CN101627053A - Pan-cell surface receptor- specific therapeutics - Google Patents

Abstract

Provided are pan-cell surface receptor- specific therapeutics, methods for preparing them and methods of treatment using them. Among the pan-cell surface receptor- specific therapeutics are pan-HER- specific therapeutics that interact with at least two different HER receptor ligands and/or dimerize with or interact with two or more HER cell surface receptors. By virtue of these properties, the therapeutics modulate the activity of at least two cell surface receptors and are useful for therapeutic purposes.

Description

The therapeutical agent that general cell surface receptor is special

Related application

[0001] present patent application requires the U.S. Provisional Application series number 60/813 of submission on June 12nd, 2006,260, the U.S. Provisional Application series number of submitting on September 29th, 2,006 60/848, the right of priority of the U.S. Provisional Application series number 60/848,941 that on January 5th, 542 and 2007 submitted to.

The theme of every related application quoting [0002] and the complete this paper of incorporating into of related sequence table mode by reference thereof are as a reference.

Invention field

[0003] the invention provides the special therapeutical agent of general cell surface receptor (pan-cell surface receptor), comprise the special therapeutical agent of general HER (pan-HER), and preparation and use their method.

Background of invention

[0004] the cell signaling approach relate to interaction born of the same parents be outer to transmit, the network of the molecule (comprising polypeptide and small molecules) of intercellular and intracellular signal.This classpath interacts, and signal is passed to next member from a member of signal transduction path.Adjusting to a member in the signal transduction path can be transmitted by signal transduction pathway, causes regulating other approach member active and produces the adjusting consequence of such signal transduction, as influence phenotype and cell or body replying signal.Disease and illness can relate to the dysregulation of signal transduction pathway regulating effect (misregulated) or change.The approach that a purpose of drug development is this type of dysregulation of target is to recover more normal adjusting in signal transduction pathway.

[0005] receptor tyrosine kinase (RTKs) is the cell signal molecule family that belongs in the polypeptide that participates in multiple signal transduction pathway.RTKs (comprises embryo's generation, cell fission, propagation, breaks up, divides a word with a hyphen at the end of a line and metabolism) in the various kinds of cell process and plays a role.RTKs can be activated by part.Next this Class Activation causes receptor dimerizationization or the oligomerization condition as follow-up activation signals pathway usually.The activation of signal transduction path such as causing autocrine cell signaling approach or paracrine cell signal transduction path by for example activating the second messenger, causes the specific biological effect.Ligand specificity at RTKs is bonded to related acceptor (cognate receptor).

[0006] RTKs also participates in numerous lysises or plays a role therein, and described lysis comprises cancer, autoimmune disease and other chronic disease (see, for example, Hynes (2005) NatureReviews Cancer 5:341-35).Wherein the cancer that involved wherein of RTKs comprises mammary cancer and the derivative tumour of colorectal carcinoma, cancer of the stomach, neurospongioma and mesoderm (mesodermal-derivedtumor).In several cancers, noticed the dysregulation of RTKs.For example, mammary cancer may be expressed relevant with the amplification of p185-HER2.RTKs is also relevant with eye disease, comprises diabetic retinopathy and macular degeneration.RTKs is also relevant with the adjusting approach that participates in vasculogenesis (comprising that physiological vascularization and tumor vessel form).The adjusting that RTKs also participates in cell proliferation, divides a word with a hyphen at the end of a line and survives.

[0007] with the RTKs of disease-related in, HER (people EGFR family is called ErbB or EGFR again) receptor family (seeing that for example, Hynes etc. (2005) Nature Reviews Cancer5:341-354 is to its discussion in the cancer effect) is arranged.These acceptors (being called the I receptoroid) comprise HER1/EGFR, HER2, HER3 and HER4.Name changes: HER1 is also referred to as EGFR and ERBB1; HER2 is also referred to as ERBB2 and NEU; HER3 is also referred to as ERBB3; And HER4 is also referred to as ERBB4.Whole members of this family have the outer ligand binding domain of born of the same parents, the single structural domain of striding the film district and containing cytoplasmic tyrosine kinase.HERs is expressed in the multiple tissue of epithelium, mesenchyme and neurone origin.

[0008] under normal physiological conditions, the activation of HERs is controlled by the spatial and temporal expression of its part, and wherein said part is the EGF family member in the somatomedin.The zygotic induction of part forms acceptor homodimer and heterodimer, and this causes inherent kinase domain to activate, and causes the phosphorylation on the specific tyrosine residues in the kytoplasm tail, and this finally causes the intracellular signal pathway to activate.

[0009] each acceptor in verified these acceptors all in cancer, play a role (see, for example, people such as Slamon (1989) Science 244:707-712; People such as Bazley (2005) Endocr.Relat.Cancer Jul12 Suppl.1:S17-S27).For example, HER1 (ErbB1) and HER2 (ErbB2) participate in the development and the pathology of multiple human cancer; And the change in these acceptors with spread disease more rapidly and follow the disease-related of bad clinical consequences.Following table has been summed up HER receptor family member and the effect of related part in some cancer thereof.

* (2001) Mol.Cell.Biol. such as Yarden, 2:127

[0010] because of the effect of HER acceptor in cancer and other disease, so the HER acceptor becomes the treatment target.Have the anti-HER therapeutical agent of two classes: target is called the antibody of the ectodomain (or extracellular domain (ectodomain)) of ECD in this article; With the small molecules tyrosine kinase inhibitor.Anti-HER medicine shows limited effectiveness and limited replying the time length.For example, He Sai

(Herceptin (Trastuzimab)) is the humanization form of mouse monoclonal antibody and the ectodomain of target HER2.Validity needs the high expression level (at least 3 times to 5 times overexpressions) of HER2.Therefore, be lower than 25% patient with breast cancer and satisfy the condition of receiving treatment.In this colony, (Piccart-Gebhart etc. 2005 to the treatment no response for the patient of huge ratio; Romond etc. 2005).In addition, the small molecules tyrosine kinase inhibitor often lacks specificity.Therefore, except highly expressing the preliminary election patient of HER2, be 10-15% with anti-HER agent, antibody or the viewed effectiveness of small molecules tyrosine kinase inhibitor of single target with Trastuzumab combination chemotherapy treatment.

[0011] because the limited validity of available therapy, so still need to develop the alternate strategies that is used to tackle these targets.Therefore, belong to the alternate strategies that providing of this paper target is used for target HER receptor family, comprise providing than anti-HER antibody and the more effective therapeutical agent of small molecules.

The invention summary

[0012] as the part of this specification sheets, has the sequence list of using as part of the present invention.Described sequence is incorporated into as the part of this specification sheets.

[0013] provides therapeutical agent and candidate therapeutic agent and the method that is used to identify or find candidate therapeutic agent herein.The invention provides the methods of treatment of using this type of therapeutical agent.Described therapeutical agent is designed to general cell surface receptor therapeutical agent, because they specifically target more than a kind of cell surface receptor, as by combining with part and/or interacting, as long as regulated more than a kind of activity of cell surface receptor with one or more cell surface receptors at one or more acceptors.Described therapeutical agent comprises target those therapeutical agents more than those therapeutical agents of a kind of HER acceptor and one or more HER acceptors of target and other acceptor the HER acceptor of anti-HER therapy resistance development (as help or participate in).In specific embodiments, design described therapeutical agent and candidate therapeutic agent and be intended to solve problem like this, comprise limited effect, resistance and the relevant problem of the anti-HER therapeutical agent validity of restriction take place.

[0014] provides the ectodomain (ECD) of two kinds of cell surface receptors or the polymer of its part herein.This polymeric component comprises an ECD polypeptide and the 2nd ECD polypeptide, and wherein first and second polypeptide directly or through joint are connected with the multimerization structural domain respectively indirectly.In the polymer that provides in the text, first chimeric polyeptides can be the total length ECD of HER1; Or first chimeric polyeptides can contain HER1, HER2, the ECD of HER3 or HER4 less than total length, wherein this ECD part enough parts of containing subdomain I and III at least combine with the part with the HER acceptor, and enough parts of ECD and cell surface receptor dimerization, it comprises enough parts of subdomain II, if all or part of of ECD is not from HER2, ECD all or part of from HER2 in this case, structural domain IV to small part, usually enough parts of module (modules) 2-5 of structural domain IV must exist to realize the dimerization of HER2ECD.Second component of described polypeptide is second chimeric polyeptides, its contain at least cell surface receptor (CSR) ECD enough parts with combine with part and/or with the cell surface receptor dimerization.The CSR of second chimeric polyeptides can be any ECD or its part or the CSR that wants.Yet if first chimeric polyeptides is total length HER1ECD, second chimeric polyeptides can not be total length HER2, although total length HER1 can make up with the HER2 of brachymemma, as long as the HER2 of described brachymemma contains enough parts of structural domain IV to realize dimerization.The first and second chimeric ECD polypeptide form polymer by the interaction of their multimerization structural domain.When comparing with first chimeric polyeptides or its homodimer, gained polymer provided herein combine with extra part and/or with except that first chimeric polyeptides or its homodimer also with more kinds of cell surface receptor dimerizations.

[0015] in other polymer, one of at least person of ECD structural domain or its part comprises sudden change, wherein compares with the form that lacks this sudden change, and described sudden change changes part keying action or other activity.In this type of polymer, the 2nd ECD part can be identical ECD structural domain, wild-type or mutant form, maybe can be the ECD from any other cell surface receptor.As mentioned, each monomeric ECD or its part are directly or be connected with the multimerization structural domain through joint ground or be connected with the 2nd ECD or its part.This type of polymeric example is the polymer that contains at least a HER1ECD, and wherein said HER1ECD contains such sudden change in subdomain III, and it increases the avidity to the part except that EGF.This increase of avidity is at least 10 times, general 100,1000,10 ⁴, 10 ⁵, 10 ⁶Or more times.

The polymer of the ECD (as the reformed ECD of its ligand binding affinity or a plurality of ECD) that contains modification [0016] especially, also is provided.For example, the EGFR1 that activated by EGF and not stimulated by NRG-2 β has modified through like this, thereby these two kinds of parts and EGFR ECD interact to promote receptor dimerizationization/receptor signal effect (to see, Gilmore etc. (2006) Biochem J.396:79-88, this author confirms that NRG2 β is the stimulator of the EFGR mutant stronger than wild-type).The sequence of the EGFR of exemplary modification (being EGFR-S442F) is described in SEQ ID No.414, and wherein ECD begins at the 25th amino acids place.Can be in the polymer that this paper provided and in mosaic that this paper provided and other general cell surface therapeutical agent, use such ECD (25-645 of SEQ ID No.414; When referring to the ECD sequence that comprises initial 25 amino acid signal sequences, the position of described modification is at locus 442 places; And when referring to mature form, the position of described modification is at locus 418 places) or the corresponding section of its part or its allelic variant or species variant, its enough at least part that contain structural domain I-III is combine (or the domain II I that modifies at least enough partly to combine with NRG-2 β) with EGFR1 and NRG-2 β.Can modify ECD that this paper provides or well known by persons skilled in the art changing the part binding specificity, as have with the modification that is exemplified and modify accordingly.From EGFR-S442F and from the ECD among other ECD, especially when with multimerization structural domain (as the Fc structural domain) when being connected, can be used as general cell surface receptor therapeutical agent, wherein said other ECD is modified and ECD to the special ligand interaction of different ECD.The ECD of these modifications can use in described in this article whole embodiments.Therefore, provide the same polymer (homo-multimer) with modified ECD at least two kinds of ligand interactions, acceptor herein, wherein every kind of part and different wild-type ECD interactions.

[0017] polymer provided herein can be a kind of like this polymer, and wherein one of first and second chimeric polyeptides person or the ECD of these two are such heterozygosis ECD, and it contains the subdomain from least 2 kinds of different cell surface receptor ECD.Also comprise such polymer herein, wherein first chimeric polyeptides can contain the ECD less than total length of HER2, HER3 or HER4.The most frequently, first chimeric polyeptides contains the ECD less than total length of HER3 or HER4.

[0018] in addition, this paper provides the ECD of second polypeptide in the polymer partly to comprise those ECD parts, and the ECD of wherein said second polypeptide part is not HER1, but contains ECD all or part of of another kind of CSR.In some cases, other ECD partly comprises the ECD structural domain of second chimeric polyeptides wherein those ECD parts from HER3 or HER4.

[0019] also comprise those ECD polymers in ECD polymer provided herein, wherein second chimeric polyeptides comprises the ECD polypeptide as total length ECD.Perhaps, the ECD structural domain of second chimeric polyeptides be brachymemma and enough parts that contain subdomain I, II and III at least with combine with its part and with the cell surface receptor dimerization.In some cases, the brachymemma ECD structural domain of second chimeric polyeptides comprises that enough parts of structural domain I and III are with binding partner.In other cases, the brachymemma ECD structural domain of second chimeric polyeptides enough parts of comprising this ECD with the cell surface receptor dimerization.

[0020] also comprises the polymer that contains such ECD structural domain, wherein compare, modified described ECD structural domain to change this ECD or to contain part keying action or other activity of the total length acceptor of this ECD with the ECD or the total length acceptor of unmodified.Change and comprise elimination or add the part keying action.For example, ECD compares with unmodified, can modify this ECD to combine with extra part.This modification comprises the corresponding position of modifying S442 (for example, SEQ ID.No.2) or HER acceptor, thereby this ECD is bonded to the part (as NRG-2 β) of HER3 and the part (as EGF) of HER1.

[0021] these polymers can comprise from HER1 with from ECD or its part of HER3 or HER4, thus the ligand interaction of the polymer of gained and at least two kinds, three kinds, four kinds, five kinds, six kinds or seven kinds HER acceptors.Comprise dimer in the described polymer.The multimerization structural domain comprises any multimerization structural domain well known by persons skilled in the art, and any multimerization structural domain that comprises above or hereinafter list is as Fc structural domain or its variant.

[0022] the multimerization structural domain of first and second polypeptide in the polymer provided herein comprises from immunoglobulin (Ig) constant domain (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond, stablize any multimerization structural domain in the middle of hole projection (proturberance-into-cavity) and compensatory hole (the compensatory cavity) of going into of polymeric same or similar size with formation.In some embodiments, described multimerization structural domain is Fc structural domain or its variant of realizing multimerization.The Fc structural domain can comprise from IgG, IgM or IgE from any immunoglobulin molecules.

[0023] common, for polymer provided herein, by its second chimeric polyeptides and/or to produce polymeric cell surface receptor (CSR) or cell surface protein by its dimerization be related acceptor at this polymeric ECD or its part of deriving.The example of CSR comprises HER2, HER3, HER4, IGF1-R, VEGFR, FGFR, TNFR, PDGFR, MET, Tie (being Tie-1 or TEK (Tie-2)), RAGE, Eph acceptor and TXi Baoshouti.In some embodiments, the ECD of second chimeric polyeptides is from VEGFR1, FGFR2, FGFR4, IGF1-R or Tie1.In other cases, the ECD of second chimeric polyeptides or its part are directly or the intron fusion proteins (intron fusion protein) that is connected with the multimerization structural domain indirectly through joint.In some cases, this intron fusion proteins is that Hess reaches spit of fland (Herstain).In one aspect, polymer provided herein combines with at least 7 different ligands.In some embodiments, it is another kind of receptor tyrosine kinase (RTK) that this paper provides polymeric second chimeric polyeptides, and it is not ECD all or part of of HER1.

[0024] this kind ECD polymer can with any HER part be EGF, TGF-α, amphiregulin, HB-EGF, β-tunicin, epiregulin and with arbitrary other ligand interaction of ECD bonded of cell surface receptor except that HER1.For example, described other part can comprise neuregulin, as in neuregulin-1, neuregulin-2, neuregulin-3 and the neuregulin-4 any.

[0025] in some instances, polymer provided herein comprises that polypeptide like this is as first chimeric polyeptides, this polypeptide contains i) from the total length ECD of HER1 acceptor, or ii) its part, described total length ECD or its part are enough to binding partner and/or dimerization, and comprising ECD all or part of of HER3 or HER4 as second chimeric polyeptides, all or part of being enough to of described ECD combines and/or dimerization with part.

[0026] any polymer that provides herein comprises the component chimeric polyeptides that is connected with the multimerization structural domain, and wherein said multimerization structural domain can be immunoglobulin (Ig) constant domain (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, form the free sulfhydryl groups of intermolecular disulfide bond between two molecules and form in hole projection and the compensatory hole any of going into of stablizing polymeric same or similar size.This type of polymer is by their interaction of multimerization structural domain, and with formula configuration (back-to-backconfiguration) orientation back-to-back, the ECD of two kinds of chimeric polyeptides can be used for and the cell surface receptor dimerization in described configuration.In an example, this multimerization structural domain is the Fc structural domain.The Fc structural domain can be from any immunoglobulin molecules, as from IgG, IgM or IgE.

[0027] in the middle of polymer provided herein, comprises those polymers with at least two kinds of chimeric polyeptides.In an example, polymer comprises the polymer with at least two kinds of chimeric polyeptides, and wherein first chimeric polyeptides contains all or part of of HER1 and second chimeric polyeptides contains all or part of of HER3 or HER4.

[0028] comprises also that in the middle of polymer provided herein wherein one of component chimeric polyeptides (constituentchimeric polypeptides) is those polymers of fusion polypeptide.In some embodiments, first chimeric polyeptides and second chimeric polyeptides all are fusion polypeptide.In other example, component chimeric polyeptides peptide forms by chemically conjugated effect.In one embodiment, first chimeric polyeptides and second chimeric polyeptides the two all form by chemically conjugated effect.In other example, one of at least multimerization structural domain directly is connected with ECD in the described chimeric polyeptides.Perhaps, the multimerization structural domain of one of described chimeric polyeptides is connected with the ECD polypeptide through joint.In some embodiments of this situation, the multimerization structural domain of each polypeptide in first and second chimeric polyeptides divides other ECD to be connected through joint and each.Described joint can be chemical joint or peptide linker.

[0029] polymer provided herein can be a heterodimer.This heterodimer can be such heterodimer, and wherein the component chimeric polyeptides is in formula configuration back-to-back, thereby the ECD in every kind of chimeric polyeptides can be used for and the cell surface receptor dimerization.

[0030] provides such heteromultimeric herein, it comprises from the ectodomain (ECD) of a kind of HER acceptor (being HER1, HER2, HER3 or HER4) with from the ECD of second acceptor, thereby among the described ECD one of at least the person be HER ECD and subdomain I, the II that contains ECD and III and part (comprising module 1 at least) thereof, but do not contain the whole of subdomain IV.In this heteromultimeric, the ECD of first and second acceptors is different.In some cases, the ECD of first and second acceptors all is HER ECD.Therefore, heteromultimeric provided herein comprises that wherein a kind of HER is HER1 and all the other HER are heteromultimerics of HER3 or HER4.In other cases, the ECD of second acceptor is from cell surface receptor.The dimerization arm of the ECD of first or second acceptor in described heteromultimeric can be used for the dimerization with cell surface receptor.

These heteromultimerics are provided in the middle of the heteromultimeric that [0031] provides herein, and wherein each ECD directly or through joint is connected with the multimerization structural domain indirectly, thereby the multimerization domain interaction of at least two ECD is to form heteromultimeric.The multimerization structural domain of each ECD in this heteromultimeric comprises immunoglobulin (Ig) constant domain (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, form the free sulfhydryl groups of intermolecular disulfide bond between two molecules and form in hole projection and the compensatory hole any of going into of stablizing polymeric same or similar size.In some embodiments, described multimerization structural domain is the Fc structural domain.This Fc structural domain can comprise from IgG, IgM or IgE from any immunoglobulin molecules.

[0032] cell surface receptor (CSR) of second acceptor of heteromultimeric provided herein is at as the ECD of component in the heteromultimeric or the related acceptor of its part.The example of CSR comprises HER2, HER3, HER4, IGF1-R, VEGFR, FGFR, TNFR, PDGFR, MET, Tie (being Tie-1 or Tie-2 (TEK)), RAGE and EPH acceptor or TXi Baoshouti.In some embodiments, CSR is arbitrary VEGFR1, FGFR2, FGFR4, IGF1-R or Tie-1.

[0033] also considered such heteromultimeric, wherein structural domain or its part from ECD contains change part keying action or specificity or other active sudden change at described structural domain.Compare with the ECD or the total length acceptor of unmodified, this sudden change changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD, thus the part keying action or the specificity of described heteromultimeric display change.The example of this type of heteromultimeric is such heteromultimeric, it comprise modified with two kinds of parts (as HER1 and HER3 part) bonded HER1ECD.For example, the corresponding position has changed the part keying action in S442 modification HER ECD or the modification HER acceptor by replacing with for example F.This type of is modified and produces and the interactional HER1ECD of NRG-2 β.This type of heteromultimeric can contain from HER1 with from ECD or its part of HER3 or HER4, thus the ECD of gained can with at least two or more as three, four, five, six and the ligand interaction of seven kind of HER acceptor.

[0034] provides such heterozygosis ECD herein, each heterozygosis ECD contains the I of structural domain at least of the ECD of one or more CSR, II and III's is all or part of, thereby enough parts (it comprises enough parts of domain II) of the ECD that at least two described structural domains contain cell surface receptor from the ECD and the described heterozygosis ECD of different cell surface receptors, when being intended to described heterozygosis ECD and being connected and the cell surface receptor dimerization with the multimerization structural domain, and/or contain enough parts of ligand binding domains, be intended to ligand interaction with the ECD of therefrom derive ECD structural domain or its part.In some embodiments, described cell surface receptor is the member of HER family.Therefore, for example, structural domain I from HER1, domain II from HER2 and domain II I from HER3.In another embodiment, structural domain I and III come to contain among the comfortable domain II I ECD of sudden change, and wherein said sudden change can combine domain II I with the part of HER3 or HER4.

[0035] described heterozygosis ECD comprises for example these heterozygosis ECD, and it contains from the subdomain of ECD like this or its part, and wherein said ECD contains change part keying action or specific sudden change in described subdomain.The example of this type of sudden change is those sudden changes above and hereinafter described, as modification to HER1, thus the HER1 that modifies and two or more parts (as EGF and NRG-2 β) interaction.

[0036] chimeric polyeptides that also provides this paper that heterozygosis ECD is provided herein, wherein said heterozygosis ECD directly or through joint ground are connected with the multimerization structural domain.The multimerization structural domain comprises immunoglobulin (Ig) constant domain (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, form the free sulfhydryl groups of intermolecular disulfide bond between two molecules and form in hole projection and the compensatory hole any of going into of stablizing polymeric same or similar size.In some cases, described multimerization structural domain is the Fc structural domain.The Fc structural domain can comprise from IgG, IgM or IgE from any immunoglobulin molecules.The polymer that forms between at least two chimeric heterozygosis ECD polypeptide that are provided at this paper herein and provided.

[0037] provides such heteromultimeric herein, it contains from all or part of of the ECD of HER1 and all or part of from the ECD of HER3 or HER4, to such an extent as to if described heteromultimeric contains the brachymemma part of the ECD of HER1, HER3 or HER4, then described brachymemma part comprises subdomain I, II and III at least.

[0038] provide herein contain be connected with the multimerization structural domain enough be used for part in conjunction with and/or the ECD of dimerization or the chimeric polyeptides of its part.The ECD of the chimeric polyeptides that this paper provided or its part can be from any or its modified forms among HER2, HER3 or the HER4ECD.This type of example is: HER2-530 (SEQ ID NO:14), HER2-595 (SEQ ID NO:16), HER2-650 (SEQ ID NO:18), Her3-500 (SEQ ID NO:20), p85Her3 (SEQ IDNO:22), HER3-519 (SEQ ID NO:24), HER3-621 (SEQ ID NO:26), HER4-485 (SEQ ID NO:28), HER4-522 (SEQ ID NO:30), HER4-650 (SEQID NO:32), at arbitrary SEQ ID NO:32,34,127,141,146,159 and 54-125 described in polypeptide and the allelic variant of any aforementioned ECD and species variant with and modified forms, (seen to change active form as modifying, for example, the 25-645 position residue of SEQ ID No.414 or it comprise the part of residue 442F, wherein said SEQ ID No.414 has provided the sequence of the HER1 (EGFR1) that modifies, and wherein the S at 442 places is replaced to produce and NRG2 β and EGF bonded ECD by F).Such heteromultimeric also is provided, it contains from two or more following chimeric polyeptides: arbitrary HER1-501 (SEQ ID NO:10), HER1-621 (SEQ ID NO:12), HER1S442F (SEQ ID No.414,25-645 position residue), or any aforementioned HER1 polypeptide mesopodium is enough in the part of (at the HER1S442F's that contains the S442F sudden change) part combination and/or receptor dimerizationization, HER2-530 (SEQ ID NO:14), HER2-595 (SEQ ID NO:16), HER2-650 (SEQ ID NO:18), Her3-500 (SEQ ID NO:20), p85Her3 (SEQ IDNO:22), HER3-519 (SEQ ID NO:24), HER3-621 (SEQ ID NO:26), HER4-485 (SEQ ID NO:28), HER4-522 (SEQ ID NO:30), HER4-650 (SEQID NO:32), at arbitrary SEQ ID NO:32,34,127,141,146,159 and 54-125 shown in polypeptide, with allelic variant and the species variant of any aforementioned ECD, ECD or its part in the wherein said heteromultimeric directly or through joint are connected with the multimerization structural domain indirectly.

[0039] provides such chimeric polyeptides, it contains ECD or its part of the HER1 acceptor that is connected with the multimerization structural domain, and as arbitrary above listed those, wherein ECD or its part comprise modification, thereby compare with ECD or its part of unmodified, this ECD combines with extra ligand.The example of this type of polypeptide is the chimeric polyeptides that is connected with the multimerization structural domain, this chimeric polyeptides contains from all or part of of the continuous amino acid sequence of the 25-645 position residue of SEQ ID No.414 or has the sudden change that comprises change part keying action at least about 70,80,90,95% sequence identity and in the corresponding position, the 442nd position with SEQ ID No.414 with it, as Ser to Phe.Change to the part keying action comprises such modification, thereby the ECD of HER1 also combines with HER3 part (as NRG-2 β).For example, such chimeric polyeptides, its enough part of ECD that contain the multimerization structural domain and modified HER1 is to interact with EGF and NRG-2 β.

[0040] chimeric polyeptides in polymer and heteromultimeric comprises the chimeric polyeptides that contains the multimerization structural domain, wherein said multimerization structural domain directly or through joint is connected with polypeptide or its part shown in the 25-645 amino acids of SEQ IDNo.414 indirectly, and wherein said polypeptide or its part are enough to realize the part keying action with at least 2 kinds of different ligands.The present invention also provides these chimeric polyeptides.

[0041] in some embodiments, the multimerization structural domain of chimeric polyeptides or heteromultimeric can be constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond, stablize in hole projection and the compensatory hole any of going into of dimeric same or similar size with formation, thereby the chimeric polyeptides in heteromultimeric interacts with formula configuration back-to-back, and the ECD of these two kinds of chimeric polyeptides can be used for the dimerization with cell surface receptor thus.In some cases, this multimerization structural domain is the Fc structural domain.The Fc structural domain can comprise IgG, IgM or IgE from any immunoglobulin molecules.

[0042] also provide isolated polypeptide herein, it contains the amino acid residue sequence shown in arbitrary SEQ ID NO:127,141,146,153,155,157,159,297 or 299.This isolated polypeptide can be connected with the multimerization structural domain to produce chimeric polyeptides.The heteromultimeric that contains chimeric polyeptides also is provided, and wherein said chimeric polyeptides has the aminoacid sequence shown in arbitrary SEQ ID NO:127,141,146,153,155,157,159,297 or 299 and the sequence of multimerization structural domain.Described heteromultimeric can contain enough be used for part in conjunction with and/or the HERECD of receptor dimerizationization or its part as second polypeptide.

[0043] provide nucleic acid molecule herein, its coding chimeric polyeptides or at least a chimeric polyeptides in polymer that this paper provides or heteromultimeric that this paper provided comprise the heterozygosis ECD that this paper provides).The carrier that contains described nucleic acid molecule is provided herein.Also provide and contain the cell of described carrier herein.

[0044] provide pharmaceutical composition herein, polymer, heteromultimeric or chimeric polyeptides that provides herein is provided for it, or coding property nucleic acid molecule.The pharmaceutical composition that contains isolated cell also is provided, and wherein said isolated cells contains nucleic acid provided herein or carrier provided herein.In some embodiments, the preparation of this pharmaceutical composition is used for single dose and uses.In some cases, this pharmaceutical composition also can be prepared and be used for limitation (local), local (topical) or systemic administration.

[0045] provide herein by using the method for described any medicine composite for curing disease or illness herein, disease of being treated or illness comprise cancer, inflammatory diseases, vasculogenesis disease or excessively proliferative disease.The example of cancer comprises carcinoma of the pancreas, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer, kidney or mammary cancer.Comprise proliferative disease in the disease to be treated.The example of proliferative disease comprises those proliferative diseases that relate to smooth muscle cell proliferation and/or divide a word with a hyphen at the end of a line, perhaps preceding ophthalmic, diabetic retinopathy or psoriatic.Other example disease to be treated comprises restenosis, illness in eye, narrow, atherosclerosis, because of hypertension, the urinary bladder disease and the obstructive respiratory disease of blood vessel due to thickening.Other example disease comprises and the disease or the illness that are exposed to one or more neuregulins (" NRG ") (as NRG1 (comprising I, II and III type), NRG2, NRG3 and/or NRG4) relevant (for example caused by it or because of its increase the weight of).The example of NRG relative disease comprises neurological disorder or neuromuscular disease, comprises schizophrenia and alzheimer's disease.

[0046] provides herein by using and treat method for cancer with described herein any pharmaceutical composition of another kind of carcinostatic agent associating.Described carcinostatic agent comprises radiation and/or chemotherapeutic.In an example, carcinostatic agent comprises tyrosine kinase inhibitor or antibody.The example of carcinostatic agent comprises quinazoline kinase inhibitor, antisense or siRNA or other double stranded rna molecule, with the antibody of HER acceptor interaction, put together antibody or cytotoxin in radionuclide.Other exemplary carcinostatic agent comprises Gefitinib (Gefitinib), lapatinibditosylate (Tykerb), handkerchief Buddhist nun monoclonal antibody (Panitumumab), erlotinib (Eroltinib), Cetuximab (Cetuximab), Herceptin (Trastuzimab), imatinib (Imatinib), platinum complex (platinum complex) or nucleoside analog.

[0047] provides the method for the disease of treatment HER mediation herein, comprise that test suffers from the experimenter of described disease to identify which HER expression of receptor or overexpression and based on this result, to select the polymer of at least a, general two kinds of HER acceptors of target.In one embodiment, described disease is a cancer.The example of cancer comprises carcinoma of the pancreas, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer or mammary cancer.

[0048] provides herein and have arbitrary polypeptide at aminoacid sequence shown in SEQ ID NO:54-125 or 405.

[0049] provides herein by mode like this and identify method with the candidate therapeutic molecule of HER acceptor interaction, promptly at first combine with participation dimerization, part in the III based on domain II and IV or I and/or constraint effect (tethering) in the zone of arbitrary effect, the set of test molecule or its is contacted to about 50 amino acid or 50 amino acid whose polypeptide at the most with containing at least about 6 amino acid or 6 amino acid, and identify subsequently and selection and the interactional any test molecule of one or more described polypeptide.In one embodiment, comprise described polypeptide in a library, wherein this library is based on the polypeptides in combination library of HER acceptor.The test molecule polypeptide example that can contact with it comprises any polypeptide with aminoacid sequence shown in arbitrary SEQ ID NO:54-125 and has the arbitrary portion of the polypeptide of 4,5,6,8,10,12 of aforementioned SEQ ID NO or more a plurality of amino-acid residues, or SEQ ID NO:405 and have 6,8,10,12,14,15,18,20,25,30,35,40,45 of SEQ ID NO:405 or 50 or the part of more a plurality of amino-acid residues.Have these libraries in the library of molecule, it contains at the polypeptide on the solid support or on the virus surface.In an example, in phage display library, comprise described polypeptide.

[0050] in one embodiment, the test molecule is a molecular library.Therefore, in an example, the test molecule comprises those molecules in phage display library.In another embodiment, described molecule is small molecules organic compound or polypeptide.

[0051] in method provided herein, selected with structural domain I and/or domain II I or with domain II or with structural domain IV bonded test molecule.Aspect of this method, produced heterodimer with two or more polypeptide test molecules of having identified, a kind of peptide of wherein said heterodimer combines with domain II and another kind of peptide combines with structural domain IV.

[0052] provide isolated antibody herein, it interacts with any polypeptide that has at aminoacid sequence shown in arbitrary SEQ ID NO:54-125 or 405.In one embodiment, this antibody is the interactional polyclonal antibody of two or more polypeptide that is provided with this paper.In some instances, this antibody is receptabody dimer or polymer, and it contains be connected with the multimerization structural domain separately at least 2 kinds not homopolypeptides.Described multimerization structural domain is constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, form the free sulfhydryl groups of intermolecular disulfide bond between two molecules and form in hole projection and the compensatory hole any of going into of stablizing polymeric same or similar size.In an example, this multimerization structural domain is the Fc structural domain.Described Fc structural domain can be from any immunoglobulin molecules, as from IgG, IgM or IgE.

[0053] be these heteromultimerics in described heteromultimeric, wherein the subdomain of ECD or its part contain at described structural domain and change part keying action or specificity or other active sudden change.For example, compare with the ECD or the total length acceptor of unmodified, this sudden change changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD, thus the part keying action or the specificity of this heteromultimeric display change.This type of modification comprises elimination or adds or strengthen any modification of certain activity (as combining with extra ligand, as the interaction of the ECD of HER1 and the part of HER3 (as the NRG-2 beta ligands)).The example of this type of modification is such modification, and it is corresponding in the modification (as the S442F of HER1) of S442 position or in the modification of the corresponding position of HER acceptor.The ECD of gained and at least two kinds of parts, the part of HER1 (as ligands, EGF) and second part (as NRG-2 β) that is used for HER3 in conjunction with or interaction.

[0054] these heteromultimerics can contain from HER1 with from ECD or its part of HER3 or HER4, thus the heterozygote of gained can with the ligand interaction of at least three kinds of HER acceptors.These heteromultimerics can contain the multimerization structural domain, as described herein or any multimerization structural domain well known by persons skilled in the art, and as Fc multimerization structural domain or its variant (being the variant that its T cell interaction is changed).

[0055] the present invention also provides composition, it comprises heteromultimeric and with polymeric mixture, wherein said heteromultimeric comprises from a kind of ECD of HER1 or its part with from another kind of ECD or its part of HER3, and wherein saidly comprises from the ECD of HER1 or its part or from ECD or its part of HER3 with polymer.In some respects, described HER1 part is being strengthened aspect part keying action and/or the biologic activity.In others, described HER3 part is being strengthened aspect part keying action and/or the biologic activity.In yet another aspect, HER1 part and HER3 part are all being strengthened aspect part keying action and/or the biologic activity.

[0056] the present invention also provides pharmaceutical composition, and it includes the composition that part (topical), oral, whole body or limitation (local) are used that is used for through preparation mentioned above.

[0057] in yet another aspect, the invention provides the method that is used for the treatment of cancer, inflammatory diseases, vasculogenesis disease or excessively proliferative disease, comprise the treatment significant quantity of using above listed composition.In some respects, cancer is pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer, kidney or mammary cancer.In others, described disease is a proliferative disease.In others, described proliferative disease comprises the propagation of smooth muscle cell and/or divides a word with a hyphen at the end of a line or preceding ophthalmic or diabetic retinopathy or psoriatic.In others, described disease is restenosis, illness in eye, narrow, atherosclerosis, because of hypertension, the urinary bladder disease and the obstructive respiratory disease (obstructive airway diseases) of blood vessel due to thickening.

The accompanying drawing summary

[0058] because to interact be dynamic, thus shown in amino acid position as a reference and give an example.Shown in position reflection change reach 2,3,4,5 or more a plurality of amino acid whose series of genes seat.Variation also is present in allelic variant and the species variant.Those skilled in the art can identify corresponding sequence by visual comparison or other relative method (comprising obtainable easily algorithm and software).

[0059] Fig. 1 (a) describes people EGF acceptor 1 (HER1; ErbB1; EGFR) synoptic diagram and locus is described various features with reference to HER1, but this class formation is also guarded in other family member (being HER2,3,4).HER (ErbB) family member's ECD contains four subdomains, called after structural domain I (L1), II (S1), III (L2) and IV (S2).Subdomain I cooperates to be used for the part combination with III; Domain II contains the needed sequence of dimerization (' dimerization arm '); And structural domain IV contains the sequence (HER2 exception, it does not take to fetter conformation) of constraint domain II/IV.Represent by single box at the little module that domain II is connected with disulfide linkage among the IV.Shown the β-hair clip in the domain II/ring (being called the dimerization arm again) (corresponding to 240-260 amino acids of the ripe HER1 of total length).Shown the shorter β-hair clip/ring (corresponding to 561-569 amino acids and the 572-585 amino acids of the ripe HER1 of total length) that promotes constraint among the structural domain IV.Marked some amino-acid residue that participates in receptor dimerizationization and/or constraint effect in the ring zone.HER total length acceptor also contains membrane spaning domain (adding the shadow zone), nearly film (JM) structural domain, kinase domain and kytoplasm tail (CT).

[0060] Fig. 1 (b) has described part inductive HER dimerization mechanism.Structural domain I, II, III and IV have drawn.The HER acceptor of most of (about 95%) is with wherein constraint conformation (tethered conformation) existence of domain II and IV formation intramolecular interaction, and the 5% monomer shape acceptor of all the other on the cell surface is in non-constraint configuration or the open configuration.Part (E) combines with the structural domain I and/or the III of HER family receptors.The part keying action is stablized non-constraint conformation, and the dimerization arm in this conformation in the domain II exposes.Regional interaction in the dimerization arm of domain II and the domain II of another HER family receptors is to produce homodimer and heterodimer.The activation that the part keying action of HER acceptor and dimerization are induced inherent kinase domain causes phosphorylation on the specific tyrosine residues and follow-up downstream signal conduction in the kytoplasm tail.

[0061] comparison result and the structural domain organizational form of HER1 (EGFR) the ECD isoform (isoform) of mature form (lacking signal sequence) when comparing of Fig. 2 (a) description and length EGF R (NP_005219 is corresponding to the 25-1210 amino acids of SEQ ID NO:2).The HER1 that is compared (EGFR) ECD isoform (lacking signal sequence) comprises HF100 (SEQ IDNO:12), HF110 (SEQ ID NO:10), HF120 (ERRP, SEQ ID NO:34), HER1 (EGFR) isoform b (NP_958439,25-628 amino acids corresponding to SEQ ID NO:12), HER1 (EGFR) isoform c (NP_958440,25-405 amino acids corresponding to SEQ ID NO:133) and HER1 (EGFR) isoform d (NP_958441 is corresponding to the 25-705 amino acids of SEQ IDNO:131).Structural domain I (corresponding to the 1-165 amino acids of the ripe HER1 of total length (EGFR)) and domain II I (corresponding to the 313-481 amino acids of the ripe HER1 of total length (EGFR)) show with runic.Domain II (corresponding to the 166-312 amino acids of the ripe HER1 of total length (EGFR)) and structural domain IV (corresponding to the 482-621 amino acids of the ripe HER1 of total length (EGFR)) show with conventional font, and highlight the halfcystine module.The non-ECD part of the ripe HER1 of total length (EGFR) shows with light gray.Do not show that with aminoacid sequence among the ripe total length HER1 (EGFR) amino acid of coupling describes with italic.

[0062] comparison and the structural domain organizational form of the HER2ECD isoform of mature form (lacking signal sequence) when comparing of Fig. 2 (b) description and total length HER2 (AAA75493.1 is corresponding to the 23-1255 amino acids of SEQ ID NO:4).The HER2ECD isoform of being compared (lacking signal sequence) comprises HF200 (SEQ ID NO:18), ErbB2.1e (corresponding to the 23-633 amino acids of SEQ ID NO:137), HF210 (SEQ ID NO:16), HF220 (SEQ ID NO:14), ErbB2.1d (corresponding to the 25-680 amino acids of SEQ ID NO:136), ErbB2.1f (corresponding to the 23-575 amino acids of SEQ ID NO:138), HER2-int11 (corresponding to the 23-438 amino acids of SEQ IDNO:141), Hess reaches spit of fland (herstatin) (AAD56009 is corresponding to the 23-419 amino acids of SEQ ID NO:135) and ErbB2.a (corresponding to the 23-90 amino acids of SEQ IDNO:139).Structural domain I (corresponding to the 1-172 amino acids of the ripe HER2 of total length) and domain II I (corresponding to the 320-488 amino acids of the ripe HER2 of total length) show with runic.Domain II (corresponding to the 173-319 amino acids of the ripe HER2 of total length) and structural domain IV (corresponding to the 489-628 amino acids of the ripe HER2 of total length) show with conventional font, and highlight the halfcystine module.The non-ECD part of the ripe HER2 of total length (EGFR) shows with light gray.Do not show that with aminoacid sequence among the ripe total length HER2 amino acid of coupling described by italic.

[0063] comparison result and the structural domain organizational form of the HER3ECD isoform of mature form (lacking signal sequence) when comparing of Fig. 2 (c) description and total length HER3 (NP_001973.1 is corresponding to the 20-1342 amino acids of SEQ ID NO:6).The HER3ECD isoform of being compared (lacking signal sequence) comprises HF300 (SEQ ID NO:26), HF310 (SEQ ID NO:20), p85HER3 (corresponding to the 20-562 amino acids of SEQ ID NO:22), HER3-519 (SEQ ID NO:24), HER3 isoform (AAH02706,20-331 amino acids corresponding to SEQ ID NO:143), HER3-int10 (corresponding to the 20-403 amino acids of SEQ ID NO:146), p75sHER3 (corresponding to the 20-534 amino acids of SEQ ID NO:150), HER3-int11 (corresponding to the 20-425 amino acids of SEQ IDNO:148), p45sHER3 (corresponding to the 20-331 amino acids of SEQ ID NO:149), p50sHER3 (corresponding to the 20-400 amino acids of SEQ ID NO:151) and HER3 isoform 2 (P21860-2 is corresponding to the 20-183 amino acids of SEQ ID NO:144).Structural domain I (corresponding to the 1-159 amino acids of the ripe HER3 of total length) and domain II I (corresponding to the 312-480 amino acids of the ripe HER3 of total length) show with runic.Domain II (corresponding to the 160-311 amino acids of the ripe HER3 of total length) and structural domain IV (corresponding to the 481-621 amino acids of the ripe HER3 of total length) show with conventional font, and highlight the halfcystine module.The non-ECD part of the ripe HER3 of total length shows with light gray.Do not show that with aminoacid sequence among the ripe total length HER3 amino acid of coupling described by italic.

[0064] comparison and the structural domain organizational form of HER4 (ErbB4) ECD of mature form (lacking signal sequence) when comparing of Fig. 2 (d) description and HER4 (ErbB4) (NP_005226 is corresponding to the 26-1308 amino acids of SEQ ID NO:8).The ErbB4ECD isoform of being compared (lacking signal sequence) comprises ErbB4-522 (SEQ ID NO:30), HF400 (SEQ IDNO:32), ErbB4-int11 (corresponding to the 26-430 amino acids of SEQ ID NO:157), ErbB4-int12 (corresponding to the 26-506 amino acids of SEQ ID NO:159), HF410 (SEQID NO:28), ErbB4-int9 (corresponding to the 26-391 amino acids of SEQ ID NO:153) and ErbB4-int10 (corresponding to the 26-421 amino acids of SEQ ID NO:155).Structural domain I (corresponding to the 1-163 amino acids of the ripe ErbB4 of total length) and domain II I (corresponding to the 309-477 amino acids of the ripe ErbB4 of total length) show with runic.Domain II (corresponding to the 164-308 amino acids of the ripe ErbB4 of total length) and structural domain IV (corresponding to the 478-625 amino acids of the ripe ErbB4 of total length) show with conventional font, and highlight the halfcystine module.The non-ECD part of the ripe HER1 of total length (EGFR) shows with light gray.Do not show that with aminoacid sequence among the ripe total length ErbB4 amino acid of coupling described by italic.

[0065] Fig. 3 (a) shows observed synergetic property growth inhibitory effect when MDA MB 468 cells are handled with RB200h and tyrosine kinase inhibitor AG825.

[0066] Fig. 3 (b) shows observed synergetic property growth inhibitory effect when A 431 cells are handled with RB200h and Gefitinib (Gefitinib) (Iressa (Iressa)).

[0067] Fig. 4 shows the synoptic diagram of general Her part trap (Pan-Her ligand trap) RB200h.

[0068] Fig. 5 shows the purity of transferring albumen (hermodulin) construct (RB600, HFD 100, HDF300 and RB200h) as the HER that analyzes by reversed-phase HPLC.

[0069] Fig. 6 a shows that the reservation of through engineering approaches dimer is right ¹²⁵I-EGF and ¹²⁵The specificity of I-HRG β: swimming lane 1:HFD100=HER1-621/Fc, swimming lane 2:HFD200=HER2-628/Fc, swimming lane 3:HFD300=HER3-621/Fc and swimming lane 4:HFD400=HER4-625/Fc.

It is right that Fig. 6 b shows that the through engineering approaches dimer of RB200h keeps ¹²⁵I-EGF and ¹²⁵The specificity of I-HRG1 β 1.

[0070] Fig. 7 a shows and RB200h bonded EU-NRG1 β 1.

[0071] Fig. 7 b shows that EU-EGF combines with RB200h.

[0072] Fig. 7 c shows that other HER part competes the Eu-EGF bonded.

[0073] Fig. 7 d shows that other HER part competes the Eu-NRG1-b1 bonded.

[0074] Fig. 8 a-c shows that RB200h, Trastuzumab (Herceptin) or Erbitux (Erbitux) suppress the HER family protein phosphorylation of EGF ligand stimulation in A431 epidermoid carcinoma cell (A431 epidermoid cancer cell).

[0075] Fig. 8 d-f shows that RB200h, Trastuzumab or Erbitux suppress the HER family protein phosphorylation of NRG1 β 1 ligand stimulation in A431 epidermoid carcinoma cell.

[0076] Fig. 9 a-c shows that RB200h, Trastuzumab or Erbitux suppress the HER family protein phosphorylation of EGF ligand stimulation in the ZR-75-1 breast cancer cell.

[0077] Fig. 9 d-f shows that RB200h, Trastuzumab or Erbitux suppress the HER family protein phosphorylation of NRG1 β 1 ligand stimulation in the ZR-75-1 breast cancer cell.

Figure 10 a shows that RB600 is suppressing stronger aspect the receptor phosphorylation of EGF stimulation than RB200h.

[0078] Figure 10 b shows that RB600 is suppressing stronger aspect the receptor phosphorylation of NRG1 β 1 stimulation than RB200h.

[0079] Figure 11 a shows that RB200h suppresses the tumour cell A431 cell proliferation of cultivating.

[0080] Figure 11 b shows that RB200h suppresses the tumour cell of cultivating: the MDA-MB-468 breast cancer cell.

[0081] Figure 12 a-b show RB200h suppress ZR-75-1 (Figure 11 a) and the ligand stimulation of A549 (Figure 11 b) tumour cell and the soft agar colony that do not stimulate grow.

[0082] Figure 13 a shows that RB200h suppresses to induce breast cancer cell propagation by EGF inductive part.

[0083] Figure 13 b shows that RB200h suppresses to induce breast cancer cell propagation by NRG1 β 1 inductive part.

[0084] Figure 13 c shows that RB200h suppresses to induce breast cancer cell propagation by LPA inductive part.

[0085] Figure 14 a shows that RB200h suppresses to induce breast cancer cell propagation by the part of EGF inductive SUM149 breast cancer cell.

[0086] Figure 14 b shows that RB200h suppresses to induce breast cancer cell propagation by the part of LPA inductive SUM149 breast cancer cell.

[0087] Figure 15 a-d shows the synergetic property growth-inhibiting of RB200h and tyrosine kinase inhibitor: AG-825, Gefitinib and erlotinib (Erlotinib).

[0088] Figure 16 shows the synergetic property growth-inhibiting of RB200h and tyrosine kinase inhibitor Gefitinib.

[0089] Figure 17 shows that RB200h and AG 825 tyrosine kinase inhibitors have the synergetic property antiproliferative effect.

[0090] Figure 18 shows that RB200h and Iressa produce strong collaborative antiproliferative and reply in A431 epidermal carcinoma cell.

[0091] Figure 19 shows the synergy between the RB200h and Iressa in the BT474 breast cancer cell.

[0092] Figure 20 shows the treatment evaluation of RB200h in the subcutaneous model of A431.The mean tumour volume of subcutaneous A431 tumour in the nude mouse.Beginning administration on the 10th.Two factor variance analyses (Two-way ANOVA with Bonferroni ' s posttest) with the combination of Bonferroni inspecting rear test.In this figure, the * statistical significance is represented p＜0.05, and * * represents p＜0.01, and * * * represents p＜0.001.

[0093] Figure 21 shows the method synoptic diagram that is used for producing from HFD100 by PCR the HFD100 mutant.

[0094] Figure 22 shows HFD100-T39S (Figure 22 a), HB-EGF (Figure 22 b) and TGF-α (Figure 22 c) have enhanced avidity to EGF.

[0095] Figure 23 shows binding affinity and the correlated expression level of HFD100 mutant to EGF, HB-EGF and TGF-α.

[0096] Figure 24 shows the mean body weight (figure A) and the whole last gross tumor volume (figure B) of preliminary toxicity research.

[0097] Figure 25 shows the mean tumour volume of subcutaneous A431 tumour in the nude mouse.Beginning administration on the 10th.Use two factor variance analytical calculation statistical significance * p＜0.05, * * p＜0.01, * * * p＜0.001 with the combination of Bonferroni inspecting rear test.

[0098] Figure 26 shows the average tumor weight of subcutaneous A431 tumour.Use one-way analysis of variance (One way ANOVA) counting statistics significance.

[0099] Figure 27 shows the mouse body weight during treatment is studied.

Detailed Description Of The Invention

A. definition

B. the special therapeutic agent of general cell surface receptor

C.HER acceptor and other cell surface receptor structure and activity

1.HER1ECD structure and domain organizational form

2.HER2ECD structure and domain organizational form

3.HER3ECD structure and domain organizational form

4.HER4ECD structure and domain organizational form

5.HER family's part, ligand specificity and ligand-mediated receptor activation.

6. the generation of dimerization and constraint and active homodimer and heterodimer

7.HER family receptors is active

A. cell proliferation

B. cell survival

C. Angiogenesis

D. divide a word with a hyphen at the end of a line and invade

8. other CSR ECD

A.VEGFR1 (Flt-1) and VEGFR2 (KDR)

b.FGFR1-FGFR4

c.IGF 1R

D.RAGE and other CSR

The polymeric component of D.ECD and the polymeric formation of ECD

1.ECD polypeptide

The total length ECD of a.HER family

i.HER1ECD

ii.HER2ECD

iii.HER3ECD

iv.HER4ECD

The ECD of b.HER family brachymemma

I. the HER1ECD of brachymemma

Ii. the HER2ECD of brachymemma

Iii. the HER3ECD of brachymemma

Iv. the HER4ECD of brachymemma

C. heterozygosis ECD

D. other CSR or RTK ECD or its part

E. the polypeptide isoform of alternative splicing

2. polymeric formation

A. peptide linker

B. Heterobifunctional bridging agent

C. polypeptide multimerization domain

I. immunoglobulin domains

(a) .Fc domain

(b). enter hole projection (being knuckle and hole (knobs and holes))

Ii. leucine zipper

(a) fos and jun

(b)GCN4

Ii. other multimerization domain

(a)R/PKA-AD/AKAP

3 chimeric ECD polypeptide

A. exemplary chimeric HER ECD polypeptide

The E.ECD polymer

A. the ECD's of total length HER1ECD and another CSR is all or part of

B. the ECD component of two or more brachymemmas

C. heterozygosis ECD polymer

D. identical or from identical CSR deutero-ECD component

F. produce the method and the ECD polymer that produces gained of the nucleic acid of the chimeric ECD polypeptide fusions of coding

1. synthetic gene and polypeptide

2. clone and the method for separating the ECD polypeptide

3. produce and the chimeric method of clone's ECD polypeptide

4. expression system

A. prokaryotic expression

B. yeast

C. insect cell

D. mammalian cell

E. plant

5. the method for transfection and conversion

6.ECD polypeptide, chimeric polyeptides and polymeric recovery of gained ECD and purifying

G. assess or the active assay method of monitoring ECD polymer

1. kinases/phosphorylation assay method

2. compound/dimerization

3. part combined techniques

4. cell proliferating determining method

5. cell disease model determination method

6. animal model

H.ECD polymer and ECD polymer preparation of compositions, preparation and use

I. use the illustrative methods of ECD polymer treatment

1.HER the disease or the illness of mediation

A. cancer

B. vasculogenesis

C. the relevant disease of neuregulin

D. the relevant disease and the patient's condition of smooth muscle proliferation

2.RTK the illness or the disease of mediation

A. vasculogenesis related Ophthalmopathy

B. vasculogenesis dependency atherosclerosis

C. other vasculogenesis dependency treatment

D. cancer

3. the disease or the illness of other CSR mediation

4. select the polymeric ECD polypeptide fraction of ECD

5. the patient selects

6. conjoint therapy

J. be used to identify, screen and produce the method for general HER therapeutical agent

1. the target of general HER therapeutical agent

2. identify the screening method of general HER therapeutical agent

A. phage display

I. peptide library

Ii. polymer polypeptide (heterodimer peptide)

B. exemplary screening assay method

K. embodiment

A. definition

[0100] unless otherwise defined, whole term used herein and scientific terminology have the equivalent with those skilled in the art in the invention's common sense.Unless otherwise indicated, whole patents, present patent application, disclosed application and publication, GENBANK sequence, website and other the disclosed material mode of mentioning in the whole text in this paper is open by reference incorporated in full.Term at this paper exists under the situation of various definitions, and those definition in this part are preferential.In Referral URL or other project such as identifier or address, be to be understood that this class identifier can change and the internet on the specifying information conversion of can coming and going, but equivalent information is known and can obtains easily, as passing through retrieval internet and/or suitable database.Its operability and public who quotes the expression this type of information is popularized.

[0101] as used herein, " general cell surface receptor therapeutical agent " or " therapeutical agent that general cell surface receptor is special " are to regulate the active molecule of two or more cell surface receptors, comprise compound and small molecules based on peptide.

[0102] as used herein, " general HER therapeutical agent " or " therapeutical agent that general HER is special " are the general cell surface receptor therapeutical agents (molecule comprises compound and small molecules based on peptide) that can regulate two or more HER (ErbB) receptor active.Usually, at least 2 kinds of different HER acceptors of general HER therapeutical agent target, as by the part keying action and/or with the interaction of acceptor.

[0103] as used herein, carcinostatic agent comprises any cancer therapy and is used for the medicine of described therapy and comprises radiotherapy, surgical operation, anticancer compound (comprising small molecules), chemotherapeutic (as cis-platinum and gencytinbine) and monoclonal antibody.

[0104] as used herein, cell surface receptor is a kind of protein, and it is expressed on the cell surface and generally comprises membrane spaning domain or make it be anchored into the other parts of cell surface.As acceptor, it combines with the part of mediation or participation cell surface receptor activity (as signal transduction or part internalization).Cell surface receptor includes but not limited to single transmembrane receptor and g protein coupled receptor.Receptor tyrosine kinase also belongs to the row of this type of cell surface receptor as growth factor receptors.

[0105] as used herein, structural domain refers to the part like this (having 3 or more a plurality of, common 5 or 7 or more a plurality of amino acid whose sequence) of polypeptide, and this part is structurally and/or can distinguishes on the function or definable.For example, structural domain comprises those parts that can form independent pleated sheet structure and/or be discerned by functionally active (as kinase activity) in the protein of being made up of one or more structural motifs (for example the α spiral that is connected by the ring district and/or the combination of βZhe Die chain).Protein can have one or more than one different structure territory.For example, structural domain can be by identifying, define or distinguish with relevant family member's sequence homology (as the homology and the motif of definition ectodomain).In another example, structural domain can by its function (as by enzymic activity (for example kinase activity)) or with the ability of bio-molecular interaction (as DNA combine, part combination and dimerization) distinguish.Structural domain is Presentation Function or activity independently, thus independently or the structural domain that merges with another molecule can show activity, for example proteolytic activity or part keying action.Structural domain can be from the linear aminoacid sequence of polypeptide or non-linear aminoacid sequence.Many polypeptide contain a plurality of structural domains, for example, figure 1 illustrates the structural domain structure of HER1 (EGFR): it comprises ECD, membrane spaning domain, nearly membrane structure territory, kinase domain and carboxyl terminal cytoplasmic structure territory.For HER1 (EGFR), described ECD comprises 4 subdomains, is called I (or L1), II (or S1), III (or L2) and IV (or S2)." L " subdomain (I and III) participates in ligand interaction, and II (S1) and IV (S2) structural domain interact through constraint district (tethering region); Subdomain II (S1) comprises the dimerization ring.Those skilled in the art are familiar with structural domain and can be by identifying them with the structural homology of other this type of structural domain and/or function homology.

[0106] as used herein, the cytoplasmic structure territory is the structural domain that participates in signal transduction.

[0107] as used herein, ectodomain (ECD) is the part like this of cell surface receptor, and it is present on the surface of acceptor and comprises the ligand-binding site point.For this paper purpose, ECD comprises any molecule or its part that contains ECD when referring to ECD, as long as this ECD polypeptide does not contain the relevant any continuous sequence of another kind of structural domain (being membrane spaning domain, protein kinase structural domain or other structural domain) with related acceptor.Therefore, for example, the ECD polypeptide comprises the alternative splicing isoform of CSR, and wherein said isoform has the ECD of containing part, but lack any other structural domain of related CSR, and have the uncorrelated or unmatched additional sequences of another kind of structural domain sequence with related CSR.These additional sequences can be the sequences (intron-encoded sequences) of coding intron, as occurring in the intron fusion proteins isoform.Usually, described additional sequences does not suppress or does not disturb the part keying action and/or the receptor dimerization activity of CSR ECD polypeptide.The ECD polypeptide also comprises heterozygosis ECD.

[0108] as used herein, heterozygosis ECD refers to such ECD, and it contains the part from the ECD of different cell surface receptors.Usually, heterozygosis ECD contains at least two ECD subdomains from different cell surface receptors.

[0109] as used herein, chimeric polyeptides refers to contain the polypeptide of part like this, and wherein said part is from least 2 kinds of homopolypeptide or single peptide species two contiguous sectionss not.Therefore, chimeric polyeptides generally comprises from all or part of amino acid residue sequence of a peptide species with from the another kind all or part of aminoacid sequence of homopolypeptide not.These two parts can connect directly or indirectly and can by intensity be enough under equilibrium conditions with physiological condition under (as etc. open in pH 7 buffer saline) keep most as described in peptide bond, other covalent linkage or other noncovalent interaction of integrity of chimeric polyeptides be connected.For this paper purpose, chimeric polyeptides comprises these polypeptide, partly all or part of of its ECD that contains the CSR that directly or indirectly is connected with the multimerization structural domain.Chimeric polyeptides also can comprise additional sequences, for example the epi-position label.

[0110] as used herein, fusion constructs refers to contain from the encoding sequence of a nucleic acid molecule with from the nucleic acid molecule of the encoding sequence of another nucleic acid molecule, wherein said encoding sequence is arranged in same open reading-frame (ORF), when thereby this fusion constructs is transcribed in host cell and translated, produce and contain two proteinic protein.These two molecules can in described construct be adjacent or by contain 1,2,3 or more, generally be less than 10,9,8,7,6 amino acid whose joint polypeptide and separate.By fusion constructs encoded protein matter products known as fusion polypeptide.Transcribed spacer can be encoded and be changed the polypeptide of this fusion polypeptide characteristic (as transporting in solubleness or the born of the same parents).

[0111] as used herein, fusion rotein refer to contain from more than two protein or peptide two or more directly or the chimeric protein of the part that connects indirectly through peptide bond.

[0112] as used herein, the multimerization structural domain refers to promote the interactional aminoacid sequence of stablizing of a peptide molecule and another peptide molecule that contains complementary multimerization structural domain (complementary multimerization domain), it can be identical or different multimerization structural domain, to form stable polymer with first structural domain.Usually, polypeptide is connected with the multimerization structural domain directly or indirectly.The multimerization structural domain of example comprise immunoglobulin sequences or its part, leucine zipper, hydrophobic region, hydrophilic area, compatible protein matter-protein interaction structural domain (as, but be not limited to R subunit and anchoring structure territory (AD) of PKA), between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection (that is, going into the knuckle (knobs into holes) of hole) and compensatory hole.This multimerization structural domain for example can be a constant region for immunoglobulin.Immunoglobulin sequences can be the immunoglobulin (Ig) constant domain, as Fc structural domain or its part from IgG1, IgG2, IgG3 or IgG4 hypotype, IgA, IgE, IgD and IgM.

[0113] as used herein, (also being called in this article into the hole projection) refers to the specific multimerization structural domain of through engineering approaches " to go into the knuckle of hole ", thereby this type of structural domain between the two and/or the steric interaction among the many persons of this type of structural domain not only promote the stable phase mutual effect, also promote heterodimer (or polymer) from monomer mixture, to have precedence over homodimer (or same polymer) and form.This can for example realize by making up projection and hole.Can make up projection by the small volume amino acid side chain of replacing from the first polypeptide interface with volume larger side chain (for example tyrosine or tryptophane).By randomly on the second polypeptide interface, produce big or small same or analogous compensatory " hole " with less amino acid side chain (for example L-Ala or Threonine) the replacement large volume amino acid side chain of volume corresponding to described projection.

[0114] as used herein, complementary multimerization structural domain refers to so two or more multimerization structural domains, and they interact to form the stable polymer of the polypeptide that is connected with each this structural domain.Complementary multimerization structural domain can be the member of same structure territory or structural domain family, as Fc district, leucine zipper and knuckle and hole).

[0115] as used herein, " Fc " or " Fc district " or " Fc structural domain " refer to such polypeptide, and it contains the constant region of heavy chain of antibody, but do not comprise first constant region of immunoglobulin domains.Therefore, Fc refers to last three constant regions of the immunoglobulin domains of latter two constant region of immunoglobulin domains of IgA, IgD and IgE or IgE and IgM.Randomly, the Fc structural domain can be included in all or part of of the N-terminal flexible hinge of these structural domains.For IgA and IgM, Fc can comprise the J chain.For the exemplary Fc structural domain of IgG, Fc contains immunoglobulin domains C γ 2 and C γ 3, and the hinge between C γ 1 and C γ 2 all or part of randomly.The border in Fc district can change, but usually, comprises at least a portion of hinge area.The exemplary sequence of IgG Fc structural domain is described in SEQ ID NO:167.In addition, Fc also comprises any allelic variant or species variant or any variation or modified forms, as changing and the keying action of FcR or any variant or the modified forms of the effector function that change Fc mediates.The exemplary sequence of other Fc structural domain (the Fc structural domain that comprises modification) is described in SEQ ID NO:168 or 169.

[0116] as used herein, " Fc mosaic " refers to the chimeric polyeptides that wherein one or more polypeptide directly or indirectly are connected with Fc district or derivatives thereof.Generally, the combination of Fc mosaic has the Fc district and the another kind of polypeptide (for example ECD polypeptide) of immunoglobulin (Ig).The derivative of Fc polypeptide or modified Fc polypeptide are well known by persons skilled in the art.

[0117] as used herein, the polypeptide that contains at least two chimeric polyeptides that comprise ECD part and multimerization structural domain is also referred to as " ECD polymer " (being called again with polymer or heteromultimeric or homodimer or heterodimer).The multimerization structural domain is derived under the situation of antibody or its part therein, and this polypeptide can be called immunoadhesin (immunoadhesins) or receptabody dimer or polymer.Described polymeric composition polypeptide is also referred to as chimeric polyeptides in this article.The multimerization structural domain can be direct or indirect with being connected of ECD and can use the recombinant nucleic acid method to realize to produce fusion rotein.Can use the chemical coupling method to realize connecting, as using Heterobifunctional reagent.Exemplary coupling agent comprises N-succinimido-3-(2-pyridine dimercapto) propionic ester (SPDP); imino-sulfane (iminothiolane) (IT); the difunctionality derivative of imido-ester (imidoester) (as diimine for the dimethyl adipate hydrochloride); active ester (as disuccinimidyl suberate); aldehyde (as glutaraldehyde); two-triazo-compound (as two-(to the triazobenzene formyl radical) hexanediamines); two-diazo compound derivative (as two-(right-the diazobenzene formyl radical)-quadrols); vulcabond is (as toluene 2; the 6-vulcabond) and the dual-active fluorine cpd (as 1; 5-two fluoro-2, the 4-dinitrobenzene).

[0118] as used herein, antibody refers to have the immunoglobulin molecules of specific amino acids sequence, and wherein said specific amino acids recognition sequence is unique specific antigen to its target.Immunoglobulin (Ig) is the glycoprotein that is shown as " Y " shape molecule on the structure, and described molecule contains (from arbitrary class among 5 class heavy chain: γ, δ, α, μ, the ε) two the identical heavy chains and two identical light chains that connected by disulfide linkage.Every heavy chain has constant region (C _H), it is identical for of a sort all immunoglobulins, and variable region (V _H), it serves as antigen binding site and is different according to antigen-specific and between immunoglobulin (Ig).Heavy chain γ, δ, α have by three structural domain (C _H1, C _H2 and C _H3) constant region of Zu Chenging and have hinge area, and the constant region of heavy chain μ, ε is by four structural domain (C _H1, C _H2, C _H3, C _H4) form.Light chain has a constant domain (C _L) and a variable domains (V _L).For this paper purpose, the appellation of antagonist refers to such molecule, and it contains all or part of of immunoglobulin molecules, and it contains one or more structural domains of immunoglobulin molecules.For example, the Fab fragment is the part of antibody molecule, and its constant domain and variable domains by every heavy chain and every light chain is formed.The Fc fragment by 2-3 constant domain of heavy chain and randomly all or part of (class that depends on antibody) of hinge area form.Therefore, the appellation of antagonist refers to polyclonal antibody, monoclonal antibody or contains any molecule of antibody moiety, for example receptabody dimer or polymer, wherein the multimerization structural domain that two peptide species (i.e. the ECD of at least two CSR or its part) are linked together is antibody or its part (as the Fc fragment).

[0119] as used herein, monoclonal antibody refers to have the antibody of high degree of specificity, and this antibody produces by the clone by the single hybrid cell due to B cell and the tumour cell fusion in the laboratory.

[0120] as used herein, put together connection, pairing or the joint that refers to two or more molecules.For example, identical or different two or more polypeptide (or its fragment, structural domain or active part) can link together, or polypeptide (or its fragment, structural domain or active part) can be connected with synthetic molecules or chemical molecular or other parts.The joint of two or more molecules can be undertaken by directly connecting (being connected with the nucleotide sequence of another polypeptide of coding as the nucleotide sequence by the polypeptide of will encoding), maybe can be indirect, as non-covalent or covalent coupling effect by a molecule and another molecule.For example, puting together of two or more molecules or polypeptide can connect realization by chemistry.

[0121] as used herein, " label " or " epi-position label " refers to be added into usually the aminoacid sequence of polypeptide aminoterminal or carboxyl terminal.Comprise the label that merges with polypeptide and can be beneficial to peptide purification and/or detection.Usually, label or label polypeptide refer to such polypeptide, and it has enough residues maybe can serve detection or purifying to provide by the epi-position of antibody recognition, but are enough short, thereby it does not disturb the activity of the chimeric polyeptides that is attached thereto.The label polypeptide generally is enough unique, thus with label polypeptid specificity bonded antibody basically not with the polypeptide that is connected in this label polypeptide in the epi-position cross reaction.Suitable label polypeptide has at least 5 or 6 amino-acid residues usually, and generally has about 8-50 amino-acid residue, and common have a 9-30 residue.Label can be connected with the one or more chimeric polyeptides in the polymer and allow to detect or this polymer of recovery from sample or mixture.This type of label be know and can synthesize easily and design.The example tag polypeptide comprises those label polypeptide that are used for affinity purification and comprises His label, influenza hemagglutinin (HA) label polypeptide and antibody 12CA5 thereof (Field etc. (1988) Mol Cell.Biol.5:2159-2165); C-myc label and 8F9 thereof, 3C7,6E10, G4, B7 and 9E10 antibody (are seen, (1985) Molecularand Cellular Biology 5:3610-3616 such as Evan) and herpes simplex virus glycoprotein D (gD) label and antibody (Paborsky etc. (1990) Protein Engineering5:547-553 (1990) thereof for example.

[0122] as used herein, the fusion tag polypeptide refers to contain the chimeric polyeptides of the ECD polypeptide that merges with the label polypeptide.

[0123] as used herein, the constraint effect refers to the interaction between two structural domains of acceptor monomer, thereby this monomer is not so that it more is easy for interactional conformation exists.For example, subdomain II (S1) can with subdomain IV (S2) domain interaction among HER1, HER3 and the HER4, form the non-activity structure carry the baby.When being in bound state, acceptor or its isoform be more difficult to be used for or to be not useable for dimerization and/or receptors bind.The ECD of the HER1 of monomeric form, HER3 and HER4 exists with the constraint form, thereby shows than the lower ligand affinity of non-constraint form.The HER2 that lacks some residue in subdomain IV exists with non-constraint form and can be used for and the dimerization of HER1, HER3 and HER4.With after the form of constraint (monomer) combines, this bound interaction is released and this ECD (or acceptor) is in the conformation that can be used for dimerization at part, and it relates to the interaction between the domain II of two ECD.

[0124] as used herein, mention the activity of regulating CSR or HER acceptor herein, any activity (as part keying action or other signal transduction related activity) that means this acceptor is changed.

[0125] as used herein, the formula configuration refers to two ECD configurations like this back-to-back, thereby each ECD can be used for the dimerization with cell surface receptor.When being in back-to-back the formula configuration, each ECD part of chimeric polyeptides that contains multimerization structural domain orientation like this after forming the ECD polymer, thus each ECD or its part can be used for the dimerization with cell surface receptor.

Dimer when [0126] as used herein, referring to two chimeric polyeptides and dimerization mean the interaction between these two chimeric polyeptides.When appropriate dimerization, can be used for dimerization with cell surface receptor at each chimeric polyeptides or the described chimeric polyeptides ECD in one of at least.

[0127] as used herein, " with the dimerization of cell surface receptor " phalangeal cell surface receptor and this paper provide in the polymer ECD or with the interaction of another kind of cell surface receptor." dimer " of this language indication or " dimerization " will be conspicuous from context.

[0128] as used herein, " polypeptide that comprises structural domain " refers to such polypeptide, and it contains the complete structure territory relevant for the corresponding construction territory of related acceptor.This complete structure territory is determined with reference to the definition in the described ad hoc structure territory in the related polypeptide.For example, the acceptor isoform that comprises structural domain refers to such isoform, its contain with related acceptor in the corresponding structure territory, complete structure territory found.If related acceptor for example contains 21 transmembrane amino acid structural domains between 400-420 amino acids position, the acceptor isoform that then comprises this membrane spaning domain contains 21 amino acid structure territories that have essence identity with these 21 amino acid structure territories of related acceptor.When essence identity refers to compare with the structural domain of related acceptor, can contain allelic variation and the conservative structural domain of replacing.When comparing with the structural domain of related acceptor, identical in fact structural domain does not have amino acid whose disappearance, non-conservation is replaced or insertion.

[0129] as used herein, allelic variant or allelic variation refer to by with the polypeptide with reference to the different coded by said gene of form (promptly by allelotrope coding) of gene.Generally, gene with reference to colony or single wild-type form and/or the super advantage form with reference to polypeptide in member of form coding from certain species.Usually, be included between two species and between a plurality of species the allelic variant of variant general with have at least 80%, 90% or bigger amino acid identity from the wild-type of same species and/or super advantage form; Whether the degree of identity depends on gene and is to compare between species or in species.Usually, the wild-type and/or the super advantage form of an allelic variant and a peptide species have at least about 80%, 85%, 90% or 95% identity or bigger in planting, and comprise 96%, 97%, 98%, 99% or bigger identity.

[0130] as used herein, the species variant refer between two species with identical variant polypeptides between a plurality of species.Normally, between species variant with have at least about 60%, 70%, 80%, 85%, 90% or 95% identity or bigger identity from the wild-type and/or the super advantage form of another species, comprise and the wild-type of a peptide species and/or 96%, 97%, 98%, 99% or bigger identity of super advantage form.

[0131] as used herein, modify the sequence of Nucleotide in the aminoacid sequence refer to modified polypeptide or the nucleic acid molecule and comprise disappearance, insertion and the replacement of amino acid and Nucleotide respectively.

[0132] as used herein, open reading-frame (ORF) refers to Nucleotide or the ribonucleoside acid sequence in the nucleic acid molecule, its encoding function polypeptide or its part (generally at least about 50 amino acid).Open reading-frame (ORF) can encode full-length polypeptide or its part.Open reading-frame (ORF) can produce by connecting one or more exons or exon and intron effectively, and the whole or portion that this moment, terminator codon was arranged in this intron and this intron is positioned at the mRNA that transcribes.

[0133] as used herein, polypeptide refers to covalently bound two or more amino acid.Term " polypeptide " and " protein " use in basis convertibly.

[0134] as used herein, brachymemma or shortening are with regard to nucleic acid molecule or proteinic shortening, be meant the sequence of aminoacid sequence residue in the sequence of Nucleotide in the nucleic acid molecule or ribonucleotide or the polypeptide, compare with the wild-type or the super advantage form of this protein or nucleic acid molecule, the former is less than total length.

[0135] as used herein, reference gene refers to can be used for drawing the gene of intron and exon position in the gene.Reference gene can be as with the gene order comparison of expressing to draw genomic dna or its part of intron and exon position in the gene.Reference gene also can be the gene of the wild-type or the super advantage form of coded polypeptide.

[0136] as used herein, the family of protein or gene or relevant family refer to have each other a histone matter or a gene of homology and/or structural similarity and/or functional similarity respectively.

[0137] as used herein, the premature termination codon is such terminator codon, and it is used for producing or creating the terminator codon appearance before of proteinic total length form (as the wild-type or the super advantage form of polypeptide) in the open reading-frame (ORF) of nucleic acid molecule.The appearance of premature termination codon can be for example alternative splicing and results of mutation.

[0138] as used herein, kinases is the protein of catalytic molecular phosphorylation, and wherein said molecule generally is a biomolecules, comprises macromole and small molecules, and for example, this molecule can be small molecules or protein.Phosphorylation comprises autophosphorylation.Some kinases have the composing type kinase activity.Other kinases need to activate.For example, many kinases of participation signal transduction are phosphorylations.Phosphorylation activates the activity of kinases to another biomolecules in the approach.Some kinases are regulated by the variation of protein structure and/or with another interaction of molecules.For example, proteinic compound or molecule can activate or suppress kinase activity with kinase whose the combination.

[0139] as used herein, adjusting and regulating effect (modulation) refer to the active variation of molecule (as protein).The activity of example includes but not limited to biologic activity, as signal transduction.Adjusting can comprise that active increase (i.e. rise effect or agonist activity) or active minimizing (i.e. downward modulation effect or restraining effect) or active any other change (as the variation of periodicity, frequency, time length, kinetics or other parameter).Adjusting can be environmental factor dependence and regulate generally than specified state, for example, wild-type protein, be in the protein of composing type state or expressed protein in specified cell type or condition.

[0140] as used herein, inhibition and restraining effect refer to respect to suppressing active active the reduction.

[0141] as used herein, composition refers to any mixture.It can be solution, suspension, liquid, mashed prod, hydrate, non-hydrate or its arbitrary combination.

[0142] as used herein, combination refers between two article (item) or any associating between a plurality of article.Combination can be two or more independently article, as two compositions or two set, can be its mixture, as single mixture or its any variation of two or more article.The key element of combination normally is relevant or related on the function.Test kit is the combination of packing, and it randomly comprises the specification sheets that uses combination or its key element.

[0143] as used herein, effect of drugs or result of treatment refer to when use the intention be used for the treatment of disease or illness or be used to improve the medicine of its symptom after observed effect.

[0144] as used herein, vasculogenesis refers to form neovascularity from existing blood vessel; Neovascularization (neovascularization) finger-type becomes neovascularity.The physiological vasculogenesis is subjected to strict adjusting and essential to regeneration and embryonic development.After birth and the intra vitam of growing up, vasculogenesis be present in wound repair and the muscle of practicing in and be limited to a few days or several weeks usually.On the contrary, pathologic vessels generates (or abnormal vascular generation) can continue several months or several years, for example support entity tumour and leukemia development.It provides passage for inflammatory cell enters chronic inflammatory diseases position (for example Crohn disease and chronic cystitis).It is the most commonly encountered diseases that loses one's sight because of; It destroys cartilage and helps the growth of atherosclerotic plaque and hemorrhage in rheumatoid arthritis.It causes intraperitoneal hemorrhage in the film dystopy in uterus.Tumor growth is an angiogenesis-dependent.Tumour stimulates the factor of vasculogenesis to enlist himself blood supply by discharging.This type of factor comprises VEGF, FGF, PDGF, TGF-β, Tek, EPHA2, AGE and other etc.AGE-RAGE interacts can to rely on through the VEGF genetic transcription activation of the NF-κ B and the AP-1 factor and excites vasculogenesis.VEGF excessive generation in a large amount of human cancers (comprising mammary cancer, lung cancer, colorectal carcinoma).

