments, ABP dimer of the present invention can be regulated the dimerization effect of ABP receptor.In other embodiments, ABP dimer of the present invention or polymer can serve as ABP receptor antagonist, agonist or regulator.

In certain embodiments, be present in one or more ABP that contain among dimer or the polymeric ABP and comprise the non-naturally encoded amino acids that is connected in water-soluble polymer.In certain embodiments, ABP directly connects, and includes, but is not limited to connect through Asn-Lys amido link or Cys-Cys disulfide bond.In certain embodiments, connect ABP and/or connect non-ABP polypeptide and can comprise different non-naturally encoded amino acids helping the dimerization effect, its include, but is not limited in the non-naturally encoded amino acids of an ABP alkynes can with nitrine in second non-naturally encoded amino acids of the 2nd ABP through Huisgen[3+2] cycloaddition reaction and combining.Perhaps, an ABP and/or comprise contain the ketone non-naturally encoded amino acids connect non-ABP polypeptide and can combine with the 2nd ABP polypeptide that comprises the non-naturally encoded amino acids that contains azanol, and polypeptide reacts by forming corresponding oxime.

Perhaps, two ABP and/or connect non-ABP polypeptide and connect through connexon.Any Heterobifunctional or with the difunctionality connexon can be used for connecting two ABP that can have identical or different main sequence and/or connect non-ABP polypeptide.In some cases, be used for ABP and/or to connect the connexon that non-ABP polypeptide is strapped in together can be difunctionality PEG reagent.Connexon can have the molecular weight or the molecular length of wide scope.Can use big or concern or conformation with requisite space between the be connected entity so that ABP to be provided than the small-molecular weight connexon.Have long or also can be used to provide desired spacing or flexibility between ABP and the connection entity than the connexon of short molecule length.Similarly, before or after ABP arrives its target, the connexon with given shape or conformation can be used for to ABP or connect entity and give given shape or conformation.Can be selected to regulate the release of ABP at desired conditions or non-ABP polypeptide being present in each terminal functional group of connexon.Between ABP and the connection entity optimization of spatial relationship can to molecule provide new, through regulate or desirable characteristics.

In certain embodiments, the invention provides the water solublity difunctionality connexon with dumbbell structure, it comprises: a) nitrine, alkynes, hydrazine, hydrazides, the azanol at least the first end of polymer backbone or contain carbonyl moiety; And b) at least one second functional group on second end of polymer backbone.Second functional group can be identical or different with first functional group.In certain embodiments, a reactivity is not had to first functional group in second functional group.In certain embodiments, the invention provides the water soluble compound of at least one arm that comprises the branched chain molecule structure.For instance, the branched chain molecule structure can be dendroid.

In certain embodiments, the invention provides comprise one or more by with the polymer of the formed ABP of water-soluble active polymer reaction, described water-soluble polymer has following structure:

R-(CH ₂CH ₂O) _n-O-(CH ₂) _m-X，

Wherein n is about 5 to 3,000, and m is 2-10, and X can or contain carbonyl moiety for nitrine, alkynes, hydrazine, hydrazides, aminooxy group, azanol, acetyl group, and R is END CAPPED GROUP, functional group or leaving group that can be identical or different with X.For instance, R can be for being selected from the functional group that is made of group following each thing: hydroxyl; protected hydroxyl; alkoxyl; N-hydroxy-succinamide base ester; 1-benzotriazole base ester; N-hydroxy-succinamide base carbonic ester; 1-benzotriazole base carbonic ester; acetal; aldehyde; aldehyde hydrate; thiazolinyl; acrylate; methacrylate; acrylamide; active sulfone; amine; aminooxy group; protected amine; hydrazides; protected hydrazides; protected mercaptan; carboxylic acid; protected carboxylic acid; isocyanates; isothiocyanate; maleimide; vinyl sulfone; two thiopyridines; vinylpyridine; iodoacetamide; epoxide; the Biformyl class; diketone; methanesulfonates; tosylate and three fluoro ethane sulfonic acid esters; alkene and ketone.

XII. measure the affinity of ABP activity and ABP and ABP antigen or binding partners

Can in vitro or in vivo test with standard and measure the ABP activity.For instance, can use in conjunction with the cell of ABP or cell line (cell that includes, but is not limited to contain the cell of natural A BP antigen or binding partners or produce reorganization ABP antigen or binding partners) and monitor the ABP combination.For non-PEGization that comprises alpha-non-natural amino acid or PEGization antigen-binding polypeptides, known technology that can be by technical field under using (BIAcore for example ^TMBiosensor (Pharmacia)) measures the affinity of ABP and its antigen or binding partners.

No matter use which kind of method to produce ABP, all ABP is carried out biological activity test.If suitably, then can carry out the tritiated thymidine test to determine degree of active cell division.Yet, also can use other biological test to determine required activity.For example measure the biological test that suppresses antigen biological activity (for example enzymatic activity, proliferation activity or metabolic activity) ability and also indicate the ABP activity.Other is in vitro tested and also can be used for determining biological activity.In general, look antigen biological activity needs, biological activity test is tackled required result and is analyzed, active increase of biological example or minimizing (with not changing ABP and comparing), different biological activity (with not changing ABP and comparing), receptor affinity analysis, conformation or structural change or serum half-life analysis.

Above also non-exhaustive about the list of references compilation of test method, and one of ordinary skill in the art should understand the test that other can be used for testing required final result.

XIII. measure usefulness, functional in vivo half-life and pharmacokinetic parameter

An importance of the present invention is by making or do not make ABP and water-soluble polymer part to combine to make up the biological half-life of the prolongation that ABP obtains.The quick reduction of ABP serum-concentration makes to be estimated significant with the biological respinse of combination and non-binding ABP and variant treatment thereof.Combination of the present invention and non-binding ABP and variant thereof preferably also have the serum half-life of prolongation after the intravenous dispensing, make it possible to measure by (for example) ELISA method or by primary screening test.As described hereinly carry out the in vivo measurement of biological half-life.

Can in normal Sprague-Dawley male rat (N=5 animal of every treatment group), estimate the pharmacokinetic parameter of the antigen-binding polypeptides that comprises non-naturally encoded amino acids.Animal can be accepted the single dose of 25 micrograms/rat (intravenous) or 50 micrograms/rat (subcutaneous), and according to the about 5-7 of a predetermined time-histories collection blood sample, the common covering antigen-binding polypeptides that comprises non-naturally encoded amino acids 4 days that is not incorporated into the antigen-binding polypeptides that comprises non-naturally encoded amino acids 6 hours of water-soluble polymer and is incorporated into water-soluble polymer.Fully studied the ABP pharmacokinetic data of some kinds of apoplexy due to endogenous wind, and it directly can have been compared with the data that ABP obtained that comprise non-naturally encoded amino acids.

Can measure specific activity by the known various tests of affiliated technical field according to ABP of the present invention.Can be by described herein or institute's reference or the known method test of one of ordinary skill in the art acquisition and purified ABP mutein or its segmental biological activity according to the present invention.

XIV. offer medicine and medical composition

Polypeptide of the present invention or albumen (include, but is not limited to ABP, synzyme, comprise albumen of one or more alpha-non-natural amino acids or the like) are used for the treatment of purposes according to circumstances, include, but is not limited to combine with suitable medical supporting agent.For instance, described compositions comprises the chemical compound for the treatment of effective dose, pharmaceutically acceptable supporting agent or excipient.Described supporting agent or excipient include, but is not limited to saline, buffer saline, dextrose, water, glycerol, ethanol and/or its combination.Dosage form should cooperate the dispensing pattern.In general, throw with proteic method and known, and be applicable to throwing and polypeptide of the present invention by affiliated technical field.

Well-known process according to affiliated technical field, test comprises the therapeutic combination of one or more polypeptide of the present invention in one or more suitable in vitro and/or in vivo infected animal models according to circumstances, to confirm effect, tissue metabolism and to assess dosage.Specifically, beginning can be determined dosage by activity, stability or other the suitable module (including, but is not limited to more modified ABP and natural amino acid ABP to comprise one or more alpha-non-natural amino acids) that compare non-natural and natural amino acid congener, promptly in correlation test.

By any being generally used for molecule is introduced to contact the dispensing of blood or histiocytic approach fully.With any suitable method, throw and non-natural amino acid polypeptides of the present invention with one or more pharmaceutically acceptable supporting agents according to circumstances.Can get to the come into operation appropriate method of polypeptide described in the content of the present invention of patient, although and can use more than one approach to throw and particular composition, particular approach can provide usually than more timely, more effective effect of another approach or reaction.

On the part by throw and particular composition and determine pharmaceutically acceptable supporting agent by the ad hoc approach that is used to throw with compositions.Therefore, the dosage forms that has multiple medical composition of the present invention.

Can throw and peptide composition by many approach, include, but is not limited to per os, through intravenous, through intraperitoneal, through intramuscular, through skin, through subcutaneous, through local, through the Sublingual or the per rectum means.Also can throw and comprise modified or the compositions of modified non-natural amino acid polypeptides not through liposome.It is known that described dosing way and appropriate dosage forms are generally one of ordinary skill in the art.

Also the ABP that comprises alpha-non-natural amino acid can be combined separately or with other suitable ingredients and make by sucking the aerosol of offeing medicine (be its can by " atomizing ").Aerosol can place pressurization can accept propellant, for example dichlorodifluoromethane, propane, nitrogen or the like.

Be applicable to that the dosage form by dispensing outside (for example) intraarticular (in the joint), intravenous, intramuscular, Intradermal, intraperitoneal and the subcutaneous route intestinal comprises aqueous and non-aqueous isotonic sterile injection solution (can contain antioxidant, buffer, antibacterial and make preparation and the isoosmotic solute of expection receiver's blood) and aqueous and non-aqueous sterile suspensions (can comprise suspending agent, solubilizing agent, thickening agent, stabilizing agent and antiseptic).Preparation through encapsulation ABP can be kept in unit dose or the multiple dose sealed container, for example ampoule and bottle.

Outer dispensing of intestinal and intravenous dispensing are preferred medication administration method.Specifically, the dosing way (include, but is not limited to be generally used for EPO, GH, ABP, G-CSF, GM-CSF, IFN, interleukin, antibody and/or any other medicine and pharmacology and transmit albumen) of natural amino acid congener therapy and preferred dispensing and the allotment approach that present used dosage form provides polypeptide of the present invention have been used for.

In content of the present invention, throwing is enough to have useful therapeutic response in time in the patient with patient's dosage, perhaps includes, but is not limited to suppress pathogenic infection, or depends on other suitable active of application.By the effect of specific support or preparation and activity, stability or serum half-life and the conditions of patients of used non-natural amino acid polypeptides, and patient's to be treated body weight or surface area are determined dosage.Existence, the nature and extent of any adverse side effect that also can be by following throwing and specific support, preparation etc. in the particular patient are determined the dosage size.

In determining treatment or prevent disease (including, but is not limited to cancer, genetic diseases, diabetes, AIDS or the like), wait to throw and carrier or the effective dose of preparation in, doctor's evaluation cycle plasma content, preparation toxicity, disease process and/or (when relevant) anti-non-natural amino acid polypeptides production of antibodies.

For instance, throw with the dosage of 70 kg of patient and treat in the proteic dosage range being equal to present use usually, the described dosage of scalable changes to obtain compositions related activity or serum half-life.Carrier of the present invention can comprise throwing and antibody, throwing and vaccine, throwing and cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, biological response modifier or the like by any known conventional therapy supplement therapy condition.

For dispensing, can include, but is not limited to be suitable for patient's quality and general health in LD-50 or ED-50 and/or throwing and preparation of the present invention under the determined speed of the side effect of any alpha-non-natural amino acid observed under the various concentration by related preparations.Can finish dispensing through single dose or separate doses.

If the patient through preparation transfusion generates heat, shiver with cold or myalgia, then he should accept the medicine of aspirin (aspirin), ibuprofen (ibuprofen), acetaminophen (acetaminophen) or other pain management/fever of suitable dosage.The patient who stands infusion reaction (for example heating, myalgia and shiver with cold) was premedicated at the aspirin of will infusing, acetaminophen or (including, but is not limited to) diphenhydramine before in 30 minutes.Pethidine (meperidine) is used for not rapidly in response to antipyretic and antihistaminic even more serious shiver with cold and myalgia.The cell transfusion is slowed down or stopped to the order of severity that depends on reaction.

Can directly throw with human antigen of the present invention the mammal person under inspection and combine polypeptide.By any approach dispensing that is generally used for introducing ABP to the person under inspection.Although any under the stable condition the most suitable way should depend on the character and the order of severity of the state of an illness to be treated, but comprise according to the ABP compositions of the embodiment of the invention and to be applicable to per os, per rectum, through the part, through sucking (including, but is not limited to pass through aerosol), through buccal (including, but is not limited to the Sublingual), transvaginal, intestinal is outer (to be included, but is not limited to subcutaneous, intramuscular, Intradermal, intraarticular, in the pleura, intraperitoneal, in the brain, intra-arterial or intravenous), the compositions of local (be skin and mucomembranous surface, comprise airway surface) and percutaneous dispensing.Can the part or the mode of general offer medicine.Compound formulation can be kept in unit dose or the multiple dose sealed container, for example ampoule and bottle.Can prepare be unit dose injection form (including, but is not limited to solution, suspension or emulsion) with the blended ABP of the present invention of pharmaceutically acceptable supporting agent.Also can be by continuously transfusion (use includes, but is not limited to micropump, for example osmotic pumps), single fast injection or the sustained-release storage type preparation ABP of the present invention that comes into operation.

The dosage form that is applicable to dispensing comprise aqueous and non-aqueous solution, etc. ooze sterile solution (can contain antioxidant, buffer, antibacterial and make the isoosmotic solute of preparation) and aqueous and non-aqueous sterile suspensions (can comprise suspending agent, solubilizing agent, thickening agent, stabilizing agent and antiseptic).Can be by previous sterilized powder, granule and pharmaceutical tablet solution and the suspension of describing kind.

Medical composition of the present invention can comprise pharmaceutically acceptable supporting agent.On the part by throw and particular composition and determine pharmaceutically acceptable supporting agent by the ad hoc approach that is used to throw with compositions.Therefore, exist the multiple dosage form that is suitable for medical composition of the present invention (comprising optionally pharmaceutically acceptable supporting agent, excipient or stabilizing agent) (referring to, Remington ' s Pharmaceutical Sciences for example, the 17th edition .1985).

Suitable supporting agent comprises and contains phosphate, borate, HEPES, citrate and other organic acid buffer; The antioxidant that comprises ascorbic acid; Low-molecular-weight (being less than about 10 residues) polypeptide; Albumen, for example serum albumin, gelatin or immunoglobulin; Hydrophilic polymer, for example polyvinylpyrrolidone; Aminoacid, for example glycine, glutamine, agedoite, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose or dextrin; Chelating agen, for example EDTA; Bivalent metal ion, for example zinc, cobalt or copper; Sugar alcohol, for example mannitol or sorbitol; Salify counter ion, for example sodium; And/or non-ionic surface active agent, for example Tween ^TM, Pluronics ^TMOr PEG.

Can throw and ABP of the present invention by sustained release system or as a sustained release system part, comprise the ABP that is connected with the water-soluble polymer of for example PEG.Sustained-release composition comprises that (including, but is not limited to) is the semipermeable polymers substrate of shaping particulate forms, includes, but is not limited to thin film or microcapsule.Lasting release matrix comprises biocompatible substance, for example poly-(2-hydroxyethyl methacrylate) (people such as Langer, J.Biomed.Mater.Res., 15:167-277 (1981); Langer, Chem.Tech., 12:98-105 (1982)), ethene-vinyl acetate people such as (, the same) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988), polylactic acid (United States Patent (USP) the 3rd, 773, No. 919; EP 58,481), polyglycolic acid, lactic acid/glycolic acid copolymer, poly-anhydride, L-glutamic acid and γ-ethyl-L-glutamate copolymer (people such as U.Sidman, Biopolymers, 22,547-556 (1983)), poly-(former) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate (chondroitin sulfate), carboxylic acid, fatty acid, phospholipid, polysaccharide, nucleic acid, polyamino acid, aminoacid (phenylalanine for example, tyrosine, isoleucine), polynucleotide, polyvinyl propylene (polyvinyl propylene), polyvinylpyrrolidone and silicone.Sustained-release composition comprises that also lipid catches chemical compound (liposomally entrapped compound).The liposome that can contain chemical compound by known method preparation itself: DE 3,218, and 121; People such as Epstein, Proc.Natl Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; With EP 102,324.Used list of references and patent are all incorporated this paper by reference into.

