CN101616934A - Suppress DR6 antibody and the purposes in treatment neuroscience illness thereof of DR6 in conjunction with APP - Google Patents

Abstract

The method and composition that comprises the DR6 antagonist that is used for the treatment of neuroscience illness (comprising Alzheimer's) is provided.The DR6 antagonist comprises anti-APP antibody, anti-DR6 antibody, DR6 immunoadhesin and the DR6 variant (and fusion rotein) of the growth, regeneration or the survival that strengthen mammalian nervous unit cell or tissue.

Description

Suppress DR6 antibody and the purposes in treatment neuroscience illness thereof of DR6 in conjunction with APP

Cross reference to related application

The application is the non-provisional application of submitting to according to 37CFR 1.53 (b) (1), require the provisional application submitted on December 22nd, 2006 number 60/871 according to 35USC 119 (e), the provisional application of submitting on February 12nd, 528 and 2007 number 60/900,848 right of priority is by addressing its content income this paper.

Invention field

The present invention relates generally to use the method for DR6 antagonist for treating neuroscience illness and in these class methods useful DR6 antagonist composition, described DR6 antagonist for example suppresses the combination between the related part APP with it of DR6.In optional embodiments, use DR6 antagonist (such as DR6 receptor antibody, DR6 acceptor variant, DR6 receptor immunoadhesins or APP antibody) to treat neuroscience illness (comprising the treatment of Alzheimer's).

Background of invention

Multiple part and the acceptor that belongs to tumour necrosis factor (TNF) superfamily identified in this area.Tumor necrosis factor-alpha (" TNF-α ") is arranged in these parts, tumour necrosis factor-β (" TNF-β " or " lymphotoxin-α "), lymphotoxin-β (" LT-β "), the CD30 part, the CD27 part, the CD40 part, the OX-40 part, the 4-1BB part, LIGHT, Apo-1 part (being also referred to as Fas part or CD95 part), Apo-2 part (being also referred to as Apo2L or TRAIL), Apo-3 part (being also referred to as TWEAK), APRIL, the OPG part (is also referred to as the RANK part, ODF or TRANCE) and TALL-1 (be also referred to as BlyS, BAFF or THANK) (referring to for example Ashkenazi, Nature Review, 2:420-430 (2002); Ashkenaziand Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit, Curr.Opin.CellBiol., 11:255-260 (2000); Golstein, Curr.Biol., 7:750-753 (1997); Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley etc., Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404 (1995); Schmid etc., Proc.Natl.Acad.Sci., 83:1881 (1986); Dealtry etc., Eur.J.Immunol., 17:689 (1987); Pitti etc., J.Biol.Chem., 271:12687-12690 (1996); Wiley etc., Immunity, 3:673-682 (1995); Browning etc., Cell, 72:847-856 (1993); Armitage etc., Nature, 357:80-82 (1992); WO 97/01633, is disclosed on January 16th, 1997; WO 97/25428, is disclosed on July 17th, 1997; Marsters etc., Curr.Biol., 8:525-528 (1998); Chicheportiche etc., Biol.Chem., 272:32401-32410 (1997); Hahne etc., J.Exp.Med., 188:1185-1190 (1998); WO 98/28426, is disclosed on July 2nd, 1998; WO 98/46751, is disclosed on October 22nd, 1998; WO 98/18921, is disclosed on May 7th, 1998; Moore etc., Science, 285:260-263 (1999); Shu etc., J.Leukocyte Biol., 65:680 (1999); Schneider etc., J.Exp.Med., 189:1747-1756 (1999); Mukhopadhyay etc., J.Biol.Chem., 274:15978-15981 (1999)).

Begin by them and specific cells receptors bind by bringing out normally of replying of the ligand-mediated various kinds of cell of these TNF families.In the TNF receptor superfamily member who has identified up to now, comprise TNFR1, TNFR2, p75-NGFR, TACI, GITR, CD27, OX-40, CD30, CD40, HVEM, Fas (being also referred to as Apo-1 or CD95), DR4 (being also referred to as TRAIL-R1), DR5 (being also referred to as Apo-2 or TRAIL-R2), DR6 (is also referred to as TR9, be also referred to as TNF receptor superfamily member 21 or TNFRSF21 in the literature), DcR1, DcR2, protect bone protein (OPG), RANK and Apo-3 (being also referred to as DR3 or TRAMP) are (referring to for example Ashkenazi, Nature Reviews, 2:420-430 (2002); Ashkenazi and Dixit, Science, 281:1305-1308 (1998); Ashkenazi and Dixit, Curr.Opin.Cell Biol., 11:255-260 (2000); Golstein, Curr.Biol., 7:750-753 (1997); Wallach, Cytokine Reference, Academic Press, 2000, pages 377-411; Locksley etc., Cell, 104:487-501 (2001); Gruss and Dower, Blood, 85:3378-3404 (1995); Hohman etc., J.Biol.Chem., 264:14927-14934 (1989); Brockhaus etc., Proc.Natl.Acad.Sci., 87:3127-3131 (1990); EP 417,563, are disclosed on March 20th, 1991; Loetscher etc., Cell, 61:351 (1990); Schall etc., Cell, 61:361 (1990); Smith etc., Science, 248:1019-1023 (1990); Lewis etc., Proc.Natl.Acad.Sci., 88:2830-2834 (1991); Goodwin etc., Mol.Cell.Biol., 11:3020-3026 (1991); Stamenkovic etc., EMBO J., 8:1403-1410 (1989); Mallett etc., EMBO J., 9:1063-1068 (1990); Anderson etc., Nature, 390:175-179 (1997); Chicheportiche etc., J.Biol.Chem., 272:32401-32410 (1997); Pan etc., Science, 276:111-113 (1997); Pan etc., Science, 277:815-818 (1997); Sheridan etc., Science, 277:818-821 (1997); Degli-Esposti etc., J.Exp.Med., 186:1165-1170 (1997); Marsters etc., Curr.Biol., 7:1003-1006 (1997); Tsuda etc., BBRC, 234:137-142 (1997); Nocentini etc., Proc.Natl.Acad.Sci., 94:6216-6221 (1997); VonBulow etc., Science, 278:138-141 (1997); Johnson etc., Cell, 47:545-554 (1986); Radeke etc., Nature, 325:593-597 (1987); Pan etc., FEBS Lett., 431:351-356 (1998)).

These TNF receptor family members of great majority share the typical structure of cell surface receptor, comprise extracellular region, stride film district and intracellular region, and the then natural discovery of other member is to lack the soluble protein of striding film and born of the same parents' intracellular domain.Born of the same parents' outside part of typical case TNFR comprises terminal initial a plurality of repetition aminoacid sequence patterns that are rich in halfcystine structure territory (CRD) from NH2-.