[0145] as used herein, angiogenic disease (or relevant disease takes place blood vessel) is that balance that its medium vessels takes place is changed or its time is controlled reformed disease.Angiogenic disease comprises those diseases of the change (as undesired vascularization) that the blood vessel generation wherein occurs.This type of disease includes but not limited to cell proliferative diseases, comprise cancer, diabetic retinopathy and other diabetic complication, inflammatory diseases, endometriosis and other disease, wherein excessively vascularization is the lysis part of (comprising those above-mentioned lysises).

[0146] as used herein, disease that HER (ErbB) is relevant or the receptor-mediated disease of HER are that wherein HER acceptor and/or part relate to any disease, the patient's condition or the illness of some aspects of nosetiology, pathology or its development.Especially, relate to expression or overexpression or the activity that comprises HER receptor family member for example or part.Disease includes but not limited to proliferative disease, comprise cancer, such as but not limited to pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer or mammary cancer.Other patient's condition comprises those diseases that relate to cell proliferation and/or divide a word with a hyphen at the end of a line, comprise those diseases relate to pathologic inflammatory reaction, non-pernicious excessively proliferative disease such as illness in eye, tetter, smooth muscle cell proliferation and/or divide a word with a hyphen at the end of a line due to the patient's condition such as the flesh of narrow (comprising restenosis), atherosclerosis, bladder, heart or other muscle thicken (muscle thickening), endometriosis or rheumatoid arthritis.

[0147] as used herein, treatment means any way that wherein improves or change valuably symptom or other sign of the patient's condition, illness or disease.

[0148] as used herein, result of treatment means the effect due to the experimenter treatment, and wherein said treatment changes, generally improves or palliate a disease or symptom or the cure diseases or the illness of illness.The treatment significant quantity refers to produce the amount of composition, molecule or the compound of result of treatment after being applied to the experimenter.

[0149] as used herein, term " experimenter " refers to comprise Mammals by animal, as the mankind.

[0150] as used herein, " patient " refers to people experimenter.

[0151] as used herein, " individuality " can be the experimenter.

[0152] as used herein, normal level or value can define according to multiple mode well known by persons skilled in the art.Generally speaking, normal level refers to the expression level of in health population scope CSR or CSR part.Normal level (or reference level) is based on health volunteer's measuring result, as originate from appointment (being blood, serum, tissue or other source).Normal level often is called as " normal range ", and it refers generally to the value scope of the median 95% of health population.Reference value can be used interchangeably with normal level in this article, but can be according to experimenter or source and different with normal level.For example, the normal level of CSR or part can be different between the patient with 50 years old 2 years old patient.Therefore, reference level generally depend on the normal level of the specific part of colony.Therefore, for this paper purpose, normal level or reference level be can with test the patient preassigned or the contrast that compare.

[0153] as used herein, the level of rising refers to increase above the CSR of normal level or reference level or any expression level of CSR part.The expression in the test experimenter of CSR or CSR part can be compared with the normal level or the control level of CSR or part, to determine whether this level raises.

[0154] as used herein, activity refers to the function of biomolecules such as polypeptide or plays function or its interactional variation that this type of active example (but non-limiting) is: compound, dimerization, multimerization, kinase activity or other enzymic activity or catalytic activity that acceptor is relevant, the protease activity that acceptor is relevant, phosphorylation, dephosphorylation, autophosphorylation, ability with other molecule formation complex body, the part keying action, catalytic activity or enzymic activity, activate (comprise and self activate and activate other polypeptide), the function that suppresses or regulate another molecule, stimulate or inhibition signal transduction and/or cell response such as cell proliferation, divide a word with a hyphen at the end of a line, differentiation and growth, degraded, the film location, film combination and tumour take place.Active can the assessment by described assay method herein with by any suitable assay method well known by persons skilled in the art, described assay method includes but not limited to assay method (being included at the assay method in the animal model of specified disease) in external test method (comprising the assay method based on cell), the body.

[0155] as used herein, the two or more molecules of compound finger (as two protein molecules) form the interaction of complex body.This interaction can be undertaken by non covalent bond and/or covalent linkage, and includes but not limited to hydrophobic and electrostatic interaction, Van der Waals for and hydrogen bond.Usually, protein-protein interaction relates to hydrophobic interaction and hydrogen bond.Compoundly can be subjected to envrionment conditions (as temperature, pH, ionic strength and pressure with protein concn) influence.

[0156] as used herein, dimerization refers to the interaction of two molecules (as two acceptor molecules).Dimerization comprises wherein two interactional same dimerizations of same molecular.Dimerization also comprises wherein two interactional different dimerizations of differing molecular.Generally speaking, dimerization relates to two molecules interact with each other by the interaction of contained dimerization structural domain or multimerization structural domain in each molecule.Similarly, multimerization refers to that a plurality of molecules form dimer, tripolymer or the more interaction of high-order oligomer, and wherein said molecule is a same type or different.

[0157] dimerization that relates to two chimeric polyeptides refers to the dimerization that occurs by interacting between the multimerization structural domain of each chimeric polyeptides.Receptor dimerizationization refers to cause between two acceptors the dimerization of receptor activation, or acceptor and can and the ECD of this receptor dimerization part (as the ECD polymer) between dimerization, this dimerization will be regulated the activation of this receptor subsequently.

[0158] as used herein, research and the experiment that method (in silico) refers to use a computer and carries out on the silicon chip.Method includes but not limited to that molecule Modeling Research, biomolecules butt joint experiment (dockingexperiments) and void present (virtual representation) molecular structure and/or process (as interaction of molecules) on the silicon chip.

[0159] as used herein, biological sample refers to any sample of obtaining from live body or viral source or other macromole and biomolecules, and comprises any cell type or the tissue that therefrom can obtain nucleic acid or protein or other macromolecular experimenter.This biological sample can be sample or the sample through processing that directly obtains from biological origin.For example, the isolating nucleic acid of amplification constitutes biological sample.Biological sample includes but not limited to body fluid such as blood, blood plasma, serum, cerebrospinal fluid, synovial membrane liquid, urine and sweat, from the tissue of animal and plant and organ and from deutero-processing sample wherein.Also comprise soil sample and water sample and other environmental samples, virus, bacterium, fungi, algae, protobiont and its component.

[0160] as used herein, term " nucleic acid " refers to strand and/or double-stranded polynucleotide, as analogue or the derivative of thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA) and RNA or DNA.Term " nucleic acid " also comprises the analogue of nucleic acid, as peptide nucleic acid(PNA) (PNA), phosphorothioate dna and this type of other analogue and derivative or its combination.Nucleic acid can refer to polynucleotide, as thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA).This term comprises that also derivative, variant and analogue, strand (justice or antisense are arranged) and the double-stranded polynucleotide of the RNA that is made up of nucleotide analog or DNA are as equivalent.Deoxyribonucleotide comprises Desoxyadenosine, Deoxyribose cytidine, pancreatic desoxyribonuclease and deoxythymidine.For RNA, the uridylic base is a uridine.

[0161] as used herein, term " polynucleotide " refers to contain the Nucleotide of at least two connections or the oligomer or the polymkeric substance of nucleotide derivative, comprise thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA) and DNA derivative or RNA derivative, described derivative contains for example nucleotide analog or " main chain " key except that the phosphodiester key, for example tricresyl phosphate ester bond, phosphamide key, phosphorothioate bond, thioester bond or peptide bond (peptide nucleic acid(PNA)).Term " oligonucleotide " also uses with the free burial ground for the destitute with " polynucleotide " in this article basically, although those skilled in the art recognize that oligonucleotide (for example PCR primer) is usually less than about 50-100 length of nucleotides.

[0162] polynucleotide comprise nucleotide analog, for example comprise, allow the Nucleotide of the quality modification of polynucleotide mass discrepancy; The Nucleotide that contains the detectable (as fluorescence, radiation, luminous or chemiluminescent labels) that allows the detection polynucleotide; Or contain and promote polynucleotide to be fixed to the reactive group of solid support as the Nucleotide on (vitamin H or sulfydryl); One or more main chain keys of cutting that polynucleotide also can contain selective (as chemistry, enzyme or photodissociation).For example, polynucleotide can comprise one or more deoxyribonucleotides, after connect one or more ribonucleotides, can be thereafter one or more deoxyribonucleotides, this kind sequence can be cut by alkaline hydrolysis at ribonucleoside acid sequence place.Polynucleotide also can contain one or more keys of relative tolerance cutting, for example, a kind of chimeric oligonucleotide primer, it can comprise Nucleotide and at least one Nucleotide at 3 ' end that connects by the peptide nucleic acid(PNA) key, and wherein said Nucleotide at 3 ' end is connected by phosphodiester bond or other appropriate key and can be by polymerase extension.The peptide nucleic acid(PNA) molecule can use well-known process preparation (see, for example, people such as Weiler, Nucleic acids Res.25:2792-2799 (1997)).

[0163] as used herein, oligonucleotide refers to comprise DNA, RNA, nucleic acid analog (as PNA) and combination of polymers thereof.For this paper purpose, primer and probe are the oligonucleotide of single stranded oligonucleotide or part strand.

[0164] as used herein, synthetic when for example relating to synthetic nucleic acid molecule or synthetic gene or synthetic peptide, refer to by recombination method and/or the nucleic acid molecule or the peptide molecule that produce by chemical synthesis process.

[0165] as used herein, by recombination method or use recombinant DNA method to produce to mean and use the molecular biology method of knowing to express protein by the dna encoding of being cloned.

[0166] as used herein, term " carrier (vector) " refers to transport the nucleic acid molecule of another nucleic acid that has been attached thereto.One type carrier is an episomal vector, promptly can be at the nucleic acid of extrachromosomal replication.Carrier comprises can self-replacation and/or express those carriers of connected nucleic acid.Can instruct with the carrier of its genetic expression that effectively is connected and be called " expression vector " in this article.Generally speaking, expression vector usually is " plasmid " form, the circular double stranded DNA ring that wherein said plasmid does not normally combine with karyomit(e) under its carrier format.Use " plasmid " and " carrier " interchangeably, because plasmid is the carrier format of the most normal use.This type of other carrier format that plays equivalent function is known in the art hereinafter.

[0167] as used herein, phrase " effectively connection " with regard to nucleotide sequence, means nucleic acid molecule or its sections usually and covalently connects into one section nucleic acid, as DNA or RNA, no matter is single stranded form or double chain form.Described sections needs not to be adjacency, but two or more component so near, thereby described component is in and allows in the relation that they play a role in the expection mode.For example, the sections of RNA (exon) can be by effectively connecting to form a single RNA molecule as splicing.In another example, the DNA sections can effectively connect, thereby control on a control sections or adjusting sequence allow the expression of other sections or duplicate or other this type of control.Therefore, under the situation that regulatory region effectively is connected with reporter molecule or any other polynucleotide connect, perhaps under the situation that reporter molecule or any polynucleotide are effective with regulatory region is connected, the expression of described polynucleotide/reporter molecule is influenced by this regulatory region or (for example controls, regulated or changed, as increasing or reducing).For genetic expression, the sequence of Nucleotide in such a manner with regulate sequence and be connected, thereby when suitable molecular signal (as transcription activating protein) with as described in the adjusting sequence control or permission genetic expression when combining.Heterologous nucleic acids (as DNA) effectively is connected with effect sequence (as promotor, enhanser, Transcription Termination site and translation termination site and other signal sequence) with the adjusting sequence of Nucleotide, refers to this kind DNA relation between the class nucleotide sequence therewith.For example, effective physical relation that refers between DNA and the promotor that is connected of allogeneic dna sequence DNA and promotor, thereby transcribing by RNA polymerase of this kind DNA starts from described promotor, and the described DNA in the open reading-frame (ORF) is discerned, is incorporated into and transcribed to wherein said RNA polymerase specifically.

[0168] as used herein, heterologous nucleic acids and the adjusting sequence of Nucleotide and effective connection of effect sequence (as promotor, enhanser, Transcription Termination site and translation termination site and other signal sequence), refer to described nucleic acid (as DNA) and as described in relation between the nucleotide sequence.For example, allogeneic dna sequence DNA with effective connection of promotor refer to this DNA physical relation like this between the promotor therewith, thereby transcribing by RNA polymerase of described DNA starts from described promotor, and described DNA is discerned, is incorporated into and transcribed to wherein said RNA polymerase specifically.Therefore, effectively connection or effective in conjunction with referring to nucleic acid (as DNA) and the adjusting sequence of Nucleotide and the functional relationship of effect sequence (as promotor, enhanser, Transcription Termination site and translation termination site and other signal sequence).For optimization expression and/or in-vitro transcription, may must will remove, add or change partly extra, initial (the being starting point) codon of potential unaccommodated alternate translation of 5 ' untranslated of clone or may on transcriptional level or translation skill, disturb or reduce other sequence of expression to eliminate.Alternatively, and total ribosome bind site (see, for example, Kozak J.Biol.Chem.266:19867-19870 (1991)) 5 of initiator codon ' insertion can be close to and expression can be strengthened.Can determine to experience requirement (or needs) to this type of modification.

[0169] as used herein, term when referring to polypeptide " effectively connects ", for example when in the context of phrase " at least one subdomain of cell surface receptor or its part effectively are connected to another subdomain or its part ", using, mean these two aminoacid sequences and connect to form single amino acid residue sequence by the peptide bond between the amino-acid residue of end in each sequence.

[0170] as used herein, phrase used when referring to generation polypeptide (as isoform and intron fusion proteins) " produces from nucleic acid ", comprises really producing peptide molecule by the translation nucleic acid molecule and producing polypeptide.

[0171] as used herein, the generation when referring to polypeptide refers to the expression and the recovery of expressed protein (recyclable or separable expressed protein).Can influence factor that protein produces and comprise that selected expression system and host cell, cell culture condition, host cell detect proteinic ability to proteinic secretion with for the purifying purpose.Proteinic generation can for example be secreted in cell culture medium by evaluating protein matter and be monitored.

[0172] as used herein, secretion finger protein matter is so as to being transported to extracellular environment or being transported to the process of periplasmic space under the situation of gram negative bacterium.Usually, secretion takes place by cell internal secretion approach, and for example in eukaryotic cell, this relates to endoplasmic reticulum and golgi body.

[0173] as used herein, with regard to regard to the molecule (as nucleic acid molecule or polypeptide) of different plant species, homologous is meant corresponding molecule (being the species variants).This quasi-molecule generally is similar and total usually about 45% sequence identity or homology.Those skilled in the art can identify the homologue (homologs) between the species.

[0174] as used herein, heterologous nucleic acids is such nucleic acid, its cell of can't help to express this nucleic acid usually produces in vivo, or by this cell generation, but be in different genes seat place or differently expressed, perhaps mediation or coding are transcribed, are translated by influence or other controllability Biochemical processes change the mesosome that endogenous nucleic acid (as DNA) is expressed.Heterologous nucleic acids is not endogenic for the cell that imports this nucleic acid usually, but obtains or preparation synthetically from another kind of cell.Heterologous nucleic acids can be endogenic, but is to express reformed nucleic acid from the expression of different genes seat or its.Usually, although be not inevitably, this type of nucleic acid encoding under normal circumstances cell does not produce or expresses RNA and the protein of the cell of described nucleic acid not according to the generation of the same way as in this cell.Heterologous nucleic acids (as DNA) may also be referred to as exogenous nucleic acid (as DNA).Therefore, heterologous nucleic acids or exogenous nucleic acid comprise not with the identical direction of existing corresponding nucleic acid molecule (as DNA) in the genome and the nucleic acid molecule of position existence.It also can refer to the nucleic acid molecule from another kind of biology or species (being external source).Heterologous nucleic acids can refer to a part of this molecule with regard to isolated nucleic acid molecule, its from difference source or always since then the locus of another part of molecule derive.The example of allos secretion signal comprises not any presequence (being signal sequence) or the preceding former sequence as the endogenous signal sequence of coded molecule, for example, but be not limited to former sequence before the tPA, preprogastrin sequence and any other sequence well known by persons skilled in the art.

[0175] similarly, with regard to the part of polypeptide, the allogenic part of comparing with rest part in the chimeric polyeptides that is meant.Therefore, in heterozygosis ECD, wherein said heterozygosis ECD contains subdomain I from HER1, from the subdomain II of HER2 with from the subdomain III of HER3, and every kind of subdomain is a heterology for each other subdomain.

[0176] heterologous molecule can be derived from different genetic origins or species.Therefore, comprise any molecule that contains sequence like this with specific CSRECD or the allogenic molecule of its isoform, wherein said sequence is not from described CSR ECD or its isoform is derived or it is endogenous to be not with it.The example of heterologous molecule comprises all or part of from secretion signal, label such as the fusion tag of the not homopolypeptide of identical or different species or marker or any other molecule.Heterologous molecule can merge to produce fusion molecule or chimeric molecule or can chemically connect through covalently or non-covalently being connected with purpose nucleic acid or peptide sequence.

[0177] as used herein, the allos secretion signal refers to the signal sequence from polypeptide from identical or different species, and wherein said signal sequence is different with the endogenous signal sequence on sequence.The allos secretion signal can use in the host cell of this allos secretion signal of deriving, or it can use in the host cell different with the cell of this signal sequence of deriving.

[0178] as used herein, the active part of polypeptide as with regard to the active part of ECD, refers to have the part of active polypeptide.

[0179] as used herein, protein purification refers to the process of isolated protein, and as the process of isolated protein from homogenate, wherein said homogenate can contain cell and structural constituent, comprises DNA, cytolemma and other protein.Protein can for example pass through pH gradient gel or ion exchange column, carry out purifying according to its size or molecular weight by the size exclusion chromatography method or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analytical method or according to its hydrophobicity by making their according to its iso-electric point according to any-mode in the multiple mode well known by persons skilled in the art.Other purification technique includes but not limited to the precipitator method or affinity chromatography, comprises immune-affinity chromatography and other method that comprises the combination of arbitrary these methods.In addition, can promote purifying as the detectable label that is used for the histidine-tagged of affinity purification or be used to identify by on molecule, comprising label.

[0180] as used herein, " separation " represents that this molecule differs from the state that this molecule exists because of manual operation in its natural surroundings with regard to molecule (as nucleic acid molecule, oligonucleotide, polypeptide or antibody).For example, think and produce or the molecule that comprised therein is " isolating " by recombinant host cell.Similarly, think from its natural origin or recombinant host cell that part or purifying or that produce by synthetic method basically molecule are " isolating ".Use according to expection, isolating molecule can exist in any form, as existing in animal, cell or its extract; The dehydration, in steam, solution or suspension; Or be fixed on the solid support.

[0181] as used herein, pure basically polypeptide or isolated polypeptide (or other molecule) be commutative use and mean described polypeptide and from the source, be purified or have as by the detected sample homogeneity of chromatographic technique or this type of other technology (as use the SDS-PAGE of coomassie orchid for example or argentation at non-reduced or reductive condition).Homogeneity means and other proteinic mixing less than about 5% or less than 5% of source usually.

[0182] as used herein, detection comprises that permission shows (by eyes or equipment) method of protein.Can utilize and protein can be manifested the special antibody of protein.Also can merge with the label or tag thing that comprises the epi-position label and promote proteinic detection by protein.

[0183] as used herein, marker refers to detectable compound or composition, its directly or indirectly with conjugation of polypeptides, thereby produce the polypeptide of mark.Marker can be itself detectable (for example radioisotopic tracer or fluorescent marker); Or under the situation of enzyme labelling thing, can the catalytic substrate compound or the detected chemical transformation of composition.The limiting examples of marker comprise living fluorescence (fluorogenic) partly, green fluorescent protein or luciferase.

[0184] as used herein, expression refers to such process, and wherein the information of coded by said gene changes into the works that exists and play a role in cell.The gene of expressing comprises that those are transcribed into mRNA and translate into proteinic gene subsequently and those are transcribed into RNA but do not translate into proteinic gene (for example transfer RNA (tRNA) and ribosome-RNA(rRNA)).For this paper purpose, expressed protein can be trapped in the cell, as is trapped in the tenuigenin, maybe can come out from emiocytosis.

[0185] as used herein, promoter region refers to the DNA part of gene, and wherein said DNA partly controls with it effectively transcribing of the DNA of connection.Promoter region comprises the specific dna sequence that enough is used for RNA polymerase identification, combination and transcription initiation.This part of promoter region is called promotor.In addition, promoter region comprises the identification of adjusting RNA polymerase, combination and the active sequence of transcription initiation.These sequences can be cis acting maybe can respond to trans-acting factor.According to the essence of regulating effect, promotor can be composing type or be subjected to adjustment type.

[0186] as used herein, regulatory region means the cis acting nucleotide sequence of forward or the effective genetic expression that connects of negative sense ground influence.Regulatory region comprises gives the nucleotide sequence that gene induced type is expressed (promptly needing to be used to increase the material or the stimulator of transcribing).When inductor exists or is in the concentration of rising, can increase genetic expression.Regulatory region also comprises and causes genetic expression to check the sequence of (be material or stimulator reduce transcribe).When repressor exists or is in the concentration of rising, can reduce genetic expression.Biologic activity in known regulatory region influence, adjusting or the control volume comprises cell proliferation, cell growth and death, cytodifferentiation and immunomodulatory.General and one or more trans-acting protein binding of regulatory region, this causes that genetic transcription increases or reduces.

[0187] example in generegulation district is promotor and enhanser.Promotor be positioned at transcribe or translation initiation site near sequence, generally be positioned at translation initiation site 5 '.Promotor is usually located in the 1Kb scope of translation initiation site, but can be positioned at the farther place, for example, 2Kb, 3Kb, 4Kb, 5Kb or farther, as many as comprises 10Kb.Known to being arranged in 5 of gene ' or 3 ' time or be positioned at exon or intron or during for they a part of, enhanser influences genetic expression.Enhanser also can with gene be separated by obviously apart from the time play a role for example distance about 3Kb, 5Kb, 7Kb, 10Kb, 15Kb or farther.

[0188] except that promoter region, regulatory region also comprises the sequence, the intron splicing signal that are beneficial to translation, keeps the correct open reading-frame (ORF) of gene to allow to meet the sequence of open reading-frame (ORF) ground translation mRNA, and terminator codon, leader sequence and fusion partner (partner) sequence, be used to produce polygene or polycistronic internal ribosome binding site (IRES) element, courier (messages), the polyadenylation signal and the terminator codon of the correct polyadenylation of goal gene transcript are provided, and can randomly be contained in the expression vector.

[0189] as used herein, " amino acid " that exists in the multiple amino acids sequence that this paper occurs is determined according to its trigram known or single-letter abbreviation (seeing Table 2).The Nucleotide that exists in multiple dna fragmentation provides with the usual standard single-letter name of using in this area.

[0190] as used herein, " amino-acid residue " refers to formed amino acid behind peptide bond place chemical digestion (hydrolysis) polypeptide.Described herein amino-acid residue normally is " L " isomeric forms.The residue that is in " D " isomeric forms can be replaced any L-amino-acid residue, as long as polypeptide keeps the functional performance of wanting.NH2 refers to the free amine group in the existence of the N-terminal place of polypeptide.COOH refers to the free carboxy in the existence of the C-terminal place of polypeptide.With J.Biol.Chem., that describes among the 243:3552-59 (1969) reaches at 37C.F.R, and accepted standard polypeptide nomenclature is consistent among the § § .1.821-1.822, and the abbreviation of amino-acid residue is shown in table 2:

Table 2-correspondence table

[0191] full sequence of this paper amino-acid residue of explaining by formula has in the from left to right direction of aminoterminal to the conventional direction of carboxyl terminal.In addition, phrase " amino-acid residue " is defined as comprises amino acid, modified amino acid, alpha-non-natural amino acid and rare amino acid of listing in the correspondence table.In addition, should be understood that the straight sea line of weak point at the beginning of amino acid residue sequence and place, end represent with other sequence that contains one or more amino-acid residues or with amino end group such as NH ₂Or the peptide bond that is connected with carboxyl end group such as COOH.

[0192] in peptide or protein, amino acid whose suitable conservative replacement is well known by persons skilled in the art and normally can carries out and do not change the activity of gained molecule.Those skilled in the art will know that, generally speaking, single amino acids replacement in the nonessential region of polypeptide does not change biologic activity basically and (sees, for example, Molecular Biology of the Gene such as Watson, the 4th edition, 1987, The Benjamin/Cummings Pub.co., the 224th page).

[0193] this type of replacement can be for example according to carrying out in those replacements described in the following table 3:

Table 3

Initial residue	Conservative property is replaced
		Ala(A)	Gly；Ser
Arg(R)	Lys
		Asn(N)	Gln；His
Cys(C)	Ser
		Gln(Q)	Asn
Glu(E)	Asp
		Gly(G)	Ala；Pro
His(H)	Asn；Gln
		Ile(I)	Leu；Val
Leu(L)	Ile；Val
		Lys(K)	Arg；Gln；Glu
Met(M)	Leu；Tyr；Ile
		Phe(F)	Met；Leu；Tyr
Ser(S)	Thr
		Thr(T)	Ser

Trp(W)	Tyr
		Tyr(Y)	Trp；Phe
Val(V)	Ile；Leu

Other replacement (comprise non-conservation change) also be allow and can experience ground or replace according to other known conservative property or non-conservation and to determine.

[0194] as used herein, peptide mimics is the conformation of biologic activity form of simulation particular peptide and the compound of some stereochemistry feature.Generally speaking, the designed peptide stand-in are intended to some advantageous feature of simulated compound, do not cause forfeiture of biologic activity conformation and the unfavorable characteristic of key destructive but do not simulate, as flexibility.Peptide mimics can prepare from bioactive compounds contributive some group of unfavorable characteristic or key by replacing with bioisostere (bioisostere).Bioisostere is well known by persons skilled in the art.For example, methylene radical bioisostere CH2S has replaced as the acid amides in the ICI154129 N,N-bisallyl-Tyr-Gly-Gly-and (has seen, for example, the Chemistry and Biochemistry of Amino Acids that Spatola (1983) edits at Weinstein, Peptides, and Proteins the 7th volume 267-357 page or leaf, Marcel Dekker, New York).It can Orally administered morphine be compound as the peptide mimics of endorphin.For this paper purpose, cyclic peptide belongs to peptide mimics, and described cyclic peptide is the polypeptide that wherein one or more peptide bonds are replaced by stand-in.Heteromultimeric that provides herein and polymer and heterozygosis ECD and chimeric polyeptides can be modified by replacing key with stand-in, and this quasi-molecule is provided herein.

[0195] as used herein, " similarity " between two protein or the nucleic acid refers between the described proteinic aminoacid sequence or the dependency between the nucleotide sequence of described nucleic acid.Similarity can be based on sequence and the wherein identity of contained residue and/or the degree of homology.Be used for that the similarity degree methods is well known by persons skilled in the art between evaluating protein matter or the nucleic acid.For example, in a kind of method of assessment sequence similarity, compare two aminoacid sequences or nucleotide sequence in such a manner, thereby between described sequence, produce the identity of maximum horizontal." identity " refers to the degree that aminoacid sequence or nucleotide sequence do not make a variation.The comparison of aminoacid sequence and in a way the comparison of nucleotide sequence also can consider the common replacement of conservative property difference and/or amino acid (or Nucleotide).Conservative property difference is those differences that keep the physical-chemical characteristic of related residue.Comparison can be (compare the sequence that is compared and comprise whole residues in the whole sequence length range) of overall importance or partial (only comparison comprises the sequence part of similar area).

[0196] " identity " itself has implication known in the art and can use disclosed technique computes (to see, for example: Computational Molecular Biology, Lesk, A.M., editor, OxfordUniversity Press, New York, 1988; Biocomputing:Informatics andGenome Projects, Smith, D.W., editor, Academic Press, New York, 1993; Computer Analysis of Sequence Data, I portion, Griffin, A.M. and Griffin, H.G., editor, Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; With SequenceAnalysis Primer, Gribskov, M. and Devereux, J., editor, M Stockton Press, New York, 1991).Although exist numerous methods to measure identity between two polynucleotide sequences or the peptide sequence, yet term " identity " is the technician (Carillo, H. and Lipton, D., the SIAM J Applied Math 48:1073 (1988)) that know.

[0197] as used herein, along the sequence identity that the total length of each SEQ ID is compared with the total length of isoform, refer to compare along its total length with specifying the defined reference polypeptide of SEQ ID, the isoform polypeptide is along the identity percentage ratio of the aminoacid sequence of its total length.For example, if polypeptide A has 100 amino acid and polypeptide B has 95 amino acid identical with polypeptide A 1-95 amino acids, then compare with the total length of polypeptide B, when along the total length comparative sequences identity of polypeptide A, polypeptide B has 95% identity.Generally, when isoform polypeptide or reference polypeptide are when lacking the mature polypeptide of signal sequence, along total length (except that described signal sequence part) the comparative sequences identity of described polypeptide.For example, if isoform lacks signal peptide, but reference polypeptide contains signal peptide, then for determining the signal sequence part of relatively getting rid of this reference polypeptide of sequence identity along these two polypeptide total lengths.As discussed below and it be known to those skilled in the art that the multiple program and the method that are used to assess identity are well known by persons skilled in the art.For example, when considering whole length, can utilize the best comparison and the identity that find two sequences as the overall comparison method of using Needleman-Wunsch overall comparison algorithm.High-caliber identity (as 90% or 95% identity) can be determined easily without software.

[0198] as used herein, homology (with regard to nucleotide sequence and/or aminoacid sequence) mean approximately sequence homology more than or equal to 25%, generally more than or equal to 25%, 40%, 60%, 70%, 80%, 85%, 90% or 95%, 90% or 95% sequence homology; Can specify accurate percentage ratio as required.For this paper purpose, term " homology " and " identity " are usually used convertibly, unless otherwise indicated.Generally speaking, be definite homology or identity percentage ratio, aligned sequences like this, thus the coupling that obtains highest level (is seen, for example: Computational MolecularBiology, Lesk, A.M. edits, Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D.W., editor, Academic Press, New York, 1993; Computer Analysis of Sequence Data, I portion, Griffin, A.M., and Griffin, H.G., editor, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; With Sequence Analysis Primer, Gribskov, M. and Devereux, J., editor, M Stockton Press, New York, 1991; Carillo etc. (1988) SIAM J AppliedMath 48:1073).By sequence homology, the number of conserved amino acid is determined by standard alignment algorithm program, and can be used with the acquiescence gap penalty that every supplier sets up.The homologous nucleic acid molecule is generally hybridized along the whole length of purpose nucleic acid with medium severity or height severity in fact.Also considered such nucleic acid molecule, it contains the degenerate codon of replacing codon in the crossability nucleic acid molecule.

[0199] no matter whether any two kinds of nucleic acid molecule have at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, the nucleotide sequence of 98% or 99% " identical " or " homology ", can use known computerized algorithm as " FASTA " program, use for example default parameters in (1988) Proc.Natl.Acad.Sci USA 85:2444 such as Pearson, determine that " identity " or " homology " (other program comprises GCG routine package (Devereux, J. wait Nucleic Acids Research12 (I): 387 (1984), BLASTP, BLASTN, FASTA (Atschul, S.F. etc., J MolecBiol 215:403 (1990); Guide to Huge Computers, Martin J.Bishop, editor, Academic Press, San Diego, 1994 and (1988) SIAM J Applied Math48:1073 such as Carillo)).For example, can use the BLAST function at national biomolecule information database center to determine identity.Other commerciality or public's available program comprise DNAStar " MegAlign " program (Madison is WI) with the Genetics Computer Group of winconsin university (UWG) " Gap " program (Madison WI)).Can be by (for example for example using the GAP computer program, Needleman etc. (1970) J.Mol.Biol.48:443, it improves homology or the identity percentage ratio that ((1981) Adv.Appl.Math.2:482) comparative sequences information is determined protein and/or nucleic acid molecule by Smith and Waterman.In brief, the GAP program is defined as the number of the similar symbol of comparing (being Nucleotide or amino acid) divided by the total number of symbols in the shorter sequence of two sequences with similarity.The default parameters that is used for the GAP program can comprise: write as Schwartz and Dayhoff (1), ATLAS OFPROTEIN SEQUENCE AND STRUCTURE, national biomedical research foundation, 353-358 page or leaf (1979) is described, the monobasic comparator matrix of Gribskov etc. (1986) Nucl.Acids Res.14:6745 (containing value 1 that is useful on identity and the value 0 that is used for nonidentity) and weighting comparator matrix; (2) be used for the point penalty 3.0 in each room and be used for the extra point penalty 0.10 of each symbol in each room; (3) to the no point penalty in terminal room.

[0200] therefore, as used herein, term " identity " or the comparison of " homology " representative between test and reference polypeptide or polynucleotide.

[0201] as used herein, term at least " 90% same in " refer to identity percentage ratio 90-99.99 with respect to reference nucleic acid sequence or aminoacid sequence.90% or higher level on identity represent such fact, promptly with 100 amino acid whose test polypeptide length and reference polypeptide length relatively for purpose supposition for example.It is different with the amino acid of reference polypeptide to be no more than (promptly 100 in 10) amino acid of 10% in the test polypeptide.Can carry out similar comparison at the test polynucleotide with between with reference to polynucleotide.This type of difference can be expressed as point mutation in the whole length range that is randomly dispersed in aminoacid sequence or they are can bunch collection different at the most in one or more positions that can allow maximum value (for example 10/100 amino acid difference (about 90% identity)) in length.With different definition is that nucleic acid or amino acid are replaced, inserted or disappearance.On homology that is higher than about 85-90% or identity level, this result should not rely on program and room parameter setting; Can assess high-caliber identity like this easily, often need not depended software and so assess by artificial comparison.

[0202] as used herein, the sequence of comparison refers to utilize homology (similarity and/or identity) to compare corresponding position in Nucleotide or the amino acid whose sequence.Generally speaking, reach 50% or two or more sequence alignments of higher identity with relevant.The sequence set of comparison refers to 2 of corresponding position comparison or a plurality of sequence and can comprise with respect to genomic dna sequence and comparing from RNA such as EST and other cDNA deutero-sequence.

[0203] as used herein, the polypeptide that comprises the specific amino acids percentage ratio described in the reference polypeptide is meant consecutive identical amino acid whose ratio common between polypeptide and the reference polypeptide.For example, comprise 70% and mean this reference polypeptide contains the aminoacid sequence of describing in SEQ ID NO:XX at least 103 continuous amino acids at amino acid whose isoform described in the reference polypeptide (it has the aminoacid sequence of describing in 147 amino acid whose SEQ ID NO:XX).

[0204] as used herein, " primer " refers to contain 2 or a plurality of, common oligonucleotide more than 3 deoxyribonucleotides or ribonucleotide, can start the synthetic of primer extension product from described primer.Primer can be used as under suitable condition (for example having 4 kinds of different nucleoside triphosphates and polymerizing agent such as archaeal dna polymerase, RNA polymerase or reversed transcriptive enzyme), in the suitable damping fluid and a starting point in the DNA building-up process that the suitable temp top plate instructs play a role.Help the synthetic experiment condition to comprise and have nucleoside triphosphate and the reagent such as the archaeal dna polymerase that are used for polymerization and extension, and suitable buffer, temperature and pH.Some nucleic acid molecule can be used as " probe " and plays a role as " primer ".Yet primer has the 3 ' hydroxyl that is used to extend.Primer can be used for several different methods, and described method comprises for example polymerase chain reaction (PCR), reversed transcriptive enzyme (RT)-PCR, RNAPCR, LCR, multiple PCR, panhandle formula PCR, catch PCR, express PCR, 3 ' and 5 ' RACE, original position PCR, PCR and other amplification method of ligation mediation.

[0205] as used herein, " primer to " refers to one group of primer, it comprise 5 ' (upstream) primer that the sequence 5 to be amplified with (for example passing through PCR) ' end is hybridized and with 3 ' (downstream) primers of the complementary sequence hybridization of this sequence to be amplified 3 ' end.

[0206] as used herein, " specific hybrid " refers to complementary base pairing effect and the target nucleic acid molecule annealing by nucleic acid molecule (for example oligonucleotide).Those skilled in the art know influences the external of specific hybrid and body intrinsic parameter, as the length and the composition of specific molecular.Parameter to external hybridization particularly important also comprises annealing temperature and wash temperature, damping fluid composition and salt concn.The exemplary wash conditions that is used for removing the nucleic acid molecule of non-specific binding under high severity is 0.1 * SSPE, 0.1%SDS, 65 ℃ and be 0.2 * SSPE under medium severity, 0.1%SDS, 50 ℃.The stringency of equivalence is known in the art.The technician can adjust these parameters easily to realize nucleic acid molecule and the specific hybrid that is suitable for the target nucleic acid molecule of application-specific.

[0207] as used herein, significant quantity is for the amount of preventing, cure, alleviate, stop or partly stop the necessary therapeutical agent of symptom of disease or illness.

[0208] as used herein, unit dosage form refers to be applicable to humans and animals experimenter and the unit of packing separately that physically separates, and this point is known in the art.

[0209] as used herein, single-dose preparations refers to the preparation that is used for directly using.

[0210] as used herein, singulative " ", " a kind of " and " being somebody's turn to do " comprise that plural number censures, unless that context is clearly stated is really not so.Therefore, for example the denotion of the compound that comprises " ectodomain " is comprised the compound with one or more ectodomains.

[0211] as used herein, scope and amount can be expressed as " pact " certain occurrence or scope." pact " also comprises exact amount.Therefore, " about 5 bases " mean " about 5 bases " and also refer to " 5 bases ".

[0212] as used herein, " optional " or " randomly " means incident or the situation described subsequently and occurs or do not occur, and this description comprises situation that wherein said incident or situation occur and its absent variable situation wherein.For example, to mean this group not replaced or replaced for optional alternate group.

B. the special therapeutical agent of general cell surface receptor

[0213] provide such compound herein, it is and one or more, general two or more cell surface receptors such as HER family member especially HER1, HER3 and HER4, IGF-1R (IGF-1R or IGF1R), especially IFG1R and interactional therapeutical agent of vascular endothelial growth factor receptor (VEGFR) family member or candidate therapeutic agent.At least one that these therapeutical agents and candidate therapeutic agent are cooperated in activating the disease approach by target specifically or a plurality of acceptor and/or its part work.The problem that this type of therapeutical agent overcomes or solution is relevant with the therapeutical agent of target single receptor.

[0214] for example, use anti-HER medicine such as He Sai

The problem of (Herceptin) is because limited effect due to the HER2 overexpression only occurs in the subgroup of mammary cancer, and limited replying the time length, and its reason is may develop at the resistance of this medicine, as by activating other acceptor development.For the acceptor with non-HER family member is that the observed drug of target is to similar problem.He Sai

The mechanism of drug resistance of (Herceptin) is other HER family member coexpression.Chemical sproof other mechanism comprises the IGF-1R coexpression; The HER2 of metalloprotease mediation activates (by " pruning (clippling) " ectodomain and rise PI3K-AKT (phosphatidylinositol-3-kinase-protein kinase B) approach, this often by lose PTEN (Phosphoric acid esterase and tensin homologue, wherein this albumen suddenlys change in cancer; Referring to (2006) Cancer Lett.8:123-38 such as for example Nahta; Hynes etc. (2005) Nature Reviews Cancer 5:341-354) mediates.The mechanism of drug resistance of HER1/EGFR therapeutical agent is similar to He Sai

Those mechanism of drug resistance of (Herceptin).The patient of data presentation 60% (88/145 patient) expresses one or both HER family members; Three kinds of HER family members of 18.6% (27/145) coexpression.These data also show cumulative bad expression of receptor advance notice serious many diseases (p＜0.0001).Two kinds of HER family members of the mammary cancer coexpression of other data sheet express contract 40%.The frequency of HER family member coexpression and the frequency in mammary cancer are comparable in other cancer, estimate that about 50% patient of as many as expresses HERs simultaneously, therefore and can tolerate the therapeutical agent of the single object of target (single agent), due to this other cellular pathways that may stimulate cellular proliferation because of constitutive activation AKT (protein kinase B) and constitutive activation (Hynes etc. (2005) NatureReviews Cancer 5:341-354).Coexpression also causes and induces Survivin (a kind of anti-apoptosis factor in the time of the HER family member; Xia etc. (2006) Oncogene24:6213-6221) and mediation be created in different somatomedins important in the tumor development (vascular endothelial growth factor for example; VEGF).

[0215] conclusion that obtains here is: the resistance at the therapeutical agent of any specific HER often mediates by all the other HER family members' expression or by relevant receptor tyrosine kinase such as the expression of IGF1R, VEGFR, FGFR etc.For example, IGF-1R is by directly suppressing He Sai with the HER2 heterodimerization

The activity of (Herceptin) (Nahta etc. (2006) CancerLett.8:123-38).

[0216] except that common overexpression, the frequency of any specific HER family member overexpression is different between cancer.This paper finds that the modal member of overexpression is HER1 and HER3 in the HER family, and the least common member of overexpression is HER4.TGF-α is the part of common expression.Following table provides the sickness rate of estimation and the estimation distribution of HER family member overexpression frequency (to measure from literature reference; All data is based on immunohistochemistry):

Table 4: patient's percentage ratio of overexpression

Disease

Mortality ratio * * (U.S.)

HER1

HER2

HER3

HER4

NSCLC＊	113,000	60	20-50	84	pos
						Mammary cancer	40,580	16	25	18	12
Colorectal carcinoma	56,730	70	pos	50
						Carcinoma of the pancreas	31,270	33	25	50	pos
Liver cancer	14,270	68	21	84	61
						The stomach esophageal carcinoma	24,000	30-50	10-20	81	pos

* nonsmall-cell lung cancer

True and the data (Cancer Facts and Figures) of * cancer, 2003

[0217] HER family member coexpression is challenged to treatment, and wherein said coexpression causes shortage to be replied or the resistance development by alternative HER family member's compensatory rise.Recognize observations like this herein: different HER family members help tumor development and progress with overlapping and synergetic property mode, and utilize the providing based on the expression of receptor in the tumour of this observations can design the therapeutical agent that is intended to avoid the resistance problem and can be designed for specific tumors in this article.Therapeutical agent that this paper provided and candidate therapeutic agent by target at least a or various kinds of cell surface receptor, generally two or more cell surface receptors such as multiple HER family member and/or target participate in or chemical sproof HER family member and any other cell surface receptor of relating to the medicine of guiding single cell surface receptor addresses these problems, and comprise those problems and the other problem determined herein.

[0218], is provided for numerous treatments site of target and intervention herein based on structure, function and the interaction of HER family member and other cell surface receptor.These treatment sites comprise zone that participates in part bonded acceptor zone and participation receptor dimerizationization and the zone that participates in the constraint effect.These zones can be simultaneously in multiple acceptor by target, thereby the part that a kind of therapeutical agent disturbs two or more acceptors in conjunction with and/or receptor dimerizationization.Several method and candidate therapeutic molecule are provided herein.

[0219] provides the method that is used for receptor targeted zone (structural domain that comprises responsible dimerization, part combination and/or constraint effect).Especially, receptor dimerizationization is by interacting in the therapeutical agent blocking-up of a plurality of acceptors.These therapeutical agents comprise the heteromultimeric that this paper provides and hereinafter describe in detail.

[0220] in addition, be provided for producing and the interactional therapeutical agent of target region.For example, target subdomain II and IV are with the interference receptor dimerizationization and/or with stable or promotion constraint effect.As the first step in these methods, as by the phage display back-and-forth method, identified respectively and DII and IV homologous region specificity bonded peptide.Subsequently, identify in conjunction with the suitable high-affinity peptide counterpart (peptide pairs) of DII and IV and use one of methods availalbe to make up heterodimer as chemosynthesis or PEGization method.Can keep himself inhibition configuration that is in of acceptor when identifying securely in conjunction with the high-affinity heterodimer peptide of DII and IV.In addition, domain II that selected peptide binding substances can target HER family receptors and the homologous region among the structural domain IV.Make the peptide of target in this way can be crosslinked between structural domain constraint, among the non-activity HER family member zone (for example, stablize the DII/IV interaction); Or can be, thereby spatially suppress the ability of this receptor dimerization in conjunction with (for example on the DII of single receptor) different loci.

[0221] also provides the method for using with a plurality of part bonded therapeutical agent target parts.The part that can screen acceptor is to identify and its bonded molecule.Can produce the heteromultimeric that contains two or more these quasi-molecules.

[0222] is provided for making the stable method of constraint conformation of acceptor.HER1,3 and 4 exists with constraint form and opening mode.Constraint acts on subdomain II and IV interaction back forms.In this form, (among the DII) main dimerization arm can not and thereby can not form receptor dimer or heterodimer with other acceptor interaction.The HER acceptor of estimation on cell surface (the HER2 exception thinks that its composing type ground ' is ready for use on dimerization ') is present on the cell when using ligand stimulation (even if) in about 95% time with the constraint form.Stablize the constraint form of acceptor, thereby it can not take open configuration, this has suppressed receptor active.

[0223] therefore, provide or the whole above mentioned consideration item and the therapeutical agent and the method for problem of solving herein.The a plurality of acceptors of target, HER family member's therapeutical agent especially are provided herein.In particular, provide herein general cell surface receptor therapeutical agent (comprising general HER therapeutical agent), be used to produce and the disease using this type of therapeutical agent to treat to relate to HER family receptors and part thereof and the method for illness.The screening assay method that also is provided for identifying the method for general Her therapeutical agent candidate molecules and is used for them.These class methods are described in the J of this paper part and embodiment.

[0224] in some embodiments, design the special therapeutical agent of general cell surface receptor and be intended to the ligand interaction of one or more acceptors and/or be intended to and one or more acceptor interactions, to regulate the activity of (suppressing usually) two above acceptors.This realizes that by forming from two or more ECD or its segmental heteromultimeric of at least a HER and another RTK or other CSR wherein said other CSR can be or can not be the member of HER family.In particular, at least a ECD from HER acceptor and the part that comprises structural domain I, II and III at least with allow part in conjunction with and with the cell surface receptor dimerization.The described heteromultimeric of general connection like this, thus the dimerization structural domain is in and the interactional position of cell surface receptor.Generally, described ECD can comprise the multimerization structural domain that promotes two or more ECD dimerizations or multimerization.In ECD, comprise and containing from two or more heterozygosis ECD of the structural domain of isoacceptor not.

[0225] at least a ECD in heteromultimeric contains structural domain I-III and contains enough parts of structural domain IV as required, thereby described heteromultimeric and ligand interaction and/or can be used for and the cell surface receptor dimerization, to such an extent as to described heteromultimeric is regulated the activity of at least two kinds of cell surface receptors.At least two kinds of cell surface receptors generally include at least a HER receptor family member.

[0226] contain can be by connecting acceptor from the general Her therapeutical agent of at least two kinds of ECD of two kinds of different HER family members or its part ectodomain part (as being similar to Trastuzumab and Erbitux) and/or by block two or more members' of HER family activity in conjunction with the part that can activate one or more acceptors.General Her therapeutical agent is regulated two or more cell surface receptors activity of (comprising at least a cell surface receptor as the HER acceptor).

[0227] also provide such polymer, two or more of wherein said ECD are receptor-derived from identical HER.Yet in this type of polymeric dimer, described ECD contains different ECD parts.

[0228] with the lower section their method of exemplary treatment agent, generation, screening and use is described.

C.HER acceptor and other cell surface receptor structure and activity

[0229] provides the polymer that contains from the ECD of different cell surface receptors (the HER family member who comprises acceptor) herein.This polymer comprises the receptor domain that is connected with the multimerization structural domain and the combination of subdomain.Design as this type of ECD polymer that provides herein are provided, are favourable to the understanding of receptor structure and function.This part provides this class description.

[0230] receptor tyrosine kinase is to participate in embryo's generation, cell growth and break up and participate in several lysises comprising the so various disease such as the huge cell signal molecule family (summary is seen Hynes and Lane (2005) Nat Rev Cancer 5:341-54) of cancer, autoimmune disease and other chronic human diseases.What characterize fullest in these receptor tyrosine kinases is people EGF receptor family (HER) in the receptor tyrosine kinase.These acceptors are called the I receptoroid.Acceptor HER family belongs to receptor tyrosine kinase (RTK) family, and has protein tyrosine kinase activity (except that HER3; Summary, referring to for example, Jorissen etc. (2003) Exptl.Cell Res.284:31-53; Dawson etc. (2005) Mol.Cell Biol.25:7734-7742, it has described nomenclature used herein; With (2005) Endocrine Related-Cancer 12:S17-S27 such as Bazley).The acceptor gene that has 4 kinds of coding HER family members: HER1 (EGFR or ErbB1), HER2 (or c-erbB-2 or ErbB2 or NEU), HER3 (c-erbB3 or ErbB3) and HER4 (c-erbB4 or ErbB4).Described encoding gene can be through alternative splicing to produce numerous variants, comprises truncated variant and as the variant of intron fusion proteins.In the described acceptor some in normal development, break up, divide a word with a hyphen at the end of a line, play a role in wound healing and the apoptosis, this is essential active.Abnormal function and activity play a role in various disease states (comprising cancer).

[0231] sequence of exemplary people HER family receptors is described in SEQ ID NO:2 (HER1), 4 (HER2), 6 (HER3) and 8 (HER4) and nucleotide sequence coded by described in the SEQ ID NO:1,3,5 and 7 respectively.Generally, the HER polypeptide of coding experience translation post-treatment process lacks the mature polypeptide of signal sequence with generation.Set forth the aminoacid sequence of ripe full-length polypeptide and they have been separated the description that is right at Fig. 2 (A)-(D) and respective drawings.For this paper purpose, the amino acid numbering in describing exemplary HER family receptors, its ECD part or ECD isoform is with reference to the numbering of mature polypeptide, and is really not so unless stated otherwise.In addition, being used for the amino acid position of description scheme tract tissue form is intended to illustrative explanation, and the scope of the embodiment that provides is provided.Be to be understood that polypeptide and description to its structural domain are based on to the homology analysis of similar acceptor and comparison and deutero-in theory.Therefore, accurate location can change, and every kind of acceptor is needed not to be identical.

[0232] as shown in Figure 1, each member of HER family has common structural domain organizational form, and it comprises and contains about 620 amino acid whose ectodomains parts (ECD or ectodomain or extracellular domain), membrane spaning domain and cytoplasmic tyrosine kinase structural domain.ECD partly shows four subdomains, called after I (L1), II (S1), III (L2) and IV (S2).Sequence identity between total length HER family is 37% for HER1 (EGFR) and HER3 and is 49% for HER1 and HER2, the degree difference of sequence identity between while every kind of structural domain.For example, the sequence identity of tyrosine kinase domain the highest (about 59-81%), and the identity of carboxyl terminal structural domain minimum (about 12-31%).In ECD structural domain inside, the sequence identity of subdomain I and III total about 37% and domain II and IV be homologous and total about 17% sequence identity (Ferguson etc. (2003) Mol.Cell, 11:507-517).

[0233] subdomain I and III are called the L structural domain again, and constitute 2 lobate ligand-binding site points.Each L structural domain all contains the six corner strand right hand β spirals that form the ladle sample structure, and wherein said ladle sample structure is covered by a α spiral at each end.Part is combined between L1 and the L2 structural domain.

[0234] subdomain II and IV are called not only S structural domain or abundant (CR) structural domain (but also being called Fu Lin (furin) sample repeating structure territory) of halfcystine and constitute the abundant district of halfcystine.This Cys is made up of the little module that a series of disulfide linkage that form rod shaped structure connect in abundant zone.There is two types disulfide linkage link block in each structural domain: the C1 disulfide linkage, wherein single disulfide linkage has retrained interleaving property bow sample ring, with the C2 disulfide linkage, wherein two disulfide linkage with Cys1-Cys3 be connected with the Cys2-Cys4 pattern 4 continuous halfcystines with form the knuckle spline structure (Ferguson etc. (2003) Molecular.Cell, 11:507-517).Domain II contains three continuous C2 modules, follow-up 5 C1 modules, and structural domain IV contains 7 modules, and wherein two is the C1 module, follow-up C2 module, 2 C1 modules and another C2 module.

[0235] generally speaking, the intramolecularly of domain II and IV mediation HER structure contacts with intermolecular and contacts.For example, intramolecular interaction is taking place in a process that is called " constraint " between subdomain I and the IV, and β-ring enriches outstanding (see figure 1) the module from the 5th Cys in described process.This β-ring with from the equivalence in structural domain IV inner module 5 and the module 6 but interact than circlet.The interaction of domain II and IV is further contributed by hydrogen bond between these two zones and carbohydrate and is stablized.In addition, the side chain of the amino-acid residue corresponding with Y246 in the domain II of HER1 with structural domain IV in the side chain of the corresponding amino-acid residue of D563 and K585 form hydrogen bond.In table 5, describe the important corresponding amino-acid residue in the ECD of ripe HER family receptors of contact between mediation domain II/IV.Do not have the interaction between domain II and the IV in HER2, partly cause is to compare with all the other HER family members, has non-conservative amino acid residues (i.e. the residue of representing with italic in the table 5).

[0236] also have molecular interaction and permission acceptor-acceptor interaction, wherein said acceptor-acceptor interaction is to be subjected to the same dimerization and the different dimerization of body characteristics necessary as active HER.In fact, mentioned above in the module 5 of domain II same a kind of ring of mediation constraint effect responsible to dimerization.This ring often is called " dimerization arm ".Amino-acid residue corresponding to Y246 also is important in promoting the needed molecular interaction of dimerization.

[0237] the HER family receptors also comprise stride film (TM) structural domain (according to difference be reported in the 621st, 622 or 626-644 or 647 residue places begin) and cytoplasmic structure territory.Described membrane spaning domain connects the endochylema film of grappling this receptor and generally includes hydrophobic residue.Generally, constitute the residue formation alpha-helix of membrane spaning domain.

[0238] nearly film (JM) structural domain as zone between membrane spaning domain and kinase domain plays multiple regulatory function, for example, in downward modulation effect and ligand dependent internalization incident, the polarization cell substrate outside sorting of (for example EGFR) and with protein such as eps8 and calmodulin associating.In addition, the JM structural domain plays a role in the acceptor transportation and needs (together with membrane spaning domain) to be used for targeting EGFR to the after birth caveolae.

[0239] tyrosine kinase domain is total conservative catalytic core of receptor tyrosine kinase (RTKs) and the transphosphorylation of being responsible for mediating the carboxyl terminal tyrosine residues that exists in the carboxyl terminal structural domain.The activation of tyrosine kinase domain occurs after because of part and receptors bind institute inductive conformational change.

[0240] carboxyl terminal (CT) structural domain contains its phosphorylation tyrosine residues of conditioning signal transduction then.Each HER family member's described tyrosine residues and near amino acid and multiple second messenger interact to regulate specific biologically and biochemical reaction.For example, the second messenger of containing SH2 (src homology-2) structure for example or PTB structural domain discern that phosphorylation " is stopped site (docking sites) " and with acceptor interaction to be delivered in the signal received at this receptor place to tenuigenin or nucleus by interacting with other signal component.Also have several serine/threonine residues, wherein the phosphorylation to these residues influences receptor down-regulated effect and endocytosis process.984-996 position residue (Fig. 1) in the EGFR carboxyl terminal is served as the Actin muscle binding site and participate in bigger receptor oligomers formation and/or acceptor bunch collection effect after part is activated kinase domain.

1.HER1ECD structure and structural domain organizational form

[0241] the structural domain organizational form of ripe ECD of a kind of total length of description and multiple HER1ECD isoform in Fig. 2 (A).Born of the same parents' outside part of HER1 comprises the 1-621 position residue of ripe HER1 acceptor and contains subdomain I (1-165 amino acids residue), II (166-313 amino acids residue), III (314-481 amino acids residue) and IV (482-621 amino acids residue).Three structural domains of the I of HER1, II and III structural domain and I type IGF-1 (IGF-1R, referring to for example, Garret etc. (2002) Cell, 110:763-773) have structural homology and sequence homology.Be similar to IGF-1R, L structural domain (being structural domain I and III) has at each end by six corner β spirane structures of α spiral and disulfide linkage covering.When comparing with IGF-1, the HER1 sequence includes the aminoacid insertion thing that helps these biological chemical structures, and combination is important to wherein said biological chemical structure to HER1 mediation part.Comprise V-arrangement projection (V-shaped excursion) (8-18 position residue) in these biological chemical structures, this V-arrangement projection is positioned at the big βZhe Die top of structural domain I to form the major portion of part bonding interface.In domain II I, corresponding zone forms and also participates in a ring of part bonded (316-326 position residue).The 3rd insertion zone (351-369 position residue) that exists among the domain II I is an extra loop in second corner of domain II I.This ring be the epi-position that prevents the multiple antibody of part bonded (be LA22, LA58 and LA90, referring to for example, Wu etc. (1989) J Biol Chem., 264:17469-17475).In addition, the ring of other in the 4th corner of β helical spiral pipe participates in the part keying action.

[0242] TGF-α (a kind of part of HER1) interacts with the big βZhe Die of two L structural domains (I and III) of an acceptor molecule.Similarly, ligands, EGF also interacts with two structural domain I and the III of HER1, though think the interaction of EGF and domain II I be EGF main binding site (Kim etc. (2002) FEBS, 269:2323-2329).Crosslinked research has determined that the aminoterminal of EGF part and carboxyl terminal part interact with the structural domain I and the III of HER1 acceptor respectively.In domain II I, combine with EGF by interacting to participate in mediating with the Arg45 of people EGF with the corresponding amino acid Gly441 of ripe total length HER1.40kDa HER1 fragment with 202 amino acid (corresponding to the 302-503 amino acids of ripe HER1 polypeptide) is enough to keep the whole ligand binding capacities of HER1 to EGF.Described have only some residues (Kohda etc. (1993) JBC 268:1976) that 202 amino acid whose parts contain each structural domain among the whole and domain II of domain II I and the structural domain IV.

[0243] domain II of EGFR contains the module that 8 disulfide linkage connect.Domain II and structural domain I and III act on mutually.Take place with contacting of domain II I, and module 7 and 8 has flexibility to a certain degree, thereby its effect is to produce hinge in the no part form of EGFR molecule by module 6 and 7.Big ordered ring forms and directly stretches out away from the ligand-binding site point from the module 5 of domain II.This ring is corresponding to 240-260 position residue (also being described as 242-259 position residue) and contain antiparallel β-zonal structure.This ring (being called the dimerization arm again) is important for reporter molecule interphase interaction and mediation acceptor-acceptor contact.In the non-activity conformation of HER1 or " constraint " in the conformation, this ring is by inserting and helping the intramolecular interaction (see figure 1) respectively between the 561-569 of ripe total length ECD and the similar ring structure in the corresponding module 5 of 572-585 amino acids and 6.In last table 5, described and helped the interactional amino-acid residue of domain II/IV.

[0244] disappearance of domain II ring has been cancelled the ability of HER1ECD dimerization, therefore shows its importance aspect the promotion molecular interaction.Dimerization mediates by stretching out the domain II of crossing second HER molecule in the space of this ring between structural domain I, II and III.For example, contact is produced by 229-239,262-278 on the concave surface of domain II and 282-288 position residue in dimerization arm 244-253 position residue and the 2nd HER molecule.Gly264 in Tyr246 in the domain II and the 2nd HER molecule and Cys283 residue form hydrogen bond, and the benzyl ring of Tyr246 also interacts with the Ser262 and the Ser282 of adjacent molecule.Comprise Tyr251 and Phe263, Gly264, Tyr275 and Arg285 in the domain II of EGFR and other amino acid contact between another HER molecule; Pro248 and Phe230 and Ala265; Met253 and Thr278; With Tyr251 and Arg285.In addition, to be under the suitable conformation be important for keeping this ring for Asn247 and Asn256.These residues of the overwhelming majority are conservative between the HER family member and play a role similarly between the HER family receptors.In addition, proline residue is present in arbitrary position of these interior the 243rd, 248,255 and 257 positions of ring of HER family receptors, and HER3 contains 3 proline(Pro) simultaneously.Proline residue is further stablized this ring conformation.For example, HER1 contains proline(Pro) in the 248th and 257 positions.

[0245] except structural domain IV (module 5 and 6) participates in constraint non-activity HER1 molecule, as if also need HER1 structural domain IV module 1 to small part to keep the structural integrity of active HER1 molecule.For example, as mentioned, the 40kDa proteolytic fragments of HER1 (its contain the whole and domain II of domain II I and the part of IV) keeps ligand binding capacity completely.The part of the structural domain IV that exists in this molecule comprises the whole of module 1 corresponding to the 482-503 amino acids.With Trp492 is corresponding in the ripe HER1 molecule amino acid by with domain II I in hydrophobic pocket interact and in keeping the HER1 stability of molecule, play a role.Contain whole structural domain I, II and III but lack the reorganization HER1 molecule of whole structural domain IV can not binding partner (corresponding to the 1-476 amino acids of ripe HER1, referring to for example, Elleman etc. (2001) Biochemistry 40:8930-8939).Therefore, module 1 all or part of at least that for the ligand binding capacity of HER1, as if needs structural domain IV.For part combination and signal conduction, the remainder of structural domain IV is droppable.For example, the normal part combination of HER1 and signal transport properties are present in a kind of HER1 molecule of the 521-603 position residue of losing ripe HER1 polypeptide.

2.HER2ECD structure and structural domain organizational form

[0246] the structural domain organizational form of ripe HER2ECD of a kind of total length of description and multiple HER2ECD isoform in Fig. 2 (B).Born of the same parents' outside part of HER2 comprises the 1-628 position residue of ripe HER2 acceptor and contains subdomain I (1-172 amino acids residue), II (173-319 amino acids residue), III (320-488 amino acids residue) and IV (489-628 amino acids residue).Although have similar structural domain organizational form, yet the crystal analysis of HER2 has been confirmed that HER2 does not possess the same intramolecular interaction of " constraint ", non-activity constitutional features as all the other HER family members'.In other words, the ring in the module 5 of domain II does not interact with the residue of structural domain IV.Last table 5 has been described the amino acid that contacts between the guide structure territory II/IV of HER family receptors intermediary, and has provided those not conservative in HER2 amino acid.For example, conservative Gly residue is replaced by a proline(Pro) in HER2 respectively in the 564th, 563 and 561 positions of HER1, HER3 and HER4.This proline residue spatially is suppressed at observed interaction in all the other HER family receptors (being that the Gly residue interacts with corresponding HER3 amino acid Phe251).Therefore, because sequence difference, HER2 does not exist with " constraint ", non-activity state, and composing type ground exists with activity conformation, and wherein the dimerization arm of HER2 in domain II exposes.

[0247] although only have the 33-44% sequence homology between the HER family receptors, yet domain II dimerization arm is comprising that between whole HER family receptors of HER2 be the function high conservative.In HER2, this dimerization arm is corresponding to the 246-267 amino acids residue of ripe HER2.Because HER2 always exists with active, non-constraint conformation, expose its dimerization arm, so HER2 is all the other HER family members' a preferred heterodimer mating partner.Yet HER2 does not form homodimer.Can not form the reason Coulomb repulsion effect seemingly of homodimer, because the pocket of this dimerization arm contact all is electronegativity among the dimerization arm of HER2 and the HER2.The high electronegativity of HER2 can be owing to comparing with all the other HER family members, and acidity in HER2 and alkaline residue number are more.Yet when HER2 in cell during overexpression, it can same dimerization.Hydrophobic region behind the overexpression in the observed carboxyl terminal structural domain that relates to HER2 with dimerization is especially for observed part dependent/non-dependent multimerization (Garret etc. (2003) Mol.Cell, 11 behind the acceptor overexpression; 495-505).

[0248] in addition, different with other HER family receptors, HER2 does not combine with part.This can not binding partner a reason be ligand binding domains I and III closely near and relative direction.In HER2, opposed structural domain I causes quite a large amount of to each other direct contacts with III.In this conformation, part can not combine with any potential binding site because each binding site sealed by opposed ligand binding domains (Garret etc. (2003) Molecular Cell, 11:495-505).In addition, compare with other HER family member, HER2 contains the sequence difference that can suppress ligand interaction in the part bonding interface of structural domain I and III.For example, Arg12 (corresponding to the Thr15 among the HER1, the Ser18 among the HER3, the Ser12 among the HER4) is different with the corresponding residue at place, equivalent site among all the other HER family members with Pro14 (corresponding to the Leu17 among the HER1, the Thr20 among the HER3, the Leu14 among the HER4).These residues are the parts that form the V-arrangement projection of stretch-like βZhe Die with part, and disturb the ability of HER2 binding partner.Other sequence difference in structural domain I and III also causes HER2 to combine with part.

3.HER3ECD structure and structural domain organizational form

[0249] the structural domain organizational form of ripe HER3ECD of a kind of total length of description and multiple HER3ECD isoform in Fig. 2 (C).Born of the same parents' outside part of HER3 comprises the 1-621 position residue of ripe HER3 acceptor and contains subdomain I (1-166 amino acids residue), II (167-311 amino acids residue), III (312-480 amino acids residue) and IV (481-621 amino acids residue).As other HER family receptors, structural domain I, the II of HER3 and the structure of III can superpose with IGF-1R, and show the numerous same structure features as other HER acceptor.For example, the structural domain I of HER3 and III show β-spirane structure that the stretching, extension tumor-necrosis factor glycoproteins of the module that is contained disulfide linkage is interrupted.Handiness between the structural domain that has high level between domain II and the III, and IGF-1R does not show this point.In addition, HER3 indicating characteristic beta-hairpin loop or dimerization arm (corresponding to the 242-259 amino acids of HER3) in domain II.This beta-hairpin loop provides with the intramolecularly of conserved residues among the structural domain IV and contacts, and produces the HER3 structure of closure or non-activity.Provided important residue in this constraint interacts in the last table 5, and these residues comprise the interaction of Y246 and D562 and K583, F251 and G563 and Q252 and H565.Behind binding partner, conformational change is redirected structural domain I and III, makes the dimerization arm be exposed to allow receptor dimerizationization from the structure of constraint.

[0250] unlike other HER family receptors, HER3 does not have functional kinase domain.4 amino-acid residues of guarding between whole protein tyrosine kinases cause the imbalance of HER3 kinase function in the change kinases zone.Yet, HER3 in its carboxyl terminal structural domain, keep tyrosine residues and suitably activate and transphosphorylation after can inducing cell signal conduction.Thereby the same dimerization of HER3 can not be supported the linear signal conduction.For HER3, preferential dimerization mating partner is HER2.

4.HER4ECD structure and structural domain organizational form

[0251] the structural domain organizational form of ripe HER4ECD of a kind of total length of description and multiple HER4ECD isoform in Fig. 2 (D).Born of the same parents' outside part of HER4 comprises the 1-625 position residue of ripe HER4 acceptor and contains subdomain I (1-163 amino acids residue), II (164-308 amino acids residue), III (309-477 amino acids residue) and IV (478-625 amino acids residue).HER4 is the most similar to HER1, because as HER1, HER4 can binding partner and showed kinase activity.Have such structural domain organizational form in HER4, it comprises that existence is to constraint effect and all important dimerization arm of dimerization.Last table 5 has been summarized in domain II and IV the conserved residues that HER4 is locked under the non-activity state.Corresponding dimerization arm in HER1 is corresponding to the 237-258 amino acids residue of HER4.With regard to ligand binding domains I and III, structural domain I is responsible for the primary structure territory that part neuregulin (NRG) is bonded to HER4.The aminoterminal residue (Kim etc. (2002) Eur.J.Biochem269:2323-2329) of the structural domain I identification NRG of HER4.

[0252] total length HER4 expression of receptor is four kinds of alternative splicing isoforms.Two kinds of differences (being CYT-1 and CYT-2) in the kytoplasm tail in the alternative splicing isoform, and all the other two kinds differences (being JM-a and JM-b) in membrane-proximal region.The result of alternative splicing produces the isoform with unlike signal transmissibility.For example, the CYT-1 isoform contains an extra exon, and it contains the non-existent extra stop site (being SH2) that is used for signaling molecule in the CYT-2 isoform.In addition, described JM isoform at them to proteolytic enzyme cutting for example by difference aspect the susceptibility of tumor necrosis factor-alpha saccharase (TACE) cutting.

5.HER family's part, ligand specificity and ligand-mediated receptor activation

[0253] acceptor ErbB (HER) family member's activity needs the part combination with dimerization, and this causes catalytic activity, finally causes signal transduction.There are several HER ligands specifics belong to ligands, EGF family respectively (referring to for example, table 6).All the EGF part has EGF spline structure territory, and this structural domain is a 45-55 amino acid motif with six halfcystines, and wherein said six halfcystines interact to form by 3 covalently bound rings of disulfide linkage.This zone is important to the binding specificity of giving the HER part.Other structural motif in the EGF part comprises immunoglobulin like domain, heparin binding site and glycosylation site.Usually, part be expressed as at first need proteolytic cleavage with in solution, realize active and/or with the protein bound film anchorin of cell surface HER.This split requirement plays the effect of control part availability and receptor activation.Participate in proteolytic enzyme that the EGF part disseminates and for example comprise those proteolytic enzyme from metalloprotein enzyme family (comprising integrin and metalloprotease (ADAM) family) and matrix metalloproteinase (MMP) family.The generation of EGF part is regulated in the activation of g protein coupled receptor (GPCR).In cancer, the GPCR signal conduction imbalance in the tumour is preponderated relevant with the constitutive activation of HER acceptor with the EGF part.

[0254] table 6 has been listed part and the receptor-specific thereof of knowing and characterize the row of fullest in these parts most.Based on the acceptor preference, part is divided into three groups (being summarized as the 1-3 group in the following table).These parts all do not combine with HER2, wherein each the member's different dimerization among HER2 and all the other family members.In following table, the alternative title of cytokine neuregulin (NRG) family comprises that Neu differentiation factor, NDF or Her regulate albumen (heregulin; HRG).The neuregulin of part/Her adjusting protein family is NRG-1, NRG-2, NRG-3 or the NRG-4 gene deutero-structural dependence somatomedin from alternative splicing.For example, exist from least 14 kinds of soluble transmembrane protein isoforms of NRG-1 gene deutero-.The proteolysis processing of striding film NRG-1 isoform ectodomain discharges soluble growth factor.HRG-1 β is one of these soluble growth factor and to contain directly combining with HER3 and HER4 be essential Ig structural domain and EGF spline structure territory.The recombinant human HRG-1 β that only contains the EGF structural domain of Her adjusting albumen β is enough to combination and activates the HER acceptor.The another kind of isoform of NRG-1 gene is HRG1-α.HRG α to the binding affinity of HER3 and HER4 than HRG β a little less than 100 times (Jones etc. (1999) FEBS Letters, 447:227-231).There are at least two kinds of NRG-2 isoforms, are called NRG2-α and NRG2-β.NRG2-α and NRG2-β all are HER3 agonists and stimulate HER3 signal conduction.NRG2 β also is the agonist of HER4, but NRG2-α is not the strong stimulator of HER4 tyrosine phosphorylation or signal conduction.The isoform that does not have other NRG-3 that has reported and NRG-4.

[0255] since exist considerably beyond 15 kinds can different EGF parts with HER family member bonded, thereby control and to regulate HER family signal conduction be complicated.The tissue specific expression that in the factor of this sophisticated signal of adjusting system, comprises receptors ligand.For example, be expressed in to the NRG advantage in the organa parenchymatosum and embryo maincenter and peripheral nervous system in.In addition, though part generally can combine with monomeric acceptor, yet they can not activate monomeric acceptor.On the contrary, compare with monomeric acceptor, activation that the dimer of acceptor forms and final HER mediates and signal conduction are driven by the more high stability of the complex body of two HER acceptors and a part.Combine the conformational change that not only mediates monomeric acceptor with the part of monomeric acceptor allowing acceptor with dimerization or different dimerization (seeing below), in case and part also after the dimer acceptor forms, make it stable.Therefore, for activation, multiple dimer counterpart depends on acceptor density and ligand concentration.Therefore, the activation of HER is subjected to the room and time of its part to express control.

6. the generation of dimerization and constraint and active homodimer and heterodimer

[0256] control HER family receptors activated mechanism depends on the part combination and the inducing of conformational change in acceptor.Generally, between the inactive form of HER acceptor and activity form, there is balance.At least under the example of HER1, about 95% is present on the cell surface with constraint form or inactive form; And only 5% is in activity form.

[0257] when the bonded part lacks, in monomeric acceptor, the dimerization arm in the domain II because of with same molecular in subdomain IV interact and to be embedded in the intramolecularly lashing (tether), thereby receptor autophosphorylation is suppressed.Therefore, under the normal circumstances, except that HER2, all HER acceptors are in the conformation of non-activity or " constraint ".The conformation of constraint is the closed form of acceptor, and wherein said closed form prevents that this receptor and other HER family member from interacting.Under the normal circumstances, in this conformation, keep ligand binding domains I and III to be far apart separately.For whole HER family receptors also is so, except that HER2.For HER2, even if as monomeric acceptor, structural domain I and III structurally near and spatially suppress combining of part and this zone.Therefore, HER2 can not combine with part, and always makes the dimerization arm exposure of HER2 and prepare to be used for promotion and another HER family receptors dimerization at any time.

[0258] part of HER acceptor molecule is induced dimerization to induce receptor activation and the normal downstream signal mechanism of action of acceptor HER family is provided.Activity part and structural domain I and/or III interact, and promote the rearrangement among the ECD, cause fettering the exposure of conformation opening and dimerization arm.The bonded part is fixed the relative position of structural domain I and III, forces them to rotate (for HER1, about 130 °).Intramolecularly domain II/IV connection or constraint broken in this rearrangement, and discharges the dimerization arm, thereby this dimerization arm can participate in molecular interaction." opening " of this generation acceptor or activity conformation also have the ability and other HER family member dimerization molecule.HER2 always is in open conformation, even if also be like this as monomer.Therefore, even if lack part, yet HER2 can with another HER family member dimerization, though HER2 not with self dimerization, unless overexpression.In open configuration, dimerization arm (see figure 1) is stretched out from domain II and can be interacted by the pocket of noncovalent interaction (as preferendum and hydrophobic interaction, Van der Waals interaction and hydrogen bond action) with the base portion that is arranged in the second acceptor domain II dimerization ring.Sudden change in the dimerization ring can cause the composing type dimerization, and verified this composing type dimerization inducing cell transforms (Bazley etc. (2005) Endocrine-Related Cancer12:S17-S27) under the situation of HER2.Between subdomain ring II and subdomain I and II, exist and contact.Also can form more the high-grade structure such as the different tetramer (see, for example, Jorissen etc. (2003) Exptl.Cell Res.284:31-53).

[0259] only the dimerization arm is not enough to take place dimerization.Extra interaction (comprise domain II/III interact) turns with stablizing (see, for example, Dawson etc. (2005) Mol.Cell.Biol.25:7734-7742) receptor dimerization into.As discussed above, though dimerization arm high conservative between HER1,2,3 and 4, HER2 can not form homodimer.For HER1, module 6 is for providing extra complementary the interaction (comprising D279 and H280) of oneself with dimerization.Module 7 participates in the HER2/HER3 different dimerization.These residues conservative between whole four kinds of HER acceptors (see, for example, Dawson etc. (2005) Mol.Cell.Biol.25:7734-7742).

7.HER family receptors activity

[0260] HER is expressed in the multiple tissue of epithelium, mesenchyme and neurone origin and regulates growth, survival, propagation and differentiation.Under normal physiological conditions, the activation of HER is expressed control by the room and time of its part, and wherein said part is member's (seeing above) of somatomedin EGF family.The activation of the zygotic induction acceptor homodimer of part and HER acceptor and heterodimer formation and inherent kinase domain causes phosphorylation on the specific tyrosine residues in the kytoplasm tail.The residue of these phosphorylations serves as the proteic stop of series of effects site, wherein convenes described effect protein to cause the activation of intracellular signal pathway.For example, phosphatidyl-inositol 3-kinase (PI3K)-AKT approach is upset by p85 joint subunit to the acceptor of convening PI3K.Mitogen activated protein kinase (MAPK) approach activates to acceptor by convening growth factor receptor binding protein precursor 2 (GRB2) or SHC.

[0261] activation of every kind of acceptor differs from one another aspect several.For example, HER2 does not have corresponding somatomedin part, and HER3 does not have the fully tyrosine kinase activity of definition.These two kinds of acceptors depend on other member usually altogether so that the signal conduction can take place, although as HER2 fully during overexpression, HER2 can under no co-receptor (co-receptor) or part the signal conduction take place effectively.On the contrary, the HER3 homodimer is complete non-activity, and reason is the defective kinase activity of tyrosine kinase domain.Generally, the signal conduction more effectively takes place than HER homodimer in the HER heterodimer.This is because heterodimer provides from two kinds of different kytoplasm tails of isoacceptor not, thereby is provided for convening the extra Tyrosine O-phosphate residue and the different phosphate acidifying pattern of different effect molecule.Therefore, the HER different dimerization is can be so as to making the mechanism of amplification of signal and diversified signal.The HER2/HER3 heterodimer is the strongest receptor signal effect counterpart.There is several reasons in the effectiveness increase of HER2/HER3 heterodimer.At first, HER2 and HER3 and various signal transduction path coupling, are included in mitogen activated protein kinase (MAPK) approach important in the cell proliferation and regulate cell survival and phosphatidyl-inositol 3-kinase (the PI3K)/Akt approach of anti-apoptotic signal at described signal transduction path.In addition, the HER2/HER3 heterodimer also has the signal conduction of prolongation, and its reason is the efficient recirculation of acceptor and the poor efficiency downward modulation effect that cell surface receptor is expressed.