Can prepare liposome by (for example) method hereinafter described and catch ABP:DE 3,218,121; People such as Epstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; With EP 102,324.Liposome composition and size be for knowing, and can be rule of thumb easily definite by one of ordinary skill in the art.Some liposome case descriptions are in following document: people such as Park JW for example, Proc.Natl.Acad.Sci.USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (editor): MEDICALApplications of Liposomes (1998); People such as Drammond DC, Liposomal drug deliverysystems for cancer therapy, in Teicher B (editor): Cancer Drug Discovery andDevelopment (2002); People such as Park JW, Clin.Cancer Res.8:1172-1181 (2002); People such as Nielsen TJB, Biochim.Biophys.Ada 1591 (1-3): 109-118 (2002); People such as Mamot C, Cancer Res.63:3154-3161 (2003).Used list of references and patent are all incorporated this paper by reference into.

In content of the present invention, throw dosage with the patient and should be enough to cause among the person under inspection in time useful reaction.In general, every dosage intestinal is outer throw and total medicine and pharmacology effective dose of ABP of the present invention be about 0.01 microgram/kg/day to about 100 microgram/kilograms, or about 0.05 mg/kg about 1 mg/kg weight in patients extremely, but this is treated decision influence.Administration frequency is also treated decision influence, and can than to be proposed to be used in human commercially available ABP goods frequency higher or lower.In general, can throw and PEGization antigen-binding polypeptides of the present invention by any above-mentioned dosing way.

XV. the therapeutic use of antigen-binding polypeptides of the present invention

ABP polypeptide of the present invention can be used for treating many diseases.The medical composition that contains ABP can be deployed into effectively by the concentration of various means to the human patients dispensing that suffers disease, wherein said disease (for example may be subjected to ABP agonist or antagonist, but be not limited to, use antiproliferative, anti-inflammatory agent or antivirin) influence, it is independent or as the part of the state of an illness or disease.The average magnitude of ABP can be different, and specifically, should be based on the suggestion and the prescription of qualified physicians.The accurate amount of ABP is subjected to following factor affecting to the person under inspection: other composition in accurate state of an illness type for example to be treated, the status of patient of being treated and the compositions.The present invention also provides the another kind of activating agent of throwing with the treatment effective dose, for example anti-cancer chemotherapeutic agents.One of ordinary skill in the art can determine its dosage easily based on the ABP therapy.

Example

Provide following example to describe, rather than will limit the invention of being advocated.

Example 1

This case description is used for selecting non-naturally encoded amino acids is incorporated into a group of many potential criterion group of the preferred sites of ABP.

This example confirms how to select to be used in the antigen-binding polypeptides introduce the preferred sites of non-naturally encoded amino acids.Use comprises secondary, three grades or the quarternary structure of the three dimensional structure of two ABP molecules or ABP and determines the optimum position that one or more non-naturally encoded amino acids can be introduced.

Following standard is used to estimate each position that is used to introduce non-naturally encoded amino acids: residue (a) should not disturb based on the ABP of three dimensional structure structural analysis or the secondary of ABP, the combination of three grades or quarternary structure, (b) influence that not suddenlyd change by alanine or homologue scanning, (c) should the surface expose and show and the minimum Van der Waals force of residue (van der Waals) or hydrogen bond are done mutually on every side, (d) can be on one or more exposures of ABP, (e) can for the 2nd ABP or other molecule or the juxtaposed ABP of its fragment site, (f) in the ABP variant, should lack or variable, (g) should produce conservative after replacing it with non-naturally encoded amino acids changes, (h) by optionally changing the flexibility or rigidity of complete structure, ABP self be can regulate or dimer or the polymeric conformation of one or more ABP comprised, (i) be found in high flexibility district or the structural rigidity district and

(j) be found in and maybe can not see in the complementary determining region (CDR).In addition, adopt Cx program people Bioinformatics such as (, 18, the 980 pages) Pintar that the ABP molecule is further calculated to estimate the projecting degree of each albumen atom.Therefore, in certain embodiments, non-naturally encoded amino acids is replaced in one or more positions of (but being not limited to) ABP.

Example 2

This example describes the clone in detail and comprises the ABP of non-naturally encoded amino acids at expression in escherichia coli.

The translation system of introducing that will comprise quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) is used to express the ABP that contains non-naturally encoded amino acids.O-RS preferably uses non-naturally encoded amino acids aminoacylation O-tRNA.Translation system is again with among the ABP of non-naturally encoded amino acids insertion in response to coded selection codon.

Table 2:O-RS and O-tRNA sequence

SEQ?ID?NO：1	Methane coccus mtRNA CUA Tyr	tRNA
			SEQ?ID?NO：2	HLAD03; Optimization amber suppressor tRNA	tRNA
SEQ?ID?NO：3	HL325A; Optimization AGGA frame shift suppressor tRNA	tRNA
			SEQ?ED?NO：4	Be used to incorporate into aminoacyl tRNA synthetase to propylhomoserin p-Az-PheRS (6) in azido-L-benzene	RS
SEQ?ID?NO：5	Be used to incorporate into aminoacyl tRNA synthetase to benzoyl-L-phenylalanine p-BpaRS (1)	RS
			SEQ?ID?NO：6	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine Propargyl-PheRS	RS
SEQ?ID?NO：7	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine Propargyl-PheRS	RS
			SEQ?ID?NO：8	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine Propargyl-PheRS	RS
SEQ?ID?NO：9	Be used to incorporate into aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS (1)	RS
			SEQ?ID?NO：10	Be used to incorporate into aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS (3)	RS
SEQ?ID?NO：11	Be used to incorporate into aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS (4)	RS
			SEQ?ID?NO：12	Be used to incorporate into aminoacyl tRNA synthetase to azido-phenylalanine p-Az-PheRS (2)	RS
SEQ?ID?NO：13	Be used to incorporate into aminoacyl tRNA synthetase to acetyl group-phenylalanine (LW1)	RS
			SEQ?ID?NO：14	Be used to incorporate into aminoacyl tRNA synthetase to acetyl group-phenylpropyl alcohol hydracid (LW5)	RS
SEQ?ID?NO：15	Be used to incorporate into aminoacyl tRNA synthetase to acetyl group-phenylalanine (LW6)	RS
			SEQ?ID?NO：16	Be used to incorporate into aminoacyl tRNA synthetase to azido-phenylalanine (AzPheRS-5)	RS
SEQ?ID?NO：17	Be used to incorporate into aminoacyl tRNA synthetase to propylhomoserin in azido-benzene (AzPheRS-6)	RS

Allow the non-naturally encoded amino acids locus specificity is incorporated among the ABP with containing modified ABP gene and quadrature aminoacyl tRNA synthetase/tRNA plasmid transformation escherichia coli to (being specific to required non-naturally encoded amino acids).In the culture medium that is containing the specific non-naturally encoded amino acids of 0.01-100mM under 37 ℃, grow through the transformed into escherichia coli high fidelity and express modified ABP effectively.Producing His label (His-tagged) ABP that contains non-naturally encoded amino acids by e. coli host cell is inclusion body or aggregation.The dissolving aggregation, and under the degeneration condition in the 6M guanidine hydrochloride affinity purification.By under 4 ℃ at 50mM TRIS-HCl (pH 8.0), 40 μ M CuSO ₄Carry out again folding with dialysis overnight among 2% (w/v) Sarkosyl.Subsequently with described material to 20mM TRIS-HCl (pH 8.0), 100mM NaCl, 2mM CaCl ₂Dialysis then removes His label (His-tag).Referring to people such as Boissel, (1993) 268:15983-93.The method of purification ABP is that affiliated technical field is known, and is confirmed by SDS-PAGE, Western engram analysis or electron spray-ionizing ion trap mass spectrometry method or the like.

Expression construct

Pericentral siphon scFv-108: variable region (VL and VH) (United States Patent (USP) the 6th, 217, No. 866, it the incorporates this paper by reference into) clone of EGFR monoclonal antibody specific mAb108 is had (GGGGS) that is positioned at yeast BGL2 (C7) pericentral siphon targeting sequencing downstream ₄The scFv fragment of connexon sequence (Humphreys, people Protein Expr Purif such as DP in November, 2000; 20 (2): 252-64).The downstream that the epitope sequence that will be discerned by c-myc antibody and 6X-His label are cloned into the VL territory.Under the control of inducible promoters, the variant that contains succinum termination codon (TAG) in wild type scFv-108 construct and the VL territory (is seen Fig. 2, A) cloned in the coli expression carrier.This plasmid also constitutive expression from the amber suppressor tyrosyl-tRNA of methane coccus ^Tyr/CUA(Mj tRNA ^Tyr/CUA).Indicate the position of succinum termination codon.Constructed construct is shown in SEQ ID NO:18 (nucleotide sequence) and SEQ ID NO:19 (translation protein sequence).Construct is described in table 3 and 4.

Table 3:

Sequence	Position (SEQ ID NO:18)
		The C7 conductor	?15-83
VH	?84-446
		The GlySer connexon	?447-506
VL	?507-827
		Myc	?843-887
His	?888-905
		TAA	?906-908

Table 4:

Sequence	Position (SEQ ID NO:19)
		The C7 conductor	?1-23
VH	?24-144
		The GlySer connexon	?145-164

?VL	165-276
		?Mvc	277-291
?His	292-297

Cytoplasm scFv-108: under the control of T7 promoter, VH-connexon-VL (VH-GlySer-VL) clone that will contain N end MetGly-sequence and 6X-His sequence advances in the expression vector (to see Fig. 2, B).Indicate the position of succinum termination codon and PEGization residue.Constructed construct is shown in SEQ ID NO:20 (nucleotide sequence) and SEQ ID NO:21 (translation protein sequence).Construct is described in the table 5.

Table 5:

Sequence	Position (SEQ ID NO:20)
		His	?3-26
VH	?27-389
		The GlySer connexon	?390-449
VL	?450-767

Fab-108: VL and the VH sequence clone of mAb108 are advanced among the pFT3, and pFT3 is the constant plasmid with the CH1 territory of a kind of coding g3 (VL) and STII (VH) pericentral siphon targeting sequencing and human κ.The C end in CH1 territory contains the 6-His label and then promotes purification.Amber mutation is introduced the CH1 territory, and whole bicistronic mRNA cascade is cloned into constitutive expression from the amber suppressor tyrosyl-tRNA of methane coccus ^Tyr/CUA(MjtRNA ^Tyr/CUA) expression plasmid in.Show two ShineDelgamo sequences (SD) that drive the translation in Fab fragment VL and VH territory.Constructed construct is shown in SEQ ID NO:22 (nucleotide sequence) and SEQ ID NO:23 and 24 (the translation protein sequence of the VL κ chain of Fab 108 and the VH-CH1 chain of Fab108).Construct is described in the table 6.

Table 6:

Sequence	Position (SEQ ID NO:22)
		The g3 conductor	?15-68
VL	?81-416
		The K chain	?432-755
The STII conductor	?788-856
		VH	?875-1237
The CH1 territory	?1247-1543
		His	?1544-1561

Pericentral siphon scFv-4D5: with the variable region (VL and VH) of HER2 monoclonal antibody specific mAb-4D5 (Carter, people such as P., Biotechnology (N Y) .1992 February; 10 (2): 163-7) clone is the scFv fragment in yeast BGL2 (C7) pericentral siphon targeting sequencing downstream.At the C of VL sequence end (scFv-4D5-His; Fig. 2 is D) or at the N in VH territory end (His-scFv-4D5; Fig. 2, E) clone 6X-His label.Wild type scFv-4D5 construct and GlySer are connected variant that subdomain contains succinum termination codon (TAG) clone into constitutive expression from the amber suppressor tyrosyl-tRNA of methane coccus ^Tyr/CUA(MjtRNA ^Tyr/CUA) coli expression carrier.Constructed 6X-His C end construct is shown in SEQ ID NO:25 (nucleotide sequence) and SEQ ID NO:26 (translation protein sequence).Constructed 6X-His N end construct is shown in SEQ ID NO:27 (nucleotide sequence) and SEQ ID NO:28 (translation protein sequence).6X-His C end construct is described in the table 7, and 6X-His N end construct is set forth in the table 8.

Table 7:

Sequence	Position (SEQ ID NO:25)
		The C7 conductor	?15-83
VH	?84-443
		The GlySer connexon	?444-503
VL	?504-824
		His	?825-848

Table 8

Sequence	Position (SEQ ID NO:27)
		The C7 conductor	?15-83
His	?84-107
		VH	?108-470
The GlySer connexon	?471-530
		VL	?531-854

Fab-4D5: the VL of mAb 4D5 and VH sequence sub-clone are advanced to encode among the plasmid pFT3 in the constant and CH1 territory of g3 and STII pericentral siphon targeting sequencing and human κ, and advance the amber suppressor tyrosyl-tRNA of constitutive expression from the methane coccus with rear clone ^Tyr/CUA(Mj tRNA ^Tyr/CUA) expression plasmid.Fig. 2, F shows the cistron that is used to express Fab-4D5.Amber mutation is introduced 139 of the lysines in the CH1 territory of Fab 4D5.K142 among the corresponding Fab 108 of this lysine.Be structured in CH1 territory C end by overlapping PCR (Fab-4D5-cys) and contain extra cysteine residues (THT CAA) Fab-4D5 construct.Constructed construct is shown in SEQ ID NO:29 (nucleotide sequence) and SEQ ID NO:30 and 31 (the translation protein sequence of the VL κ chain of Fab 4D5 and the VH-CH1 chain of Fab 4D5).Construct is described in the table 9.

Table 9:

Sequence	The position
		The g3 conductor	?1-54
VL	?67-386
		The K chain	?403-726
The STII conductor	?759-827
		VH	?846-1205
The CH1 territory	?1215-1511
		His	?1512-1529

Expression/inhibition

With acetyl group-phenylalanine (pAcF) is suppressed: the known standard test scheme of technical field is finished the inhibition of amber mutation in the escherichia coli under using.In simple terms; for the pericentral siphon of reaching antibody fragment in the escherichia coli (scFv and Fab) suppresses, the expression vector establishment body is transformed in the e. coli host cell with the plasmid from quadrature tyrosyl--tRNA-synzyme (MjTyrRS) of methane coccus of encoding.Bacterial cultures overnight is diluted in the vibration flask that contains LB culture medium (Luria-Bertani) or Superbroth at 1: 100, and under 37 ℃, grows to OD and be approximately 0.8.Induce Fab and scFv to express, by adding acetyl group-phenylalanine (pAcF) to 4mM final concentration is suppressed amber codon simultaneously.Incubation culture overnight under 25 ℃.Carry out the expression of wild type (shortage amber codon) scFv and Fab (comprising Fab-4D5-cys) under the same conditions.Reach in a similar manner the segmental expression of Cytoplasm scFv/inhibition (Fig. 2, B).

Suppress with aa9.2: reach with pAcF derivant (aa9.2) in the mode similar and suppress amber mutation, except used quadrature tyrosyl-from the methane coccus-tRNA-synzyme (MjTyrRS) is specific to this aminoacid to pAcF.Finish inhibition by when inducing, adding aa9.2 (4mM).

Protein extraction and purification

Centrifugal collecting cell, and be resuspended in the pericentral siphon buffer release liquid (50mMNaPO that is supplemented with 100 μ g/ml lysozyme ₄, 20% sucrose, 1mM EDTA, pH 8.0) in, and cultivated 30 minutes on ice.After centrifugal, the antibody fragment in the supernatant is fixed on ProBind globule (Invitrogen by its His label; Carlsbad, CA) on, with binding buffer liquid thorough washing, and then will be molten from coming out from globule through bonded fragment with the 0.5M imidazoles.Purified fragment is dialysed to store buffer liquid (50mM HEPES, 150mM NaCl, 10% glycerol, 5% sucrose, pH 7.8).Analyze for the segmental small-scale of the scFv that in Cytoplasm, expresses, centrifugal collection escherichia coli from the 15ml culture, and be resuspended in dissolving buffer (B-PER, the Pierce Biotechnology that 1ml is supplemented with 10 μ g/ml DNase; Rockford, IL) in.In 37 ℃ of following incubation mixture 30 minutes, at Protein Loading buffer (Invitrogen; Carlsbad, CA) middle dilution is 1 times, and analyzes by SDS-PAGE.