About the summary of TNF acceptor and ligand family generally referring to for example Wallach, CytokineReference, Academic Press, 2000, page or leaf 377-411; Locksley etc., Cell, 104:487-501 (2001); Ware, Cytokine﹠amp; Growth Factor Reviews, 14:181-184 (2003); Liu etc., Immunity, 15 (1): 23-34 (2001); And Bossen etc., J Biol Chem.281 (20): 13964-71 (2006).

Be called the DR6 acceptor and (be also referred to as " TR9 " in the literature; Be also referred to as TNF receptor superfamily member 21 or TNFRSF21 in the literature) the TNFR family member be described to I type transmembrane receptor, it has motif and a kytoplasm death domain structure (Pan etc. that four born of the same parents are rich in halfcystine outward, FEBSLett., 431:351-356 (1998); Also can be referring to United States Patent (USP) 6,358,508; 6,667,390; 6,919,078; 6,949,358).Having reported that DR6 crossing in some transfectional cell series expressed causes the two activation (Pan etc., FEBS Letters, 431:351-356 (1998)) of apoptosis and NF-kB and JNK.In DR6 deficient mice model, the T cell is being subjected to substantive damage aspect the JNK activation, and when attacking DR6 (/-) mouse with proteantigen, find their T cell hyperproliferation and show deep polarization (and the Th1 differentiation is not subjected to equal the influence) (Zhao etc. that lead to Th2 and reply, J.Exp.Med., 194:1441-1448 (2001)).Also reported external target destruction and caused enhanced t helper cell 2 (Th2) differentiation (Zhao etc. see above) DR6.DR6 antagonist or the antagonist various uses in the cell-mediated illness of regulation and control B is recorded in the US 2005/0069540 that announced on March 31st, 2005.

DR6 acceptor may in the airway inflammation in regulating OVA inductive mouse asthmatic model, play a role (Venkataraman etc., Immunol.Lett., 106:42-47 (2006)).

Use myelin oligodendrocyte glycoprotein (MOG (35-55)) inductive experimental autoimmune encephalomyelitis model, find to compare with wild-type (WT) littermate, DR6-/-mouse is to the outbreak of CNS disease and make progress that the two highly has resistance.So, DR6 may involve regulate experimental autoimmune encephalomyelitis inducing and make progress in leukocyte infiltration and function (Schmidt etc., J.Immunol., 175:2286-2292 (2005)).

Have biologic activity miscellaneous and characteristic though identified a plurality of tnf ligands and receptor family member, this type of part seldom and acceptor have report to involve the neuroscience correlation function.For example, the WO2004/071528 that announced on August 26th, 2004 has put down in writing and has suppressed CD95 (Fas) ligand/receptor mixture treat Spinal injury in mouse model.

Summary of the invention

In embodiments of the invention, provide isolating death receptor 6 (" DR6 ") antagonist.Some embodiment inhibition of antagonist disclosed herein or the interaction between its related part of blocking-up DR6 and one or more.In preferred embodiments, DR6 antagonist disclosed herein suppress or the blocking-up related part amyloid precursor protein of DR6 (" APP ") with it between interaction.The embodiment of DR6 antagonist can comprise antibody, such as DR6 or APP antibody.This type of DR6 antagonistic antibodies can be for example monoclonal antibody, chimeric antibody, humanized antibody or people's antibody.In certain embodiments of the invention, the DR6 antagonist can comprise anti-DR6 antibody, and it is in conjunction with DR6 ectodomain polypeptide or its fragment, and optional can be in conjunction with the DR6 polypeptide that comprises Figure 1A amino acid/11-349 or 42-349.Perhaps, the DR6 antagonist can comprise anti-APP antibody, and it is in conjunction with the APP polypeptide, and optional be can be in conjunction with the APP polypeptide that comprises Figure 1B (SEQ ID NO:6) amino acid 66-81.The DR6 antagonist also comprises DR6 immunoadhesin, DR6 variant, DR6 fragment, its covalent modification form or its fusion rotein, and the small molecules antagonist.For example, the DR6 antagonist can comprise PEGization DR6 or merge to the solubility ectodomain form DR6 of heterologous sequence (such as epi-position label, antibody fragment (such as people Fc) or leucine zipper).

Exemplary embodiment of the present invention also comprise suppress or blocking-up DR6 to the bonded method of APP, be included under the condition that DR6 is suppressed the combination of APP DR6 polypeptide and/or APP polypeptide be exposed to one or more DR6 antagonists.Employed typical DR6 antagonist comprises the antibody in conjunction with DR6 or APP in these class methods, and solubility DR6 polypeptide.Optional is to select employed DR6 antagonist in these methods by observing the bonded ability that suppresses between DR6 and the APP.In certain embodiments of the invention, use these class methods to come for example in vitro tissue is cultivated, to suppress the growth and/or the survival of the apoptosis and/or the enhancing neuronal cell of neuronal cell.Described method contains the combination of the DR6 antagonist of the DR6 antagonist molecules that uses single type or two or more types.

Embodiment of the present invention also provide neuronal cell or the growth of tissue or the method for regeneration or survival that is used for strengthening Mammals, comprise the DR6 antagonist to the administration significant quantity.In optional embodiments, the growth that being applied in the described Mammals of DR6 antagonist strengthens neuronal cell or tissue and the necrocytosis and the sex change (degeneration) of block nerves unit cell or tissue.Neuronal cell or tissue can comprise for example motor neuron, Sensory neurone, commissural neuron, aixs cylinder, little colloid and/or oligodendrocyte.In some embodiment of the present invention, employed DR6 antagonist can comprise in conjunction with APP and suppresses its antibody in conjunction with the ability of DR6 in these class methods.In other embodiments of the present invention, employed DR6 antagonist can comprise in conjunction with DR6 and suppresses its antibody in conjunction with the ability of APP in these class methods.Perhaps, the DR6 antagonist can comprise the DR6 immunoadhesin, the be connected to nonprotein character polymkeric substance DR6 polypeptide or the DR6 polypeptide variants of (it is selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene).The DR6 immunoadhesin that is adopted in the described method can comprise the solubility DR6 acceptor that merges to immunoglobulin fc region.In addition, DR6 antagonist of the present invention can comprise small molecules.

Embodiment of the present invention also provide the method that is used for the treatment of the neuroscience illness, comprise the DR6 antagonist to the administration significant quantity.In optional embodiments, described method comprises the Alzheimer's in the treatment Mammals.In these class methods employed DR6 antagonist can comprise in conjunction with APP and suppress its in conjunction with the ability of DR6 antibody.The DR6 antagonist also can comprise DR6 antibody.Perhaps, the DR6 antagonist can comprise the DR6 immunoadhesin, be connected to nonprotein character polymkeric substance DR6 polypeptide, DR6 antibody or the DR6 variant of (it is selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene).The DR6 immunoadhesin that is adopted in the described method can comprise the solubility DR6 acceptor that merges to immunoglobulin fc region.The anti-DR6 antibody that is adopted in the described method can be in conjunction with the DR6 acceptor that comprises Figure 1A amino acid/11-349 or 42-349.