[0262] verified each HER acceptor the various kinds of cell process comprise cytodifferentiation, cell proliferation, cell survival, vasculogenesis and divide a word with a hyphen at the end of a line and invade in play a role.The HER acceptor is the essential mesosome of cell proliferation and differentiation in embryo of growing and adult tissue, but their inappropriate activation is relevant with seriousness with other advancing of disease with multiple cancer (comprising for example mammary cancer, colorectal carcinoma and prostate cancer).There are the numerous mechanism of the inappropriate activated of disease-related HER acceptor that influence.Comprise in these mechanism that the gene amplification that for example causes the acceptor overexpression is unusual or it is unusual to transcribe, transgenation and because of the autocrine stimulation effect due to the excessive generation of HER part.Therefore, target HER acceptor, for example the target HER acceptor by the general therapeutical agent that provides herein is can be so as to regulating these processes to treat the disease relevant with unsuitable HER signal conduction or the mechanism of illness.Following process belongs to this type of the active and corresponding cell processes by the mediation of HER receptor signal conduction.These processes, cell proliferation, cell survival, vasculogenesis and cell migration and intrusion are tumorigenic signs.These processes also can be carried out external monitoring, as describing in the G part, to assess the feasibility of this type of therapeutical agent.

A. cell proliferation

[0263] HER receptor signal conduction plays a role in regulating propagation by control cell cycle check point (checkpoint).For example, the overexpression dysregulation G1-S of HER2 conversion and driving cell proliferation.The level that causes protein c-Myc and cyclin D increase by HER2 inductive strong signal.These proteinic each all play chelating protein p27, wherein said p27 is the cyclin kinase inhibitor.Cyclin E-CDK2 mediation enters the cell cycle.The chelating of p27 is prevented that p27 from combining with cyclin E-CDK2 suppressing its activity, and therefore cause uncontrolled cell proliferation.Inhibition to the effect of HER2 signal causes MAPK and the downward modulation of P13K/AKT approach, and this reduces the level of c-Myc and cyclin D.This allows not, and compound p27 is bonded to cyclin E-CDK2 and the cell proliferation of the deactivation latter to prevent to continue.

B. cell survival

[0264] the HER family receptors is regulated cell survival by regulating the effect protein that participates in inherent apoptosis pathway.For example, because of the cell survival due to the effect of HER signal mediates by the PI3K/AKT approach, wherein said PI3K/AKT approach is a target with the substrate that suppresses pro apoptotic protein BAD and Caspase 9.In addition, comprise also by the target substrate of AKT phosphorylation and to suppress for example transcription factor expressed of FAS part and other transcription factor (being NF-κ B) of several short apoptogenes that wherein said other transcription factor raises for example level of BCL-XL of short survivin.

C. vasculogenesis

[0265] HER signal conduction is induced for example vascular endothelial growth factor (VEGF) expression of multiple short angiogenesis factor.For example, the activation-inducing of HER1 produces VEGF.In addition, the overexpression of HER2 is relevant with the increase generation of VEGF in colorectal carcinoma, carcinoma of the pancreas, cancer of the stomach, mammary cancer, renal cell carcinoma and the nonsmall-cell lung cancer.The vasculogenesis effect of VEGF by its in conjunction with and activate two kinds and be expressed in the associated receptor (being VEGFR-1 and VEGFR-2) on the endotheliocyte and to grow (being vasculogenesis) at neovascularity relevant with effect during blood vessel is kept or the prematurity blood vessel is survived with it.Vasculogenesis plays a role in tumour takes place.

D. divide a word with a hyphen at the end of a line and invade

[0266] to the stimulation of HER signal conduction also mediated cell mobility and the many aspects of dividing a word with a hyphen at the end of a line, wherein said cell mobility and dividing a word with a hyphen at the end of a line is playing a significant role during embryonic development, the wound healing and at tumor growth with in shifting.The cell movement reaction can activate back institute inductive signal transduction path at HER widely by type and start.For example, relevant with HER1 inductive cell migration by HER1 to the activation of PLC γ dependent pathway, because the inhibition blocking-up EGF inductive cell of this kind of enzyme is moved (Jorissen etc. (2003) Exp.Cell Res.284:31-53).The cell migration mechanism of EGF mediation has been with to stimulate actin cytoskeleton to reset related, and its reason is that the release Actin muscle modified protein that mediates of PLC-γ (is gelsolin, profilin, Actin muscle Cofilin (cofolin) and CapG).MAPK also plays a role in the cell mobility of HER mediation, for example, and by regulating the integrin level.Participating in the cell migration of HER mediation and other signal transduction path or the effector molecule of mobility comprises PI3-K and participates in the film crumple and downstream effect molecule Rac that pseudopodium forms and the Rho that participates in cell rounding and cortex actin polymerization.

[0267] in addition, divide a word with a hyphen at the end of a line by HER signal conduction inductive and express relevant with the increase of the matrix metalloproteinase (MMP) of cutting extracellular matrix component with invading also.For example, related with tumour cell intrusion and generation proteolytic activity due to predisposition is led MMP-2 and MMP-9 by neuregulin to the stimulation of HER3 and HER4.

8. other CSR ECD

[0268] except target HER family member, any other cell surface receptor (CSR) or its part that the therapeutical agent that provides herein is intended to go back target involved in diseases process also can be provided, and described lysis includes but not limited to tumour generation, vasculogenesis or inflammatory diseases.In particular, all the other ECD are from the chemical sproof acceptor of therapeutical agent that participates in or relate to a kind of acceptor of development target.

[0269] usually, this kind CSR is receptor tyrosine kinase (RTK).Normally, this kind therapeutical agent contains is enough to ligand interaction and/or prevents ECD or its part of the CSR of receptor dimerizationization.The example of RTKs includes but not limited to Urogastron (EGF) acceptor (as discussed above), platelet-derived somatomedin (PDGF) acceptor, fibroblast growth factor (FGF) acceptor, rhIGF-1 (IGF) acceptor, nerve growth factor (NGF) acceptor, vascular endothelial growth factor (VEGF) acceptor, join the albumen acceptor of (being called Eph) at liver, pHGF (HGF) acceptor (being called MET), TIE/Tie-1 or TEK/Tie-2 (acceptor of angiogenesis factor-1), coil basic mycoprotein (discoidin) domain receptor (DDR) and other RTKs, as Tyro3/Axl.Can include but not limited to TNFR (be TNFR1, TNFR2, CD27,4-1BB, OX40, HVEM, Lt β R, CD30, GITR, CD40 and other etc.) or RAGE as other CSR of therapeutical agent with ECD part.Table 7 is listed exemplary CSR and has been described and constitutes the amino acid of the ECD of polypeptide separately.The exemplary sequence of RTKs and other CSRs and coded amino acid are being described among the SEQ ID NO:193-262 arbitrarily.

[0270] ectodomain that comprises the RTKs of growth factor receptors is made of the modular structure territory of various classification, and wherein said modular structure territory includes but not limited to that fibronectin III type structural domain, halfcystine enrich structural domain, Urogastron structural domain and immunoglobulin (Ig) (Ig) spline structure territory.For many RTKs, part keying action (referring to for example, Wiesmann etc. (2000) J Mol.Med.78:247-260) is responsible in Ig spline structure territory.80-110 the residue that forms two antiparallel beta sheets that contain 3-5 bar beta chain generally contained in Ig spline structure territory, and described beta sheet is connected by disulfide linkage in some cases simultaneously.According to the beta chain number, Ig spline structure territory is divided into four classes: V (variable), I (centre) and C1 and C2 (constant).For example, the structural domain of C2 class contains the minimized number beta chain, contains 4 beta chains and contains 4 beta chains at second beta sheet at first beta sheet.Table 8 is described exemplary RTK family member and and their the bonded parts that contains Ig spline structure territory.

[0271] the following discussion is intended to for example.Be to be understood that and in heteromultimeric, make up, particularly with HER ECD or the combination of its part for part combination and/or the needed ECD of dimerization or its part.

(a) VEGFR1 (Flt-1) and VEGFR2 (KDR)

[0272] VEGFR1 and VEGFR2 combine with VEGF and play a role in the reaction of VEGF-inductive vasculogenesis.Endothelial cell morphology needs VEGFR1, and VEGFR2 plays a role in mitotic division takes place.The ECD structure of VEGFR1 and VEGFR2 all contains 7 Ig spline structure territories, and these two kinds of acceptors combine with VEGF similarly, though VEGFR1 also combines with part PIGF.Therefore, as if on function different are the tyrosine-kinase enzyme sequence signal transduction characteristic different with them in the born of the same parents of acceptor between VEGFR1 and the VEGFR2.Associated receptor VEGFR3 also contains 7 Ig spline structure territories, but does not combine with VEGF.Sequence for the VEGFR1 described in the SEQ IDNO:254, the one Ig spline structure territory is corresponding to the 32-123 amino acids, the 2nd Ig spline structure territory is corresponding to the 151-214 amino acids, the 3rd Ig spline structure territory is corresponding to the 230-327 amino acids, the 4th Ig spline structure territory is corresponding to the 335-421 amino acids, the 5th Ig spline structure territory is corresponding to the 428-553 amino acids, the 6th Ig spline structure territory is corresponding to the 556-654 amino acids, and the 7th Ig spline structure territory is corresponding to the 661-747 amino acids.Sequence for the VEGFR2 described in the SEQ ID NO:256, the one Ig spline structure territory is corresponding to the 46-110 amino acids, the 2nd Ig spline structure territory is corresponding to the 141-207 amino acids, the 3rd Ig spline structure territory is corresponding to the 224-320 amino acids, the 4th Ig spline structure territory is corresponding to the 328-414 amino acids, the 5th Ig spline structure territory is corresponding to the 421-548 amino acids, the 6th Ig spline structure territory corresponding to 551-660 amino acids and the 7th Ig spline structure territory corresponding to the 667-753 amino acids.

[0273] for VEGFR1, the 2nd Ig spline structure territory (structural domain 2) decision part keying action and specificity are because this structural domain of disappearance has been cancelled the ability (Smyth etc. (1996) EMBO J.15:4919-4927) of this receptor in conjunction with VEGF from VEGFR1ECD.Lack all the other structural domains and only reduce and the combining of VEGF, but do not cancel this combination.Yet independent structural domain 2 is not enough in conjunction with VEGF.Structural domain 1 and 2 or structural domain 2 and 3 do not show yet and combine with VEGF, or only show and combine with VEGF is faint.Only contain structural domain 1,2 among the VEGFR1 and partly have the avidity to VEGF identical in fact with total length VEGFR1 with 3 ECD.

(b)FGFR1-FGFR4

[0274] ECD of FGFR contains three Ig spline structure territories.For example, sequence for the FGFR2 described in the SEQ ID NO:236, the one Ig spline structure territory is corresponding to the 39-125 amino acids, the 2nd Ig spline structure territory corresponding to 154-247 amino acids and the 3rd Ig spline structure territory corresponding to the 256-358 amino acids.4 kinds of FGFR that existence is produced by alternative splicing.Each FGFR is activated by part subgroup (belonging to 19 kinds of relevant FGF parts) at least and the alternative splicing in Ig domain II I can significantly change some part specificity (Chellaiah etc. (1999) JBC, 274:34785-34794).Therefore, the main ligand-binding site point of FGF part generally is arranged in different Ig spline structure territories, be usually located at most in structural domain 2 and the structural domain 3 (Cheon etc. (1994) PNAS, 91:989-993).For example, 3 sudden changes of structural domain among the FGFR2 suppress and the combining of FGF2, and do not influence and the combining of FGF1 and FGF7.In addition, determined that with chimeric FGFR molecular studies FGF1 combines with structural domain 2 or structural domain 3; FGF2 preferentially discerns the far-end sequence of the FGFR1 that contains Ig structural domain 2 and 3; The sequence of FGF8 identification Ig structural domain 2 or FGFR3 aminoterminal and carboxyl terminal; And the FGF9 keying action depends on Ig structural domain 2 N-terminal sequences among the FGFR3 and this sequence and comprises Ig structural domain 2 among the FGFR3, and do not need structural domain 3 (Chellaiah etc. (1999) JBC, 274:34785-34794).For combining of FGF and FGFR, ligand binding affinity is optimized in the existence of heparin.

c)IGF-1R

[0275] example of RTK acceptor is IGF-1R.Insulin Receptor Family contains the homologous tyrosine kinase receptor, comprises insulin receptor (IR), type-1 insulin like growth factor acceptor (IFG1R) and insulin receptor associated receptor.IR all synthesizes as the wall scroll polypeptide chain with IGF-1R and is cut two different chains that are connected by disulfide linkage to produce by proteolytic enzyme, is called α and β.The α chain is the born of the same parents' outside part and the binding partner of this receptor, and the β chain has extracellular region, a single and strides tyrosine kinase domain in the born of the same parents of film sections and one mediation signal transduction behind binding partner.Born of the same parents' outside part of IGF-1R has 6 structurally different structural domains.Three structural domains and HER ectodomain I-III homology also are that L1 (corresponding to the 51-61 amino acids of SEQ ID NO:260), halfcystine enrich structural domain (corresponding to the 175-333 amino acids of SEQ ID NO:260) and L2 (corresponding to the 352-467 amino acids of SEQ IDNO:260).Minimum ligand binding moiety and mediation that these three structural domains form this receptor combine with the low-affinity of Regular Insulin.The carboxyl terminal of L2 structural domain is the outer fibronectin 3 pattern pieces of three born of the same parents, be the module (corresponding to the 489-587 amino acids of SEQ ID NO:260) in the α chain, a module (corresponding to the 611-703 amino acids of SEQ ID NO:260) and the 3rd module in the β chain (corresponding to the 831-926 amino acids of SEQ ID NO:260) in the alpha-beta link block.α is connected to form complete (α β) by disulfide linkage with β chain formation α β heterodimer and two heterodimers ₂Acceptor.With regard to the HER family receptors, need part in conjunction with to activate this receptor and to induce the transphosphorylation in cytoplasmic structure territory.The activation of IGF-1R relates to cell growth, conversion and apoptosis.

(d) RAGE and other CSR

[0276] also conceived other CSR ECD herein, comprised that those ECD (seeing co-pending U. S. application series number 11/429,090) from RAGE CSRs and the reference of wherein quoting described RAGE CSR and exemplary ECD and CSR isoform.Last table 7 has also been described the sequence of total length RAGE and its ECD part.

Polymeric component of D.ECD and the polymeric formation of ECD

[0277] the ECD heteromultimeric comprises at least 2 kinds of different ECD or its part that is used for part combination and/or dimerization.In the exemplary of this paper, at least a among the component ECD is the HER ECD that is used for part combination and/or dimerization, usually at least a ECD or its part among the HER1,3 or 4.Usually, among the ECD at least two kinds from HER, particularly from HER1 and HER3 or HER4.Other ECD comprises such ECD, and it is from other CSR, and RTKs normally particularly with tumour generation or vasculogenesis or the relevant any CSR of inflammatory diseases, and generally is the relevant any CSR of resistance with the medicine of target single cell surface receptor.The ECD polypeptide also can be the heterozygosis ECD molecule that contains from the structural domain of two or more CSR.ECD in heteromultimeric connects, thereby forms polymer, heterodimer at least.Conceived permission or caused ECD to interact to form any connection of heteromultimeric, thereby the multimeric molecule of gained with at one of related acceptor of this ECD or whole ligand interaction and/or with one of described related acceptor or other interaction acceptor or the two interaction, to suppress dimerization.This type of connection can be based on any stable connection of covalency and noncovalent interaction.

1.ECD polypeptide

[0278] the ECD polypeptide that is used for producing the ECD polymer that this paper provides can be for example ECD all or part of of any RTK of CSR, or it contains any part of ECD.Generally, unless described ECD is all or part of of HER2, otherwise the ECD of gained keeps the ability of its binding partner.In addition, the ECD that belongs to HER family (for example all or part of HER1, HER2, HER3 or HER4) generally also keeps the ability of itself and HER family receptors (comprising total length HER family receptors) dimerization.Therefore, when the polymer mating partner was HER ECD, then this HER ECD polypeptide portion enough at least parts of comprising subdomain I and subdomain III were used for dimerization with enough parts of binding partner and subdomain II.Normally, this HER ECD also contain subdomain IV module 1 to small part.The remainder of subdomain IV is chosen wantonly.

(a) the total length ECD of HER family

[0279] the ECD polypeptide that contains in HER polymer provided herein can be the total length ECD of HER polypeptide.For the HER polypeptide, described HER ECD contains that be enough to can binding partner and structural domain I, the II, III and the IV that are enough to mediate with related or relevant HER family receptors dimerization.HER ECD polypeptide is also included within allelic variant or species variant or other known variant in the ECD part of HER polypeptide, if gained HER ECD polypeptide keep its with the part combination and/or with the ability of related acceptor or relevant HER family receptors dimerization.

(i)HER1ECD

[0280] total length HER1ECD polypeptide can be used in the polymeric formation of ECD that this paper provides.This total length HER1ECD contains the 1-621 amino acids residue (HER1-621 of ripe HER1 acceptor; HF100).The nucleotides sequence of HF100 molecule is listed among the SEQ ID NO:11 to be described and coding has total length HER1ECD polypeptide at aminoacid sequence shown in the SEQ ID NO:12.Total length HER1ECD polypeptide comprises when comparing with exemplary HER1ECD polypeptide shown in the SEQ ID NO:12 having any total length HER1ECD polypeptide of one or more variations in aminoacid sequence.The example of the variation in the HER1 polypeptide be with precursor HER1 polypeptide as shown in SEQ ID NO:263 in the corresponding any variation of any allelic variant.Exemplary variation in HER1 total length ECD polypeptide comprise with SEQ ID NO:12 in R74Q, P242R, R497K or C604S in any one or a plurality of corresponding any one or a plurality of variation.

(ii)HER2ECD

[0281] ECD polymer provided herein also can contain such total length HER2ECD polypeptide, and it contains the 1-628 amino acids residue (HER2-650 of ripe HER2 acceptor; HF200).The nucleotides sequence of HF200 molecule is listed among the SEQ ID NO:17 to be described and coding has total length HER2ECD polypeptide at aminoacid sequence shown in the SEQID NO:18.Total length HER2ECD polypeptide comprises when comparing with exemplary HER2ECD polypeptide shown in the SEQ ID NO:18 having any total length HER2ECD polypeptide of one or more variations in aminoacid sequence.The example of the variation in the HER2 polypeptide be with precursor HER2 polypeptide shown in SEQ ID NO:264 in the corresponding any variation of any allelic variant.Exemplary variation in HER2 total length ECD polypeptide comprises corresponding any one or a plurality of variation with any one or a plurality of W430C shown in SEQ ID NO:18.

(iii)HER3ECD

[0282] in another example, total length HER3ECD polypeptide can be used in the polymeric formation of ECD that this paper provides.This HER3ECD polypeptide contains the 1-621 amino acids residue (HER3-621 of ripe HER3 acceptor; HF300).The nucleotides sequence of HF300 molecule is listed among the SEQID NO:25 to be described and coding has total length HER3ECD polypeptide at aminoacid sequence described in the SEQ ID NO:26.Total length HER3ECD polypeptide comprises when comparing with exemplary HER3ECD polypeptide described in the SEQ ID NO:26 having any total length HER3ECD polypeptide of one or more variations in aminoacid sequence.The example of the variation in the HER3 polypeptide be with precursor HER3 polypeptide shown in SEQ IDNO:265 in the corresponding any variation of any allelic variant.Exemplary variation in HER3 total length ECD polypeptide comprise with SEQ ID NO:26 in any one or a plurality of G541E corresponding any one or a plurality of variation.

(iv)HER4ECD

[0283] ECD polymer provided herein also can contain such total length HER4ECD polypeptide, and it contains the 1-625 amino acids residue (HER4-650 of ripe HER4 acceptor; HF400).The nucleotides sequence of HF400 molecule is listed among the SEQ ID NO:31 to be described and coding has total length HER4ECD polypeptide at aminoacid sequence described in the SEQID NO:32.Total length HER4ECD polypeptide comprises when comparing with exemplary HER4ECD polypeptide described in the SEQ ID NO:32 having any total length HER4ECD polypeptide of one or more variations in aminoacid sequence.The example of the variation in the HER4 polypeptide be with precursor HER4 polypeptide shown in SEQ ID NO:266 in the corresponding any variation of any allelic variant.Exemplary variation in HER4 total length ECD polypeptide comprises and aminoacid sequence shown in SEQ ID NO:32 corresponding any one or a plurality of amino acid variation.

(b) the brachymemma ECD of HER family

[0284] the ECD polypeptide that is contained in HER polymer provided herein can be the brachymemma ECD of HER polypeptide.For the HER polypeptide of brachymemma, enough parts that described HER ECD generally contains structural domain I and III with enough parts of binding partner and domain II with the mediation receptor dimerizationization.Usually, the HER ECD of brachymemma also contains at least a portion of module 1 of structural domain IV for example to stablize this molecule.Any remainder of structural domain IV is chosen wantonly.In addition, the ECD polypeptide of brachymemma also can comprise the additional sequences that is not this HER ECD part, as long as described additional sequences does not suppress or do not disturb the part keying action and/or the receptor dimerizationization of described HER ECD polypeptide.For example, the ECD polypeptide of brachymemma can comprise the polypeptide that produces by alternative splicing, for example, but is not limited to contain the polypeptide of intron amino acids coding.The HER ECD polypeptide of brachymemma is also included within allelic variant or species variant or other known variant in the ECD part of brachymemma HER polypeptide, if the brachymemma HER ECD polypeptide of gained keep its with the part combination and/or with the ability of related acceptor or relevant HER family receptors dimerization.

(i) HER1ECD of brachymemma

[0285] in an example, can be used in the 1-501 amino acids residue (HER1-501 that brachymemma HER1ECD polypeptide in the ECD polymer provided herein contains ripe HER1 acceptor; HF110).The nucleotides sequence of HF110 molecule is listed among the SEQ ID NO:9 to be described and coding has brachymemma HER1ECD polypeptide at aminoacid sequence described in the SEQ ID NO:10.HF110 contains module 1 whole of the whole and structural domain IV of structural domain I, the II of relevant HER1ECD and III.

[0286] also having conceived what be used for the ECD polymer is the brachymemma HER1ECD polypeptide that produces because of alternative splicing.Any isoform known in the art or that describe or provide as intron fusion proteins below this paper in related U.S. patent publication number US 2005-0239088 is provided this type of isoform.A kind of so exemplary brachymemma HER1ECD polypeptide is by EGFR isoform b (NP_958439 nucleotide sequence coded described in the SEQ ID NO:128; SEQ ID NO:129).The HER1ECD polypeptide of this brachymemma has 628 amino acid (comprising the signal peptide corresponding to 1-24 amino acids residue) and contains a non-existent additional amino acid in related HER1ECD at its carboxyl terminal.The mature form of the HER1ECD polypeptide of the precursor brachymemma of describing in SEQ ID NO:129 (not comprising signal sequence) has 604 amino acid lengths, as describing among Fig. 2 (A), and contain the almost whole of structural domain I, the II of a kind of related HER1ECD and III and structural domain IV, and to and comprise the module 7 of the overwhelming majority.In other example, the HER1ECD polypeptide of brachymemma can comprise by EGFR isoform d (NP_958441 nucleotide sequence coded described in the SEQ ID NO:130; SEQ ID NO:131).The HER1ECD polypeptide of this brachymemma has 705 amino acid (comprising the signal peptide corresponding to 1-24 amino acids residue) and contains 76 non-existent additional amino acids in related HER1ECD at its carboxyl terminal.The mature form of the HER1ECD polypeptide of the precursor brachymemma of describing in SEQ ID NO:131 (not comprising signal sequence) has 681 amino acid lengths, as describing among Fig. 2 (A), and the major part that contains structural domain I, II and III and the structural domain IV of relevant HER1ECD comprises and to the major part of module 7 and module 7.

[0287] the HER1ECD polypeptide of brachymemma comprises, when for example the exemplary brachymemma HER1ECD polypeptide described in the SEQ ID NO:10,129 or 131 is compared, has the HER1ECD polypeptide of any brachymemma of one or more variations in aminoacid sequence.The example of the variation in the HER1 polypeptide be with precursor HER1 polypeptide shown in SEQ ID NO:263 in the corresponding any variation of any allelic variant.Exemplary variation in the HER1ECD of brachymemma polypeptide comprise with SEQ IDNO:10 in R74Q, P242R or R497K in any one or a plurality of corresponding any one or a plurality of variation.Exemplary variation also can comprise and have in the brachymemma HER1 polypeptide of aminoacid sequence shown in SEQ ID NO:129 or 131 R98Q, P266R, R521K, C628S or V674I corresponding any one or a plurality of amino acid variation.

The (ii) HER2ECD of brachymemma

[0288] the ECD polymer also can contain the HER2ECD polypeptide of brachymemma.For example, the brachymemma HER2ECD polypeptide (HER2-595 that contains the 1-573 amino acids residue of ripe HER2 acceptor; HF210) can in the polymeric formation of ECD, use.The nucleotides sequence of HF210 molecule is listed among the SEQ ID NO:15 to be described and coding has brachymemma HER2ECD polypeptide at aminoacid sequence described in the SEQ ID NO:16.HF210 comprises the whole of structural domain I, the II of related HER2ECD and III, and and to and comprise the part of the module 5 of structural domain IV.The brachymemma HER2ECD polypeptide (HER2-530 of the 1-508 amino acids residue that contains ripe Her2 acceptor also is provided herein; HF220) as the multimerization mating partner.The nucleotides sequence of HF220 is listed among the SEQID NO:13 to be described and coding has brachymemma HER2ECD polypeptide at aminoacid sequence described in the SEQ ID NO:14.HF220 comprises the whole of structural domain I, the II of related HER2 acceptor and III, and and to and comprise module 1 whole of structural domain IV.

[0289] also having conceived what be used for the ECD polymer is the brachymemma HER2ECD polypeptide that produces because of alternative splicing.Any isoform known in the art or that describe or provide as intron fusion proteins below this paper in related U.S. patent publication number US 2005-0239088 is provided this type of isoform.A kind of exemplary brachymemma HER2ECD polypeptide like this is the ErbB2.1e with aminoacid sequence described in the SEQ ID NO:137.The HER2ECD polypeptide of this brachymemma has 633 amino acid, comprises the signal peptide corresponding to 1-22 amino acids residue.The mature form of the HER2ECD polypeptide of the precursor brachymemma of describing in SEQ ID NO:137 (not comprising signal sequence) has 611 amino acid lengths, as describing among Fig. 2 (B), and contain the almost whole of structural domain I, the II of relevant HER2ECD and III and structural domain IV, and to and comprise the major part of module 7.In extra example, the HER2ECD polypeptide of brachymemma is the ErbB2.1d with aminoacid sequence described in the SEQ ID NO:136.The HER2ECD polypeptide of this brachymemma has 680 amino acid, comprises the signal peptide corresponding to 1-24 amino acids residue, and wherein this signal peptide is compared with the signal peptide among the related HER2 shown in the SEQ ID NO:4, contains two aminoacid insertion things.ErbB2.1d also contains 30 non-existent additional amino acids in related HER1ECD at its carboxyl terminal.The mature form of the HER2ECD polypeptide of the precursor brachymemma of describing in SEQ IDNO:136 (not comprising signal sequence) has 656 amino acid lengths, as describing among Fig. 2 (B), and structural domain I, the II and the III that contain relevant HER2ECD, and the major part of structural domain IV, comprise that module 1-7's is whole.

[0290] the HER2ECD polypeptide of brachymemma comprises, when for example SEQ ID NO:14,16,136 compares with the exemplary brachymemma HER2ECD polypeptide described in 137, has the HER2ECD polypeptide of any brachymemma of one or more variations in aminoacid sequence.The example of the variation in the HER2 polypeptide be with precursor HER2 polypeptide shown in SEQ ID NO:264 in the corresponding any variation of any allelic variant.Exemplary variation in the HER2ECD of brachymemma polypeptide comprise with SEQID NO:14 or 16 in W430C corresponding any one or a plurality of variation.Exemplary variation also can comprise and have respectively described in SEQ ID NO:137 or 136 corresponding any one of W452C in the brachymemma HER2 polypeptide of aminoacid sequence or W454C or a plurality of amino acid variation.

The (iii) HER3ECD of brachymemma

[0291] the ECD polymer also can contain such brachymemma HER3ECD polypeptide (HER3-500; HF310), it contains the 1-500 amino acids residue of ripe HER3 acceptor.The nucleotides sequence of HF310 molecule is listed in to describe among the SEQ ID NO:19 and encode has the polypeptide at the brachymemma HER3ECD of aminoacid sequence described in the SEQID NO:20.HF310 comprises the whole of structural domain I, the II of related HER3ECD and III, and and to and comprise the part of the module 1 of structural domain IV.In another example, the ECD polymer can contain such brachymemma HER3ECD polypeptide (HER3-519), and it contains the 1-519 amino acids residue (HER3-519) of ripe HER3 acceptor.The nucleotides sequence of HER3-519 is listed among the SEQ ID NO:23 to be described and coding has brachymemma HER3ECD polypeptide at aminoacid sequence described in the SEQ ID NO:24.HER3-519 comprises the whole of structural domain I, the II of related HER3ECD and III, and and to and comprise the part of the module 3 of structural domain IV.

[0292] also having conceived what be used for the ECD polymer is the brachymemma HER3ECD polypeptide that produces because of alternative splicing.Any isoform known in the art or that describe or provide as intron fusion proteins below this paper in related U.S. patent publication number US 2005-0239088 is provided this type of isoform.A kind of so exemplary brachymemma HER3ECD polypeptide is to describe in SEQ ID NO:22 and by nucleotide sequence coded p85HER3 described in the SEQ ID NO:21.The HER3ECD polypeptide of this brachymemma has 562 amino acid, comprises the signal peptide corresponding to 1-19 amino acids residue, and contains 24 non-existent additional amino acids in related HER3ECD at its carboxyl terminal.The mature form of the HER3ECD polypeptide of the precursor brachymemma of describing in SEQ ID NO:22 (not comprising signal sequence) is 543 amino acid lengths, as describing among Fig. 2 (C), and contain structural domain I, the II of relevant HER3ECD and III and and to and comprise the part of the module 3 of structural domain IV.

[0293] the HER3ECD polypeptide of brachymemma comprises, when for example SEQ ID NO:14,16,136 compares with the exemplary brachymemma HER3ECD polypeptide described in 137, has the HER3ECD polypeptide of any brachymemma of one or more variations in aminoacid sequence.The example of the variation in the HER3 polypeptide be with precursor HER3 polypeptide shown in SEQ ID NO:265 in the corresponding any variation of any allelic variant.

The (iv) HER4ECD of brachymemma

[0294] in addition, can form the ECD polymer of the HER4ECD that contains brachymemma.A kind of exemplary brachymemma HER4ECD polypeptide contains the 1-522 amino acids residue (HER4-522) of ripe HER4 acceptor.The nucleotides sequence of HER4-522 molecule is listed among the SEQ ID NO:29 to be described and coding has brachymemma HER4ECD polypeptide at aminoacid sequence described in the SEQ ID NO:30.HER4-522 comprises the whole of structural domain I, the II of related HER3ECD and III, and comprise and to and comprise the module 1 of structural domain IV.The HER4ECD polypeptide of another kind of exemplary brachymemma contains the 1-460 amino acids residue (HF410 of ripe HER4 acceptor; HER4-485).The nucleotides sequence of HF410 is listed among the SEQ ID NO:27 to be described and coding has brachymemma HER4ECD polypeptide at aminoacid sequence described in the SEQ ID NO:28.HF410 comprises the major part of whole and domain II I of structural domain I, the II of related HER4ECD.

[0295] also having conceived what be used for the ECD polymer is the brachymemma HER4ECD polypeptide that produces because of alternative splicing.Any isoform known in the art or that describe or provide as intron fusion proteins below this paper in related U.S. patent publication number US 2005-0239088 is provided this type of isoform.A kind of so exemplary brachymemma HER4ECD polypeptide is to describe in SEQ ID NO:159 and by nucleotide sequence coded ErbB4_int12 described in the SEQ ID NO:158.The HER4ECD polypeptide of this brachymemma is 506 amino acid, comprises the signal peptide corresponding to 1-25 amino acids residue, and contains 10 non-existent additional amino acids in related HER4ECD at its carboxyl terminal.Described additional amino acid is by the part coding that keeps as the HER4 gene intron 12 of alternative splicing product.The mature form of the HER4ECD polypeptide of the precursor brachymemma of describing in SEQ ID NO:159 (not comprising signal sequence) is 481 amino acid lengths, as describing among Fig. 2 (D), and the structural domain I, the II that contain relevant HER4ECD, and the major part of domain II I.

[0296] the HER4ECD polypeptide of brachymemma comprises, when for example SEQ ID NO:28,30 compares with the exemplary brachymemma HER4ECD polypeptide described in 159, has the HER4ECD polypeptide of any brachymemma of one or more variations in aminoacid sequence.The example of the variation in the HER3 polypeptide be with precursor HER4 polypeptide described in SEQ ID NO:266 in the corresponding any variation of any allelic variant.

(c) heterozygosis ECD

[0297] provides heterozygosis ECD or its part that contains from the subdomain of two or more HER acceptors herein.Normally, heterozygosis ECD contains the whole of the structural domain I of one or more HER acceptors or III or enough parts combining with part, with the whole of domain II or enough parts to mediate from receptor dimerizationization identical or another HER ECD.Therefore, heterozygosis ECD molecule can contain the part of whole HER ECD of family, normally at least a portion of the part of three ECD of HER family and two ECD of HER family.Usually, this type of ECD comprises from the subdomain II of HER2 and comprises such subdomain I and III that it can be from identical or different acceptor, from ErbB1,3 or 4.So select each subdomain part, thus the ECD dimerization of gained and combine with at least a part, and can with two or more (different) part combinations.Therefore, so choice structure territory combination, thus it combines with at least a part, and can combine with two kinds of parts, and this structural domain combination comprises that also enough parts of subdomain II are used for dimerization.The example of this type of heterozygote is such monomer heterozygosis ECD, and it contains subdomain I from HER3 or HER4, from the subdomain II of HER2 with from the subdomain III of HER1.For example, provide such heterozygosis ECD, it contains subdomain I from ErbB3, from the subdomain II of ErbB2 with from the subdomain III of ErbB1.HRG be bonded to HER3 or HER4 (subdomain I) and EGF will be main with the subdomain III interaction of HER1 (see, for example, Singer etc. (2001) J.Biol.Chem.276:44266-44274; Kim etc. (2002) Eur.J.Biochem.269:2323-2329). therefore, described heterozygote combines (see, for example, Singer etc. (2001) J.Biol.Chem.276:44266-44274) with at least two kinds of parts.In addition, after adding the multimerization structural domain and forming chimeric polymer, the chimeric molecule of gained can different HER acceptors with at least two kinds and at least 2 kinds of different ligands interactions.

(d) other CSR or RTK ECD or its part

[0298] other ECD polypeptide comprises being used in any ECD part or its fragment of the CSR that is enough to binding partner or other RTK in the polymeric formation of ECD that this paper provides.Generally, this type of CSR ECD or its part are the ECD of the chemical sproof CSR of the ECD of the etiologic etiological any CSR of involved in diseases and/or the medicine that participates in target single cell surface receptor.Describe exemplary CSR or RTK acceptor in table 7, this table points out that also each divides other acceptor ECD part separately.Therefore, conceived any total length ECD described in table 7 as herein multimerization mating partner.Also conceived in the part of the total length ECD of any CSR described in the table 7 or fragment as the multimerization mating partner, if described part or fragment keep its binding partner and/or with the ability of related receptor dimerizationization.For example, the part of VEGFR ECD (as VEGFR1) or fragment contain enough parts of Ig-structural domain 1,2 and 3 at least to combine with part.In another example, the part of FGFR ECD (as arbitrary FGFR1-4) or fragment contain Ig-structural domain 2 and 3 at least enough parts with binding partner.In additional examples, the part of IGF-1R ECD or fragment contain the L1 structural domain at least, halfcystine enriches enough parts of structural domain and L2 structural domain to combine and/or to mediate receptor dimerizationization with part.

(c) the polypeptide isoform of alternative splicing

[0299] other ECD polypeptide that is used in the polymeric formation of ECD that this paper provides comprises any isoform, and it contains ECD part or the fragment of CSR and randomly contains the unmatched additional amino acid of structural domain sequence with related acceptor.This type of ECD polypeptide comprises CSR or other RTKs of for example alternative splicing.Generally, contain ECD polypeptide isoform binding partner and/or with the cell surface receptor dimerization.The isoform of alternative splicing comprises the isoform that for example is detained those alternative splicings that produce because of exon extension, exon insertion, Exon deletion, exon brachymemma or intron.The isoform of this type of alternative splicing is known in the artly (for example to see Application No. 6,414,130; Laid-open U.S. Patents application number US2005/0239088, US2004/0022785A1, US20050123538; Disclosed international patent application no WO0044403, WO0161356 and WO0214470) and describe in each at SEQ ID NO:22,129,131,133,135,136,137,138,139,143,144,149,150,151,301-399 and 408-413.For example, the isoform of alternative splicing comprises the HER1 isoform, includes but not limited to the IDNO:129 at SEQ, any HER1 isoform of describing in 131 or 133; The HER2 isoform includes but not limited to that the Hess of describing reaches spit of fland or its variant in SEQ ID NO:135 or any item of 385-399; Or the isoform of other alternative splicing, include but not limited to isoform in any alternative splicing described in SEQ ID NO:136-139 or the 408-413; The HER3 isoform includes but not limited at any HER3 isoform described in the SEQ IDNO:22,143,144,149,150 or 151).

[0300] isoform of alternative splicing also can comprise other isoform of HER1 gene.HER1 gene (SEQ ID NO:400) is made up of 27 28 exons that intron cut off.Be provided as in this article in the exemplary genome sequence of HER1 of SEQ ID NO:400, exons 1 comprises 1-254 position Nucleotide, comprise 5 '-non-translational region.Initiator codon begins at the 167th Nucleotide place.Introne 1 comprises 255-614 position Nucleotide; Exon 2 comprises 615-766 position Nucleotide; Intron 2 comprises 767-1126 position Nucleotide; Exon 3 comprises 1127-1310 position Nucleotide; Introne 3 comprises 1311-1670 position Nucleotide; Exon 4 comprises 1671-1805 position Nucleotide; Intron 4 comprises 1806-2165 position Nucleotide; Exon 5 comprises 2166-2234 position Nucleotide; Intron 5 comprises 2235-2594 position Nucleotide; Exon 6 comprises 2595-2713 position Nucleotide; Intron 6 comprises 2714-3073 position Nucleotide; Exon 7 comprises 3074-3215 position Nucleotide; Intron 7 comprises 3216-3575 position Nucleotide; Exon 8 comprises 3576-3692 position Nucleotide; Intron 8 comprises 3693-4052 position Nucleotide; Exon 9 comprises 4043-4179 position Nucleotide; Intron 9 comprises 4180-4539 position Nucleotide; Exons 10 comprises 4540-4613 position Nucleotide; Introne 10 comprises 4614-4973 position Nucleotide; Exons 11 comprises 4974-5063 position Nucleotide; Introne 11 comprises 5064-5423 position Nucleotide; Exons 12 comprises 5424-5623 position Nucleotide; Introne 12 comprises 5624-5983 position Nucleotide; Exons 13 comprises 5984-6116 position Nucleotide; Introne 13 comprises 6117-6476 position Nucleotide; Exons 14 comprises 6477-6567 position Nucleotide; Introne 14 comprises 6568-6927 position Nucleotide; Exons 15 comprises 6928-7085 position Nucleotide; Introne 15 comprises 7086-7445 position Nucleotide; Exons 16 comprises 7446-7484 position Nucleotide; Introne 16 comprises 7485-7844 position Nucleotide; Exons 17 comprises 7845-7988 position Nucleotide; Introne 17 comprises 7987-8346 position Nucleotide; Exons 18 comprises 8347-8469 position Nucleotide; Introne 18 comprises 8470-8829 position Nucleotide; Exons 19 comprises 8830-8295 position Nucleotide; Introne 19 comprises 8929-9288 position Nucleotide; Extron 20 comprises 9289-9474 position Nucleotide; Intron 20 comprises 9475-9834 position Nucleotide; Exon 21 comprises 9835-9990 position Nucleotide; Intron 21 comprises 9991-10350 position Nucleotide; Exon 22 comprises 10351-10426 position Nucleotide; Intron 22 comprises 10427-10786 position Nucleotide; Exon 23 comprises 10787-10933 position Nucleotide; Intron 23 comprises 10934-11293 position Nucleotide; Exon 24 comprises 11294-11391 position Nucleotide; Intron 24 comprises 11392-11751 position Nucleotide; Exon 25 comprises 11752-11919 position Nucleotide; Intron 26 comprises 11920-12279 position Nucleotide; Exon 26 comprises 12280-12327 position Nucleotide; Intron 26 comprises 12328-12687 position Nucleotide; Exon 27 comprises 12688-12796 position Nucleotide; Intron 27 comprises 12797-13156 position Nucleotide; And exon 28 comprises 13157-15233 position Nucleotide.Terminator codon in the exon 28 begins at the 13516th nucleotide position place and the remainder of exon 28 comprises 3 '-non-translational region.After the RNA montage and removing described intron, the primary transcript of HER1 contains exons 1-28 and encodes 1210 amino acid whose polypeptide (SEQID NO:2).The isoform of the alternative splicing of HER1 gene is described in embodiment 10 and is set forth, and comprises the isoform of the intron sequences with reservation.The sequence of this exemplary HER1 isoform is described in SEQ ID NO:126, and coding has the polypeptide of aminoacid sequence described in the SEQ ID NO:127.

[0301] isoform of alternative splicing also can comprise other isoform of HER2 gene.HER2 gene (SEQ ID NO:401) is made up of 26 27 exons that intron cut off.Be provided as in this article in the exemplary genome sequence of HER of SEQ ID NO:401, exons 1 comprises 181-349 position Nucleotide, comprise 5 '-non-translational region.Initiator codon begins at the 277th Nucleotide place.Introne 1 comprises 350-709 position Nucleotide; Exon 2 comprises 710-861 position Nucleotide; Intron 2 comprises 862-1221 position Nucleotide; Exon 3 comprises 1222-1435; Introne 3 comprises 1436-1795 position Nucleotide; Exon 4 comprises 1796-1930 position Nucleotide; Intron 4 comprises 1931-2290 position Nucleotide; Exon 5 comprises 2291-2359 position Nucleotide; Intron 5 comprises 2360-2719 position Nucleotide; Exon 6 comprises 2720-2835 position Nucleotide; Intron 6 comprises 2836-3195 position Nucleotide; Exon 7 comprises 3196-3337 position Nucleotide; Intron 7 comprises 3338-3697 position Nucleotide; Exon 8 comprises 3698-3817 position Nucleotide; Intron 8 comprises 3818-4177 position Nucleotide; Exon 9 comprises 4178-4304 position Nucleotide; Intron 9 comprises 4305-4664 position Nucleotide; Exons 10 comprises 4665-4738 position Nucleotide; Introne 10 comprises 4739-5098 position Nucleotide; Exons 11 comprises 5099-5189 position Nucleotide; Introne 11 comprises 5190-5549 position Nucleotide; Exons 12 comprises 5550-5749 position Nucleotide; Introne 12 comprises 5750-6109 position Nucleotide; Exons 13 comprises 6110-6242 position Nucleotide; Introne 13 comprises 6243-6602 position Nucleotide; Exons 14 comprises 6603-6696 position Nucleotide; Introne 14 comprises 6694-7053 position Nucleotide; Exons 15 comprises 7054-7214 position Nucleotide; Introne 15 comprises 7215-7574 position Nucleotide; Exons 16 comprises 7575-7622 position Nucleotide; Introne 16 comprises 7623-7982 position Nucleotide; Exons 17 comprises 7983-8121 position Nucleotide; Introne 17 comprises 8122-8481 position Nucleotide; Exons 18 comprises 8482-8604 position Nucleotide; Introne 18 comprises 8605-8964 position Nucleotide; Exons 19 comprises 8695-9067 position Nucleotide; Introne 19 comprises 9068-9427 position Nucleotide; Extron 20 comprises 9428-9610 position Nucleotide; Intron 20 comprises 9611-9970 position Nucleotide; Exon 21 comprises 9971-10126 position Nucleotide; Intron 21 comprises 10127-10486 position Nucleotide; Exon 22 comprises 10487-10562 position Nucleotide; Intron 22 comprises 10563-10922 position Nucleotide; Exon 23 comprises 10923-11069 position Nucleotide; Intron 23 comprises 11070-11429 position Nucleotide; Exon 24 comprises 11430-11527 position Nucleotide; Intron 24 comprises 11528-11887 position Nucleotide; Exon 25 comprises 11888-12076 position Nucleotide; Intron 26 comprises 12077-12436 position Nucleotide; Exon 26 comprises 12437-12689 position Nucleotide; Intron 26 comprises that 12690-13049 position Nucleotide and exon 27 comprise 13050-14018 position Nucleotide.Terminator codon in the exon 27 begins at the 13403rd nucleotide position place and the remainder of exon 27 comprises 3 '-non-translational region.After the RNA montage and removing described intron, the primary transcript of HER2 contains exons 1-27 and 1255 the amino acid whose polypeptide of encoding (SEQ ID NO:4).The isoform of the alternative splicing of HER2 gene is described in embodiment 10 and is set forth, and comprises having those isoforms that keep intron sequences.The sequence of so exemplary HER2 isoform is described in SEQ ID NO:140, and coding has the polypeptide of aminoacid sequence described in the SEQ ID NO:141.

[0302] isoform of alternative splicing also can comprise other isoform of HER3 gene.HER3 gene (SEQ ID NO:402) is made up of 27 28 exons that intron cut off.Be provided as in this article in the exemplary genome sequence of HER3 of SEQ ID NO:402, exons 1 comprises 181-460 position Nucleotide, comprise 5 '-non-translational region.Initiator codon begins at the 379th Nucleotide place.Introne 1 comprises 461-820 position Nucleotide; Exon 2 comprises 821-972 position Nucleotide; Intron 2 comprises 973-1332 position Nucleotide; Exon 3 comprises 1333-1519 position Nucleotide; Introne 3 comprises 1520-1879 position Nucleotide; Exon 4 comprises 1880-2005 position Nucleotide; Intron 4 comprises 2006-2365 position Nucleotide; Exon 5 comprises 2366-2431 position Nucleotide; Intron 5 comprises 2432-2791 position Nucleotide; Exon 6 comprises 2792-2910 position Nucleotide; Intron 6 comprises 2911-3270 position Nucleotide; Exon 7 comprises 3237-3412 position Nucleotide; Intron 7 comprises 3413-3772 position Nucleotide; Exon 8 comprises 3773-3886 position Nucleotide; Intron 8 comprises 3887-4246 position Nucleotide; Exon 9 comprises 4247-4367 position Nucleotide; Intron 9 comprises 4368-4727 position Nucleotide; Exons 10 comprises 4728-4801 position Nucleotide; Introne 10 comprises 4802-5161 position Nucleotide; Exons 11 comprises 5162-5252 position Nucleotide; Introne 11 comprises 5253-5612 position Nucleotide; Exons 12 comprises 5613-5818 position Nucleotide; Introne 12 comprises 5819-6178 position Nucleotide; Exons 13 comprises 6179-6311 position Nucleotide; Introne 13 comprises 6312-6671 position Nucleotide; Exons 14 comprises 6672-6762 position Nucleotide; Introne 14 comprises 6763-7122 position Nucleotide; Exons 15 comprises 7123-7277 position Nucleotide; Introne 15 comprises 7278-7637 position Nucleotide; Exons 16 comprises 7638-7691 position Nucleotide; Introne 16 comprises 7692-8051 position Nucleotide; Exons 17 comprises 8052-8193 position Nucleotide; Introne 17 comprises 8194-8553 position Nucleotide; Exons 18 comprises 8554-8673 position Nucleotide; Introne 18 comprises 8674-9033 position Nucleotide; Exons 19 comprises 9034-9132 position Nucleotide; Introne 19 comprises 9133-9492 position Nucleotide; Extron 20 comprises 9493-9678 position Nucleotide; Intron 20 comprises 9679-10038 position Nucleotide; Exon 21 comprises 10039-10194 position Nucleotide; Intron 21 comprises 10195-10554 position Nucleotide; Exon 22 comprises 10555-10630 position Nucleotide; Intron 22 comprises 10631-10990 position Nucleotide; Exon 23 comprises 10991-11137 position Nucleotide; Intron 23 comprises 11138-11497 position Nucleotide; Exon 24 comprises 11498-11595 position Nucleotide; Intron 24 comprises 11596-11955 position Nucleotide; Exon 25 comprises 11956-12147 position Nucleotide; Intron 26 comprises 12148-12507 position Nucleotide; Exon 26 comprises 12508-12579 position Nucleotide; Intron 26 comprises 12580-12939 position Nucleotide; Exon 27 comprises 12940-13240 position Nucleotide; Intron 27 comprises that 13241-13600 position Nucleotide and exon 28 comprise 13601-14875 position Nucleotide.Terminator codon in the exon 28 begins at the 14125th nucleotide position place and the remainder of exon 28 comprises 3 '-non-translational region.After the RNA montage and removing described intron, the primary transcript of ErbB3 contains exons 1-28 and encodes 1342 amino acid whose polypeptide (SEQID NO:6).The isoform of the alternative splicing of HER3 gene is described in embodiment 10 and is set forth, and comprises having those isoforms that keep intron sequences.The sequence of exemplary HER3 isoform is like this described in SEQ ID NO:145 and 147, and coding has the polypeptide of aminoacid sequence described in SEQ ID NO:146 and 148 respectively.

[0303] isoform of alternative splicing also can comprise other isoform of HER4 gene.HER4 gene (SEQ ID NO:403) is made up of 27 28 exons that intron cut off.Be provided as in this article in the exemplary genome sequence of HER4 of SEQ ID NO:403, exons 1 comprises 181-295 position Nucleotide, comprise 5 '-non-translational region.Initiator codon begins at the 215th Nucleotide place.Introne 1 comprises 296-655 position Nucleotide; Exon 2 comprises 656-807 position Nucleotide; Intron 2 comprises 808-1167 position Nucleotide; Exon 3 comprises 1168-1354 position Nucleotide; Introne 3 comprises 1355-1714 position Nucleotide; Exon 4 comprises 1715-1849 position Nucleotide; Intron 4 comprises 1850-2209 position Nucleotide; Exon 5 comprises 2210-2275 position Nucleotide; Intron 5 comprises 2276-2635 position Nucleotide; Exon 6 comprises 2636-2754 position Nucleotide; Intron 6 comprises 2755-3114 position Nucleotide; Exon 7 comprises 3115-3256 position Nucleotide; Intron 7 comprises 3257-3616 position Nucleotide; Exon 8 comprises 3617-3730 position Nucleotide; Intron 8 comprises 3731-4090 position Nucleotide; Exon 9 comprises 4091-4217 position Nucleotide; Intron 9 comprises 4218-4577 position Nucleotide; Exons 10 comprises 4578-4651 position Nucleotide; Introne 10 comprises 4652-5011 position Nucleotide; Exons 11 comprises 5012-5102 position Nucleotide; Introne 11 comprises 5103-5462 position Nucleotide; Exons 12 comprises 5463-5662 position Nucleotide; Introne 12 comprises 5663-6022 position Nucleotide; Exons 13 comprises 6023-6155 position Nucleotide; Introne 13 comprises 6156-6515 position Nucleotide; Exons 14 comprises 6516-6609 position Nucleotide; Introne 14 comprises 6610-6969 position Nucleotide; Exons 15 comprises 6970-7124 position Nucleotide; Introne 15 comprises 7125-7484 position Nucleotide; Exons 16 comprises 7485-7559 position Nucleotide; Introne 16 comprises 7560-7919 position Nucleotide; Exons 17 comprises 7920-8052 position Nucleotide; Introne 17 comprises 8053-8412 position Nucleotide; Exons 18 comprises 8413-8535 position Nucleotide; Introne 18 comprises 8536-8895 position Nucleotide; Exons 19 comprises 8896-8994 position Nucleotide; Introne 19 comprises 8995-9354 position Nucleotide; Extron 20 comprises 9355-9540 position Nucleotide; Intron 20 comprises 9541-9900 position Nucleotide; Exon 21 comprises 9901-10056 position Nucleotide; Intron 21 comprises 10057-10416 position Nucleotide; Exon 22 comprises 10417-10492 position Nucleotide; Intron 22 comprises 10493-10852 position Nucleotide; Exon 23 comprises 10853-10999 position Nucleotide; Intron 23 comprises 11000-11359 position Nucleotide; Exon 24 comprises 11360-11457 position Nucleotide; Intron 24 comprises 11458-11817 position Nucleotide; Exon 25 comprises 11818-11988 position Nucleotide; Intron 26 comprises 11989-12348 position Nucleotide; Exon 26 comprises 12349-12396 position Nucleotide; Intron 26 comprises 12397-12756 position Nucleotide; Exon 27 comprises 12757-13054 position Nucleotide; Intron 27 comprises that 13055-13414 position Nucleotide and exon 28 comprise 13415-15385 position Nucleotide.Terminator codon in the exon 28 begins at the 13858th nucleotide position place and the remainder of exon 28 comprises 3 '-non-translational region.After the RNA montage and removing described intron, the primary transcript of HER4 contains exons 1-28 and encodes 1308 amino acid whose polypeptide (SEQID NO:8).The isoform of the alternative splicing of HER4 gene is described in embodiment 10 and is set forth, and comprises having those isoforms that keep intron sequences.The sequence of exemplary HER4 isoform is like this described in SEQ ID NO:152,154,156 or 158, and coding has the polypeptide of aminoacid sequence described in the SEQ ID NO:153,155,157 or 159 respectively.

[0304] isoform of alternative splicing also can comprise the isoform of IGF-1R gene.IGF1-R gene (SEQ ID NO:404) is made up of 20 21 exons that intron cut off.Be provided as in this article in the exemplary genome sequence of IGF1-R of SEQ ID NO:404, exons 1 comprises 181-306 position Nucleotide, comprise 5 '-non-translational region.Initiator codon begins at the 213rd Nucleotide place.Introne 1 comprises 307-666 position Nucleotide; Exon 2 comprises 667-1212 position Nucleotide; Intron 2 comprises 1213-1572 position Nucleotide; Exon 3 comprises 1573-1884 position Nucleotide; Introne 3 comprises 1885-2255 position Nucleotide; Exon 4 comprises 2256-2394 position Nucleotide; Intron 4 comprises 2395-2754 position Nucleotide; Exon 5 comprises 2755-2899 position Nucleotide; Intron 5 comprises 2990-3259 position Nucleotide; Exon 6 comprises 3260-3474 position Nucleotide; Intron 6 comprises 3475-3834 position Nucleotide; Exon 7 comprises 3835-3961 position Nucleotide; Intron 7 comprises 3962-4321 position Nucleotide; Exon 8 comprises 4322-4560 position Nucleotide; Intron 8 comprises 4561-4920 position Nucleotide; Exon 9 comprises 4921-5088 position Nucleotide; Intron 9 comprises 5089-5448 position Nucleotide; Exons 10 comprises 5449-5653 position Nucleotide; Introne 10 comprises 5654-6013 position Nucleotide; Exons 11 comprises 6014-6297 position Nucleotide; Introne 11 comprises 6298-6657 position Nucleotide; Exons 12 comprises 6658-6794 position Nucleotide; Introne 12 comprises 6795-7154 position Nucleotide; Exons 13 comprises 7155-7314 position Nucleotide; Introne 13 comprises 7315-7674 position Nucleotide; Exons 14 comprises 7675-7777 position Nucleotide; Introne 14 comprises 7778-8137 position Nucleotide; Exons 15 comprises 8138-8208 position Nucleotide; Introne 15 comprises 8209-8568 position Nucleotide; Exons 16 comprises 8569-8798 position Nucleotide; Introne 16 comprises 8799-9158 position Nucleotide; Exons 17 comprises 9159-9269 position Nucleotide; Introne 17 comprises 9270-9629 position Nucleotide; Exons 18 comprises 9630-9789 position Nucleotide; Introne 18 comprises 9790-10149 position Nucleotide; Exons 19 comprises 10150-10279 position Nucleotide; Introne 19 comprises 10280-10639 position Nucleotide; Extron 20 comprises 10640-10774 position Nucleotide; Intron 20 comprises that 10775-11134 position Nucleotide and exon 21 comprise 11135-12356 position Nucleotide.Terminator codon in the exon 21 begins at the 11514th nucleotide position place and the remainder of exon 21 comprises 3 '-non-translational region.After the RNA montage and removing described intron, the primary transcript of IGF1-R contains exons 1-21 and 1367 the amino acid whose polypeptide of encoding (SEQ ID NO:290).The isoform of the alternative splicing of IGF1-R gene is described in embodiment 11 and is set forth, and comprises having those isoforms that keep intron sequences.The sequence of exemplary IGF1-R isoform is like this described in SEQ ID NO:297 or 299, and coding has the polypeptide of aminoacid sequence described in SEQ ID NO:298 or 300 respectively.

[0305] provides in this article and the isoform of the alternative splicing of HER1, the HER2, HER3, HER4 and the IGF1-R that describe in SEQ ID NO:127,141,146,148,153,155,157,159,298 or 300 can be used in the polymeric formation of ECD that this paper provides.Alternatively, described isoform can use with the related receptor-mediated any disease of treatment by them individually or with any other isoform in combination.The example of this type of disease is that any vasculogenesis disease, tumour generate disease or inflammatory diseases, and especially cancer is that those skilled in the art are known also as described herein.

2.ECD polymeric formation

[0306] ECD polymer comprises HER ECD polymer, can be the polymer of the acceptor ECD of covalently bound, non-covalent connection or chemistry connection, to form dimer, tripolymer or bigger polymer.In some cases, polymer can form by two or more ECD polypeptide dimerizations.Multimerization between two ECD polypeptide can be spontaneous, or can take place because of the pressure keyed jointing of two or more polypeptide.In an example, polymer can be connected by formed disulfide linkage between the cysteine residues on the different ECD polypeptide.Polymer can comprise through the ECD polypeptide be connected with peptide moiety that covalently or non-covalently interacts and merging to soluble polypeptide in another example.This type of peptide can be peptide linker (spacer peptide) or have the peptide that promotes the multimerization characteristic.In other example, polymer can connect for example by using the Heterobifunctional joint to form by chemistry between two polypeptide.

A. peptide linker

[0307] peptide linker can be used for producing the polypeptide polymer, for example the polymer when the multimerization mating partner ECD that is the HER family receptors all or part of.In an example, peptide linker can be blended in the C-terminal of first polypeptide and the N-terminal of second polypeptide.This structure can repeat repeatedly, thus at least 1, preferred 2,3,4 or more a plurality of soluble polypeptide be connected to each other by peptide linker at its associated end place.For example, the polymer polypeptide can have sequence Z ₁-X-Z ₂, Z wherein ₁And Z ₂Be respectively all or part of sequence of ECD of cell surface polypeptide and the sequence that X is peptide linker.In some cases, Z ₁And/or Z ₂Be ECD all or part of of HER family receptors.In another example, Z ₁And Z ₂Be identical or they are different.In another example, described polypeptide has sequence Z ₁-X-Z ₂(-X-Z) _n, wherein " n " is arbitrary integer, promptly normally 1 or 2.

[0308] usually, the length of described peptide linker is enough to allow soluble E CD polypeptide and contiguous soluble E CD polypeptide to form key.The example of peptide linker comprises-Gly-Gly-, GGGGG (SEQ IDNO:273), GGGGS or (GGGGS) _n(SEQ ID NO:174), SSSSG or (SSSSG) n (SEQ ID NO:187), GKSSGSGSESKS (SEQ ID NO:175), GGSTSGSGKSSEGKG (SEQ ID NO:176), GSTSGSGKSSSEGSGSTKG (SEQ ID NO:177), GSTSGSGKPGSGEGSTKG (SEQ ID NO:178), EGKS S GS GS ESKEF (SEQ ID NO:179) or AlaAlaProAla or (AlaAlaProAla) _n(SEQ IDNO:188), wherein n is 1-6, as 1,2,3 or 4.Exemplary adapter comprises:

(1) Gly of band NcoI end ₄Ser SEQ ID NO.189

CCATGGGCGGCGGCGGCTCTGCCATGG

(2) (the Gly of band NcoI end ₄Ser) ₂SEQ ID NO.190

CCATGGGCGGCGGCGGCTCTGGCGGCGGCGGCTCTGCCATGG

(3) (the Ser of band NcoI end ₄Gly) ₄SEQ ID NO.191

CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGCGCCATGG

(4) (the Ser of band NcoI end ₄Gly) ₂SEQ ID NO.192

CCATGGCCTCGTCGTCGTCGGGCTCGTCGTCGTCGGGCGCCATGG

[0309] connection portion is described among (1996) Biochemistry35:545-553 such as Whitlow etc. (1993) Protein Engineering6:989-995 and Newton for example at (1988) PNAS 85:5879-5883 such as Huston.Other appropriate peptide joint is included in U.S. Patent number 4,751, and 180 or 4,935, those peptide linkers arbitrarily described in 233, described patent mode is by reference incorporated this paper into.Use any suitable routine techniques, can between the open reading-frame (ORF) identical with the polynucleotide of coding soluble E CD polypeptide or within insert the polynucleotide of the required peptide linker of coding.In an example, fusion polypeptide has the soluble E CD polypeptide of being separated by peptide linker from 2-4, comprises it being an all or part of soluble E CD polypeptide of HER ECD polypeptide.

B. Heterobifunctional linking agent

[0310] practice midwifery described connection of living heteromultimeric fusion polypeptide of a kind of ECD polypeptide and another kind of ECD polypeptide chain can be direct or indirect.For example, the connection of two or more ECD polypeptide can realize or promoted by the Heterobifunctional joint by chemical bonding, as known in the art or provide herein like that.

[0311] those skilled in the art known be used between amino and sulfydryl forming covalent linkage and be used for sulfydryl import the agent of proteinic numerous Heterobifunctional sexual intercourse joint-trial (see, for example, PIERCECATALOG, ImmunoTechnology Catalog﹠amp; Handbook, 1992-1993, the document has been described the preparation and the purposes of this type of reagent and the commercial source of this type of reagent is provided; Also referring to, for example, Cumber etc. (1992) Bioconjugate Chem.3:397-401; Thorpe etc. (1987) Cancer Res.47:5924-5931; Gordon etc. (1987) Proc.Natl.Acad Sci.84:308-312; Walden etc. (1986) J.Mol.Cell Immunol.2:191-197; Carlsson etc. (1978) Biochem.J.173:723-737; Mahan etc. (1987) Anal.Biochem.162:163-170; Wawryznaczak etc. (1992) Br.J.Cancer 66:361-366; Fattom etc. (1992) Infection﹠amp; Immun.60:584-589).These reagent can be used between the carboxyl terminal part of the aminoterminal part of an ECD polypeptide and another ECD polypeptide or form covalent linkage between each these part and joint.These reagent include but not limited to: N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP; The disulphide joint); Sulfosuccinimide base 6-[3-(2-pyridyl dithio) propionamido-] and capronate (sulfo group-LC-SPDP); Succinimido oxygen carbonyl-α-Jia Bianji thiosulfates (SMBT, steric hindrance pyrosulphate joint); Succinimido 6-[3-(2-pyridyl dithio) propionamido-] capronate (LC-SPDP); Sulfosuccinimide base 4-(N-maleimide ylmethyl) hexanaphthene-1-carboxylicesters (sulfo group-SMCC); Succinimido 3-(2-pyridyl dithio) butyric ester (SPDB; Steric hindrance disulfide linkage joint); Sulfosuccinimide base 2-(7-azido--4-methylcoumarin-3-ethanamide) ethyl-1,3 '-dithio propionic ester (SAED); Sulfosuccinimide base 7-azido--4-methylcoumarin-3-acetic ester (SAMCA); Sulfosuccinimide base-6-[Alpha-Methyl-α-(2-pyridyl dithio) toluyl amido] and capronate (sulfo group-LC-SMPT); 1,4-two-[3 '-(2 '-the pyridyl dithio) propionamido-] butane (DPDPB); 4-succinimido oxygen carbonyl-Alpha-Methyl-α-(2-pyridylthio) toluene (SMPT, steric hindrance pyrosulphate joint); Sulfosuccinimide base-6-[Alpha-Methyl-α-(2-pyridyl dithio) toluyl amido] and capronate (sulfo group-LC-SMPT); Between-dimaleoyl imino benzoyl-N-hydroxyl-succinimide ester (MBS); Between-dimaleoyl imino benzoyl-N-hydroxyl-sulfosuccinimide ester (sulfo group-MBS); N-succinimido (4-iodo acetyl) Aminobenzoate (SIAB; The thioether joint); Sulfosuccinimide base-(4-iodo acetyl) Aminobenzoate (sulfo group-SIAB); Succinimido-4-(right-the dimaleoyl imino phenyl) butyric ester (SMPB); Sulfosuccinimide base 4-(right-the dimaleoyl imino phenyl) butyric ester (sulfo group-SMPB); Azido benzoyl base hydrazides (azidobenzoyl hydrazide) (ABH).These joints for example can be used in combination as increasing flexibility or solubleness or providing or eliminate sterically hindered those peptide linkers with peptide linker.Can use a peptide molecule any other joint that is used to connect well known by persons skilled in the art to another molecule.Overall characteristic is such, and promptly the gained molecule is that biocompatible (be used to be applied to animal, comprise the people) and gained molecule are the active heteromultimeric molecules of regulating cell surface molecule such as HER or other cell surface molecule or acceptor.

C. polypeptide multimerization structural domain

[0312] interaction of two or more polypeptide can be by directly or indirectly being connected and being promoted with any part of formation rock steady structure or other polypeptide with itself can interacting.For example, discrete encoded polypeptides chain can connect by multimerization, and the multimerization of wherein said polypeptide is mediated by the multimerization structural domain.Generally, provide this multimerization structural domain to be used between first chimeric polyeptides and second chimeric polyeptides, forming stable protein-protein interaction.Chimeric polyeptides for example comprises the nucleic acid (directly or indirectly) of the nucleic acid partly of ECD in the coded polypeptide with coding multimerization structural domain is connected.Generally, at least a multimerization mating partner is the nucleic acid of the direct or indirect all or part of HER ECD that is connected with the multimerization structural domain of coding.Can divide other chimeric polyeptides to produce from coexpression with polymer or heteromultimeric polypeptides.First and second chimeric polyeptides can be identical or different.

[0313] common, the multimerization structural domain comprises arbitrary structural domain that can form stable protein-protein interaction.Described multimerization structural domain can be by immunoglobulin sequences, leucine zipper, hydrophobic region, hydrophilic area, the free sulfhydryl groups of shape group intermolecular disulfide bond interacts between with the chimeric molecule of polymer or heteromultimeric.In addition, the multimerization structural domain can comprise such aminoacid sequence, and it comprises and the aminoacid sequence complementary projection that contains hole, as for example describing in the U.S. Patent Application Serial 08/399,106.Can design this multimerization zone, thereby steric interaction not only promotes stable interaction, also further promote heterodimer from the mixture of chimeric monomer, to have precedence over homodimer and form.Usually, make up projection by the small volume amino acid side chain of replacing from the first polypeptide interface with volume larger side chain (for example tyrosine or tryptophane).Optional real estate is given birth to big or small same or analogous compensatory " hole " with described projection on the second polypeptide interface by replacing the large volume amino acid side chain with the less amino acid side chain (for example L-Ala or Threonine) of volume.

[0314] but chimeric polyeptides of ECD arbitrarily that for example provides herein of ECD chimeric polyeptides can be at any position generally is connected with the aminoterminal or the carboxyl terminal of multimerization structural domain through its aminoterminal or carboxyl terminal, with the formation chimeric polyeptides.Connection can be direct or indirect through joint.In addition, chimeric polyeptides can be that fusion rotein maybe can be by chemical bonding as forming through covalently or non-covalently interacting.For example, when preparation contained the chimeric polyeptides of multimerization structural domain, the nucleic acid of all or part of ECD of coded polypeptide can directly or indirectly or be chosen the nucleic acid that effectively is connected to coding multimerization structural domain sequence through the joint design territory wantonly.Generally, the construct chimeric protein that the carboxyl terminal of ECD polypeptide wherein is connected with multimerization structural domain aminoterminal of encoding.In some cases, the construct chimeric protein that the aminoterminal of ECD polypeptide wherein is connected with multimerization structural domain aminoterminal or carboxyl terminal of can encoding.

[0315] the polypeptide polymer contains by two identical or different ECD polypeptide directly or indirectly are connected two chimeric proteins that produce with the multimerization structural domain.In some instances, when described multimerization structural domain is polypeptide, with the suitable expression vector of genetic fusant insertion of coding ECD-multimerization structural domain chimeric polyeptides.The ECD-multimerization structural domain chimeric protein of gained can be expressed in in the described recombinant expression vector transformed host cells, and allows to be assembled into polymer, and wherein the multimerization domain interaction is to form the multivalence polypeptide.The chemical bonding of multimerization structural domain and ECD polypeptide can use Heterobifunctional joint as discussed above to realize.

[0316] chimeric polyeptides of gained and by its polymer that forms can by as any suitable method purifying that hereinafter describes in detail in the F part, for example by affinity chromatography through A albumen post or G albumen post.Two kinds of nucleic acid molecule at the different ECD chimeric polyeptides of will encoding are converted under the situation of cell, will the formation of homodimer and heterodimer occur.Thereby can regulate the condition that is used to express supports heterodimer to form better than homodimer formation.

I. immunoglobulin domains

[0317] the multimerization structural domain comprises such multimerization structural domain, and it comprises and can react with the multimerization structural domain of additional amino acid sequence to form the free sulfhydryl groups part of intermolecular disulfide bond.For example, the multimerization structural domain can comprise as the part from the immunoglobulin molecules of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgM and IgE.Normally, this part is constant region for immunoglobulin (Fc).Described and prepare such fusion rotein, it contains the soluble E CD polypeptide that a plurality of parts (comprising the Fc structural domain) with the derivative polypeptide of antibody merge, referring to for example, and Ashkenazi etc. (1991) PNAS 88:10535; Byrn etc. (1990) Nature, 344:677; And (1992) " Construction of Immnoglobulin Fusion Proteins " such as Hollenbaugh and Aruffo are at Current Protocols in Immunology, Suppl.4,10.19.1-10.19.11 page or leaf.

[0318] antibody and specific antigens in conjunction with and contain by disulfide linkage covalently bound two identical heavy chains and two identical light chains.Heavy chain and light chain all contain the variable region of conjugated antigen, also contain constant (C) district.In each chain, a kind of structural domain (V) has the variable amino acid sequence of the antibodies specific that depends on molecule.Another kind of structural domain (C) has the sequence of quite stable common between the molecule of identical category.Described structural domain is numbered from N-terminal successively.For example, the IgG light chain by from aminoterminal to carboxyl terminal with V _L-C _LTwo immunoglobulin domains that are linked in sequence are formed, and are called light chain variable structural domain and light chain constant domain respectively.The IgG heavy chain by from aminoterminal to carboxyl terminal with V _H-C _H1-C _H2-C _H3 four immunoglobulin domains that are linked in sequence (being called variable heavy chain structural domain, constant heavy chain structural domain 1, constant heavy chain structural domain 2 and constant heavy chain structural domain 3) are formed.The gained antibody molecule is a kind of four chain molecules, and wherein every heavy chain is connected to a light chain by disulfide linkage, and two heavy chains are connected to each other by disulfide linkage.The connection of heavy chain is by flex region (the being called hinge area) mediation of heavy chain.The fragment of antibody molecule can for example produce by the enzyme cutting.For example, behind proteolytic cleavage, be that the Fc structural domain separates with two Fab districts (part that promptly contains the variable region) with the dimer of CH by papoid.

[0319] in the mankind, there are 5 kinds of antibody isotypes of classifying based on heavy chain of antibody, be called delta (δ), gamma (γ), Miao (μ) and alpha (α) and dust and sprinkle Shillong (ε), produce antibody I gD, IgG, IgM, IgA and IgE class respectively.IgA and IgG class contain subclass IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.Sequence difference between the heavy chain immunoglobulin causes different a plurality of isotypes aspect for example C-structure territory number, hinge area existence and interchain disulfide bond number and position.For example, IgM and IgE heavy chain contain the extra C-structure territory (C4) of replacing hinge area.The Fc district of IgG, IgD and IgA is paired with each other by its C γ 3, C δ 3 and C α 3 structural domains, and the Fc district of IgM and IgE passes through its C μ 4 and C ε 4 structural domains and dimerization.IgM and IgA form the polymer structure of 10 and 4 antigen binding sites of tool respectively.

[0320] the ECD immunoglobulin chimeric polypeptide that provides herein comprises the total length immunoglobulin polypeptides.Alternatively, described immunoglobulin polypeptides promptly contains heavy chain, light chain, Fab, Fab2, Fv or Fc less than total length.In an example, ECD immunoglobulin chimeric polypeptide is assembled into monomer or heteromultimeric or with polymer and be assembled into the dimer or the tetramer especially.The chain or the elementary cell that can utilization structure change are assembled monomer and heteromultimeric and same polymer.For example, the ECD polypeptide can be blended in all or part of of immunoglobulin molecules, and the latter comprises the C of immunoglobulin molecules _H, C _L, V _H, or V _LAll or part of (see, for example, Application No. 5,116,964) of structural domain.Chimeric ECD polypeptide can produce justacrine by the mammalian cell that transforms through suitable nucleic acid molecule easily.Secreted form comprises that ECD polypeptide wherein is present in those in the heavy chain homodimer; Light chain monomer or dimer; The heavy chain and the different tetramer of light chain that merge of ECD polypeptide and one or more light chain or heavy chain wherein, comprise wherein reach to and comprise that whole four kinds of variable region analogues are by the different tetramer of metathetical.In some instances, a kind of or can be converted in the host cell to produce polymer more than a kind of nucleic acid fusion molecule, wherein said polymeric ECD partly is identical or different.In some instances, there is non--ECD peptide light chain-variable region of heavy chain spline structure territory, thereby produces Heterobifunctional antibody.In some instances, can make chimeric polyeptides and the meromixis that lacks the immunoglobulin molecules of hinge disulphide, wherein non-covalent the or covalent interaction of two ECD polypeptide portions makes this molecular tie become homodimer or heterodimer.

(a) Fc structural domain

[0321] general, the immunoglobulin part of ECD chimeric protein comprises the heavy chain of immunoglobulin polypeptides, is the constant domain of heavy chain the most commonly.The exemplary sequence of the CH of human IgG hypotype is described in SEQ ID NO:163 (IgG1), SEQ ID NO:164 (IgG2), SEQ IDNO:165 (IgG3) and SEQ ID NO:166 (IgG4).For example, for the exemplary CH described in the SEQ IDNO:163, C _H1 structural domain is corresponding to the 1-98 amino acids, and hinge area is corresponding to the 90-110 amino acids, C _H2 structural domains are corresponding to 111-223 amino acids and C _H3 structural domains are corresponding to the 224-330 amino acids.

[0322] in an example, the immunoglobulin polypeptides chimeric protein can comprise the Fc district of immunoglobulin polypeptides.Generally, this fusions remains to the C of constant region of hinge, the heavy chain immunoglobulin of rare functionally active _H2 and C _H3 structural domains.For example, the total length Fc sequence of IgG1 is included in the 99-330 amino acids of the sequence described in the SEQ ID NO:163.The exemplary Fc sequence of hIgG1 is described in SEQ ID NO:167 and is contained almost all hinge sequences and the C described in SEQ ID NO:163 corresponding to the 100-110 amino acids of SEQ ID NO:163 _H2 and C _HThe complete sequence of 3 structural domains.The Fc polypeptide of another kind of example is described in PCT application WO93/10151, and is the single chain polypeptide that extends to the natural carboxyl terminal in human IgG1's antibody (SEQ IDNO:168) Fc district from the aminoterminal hinge area.The accurate position that produce to connect is not critical: concrete position be know and can select with the biologic activity of optimizing the ECD polypeptide, secretion or in conjunction with feature.For example, other exemplary Fc peptide sequence is in the amino acid C109 of sequence described in the SEQ ID NO:163 or the beginning of P113 place (referring to for example, US 2006/0024298).

[0323] except hIgG1Fc, also can in ECD chimeric polyeptides provided herein, comprise other Fc district, for example, make under the minimized situation of effector function that mediates by Fc/Fc γ R interaction in intention, can conceive with the IgG isotype (for example Fc of IgG2 or IgG4) of seldom convening complement and effector cell and merge.In addition, the Fc fusions can contain the immunoglobulin sequences of being encoded by such immunoglobulin gene substantially, wherein said immunoglobulin gene belongs to antibody classification arbitrarily, includes but not limited to IgG (comprising people's subclass IgG1, IgG2, IgG3 or IgG4), IgA (comprising people's subclass IgA1 and IgA2), IgD, IgE and the IgM classification of antibody.In addition, can use joint with Fc covalently bound to another polypeptide to produce the Fc mosaic.