Fig. 3, A show the inhibition of amber mutation in second serine in the GlySer connexon (S131Am), and show the scFv of the corresponding pAcF of containing purification (Fig. 3, B).As Fig. 3, the engram analysis of Western shown in the A proves, when cell is grown in LB or Superbroth culture medium, needs pAcF to stop mutation to suppress succinum.The existence of pAcF does not influence the expression (WTscFv-108) of the scFv that lacks TAG termination codon.Fig. 3, B shows by fixing metal affinity chromatography (IMAC) purification pAcF-scFv 108-(S131).The estimated output that contains the scFv of pAcF is 1.5mg/L.By the segmental position of arrow indication scFv.Following loading coomassie gel: swimming lane 1-scFv contrasts (1.7 μ g); Swimming lane 2-IMAC is pre-in conjunction with (20 μ l/70ml); Swimming lane 3-IMAC sky (20 μ l/70ml); Swimming lane 4-IMAC eluting (5 μ l/1.3ml); Swimming lane 5-NAP10 buffer-exchanged (10 μ l/1.5ml); Behind the swimming lane 6-IMAC globule eluting; Swimming lane 7-scFv contrasts (3.4 μ g).

The inhibition of amber mutation in the VL chain (L156) in during Fig. 4 showed cell matter expression scFv.Output is＞the 100mg/L culture of Escherichia coli, and termination codon suppress the to place one's entire reliance upon existence of pAcF.Arrow indication total length scFv.The solid circles indication is the product through truncate at the amber codon place.

PEGization/the dimerization of antibody fragment (1)

PEGization: in reaction buffer (100mM NaOAc, 150mM NaCl, 1mM EDTA, pH 4.0), about 1mg pAcF-scFv-108 albumen is concentrated into 50 μ l final volume.With 100 μ l final volume with simple function (azanol) the 5K PEG (balance in reaction buffer) of 100 times of molar excess in 28 ℃ of following incubation reaction mixture 32 hours.Estimate the PEGization material after the gel electrophoresis, and be directly used in cell in conjunction with in the test.

Dimerization: similar process is used for the scFv-108 fragment that dimerization contains pAcF.In simple terms, the scFv fragment of the initial pAcF of containing is concentrated in reaction buffer＞5 μ g/ μ l, and uses subsequently and the bonded PEG connexon of difunctionality azanol (364Da) incubation.Dialysis removes unreacted PEG, and adds pAcF-scFv fragment (1 mole of albumen: PE) of fresh aliquot in mixture.Incubation mixture 32 hours again under 28 ℃ subsequently.Dimer is loaded on 20mM sodium acetate (pH 4.0) equilibrated cation exchange columns (SP-5PW), and with NaCl gradient (0-0.4M) eluting.

Fig. 5 shows segmental PEGization of pAcF-scFv-108 and dimerization.Fig. 5, A shows the PEGization (5K) of pAcF-scFv-108-(L156) and pAcF-scFv-108-(S136), and the dimerization of pAcF-scFv-108-(S136).Following loading gel: swimming lane 1-pAcF-scFv-108-(L156) (5KPEG); Swimming lane 2-pAcF-scFv-108-(S136) (5K PEG); The dimerization of swimming lane 3-pAcF-scFv-108-(S136) (364da PEG) connexon; The dimerization of swimming lane 4-pAcF-scFv-108-(S136); Do not remove connexon after the one PEGization reaction.Indicate single PEGization scFv fragment and the dimeric position of scFv-108-(S136) by single arrow and double-head arrow respectively.No dimerization proof scFv and coupling in the swimming lane 4 without intermolecular disulfide bond forms.Fig. 5, C shows PEG and the segmental existence that combines the pAcF that places one's entire reliance upon of scFv.Do not observe the segmental PEGization of WT scFv.Following loading gel: swimming lane 1-WT scFv 108 contrasts; Swimming lane 2-scFvWT, in reaction buffer, no PEG, 16 hours; Swimming lane 3-scFv WT+5K PEG, in reaction buffer, 16 hours.

The SDSPAGE that Fig. 6 is presented at elution fraction collected during the cation-exchange chromatography of scFv homodimer analyzes.Use difunctionality azanol 364 dalton PEG connexon homotype dimerization pAcF-scFv108-(S131) fragments.Collect elution fraction during difference during cation-exchange chromatography in the NaCl gradient (0-0.4M).Following loading gel: swimming lane 1-indicates; Swimming lane 2-pAcF-scFv108 (S131)-X-pAcF-scFv108 (S131) elution fraction #1; Swimming lane 3-pAcF-scFv108 (S131)-X-pAcF-scFv108 (S131) elution fraction #2; Swimming lane 4-pAcF-scFv108 (S131)-X-pAcF-scFvl08 (S131) elution fraction #3.

Fig. 8, A show that pAcF and the segmental SDS PAGE of pAcF-PEGization Fab analyze.Be presented at the Fab-108 fragment that K142, T204 and K219 place are modified, and the efficient of PEGization is locus specificity.The segmental PEGization of Fab that use contains pAcF through the bonded 5K PEG of azanol.

Figure 10, A and B show C end (pAcF-scFv-4D5-His (S133); Figure 10, A) or N end (pAcF-His-scFv-4D5 (S139); Figure 10, the B) inhibition of amber mutation in segmental GlySer connexon second serine of scFv-4D.Induce the proteic expression of scFv by adding 0.02% arabinose, last 5 hours or (16 hours) overnight.Add the inhibition that aa9.2 (4mM) finishes amber mutation by following.Arrow is indicated downtrod product, and solid circles indication truncated protein.Reach inhibition output (1.5mg/L) greater than 50%.Indication is mounted with the contrast swimming lane (C) of scFv 108, and the ratio scFv-4D5 that it runs is slightly high.

Express Fab fragment pAcF-Fab-4D5-(K139) and Fab-4D5-cys, and purification in the same manner.Figure 11, A are presented at the sample that SDS-PAGE analyzed under reduction and the non-reduced condition.Fab fragment output is as follows: pAcF-Fab-4D5-(K139) is 0.37mg/L/OD (final OD ₆₀₀=3.14), and Fab-4D5-cys is 0.23mg/L/OD (final OD ₆₀₀=3.26).Figure 11, B shows the Figure 11 that uses anti-His antibody, the Western trace of sample shown in the A (5 μ l).Under non-reduced condition, make sample leakage of electricity swimming.The poly VH-CH1 complex of arrow indication Fab-4D5-cys construct.Do not find to have the polycomplex of pAcF-Fab-4D5-(K139).

PEGization/the dimerization of antibody fragment (2)

PEGization: at natural response buffer (20mM NaOAc, 150mM NaCl, 1mM EDTA, pH4.0) in and in the reaction of degeneration buffer (20mM NaOAc, 150mM NaCl, 1mM EDTA, 8M carbamide, pH 4.0) the pAcF-scFv-108 albumen of about 1mg is concentrated into 50 μ l final volume.With 100 μ l final volume with simple function (azanol) the 5K PEG (balance in the respective reaction buffer) of 100 times of molar excess in 28 ℃ of following incubation reaction mixture 32 hours.After 16 hours,, and be directly used in cell in conjunction with test by SDS-PAGE evaluation response mixture.

Polypeptide can have one or more advantages with combining of connexon, polymer, bioactive molecule or other molecule under the degeneration condition.These advantages include, but is not limited to make in conjunction with easier owing to the improvement accessibility of reactive group; With do not compare in conjunction with polypeptide, through easier to be folding again in conjunction with polypeptide; With compare with available peptide concentration under non-degeneration condition, can use the higher concentration polypeptide to be used for combination.For instance, if polypeptide is unstable and can not highly concentrate to be used for association reaction, then may need the degeneration condition.Yet, with standard based on cysteine combine chemistry (for example maleimide chemistry) or based on the reaction of the chemistry (for example maleimide chemistry) of lysine after, under the degeneration condition, can produce in the polypeptide and the bonded non-required and/or unexpected site of one or more cysteine, lysine or other aminoacid in conjunction with polypeptide.Described bonded non-required and/or unexpected site may influence through the activity in conjunction with polypeptide.On the other hand, be the part of non-naturally encoded amino acids owing to relate to the reactive group of association reaction, so polypeptide (ABP that for example comprises one or more non-naturally encoded amino acids) can be with site-specific mode combination under the degeneration condition.Therefore, can develop under the degeneration condition available from bonded advantage by means of the polypeptide that comprises one or more non-naturally encoded amino acids.

Fig. 5 B shows that the SDS-PAGE of PEGization pAcF-scFv-(S136) and control sample analyzes.Following loading gel: swimming lane 1-pAcF-scFv-(S136); Swimming lane 2-pAcF-scFv-(S136) was 28 ℃ of following incubations 16 hours; Swimming lane 3-pAcF-scFv-(S136), 5K PEG, 28 ℃ of following incubations 16 hours, natural endowment; Swimming lane 4-pAcF-scFv-(S136), 5K PEG, 28 ℃ of following incubations 16 hours, the degeneration condition.Arrow indication scFv and PEGization scFv.Fig. 5 C shows, PEG and the segmental existence that combines the pAcF that places one's entire reliance upon of scFv.Do not observe the segmental PEGization of WT scFv.Following loading gel: swimming lane 1-WT scFv 108 contrasts; Swimming lane 2-scFv WT, in reaction buffer, no PEG, 16 hours; Swimming lane 3-scFv WT+5KPEG, in reaction buffer, 16 hours.

Continuous dimerization: in simple terms, the scFv fragment of the initial pAcF of containing is concentrated into 10mg/ml in reaction buffer, and use subsequently 100 times excessive in the bonded PEG connexon of difunctionality azanol (364Da) incubation.In 28 ℃ of following incubation reaction mixture 16 hours.Dialysis removes unreacted PEG, and adds pAcF-scFv fragment (1 mole of albumen: PE) of fresh aliquot in mixture.Incubation mixture 32 hours again under 28 ℃ subsequently.See Figure 13.Dimer is loaded on the equilibrated cation exchange column of 20mM sodium acetate (pH 4.0) (SP-5PW, 20 microns), and with NaCl gradient (0-0.4M) eluting.

Figure 14, A and B show that the irreducibility of dimerization sample and reproducibility SDS PAGE analyze.Following loading gel: swimming lane 1-has the final scFv dimerization reactant mixture of 364 Da PEG difunctionality connexons; Swimming lane 2-does not have the final scFv dimerization reaction control mixture of 364 Da PEG difunctionality connexons; Swimming lane 3-scFv.Arrow indication scFv dimer and scFv monomer.Only in the presence of the difunctionality connexon, synthesize the scFv dimer.No dimerization proof scFv and coupling in the swimming lane 2 without intermolecular disulfide bond forms.Owing to do not observe difference between the sample that reproducibility and irreducibility SDS PAGE gel are analyzed, so the existence of difunctionality connexon does not help intermolecular disulfide bond formation.

Dimer purification: use strong cat ion exchange column (SP-5PW, 20 microns) purification dimerization reactant mixture.Buffer A: 20mM NaOAc, pH 4.0; Buffer B: 20mM NaOAc, 1M NaCl, pH4.0.With 40%B eluting scFv dimer.Purified dimeric SDS PAGE analyzes (Figure 15) and shows that dimeric purity is approximately 90% after the column purification.

Fig. 9 shows the example of Heterobifunctional ABP of the present invention.Based on the known crystal structure that two different antibodies molecules (for example He Saiting and Omnitarg) of the different epitopes that are incorporated into same antigen (for example ErbB2) are measured, differentiate the specific amino acid position, cause it to meet specific required choice criteria.The required choice criteria of amino acid position is included in the locational relative proximity of one or more specific amino acid of each molecule in this example.Need described amino acid position to use link molecule to form Heterobifunctional molecule shown in Figure 9.Following table 10 shows standard compliant specific amino acid position on each molecule, as the link molecule that can be used for forming Heterobifunctional ABP.One of ordinary skill in the art will be appreciated that described tabulation by no means for detailed only for illustrative, and all amino acid positions that meet specific required choice criteria all are applicable to the present invention.Alpha-non-natural amino acid of the present invention can be in each molecule one or more these positions through replacing, to be provided for the chemical functional group that connexon connects.Multiple other choice criteria can be used to also differentiate that amino acid position is to meet required standard, include, but is not limited to the proximity between the identical or different molecule, conformation change is regulated, between the ABP or be connected in distance adjustment between the molecule of ABP, connexon length or shape, the surface exposes, and part is in conjunction with adjustment of features, receptor dimerization adjusting or the like.

Table 10
		Potential key site (He Saiting heavy chain-Pa trastuzumab light chain)	Distance (_)
Asn211-Asp28	15.4A
		Asn211-Gly68	15.8A
Asn211-Ser30	15.8A
		Asn211-Ser67	17.8A
Lys213-Asp28	18.0A
		Asn211-Thr69	18.1A
Lys213-Gly68	18.2A
		Lys208-Ser30	19.2A
Lys213-Thr69	20.1A
		Lys208-Ser67	20.6A
Lys213-Asp70	21.8A
		Thr123-Tyr92	24.9A
Ser122-Tyr92	26.2A
		Gln13-Tyr92	27.9A

Based among the HIV-1 and human Fab 4E10 and the proteic mutual work of HIV gp41env, discriminating specific amino acid position makes it meet specific required choice criteria.The required choice criteria of amino acid position comprises and can be used for T-20 peptide and the bonded residue of Fab in this example, cause combining of T-20 and gp41 taken place, and the combining and the identification of 4E10 complementary determining region (CDR) of not negative influence 4E10 complementary determining region (CDR) and gp41.T-20 (being also referred to as DP-178) suppresses HIV and enters cell by serving as viral fusion inhibitor.Figure 12 shows among the HIV with simulation g41 peptide and human Fab 4E10.Show and be used for the potential residue that the T-20 peptide connects.The potential residue that is used to incorporate into non-naturally encoded amino acids includes, but is not limited to the Gln64 heavy chain of Fab, the Glul light chain of Fab and the Gln27 light chain of Fab.One of ordinary skill in the art will be appreciated that described tabulation by no means for detailed only for illustrative, and all amino acid positions that meet specific required choice criteria all are applicable to the present invention.Multiple other choice criteria can be used to also differentiate that amino acid position is to meet required standard, include, but is not limited to the proximity between the identical or different molecule, conformation change is regulated, between the ABP or be connected in distance adjustment between the molecule of ABP, forgiving of connexon, connexon length or shape, the surface exposes, part is in conjunction with adjustment of features, receptor dimerization adjusting or the like.

Example 3

This example describe in detail contain the introducing of carbonylamino acid and subsequently with the reaction that contains aminooxy group PEG.

This example demonstration produces incorporates the method that the antigen-binding polypeptides that contains the ketone non-naturally encoded amino acids is arranged into, and wherein said antigen-binding polypeptides is subsequently with about 5, and 000MW contains aminooxy group PEG reaction.Replace by non-naturally encoded amino acids respectively according to each residue that the standard of example 1 is differentiated with following structure:

The sequence that is used for incorporating ABP into to acetyl group-phenylalanine locus specificity is (muttRNA, the M.jannaschii mtRNA of the SEQ ID NO:1 described in the example 2 above ^Tyr _CUA) and 13,14 or 15 (TyrRS LW1,5 or 6).