Embodiment of the present invention also comprise be used to diagnose suffer from the neuroscience illness or to the patient's of neuroscience illness susceptible method, comprise from described patient and obtain sample and described sample test is had the existence of the DR6 polypeptide variants of the peptide sequence different with the DR6 peptide sequence of SEQID NO:1.Be typically, in these class methods, described polypeptide variants is accredited as to have and the avidity to APP polypeptide different to the observed avidity of DR6 peptide sequence of SEQ ID NO:1.

Embodiment of the present invention also provide and have been used to identify the method for inhibition DR6 to the bonded molecules of interest of APP.These class methods can be included in combination DR6 and APP in the situation that has or do not exist molecules of interest; Detect then in having the situation of described molecules of interest the inhibition of DR6 in conjunction with APP.Optional is to use the mammalian cell of expressing DR6 on cell surface to implement this class methods; And comprise that further detection activates DR6 or the inhibition of signal conduction.Embodiment of the present invention further comprise the molecule of identifying by these class methods.Optional is, molecules of interest be in conjunction with APP antibody, in conjunction with antibody or the solubility DR6 polypeptide of DR6.

Embodiment of the present invention also provide can specificity in conjunction with APP part, DR6 acceptor and/or can regulation and control relevant biologic activity and useful antibody in the various neuroscience treatment of conditions with DR6 and/or its part and/or co-receptor (co-receptor).In specific embodiment, provide the antibody (hereinafter in embodiment further describe) of specificity in conjunction with the ectodomain sequence of DR6 polypeptide.Typical antibody is those in conjunction with APP or DR6's and the bonded ability that suppresses between DR6 and the APP carried out further selection.Optional is that antibody is monoclonal antibody.Optional is that monoclonal antibody comprises by hybridoma excretory 3F4.4.8, the 4B6.9.7 or the 1E5.5.7 antibody that are preserved in ATCC respectively with numbering PTA-8095, PTA-8094 or PTA-8096.

Also provide in conjunction with the 3F4.4.8, the 4B6.9.7 that generate with the hybridoma cell line of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively or 1E5.5.7 monoclonal antibody in conjunction with the antibody of epi-position identical epi-position.On the one hand, the present invention pay close attention to demonstration identical with antibody 3F4.4.8,4B6.9.7 or 1E5.5.7 at least to the avidity of DR6 and/or show at least the biologic activity identical and/or the anti-DR6 antibody of effectiveness with antibody 3F4.4.8,4B6.9.7 or 1E5.5.7, comprise 3F4.4.8,4B6.9.7 or 1E5.5.7 antibody.

In other specific embodiment, the hybridoma cell line that generates monoclonal antibody 3F4.4.8,4B6.9.7 or 1E5.5.7 and be preserved in ATCC respectively with numbering PTA-8095, PTA-8094 or PTA-8096 is provided, and by hybridoma excretory monoclonal antibody 3F4.4.8, the 4B6.9.7 or the 1E5.5.7 that are preserved in ATCC respectively with numbering PTA-8095, PTA-8094 or PTA-8096.

Isolating anti-DR6 monoclonal antibody also is provided, has comprised in conjunction with DR6 polypeptide and competitive inhibition by the antibody of the monoclonal antibody that generates with the hybridoma of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively in conjunction with described DR6 polypeptide.Chimeric or humanized anti-DR6 antibody also are provided, and its specificity is in conjunction with the DR6 polypeptide and comprise (a) freedom is preserved in ATCC hybridoma excretory 3F4.4.8,4B6.9.7 or 1E5.5.7 antibody deutero-sequence with numbering PTA-8095, PTA-8094 or PTA-8096 respectively.Optional is that this antibody-like can comprise from 3F4.4.8,4B6.9.7 or 1E5.5.7 antibody deutero-heavy chain, light chain or variable region.

Another aspect, the present invention pay close attention to the coding anti-DR6 antibody of this paper or antibody fragment isolated nucleic acid molecule, comprise the carrier of this type of nucleic acid molecule, the method that comprises the host cell of this type of nucleic acid molecule and be used to produce this paper antibody and antibody fragment.

The invention further relates to the composition that comprises defined DR6 antagonist and carrier herein.Carrier can be a pharmaceutical acceptable carrier, and composition can further comprise other medicament.

On the other hand, the present invention pays close attention to goods, and it comprises container and be contained in the interior composition of described container that wherein said composition comprises DR6 antagonist of the present invention.Goods can further comprise about external or use the explanation of DR6 antagonist in vivo.In a preferred embodiment, the neuroscience treatment of conditions is paid close attention in described explanation.

In a related aspect, embodiment of the present invention comprise test kit, and it comprises the label on first container, the described container and is contained in the interior composition of described container.In this type of test kit, described composition comprises the DR6 antagonist of the apoptosis in the mammalian nervous unit cell of at least a type of effective treatment, the described label on the described container or be included in package insert in the described container and indicate described composition to can be used for suppressing apoptosis in the mammalian nervous unit cell of at least a type.Optional is that test kit comprises other parts (element), can accept second container of buffer reagent such as pharmacy is housed; And/or about using described DR6 antagonist to suppress the explanation of the apoptosis in the mammalian nervous unit cell of at least a type.

The present invention further provides the purposes that DR6 antagonist described herein and composition are used to prepare or make medicine, described medicine is used for the treatment of the neuroscience illness in the Mammals, comprises being used for the treatment of Alzheimer's.

The accompanying drawing summary

The nucleotide sequence (Figure 1A-1, SEQ ID NO:2), its derivation aminoacid sequence (Figure 1A-2, SEQ ID NO:1) that Figure 1A has shown people DR6cDNA with and the synoptic diagram (Figure 1A-3) of structural domain system structure.In the DR6 synoptic diagram, indicated the structural domain border, comprise the signal peptide of inferring, structural domain motif, membrane spaning domain and the death domain that is rich in halfcystine.In this synoptic diagram, the border, structural territory of structural domain motif, membrane spaning domain and the death domain that indicated the signal peptide of inferring, is rich in halfcystine.Figure 1B has shown the nucleotide sequence (Figure 1B-1, SEQ ID NO:5) and the derivation aminoacid sequence (Figure 1B-2, SEQ ID NO:6) thereof of cDNA of 695 isoforms of people's amyloid precursor protein (APP).Fig. 1 C has shown the aminoacid sequence (SEQ ID NO:7) of 751 isoforms of people's amyloid precursor protein.Fig. 1 D has shown the nucleotide sequence (Fig. 1 D-1, SEQ ID NO:8) and the derivation aminoacid sequence (Fig. 1 D-2, SEQ ID NO:9) thereof of cDNA of 770 isoforms of people's amyloid precursor protein (APP).Referring to for example UniProtKB/Swiss-prot clauses and subclauses P05067 and relevant disclosure, comprise (the http://expasy.org/uniprot/P05067) that relate separately to isoform ID P05067-1, isoform ID P05067-4 and isoform IDP05067-8.