[0324] also conceived the mosaic that modified Fc structural domain is used for having the ECD polypeptide herein, referring to for example U.S. Patent Publication No. US 2006/0024298; Exemplary modification with International Patent Publication No. WO 2005/063816.In some instances, the Fc district is such, thereby it has (being higher or lower) effector function of change than the Fc district of wild-type heavy chain immunoglobulin.The Fc district of antibody and numerous Fc acceptor and ligand interaction are given a series of important function sexualities, are called effector function.The Fc effector function comprises that for example Fc receptors bind effect, complement fixation(CF) and T cell exhaust active (referring to for example, U.S. Patent number 6,136,310).Measure the T cell and exhaust that the method for activity, Fc effector function and antibody stability is known in the art.For example, the Fc district of IgG molecule and Fc γ R interact.These Fc γ R expression of receptor are in the panimmunity cell, and wherein said immunocyte comprises for example monocyte, scavenger cell, neutrophilic granulocyte, dendritic cell, eosinophilic granulocyte, mastocyte, thrombocyte, B cell, large granular lymphocyte, Langerhans cell, natural killer cell (NK) and gamma delta T cells.The position of these effector cells to conjugated antigen convened in the formation of Fc/Fc γ R complex body, usually this causes signal conduction incident and important follow-up immunization to be replied in cell, attacks as discharging inflammatory mediator, B cell activation, endocytosis, phagolysis and cytotoxicity.The ability of mediated cell toxicity and engulfing property effector function is that antibody is so as to destroying the potential mechanism of target cell.The cytotoxic cell of expressing Fc γ R is to the identification of binding antibody and splitting action are called the cytotoxicity (ADCC) of antibody dependent cellular mediation on the target cell.Other Fc acceptor at the different antibodies isotype comprises Fc ε R (IgE), Fc α R (IgA) and Fc μ R (IgM).

[0325] therefore, the Fc structural domain of modification can have the avidity of change, includes but not limited to the avidity that the Fc acceptor is increased or the avidity of reduction or do not have avidity.For example, different IgG subclass has different avidity to Fc γ R, and wherein IgG1 and IgG3 generally combine with this receptor better than IgG2 and IgG4 essence.In addition, the different effector function of different Fc γ R mediations.Fc γ R1, Fc γ RIIa/c and Fc γ RIIIa are the positive regulators of the immunocomplex effect of triggering the activation, and it is characterized in that having the born of the same parents intracellular domain of immunity receptor based on the activation motif (ITAM) of tyrosine.Yet Fc γ RIIb has immunity receptor based on the inhibition motif (ITIM) of tyrosine and thereby be inhibition.Changing the Fc district can regulate by Fc structural domain inductive effector function the avidity of acceptor.

[0326] in an example, use to optimizing to combine and better mediate for example Fc district modified of ADCC of effector function with some Fc γ R.The G20S with exemplary Fc sequence described in the SEQ IDNO:167 can be contained in the Fc district of this type of modification, G20A, S23D, S23E, S23N, S23Q, S23T, K30H, K30Y, D33Y, R39Y, E42Y, T44H, V48I, S51E, H52D, E56Y, E56I, E56H, K58E, G65D, E67L, E67H, S82A, S82D, S88T, S108G, S108I, K110T, K110E, K110D, A111D, A114Y, A114L, A114I, I116D, I116E, I116N, I116Q, E117Y, E117A, K118T, K118F, any one of K118A and P180L or multiple corresponding modification or its combination.The modified Fc that contains these sudden changes can have with FcR for example activation receptor Fc γ RIIIa the enhanced keying action and/or can have with the keying action that weakens of inhibition acceptor Fc γ RIIb (referring to for example, US2006/0024298).Modification can be beneficial in the patient to have Fc district with the enhancing keying action of FcR that to destroy aspect the cancer cells be more effective, even if also be like this when being connected with the ECD polypeptide.Exist the antibody may mechanism, comprise by the antiproliferative effect of blocking required growth pathway, the intracellular signal conduction that causes apoptosis, enhanced receptor down-regulated and/or turnover, ADCC with by promoting adaptive immune response so as to the numerous of destroyed tumor cell.

[0327] in another example, also known multiple Fc mutant with reduction or elimination and the replacement of Fc γ R bonded.This type of mutain is used for the situation that needs reduce or eliminate the effector function that is mediated by Fc.This is needing antagonism rather than is killing under the situation of the cell that carries target antigen often like this.The example of this kind Fc is at U.S. Patent number 5,457, the Fc mutain of describing and providing in SEQ ID NO:169 in 035.The aminoacid sequence of this mutain is identical with the Fc sequence described in the SEQ ID NO:168, and except the 19th amino acids being become Ala from Leu, the 20th amino acids becomes Glu and the 22nd amino acids becomes outside the Ala from Gly from Leu.Can for example in the exemplary Fc sequence described in the SEQ ID NO:167, produce similar sudden change in any Fc sequence.This mutain shows the avidity that the Fc acceptor is reduced.

[0328] in some cases, the ECD polypeptide Fc chimeric protein that provides herein can be modified to strengthen and the combining of complement proteins C1q.Except interacting with FcR, Fc also interacts with the cytotoxicity (CDC) of mediation complement dependence with complement proteins C1q.C1q and serine protease C1r and C1s form complex body to form the C1 complex body.C1q can be in conjunction with 6 antibody, are enough to the activating complement cascade although combine with 2 IgG.Be similar to the interaction of Fc and FcR, different I gG subclass has different avidity to C1q, and IgG1 and IgG3 are generally than IgG2 and IgG4 combination in fact better.Therefore, have the modified Fc mediation enhanced CDC of the keying action that increases with C1q, this is that antibody is so as to promoting that a kind of of destroyed tumor cell may mechanism.Increase and the exemplary modification of C1q bonded include but not limited to K110W, K110Y and the corresponding modification of E117S among amino acid modified and the SEQ IDNO:167 in the Fc district.

[0329] in extra example, can use the Fc district of on the keying action of itself and FcRn, being modified, thereby improve the pharmacokinetics of ECD-Fc chimeric polyeptides.FcRn is neonatal FcR, during the recirculation blood back flows from the endocytosis body in conjunction with the antibody that can make endocytosis with it.This process is because of the large size of full-length molecule, when preventing that the filtering effect of kidney from combining, produces the favourable antibody serum half life in 1-3 week.Fc plays a role in the antibody transportation with combining also of FcRn.In Fc protein, be used for strengthening with the exemplary modification of FcRn bonded and comprise modification and SEQ ID NO:267 T34Q, T34E, M212L and the corresponding amino acid of M212F.

[0330] general, the polypeptide polymer is the dimer that directly or indirectly is connected to two kinds of chimeric proteins that the Fc polypeptide produces by two identical or different ECD polypeptide.In some instances, the fusion gene with coding ECD-Fc chimeric protein inserts suitable expression vector.The ECD-Fc chimeric protein of gained can be expressed in in the described recombinant expression vector transformed host cells, and allows extremely to be similar to antibody molecule and assemble like that, wherein forms interchain disulfide bond to produce divalence ECD polypeptide between the Fc part.Generally, host cell and expression system are to allow the corresponding amino acid whose mammalian expression system of N81 among glycosylation and the SEQ ID NO:167.Glycosylation in this position is important for stablizing Fc protein.Do not considering under the glycosylated situation in this position, also can to use other host cell.

[0331] chimeric polyeptides that contains Fc part of gained and can be by affinity chromatography purifying through A albumen post or G albumen post by its polymer that forms.Two kinds of nucleic acid at the different ECD chimeric polyeptides of will encoding are transformed under the situation of cell, and the formation of heterodimer must realize in the biological chemistry mode, also will be expressed as the homodimer that disulfide linkage connects because carry the ECD chimeric molecule of Fc structural domain.Therefore, do not influence helping to destroy interchain disulfide bond under the condition of intrachain disulfide bond and can reduce homodimer.Generally, the chimeric monomer that will have an outside part born of the same parents not mix with equimolar amount and oxidation to form the mixture of homodimer and heterodimer.The component of this mixture is separated by chromatographic technique.Alternatively, can and express such ECD fusion molecule by the genetically engineered design and form this type of heterodimer with being partial to, wherein said ECD fusion molecule contains the ECD polypeptide, after connect the Fc-structural domain of hIgG, follow by c-jun or c-fos leucine zipper (seeing below).Because leucine zipper advantage ground forms heterodimer, so they can be used for driving the formation of described heterodimer as required.Also can design the ECD chimeric polyeptides that contains the Fc district with the label that comprises the tool metallo-chelate or the label of other epi-position.This tagged structural domain can be used for fast purifying by the metallo-chelate chromatography and/or by antibody, to allow the detection of active removing/blocking effect in western blotting thing, immunoprecipitate or the bioassay method.

(b). go into hole projection (being knuckle and hole)

[0332] in one aspect, design ECD polymer promotes different oligomerization to surpass the interface with oligomerization to contain between first chimeric polyeptides and second chimeric polyeptides.Generally, one of first and second ECD chimeric polyeptides or the two multimerization structural domain are modified antibody fragments, thereby the interface of this antibody molecule is modified and/or promote different dimerization.In some cases, antibody molecule is modified Fc district.Therefore, modification comprises importing projection to a Fc polypeptide and imports hole to the two Fc polypeptide, and is compound with first and second chimeric ECD polypeptide that promotion contains Fc thereby described projection can be arranged in this hole.

[0333] general, the stable phase mutual effect of first chimeric polyeptides and second chimeric polyeptides is the interfacial interaction by identical or different multimerization structural domain, and wherein said multimerization structural domain contains the C of immunoglobulin (Ig) constant domain _HEnough parts of 3 structural domains.A plurality of structured datas and performance data show that the heavy chain of antibody combination is by C _H3 structural domains instruct.For example, X-ray crystallography has shown that the intermolecular combination between human IgG1's heavy chain is included in C in the Fc district _HProtein/protein interaction widely between 3 structural domains, and glycosylation C _H2 structural domains are by its carbohydrate and interact (Deisenhofer etc. (1981) Biochem.20:2361).In addition, there is disulfide linkage between two heavy chains, efficiently forms during their antibody expressions in mammalian cell, unless the described heavy chain of brachymemma and remove C _H2 and C _H3 structural domains (King etc. (1992) Biochem.J.281:317).Therefore as if, heavy chain assembling promotes disulfide linkage to form, but non-vice versa.To C _HThe heteromultimeric that the Interface Design of 3 structural domains promotes to contain different heavy chains form and hinder corresponding with the polymer assembling (referring to for example, U.S. Patent number 5,731,168; International Patent Application WO 98/50431 and WO 2005/063816; Ridgway etc. (1996) Protein Engineering, 9:617-621).

[0334] therefore, ECD polymer provided herein can form between the interface of the first and second chimeric ECD polypeptide, and wherein the multimerization structural domain of first polypeptide contains the C of Fc structural domain at least _HEnough parts at 3 interfaces, described part have been subjected to modifying containing projection, and the multimerization structural domain of second polypeptide contains the C of Fc structural domain at least _HEnough parts at 3 interfaces, described part have been subjected to modifying to contain the hole.Modified C _HWhole or enough parts at 3 interfaces can be from IgG, IgA, IgD, IgE or IgM immunoglobulin (Ig).At U.S. Patent number 5,731, panimmunity globulin molecule C is described in 168 _HThe interface residue of target is modified in conduct in 3 structural domains.Usually, the multimerization structural domain is from IgG antibody IgG1 deutero-C for example _HWhole or enough parts of 3 structural domains.

[0335] replace and/or modified amino acid with the target amino acid that in polypeptide, produces projection or hole generally be with the second polypeptide interface in one or more amino acid interface amino acid of interacting or contacting.Be subjected to modify to contain amino acid whose first polypeptide of projection and comprise and replace natural or amino acid originally with amino acid like this, wherein said amino acid has at least one side chain of stretching out and thereby can be positioned in the compensatory hole in the second polypeptide adjacent interfaces from the first polypeptide interface.In most cases, replacing amino acid is the amino acid that has larger side chain volume than amino-acid residue originally.Those skilled in the art will know that the characteristic of how determining and/or assessing amino-acid residue replaces those amino acid that amino acid is intended to produce projection to identify as ideal.Usually, the replacement residue that is used to form projection is naturally occurring amino-acid residue and comprises for example arginine (R), phenylalanine (F), tyrosine (Y) or tryptophane (W).In some instances, the residue originally that is used to replace of identifying is the amino-acid residue with small volume side chain, for example L-Ala, l-asparagine, aspartic acid, glycine, Serine, Threonine or Xie Ansuan.

[0336] being modified with second polypeptide that contains the hole is to comprise with amino acid like this replacing natural or originally amino acid whose polypeptide, and wherein said amino acid has from least one side chain of second polypeptide interface withdrawal and thereby can admit complementary protuberance from the first polypeptide interface.In most cases, replacing amino acid is the amino acid that has smaller side chain volume than amino-acid residue originally.Those skilled in the art will know that the characteristic of how determining and/or assessing amino-acid residue replaces those amino acid that amino acid is used to form the hole to identify as ideal.Usually, the replacement residue that is used to form the hole is naturally occurring amino-acid residue and comprises that for example L-Ala (A), Serine (S), Threonine (T) and Xie Ansuan are (v).In some instances, identify that the amino acid originally that is used to replace is the amino acid with large volume side chain, for example tyrosine, arginine, phenylalanine or tryptophane.

[0337] for example, human IgG1's C _H3 interfaces relate to 16 residues on each structural domain that is positioned on four antiparallel beta chains, and it buries 1090 from each surface

(referring to for example, Deisenhofer etc. (1981) Biochemistry, 20:2361-2370; Miller etc. (1990) J.Mol.Biol, 216,965-973; Ridgway etc. (1996) Prot.Engin., 9:617-621; United States Patent (USP) 5,731,168).For example in United States Patent (USP) 5,731,168; International Patent Application WO 98/50431 and WO2005/063816; With (1996) Prot.Engin. such as Ridgway, the C that produces projection or hole is described among the 9:617-621 _H3 structural domains are modified.For example, at C _HThe modification that produces projection or hole in 3 structural domains can be interface amino acid Q230, V231, Y232, T233, L234, V246, S247, L248, T249, C250, L251, V252, K253, G254, F255, Y256, K275, T276, T277, P278, V279, L280, D281, G285, S286, F287, F288, L289, Y290, S291, K292, L293, T294 and the corresponding arbitrary amino acid of V295 of sequence described in replacement and the SEQ ID NO:163.In some instances, at C _HProduce the general target of modification localized residue on two central antiparallel beta chains in projection or hole in 3 structural domains.Purpose is to make the projection of generation can be by charging on every side solvent and admitted rather than by mating partner C _HThe risk minimization that admit in compensatory hole in 3 structural domains.The example of this type of modification comprises for example replaces and interface amino acid T249, L251, P278, F288, Y290 and the corresponding arbitrary amino acid of K292.Be used at C _HThe amino acid ligand of modifying in the 3 structural domain interfaces to produce projection/hole interaction comprises modification T249 and Y290 and F288 and T277 to example.For example, modification can comprise T249Y and Y290T; T249W and Y290A; F288A and T277W; F288W and T277S; With Y290T and T249Y.

[0338] in some instances, can produce more than a kind of interfacial interaction.For example, modification also comprises two or more modifications that for example produce two or more modifications of projection and produce the hole in first polypeptide in second polypeptide.The example of this type of modification for example comprises, modifies T249Y in first polypeptide and T277W and the Y290T in F288A and modification second polypeptide; Modify T277W in first polypeptide and T277S and the Y290A in F288W and modification second polypeptide; Or modify F288A in first polypeptide and T249W and the T277S in Y290A and modification second polypeptide.

[0339] described other multimerization structural domain herein just comprises all or part of of any immunoglobulin molecules or its variant, as Fc structural domain or its variant, contains C _HThe Fc variant that 3 projections/hole is modified can be connected with any position of ECD polypeptide, but generally through its aminoterminal or carboxyl terminal, is connected to form chimeric polyeptides with the aminoterminal or the carboxyl terminal of the first and/or the 2nd ECD polypeptide.Connection can be direct or indirect through joint.In addition, this chimeric polyeptides can be that fusion rotein maybe can connect as forming by covalently or non-covalently interacting by chemistry.Usually, knuckle and hole molecule are connected to by coexpression and contain C _HThe one ECD polypeptide of 3 projection-decorated Fc variants and be connected to and contain C _HThe 2nd ECD polypeptide of the Fc variant that 3 holes are modified and producing.

Ii. leucine zipper

[0340] the polymeric another kind of method of preparation ECD polypeptide relates to the use leucine zipper motif.Leucine zipper is the peptide that promotes wherein to exist the protein multimerization of leucine zipper.Generally, leucine zipper is such term, and it is used to refer to the repetition that contains 4-5 leucine residue seven collectives (heptad) motif that exists as the conserved domain of some protein.Leucine zipper is folded into short, parallel curling spiral, and thinks and be responsible for making the wherein protein oligomerization of leucine zipper formation structural domain.In several DNA are conjugated protein, identify leucine zipper (referring to for example, Landschulz etc. (1988) Science 240:1759) at first, and in multiple proteins, find them so far.In known leucine zipper, the natural peptide and the derivative thereof of existing of dimerization or trimerizing arranged.The reorganization chimeric protein that contains the ECD polypeptide that directly or indirectly is connected with the leucine zipper peptide can be expressed in proper host cell, and the ECD polypeptide polymer that forms can reclaim from culture supernatant.

[0341] leucine zipper motif be folded into short, parallel curling spiral (O ' Shea etc. (1991) Science, 254:539).The overall structure of this parallel coiled coil be characterized by at first " knuckle-go into-hole " obturator (packing) that proposes in nineteen fifty-three by Crick (Acta Crystallogr., 6:689).The dimer that is formed by leucine zipper motif is by (abcdefg) by name _nSeven collective's tumor-necrosis factor glycoproteinss stable (referring to for example, McLachlan and Stewart (1978) J.MoI.Biol.98:293), residue a and d hydrophobic residue normally wherein, and d is a linable leucine on the similar face of spiral.The residue of oppositely charged appears at position g and e place usually.Therefore, in the parallel coiled coil that forms from two leucine zipper motifs, " knuckle " that is formed by the hydrophobic side chain of first spiral is filled in " hole " between the side chain that is formed at second spiral.

[0342] big hydrophobic stabilization energy to be provided and to form for dimer be important (Krystek etc. (1991) Int.J.Peptide Res.38:229) to the leucine residue at position d place.Hydrophobic stabilization energy provides main impellent to be used for forming coiled coil from the spirrillum monomer.Electrostatic interacts and also helps the stoichiometry and the geometry of coiled coil.

(a) fos and jun

[0343] two kind of consideration convey albumen fos and jun show leucine zipper motif, and the gene product of mouse proto-oncogene c-myc also is like this.(DNA combination) is essential to leucine zipper motif for these proteinic biologic activity.The product of karyon oncogene fos and jun contains leucine zipper motif (O ' Shea etc. (1989) Science, the 245:646 that preference ground forms heterodimer; Turner and Tijian etc. (1989) Science, 243:1689).For example, the leucine zipper motif of human transcription factor c-jun and c-fos verified with 1: 1 stoichiometry form stable heterodimer (referring to for example, Busch and Sassone-Corsi (1990) Trends Genetics, 6:36-40; Gentz etc. (1989) Science, 243:1695-1699).Though the verified jun-jun homodimer that also forms, yet their stability is lower about 1000 times than jun-fos heterodimer.

[0344] therefore, generally speaking, use jun-fos combination results ECD polypeptide provided herein polymer.Normally, the leucine zipper motif of c-jun or c-fos is by the carboxyl terminal of genetically engineered fusion gene with the ECD that meets the open reading-frame (ORF) mode and merge at polypeptide.The exemplary amino acid sequence of c-jun and c-fos leucine zipper is described in SEQ ID NO:170 and 171 respectively.In some cases, the sequence of leucine zipper can be for example allows cysteine residues that disulfide linkage forms or adds the tyrosine residues of being convenient to measure peptide concentration at carboxyl terminal and modify by adding.Coded amino acid whose this type of exemplary sequence of modified c-jun and c-fos leucine zipper is described in SEQ IDNO:172 and 173 respectively.In addition, the ECD polypeptide can be directly maybe to use flexible joint structural domain such as IgG hinge area or the small volume amino acid under multiple length and combination such as other peptide linker of glycine, Serine, Threonine or L-Ala to carry out with being connected of leucine zipper.In some cases, leucine zipper can be by carrying out with the sequence fusion of proteins encoded enzyme cleavage site such as zymoplasm cleavage site with separating of encoded polypeptides carboxyl terminal.In addition, described chimeric protein can tag, for example add 6 * histidine-tagged, with allow by metallo-chelate chromatography and/or the epi-position by can obtaining antibody for example the myc label carry out fast purifying, to allow detection to active removing/blocking effect in western blotting thing, immunoprecipitate or the bioassay method.

(b).GCN4

[0345] leucine zipper motif also exists in a kind of nucleoprotein, and the transcriptional activator that wherein said nucleoprotein is totally controlled (GCN4) gene family as nitrogen metabolism in the participation yeast saccharomyces cerevisiae (S.cerevisiae) plays a role.This protein can dimerization and in conjunction with the promoter sequence that contains the GCN4 recognition sequence, thus when nitrogen is removed activated transcription.The exemplary sequence that can form the GCN4 leucine zipper of dimeric complexes is described in SEQ ID NO:180.

[0346] residue a that has been found that at the synthetic peptide of representing the GCN4 leucine zipper motif and the amino acid among the d are replaced the oligomerization characteristic that (amino acid that promptly is being described as in the sequence of SEQ ID NO:180 is replaced) changed leucine zipper motif.For example, when whole residues at position a place made Isoleucine into, leucine zipper still formed parallel dimer.When changing except that this, when whole leucine residues at d place, position made Isoleucine in addition, the peptide of generation is the parallel coiled coil of spontaneous formation tripolymer in solution.The exemplary sequence that can form trimerical this GNC4 leucine zipper motif is described in SEQ ID NO:181.Produce the peptide that four dimerizations take place with the Isoleucine replacement at whole amino acid at position d place and whole amino acid of replacing at position a place with leucine.This exemplary sequence that can form tetrameric GNC4 leucine zipper motif is described in SEQ ID NO:182.The peptide that contains these replacements still is called leucine zipper motif, forms mechanism and conventional leucine zipper motif because it is believed that oligomer, and as indicated above is identical with the GCN4 that describes in SEQ IDNO:180.

Iii. other multimerization structural domain

[0347] other multimerization structural domain is well known by persons skilled in the art, and is any multimerization structural domain of protein-protein interaction of being convenient to produce respectively and be expressed as two or more polypeptide of ECD fusions.The example that can be used to provide other multimerization structural domain of protein-protein interaction between two chimeric polyeptides includes but not limited to barnase-barstar module (referring to for example, Deyev etc. (2003) Nat.Biotechnol.21:1486-1492); Select particular proteins structural domain (referring to for example, (1998) FEBS Lett.422:259-264 such as Terskikh etc. (1997) PNAS 94:1663-1668 and Muller); Select specific peptide motif (referring to for example, (1998) FEBS Lett.432:45-49 such as (1996) J.Biol.Chem.271:7630-7634 such as de Kruif and Muller); With use disulphide bridges with enhanced stability ((2000) ProteinEng.13:725-734 such as (1996) J.Biol.Chem.271:7630-7634 such as deKruif and Schmiedl).The example of the multimerization structural domain of another type is a kind of multimerization structural domain that wherein promotes multimerization by the protein-protein interaction between the different subunit polypeptides, as hereinafter to the interactional description of PKA/AKAP.

(a).R/PKA-AD/AKAP

[0348] heteromultimeric ECD polypeptide also can utilize the adjusting subunit (R) and A kinases anchorin (AKAP of cAMP deopendent protein kinase (PKA), referring to for example, Rossi etc. (2006) PNAS103:6841-6846) protein-protein interaction between the anchoring structure territory (AD) and producing.Have two types R subunit (RI and RII) in PKA, every kind of subunit has α and β isoform.The R subunit exists as dimer, and for RII, and the dimerization structural domain is positioned at 44 aminoterminal residues (referring to for example, SEQ ID NO:183).AKAP interacts in the R of PKA subunit to regulate its activity by the interaction of their AD structural domain.AKAP only combines with dimer shape R subunit.For example, for people RII α, AD combines with the hydrophobic surface that forms from 23 aminoterminal residues.The exemplary sequence of AD is the AD1 that describes in SEQ ID NO:184, and it is from one 17 amino acid residue sequence of AKAP-IS (optimization is used for a kind of synthetic peptide of RII selective binding) deutero-.Therefore, can produce heteromultimeric ECD polypeptide by the nucleic acid of (directly or indirectly) connection coding ECD polypeptide (as HER ECD polypeptide) and the nucleic acid of coding R subunit sequence (being SEQ ID NO:183).This produces the homodimer molecule, and reason is the spontaneous formation of R subunit realization dimer.With series system, can the nucleic acid by another ECD polypeptide of will encoding be connected and produce another ECD polypeptide fusions with the nucleic acid of coding AD sequence.Behind these two assemblies of coexpression, behind the chimeric assembly of the described ECD of cotransfection in host cell, dimer shape R subunit provides the stop site for being bonded to the AD sequence, produces the heteromultimeric molecule.This binding events can for example disulfide linkage be further stable by covalent linkage.In some instances, the flexible joint residue can merge between the nucleic acid and coding multimerization structural domain of coding ECD polypeptide.In another example, the nucleic acid of coding ECD polypeptide can be merged the nucleic acid to the R subunit of encoding, wherein said R subunit contains and incorporates near the R subunit N-terminal so that covalently bound cysteine residues (referring to for example, SEQID NO:185).Similarly, the nucleic acid of coding mating partner ECD polypeptide can be merged the nucleic acid to the AD subunit of encoding, wherein said AD subunit also contains the cysteine residues of incorporating AD N-terminal and C-terminal into (referring to for example, SEQ ID NO:186).

3. chimeric ECD polypeptide

[0349] preparation is used to form the polymeric chimeric ECD polypeptide of ECD as described herein.Chimeric ECD polypeptide generally contains ECD all or part of of the CSR that directly or indirectly is connected with the multimerization structural domain.Exemplary multimerization structural domain is described herein any multimerization structural domain, includes but not limited to that immunoglobulin sequences (being constant region (Fc)), leucine zipper, compatible protein matter-protein interaction structural domain, coiled coil motif, spiral ring motif, complementary hydrophobic region, complementary hydrophilic area, size are same or analogous go into hole projection and compensatory hole and be enough to form to stablize polymeric any other multimerization structural domain.In order to allow to form multimeric molecule, the multimerization structural domain is identical or complementary between first chimeric polyeptides and second chimeric polyeptides.In case express, divide the monomer of other chimeric ECD polypeptide stably to connect to form polymer ECD polypeptide by described multimerization structural domain.

[0350] any ECD part of CSR can be used as the polymer mating partner.For example, can use any ECD mentioned above or at SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,34,127,129,131,133,135,136,137,138,139,141,143,144,146,148,149,150,151,153,155,157,159,298,200 or 301-399 those ECD described in arbitrarily or any ECD part of CSR, comprise FGFR, VEGFR, the ECD of IGF1-R and its splice variant, as describing at table 7 and at SEQID NO:194,196,198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246,248,250,252,254,256,258, the ECD of any CSR described in 260 or 262 any partly produces chimeric ECD polypeptide, and all or part of and multimerization structural domain of wherein said ECD polypeptide is connected.Generally, at least one of described ECD part, but two sometimes is all or part of (being all or part of of HER1, HER2, HER3 or HER4 molecule) of the HER family receptors that is enough to binding partner and/or dimerization that is connected with the multimerization structural domain.Describe hereinbefore and provide in arbitrarily as the example of the ECD of the HER family receptors of multimerization mating partner or its part at SEQ ID NO:10,12,14,16,18,20,22,24,26,28,30,32,129,131,136,137 and 159.In some instances, in the polymer mating partner one of at least the person be ECD all or part of of HER1 acceptor.For example, the example of polymer HER ECD polypeptide is the polymer that forms between the ECD of HER1/HER3 or HER1/HER4 or its part.In addition, be used in the chimeric ECD polypeptide that forms in the ECD polymer and can comprise the heterozygosis ECD polypeptide that is connected with the multimerization structural domain.

[0351] in an example, the ECD chimeric polyeptides comprises the ECD polypeptide directly or indirectly is connected with sequence from immunoglobulin molecules.In an example, the multimerization component is the immunoglobulin (Ig) derived structure territory from human IgG, IgM, IgD, IgM or IgA, or from other animal, includes but not limited to mouse, comparable immunoglobulin domains.In other example, the multimerization component is selected from any Fc structural domain of IgG, IgG heavy chain and IgG light chain.Generally, use the Fc structural domain of IgG, and it the IgG isotype be can be selected from, IgG1, IgG2, IgG3 and IgG4 comprised, and any allotype (allotype) in each isotype group.In most of the cases, the Fc structural domain is the Fc structural domain or derivatives thereof of IgG1, and it can be for the described characteristic of specifically wanting herein and modifies.The most common C that contains hinge area of Fc part to small part and heavy chain immunoglobulin _H2 and C _H3 structural domains.Exemplary Fc sequence as the multimerization component is described in SEQ IDNO:167, but for example depends on the length of used hinge fraction in the Fc sequence, and other Fc sequence is known.Generally, the ECD polypeptide is undertaken by directly being connected with the fusion of Fc sequence.But also can be by connecting indirectly as being undertaken by peptide linker or chemical joint (comprising the Heterobifunctional linking agent).Usually, the aminoterminal ECD of CSR (comprising any HER family receptors) or its part partly and as required merge with peptide linker and/or epi-position label at carboxyl terminal and human IgG1's Fc.

A. exemplary chimeric HER ECD polypeptide

[0352] comprise so any chimeric polyeptides for being used in the chimeric polyeptides that comprises in the polymeric formation of ECD that this paper provides, it contains the total length ECD of HER1 or its brachymemma part and Fc multimerization component and randomly contains epi-position label such as the c-myc or histidine-tagged that is useful on purifying and/or detects described HER1ECD chimeric polyeptides.Exemplary HER1-Fc chimeric polyeptides is described respectively in SEQ ID NO:38 and 40 and is encoded respectively by the nucleotide sequence described in SEQ ID NO:37 and 39.For example, the exemplary HER1-Fc chimeric polyeptides (HF110-Fc that describes as SEQ ID NO:38; HER1-501/Fc; HFD110) contain brachymemma ECD sequence (corresponding to the 1-501 amino acids of SEQ ID NO:38) at the HER1 described in the SEQ ID NO:10, this brachymemma ECD sequence effectively is connected in the sequence (corresponding to the 502-503 amino acids) that contains the restricted joint of XhoI at the aminoterminal place), the sequence (corresponding to the 509-739 amino acids) of peptide linker sequence (corresponding to the 504-508 amino acids) and Fc multimerization component.In another example, the exemplary HER1-Fc chimeric polyeptides (HF100-Fc that describes as SEQ IDNO:40; HER1-621/Fc; HFD100) contain sequence (corresponding to the 627-857 amino acids) in total length ECD sequence (corresponding to the 1-621 amino acids of SEQ ID NO:40), peptide linker sequence (corresponding to the 622-626 amino acids) and the Fc multimerization component of the HER1 described in the SEQ ID NO:12.In addition, HER1-Fc molecule (comprising for example exemplary HF110-Fc and HF100-Fc molecule) can randomly contain the epi-position label.For example, also can randomly comprise myc epi-position set of tags (corresponding to the 740-749 amino acids of SEQ ID NO:38) at the exemplary HF110-Fc molecule described in the SEQ ID NO:38.In another example, also can randomly comprise His epi-position label or other label (being HFD100T) at the HF100-Fc molecule described in the SEQ ID NO:40.The total length ECD sequence (corresponding to the 1-621 amino acids of SEQ IDNO:406) of HER1 is described and contained to exemplary HFD100T molecule in SEQ ID NO:406, this total length ECD sequence effectively is connected in the sequence (corresponding to the 622-623 amino acids) that contains the Xbal joint at the aminoterminal place), peptide linker sequence (corresponding to the 624-627 amino acids), the sequence (corresponding to the 628-858 amino acids) of Fc multimerization component, sequence (corresponding to the 859-860 amino acids) and the 6 * histidine-tagged sequence (corresponding to the 861-866 amino acids of SEQ ID NO:406) that contains the AgeI joint.

[0353] comprise so any chimeric polyeptides for being used in the chimeric polyeptides that comprises in the polymeric formation of ECD that this paper provides, it contains the total length ECD of HER2 or its brachymemma part and Fc multimerization component and randomly contains epi-position label such as the c-myc label or histidine-tagged that is useful on purifying and/or detects described HER2ECD chimeric polyeptides.Exemplary HER2-Fc chimeric polyeptides is described in SEQ IDNO:42 and nucleotide sequence coded by described in the SEQ ID NO:41.Exemplary HER2-Fc chimeric polyeptides (HF200-Fc as SEQID NO:40 description; HER2-650/Fc; HFD200) contain total length ECD sequence (corresponding to the 1-628 amino acids of SEQ ID NO:42) at the HER2 described in the SEQ ID NO:18, this total length ECD sequence effectively is connected in the sequence (corresponding to the 629-633 amino acids) that contains the peptide linker sequence and the sequence (corresponding to the 634-864 amino acids) of Fc multimerization component at the aminoterminal place.In addition, HER2-Fc molecule (comprising for example exemplary HF200-Fc molecule) can randomly contain the epi-position label.

[0354] comprise so any chimeric polyeptides for being used in the chimeric polyeptides that comprises in the polymeric formation of ECD that this paper provides, it contains the total length ECD of HER3 or its brachymemma part and Fc multimerization component and randomly contains epi-position label such as the c-myc label or histidine-tagged that is useful on purifying and/or detects described HER3ECD chimeric polyeptides.Exemplary HER3-Fc chimeric polyeptides is described respectively in SEQ IDNO:44 and 46 and is encoded respectively by the nucleotide sequence described in SEQ ID NO:43 and 45.For example, the exemplary HER3-Fc chimeric polyeptides (HF310-Fc as describing among the SEQ ID NO:44; HER3-500/Fc; HFD310) contain brachymemma ECD sequence (corresponding to the 1-500 amino acids of SEQ ID NO:44) at the HER3 described in the SEQ ID NO:20, this brachymemma ECD sequence effectively is connected in the sequence (corresponding to the 501-505 amino acids) that contains the peptide linker sequence and the sequence (corresponding to the 506-736 amino acids) of Fc multimerization component at the aminoterminal place.In another example, the exemplary HER3-Fc chimeric polyeptides (HF300-Fc as describing among the SEQ ID NO:46; HER3-621/Fc; HFD300) contain total length ECD sequence (corresponding to the 1-621 amino acids of SEQ ID NO:46) at the HER3 described in the SEQ ID NO:26, this total length ECD sequence effectively is connected in the sequence (corresponding to the 622-626 amino acids) that contains the peptide linker sequence and the sequence (corresponding to the 627-857 amino acids) of Fc multimerization component at the aminoterminal place.In addition, HER3-Fc molecule (comprising for example exemplary HF310-Fc and HF300-Fc molecule) can randomly contain the epi-position label.

[0355] comprise so any chimeric polyeptides for being used in the chimeric polyeptides that comprises in the polymeric formation of ECD that this paper provides, it contains the total length ECD of HER4 or its brachymemma part and Fc multimerization component and randomly contains epi-position label such as the c-myc or histidine-tagged that is useful on purifying and/or detects described HER4ECD chimeric polyeptides.Exemplary HER4-Fc chimeric polyeptides is described in SEQ ID NO:48 and nucleotide sequence coded by described in the SEQ ID NO:47.As the exemplary HER4-Fc chimeric polyeptides (HF400-Fc that describes among the SEQ ID NO:48; HER4-650/Fc; HFD400) contain total length ECD sequence (corresponding to the 1-625 amino acids of SEQ IDNO:48) at the HER4 described in the SEQ ID NO:32, this total length ECD sequence effectively is connected in the sequence (corresponding to the 626-630 amino acids) that contains the peptide linker sequence and the sequence (corresponding to the 631-861 amino acids) of Fc multimerization component at the aminoterminal place.In addition, HER4-Fc molecule (comprising for example exemplary HF400-Fc molecule) can randomly contain the epi-position label.

The E.ECD polymer

[0356] the ECD polymer that provides herein contain by its separately the multimerization domain interaction and at least two ECD polypeptide of stable bond.The ECD polymer can be same polymer, but modal be the different heteromultimeric of wherein polymeric ECD polypeptide fraction.The ECD heteromultimeric is general acceptor therapeutical agent, comprises general HER therapeutical agent.Several epi-positions on the ECD polymer target HER family member.Therefore, the ECD multimeric molecule of gained is regulated, and suppresses the activity of two or more cognations or interaction CSR usually.Adjusting can be by carrying out with one or more ligand interactions and/or by the dimerization with the related acceptor of total length or other interaction CSR.Therefore, one or more parts of each polypeptide in described polymer ECD polypeptide and each the ECD polypeptide, usually two or more parts in conjunction with and/or with cell surface on related acceptor or interaction receptor dimerizationization.Therefore, the ECD polypeptide polymer of gained is as the antagonist of related CSR.This type of antagonist is used for the treatment of because of the part combination of related acceptor and/or the disease due to activating.

[0357] the most common inactive form that is in of HER family receptors, only having at the most, 5% the film HER molecule of striding is in active configuration.Under the normal circumstances, for total length HER acceptor, the mechanism that the decision inactive form is converted to activity form is the part keying action.The part keying action makes acceptor molecule direction reorientation, forces the dimerization arm to become the conformation have with another kind of HER molecule dimerization potentiality from the constraint conformation transition.All or part of and the multimerization structural domain of ectodomain that can be by forcing the HER molecule as but be not limited to the activity form that Fc fragment dimerization is simulated the HER molecule.Therefore, the fusion of HER ECD and multimerization structural domain forces this HER molecule to take the part dependent/non-dependent to activate (being non-constraint) conformation, is similar to the HER2 molecule of constitutive activation.For example, when described multimerization structural domain is under the situation of Fc molecule, chimeric polyeptides is expressed can be produced as homodimer, and wherein the interaction by the Fc structural domain forces dimerization between two expressed monomer polypeptide.In some cases, this homodimer is compared with the monomeric form of this ECD, can cause the characteristic of improving of ECD polypeptide.In an example, HER ECD can produce the high-affinity receptor complex body that can have high ligand binding affinity all or part of and being connected of Fc multimerization structural domain, and the monomeric form of this ECD can not binding partner.For example, as described in example 4 above, the monomer ECD molecule that contains the complete ECD (i.e. 1-621 amino acids) of ripe HER1 acceptor only shows with the minimum of EGF and combines.When this ECD polypeptide was connected with Fc multimerization structural domain, homodimer HER1ECD molecule and EGF bonded ability obviously increased.

[0358] proposes to utilize this same mechanism to stablize the heteromultimeric of CSR molecule, be used to produce the high-affinity receptor therapeutical agent of general acceptor ECD polymer (comprising general HER ECD polymer) as wide spectrum.

[0359] therefore, belonging to general acceptor therapeutical agent active is to have the high-affinity soluble receptors complex body of avidity more than a kind of part.Therefore, general acceptor polymer can be as the part trap with isolated part (comprising the somatomedin part).Can be that known and polymeric ECD polypeptide combines or interactional those parts by the isolated part of described ECD polymer.When the polymeric component of ECD contains under the situation of the one or more all or part of ECD that are enough to binding partner in the HER molecule, this ECD polymer can completely cut off potentially any one or multiple in the ligand combination described in the table 6.For example, if described polymer is the combination of HER1 and HER4, then at least 10 kinds of different ligands can be targets.Perhaps, if described polymer is the combination of HER1 and HER3, then following any one or multiple part can be isolated by this multimeric molecule, and wherein said part comprises that EGF, amphiregulin, TGF-α, β tunicin, heparin are in conjunction with EGF, epiregulin or neuregulin 1 or 2 (Her regulates albumen 1 or 2).Therefore, at one of polymeric ECD polypeptide fraction is HER molecule (for example HER1) and all the other components are under all or part of certain situation of another CSR, this ECD polymer can with at least 7 kinds of ligand interactions, 6 kinds in the wherein said part is by the part of the ECD identification of HER1, is discerned by mating partner ECD polypeptide and remain one or more parts.Extra ligand can be the involved in diseases process (as but be not limited to proliferative disease, vasculogenesis disease or inflammatory diseases) somatomedin or other ligand molecular.The example of this type of part comprises VEGF, FGF, Regular Insulin, HGF, angiogenesis factor and other part.In extra example, can design the ECD polymer that from the combination of one or more heterozygosis ECD polypeptide, produces, thus this ECD polymer contain at 2 kinds, 3 kinds or how to enough ligand binding moieties of 4 kinds of different CSR and therefore have make 2,3,4,5,6,7,8,9,10,11,12,13,14, ability that 15 kind or more kinds of part isolate from its total length CSR separately.

[0360] the ECD polymer that provides herein also can be by carrying out with cognation or interaction transmembrane receptor direct interaction to the adjusting of CSR.For example, by receptor dimerizationization together producing total length homodimer acceptor and total length heterodimer acceptor, thereby make and convene and the catalytic afterbody self-phosphorylation of downstream signal effect and activate almost all RTK acceptors in order to effector.For example, amplify and diversified a kind of mechanism as HER signal conduction, the HER acceptor is with multiple combination generation dimerization.Observed whole combinations of total length HER acceptor, wherein HER2 is as modal dimerization mating partner.Therefore, to CSR, especially any interference of RTKs (comprising HER) dimerization ability will destroy receptor-mediated signal conduction.The example that can destroy the molecule of CSR dimerization is ECD polymer, HER ECD polymer especially.Because of merge the polymeric activation high-affinity of HER ECD form indication a kind of " back-to-back " conformation that (for example merging with Fc albumen) produces with the multimerization structural domain, wherein whether no matter by the part combination, the dimerization arm in described " back-to-back " conformation representative structure territory II is in the configuration that interacts in transmembrane receptor.This interaction can disturb total length HER acceptor striding the ability of place, film district with another total length HER acceptor coupling, thereby suppresses the activation of this receptor.Other CSR ECD (comprising other RTK ECD polymer) that disturbs related receptor dimerizationization similar interaction and restraining effect have been conceived.Therefore, remove isolated part or replace, general acceptor polymer provided herein can with one or more receptor dimerizationizations to suppress its activity.As described below, the activity of transmembrane receptor can be assessed by assay method (including but not limited to phosphorylation method or cell proliferation method).

[0361] general, the ECD polymer is a dimer, but also can be tripolymer or bigger polymer, and this depends on for example for forming the selected multimerization structural domain of polymer.For example, the Fc structural domain will produce dimer molecule.In addition, the multimerization structural domain as leucine zipper also produces dimer ECD molecule usually.Yet the variant form of leucine zipper for example variant GCN4 can be used for producing tripolymer or bigger polymer.Under the situation that needs high-order multimerization structural domain, can select the multimerization structural domain in view of the above.Those skilled in the art are provided by the structure organization form of exemplary multimerization structural domain (as any multimerization structural domain that provides herein).

A. the ECD's of total length HER1ECD and another CSR is all or part of

[0362] provide a kind of ECD polymer herein, its total length ECD that contains the HER1 that is connected with the multimerization structural domain is all or part of as second polypeptide as the ECD's of first polypeptide and another CSR that also is connected with the multimerization structural domain.The multimerization structural domain of first and second polypeptide can be identical or different, but under the different situation of multimerization structural domain, described multimerization structural domain is complementary, to allow stable protein-protein interaction between the polymer component.The example of total length HER1ECD polypeptide is HF100 or its allelic variant or species variant, and wherein said HF100 comprises the 1-621 amino acids as the ripe HER1 acceptor of describing among the SEQ ID NO:12.The ECD of second polypeptide can be ECD all or part of of any CSR any CSR, that relate in particular to lysis, and wherein said lysis relates to propagation, vasculogenesis or inflammation, as long as this ECD polypeptide is not a total length HER2 molecule.Yet the ECD of second polypeptide can be the part that is enough in the HER2 molecule with the ECD of other HER molecule dimerization.The example of the HER2ECD polypeptide of brachymemma is included in HF220 molecule of describing among the SEQ ID NO:18 and HF210 molecule and the allelic variant of describing thereof in SEQ IDNO:16.In some cases, the ECD polymer that preferably contains the HER2 molecule HF210 of total length HER1 molecule and brachymemma, because the module 2-5 that exists in the subdomain IV of this brachymemma HER2 molecule influences the dimerization ability of this brachymemma HER2 molecule, as describing among the embodiment 5.

[0363] contain total length HER1ECD as the ECD polymer of first polypeptide can be with the ECD of HER3 or HER4 acceptor all or part of as its second polypeptide fraction.A kind of so specifically ECD polymer of this kind ECD polymer, it has in conjunction with the ability from two or more parts in EGF, amphiregulin, TGF-α, β tunicin, heparin associativity EGF or epiregulin and one or more neuregulins.This peptide species also can with any one or multiple dimerization of HER acceptor.For example, has person's's (comprising its any isoform) the ability arbitrarily that combines among the neuregulin 1-4 with the ECD polymer of all or part of combination of HER4ECD polypeptide.The polymeric example of this kind ECD is a kind of like this ECD polymer, wherein this polymeric first polypeptide be the total length HER1ECD (i.e. the HF100 that in SEQ ID NO:12, describes or its allelic variant) and second polypeptide be can binding partner brachymemma HER4 polypeptide, for example, but be not limited to HF410 molecule or its allelic variant in SEQID NO:28, described.The polymeric HER4 part of this ECD also can be a kind of like this total length HER4 molecule, and it contains the complete ECD part (being HF400) just like the ripe HER4 acceptor of describing in SEQ ID NO:32.In some instances, the multimerization of HER1ECD and all or part of HER4ECD is mediated by the multimerization structural domain.For example, the exemplary chimeric polyeptides of can coexpression in SEQ ID NO:40, describing (HF100-Fc or as in SEQ ID NO:406, describe its add the epi-position label form) and the chimeric polyeptides (HF400-Fc) in SEQ ID NO:48, described) to produce multimeric molecule.

[0364] yet, usually, the all or part of associating of total length HER1ECD polypeptide and HER3ECD polypeptide in polymer, thus the polymer of gained have in conjunction with in neuregulin 1 or 2 arbitrarily person's (comprising its any isoform) and/or with cell surface on the ability of one or more HER receptor dimerizationizations arbitrarily.The polymeric example of this kind ECD is so a kind of ECD polymer, and wherein this polymeric first polypeptide is that total length HER1ECD and this polymeric second polypeptide are all or part of of HER3 polypeptide.HER1 and HER3 are two kinds of acceptors in the acceptor of modal overexpression.Therefore, the ECD polymer of HER1 and HER3 has such ability, promptly catch part with the receptors bind of two kinds of modal overexpressions, ignore and HER4 (being neuregulin 3 and neuregulin 4) some parts of bonded wherein said HER4 does not confirm to have extensive activity in cancer (Barnes etc. (2005) Clin Cancer Res 11:2163-8 simultaneously; Srinivasan etc. (1998) J Pathol.185:236-45).

[0365] in an example, as second polypeptide, wherein every peptide species is connected with the multimerization structural domain HER1ECD that the ECD polymer of HER1ECD and HER3ECD can comprise total length as the HER3ECD polypeptide of first polypeptide and brachymemma.As mentioned, the example of total length HER1 molecule is HF100 molecule (SEQ ID NO:12) or its allelic variant.Conceived brachymemma HER3ECD polypeptide arbitrarily, as long as its keeps the ability in conjunction with any one or multiple and/or dimerization in neuregulin 1 or 2 isoforms.The example of the HER3ECD polypeptide of this brachymemma is included in the HF310 that describes among the SEQ ID NO:20, the p85HER3 that describes or the ErbB3-519 that describes or its allelic variant in SEQ ID NO:24 in SEQ ID NO:22.For example, the exemplary chimeric polyeptides of can coexpression in SEQ ID NO:40, describing (HF100-Fc, or as in SEQ IDNO:406, describe its add the epi-position label form) and the exemplary chimeric polyeptides (HF310-Fc) in SEQ ID NO:44, described to produce multimeric molecule.

[0366] in another example, as second polypeptide, wherein every peptide species is connected with the multimerization structural domain HER1ECD (as HF100 molecule (SEQ ID NO:12)) that the ECD polymer of HER1ECD and HER3ECD can comprise total length as first polypeptide and total length HER3ECD molecule.A kind of exemplary total length HER3ECD molecule comprises the 1-621 amino acids as the ripe HER3 total length acceptor of describing in SEQ ID NO:26 (HF300).The total length ECD polymer of HER1/HER3 can divide other multimerization domain interaction to connect by it.The multimerization structural domain of the first total length HER1ECD polypeptide and the 2nd HER3ECD polypeptide can be identical or different, but under the different situation of multimerization structural domain, described multimerization structural domain is complementary, to allow stable protein-protein interaction between the polymer component.In an example, first and second polypeptide are connected with Fc fragment (such as but not limited to IgG1 Fc fragment) separately.The total length HER1 that is connected with the Fc fragment and the example of HER3ECD chimeric polyeptides are described in SEQ ID NO:40 or SEQ ID NO:46 respectively.Therefore, the HER1/HER3ECD polymer can form behind such nucleotide sequence coexpression, wherein said nucleic acid sequence encoding have SEQ IDNO:40 (or as in SEQ ID NO:406, describe its add the epi-position label form) and SEQ IDNO:46 (or add epi-position label form as its that in SEQ ID NO:407, describe) described in the polypeptide or the allelic variant of aminoacid sequence.In addition, as required, can contain the epi-position label (as histidine-tagged c-myc) of interpolation in one of the sequence of the chimeric polyeptides described in SEQ ID NO:40 or the SEQ IDNO:46 or both, it can incorporate the HER1/HER3ECD polymer of gained subsequently into.For example, can produce such polymer, one or both chimeric polyeptides wherein have the aminoacid sequence described in SEQ IDNO:406 and/or the SEQ ID NO:407.

[0367] in addition, the CSR ECD polypeptide that can to combine to form polymeric second polypeptide of ECD with total length HER1ECD can be random length combines with part and/or the ability of dimerization as long as this 2nd ECD polypeptide keeps.Can be in polymer include but not limited to any other CSR all or part of of VEGFR1 or 2, FGFR1-4, IGF1-R, Tie-1, Tie-2, MET, PDGFRA or B, PDGFRB, Epha1-8, TNFR, RAGE or involved in diseases process with the exemplary ECD polypeptide of total length HER1ECD polypeptides in combination, wherein said lysis is feature with propagation, vasculogenesis or inflammatory component.The exemplary sequence of the total length ECD polypeptide of exemplary CSR is described in table 7.To as described in some RTKs that exemplify, its part that is enough to binding partner is known in the art as herein.If unknown, then can be in conjunction with needed subdomain based on the comparison associated receptor and/or by using recombinant DNA technology to determine on experience ground together with the part binding assay for part.Can determine wherein said drug targeting single cell surface receptor to the resistance contribution experience ground of medicine based on disease to be treated and/or based on CSR for being used in other CSR and the ECD part thereof conceived in the polymer with total length HER1ECD polypeptide.In addition, the alternative splicing isoform of CSR can be used in the polymer with total length HER1ECD polypeptide arbitrarily.The example of these alternative splicing isoforms is as described in example 11 above and is described as the IGF-1R isoform of SEQ ID NO:298-300.Other CSR isoform that can be used in the ECD polymer is described in the item arbitrarily at SEQ IDNO:301-384.

B. the ECD component of two or more brachymemmas

[0368] also be provided at the ECD multimeric molecule that forms between the ECD part of two or more brachymemmas of any CSR ECD herein, wherein said CSR's is the HER molecule that shortens one of at least.Generally, one of at least (the ECD parts of general two brachymemmas) of brachymemma ECD part be enough to binding partner and/or with the CSR dimerization, unless described brachymemma ECD polypeptide is derived from HER2, in this case this polypeptide portion must be at least can with another cell surface receptor dimerization.This molecule can be by regulating, and normally suppresses one or more HER acceptors and/or another CSR and work as general acceptor therapeutical agent.Regulating effect can be by isolated part and/or by carrying out with described CSR dimerization.In some instances, each in first and second polypeptide fraction can directly connect or connect indirectly through the multimerization structural domain.The multimerization structural domain of first and second polypeptide can be identical or different, but under the different situation of multimerization structural domain, described multimerization structural domain is complementary, to allow stable protein-protein interaction between the polymer component.Described ECD polymer can form between the HER of two shortenings polypeptide, and wherein said shortening HER polypeptide generally is the brachymemma ECD polypeptide that keeps its part combination and/or dimerization ability in the different HER acceptors.Those skilled in the art can determine to be ready to use in the part that produces the polymeric HER molecule of described ECD, thereby at least one (HER polypeptide of general two shortenings) keep the ability of their binding partners and/or dimerization.For example, normally, the HER1 of brachymemma, 2 or 3 molecules contain in the enough parts, subdomain II of binding partner among subdomain I and the III and are used for enough parts of dimerization and the module I at least of subdomain IV.The HER2 molecule of brachymemma contains the 2-5 of module at least of the enough at least parts of subdomain I, II and III and subdomain IV usually with dimerization.

[0369] conceived the arbitrary combination of the brachymemma HER ECD that is used in the heterozygosis ECD polymer.For example, brachymemma HER1ECD polypeptide can with HER2, HER3 or the HER4 polypeptides in combination of brachymemma; The HER2ECD polypeptide of brachymemma can with the HER3 or the HER4ECD polypeptides in combination of brachymemma; Can make up with the HER4ECD of brachymemma with the HER3 polypeptide of brachymemma.The example of the HER polypeptide of brachymemma comprises the described HER of brachymemma arbitrarily polypeptide herein, has for example described brachymemma HER polypeptide arbitrarily in SEQ ID NO:10,14,16,20,24,28,30,34; The alternative splicing variant of HER acceptor, for example any alternative splicing variant of in SEQ ID NO:22,127,129,131,133,135,136,137,138,139,141,143,144,146,148,149,150,151,153,155,157 or 159, describing; Or its any allelic variant or species variant.In an example, Hess reach spit of fland molecule or its variant (as describing in arbitrarily) at SEQ ID NO:135 or 385-399 can with any other brachymemma ECD HER polypeptides in combination.In an example, as second polypeptide, wherein every peptide species is connected with the multimerization structural domain part that the ECD polymer can comprise HER1ECD as the part of first polypeptide and HER3ECD polypeptide.The example of the HER1 molecule of brachymemma is HF110 (SEQ ID NO:10) or its allelic variant.The example of the HER3 molecule of brachymemma is HF310 (SEQ ID NO:20), p85-HER3 (SEQ ID NO:22), or ErbB3-519 (SEQ ID NO:24) or its allelic variant.For example, the exemplary chimeric polyeptides (HER1-501/Fc that can coexpression in SEQ IDNO:38, describes; HFD110 is with or without the c-myc label) and the chimeric polyeptides (HER3-500/Fc that in SEQ ID NO:44, describes; HFD310) to produce multimeric molecule as the HER1/HER3ECD heteromultimeric of brachymemma.

[0370] in other example, the HERECD polypeptide that ECD polymer provided herein can contain brachymemma is as first polypeptide and be not that the another kind of brachymemma CSR ECD polypeptide of acceptor HER family is as second polypeptide.As above, the HER ECD polypeptide of brachymemma can be the part of the ECD of HER1, HER2, HER3 or HER4 acceptor, if polymeric at least one polypeptide fraction (general two polypeptide fraction) combines with part and/or with the transmembrane receptor dimerization.The HER family receptors of exemplary brachymemma includes but not limited at SEQ ID NO:10,14,16,20,22,24,26,28,30,34,127,129,131,133,135,136,137,138,139,141,143,144,146,148,149,150,151,153,155,157,159 or arbitrarily brachymemma HER family receptors or its any allelic variant or the species variant of 385-399 described in arbitrarily.Chimeric ECD polypeptide can comprise ECD polypeptide all or part of of another cell surface receptor that is connected with the multimerization structural domain.Brachymemma ECD CSR is combined in to obtain herein conceiving with formation and has the ECD polymer that shortens HER ECD polypeptide arbitrarily, and can determine based on following aspect experience ground: disease to be treated, divide the contribution of other CSR to this disease, at the known ligand of this CSR, CSR chemical sproof contribution and other factors, wherein said drug targeting single cell surface receptor to medicine.IGF-R1, VEGFR (being VEGFR1 or VEGFR2), FGFR (being FGFR1, FGFR2, FGFR3 or FGFR4), TNFR, PDGFRA or PDGFRB, MET, Tie (Tie-1 or Tie-2), Eph acceptor or RAGE are above being described and included but not limited to the example of CSR.The exemplary sequence of the total length ECD polypeptide of exemplary CSR is described in table 7.To as described in some RTKs that exemplify, its part that is enough to binding partner is known in the art as herein.If unknown, then can be in conjunction with needed subdomain based on the comparison associated receptor and/or by using recombinant DNA technology to determine on experience ground together with the part binding assay for part.In addition, the alternative splicing isoform of CSR can be used in the polymer arbitrarily.The example of these alternative splicing isoforms is the isoforms as the IGF-1R that describes among the embodiment 11 and describe as SEQ ID NO:298-300.Other CSR isoform that can be used in the ECD polymer is described in the item arbitrarily at SEQ ID NO:301-384.

C. heterozygosis ECD polymer

[0371] provides such ECD polymer herein, one of wherein said polymeric chimeric ECD polypeptide or both are such heterozygosis ECD molecules, and it contains ECD ligand binding domains and/or the dimerization structural domain partly from any two or more CSR that is connected with the multimerization structural domain.This type of heterozygosis ECD molecule is as discussed herein above.For example, a kind of such heterozygosis ECD polypeptide contain from the subdomain II of HER2 and from identical or different acceptor, from HER1,3 or 4 subdomain I and III.Other combination of heterozygosis ECD can be based on the activity of the known subdomain of relevant CSR and experience ground is determined.Generally, at least one subdomain of a kind of heterozygote is given gained ECD polymeric dimerization ability in the described ECD heterozygote.Two or more identical or different heterozygosis ECD molecules can link together directly or indirectly.In an example, the fusion that heterozygosis ECD molecule can be by the first heterozygosis ECD polypeptide and multimerization structural domain and the second heterozygosis ECD polypeptide are connected with fusion with a kind of or complementary multimerization structural domain.Heterozygosis ECD is polymeric to be realized after being formed on each own coding nucleic acid of coexpression first and second polypeptide.

[0372] in addition, can form such ECD polymer, in the wherein said polymeric polypeptide only a polypeptide be that the heterozygosis ECD and second polypeptide are all or part of of any other CSR molecule, ECD polypeptide of total length arbitrarily for example mentioned above or the ECD of brachymemma arbitrarily polypeptide mentioned above.Generally, another CSR ECD polypeptide is alternative splicing isoform or its allelic variant of all or part of, the HER family receptors of HER family receptors.Other CSR except that the HER family receptors can and can suitably select wherein said drug targeting single cell surface receptor based on the chemical sproof cognation of disease to be treated and/or described CSR and medicine with heterozygosis ECD combination.

D. identical or from identical CSR deutero-ECD component

[0373] also provide herein by isolated part and/or by with the direct interaction of related CSR or other interaction CSR at least a, same polymer or the heteromultimerics regulated of two or more CSR sometimes.This type of ECD polymer can be the same polymer of an ECD polypeptide that is connected with the multimerization structural domain and the 2nd ECD polypeptide that is connected with the multimerization structural domain, generally be homodimer that wherein said first and second polypeptide are identical.Perhaps, this type of ECD polymer can be such heteromultimeric, wherein the first and second ECD polypeptide each derive since identical related CSR, but they are different.Generally, but be not total be identical or under same receptor deutero-situation so in described ECD component, only singly plant the activity of acceptor by target.For example, in some cases, a kind of ECD polymer is to be used to regulate the active candidate therapeutic agent of total length IGF1-R at least, wherein said ECD polymer with total length IGF1-R ECD (promptly corresponding to SEQ IDNO:260 31-935 amino acids) as first polypeptide and with the polypeptide identical or its brachymemma polypeptide or isoform with first polypeptide as second polypeptide.In another example, containing Hess, to reach the same polymer of spit of fland and/or another kind of HER2ECD component or heteromultimeric be for example by directly and the total length HER1 on the cell surface, the material standed for of that HER3 or HER4 acceptor interaction and being used to regulated is at least a (but generally two or more) CSR.

F. produce the method and the ECD polymer that produces gained of the nucleic acid of the chimeric ECD polypeptide fusions of coding

[0374] can use at ECD, its part, especially enough be used for part in conjunction with and/or the part of receptor dimerizationization and alternative splicing part between produce any usability methods of chimeric polyeptides.Similarly, can realize by any method known to those skilled in the art forming polymer from chimeric polyeptides.As described, described polymer generally comprises from least a HER family member (generally being HER1 or HER3 or HER4) and the 2nd HER family member's ECD and/or from CSR (as IGF1-R, VEGFR and FGFR) or from the ECD of other acceptor that relates to tumour generation or inflammatory or other lysis.

[0375] be provided for producing the illustrative methods of the nucleic acid molecule of coding ECD chimeric polyeptides, wherein said ECD chimeric polyeptides comprises the ECD polypeptide that is connected with multimerization structural domain described herein directly or indirectly.These class methods comprise the external synthetic method that is used for nucleic acid molecule, make up method and external connection is isolating and/or synthetic nucleic acid fragment method as PCR, synthetic gene.Can be by the cloning process separation of C SR nucleic acid molecule of (comprising HER family receptors or other RTKs), wherein said cloning process comprises that pcr amplification is from the RNA of cellular segregation and DNA and by screening by hybridization nucleic acid molecule library and/or expression screening method.

[0376] ECD polypeptide or its part can use synthetic method in the external and body to produce from the nucleic acid molecule of coding ECD polypeptide.ECD polymer (it contains being connected of one or more chimeric ECD polypeptide (for example ECD-Fc protein blend compound) or ECD and any other multimerization structural domain) can be expressed the back and be produced in being fit to produce the polymeric any biology of ECD polypeptide of aequum and desired form, wherein said ECD polypeptide polymer be use and treat needed.Expressive host comprises protokaryon and eukaryote, as intestinal bacteria (E.coli), yeast, plant, insect cell, mammalian cell (comprising the human cell line) and transgenic animal.ECD polypeptide or ECD polypeptide polymer also can separate from express described ECD polypeptide or the polymeric cell of ECD polypeptide and biology, described cell and the biological cell that wherein is recombinantly produced the ECD polypeptide and biological and wherein those cells and the biology of synthetic isoform (as the isoform by alternative splicing incident generation genome encoding) without recombinant means of comprising.

1. synthetic gene and synthetic polypeptide

[0377] nucleic acid molecule of coding ECD polypeptide can pass through method known to those skilled in the art, uses the synthetic gene synthesis method synthetic.In these class methods, the peptide sequence of ECD is carried out " replying translation (back-translated) " to produce one or more nucleic acid molecule of coding ECD or its part.The nucleic acid molecule of described answer translation is synthesized one or more dna fragmentations subsequently as by use automated DNA synthetic technology.Connect described fragment subsequently effectively to form the nucleic acid molecule of coding ECD polypeptide.Chimeric ECD polypeptide can be by the ECD polypeptide of will encoding nucleic acid molecule and extra nucleic acid molecule (as other nucleic acid of any nucleic acid molecule of coding multimerization structural domain or coding epi-position or fusion tag, be used to regulate adjusting sequence, carrier and other peptide coding nucleic acid molecule of transcribing and translating) be connected and produce.ECD coding property nucleic acid molecule also can be effectively with other fusion tag or marker as being used for the spike purposes, comprise that the radio-labeled thing partly is connected with fluorescence.

[0378] replying translation process uses genetic codon to obtain the nucleotide gene sequence at any desired polypeptides (as the ECD polypeptide).Genetic codon has degeneracy, and 64 kinds of codons are specified 20 seed amino acids and 3 kinds of terminator codons.This degeneracy provides nucleic acid design and aborning handiness, for example allows to incorporate restriction site into so that connect nucleic acid fragment and/or settle unique identity flag sequence in every kind of synthetic fragment.The degeneracy of genetic codon also allows the designing nucleic acid molecule to avoid undesired nucleotide sequence, comprises undesired restriction site, donor splicing site or receptor site or can be detrimental to other nucleotide sequence of efficient translation.In addition, biological GC and the AT nucleotide proportion of having a liking for use of specific cryptosystem and/or definition sometimes partially.Therefore, the degeneracy of genetic codon allows to be designed to express the nucleic acid molecule that is customized in particular organisms or category.In addition, can optimize the nucleic acid molecule that (or unoptimizable) is designed for different expression levels based on sequence.Replying translation is undertaken by the codon of selecting coded polypeptide.This class process can use genetic code sublist and peptide sequence to carry out with manual type.Perhaps, the program (comprising the software that can openly obtain) that can use a computer produces the nucleotide sequence of replying translation.

[0379] for synthetic nucleic acid molecule of replying translation, can use this area to be used for any methods availalbe of nucleic acid synthetic.For example, the corresponding various oligonucleotide of fragment of the nucleotide sequence of and coding ECD synthetic and in renaturation or hybridization, mixing by the standard automatic mode.This class oligonucleotide is so synthetic, thereby renaturation process causes gene to utilize complementary sequence formed plyability strand overhang of when pairing (about 100 length of nucleotides usually) by described oligonucleotide being made by manufacturers or users.Utilize the single Nucleotide " otch " among ligation (for example phage T4DNA ligase enzyme) the sealing duplex DNA.The restriction endonuclease joint sequence can for example be used for synthetic gene is inserted in arbitrary carrier of the multiple recombinant DNA carrier that is applicable to protein expression subsequently.In another kind of similar methods, prepare a series of plyability oligonucleotide by the oligonucleotide chemical synthesis.The renaturation of these oligonucleotide produces notched dna structure.Can use the catalytic DNA of enzyme (as dna polymerase i) to synthesize and fill and lead up these breach, and the use ligation seals any otch in the duplex structure.Can use PCR and/or other DNA cloning technology formed linear DNA duplex that increases.

[0380] extra nucleotide sequence can be connected with the nucleic acid molecule of coding ECD, thereby generation ECD fusions, comprise containing and be intended to clone synthetic gene to the joint sequence that carries intravital restriction endonuclease site, wherein said carrier for example is protein expression carrier or the carrier that is designed for the dna sequence dna of amplification coding core protein.In addition, the extra nucleotide sequence of having described functional DNA elements can be effectively be connected with the nucleic acid molecule of coding ECD.The example of this type of sequence includes but not limited to design and is intended to promote that intracellular protein expression promoter sequence or design are intended to promote the precursor sequence of protein secreting.Other example of the nucleotide sequence that can effectively be connected with the nucleic acid molecule of coding ECD comprises the sequence of being convenient to peptide purification and/or detection.For example, fusion tag such as epi-position label or fluorescence part can merge with isoform or be connected.Extra nucleotide sequence also can be connected with the nucleic acid molecule of coding ECD as the sequence of describing the protein bound zone.This type of zone includes but not limited to promote to absorb the sequence of ECD polypeptide to the pharmacokinetics of particular target cell or enhancing synthetic gene.

[0381] the ECD polypeptide also can use the automated peptide synthesis synthesis method synthetic.The peptide sequence that (in silico) generates on clone's property and/or the silicon chip can synthesize piecemeal and chemically couple together subsequently.Perhaps, it is synthetic that chimeric molecule can be used as single polypeptide.This type of polypeptide subsequently can described in this article assay method and treatment use middle use.

2. clone and the method for separating the ECD polypeptide

[0382[can use known in the art being used to clone the nucleic acid molecule of cloning or separate the ECD that encodes with any methods availalbe of isolated nucleic acid molecule, comprises the nucleic acid molecule of coding ECD fusions.These class methods comprise the pcr amplification method and the library screening method of nucleic acid, comprise the nucleic acid hybridization sieve method, based on the sieve method of antibody with based on active sieve method.

[0383] also can use the library screening method to separate the nucleic acid molecule of coding ECD polypeptide.For example, can by with the making nucleic acid molecular hybridization screening of coding ECD polypeptide or its part as the representative of the cDNA nucleic acid library of expressed rna transcript.For example, the nucleotide sequence of the part (for example part of the module 1 of the structural domain IV of the ECD of HER family) of coding ECD polypeptide can be used for the molecule that contains structural domain IV based on screening with the hybridization of homologous sequence.

[0384] the expression library sieve method can be used for separating the nucleic acid molecule of coding ECD polypeptide.For example, expression library can be used the antibody screening of the part of specific ECD of identification or ECD.Can obtain and/or prepare such antibody, its specifically in conjunction with the ECD polypeptide or in ECD contained zone or peptide.Can be used for screening the expression library of the nucleic acid molecule that contains coding ECD (as the ECD of HER family receptors) specifically in conjunction with the antibody of ECD polypeptide.The method of preparation and separation antibody (comprising polyclone and monoclonal antibody and fragment thereof) is well known in the art.Preparation also is well known in the art with the method for separating recombinant antibodies and synthetic antibody.For example, this antibody-like can use the solid phase method of peptide synthesis to make up maybe can to use in specificity generation of recombinating in conjunction with the nucleotide sequence of the antigen binding site in the antibody of candidate's polypeptide and amino acid sequence information.Antibody also can contain combinatorial library variable heavy chain and variable light chain or that contain its antigen-binding portion thereof by screening and obtain.The method of preparation, separation and use polyclone, mono-clonal and non-natural antibody is for example write (2001) " Antibody Engineering " Springer Verlag at Kontermann and Dubel; Howard and Bethell write (2001) " Basic Methods in Antibody Production and Characterization " CRC Press; And O ' Brien and Aitkin write among (2001) " Antibody Phage Display " Humana Press and summarize.This antibody-like also can be used for screening the existence of ECD polypeptide, for example is intended to detect ECD polypeptide expression in cell, tissue or extract.

[0385] method that is used for amplification of nucleic acid can be used for separating the nucleic acid molecule of coding ECD polypeptide, for example comprises polymerase chain reaction (PCR) method.The material that contains nucleic acid can be as the starting material of the nucleic acid molecule that therefrom can be separated to coding ECD.For example, can in amplification method, use DNA and mRNA prepared product, cell extract, tissue extract, body fluid sample (for example blood, serum, saliva), from healthy or ill experimenter's sample.Nucleic acid library also can be used as raw-material source.Can design the primer of the ECD molecule that is intended to increase.For example, can be based on designing primer from the expressed sequence that wherein produces the ECD molecule.Can translate and the design primer based on the answer of ECD aminoacid sequence.Can check order and confirm the ECD that encodes the nucleic acid molecule that produces by amplification.

3. produce and the chimeric method of clone's ECD polypeptide

[0386] chimeric protein is to comprise from difference or heterologous protein or two or more regional polypeptide of peptide deutero-.Chimeric protein can contain several sequences, comprises signal peptide sequence, one or more are at the sequence of the ECD of CSR (as the HER family receptors) or its part and any other heterologous sequence (as joint sequence, multimerization structural domain sequence (be Fc structural domain, leucine zipper or form polymeric other sequence)) and/or in order to the epi-position label that promotes protein purification or the sequence of other parts.For example, the ECD polypeptide can be connected directly to another polypeptide (being another ECD polypeptide or its part and/or multimerization structural domain) to form fusion rotein.Perhaps, described protein can separate the distance that one section sufficient to guarantee protein forms correct secondary structure and tertiary structure.Suitable joint sequence (1) will be taked the flexible extensions conformation, (2) will not show formation may with the tendency of the interactional orderly two-level structure in functional structure territory of fusion polypeptide, and (3) will have the minimum hydrophobic character or the charge characteristic that can promote with the functional protein domain interaction.The exemplary adapter sequence is being discussed above and it generally includes those joint sequences that contain Gly, Asn or Ser or other neutral amino acids (comprising Thr or Ala).Usually, connect ECD part and heterologous sequence by aforesaid recombinant DNA technology.Perhaps, heterologous sequence can covalently partly be connected with ECD by Heterobifunctional linking agent (any as described herein Heterobifunctional linking agent).

[0387] normally, the chimeric polyeptides that ECD fusion molecule coding is such, it has all or part of of the ECD that is enough to binding partner among the CSR, and all or part of and heterologous polypeptide (as the multimerization structural domain) of being convenient to polymer formation of described ECD is connected.

[0388] in addition, the ECD polypeptide also can directly or indirectly be connected with one or more other heterologous sequences.For example, the ECD chimeric polyeptides can comprise also and a label polypeptide fusion that wherein said label polypeptide provides the anti-tag antibody epi-position of selective binding with it.It is useful that this type of of ECD polypeptide fusions adds the epi-position label form, because can use the existence that detects this form at the traget antibody of this label polypeptide.The providing of this epi-position label allows affinity purification this ECD fusion polypeptide of purifying easily by using anti-tag antibody.

[0389] chimeric protein can use enzyme to cut and from the routine techniques preparation of the sequence junction fragment wanted.For example, the sequence of wanting can use oligonucleotide synthesizer synthetic separate from the DNA that produces described proteinic parental cell by the Restriction Enzyme digestion of appropriateness or by genomic dna being carried out PCR with suitable primer from the target source as the acquisition of originating of cell, tissue, carrier or other target.In an example, can by as continuously round ground connect by the DNA target sequence of pcr amplification to the through engineering approaches recombination site place of carrier and produce the ECD chimeric sequences.For example, can use primer to carry out pcr amplification, wherein said primer and corresponding chain hybridization and be distributed in purpose zone flank in the target DNA at the nucleotide sequence of one or more ECD polypeptide, fusion tag and/or multimerization structural domain sequence.Can use the cell or tissue of known expression target DNA molecule or other sources, the carrier of sequence that perhaps contains the target DNA molecule is as the initial product of pcr amplification incident.Pcr amplification product can subclone to carrier be used for the reorganization operation of further sequence, for example be intended to produce with carrier in another nucleotide sequence of having contained merge, or be used to express target molecule.

[0390] also can design effective connection that PCR primer used in the pcr amplification is intended to promote nucleotide sequence.For example, can add non-template complementary 5 ' extension with operation after allowing the multiple amplification of PCR product to primer, and not remarkably influenced amplification itself.For example, these 5 ' extensions can comprise restriction site, promoter sequence, Restriction Enzyme joint sequence, proteolytic enzyme cutting site sequence or epi-position sequence label.In an example, for the purpose that produces fusion sequence, the sequence that can incorporate primer into comprises, for example, the sequence of coding myc epi-position label or other little epi-position label, thus amplification PCR products contains the fusions of purpose nucleotide sequence and epi-position label effectively.

[0391] in another example, the Restriction Enzyme site is incorporated primer into and can be promoted the amplified production subclone to the carrier that contains compatible restriction site, as by being provided for connecting the sticking end of nucleotide sequence.Product subclone to the single carrier of multiplex PCR amplification can be used as effective connection or merges the strategy use of different IPs acid sequence.The example that can be merged in the Restriction Enzyme site of primer sequence can include but not limited to Xho I restriction site (CTCGAG, SEQ ID NO:267), NheI restriction site (GCTAGC, SEQ ID NO:268), Not I restriction site (GCGGCCGC, SEQ ID NO:269), EcoRI restriction site (GAATTC, SEQ ID NO:270), AgeI site (ACCGGT, SEQ ID NO:271) or Xba I restriction site (TCTAGA, SEQ IDNO:272).Be used for PCR product subclone to other method of carrier and comprise flat terminal cloning, TA cloning, the cloning that does not rely on connection and vivo clone method.

[0392] the effective Restriction Enzyme of generation site need be used for subclone subsequently with compatible Restriction Enzyme digestion PCR fragment to expose sticking end or then to expose flat end for some Restriction Enzyme sites in primer.In primer, exist several factors to wait to consider during design limit enzyme site, thereby this Restriction Enzyme site keep the consistency to Restriction Enzyme.At first, the efficient that 2-6 extra base can greatly increase the digest amplification product is added in the designed restriction site upstream in the PCR primer.Can be used for improving Restriction Enzyme to other method of Restriction Enzyme site digestion comprise the Proteinase K facture of any thermostability polysaccharase of removing DNA capable of blocking, with the terminal smoothing method of Ke Lienuo or T4DNA polysaccharase and/or add spermidine.After also can being included in amplification, the alternative approach that is used to improve the digestive efficiency of PCR product makes the fragment concatemerization.This realizes by the PCR product (if described primer also not phosphorylation) of at first handling purifying with the T4 polynucleotide kinase.If make the heat-stabilised poly synthase such as the Pfu of apparatus corrective action, end flush endization then, or if use the enzyme such as the Taq of no corrective action, then amplification PCR products can be with the processing of T4DNA polysaccharase so that described terminal smoothing.The PCR product can connect with the T4DNA ligase enzyme.This removes the Restriction Enzyme site effectively and allows efficient digestion from described segmental end.

[0393] at the PCR product subclone that will contain the Restriction Enzyme site that has exposed to carrier, for example be intended to create before the fusion with aim sequence, sometimes need will digestion the PCR product separate with those PCR products that cut not yet.In this type of example, can before carrying out PCR, add fluorescence labels at primer 5 ' end.This allows to identify the product of digestion, because those products that successfully digested will will be lost fluorescent marker after digestion.

[0394] in some cases, use amplification PCR products can cause in fusion protein product, incorporating into the Restriction Enzyme joint sequence, wherein said amplification PCR products contain be useful on subsequently subclone to carrier to produce the restriction site of fusion sequence.Usually, this type of joint sequence is short and small and does not destroy the function of polypeptide, as long as this sequence is effectively to connect.