In case modified, comprise contain carbonylamino acid the ABP variant promptly with following form contain aminooxy group PEG derivatives reaction: R-PEG (N)-O-(CH ₂) _n-O-NH ₂,

Wherein R is a methyl, n be 3 and N be approximately 5,000MW.To be dissolved in 25mM MES (SigmaChemical; St.Louis; MO) (pH 6.0), 25mM Hepes (Sigma Chemical; St.Louis; MO) (pH 7.0) or be dissolved in 10mM sodium acetate (Sigma Chemical; St.Louis; MO) the purified ABP (10mg/mL) that contains acetyl phenyl alanine in (pH 4.5) reacts with 10 to 100 times of excessive aminooxy group PEG that contain; and then at room temperature stir 10-16 hour (Jencks; W.J.Am.Chem.Soc.1959; 81, the 475 pages).PEG-ABP is diluted in the suitable buffer subsequently, carries out purification and analysis immediately.

Example 4

Combine with the PEG that forms by the azanol group that is connected in PEG through amido link.

The PEG reagent that process described in the use-case 3 will have a following structure is coupled to and contains the ketone non-naturally encoded amino acids:

R-PEG(N)-O-(CH ₂) ₂-NH-C(O)(CH ₂) _n-O-NH ₂

R=methyl wherein, n=4 and N are approximately 20,000MW.Described in reaction, purification and analysis condition such as the example 3.

Example 5

This example describes in detail to be introduced two unique non-naturally encoded amino acids among the ABP.

The demonstration of this example produces the method for incorporating the antigen-binding polypeptides that non-naturally encoded amino acids is arranged into, and wherein said non-naturally encoded amino acids comprises ketone two positions of being differentiated according to example 1, wherein X ^*The expression non-naturally encoded amino acids.Described in example 1 and 2, prepare antigen-binding polypeptides, except two in nucleic acid unique site are introduced the suppressor gene codon.

Example 6

This example describes antigen-binding polypeptides in detail and contains combining and subsequently in-situ reducing of hydrazides PEG.

Preparation is incorporated into according to the process described in example 2 and 3 antigen-binding polypeptides that contains carbonylamino acid.In case after modifying, the hydrazides PEG that contains with following structure promptly combines with ABP:

R-PEG(N)-O-(CH ₂) ₂-NH-C(O)(CH ₂) _n-X-NH-NH ₂

R=methyl wherein, n=2 and N=10,000MW and X are carbonyl (C=O).To be dissolved in 25mM MES (Sigma Chemical; St.Louis; MO) (pH 6.0), be dissolved in 25mM Hepes (Sigma Chemical; St.Louis; MO) (pH 7.0) or be dissolved in 10mM sodium acetate (Sigma Chemical; St.Louis, MO) the purified ABP (0.1-10mg/mL) that contains acetyl phenyl alanine in (pH 4.5) reacts with 1 to 100 times of excessive hydrazides PEG that contains, and by adding 1M raw material NaCNBH ₃(Sigma Chemical, St.Louis MO) (is dissolved in H ₂O) come the corresponding hydrazone of in-situ reducing to the 10-50mM final concentration.4 ℃ were reacted 18-24 hour to RT in the dark.(Sigma Chemical, St.Louis MO) come cessation reaction to 50mM Tris final concentration, perhaps reaction are diluted in the suitable buffer to be used for purification immediately by add 1M Tris pH 7.6 times.

Example 7

This example describes in detail will contain among the alkynyl amino acid introducing ABP and with the mPEG-nitrine and carries out derivatization.

Any residue of being differentiated according to example 1 is replaced through following non-naturally encoded amino acids:

Being used for locus specificity incorporates into the sequence of propargyl-tyrosine is (muttRNA, the Mjannaschii mtRNA of the SEQ IDNO:1 described in the example 2 above _CUA ^Tyr) and 6,7 or 8.Contain the antigen-binding polypeptides of propargyl tyrosine and condition purification described in the use-case 3 at expression in escherichia coli.

(the purified ABP (0.1-10mg/mL) that contains propargyl-tyrosine pH=8) and 10 to 1000 times of excessive nitrine PEG that contain are added in the reactant mixture for 100mM sodium phosphate, 0.15M NaCl will to be dissolved in the PB buffer.Subsequently with the CuSO of catalytic amount ₄Be added in the reactant mixture with the Cu line.After incubation (included, but is not limited to room temperature or 37 ℃ about 4 hours down, or 4 ℃ overnight down) mixture, add H ₂O, and mixture filtered through dialyzer.Add being used for by (including, but is not limited to) and process analysis sample similar described in the example 3.

In this example, PEG has following structure:

R-PEG(N)-O-(CH ₂) ₂-NH-C(O)(CH ₂) _n-N ₃

R=methyl wherein, n=4 and N are approximately 10,000MW.

Example 8

This example describes in detail by propargyl tyrosine and replaces big among the ABP, hydrophobic amino acid.

As described in example 7, Phe, the Trp or the Tyr residue that are present in the ABP sequence are replaced by following non-naturally encoded amino acids.

In case after modifying, PEG promptly is connected in and comprises the ABP variant that contains alkynyl amino acid.PEG has following structure: Me-PEG (N)-O-(CH ₂) ₂-N ₃,

And coupling process will be followed described in the example 7.This can produce the ABP variant that comprises non-naturally encoded amino acids, described non-naturally encoded amino acids and a kind of natural generation, the big roughly same coordination of hydrophobic amino acid (isosteric), and the unique site in polypeptide is modified through the PEG derivant.

Example 9

This example describes the generation of the homodimer, heterodimer, homology polymer or the heteromultimers that are separated by one or more PEG connexons in detail.

The difunctionality PEG derivatives reaction that contains alkynes ABP variant and following form that is produced in the example 7:

N ₃-(CH ₂) _n-C(O)-NH-(CH ₂) ₂-O-PEG(N)-O-(CH ₂) ₂-NH-C(O)-(CH ₂) _n-N ₃，

(wherein n be 4 and PEG have be approximately 5,000 average MW) to produce corresponding A BP homodimer, wherein two ABP molecules by on the PEG physical location separately.Antigen-binding polypeptides can be coupled to one or more other polypeptide in a similar manner to form heterodimer, homology polymer or heteromultimers.Described in example 7 and 3, carry out coupling, purification and analysis.

Example 10

This example describes in detail sugar moieties and ABP coupling.

Described in example 3, one or more amino acid residues of ABP are replaced by following non-naturally encoded amino acids.

In case modified, comprise the ABP variant that contains carbonylamino acid and promptly be connected the reaction of aminooxy group analog with the β of N-acetyl glucosamine (GlcNAc).ABP variant (10mg/mL) is mixed in 100mM sodium acetate water buffer (pH 5.5) with aminooxy group sugar (21mM), and 37 ℃ of following incubations 7 to 26 hours.By being used in UDP-galactose (16mM) and the β-1 in the 150mM HEPES buffer (pH 7.4) at ambient temperature; 4-galactose acyltransferase (galacytosyltransferase) (0.4 units per ml) incubation is coupled to first sugar (people such as Schanbacher through the bonded ABP of sugar (5mg/mL) with the second carbohydrase method; J.Biol.Chem.1970; 245,5057-5061).

Example 11

This example describes the generation of PEGization ABP antagonist in detail.

Described in example 3, one or an ABP amino acid residue are replaced by following non-naturally encoded amino acids.

In case modified, comprise contain carbonylamino acid the ABP variant promptly with following form contain aminooxy group PEG derivatives reaction:

R-PEG(N)-O-(CH ₂) _n-O-NH ₂，

(wherein R is a methyl, n be 4 and N be 20,000MW) comprise the ABP antagonist of non-naturally encoded amino acids with generation, the single site of wherein said non-naturally encoded amino acids in polypeptide modified through the PEG derivant.Carry out coupling, purification and analysis as example 3.

Example 12

Wherein the ABP molecule is the generation through direct-connected ABP homodimer, heterodimer, homology polymer and heteromultimers

Comprise the ABP variant that contains alkynyl amino acid and can directly be coupled to comprise and contain amino acid whose another ABP variant of azido, each ABP variant is replaced including (but not limited to) the non-naturally encoded amino acids in the site described in the example 10.This can produce corresponding A BP homodimer, and wherein two ABP variant physics link to each other.Antigen-binding polypeptides can be coupled to one or more other polypeptide in a similar manner to form heterodimer, homology polymer or heteromultimers.Carry out coupling, purification and analysis as example 3,6 and 7.

Example 13

A. B

Ployalkylene glycol (P-OH) forms ether (B) with alkyl halide (A) reaction.In these chemical compounds, n is 1 to 9 integer, and R ' can be straight or branched, saturated or unsaturated C1 to C20 alkyl or assorted alkyl.R ' also can be for the assorted alkyl of the saturated or unsaturated cycloalkyl of C3 to C7 or ring-type, be substituted or be unsubstituted aryl or heteroaryl, or is substituted or is unsubstituted alkaryl (alkyl is the saturated or unsaturated alkyl of C1 to C20) or assorted alkaryl.PEG-OH is generally has 800 to 40,000 dalton (Da) molecular weight polyethylene glycol (PEG) or mono methoxy polyethylene glycol (mPEG).

Example 14

(12mg 0.5mmol) handles and has 20, the mPEG-OH of 000Da molecular weight (mPEG-OH 20kDa to be used in NaH among the THF (35mL); 2.0g, 0.1mmol, Sunbio).In described solution, add subsequently and be dissolved in formed 80 weight % propargyl bromide solution in the dimethylbenzene (50 equivalents Aldrich) and the KI of catalytic amount, and reflux the gained mixture heated 2 hours for 0.56mL, 5mmol.Add entry (1mL) subsequently, and move down in vacuum and to desolventize.In residue, add CH ₂Cl ₂(25mL), and separate organic layer, through anhydrous Na ₂SO ₄Drying, and volume is reduced to about 2mL.With this CH ₂Cl ₂Solution dropwise is added in the ether (150mL).Collect the gained precipitate, with some parts of cold diethyl ether washings, and dry, obtain propargyl-O-PEG.

Example 15

(12mg 0.5mmol) handles and to have 20, the mPEG-OH of 000Da molecular weight (mPEG-OH 20kDa to be used for the NaH of THF (35mL); 2.0g, 0.1mmol, Sunbio).(0.53mL, 5mmol is Aldrich) with catalytic amount KI to add 50 equivalent 5-bromo-1-pentynes subsequently in mixture.With the gained mixture heated to refluxing 16 hours.Add entry (1mL) subsequently, and move down in vacuum and to desolventize.In residue, add CH ₂Cl ₂(25mL), and the separation organic layer, anhydrous Na ₂SO ₄Drying, and volume is reduced to about 2mL.With this CH ₂Cl ₂Solution dropwise is added in the ether (150mL).Collect the gained precipitate, with some parts of cold diethyl ether washings, and drying obtains corresponding alkynes.5-chloro-1-pentyne can be used in the similar reaction.

Example 16

(1)

(2)

(3)

(4)

To 3-hydroxy-benzyl alcohol (2.4g, 20mmol) at first add Powdered sodium hydroxide (1.5g in the solution in THF (50mL) and water (2.5mL), 37.5mmol), then add be dissolved in formed 80 weight % propargyl bromide solution in the dimethylbenzene (3.36mL, 30mmol).Reactant mixture is heated to backflow 6 hours.In mixture, add 10% citric acid (2.5mL), and move down in vacuum and to desolventize.With ethyl acetate (3 * 15mL) extracted residues, and the organic layer after merging with saturated NaCl solution (10mL) washing, MgSO ₄Dry and the concentrated 3-alkynes propoxyl group benzylalcohol that obtains.

Under 0 ℃ with methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL 20mmol) adds chemical compound 3 (2.0g, CH 11.0mmol) ₂Cl ₂Solution, and reaction placed refrigerator 16 hours.Usually reaction generates light yellow oily methanesulfonates.With described oil (2.4g 9.2mmol) is dissolved among the THF (20mL), and add LiBr (2.0g, 23.0mmol).Reactant mixture is heated to backflow 1 hour, and then is cooled to room temperature.In mixture, add entry (2.5mL), and move down in vacuum and to desolventize.With ethyl acetate (3 * 15mL) extracted residues, and the organic layer after merging with saturated NaCl solution (10mL) washing, anhydrous Na ₂SO ₄Dry also concentrating obtains required bromide.

With mPEG-OH 20kDa (1.0g, 0.05mmol Sunbio) are dissolved among the THF (20mL), and in ice bath cooling solution.(6mg, 0.25mmol), (2.55g is 11.4mmol) with catalytic amount KI then to add the above-mentioned bromide that obtains to follow vigorous stirring to add NaH in several minutes.Remove cooling bath, and with the gained mixture heated to refluxing 12 hours.In mixture, add entry (1.0mL), and move down in vacuum and to desolventize.In residue, add CH ₂Cl ₂(25mL), and the separation organic layer, anhydrous Na ₂SO ₄Drying, and volume is reduced to about 2mL.Dropwise add and generate white depositions in the diethyl ether solution (150mL), collection white precipitate deposits yields PEG derivant.

Example 17

As indicated above, also can obtain to contain poly-(ethylene glycol) polymer of terminal alkynes by poly-(ethylene glycol) polymer that will contain functional end-group and the reactive molecule coupling that contains alkynes functional group.N is between 1 and 10.R ' can be the little alkyl of H or C1 to C4.

Example 18

(1)

(2)

(2.943g 3.0mmol) is dissolved in CH with the 4-pentinoic acid ₂Cl ₂(25mL).Add N-hydroxy-succinamide (3.80g, 3.3mmol) and DCC (4.66g, 3.0mmol), and agitating solution at room temperature overnight.With the thick NHS ester 7 of gained without being used for following reaction under being further purified.

To have 5, the mPEG-NH of 000Da molecular weight ₂(mPEG-NH ₂, 1g Sunbio) is dissolved among the THF (50mL), and mixture is cooled to 4 ℃.Follow vigorous stirring to pursue part to add a NHS ester 7 (400mg, 0.4mmol).Mixture was stirred 3 hours, make it be warmed to room temperature simultaneously.Add entry (2mL) subsequently, and move down in vacuum and to desolventize.In residue, add CH ₂Cl ₂(50mL), and the separation organic layer, anhydrous Na ₂SO ₄Drying, and volume is reduced to about 2mL.With this CH ₂Cl ₂Solution dropwise is added in the ether (150mL).Collect the gained precipitate, and dry in a vacuum.

Example 19

This example is the preparation about the methane sulfonyl ester of poly-(ethylene glycol), and the sulfonyl methane ester of poly-(ethylene glycol) also can be called the methane sulfonate or the methanesulfonates of poly-(ethylene glycol).Can prepare corresponding tosylate and halogenide by similar process.

Under nitrogen will in the mPEG-OH in the 150mL toluene (MW=3,400,25g, 10mmol) azeotropic distillation is 2 hours, and solution is cooled to room temperature.In solution, add the anhydrous CH of 40mL ₂Cl ₂With 2.1mL anhydrous triethylamine (15mmol).Cooling solution in ice bath, and dropwise add 1.2mL through distillation methane sulfonyl chloride (15mmol).Agitating solution overnight under the room temperature under nitrogen, and come cessation reaction by adding the 2mL dehydrated alcohol.The vaporising under vacuum mixture mainly is a solvent except that toluene to remove, and filters, and concentrates under vacuum, and then precipitates in the 100mL ether.With several parts of cold diethyl ether washing filter liquors, and dry in a vacuum to generate methanesulfonates.

(20g 8mmol) is dissolved among the 75ml THF, and solution is cooled to 4 ℃ with methanesulfonates.In cold soln, add Hydrazoic acid,sodium salt (1.56g, 24mmol).Under nitrogen, reaction is heated to and refluxed 2 hours.Evaporating solvent subsequently, and use CH ₂Cl ₂(50mL) dilution residue.With organic part of NaCl solution washing, and use anhydrous MgSO ₄Dry.Volume is reduced to 20ml, and be settled out product by adding in the cold absolute ether of 150ml.

Example 20

(1)

(2)

(3)

Can use United States Patent (USP) 5,998, method described in 595 generates 4-azido benzylalcohol, and described patent is incorporated this paper by reference into.Under 0 ℃ with methane sulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL 20mmol) joins 4-azido benzylalcohol (1.75g, CH 11.0mmol) ₂Cl ₂In the solution, and reaction placed refrigerator 16 hours.Usually reaction generates light yellow oily methanesulfonates.Described oil (9.2mmol) is dissolved among the THF (20mL), and adds LiBr (2.0g, 23.0 mmol).Reactant mixture is heated to backflow 1 hour, and is cooled to room temperature subsequently.In mixture, add entry (2.5mL), and move down in vacuum and to desolventize.With ethyl acetate (3 * 15mL) extracted residues, and the organic layer after merging with saturated NaCl solution (10mL) washing, anhydrous Na ₂SO ₄Dry also concentrating obtains required bromide.