Fig. 2 A has shown that DR6 is strongly expressed in developing central nerve system at etap E10.5-E12.5, comprises motion and the commissural neuron and the dorsal root ganglion neurons of spinal cord.Fig. 2 B has shown the DR6 albumen of expressing on aixs cylinder and cell main body.Fig. 2 C has shown the DR6 mRNA that expresses in the neurone in differentiation.

Fig. 3 has shown the synoptic diagram of axonal degeneration and neuronal cell death in the dorsal part spinal cord explant survival assay method; Having indicated by electroporation disturbs the siRNA agent to import in embryo's commissural neuron with the plasmid of expressing GFP RNA.

Fig. 4 A illustrates the inhibition that siRNA is expressed DR6 in dorsal part spinal cord survival assay method and blocks the Colaesce axonal degeneration and stop neuronal cell death.Fig. 4 B has shown that RNAi resistance DR6cDNA saves the sex change phenotype by DR6siRNA blocked.

Fig. 5 has shown that antagonism DR6 antibody helps blocking-up axonal degeneration and neuronal cell death in dorsal part spinal cord survival assay method.

Fig. 6 provides neuronic schematic diagram of mechanism and photo, has shown that in explant survival assay method the downward modulation of the DR6 downstream intracellular signal conduction that the pharmacology restraining effect by the terminal kinases of c-Jun N-(JNK) causes stops axonal degeneration and neuronal cell death.

Fig. 7 has shown that antagonism DR6 antibody is to the neuroprotective effect of the survival of spinal motor and relay cell in exsomatize (ex vivo) full embryo culture thing.

Fig. 8 provides with the photo through the painted E15.5 spinal cord of the Caspase-3 antibody mediated immunity cervical part of esophagus section of cutting, causes DR6 to lack the reduction of neuronal cell death in no embryo's spinal cord and the dorsal root ganglion to show losing of DR6.

Fig. 9 A shown from express through the E15.5 DR6 KO embryo's of the Caspase-3 of cutting neuronal cell quantitatively, its show DR6 lack neuronal cell among the no embryo dead with DR6+/-littermate contrasts (DR6 heterozygosis) and compares reduction about 50%.Fig. 9 B provides the photo of cell, has shown that DR6 is that the kinematic axis mutagenicity is needed, as normal in the situation that has and do not exist the neurotrophic somatomedin and DR6 knock-out mice comparison confirmed.Fig. 9 C provides the photo of cell, has shown that the axonal degeneration that damage is brought out has been delayed in the DR6 knock-out mice.

Figure 10 A provides neuronic photo, has shown that anti-DR6 antibody suppresses in the bereft neurone of various nutritional factor because the axonal degeneration that caused is broken off in neural factor (NGF) supply.Figure 10 B provides the further picture data from the TUNEL dyeing video picture of apoptotic cell main body in Colaesce, sensation and the motor neuron, and it has shown that anti-DR6 antibody suppresses the bereft neuronic sex change of nutritional factor miscellaneous.

Figure 11 A provides the photo of commissural neuron, has shown that DR6-Fc can postpone the Colaesce axonal degeneration.Figure 11 B provides the photo of Sensory neurone, has shown that DR6-Fc can postpone the sensation neurite sex change of being broken off bringing out by the NGF supply.

Figure 12 A provides neuronic photo, uses DR6-AP to show the DR6 binding site on the aixs cylinder.Figure 12 B provides the neuronic photo in the situation that has and do not exist NGF, has shown that depriving back DR6 ligand-binding site point at NGF loses from aixs cylinder.The photo that Figure 12 C provides the BAX that is in etap E12.5 to lack the research of numbness aixs cylinder has shown that beta-secretase (BACE) inhibitor can block the NGF supply and break off back DR6-AP binding site from the disappearance of feeling aixs cylinder.

Figure 13 A provides the photo of the data that obtain from multiple Western trace rules, wherein use DR6-AP (upper left) or anti-N-APP antibody (upper right) to detect polypeptide from neuronal cell and following polypeptide: (1) is selected in conjunction with the ability of DR6 because of it; (following, " DR6-ECD is drop-down ") that detect with anti-N-APP antibody (2) then.These data are accredited as DR6 extracellular domain binding partner with amyloid precursor protein (APP).Figure 13 B provides the photo of the data that obtain from multiple Blot experiment, and it allows the DR6 part (comprising the APP polypeptide) that manifests in the aixs cylinder conditioning substratum of detecting with DR6-AP.These trace data have been identified many APP polypeptide, comprise the terminal APP of the N-that is positioned at 35kDa and C99-APP and C83/C89APP polypeptide.

Figure 14 A provides neuronic photo, has shown that the APP extracellular domain comes off after NGF deprives soon.Figure 14 B provides the photo of cell, has shown that the DR6 extracellular domain is in conjunction with APP that culturing cell generated.Figure 14 C provides the photo of cell, has shown that DR6 is the N-APP principal recipient on the sensation neurite, and the APP binding site is significantly subdued in the neuronal cell of the scarce no mouse of DR6.Figure 14 D provides the photo of cell, has shown that the DR6 function blocking antibodies destroys the interaction between DR6 extracellular domain and the N-APP.

Figure 15 A provides neuronic photo, has shown in Colaesce aixs cylinder assay method to block axonal degeneration at the polyclonal antibody of the terminal APP of N-.Figure 15 B provides neuronic photo, has shown that the polyclonal antibody of the terminal APP of diagnosis N-and the anti-APP monoclonal antibody of 22C11 suppress to eliminate the local axonal degeneration of inductive by NGF.Figure 15 C provides neuronic photo, has shown that interpolation N-APP can save the axonal degeneration of being blocked by the active restraining effect of beta-secretase (BACE).Figure 15 D provides neuronic photo, has shown that the APP elimination that realizes by RNAi makes neuronal cell to the dead sensitization of N-APP inductive.

Figure 16 A provides neuronic photo, has shown that the DR6 function is that N-APP inductive axonal degeneration is needed, but is not that the sex change that triggers of A β is needed.Figure 16 B provides neuronic photo, has shown that function interdiction DR6 antibody fails to block the axonal degeneration that A β triggers.