[0395] can provide this nucleic acid molecule with the carrier format of the nucleic acid molecule that comprises coding ECD chimeric polyeptides.An example of this kind carrier is a plasmid.Multiple expression vector is can obtain and well known by persons skilled in the art, and can be used to express ECD polypeptide (comprising chimeric ECD polypeptide).The influence that the selection of expression vector can be selected by host expression system.Generally speaking, expression vector can comprise transcripting promoter and randomly comprise enhanser, translation signals and Transcription Termination and translation termination signal.The expression vector that is used for stable conversion generally has the selective marker that allows to select and keep transformant.In some cases, replication orgin can be used for amplifying the copy number of carrier.In addition, numerous expression vectors provide carboxyl terminal or the aminoterminal epi-position label that adjoins multiple clone site, thereby any gained protein of this vector expression will have to meet the open reading-frame (ORF) mode and insert the epi-position label of peptide sequence certainly.Exemplary expression carrier with epi-position label of insertion is pcDNA/myc-His mammalian expression vector (Invitrogen, SEQ ID NO:161).Therefore, for example the ECD polypeptide from this vector expression causes containing the histidine-tagged polypeptide expression of carboxyl terminal myc-, and wherein said myc-is histidine-tagged to have at the aminoacid sequence described in the SEQ ID NO:162.Therefore, can express histidine-tagged any ECD polypeptide or its part of band myc-.This type of the exemplary polypeptide that contains label is described in an embodiment and with " T " indication, for example, HER1-621 (T) molecule is to connect the histidine-tagged polypeptide of carboxyl terminal myc-after containing total length HER1ECD.The exemplary sequence of the ECD polypeptide that contains the epi-position sequence label that provides is herein described in SEQ ID NO:274 and 275.Can produce by any method known to those skilled in the art contain the epi-position label (as, but be not limited to the histidine-tagged combination of c-myc/ described in c-myc label, the histidine-tagged or SEQ ID NO:162) any ECD polypeptide or its brachymemma part.

4. expression system

[0396] will encode DNA transfection to the host cell of chimeric polyeptides (as any chimeric polyeptides that provides herein) is used for expressing.Needing under wherein the certain situation of ECD polymer polypeptide by multimerization structural domain mediation multimerization, the DNA transformed host cell that divides other chimeric ECD molecule with coding, other chimeric ECD molecule of wherein said branch will produce polymer, select described host cell to be intended to and can to assemble out this other chain of polymeric branch in required mode best simultaneously.The assembling of other monomer polypeptide of branch is promoted that by the interaction of every kind of corresponding multimerization structural domain wherein described corresponding multimerization structural domain is identical or complementary between chimeric ECD polypeptide.Under the situation as one or both ECD parts of this polymer polypeptide, so select the multimerization structural domain, thereby monomeric assembling makes the dimerization arm of HER molecule depart from mating partner multimeric molecule orientation at HER family receptors ECD or its part.This orientation be called " back-to-back " and guarantee this dimerization arm can near be used for cell surface on related HER dimerization.

[0397] ECD polypeptide comprises chimeric ECD polypeptide, can in any biology of the polypeptide that be fit to produce aequum or desired form, express, wherein said polypeptide be use and treat needed.Normally, can carry out through engineering approaches is suitable with expressing heterologous DNA and any cell type with Secretory Pathway.Expressive host comprises protokaryon and eukaryote, as intestinal bacteria, yeast, plant, insect cell, mammalian cell (comprising the human cell line) and transgenic animal.Expressive host can its protein produce level and posttranslational modification type that expressed protein is taken place aspect difference.Can be to the selection of expressive host based on these factors and other factors as consideration, the production cost of management and security with whether need purifying and purification process and carrying out.

A. prokaryotic expression

[0398] prokaryotic organism especially intestinal bacteria the system that is used to produce a large amount of protein (as ECD polypeptide and the ECD polypeptide fusions that provides) herein is provided.Also can use other microorganism strains, as bacillus (bacilli) for example a plurality of species or other bacterial isolates of withered grass bar genus bacillus (Bacillus subtilis), Rhodopseudomonas (Pseudomonas).The conversion of bacterium (comprising intestinal bacteria) is a simple and rapid technology well known to those skilled in the art.In this type of prokaryotic system, the normal use contained from the species deutero-replication site compatible with the host and the plasmid vector of control sequence.For example, comprise PBR322, pUC18, pBAD and derivative thereof for colibacillary common carrier.(it contains the promotor that is useful on transcription initiation to protokaryon control sequence commonly used, randomly have operon and ribosome bind site sequence) comprise promotor commonly used, as β-Nei Xiananmei (penicillinase) and lactose (lac) promoter systems, tryptophane (trp) promoter systems, pectinose promotor and λ deutero-P1 promotor and N-gene ribosome bind site.Yet, can use any available promoter systems compatible with prokaryotic organism.Be used for colibacillary expression vector and can contain inducible promoter, this type of promotor is used to induce high-caliber protein expression with being used for expressing host cell to be shown some toxic protein.The example of inducible promoter comprises lac promotor, trp promotor, heterozygosis tac promotor, T7 and SP6RNA promotor and temperature regulation type λ PL promotor.

[0399] the ECD polypeptide can be expressed in colibacillary kytoplasm environment.Tenuigenin is reductibility environment and for some molecule, and this may cause insoluble inclusion body to form.Can use reductive agent to come solubilising protein again as two sulphur threoses and beta-mercaptoethanol and denaturing agent such as HCl guanidine and urea.Alternative approach is to express ECD polypeptide (comprising ECD polypeptide fusions) in the bacterium periplasmic space, and wherein said periplasmic space provides well-oxygenated environment and chaperone sample and disulfide bond isomerase and can cause the generation of soluble protein.In some instances, be used for the precursor of bacterium or signal sequence (comprising OmpA, OmpF, PelB) or other precursor sequence, merge, instruct this protein to pericentral siphon with treating expressed protein as by replacing the endogenous precursor sequence.Remove described leading peptide by the signal peptide of pericentral siphon inside subsequently.The example of pericentral siphon guidance quality precursor sequence or leader sequence comprises from the pelB leader sequence of pectate lyase with from alkaline phosphatase gene deutero-leader sequence.In some cases, periplasmic expression allows expressed protein to leak in the substratum.Proteinic secretion allows purifying from culture supernatant fast and simply.Can from pericentral siphon, obtain not excretory protein by the infiltration cracking process.Be similar to kytoplasm and express, in some cases, protein can become can not dissolve and can use denaturing agent and reductive agent to promote solubilising to reach again and fold.Inducing temperature and growth temperature also can influence expression level and solubleness, the general temperature of using between 25 ℃ and 37C.Generally, bacterium produces non-glycosylated protein matter.Therefore, if proteinic function needs glycosylation, can from host cell, add glycosylation in external mode behind the purifying.

B. yeast

[0400] yeast such as yeast saccharomyces cerevisiae (Saccharomyces cerevisae), schizosaccharomyces pombe (Schizosaccharomyces pombe), separate fat Ye Shi yeast (Yarrowia lipolytica), lactic acid Crewe tie up this yeast (Kluyveromyces lactis) and pichia pastoris phaff (Pichia pastoris) be can be used to produce the ECD polypeptide know the yeast expression host.Yeast can be with the episomal replication carrier or by being transformed by the stable chromosomal integration effect due to the homologous recombination.Generally, use inducible promoter to come regulatory gene to express.The example of this type of promotor comprises GAL1, GAL7 and GAL5 and metallothionein promoter such as CUP1, AOX1 or other pichia spp or other Yeast promoter.Other Yeast promoter comprises the promotor that is used for glycolytic ferment, for example, and kinase whose those promotors of 3-phoshoglyceric acid that obtain from Yep13 or from those promotors of enolase gene or Leu2 gene.Expression vector often comprises that selective marker such as LEU2, TRP1, HIS3 and URA3 are to select and to keep the DNA of conversion.The exemplary expression carrier system that is used for yeast is to allow by being grown in the POT1 carrier system of selecting transformant in the substratum that contains glucose (referring to for example, U.S. Patent number 4,931,373).Expressed protein solubility often in yeast.Can improve expression levels and solubleness with chaperone such as Bip and protein disulfide isomerase coexpression.In addition, can use the secreting signal peptide fusions to instruct expressed protein secretion in yeast, described secreting signal peptide fusions for example promotes any other allos of polypeptide excretory or homology precursor sequence from the yeast mating type α-factor secretion signal thing of yeast saccharomyces cerevisiae and with the fusions of yeast cell surface albumen (acceptor or Arxulaadeninivorans glucoamylase are sticked in mating as Aga2p) or in yeast.Can design proteolytic enzyme for example Kex-2 proteolytic enzyme cutting site when Secretory Pathway leaves, from this polypeptide, to remove fusion sequence at expressed polypeptide.Yeast also can be in the place's glycosylation of Asn-X-Ser/Thr motif.

C. insect cell

[0401] insect cell especially utilizes the insect cell of baculovirus expression to be used for express polypeptide, as the ECD polypeptide, comprises ECD polypeptide fusions.The high-caliber protein of insect cell expression and can be embodied as the used most of posttranslational modification of higher eucaryote.Baculovirus has limited host range, and this has improved security and has reduced the misgivings of authority to eukaryotic expression.Common expression vector is used for the promotor of high level expression, as baculovirus polyhedrin body protein promotor.Normally used rhabdovirus system comprises baculovirus (as autographa california nuclear polyhedrosis virus AcNPV) and Bombyx mori nuclear polyhydrosis virus (BmNPV) and insect cell line, as from the meadow greedy noctuid (Spodoptera frugiperda) deutero-Sf9, America one star mythimna separata (Pseudaletiaunipuncta) (A7S) and black lode gold Ursula butterfly (Danaus plexippus) (DpN1).For high level expression, merge in the nucleotide sequence of molecule to be expressed next-door neighbour baculovirus polyhedrin body protein initiator codon downstream.The Mammals secretion signal is accurately processed in insect cell and can be used for expressed protein is secreted to substratum.For example, the precursor sequence of mammalian tissues plasminogen activator promotes insect cell expression justacrine protein.In addition, clone America one star mythimna separata (A7S) produces the protein with glycosylation pattern similar to the mammal cell line system with black lode gold Ursula butterfly (DpN1).

[0402] the alternative expression system in the insect cell is to use the cell of stable conversion.(Aedes albopictus (Aedes albopictus) can be used for expressing for clone such as Schnieder2 (S2) and Kc cell (drosophila melanogaster (Drosophila melanogaster)) and C7 cell.The fruit bat metallothionein promoter can be induced high level expression in the presence of inducing with the heavy metal of cadmium or copper.Generally by using selective marker such as Xin Meisu and Totomycin to keep expression vector.

D. mammalian cell

[0403] mammiferous expression system can be used for expressing the ECD polypeptide, and the ECD polypeptide fusions that provides herein is provided.Expression construct can by virus infection as by use adenovirus carrier or by direct DNA transfer method as being transferred to mammalian cell by conventional infection protocol (comprising liposome, calcium phosphate, DEAE-dextran) with by physical method such as electroporation and micro-injection.Exemplary expression carrier comprises for example pcDNA3.1/myc-His (Invitrogen, SEQ ID NO:161).The expression vector that is used for mammalian cell generally comprises mRNA cap site, TATA box, translation initiation sequence (Kozak consensus sequence) and polyadenylation element.Examples of such carriers often comprises transcripting promoter-enhanser (for example SV40 promoter-enhancer), human cytomegalic inclusion disease virus (CMV) promotor (as the hCMV-MIE promoter-enhancer) and Rous sarcoma virus (RSV) long terminal repeat or other viral promotors that is used for high level expression, as from polyomavirus, adenovirus II, bovine papilloma virus or those viral promotors of avian sarcomata virus deutero-.Other suitable mammalian promoter comprises beta-actin promoter-enhancer and human metal thioalbumen II promotor.These promoter-enhancers have activity in the various kinds of cell type.Tissue-type and cellular type promotor and enhancing subarea also can be used for expressing.Exemplary promotor/enhancing subarea includes but not limited to those controlling elementss from gene such as elastoser I, Regular Insulin, immunoglobulin (Ig), mouse mammary tumor virus, albumin, alpha-fetoprotein, alpha1 Anti-trypsin, beta globin, myelin basic protein, myosin light chain 2 and gonadotropin releasing hormone gene.Selective marker can be used for selecting and keeping the cell with expression construct.The example of selectable marker gene includes but not limited to hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, Glucosaminitol phosphotransferase, Tetrahydrofolate dehydrogenase and thymidine kinase.Can instruct protein to be expressed on the cell surface with the fusion of cell surface signaling molecule such as TCR-ζ and Fc ε RI-γ with active condition.

[0404] numerous clones can be used for the Mammals expression, comprise mouse, rat, people, monkey, chicken and hamster cell.Exemplary cells system includes but not limited to CHO, Balb/3T3, HeLa, MT2, mouse NS0 (non-secretory) and other myeloma cell line, hybridoma and allosome hybridoma cell line, lymphocyte, inoblast, Sp2/0, COS, NIH3T3, HEK293,293T, 293S, 2B8 and HKB cell.Clone also can be adapted to serum free medium, and the latter promotes albumen purifying secreted from cell culture medium.A kind of example of clone like this is serum-free EBNA-1 clone (Pham etc. (2003) Biotechnol Bioeng.84:332-42).

E. plant

[0405] transgenic plant cells and plant can be used for expressing the ECD polypeptide.General direct DNA transfer method (being transferred to protoplastis as little projectile body blast technique and PEG mediation) and the conversion method that mediates with Agrobacterium (agrobacterium) of using of expression construct is transferred in the plant.Expression vector can comprise promotor and enhancer sequence, Transcription Termination element and translation controlling elements.Expression vector and transformation technology are distinguished between dicotyledons host such as Arabidopis thaliana (Arabidopsis) and tobacco (tobacco) and monocotyledons host such as corn and rice usually.The example of the plant promoter that is used to express comprises cauliflower mosaic virus promoter, nopaline synthase promoter, ribose bisphosphate carboxylase promotor and ubiquitin and UBQ3 promotor.Selective marker such as Totomycin, phosphomannose isomerase and neomycin phosphotransferase are commonly used to promote to select and keep cell transformed.The plant transformed cell can maintain in the cultivation as cell, agglomeration body (callus) or the complete plant of regeneration.Transgenic plant cells also can be included as the algae that produces the CSR isoform and design (referring to for example, Mayfield etc. (2003) PNAS 100:438-442).Because plant has the glycosylation pattern different with mammalian cell, so this can influence the selection of the CSR isoform that is produced among these hosts.

5. the method for transfection and conversion

[0406] conversion of host cell or transfection use the standard technique that is suitable for selected host cell to realize.Transfection method is well known by persons skilled in the art, for example calcium phosphate method and electroporation, and use the commercially available cation lipid reagent that promotes transfection, as Lipofectamine ^TM, Lipofectamine ^TM2000 or

(Invitrogen, Carlsbad CA).According to used host cell, use the standard technique that is suitable for this type of cell to transform.For example use the calcium facture of calcium chloride or prokaryotic organism or other cell that electroporation is generally used for containing a large amount of cell walls barriers.Agrobacterium tumefaciens (Agrobacterium tumefaciens) infection method is used to transform the certain plants cell.Mammalian cell for there not being this type of cell walls can use the calcium phosphate precipitation method.To vegetable cell (referring to for example, Shaw etc. (1983) Gene, 23:315, WO89/05859), mammalian cell (referring to for example, U.S. Patent number 4,399,216, Methods inEnzymolog. such as Keown, (1990) 185:527; Mansour etc. (1988) Nature 336:348) or yeast cell (referring to (1977) J Bact (1977) 130:946 such as for example Val Solingen, Hsiao etc. (1979) Proc.Natl.Acad.Sci 76:3829) has described the general introduction aspect that transforms.Being used to import DNA includes but not limited to examine microinjection, electroporation, bacterium protoplastis and intact cell fusion method or uses polycation to other method of host cell, as 1,5-dimethyl-1, poly-Methobromide of 5-phenodiazine 11 methylene radical or poly ornithine.

6.ECD polypeptide, chimeric polyeptides and polymeric recovery of gained ECD and purifying

[0407] can use multiple technologies well known in the art to separate ECD polypeptide and chimeric ECD polypeptide, comprise ECD polypeptide polymer.Those skilled in the art can be easily according to becoming known for isolated polypeptide and method of protein so that obtain one of isolated polypeptide that this paper provided or protein.These methods include but not limited to immunochromatographic method, HPLC, size exclusion chromatography method and ion exchange chromatography.The example of ion exchange chromatography comprises negatively charged ion and base exchange method and comprises use DEAE sepharose, DEAE sephadex, CM sepharose, SP sepharose or any other similar pillar well known by persons skilled in the art.Can use at the antibody of epi-position label in the chimeric ECD polypeptide or at the antibody of this ECD polypeptide and promote to separate ECD polypeptide or ECD polymer polypeptide and they are separated by immuno-precipitation subsequently and separate by SDS-polyacrylamide gel electrophoresis (PAGE) from cell culture medium or from the cracked cell.Perhaps, ECD polypeptide or chimeric ECD polypeptide (comprising the ECD polymer) can by polypeptid specificity antibody and ECD polypeptide in conjunction with and/or subsequently this antibody and A albumen or G albumen sepharose post in conjunction with and from this post this protein of wash-out separated.The purifying of ECD polypeptide also can comprise to be used with the affinity column of the immobilization of reagents of protein bound or affine pearl and carries out one or more post steps subsequently with the described protein of wash-out from this wedding agent.The example of affinity reagent comprises concanavalin A-agarose, heparin-Toyopearl or Cibacrom blue 3Ga sepharose.Also can be by using the hydrophobic interaction chromatography method protein purification of this resinoid such as phenyl ether, butyl ether or propyl ether.

[0408] in some instances, can use the chimeric ECD polypeptide of immune-affinity chromatography purifying.In this type of example, the ECD polypeptide can be expressed as fusion rotein, it has epi-position label as described herein, includes but not limited to maltose binding protein (MBP), glutathione-S-transferase (GST) or Trx (TRX), myc label and/or histidine-tagged.Be used to express and the test kit of this type of fusion rotein of purifying can commercially (Beverly, Mass.), (Piscataway, NJ.), Invitrogen and other company obtain Pharmacia from New England BioLab.Protein also can merge also subsequently by using the specific antibody at this epi-position to carry out purifying with label.In some instances, can use with reagent place's fixed affinity column or affine pearl and come purifying ECD polypeptide fusions in conjunction with the epi-position label.For example, binding reagents can comprise with the interactional gsh of GST epi-position label, with the interactional immobilization metal affinity reagent of polyhistidyl label (as Cu ²⁺Or Ni ²⁺), anti-epitope antibodies (as anti-myc antibody) and/or can be fixed to and be used for proteic any other reagent of the chimeric ECD of purifying on post or the pearl.

[0409] contain under the situation of Fc structural domain or its mixture at same polymer that needs purifying or heteromultimeric molecule, can use those skilled in the art known and as the method that describes in detail among the embodiment reclaim or purifying as described in molecule.When host cell coexpression coding contains the nucleic acid of the nucleic acid of first polypeptide of Fc structural domain and second polypeptide that coding also contains the Fc structural domain, the expression molecule of gained will form as the homodimer of first polypeptide, the homodimer of second polypeptide and the heterodimer of first and second polypeptide, and wherein every kind of dimer connects by Fc multimerization domain interaction.The combination of homodimer and heterodimer can be from substratum be reclaimed as the excretory polypeptide, though it also can be reclaimed from the host cell lysate when directly producing in band signal sequence not.If described is membrane-bound with polymer or heteromultimeric, then can use suitable washing agent solution (for example Triton-X 100) to discharge described with polymer or heteromultimeric from film.

[0410] can have the homodimer or the heterodimer of antibody constant domain or its mixture easily by several different methods purifying from conditioned medium, thereby separate with other particulate state cell relic or impurity protein, described method includes but not limited to hydroxyapatite chromatography method, gel electrophoresis, dialysis method or affinity chromatography.Has C at polymer _HUnder the situation of 3 structural domains, BakerbondABX ^TM(J.T.Baker, Phillipsburg NJ.) are used for purifying to resin.According to polypeptide to be recycled, also available is other technology that is used for protein purification, as ion-exchange column fractionation, ethanol sedimentation, reversed-phase HPLC, chromatography on the silicon-dioxide, on heparin sepharose chromatography, go up chromatography, chromatographic focusing, SDS-PAGE and ammonium sulfate precipitation method at negatively charged ion or Zeo-karb (as the poly aspartic acid post).

[0411] in addition, can also use A albumen or G albumen.A albumen depends on species as the suitability of affinity ligand and the isotype of used immunoglobulin Fc domain in mosaic.A albumen can be used for purifying based on the immunoadhesin (Lindmark etc. (1983) J.Immunol.Meth.62:1-13) based on people γ 1, γ 2 or γ 4 heavy chains.Recommendation G albumen is used for whole mouse isotypes and is used for people γ 3 (Guss etc. (1986) EMBO J.5:1567-1575).Affinity ligand such as A albumen or G albumen or can often be agarose with the matrix of interactional other affinity ligand of multimeric molecule, but other matrix is available.The matrix of mechanically stable such as controllable bore diameter glass or poly-(vinylbenzene-divinyl) benzene provide than using attainable faster flow velocity of agarose and shorter process period.Immunoadhesin molecule and A albumen affinity column or G albumen affinity column bonded conditionally complete are species and isotype decision by the feature of Fc structural domain.Usually, when selecting correct part, directly produce efficient keying action from the nutrient solution (unconditioned culture fluid) of unconditional restriction.The binding molecule that contains ECD-Fc can or contain wash-out in the neutral pH damping fluid of gentle chaotropic salt at acid pH (3.0 or be higher than 3.0).In addition or in addition, binding molecule can be with excessive IgG wash-out.As required, the molecule of wash-out can be neutralized to alkaline pH.The purifying molecule of gained contains purifying (generally greater than 95%) with polymer and heteromultimeric.

[0412] several factors can be used for enrichment heteromultimeric molecule and break away from homodimer, and this includes but not limited to use the anti-epi-position tag antibody or the receptor specific antibody of a kind of chimeric polyeptides component of only discerning multimeric molecule.For example, one of chimeric polyeptides can merge with epi-position label (being c-myc or His).Therefore, for example according to according to used multimerization structural domain, use A albumen affinity column or other preliminary purification method purifying after, the molecule of purifying can use second affinity column or the further enrichment of other matrix.For example, any wedding agent can be fixed to affinity column or affine pearl to be further purified the ECD polymer.This example is that the metal affinity agent is as being used for the Ni of nickel affinity metal chromatography column ²⁺Immobilization.At first chimeric polyeptides only under the situation by second affinity column identification, can flush away contain the homodimer of second chimeric polyeptides, only stay the homodimer of first polypeptide and the heterodimer of first and second polypeptide.Further continuous affine step can be used for the purifying heteromultimeric.This type of further affine step is included on affinity column or other matrix fixingly only discerns second chimeric polyeptides that exists in the heteromultimeric but nonrecognition is remaining with polymeric antireceptor antibody or part.For example, embodiment 3 describes and uses the EGF affinity columns as final purification step, use subsequently preparation type SEC post to remove any excess ligand purifying HER1/HER3ECD polymer.As final enriching method, can be according to the polymeric component of ECD, use any wedding agent, part or the antireceptor antibody of for example discerning a kind of component in the ECD polymer and the similar affinity column of experience ground design.

[0413] in addition, can adopt one or more RPLCs (RP-HPLC) step, use hydrophobicity RP-HPLC medium (silica gel that for example, has pending methyl group or other aliphatic group) to be further purified protein.Also can use some or all the aforementioned purification steps in various combinations that the isolating recombinant protein of homogeneous basically is provided.

[0414] before purifying, the conditioned medium that contains secretion property ECD polypeptide (comprising chimeric ECD polypeptide and/or ECD polymer) can be purified and/or concentrates.Purification can be undertaken by centrifugal, subsequent filtration.Concentrate and to carry out by any method known to those skilled in the art, for example, use the tangential flow film or use stirring-type cell system filter.Various molecular weights (MW) is separated cutoff value can be used for concentration process.For example, can use 10,000MW separates cutoff value.Embodiment has described the several different methods of the mixture of the heteromultimeric of purifying HER1/HER3 (for example, Rb200 and Rb200h) and same polymer of purifying (HER1/HER1 and HER3/HER3) and heteromultimeric (HER1/HER3) in detail.Therefore, in one aspect, the invention provides comprise heteromultimeric with the composition of polymeric mixture, wherein said heteromultimeric comprises from the ECD of HER1 or its part with from another kind of ECD or its part of HER3, and wherein saidly comprises from the ECD of HER1 or its part with from ECD or its part of HER3 with polymer.This mixture can have three kinds of polymer components that are in arbitrary proportion.In some cases, the ratio of three kinds of polymer components depends on the type of used expression system.In one embodiment, the ratio of three kinds of polymer components each other approximately equates.

G. assess or the active assay method of monitoring ECD polymer

[0415] normally, the ECD polymer regulate one or more, general one or more biologic activity of two or more related CSR or other interaction CSR.The interior assay method of external and body can be used for monitoring the polymeric biologic activity of ECD.Provide in the exemplary external and body assay method to assess RTK ECD polymer, the polymeric biologic activity of HER ECD especially herein.Numerous assay methods can be used for other CSR ECD polymer.In addition, the multiple assay method that is used for the biologic activity of CSR is well known by persons skilled in the art, and can select the active any assay method of the concrete CSR of known assessment according to ECD polymer to be tested.Test ECD polymer to the assay method of RTK activity influence include but not limited to kinase assay, with dimerization and different dimerization assay method, protein: phenotype test method in protein interaction assay method, structure determination method, cell signaling effect assay method and the body.Assay method also comprises the use animal model, comprises the disease model that wherein can observe and/or measure biologic activity.The dose response curve of ECD polymer in this type of assay method can be used for assessing to the regulating effect of biologic activity and also be used for the polymeric treatment significant quantity of ECD that is identified for using.Exemplary assay method is described below.

1. kinases/phosphorylation assay method

[0416] can be directly and detect and/or measure kinase activity indirectly.For example, the antibody of anti-Tyrosine O-phosphate can be used for detecting the phosphorylation of RTK.For example, the activation of the tyrosine kinase activity of RTK can be measured in the presence of the part at RTK.Transphosphorylation can detect by anti-phosphotyrosine antibody.Can measure and/or detect transphosphorylation in the existence of ECD polymer or not, thereby measure the ability that the ECD polymer is regulated the RTK transphosphorylation.In brief, the cell of expression RTK can be exposed to the ECD polymer and handle with part.Be loaded on the polyacrylamide gel, separate and transfer on the film with lysis and with protein extract (intact cell extract or fractionated extracts), as be used for western blotting by electrophoresis.With the immuno-precipitation of anti-RTK antibody also can be used for to the RTK protein fractionation with separate, carry out gel electrophoresis and western blotting subsequently.Described film can be surveyed and survey with detecting the proteinic anti-RTK antibody of whole RTK with the anti-phosphotyrosine antibody that detects phosphorylation.Control cells (as the cell of not expressing the RTK isoform and the cell that is not exposed to part) can be accepted identical process and handle and be used for comparison.

[0417] also can directly measure tyrosine phosphorylation, as pass through mass spectroscopy.For example, the ECD polymer can be by with the ECD polymer processes complete cell of multiple concentration and measure to the RTK activated influence and measure to the influence of RTK phosphorylation state, and described RTK can separate and be used for analytical reagent composition with trysinization with the generation peptide fragment by immuno-precipitation.The peptide mass spectroscopy is being used for quantification of protein mensuration tyrosine phosphorylation degree methods of fully setting up; The phosphorylation of tyrosine has increased the peptide ionic quality that contains Tyrosine O-phosphate, and this peptide separates with unphosphorylated peptide easily by mass spectroscopy.

[0418] for example, known tyrosine-1139 and tyrosine-the 1248th in HER2RTK, self-phosphorylation.Can determine or predict the peptide of trysinization based on peptide sequence experience ground, for example by using the ExPASy-PeptideMass program.Can from the peptide mass-spectrometric data that contains these tyrosine, determine the phosphorylation degree of tyrosine-1139 and tyrosine-1248.This type of assay method can be used for assessing the autophosphorylation degree of RTK and the ability that the ECD polymer is regulated the RTK transphosphorylation.

2. compound/dimerization

[0419] can detect and/or measure compound action, as RTKs and ECD polymer dimerization.For example, isolated polypeptide can be mixed, carry out gel electrophoresis and western blotting.RTKs and/or ECD polymer also can be added into cell and cell extract (as intact cell or fractionated extract), and can carry out gel electrophoresis and western blotting.The antibody of discerning described polypeptide can be used for detecting the existence of monomer, dimer and other complex form.Perhaps, can be in described assay method the RTKs of certification mark and/or the ECD polymer of mark.This type of assay method can be used for comparison the ECD polymer exist or not in the presence of the same dimerization of RTK or the different dimerization of two or more RTK.Also can carry out the ability of assay method with assessment ECD polymer and RTK dimerization.For example, can assess the different dimerization ability of HER ECD polymer and HER1, HER2, HER3 and HER4.In addition, can assess the ECD polymer to the ability of RTK with dimerization or different dimerization capacity adjustment.For example, can assess the different dimerization ability that HER ECD polymer is regulated HER2 and HER1, HER3 or HER4 and other combination.

[0420] in another example, can carry out molecular size exclusion analytical method.Carry out molecular size exclusion method with specific size-exclusion column, and molecule and one group of reference standard product of wash-out are compared.Molecule can use individually or can with another kind of molecular combinations.For example, any RTK polypeptide, chimeric polyeptides or ECD polymer can be applied to size-exclusion column.As described in example 4 above, can determine elution volume and to every kind of molecular computing molecular weight.Perhaps, can use two or more polypeptide and assessment elution curve altogether to determine whether these two or more polypeptide or molecule can form the oligomer molecule.

3. part keying action

[0421] common, RTK is in conjunction with one or more parts.The part keying action is regulated the active of this receptor and thereby is regulated signal conduction in the signal transduction pathway for example.Can measure in the presence of the ECD polymer that part and ECD are polymeric to be combined and the part keying action of RTK.For example, tagged ligand such as radio-labeled part can be added into purifying or partially purified RTK with not existing in the existence of ECD polymer under (contrast).Immuno-precipitation and radioactivity method of masurement can be used for quantitatively the ECD polymer exist and not in the presence of with the quantity of RTK bonded part.Also can assess the polymeric part keying action of ECD,, determine quantity by ECD polymer institute bonded tagged ligand as by the ECD polymer being hatched with tagged ligand and for example comparing with wild-type or the advantage form institute bonded quantity of corresponding RTK.

4. cell proliferating determining method

[0422] for example VEGFR, HER family receptors and other growth factor receptors participate in cell proliferation to numerous RTK.Can measure the influence of ECD polymer on cell proliferation.Cell to be tested is generally expressed target RTK acceptor.For example, part can be added into the cell of expressing RTK.The ECD polymer can be before part adds, simultaneously or be added into these cells and the influence of measuring on cell proliferation afterwards.The level of cell proliferation can be carried out optimum density and measure and assessed subsequently by with dyestuff such as Alamar indigo plant or Viola crystallina or other similar dye marker.MTT[3-(4,5-dimethylthiazole-2-yl)-2,5-phenylbenzene tetrazolium bromide] also can be used for assessing cell proliferation.Use MTT as propagation reagent, it cuts the tetrazole ring of faint yellow MTT based on the mitochondrial dehydrogenase from viable cell and forms mazarine Jia Za crystalline ability, and wherein said Jia Za crystal accumulation is in healthy cell, because its impermeable cytolemma.Cause this crystalline to discharge and solubilising by the solubilising that adds the washing agent pair cell.Can quantitatively directly be proportional to proliferative cell number purpose color alive by the spectrophotometer measurement means.Therefore, selected cell and ECD polymer in part exist or not in the presence of hatch after, MTT can be added into this cell, can use washing agent pair cell solubilising, and in 570nm place reading absorbancy.Perhaps, cell can be before proliferation experiment with the radio-labeled thing as ³H-tritium or other fluorescent mark such as CFSE preliminary making.

5. cell disease model determination method

[0423] can use and be derived from disease or illness and maybe can regulate with the cell of simulation disease or illness and measure and/or detect the polymeric effect of ECD.Add the ECD polymer or it is expressed and measure or detect phenotype in cell, and be not exposed to or do not express the polymeric cell comparison of ECD.This type of assay method can be used for measuring effect, comprises the effect of on cell proliferation, transfer, inflammation, vasculogenesis, pathogenic infection and bone resorption.

[0424] for example, can in vasculogenesis, measure the polymeric effect of ECD.For example, can use the external tubule that causes by endotheliocyte such as Human umbilical vein endothelial cells (HUVEC) to form as assay method to measure vasculogenesis and to the effectiveness of vasculogenesis.Generating the mensuration system variable ECD polymer of interpolation quantity to extracorporeal blood vessel is a kind of method that is applicable to screening as the polymeric validity of ECD of vasculogenesis instrumentality.

6. animal model

[0425] animal model can be used for assessing the polymeric effect of ECD.For example, can measure the ECD polymer to cancer cell multiplication, divide a word with a hyphen at the end of a line and the influence of invasiveness.In a kind of like this assay method, after vitro culture,, be suspended in the suitable damping fluid and be injected to mouse (for example being injected to veutro and the shoulder of model mice such as Balb/c nude mouse) cancer cells such as ovarian cancer cell trysinization.Before cancer cells is applied to mouse by any suitable route of administration (promptly subcutaneous, intravenously, intraperitoneal and other approach), simultaneously or afterwards, mouse is used the ECD polymer altogether.Monitor tumor growth in time.Can carry out similar assay method with animal model with other cell type.For example, mouse lung cancer (LLC) cell and C57BL/6 mouse and SCID mouse.Tumor growth can not compare with using the polymeric mouse of ECD, or compares with the mouse of polymeric respective associated acceptor of ECD or interaction acceptor defect.

[0426] in another example, can use the effect of assay method (as cornea micro-capsule bag assay method (cornealmicropocket assay)) assessment ECD polymer to eye disease.In brief, before assay method, mouse imposes ECD polymer (or contrast) 2-3 day by injection.Subsequently, with mouse anesthesia, and the granule of part such as VEGF or other somatomedin part implanted eye's cornea micro-capsule bag.Measure neovascularization subsequently, for example back 5 days in implantation.Subsequently compared with the control, assessment ECD polymer is to the effect of vasculogenesis.

[0427] any animal model known in the art can be used for assessing ECD polymer such as the polymeric effect of HER, wherein said animal model comprises transgenic mice (as the humanization transgene mouse model), as express the atherosclerosis mouse of DR and DQ major histocompatibility complex II molecule, it can comprise the model (Gregersen etc. (2004) TissueAntigens 63 (5): 383-94)) of rheumatoid arthritis, celiaca, multiple sclerosis and insulin-dependent diabetes as for example autoimmune disease; Can be as the lipophorin-E deficient mice (ApoE of Atherosclerosis Model ^-/-); Can be as for example inflammatory bowel and Crohn disease (Scheinin (2003) Clin.Exp.Immunol.133 (1): 38-43) the IL-10 knock-out mice of model; And Alzheimer disease model, as the transgenic mice of the amyloid precursor protein of overexpression sudden change with express the mouse of the chain PS1 of family's autosomal dominant.Animal model also comprises is induced or is handled to show the animal of disease, as the EAE inductive animal as the multiple sclerosis model.

H.ECD polymer and ECD polymer preparation of compositions, preparation and use

[0428] can prepare ECD polymer and ECD polymer composition (comprising HER ECD polymer and HER ECD polymer composition) is used for using by any approach well known by persons skilled in the art, described approach comprises intramuscular, intravenously, intracutaneous, peritoneal injection, subcutaneous, epidural, intranasal, per os, rectum, part, imbedibility, mouthful cheek (for example, hypogloeeis) and applied dermally or any approach.The ECD polymer can be used by any approach that makes things convenient for, for example by the injection of infusion or bolus, by through epithelium or mucocutaneous lining (for example oral mucous membrane, rectum with intestinal mucosa etc.) absorption and can with other biologically active drug successively, intermittent type or in identical composition, use.Use to be limitation, part or general according to therapentic part.Topical application can be for example to the zone that needs treatment by (but being not limited to) surgery intra-operative local infusion, topical application (for example after surgical operation with wound dressings combination), by inject, by conduit, by suppository or pass through implant and realize.Use also and can comprise controlled release system, comprise the release (as passing through pump) of controlled release preparation and device control.The most suitable approach under arbitrary given situation will depend on disease or the character and the seriousness of the patient's condition and the character that depends on used concrete composition for the treatment of.

It is [0429] multiple that to send delivery system be known and can be used for using the ECD polymer, as, but be not limited to, the packing in the liposome, micropartical, micro-capsule, the reconstitution cell that can express described compound, receptor mediated endocytosis and send the coding the polymeric nucleic acid molecule of ECD (as the retrovirus delivery system).

[0430] can prepare and contain the polymeric pharmaceutical composition of ECD, usually, consider authority's approval or preparing pharmaceutically useful composition according to the pharmacopeia that is used for animal and human's class of extensive approval.Pharmaceutical composition can comprise that the ECD polymer reaches and its carrier of using jointly (carrier), as thinner, adjuvant, vehicle or carrier (vehicle).This type of pharmaceutical carrier can be a sterile liquid, Ru Shui and oil, and the latter comprises those oil in petroleum source, animal source, plant-sourced or synthetic source, as peanut oil, soybean oil, mineral oil and sesame oil.When intravenously drug administration composition, water is common carrier.Also can use salt brine solution and dextrose and aqueous glycerin solution as liquid vehicle, be particularly useful for injectable solutions.Composition can contain with activeconstituents: thinner such as lactose, sucrose, Lin Suanergai or carboxymethyl cellulose; Lubricant such as Magnesium Stearate, calcium stearate and talcum; With tackiness agent such as starch, natural gum such as acacia gum, gelatin, glucose, molasses, polyvinylpyrrolidone, Mierocrystalline cellulose and derivative thereof, polyvidone, polyvinylpolypyrrolidone and this type of other tackiness agent well known by persons skilled in the art.Suitable pharmaceutical excipient comprises starch, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium-chlor, skimmed milk powder, glycerine, propylene, ethylene glycol, water and ethanol.As required, composition also can contain a small amount of wetting agent or emulsifying agent or pH buffer reagent, for example, acetate, Trisodium Citrate, cyclodextrin derivative, sorbitan mono-laurate, acetate trolamine sodium (triethanolamine sodiumacetate), triethanolamine oleate and this type of other material.These compositions can adopt solution, suspensoid, emulsion, tablet, pill, capsule, powder and sustained release preparation form.Composition can be formulated as suppository, has conventional tackiness agent and carrier such as triacylglycerol.Oral preparations can comprise standard vector, as other N.F,USP MANNITOL of pharmaceutical grade, lactose, starch, Magnesium Stearate, soluble saccharin, Mierocrystalline cellulose, magnesiumcarbonate and this type of other material.Suitable pharmaceutical carrier example is described in " Remington ' s Pharmaceutical Sciences " of E.W.Martin.This based composition contains the compound for the treatment of significant quantity, and wherein said compound is in purified form usually, also has the carrier of appropriate amount, thereby is provided for being applied to patient's suitable form.Described preparation should be fit to mode of administration.

[0431] preparation is provided for being applied to the human and animal with unit dosage form, described unit dosage form for example is tablet, capsule, pill, powder, granule, sterile parenteral is with solution or suspensoid and oral solution or oral suspensions and contain the described compound of appropriate amount and the oil of pharmaceutically acceptable derivative thereof: aqueous emulsion.Medicinal therapeutical active compound and derivative thereof are generally prepared and are used with unit dosage form or multiple doses form.Unit dosage form refers to as known in the art as used in this article, is applicable to humans and animals experimenter and the unit of packing separately that physically separates.Each unitary dose contains the therapeutical active compound of the predetermined amount that is enough to produce required result of treatment together with needed pharmaceutical carrier, carrier (vehicle) or thinner.The example of unit dosage form comprises the tablet or the capsule of ampoule and syringe and independent packing.Unit dosage form can be used with the part of unit dosage form or the mode of a plurality of unit dosage forms.The multiple doses form is a plurality of same unit dosage forms to be administered of the unit dosage form to separate that are packaged in the single container.The example of multiple doses form comprises phial dress, bottled tablet or capsule or product off-type bottle or gallon type bottle.Therefore, the multiple doses form is indiscrete a plurality of unitary doses in packing material.

[0432] can prepare and contain the 0.005%-100% activeconstituents and supply as equilibrated formulation or composition with non-toxic carrier simultaneously.For Orally administered, pharmaceutical composition for example can be taked tablet or the Capsule form by the ordinary method preparation, wherein said tablet or capsule contain pharmaceutically acceptable vehicle such as tackiness agent (for example, pregelatinized W-Gum, polyvinylpyrrolidone or Vltra tears); Weighting agent (for example, lactose, Microcrystalline Cellulose or secondary calcium phosphate); Lubricant (for example, Magnesium Stearate, talcum or silicon-dioxide); Disintegrating agent (for example, yam starch or sodium starch glycolate); Or wetting agent (for example, sodium lauryl sulphate).Described tablet can be by well known method dressing.

[0433] medicinal products also can be a liquid form, for example, and solution, syrup or suspensoid, or can be used as with pharmaceutical product existence preceding and water or other suitable carrier (vehicle) reconstruct.This type of flowing product can be by ordinary method and pharmaceutically acceptable additive such as suspension agent (for example, sorbitol syrups, derivatived cellulose or hydrogenation edible fat); Emulsifying agent (for example, Yelkin TTS or Sudan Gum-arabic); Nonaqueous carrier (vehicle) (for example, Prunus amygdalus oil, oily ester or fractionated vegetables oil); And sanitas (for example, methyl p-hydroxybenzoate or propyl ester or Sorbic Acid) prepares together.

[0434] can provide be suitable for rectal administration preparation as unitary dose suppository.These suppositorys can be by mixing described active compound and one or more conventional solid carriers (for example theobroma oil) and the gained mixture forming being prepared.

[0435] is suitable for the preparation that the part is applied to skin or eye and comprises paste, ointment, lotion, paste, gelifying agent, sprays, aerosol agent and finish.Exemplary carrier comprises Vaseline, lanolin, polyoxyethylene glycol, pure and mild its two or more combination.The thickening material of 0.05-15,20,25 weight percentage also can be contained in topical preparation, and described thickening material is selected from Vltra tears, methylcellulose gum, polyvinylpyrrolidone, polyvinyl alcohol, poly-(aklylene glycol), poly-hydroxyalkyl, (methyl) acrylate or poly-(methyl) acrylamide.Topical preparation often uses by instillation or is applied to conjunctival sac as paste.It also can be used for the lavation of eye, facial sinus and external auditory meatus or lubricate.It also can be injected to anterior chamber of the eye or other position.The topical preparation that is in liquid state also can exist from the wetting ability three-dimensional polymer matrix of the rectangular or cornea contact lens form that wherein discharges in active ingredient.

[0436] for using by suction, compound used herein can use suitable propelling agent, send with the aerosol spray form of presenting from supercharging packing material or atomizer, wherein said propelling agent for example is Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas.Under the situation of pressurized aerosol agent, unitary dose can be determined by the valve of sending number of computations is provided.Can prepare the capsule and the cartridge case that are used in for example gelatin in sucker or the insufflator, it contains the powder mixture of described compound and suitable powder matrix (as lactose or starch).

[0437] is suitable for the preparation that a mouthful cheek (hypogloeeis) uses and comprises the lozenge that for example in matrix (normally sucrose and Sudan Gum-arabic or tragakanta), contains active compound with seasonings; With the pastille that in inert base (as gelatin and glycerine or sucrose and Sudan Gum-arabic), contains compound.

[0438] can prepare the polymeric pharmaceutical composition of ECD and be used for making parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation that is used to inject can exist with the sanitas that adds with unit dosage form (for example, at ampoule or in multi-dose container).Said composition can be suspensoid, solution or the emulsion in oil or water quality carrier (vehicle), and can contain the preparation material, as suspension agent, stablizer or dispersion agent.Perhaps, activeconstituents can be in powder type so that with preceding and suitable carrier (vehicle) (as pyrogen-free aqua sterilisa or other solvent) reconstruct.

[0439] preparation that is suitable for applied dermally can be used as patch separately and exists, and wherein said patch is fit to keep contacting closely the time durations of prolongation with receptor's epidermis.This type of patch contains active compound suitably, and wherein this active compound exists with the randomly buffered aqueous solution of for example 0.1-0.2M concentration.The preparation that is suitable for applied dermally also can be sent (seeing that for example, Pharmaceutical Research 3 (6), 318 (1986)) by iontherapy and generally take the form of the randomly buffered aqueous solution of active compound.

[0440] pharmaceutical composition also can use by controlled release means and/or controlled-release delivery device (see, for example, U.S. Patent number 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; In 5,674,533 and 5,733,566).

[0441] in certain embodiments, the liposome and/or the particle of receiving also can be used use with the ECD polymer.Liposome forms (being called multilamellar liposome (MLV) again) from be scattered in water-bearing media and in the phosphatide of the double-deck vesicle in the common center of spontaneous formation multilayer.The diameter of MLV is 25nm-4 μ m normally.Supersound process to MLV causes forming diameter 200-500

The little unilamellar liposome (SUV) that in kernel, contains the aqueous solution.

[0442] in the time of in being dispersed in water, according to the molar ratio of lipid and water, phosphatide can form the multiple structure except that liposome.On low ratio, liposome forms.The physical features of liposome depends on the existence of pH, ionic strength and divalent cation.Liposome can show ionic and low permeability polar material, but on the temperature that raises, the phase transformation of liposome permeability take place obviously to improve.This phase transformation relates to the lower slightly structure (being called fluid state) of order that changes over loose accumulation from closelypacked ordered structure (being called gel state).This situation takes place at characteristic transformation temperature place and causes the permeability of ion, carbohydrate and medicine is increased.

[0443] liposome is by following different mechanisms and cell interaction: by the endocytosis of engulfing sexual cell such as scavenger cell and neutrophilic granulocyte of reticuloendothelial system; Be adsorbed to cell surface by nonspecific weak hydrophobic force or electrostatic force or by interacting with cell surface component specificity; Merge by the lipid bilayer insertion endochylema film of liposome and with the endochylema film, lipid release body content is to tenuigenin simultaneously; And the lipid by shifting liposome is to cytolemma or ubcellular film, and perhaps vice versa, but do not have any combination of liposome content.It is effectively that variable element plastid prescription can change any mechanism, although can play a role in the identical time more than a kind of mechanism.

[0444] nanocapsule (nanocapsule) but can stablize usually and repetitive mode catch compound.For avoiding the side effect due to the intracellular polymer overload, can use this type of ultrafine particle of polymer design (about 0.1 micron of diameter) of degradable in vivo.Conceived the biodegradable Polyalkylcyanoacrylanano that meets these requirements and received particle and be used for herein, and this type of particle can be to make easily.

[0445] application process can be used for reducing the exposure of ECD polymer to degradation process such as proteasome degradation and the immunology intervention procedure due to antigenicity and the immunogenic response.The example of these class methods is included in the treatment site topical application.The ECD polymer also can be subjected to modifying to regulate serum stability and half life and to reduce immunogenicity.This type of modification can be undertaken and comprises and add molecule to ECD polymer (as PEGization) and interpolation carrier proteins (as serum albumin) and glycosylation (Raju etc. (2001) Biochemistry 40 (3): 8868-76 by any method known in the art; (2001) Am J Hematol.66 (4) such as van DerAuwera: 245-51).In addition, partly regulate serum stability and half life because of the polymeric Fc of formed those ECD of multimerization between the Fc.

[0446] it is reported that the PEGization (pegylation) of therapeutical agent improves the tolerance to protease cracking; Increase the blood plasma half life and reduce antigenicity and immunogenicity.The methodological example of PEG be known in the art (referring to for example, Lu and Felix, Int.J.Peptide Protein Res., 43:127-138,1994; Lu and Felix etc., Peptide Res., 6:142-6,1993; Felix etc., Int.J.Peptide Res., 46:253-64,1995; J.Biol.Chem. such as Benhar, 269:13398-404,1994; Brumeanu etc., J Immunol, 154:3088-95,1995; Also referring to (2003) Adv.Drug Deliv.Rev.55 (10): (2003) Pharmacotherapy 23 such as 1261-77 and Molineux (8 Pt 2): 3S-8S) such as Calieeti.During PEGization also can be used in and send in the body of nucleic acid molecule.For example, the PEGization of adenovirus can increase stability and transgenosis (see, for example, Cheng etc. (2003) Pharm.Res.20 (9): 1444-51).

[0447] required blood levels can be kept by the continuous infusion active substance, as being determined by blood levels.Should be understood that because toxicity or marrow, liver or kidney dysfunction the doctor in charge will know how to stop and when stop, interruption or adjustment of treatment be to than low dosage.Conversely, reply insufficient (eliminating toxic side effects) if face urine, then the doctor in charge will know that how and when adjustment of treatment is to higher level, for example by per os, through lung, parenteral (intramuscular, intraperitoneal, intravenously (IV) or subcutaneous injection), suck (by the fine powder preparation), use through skin, intranasal, vagina, rectum or sublingual administration approach, and described active substance preparation (can be seen for the formulation that is fit to every kind of route of administration, for example, international pct application WO 93/25221 and WO 94/17784 and european patent application 613,683).

[0448] comprise the ECD polymer with such amount in pharmaceutically acceptable carrier, wherein said amount is enough to the patient of treatment is produced the useful effect of treatment, and does not have adverse side effect.Treatment effective concentration can be determined to experience by test compounds in system's (as assay method that provides herein) in known external and body.

[0449] concentration of ECD polymer in composition will depend on absorption, inactivation, the discharge rate of this complex body, physicochemical characteristic, dosage regimen and the amount of being used and the other factors well known by persons skilled in the art of this complex body.

[0450] can determine by standard clinical techniques to be administered with treatment disease or the illness polymeric amount of ECD of cancer, autoimmune disease and infection for example.In addition, can use external test method and animal model to help determine the optimal dose scope.Exact dosage desired (it can be determined to experience) can be depending on route of administration and severity of disease.The suitable dose scope that is used to use can be that about 0.01pg/kg body weight to 1mg/kg body weight and the more common 0.05mg/kg to 200mg/kg of being ECD polymer compares weight in patients.

[0451] the ECD polymer can disposable employedly maybe can be divided into numerous more low dose of to use pitch time.The ECD polymer can be during treatment time (for example through a few hours, a few days, several weeks or several months) use with one or more dosage.In some cases, continuous administration is useful.Be to be understood that exact dosage desired and treatment time length and disease-related to be treated and can use known testing method or determine to experience by extrapolation in body or the experiment in vitro data.Should be understood that concentration and dose value also can be with waiting that the seriousness of alleviating illness changes.It should also be understood that for concrete experimenter, the specific administration scheme should be adjusted in time according to individual need and the professional judgement of using or supervise the personnel that use said composition, and described herein concentration range only is exemplary and be not intended to limit composition and contain the scope or the purposes of the associating of these compositions.

I. use the illustrative methods of ECD polymer treatment

[0452] provides the method for the treatment of the disease and the patient's condition with ECD polymer and the polymeric mixture of ECD herein.ECD polymer (comprising HER ECD polymer) can be used in the multiple disease and the patient's condition that treatment relates to CSR (comprise RTK and especially HER protein family (comprising described those HER protein families herein)).CSR signal conduction relates to the nosetiology of the multiple disease and the patient's condition and has conceived any this type of disease or illness is treated by ECD polymer provided herein.Use the polymeric treatment of ECD that this paper provides to include but not limited to treat the vasculogenesis relevant disease and the patient's condition, comprise illness in eye, atherosclerosis, cancer and blood vessel injury; Neurodegenerative disease comprises alzheimer's disease; The inflammatory diseases and the patient's condition comprise atherosclerosis; The disease relevant with cell proliferation and the patient's condition comprise cancer; The smooth muscle cell relative disease; With the various autoimmune disease.RTK mediates property, especially HER mediates the property disease and the patient's condition, has described exemplary therapy and preclinical study for treating by the ECD polymer.Exemplary therapy to other CSR mediation property disease and the patient's condition (for example, but be not limited to the RAGE mediation property disease and the patient's condition) has also been described.This class description only is intended to example and is not limited to concrete ECD polymer.Treatment can realize by the preparation that suitable pathways is used molecule, wherein said molecule can be used as the polypeptide in the composition and provides and do and can be connected with directed agents to be used for targeted delivery, or in delivery vector (vehicle) (as liposome) capsulation, or send as naked nucleic acid or in carrier.Concrete therapy and dosage can be determined by those skilled in the art.Consideration in the therapy assessment comprises whether disease to be treated, this severity of disease and process, this molecule are that prevention is still for therapeutic purpose are used, previous therapy, patient's clinical history and treatment replied and doctor in charge's consideration is handled.

1.HER the disease or the illness of mediation

[0453] any disease, the patient's condition or the illness that relate in being HER acceptor and/or part aspect some of nosetiology, pathology or its development of disease that HER (ErbB) is relevant or the receptor-mediated disease of HER.Relate in particular to the expression or overexpression or the activity that comprise HER receptor family member for example or part.Disease includes but not limited to proliferative disease, described proliferative disease for example comprises cancer, but is not limited to pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer or mammary cancer.Other disease comprises and relates to cell proliferation and/or divide a word with a hyphen at the end of a line, comprises the cell proliferation that relates to the pathologic inflammatory reaction and/or those diseases of dividing a word with a hyphen at the end of a line; Non-pernicious excessively proliferative disease as illness in eye, tetter, because of smooth muscle cell proliferation and/or the disease due to dividing a word with a hyphen at the end of a line, as narrow, comprises restenosis; Atherosclerosis; The flesh of bladder, heart or other muscle thickens; Endometriosis or rheumatoid arthritis.Can provide other disease of HER ECD polymer treatment to comprise with this paper, include but not limited to aggressiveness, growth retardation, schizophrenia, shock, Parkinson's disease, alzheimer's disease, congestive cardiomyopathy, preeclampsia, nervous system disorders and heart failure by HER family receptors or its ligand-mediated any disease or illness.The example of this type of disease or treatment is described below.

A. cancer

[0454] as discussed, the HER family receptors usually is expressed in the various human carcinoid, and its expression is related with the pathology of multiple cancer.For example, the overactivity of HER signal conduction or dysregulation can cause with tumour relevant abnormal cells taking place and activate, and comprise cell proliferation, vasculogenesis and divide a word with a hyphen at the end of a line and invade.Several mechanism can be explained the dysregulation of the HER family receptors signal conduction that occurs in cancer, wherein said mechanism includes but not limited to the excessive generation of part, the excessive generation of acceptor or the constitutive activation of acceptor.Because the effect in cancer and other disease is so the HER acceptor becomes the treatment target.Yet HER family member's coexpression often causes replying of this type of therapy lacked or cause the tolerance development by alternative HER family member's compensatory rise.Therefore, the HER ECD polymer that provides herein can be with the alternative therapy of doing cancer, is being in feature or the associated cancer with two or more cell surface receptor coexpressions especially.

[0455] the ECD polymer that contains all or part of HER1, HER2, HER3 or HER4ECD can use in cancer therapy.In one aspect, the invention provides the method that is used for treating broad variety cancer, inflammatory diseases, vasculogenesis disease or excessively proliferative disease by the pharmaceutical composition of administering therapeutic significant quantity, wherein said pharmaceutical composition comprises heteromultimeric and with polymeric mixture, wherein said heteromultimeric comprises from the ECD of HER1 or its part with from another ECD or its part of HER3, and wherein saidly comprises from the ECD of HER1 or its part or from ECD or its part of HER3 with polymer.In some cases, cancer is pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer, kidney or mammary cancer.In other cases, the disease of being treated is a proliferative disease.The limiting examples of this type of proliferative disease comprises the propagation of smooth muscle cell and/or divides a word with a hyphen at the end of a line or preceding ophthalmic or diabetic retinopathy or psoriatic.In other cases, the disease of being treated be restenosis, illness in eye, narrow, atherosclerosis, because of hypertension, the urinary bladder disease and the obstructive respiratory disease of blood vessel due to thickening.

[0456] cancer example to be treated herein includes but not limited to cancer knurl, lymphoma, blastoma, sarcoma and leukemia or lymph sample malignant tumour (lymphoid malignancy).Other example of this carcinoid comprises squamous cell carcinoma (for example squamous cell cancer), lung cancer (comprises small cell lung cancer, nonsmall-cell lung cancer, adenocarcinoma of lung and lung squamous cancer), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach (comprising gastrointestinal cancer), carcinoma of the pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer (hepatoma), mammary cancer, colorectal carcinoma, the rectum cancer, renal cell carcinoma, the esophageal carcinoma, neurospongioma, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer (hepatic carcinoma), anus cancer, penile cancer and head and neck cancer.Can comprise hormone antagonist compound, cardioprotectant, carcinostatic agent such as chemotherapeutics and growth inhibitor and any as described herein other medicines with the conjoint therapy that HER ECD polymer uses.

[0457] cancer of available HER ECD polymer treatment is normally expressed at least a HER acceptor, general cancer more than a kind of HER acceptor.This carcinoid can be by being used to detect known in the art any method evaluation that HER expresses.For example, HER2 expresses and can use obtainable diagnosis/prognosis assay method (comprising HERCEPTEST.RTM. (Dako)) to assess.Paraffin-embedded tissue section from tumor biopsy is carried out the IHC assay method and met HER2 protein staining density criterion.The tumour that meets less than threshold value can be characterized by not overexpression HER2, and those tumours that have more than or equal to threshold value can be characterized by overexpression HER2.In an embodiment of treatment, the tumour of overexpression HER2 is evaluated as the candidate of usefulness HER ECD polymer (as any HER ECD polymer provided herein) treatment.

B. vasculogenesis

[0458] vasculogenesis relates to from existing blood vessel modulated and saves land and form the process of neovascularity, and this process is usually nourished tumour and promoted metastasis of cancer.The generation of VEGF is the factor of vasculogenesis and the cancer cells necessity of dividing a word with a hyphen at the end of a line.Numerous factors are induced vegf expression, comprise EGF and TGF-signal conduction by the HER family receptors.In fact, HER1 and HER2 all be the cancer associated gene that in vasculogenesis, relates to (Yance etc. (2006) Int.Can.Ther., 5:9-29).The HER family receptors also is expressed on the endotheliocyte to otherness.For example, on normal endothelial cell, HER2, HER3 and HER4 express, and on tumour deutero-endotheliocyte, HER1, HER2 and HER4 express (Amin etc. (2006) Cancer Res.66:2173-80).Therefore, as with normal cell is compared, tumour deutero-endotheliocyte has been lost that HER3 expresses and has been obtained HER1 and express, this with endotheliocyte VEGF produce and the promotion vasculogenesis in reactive consistent to EGF.

[0459] target HER family receptors (as by ECD polymer provided herein) can be used the therapy of doing vasculogenesis.Can use external or body in assay method assess of the influence of ECD polymer to vasculogenesis.For example, can test person mammary cancer deutero-MDA-MB-231 cell (its secretion angiogenesis factor VEGF) whether can antagonizing vessel generate the generation of the factor to determine the ECD polymer.In addition, can by analyst's huve cell (HUVEC) propagation test the ECD polymer exist or not in the presence of or in the presence of the recombinant vascular generation factor, be created in the activity of the angiogenesis factor in the supernatant liquor of these cells.Whether the situation that the HUVEC that can relatively breed mixes [3H]-thymidine reduces with the propagation of determining HUVEC in the presence of the ECD polymer.

C. the relevant disease of neuregulin

[0460] neuregulin (NRG) is one group of complicated part (NRG1-4) by four different genes codings.Think that some molecules in these molecules have activity striding under the film precursor forms, as comprise the free ligand of NRG ectodomain.NRG stride form membrane and free form is brought into play its biological effect by HER 1-4 acceptor.These parts play a role in the cell interaction of neuromuscular synapse growth, neurone-spongiocyte interaction and adjusting heart development and function.Can be used for the treatment of and the disease that is exposed at least a NRG relevant (for example cause or because of its increase the weight of) from the ectodomain deutero-therapeutical agent of HER 1-4 monomer molecule, homodimer molecule and the heterodimer molecule of the ligand binding domains of HER family (as contain), as neurological disorder or neuromuscular disease by it.In one embodiment, described disease is relevant with HER4 bonded NRG1 (I, the II and the III type that comprise NRG1) with HER3.The example of NRG relative disease that can be by HER ECD therapeutical agent as described herein treatment includes but not limited to alzheimer's disease and schizophrenia.

[0461] wherein the NRG example that is conditioned unusual neurological disorder can be alzheimer's disease (Chaudhury etc. (2003) J Neuropathol Exp Neurol 62:42-54).Chaudhury etc. have checked NRG1 and kinase whose expression of erbB and distribution in the hippocampus that is derived from cognitive normal old human patients with Alzheimer disease and dual transgenic mice, wherein said dual transgenic mice performance alzheimer's disease phenotype.The two expression of NRG-1 and erbB4 is all relevant with active cells element specificity in the neural patch, prompting autocrine and/or paracrine interaction.HER ECD polymer can be used for treating alzheimer's disease and related conditions as described herein.Multiple mouse model can be used for people's alzheimer's disease, and described mouse model comprises the transgenic mice of overexpression sudden change amyloid precursor protein and expresses the mouse of the chain PS1 of family's autosomal dominant and the mouse of two kinds of protein of expression (PS1M146L/APPK670N:M671L).The Alzheimer model is for example treated by injection HER ECD polymer.The development of patch can use staining and antibody response assay method to assess for example by observing the neural patch in hippocampus, entorhinal cortex and the brain cortex.

[0462] schizophrenia is still nervous system disorders serious and that can't solve basically.The world population of estimation 1% suffers from serious behavior, emotionality and the cognitive impairment as this sick feature.At present, think that the symptom that lacks molecule marker helps diagnosis.The evidence of getting in touch between NRG and the schizophrenia is at first proposed by (2002) Am J Hum Genet 71:877-892 such as Stefannson.Recently data have shown that the NRG1 transcript level of increase appears at (Hashimoto etc. (2004) Mol Psychiatry 9:299-307 in schizophreniac's the prefrontal cortex and peripheral leukocytes; Petryshen etc. (2005) Mol Psychiatry 10:366-74).Get in touch the long time-histories potential difference relevant (Kwon etc. (2005) J Neurosci25:9378-83) that may reverse some nerve synapse between NRG1 and the schizophrenia with NRG1.HER ECD polymer as described herein can be used for treating schizophrenia.

D. the relevant disease and the patient's condition of smooth muscle proliferation

[0463] HER ECD polymer can be used for the treatment of the multiple disease and the patient's condition that relates to smooth muscle cell proliferation in the Mammals (as the mankind).Example is that treatment relates to heart trouble, atherosclerosis and the hypertension that inner membrance hyperplasia (as angiostenosis, because of the restenosis due to angioplasty or surgical operation or the support implant) is bred and caused to vascular smooth muscle cell (VSMC).In this type of patient's condition, various kinds of cell and discharge cytokine interaction play a role with autocrine, paracrine or closely secretion mode, this causes VSMC to travel to impaired inner membrance from its normal position in the middle level.The VSMC hyper-proliferative of dividing a word with a hyphen at the end of a line also causes intimal thickening, and this causes the narrow or inaccessible of blood vessel.This problem is assembled at the damage location place because of thrombocyte and deposition increases the weight of.α-zymoplasm (a kind of multi-functional serine protease) is to concentrate and stimulate VSMC propagation in damaged portion.After this receptor activated, VSMC produced the multiple autocrine somatomedin of justacrine, comprises PDGF-AA, HB-EGF and TGF.EGFR participates in the signal transduction cascade, and the latter finally causes inoblast and VSMC to divide a word with a hyphen at the end of a line and breeds, and causes stimulating the VSMC secretion that endotheliocyte is had the multiple factor of mitogen effect and the chemotactic response in the inducing endothelial cell.Can be used for regulating this type of signal conduction and reply with the polymeric treatment of HER ECD.

[0464] HER ECD polymer, as contain all or part of HER ECD heteromultimeric of one of HER2 and HER3 or both ECD, can be used for treating HER (as HER2 and HER3) wherein and regulate the bladder SMC patient's condition of (as thickening responding to the The bladder wall that occurs when influencing the obstruction of lower urinary tract syndrome).HER ECD polymer can be in control smooth muscle of bladder cell proliferation and is therefore used in prevention or treatment urethral obstruction syndrome.

[0465] HER ECD polymer can be used for treating the obstructive respiratory disease with the basic pathology that relates to smooth muscle cell proliferation.A kind of example is the asthma of performance airway inflammation and bronchostenosis.Confirmed that EGF stimulates people's air flue SMC to breed and can be a factor of air flue SMC pathologic propagation in the participation obstructive respiratory disease.HER ECD polymer can be used for regulating HER1 to the effect of EGF with reply.

2.RTK the disease or the illness of mediation

A. vasculogenesis related Ophthalmopathy

[0466] ECD polymer, include but not limited to contain one or more ECD of VEGFR, PDGFR, TIE/TEK, FGF, EGFR and EphA or those ECD polymers of its part, can be used for the treatment of (comprising the illness in eye that relates to neovascularization) of the vasculogenesis related Ophthalmopathy and the patient's condition.The feature of eye neovascularity disease is that neovascularity is invaded ocular structure (as retina or cornea).This be the most commonly encountered diseases that loses one's sight because of and participate in about 20% illness in eye.In age-related macular degeneration, relevant visual problem is caused through damaged inside growth in the Bruch's membrane and the propagation of the vascular tissue below retinal pigment epithelium by choroidal capillary.Vasculogenesis damage is also relevant with Terry's sign disease with diabetic retinopathy, retinopathy of prematurity, corneal transplantation, neovascular glaucoma.Other disease relevant with the cornea neovascularization includes but not limited to epidemic keratoconjunctivitis, vitamin(e) A deficiency, the overwear vt. of cornea contact lens, atopy keratitis, top corneal limbus keratitis, pteryium keratitis sicca, sjogren syndrome, acne erythematosa, phylectenulosis, syphilis, tubercle bacillus affection, the lipid sex change, the chemicals burn, bacterial canker, mycotic ulcer, herpes simplex infections, the belt-shaped blister virus infection, protozoan infection, the Ka Boqi sarcoma, silkworm erosion property keratitis, the sex change of Terrien edge of cornea, edge angling (marginalkeratolysis), rheumatoid arthritis, the general lupus, polyarteritis, wound, the Wegener granuloma, scleritis, Stevens Johnson syndrome (Stevens Johnson disease), pemphigus radial keratotomy and corneal transplantation rejection (corneal graph rejection).Include but not limited to diabetic retinopathy with the disease that retina/the choroid neovascularization is relevant, macular degeneration, herrik syndrome, sarcoidosis, syphilis, nevus elasticus, eczematoid carcinoma, vein obstruction, obstruction of artery, the carotid artery obstruction disease, chronic hyalitis, mycobacterium tuberculosis infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, eales disease, behcet disease, cause the retinitis and uvaeformis infection, intend ocular histoplasmosis, best's disease, myopia, look recessed, the Stargart disease, pars planitis, chronic retina peels off, hyperviscosity syndrome, toxoplasmosis, wound and laser surgery infectious-related complication.Other disease includes but not limited to disease relevant with rubescent (neovascularization of anterior chamber angle (neovascularization of the angle)) and the disease that is caused by fiber blood vessel or fibrous tissue abnormality proliferation (the whole forms that comprise the proliferative vitreoretinopathy).

[0467] as described herein, can in animal model, (for example in cornea implant) assess the result of treatment (as in treatment illness in eye) of ECD polymer to vasculogenesis.For example, can in nude mouse model (as the rat C6 neurospongioma of the epiderm-like A431 tumour in nude mouse and VEGF transduction of in nude mouse, implanting or PIGF transduction), assess as regulating effect to vasculogenesis by the RTK mediation.The ECD polymer can be used as protein and carries out part or systemic injection.Can more subject contrast and be subjected to tumour between the model of ECD polymer treatment to observe the tumor suppression phenotype, comprise that bad vascularization and pale tumour, necrosis, hyperplasia reduce and apoptosis of tumor cells increases.

[0468] can be to be the illness in eye of feature with the illness in eye example of the ECD heteromultimeric treatment that contains all or part of TIE/TEK ECD, include but not limited to diabetic retinopathy (major complications of diabetes) with the eye neovascularization, retinopathy of prematurity (this crushing illness in eye that often causes chronic visual problem and have a high blind risk is a kind of severe complication during the premature infant nurses), neovascular glaucoma, retinoblastoma, neovascular glaucoma, rubescent, uveitis, macular degeneration and corneal graft neovascularization.Other ocular inflammatory disease, eye neoplasms and the disease relevant with choroid or iris neovascularization also can be treated with TIE/TEK ECD polymer.

[0469] the ECD heteromultimeric that contains all or part of PDGFR ECD also can be used in the treatment proliferative vitreoretinopathy.Rabbit conjunctiva inoblast (RCF) can be injected in the vitreum part of the eye in the rabbit animal model for example, injects about 1 * 10 by gas vitreomy ⁵Individual RCF.Can inject the ECD polymer that limitation or general are used on same.Can for example after surgical operation, observe effect 2-4 week to the propagation vitreoretinopathy, as the symptom that palliates a disease.

[0470] the ECD heteromultimeric that contains all or part of EphA ECD can be used for treating disease or the illness (in illness in eye) with dysregulation and/or unfavorable vasculogenesis.For example, the effect that can join albumin A-1 inductive vasculogenesis at animal model (in the mouse cornea model) assessment EphA ECD polymer to liver.To only contain liver joins albumin A-1 or also has the polymeric Hydron granule of ECD to implant the mouse cornea.Carried out visual inspection to observe the inhibition of ECD polymer or to reduce vasculogenesis the same day after implantation.

B. vasculogenesis dependency atherosclerosis

[0471] RTK ECD polymer, one of for example contain among all or part of ECD of VEGFR1 (Flt-1) or TIE/TEK or both ECD heteromultimerics can be used for treating the vasculogenesis patient's condition relevant with atherosclerosis, as the neovascularization of atherosclerotic plaque.The verified patch that forms in vasoganglion has the vasculogenesis stimulating activity.Vegf expression in the damage of human coronary artery's congee sample is relevant with the atherosis process of human coronary artery.

[0472] animal model can be used for assessing the ECD polymer in atherosclerosis therapy.Lipophorin-E deficient mice (ApoE ^-/-) easily suffer from atherosclerosis.This type of mouse continues for some time (as continuing for 5 weeks) by injection ECD polymer (for example VEGFR ECD polymer) and is treated, since the 5th, 10 and 20 ages in week.The damage at aortic root place is at contrast ApoE ^-/-Mouse and the ApoE that treats through isoform ^-/-Assess minimizing between the mouse with the mouse medium sized artery sclerosis damage of observing the isoform treatment.

C. other vasculogenesis dependency therapy

[0473] RTK ECD polymer, the ECD heteromultimeric that for example contains all or part of VEGFR ECD and all or part of EphA ECD also can be used for treating vasculogenesis and the inflammatory dependency patient's condition such as synovial cell's propagation, inflammatory cell infiltration, cartilage damages and pannus forms, as appearance in the rheumatoid arthritis (RA).The autoimmunity model that can use II collagen type inductive sacroiliitis (as inductive multi-joint sacroiliitis in the mouse) is as people RA model.Can be to observing alleviating of arthritic symptom (comprising pawl swelling, erythema and ankylosis) with the mouse of ECD polymer (as by local injection protein) treatment.Also can observe alleviating of synovial membrane vasculogenesis and synovial membrane inflammation.Vasculogenesis plays a significant role in the pannus of RA forms and keeps.The ECD polymer can be used to regulate vasculogenesis separately or with other isoform and other therapies in combination.For example, angiogenesis inhibitor can be used for the treatment of RA in combination with the ECD polymer.Exemplary angiogenesis inhibitor includes but not limited to angiostatin (angiostatin), angiogenesis inhibitor Antithrombin III, cancer chalone (canstatin), cartilage derive inhibition, CH-296, IL-12, angiostatin (vasculostatin) and other angiogenesis inhibitor known in the art (referring to for example, Paleolog etc. (2002) Arthritis Research Therapy 4 (supp 3) S81-S90).

[0474] is fit to comprise vascular tumor with other vasculogenesis dependency patient's condition of ECD polymer (comprising for example VEGFR ECD polymer) treatment.One of modal vasculogenesis disease of children is a vascular tumor.In most of cases, this tumour is benign and need not to get involved and disappear.In cases with severe more, this tumor development becomes huge spongy and infiltration form and causes clinical complication.Angiomatous whole body form-angiomatosis has high mortality.The numerous vascular tumor cases with currently used therapeutical agent treatment can not be treated or be difficult to existence.

[0475] ECD polymer (as VEGFR ECD polymer) can be used in the treatment of this type of disease and the patient's condition, and wherein vasculogenesis is the reason of damaging in spider angioma (Osler-Weber-Rendudisease) as hereditary hemorrhagic or the hereditary hemorrhagic telangiectasia.This disease is a kind of heredopathia, it is characterized in that a plurality of little vascular tumors, blood or limph-vascular tumor.Vascular tumor is present in skin and the mucous membrane, often with nosebleed epistaxis (nasal bleeding) or gastrointestinal hemorrhage and sometimes with lung and liver arterio venous fistula.With disadvantageous vascular permeability is that the disease and the patient's condition of feature also can be treated by the ECD polymer.These diseases comprise the oedema relevant with cerebral tumor, ascites, meigs' syndrome, pneumonia, nephrotic syndrome, pericardial effusion and the hydrothorax of being correlated with malignant tumour.

[0476] vasculogenesis also participates in normal physiological processes as regeneration and wound healing.Vasculogenesis is the important step in the ovulation and also is important step in the after fertilization embryonic implantation.Regulating vasculogenesis by ECD polymer (as contain all or part of VEGFR ECD ECD heteromultimeric) can be used for inducing amenorrhoea, blocking-up ovulation and prevent embryonic implantation.The ECD polymer also can be used in the surgical procedure.For example, in wound healing, excessively repair or proliferation of fibrous tissue can be the harmful side effect of surgical procedure and can cause or increase the weight of because of vasculogenesis.Adhesion is operating common complication and causes problem, as small intestinal obstruction.

[0477] being used for the treatment of the relevant disease of vasculogenesis and the RTK ECD polymer of the patient's condition also can be used in the conjoint therapy, as with anti-angiogenic medicaments, with RTK dependency approach in other signaling molecule interact, comprise the molecule associating of regulating VEGFR part or other somatomedin part.For example, confirmed that known antirheumatic Bucillamine (BUC) comprises that in its mechanism of action suppressing the synovial cell produces VEGF.Produce VEGF by suppressing the synovial cell, thereby suppress arthritis synovial membrane medium vessels generation and synovial hyperplasia and mediate the antirheumatic effect of BUC.Multiple mechanism of action and site that the polymeric conjoint therapy of this type of medicine and EGF can be provided for treating.

D. cancer

[0478] RTK isoform as isoform TIE/TEK, VEGFR, MET and FGFR, can be used for the treatment of cancer.RTK isoform (including but not limited to VEGFR isoform such as Flt1 isoform, FGFR isoform such as FGFR4 isoform and EphA1 isoform) can be used for treating cancer.The example of cancer to be treated herein includes but not limited to cancer knurl, lymphoma, blastoma, sarcoma and leukemia or lymph sample malignant tumour.The additional examples of this carcinoid comprises squamous cell carcinoma (for example squamous cell cancer), lung cancer (comprises small cell lung cancer, nonsmall-cell lung cancer, adenocarcinoma of lung and lung squamous cancer), peritoneal cancer, hepatocellular carcinoma, cancer of the stomach comprises gastrointestinal cancer, carcinoma of the pancreas, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer (hepatoma), mammary cancer, colorectal carcinoma, the rectum cancer, colorectal carcinoma, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney (kidney) or kidney (renal) cancer, prostate cancer, carcinoma vulvae, thyroid carcinoma, liver cancer (hepatic carcinoma), anus cancer, penile cancer and head and neck cancer.

[0479] for example, the ECD heteromultimeric that contains all or part of TIE/TEK ECD can be used for the treatment (as by regulating the cancer-related vasculogenesis) of cancer.Vascularization participates in regulating cancer growth and diffusion.For example, suppress vasculogenesis and suppressed entity tumor growth and expansion with the inhibition neovascularization.Confirmed that Tie/Tek acceptor (as Tie2) influences the vascular development in healthy tissues and the cancerous tissue.TIE/TEK ECD polymer can be as the inhibition of tumor-blood-vessel growth.Can be in animal model monitor effect, wherein described TIE/TEK ECD polymer is formulated as with the surgery modus operandi and implants the condition medium (conditioned media) in the hydron granule of rat cornea micro-capsule bag or be formulated as the protein purification (for example 100 μ g/ dosage) that is applied to window chamber (window chamber) vasculogenesis by handle the rat cornea with TIE/TEK ECD polymer.For example, the rat model F344 rat of no blood vessel cornea (as have) can be united with the condition medium of tumour cell and used or generate with induction of vascular by the window chamber of implantation tumour fragment to eye.Cornea can carry out histological inspection to be suppressed with the condition medium inductive vasculogenesis that detects by tumour cell.TIE/TEK ECD polymer also can be used for treating the malignant tumour and the transitivity patient's condition, as the entity tumour, comprises primary and transitivity sarcoma and cancer.

[0480] the ECD heteromultimeric that contains all or part of FGFR4ECD can be used for treating cancer, for example pituitary tumor.Animal model can be used for the progression of anthropomorphic dummy's pituitary tumor.For example, the FGFR that the aminoterminal of expressing in transgenic mice shortens form is that ptd-FGFR4 has reappeared pituitary tumor generation (Ezzat etc. (2002) J Clin.Invest.109:69-78), is included in to prolong and the pituitary adenoma formation of huge hyperplasia under not existing.The FGFR4ECD polymer can be applied to the ptd-FGFR4 mouse and hypophysis is constructed and tumour progression process and control mice comparison.