Be used for THF (35mL) NaH (12mg, 0.5mmol) handle mPEG-OH 20kDa (2.0 g, 0.1mmol, Sunbio), and in mixture, add bromide (3.32g, 15mmol) and catalytic amount KI.With the gained mixture heated to refluxing 12 hours.In mixture, add entry (1.0mL), and move down in vacuum and to desolventize.In residue, add CH ₂Cl ₂(25mL), and the separation organic layer, anhydrous Na ₂SO ₄Drying, and volume is reduced to about 2mL.Dropwise add and generate precipitate, collecting precipitation deposits yields mPEG-O-CH in the diethyl ether solution (150mL) ₂-C ₆H ₄-N ₃

Example 21

With NH ₂-PEG-O-CH ₂CH ₂CO ₂(MW 3, and 400Da 2.0g) is dissolved in NaHCO for H ₃(10mL) in the saturated aqueous solution, and solution is cooled to 0 ℃.Follow vigorous stirring to add 3-azido-1-N-N-Hydroxysuccinimide base propionic ester (5 equivalent).After 3 hours, add 20mL H ₂O, and restir mixture 45 minutes at room temperature.Use 0.5N H ₂SO ₄PH value is adjusted to 3, and adds NaCl to about 15 weight % concentration.Use CH ₂Cl ₂(100mL * 3) extractive reaction mixture, Na ₂SO ₄Dry and concentrated.After the cold diethyl ether precipitation, filter and collect product, and drying generates ω-carboxyl-nitrine PEG derivant under vacuum.

Example 22

Follow vigorous stirring, to as the known preparation of affiliated technical field and be cooled in-78 ℃ the THF solution of ethinylation lithium (4 equivalent) and dropwise add the mPEG-OMs solution that is dissolved among the THF.After 3 hours, make reaction be warmed to room temperature, and stop by adding the 1mL butanols.Add 20mL H subsequently ₂O, and restir mixture 45 minutes at room temperature.Use 0.5N H ₂SO ₄PH value is adjusted to 3, and adds NaCl to about 15 weight % concentration.Use CH ₂Cl ₂(100mL * 3) extractive reaction mixture, Na ₂SO ₄Dry and concentrated.After the cold diethyl ether precipitation, filter and collect product, and drying generates 1-(fourth-3-alkynyloxy group)-methoxy poly (ethylene glycol) (mPEG) under vacuum.

Example 23

Using the method described in the following document will contain nitrine optionally incorporates in the albumen with the amino acid sites that contains acetylene: people such as L.Wang, (2001), Science292:498-500, people such as J.W.Chin, Science301:964-7 (2003)), people such as J.W.Chin, (2002), Journal of the American Chemical Society124:9026-9027; J.W.Chin and P.G.Schultz, (2002), Chem Bio Chem11:1135-1137; People such as J.W.Chin, (2002), PNAS United States of America99:11020-11024: and L.Wang and P.G.Schultz, (2002), Chem.Comm..1-10.In case incorporate into after the aminoacid, promptly at 2mM PEG derivant, 1mM CuSO ₄Exist the 0.01mM albumen that under 37 ℃, is used for phosphate buffer (PB) (pH 8) down to carry out cycloaddition reaction 4 hours with about 1mg Cu line.

Example 24

This case description is measured the active in vitro and in vivo method of the ABP and the PEGization ABP that comprise non-naturally encoded amino acids.

Cell is in conjunction with test

Have or do not have various concentration (volume: 10 μ l) in the presence of un-marked ABP, ABP or the negative control, and ¹²⁵I-ABP (about 100,000cpm or 1ng) exists down, under 0 ℃ in PBS/1%BSA (100 μ l) cultured cell (3 * 10 ⁶) 90 minutes (cumulative volumes: 120 μ l), and once repeat.Subsequently cell is resuspended among the ice-cold FCS of 200 μ l in the 350 μ l plastic centrifuge tubes, and layering, and centrifugal (1000g; 1 minute).Collect centrifugal fritter by the end that cuts away centrifuge tube, and on gamma counter (Packard), count centrifugal fritter and supernatant respectively respectively.

Specificity is deducted 100 times of combinations (non-specific binding) in the presence of the excessive un-marked ABP (cpm) in conjunction with the total binding (double meansigma methods) that (cpm) is defined as in the presence of uncontested person.Employed each cell type is measured non-specific binding.Use same ¹²⁵The I-ABP preparation is not experimentizing on the same day, and should show internal consistency.Un-marked natural A BP or ABP, rather than negative control suppresses combination in the dose dependent mode.The ABP competition is in conjunction with natural ¹²⁵The ability of I-ABP shows that receptor is discerned this two kinds of forms equally.

For carry out the combination test with scFv-108, collecting cell after handling with insulin is resuspended in FACS buffer (PBS, 2%FBS, 0.01%NaN ₃) in, and be inoculated in microtitration plate (3 * 10 at the bottom of 96 hole circles subsequently ⁵Cells/well) in.With variable concentrations wild type or the scFv-108 fragment that contains pAcF cultured cell on ice 30 minutes.Remove not in conjunction with scFv albumen by centrifugal after scouring (repeating 2-3 time).Use mAb-108 (ATCC#HB 9764) at 7.5nM (EC subsequently ₈₀) cultured cell 30 minutes under the concentration.After twice washing, use anti-mouse antibodies (the 10 μ g/ml) cultured cell 30 minutes of APC labelling (allophycocyanin (allophycocyanin)) on ice.Twice washed cell is resuspended in cell in the FACS buffer that is supplemented with propidium iodide (propidium iodine) (0.5 μ g/ml), and analyzes by flow cytometry to remove after the second antibody.For carry out the combination test with Fab-108, testing the Fab-108 cultured cell of using incremental change under used the same terms with scFv-108.Use anti-His antibody, then use the anti-mice second antibody of APC labelling to detect the Fab combination.

Fig. 7, the A-C demonstration contains the competitive binding curve to the A431 cell of the scFv albumen of acetyl group-phenylalanine (pAcF) or pAcF and PEG and WT scFv and expression EGF receptor.After removing not in conjunction with scFv, washing, and as indicated abovely handles cell with mAb108 with the scFv albumen cultured cell of various concentration.All albumen are all expressed in pericentral siphon.Table 11 has been summed up modified scFv (pAcF is with pAcF and PEG) combining with respect to wild type scFv:

Table 11
		scFv108	IC50
WT
		1×(8.1nm)
Ser131pAcF			1.3×
	Ser131pAcF-5K?PEG	5.0×
Ser136pAcF			1.6×
	Ser136pAcF-5K?PEG	6.3×
His144pAcF			2.6×
	Leu156pAcF	1.8×
Leu156pAcF-5K?PEG			2.9×
	Tyr190pAcF	2.1×
Ser193pAcF			1.8×
	Lys248pAcF	2.1×
Ser259pAcF			0.5×

Fig. 8, B-D show the combining of A431 cell of the Fab-108 fragment contain pAcF or pAcF-PEG and WT Fab and expression EGF receptor.The segmental PEGization of Fab causes the minimizing of fragment and EGF receptor affinity minimum degree.Table 12 shows the combination of modified Fab fragment with respect to wild type Fab.In conjunction with condition as mentioned before.

Table 12
		Fab108	EC50
WT
		1×(4.0nM)
Lys142pAcF			1.7×
	Lys142pAcF-5 K?PEG	2.8× ?
Thr204pAcF			1.7×
	Thr204pAcF-5 K?PEG	1.8× ?
Lys?219pAcF			2.0×

The in vivo research of PEGization ABP

To mice or rat throwing and PEG-ABP, not modified ABP and buffering solution.The result will show with not modified ABP and compare the splendid activity of PEGization ABP of the present invention and the half-life of prolongation.

Measurement through in vivo half-life of combination and non-binding ABP and variant thereof

Use male Sprague Dawley rat (about 7 ages in week).Offeing medicine the same day, measure the weight of each animal.Every kg body weight 100 μ g are non-binding and in the tail vein of intravenous injection to three rat of hanging oneself in conjunction with the ABP sample.Injection back 1 minute, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours, under CO2 anesthesia, extract 500 μ l blood out from every rat.Blood sample is stored at room temperature 1.5 hours, then centrifugal (4 ℃, 18000 * g 5 minutes) separation of serum.Blood serum sample is stored under-80 ℃ until analyzing the same day.

By melt the amount that the sample activity in vitro test of ABP afterwards comes active A BP in the quantitative blood serum sample on ice.

Example 31

Comprise the safety of PEGization ABP of non-naturally encoded amino acids and/or the human clinical trial of effect

The purpose comparison through subcutaneous throwing with PEGization recombined human ABP that comprises non-naturally encoded amino acids and the commercial articles that is specific to identical target antigen (Herceptin_ for example, Bexxar_, Campath_, CEA-Scan_, Enbrel_, Erbitux_, Humira_, Myoscint_, Prostascint_, Raptiva_, Remicade_, ReoPro_, Rituxan_, Simulect_, Synagis_, Verluma_, Xolair_, Zenapax_, Zevalin_ or Avastin_) safety and pharmacokinetic property.

This research of patient recruited body weight between 18 20-40 ages in year and the 60-90kg the healthy volunteer.These persons under inspection do not have any clinical significantly unusual hematology or experiment value and the screening of negative urotoxy, HIV screening and the hepatitis B surface antigen of serum chemistry.They should not have any following sign: hypertension; Any constitutional blood medical history; Remarkable liver, kidney, cardiovascular, gastrointestinal, genitourinary system, metabolic system, nervous system disease history; Anemia or tic medical history; Known to antibacterial or mammal derivative products, PEG or human serum albumin's allergy; Custom and the serious beverage that contains caffeine that relies on; Participate in any other clinical trial or blood transfusion or donate blood in research registration 30 days; Be exposed to ABP in research registration in three months; Suffers from disease in research registration in 7 days; And before research, have remarkable abnormal conditions in the health check-up or in the clinical experiment assessment in 14 days in research registration.Can assess safety to all persons under inspection, and be used for pharmacokinetic analysis according to process collection whole blood gleanings.All researchs are to carry out under the approval of Ethics Committee of mechanism and patient's agreement.

Research design is in healthy male volunteers the I phase of being carried out, single center, open-label, at random, two stages intersected (two-period crossover) research.18 persons under inspection are divided into two treatment der group (9 person under inspection/groups) at random.Through two individually dosed stages, use isodose PEGization ABP and the selected commercial articles that comprises non-naturally encoded amino acids to throw and ABP in the mode of subcutaneous fast injection at the top thigh.Illustrated in the dispensing dosage of commercial articles and frequency such as the packaging label.To use additional administration, administration frequency or other desired parameters of commercial articles to be increased in this research by comprising into additional person under inspection's group.Each administration stage was separated by 14 days cleaning phases (washout period).The person under inspection was limited to the research center in 72 hours at least 12 hours to administration before each administration stage in two administration stages, rather than between the administration stage.If also have additional administration, frequency or other parameter that to test PEGization ABP, then can add other person under inspection's groups.The experimental preparation of ABP is the PEGization ABP that comprises non-naturally encoded amino acids.

Blood specimen collection by throw with ABP before and directly puncture vein afterwards and extract continuous blood out.The venous blood sample (5mL) that is used to measure serum ABP concentration is to obtain in about 30 minutes and 1,2,5,8,12,15,18,24,30,36,48,60 and 72 hour in about 30,20 and 10 minutes (3 baseline sample) before the administration and after administration.Each blood serum sample is divided into two aliquots.All blood serum samples are stored under-20 ℃.Blood serum sample placed on the dry ice load and transport.Before the first administration at once in first day, before the 4th day morning, the 16th day administration at once and carry out quick clinical experiment test (hematology, serum chemistry and urinalysis) the 19th day morning.

Bioanalytical method radioimmunoassay (RA) or ELISA test kit process are used to measure serum ABP concentration.

Safety is determined at before each time administration at once (the 1st day and the 16th day) and 6,24,48 and 72 hour record vital signs after each time administration.Incidence rate and the type that is based on adverse events measured in safety, and the clinical experiment test departs from the variation of baseline.In addition, estimate in vital signs measurement results (comprising blood pressure) and the physical examination result with study before the variation compared.

Data analysis deducts average baselining ABP concentration by value after each administration and comes at serum concentration value after the baseline ABP concentration correction administration before the administration, and wherein said average baselining ABP concentration is determined by the meansigma methods of the ABP content of 30,20 and 10 minutes three collected duplicate samples before the calculating administration.If serum ABP concentration is lower than the quantitative levels of this mensuration before the administration, then these data are got rid of outside the calculating of meansigma methods.By determining pharmacokinetic parameter at the serum-concentration data of baseline ABP concentration correction.Calculate pharmacokinetic parameter by using the model independent solution of latest edition BIOAVL software on Digital Equipment Corporation VAX 8600 computer systems.Determine following pharmacokinetic parameter: peak serum concentration (C _Max); Reach the time (t of peak serum concentration _Max); Under the concentration-time curve (AUC) that is then calculated by linear trapezoid method from 0 area to last blood sampling time (AUC0-72); With eliminate the half-life (t the latter stage of being calculated from the elimination factor constant _1/2).Latter stage by logarithm-linear concentration-time curve, the linear regression at consecutive numbers strong point was estimated the elimination factor constant in the linear zone.Calculate meansigma methods, standard deviation (SD) and the coefficient of variation (CV) of the pharmacokinetic parameter of each treatment.The ratio of calculating parameter meansigma methods (keeping preparation/non-reservation preparation).

The incidence rate equal distribution of adverse events in the whole treatment group of safety results.There is not any clinical significant change that departs from clinical experiment test or blood pressure before baseline or the research, and compares no significant change before physical examination result and vital signs measurement result and the research.The safety class of a curve of two treatment groups seemingly.

Pharmacokinetics result is during with each Measuring Time, and average serum ABP concentration-time curve (not to the correction of baseline ABP content) that one or more that accept single dose among all 18 persons under inspection are specific to after the commercial articles of identical target antigen compare with the PEGization ABP that comprises non-naturally encoded amino acids.All persons under inspection all should have baseline ABP concentration before the administration in the normal physiological scope.From the serum data of average baselining ABP concentration correction before the administration are determined pharmacokinetic parameter, and definite C _MaxAnd t _MaxSelected clinical comparison person's average t _MaxSignificantly be shorter than the t of the PEGization ABP that comprises non-naturally encoded amino acids _MaxCompare with the terminal half-life of the PEGization ABP that comprises non-naturally encoded amino acids, the terminal half-life of the commercially available ABP goods of being tested is significantly shorter.

Although this research is to carry out in the healthy male person under inspection, similar absorption feature and safety curve also should be desired in other patient colonies; The sex patient who for example suffers from cancer or chronic renal failure, department of pediatrics patients with renal failure, the patient that patient in the lean type storage blood project (autologous predeposit program) or arrangement are chosen date for operation.

In a word, the PEGization ABP that comprises non-naturally encoded amino acids of subcutaneous throwing and single dose will be safe, and fully be tolerated by the healthy male person under inspection.Based on the comparison of adverse events incidence rate, clinical experiment value, vital signs and physical examination result, the ABP of commercial form is equal to the safety curve that comprises the PEGization ABP of non-naturally encoded amino acids.The PEGization ABP that comprises non-naturally encoded amino acids may provide very big clinical efficacy to patient and health care healthcare givers.

Should be appreciated that, example as herein described and embodiment only are the illustrative purpose, and one of ordinary skill in the art are to making various modifications or variation according to described content, and these modifications and variations are included in the scope of the spirit and scope of the application's case and appended claims.Open case, patent and the patent application case that all this paper quoted all is incorporated herein by reference in full with all purposes.

Other embodiments of the invention and alternate embodiment

1. antigen-binding polypeptides that comprises one or more non-naturally encoded amino acids.

2. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides comprises one or more post translational modifications.

3. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides is connected in connexon, polymer or bioactive molecule.

4. antigen-binding polypeptides according to claim 3, wherein said polypeptide chain is connected to water-soluble polymer.

5. antigen-binding polypeptides according to claim 1, wherein said polypeptide chain are connected to double functional copolymer, difunctionality connexon or at least one other antigen-binding polypeptides.

6. antigen-binding polypeptides according to claim 5, wherein said difunctionality connexon or polymer are connected in second polypeptide.

7. antigen-binding polypeptides according to claim 6, wherein said second polypeptide is an antigen-binding polypeptides.

8. antigen-binding polypeptides according to claim 6, wherein said second polypeptide is non-antigen-binding polypeptides.

9. antigen-binding polypeptides according to claim 4, wherein said water-soluble polymer comprise poly-(ethylene glycol) part.

10. antigen-binding polypeptides according to claim 5, wherein said double functional copolymer is poly-(ethylene glycol) part.

11. antigen-binding polypeptides according to claim 4, wherein said water-soluble polymer is connected in the non-naturally encoded amino acids that is present in the described antigen-binding polypeptides.

12. antigen-binding polypeptides according to claim 1, it comprises at least two aminoacid that are connected in the water-soluble polymer that comprises poly-(ethylene glycol) part.

13. antigen-binding polypeptides according to claim 12, wherein at least one aminoacid that is connected in described water-soluble polymer is the non-natural coded amino acid.

Replace, add or disappearance 14. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides comprise one or more aminoacid of regulating described antigen-binding polypeptides and ABP receptor or antigenic affinity.

15. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides comprise one or more regulate described antigen-binding polypeptides stability, in recombinant host cell or in vitro synthetic expression, immunogenicity, protease resistant, tissue or organ specificity or deliquescent aminoacid replace, add or disappearance.

16. antigen-binding polypeptides according to claim 1, wherein said non-naturally encoded amino acids has reactivity to connexon, polymer or bioactive molecule, and described connexon, polymer or bioactive molecule are to any 20 kinds of common amino acid anergyes in the described polypeptide.

17. antigen-binding polypeptides according to claim 1, wherein said non-naturally encoded amino acids comprise carbonyl, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.

18. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises carbonyl.

19. antigen-binding polypeptides according to claim 18, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R ₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R ₃For H, aminoacid, polypeptide or amino terminal are modified base, and R ₄For H, aminoacid, polypeptide or carboxyl terminal are modified base.

20. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises aminooxy group.

21. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises hydrazide group.

22. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises diazanyl.

23. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises amino urea groups.

24. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises azido.

25. antigen-binding polypeptides according to claim 24, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.

26. antigen-binding polypeptides according to claim 17, wherein said non-naturally encoded amino acids comprises alkynyl.

27. antigen-binding polypeptides according to claim 26, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R ₁For alkyl, aryl, be substituted alkyl or be substituted aryl; X is O, N, S or does not exist; M is 0-10; R ₂For H, aminoacid, polypeptide or amino terminal are modified base, and R ₃For H, aminoacid, polypeptide or carboxyl terminal are modified base.

28. antigen-binding polypeptides according to claim 4, wherein said water-soluble polymer have the molecular weight between about 0.1kDa and the about 100kDa.

29. antigen-binding polypeptides according to claim 28, wherein said water-soluble polymer have the molecular weight between about 0.1kDa and the about 50kDa.

30. antigen-binding polypeptides according to claim 4, it is by making the water-soluble polymer reaction that comprises the antigen-binding polypeptides that contains carbonylamino acid and comprise aminooxy group, diazanyl, hydrazide group or amino urea groups and making.

31. antigen-binding polypeptides according to claim 30, wherein said aminooxy group, diazanyl, hydrazide group or amino urea groups are connected in described water-soluble polymer through amido link.

32. antigen-binding polypeptides according to claim 4, it is by making the water-soluble polymer that comprises carbonyl make with the polypeptide reaction that comprises non-naturally encoded amino acids, and described non-naturally encoded amino acids comprises aminooxy group, diazanyl, hydrazide group or amino urea groups.

33. antigen-binding polypeptides according to claim 4, it comprises the antigen-binding polypeptides that contains alkynyl amino acid with the water-soluble polymer reaction that comprises the nitrine part and make by making.

34. antigen-binding polypeptides according to claim 4, it contains the amino acid whose antigen-binding polypeptides of nitrine with the water-soluble polymer reaction that comprises alkynyl moiety and make by making to comprise.

35. antigen-binding polypeptides according to claim 17, wherein said azido or alkynyl are connected in water-soluble polymer through amido link.

36. antigen-binding polypeptides according to claim 4, wherein said water-soluble polymer are side chain or multiarm polymers.

37. antigen-binding polypeptides according to claim 36, each branch of wherein said water-soluble polymer has the molecular weight between about 1kDa and the about 100kDa.

38. antigen-binding polypeptides according to claim 1, wherein said polypeptide are the antigen-binding polypeptides antagonisies.

39. according to the described antigen-binding polypeptides of claim 38, wherein said polypeptide comprises one or more post translational modifications, connexon, polymer or bioactive molecule.

40. according to the described antigen-binding polypeptides of claim 39, wherein said polymer comprises and is selected from the part of being made of group water-soluble polymer and poly-(ethylene glycol).

41. antigen-binding polypeptides according to claim 1, wherein said non-naturally encoded amino acids comprises sugar moieties.

42. antigen-binding polypeptides according to claim 3, wherein said connexon, polymer or bioactive molecule are connected in described polypeptide through sugar moieties.

43. an isolating nucleic acid, it is included in the polynucleotide of the polymerized nucleoside acid hybridization that combines polypeptide under the rigorous condition with coding for antigens, and wherein said polynucleotide comprises at least one and selects codon.

44. according to the described isolating nucleic acid of claim 43, wherein said selection codon is selected from by amber codon, ochre codon, opal codon, unique codon, rare codon and the molecular group of four base passwords.

45. a method for preparing antigen-binding polypeptides according to claim 3, described method comprise the separation antigen-binding polypeptides that comprises non-naturally encoded amino acids is contacted with the connexon, polymer or the bioactive molecule that comprise the part of reacting with described non-naturally encoded amino acids.

46. according to the described method of claim 45, wherein said polymer comprises and is selected from the part of being made of group water-soluble polymer and poly-(ethylene glycol).

47. according to the described method of claim 45, wherein said non-naturally encoded amino acids comprises carbonyl, aminooxy group, diazanyl, hydrazide group, amino urea groups, azido or alkynyl.

48. according to the described method of claim 45, wherein said non-naturally encoded amino acids comprises carbonyl moiety, and described connexon, polymer or bioactive molecule comprise aminooxy group, hydrazine, hydrazides or semicarbazides part.

49. according to the described method of claim 48, wherein said aminooxy group, hydrazine, hydrazides or semicarbazides part are connected in described connexon, polymer or bioactive molecule through amido link.

50. according to the described method of claim 45, wherein said non-naturally encoded amino acids comprises alkynyl moiety, and described connexon, polymer or bioactive molecule comprise the nitrine part.

51. according to the described method of claim 45, wherein said non-naturally encoded amino acids comprises the nitrine part, and described connexon, polymer or bioactive molecule comprise alkynyl moiety.

52. according to the described method of claim 47, wherein said nitrine part or alkynyl moiety are connected in connexon, polymer or bioactive molecule through amido link.

53. according to the described method of claim 46, wherein said poly-(ethylene glycol) part has the mean molecule quantity between about 0.1kDa and the about 100kDa.

54. according to the described method of claim 46, wherein said poly-(ethylene glycol) part is side chain or multiarm polymers.

55. a compositions, it comprises antigen-binding polypeptides according to claim 1 and pharmaceutically acceptable supporting agent.

56. according to the described compositions of claim 55, wherein said non-naturally encoded amino acids is connected in water-soluble polymer.

57. a treatment suffers from the patient's who is subjected to the disease that ABP regulates method, described method comprise to described patient throw with the treatment effective dose according to the described compositions of claim 55.

58. a cell, it comprises according to the described nucleic acid of claim 43.

59. according to the described cell of claim 58, wherein said cell comprises quadrature tRNA synzyme or quadrature tRNA.

60. method for preparing the antigen-binding polypeptides that comprises non-naturally encoded amino acids, described method is included in and allows cultured cell under the condition that the described antigen-binding polypeptides that comprises non-naturally encoded amino acids expresses, described cell to comprise coding for antigens in conjunction with polypeptide and comprise polynucleotide, quadrature RNA synzyme and the quadrature tRNA that selects codon; With the described antigen-binding polypeptides of purification.

61. comprising with one or more non-naturally encoded amino acids, a method of regulating antigen-binding polypeptides serum half-life or circulation time, described method replace any one or natural generation aminoacid more than one in the described antigen-binding polypeptides.

62. the antigen-binding polypeptides by the polymerized nucleoside acid encoding, wherein said polynucleotide comprises the selection codon, and wherein said polynucleotide comprises at least one non-naturally encoded amino acids.

63. according to the described antigen-binding polypeptides of claim 62, wherein said non-naturally encoded amino acids is connected in connexon, polymer, water-soluble polymer or bioactive molecule.

64. according to the described antigen-binding polypeptides of claim 63, wherein said water-soluble polymer comprises poly-(ethylene glycol) part.

65. according to the described antigen-binding polypeptides of claim 62, wherein said non-naturally encoded amino acids comprises carbonyl, aminooxy group, hydrazide group, diazanyl, amino urea groups, azido or alkynyl.

66. according to the described antigen-binding polypeptides of claim 64, wherein said poly-(ethylene glycol) part has the molecular weight between about 0.1kDa and the about 100kDa.

67. according to the described antigen-binding polypeptides of claim 64, wherein said poly-(ethylene glycol) part is side chain or multiarm polymers.

68. according to the described antigen-binding polypeptides of claim 67, wherein said poly-(ethylene glycol) part has the molecular weight between about 1kDa and the about 100kDa.

69. a compositions, it comprises according to described antigen-binding polypeptides of claim 62 and pharmaceutically acceptable supporting agent.

70. bispecific ABP who comprises an interconnective ABP and the 2nd ABP, a wherein said ABP is incorporated into different epitopes with described the 2nd ABP specificity, a wherein said ABP has the binding specificity at least one epitope on first antigen, and described the 2nd ABP has being different from the binding specificity of second epitope of described first epitope on first antigen or second antigen, and wherein said bispecific ABP comprises at least one non-naturally encoded amino acids.

71. according to the described bispecific ABP of claim 70, a wherein said ABP is connected by connexon with described the 2nd ABP.

72. according to the described bispecific ABP of claim 71, wherein said connexon is the peptide connexon.

73. according to the described bispecific ABP of claim 72, wherein said connexon is the peptide connexon that lacks the protein cleavage site.

74. according to the described bispecific ABP of claim 70, a wherein said ABP is strand ABP, and described the 2nd ABP is strand ABP, and a described ABP is coupled to described the 2nd ABP by the peptide connexon.

75. a compositions, it comprises according to described bispecific ABP of claim 70 and pharmaceutically acceptable supporting agent.

76. a method for the treatment of the disease or the state of an illness, described method comprise to the patient that these needs are arranged throw with the treatment effective dose according to the described compositions of claim 75.

77. an ABP who comprises according to the described bispecific ABP of claim 70, described ABP is coupled to bioactive molecule.

78. according to the described ABP of claim 77, wherein said bioactive molecule is to be selected from the group that is made up of cytotoxin, labelling, radionuclide, medicine, liposome, part and ABP.

79. according to the described ABP of claim 77, wherein said ABP is a fusion rotein.

80. one kind is detected the method for expressing one or more antigenic cell or tissues, described method comprises: make cell or tissue and be connected in contacting according to the described ABP of claim 70 of detectable label; With the described labelling of detection, wherein express one or more existence by the bonded antigenic cell or tissue of ABP with the detection indication of the associating described labelling of cell or tissue.

81. 0 described method according to Claim 8, wherein said detectable label is to be selected from the group that is made up of gamma ray emitter, positron emitter, MRI labelling and fluorescent labeling.

82. 0 described method according to Claim 8, wherein said detectable label is a gamma ray emitter, and described detection comprises and uses the γ camera imaging.

83. 0 described method according to Claim 8, wherein said detectable label is a positron emitter, and described detection comprises with positron emission computerized tomography method (positron emission tomography, PET) imaging.

84. 0 described method according to Claim 8, wherein said detectable label is the MRI labelling, and described detection comprises with nuclear magnetic resonance and detects.

85. an antigen-binding polypeptides, it comprises the water-soluble polymer that is connected in the single amino acid place of described antigen-binding polypeptides through covalent bond.

86. 5 described antigen-binding polypeptides according to Claim 8, wherein said water-soluble polymer comprises poly-(ethylene glycol) part.

87. 5 described antigen-binding polypeptides according to Claim 8, the wherein said aminoacid that is covalently attached to described water-soluble polymer is the non-natural coded amino acid.

88. an antigen-binding polypeptides that comprises at least one connexon, polymer or bioactive molecule, wherein said connexon, polymer or bioactive molecule are connected in described polypeptide by the functional group that incorporates the non-naturally encoded amino acids of described polypeptide through ribosome into.

89. 8 described antigen-binding polypeptides according to Claim 8, wherein said antigen-binding polypeptides is through single PEGization.

90. an antigen-binding polypeptides that comprises the connexon, polymer or the bioactive molecule that are connected in one or more non-naturally encoded amino acids, wherein said non-naturally encoded amino acids is incorporated preliminary election site in the described polypeptide into through ribosome.

91. according to the described antigen-binding polypeptides of claim 90, wherein said antigen-binding polypeptides comprises described connexon, polymer or a bioactive molecule.

Replace, add or disappearance 92. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides comprise one or more aminoacid of regulating described antigen-binding polypeptides serum half-life or circulation time.

93. regulate the immunogenic method of antigen-binding polypeptides for one kind, described method comprises with one or more non-naturally encoded amino acids replaces any one or natural generation aminoacid more than one in the described antigen-binding polypeptides.

94. according to the described isolating nucleic acid of claim 43, the sequence of wherein said isolating nucleic acid is to be selected from by SEQID NO:18,20,22,25,27 and 29 or the group that forms of its fragment.

95. an antigen-binding polypeptides, wherein said polypeptide are to be selected from by SEQ ID NO:19,21,23,24,26,28,30,31 or the group that forms of its fragment.

96. antigen-binding polypeptides according to claim 3, wherein said antigen-binding polypeptides are to be connected in described connexon, polymer or bioactive molecule under the degeneration condition.

Sequence table

<110>Cho，Ho?Sung

Daniel，Thomas?0.

Wilson，Troy?E.

cuj?ec，Thomas?P.

Tian，Feng

Hays，Anna-Maria

Kimmel，Bruce?E.