Figure 17 A provides neuronic photo, shown that the inhibition to JNK and upstream Caspase-8 has postponed axonal degeneration, but downstream Caspase-3 is quite different.Figure 17 B provides the photo from the motor neuron of E12.5 explant culture, shown that Caspase-3 brings into play function in the cell main body, and Caspase-6 is brought into play function in aixs cylinder.Figure 17 C provides the photo of Sensory neurone, is not that axonal degeneration is needed though shown Caspase-3, and BAX is needed.Figure 17 D provides the photo of commissural neuron, shown that Caspase-3 brings into play function in the cell main body, and Caspase-6 is brought into play function in aixs cylinder.

Detailed Description Of The Invention

The technology of describing herein or mentioning and rules have generally obtained fully understanding of those skilled in the art, and usually utilize conventional method to be adopted, molecular cloning method such as for example extensive use, be recorded in Sambrook et al., the 2nd edition (1989) Cold Spring of Molecular Cloning:A Laboratory Manual Harbor Laboratory Press, Cold Spring Harbor, N.Y. In the time of suitably, involve the rules of using commercial reagent box and reagent and generally carry out according to scheme and/or the parameter of manufacturer's regulation, except as otherwise noted.

Before describing method of the present invention and determination method, be to be understood that to the invention is not restricted to described concrete grammar, scheme, clone, animal species or genus, construction and reagent, because they can change certainly to some extent. It should also be understood that term used herein just in order to describe specific embodiments, is not that intention limits the scope of the invention, Cai only have claims that the scope of the invention is limited.

Must be noted that singulative "/kind ", " being somebody's turn to do " and " described " etc. comprise plural indication thing, unless clear is arranged in addition when being used for this paper and claims. So, for example, mention " place hereditary change " and comprise this type of change of many places, mention " a kind of probe " and comprise one or more probes and the equivalent that those skilled in the art will know that thereof, like that. All numerical value (such as amino acid 22-81,1-354 etc.) were interpreted as with term " about " and modify described in specification and related right required.

All publications of mentioning herein are collected herein by reference, to disclose and to describe method and/or the material relevant with quoted publication. Quoting the publication of quoting herein is because they are disclosed before the application's submission day. Any literal herein all should not be construed as admits that the present inventor does not have qualification to rely on more priority date early or formerly invention day and early than described publication. In addition, actual deliver day may with shown different, need independent verification.

I. definition

Term " amyloid precursor protein " or " APP " comprise by the coded multiple polypeptides isoform (isoform) of APP premessenger RNA, APP695, APP751 and App770 isoform shown in for example Figure 1B-1D distinguishes (the alternative splicing transcript from the APP premessenger RNA is translated the isoform that obtains), and processing part after the translation of APP isoform. As known in the art, transcribe from app gene the APP premessenger RNA experience extron alternative splicing that obtains with produce multiple isoform (referring to such as Sandbrink etc., Ann NY Acad.Sci.777:281-287 (1996); And the information relevant with PubMed NCBI albumen numbering P05067). This variable extron montage produce 695,751 and 770 amino acid whose three kinds of main isoforms (referring to such as Kang etc., Nature 325:733-736 (1987); Kitaguchi etc., Nature 331:530-532 (1988); Ponte etc., Nature 331:525-527 (1988); And Tanzi etc., Nature 331:528-532 (1988)). Two kinds of (App in these isoforms₇₅₁And APP₇₇₀) comprise the insert of 56 residues, serine protease inhibitor (KPI) the height homology of itself and Kunitz family and all over expressing. On the contrary, the shorter isoform APP that lacks the KPI motif₆₉₅Mainly (for example in neuron and Deiter's cells) expresses in nervous system, and be often referred to as for this reason " neuron APP " (referring to such as Tanzi etc., Science 235:880-884 (1988); Neve etc., Neuron 1:669-677 (1988); And Haas etc., J. Neurosci 11:3783-3793 (1991)). The rear processing event of the great translation of each isoform of APP (comprising 695,751 and 770) experience (referring to such as Esch etc., 1990 Science 248:1122-1124; Sisodia etc., 1990 Science 248:492-495). For example, each these isoform is subject to the cutting of multiple secretase and/or secretase compound, and this event produces the APP fragment, comprises the terminal secrete polypeptide (sAPP α and sAPP β) of the N-that comprises the APP extracellular domain. The cutting of being undertaken by alpha-secretase enzyme or beta-secretase causes respectively generation and the born of the same parents of the terminal APP polypeptide sAPP α of solubility N-and sAPP β to discharge outward, and the C-terminal fragment C83 of corresponding membrane grappling and the reservation of C99. Gamma-secretase produces the P3 polypeptide to the following process of C83. This is main secretory pathway, and is not produce amyloid. Perhaps, senilism albumen/stay protein mediated gamma-secretase to processing release amyloid beta polypeptides, amyloid-β 40 (A β 40) and the amyloid-β 42 (A β 42) of C99, be the main component of amyloid patch, and cytotoxicity C-terminal fragment γ-CTF (50), γ-CTF (57) and γ-CTF (59). Point out on evidence the relative importance of every kind of cutting event to depend on cell type. For example, preferentially by alpha-secretase enzymatic pathway processing APP, it cuts APP to non-neuronal cell in A β sequence, get rid of thus A β formation (referring to such as Esch etc., 1990 Science 248:1122-1124; Sisodia etc., 1990 Science 248:492-495). On the contrary, neuronal cell is by β-secretase approach processing APP₆₉₅Much bigger part, this produces complete A β by the else associating of at least two kinds of enzymes is active. In neuronal cell, beta-secretase is at the amino terminal cutting APP in A beta structure territory₆₉₅, discharge a kind of different N-terminal fragment (sAPP β). In addition, gamma-secretase is at another site of carboxyl terminal cutting APP, produce long 40 (A β 40) or 42 (A β 42) amino acid whose A β kinds (referring to such as Seubert etc., 1993 Nature 361:260-263; Suzuki etc., 1994 Science 264:1336-1340; And Turner etc., 1996 J.Biol.Chem.271:8966-8970).

Term " APP ", " APP albumen " and " APP polypeptide " are contained natural A PP sequence and APP variant and processing fragment thereof when being used for this paper. These terms are encompassed in and express APP in the multiple mammal (comprising the people). APP can be endogenous expression, as organize in various human in the pedigree natural generation, perhaps can express by restructuring or synthetic method. " native sequences APP " comprises and the polypeptide that has same acid sequence derived from natural APP (for example 695,751 and 770 isoform and processing parts thereof). So, native sequences APP can have the amino acid sequence from the natural APP of existence of any mammal (comprising the people). This type of native sequences APP can separate from nature, perhaps can generate by restructuring or synthesizing mean. Naturally occurring processing and/or secreted form (for example comprising for example soluble form of ectodomain sequence), natural variant form (for example alternative splicing and/or proteolysis form processing) and the natural allelic variant that exists of existing clearly contained in term " native sequences APP ". The APP variant can comprise fragment or the deletion mutant of native sequences APP.