3. the disease or the illness of other CSR mediation

[0481] also provide herein with contain at least non-RTK CSR (as, but be not limited to TNFR or RAGE) as the ECD heteromultimeric of one of component treatment disease.For example, the ECD polymer that contains all or part of ECD of RAGE at least can be used for treating the diabetes relevant disease and the patient's condition, comprises periodontopathy, autoimmune disease and vascular disease and Tubulointerstitial disease.Use the polymeric therapy of RAGEECD also to comprise treatment eye disease (comprising macular degeneration), cardiovascular disease, neurodegenerative disease (comprising Alzheimer's disease), inflammatory diseases (comprising rheumatoid arthritis) and disease and the patient's condition (comprise cancer) relevant with hyperplasia.In another example, at least the ECD polymer that contains all or part of ECD of acceptor TNFR family can be used for treating rheumatoid arthritis, Crohn disease, autoimmune disease, rheumatosis, inflammatory bowel, Alzheimer's disease and other diseases, especially inflammatory diseases.

4.ECD the selection of polymeric ECD polypeptide fraction

[0482] when which kind of ECD multimeric molecule of decision is used for the treatment of selected disease, determines that the polymeric component of ECD is a Consideration.Can determine experience that several factors are designed for the ECD heteromultimeric of treatment disease or illness with routine.At first, should determine disease to be treated.Generally, this kind disease is the disease that shows tolerance single receptor target Sex therapy, and for example reason is the overexpression to the contributive multiple CSR of described D Ety (comprise RTK and especially HER).Secondly, can determine to participate in one or more CSR of this D Ety or the part of CSR.This type of CSR or part can be the polymeric targets of designed ECD, thereby design this ECD poly so that regulate the activity of (the general inhibition) described CSR or its part.Therefore, the ECD polymer should contain target CSR be enough to the ECD of this CSR dimerization all or part of as a kind of component and/or contain and be enough to all or part of of the ECD that combines with the part of described target CRS.Those skilled in the art will know that and maybe can identify etiologic etiological CSR (comprising RTK or HER family receptors) and/or its part that participates in selected disease.For example, the contribution of CSR to some exemplary diseases and illness above described.The 3rd, can determine to be enough among the described ECD binding partner and/or with the integral part of cognation or interaction receptor dimerizationization.This type of part of exemplary ECD molecule is that described at this paper or known or can determines by those skilled in the art are customary, for example, and based on the associated receptor comparison and/or by using recombinant DNA technology together with the part binding assay.At least all or part of of the ECD of two or more target CSR that identified can connect directly or indirectly to form polymer, for example by being connected with the multimerization structural domain separately.In some cases, according to the method that is used for connecting various integral parts, described polymer can be dimer or high-order polymer.Gained ECD polymer then is the candidate therapeutic agent that is used for the treatment of selected disease.

[0483] for example, HER acceptor (for example HER1) relates to multiple cancer, and described cancer includes but not limited to wherein those cancers of HER1 overexpression (being colorectal carcinoma, head and neck cancer, prostate cancer, carcinoma of the pancreas, liver cancer, lung cancer, renal cell carcinoma, mammary cancer, esophagus cancer, ovarian cancer, cervical cancer/uterus carcinoma, neurospongioma cancer, bladder cancer and other cancers).Therefore, can design such ECD polymer, it has all or part of as a kind of component of HER1ECD, is intended to a kind of mechanism of target HER1 signal conduction as the treatment cancer.In the design of heteromultimeric, can identify the another kind of CSR molecule that also relates to selected disease and used as second polypeptide fraction of described heteromultimeric.For example, other HER acceptor and its part overexpression in or relate in the multiple cancer.For example, as HER1, the HER3 overexpression is in mammary cancer, colorectal carcinoma, carcinoma of the pancreas, liver cancer and esophagus cancer.Therefore, the candidate ECD therapeutical agent that is used for the treatment of multiple cancer will be a kind of like this ECD therapeutical agent, and it is the heteromultimeric of all or part of ECD of all or part of ECD of HER1 and HER2.In second example, selected disease can be the vasculogenesis disease.Those skilled in the art will know that VEGFR1 and RAGE all participate in the nosetiology of vasculogenesis.Therefore, can design such heteromultimeric as candidate therapeutic agent, it contains ECD all or part of of all or part of and RAGE of the ECD of VEGFR1.

5. patient's selection

[0484] as mentioned previously, multiple disease and illness because of activate CSR inadequately, especially the HER family receptors causes, its reason is for example excessive generation, the excessive generation of acceptor or the constitutive activation of acceptor of part.The patient can be based on the correlated expression of the CSR of medicine or molecule institute target or part to often replying of medicine or molecule (as the ECD polymer that provides herein) and is predicted.Can.Therefore, as required, before treatment disease or illness, the part or the CSR that can analyze the patient express to select such patient, estimate that wherein described patient is for having the enhanced responsiveness with the polymeric therapy of ECD provided by the present invention.For example, if at least a HER1 acceptor of ECD polymer therapeutical agent target, the HER1 that can analyze the patient expresses.In another example, if known disease to be treated is mediated by ligands specific, then before the ECD polymer treatment with this part of target, can analyze patient's described ligand expression.Can be with the expression and check sample or normal sample comparison of part or CSR in patient's sample (being blood, serum, tumour, tissue, cell or other source), be intended to select CSR with rising or those patients of ligand level.Selected patient like this can guarantee estimating that most probable responds to those patient subgroups treatments of given therapeutical agent.

[0485] in one aspect, can in the patient, assess the expression of CSR.In an example, can in diagnostic or prognostic assay method, (for example pass through the immunohistochemistry assay method by estimating the proteic increase level of CSR that on tissue or cell surface, exists; IHC) determine to express.Alternatively, or in addition, the level of the nucleic acid of coding CSR can for example be passed through fluorescence in situ hybridization (FISH in the cell; See WO 98/45479), southern blotting technique method or polymerase chain reaction (PCR) (as real-time quantitative PCR (RT-PCR)) estimate.In addition, the overexpression of CSR can (for example be seen U.S. Patent number 4,933,294 by measure the antigen (for example solubility CSR) that disseminates in biological fluid (as serum); WO91/05264; 5,401,638; Sias etc. (1990) J Immunol.Methods 132:73-80) estimates.In another kind of assay method, can and make this cellular exposure from patient's isolated cell, and can analyze the keying action of this antibody and cell in CSR specific antibody with detectable (for example radio isotope or fluorescent mark substance markers) optional markings.In another example, patient's this cell can be exposed to the keying action of a kind of antibody and this antibody in vivo can so be assessed, promptly by external scan method that is used for radioactivity or the living tissue sample that obtains from the patient who before was exposed to this antibody by analysis.Any other assay method well known by persons skilled in the art can be used for determining the CSR level among the patient, and described assay method for example is, but is not limited to immunoblotting, enzyme-linked immunosorbent assay (ELISA) and other method.In some cases, the patient who selects to have the phosphorylation form expression of receptor and increase can be used for especially identifying those patient's subgroups of the activated receptor level with rising.The multiple assay method of detection known in the art CSR phosphorylation includes but not limited to for example use the immunoblotting or the ELISA of anti-phosphotyrosine antibody or anti-phosphoric acid (anti-phospho) specific C SR antibody.

[0486] in some cases, can determine that the level of CSR part is as an index selecting the patient.For example, patient's tissue or the ligand level in the tumour can be used immunohistochemical method (IHC, referring to for example, Scher etc. (1995) Clin.Cancer Research 1:545-550) determines.Alternatively, or in addition, the ligand level in sample, tissue, tumour or other source can be determined according to any method that is used to detect white matter or coding property nucleic acid.The example of this method is ELISA, PCR (comprising RT-PCR), flow cytometry, FISH, southern blotting technique method and other method.In addition, as mentioned, the part of CSR can use the in-vivo diagnostic assay method to assess, and for example carries out mark and externally scans this patient with marker as described in locating by using with molecules detected bonded molecule (as antibody) and with detectable (being the radio-labeled thing).For example, can be at patient's sample as in serum, using the standard ELISA method (is commercially available ELISA test kit, as from R﹠amp; DSystems) or by immunohistochemical method and micro-array tissue analyze formalin fixed former generation tumour section in the part of HER family receptors, as TGF-α, EGF or amphiregulin (referring to for example, Ishikawa etc. (2005) Cancer Res.65:9176).In another example, RT-PCR can be used for being evaluated in patient's cell sample, as patient's tumour cell (Mahtouk etc. (2005) Oncogene, 24:3512-3524) or the ligand expression in blood, marrow or the lymphoglandula (as at the monocyte that separates from wherein).

6. conjoint therapy

[0487] the ECD polymer is (as RTK ECD polymer, comprise HER ECD polymer) can unite with every kind of other polymer and use or used with treating disease and other existing medicine of the patient's condition and the mixture of therapeutical agent as them, and have additivity or synergetic property result of treatment.For example, numerous as described herein ECD polymers can be used for treating the vasculogenesis dependency patient's condition and disease and/or control tumor proliferation.This type of therapy can be united angiogenesis inhibitor and/or antitumor drug and/or therapeutical agent and be carried out.Be used for the molecule that the angiogenesis inhibitor of conjoint therapy and/or antitumor drug and/or therapeutical agent comprise the tyrosine kinase inhibitor that can be used in conjoint therapy and can regulate the tyrosine kinase signal transduction, include but not limited to 4-amino-pyrroles also [2,3-d] pyrimidine is (referring to for example, U.S. Patent number 5,639,757) and quinazoline compound and composition (for example, U.S. Patent number 5,792,771.Other compound that is used in the conjoint therapy comprises steroid such as vasculogenesis inhibition 4, the steroid of 9 (11)-steroid and C21-oxidation, angiostatin, endostatin, angiostatin, cancer chalone (canstatin) and mammary gland silk press down albumen (maspin), angiogenesis factor, bacterial polysaccharides CM101 and antibody LM609 (U.S. Patent number 5,753,230), thrombospondin (TSP-I), platelet factor 4 (PF4), Interferon, rabbit, the metalloprotein enzyme inhibitor, pharmaceutical agents (comprises AGM-1470/TNP-470, Thalidomide and carboxylic amine triazole (CAI)), cortisone (as in the presence of heparin or heparin fragment), the anti-factor of invading, vitamin A acid and taxol (U.S. Patent number 5,716,981; Mode is by reference incorporated this paper into), extracts of shark cartilage, negatively charged ion polymeric amide or polyureas oligomer, Oxoindole derivative, derivatives of estradiol and thiazole and pyrimidine derivatives.

[0488] treatment cancer (cancer that comprises treatment overexpression HER) can comprise the conjoint therapy that uses carcinostatic agent, described carcinostatic agent for example is anti-HER antibody, small molecules tyrosine-kinase enzyme inhibitor, antisense oligonucleotide, at the vaccine or the immunoconjugates (promptly being coupled to radio isotope or cytotoxic antibody) of HER/ part.The example of this type of carcinostatic agent comprises Gefitinib, lapatinibditosylate, handkerchief Buddhist nun monoclonal antibody, erlotinib, Cetuximab, Herceptin, imatinib, platinum complex or nucleoside analog.Other carcinostatic agent comprises radioactive method or chemotherapeutic and/or growth inhibitor, comprises the mixture of using different chemotherapeutic agents altogether.The example of cytotoxic agent or chemotherapeutic comprises for example taxanes (as taxol and docetaxel) and anthracycline antibiotics, Dx/Zorubicin, Carubicin, daunorubicin, aminopterin, methotrexate, methopterin-A, dichioromethotrexate, ametycin, porfiromycin, 5 FU 5 fluorouracil, Ismipur, cytarabin, podophyllotoxin or podophyllotoxin derivative such as etoposide or phosphoric acid etoposide, melphalan, vinealeucoblastine(VLB), vincristine(VCR), leurosidine, vindesine, vinleurosine, maytansinol, ebomycin A or B, taxotere, taxol etc.This type of other therapeutical agent comprises estramustine, cis-platinum, combretastatin and analogue and endoxan.Preparation and dosage regimen at this type of chemotherapeutic can or as be determined to use by skilled practitioner according to manufacturer specification.The preparation and the dosage regimen of this type of chemotherapeutic are also write at ChemotherapyService, M.C.Perry, Williams﹠amp; Wilkins describes among the Baltimore, Md. (1992).

[0489] other compound can use with the ECD polymer in conjoint therapy.The hormone antagonist compound can for example use with the ECD polymer in conjoint therapy.This type of examples for compounds comprises the estrogen antagonist compound on the known dose that is in this quasi-molecule, as tamoxifen; Mifepristone is as onapristone; And androgen antagonist, as flutamide.It also can be useful using cardioprotectant (prevent or reduce may the myocardial dysfunction relevant with therapy) or one or more cytokines altogether.Except that above-mentioned treatment plan, the patient can accept surgery and extract cancer cells method and/or radiotherapy.

[0490] conjoint therapy can increase the validity of treatment, and produces synergistic effect in some cases, thereby the additivity effect of the independent therapy of this Combined Ration is more effective.For example, can show coordinate repression, promptly than the bigger growth inhibitory effect of additivity associating that uses two kinds of medicines respectively to growth of tumour cell with chemotherapeutics (as tyrosine kinase inhibitor) and the polymeric conjoint therapy of ECD as described herein.

[0491] adjuvant and other immunomodulator can be united use with the ECD polymer in the treatment cancer, for example are intended to increase the immunne response at tumour cell.The example of adjuvant includes but not limited to DNA of bacteria, attenuation tubercule bacillus cell (BCG; Bacillus-Calmette-Guerin) nucleic acid moiety, from the genomic synthetic property oligonucleotide of BCG with contain synthetic property oligonucleotide (the CpG ODN of CpG motif; Wooldridge etc. (1997) Blood 89:2994-2998), LEVAMISOLE HCL, aluminium hydroxide (Alum), BCG, Freund's incomplete adjuvant (IFA), QS-21 (plant derived immunopotentiating agent), keyhole limpet hemocyanin (KLH) and dinitrophenol(DNP) (DNP).The example of immunomodulator includes but not limited to cytokine such as interleukin-(IL-2 for example, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-1 α, IL-1 β and IL-1RA), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor, tumour inhibitor M, erythropoietin, leukaemia inhibitory factor (LIF), Interferon, rabbit, B7.1 (being also referred to as CD80), B7.2 (is also referred to as B70, CD86), TNF family member (TNF-α, TNF-β, LT-β, the CD40 part, the Fas part, the CD27 part, the CD30 part, 4-1BBL, Trail) and MIF, Interferon, rabbit, cytokine class such as IL-2 and IL-12; And chemotherapeutics class such as methotrexate and Chlorambucil.

[0492] embodiment shows except using existing medicine, also uses the heteromultimeric of various ways and heteromultimeric and has produced the synergetic property result with polymeric mixture.

J. be used to identify, screen and produce the method for general HER therapeutical agent

[0493] the ECD polymer except providing herein can also be identified the general HER therapeutical agent of other candidate's property.This paper provides the method for identifying general HER therapeutical agent and at their screening assay method.The design of described method be intended to by mode like this determine target ECD subdomain disturb part in conjunction with and/or the molecule of receptor dimerizationization and/or constraint, promptly be tested and appraised with at molecule (as small molecules and polypeptide) more than these the active regional interactions of participation on a kind of HER receptor family member.This type of therapeutical agent does not possess simultaneously several members of the multiple coexpression of HER acceptor in the target HER family.

1. at the target of general HER therapeutical agent

[0494] for this type of general HER treatment molecule of design, certified similar epi-position or conservative region have been identified as participating in given activity.For example, identified that the zone of participation constraint effect is to filter out candidate molecules stable or promotion constraint effect; Identified that participation part bonded zone is to filter out interference part and the interactional material standed for of two or more HER family members.And identified the zone that participates in dimerization.

[0495] described zone is based on this receptor family crystalline structure data and evaluation in view of the above.For example, at acceptor as designing the antagonism therapeutical agent in this respect, whether wherein said aspect decision acceptor is in non-activity or activity conformation is arranged, so that the activation form of preference ground receptor targeted, wherein said receptor activation form constitutes the about 5%HER family receptors on the cell surface.Example by this type of structural constituent of crystalline structure prediction for example comprises, keeps acceptor and is in and carries the baby or the structural constituent of non-activity state, promote the structural constituent of dimerization and/or promote part bonded structural constituent.Each conduct of hereinafter describing these structural constituents is used to design the potential target of general HER therapeutical agent.

[0496] for example, the zone in subdomain II (D II) and IV (D IV) participates in the constraint effect and participates in receptor dimerizationization.Can identify that conservative region is to filter out inhibition more than a kind of candidate compound of HER family member dimerization and/or make lashing (tether) stable or make the crosslinked candidate compound with stable constraint conformation of structural domain.Exemplify in an embodiment from this type of of several HER family members and identified polypeptide.

[0497] for this method, identified the homeopeptide sequence in each target structure territory district in every kind of HER acceptor (HER1, HER2, HER3 and HER4).In some instances, also can compare homology zone in IGF1-R and other cell surface receptor to identify the potential target sequence.Generally, the target sequence of so deriving, promptly by using aminoacid sequence and the modeling from crystalline structure in one or more HER acceptors (generally being HER1 and/or HER3), the sequence of having identified subsequently and other HER family receptors are compared, and choose the most conservative sequence.Also identified the corresponding sequence in other HER acceptor.Can for example use the phage display method to identify conjugated protein at these target sequences.Can enrichment described conjugated protein with identify combine with one or more of a little zones and 1) suppress part in conjunction with, 2) suppress acceptor unite become dimer or heterodimer and/or 3) inhibition separates that to fetter those that react (untethering reaction) (promptly activating the HER molecule) conjugated protein.In some cases, the avidity of the peptide of being identified can crosslinked because of two or more peptides (promptly producing the peptide heterodimer) increase, thereby crosslinked peptide folds with combine and prevent this minute subsolution with two of a part zones.Described crosslinked peptide can be that the peptide of different epi-positions in the identification same structure territory or they can be the peptides of different epi-positions in the identification different structure territory.For example approaching because of domain II in the constraint conformation of HER acceptor and IV, so the peptide of an epi-position can be crosslinked to suppress the constraint of separating to the constraint conformation with the peptide of an epi-position in the recognition structure territory IV constraint district among the II of recognition structure territory.

[0498] in an example, designing general HER therapeutic antagonist is intended to be in self inhibition configuration by stoping dimerization to lock acceptor.Therefore, zone and/or the zone in structural domain IV in domain II can be used as target.For example, zone in the dimerization arm of domain II or the zone around this dimerization arm can be used as the target that prevents dimerization and the associating of HER family receptors.In another example, the zone in structural domain IV can be used as the target that prevents homology regional combination among dimerization arm and the structural domain IV, is at acceptor wherein that constraint is described during conformation unites generation.Therefore, can identify the antagonist that combines also thereby spatially suppress this receptor dimerization ability with for example different loci on the domain II of single receptor, as peptide or other molecule (as antibody) or other small molecules therapeutical agent of being identified by the phage display method.Based on and HER3 in domain II or structural domain IV comparison result and be presented between the HER family member conservative target epitope regions can be as immunogen to produce at these regional antibody, maybe can be as the target substrate so that with display technique of bacteriophage enrichment for example at the peptide binding substances in these sites.Embodiment 8 has described the evaluation to exemplary homology target epi-position, and wherein said homology target epi-position is also described (domain II epi-position) or described (structural domain IV epi-position) in the item arbitrarily at SEQ ID NO:94-125 in the item arbitrarily at SEQ ID NO:62-93.In addition, embodiment 5 has described the exemplary area (describing) that participates in dimerization among the HER2 in SEQ ID NO:405.Therefore, for example, the phage display method can be used for identifying different loci bonded peptide in the homology zone with domain II and/or structural domain IV, wherein said homology zone can be respectively combines with zone among domain II and/or the structural domain IV, to keep acceptor and be in self and suppress configuration by suppressing dimerization.The peptide binding substances that avidity is higher can make by the peptide heterodimer that produces as this paper the following stated.The advantage of this method is that it is at the non-constraint form acceptor that only accounts for HER acceptor about 5% on the cell surface, therefore, those acceptor subgroups of signal effect take place in the therapeutical agent of gained only target actively, rather than 95% on the cell surface carries the baby and the acceptor of non-activity.This will improve effective target practice of acceptor and reduce drug dose, reach about 15-20 doubly because the target sum reduces.

[0499] in another example, domain II can be used as target with similar homology zone on the structural domain IV, makes the stable general HER treatment antagonist of constraint conformation of HER acceptor with generation.The inactive form (promptly about 95% HER cell surface receptor) of this type of therapeutical agent target HER acceptor also prevents that them from taking the ability of activity conformation.The feasibility of this method obtains the support of crystalline structure data, and wherein said crystalline structure data presentation is the close interaction between domain II and the IV in the non-constraint of HER acceptor or inactive form.The crystalline structure of the ECD of HER1 and HER3 is presented at before the ligand stimulation, and acceptor is kept to be on cell surface and self suppressed or the constraint configuration.In this configuration, dimerization arm in domain II and the intramolecularly specificity contiguity constraint between the homology zone among the structural domain IV relative orientation of two zones of responsible part bonded (they being structural domain I and III), thereby structural domain I can not contact part simultaneously with III.These constitutional featuress show if can the acceptor locking be in the constraint configuration that self suppresses, then can stop ligand dependent HER receptor activation.The described sequence of close indication of domain II and IV sequence can be crosslinked because it is approaching closely.Therefore, can target in domain II and IV, reach as mentioned described in the embodiment 8 and SEQID NO:62-93 arbitrarily described in (domain II epi-position) with SEQ ID NO:94-125 arbitrarily described in the identical epitope regions of (structural domain IV epi-position).For this method, selected the peptide binding substances for example by phage display methodology identified of target in HER family receptors domain II and structural domain IV homology zone in these two.If use method as described herein to make two kinds of peptide dimerizations, wherein a kind of peptide binding domains II and another kind of peptide binding domains IV, then described peptide can and the non-activity HER family member of constraint in structural domain between zone (for example interaction of rock steady structure territory II and IV) crosslinked.Therefore, the receptors bind of gained antagonist molecules and constraint form, and with this constraint form " locking " on the spot, thereby stop high-affinity, the non-constraint form of formation acceptor.

[0500] in another example, the general HER therapeutical agent target that can be identified by methods described herein of the ligand binding region in structural domain I and III.As above, can identify to participate in part bonded homology target region between the HER family receptors.For example, participate in part bonded HER1 the zone can by determine with the crystalline structure of TGF-α compound HER1 (Garrett etc. (2002) Cell, 110:763-773).This crystalline structure can be taken from and have 1D, the PDB Protein Data Bank of 1MOX.Multiple ratio that can be by HER1, HER2, HER3 and HER4 is to determining the homology zone in other HER family receptors.Embodiment 7 describes the zone of being identified by this comparison, and the sequence of being compared is described in the item arbitrarily at SEQ ID NO:54-61.These sequences can be for example by combined peptide library, display technique of bacteriophage or by the polyclone method practice shooting (referring to for example, Haurum and Bregenholt (2005) IDrugs, 8:404-409).Expect that the general HER therapeutical agent of class methods evaluation thus will suppress multiple part and multiple HER receptors bind by the site among blocking-up structure territory I and/or the III (as by the steric inhibition effect).This kind therapeutical agent is the HER acceptor of target non-activity, and suppresses the ability that they take activity conformation, and wherein said activity conformation only just occurs behind binding partner.

2. identify the screening method of general HER therapeutical agent

[0501] provides the method for target of identifying herein more than a kind of general HER therapeutical agent of HER family receptors.Screened elements collection.This type of set for example comprises that small molecules organic compound and other biomolecules comprise peptide, carbohydrate, lipid acid, steroid, purine, pyrimidine, derivative, analog or its combination.In an example, described set is screened at the polypeptide of having identified, the wherein said polypeptide of having identified is conservative between receptor family, and participates in given activity.

[0502] polypeptide of having identified also can screen with evaluation and any several different methods of those molecules of the interaction of molecules of being identified by being used to screen molecular library.For example, candidate's general HER therapeutical agent can be identified by phage display method institute deutero-peptide.This type of peptide of enrichment is with the conserved sequence element between evaluation and the HER receptor family as discussed above (promptly at any or multiple peptide epitopes described in SEQ ID NO:54-125 or the 405 any items) those peptides of bonded.

A. phage display method

[0503] display technique of bacteriophage of fully setting up comprises that generation is illustrated in library or the peptide on the phage.These libraries or peptide for example can contain nearly 10 ¹⁰Plant different peptides, therefore be better than numerous small molecules combinatorial library.Peptide (often having 7-20 or more a plurality of amino acid) can be a high degree of specificity with the interaction of protein target, sometimes than small molecules more high specific is arranged.Peptide can be modified to strengthen its treatment and be renderd a service.For example, can reduce serum short stay and the rapid filtering of kidney by PEGization or with other serum protein (merging) as albumin.PEGization not only increases the serum setup, also can reduce immunogenicity.In addition, can be by connecting the non-overlapped peptide of two or more synergetic propertyes improves peptide and protein target with the heterodimer binding substances that forms high-affinity avidity.

[0504] phage display method and this type of other method can be used according to different modes.At first, the polypeptide that this paper identified can screen at the library of the polypeptide of having showed, to identify the those polypeptides that can be used as the general HER therapeutical agent of candidate in the library.Perhaps, can show the peptide identified herein and at the library screening of small molecules and other polypeptide to identify general HER therapeutical agent candidate.

I. peptide library

[0505] peptide library that produces and screen in method provided herein is used to provide the new part of HER family receptors and produces general HER therapeutical agent.Can according to the method that describes in detail herein and the common obtainable method design of those skilled in the art and elutriation peptide library (referring to for example, U.S. Patent number 5,723,286 and Application No. US20040023887).In one aspect, (for example, can use commercially available phage display library

Or GRAB DGIBioTechnologies, Inc., Edison, NJ.; Peptide library or the 7-amino acid and the linear library of 12-amino acid of the constraint of C7C disulfide linkage, New England Biolabs).In yet another aspect, can prepare oligonucleotide library and be inserted into the appropriate carrier that is used for the peptide expression according to methods known in the art.For example, can use the carrier of coding phage structural protein, the come-at-able phage albumen of optimized encoding such as phage capsid protein (coat protein).Although those skilled in the art understand and can use multiple phage, but carrier generally is or derived from filobactivirus, for example f1, fd, Pf1, M13 and other phage.Especially, and the extensively description in the literature of fd-tet carrier (see, for example, Zacher etc. (1980) Gene 9:127-140; Smith etc. (1985), Science 228:1315-1317; Parmley and Smith (1988) Gene, 73:305-318).

[0506] select or make up the cloning site of phage vector with 5 ' zone of containing the gene that is arranged in coding phage structural protein, thus this peptide accessible acceptor in as the avidity enriching method of this paper the following stated.The phage structural protein are capsid protein normally.Suitable capsid protein example is pili (pill).Suitable carriers can allow the oligonucleotide sequence of directed cloning encoded peptide, thereby described peptide is expressed in the aminoterminal place of ripe capsid protein or in the scope apart from about 100 amino-acid residues of this aminoterminal.Generally, this capsid protein is expressed as the preceding albumen with leader sequence.

[0507] general, so insert oligonucleotide library, thereby the aminoterminal of bacteriophage coat protein (outer protein) of processing is first residue of this peptide, promptly between the part of 3 of the sequence of coding leader protein ' terminal and 5 of the proteinic sequence of encoding mature ' terminal or this 5 ' end.Contain the oligonucleotide of library member's Variable Area (with any transcribed spacer, as discussed below) to the selected cloning site and make up oligonucleotide library by the clone.Use known recombinant DNA technology (to see usually, Sambrook etc. (1989) Molecular Cloning, A Laboratory Manual, second edition., Cold SpringHarbor Laboratory Press, Cold SpringHarbor, N.Y.), can make up such oligonucleotide, it is 1 years old) remove undesired restriction site and add the restriction site of wanting; 2) the appropriate part (for example correct signal peptidase site) of any sequence of being removed of reconstruct; 3) if exist, insert the transcribed spacer residue; And/or 4) as required, proofread and correct the translation open reading-frame (ORF), to produce activated infectious phage.

[0508] middle body of oligonucleotide contains one or more HER family receptors epi-position binding sequences and the optional transcribed spacer sequence that contains usually.Described sequence final as the aminoterminal with ripe capsid protein merge or with its inner peptide (possessing or do not possess transcribed spacer) that merges be expressed in the outside of assembling phage particle, can be near on the surface.The big young pathbreaker in library according to variable cipher subnumber order and thereby change according to the size of peptide, this wants.Usually, this library will have at least about 10 ⁶Individual member, usually at least 10 ⁷Individual and general 10 ⁸Individual or more a plurality of members.Carry intravital oligonucleotide set for producing the codon that forms a series of coding random amino acid set and finally being cloned into, use a kind of codon motif, as (NNK) _x, wherein N can be A, C, G or T (name first-class mole), K is that G or T (the first-class mole of name) and x generally are to up to about 5,6,7,8 or bigger numeral, thereby the library that produces pentapeptide, six peptides, seven peptides and octapeptide or bigger peptide.The 3rd position also can be G or C, called after " S ".Therefore, NNK or NNS coding 1) amino acid of all categories; Coding 2) only a kind of terminator codon; And 3) codon bias was reduced to 3: 1 from 6: 1.

[0509] should be appreciated that the size in the library of generation may become the limiting factor in clone's process under the situation of long peptide.Known in the art in suitable recombinant vectors from the oligonucleotide mixture expression of peptides that generates at random (see, for example, Oliphant etc., Gene 44:177-183).For example, codon motif (NNK) ₆Produce 32 codons: corresponding to each amino acid whose codon in 12 seed amino acids, corresponding to each amino acid whose two codon in 5 seed amino acids, corresponding to each amino acid whose three codon and (amber) terminator codon in 3 seed amino acids.Though the identical reasonable codon that this motif produces with the available standards oligonucleotide synthesis method is obtained distributes, but it causes the bias at the peptide that contains the sub-residue of single password.Particularly, the full set of one six codon contains a kind of sequence, and every kind of peptide that this sequence encoding only is made of the sub-amino acid of single password is planted sequence yet contain 729 (36), and described sequence encoding only has the amino acid whose every kind of peptide of three codons.

[0510] a kind of be intended to make at the minimized alternative approach of the bias of the sub-residue of single password comprise 0 kind of activatory trinucleotide of Synthetic 2, wherein every kind of trinucleotide is represented the codon of one of the amino acid of 20 kinds of genetic codings.These trinucleotides are synthetic by ordinary method; take off from upholder; keep base and 5-OH blocking group simultaneously; and by the method that is used to activate the monokaryon glycosides add 3 ' O-phosphoramidite (and protect phosphoric acid ester with the b-cyanoethyl) and activated (generally referring to; McBride and Caruthers; 1983, Tetrahedron Letters 22:245).Use prepares the few codon of degeneracy as these tripolymers of structural unit.Described tripolymer is mixed with the molar ratio wanted and be placed in the synthesizer.This ratio is normally roughly equimolar, but can be adjustable inequality proportion, to obtain by the extremely not enough representative of some amino acid whose excessive representative of degeneracy oligonucleotide collective encoding.Basically as to utilize activation monokaryon glycosides as carry out as described in the conventional synthesis method of structural unit the tripolymer condensation with form few codon (see, for example, Atkinson and Smith, 1984, OligonucleotideSynthesis, M.J.Gait edits, the 35-82 page or leaf).This method produces the oligonucleotide colony that is used to clone, can the encode possible peptide sequence of (or adjustable do not wait distribution) of equal distribution of described oligonucleotide colony.Advantageously, this method can be used in producing long peptide sequence, because by (NNK) ₆The bias scope that motif causes reaches 3 times with each extra amino-acid residue increase.

[0511] when the codon motif be aforesaid (NNK) _xThe time and when x equals 8, have 2.6 * 10 ¹⁰Plant possible octapeptide.May be difficult to produce the library of containing most of described octapeptide.Therefore, can be by using many samplings that realize to about 10% possible sequence construct subgroup library described octapeptide, the subgroup of wherein screening recombinant phage particle subsequently.As required, be the diversity in expansion subgroup library, the phage subgroup of recovery can be carried out mutagenesis and be accepted the screening of subsequent passes.This mutagenesis step can be carried out according to two kinds of common modes: the Variable Area of the phage of reclaiming can carry out mutagenic treatment, or extra variable amino acid can be added into the zone of closing on original variable sequence.

[0512] for make find in the elutriation round in early days in conjunction with bioactive peptide (being binding substances) variation, can be with the positive bacteriophage order-checking to determine the homogeneity of described bioactive peptide.Subsequently can be according to these peptide sequence synthetic oligonucleotides.Building-up process is carried out to cause the one-level oligonucleotide sequence slightly to make a variation with mixing low-level whole base on each step.The mixture of this (slightly) degeneracy oligonucleotide can be cloned into affine phage by method known to those skilled in the art subsequently.This method has produced the controlled variation as the system of the original peptide sequence of secondary library part.Therefore yet this need should check order to each positive bacteriophage before mutagenesis, and described positive bacteriophage is used to enlarge the diversity of the minority phage of having reclaimed.

What [0513] cause that the diversified alternative approach of selected phage allows phage that mutagenesis reclaims compiles thing or subgroup.According to this method, compile the phage of from the elutriation process, reclaiming and separate single stranded DNA.This DNA is by carrying out mutagenesis with handling as nitrous acid, formic acid or hydrazine.These processing produce multiple damage to DNA.Duplicate the DNA of this damage subsequently with reversed transcriptive enzyme, wherein said reversed transcriptive enzyme mixes base mistakenly when running into damage location.By isolate the sections of the sequence that contains coding receptor binding peptide with the cutting of restriction nuclease enzyme, wherein said restriction nuclease enzyme is specific for the site that is distributed in the peptide-coding sequence flank subsequently.The sections of this mutagenic treatment is cloned into int carrier DNA subsequently again, this DNA is converted in the cell, and produces the secondary library according to currently known methods.Mutafacient system commonly used is that known in the art (referring to for example, Myers etc. 1985, Nucl.Acids Res.13:3131-3145; Myers etc. 1985, Science 229:242-246; Myers, 1989, Current Protocols in Molecular Bioglogy I volume, 8.3.1-8.3.6, editors such as F.Ausubel, J.Wiley and Sons, New York).

[0514] in another kind of common method, can use several different methods to peptide or find that activated peptide adds amino acid.In an example, be determined at the sequence and the synthetic sequence of being measured and the new oligonucleotide that is close to the degeneracy sequence of comprising of selected peptide in the early stage elutriation respectively.With these new oligonucleotide of rear clone to produce the secondary library.Perhaps, can use to the phage of carrying peptide and compile the method that thing adds the 2nd HER binding sequence.According to a kind of method, restriction site is settled near a HER binding sequence.Preferably, enzyme should be in its recognition sequence outside cutting.This recognition site can with first binding sequence at a distance of several bases.For inserting the 2nd HER binding sequence, phage DNA compiled thing digestion and by mend the in addition terminal smoothing of flat overhang with Klenow fragment.The degeneracy synthetic double chain oligonucleotide of terminal smoothing is connected with this site subsequently to produce second binding sequence near first binding sequence.Subsequently as preceding amplification and screen this secondary library.

[0515] in some cases, suitable is synthetic longer peptide is with in conjunction with some acceptor, and in other cases, that wants provides the peptide with two or more HER binding sequences that separated by transcribed spacer (for example joint) residue.For example, binding sequence can be separated by transcribed spacer, and wherein said transcribed spacer makes the peptide zone be presented to acceptor by different way.Distance between the land may be as little to 1 residue, or 2-20 residue or many at least 100 residues at least.Preferred transcribed spacer is 3,6,9,12,15 or 18 residue length.For surveying big binding site or series connection property binding site (for example, in epi-position on the domain II and the epi-position on structural domain IV), the land can the transcribed spacer of 20-30 amino-acid residue separates by containing nearly.When existing, the number of transcribed spacer residue generally is at least 2 residues, and usually is less than 20 residues.

[0516] oligonucleotide library can have the binding sequence that is separated by transcribed spacer (for example joint), and therefore can be represented by following formula: (NNK) _y-(abc) _n-(NNK) _zWherein N and K such as previous definition (noting to substitute K) as the S of previous definition, and y+z equals about 5,6,7,8 or bigger numeral, a, b and the identical or different Nucleotide of c representative, comprise the amino acid whose codon in encoded interval district, n is many to about 3,6,9 or 12 amino acid or more a plurality of amino acid.The transcribed spacer residue can be some flexibility, comprise few glycine or widow-glycine-glycine-Serine, for example aim to provide the interactional diversity ability in site in library structural domain and the big binding site, wherein said big binding site is relative free because of connecting with phage albumen.Rigidity transcribed spacer (for example few proline(Pro)) also can insert individually or with other transcribed spacer (comprising the glycine transcribed spacer) combination.May need to make the HER binding sequence mutually near and use a transcribed spacer so that described binding sequence is directed each other, for example by (as what can provide as the transcribed spacer of glycine-proline(Pro)-glycine by sequence) corner is provided between two kinds of sequences.In order to increase the stability of this corner, may want or must add cysteine residues at one of two ends of every kind of variable region or two ends.Described cysteine residues forms disulphide bridges subsequently so that the variable region is fixed together in a ring, and also can simulate the effect of cyclic peptide in this mode.Those skilled in the art understand also can use multiple other type covalently bound and are used for cyclisation.

[0517] one of transcribed spacer residue as indicated above two ends that also can be positioned at the HER binding sequence are located or two ends.For example, can design the cyclic peptide that does not have the property inserted transcribed spacer by two ends that make cysteine residues be positioned at peptide.As indicated above, flexible spacer district (for example few glycine) can promote peptide and selected acceptor interaction.Alternatively, the rigidity transcribed spacer can be so that peptide presents as the end that is located on rigid arm, and wherein the number of residue (for example proline residue) not only determines arm lengths, also determines the direction like this of this arm, and wherein peptide is directed on described direction.Can use the hydrophilic transcribed spacer formed by electrically charged or uncharged hydrophilic amino acid (for example Thr, His, Asn, Gln, Arg, Glu, Asp, Met, Lys) or present this peptide to the receptor binding site under multiple local environment by the hydrophobic transcribed spacer that hydrophobic amino acid (for example Phe, Leu, Ile, Gly, Val, Ala) is formed.

[0518] it should be noted that some peptides can cause the infectious serious defect of its carrier phage because of its size and/or sequence.This causes phage to be lost from colony during infecting and increasing after every round elutriation.In order at utmost to reduce the problem infectious relevant with defective, the DNA that the phage from wash-out can be prepared is converted into the suitable host cells (for example intestinal bacteria), preferably transform and (see by electroporation, for example, Dower etc., Nucl.Acids Res.16:6127-6145) or the chemical process of knowing carry out.Cell culture is continued one period that is enough to the presentation markup thing, and as to DNA conversion select to be selected like that.The amplification bacterium colony, and be used for affine enrichment according to the results of establishment method phage.The phage of identifying in affine enrichment can be increased by infecting to host cell again.Select the transformant of success by growth in suitable microbiotic (as tsiklomitsin or penbritin).This can carry out on the solid medium or in the liquid growth medium.

[0519] for being grown on the solid medium, with cell with high-density (about 10 ⁸-10 ⁹The every m of individual transformant ²) cultivate and for example to contain on the big surface of selective antibiotic L-agar, to form the lawn that converges basically.From then on cell and outstanding phage scraped on the surface and prepare phage and be used for first round elutriation (seeing for example Parmley and Smith, 1988, Gene 73:305-318).For being grown in the liquid culture, can be with cell culture about 10 times or more times multiplication of process in L-meat soup and microbiotic.By standard method results phage (see Sambrook etc., 1989, Molecular Cloning, second edition).Growth in liquid culture may the Yin Wenku size but more easily, and during the growth on the solid medium can provide amplification procedure less deflection probability.

[0520] for required clone's affine enrichment, incites somebody to action common about 10 ³-10 ⁴(a library equivalent is one of every kind of recombinant chou to individual library equivalent; Contain 10 ⁹10 of individual member library ⁴Individual equivalent is 10 ⁹* 10 ⁴=10 ¹³Individual phage), but generally at least 10 ²Individual library equivalent arrives about 10 more ⁵-10 ⁶Individual library equivalent and acceptor (or its part) are hatched, and wherein seek the peptide of wanting at described acceptor.Described acceptor is in one of several forms that are suitable for affine enrichment.In an example, this receptor is fixed on surface or the particle, and the phage library that carries peptide is subsequently according to methods known in the art elutriation on the fixed acceptor.For example, this acceptor can be expressed on the cell surface of monolayer cell (due to because of transfection, or utilizing the cell of the described suitable acceptor of natural expression).In addition, the ECD of HER molecule part can be connected with the Fc structural domain and select and can carry out at the HER-Fc complex body that is fixed on the A albumen agarose.In this kind example, can remove phage display library at irrelevant Fc fusion rotein-A (or G) albumen agarose complex body.In an alternatives, acceptor and discernible part (it can be connected through a lashing) combination.The specific examples of this part is a vitamin H.With the acceptor so modified and phage library is hatched and keying action takes place in solution with two kinds of reactants.The complex body of gained with after biotin moiety combine with streptavidin or avidin.Streptavidin can be fixed on surface (as plastic board) and go up or be fixed on the particle, and complex body (phage/peptide/acceptor/biotin/streptavidin) is retained to physical property in this case; Or streptavidin can use the fluorophore mark, for example, is intended to the active phage/peptide of mark to detect by sorting method (for example on the fluorescence-activated cell sorting device) and/or to separate.

[0521] subsequently can be by elutriation promotes the enrichment of associativity phage to how specified target (for example epitope regions of identifying among the subdomain I-IV arbitrarily).Therefore, for example, according to the purpose subdomain of HER molecule (for example SEQ ID NO:54-61 arbitrarily in (subdomain I and III), at SEQ ID NO:62-93 arbitrarily in (subdomain II) and/or at SEQ ID NO:94-125 or 405 any one described in (subdomain IV) or multiple purpose subdomain arbitrarily), can further screen positive phage clones at various synthetic peptides.Phage can carry out enrichment at the various synthetic peptide described in SEQ ID NO:54-125 or the 405 any items.This enrichment will allow the phage binding site on definite HER family receptors.For identifying those molecules as general HER therapeutical agent, also can promptly carry out follow-up screening on the HER-Fc-A albumen agarose complex body or on the monolayer cell of other HER acceptor of expression, with evaluation and more than a kind of those molecules of HER family receptors bonded at other HER family receptors.

[0522] in each step, removes because of non-specific interaction and HER family receptors bonded phage by washing.Determine the required washing degree and the severity of every kind of purpose acceptor/peptide.Can be by regulating that associativity is hatched and the condition of subsequent wash produces to a certain degree control to the peptide that reclaims in conjunction with feature.The volume of temperature, pH, ionic strength, divalent cation concentration and washing and time length will select this receptor is had the peptide of specified range avidity.Selection based on slow dissociation rate is the most practical method, and wherein said slow dissociation rate indicates high-affinity usually.This can be by continuing to hatch in the presence of the free ligand of saturation capacity or being undertaken by the volume, number of times and the time span that increase washing.In each case, prevent the combination again of dissociated peptide-phage, and increase in time, be recovered to the more and more higher peptide-phage of avidity.Can adjust combination and washing flow extraly to find the peptide of bind receptor under given conditions.In case known some avidity of acceptor molecule and the specific peptide sequence given then can be set up diversity around this binding motif.For example, variable peptide zone can be placed on one of two ends or two ends of the sequence of identifying.Known array can be determined from document or can be derived from the elutriation of early stage round.

Ii. polymer polypeptide (heterodimer peptide)

[0523] can prepare polymer polypeptide (part) by the covalently bound aminoacid sequence that two or more identify the binding peptide of (identifying as using display technique of bacteriophage).According to the intended purposes of multivalent ligand, can merge with the HER molecule on identical or different structural domain site bonded polypeptide to form individual molecule.Make up multivalent ligand with the different subdomains of not isoacceptor or acceptor on identical or corresponding site in conjunction with the time, the aminoacid sequence that is used for this receptor bonded peptide part can be identical or different, condition is that then they all combine with same loci if use the different aminoacids sequence.The cell surface specific polypeptide that can prepare other similarly.

[0524] the multivalence polypeptide can be by expressing respectively and each site bonded aminoacid sequence and subsequently that they are together covalently bound, or prepare by described multivalent ligand is expressed as the single amino acid sequence, wherein said single amino acid sequence portion within it contains the combination that is useful on the bonded specific amino acid.And the merging of the different loci bonded amino acid polypeptide in the subdomain or between the subdomain can be used for producing such molecule, and described molecule is that the higher peptide part of avidity maybe can make the different subdomains on the HER acceptor crosslinked together.

[0525] no matter produce or produce by conventional interconnection technique by recombinant gene expression, multiple polypeptides can be passed through the different joint coupling of length.Express under institute's catenation sequence and the situation based on about 4 dusts of average amino acid length on reorganization ground, the joint that is used to connect two seed amino acid sequences generally is about 3 to about 12 amino acid lengths.Can be adjusted in the flexible degree of the joint between the aminoacid sequence by the amino acid of selecting to be used for to make up joint.Glycine is used to produce flexibility, the relative joint that does not limit with the combination of Serine.Can in catenation sequence, use amino acid and make up the higher joint of rigidity with more complicated side chain.

[0526] in an example, the prepared product of polymer construct comprises one or more binding peptides.For example, because of being biotinylated and compound to form the tetramer construct with avidin, streptavidin or neutravidin with combining the peptide that target identified by the phage display method.These tetramer constructs are hatched with target or its part (for example express the cell of the HER target of wanting and do not express the cell of described target) subsequently, and the keying action of detection and described tetramer construct.Can use any detection method known in the art to detect keying action.For example, in order to detect keying action, avidin, streptavidin or neutravidin can be puted together with detectable label (for example radio-labeled thing, fluorescent marker or the enzyme labelling thing of colour-change takes place, as HRP (horseradish peroxidase), TMB (tetramethyl benzidine) or alkaline phosphatase).Randomly can in the presence of serum, screen multimeric complexes.Therefore, this assay method can also be used to assessing rapidly serum to peptide and the influence of target bonded.

[0527] the biotinylation peptide is preferably compound with neutravidin-HRP (neutravidin-HRP).The non-specific low keying action of neutravidin demonstration and the molecule except other surrogate, its reason is to lack the sugar moieties of binding lectin and lacks in the neutravidin RYD structural domain in conjunction with the cell adhesion acceptor that (referring to for example, Hiller etc. (1987) Biochem J.248:167-171; Alon etc. (1990) Biochem.Biophys.Res.Commum., 170:236-41).

[0528] use of vitamin H/avidin complex body allows relatively easily to prepare the tetramer construct that contains the different binding peptides of 1-4 kind.In addition, can by comprise with identical target on two or more targets of different epi-position bonded part increase the avidity and the avidity of target construct.Described herein screening assay method can be used to identify the combination in conjunction with polypeptide, and wherein said have the avidity of increase and/or crosslinked different subdomain (being intended to stable constraint conformation) when comprising in conjunction with polypeptide in this type of polymer construct.

B. computer assisted optimization

[0529] the another kind of method that can be used to identify the general HER treatment molecule of pharmaceutical active is to use computer assisted optimisation technique to produce more may suddenling change of high-affinity to find at part.Embodiment provides the relevant guidance that how may use this type of computer assisted optimisation technique.For example, can produce in this manner to part have HER1, the HER2, HER3 or the HER4 that strengthen keying action and as component with produce heteromultimeric, with polymer and composition thereof.

C. exemplary screening assay method

[0530] also provides the screening assay method of identifying the general HER treatment molecule that pharmaceutical active is arranged herein.Can use at the active known assay method of specific cells surface receptor and identify general cell surface specific molecular similarly.

[0531] general treatment molecule for example comprises 1) peptide (as soluble peptide, it comprises the fusogenic peptide of band Ig tail) and the member of random peptide library (see that for example, Lam etc. 1991, Nature 354:82-84; Houghten etc. 1991, Nature 354:84-86) and the derivative molecular library of combinatorial chemistry made by D-and/or L-configuration amino acid; 2) phospho-peptide (for example, the member in the directed phospho-peptide of randomness and partial degeneracy library, referring to for example Songyang etc., 1993, Cell, 72:767-778); 3) antibody (for example Fab of polyclonal antibody, monoclonal antibody, humanized antibody, antiidiotypic antibody, chimeric antibody and single-chain antibody and antibody, F (ab ') ₂, Fab expression library fragment and antibody the epi-position binding fragment); With 4) organic and inorganic molecules.Example molecule is the peptide part of identifying from display technique of bacteriophage, as discussed herein above.

[0532] the test molecule also can comprise numerous chemical classifications, although they generally are organic molecules, preferably have greater than 50 and less than the small molecules organic compound of 2500 Dalton molecular weights.This quasi-molecule can comprise for structurally with protein interaction, the required functional group of hydrogen bond action especially, and generally comprise amino, carbonyl, hydroxyl or carboxyl at least, preferably comprise at least two kinds of chemical functional groups.Described molecule often comprises ring carbon or heterocycle structure and/or aromatics or the many aromatic structures that replaces with one or more above-mentioned functional groups.Described molecule can be available from numerous sources (library that comprises synthetic compound or natural compounds) widely.The synthetic compound library can be commercially from for example Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, NJ.), Brandon Associates (Merrimack, N.H.) and Microsource (New Milford Conn.) obtains.Rare chemical library can be from Aldrich Chemical Company, and (Milwaukee Wis.) obtains Inc..The natural compounds library that comprises bacterium, fungi, plant or animal extracts for example can be from Pan Laboratories (Bothell, Wash.) acquisition.In addition, numerous methods can be used for the organic compound and the biomolecules of synthetic at random and directed synthetic numerous types, comprise and express the randomization oligonucleotide.

[0533] alternatively, can produce the natural compounds library that is in bacterium, fungi, plant and animal form of extract easily.The method that is used for the synthetic molecules library be can obtain easily (see that for example, DeWitt etc. 1993, Proc.Natl.Acad.Sci USA 90:6909; Erb etc. 1994, Proc.Natl.Acad.Sci USA 91:11422; Zuckermann etc. 1994, J.Med.Chem.37:2678; Cho etc. 1993, Science 261:1303; Carell etc. 1994, Angew.Chem.Int.Ed.Engl.33:2059; Carell etc. 1994, Angew.Chem.Int.Ed.Engl.33:2061; With Gallop etc. 1994, J.Med.Chem.37:1233).In addition, natural compounds or synthetic property library of compounds and compound can be modified easily by conventional chemical method, physical method and biochemical method (to be seen, for example, Blondelle etc. 1996, Trends in Biotech.14:60) and can be used for producing combinatorial library.In another approach, the previous pharmaceutical agents of identifying can carry out orientation or chemically modified at random, as acidylate, alkylation, esterification, amidation (amidification), and can screen HER to analogue and regulate active.

[0534] the numerous methods that are used to produce combinatorial library are known in the art, comprise relating to biology library method; Parallel solid phase of space addressable or liquid phase library method; Need go the synthetic property library method of flatung; " a kind of compound of a pearl " library method; And those methods of using the synthetic library method of affinity chromatography selection.Biology library method is limited to polypeptide or peptide library, and other four kinds of methods are applicable to polypeptide, peptide, non-peptide oligomer or micromolecular compound library (K.S.Lam, 1997, Anticancer DrugDes.12:145).

[0535] library can be in solution be used for determining that by this area is known usually the method whether part is combined on the common binding site competitively screens.These class methods can comprise screening in solution (for example, Houghten, 1992, Biotechniques 13:412-421) (Lam or on pearl, 1991, Nature 354:82-84), (Fodor on chip, 1993, Nature 364:555-556), (Ladner, U.S. Patent number 5,223 on bacterium or spore, 409), (Cull etc. 1992 on plasmid, Proc.Natl.Acad.Sci USA 89:1865-1869) (Scott and Smith, 1990, Science 249:386-390 or on phage; Devlin, 1990, Science 249:404-406; Cwirla etc., 1990, Proc.Nat.Acad.Sci USA 97:6378-6382; Felici, 1991, J.Mol.Biol.222:301-310; Ladner sees above) the library.Any described library (comprising its any test molecule) can contact with all or part of (as that identify in subdomain I, II, III or IV and any part HER epitope regions of describing in arbitrarily at SEQ ID NO:54-125) of HER molecule and can the evaluation test molecule and the interaction of HER ECD or its part.Can identify the general HER therapeutical agent of the interactional candidate of at least a or multiple epitope regions in demonstration and the described epitope regions.This type of general HER therapeutical agent also will show the interaction with at least a or multiple total length HER molecule or its ECD part, general at least two kinds or at least three kinds HER molecules.

[0536] be under the situation of binding assay in described screening assay method, all or part of or its HER ECD of HER all or part of (as any or the test molecule in the peptide epitopes of describing in 405 at SEQ ID NO:54-125) can be connected with marker, and wherein said marker can provide detectable signal directly or indirectly.Multiple marker comprises radio isotope, fluorescence molecule, chemiluminescent molecule, enzyme, specific binding molecules, particle (for example magnetic particle) etc.Specific binding molecules comprises counterpart, as vitamin H and streptavidin, digoxin and anti-digoxin (antidigoxin) etc.For the specific combination member, complementary member is usually according to currently known methods, with providing the molecule of detection effect to carry out mark.Can in the screening assay method, comprise multiple other reagent.These reagent comprise and can be used for promoting best protein-protein bound effect or minimizing and/or reduce non-specific or the interactional reagent of background, as salt, neutral protein matter (for example albumin), washing agent etc.Can use the reagent that improves assay method efficient, as protease inhibitor, nuclease inhibition or antimicrobial drug.Described component is added to produce necessary keying action with any order.On any temperature that promotes optimum activity, generally on 40 ℃, hatch at 4 ℃.Select incubation time according to optimum activity, but also can be to incubation time optimization to promote high flux screening rapidly.Usually, 0.1-1 hour is enough.Generally speaking, the multiple mixture of measuring is to move abreast with acquisition with different test substances concentration to reply at the difference of these concentration.Generally, a concentration of these concentration plays the negative control effect, promptly is in zero-dose or is lower than detection level.

[0537] in an example, can be as indicated above at phage display library screening and HER acceptor molecule or its part bonded part.Disclose and made up and analyze the details in these libraries and be used for biological elutriation and (see that for example, WO 96/04557 with the basic skills of selecting binding substances; Mandecki etc. 1997, Display Technologies--Novel Targets and Strategies, P.Guttry (editor), International Business Communications, Inc.Southborogh, Mass, 231-254 page or leaf; Ravera etc. 1998, Oncogene 16:1993-1999; Scott and Smith, 1990, Science 249:386-390); Grihalde etc., 1995, Gene 166:187-195; Chen etc. 1996, Proc.Natl.Acad.Sci USA 93:1997-2001; Kay etc. 1993, Gene128:59-65; Carcamo etc. 1998, Proc.Natl.Acad.Sci USA 95:11146-11151; Hoogenboom, 1997, Trends Biotechnol.15:62-70; Rader and Barbas, 1997, Curr.Opin.Biotechnol.8:503-508; Described whole document mode is by reference incorporated this paper into).

[0538] at known pharmaceutically active compound and the simulation thing is a kind of based on " guide (lead) " compound and the currently known methods of developing drugs.When active compound is difficult to synthetic or its synthetic expensive or this compound is not suitable for specific application process, for example, peptide is unfavorable active substance usually for oral compositions, because they are easy to be degraded rapidly by the proteolytic enzyme in the digestive tube, the method for described developing drugs is desirable.Usually utilize stand-in designs, synthetic and test to avoid screening molecule on a large scale at certain target characteristic.

[0539] when being derived from the stand-in of the compound with given target characteristic, design has the common several steps that adopts.At first, determine in decision it is the specific part of crucial and/or important this compound aspect the described target characteristic.Under the situation of peptide, this can be undertaken by the amino-acid residue (for example by replacing each residue successively) that systematically changes in this peptide.Constitute those parts of active zone of this compound or " pharmacophore " that residue is called this compound.

[0540] in a single day found pharmacophore, then according to the physical property (for example stereochemistry, combination, size and/or electric charge) of this pharmacophore, the data (for example spectroscopic techniques, X ray diffracting data and NMR) of using a series of sources are to its structural modeling.Can in modeling process, use computational analytical method, similarity graphing method (it is to the electric charge and/or the modeling volume of pharmacophore, but not interatomic bond effect) and other technology.In the changing form of described method, to the three-dimensional structure modeling of part and binding partners thereof.Especially part and/or binding partners in conjunction with the time change under the situation of conformation, this can be particularly useful, thereby allows this model that this point is considered in the design of stand-in.Select template molecule subsequently, and the chemical group of this pharmacophore of simulation can be transplanted on this template.Can select template molecule and the chemical group of being transplanted on this template molecule easily, thereby stand-in are synthetic easily, this is that pharmacy is acceptable, not the biologic activity of degradation in vivo and reservation lead compound.Which kind of degree the stand-in that found of screening suppress described target characteristic to the degree of determining their display target characteristics or they subsequently.Can implement subsequently further to optimize or modify to obtain to be used for one or more final stand-in of body build-in test or clinical trial.

[0541] can test the general HER therapeutical agent that in method mentioned above, identifies and functionally regulate the active ability of one or more HER.This type of activity is well known by persons skilled in the art and is above describing in the G part.The example of this type of assay method comprises part combination, cell proliferation, cells phosphorylation and compound/dimerization.Therefore, can further test herein because of combining the general HER therapeutical agent of any candidate who is accredited as material standed for based on high-affinity in the screening assay method to determine whether this candidate therapeutic agent has general HER therapeutical agent characteristic, promptly at HER activated rejection characteristic with HER molecule or its part.For example, best the general HER therapeutical agent of the dimerization arm of target in domain II will suppress HER molecule and self or with the dimerization of other HER family molecule.Similarly, lacking under the situation of dimerization, expecting that also this candidate therapeutic agent will suppress the ability of HER molecule inducing cell phosphorylation or cell proliferation when being subjected to suit ligand stimulation.In another example, the general HER therapeutical agent that plays the effect (for example by crosslinking structure territory II and IV) that makes lashing stable will suppress the ability that the HER molecular conversion becomes state of activation.Therefore, can test the ability that the general HER therapeutical agent of this candidate is regulated (the general inhibition) dimerization or cell activation, institute's stimulated cell proliferation or cells phosphorylation were assessed under wherein said cell activation was existed by part.In extra example, can test the general HER therapeutical agent of candidate by the keying action of analyzing any one or multiple HER family and part and suppress part bonded ability, wherein said part includes but not limited to EGF, amphiregulin, TGF-α or any neuregulin (being HRG β).The general HER therapeutical agent of being identified will be regulated (general suppress) one or more HER mediation property activity mentioned above at least two kinds of HER acceptors.

K. embodiment

[0542] property purpose and comprise following examples presented for purpose of illustration only, and be not intended to limit the scope of the invention.

The clone of embodiment 1HER ectodomain

[0543] cloned and expressed the multiple HER derivative of all or part of ectodomain that contains the HER molecule.

A. clone HER ECD derivative

[0544] the following clone of HER1-621 (SEQ ID NO:12): (the 1-621 amino acids of the aminoacid sequence of total length HER1 acceptor (obtains from Gail Clinton with ectodomain; SEQ IDNO:2)) carry out pcr amplification and by the KpnI-XhoI restriction site and subclone to pcDNA3.1Myc-His carrier (Invitrogen; Also referring to the sequence SEQ ID No.161 of pcDNA3.1 Myc-His) to produce the pcDNA/HER1-621-myc-His carrier.

[0545] the following clone of HER3-621 (SEQ ID NO:26): ectodomain (the 1-621 amino acids of the aminoacid sequence of total length HER3 acceptor (seeing SEQ ID NO:6)) carried out pcr amplification and by the KpnI-Xbal restriction site and subclone to the pcDNA3.1Myc-His carrier to produce the pcDNA/HER3-621-myc-His carrier.

[0546] cloned extra ECD derivative.Their design and respective coding nucleic acid and amino acid sequence coded sign are described in following table:

[0547] Fig. 2 (A)-2 (D) has described these and has cloned each and the comparison result of its respective associated acceptor in the isoform

B. protein expression and secretion

[0548] be in people's cell, to express HER ECD derivative, with human embryo kidney (HEK) 293T cell with 2 * 10 ⁶Individual cells/well is seeded in the 6 hole flat boards and is maintained in the Eagle substratum (DMEM) and 10% foetal calf serum (Invitrogen) of Dulbecco improvement.Use LipofectAMINE 2000 (Invitrogen), according to the manufacturer specification transfectional cell.On transfection same day, 5 μ g plasmid DNA are mixed with 15 μ l LipofectAMINE 2000 in 0.5ml serum-free DMEM.Mixture incubated at room 20 minutes, is added on the cell subsequently.With cell at 37 ℃ in CO ₂Hatched in the incubator 48 hours.Be the protein secreting effect of research HER ECD derivative, collection condition substratum 48 hours.By on the SDS-polyacrylamide gel, separating, using the immunoblotting of anti-His antibody (Qiagen) to analyze this conditioned medium subsequently.With antibody dilution in 1: 5000.

[0549] to assesses the secretion of every kind of HER ECD derivative from the substratum of people's cell of cultivating.In following table 10, describe relatively HER ECD derivative excretory.

Embodiment 2HER-Fc merges prepared product and protein expression

A. the segmental clone of human IgG1's Fc

[0550] use following forward and reverse primer to the Fc fragment compiling pcr amplification the thing from strand cDNA and go out the human IgG1 (in SEQ ID NO:167, describe, and corresponding to the amino acid Pro100-Lys330 of the aminoacid sequence described in the SEQ ID NO:163):

5′CCCAAATCTTGTGACAAAACTACTC3′(SEQIDNO：49)

5′TTTACCCGGGGACAGGGAG3′(SEQIDNO：50)

The PCR fragment through gel-purified and subclone to pDrive cloning vector (Qiagen PCR clones test kit, Qiagen, Valencai CA, SEQ ID NO:160) to produce pDrive/IgG1Fc.

The fusion of B.Fc and HER ectodomain

[0551] the following clone of HER1-621/Fc (SEQ ID NO:40): the pcDNA/HER1-621-myc-His carrier carries out restrictive diges-tion with XhoI and AgeI.Use Qiagen gel-purified test kit (Qiagen) plasmid that purifying cut.Use following primer to go out human IgG1 Fc fragment from pDrive/IgG1Fc carrier pcr amplification:

5 ' ATTA CTCGAG GGA CGA ATG GAC CCC AAA TCT TGT GACAAA ACT C 3 ' (containing the XhoI site, SEQ ID NO:51)

5 ' ACTT ACCGGT TTT ACC CGG GGA CAG GGA G 3 ' (containing the AgeI site, SEQ ID NO:52).

Through the Fc of pcr amplification fragment with XhoI and AgeI digestion and be connected in the pcDNA/HER1-621-myc-His carrier of digestion.

[0552] the following clone of HER3-621/Fc (SEQ ID NO:46): the pcDNA/HER3-621-myc-His carrier carries out restrictive diges-tion with XbaI and AgeI.The plasmid that uses Qiagen gel-purified test kit purifying to cut.Go out human IgG1 Fc fragment with following primer pcr amplification from pDrive/IgG1Fc:

5 ' ATTA TCTAGA GGA CGA ATG GAC CCC AAA TCT TGTGAC AAA ACT C (containing the XbaI site, SEQ ID NO:53)

5 ' ACTT ACCGGT TTT ACC CGG GGA CAG GGA G 3 ' (containing the AgeI site, SEQ ID NO:52).

[0553] through the Fc of pcr amplification fragment with XbaI and AgeI digestion and be connected in the pcDNA/HER3-621-myc-His carrier of digestion.

[0554] prepares other fusion constructs similarly.Whole gained fusion constructs are verified by dna sequencing.Exemplary Fc fusion protein construct is described in the following table:

C. protein expression and secretion

[0555],, uses Lipofectamine2000 (Invitrogen) transfection HER ECD Fc fusion constructs (HER1-621/Fc respectively as describing among the embodiment 1 for producing the HER-Fc chimeric protein; HER3-621/Fc; HER2-650/Fc; HER4-650/Fc) to the 293T cell.48 hours collection condition substratum after transfection.The conditioned medium (20 μ l) that on the denatured protein gel, separates equivalent.Survey the western blotting thing with check protein expression and secretion with anti-His (Qiagen) antibody or anti-Fc (Sigma) antibody.In following table 12, describe relatively HER ECD derivative excretory.

[0556] for producing the polymer of HER1 and HER3, use Lipofectamine2000 (Invitrogen) according to manufacturer specification, respectively with HER Fc fusion constructs (HER1-621/Fc and HER3-621/Fc) cotransfection to the 293T cell.Conditioned medium collection in 48 hours after transfection from each transfection.The conditioned medium (20 μ l) that on the denatured protein gel, separates equivalent.Survey the western blotting thing with check protein expression and secretion with anti-His (Qiagen) antibody or anti-Fc (Sigma) antibody.

[0557] (be called HFD100/300H (total length HER1ECD is connected with total length HER3ECD through the Fc structural domain) again, the construct of Her1 and Her3 was with 1: 3 (Her1: Her3) ratio cotransfection in order to express heterodimer RB200h.After 5 hours, substratum is replaced by DMEM+1%FBS (low IgG) in transfection.Collect the first condition substratum after 4 days in transfection, feed supplement subsequently and carry out the second time and collect.

[0558] use the Chinese hamster ovary celI and the HEK 293T cell that are adapted to serum free medium (FreeStyle 293) before to implement the suspension cell protein expression.With HEK 293T cell with 1 * 10 ⁶Individual cell/ml is inoculated among the WaveBioReactor with Freestyle 293 substratum (Invitrogen).Next day, linear PEI (Polysciences): the DNA of 25kD that uses 1: 2 ratio with HER ECD construct (HER1-621/Fc and HER3-621/Fc) transfection to the 293T cell.For expressing heterodimer RB200h, the construct of Her1 and Her3 was with 1: 3 (Her1: Her3) ratio cotransfection., after 5 hours culture volume is doubled in transfection.Monitoring viable cell and protein every day produces.In transfection after 6 days, the collection condition substratum.

The purifying of embodiment 3HER (HF) derivative and HER-Fc (HFD) molecule

[0559] whole HF molecules of band suffix " T " contain carboxyl terminal 6-Histidine tail and are used for the metal affinity purification.Use the affine metal chromatography of Ni, utilize all these molecules of preparation type size exclusion chromatography method (SEC) purifying subsequently.At first, will contain the conditioned medium (CM) of secretor type HF molecule by centrifugal (30 minutes, 10K rev/min) purification and subsequent filtration (0.3 micron).(Pau Corporation, Ann Arbor MI) concentrate the CM that purifies 4 times to produce final volume to about 400ml to use Pall tangential flow thickener subsequently.

[0560] make CM reach 50mM NaPO by adding 10 * Ni-NTA sample-loading buffer ₄(pH8.0) and 350mM NaCl.Subsequently solution was loaded into buffer A (buffer A: 50mM NaPO with flow velocity 0.6ml/ minute ₄(pH 8), 350mM NaCl) the affine metal chromatography column of 1.5ml nickel of pre-equilibration (Ni-NTA Agarose, Qiagen, Germany) on.After loading, wash this post with buffer A and do not stay unconjugated protein until absorbancy indication at the 280nm place.Pass through the molecule such as HF such as gradient elution such as degree such as grade of buffer A+150mM imidazoles subsequently.Merge and to contain the peak fraction of HF molecule and to be concentrated into 1ml, be loaded on subsequently preparation type SEC post (Superose 12 10/300GL, Amersham Biosciences, Sweden) on.By immunoblotting, (Finland) evaluation contains the monomeric peak of HF fraction to the mouse anti His6-tag antibody of puting together with horseradish peroxidase for HyTest Ltd., Turku.The amino acid sequencing of implementing the HF molecule is to confirm every kind of molecule.

[0561] the HFD100/HFD300T heterodimer is the Fc fusions of HFD100 and HFD300T.The transient transfection that produces this molecule also produces the homodimer of HFD100 by name and HFD300T.(Ni-NTA Agarose, Qiagen Germany) carry out purifying, and 6-is histidine-tagged because HF300T contains carboxyl terminal by the Ni-NTA affinity chromatography for HFD300T homodimer and HFD100/HFD300T heterodimer.Conditioned medium (CM) purifies as mentioned above and concentrates.The CM of gained is loaded into 1.5ml A albumen post, and (nProtein A Sepharose 4 Fast Flow, AmershamBiosciences Sweden) go up and with immune pure IgG elution buffer (Pierce, Rockford, IL) wash-out.Behind wash-out, by adding in the 50 μ l 1M tris-HCl damping fluids (pH 8.0) and fraction.Merge and contain proteic fraction and make solution reach 50mM NaPO4 (pH 8.0) and 350mMNaCl.With this merging thing be loaded into the affine metal chromatography column of 1.5ml nickel with the buffer A pre-equilibration (Ni-NTA Agarose, Qiagen, Germany).Collection contains the effluent (effluent liquid) of HFD100 homodimer.After the buffer A washing, with the gradient elution HFD300T homodimer such as degree such as grade and the HFD100/HFD300T heterodimer albumen of buffer A+150mM imidazoles.

[0562] by use 3ml CNBr activatory Sepharose 4 Fast F1ow pearls (Amersham Biosciences, Finland), with 10mg EGF (R﹠amp; D Systems, Minneapolis MN) covalently is connected to sepharose (sepharose) solid support and produces 10ml EGF affinity column.Merging is from the peak fraction of Ni-NTA elutriant and chromatography on described EGF affinity column immediately.Collect in the effluent liquid and the corresponding peak of HFD300T.With IgG elution buffer wash-out HFD100/HFD300T heterodimer, and merging contains proteinic fraction and (Superose 12 10/300GL, Amersham Biosciences Sweden) go up chromatography at preparation type SEC post immediately.This step is removed any EGF that institute's wash-out goes out during EGF affinity column step.The fraction that will contain the HFD100/300T of purifying neutralizes, damping fluid is changed into PBS and (Millipore, Billerica MA) concentrate with 30kD cutoff value Amicon centrifuging post with 50 μ l 1M tris damping fluids (pH 8.0).

[0563] by mode purifying RB600 like this, promptly the transfectional cell from embodiment 2 is obtained conditioned medium and by purifying in centrifugal 15 minutes at 12,000 * g at 4 ℃, with after 3 μ mVersapore 3000T filter (Pall Corporation, East Hills NY) filters.With the conditioned medium that purifies through the Ultrasette of 30kDa cutoff value Screen Channal tangential flow filtration device (Pall Corporation, East Hills, NY) concentrate 10 times and be applied to MabSelect SuRe affinity column (GE Healthcare Biosciences AB, Sweden).This post is with thoroughly washing and with IgG elution buffer (Pierce Biotechnology Inc., Rockville, IL) wash-out of the PBS that contains 0.1% (v/v) TX-114.The fraction of wash-out is neutralized to pH 8.0 with 1M Tris-HCL immediately.

[0564], forms by RB200h heterodimer and HFD100 and HFD300h homodimer from the protein that contains Fc of MabSelect SuRe affinity column wash-out in this stage.This mixture of RB200h heterodimer and HFD100 and HFD300h homodimer is called RB600.To after PBS (RB600) dialysis, this homodimer/heterodimer mixture directly uses as mixture, or as starting material in order to be further purified RB200h (the total length HER1ECD that is connected with total length HER3ECD through the Fc structural domain; Be called HFD1000/HFD300H again).The structure of RB200h is shown in Fig. 4.

Purity check

[0565] the operational analysis reversed-phase HPLC is determined lipidated protein.Use is applied to the analytical C4 post (150 * 46mm from Kromasil of AKTA:Purifier System (GE-Healthcare); 5mm; 100A) carry out proteinic reversed-phase HPLC.Buffer A is made of the 0.1%TFA in water (v/v) and buffer B contains the 25%2-propyl alcohol; 75% acetonitrile; 0.1%TFA (v/v).Generally, the linear gradient that loads 50-100mg protein and use 5-95% buffer B was come eluted sample (flow velocity=0.5ml/ minute; Gradient=6%/minute).

[0566] under the condition of this system, at first wash-out contains the homodimer of 2 erbB3 chains, follow by heterodimer (RB200h), and be the erbB1 homodimer then.Using two kinds of methods to carry out the peak specifies.At first, use standard substance of purifying from single cells transfected (a kind of polypeptide chain of only encoding) is identified homodimer peak (see figure 5).Secondly, submission is carried out aminoterminal order-checking (Stanford:PAN facility) to verify designated result (data not shown) originally from the fraction at each peak.

[0567] purification scheme is used the combination of A albumen post, Ni-sepharose post and EGFR-Affybody post.Judge that by SDS PAGE and reversed-phase HPLC the RB200h of purifying has＞90% purity.Show that as analytical reversed-phase HPLC chromatogram column technique RB200h (total length HER 1 ECD that is connected with total length HER 3 ECD through the Fc structural domain) is pure, have be no more than 10% HFD100 and uniting of HFD 300 mix (Fig. 5).

Embodiment 4HER ECD or HER-Fc combine with part

A.HER ECD derivative combines with Urogastron (EGF's)

[0568] ectodomain of HER1 (HER1), HER2, HER3 and HER4 is blended in people Fc (seeing embodiment 1 and 2) to produce chimeric polyeptides.HER ECD (HER-T) or HER-Fc obtain from the conditioned medium from cell, and wherein said cell is with related vector transfection ( embodiment 1 and 2 sees above).Supernatant liquor is from the 293T cell harvesting with Related cDNAs construct transient transfection.The following combination of determining radiolabeled EGF (Amersham) to the supernatant liquor that contains HER1-621/Fc, HER2-650/Fc, HER3-621/Fc, HER4-650/Fc, HER1-501/Fc, HER1-621 (T), HER1-501 (T): by with 20 μ l supernatant liquors and 5nM in Hepes damping fluid (pH 7.5) ¹²⁵I-EGF exists or does not exist down in mixed at room temperature enforcement in 2 hours combination in 1000 * excessive cold EGF.When binding assay finishes, add chemically crosslinked joint BS ³(Pierce) with crosslinked bonded molecule.Sample separated on the SDS-PAGE gel and expose film and be used for detecting.Normalization method and HER molecule bonded are estimated ¹²⁵I to etc. volumetric molar concentration.The result shows ¹²⁵I-EGF only combines with the HER1 derivative, and does not detect ¹²⁵I-EGF and HER2-650/Fc (HFD200), HER3-650/Fc (HFD300) or with the combining of HER4-650/Fc (HDD400). ¹²⁵I-EGF is competed by excessive cold EGF with combining of HER1-621/Fc (HFD100).

[0569] uses anti-HER1 antibody (R﹠amp; D Systems) carry out western blotting, the optical density assay method is assessed the level relatively of HER1-derivative then, and is used for normalization method and every kind of proteinic part keying action subsequently.It is right that the result shows that HER1-621/Fc (HFD100) has than HER1-501/Fc (HFD110) and HER1-501 (HF110) ¹²⁵The bigger binding affinity of I-EGF, and the total length HER1ECD (HER1-621 of the no Fc of ratio; HF100) has much bigger binding affinity.Prove that hereinafter the Fc fusions forms dimer after expression.Therefore, these part keying actions result shows that the fusion/dimerization effect that is partly mediated by Fc has recovered the high-affinity keying action of the total length ECD of HER1, and it surpasses the avidity keying action of HER1-501 monomer molecule.

[0570] other experiment confirm is compared with HF100, HFD100 (HER-621/Fc) and HFD110 (HER1-501/Fc) show with ¹²⁵The combination that the I-EGF part obviously increases, and HF110 do not show with ¹²⁵The detected combination of I-EGF.In addition, as expected, data show HER1/HER3 (HFD100/HFD300) heterodimer than HF100 and HF110 obviously more with ¹²⁵The I-EGF combination, but than HFD100 or HFD110 homodimer less with ¹²⁵The I-EGF combination.

B.HER ECD derivative is regulated combining of albumen (HRG) with Her

[0571] using as mentioned, A implements HER ECD derivative and regulates proteic the combination with Her partly to the described similar assay method of EGF keying action.In brief, from 293T cell, collect supernatant liquor, the HFD110/HFD310 heteromultimeric of wherein said DNA construct coding HF300 (HER3-621), HF310 (HER3-501), HFD300 (HER3-621/Fc), HFD310 (HER3-501/Fc) and purifying (HF110 that the Fc fragment by IgG1 is connected and the construct of HF310) with cDNA construct transient transfection.By supernatant liquor (from 2.5 μ l-20 μ l supernatant liquors) and 5nM with increasing amount ¹²⁵I-HRG implemented combination in 2 hours in mixed at room temperature in the cumulative volume of 20 μ l Hepes damping fluids (pH 7.5).When binding assay finishes, add 1mM BS ₃With crosslinked bonded molecule.Separation and combination reactant on the SDS-PAGE gel.Described protein gel is dry and be exposed to film 2 hours and 6 hours.

[0572] result shows that whole derivatives of being tested are to a certain degree in conjunction with HRG, although level is different.For whole derivatives of being tested, keying action is a dose-dependently, and observes the maximum combined effect when 20 μ l supernatant liquors.Use anti-HER3 antibody (R﹠amp; D Systems) the parallel Western blotting that carries out, the optical density assay method is assessed the level relatively of HER3-derivative and is used for subsequently based on the binding site number that equates and normalization method and every kind of proteinic part keying action then, and it is of equal value for anti-HER3 binding site.After this normalization method, the result confirms that HRG shows and the minimum keying action of HF300 molecule, compare with all the other derivatives of test, and only be about 10% of its keying action.After normalization method, HF310, HFD300, HFD310 and HFD110/HFD310 all show the equivalent keying action with HRG.