Ho，Lillian

＜120〉novel antigen-binding polypeptides and its purposes

<130>AMBX-003?8.00PCT

<150>60/581,334

<151>2004-06-18

<150>60/648,222

<151>2005-01-28

<150>60/654,018

<151>2005-02-17

<160>31

<170>PatentIn?version?3.3

<210>1

<211>77

<212>DNA

＜213〉methane coccus

<400>1

ccggcggtag?ttcagcaggg?cagaacggcg?gactctaaat?ccgcatggcg?ctggttcaaa 60

tccggcccgc?cggacca 77

<210>2

<211>88

<212>DNA

＜213〉halophilic bacteria NRC-1 (Halobacterium sp.NRc-1)

<400>2

cccagggtag?ccaagctcgg?ccaacggcga?cggactctaa?atccgttctc?gtaggagttc 60

gagggttcga?atcccttccc?tgggacca 88

<210>3

<211>89

<212>DNA

＜213〉halophilic bacteria NRc-1 (Halobacterium sp.NRc-1)

<400>3

gcgagggtag?ccaagctcgg?ccaacggcga?cggacttcct?aatccgttct?cgtaggagtt 60

cgagggttcg?aatccctccc?ctcgcacca 89

<210>4

<211>306

<212>PRT

＜213〉methane coccus i

<400>4

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?GIn

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Thr?Tyr?Tyr

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ie

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>5

<211>306

<212>PRT

＜213〉methane coccus

<400>5

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Se

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Thr?Ser?His

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>6

<211>305

<212>PRT

＜213〉methane coccus

<400>6

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Pro?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Ala?Ile?Tyr

145 150 155 160

Leu?Ala?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

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

260 265 270

Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>7

<211>305

<212>PRT

＜213〉methane coccus

<400>7

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 l0 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 SO

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Pro?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Ile?Pro?Tyr

145 150 155 160

Leu?Pro?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

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

260 265 270

Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>8

<211>305

<212>PRT

＜213〉methane coccus

<400>8

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Lys?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Ala?Ile?Tyr

145 150 155 160

Leu?Ala?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

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

260 265 270

Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>9

<211>306

<212>PRT

＜213〉methane coccus

<400>9

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Asn?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Pro?Leu?His

145 150 155 160

Tyr?Gin?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>10

<211>306

<212>PRT

＜213〉methane coccus

<400>10

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Pro?Leu?His

145 150 155 160

Tyr?Gin?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>11

<211>306

<212>PRT

＜213〉methane coccus

<400>11

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Pro?Val?His

145 150 155 160

Tyr?Gin?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>12

<211>306

<212>PRT

＜213〉methane coccus

<400>12

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Pro?Ser?His

145 150 155 160

Tyr?Gin?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>13

<211>306

<212>PRT

＜213〉methane coccus

<400>13

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Glu?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Gly?Cys?His

145 150 155 160

Tyr?Arg?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>14

<211>306

<212>PRT

＜213〉methane coccus

<400>14

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Glu?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Gly?Thr?His

145 150 155 160

Tyr?Arg?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>15

<211>306

<212>PRT

＜213〉methane coccus

<400>15

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile

Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Glu?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Gly?Gly?His

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ile?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>16

<211>306

<212>PRT

＜213〉methane coccus

<400>16

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Arg?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Val?Ile?His

145 150 155 160

Tyr?Asp?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>17

<211>306

<212>PRT

＜213〉methane coccus

<400>17

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Iyr?Leu?Gin

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gin?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gin?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gin?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gin?Val?Asn?Thr?Tyr?Tyr

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gin?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu?305

<210>18

<211>920

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>18

accacaggtc?tcccatgcga?ttcagtacga?cgctggcaac?ggccgctacg?gcactcttct 60

ttactgcaag?tcaggtaagc?gcgcaggttc?agctgcagca?gtctggagct?gagctgatga 120

agcctggggc?ctcagtgaag?atatcctgca?aggctactgg?ctacacattc?agtagttact 180

ggatagagtg?ggtaaagcag?aggcctggac?atggccttga?gtggattgga?gagattttac 240

cgggaagtaa?aaaaactaac?tacaatgaga?agttcaaggg?aaaggccaca?ttcactgcag 300

atacatcctc?caacacagcc?tacatgcaat?ttagcagcct?gacatctgag?gactctgccg 360

tctattactg?tgcaagatat?tactatagga?acgacgacta?tggtatggac?tactggggtc 420

aaggaacctc?agtcaccgtc?tcctcaggcg?gcggcggctc?cggtggtggt?ggatctggtg 480

ggggcggcag?cggcggtggc?ggcagtgaaa?tccacatgac?acagactaca?tcctccctgt 540

ctgcctctct?gggagacaga?gtcaccatca?gttgcagtgc?aagtcaggac?atcaggaatt 600

atttaaactg?gtatcagcag?aaacctgatg?gaactgttaa?actcctgatc?tattacacat 660

caactttaca?ttcaggagtc?ccatcaaggt?tcagtggcag?cgggtctggg?acagattatt 720

ctctcaccat?cagcaacctg?gaacctgaag?atattgccac?ttattattgt?cagcagtata 780

gtaagattcc?gtacacgttc?acagggggga?ccaagctgga?aataaaacgg?gctgatgctg 840

cagaacaaaa?actcatctca?gaagaggatc?tgaatagcgc?cgtcgaccat?catcatcacc 900

atcattaagg?taccaaccaa 920

<210>19

<211>297

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>19

Met?Arg?Phe?Ser?Thr?Thr?Leu?Ala?Thr?Ala?Ala?Thr?Ala?Leu?Phe?Phe

1 5 10 15

Thr?Ala?Ser?Gin?Val?Ser?Ala?Gin?Val?Gin?Leu?Gin?Gin?Ser?Gly?Ala

20 25 30

Glu?Leu?Met?Lys?Pro?Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Thr

35 40 45

Gly?Tyr?Thr?Phe?Ser?Ser?Tyr?Trp?Ile?Glu?Trp?Val?Lys?Gin?Arg?Pro

50 55 60

Gly?His?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile?Leu?Pro?Gly?Ser?Lys?Lys

65 70 75 80

Thr?Asn?Tyr?Asn?Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Phe?Thr?Ala?Asp

85 90 95

Thr?Ser?Ser?Asn?Thr?Ala?Tyr?Met?Gin?Phe?Ser?Ser?Leu?Thr?Ser?Glu

100 105 110

Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Tyr?Tyr?Tyr?Arg?Asn?Asp?Asp

115 120 125

Tyr?Gly?Met?Asp?Tyr?Trp?Gly?Gin?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser

130 135 140

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly

145 150 155 160

Gly?Gly?Gly?Ser?Glu?Ile?His?Met?Thr?Gin?Thr?Thr?Ser?Ser?Leu?Ser

165 170 175

Ala?Ser?Leu?Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Ser?Ala?Ser?Gin?Asp

180 185 190

Ile?Arg?Asn?Tyr?Leu?Asn?Trp?Tyr?Gin?Gin?Lys?Pro?Asp?Gly?Thr?Val

195 200 205

Lys?Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Thr?Leu?His?Ser?Gly?Val?Pro?Ser

210 215 220

Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile?Ser

225 230 235 240

Asn?Leu?Glu?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gin?Gin?Tyr?Ser

245 250 255

Lys?Ile?Pro?Tyr?Thr?Phe?Thr?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys?Arg

260 265 270

Ala?Asp?Ala?Ala?Glu?Gin?Lys?Leu?Ile?Ser?Glu?Glu?Asp?Leu?Asn?Ser

275 280 285

Ala?Val?Asp?His?His?His?His?His?His

290 295

<210>20

<211>779

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>20

ccatgggcca?ccaccaccac?caccaccagg?ttcagctgca?gcagtctgga?gctgagctga 60

tgaagcctgg?ggcctcagtg?aagatatcct?gcaaggctac?tggctacaca?ttcagtagtt 120

actggataga?gtgggtaaag?cagaggcctg?gacatggcct?tgagtggatt?ggagagattt 180

taccgggaag?taaaaaaact?aactacaatg?agaagttcaa?gggaaaggcc?acattcactg 240

cagatacatc?ctccaacaca?gcctacatgc?aatttagcag?cctgacatct?gaggactctg 300

ccgtctatta?ctgtgcaaga?tattsctata?ggaacgacga?ctatggtatg?gactactggg 360

gtcaaggaac?ctcagtcacc?gtctcctcag?gcggcggcgg?ctccggtggt?ggtggatctg 420

gtgggggcgg?cagcggcggt?ggcggcagtg?aaatccacat?gacacagact?acatcctccc 480

tgtctgcctc?tctgggagac?agagtcacca?tcagttgcag?tgcaagtcag?gacatcagga 540

attatttaaa?ctggtatcag?cagaaacctg?atggaactgt?taaactcctg?atctattacs 600

catcaacttt?acattcagga?gtcccatcaa?ggttcagtgg?cagcgggtct?gggacagatt 660

attctctcac?catcagcaac?ctggaacctg?aagatattgc?cacttattat?tgtcagcagt 720

atagtaagat?tccgtacacg?ttcacagggg?ggaccaagct?ggaaatataa?aggtaccaa 779

<210>21

<211>255

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>21

Met?Gly?His?His?His?His?His?His?Gin?Val?Gin?Leu?Gin?Gin?Ser?Gly

1 5 10 15

Ala?Glu?Leu?Met?Lys?Pro?Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala

20 25 30

Thr?Gly?Tyr?Thr?Phe?Ser?Ser?Tyr?Trp?Ile?Glu?Trp?Val?Lys?Gin?Arg

35 40 45

Pro?Gly?His?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile?Leu?Pro?Gly?Ser?Lys

50 55 60

Lys?Thr?Asn?Tyr?Asn?Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Phe?Thr?Ala

65 70 75 80

Asp?Thr?Ser?Ser?Asn?Thr?Ala?Tyr?Met?Gin?Phe?Ser?Ser?Leu?Thr?Ser

85 90 95

Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Tyr?Tyr?Tyr?Arg?Asn?Asp

100 105 110

Asp?Tyr?Gly?Met?Asp?Tyr?Trp?Gly?Gin?Gly?Thr?Ser?Val?Thr?Val?Ser

115 120 125

Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser

130 135 140

Gly?Gly?Gly?Gly?Ser?Glu?Ile?His?Met?Thr?Gin?Thr?Thr?Ser?Ser?Leu

145 150 155 160

Ser?Ala?Ser?Leu?Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Ser?Ala?Ser?Gin

165 170 175

Asp?Ile?Arg?Asn?Tyr?Leu?Asn?Trp?Tyr?Gin?Gin?Lys?Pro?Asp?Gly?Thr

180 185 190

Val?Lys?Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Thr?Leu?His?Ser?Gly?Val?Pro

195 200 205

Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr?Ile

210 215 220

Ser?Asn?Leu?Glu?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gin?Gin?Tyr

225 230 235 240

Ser?Lys?Ile?Pro?Tyr?Thr?Phe?Thr?Gly?Gly?Thr?Lys?Leu?Glu?Ile

245 250 255

<210>22

<211>1578

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>22

accaccggtc?tcccatgaaa?aaactgctgt?tcgcgattcc?gctggtggtg?ccgttctata 60

gccatagcac?catggagctc?gaaatccaca?tgacacagac?tacatcctcc?ctgtctgcct 120

ctctgggaga?cagagtcacc?atcagttgca?gtgcaagtca?ggacatcagg?aattatttaa 180

actggtatca?gcagaaacct?gatggaactg?ttaaactcct?gatctattac?acatcaactt 240

tacattcagg?agtcccatca?aggttcagtg?gcagcgggtc?tgggacagat?tattctctca 300

ccatcagcaa?cctggaacct?gaagatattg?ccacttatta?ttgtcagcag?tatagtaaga 360

ttccgtacac?gttcacaggg?gggaccaagc?tggaaataaa?acgggctgat?gctgcaaagc 420

ttgagatcaa?acgaactgtg?gctgcaccat?ctgtcttcat?cttcccgcca?tctgatgagc 480

agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa?taacttctat?cccagagagg 540

ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg?taactcccag?gagagtgtca 600

cagagcagga?cagcaaggac?agcacctaca?gcctcagcag?caccctgacg?ctgagcaaag 660

cagactacga?gaaacacaaa?gtctacgcct?gcgaagtcac?ccatcagggc?ctgtcctcgc 720

ccgtcacaaa?gagcttcaac?aggggagagt?gttaagctgg?ggatcctcta?gaggttgagg 780

tgattttatg?aaaaagaata?tcgcatttct?tcttgcatct?atgttcgttt?tttctattgc 840

tacaaacgcg?tacgctcagg?ttcagctgct?cgagcaggtt?cagctgcagc?agtctggagc 900

tgagctgatg?aagcctgggg?cctcagtgaa?gatatcctgc?aaggctactg?gctacacatt 960

cagtagttac?tggatagagt?gggtaaagca?gaggcctgga?catggccttg?agtggattgg 1020

agagatttta?ccgggaagta?aaaaaactaa?ctacaatgag?aagttcaagg?gaaaggccac 1080

attcactgca?gatacatcct?ccaacacagc?ctacatgcaa?tttagcagcc?tgacatctga 1140

ggactctgcc?gtctattact?gtgcaagata?ttactatagg?aacgacgact?atggtatgga 1200

ctactggggt?caaggaacct?cagtcaccgt?ctcctcagcg?ggcccatcgg?tcttccccct 1260

ggcaccctcc?tccaagagca?cctctggggg?cacagcggcc?ctgggctgcc?tggtcaagga 1320

ctacttcccc?gaaccggtga?cggtgtcgtg?gaactcaggc?gccctgacca?gcggcgtgca 1380

caccttcccg?gctgtcctac?agtcctcagg?actctactcc?ctcagcagcg?tggtgactgt 1440

gccctctagc?agcttgggca?cccagaccta?catctgcaac?gtgaatcaca?agcccagcaa 1500

caccaaggtg?gacaagaaag?ttgagcccaa?atcttgtgac?aaacatcatc?atcatcatca 1560

ttagggtacc?accataac 1578

<210>23

<211>246

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>23

Met?Lys?Lys?Leu?Leu?Phe?Ala?Ile?Pro?Leu?Val?Val?Pro?Phe?Tyr?Ser

1 5 10 15

His?Ser?Thr?Met?Glu?Leu?Glu?Ile?His?Met?Thr?Gin?Thr?Thr?Ser?Ser

20 25 30

Leu?Ser?Ala?Ser?Leu?Gly?Asp?Arg?Val?Thr?Ile?Ser?Cys?Ser?Ala?Ser

35 40 45

Gin?Asp?Ile?Arg?Asn?Tyr?Leu?Asn?Trp?Tyr?Gin?Gin?Lys?Pro?Asp?Gly

50 55 60

Thr?Val?Lys?Leu?Leu?Ile?Tyr?Tyr?Thr?Ser?Thr?Leu?His?Ser?Gly?Val

65 70 75 80

Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Tyr?Ser?Leu?Thr

85 90 95

Ile?Ser?Asn?Leu?Glu?Pro?Glu?Asp?Ile?Ala?Thr?Tyr?Tyr?Cys?Gin?Gin

100 105 110

Tyr?Ser?Lys?Ile?Pro?Tyr?Thr?Phe?Thr?Gly?Gly?Thr?Lys?Leu?Glu?Ile

115 120 125

Lys?Arg?Ala?Asp?Ala?Ala?Lys?Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala

130 135 140

Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gin?Leu?Lys?Ser?Gly

145 150 155 160

Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala

165 170 175

Lys?Val?Gin?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gin?Ser?Gly?Asn?Ser?Gin

180 185 190

Glu?Ser?Val?Thr?Glu?Gin?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser

195 200 205

Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr

210 215 220

Ala?Cys?Glu?Val?Thr?His?Gin?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser

225 230 235 240

Phe?Asn?Arg?Gly?Glu?Cys

245

<210>24

<211>235

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>24

Gin?Val?Gin?Leu?Leu?Glu?Gin?Val?Gin?Leu?Gin?Gin?Ser?Gly?Ala?Glu

1 5 10 15

Leu?Met?Lys?Pro?Gly?Ala?Ser?Val?Lys?Ile?Ser?Cys?Lys?Ala?Thr?Gly

20 25 30

Tyr?Thr?Phe?Ser?Ser?Tyr?Trp?Ile?Glu?Trp?Val?Lys?Gin?Arg?Pro?Gly

35 40 45

His?Gly?Leu?Glu?Trp?Ile?Gly?Glu?Ile?Leu?Pro?Gly?Ser?Lys?Lys?Thr

50 55 60

Asn?Tyr?Asn?Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Phe?Thr?Ala?Asp?Thr

65 70 75 80

Ser?Ser?Asn?Thr?Ala?Tyr?Met?Gin?Phe?Ser?Ser?Leu?Thr?Ser?Glu?Asp

85 90 95

Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Tyr?Tyr?Tyr?Arg?Asn?Asp?Asp?Tyr