In embodiments of the invention useful APP polypeptide comprise those above with following non-limitative example described in. Can select these illustration forms, be used for each embodiment of the present invention. In some embodiment of the present invention, the APP polypeptide comprises total length APP isoform, APP shown in Figure 1B-1D₆₉₅And/or APP₇₅₁And/or APP₇₇₀Isoform. In other embodiments of the present invention, the APP polypeptide comprises form processing after the translation of APP, for example experienced the APP polypeptide (for example solubility N-terminal fragment, such as sAPP α or sAPP β) of secretase (such as alpha-secretase enzyme, beta-secretase or gamma-secretase) cutting. In related embodiment of the present invention, can select the APP polypeptide, comprising one or more ad hoc structures territory, (referring to such as Quast etc., FASEB is J.2003 such as the terminal extracellular domain of N-; 17 (12): 1739-41), the Heparin-binding domain is (referring to such as Rossjohn etc., Nat Struct Biol.1999 Apr; 6 (4): 327-31), copper II type (referring to such as Hesse etc., FEBS Letters 349 (1): 109-116 (1994)) or Kunitz protease inhibitors domain (referring to such as Ponte etc., Nature; 331 (6156): 525-7 (1988)). In some embodiment of the present invention, the APP polypeptide comprises observes the sequence that comprises the epi-position that is subject to DR6 antagonist disclosed herein (such as antibody or DR6 immunoadhesin) identification, for example APP₆₉₅Amino acid 22-81, a kind of sequence that comprises the epi-position of monoclonal antibody 22C11 institute combination is (referring to such as Hilbich etc., Journal of Biological Chemistry, 268 (35): 26571-26577 (1993)).

In certain embodiments of the invention, the APP polypeptide does not comprise one or more ad hoc structure territory or sequences, for example do not comprise some N-end or C-end amino acid APP polypeptide (for example among the embodiment 12 disclosed people recombinate N-APP polypeptide), do not comprise the APP polypeptide (APP for example of Kunitz protease inhibitors domain₆₉₅) or do not comprise APP polypeptide (sAPP β for example, a kind of A β that do not comprise of Alzheimer's beta amyloid albumen (A β) sequence₄₀And/or A β₄₂The polypeptide of sequence) (referring to such as Bond etc., J. Struct Biol.2003 Feb; 141 (2): 156-70). In other embodiments of the present invention, employed APP polypeptide comprises one or more domains or sequence but does not comprise other domain or sequence in embodiment of the present invention, for example comprises the terminal extracellular domain (or it observes the part that is subject to DR6 antagonist (such as monoclonal antibody 22C11) combination at least) of N-but does not comprise the domain of one or more secretase cleavage site C ends or the APP polypeptide (for example sAPP α or sAPP β) of sequence (such as a beta amyloid albumen (A β) sequence).

Term " ectodomain ", " extracellular domain " or " ECD " refer to that APP is substantially free of the form in membrane spaning domain and cytoplasmic structure territory. Soluble E CD usually will have and be less than 1% described membrane spaning domain and cytoplasmic structure territory, preferably will have to be less than 0.5% described domain. Be appreciated that any membrane spaning domain for peptide identification of the present invention is to identify for the identification of the standard of this type hydrophobic domains according to this area routine. The exact boundary of membrane spaning domain can change, but most possible be that the initial arbitrary end of domain of identifying is no more than about 5 amino acid. In preferred embodiments, ECD will be comprised of (and not being membrane-bound) solubility ectodomain sequence that polypeptide does not contain membrane spaning domain and kytoplasm or born of the same parents' intracellular domain.

Term " APP variant " mean as hereinafter defined, have at least about 80% with the people APP with amino acid sequence shown in Figure 1B-1D, preferably at least about 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, most preferably at least about the APP polypeptide of 95%, 96%, 97%, 98% or 99% amino acid sequence identity, or its soluble fragments, or its solubility ectodomain. This type of variant for example comprises in that the N-of Figure 1B-1D total length or mature sequence or C-are terminal and adds or delete the APP polypeptide of one or more amino acid residues or insert or delete the APP polypeptide of one or more amino acid residues in the internal sequence of polypeptide or domain, comprise the variant from other species, but get rid of native sequences APP polypeptide.

" DR6 " or " DR6 acceptor " comprise its polynucleotides of this area indication and peptide sequence such as Figure 1A-1 to the acceptor shown in the 1A-2. The people such as Pan put down in writing the TNF receptor family member's who is called " DR6 " or " TR9 " polynucleotides and peptide sequence (Pan etc.,FEBS Lett., 431:351-356 (1998); Also can be referring to United States Patent (USP) 6,358,508; 6,667,390; 6,919,078; 6,949,358). People DR6 acceptor is 655 amino acid whose protein (seeing Figure 1A-2), and it has the burst (amino acid/11-41) of inferring, ectodomain (amino acid 42-349), membrane spaning domain (amino acid 350-369), then is cytoplasmic structure territory (amino acid 370-655). Native sequences acceptor and acceptor variant contained in term " DR6 acceptor " when being used for this paper. These terms are encompassed in the DR6 acceptor of expressing in the multiple mammal (comprising the people). The DR6 acceptor can be endogenous expression, occurs in the pedigree as organizing in various human, perhaps can express by restructuring or synthetic method. " native sequences DR6 acceptor " comprises and the polypeptide that has same acid sequence derived from natural DR6 acceptor. So, native sequences DR6 acceptor can have the amino acid sequence from the natural DR6 of the existence acceptor of any mammal (comprising the people). This type of native sequences DR6 acceptor can separate from nature, perhaps can generate by restructuring or synthesizing mean. The acceptor (for example comprising for example soluble form of ectodomain sequence) of naturally occurring brachymemma or secreted form, natural variant form (for example alternative splicing form) and the natural allelic variant that exists of existing clearly contained in term " native sequences DR6 acceptor ". The acceptor variant can comprise fragment or the deletion mutant of native sequences DR6 acceptor.

Term " ectodomain ", " extracellular domain " or " ECD " refer to that the DR6 acceptor is substantially free of the form in membrane spaning domain and cytoplasmic structure territory. Soluble E CD usually will have and be less than 1% described membrane spaning domain and/or cytoplasmic structure territory, preferably will have to be less than 0.5% described domain. Be appreciated that any membrane spaning domain for peptide identification of the present invention is to identify for the identification of the standard of this type hydrophobic domains according to this area routine. The exact boundary of membrane spaning domain can change, but most possible be that the initial arbitrary end of domain of identifying is no more than about 5 amino acid. In preferred embodiments, ECD will be comprised of (and not being membrane-bound) solubility ectodomain sequence that polypeptide does not contain membrane spaning domain and kytoplasm or born of the same parents' intracellular domain.