C. to HER derivative and Urogastron (EGF) and Her adjusting albumen (HRG β) bonded comparative analysis

[0573] by the test multiple HER derivative with ¹²⁵I-EGF (a kind of native ligand of HER1) and ¹²⁵The combination of I-HRG (a kind of native ligand of HER3 and HER4) and more described HER derivative specificity.The keying action of radiolabeled EGF and HER1-621/Fc, HER2-650/Fc, HER3-621/Fc, HER4-650/Fc mensuration as indicated above.Use as right ¹²⁵The described the same terms of the keying action of I-EGF is measured ¹²⁵The radiolabeled HRG of I combines with HER1-621/Fc, HER2-650/Fc, HER3-621/Fc, HER4-650/Fc's.Survey the western blotting thing with the comparison protein level with anti-His antibody.The result shows that radiolabeled EGF only combines with HER1-621/Fc and does not combine with other molecule of test.Radiolabeled HRG only combines with HER3-621/Fc and HER4-650/Fc molecule.

[0574] test from the conditioned medium of cell with ¹²⁵I-EGF and ¹²⁵The keying action of I-HRG, wherein said cell is with HER1-621/Fc and HER3-621/Fc (seeing embodiment 2) or HER1-501/Fc and HER3-501/Fc cotransfection.Data presentation with the cell of HER1-621/Fc:HER3-621/Fc cotransfection produce combine with radiolabeled EGF and with HRG bonded protein.

[0575] with anti-HER1 antibody and anti-HER3 (R﹠amp; D Systems) surveys the western blotting thing with the comparison protein level.The keying action of the radio-labeled part expressed proteinic amount of transfectional cell together is directly proportional, and wherein said protein comprises HER1/HER1 homodimer, HER1/HER3 heterodimer and HER3/HER3 homodimer.

[0576] HER1-621/Fc homodimer (being called HFD100) combination ¹²⁵I-EGF, and HER3-621/Fc homodimer (HFD300) and HER4-625/Fc (HFD400) combination ¹²⁵(Fig. 6 a) for I-HRG1 β 1.HER2-628/Fc (HFD200) does not show any detectable ¹²⁵I-EGF or ¹²⁵(Fig. 6 a) for I-HRG1 β 1 keying action.Data presentation HFD100, HFD200, HFD300 and HFD 400 keep its specificitys to EGF and HRG1bl (Fig. 6 a): swimming lane 1:HFD100=HER1-621/Fc, swimming lane 2:HFD200=HER2-628/Fc, swimming lane 3:HFD300=HER3-621/Fc and swimming lane 4:HFD400=HER4-625/Fc.In parallel study, the crosslinked action of these parts can be shown that this keying action is specific by its corresponding non-marked part competition.

[0577] preparation chimeric construct body HER1-621/Fc and HER3-621/Fc (being called RB200h) transfer albumen to produce general HER part associativity HER.Use ¹²⁵I-EGF or ¹²⁵I-HRG1 β 1 tests the ability of this molecule (RB200h) in conjunction with HER1 or HER3 part by crosslinked research.Data presentation RB200h is in conjunction with EGF and HRG1 β 1 (Fig. 6 b).These results of study are disclosed in mosaic HER and transfer HER1 in the albumen (RB200h) and HER3 to keep it in conjunction with the ability of part separately and the prompting RB200h general HER part binding substances as the candidate.

The formation of the dimeric structure of embodiment 5HER ectodomain and HER/Fc molecule and oligomer structure

[0578] in the activation form, the HER molecule is so that the direction that forms with the cell surface receptor dimerization presents its dimerization arm.HER derivative and Fc structural domain be connected " back-to-back " conformation of estimating the simulation activated receptor.For confirming that HER derivative and/or HER/Fc chimeric polyeptides form polymer, carry out the analysis of molecular size exclusion on HER family ectodomain polypeptide.This methodology allows to simplify the ability that the acceptor ectodomain is unified into homodimer or heterodimer of analyzing.For carrying out the analysis of molecular size exclusion, with the molecule and the comparison of reference standard thing of wash-out.Following table 13 shows used molecular mass standard substance and elution volume thereof.Smaller volume wash-out in the retention volume of post comes out, and bigger molecule comes out according to molecular weight wash-out in smaller size smaller of its increase.

[0579] uses that (Tosoh Bioscience, Montgomeryville carried out the analysis of molecular size exclusion on PA) with flow velocity 0.7ml/ minute equilibrated A TSK3000 size-exclusion column with PBS.(BioRad, Hercules CA) calibrate this post to use the gel-filtration standard substance.Their elution volume is mapped to molecular weight.By injecting the every kind molecule of 30 μ g in PBS, measure the elution volume of every kind of unknown material, and calculate their apparent molecular weight.Between injection, maintain the flow on the post.Use the typical curve of molecular weight standard thing to determine molecular weight.Table 14 summarized results:

[0580] several ectodomains of data presentation HER family have formed the polymer structure.Compound can capture ligands, and forms and stop the transmembrane receptor dimerization and thereby combine and disturb its active " vacation (mock) " dimer with transmembrane protein.

[0581] HER1-501 shows apparent molecular weight 112,170 dalton, and it is greater than estimated molecular weight 60,000 dalton; HER2-595 shows apparent molecular weight 162,000 dalton and estimated molecular weight is 67,000 dalton.HER2-530 (HF220T) does not form dimeric structure, wherein compare with HER2-595 (HF210T), described HER2-530 has lost the HER2 ectodomain sections (CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVA CAHYKDPPF, it is corresponding to the 508-573 amino acids among the SEQ ID NO:16) of cross-module 2-5 among the structural domain IV.Back one result shows that this sections of losing (or sections part) is important for dimerization.The sequence difference of two peptide species is underlined and marks with runic following.The sequence that adds shade is used label and is identical in two kinds of molecules.Because label is identical for these two kinds of molecules, thereby they do not play a role in the influence to dimerization observed.

210 (SEQ ID NO:274) with affinity tag

TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEV

QGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVT

GASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA

LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGP

LPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFE

SMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQR

CEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPES

FDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLP

DLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHH

NTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHC

WGPGPTQCVN

LESRGPFEQKLISEEDLNMHTGHHH

HHH

220 (SEQ ID NO:275) with affinity tag

TQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEV

QGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVT

GASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA

LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGP

LPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFE

SMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQR

CEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPES

FDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLP

DLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHH

NTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHC

WGPGPTQCVNLESRGPFEQKLISEEDLNMHTGHHHHHH

[0582] these data show that also HER-Fc albumen also forms the oligomer of high-order.HER1-621/Fc and HER3-621/Fc all have estimated molecular weight 180,000 dalton, and have by size exclusion chromatography observed respectively greater than 970,000 and 843,000 daltonian molecular weight.Because these assay methods are carried out in the presence of not at part, be unwanted for producing dimerization (or bigger polymkeric substance) structure so the result further confirms part.

Embodiment 6

HER acceptor propagation and phosphorylation: by the inhibition due to the HER derivative

A. the HER express spectra in the clone

[0583] analyzing the HER expression level by fluorescence-activated cell sorting method (FACS) is lip-deep relative populations with the evaluation acceptor and in various kinds of cell.Fluorescence intensity after making selected cells contacting receptor specific antibody and assessment receptor specific antibody in conjunction with cell.

[0584] cell is taken out and is resuspended among the PBS (PBS.BSA) that contains 1%BSA with 5nM EDTA from tissue culture plate.Cell in suspension with dividing other in vitro to hatch 1 hour at 4 ℃ at HER1,2,3 and 4 monoclonal antibody respectively.After hatching with first antibody, cell with cold PBS.BSA washing once.Add second antibody subsequently at mouse or human IgG (source of depending on first antibody) with fluorescence dye PE (Jackson) mark.Cell was hatched 30 minutes and washed secondary with PBS.BSA at 4 ℃.Cell is fixed and held under 4 ℃ of dark by adding Cytofix (BD-554655).Use cell sorting device (BD FACSCalibur FlowCytometer) to carry out FACS.Each clone is analyzed 10,000 cells.In every kind of clone the average fluorescent strength (MFI) of every kind of HER acceptor by MFI with BD CellQuest Pro software measurement.Scoring: ++ ++＞1000MFI, +++100-1000MFI, ++ 50-100MFI ,+＜50MFI, but have the signal that is higher than background.

[0585] table 15 is described the gained express spectra of the acceptor HER family in the various kinds of cell system.

B. cell proliferating determining method

[0586] clone MCF7, ZR75-1, ME180 buy and maintain the 10%FBS DMEM from ATCC.Cell is inoculated among the DMEM that adds 1%FBS in 96 hole flat boards with 2000 every holes of cell.Inoculate after 2-3 hour, the candidate HER ECD derivative that adds increase concentration in the presence of part (EGF or HRG β) is to culture.Cell was hatched about 72 hours at 37 ℃.Measure the cell relative density by Alamar Blue method.In PBS, prepare Alamar Blue (Sigma), it is added in the microtest plate and with flat board with 1/10 culture volume (final concentration 0.4 μ M) returns incubator with 4 μ M concentration.Read fluorescence at exciting light=530nm/ emission light=590nm place at 37 ℃ after 2-4 hour.

The result

[0587] cell proliferation data: the HFD100/300 prepared product is the thing that compiles of HFD100/100, the HFD300/300 of unknown proportion and HFD100/300 molecule.Yet this hybrid materials of data acknowledgement are carried out the ability that suppresses.The ME180 that HFD100/300 has suppressed stimulated by HRG β (5nm) breeds.The about 3nM HFD100/300 of this data presentation and have restraining effect greater than 80% at the HER1 that EGF stimulates.HF310T has suppressed to be subjected to the MCF7 propagation that HRG β stimulates (when 1 μ m about 95%).

C. based on the HER receptor phosphorylation assay method of ELISA

[0588] phosphorylation of assessment HER acceptor in based on the HER receptor phosphorylation assay method of ELISA.Various kinds of cell (A431, MCF7, SK-BR3, SK-OV3, MCF7/HER2) was carried out serum starvation about 24 hours in serum free medium.Cell uses the candidate HER ECD derivative (seeing below) of progressive concentration to handle 30 minutes at 37 ℃ subsequently.(EGF, 3nM and/or HRG β 5nM) are hatched 10 minutes to add part subsequently.After processing, cell with PBS washing once and with interpolation proteinase inhibitor and inhibitors of phosphatases (protease inhibitor cocktail group and inhibitors of phosphatases group, 100 μ l, 1 * cell lysis buffer solution (Cell Singaling) cracking Calbiochem).

[0589] cell cracking 15 minutes and cell lysate added in the 96 hole flat boards on ice, it is wherein said that dull and stereotyped (antibody is from R﹠amp with corresponding receptor-specific capture antibody; D System buys), wrap quilt in advance with manufacturer recommendation concentration (0.4-4 μ g/ml) and condition (in PBS, room temperature, spend the night).Cell lysate and capture antibody flat board were incubated at room 3 hours.Dull and stereotyped with PBST damping fluid washing 3 times.Anti-phosphotyrosine antibody clone's 4G10HRP conjugate (Upstate) was diluted among the 1%BSA.PBS and was added into described flat board with 100 μ l/ holes with 1: 1000 and continues 1 hour, to detect the specificity HER acceptor of phosphorylation on tyrosine.After PBST washing 3 times, make dull and stereotyped by adding 100 μ l substrate solutions (TMB, Sigma) colour developing and stop by 50 μ l SDS stop buffers.Measure optical density(OD) at the 650nm place by micro-plate reader (Molecular Devices, VERS Amax).

i.HER1-501

[0590] ability of test HER1-501 inhibition HER1 and HER2 phosphorylation in A431 cell and MCF7 cell.In the presence of EGF, add the HER1-501 of progressive concentration, arrive peak concentration 600nM at the most to cell.As expected, do not observe the phosphorylation of HER1 in the MCF7 cell.On the contrary, HER1 suppresses the phosphorylation of HER1 in the A431 cell in the dose-dependently mode, and IC50 is 98nM.Do not exist when comparing with this protein, it is about 60% that the maximum to the HER1 phosphorylation that is realized on 600nM HER1-501 suppresses.HER1-501 also suppresses the phosphorylation of HER2 in MCF7 and the A431 cell in the dose-dependently mode, IC50 is respectively 18nM and 42nM.Do not exist when comparing with this protein, it is about 50% that the maximum to HER2 phosphorylation in two kinds of clones of test that is realized on 600nM HER1-501 suppresses.

Ii.HER2-595 and HER2-530

[0591] in the MCF7/HER2 cell, tested the ability that HER2-595 and HER2-530 suppress HER2 and HER3 phosphorylation.In the presence of HRG, add the HER2-595 of progressive concentration or HER2-530 (0,7.4nM, 22.2nM, 66.7nM, 200nM and 600nM) to cell.Data presentation HER2-595 and HER2-530 suppress the phosphorylation of HER2 and HER3 in the dose-dependently mode; HER2-595 is more powerful.Do not exist when comparing with this protein, the maximum inhibition to HER2 and HER3 phosphorylation that is realized in the MCF7/HER2 cell by 600nMHER2-595 is about 55%, and do not exist when comparing with this protein, it is about 35% that the maximum that is realized by 600nM HER2-530 suppresses.

Iii.HER3-621 and HER3-500

[0592] in the MCF7 cell, tested the ability that HER3-621 and HER3-500 suppress the HER3 phosphorylation.In the presence of HRG, add the HER3-621 and the HER3-500 of progressive concentration, arrive peak concentration 600nM at the most to cell.Data presentation HER3-621 and HER3-500 suppress the phosphorylation of HER3 in the dose-dependently mode, though HER3-500 is more powerful.The IC50 of HER3-500 is 39nM, and the IC50 of HER3-621 is 48nM.Do not exist when comparing with this protein, it is about 78% that the maximum to HER3 phosphorylation in the MCF7 cell that is realized by 600nM HER3-500 suppresses, and does not exist when comparing with this protein, and it is about 38% that the maximum that is realized by 600nMHER3-621 suppresses.

[0593] in the SK-BR3 cell, tested the ability that HER3-621 and HER3-500 suppress HER1 and HER3 phosphorylation.In the presence of HRG, add the HER3-621 and the HER3-500 of progressive concentration, arrive peak concentration 600nM at the most to cell.In the SK-BR3 cell that stimulated by HER3-500, do not observe the phosphorylation of HER1.Be similar to the MCF7 cell, HER3-621 and HER3-500 suppress the phosphorylation of HER3 in the SK-BR3 cell in the dose-dependently mode, and HER3-500 is more powerful.Do not exist when comparing with this protein, it is about 75% that the maximum to HER3 phosphorylation in the SK-BR3 cell that is realized by 600nM HER3-500 suppresses, and does not exist when comparing with this protein, and it is about 55% that the maximum that is realized by 600nM HER3-621 suppresses.

iv.HER1-621/Fc

[0594] in the A431 cell, tested the ability that HER1-621/Fc suppresses the HER1 phosphorylation.In the presence of EGF, add the HER1-621/Fc (0.8nM-600nM) of progressive concentration to cell.HER1-621/Fc suppresses the phosphorylation of HER1 in the A431 cell in the dose-dependently mode, and IC50 is 8.8nM.At the 600nM place, HER1-621/Fc shows and almost completely to suppress the HER1 phosphorylation, do not exist when comparing with this protein, suppresses phosphorylation about 99%.

v.HER3-621/Fc

[0595] in the MCF7 cell, tested the ability that HER3-621/Fc suppresses the HER3 phosphorylation.In the presence of HRG, add the HER3-621/Fc (0.8nM-600nM) of progressive concentration to cell.HER3-621/Fc suppresses the phosphorylation of HER3 in the MCF7 cell in the dose-dependently mode.Do not exist when comparing with this protein, it is about 70% that the maximum to HER3 phosphorylation in the MCF7 cell that is realized by 600nM HER3-621/FC suppresses.

The vi.HER1-621/Fc:HER3-621/Fc mosaic.

[0596] in the A431 cell, tested the ability that HER1-621/Fc:HER3-621/Fc suppresses the HER1 phosphorylation.The conditioned medium supernatant liquor of cell of HER1-621/Fc and Her3-621/Fc cotransfection of hanging oneself carries out continuous 2 times of dilutions and is added in the cell in the presence of EGF.Recombinant protein in pure (neat) supernatant liquor is about 2 μ g/ml (about 10nM).From without the supernatant liquor of the cell of HERECD/Fc protein transfection with comparing.This result shows seldom inhibition or the unrestraint of contrast supernatant liquor performance to the HER1 phosphorylation, and observes because of a small amount of inhibition (less than 10%) due to the pure supernatant liquor.On the contrary, contain the chimeric supernatant liquor of HER1-621/Fc:HER3-621/Fc suppresses to be subjected to HER1 in the A431 cell that EGF stimulates in the dose-dependently mode phosphorylation.Not existing when comparing with this protein, is about 55% by containing that the maximum to HER1 phosphorylation in the A431 cell that the chimeric pure supernatant liquor of HER1-621/Fc:HER3-621/Fc realized suppresses.

D. the HFD100/300ECD polymer of purifying is to the inhibition of HER acceptor propagation and phosphorylation

1. phosphorylation

[0597] phosphorylation of the acceptor of assessment HER described in the C part as mentioned of the HFD100/300H by purifying.The HFD100/300H of test purifying in the SK-BR3 cell (containing the HER1-621/Fc of band His epi-position label and the ECD molecule of HER3-621/Fc) suppresses the ability of HER1 and HER3 phosphorylation.In order to assess the effect that EGF induces the HER1 phosphorylation, in the presence of EGF, add the HFD100/300H of progressive concentration 0.3nM-600nM to cell.The result shows that the HFD100/300H molecule suppresses to be subjected to the phosphorylation of HER1 in the SK-BR3 cell that EGF stimulates in the dose-dependently mode.Do not exist when comparing with this protein, it is about 60% that the maximum to HER1 phosphorylation in the SK-BR3 cell that is realized by 600nM HFD100/300H suppresses.In order to assess the effect that HRG β induces the HER3 phosphorylation, in the presence of HRG β, add the HFD100/300H of progressive concentration 0.3nM-600nM to cell.The result shows that the HFD100/300H molecule suppresses to be subjected to HER3 phosphorylation in the SK-BR3 cell that the HRG β of the about 67nM of concentration stimulates in the dose-dependently mode, and wherein the inhibition level reaches steady on this concentration.Do not exist when comparing with this protein, on concentration range 67nM-600nM HFD100/300H, realized to the SK-BR3 cell in the maximum of HER3 phosphorylation to suppress be about 65%.

[0598] HFD100/300H compares the influence and the 2C4 (being called handkerchief trastuzumab (petuzumab) again) of the phosphorylation of HER1, HER2 and HER3 in the SK-BR3 cell that is subjected to EGF or HRG β stimulation, and wherein said 2C4 is the monoclonal antibody of target HER2 dimerization structural domain.The result shows that HFD100/300H (600nM) suppresses to be subjected to phosphorylation (about 60%), the phosphorylation (about 65%) of HER2 and the phosphorylation (about 55%) of HER3 of HER1 in the SK-BR3 cell of ligand stimulation.The 2C4 monoclonal antibody suppresses the phosphorylation (about 35%) of HER2, the phosphorylation (about 65%) of HER3, but does not show detectable inhibition to the HER1 phosphorylation.Therefore, when comparing with 2C4 antibody, HFD100/300H is the general HER inhibition that can suppress HER1, HER2 and HER3 phosphorylation.

2. breed

[0599] as mentioned the HFD 100/300H of the purifying of assessment described in the B part to the influence of the cell proliferation that is subjected to the HER ligand stimulation.The result shows that the HFD 100/300 (by the A protein purification) of purifying suppresses the HT-29 cell proliferation that stimulated by EGF (3nM) or HRG (5nM) in the dose-dependently mode.Do not exist with described protein and to compare, in the presence of two kinds of parts of test, the maximum inhibition to breeding that is realized by about 200nMHFD100/300 is about 55%.The HFD100/300H (containing histidine-tagged) that has also tested purifying is to the influence of the ZR 75-1 cell proliferation that is subjected to ligand stimulation.The HFD 100/300H that the result shows purifying suppresses the ZR-75-1 cell proliferation that stimulated by HRG in the dose-dependently mode, observes about 80% maximum inhibition at about 600nM place.The HFD100/300H molecule also suppresses by many ZR-75-1 cell proliferations to about 1nM EGF stimulation in the dose-dependently mode, and wherein observed restraining effect reaches steady at the most to the concentration of about 600nMHFD100/300H the time.Do not exist when comparing with this protein, observed maximum the inhibition is about 80% when the HFD of about 1nM purifying 100/300H.

E.HER ECD derivative is to the summary of the retarding effect of HER phosphorylation

[0600] tested the ability of various exemplary HER ECD molecules in inhibiting HER phosphorylation.Summary has as a result been described in table 16.Indicating when not measuring retarding effect, be meant not experimentize.The result shows that the HER1-621/Fc:HER3-621/Fc mosaic is general HER candidate molecules.

Embodiment 7 identifies HER1, HER3, the part mating surface of HER4 and the similar sequence of HER2

[0601] identified the whole four kinds of members' of HER family roughly ligand binding region.The logical crystalline structure with TGF-α compound people EGFR (1-501 position residue) in described zone (has 1D, the PDB Protein Data Bank of 1MOX, referring to (2002) Cell such as for example Garrett, 110:763-773) the mature form HER1 (SEQ ID NO:2) of (when promptly comparing with reference SEQ ID NO, lacking signal peptide), HER2 (SEQ ID NO:4), HER3 (SEQ ID NO:6) and HER4 (SEQ ID NO:8) are determined with multiple ratio.In table 17, described among structural domain I (DI) and the domain II I (DIII) for the important amino acid whose qualification result of part keying action.Be numbered according to the proteic mature form of HER.Can combine with corresponding HER is proteic to disturb part by these aminoacid sequences of target.

Embodiment 8

Evaluation to the subdomain II (DII) and the target polypeptide among the subdomain IV (DIV) of HER family molecule

[0602] in the present embodiment, identify as peptide bound substrates (being used for for example phage display method) or as immunogen producing the successive zone from HER3 and HER1, HER2 and HER4 of polyclonal antibody, with identify can target HER family subdomain II (DII) or the molecule of subdomain IV (DIV).This quasi-molecule can serve as target dimerization structural domain and/or the target and make the general HER therapeutical agent of the stable candidate of constraint effect by interacting with DII that participates in the constraint effect and DIV sequence.

[0603] sequence of DII or DIV in the comparison HER family receptors.HER3 is the prototype of homology analysis, and the peptide that sequence is conservative is defined as DII target or DIV target.Following table 18 is described the target peptide of having identified among the DII, shows SEQ ID NO (#) in adjacent column.Following table 19 is described the target peptide of having identified among the DIV, shows SEQ ID NO (#) in adjacent column.

Embodiment 9 is by the peptide of phage display method evaluation in conjunction with the conserved residues that exposes in the HER family

[0604] the phage display method is to be used for screening and the interactional candidate therapeutic agent of target polypeptide, as those candidate therapeutic agents of identifying in embodiment 7-8 with in the method example of the target peptide through identify of SEQ ID NO:54-125 described in arbitrarily.

A. phage library is selected

[0605] phage display peptide library (the linear library of restricted ring C7C library and 7 amino acid and 12 amino acid) obtains from New England BioLabs.Get rid of phage display library at irrelevant Fc fusion rotein-A albumen (or G albumen) agarose complex body.At the phage library of people HER3-621/Fc-A albumen agarose complex body selection through getting rid of.As the HER3-621/Fc of the HER3 ectodomain that merges with IgG1 Fc district from R﹠amp; D Systens buys, or as preparation as described in the embodiment 2.The low pH damping fluid of phage usefulness (or use the synthetic peptide of selecting in the sequential element conservative from the HER3 structural domain to compile thing, and embodiment 6 and 7 sees above) wash-out.Carry out four-wheel and select, after this single plaque of picking and the back of increasing in intestinal bacteria are analyzed by phage enzyme linked immunosorbent assay (ELISA) and dna sequencing method at random.

B. phage E LISA

[0606] for implementing phage E LISA, 96 hole flat boards wrap quilt with HER3-621/Fc; Wash and seal with BSA/ sucrose damping fluid.After the sealing, various phage substratum are added in the hole and incubated at room 2 hours.Remove unconjugated phage by repetitive scrubbing.The M 13 antibody (R﹠amp that use HRP to put together; D Systems) detects the bonded phage.Further use various synthetic peptide screening positive phage clones, wherein said synthetic peptide is selected from HER3 ectodomain ( embodiment 6 and 7 sees above) conservative in HER receptor family member to determine the possible phage binding site on HER3.Similar phage combined techniques can use the monolayer cell of expressing HER3 to implement.

C. identify the peptide that is used for different dimerization

[0607] in a single day identified positive bacteriophage and determined binding peptide, then used avidin-vitamin H to interact and identify the synergetic property peptide counterpart that is applicable to different dimerization.This assay method utilizes single avidin molecule with high-affinity and the ability of specificity in conjunction with four kinds of different biotin molecules.In brief, biotinylation peptide and neutravidin-HRP were with 4: 1 mixed.This mixture was hatched 60 minutes at 4 ℃ on rot, add soft release avidin-sepharose subsequently to remove excessive peptide.Soft release avidin-sepharose precipitates by centrifuging.The supernatant liquor of gained is diluted to the needed concentration of HER3 binding assay.

Embodiment 10 is used to clone the method for other HER isoform

A. the preparation of messenger RNA(mRNA)

[0608] from Clontech (BD Biosciences, Clontech, Palo Alto, CA) and Stratagene (La Jolla CA) buys isolating mRNA from the main people types of organization of health or illing tissue or clone.Compile the mRNA of equivalent and with it as template based on the pcr amplification (RT-PCR) of reverse transcription.

B.cDNA is synthetic

[0609] with mRNA 70 ℃ of sex change 10 minutes and sudden cold in the presence of 40%DMSO on ice.The first chain cDNA is containing 10%DMSO, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl with 200ng oligomerization (dT) or 20ng random hexamer ₂, every kind of dNTP of 10mMDTT, 2mM, 5 μ g mRNA and 200 units

Reversed transcriptive enzyme (Stratagene, La Jolla, synthetic in 20 μ l reaction CA).37 ℃ hatch 1 hour after, compile from the cDNA of two reactions and with 10 RNA of unit enzyme H (Promega, Madison, WI) processing.

The C.PCR amplification

[0610] is designed for forward primer and the reverse primer that RT-PCR clones, the splice variant that is intended to clone the HER family member.(Cascade CO) selects gene specific PCR primer and by Qiagen-Operon (Richmond, CA) synthetic described primer for Molecular Biology Insights, Inc. to use Oligo 6.6 softwares.Selection be distributed in the initiator codon flank forward primer (F1, F2).(Genome Biology (2005) 6:R58) selects reverse primer (R1) (seeing Table 21) from the intron sequences of HER gene (table 20) to the method for using Hiller to describe.Each PCR reaction contains the cDNA of the 10ng reverse transcription in cumulative volume 70 μ l, 0.2 μ M F1/R1 primer mixture, 1mM Mg (OAc) ₂, 0.2mM dNTP (Amersham, Piscataway, NJ), 1 * XL-damping fluid and 0.04U/ μ lrTth archaeal dna polymerase (Applied Biosystems).The PCR condition is 94 ℃, 45 seconds, 60 ℃, and 1 minute and 68 ℃, 2 minutes, 36 circulations.Finish reaction with 68 ℃ of extension steps that continue 20 minutes.

Table 20: the list of genes that is used to clone the CSR isoform

Family	The member	Gene (SEQ ID NO.)	ntACC.#	Catalyst structure domain	SEQIDNO：	ORF	prtACC.#	SEQIDNO：
									HER	EGFR	400	NM_005228	2380-3148	1	247-3879	NP_005219	2
	ERBB2	401	NM_004448	2396-3164	3	239-4006	NP_004439	4
										ERBB3	402	NM_001982	2318-3086	5	194-4222	NP_001973	6
	ERBB4	403	NM_005235	2285-2953	7	34-3960	NP_005226	8

Table 21: the primer that is used for the PCR clone

SEQIDNO	The primer title	Sequence
			276	EGFR-F1	ATCGGGAGAGCCGGAGCGAG
277	EGFR-F2	AGCAGCGATGCGACCCTCCG
			278	EGFR-int11R1	CCAGGCTTTGGCTGTGGTCA
279	HER2-F1	ATGGGGCCGGAGCCGCAGT
			280	HER2-F2	GCACCATGGAGCTGGCGGC

281	HER2-int11R1	ATCAGGCCCCCTCTTTCTCAG
			282	HER3-F1	TCCCTTCACCCTCTGCGGA
283	HER3-F2	GCGGAGTCATGAGGGCGAA
			284	HER3-int11R1	CTGAAGATGCCATTTCCTCCATAC
285	HER3-int10R1	CAATTTATGCCAGTGGTTCACCTA
			286	HER4-F1	ATTGTCAGCACGGGATCTGAGA
287	HER4-F2	CTGAGACTTCCAAAAAATGAAGCC
			288	HER4-int12R1	AATGGGAAAAAATTTAAGTTTCTATGTT

The clone of D.PCR product and order-checking

[0611] electrophoresis PCR product on 0.8% sepharose, and with Gelstar (BioWhitaker Molecular Application, Walkersville, but MD) dyeing from the DNA of test strip.(Qiagen, Valencia CA) extract the DNA band, are connected to pDrive UA-cloning vector (Qiagen) and are transformed into the DH10B cell with the QiaQuick gel extraction kit.On the LB agar plate that contains 25 μ g/ml kantlex, 0.1mM IPTG and 60 μ g/ml X-gal, select recombinant plasmid.For each transfection, 12 bacterium colonies of picking and measure its cDNA inset size with UA carrier primer at random by PCR.Subsequently with M13 forward and reverse carrier primer to cloning two-way order-checking.The customization primer that the complete directed sequencing of relief area is striden in use carries out complete order-checking to whole clones.

E. sequential analysis

[0612] the computational analysis of alternative splicing is carried out every kind of cDNA sequence and its corresponding gene group sequence alignment by using SIM4 (a kind of computer program that is used to analyze splice variant).Only consider that the transcript with standard (for example GT-AG) D-A splice site is used for analyzing.Further study clone's (seeing below table 22) of coding HER isoform.

F. exemplary HER isoform

[0613] the exemplary HER isoform that uses described method preparation is herein described in following table 22.Provide under the nucleic acid molecule of coding HER isoform and the SEQID that its sequence marks in this table and describe.Exemplary HER isoform amino acid sequence of polypeptide is described under the SEQ ID that marks.

Table 22:HER isoform

Gene	ID	Type	Length	The primer	SEQIDNO(nt，aa)
						EGFR	HER1-int11	The intron fusions	433	EGFR-F1，EGFR-F2，EGFR-int11R1	126，127
ERBB2	HER2-int11	The intron fusions	438	HER2-F1，HER2-F2，HER2-int11R1	140，141
						ERBB3	HER3-int10	The intron fusions	403	HER3-F1，HER3-F2，HER3-int11R1	145，146
ERBB3	HER3-int11	The intron fusions	425	HER3-F1，HER3-F2，HER3-int10R1	147，148
						ERBB4	ERBB4-int12_tr	The intron fusions	506	HER4-F1，HER4-F2，HER4-int12R1	158，159
ERBB4	ERBB4_int11	The intron fusions	430		156，157
						ERBB4	ERBB4_int10	The intron fusions	421		154，155
ERBB4	ERBB4_int9	The intron fusions	391		152，153

Embodiment 11 is used to clone the method for IGF1R isoform

A. the preparation of messenger RNA(mRNA)

[0614] from Clontech (BD Biosciences, Clontech, Palo Alto, CA) and Stratagene (La Jolla CA) buys isolating mRNA from the main people types of organization of health or illing tissue or clone.Compile the mRNA of equivalent and with it as template based on the pcr amplification (RT-PCR) of reverse transcription.

B.cDNA is synthetic

[0615] with mRNA 70 ℃ of sex change 10 minutes and sudden cold in the presence of 40%DMSO on ice.The first chain cDNA is containing 10%DMSO, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl with 200ng oligomerization (dT) or 20ng random hexamer ₂, every kind of dNTP of 10mMDTT, 2mM, 5 μ g mRNA and 200 units

Reversed transcriptive enzyme (Stratagene, La Jolla, synthetic in 20 μ l reaction CA).37 ℃ hatch 1 hour after, compile from the cDNA of two reactions and with 10 RNA of unit enzyme H (Promega, Madison, WI) processing.

The C.PCR amplification

[0616] is designed for forward primer and the reverse primer that RT-PCR clones, the splice variant that is intended to clone IGF1R.(Cascade CO) selects gene specific PCR primer and by Qiagen-Operon (Richmond, CA) synthetic described primer for Molecular Biology Insights, Inc. to use Oligo 6.6 softwares.Selection be distributed in the initiator codon flank forward primer (F1, F2).(Genome Biology (2005) 6:R58) selects reverse primer (R1) (seeing Table 24) from the intron sequences of IGFR1 gene (SEQ ID NO:404, table 23) to the method for using Hiller to describe.Each PCR reaction contains the cDNA of the 10ng reverse transcription in cumulative volume 70 μ l, 0.2 μ M F1/R1 primer mixture, 1mM Mg (OAc) ₂, 0.2mMdNTP (Amersham, Piscataway, NJ), 1 * XL-damping fluid and 0.04U/ μ lrTth archaeal dna polymerase (Applied Biosystems).The PCR condition is 94 ℃, 45 seconds, 60 ℃, and 1 minute and 68 ℃, 2 minutes, 36 circulations.Finish reaction with 68 ℃ of extension steps that continue 20 minutes.

Table 23: the list of genes that is used to clone the IGF1R isoform

Protein	Genes of SEQ ID NO:	ntACC.#	SEQIDNO：	prtACC.#	SEQIDNO：
						IGF1R	404	X04434	289	CAA28030	290

Table 24: the primer that is used for the PCR clone

SEQIDNO	The primer title	Size	Sequence	The position	Tm	Length
							291	IGF1R_F1		TGAGAAAGGGAATTTCATCCC		14	65	21
292	IGF1R_F2		AGGAATGAAGTCTGGCTCCG	42	66	20
							293	IGF1R_ introne 1 0R1	2280	GGCTCCGTCTCAGTGGCTAC	2358	66	20

294	IGF1R_ introne 1 1R1	2496	CTAGGTTGTGAGGAAGGTGGC	2558	66	21
							295	IGF1R_ introne 1 2R1	2664	AGGAGGTAACCTGTGCAGTCA	2724	64	21
296	IGF1R_ introne 1 3R1	3039	ATGTAAGCCAGGTTGAAAGCA	3110	65	21

The clone of D.PCR product and order-checking

[0617] electrophoresis PCR product on 0.8% sepharose, and with Gelstar (BioWhitaker Molecular Application, Walkersville, but MD) dyeing from the DNA of test strip.(Qiagen, Valencia CA) extract the DNA band, connect into pDrive UA-cloning vector (Qiagen) and are converted into the DH10B cell with the QiaQuick gel extraction kit.On the LB agar plate that contains 25 μ g/ml kantlex, 0.1mM IPTG and 60 μ g/ml X-gal, select recombinant plasmid.For each transfection, 12 bacterium colonies of picking and measure its cDNA inset size with UA carrier primer at random by PCR.Subsequently with M13 forward and reverse carrier primer to cloning two-way order-checking.The customization primer that the complete directed sequencing of relief area is striden in use carries out complete order-checking to whole clones.

E. sequential analysis

[0618] the computational analysis of alternative splicing is carried out every kind of cDNA sequence and its corresponding gene group sequence alignment by using SIM4 (a kind of computer program that is used to analyze splice variant).Only consider that the transcript with standard (for example GT-AG) D-A splice site is used for analyzing.Further study clone's (seeing below table 25) of coding IGF1R isoform.

F. exemplary IGF1R isoform

[0619] the exemplary IGF1R isoform that uses described method preparation is herein described in following table 25.Provide nucleic acid molecule and its sequence of coding IGF1R isoform in SEQ ID NO:297 and 299 any, to describe.Exemplary IGF1R isoform amino acid sequence of polypeptide is described in SEQ ID NO:298 and 300 any.

Table 25:IGF1R isoform

Gene	ID	Type	Length	New length	The primer	SEQIDNO(nt，aa)
							IGF1R	SR024A03	The intron fusions	759	25	IGF1R_F1, IGF1R_F2; IGF1R_ introne 1 0R1	297，298
IGF1R	SR024B04	The intron fusions	831	3	IGF1R_F1, IGF1R_F2; IGF1R_ introne 1 1R1	299，300

Embodiment 12

Suppress growth of tumour cell synergistically with HER1ECD/HER3ECD heteromultimeric and tyrosine kinase inhibitor (TKI)

[0620] exponential phase growing tumors cell (buying from ATCC) is transferred to the 96 hole Microdilution flat boards with 1000 cells/well of density.(MDA MB 468 breast cancer cells are used for the experiment described in Fig. 3 a, and A 431 squamous cell cancer cells are used for the experiment described in Fig. 3 b).Allow cell attachment 24 hours and add test compound to last extent of dilution 1 eventually *: 1 μ M is used for RB200h (the total length HER1ECD that is connected with total length HER3ECD through the Fc structural domain); AG-825 (a kind of inhibition of the Tyrosylprotein kinase that HER2 is relevant with 50 μ M; Osherov etc. 1993; Fig. 3 A); Or 50 μ M Gefitinib/Iressas (kinase whose a kind of inhibition that EGFR is relevant; Herbst, 2002; Fig. 3 b).Duplicate subsequently administered compound simultaneously and carry out continuous 2 times of dilutions.

[0621] after hatching in 72 hours, cell is with phosphate buffered saline (PBS) (PBS) washing and with 0.5% violet staining in the methyl alcohol.Soft washing is dull and stereotyped and make its dried overnight in water subsequently.Be dissolved in Sorenson damping fluid (the 0.025M Trisodium Citrate in 50% ethanol, 0.025M citric acid), 0.1ml/ hole with the Viola crystallina of the protein bound of institute attached cell.In the ELISA plate reader, analyze dull and stereotyped in 540nm wavelength place.With mark mapping and the analysis (CalcuSyn of viable cell with respect to contrast; Biosoft, Cambridge, UK).

[0622] the results are shown in Fig. 3 A and Fig. 3 B from the minimum concentration of being tested.(RB200h adds AG825, Fig. 3 A from the result of the additive effect of institute's testing drug to cross the dotted line that is labeled as " combination " row and be expection; RB200h adds Iressa, Fig. 3 B).As shown in Fig. 3 A and Fig. 3 B, HER1ECD/HER3ECD heteromultimeric (RB200h) and the tyrosine kinase inhibitor tested the two one of combination show the synergetic property growth inhibitory effect, be much higher than the additive effect of this combination to growing.

[0623] this result is significant, because its meaning can be by avoiding the toxicity relevant with chemotherapeutic with the RB200h combination.Especially, the fatal toxicity that can avoid ratifying being used for the treatment of the Iressa of nonsmall-cell lung cancer therapy (is seen Http:// www.medscape.com/vievvarticle/456223).In addition, the sudden change that only about 30% Aisa people and 10% white people show EGFR/HER1, and this that to be Iressa/Gefitinib render a service is needed, and for other TKI may exist similar situation ( Http:// en.wikipedia.org/wiki/Gefitinib).Mechanism of drug resistance has also obtained describing (relevant EGFR Tyrosylprotein kinase keeps the wild-type amino acid sequence except that tumour).Belong to these mechanism row be to obtain " second site " sudden change (Pao etc. 2005) and somatomedin overexpression (Ishikawa etc. 2005).Therefore, if by with the combination of RB200h or other acceptor polymer synergistically wild phase can increase susceptibility and can avoid the toxicity relevant for toxic effectiveness with TKI, then this will cause and can patient's number that the cancer that relate to Tyrosylprotein kinase or other disease are successfully treated sharply be increased.

Embodiment 13

EGF that measures by Biacore surface plasma body resonant vibration method (Surface Plasmon Resonance) and NRG1 β 1 combine with RB200h's

[0624] in order to measure the somatomedin part, carries out in conjunction with research by Biacore to the RB200h avidity of (being called 100/300h (containing the HER1-621/Fc of band HIS epi-position label and the ECD molecule of HER3-621/Fc) again).(BIAcore AB, Uppsala Sweden) carry out the combination experiment with the bio-sensing instrument BIAcore 3000 based on surface plasma body resonant vibration at 25 ℃.Be fixed ligands, with freeze dried DNAcarrier free people EGF and HRG (R﹠amp; D Systems) is dissolved in HBP-ES damping fluid (3mM EDTA, pH 7.5, BIAcore AB for 20mM HEPES, 150mM NaCl) and be diluted to 0.2mg/ml.RB200h among the PBS is diluted to 0.2mg/ml in same buffer.Use the NHS/EDC coupling method that these molecules are fixed on the BIAcore CM5 chip.EGF or NRG1 β 1 are fixed on the Biacore chip, flow through RB200h solution subsequently.In case reach the target surface resonance of 10000 secondary response units, then use the thanomin quencher should the surface.Preparation is used for the blank flow-through cell of Total Test.

[0625] there is not mass transfer effect (mass transfer effect) with different in flow rate with injecting with different analyte concentrations with confirmation.Duplicate or three parts carry out the final measurement shown in the table 26.Data evaluation uses Scrubber (BioLogic software) to carry out by overall match (global fitting).The dissociation constant of part (Kd) is determined the ratio of part association rate from dissociation of ligand speed.These researchs disclose HER and transfer albumen RB200h to combine with EGF with Kd 24nM, and it combines (table 26) with Kd 56nM with NRG1 β 1.

Table 26. binding affinity

Molecule in the solution	Lip-deep molecule	KD(nM)
			RB200h	EGF		24
RB200h	NRG1β1						56

The EGF of embodiment 14 usefulness europium marks or NRG1 β 1 carry out the saturated in conjunction with research of RB200h

[0626] because only when fix N RG1 β 1, just may measure the keying action of HER3 part (NRG1 β 1) and RB200h, so carry out the combination research of RB200h by another kind of method-time-resolved fluorometry (DELFIA) by the Biocore method.For the HER1 ligand-binding activity, by the DELFIA method, use the part Eu-EGF of europium mark to determine that HER transfers proteic ligand-binding activity, or, on the microtiter plate of anti-IgG Fc bag quilt, determine that HER transfers proteic ligand-binding activity with Eu-NRG1 β 1 for the HER3 ligand-binding activity.RB200h is fixed on 96 orifice plates of anti-Fc bag quilt and use as Fig. 7 a and b as shown in greatly lanthanon (europium) tagged ligand (Eu-EGF or Eu-NRG1 β 1) in the concentration range determine the binding affinity of EGF or NRG1 β 1.The yellow plates in DELFIA 96 holes (PerkinElmer) are spent the night at 4 ℃ of bags with 0.5 μ g/ hole (100 μ l/ pore volume) with anti-human IgG Fc antibody (Sigma).Plate was also sealed 2 hours down in room temperature about 22 ℃ (RT) with the PBS damping fluid that contains 1%BSA, 5% sucrose and 0.01% sodium azide subsequently with PBS/0.05% polysorbas20 washed twice.After the sealing, the sucking-off damping fluid, with plate air-dry overnight at room temperature, sealing also is housed in 4 ℃ subsequently and continues one month.Measuring the same day, the plate of anti-IgG Fc bag quilt is added in the 50 μ l/ pore volumes in the DELFIA binding buffer liquid with 10ng/ hole or 20ng/ hole with DELFIA L*R lavation buffer solution (PerkinElmer) washing 3 times and HER accent albumen.Hatch 2 hours and gentle jolting at 30 ℃, after this, the part of 50 μ l europium (Eu) marks is added in the hole with multiple concentration or the following concentration shown in scheming.

[0627] for saturated in conjunction with research, multiple hole contains and is useful on 100 times of excessive non-marked parts and the Eu tagged ligand of measuring the non-specific binding effect.For routine analysis HER transfers proteic ligand-binding activity, conduct a research as mentioned above, except the Eu-EGF (being used for the total binding effect) that only uses fixed saturation concentration 30nM locates or except quantitative HER1 ligand binding capacity in the presence of 5 μ M non-marked EGF (being used for the non-specific binding effect).Similarly, for quantitative HER3 ligand binding capacity, only use 100nM Eu-NRG1 β 1 (being used for the total binding effect) or 1 (being used for the non-specific binding effect) of 10 μ M non-marked NRG1 β to exist and analyze HER accent albumen down.After adding part, hatched 2 hours and gentle jolting at 30 ℃.Subsequently, plate is placed on ice, wash 3 times to remove unconjugated part with the ice-cold DELFIA lavation buffer solution (PerkinElmer) that contains 0.02% polysorbas20 rapidly.For quantitative bonded Eu-tagged ligand, the DELFIA that adds 130 μ l/ holes strengthens solution, and incubated at room 15 minutes, (Envision, 2100 types PerkinElmer) went up to filter in the Eu temporal resolution reading down is set at the fluorescent plate readout instrument subsequently with plate.Data use list-site or two-site binding curve match software to analyze to produce K _dAnd B _MaxFor routine analysis, HER transfers proteic specific combination activity to be expressed as and flies mole (fmol) binding partner/every milligram of protein or whenever fly mole HER to transfer albumen.

[0628] HER transfer albumen RB200h in the saturable mode in conjunction with Eu-EGF or Eu-NRG1 β 1.The keying action of Eu tagged ligand can be replaced by its non-marked ligands, EGF or NRG1 β 1 separately, shows that this keying action is specific (Fig. 7 a and b).The Kd of Eu-EGF or NRG1 β 1 is about 10nM.In addition, NRG1 β 1 combines (the about 10nM of Kd) to be higher than the viewed avidity of Biacore with fixed RB200h.In a word, data presentation RB200h with high-affinity in conjunction with HER1 and HER3 part.

Embodiment 15

HER transfers albumen RB200h to suppress the HER family protein tyrosine phosphorylation of EGF and neuregulin-1 β stimulation

[0629] above embodiment confirms that HER transfers albumen RB200h in conjunction with EGF (HER1 part) and NRG1 β 1 (HER3 part).Conduct a research subsequently and the part of HER family protein tyrosine phosphorylation is induced hormesis (wherein part is EGF or NRG1 β 1) to determine whether RB200h will block.

Method

Clone and tissue culture

[0630] from American type culture collection (Manassa, VA) purchaser's colorectum gland cell system HT-29, people's lung cancer A549, cancer of the stomach NCI-N87, breast ductal cancer ZR-75-1, epidermoid carcinoma A431 and breast adenocarcinoma cell are SK-BR-3, ACHN renal carcinoma cell line, and the SUM149 cell is from Asterand.HT-29 and SK-BR-3 cell are being added the McCoys 5a (Mediatch of 10% foetal calf serum, Herndon, VA) cultivate in, NCI-N87 and ZR-75-1 cell are cultivated in the RPMI that adds 10% foetal calf serum (Mediatch), and A549 and A431 cell are cultivated in the DMEM that adds 10% foetal calf serum (Mediatch).The SUM149 cell is cultivated in the Ham ' s F-12 substratum of adding Regular Insulin (5 μ g/ml), hydrocortisone (1 μ g/ml), HEPES damping fluid (10mM) and 5% foetal calf serum.All cells in incubator in 37 ℃, containing %CO ₂With cultivate under the humidification atmosphere of 95% air.Cell goes down to posterity weekly and cultivates twice.

The Tyrosine O-phosphate ELISA that is used for the HER family protein

[0631] clone A431, A549, HT-29, N87, SK-BR-3 and ZR-75-1 cell have been tested.The A431 cell has high-caliber HER1 and low-level HER2 and HER3.With cell be suitable for its separately the density of the speed of growth be seeded in the growth medium in 96 orifice plates, general every hole 5,000-20,000 cell, and overnight incubation were carried out serum starvation 24 hours subsequently.Rest cell with 50 μ l/ holes contain 0.1%BSA DMEM (Sigma, St.Louis, MO) pre-treatment, and add the inhibition (HER transfers albumen or Trastuzumab or Erbitux) of serial dilution, and with cell at 37 ℃, 5%CO ₂Hatched 30 minutes.HER family protein phosphorylation uses somatomedin (3nM EGF or 1nM NRG-β 1) at 37 ℃, 5%CO ₂Stimulated 10 minutes.After stimulation, the plate of band cell is placed on ice, with the ice-cold PBS in 200 μ l/ holes washing 1 time and with 100 μ l/ holes contain inhibitors of phosphatases mixture I group and II organizes (EMD Biosciences, San Diego, CA) and ice-cold 1 * cell lysis buffer solution (Cell Signaling of general protease inhibitor cocktail (Sigma), Danvers, MA) about 30 minutes in cracking on ice.

[0632] in preliminary study, find that RB200h changes in the lysate that is derived from the handled cell of this HER accent albumen, and the RB200h of this level and HER1 competition combines capture antibody, but does not observe RB200h to hereinafter described the HER3 or the obvious competition of HER2 and its respective capture antibodies.By purifying described lysate and eliminate with A albumen-sepharose pearl, wherein said A albumen-sepharose pearl is in conjunction with the Fc structural domain among the RB200h, and is as mentioned below to this competition effect of HER1 capture antibody for RB200h and HER1.This point is verified by following experiment, in described experiment, will in this research, mix the cell lysate that contains HER1, HER2 and HER3 and handle with A albumen pearl subsequently by the RB200h of used maximum concentration, subsequently to HER1 or HER2 or HER3 capture antibody enforcement ELISA.

[0633] as indicated above, cell lysate and 20 μ l/ hole 50%A albumen-sepharose pearl slurries (Invitrogen from cell that RB200h handles, Carlsbad, CA) (it is balance in lysis buffer) on the plate vibrator 4 ℃ of overnight incubation, to remove RB200h.Subsequently with described pearl through centrifugal and from lysate, remove, and the supernatant liquor that does not contain RB200h impurity is used for Tyrosine O-phosphate ELISA.Being used for the HER1 of ELISA or HER2 or HER3 capture antibody plate is prepared as follows.(Thermo, Waltham's 96 hole Immulon 4HXB microtiter plates MA) are spent the night room temperature 2 hours or at 4 ℃ of bags with 100 μ l/ holes with the capture antibody as described below among the PBS.Use following anti-HER ectodomain capture antibody.Detect for HER1, anti-people EGFR antibody (#AF231,0.4 μ g/ml) is capture antibody; Detect for HER2, people's anti-ErbB capture antibody (#DYC1768,4 μ g/ml) only is used to have the research (seeing below) of RB200h; Detect for HER3, people Erb3DuoSet IC (#DYC1769,4 μ g/ml) is a capture antibody.We find that Trastuzumab combines HER2 capture antibody mentioned above (DYC 1768) with the HER2 competition, but Trastuzumab does not combine from R﹠amp with cell HER2 competition; The anti-ErbB capture antibody that is called AF1129 of D Systems.Therefore, when using Trastuzumab or C225, in institute's captured cell lysate on the anti-people ErbB2 antibody (#AF1129,2 μ g/ml), carry out HER2 and detect.All capture antibody is from R﹠amp; (Minneapolis, MN), (Equitech, Kerrville is TX) with 0.05% polysorbas20 (Fisher, Waltham, MA) sealing to be diluted in 2% bovine serum albumin of also using among the PBS among the PBS for DSystems.Jia Gong cell lysate (75 μ l) is transferred to and wraps by in every hole of plate as mentioned, 4 ℃ of overnight incubation, mix simultaneously, and subsequently with PBS (PBS-tween) washing that contains 0.05% polysorbas20 4 times.Anti-Tyrosine O-phosphate-HRP conjugate (R﹠amp with 100 μ l/ holes; D Systems) detect tyrosine phosphorylation on the HER albumen, wherein said anti-Tyrosine O-phosphate-HRP conjugate is diluted in according to manufacturer specification among the PBS that contains 2%BSA and incubated at room 2 hours.Plate is washed 4 times with the PBS-tween, and, use the termination reagent at TMB (all from Sigma) in 100 μ l/ holes to stop subsequently subsequently with 100 μ l/ hole tmb substrates colour developings.Developing time changes, thereby is 0.5-1.0 through the optical density(OD) of color plate.(Molecular Devices, Sunnyvale CA) determine at the 650nm place optical density(OD) by VERSAmax micro plate readout instrument.

The result

[0634] EGF handles the A431 cell and causes and stimulate the whole three kinds of proteic tyrosine phosphorylation of HER: HER1 to be subjected to maximal stimulation (about 10 times), follows by HER2 (4-doubly) and is HER3 (2 times) at last.Listed detecting EGF and stimulate the phosphorylation of HER1 in whole clones to reach 2 times-10 times in table 27, HER 2 phosphorylations reach 1.6 times-4 times in A431, HT-29, SK-BR-3 and the ZR-75-1 cell of test cell system of institute but it only stimulates.EGF is to induce the HER3 phosphorylation to stimulate in A431 and the SK-BR-3 cell to reach 2 times-3 times (tables 27) in institute's test cell only.RB200h through ascending-dose handles when the A431 cell, when stimulating with EGF subsequently, with only compare with the EGF stimulated cells, have the dose-dependent inhibition of whole three kinds of HER1, HER2 and the proteic tyrosine phosphorylation of HER3, this point determines that by anti-Tyrosine O-phosphate ELISA (Fig. 8 a).For the HER1 phosphorylation, the maximum reaction of observing with RB200h is about 75% inhibition, EC ₅₀Be 160nM (Fig. 8 a and table 27 and 28).Whole institutes test cell of listing in Table II is observed this retarding effect of RB200h to the phosphorylation of EGF stimulation.Yet as Trastuzumab and C225 (Erbitux), only C225 is effective equally with RB200h at other the biological substance (biolgic) of HER family, and wherein said C225 suppresses EGF and combines (table 27 and 28) with HER1.Trastuzumab does not suppress the HER protein phosphorylation that EGF stimulates with any conspicuous level, and these researchs will be in following further discussion.

Table 27: suppress HER family protein phosphorylation (PanHER index) by RB200h and other biological substance.

Table 28: by the HER family protein tyrosine phosphorylation of EGF or NRG1 β 1 stimulation in RB200h, Trastuzumab or the Erbitux inhibition tumour cell.

[0635] except stimulating the HER1 phosphorylation, EGF also causes stimulates HER2 (4 times) and HER3 (3 times) phosphorylation, the different dimerization of prompting EGF inductive HER1 and HER2 or HER3.HER2 that this EGF stimulates or HER3 phosphorylation also suppress to reach about 60% by RB200h, and (Fig. 8 a).Because somatomedin (as EGF) is induced HER family receptors protein different dimerization and induced its commentaries on classics phosphorylation of dividing other mating partner, therefore assessing certain molecule is important for the proteic inhibition effectiveness of the whole three kinds of HER that are subjected to ligand stimulation.This is undertaken by suppressing the phosphorylation data and being expressed as " panHER index ".

[0636] the average % of this index measurement HER family protein suppresses and following derivation: the panHER index equals HER and transfers albumen or another kind of reagent to (inhibition %/3 of [HER1+HER2+HER3] phosphorylation of ligand stimulation).The panHER index is 70% for the RB200h in the A431 cell that stimulated by EGF, and expression is the blocking-up EGF inductive proteic signal conduction of HER (table 27) effectively.Have in the another kind of tumor cell line ZR-75-1 breast cancer cell of medium level HER2 and HER3 having low-level HER1, HER1 and HER2 phosphorylation that RB200h suppresses EGF and stimulates reach 40 and 20% respectively, the panHER index is about 20%, and EC50 is 50-100nM (Fig. 9 a, a table 27 and 28).In the ZR-75-1 cell, the HER3 phosphorylation does not have obviously in EGF stimulation back to be increased, and therefore, RB200h does not have influence to the HER3 phosphorylation in the cell of EGF processing.

[0637] NRG1 β 1 (HER3 part) processing A431 cell causes that about 2 times of-4 times of HER3 phosphorylations stimulate.In other cell, see this level that stimulates the HER3 phosphorylation by NRG1 β 1, and the ZR-75-1 exception, NRG1 β 1 causes that about 7 times of HER3 phosphorylations stimulate therein.In most of tumour cells of research, the phosphorylation that NRG1 β 1 stimulates HER2, but only in some tumor cell line of test, observe the HER1 phosphorylation.In A431 or ZR-75-1 cell that NRG1 β 1 stimulates, RB200h causes the dose-dependent inhibition of maximum HER3 phosphorylation, and causing that maximum 60-80% suppresses, EC50 is that about 120nM and panHER index are 45-60% (Fig. 8 d and 9d, tables 27 and 28).The stimulation of 1 pair of A431 cell of NRG1 β does not cause any noticeable change of HER1 phosphorylation, does not therefore observe the influence of RB200h to HER1.On the other hand, NRG1 β 1 has handled the ZR-75-1 cytositimulation whole three kinds of HER1, HER2 and HER3 phosphorylation, and these phosphorylations are subjected to RB200h to suppress to reach 40-60%, the panHER index be about 50% and EC50 be 24-90nM, this depends on HER protein (Fig. 9 d, table 27 and 28).The phosphorylation (table 27 and 28) that other tumor cell line implementation of class is also suppressed EGF or NRG1 β 1 stimulation like research and RB200h in various tumour cells with RB200h.

[0638] tested the influence of other biological substance at the HER family protein (being C225 or Erbitux (at HER1) and Trastuzumab (at HER2)) of known adjusting HER family protein phosphorylation to the phosphorylation of EGF or NRG1 β 1 stimulation.In A431 and ZR-75-1 cell, C225 causes that the maximal dose dependency of the HER1 phosphorylation that EGF stimulates suppresses, and EC50 is about 8nM and 40-80% maximum suppression effect (Fig. 8 c and 9c, table 27 and 28).Similarly, the HER1 phosphorylation that EGF stimulates in other clone that C225 suppresses to be tested, it is renderd a service with RB200h suitable (table 27 and 28).In A431 or ZR-75-1 cell that EGF stimulates, C225 also suppresses the HER2 phosphorylation, but only is suppressed at the HER3 phosphorylation in the A431 cell, it is similar to the effect of RB200h, but compare with RB200h, C225 has low render a service (Fig. 8 c, table 27 and 28) to HER3.Yet, different with the effect of RB200h, the HER family protein phosphorylation (Fig. 8 c and 9c and table 27 and 28) that NRG1 β 1 stimulates in the arbitrary cell system that does not suppress to be tested in conjunction with the C225 of HER1.

[0639] tested the ability that Trastuzumab (it is at HER2) is regulated HER family protein tyrosine phosphorylation.In the A431 that EGF stimulates, HER2 that Trastuzumab only causes low-level (about 20%) or HER3 phosphorylation restraining effect, and in NRG1 β 1 stimulated cells, only suppress extremely about 30% low restraining effect (Fig. 8 b and e, table 27 and 28) of HER3 phosphorylation.Yet in the ZR-75-1 cell that EGF stimulates, Trastuzumab does not suppress HER family protein phosphorylation, causes also that on the contrary about 60% of HER2 phosphorylation stimulates (Fig. 9 b).Yet it is to the influence of HER2 relatively, and Trastuzumab is 50% inhibition HER3 phosphorylation after NRG1 β 1 stimulates the ZR-75-1 cell.In whole cells (A431, A549, HT29, N87, SK-BR-3 and ZR-75-1 cell) of test, observe Trastuzumab consistently and suppress the HER3 tyrosine phosphorylation to low-level 20-30%, especially in NRG1 β 1 stimulated cells.In the aforementioned clone of test, only in the A431 cell, Trastuzumab is handled the slight inhibition (about 20%) that causes the HER2 phosphorylation.In whole other clones, as mentioned, Trastuzumab is handled and is caused stimulation HER2 tyrosine phosphorylation, compare with untreated cell, and be that 10-60% stimulates.

[0640] in other clones, also carries out the similar research that relevant RB200h, Trastuzumab and C225 suppress the phosphorylation of ligand stimulation.Data are summarized in table 27 and table 28.By relatively RB200h, Trastuzumab and C225 are to the average % inhibition (panHER index) of HER family protein phosphorylation to several clones, it is being the most effective suppressing aspect the part inductive HER family protein phosphorylation that HER transfers albumen RB200h.Although C225 is effective equally with RB200h aspect the HER family protein phosphorylation that suppresses the EGF stimulation, but it be not effective aspect the HER protein phosphorylation that suppresses NRG1 β 1 stimulation.With regard to NRG1 β 1 stimulated cells, only RB200h suppresses the phosphorylation of whole HER family proteins, and Trastuzumab or C225 then deny, as being judged by panHER index (table 27 and 28).Data presentation HER transfers albumen RB200h to block the EGF (HER1 part) of whole three kinds of HER families or the tyrosine phosphorylation that NRG1 β 1 (HER3 part) stimulates, i.e. HER1, HER2 and HER3 phosphorylation, and C225 or Trastuzumab are then denied.Generally speaking, data presentation RB200h is the proteic part trap of HER1 and HER3 and has the anti-HER activity of wide spectrum.

Embodiment 16

The HER1 part and the HER3 part of various type are bonded to RB200h

[0641] conducts a research to determine whether other HER1 part except that EGF or other HER3 part except that NRG1 β 1 combine with RB200h.In these researchs, the binding ability of part is replaced with the ability of RB200h bonded Eu-EGF or Eu-NRG1 β 1 by it and is tested.Experiment is carried out as described in example 14 above.

[0642] as shown in Fig. 7 c and d, unlabelled EGF, HB-EGF, TGF-α suppress the Eu-EGF combination, show that these HER1 parts combine with RB200h.In similar research, NRG1-α, NRG1 β 3a and NRG1 β 1 inhibition Eu-NRG1 β 1 combine with RB200h's, and EGF does not then suppress, and shows that these neuregulins combine (Fig. 7 d) with RB200h.In addition, somatomedin (as with irrelevant Regular Insulin or the insulin-like growth factor-i of HER family part) do not compete and combine Eu-EGF or Eu-NRG1 β 1 (Fig. 7 c and d), show that RB200h is specifically in conjunction with HER1 part or HER3 part.This shows that RB200h can non-binding growth factor specifically, but high special ground is in conjunction with HER1 part or HER3 part.In a word, HER1 among the data presentation RB200h and HER3ECD bring into play function in ligand binding capacity as its natural counterpart.

Embodiment 17

HER1 among the RB200h and the ligand binding capacity of HER3 are separate

[0643] whether independently of one another for the ligand-binding site point on HER1 and HER3 among the research RB200h, in the presence of HER3 part (NRG1 β 1), carry out Eu-EGF and RB200h bonded competition research, otherwise and carry out Eu-NRG1 β 1 and studied by the competition that non-marked EGF competes.Experiment is implemented as described in example 14 above.

[0644] data presentation is under Eu-EGF bonded situation, and only unlabelled EGF, HB-EGF or TGF-α combine RB200h with the Eu-EGF competition, but unlabelled NRG1 β 1 does not compete.Similarly, only unlabelled NGR1 β 1 competition Eu-NRG-1 β 1 keying action, but do not compete EGF.In a word, data presentation HER1 ligand-binding site point is independent of HER3 ligand-binding site point and in conjunction with the HER1 part.Otherwise also be so, promptly HER-3 ligand-binding site point can be independent of HER1 ligand-binding site point and in conjunction with the HER3 part.

Embodiment 18

HER accent albumen is at the monolayer culture thing and suppress cell proliferation in soft agar

[0645] because RB200h in conjunction with EGF (HER1 part) and NRG1 β 1 (HER3 part) both and suppress the HER family protein tyrosine phosphorylation of factors stimulated growth, so RB200h also may suppress cell proliferation.This is by using or testing without the monolayer cell proliferation research that RB200h carries out.

[0646] soft agar colony growth measurement method is carried out based on (1987) described methods such as Hudziak, except this assay method is carried out in 24 orifice plates, wherein in described 24 orifice plates with 0.5% agarose of the 1.5ml in the substratum that contains 10% foetal calf serum as basal layer, and the top layer that contains cell is 0.5ml 0.25% agarose in 10% foetal calf serum.Compound is added into top layer.Has 5%CO at 37 ℃ ₂With implement the colony growth in the humidification incubator of 95% air.Every about 3 days, add 50 μ l/ hole sterilized waters to prevent exsiccation.Cell colony spends the night 4 ℃ of dyeing with 0.001% Viola crystallina of 1.0ml/ hole in water.Use the microscopic counting cell colony.

[0647] HER transfers albumen RB200h to suppress A431 epidermoid carcinoma and MDA-MB-468 breast cancer cell propagation in the dose-dependently mode, and EC50 is respectively 71nM and 1.4nM (Figure 11).Examination several other tumor cell lines of monolayer culture to the susceptibility of RB200h.With the tumour cell of this research extension to comprise that other is selected at random.The growth of the tumour cell of various type (comprising skin carcinoma, mammary cancer and lung carcinoma cell) is subjected to RB200h to suppress (table 29).Yet some tumor cell lines (comprising mammary cancer, lung cancer, colorectal carcinoma and stomach cancer cell) are insensitive to the growth-inhibiting effect of RB200h.

Table 29:RB 200 suppresses the propagation of the various tumor types cell in the monolayer culture

[0648] also tested the ability that RB200h suppresses not rely on the growth of adherent property by soft agar colony growth measurement method.Two kinds of tumor cell line ZR-75-1 mammary cancer and A549 lung carcinoma cell in the soft agar assay method, have been tested in monolayer growth to the growth-inhibiting effect sensitivity of RB200h.ZR-75-1 cell poor growth in soft agar, but stimulate to form colony through EGF (HER1 part) or NRG1 β 1 (HER3 part), wherein somatomedin NRG1 β is more effective, produces 9 times of stimulations, and EGF causes 3 times of stimulations of colony growth (Figure 12 is a).RB200h suppresses the ZR-75-1 cell soft agar colony growth that EGF or NRG1 β 1 stimulate, and the behavior that shows RB200h is similar to part trap at these somatomedins, and (Figure 12 a).The A549 lung carcinoma cell is easy to form colony in soft agar, but compares with handling without somatomedin, and can be subjected to NRG1 β 1 or EGF stimulates and reach 1.3 times and 1.4 times (Figure 12 b) respectively.It is much smaller that the colony growth stimulation of this level is compared to the viewed level of ZR-75-1 cell.RB200h handles the A549 cell and causes under somatomedin lacks about 65% of colony growth being suppressed (Figure 12 b).Yet the statistical significance that the colony that RB200h does not cause EGF or NRG1 β 1 to handle is grown suppresses (Figure 12 b).The result of study in back may be owing to such fact: the A549 cell forms colony easily and adds EGF or NRG1 β 1 only causes inadequate qualified (～1.3 times) hormesis of colony growth when not adding somatomedin in soft agar, therefore for the colony growth, they do not rely on these parts.In a word, data presentation RB200h suppresses cell proliferation by playing a role as somatomedin part trap with by non-part trap mechanism.

Embodiment 19 studies RB200h blocking-up EGF or NRG1 β 1 inductive cell proliferation in serum free medium

[0649] is the hypothesis of HER1 part trap or HER3 part trap for further checking R B200h, conducts a research to determine whether RB200h suppresses EGF or NRG1 β 1 stimulated cell proliferation.

In serum free medium, carry out cell proliferation research as described.According to clone, (Falcon#35-3075, BectonDickinson NJ), and cultivate spend the night (15-18 hour) subsequently in 96 hole tissue culturing plates with every hole 2000-6000 cell cover plant with cell.For the cell proliferation carried out in containing the substratum of serum research, cell is used subsequently or need not described compound treatment, and allows its growth 3-5 day.As hereinafter under the serum-free growth conditions, carrying out the influence that RB200h stimulates proliferation to somatomedin (EGF or NRG1 β 1).In serum, behind the inoculating cell, cell culture is spent the night (15-20 hour), subsequently cell is converted to serum free medium and cultivates 24-48 hour (serum starvation).Cell handle with somatomedin or LPA subsequently and with or handle and cultivation 3-5 day without RB200h.Cell proliferation is quantitative by crystal violet staining assay (Sugarman, 1987) as previously mentioned.In brief, the substratum that inclines, with cell once with PBS washing, be added on subsequently 50 μ l/ hole 0.5% (w/v) Viola crystallina dye liquors in the methyl alcohol (Sigma-Aldrich, St Loius, MO) and hatched 20 minutes.Plate washed with water 3 times and air-dry overnight subsequently.In conjunction with the dyestuff of cell with 100 μ l/ hole Sorenson damping fluids (the 25mM Trisodium Citrate in 50% ethanol) wash-out 15 minutes on the plate vibrator.This plate reads absorbancy at 540nm wavelength place subsequently on the plate readout instrument, the number of wherein said absorbancy and hole inner cell is proportional.

[0650] EGF stimulates SUM149 cell proliferation.This propagation that EGF stimulates is blocked (Figure 13 a and 14a) fully by RB200h.Handle with NRG1b1 and it is reported and respond to NRG1 β 1 (Lewis, Cancer Res.1996 such as GD, MCF-7 cell 56:1457-65) (Figure 13 b).This somatomedin causes that under serum-free condition the dose-dependently to MC7 cell proliferation stimulates.This cell proliferation that NRG1 β 1 stimulates is blocked fully by RB200h.

[0651] in a word, the antagonistic action data to the propagation of ligand stimulation show that RB200h is the part trap at HER1 part and HER3 part.

Embodiment 20

HER transfers albumen to suppress the cell proliferation of GPCR ligand stimulation

[0652] for tumour cell, the important source of somatomedin is from the GPCR part activation of ADAM metalloprotease, membrane-bound somatomedin such as amphiregulin, HB-EGF or TGF-α are striden in wherein said ADAM metalloprotease cutting, final these somatomedins (Huovila that discharges, TIBS such as AJ 2005,30:413-422).So the somatomedin that produces can be used for stimulating tumor cell proliferation with paracrine or in the autocrine mode subsequently.Because RB200h is in conjunction with HER1 part and HER3 part, it can be blocked this somatomedin source of tumour cell and cause growth of tumour cell to suppress.Use the SUM149 breast cancer cell to check this hypothesis, wherein it is reported SUM149 mammary cancer carefully be amphiregulin (AR) autocrine produce property and AR dependent cell (Willmarth, NE and Ethier, SP.J.Biol.Chem.2006,281:37728-37737).

[0653] cell proliferation is carried out as described in example 19 above.Under the serum-free growth pattern, carry out the proliferation function that GPCR ligand L PA stimulates.

[0654] cause the dose-dependently of cell proliferation to increase (Figure 13 b and 14b) with Ultrapole L (LPA) treatment S UM149 cell.This propagation that LPA stimulates is blocked (Figure 13 b and 14b) fully by RB200h, this is consistent with following viewpoint: HER transfers albumen to play a role at GPCR activated growth factor release as somatomedin part trap.

Embodiment 21HER transfers albumen and tyrosine kinase inhibitor to work in coordination with

[0655] when with the time at the kinase whose tyrosine kinase inhibitor associating of HER1 or HER2, biological substance (Biologic agent) at the HER family protein has been presented at the collaborative (Mendelsohn that replys that suppresses the cell proliferation aspect, J and Baselga, J.Semin.Oncol.2006,33:369-385).Therefore, we also implement joint study with tyrphostin (tyrphostin) AG 825 (HER2 kinase inhibitor) with RB200h and tyrosine kinase inhibitor Gefitinib (Iressa), erlotinib (Tarceva) (a kind of EGFR kinase inhibitor of FDA approval) in the monolayer cell proliferation assay.

[0656] cell proliferation research is containing blood serum medium or is carrying out in serum-free culture as described.According to clone, (Falcon#35-3075, Becton Dickinson NJ), and cultivate spend the night (15-18 hour) subsequently in 96 hole tissue culturing plates with every hole 2000-6000 cell cover plant with cell.For the cell proliferation in containing blood serum medium, carried out research, cell used subsequently or need not described compound treatment, and allow its growth 3-5 day.For the cell proliferation research of under the serum-free growth conditions, carrying out, in serum, behind the inoculating cell, cell culture is spent the night (15-20 hour), subsequently cell is converted to serum free medium and cultivates 24-48 hour (serum starvation).Compound such as RB200h, IRS, Iressa, Gefitinib, erlotinib and AG-825 use simultaneously in duplicate subsequently and carry out continuous doubling dilution.Cell proliferation is as previously mentioned by crystal violet staining assay (Sugarman, 1987) quantification.In brief, the substratum that inclines, with cell once with PBS washing, be added on subsequently 50 μ l/ hole 0.5% (w/v) Viola crystallina dye liquors in the methyl alcohol (Sigma-Aldrich, St Loius, MO) and hatched 20 minutes.Plate washed with water 3 times and air-dry overnight subsequently.In conjunction with the dyestuff of cell with 100 μ l/ hole Sorenson damping fluids (the 25mM Trisodium Citrate in 50% ethanol) wash-out 15 minutes on the plate vibrator.This plate reads absorbancy at 540nm wavelength place subsequently on the plate readout instrument, the number of wherein said absorbancy and hole inner cell is directly proportional.

[0657] in NSCLC (H1437) cell, only RB200h or only AG 825 cell growth inhibiting to low-level.These tumour cells tolerance EGFR and HER2 kinase inhibitor.The RB200h of associating and AG 825 produce obvious synergy, and (Figure 15 a).The synergy data are used for determining objectively the synergistic program of medication combined research (T-C Chou and P.Talalay by specialized designs; Trends Pharmacol.Sci 4,450-454) CalcuSyn (Biosoft, Cambridge UK) analyzes.Use this analytical data, the CalcuSyn program produces the parameter that is called association index (CI).When CI less than 1.0 the time, then have synergy between two kinds of compounds.CI is 1, and meaning exists additivity to reply and represents to exist between the compound antagonistic action greater than 1 CI.For AG-825, work in coordination with on whole concentration of being tested with uniting in NSCLC H1437 cell of RB200h, CI is that 0.20 (Figure 15 is a).

[0658] in breast cancer cell line MDA-MB-468 at the another kind of tyrosine kinase inhibitor Gefitinib of EGFR also with the RB200h high Collaboration, C.I is 0.20 (Figure 15 b).This and synergy RB200h is also observed in erlotinib, and wherein erlotinib is the another kind of EGFR kinase inhibitor (Figure 15 c) of FDA approval.In NSCLC cell H2122, also find Rb200h and erlotinib collaborative play a role (Figure 16).On the contrary, in normal cell such as Hs578Bst, RB200h does not significantly suppress cell proliferation and do not have synergy (Figure 15 d) yet between RB200h and Gefitinib.In several other tumor cell lines, also see RB200h and AG-825 or and Iressa between synergy.

[0659] Figure 17-20 demonstration is compared with RB200h or tyrosine kinase inhibitor, in the A431 cell RB200h and AG825, in the A431 cell RB200h and Iressa and in the BT474 cell serial dilution thing of RB200h and IRS bring into play the effect that suppresses cell proliferation synergistically.In some cells, synergy is an intensive, and in other cell, has faint synergy (table 30).

Table 30:RB200 and tyrosine kinase inhibitor are collaborative to heavens

[0660] in a word, the RB200h of data presentation on very low dose is collaborative with the growth inhibitory activity at HER1 or the kinase whose tyrosine kinase inhibitor of HER2 really.This hint RB200h can be used as therapeutical agent with at the kinase whose tyrosine kinase inhibitor associating of HER1 or HER2 and have its maximum purposes, be included in the purposes among those patients of these kinase inhibitor of tolerance.

Embodiment 22

HER transfers albumen RB200h to have anti-tumor in vivo in the A431 human tumor xenograft model and renders a service.

[0661] uses nude mouse, in the A431 human tumor xenograft model, render a service in the body of test RB200h.Provided used general method in the present embodiment, and some variations of used general method.

Animal

[0662] (Harlan UK) obtains mouse from suppliers.Mouse is 4-6 age in week when this research begins.In between the disinfection and isolation of mouse raising in the barrier unit, by fluorescent lighting, wherein described luminescent lamp is set to produce illumination-dark cycle (7 unlatchings in 12 hours, 19.00 point is closed), as british animal affairs (scientific procedure method) bill (United Kingdom Home OfficeAnimals (Scientific Procedures) Act) in 1986 recommendation.Be intended to keep system's adjusting room environment of 23 ± 2 ℃ of air themperature by design.Mouse stays in (Techniplast UK) in the plastics cage at experimental session with 2 or 5 groupings, and described plastics cage has the bedding that postdose crosses and is equipped with nest material and Feng Rong environment (environmental enrichment).Arbitrarily supply with the 2019 rodents foodstuffs (Harlan Teckland UK, production code member Q219DJ1R2) and the autoclaving water of aseptic radiation.

Preliminary toxicity research

[0663] every group of 2 mouse, be divided into three groups as follows: group 1: time peritoneal injection (n=2) 30mg/kg RB200h on every Wendesdays; Group 2: weekly at 1-5 day circulation peritoneal injection (n=2) 75mg/kg Iressa; With group 3: time peritoneal injection (n=2) 10mg/kg RB200h and weekly at 1-5 day circulation peritoneal injection 38mg/kg Iressa on every Wendesdays.