100 105 110

Gly?Met?Asp?Tyr?Trp?Gly?Gin?Gly?Thr?Ser?Val?Thr?Val?Ser?Ser?Ala

115 120 125

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

130 135 140

Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro

145 150 155 160

Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr

165 170 175

Phe?Pro?Ala?Val?Leu?Gin?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val

180 185 190

Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gin?Thr?Tyr?Ile?Cys?Asn

195 200 205

Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro

210 215 220

Lys?Ser?Cys?Asp?Lys?His?His?His?His?His?His

225 230 235

<210>25

<211>863

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>25

accacaggtc?tcccatgcga?ttcagtacga?cgctggcaac?ggccgctacg?gcactcttct 60

ttactgcaag?tcaggtaagc?gcggaagttc?aactggttga?atctggtggc?ggtctggtgc 120

aaccaggtgg?ttctctgcgt?ctgtcttgcg?ccgcgtccgg?tttcaacatt?aaagacacct 180

acattcactg?ggtgcgccag?gctccgggta?agggtctgga?atgggtggcc?cgcatttatc 240

caacgaatgg?ctacacccgc?tacgcggatt?ccgtgaaagg?ccgtttcacg?attagcgctg 300

ataccagcaa?gaacaccgct?tatctgcaaa?tgaacagcct?gcgtgccgaa?gacaccgccg 360

tgtactattg?tagccgttgg?ggcggtgatg?gcttttacgc?gatggactac?tggggccagg 420

gtacgctggt?cacggtttct?tctggcggcg?gcggctccgg?tggtggtgga?tctggtgggg 480

gcggcagcgg?cggtggcggc?agtgatattc?aaatgactca?atctccttcc?tctctgtctg 540

caagcgttgg?tgatcgtgtg?accatcacct?gtcgcgcatc?ccaggatgt?aaataccgcag 600

tagcgtggta?tcagcagaag?ccgggtaaag?caccaaaact?gctgatttat?tccgcgagct 660

ttctgtatag?cggcgtacca?agccgtttta?gcggttctcg?tagcggtact?gattttaccc 720

tgaccatctc?ctctctgcag?cctgaggatt?ttgcgaccta?ttattgccag?cagcattata 780

cgacgccgcc?tacgtttggc?cagggtacta?aggtggaaat?taaaagcggc?catcatcatc 840

accatcatta aggtacccaa cca 863

<210>26

<211>278

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>26

Met?Arg?Phe?Ser?Thr?Thr?Leu?Ala?Thr?Ala?Ala?Thr?Ala?Leu?Phe?Phe

1 5 10 15

Thr?Ala?Ser?Gin?Val?Ser?Ala?Glu?Val?Gin?Leu?Val?Glu?Ser?Gly?Gly

20 25 30

Gly?Leu?Val?Gin?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser

35 40 45

Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gin?Ala?Pro

50 55 60

Gly?Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr

65 70 75 80

Thr?Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp

85 90 95

Thr?Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gin?Met?Asn?Ser?Leu?Arg?Ala?Glu

100 105 110

Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr

115 120 125

Ala?Met?Asp?Tyr?Trp?Gly?Gin?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Gly

130 135 140

Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly

145 150 155 160

Gly?Gly?Ser?Asp?Ile?Gin?Met?Thr?Gin?Ser?Pro?Ser?Ser?Leu?Ser?Ala

165 170 175

Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gin?Asp?Val

180 185 190

Asn?Thr?Ala?Val?Ala?Trp?Tyr?Gin?Gin?Lys?Pro?Gly?Lys?Ala?Pro?Lys

195 200 205

Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Phe?Leu?Tyr?Ser?Gly?Val?Pro?Ser?Arg

210 215 220

Phe?Ser?Gly?Ser?Arg?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser

225 230 235 240

Leu?Gin?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gin?Gin?His?Tyr?Thr

245 250 255

Thr?Pro?Pro?Thr?Phe?Gly?Gin?Gly?Thr?Lys?Val?Glu?Ile?Lys?Ser?Gly

260 265 270

His?His?His?His?His?His

275

<210>27

<211>869

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>27

accacaggtc?tcccatgcga?ttcagtacga?cgctggcaac?ggccgctacg?gcactcttct 60

ttactgcaag?tcaggtaagc?gcgggccacc?accaccacca?ccactctggt?gaagttcaac 120

tggttgaatc?tggtggcggt?ctggtgcaac?caggtggttc?tctgcgtctg?tcttgcgccg 180

cgtccggttt?caacattaaa?gacacctaca?ttcactgggt?gcgccaggct?ccgggtaagg 240

gtctggaatg?ggtggcccgc?atttatccaa?cgaatggcta?cacccgctac?gcggattccg 300

tgaaaggccg?tttcacgatt?agcgctgata?ccagcaagaa?caccgcttat?ctgcaaatga 360

acagcctgcg?tgccgaagac?accgccgtgt?actattgtag?ccgttggggc?ggtgatggct 420

tttacgcgat?ggactactgg?ggccagggta?cgctggtcac?ggtttcttct?ggcggcggcg 480

gctccggtgg?tggtggatct?ggtgggggcg?gcagcggcgg?tggcggcagt?gatattcaaa 540

tgactcaatc?tccttcctct?ctgtctgcaa?gcgttggtga?tcgtgtgacc?atcacctgtc 600

gcgcatccca?ggatgtaaat?accgcagtag?cgtggtatca?gcagaagccg?ggtaaagcac 660

caaaactgct?gatttattcc?gcgagctttc?tgtatagcgg?cgtaccaagc?cgttttagcg 720

gttctcgtag?cggtactgat?tttaccctga?ccatctcctc?tctgcagcct?gaggattttg 780

cgacctatta?ttgccagcag?cattatacga?cgccgcctac?gtttggccag?ggtactaagg 840

tggaaattaa?aagctaaggt?acccaacca 869

<210>28

<211>280

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>28

Met?Arg?Phe?Ser?Thr?Thr?Leu?Ala?Thr?Ala?Ala?Thr?Ala?Leu?Phe?Phe

1 5 10 15

Thr?Ala?Ser?Gin?Val?Ser?Ala?Gly?His?His?His?His?His?His?Ser?Gly

20 25 30

Glu?Val?Gin?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gin?Pro?Gly?Gly

35 40 45

Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr

50 55 60

Tyr?Ile?His?Trp?Val?Arg?Gin?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val

65 70 75 80

Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr?Arg?Tyr?Ala?Asp?Ser?Val

85 90 95

Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr?Ser?Lys?Asn?Thr?Ala?Tyr

100 105 110

Leu?Gin?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys

115 120 125

Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala?Met?Asp?Tyr?Trp?Gly?Gin

130 135 140

Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly

145 150 155 160

Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Gin?Met

165 170 175

Thr?Gin?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr

180 185 190

Ile?Thr?Cys?Arg?Ala?Ser?Gin?Asp?Val?Asn?Thr?Ala?Val?Ala?Trp?Tyr

195 200 205

Gin?Gin?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser

210 215 220

Phe?Leu?Tyr?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Arg?Ser?Gly

225 230 235 240

Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gin?Pro?Glu?Asp?Phe?Ala

245 250 255

Thr?Tyr?Tyr?Cys?Gin?Gin?His?Tyr?Thr?Thr?Pro?Pro?Thr?Phe?Gly?Gin

260 265 270

Gly?Thr?Lys?Val?Glu?Ile?Lys?Ser

275 280

<210>29

<211>1538

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic construct

<400>29

atgaaaaaac?tgctgttcgc?gattccgctg?gtggtgccgt?tctatagcca?tagcaccatg 60

gagctcgata?ttcaaatgac?tcaatctcct?tcctctctgt?ctgcaagcgt?tggtgatcgt 120

gtgaccatca?cctgtcgcgc?atcccaggat?gtaaataccg?cagtagcgtg?gtatcagcag 180

aagccgggta?aagcaccaaa?actgctgatt?tattccgcga?gctttctgta?tagcggcgta 240

ccaagccgtt?ttagcggttc?tcgtagcggt?actgatttta?ccctgaccat?ctcctctctg 300

cagcctgagg?attttgcgac?ctattattgc?cagcagcatt?atacgacgcc?gcctacgttt 360

ggccagggta?ctaaggtgga?aattaaaaag?cttgagatca?aacgaactgt?ggctgcacca 420

tctgtcttca?tcttcccgcc?atctgatgag?cagttgaaat?ctggaactgc?ctctgttgtg 480

tgcctgctga?ataacttcta?tcccagagag?gccaaagtac?agtggaaggt?ggataacgcc 540

ctccaatcgg?gtaactccca?ggagagtgtc?acagagcagg?acagcaagga?cagcacctac 600

agcctcagca?gcaccctgac?gctgagcaaa?gcagactacg?agaaacacaa?agtctacgcc 660

tgcgaagtca?cccatcaggg?cctgtcctcg?cccgtcacaa?agagcttcaa?caggggagag 720

tgttaagctg?gggatcctct?agaggttgag?gtgattttat?gaaaaagaat?atcgcatttc 780

ttcttgcatc?tatgttcgtt?ttttctattg?ctacaaacgc?gtacgctcag?gttcagctgc 840

tcgaggaagt?tcaactggtt?gaatctggtg?gcggtctggt?gcaaccaggt?ggttctctgc 900

gtctgtcttg?cgccgcgtcc?ggtttcaaca?ttaaagacac?ctacattcac?tgggtgcgcc 960

aggctccggg?taagggtctg?gaatgggtgg?cccgcattta?tccaacgaat?ggctacaccc 1020

gctacgcgga?ttccgtgaaa?ggccgtttca?cgattagcgc?tgataccagc?aagaacaccg 1080

cttatctgca?aatgaacagc?ctgcgtgccg?aagacaccgc?cgtgtactat?tgtagccgtt 1140

ggggcggtga?tggcttttac?gcgatggact?actggggcca?gggtacgctg?gtcacggttt 1200

cttctggggc?ccaatcggtc?ttccccctgg?caccctcctc?caagagcacc?tctgggggca 1260

cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?gtgtcgtgga 1320

actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?tcctcaggac 1380

tctactccct?cagcagcgtg?gtgactgtgc?cctctagcag?cttgggcacc?cagacctaca 1440

tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?gagcccaaat 1500

cttgtgacaa?acatcatcat?catcatcatt?aaggtacc 1538

<210>30

<211>219

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>30

Asp?Ile?Gin?Met?Thr?Gin?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly

1 5 10 15

Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gin?Asp?Val?Asn?Thr?Ala

20 25 30

Val?Ala?Trp?Tyr?Gin?Gin?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile

35 40 45

Tyr?Ser?Ala?Ser?Phe?Leu?Tyr?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly

50 55 60

Ser?Arg?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gin?Pro

65 70 75 80

Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gin?Gin?His?Tyr?Thr?Thr?Pro?Pro

85 90 95

Thr?Phe?Gly?Gin?Gly?Thr?Lys?Val?Glu?Ile?Lys?Lys?Leu?Glu?Ile?Lys

100 105 110

Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu

115 120 125

Gin?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe

130 135 140

Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gin?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gin

145 150 155 160

Ser?Gly?Asn?Ser?Gin?Glu?Ser?Val?Thr?Glu?Gin?Asp?Ser?Lys?Asp?Ser

165 170 175

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

180 185 190

Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gin?Gly?Leu?Ser?Ser

195 200 205

Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys

210 215

<210>31

<211>234

<212>PRT

＜213〉artificial

<220>

＜223〉synthetic construct

<400>31

Gin?Val?Gin?Leu?Leu?Glu?Glu?Val?Gin?Leu?Val?Glu?Ser?Gly?Gly?Gly

1 5 10 15

Leu?Val?Gin?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly

20 25 30

Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gin?Ala?Pro?Gly

35 40 45

Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr

50 55 60

Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr

65 70 75 80

Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gin?Met?Asn?Ser?Leu?Arg?A1a?Glu?Asp

85 90 95

Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?G1y?Asp?Gly?Phe?Tyr?A1a

100 105 110

Met?Asp?Tyr?Trp?Gly?Gin?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Gly?Ala

115 120 125

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

130 135 140

Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val

145 150 155 160

Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe

165 170 175

Pro?Ala?Val?Leu?Gin?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val

180 185 190

Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gin?Thr?Tyr?Ile?Cys?Asn?Val

195 200 205

Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys

210 215 220

Ser?Cys?Asp?Lys?His?His?His?His?His?His

225 230

Claims (23)

1. antigen-binding polypeptides that comprises one or more non-naturally encoded amino acids.

2. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides comprises one or more post translational modifications.

3. antigen-binding polypeptides according to claim 1, wherein said antigen-binding polypeptides is connected in connexon, polymer or bioactive molecule.

4. isolating nucleic acid, it is included in the polynucleotide of the polymerized nucleoside acid hybridization that combines polypeptide under the rigorous condition with coding for antigens, and wherein said polynucleotide comprises at least one and selects codon.

5. method for preparing antigen-binding polypeptides according to claim 3, described method comprise makes the separation antigen-binding polypeptides that comprises non-naturally encoded amino acids contact with comprising with connexon, polymer or the bioactive molecule of the part of described non-naturally encoded amino acids reaction.

6. compositions, it comprises antigen-binding polypeptides according to claim 1 and pharmaceutically acceptable carrier.

7. a treatment suffers from the patient's who is subjected to the disease that ABP regulates method, and described method comprises the compositions according to claim 6 that described patient is dropped into the treatment effective dose.

8. cell, it comprises nucleic acid according to claim 4.

9. method for preparing the antigen-binding polypeptides that comprises non-naturally encoded amino acids, described method is included in and allows cultured cell under the condition that the described antigen-binding polypeptides that comprises non-naturally encoded amino acids expresses, described cell to comprise coding for antigens in conjunction with polypeptide and comprise polynucleotide, quadrature RNA synzyme and the quadrature tRNA that selects codon; With the described antigen-binding polypeptides of purification.

10. method of regulating antigen-binding polypeptides serum half-life or circulation time, described method comprise with one or more non-naturally encoded amino acids replaces any one or natural generation aminoacid more than one in the described antigen-binding polypeptides.

11. the antigen-binding polypeptides by the polymerized nucleoside acid encoding, wherein said polynucleotide comprises the selection codon, and wherein said polynucleotide comprises at least one non-naturally encoded amino acids.

12. a compositions, it comprises antigen-binding polypeptides according to claim 11 and pharmaceutically acceptable carrier.

13. bispecific ABP who comprises an interconnective ABP and the 2nd ABP, a wherein said ABP is incorporated into different epitopes with described the 2nd ABP specificity, a wherein said ABP has the binding specificity at least one epitope on first antigen, and described the 2nd ABP has being different from the binding specificity of second epitope of described first epitope on first antigen or second antigen, and wherein said bispecific ABP comprises at least one non-naturally encoded amino acids.

14. a compositions, it comprises bispecific ABP according to claim 13 and pharmaceutically acceptable carrier.

15. a method for the treatment of the disease or the state of an illness, described method comprise the patient that these needs are arranged is thrown and the compositions according to claim 14 for the treatment of effective dose.

16. an ABP who comprises bispecific ABP according to claim 13, described ABP is coupled to bioactive molecule.

17. one kind is detected the method for expressing one or more antigenic cell or tissues, described method comprises: cell or tissue is contacted with the ABP according to claim 13 that is connected in detectable label; With the described labelling of detection, wherein express one or more existence by the bonded antigenic cell or tissue of ABP with the detection indication of the associating described labelling of cell or tissue.

18. an antigen-binding polypeptides, it comprises the water-soluble polymer that is connected in the single amino acid place of described antigen-binding polypeptides through covalent bond.

19. an antigen-binding polypeptides that comprises at least one connexon, polymer or bioactive molecule, wherein said connexon, polymer or bioactive molecule are connected in described polypeptide by the functional group that incorporates the non-naturally encoded amino acids in the described polypeptide through ribosome into.

20. an antigen-binding polypeptides that comprises the connexon, polymer or the bioactive molecule that are connected in one or more non-naturally encoded amino acids, wherein said non-naturally encoded amino acids is incorporated preliminary election site in the polypeptide into through ribosome.

21. regulate the immunogenic method of antigen-binding polypeptides for one kind, described method comprises with one or more non-naturally encoded amino acids replaces any one or natural generation aminoacid more than one in the described antigen-binding polypeptides.

22. an antigen-binding polypeptides, wherein said polypeptide are to be selected from by SEQ ID NO:19,21,23,24,26,28,30,31 or the group that forms of its fragment.

23. antigen-binding polypeptides according to claim 3, wherein said antigen-binding polypeptides are to be connected in described connexon, polymer or bioactive molecule under the degeneration condition.

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