Term " DR6 variant " means as hereinafter defined, have at least about 80% with the people DR6 with the amino acid sequence of derivation shown in Figure 1A, preferably at least about 85%, 86%, 87%, 88%, 89%, more preferably at least about 90%, 91%, 92%, 93%, 94%, most preferably at least about the DR6 polypeptide of 95%, 96%, 97%, 98% or 99% amino acid sequence identity, or its soluble fragments, or its solubility ectodomain. This type of variant for example comprises in that the N-of Figure 1A total length or mature sequence or C-are terminal and adds or delete the DR6 polypeptide of one or more amino acid residues or insert or delete the DR6 polypeptide of one or more amino acid residues in the internal sequence of polypeptide or domain, comprise the variant from other species, but get rid of native sequences DR6 polypeptide. Optional is, the DR6 variant comprises and comprises Figure 1A amino acid/11-349 or 42-349 and the soluble form DR6 acceptor that substitutes of 10 place's conserved amino acids at the most. Preferably, this type of variant plays the DR6 antagonist, as hereinafter defined.

Term " DR6 antagonist " uses with broad sense, comprise any external, original position, in vivo or ex vivo (ex vivo) is partially or completely blocked, is suppressed or in its related part of DR6 receptors bind (preferably its related part APP), perhaps activate one or more intracellular signals in neuronal cell or the tissue or the molecule of the ability of intracellular signal pathway. For example, the DR6 antagonist can partially or completely block, suppress or in and the DR6 receptor activation cause the ability of one or more intracellular signals in the neuronal cell of apoptosis or cell death in neuronal cell or the tissue or the tissue or intracellular signal pathway. The DR6 antagonist can by number of mechanisms come partially or completely to block, suppress or in and the effect of DR6, include but not limited to by blocking-up, suppress or the related part that neutralizes to complex formation between oligomerization, DR6 acceptor and the allos coreceptor of complex formation, DR6 acceptor between the related part with it of combination, DR6 (for example APP) of DR6, related part to complex formation between the combination of DR6 acceptor/allos coreceptor compound or DR6 acceptor, allos coreceptor and related part thereof. The DR6 antagonist can be brought into play function in direct or indirect mode. The DR6 antagonist that the present invention is contained includes but not limited to APP antibody, DR6 antibody, immunoadhesin, the DR6 immunoadhesin, the DR6 fusion, covalent modification form DR6, DR6 variant and fusion thereof, or senior oligomer form DR6 (dimer, aggregation), or homopolymer or heteropolymer form DR6, little molecule (such as the pharmacology mortifier of JNK signal transduction cascade, comprising little molecule and the peptide mortifier of the terminal kinases JNK of Jun N-activity), in signal transduction pathway, bring into play the protein kinase MLK of function and the pharmacology mortifier of MKK activity in the JNK upstream, JNK is in conjunction with the pharmacology mortifier of scaffolding protein JIP-1, JNK is in conjunction with the pharmacology mortifier of its substrate (such as c-Jun or AP-1 transcription factor complex), the pharmacology mortifier of the phosphorylation in the Binding Capacity territory of its substrate of JNK mediation (such as JNK in conjunction with territory (JBD) peptide) and/or JNK and/or comprise the peptide mortifier in JNK substrate phosphorylation site, blocking-up ATP is in conjunction with the little molecule of JNK, little molecule with blocking-up Binding Capacity JNK.

In order to measure the whether partially or completely blocking-up of DR6 antagonist, suppress or in and one or more intracellular signals in DR6 receptor activation neuronal cell or the tissue or the ability of intracellular signal pathway, can implement assay method and assess the DR6 antagonist for example influence of various neuronal cells or tissue (as be shown in the examples), and at the body inner model of apoplexy/cerebral ischemia, the body inner model of neurodegenerative disease is (such as parkinsonian mouse model, the mouse model of Alzheimer's, the mouse model of amyotrophic lateral sclerosis ALS, the mouse model of Duchenne-Arandisease SMA, in the mouse/rat model (for example common carotid artery occlusion model or middle cerebral artery occlusion model) of focal and globality cerebral ischemia, or in stripped (ex vivo) full embryo culture thing.Can implement various assay methods to measure form in the known external or body, all as described below or as known in the art with document in put down in writing (referring to for example McGowan etc., TRENDS in Genetics, 22:281-289 (2006); Fleming etc., NeuroRx, 2:495-503 (2005); Wong etc., Nature Neuroscience, 5:633-639 (2002)).Be used for measuring the DR6 antagonist whether partially or completely block, suppress or and DR6 receptor activation neuronal cell or tissue in the embodiment of assay method of ability of one or more intracellular signals or intracellular signal pathway be included in the situation that has or do not exist DR6 antagonist or potential DR6 antagonist (being molecules of interest) DR6 and APP combination, in the situation that has this DR6 antagonist or potential DR6 antagonist, detect then the inhibition of DR6 in conjunction with APP.

" nucleic acid " intention comprises any DNA or RNA, for example karyomit(e) that exists in the tissue sample, plastosome, virus and/or bacterial nucleic acid.Two chains of double chain acid molecule or one of them contained in term " nucleic acid ", comprises any fragment or the part of complete nucleic acid molecule.

" gene " means in coding or transcription factor matter or regulates and control any nucleotide sequence or its part that has functional effect aspect other genetic expression.Gene can be made up of the proteinic nucleic acid of responsible encoding function fully, perhaps has only part nucleic acid to be responsible for coding or marking protein.Nucleotide sequence can comprise genetic abnormality in the exon of gene, intron, initial or terminator, promoter sequence, other regulating and controlling sequence or unique proximity.

Term " amino acid " refers to all naturally occurring L-a-amino acids.This definition intention comprises nor-leucine, ornithine and homocysteine.Amino acid indicates by single-letter or trigram and differentiates:

Asp D aspartic acid Ile I Isoleucine

Thr T Threonine Leu L leucine

Ser S Serine Tyr Y tyrosine

Glu E L-glutamic acid Phe F phenylalanine

Pro P proline(Pro) His H Histidine

Gly G glycine Lys K Methionin

Ala A L-Ala Arg R arginine

Cys C halfcystine Trp W tryptophane

Val V Xie Ansuan Gln Q glutamine

Met M methionine(Met) Asn N l-asparagine

In the accompanying drawings, can adopt some other single-letter or trigram to indicate two or more amino acid or the Nucleotide that points to and identify given position in the sequence.