Treatment is estimated

[0664] there are following 8 groups: group 1: the carrier (n=10) of time peritoneal injection RB200h on every Wendesdays, group 2: inferior on every Wendesdays peritoneal injection (n=10) 10mg/kg RB200h, group 3:(n=10) the inferior on every Wendesdays peritoneal injection of 30mg/kg RB200h, group 4: weekly at the carrier (n=10) of 1-5 day circulation peritoneal injection Iressa, group 5: weekly at 1-5 day circulation peritoneal injection (n=10) 38mg/kg Iressa, group 6: weekly at 1-5 day circulation peritoneal injection (n=10) 75mg/kg Iressa, the carrier (n=10) of group 7:RB200h and Iressa and organize 8: time peritoneal injection (n=10) 10mg/kg RB200h and weekly at 1-5 day circulation peritoneal injection 38mg/kg Iressa on every Wendesdays.

Tumour is initial

[0665] the A431 cell by PRECOS supply and at 37 ℃ in 5%CO ₂With add the wet environment lower body maintain outward contain the heat-inactivated foetal calf serum of 10% (v/v) (Sigma, Poole, RPMI1640 substratum UK) (Gibco, Paisley, UK) in.To be derived from the cell that converges (sub-confluent) individual layer in the Asia with 0.025%EDTA collect, in substratum mentioned above washed twice, and be resuspended among the sterile phosphate buffered saline pH 7.4 (PBS) and be used for using in the body.With cell with 1 * 10 ⁷Individual cell in 100 μ l volumes subcutaneous injection to mouse.

Tumor monitoring

[0666] for preliminary toxicity research, mouse is dispensed to the treatment group and treats on 5th is that per injection 150 μ l begin to continue for 2 weeks with the administration volume.For treatment research, mouse is dispensed to the treatment group, and reaches 50-100mm at mean tumour volume ³The time begin treatment and be per injection 150 with the administration volume] μ l continues 3 all administrations.Write down tumour size three times (the tumour cross-sectional area and the volumes of kind of calliper length and width and calculating) weekly and measure body weight weekly.

Put to death

[0667] keeps every mouse under study for action until execution maybe must remove mouse from this research till.Obvious as tumour size too big or any harmful effect that becomes, then put to death animal.When putting to death, mouse anesthesia (Hypnorm/Hynovel) is also taken out about 1ml blood by cardiac puncture, be processed into blood plasma, the freezing preliminary study that is used for is studied with treatment.Mouse is put to death by the S1 method of approval subsequently.For treatment research, tumour is downcut, weighed, measures and in formalin, fix.

Data and statistical analysis

[0668] with weight data, tumor growth and final tumor weight record and with form page or leaf and chart format report.Use Minitab to carry out statistical study as required.

Variation from preliminary study

[0669] preliminary toxicity research stops after 12 days in administration, so as after administration 3 hours collection plasma samples.Be used for the treatment of research by PRECOS interpolation as next additional set.Group 9:30mg/kg human IgG peritoneal injection, inferior on every Wendesdays.By PRECOS preparation in PBS 10%DMSO and the Iressa among the 5%Cremaphor.Tumour is with 2 * 10 ⁶Every mouse of individual cell causes (initiated) and is used for the treatment of research.As the requirement of institute of sponsor, RB200h and IgG are carried out intravenous administration be used for the treatment of research.Because for the first time after the administration in group 2, group 3 with organize detrimental action in 8, administration is back to the remainder that peritoneal injection is used to study.This research stopped on 26th, and reason is the ulcer of tumour in numerous mouse.

The result

[0670], as auspicious stating in the method, causing Subcutaneous tumor, and began with Rb200h and/or Iressa on 5th with the A431 cell for preliminary toxicity research.In the A431 tumor-bearing mice, do not observe detrimental action.During whole toxicity research, the body weight of mouse is still at acceptable scope (Figure 24 A).Measure gross tumor volume before execution, the average tumor size of group 1 is for very big.In the Iressa treatment group, there is inhibition (group 2 and 3) (Figure 24 B) to the tumour size.

[0671] because injection 1 * 10 ⁷The huge size of the tumour that 2 weeks occurred behind the individual cell is so reduce cell number to 2 * 10 that are used for causing tumour ⁶To increase the time range (time frame) of research.Use two batches cell and mouse (batch A and batch B) to start this research through 2 days.On 10th, the tumour mean size reached 50-100mm ³And administration begins.Make that intravenously is used into but not intraperitoneal is used for the administration of RB200h, IgG and RB200h carrier.With first batch of administration, and in group 2, group 3 with organize in the RB200h treatment mouse in 8 and observe untoward reaction.In group 3, on the used maximum concentration 30mg/kg RB200h, a mouse does not recover in this research.Observe all the other mouse and they recovered after 1 hour.Used though RB200h has before carried out intravenously, yet described RB200h batch and tumor model are different from RB200h used in this research batch and tumor model.Level of endotoxin in this batch is also lower than the level of endotoxin in previous used batch.Before intravenous administration, RB200h is warming up to 37 ℃ and to organizing 2 mouse administrations in 3 and observing.As preceding, mouse occur after 10 minutes skin red/purple and after 1 hour, recovering.Therefore medication being reverted to peritoneal injection is used to remain mouse and does not see other reaction.

[0672] monitoring tumour size and every class mean of time mapping is shown in Figure 25 A-D in the whole process of this research.These data also are summarized in following table 40.Also measured final tumor weight and every class mean is shown in Figure 26. these data also are summarized in the table 40.

[0673] compare with vehicle group (vehicle group), the independent RB200h of higher dosage (30mg/kg, group 3) significantly reduces tumor growth rate (p＜0.05, two factor variance analysis).Also 50% significantly reduce final tumor weight (p=0.016, one-way analysis of variance, Figure 26).Comparatively speaking, 10mg/kg RB200h does not significantly weaken the A431 tumor growth, although there is the trend that reduces the tumour size with about 15-20%.Provide the human IgG dosage (30mg/kg, group 9) that equates with RB200h dosage to contrast by PRECOS as protein.Do not find the influence of IgG to tumor growth.

[0674] Iressa of higher dosage (75mg/kg, group 6) significantly reduces tumor growth rate (Fig. 2, p＜0.001, two factor variance analysis), and the 38mg/kg Iressa does not compare with vehicle group (group 4).When with the treatment of 75mg/kg Iressa, final tumor weight also reduced by 69% (p=0.016, one-way analysis of variance, Figure 26).Pass in time, the carrier of Iressa (group 4) also has from the inhibition influence (Figure 25 D, p＜0.05, two factor variance analysis) of Rb200h vehicle group and reduces final tumour size 43% (Figure 26), although this is not significant.In combination, 10mg/kgRB200h and 38mg/kg Iressa (group 8) do not influence A431 growth of tumor (Figure 25 C and 26).Find that vehicle group (group 7) has reduced tumour size 30%, but find that it is not significant.

Table 40

* with the statistical significance of organizing 1

# and the statistical significance of organizing 4

[0675] the mouse body weight also this research the duration detect and progressively increase (Figure 27) as expected with the age of mouse.

Discuss

[0676] in the present embodiment purpose of these experiments be in the subcutaneous heteroplastic transplantation model of A431 assessment separately or with the effect of the RB200h of Iressa combination.It is reported the A431 epidermoid carcinoma express high-caliber EGFR and Her2 (2006.Clin Cancer Res.12 (24) such as Ono M: 7242-51) and it be used in preclinical phase evaluation (the Wakeling AE etc. of Iressa, 2002.CancerRes.62 (20): 5749-54), wherein said Iressa is the selection inhibition of EGFR tyrosine kinase domain, can be used for the treatment of NSCLC clinically in the U.S. at present.RB200h is at the tumour of expressing general Her and custom-designed part trap molecule, and therefore selects the A431 heteroplastic transplantation model with assessment RB200h.

[0677] initial preliminary toxicity research is presented in the mouse of carrying this tumour fully the tolerance intraperitoneal and uses, yet when route of administration makes intravenously into, observes untoward reaction.In the mouse of carrying ZR75-1, do not see this untoward reaction (P130:RB200h is carrying the preliminary toxicity research in the nude mouse of Subcutaneous tumor ZR75-1) with the RB200h intravenous administration.It is reported that level of endotoxin is also less than the level of endotoxin of used batch of current research.The effect of gained therefore can be owing to the variation of alternative parameter in batch prepared product of RB200h and/or tumor type.

[0678] find that 30mg/kg RB200h dosage significantly weakens the A431 tumor growth, and 10mg/kg does not significantly weaken the A431 tumor growth.Similarly, maximum dose level 75mg/kg Iressa significantly reduces tumor growth, and 38mg/kg does not significantly reduce tumor growth.When RB200h and Iressa when uniting than low dosage, do not observe weakening of tumor growth.In this research, do not unite higher dosage.Though Iressa has bigger result of treatment than RB200h, yet used Iressa dosage approaches MTD in this research, and the MTD of RB200h does not determine yet.

[0679] carries out further dosage and rise research, thereby determine the maximum therapy reaction with the MTD of definite RB200h with clinical obtainable dosage contrast.Another group experiment test RB200h influence in other model (as subcutaneous ZR75-1 (breast cancer cell line of Her2 high expression level) model and MDA-MB231 (breast cancer cell line of EGFR high expression level) bone metastasis model).The BT20 breast cancer cell line is expressed high-caliber EGFR and Her2 and therefore will be used to estimate RbB200h.

Embodiment 23

The panHER part trap that design has higher ligand binding affinity and ability: HER1ECD is based on the mutagenesis of structure

[0680] although RB200h shows high relatively binding affinity with about 10nM to HER1 or HER3 part, yet cell with the about 0.3-3nM of Kd in conjunction with HER1 part (as EGF or TGF-α), and for the HER3 part, NRG1 β 1 is about 0.1nM to 7.0nM (Holmes etc.; Slikowski etc.; Pinkas-Kramarski etc. 1996).This shows that tumour cell has the avidity higher than RB200h for HER1 or HER3 part, and wherein said RB200h is about 10nM to EGF or NRG1-β 1 bonded Kd.Therefore, the panHER part trap that the intention design is such, its avidity is higher than the viewed avidity to RB200h.This is at first undertaken by microcomputer modelling, and the disclosed eutectic structure of use and EGFR (HER1) bonded EGF is optimized the high-affinity HER1/Fc at its part.The crystalline structure (Ogiso H etc., Cell (2002) 775-787) of the complex body of human epidermal growth factor and acceptor ectodomain is used for the optimization of computer based ligand-receptor interaction.Three dimensional protein structure from structural information research cooperation laboratory Protein Data Bank (Research Collaboratory for StructuralBioinformatics (RCSB) ' s Protein Data Bank ( Http:// www.rcsb.org/pdb)).The optimization of designed ligand-receptor interaction is based on amino acid whose physics-chem characteristic and classification thereof, as electric charge, polarity, aromatic series etc.Also consider residue volume, surface-area, arriving at property of solvent etc.Use the PAM250 matrix to assist predicted amino acid to replace (W.A Pearson, Rapid and SensitiveSequence Comparison with FASTP and FASTA, in Methods inEnzymology one book, R.Doolittle edits (ISBN 0-12-182084-X, AcademicPress, San Diego) 183 (1990) 63-98; And also have M.O.Dayhoff, editor, 1978, Atlas of Prtotein Sequence and Structure, the 5th volume).

A. high-throughput mutagenesis

[0681] followingly carries out high-throughput mutagenesis: replace single amino acids by mutagenesis, make clonal expression subsequently and screen its ligand-binding activity at EGF, HB-EGF, TGF-α and amphiregulin (AR).The 39th position has mutant that Threonine to Serine replaces (being called T39S) by Modeling Research prediction generating high-affinity in HER1, to its screening and find that it is in conjunction with EGF, TGF-α and HB-EGF.This HER1/Fc T39S mutant is called HFD120.Except HFD120, several other mutant have also been produced.

[0682] uses extension enzyme (Invitrogen) and pfu polysaccharase (Stratagene) to carry out overlapping PCR and import HFD100 (template) (Figure 21 and table 31) with the point mutation that will design.

[0683] used forward primer is that EGFR-F1:5 '-AATTCGTACGACCGCCACC ATG GGA CCCTCCGGGACGGCC-3 ' and used reverse primer are EGFR650-R1:GGGGACCACTTTGTACAAGAAAGCTGGGT CTAGGA CGG GAT CTTAGG CCCA.

[0684] first round PCR:HFD100 is as pcr template.Use and extend enzyme and pfu polysaccharase, carry out PCR with primer EGFR-F1 and EGFRmu_R2.The PCR condition is 94 ℃, 2 minutes; 94 ℃ then, 45 seconds; 60 ℃, 45 seconds; 68 ℃, carried out 26 circulations in 3 minutes.For primer EGFRmu_F2 and EGFR-R1, the PCR condition is 94 ℃, 2 minutes; 94 ℃ then, 45 seconds; 60 ℃, 45 seconds; 68 ℃, carried out 26 circulations in 3 minutes.After the amplification, the PCR product is separated on 1% sepharose and use Qiagen gel-purified test kit (Qiagen) to carry out purifying.

[0685] second takes turns PCR: first round PCR product mixes with molar ratio at 1: 1.Use 9 ℃ of extension enzyme and pfu polysaccharase and following conditions, 2 minutes; 94 ℃ then, 45 seconds; 57 ℃, 45 seconds; 68 ℃, continued 8 in 3 minutes and circulate and carry out PCR.

[0686] third round PCR: second takes turns the PCR product as template.Use and extend enzyme and pfu polysaccharase, carry out PCR with primer EGFR-F1 and EGFR-R1.The PCR condition is 94 ℃, 2 minutes; 94 ℃, 45 seconds; 60 ℃, 45 seconds; 68 ℃, carried out 26 circulations in 3 minutes.The PCR product separates on 1% sepharose and uses Qiagen gel-purified test kit (Qiagen) to carry out purifying.The PCR product subclone of purifying is to the p221DONR carrier.

Table 31: the EGFR mu primer that is used for mutation analysis HFD100

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu01_R2	138	A01	CTCTGGAGGCTGAGAAAATGTTCTTCAAAAGTGCCCAACTGCG
EGFRmu02_R2	137	A02	CTCTGGAGGCTGAGAAAATGATTTTCAAAAGTGCCCAACTGCG
				EGFRmu03_R2	137	A03	CTCTGGAGGCTGAGAAAATGTTGTTCAAAAGTGCCCAACTGCG
EGFRmu04_R2	124	A04	TGAGAAAATGATCTTCAAAAGTGTTCAACTGCGTGAGCTTGTTAC
				EGFRmu05_R2	124	A05	CTCTGGAGGCTGAGAAAATGTTCTTCAAAAGTGTTCAACTGCGTGAGCTTGTTAC
EGFRmu06_R2	121	A06	AAAATGATCTTCAAAAGTGCCCACCTGCGTGAGCTTGTTACTCG
				EGFRmu07_R2	121	A07	GAAAATGATCTTCAAAAGTGCCAATCTGCGTGAGCTTGTTACTC

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu08_R2	277	A08	GAATTCGCTCCACTGTGTTGACGGCAATGAGGACATAACCAG
EGFRmu09_R2	277	A09	GAATTCGCTCCACTGTGTTGATGGCAATGAGGACATAACCAG
				EGFRmu10_R2	205	A10	AAAGATCATAATTCCTCTGCACCCAGGTAATTTCCAAATTCCCA
EGFRmu11_R2	338	A11	CTGCTAAGGCATAGGAATTTTCCCAGTACATATTTCCTCTGATGAT
				EGFRmu12_R2	342	A12	GACTGCTAAGGCATAGGAATTATCGTAGTACATATTTCCTCTGA
EGFRmu13_R2	340	B01	ACTGCTAAGGCATAGGAATTTTGGTAGTACATATTTCCTCTGATG
				EGFRmu14_R2	367	B02	GGTTTTATTTGCATCATAGTTAGCTAAGACTGCTAAGGCATAGGA
EGFRmu15_R2	367	B03	GGTTTTATTTGCATCATAGTTAGTTAAGACTGCTAAGGCATAGGA
				EGFRmu16_R2	128	B04	GGCTGAGAAAATGATCTTCAAATTTGCCCAACTGCGTGAGCTT
EGFRmu17_R2	128	B05	GGCTGAGAAAATGATCTTCAAATTGGCCCAACTGCGTGAGCTT
				EGFRmu18_R2	129	B06	GGCTGAGAAAATGATCTTCAAAAATGCCCAACTGCGTGAGCT
EGFRmu19_R2	128	B07	GGCTGAGAAAATGATCTTCAAAATCGCCCAACTGCGTGAGCTT
				EGFRmu20_R2	128	B08	GGCTGAGAAAATGATCTTCAAAATAGCCCAACTGCGTGAGCTT
EGFRmu21_R2	128	B09	GGCTGAGAAAATGATCTTCAAAACCGCCCAACTGCGTGAGCTT
				EGFRmu22_R2	128	B10	GGCTGAGAAAATGATCTTCAAAAAGGCCCAACTGCGTGAGCTT
EGFRmu23_R2	145	B11	GAACATCCTCTGGAGGCTGGCAAAATGATCTTCAAAAGTGCCCA
				EGFRmu24_R2	145	B12	TTGAACATCCTCTGGAGGCTCCAAAAATGATCTTCAAAAGTGCCC
EGFRmu25_R2	149	C01	TTATTGAACATCCTCTGGAGGGTGAGAAAATGATCTTCAAAAGTG
				EGFRmu26_R2	148	C02	TATTGAACATCCTCTGGAGGAGGAGAAAATGATCTTCAAAAGTGC
EGFRmu27_R2	145	C03	GTTATTGAACATCCTCTGGAGGAGGGCAAAATGATCTTCAAAAGTGCCC
				EGFRmu28_R2	145	C04	GTTATTGAACATCCTCTGGAGTTGGGCAAAATGATCTTCAAAAGTGCCC
EGFRmu29_R2	148	C05	TTATTGAACATCCTCTGGAGGGCGAGAAAATGATCTTCAAAAGTGC
				EGFRmu30_R2	145	C06	TTATTGAACATCCTCTGGAGGGCGTAAAAATGATCTTCAAAAGTGCCCA
EGFRmu31_R2	145	C07	TTATTGAACATCCTCTGGAGGGCGTTAAAATGATCTTCAAAAGTGCCC
				EGFRmu32_R2
	118	C08	TGATCTTCAAAAGTGCCCAACTCCGTGAGCTTGTTACTCGTGCC
				EGFRmu33_R2
	118	C09	TGATCTTCAAAAGTGCCCAACGACGTGAGCTTGTTACTCGTGC

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu34_R2
	118	C10	GATCTTCAAAAGTGCCCAACTTCGTGAGCTTGTTACTCGTGC
				EGFRmu35_R2
	118	C11	ATGATCTTCAAAAGTGCCCAAGTACGTGAGCTTGTTACTCGTG
				EGFRmu36_R2
	115	C12	CTTCAAAAGTGCCCAACTGCGAGAGCTTGTTACTCGTGCCTT
				EGFRmu37_R2	116	D01	CTTCAAAAGTGCCCAACTGCTTGAGCTTGTTACTCGTGCCTT
EGFRmu38_R2
		115	D02	CTTCAAAAGTGCCCAACTGCTCGAGCTTGTTACTCGTGCCTT
EGFRmu39_R2
		115	D03	ATCTTCAAAAGTGCCCAACTGATAGAGCTTGTTACTCGTGCC
EGFRmu40_R2
		100	D04	ACTGCGTGAGCTTGTTACTCTGGCCTTGGCAAACTTTCTTTTC
EGFRmu41_R2
		1300	D05	CGACTGCAAGAGAAAACTGACGATGTTGCTTGGTCCTGCCG
EGFRmu42_R2
		1300	D06	ACGACTGCAAGAGAAAACTGATTATGTTGCTTGGTCCTGCCG
EGFRmu43_R2					1393	D07	GCATAGCACAAATTTTTGTTTCGTGAAATTATCACATCTCCATC
	EGFRmu44_R2	1393	D08	TTTGCATAGCACAAATTTTTGTTATGTGAAATTATCACATCTCCATC
EGFRmu45_R2					146	D09	TTGAACATCCTCTGGAGGCTTTGAAAATGATCTTCAAAAGTGCC
	EGFRmu46_R2	1109	D10	AAATGCCACCGGCAGGATGCGGAGATCGCCACTGATGGA
EGFRmu47_R2					1120	D11	GAGTCACCCCTAAATGCCAGCGGCAGGATGTGGAGATCG
	EGFRmu48_R2	1126	D12	TGTGAAGGAGTCACCCCTATGTGCCACCGGCAGGATGTG
EGFRmu49_R2					1132	E01	GAGTATGTGTGAAGGAGTCAGCCCTAAATGCCACCGGCA
	EGFRmu50_R2	1132	E02	GAGTATGTGTGAAGGAGTCATTCCTAAATGCCACCGGCAG
EGFRmu51_R2					1226	E03	CCGTCCTGTTTTCAGGCCATTCCTGAATCAGCAAAAACCCT
	EGFRmu52_R2	1226	E04	CCGTCCTGTTTTCAGGCCAATCCTGAATCAGCAAAAACCCT
EGFRmu53_R2					1228	E05	GTCCGTCCTGTTTTCAGGCTCAGCCTGAATCAGCAAAAACC
	EGFRmu54_R2	1330	E06	GTAATCCCAAGGATGTTATGTCCAGGCTGACGACTGCAAGA
EGFRmu55_R2					1472	E07	CTGTTTTCACCTCTGTTGCTTTTAATTTTGGTTTTCTGACCGG
	EGFRmu56_R2	1459	E08	TCTGTTGCTTATAATTTTGGTTTCCTGACCGGAGGTCCCAAAC
EGFRmu57_R2					1459	E09	TCTGTTGCTTATAATTTTGGTTTGCTGACCGGAGGTCCCAAAC
	EGFRmu58_R2	1475	E10	GCAGCTGTTTTCACCTCTGTTTTTTATAATTTTGGTTTTCTGACCG
EGFRmu59_R2					166	E11	CCAAGGACCACCTCACAGTTTTCGAACATCCTCTGGAGGCTG

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu60_R2
	160	E12	CCACCTCACAGTTATTGAACAGCCTCTGGAGGCTGAGAAAAT
				EGFRmu61_R2
	160	F01	CCAAGGACCACCTCACAGTTTTCGTACAGCCTCTGGAGGCTGAGAAAA
				EGFRmu62_R2	127	F02	GAGGCTGAGAAAATGATCTTCAGCATCGCCCAACTGCGTGAGCTT
EGFRmu63_R2	127	F03	TCTGGAGGCTGAGAAAATGATTTTCAGCATCGCCCAACTGCGTGAGCTT
				EGFRmu64_R2
	95	F04	TGAGCTTGTTACTCGTGCCTGGGCAAACTTTCTTTTCCTCCA
				EGFRmu65_R2	283	F05	TGCAGGTTTTCCAAAGGAATTGTCGAAAATTCGTTGAGGGCAATGAGGACA
EGFRmu66_R2	314	F06	GTACATATTTCCTCTGATGATCCGCAGGTTTTCCAAAGGAATTC
				EGFRmu67_R2	329	F07	AAGGCATAGGAATTTTCGTAGACCTGAGTTCCTCTGATGATCTGCAGG
EGFRmu68_R2	364	F08	CGGTTTTATTTGCATCATAGTTTAACATGACTGCTAAGGCATAGGAAT
				EGFRmu69_R2	407	F09	CATGCAGGATTTCCTGTAAATTTGTCAGGCGCAGCTCCTTCAGTCCGG
EGFRmu70_R2	448	F10	CGTTGCACAGGGCAGGGTTCTTTTCGATCCGCACGGCGCCATGCA
				EGFRmu71_R2
	460	F11	TGCTCTCCACGTTGCACAGTTTATCGTTGTTGCTGAACCGCACG
				EGFRmu72_R2	472	F12	CCACTGGATGCTCTCCACGTGGCACAGGGCAGGGTTGTTG
EGFRmu73_R2	202	G01	AGATCATAATTCCTCTGCACAAGGACAATTTCCAAATTCCCAAGGAC
				EGFRmu74_R2	202	G02	AGAAGGAAAGATCATAATTCCTCCCCGTAAGGACAATTTCCAAATTCCCAAGGAC
EGFRmu75_R2	286	G03	GTTTTCCAAAGGAATTCGCTCAAATGTGTTGAGGGCAATGAGGA
				EGFRmu76_R2	286	G04	TGCAGGTTTTCCAAAGGAATTGACTCAAATGTGTTGAGGGCAATGAGGA
EGFRmu77_R2	283	G05	TGCAGGTTTTCCAAAGGAATTGTCGAAAATTCGTTGAGGGCAATGAGGACA
				EGFRmu78_R2	364	G06	CGGTTTTATTTGCATCATAGTTAAACATGACTGCTAAGGCATAGGAAT
EGFRmu79_R2	364	G07	CTTCAGTCCGGTTTTATTTGCATTATAGTTAAACATGACTGCTAAGGCATAGGAATT
				EGFRmu80_R2	448	G08	AGGGCAGGGTTGTTGCTGATCCGCACGGCGCCATGCA
EGFRmu81_R2	448	G09	CCACGTTGCACAGGGCAGGCTTGTTGCTGATCCGCACGGCGCCATGCA
				EGFRmu82_R2	103	G10	CCAACTGCGTGAGCTTGTTAAGCGTGCCTTGGCAAACTTTCT
EGFRmu83_R2	103	G11	CCAACTGCGAGAGCTTGTTAAGCGTGCCTTGGCAAACTTTCTT
				EGFRmu84_R2	127	G12	TCCTCTGGAGGCTGAGAAATTGATTTTCAGCATCGCCCAACTGCGTGAGCTT
EGFRmu85_R2	127	H01	CATCCTCTGGAGGCTGAGATATTGATTTTCAGCATCGCCCAACTGCGTGAGCTT

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu86_R2	124	H02	GGCTGAGAAAATGATCTTCAAAACCGTTCAACTGCGTGAGCTTGTTAC
EGFRmu87_R2	124	H03	AGGCTGAGAAAATGATCTTCATAACCGTTCAACTGCGTGAGCTTGTTAC
				EGFRmu88_R2	124	H04	GAGGCTGAGAAAATGATCTTCATAATTGTTCAACTGCGTGAGCTTGTTAC
EGFRmu89_R2	1471	H05	GCAGCTGTTTTCACCTCTGTTTTTTTGAATTTTGGTTTTCTGACCGGAG
				EGFRmu90_R2	1297	H06	CGACTGCAAGAGAAAACTGACGAACTTGCTTGGTCCTGCCGCG
EGFRmu91_R2	507	H07	ACATGTTGCTGAGAAAGTCACCCCTGACTATGTCCCGCCACT
				EGFRmu92_R2	514	H08	GTGGTTCTGGAAGTCCATCACGATCTCGGCGTCACGGTCACTGCTGACTATGTCC
EGFRmu93_R2	532	H09	GCAGCTGCCCAGGTGGTTGTCGCCTTTCACCGACATGTTGCTGAGAAAGTC
				EGFRmu94_R2
	118	H10	TGAGAAAATGATCTTCAAAAGTGTCCAAGTACGTGAGCTTGTTACTCGTGC
				EGFRmu95_R2
	115	H11	GATCTTCAAAAGTGCCCAACTCATAGAGCTTGTTACTCGTGCCTT
				EGFRmu96_R2	337	H12	GACTGCTAAGGCATAGGAATTATCGTGGTACATATTTCCTCTGATGATCTG
EGFRmu01_F2	1725	A01	CGCAGTTGGGCACTTTTGAAGAACATTTTCTCAGCCTCCAGAG
				EGFRmu02_F2	1726	A02	CGCAGTTGGGCACTTTTGAAAATCATTTTCTCAGCCTCCAGAG
EGFRmu03_F2	1726	A03	CGCAGTTGGGCACTTTTGAACAACATTTTCTCAGCCTCCAGAG
				EGFRmu04_F2	1739	A04	GTAACAAGCTCACGCAGTTGAACACTTTTGAAGATCATTTTCTCA
EGFRmu05_F2	1739	A05	GTAACAAGCTCACGCAGTTGAACACTTTTGAAGAACATTTTCTCAGCCTCCAGAG
				EGFRmu06_F2	1736	A06	CGAGTAACAAGCTCACGCAGGTGGGCACTTTTGAAGATCATTTT
EGFRmu07_F2	1736	A07	GAGTAACAAGCTCACGCAGATTGGCACTTTTGAAGATCATTTTC
				EGFRmu08_F2	1586	A08	CTGGTTATGTCCTCATTGCCGTCAACACAGTGGAGCGAATTC
EGFRmu09_F2	1586	A09	CTGGTTATGTCCTCATTGCCATCAACACAGTGGAGCGAATTC
				EGFRmu10_F2	1658	A10	TGGGAATTTGGAAATTACCTGGGTGCAGAGGAATTATGATCTTT
EGFRmu11_F2	1525	A11	ATCATCAGAGGAAATATGTACTGGGAAAATTCCTATGCCTTAGCAG
				EGFRmu12_F2	1521	A12	TCAGAGGAAATATGTACTACGATAATTCCTATGCCTTAGCAGTC
EGFRmu13_F2	1519	B01	CATCAGAGGAAATATGTACTACCAAAATTCCTATGCCTTAGCAGT
				EGFRmu14_F2	1496	B02	TCCTATGCCTTAGCAGTCTTAGCTAACTATGATGCAAATAAAACC
EGFRmu15_F2	1496	B03	TCCTATGCCTTAGCAGTCTTAACTAACTATGATGCAAATAAAACC

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu16_F2	1735	B04	AAGCTCACGCAGTTGGGCAAATTTGAAGATCATTTTCTCAGCC
EGFRmu17_F2	1735	B05	AAGCTCACGCAGTTGGGCCAATTTGAAGATCATTTTCTCAGCC
				EGFRmu18_F2	1734	B06	AGCTCACGCAGTTGGGCATTTTTGAAGATCATTTTCTCAGCC
EGFRmu19_F2	1735	B07	AAGCTCACGCAGTTGGGCGATTTTGAAGATCATTTTCTCAGCC
				EGFRmu20_F2	1735	B08	AAGCTCACGCAGTTGGGCTATTTTGAAGATCATTTTCTCAGCC
EGFRmu21_F2	1735	B09	AAGCTCACGCAGTTGGGCGGTTTTGAAGATCATTTTCTCAGCC
				EGFRmu22_F2	1735	B10	AAGCTCACGCAGTTGGGCCTTTTTGAAGATCATTTTCTCAGCC
EGFRmu23_F2	1718	B11	TGGGCACTTTTGAAGATCATTTTGCCAGCCTCCAGAGGATGTTC
				EGFRmu24_F2	1718	B12	GGGCACTTTTGAAGATCATTTTTGGAGCCTCCAGAGGATGTTCAA
EGFRmu25_F2	1722	C01	CACTTTTGAAGATCATTTTCTCACCCTCCAGAGGATGTTCAATAA
				EGFRmu26_F2	1722	C02	GCACTTTTGAAGATCATTTTCTCCTCCTCCAGAGGATGTTCAATA
EGFRmu27_F2	1718	C03	GGGCACTTTTGAAGATCATTTTGCCCTCCTCCAGAGGATGTTCAATAAC
				EGFRmu28_F2	1718	C04	GGGCACTTTTGAAGATCATTTTGCCCAACTCCAGAGGATGTTCAATAAC
EGFRmu29_F2	1722	C05	GCACTTTTGAAGATCATTTTCTCGCCCTCCAGAGGATGTTCAATAA
				EGFRmu30_F2	1718	C06	TGGGCACTTTTGAAGATCATTTTTACGCCCTCCAGAGGATGTTCAATAA
EGFRmu31F2	1718	C07	GGGCACTTTTGAAGATCATTTTAACGCCCTCCAGAGGATGTTCAATAA
				EGFRmu32_F2	1745	C08	GGCACGAGTAACAAGCTCACGGAGTTGGGCACTTTTGAAGATCA
EGFRmu33_F2	1745	C09	GCACGAGTAACAAGCTCACGTCGTTGGGCACTTTTGAAGATCA
				EGFRmu34_F2	1745	C10	GCACGAGTAACAAGCTCACGAAGTTGGGCACTTTTGAAGATC
EGFRmu35_F2	1745	C11	CACGAGTAACAAGCTCACGTACTTGGGCACTTTTGAAGATCAT
				EGFRmu36_F2	1742	C12	AAGGCACGAGTAACAAGCTCTCGCAGTTGGGCACTTTTGAAG
EGFRmu37_F2	1743	D01	AAGGCACGAGTAACAAGCTCAAGCAGTTGGGCACTTTTGAAG
				EGFRmu38_F2	1742	D02	AAGGCACGAGTAACAAGCTCGAGCAGTTGGGCACTTTTGAAG
EGFRmu39_F2	1742	D03	GGCACGAGTAACAAGCTCTATCAGTTGGGCACTTTTGAAGAT
				EGFRmu40_F2	1763	D04	GAAAAGAAAGTTTGCCAAGGCCAGAGTAACAAGCTCACGCAGT
EGFRmu41_F2	563	D05	CGGCAGGACCAAGCAACATCGTCAGTTTTCTCTTGCAGTCG

The primer title	Position bp	Hole number	Primer sequence
				EGFRmu42_F2	563	D06	CGGCAGGACCAAGCAACATAATCAGTTTTCTCTTGCAGTCGT
EGFRmu43_F2	470	D07	GATGGAGATGTGATAATTTCACGAAACAAAAATTTGTGCTATGC
				EGFRmu44_F2	470	D08	GATGGAGATGTGATAATTTCACATAACAAAAATTTGTGCTATGCAAA
EGFRmu45_F2	1717	D09	GGCACTTTTGAAGATCATTTTCAAAGCCTCCAGAGGATGTTCAA
				EGFRmu46_F2	754	D10	TCCATCAGTGGCGATCTCCGCATCCTGCCGGTGGCATTT
EGFRmu47_F2	743	D11	CGATCTCCACATCCTGCCGCTGGCATTTAGGGGTGACT
				EGFRmu48_F2	737	D12	CACATCCTGCCGGTGGCACATAGGGGTGACTCCTTCACA
EGFRmu49_F2	731	E01	TGCCGGTGGCATTTAGGGCTGACTCCTTCACACATACTC
				EGFRmu50_F2	731	E02	CTGCCGGTGGCATTTAGGAATGACTCCTTCACACATACTC
EGFRmu51_F2	637	E03	AGGGTTTTTGCTGATTCAGGAATGGCCTGAAAACAGGACGG
				EGFRmu52_F2	637	E04	AGGGTTTTTGCTGATTCAGGATTGGCCTGAAAACAGGACGG
EGFRmu53_F2
		635	E05	GGTTTTTGCTGATTCAGGCTGAGCCTGAAAACAGGACGGAC
EGFRmu54_F2
		633	E06	TCTTGCAGTCGTCAGCCTGGACATAACATCCTTGGGATTAC
EGFRmu55_F2					391	E07	CCGGTCAGAAAACCAAAATTAAAAGCAACAGAGGTGAAAACAG
	EGFRmu56_F2	404	E08	GTTTGGGACCTCCGGTCAGGAAACCAAAATTATAAGCAACAGA
EGFRmu57_F2					404	E09	GTTTGGGACCTCCGGTCAGCAAACCAAAATTATAAGCAACAGA
	EGFRmu58_F2	388	E10	CGGTCAGAAAACCAAAATTATAAAAAACAGAGGTGAAAACAGCTGC
EGFRmu59_F2					1697	E11	CAGCCTCCAGAGGATGTTCGAAAACTGTGAGGTGGTCCTTGG
	EGFRmu60_F2	1703	E12	ATTTTCTCAGCCTCCAGAGGCTGTTCAATAACTGTGAGGTGG
EGFRmu61_F2					1703	F01	TTTTCTCAGCCTCCAGAGGCTGTACGAAAACTGTGAGGTGGTCCTTGG
	EGFRmu62_F2	1736	F02	AAGCTCACGCAGTTGGGCGATGCTGAAGATCATTTTCTCAGCCTC
EGFRmu63_F2					1736	F03	AAGCTCACGCAGTTGGGCGATGCTGAAAATCATTTTCTCAGCCTCCAGA
	EGFRmu64_F2	1768	F04	TGGAGGAAAAGAAAGTTTGCCCAGGCACGAGTAACAAGCTCA
EGFRmu65_F2					1580	F05	TGTCCTCATTGCCCTCAACGAATTTTCGACAATTCCTTTGGAAAACCTGCA
	EGFRmu66_F2	1549	F06	GAATTCCTTTGGAAAACCTGCGGATCATCAGAGGAAATATGTAC
EGFRmu67_F2					1534	F07	CCTGCAGATCATCAGAGGAACTCAGGTCTACGAAAATTCCTATGCCTT
	EGFRmu68_F2	1499	F08	ATTCCTATGCCTTAGCAGTCATGTTAAACTATGATG

The primer title	Position bp	Hole number	Primer sequence
							CAAATAAAACCG
EGFRmu69_F2	1456	F09	CCGGACTGAAGGAGCTGCGCCTGACAAATTTACAGGAAATCCTGCATG
				EGFRmu70_F2	1415	F10	TGCATGGCGCCGTGCGGATCGAAAAGAACCCTGCCCTGTGCAACG
EGFRmu71_F2	1403	F11	CGTGCGGTTCAGCAACAACGATAAACTGTGCAACGTGGAGAGCA
				EGFRmu72_F2	1391	F12	CAACAACCCTGCCCTGTGCCACGTGGAGAGCATCCAGTGG
EGFRmu73_F2	1661	G01	GTCCTTGGGAATTTGGAAATTGTCCTTGTGCAGAGGAATTATGATCT
				EGFRmu74_F2	1661	G02	GTCCTTGGGAATTTGGAAATTGTCCTTACGGGGAGGAATTATGATCTTTCCTTCT
EGFRmu75_F2	1577	G03	TCCTCATTGCCCTCAACACATTTGAGCGAATTCCTTTGGAAAAC
				EGFRmu76_F2	1577	G04	TCCTCATTGCCCTCAACACATTTGAGTCAATTCCTTTGGAAAACCTGCA
EGFRmu77_F2	1580	G05	TGTCCTCATTGCCCTCAACGAATTTTCGACAATTCCTTTGGAAAACCTGCA
				EGFRmu78_F2	1499	G06	ATTCCTATGCCTTAGCAGTCATGTTTAACTATGATGCAAATAAAACCG
EGFRmu79_F2	1499	G07	AATTCCTATGCCTTAGCAGTCATGTTTAACTATAATGCAAATAAAACCGGACTGAAG
				EGFRmu80_F2	1415	G08	TGCATGGCGCCGTGCGGATCAGCAACAACCCTGCCCT
EGFRmu81_F2	1415	G09	TGCATGGCGCCGTGCGGATCAGCAACAAGCCTGCCCTGTGCAACGTGG
				EGFRmu82_F2	1760	G10	AGAAAGTTTGCCAAGGCACGCTTAACAAGCTCACGCAGTTGG
EGFRmu83_F2	1760	G11	AAGAAAGTTTGCCAAGGCACGCTTAACAAGCTCTCGCAGTTGG
				EGFRmu84_F2	1736	G12	AAGCTCACGCAGTTGGGCGATGCTGAAAATCAATTTCTCAGCCTCCAGAGGA
EGFRmu85_F2	1736	H01	AAGCTCACGCAGTTGGGCGATGCTGAAAATCAATATCTCAGCCTCCAGAGGATG
				EGFRmu86_F2	1736	H02	GTAACAAGCTCACGCAGTTGAACGGTTTTGAAGATCATTTTCTCAGCC
EGFRmu87_F2	1736	H03	GTAACAAGCTCACGCAGTTGAACGGTTATGAAGATCATTTTCTCAGCCT
				EGFRmu88_F2	1736	H04	GTAACAAGCTCACGCAGTTGAACAATTATGAAGATCATTTTCTCAGCCTC
EGFRmu89_F2	392	H05	CTCCGGTCAGAAAACCAAAATTCAAAAAAACAGAGGTGAAAACAGCTGC
				EGFRmu90_F2	566	H06	CGCGGCAGGACCAAGCAAGTTCGTCAGTTTTCTCTTGCAGTCG
EGFRmu91_F2	1356	H07	AGTGGCGGGACATAGTCAGGGGTGACTTTCTCAGCAACATGT
				EGFRmu92_F2	1349	H08	GGACATAGTCAGCAGTGACCGTGACGCCGAGATCGTGATGGACTTCCAGAACCAC
EGFRmu93_F2	1331	H09	GACTTTCTCAGCAACATGTCGGTGAAAGGCGACAACCACCTGGGCAGCTGC
				EGFRmu94_F2	1745	H10	GCACGAGTAACAAGCTCACGTACTTGGACACTTTTGAAGATCATTTTCTCA
EGFRmu95_F2	1748	H11	AAGGCACGAGTAACAAGCTCTATGAGTTGGGCACTT

The primer title	Position bp	Hole number	Primer sequence
							TTGAAGATC
EGFRmu96_F2	1526	H12	CAGATCATCAGAGGAAATATGTACCACGATAATTCCTATGCCTTAGCAGTC

[0687] attested HFD100 mutant among the pDONR221 is reacted subclone to the pcDNA3.2-DEST expression vector by LR.

B. protein expression and secretion

[0688] the transient gene expression method (Invitrogen) of using Lipofectamin 2000 to mediate according to manufacturer specification, is expressed the HFD100-mutant in the pcDNA3.2-DEST expression vector in the 293T cell.48 hours collection condition substratum after transfection.Analyze the conditioned medium of 15 μ l volumes by Western blotting.The western blotting thing is surveyed with check protein expression and secretion with anti-Fc antibody.Use Duoset people EGFR ELISA test kit (R﹠amp; D System) determines reorganization HFD100-mutant in the conditioned medium.Elisa plate is spent the night at the room temperature bag with 0.4 μ g/ml anti-egfr antibodies.The plate of bag quilt is washed 3 times in the PBS+0.05% polysorbas20, sealed 2 hours and in the PBS+0.05% polysorbas20, washed again 3 times in room temperature with PBS/1%BSA.Conditioned medium is at first with dilution in 1: 1000 and further with 1: 2 dilution proportion.The conditioned medium (CM) of dilution is applied to plate to carry out the ELISA detection according to manufacturer specification.

C. part is in conjunction with sieve method

[0689] the EGF combined techniques of EU-mark: plate is spent the night at the room temperature bag with the anti-Fc antibody of 5 μ g/ml.Behind the bag quilt, plate is washed 3 times in the PBS/0.05% polysorbas20 and sealed 2 hours in room temperature with PBS/1%BSA.After sealing, plate is washed 3 times with the PBS/0.05% polysorbas20.Recombinant protein in the conditioned medium (20ng) is with the dilution of 1 * DELFIA binding buffer liquid and be added into plate (100 μ l/ hole).Plate was incubated at room 2 hours.Ice-cold DELFIA lavation buffer solution with 120 μ l/ holes washs 3 times subsequently.Subsequently, the EU-EGF (0.5nM) in the DELFIA binding buffer liquid is added into each hole (100 μ l/ hole) and with plate incubated at room 2 hours.Plate washs 3 times (120 μ l/ hole) with ice-cold DELFIA lavation buffer solution.

[0690] DELFIA enhancing solution (110 μ l/ hole) is added into each hole, and plate was hatched 20 minutes in room temperature again.After hatching, the fluorescence that plate is differentiated with detection time by Envision (PerkinElmer) reading.

D.TGF_ and HB-EGF keying action

[0691] TGF_ELISA test kit or HB-EGF ELISA test kit (R﹠amp; D System) improvement is used for the part binding assay.Plate with the anti-Fc antibody of 1 μ g/ml (Sigma) the room temperature bag spent the night and with PBS/1%BSA room temperature sealing 2 hours.Through the plate of sealing and 20ng HFD100 mutant protein incubated at room 2 hours.Wash plate and make its room temperature respectively with the binding buffer liquid (PBS/1%BSA) in 100 μ l/ holes in 5-50nM TGF_ or 5nM HB-EGF hatched again 2 hours.Wash plate also makes it further room temperature and the biotinylated goat of 300ng/ml anti-people TGF_ antibody or the anti-people HB-EGF of biotinylated goat antibody incubation 2 hours.Subsequently streptavidin-HRP (1: 200 dilution) is added into described plate and at 20 minutes after-applied substrate solutions with colour developing.Plate by micro plate readout instrument reading to determine value at OD 650nm place.

E. result

[0692] detailed part in conjunction with research disclose HFD 120 with than the higher avidity of wild-type (HFD100) in conjunction with the HER1 part.With wild-type (HFD100) relatively, the HFD120 mutant has 2 times more high-affinity to EGF, and HB-EGF is had the avidity of 7 times of improvement and TGF-α is had avidity (Figure 22 a-c and table 32) greater than 30 times of improvement.

Table 32: binding affinity

[0693] is called the mutant of T43K/S193N/E330D/G588S, except that designed T43K sudden change, also has the sudden change that imports by random PCR.This quadruple mutant (quad mutant) has the HER1 ligand-binding activity (Figure 23) of obvious increase.This mutant is carried out systematicness to be changed to produce two kinds of other HER1 mutant, be called S193N/E330D/G588S and E330D/G588S, with wild-type (HFD100) relatively, they are all with the horizontal integration HER1 part of obvious increase: EGF, HB-EGF and TGF-α; Yet S193N/E330D/G588S produces higher protein secreting level (Figure 23) than E330D/G588S.

The panHER part trap that embodiment 24 designs have higher ligand binding affinity and ability: the HER with ligand binding capacity of increase transfers albumen

[0694] except that the HFD120 that the HER1 part is had high-affinity, (discusses among the embodiment 18 as mentioned), also be created in the heterodimer HER1/Fc:HER3/Fc construct that has the T39S sudden change in the HER1 arm, be called RB220h.This T39S sudden change is identical with the T39S sudden change among the HFD120.HFD120 expresses as the HFD100 in embodiment 2 and 3 and purifying.This HER transfers albumen also to be expressed as the mixture that comprises homodimer and heterodimer, is called RB620, and this mixture of cell expression system produces as HFD120, HFD300 and RB220h.See Table 33.RB620 expresses and as described in example 2 above as carrying out purifying among the embodiment 3 as described in RB600.

Table 33:HER transfers protein composition

The molecule title	Element
		HFD100	The Her1/Fc homodimer
HFD120	The Her1/Fc homodimer of band HER1T39S sudden change
		HFD300 (being also referred to as HFD300h)	The Her3/Fc homodimer
RB200h	The Her1/Fc-Her3/Fc heterodimer of purifying
		RB220	RB200h with enhanced Her1 component (Her1 of band T39S sudden change)
RB600 (RB-mixture)	Her1/Fc homodimer, Her3/Fc homodimer, Her1-Her3 heterodimer
		RB620	RB600 with enhanced Her1 component (Her1 of band T39S sudden change)
RB630	RB600 with enhanced Her1 component and enhanced Her3 component

[0695] HER1 ligand-binding activity or the HER3 ligand-binding activity (ability) with mutant compares with the wild-type construct.Relatively homodimer HFD100 and HFD120 (sudden change construct) or relatively RB200h and RB220h (sudden change construct), the mutant that contains the T39S sudden change in HER1 has high about 2.5 times EGF binding ability (table 34,35,38,39) than its wild type counterparts.In addition, the data presentation mixture as RB600 or as RB620, has HER1 ligands, EGF binding ability (table 35,38 and 39) better, 3 times to 10 times higher.

[0696] with regard to wild-type and mutant HER transfer proteic HER3 part (NRG1 β 1) binding ability, difference is not as viewed obvious like that to the EGF keying action.At first, heterodimer RB200h has about 1.6 times higher NRG1 β 1 binding ability than mixture RB600.NRG1 β 1 binding ability of the mixture RB620 of the heterodimer (RB220h) of sudden change or sudden change is roughly the same (seeing Table 38 and 39).Yet, find that enjoyably HER3 homodimer HFD300 only has 30% combination activity (seeing Table 36 and 37) of described heterodimer when NRG1 β 1 binding ability of heterodimer (wild-type RB200h or mutant RB220h) and HER3 homodimer (HFD300) when comparing.

Table 34: relative EGF is in conjunction with activity

Protein	Relative keying action with EGF	HFD％
			HFD100-63	1	＞99％
HFD120-1	1.8	＞99％
			RB200H-X.C	1	＜0.5％
RB220h-1	2.47	＜0.5％

Table 35: relative EGF is in conjunction with activity

Protein	Relative keying action with EGF	HFD1xx％	RB2xxh％
				RB600-1	1	64％	27％
RB200h-X.C	0.11	＜0.5％
				RB602-1	1	37％	46％
RB220h-1	0.29	＜0.5％

Table 36: relative NRG1 β 1 is in conjunction with active

Protein	Relative keying action with NRG-β 1	HFD2xx％	HFD300％
				RB600-1	1	27％	9％
RB200h-X.C	1.57	95％	5％
				RB602-1	1.68	46％	17％
RB220h-1	1.76	＞98％	＜2％

Table 37: relative NRG1 β 1 is in conjunction with active

Protein	Specific combination effect with NRG-β 1	Purity %	RB2xxh％
				HFD100-63	0.016	＞98％
HFD120-1	0.032	＞98％
				RB300-1	0.676	75％	25％

Table 38: part bonded activity specific: RB200h and RB600

	fmolEGF/fmolRB200	SD	fmolNRG/fmolRB200	SD	The ratio of EGF: NRG
						RB200h-65/67/70/72	0.153	0.008	0.302	0.013	0.508
RB220h-1	0.051	0.003	0.407	0.025	0.125
						RB620-1	0.174	0.008	0.387	0.031	0.449
RB200h-XC	0.031	0.001	0.363	0.006	0.086
						RB600-1	0.283	0.024	0.231	0.009	1.223

Table 39: part bonded activity specific: RB200h and RB600

Protein	fmolEGF/mgRB	fmolNRG/mgRB
			RB200h	0.16x10 6	1.91x10 6
RB600	1.50x10 6	1.22x10 6
			RB220h	0.27x10 6	2.14x10 6
RB620	0.91x10 6	2.04x10 6

Claims (158)

1. polymer, it comprises:

A) first chimeric polyeptides, described first chimeric polyeptides is selected from:

I) the total length ectodomain (ECD) that chimeric polyeptides, described chimeric polyeptides contain directly or be connected with the multimerization structural domain indirectly through joint from the HER1 acceptor, or

Ii) chimeric polyeptides, the HER1 that described chimeric polyeptides contains directly or is connected with the multimerization structural domain indirectly through joint, HER2, the ECD less than total length of HER3 or HER4 acceptor, wherein said ECD contains enough parts of subdomain I and/or III at least to combine with the part of described acceptor, and ECD and the enough parts cell surface receptor dimerization, described ECD and the ECD of the enough parts cell surface receptor dimerization in this chimeric polyeptides are not the enough parts that comprise subdomain II during from the HER2 acceptor, ECD in this chimeric polyeptides is during from the HER2 acceptor, then ECD also contains all or part of of structural domain IV, module 2-5 enough part or all of that comprises subdomain IV is to realize the dimerization with cell surface receptor; With

B) directly or second chimeric polyeptides that is connected with the multimerization structural domain indirectly through joint, and contain at least cell surface protein ECD enough parts with combine with the part of this cell surface protein and/or with the cell surface receptor dimerization, wherein the multimerization structural domain in first and second chimeric polyeptides is a complementary or identical, condition is that then second chimeric polyeptides does not contain the ECD from HER2 if first chimeric polyeptides is total length HER1 ECD; If perhaps second chimeric polyeptides contains the ECD from HER2, then described HER2 ECD is enough parts of comprising structural domain IV less than total length and enough parts that be used for receptor dimerizationization realizing dimerization, thereby:

Described chimeric polyeptides forms polymer; And

The polymer of gained be bonded to the extra ligand of comparing with the homodimer of first chimeric polyeptides or first chimeric polyeptides and/or with the cell surface receptor dimerization many than the homodimer of first chimeric polyeptides or first chimeric polyeptides.

2. the described polymer of claim 1, wherein one of first and second chimeric polyeptides person or the ECD of these two are heterozygosis ECD, described heterozygosis ECD contains the subdomain from least 2 kinds of different cell surface receptor ECD.

3. the described polymer of claim 1, wherein first chimeric polyeptides contains the ECD less than total length of HER2, HER3 or HER4.

4. the described polymer of claim 1, wherein first chimeric polyeptides contains the ECD less than total length of HER3 or HER4.

5. the described polymer of claim 1, it is a heteromultimeric, wherein the ECD of second chimeric polyeptides partly is from the cell surface receptor that is different from HER1.

6. the described polymer of claim 5, wherein the ECD in second chimeric polyeptides is from HER3 or HER4.

7. any described polymer among the claim 1-6, wherein the ECD structural domain of second chimeric polyeptides contains total length ECD.

8. any described polymer among the claim 1-6, wherein the ECD structural domain of second chimeric polyeptides enough parts of containing subdomain I, II and III at least with combine with its part and with the cell surface receptor dimerization.

9. any described polymer among the claim 1-6, wherein second chimeric polyeptides contains the ECD less than total length, and the enough parts that comprise structural domain I and III are to combine with its part.

10. any described polymer among the claim 1-8, wherein second chimeric polyeptides contains the ECD less than total length, and the enough parts that comprise this ECD with the cell surface receptor dimerization.

11. any described polymer among the claim 1-10, wherein the multimerization structural domain be selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection and compensatory hole.

Any described polymer among the 12 claim 1-10, wherein the multimerization structural domain is Fc structural domain or its variant of realizing multimerization.

13. the described polymer of claim 12, wherein said Fc structural domain is from IgG, IgM or IgE.

14. any described polymer among the claim 1-13, wherein cell surface receptor is the related acceptor at the subdomain of described polymeric ECD or this ECD.

15. any described polymer among claim 1-13 and the 117-126, wherein the ECD of second chimeric polyeptides is selected from HER2, HER3, HER4, IGF1-R, VEGFR, FGFR, TNFR, PDGFR, MET, Tie, RAGE, EPH acceptor and TXi Baoshouti.

16. the described polymer of claim 15, wherein the ECD of second chimeric polyeptides is selected from VEGFR1, FGFR2, FGFR4, IGF1-R and Tie1.

17. any described polymer among the claim 2-16, wherein the ECD of second chimeric polyeptides is the intron fusion proteins that is connected with the multimerization structural domain.

18. any described polymer among the claim 2-16, wherein said the 2nd ECD are to be used for the receptor dimerizationization of cell surface receptor and/or to be used for part bonded total length HER2, HER3 or HER4 or its enough part with cell surface receptor.

19. any described polymer among the claim 2-17, wherein said the 2nd ECD is from the receptor tyrosine kinase except that HER1.

20. claim 2-19,105,106 and 117-126 in any described polymer, it combines with at least three, four, five, six or seven kind of different ligands.

21. the described polymer of claim 20, wherein said part be selected from EGF, TGF-α, amphiregulin, HB-EGF, β-tunicin, epiregulin and with other part of ECD bonded of cell surface receptor except that HER1.

22. the described polymer of claim 21, wherein said other part is selected from neuregulin-1, neuregulin-2, neuregulin-3 and neuregulin-4.

23. the described polymer of claim 1, wherein:

First chimeric polyeptides contains to be enough to combine with part and/or the i of dimerization) from the total length ECD of HER1 or ii) its part; And

Second chimeric polyeptides contains to be enough to combine with part and/or the ECD of the HER3 of dimerization or HER4 all or part of.

24. claim 1-23,105,106 and 117-126 in any described polymer, wherein the multimerization structural domain in each chimeric polyeptides is selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond, go into hole projection and compensatory hole with polymeric same or similar size is stablized in formation, thereby described chimeric polyeptides is with formula configuration interaction back-to-back, and the ECD of these two chimeric polyeptides can be used in and the cell surface receptor dimerization thus.

25. the described polymer of claim 23 or claim 24, wherein said multimerization structural domain is the Fc structural domain.

26. the polymer of claim described 25, wherein said Fc structural domain is from IgG, IgM or IgE.

27. claim 1,7-14,106 and 117-122 in any described polymer, it comprises at least two kinds of chimeric polyeptides, wherein:

First chimeric polyeptides contains ECD all or part of of HER1; With

Second chimeric polyeptides contains ECD all or part of of HER3 or HER4.

28. claim 1-27,106 and 117-126 in any described polymer, wherein the component chimeric polyeptides is a fusion polypeptide.

29. claim 1-27,106 and 117-126 in any described polymer, wherein chimeric polyeptides a) and b) be fusion polypeptide.

30. claim 1-27,106 and 117-126 in any described polymer, wherein the component chimeric polyeptides forms by chemically conjugated effect.

31. claim 1-27,106 and 117-126 in any described polymer, wherein chimeric polyeptides a) and b) form by chemically conjugated effect.

32. claim 1-31,106 and 117-126 in any described polymer, wherein the multimerization structural domain of at least a chimeric polyeptides directly is connected with ECD.

33. claim 1-31,106 and 117-126 in any described polymer, wherein the multimerization structural domain of at least a chimeric polyeptides is connected with ECD through joint.

34. the described polymer of claim 32, wherein all the multimerization structural domain of component chimeric polyeptides peptide directly divides other ECD to be connected with each.

35. the described polymer of claim 33, wherein all the multimerization structural domain of component chimeric polyeptides peptide divides other ECD to be connected through joint and each.

36. the described polymer of claim 33 or claim 35, wherein said joint are chemical joint or peptide linker.

37. claim 1-36,105,106 and 117-126 in any described polymer, it is a heterodimer.

38. claim 1-37,105,106 and 117-126 in any described polymer, it is to contain to be in the heterodimer of the component chimeric polyeptides of formula configuration back-to-back, thereby the ECD in each chimeric polyeptides can be used in the dimerization with cell surface receptor.

39. heteromultimeric, it comprises:

Ectodomain (ECD) from a kind of HER acceptor; With

From the ECD of second acceptor, wherein:

One of at least be HER ECD among the described ECD and contain subdomain I, II and the part of III and subdomain IV, but do not contain the whole of subdomain IV;

Subdomain IV comprises module 1 at least; And

Described ECD is different.

40. any described heteromultimeric among claim 39 and the 127-134, wherein the 2nd ECD is from cell surface receptor.

41. any described heteromultimeric among claim 39 and the 127-134, wherein a kind of HER are HER1 and another kind of HER is HER3 or HER4.

42. claim 39-41,104 and 127-134 in any described heteromultimeric, the dimerization structural domain of at least one ECD in the wherein said heteromultimeric can be used in the dimerization with cell surface receptor.

43. any described heteromultimeric among claim 39-42 and the 127-134, wherein each ECD directly or through joint is connected with the multimerization structural domain, thereby the multimerization domain interaction of at least two ECD is to form heteromultimeric.

44. the described heteromultimeric of claim 43, wherein said multimerization structural domain are selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection and compensatory hole.

45. the described heteromultimeric of claim 43 or claim 44, wherein the multimerization structural domain is the Fc structural domain.

46. the described polymer of claim 45, wherein the Fc structural domain is from IgG, IgM or IgE.

47. any described heteromultimeric among claim 40 and the 42-46, wherein cell surface receptor is the related acceptor of ECD of described heteromultimeric or the related acceptor of this ECD subdomain.

48. any described heteromultimeric among the claim 38-47, wherein the 2nd ECD is from the acceptor that is selected from HER2, HER3, HER4, IGF1-R, VEGFR, FGFR, TNFR, PDGFR, MET, Tie, RAGE, EPH acceptor and TXi Baoshouti.

49. the described heteromultimeric of claim 48, wherein ECD is selected from VEGFR1, FGFR2, FGFR4, IGFR1 and Tie1.

50. heterozygosis ectodomain (ECD), it comprises:

The I of structural domain at least, the II of the ECD of one or more cell surface receptors and III's is all or part of, wherein:

At least two structural domains are the ECD from different cell surface receptors in the described structural domain;

This heterozygosis ECD contains enough parts from the structural domain I of one or more ECD of cell surface receptor or III with binding partner, and contain enough parts of the ECD of cell surface receptor, the enough parts that comprise domain II are with when described heterozygosis ECD is connected to the multimerization structural domain and the cell surface receptor dimerization.

51. the described heterozygosis ECD of claim 50, wherein said cell surface receptor are the members of HER family.

52. the described heterozygosis ECD of claim 50, wherein structural domain I is from HER1, and domain II is from HER2, and domain II I is from HER3.

53. chimeric polyeptides comprises directly or any described heterozygosis ECD in claim 50-52 that joint is connected with the multimerization structural domain indirectly and 135-142.

54. the described chimeric polyeptides of claim 53, wherein the multimerization structural domain be selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection and compensatory hole.

55. the described chimeric polyeptides of claim 53 or claim 54, wherein said multimerization structural domain is the Fc structural domain.

56. the described chimeric polyeptides of claim 55, wherein said Fc structural domain is from IgG, IgM or IgE.

57. polymer, it comprises any described at least two chimeric polyeptides among claim 50-56 and the 147-142.

58. heteromultimeric, it comprises:

All or part of from the ectodomain (ECD) of HER1 acceptor; With

All or part of from the ECD of HER3 or HER4 acceptor, wherein:

Described part comprises subdomain I, II and III at least.

59. nucleic acid molecule, the sequence that comprises nucleic acid, any described chimeric polyeptides or contain the heteromultimeric of this kind chimeric polyeptides among at least a chimeric polyeptides among wherein said nucleic acid encoding claim 1-49,50-53,58,96,102-106 and the 143-150 in any described heteromultimeric, claim 95,97,98,99,101, the 127-134,151 and 152, or any described heterozygosis ECD among coding claim 50-52 and the 137-142.

60. carrier, it comprises the described nucleic acid of claim 59.

61. isolated cells, it comprises described nucleic acid molecule of claim 59 or the described carrier of claim 60.

62. pharmaceutical composition, its comprise claim 1-58,95-99,101,102,104,105,106 and 117-152 in any described polymer, heteromultimeric chimeric polyeptides or polypeptide or described nucleic acid molecule of claim 59 or the described cell of claim 61.

63. the described pharmaceutical composition of claim 59, its preparation is used for being used for single dose.

64. pharmaceutical composition, use, topical application or systemic administration by limitation in order to be used for for its preparation.

65. the method for treatment cancer, inflammatory diseases, vasculogenesis disease or excessively proliferative disease comprises any described pharmaceutical composition among the claim 62-64 of administering therapeutic significant quantity.

66. the described method of claim 65, wherein said cancer are pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer, kidney or mammary cancer.

67. the described method of claim 65, wherein said disease is a proliferative disease.

68. the described method of claim 67, wherein said proliferative disease comprise the propagation of smooth muscle cell and/or divide a word with a hyphen at the end of a line, or preceding ophthalmic, or diabetic retinopathy or psoriatic.

69. the described method of claim 65, wherein said disease be restenosis, illness in eye, narrow, atherosclerosis, because of hypertension, the urinary bladder disease and the obstructive respiratory disease of blood vessel due to thickening.

70. be used for the treatment of method for cancer, comprise:

Use any described pharmaceutical composition and another kind of carcinostatic agent among the claim 62-64.

71. the described method of claim 70, wherein said carcinostatic agent are radiotherapy and/or chemotherapeutic.

72. the described method of claim 70, wherein said carcinostatic agent are tyrosine kinase inhibitor or antibody.

73. the described method of claim 72, wherein said carcinostatic agent are quinazoline kinase inhibitor, antisense or siRNA or other double stranded rna molecule, or the antibody of puting together with the antibody of HER acceptor interaction, with radionuclide or cytotoxin.

74. the described method of claim 73, wherein said carcinostatic agent are Gefitinib, lapatinibditosylate, handkerchief Buddhist nun monoclonal antibody, erlotinib, Cetuximab, Herceptin, imatinib, platinum complex or nucleoside analog.

75. the method for the receptor-mediated disease of treatment HER comprises:

Test suffers from the experimenter of described disease, to identify which HER acceptor is expressed or overexpression; With

Based on described result, select the polymer of at least two HER acceptors of target.

76. the described method of claim 75, wherein said disease is a cancer.

77. the described method of claim 76, wherein said cancer are pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer or mammary cancer.

78. polypeptide, it is selected from

CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPF(SEQIDNO.405)；

With

With SEQ ID NO.54-61, be to be used for part bonded target polypeptide.

79. be used to identify method with the candidate molecules of HER acceptor interaction;

A) make the set of test molecule or its with based on participate among domain II and IV or I and the III dimerization, part in conjunction with and/or constraint on any effect the zone extremely about 50 amino acid or 50 amino acid whose polypeptide contact at least about 6 amino acid or 6 amino acid; With

B) the interactional any test molecule of evaluation and selection and one or more described polypeptide.

80. the described method of claim 79, wherein said polypeptide comprises such library, and described library is based on the polypeptides in combination library of HER acceptor.

81. comprising, the described method of claim 79, wherein said polypeptide be selected from one or more following polypeptide:

CSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPE ADQCVACAHY KDPPF (SEQ ID NO.405) and comprise 6,8,10,12,14,15,18,20,25,30,35,40,45 or 50 or the part of more a plurality of amino-acid residues of SEQ IDNO.405;

At the polypeptide shown in the SEQ ID NO.54-61; 4, with the arbitrary polypeptide portion that comprises 5,6,8,10,12 or more a plurality of amino-acid residues.

82. the described method of claim 79, wherein said test molecule comprises the library of molecule.

83. the described method of claim 82, wherein said molecule are small molecules organic compound or polypeptide.

84. the described method of claim 80, wherein said library comprise and are illustrated on the solid support or at the polypeptide of virus surface.

85. the described method of claim 84, wherein said polypeptide comprises phage display library.

86. any described method among the claim 79-85 is wherein selected and structural domain I and/or III, perhaps tests molecule with domain II or with structural domain IV bonded.

87. the described method of claim 86 also comprises: form the heterodimer comprise two or more polypeptide, wherein a peptide species combines with domain II and another kind of polypeptide combines with structural domain IV.

88. the described method of claim 79, wherein said test molecule comprises phage display library.

89. isolated antibody, it interacts with the described polypeptide of claim 78 specifically.

90. isolating polyclonal antibody, it interacts with described two or more polypeptide of claim 78 specifically.

91. the described separation antibody of claim 89 or claim 90, it is to comprise two kinds of not synthetic property antibody of homopolypeptide, and wherein said every peptide species is connected with the multimerization structural domain.

92. the described antibody of claim 91, wherein said multimerization structural domain are to be selected from the polypeptide of going into hole projection and compensatory hole that polymeric same or similar size is stablized in constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, the free sulfhydryl groups that forms intermolecular disulfide bond between two molecules and formation.

93. the described antibody of claim 91, wherein said multimerization structural domain is the Fc structural domain.

94. the described antibody of claim 93, wherein said Fc structural domain is from IgG, IgM or IgE.

95. chimeric polyeptides, it comprises:

The ECD or its part that enough are used for part combination and/or receptor dimerizationization; With

The multimerization structural domain,

Wherein said ECD or its part are selected from HER2-530 (SEQ ID No.14), HER2-595 (SEQ ID No.16), HER2-650 (SEQ ID No.18), HER3-500 (SEQ IDNo.20), P85HER3 (SEQ ID No.22), HER3-519 (SEQ ID No.24), HER3-621 (SEQ ID No.26), HER4-485 (SEQ ID No.28), HER4-522 (SEQ IDNo.30), HER4-650 (SEQ ID No.32), HER1ECE shown in the 25-645 amino acids of SEQ ID No.414, at SEQ ID NO.32,34,127,141,146,148,159 and each of 54-125 shown in polypeptide, and the allelic variant of arbitrary aforementioned ECD and species variant.

96. heteromultimeric comprises two or more chimeric polyeptides, wherein:

ECD is selected from allelic variant or the species variant at the HER1-621 shown in HER1-501 shown in the SEQ ID No.10 and the SEQ ID No.12 or its part, the described chimeric polyeptides of claim 95 and arbitrary aforementioned polypeptide that enough is used for part combination and/or receptor dimerizationization; And

Each chimeric polyeptides all directly or through joint is connected with the multimerization structural domain indirectly.

97. described chimeric polyeptides of claim 95 or the described heteromultimeric of claim 96, wherein said multimerization structural domain is to be selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond, stablize the polypeptide of going into hole projection and compensatory hole of polymeric same or similar size with formation, described thus chimeric polyeptides interacts with formula configuration back-to-back, thereby the ECD of two kinds of chimeric polyeptides can be used for the dimerization with cell surface receptor.

98. described chimeric polyeptides of claim 97 or heteromultimeric, wherein said multimerization structural domain is the Fc structural domain.

99. described chimeric polyeptides of claim 98 or heteromultimeric, wherein said Fc structural domain is from IgG, IgM or IgE.

100. isolated polypeptide comprises as the amino-acid residue shown among the SEQ ID NO.127,141,146,148,153,155,157,159,297 and 299 each.

101. chimeric polyeptides comprises described multimerization structural domain of described polypeptide of claim 100 and claim 151 or claim 152 or polypeptide.

102. heteromultimeric, it comprises the described chimeric polyeptides of claim 101.

103. the described heteromultimeric of claim 102, it comprises second polypeptide, and described second polypeptide is HER ECD or its part that enough is used for part combination and/or receptor dimerizationization.

104. the described heteromultimeric of claim 39, wherein said two kinds of ECD all are HERECD.

105. being Hess, the described polymer of claim 17, wherein said intron fusion proteins reach spit of fland or its variant.

106. any described polymer among the claim 1-25, it comprises at least two chimeric polyeptides.

107. chimeric polyeptides comprises ECD or its part of the HER1 acceptor that is connected with the multimerization structural domain, wherein:

Described ECD or its part comprise modification, thereby compare with ECD or its part of unmodified, and this ECD combines with extra part.

108. chimeric polyeptides, comprise 25-645 amino acids all or part of of the SEQ ID No.114 that is connected with the multimerization structural domain or have sequence with it, but described sequence is in the sudden change that comprises Ser to Phe corresponding to SEQ ID No.114 the 442nd bit position place at least about 70,80,90,95% sequence identity.

109. claim 107 or 108 described chimeric polyeptides, wherein said multimerization structural domain are selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection and compensatory hole.

110. profit requires any described chimeric polyeptides power among the 107-109, wherein said multimerization structural domain is Fc structural domain or its variant of realizing multimerization.

111. the described chimeric polyeptides of claim 110, wherein said Fc structural domain is from IgG, IgM or IgE.

112. any described chimeric polyeptides among the claim 107-111, wherein said ECD is from the HER1 acceptor.

113. any described chimeric polyeptides among the claim 107-112, wherein said modification is corresponding in the S442 position of HER acceptor or the modification of corresponding position.

114. the described chimeric polyeptides of claim 113, wherein said modification is arranged in the ECD of HER1 acceptor, thereby this HER1ECD and NRG-2 β interact.

115. the described polymer of claim 114, wherein said modification are or corresponding to the S442F among the Seq.IDNo.2.

116. any described chimeric polyeptides among the claim 107-115, its enough part of ECD that comprise modified HER1 is to interact with EGF and NRG-2 β.

117. any described polymer among the claim 1-14, wherein:

Described ECD is modified ECD;

Compare with the ECD or the total length acceptor of unmodified, described modification changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD.

118. any described polymer among the claim 1-14, wherein:

Described ECD is not intended to change part keying action or other active modification.

119. the described polymer of claim 15, wherein said modification changes the part keying action.

120. the described polymer of claim 119, wherein said modification is corresponding to the S442 position of HER acceptor or the modification of corresponding position.

121. the described polymer of claim 120, wherein said modification is arranged in the ECD of HER1 acceptor, thereby HER1ECD and NRG-2 β interact.

122. the described polymer of claim 121, wherein said modification are or corresponding to the S442F among the Seq.IDNo.2.

123. any described polymer among the claim 117-122, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the ligand interaction of the polymer of gained and at least two kinds of HER acceptors.

124. any described polymer among the claim 117-122, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the ligand interaction of the polymer of gained and at least three kinds of HER acceptors.

125. any described polymer among the claim 117-124, it is a dimer.

126. any described polymer among the claim 117-125, it comprises Fc multimerization structural domain.

127. the described heteromultimeric of claim 39, wherein structural domain or its part from ECD contains change part keying action or specific sudden change in described structural domain; Compare with the ECD or the total length acceptor of unmodified, described sudden change changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD, thus the part keying action or the specificity of this heteromultimeric display change.

128. the described heteromultimeric of claim 127, wherein said modification changes the part keying action.

129. the described heteromultimeric of claim 128, wherein said modification is corresponding to the S442 position of HER acceptor or the modification of corresponding position.

130. the described heteromultimeric of claim 129, wherein said modification is arranged in the ECD of HER1 acceptor, thereby this HER1ECD and NRG-2 β interact.

131. the described heteromultimeric of claim 130, wherein said modification are or corresponding to S442F.

132. any described heteromultimeric among the claim 127-131, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the ECD of gained can with the ligand interaction of at least two kinds of HER acceptors.

133. any described heteromultimeric among the claim 127-132, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the heterozygote of gained can with the ligand interaction of at least three kinds of HER acceptors.

134. any described heteromultimeric among the claim 127-133, it comprises Fc multimerization structural domain.

135. the described heterozygosis ECD of claim 50 comprises structural domain or its part from ECD, wherein said ECD contains change part keying action or specific sudden change in described structural domain;

Compare with the ECD or the total length acceptor of unmodified, described sudden change changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD, wherein the part keying action or the specificity of this heterozygosis ECD display change.

136. the described heterozygosis ECD of claim 135, wherein said modification changes the part keying action.

137. the described heterozygosis ECD of claim 136, wherein said modification is corresponding to the S442 position of HER acceptor or the modification of corresponding position.

138. the described heterozygosis ECD of claim 137, wherein said modification is arranged in the ECD of HER1 acceptor, thereby this HER1ECD and NRG-2 β interact.

139. the described heterozygosis ECD of claim 138, wherein said modification is or corresponding to S442F.

140. any described heterozygosis ECD among the claim 135-139, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the ECD of gained can with the ligand interaction of at least two kinds of HER acceptors.

141. any described heterozygosis ECD among the claim 135-139, it comprises from HER1 with from ECD or its part of HER3 or HER4, thus the heterozygote of gained can with the ligand interaction of at least three kinds of HER acceptors.

142. any described heterozygosis ECD among the claim 135-141, it comprises Fc multimerization structural domain.

143. the described heteromultimeric of claim 58, wherein structural domain or its part from ECD contains change part keying action or specific sudden change in this structural domain;

Compare with the ECD or the total length acceptor of unmodified, described sudden change changes this ECD or contains part keying action or other activity of the total length acceptor of this ECD, thus the part keying action or the specificity of this heteromultimeric display change.

144. the described heteromultimeric of claim 143, wherein said modification changes the part keying action.

145. the described heteromultimeric of claim 144, wherein said modification is corresponding to the S442 position of HER acceptor or the modification of corresponding position.

146. the described heteromultimeric of claim 145, wherein said modification is arranged in the ECD of HER1 acceptor, thereby this HER1ECD and NRG-2 β interact.

147. the described heteromultimeric of claim 146, wherein said modification are or corresponding to S442F.

148. any described heteromultimeric among the claim 143-147 comprises from HER1 with from ECD or its part of HER3 or HER4, thus the ECD of gained can with the ligand interaction of at least two kinds of HER acceptors.

149. any described heteromultimeric among the claim 143-148 comprises from HER1 with from ECD or its part of HER3 or HER4, thus the heterozygote of gained can with the ligand interaction of at least three kinds of HER acceptors.

150. any described heteromultimeric among the claim 143-149, it comprises Fc multimerization structural domain.

151. chimeric polyeptides, it comprises the multimerization structural domain, this multimerization structural domain directly or through joint is connected with polypeptide or its part shown in the 25-645 amino acids of SEQ ID No.414 indirectly, and wherein said polypeptide or its part are enough to realize the part keying action with at least 2 kinds of different ligands.

152. the described polypeptide of claim 151, wherein said multimerization structural domain is selected from constant region for immunoglobulin (Fc), leucine zipper, complementary hydrophobic region, complementary hydrophilic area, compatible protein matter-protein interaction structural domain, between two molecules, form the free sulfhydryl groups of intermolecular disulfide bond and form stablize polymeric same or similar size go into hole projection and compensatory hole, thereby described chimeric polyeptides interacts with formula configuration back-to-back, thereby the ECD of two chimeric polyeptides all can be used in the dimerization with cell surface receptor.

153. composition, comprise heteromultimeric and with polymeric mixture, wherein said heteromultimeric comprises from a kind of ECD of HER1 or its part with from another kind of ECD or its part of HER3, and wherein saidly comprise ECD or its part, perhaps from ECD or its part of HER3 from HER1 with polymer.

154. pharmaceutical composition, it comprises preparation is to be used for the described composition of claim 153 that part, oral, whole body or limitation are used.

155. be used for the treatment of the method for cancer, inflammatory diseases, vasculogenesis disease or excessively proliferative disease, comprise the claim 153 or the 154 described compositions of administering therapeutic significant quantity.

156. the described method of claim 155, wherein said cancer are pancreas cancer, cancer of the stomach, head and neck cancer, cervical cancer, lung cancer, colorectal carcinoma, carcinoma of endometrium, prostate cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, neurospongioma, bladder cancer, kidney or mammary cancer.

157. the described method of claim 155, wherein said disease is a proliferative disease.

158. the described method of claim 157, wherein said proliferative disease comprise the propagation of smooth muscle cell and/or divide a word with a hyphen at the end of a line, or preceding ophthalmic, or diabetic retinopathy or psoriatic.

159. the described method of claim 155, wherein said disease be restenosis, illness in eye, narrow, atherosclerosis, because of hypertension, the urinary bladder disease and the obstructive respiratory disease of blood vessel due to thickening.

Images