" isolating ", when being used to describe various peptides disclosed herein or protein, mean identify and with/peptide or the protein that separate and/or reclaim by a kind of composition of its natural surroundings.The contaminative composition of peptide or proteinic natural surroundings refers to can disturb usually the material of its diagnosis or therepic use, can comprise the solute of enzyme, hormone and other protein properties or nonprotein character.In preferred embodiments, peptide or protein purification to (1) are enough to by using the rotary-cup type sequenator to obtain the N-end of 15 residues or the degree of internal amino acid sequence at least, or (2) are according to the SDS-PAGE that uses under Coomassie blue or preferred silver-colored painted irreducibility or the reductive condition, reach homogeneity, or (3) reach homogeneity according to mass spectrum or peptide mapping technology.Since at least a composition of peptide or proteinic natural surroundings can not exist, so isolating material comprises original position peptide or the protein in the reconstitution cell.Yet isolating peptide or protein prepare by at least one purification step usually.

" per-cent (%) amino acid sequence identity " about the sequence identified herein is defined as the contrast sequence and introduces breach where necessary with after obtaining largest percentage sequence identity, and with any conservative part that is considered as sequence identity that substitutes, the percentage of the amino-acid residue identical in the candidate sequence with amino-acid residue in the reference sequences.Can carry out the sequence contrast to measure the per-cent amino acid sequence identity in the multiple mode in the art technology scope.Those skilled in the art can determine to measure the correlated suitable parameter of sequence, comprise that appointment obtains the required algorithm of maximum contrast to the full length sequence that is compared.For the present invention, can use sequence comparison computer program ALIGN-2 to obtain per-cent amino acid sequence identity value, the ALIGN-2 program is write by Genentech company, its source code is submitted to (the US Copyright Office of U.S. Copyright Bureau together with customer documentation, Washington, DC, 20559), and with U.S. copyright registration TXU510087 register.The public can pass through Genentech company, and (South San Francisco CA) obtains the ALIGN-2 program.The all sequences comparative parameter is by ALIGN-2 program setting and constant.

" severity " of hybridization can be easy to determine, and calculate by rule of thumb according to probe length, wash temperature and salt concn usually by those of ordinary skills.Generally speaking, the temperature that long probe is had relatively high expectations is with correct annealing, and short probe needs lower temperature.Hybridization depends on usually when complementary strand and is present in the environment that is lower than its melting temperature(Tm) time variation DNA annealed ability again.But the expectation identity degree between probe and the hybridization sequences is high more, and spendable relative temperature is also high more.The result is infer that higher relative temperature will trend towards making reaction conditions more strict, and lesser temps to be just not strict yet.About other details and the explanation of hybridization severity, referring to Ausubel etc., CurrentProtocols in Molecular Biology, Wiley Interscience Publishers (1995).

" high stringency ", as defined herein, by following every definition: (1) adopts low ionic strength and high temperature to clean; 0.015M sodium-chlor/0.0015M Trisodium Citrate/0.1% sodium lauryl sulphate is in 50 ℃; (2) in crossover process, adopt denaturing agent; 50% (v/v) methane amide and 0.1% bovine serum albumin(BSA)/0.1%Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer pH 6.5 and 750mM sodium-chlor, the 75mM Trisodium Citrate is in 42 ℃; Or (3) adopt 50% methane amide, 5x SSC (0.75M NaCl, 0.075M Trisodium Citrate), 50mM sodium phosphate (pH 6.8), 0.1% trisodium phosphate, 5x DenhardtShi solution, the salmon sperm dna of supersound process (50 μ g/ml), 0.1%SDS, with 10% sulfuric acid dextran, in 42 ℃, and in 42 ℃ in 0.2x SSC (sodium chloride/sodium citrate) and 50% methane amide in 55 ℃ of cleanings, then in 55 ℃ of 0.1x SSC that containing EDTA, carry out high severity cleaning.

" medium stringent condition " can be as Sambrook etc., Molecular Cloning:A LaboratoryManual, New York, identify described in the Cold Spring Harbor Press (1989), be included in 37 ℃ and containing 20% methane amide, 5x SSC (150mM NaCl, the 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5x DenhardtShi solution, be incubated overnight in the solution of the salmon sperm dna that 10% sulfuric acid dextran and 20mg/ml sex change are sheared, then in about 37-50 ℃ of cleaning filter membranes in 1x SSC.The technician will recognize and adjust temperature, ionic strength etc. how where necessary to adapt to such as factors such as probe length.

Term " primer " refer to the hybridization of complementary RNA or DNA target polynucleotide and the effect by nucleotidyl transferase of serving as from the mononucleotide oligonucleotide sequence of the starting point of synthetic polyribonucleotides (as taking place the polymerase chain reaction for example) progressively.

Term " control sequence " refers to express the necessary dna sequence dna of encoding sequence that can be operatively connected in the specific host organism.For example, be suitable for procaryotic control sequence and comprise promotor, optional operator gene sequence and ribosome bind site.The known genuine karyocyte utilizes promotor, polyadenylation signal and enhanser.

If one section nucleic acid and another section nucleotide sequence are in the functional mutual relationship, then it is " can be operatively connected ".For example, participate in protein (preprotein) before the polypeptide excretory if presequence (presequence) or the DNA that secretes leading (secretory leader) are expressed as, then the DNA of it and this polypeptide can be operatively connected; If promotor or enhanser influence transcribing of encoding sequence, then it and this sequence can be operatively connected; Perhaps, if the position of ribosome bind site promotes translation, then it and encoding sequence can be operatively connected.Generally speaking, " can be operatively connected " means that continuous dna sequence dna is adjacent, and means adjacent in the leading situation of secretion and be in read state.Yet enhanser needn't be adjacent.Connection can be by realizing in the connection at restriction site place easily.If there is not this type of site, then use synthetic oligonucleotide adapter or joint according to conventional practice.

Term " marker " refers to when being used for this paper and reagent such as nucleic acid probe or direct or indirect coupling of antibody or fusion, so that detect the compound or the composition of the reagent of its institute's coupling or fusion.Marker can be self detectable (for example radioisotopic tracer or fluorescent marker), perhaps in the situation of enzyme labelling thing, but the chemically changed of detectable substrate compounds of catalysis or composition.

When being used for this paper, term " immunoadhesin " refers to antibody molecule that the effector functions of the binding specificity of heterologous protein (" adhesin ") and immunoglobulin (Ig) constant domain is joined together.Structurally, immunoadhesin comprises the antigen recognition that is different from antibody and binding site (promptly being " allos "), has the aminoacid sequence of expectation binding specificity and the fusions of immunoglobulin (Ig) constant domain sequence.The adhesin of immunoadhesin molecule partly is typically the continuous amino acid sequence of the binding site that comprises acceptor or part at least.Immunoglobulin (Ig) constant domain sequence in the immunoadhesin can obtain from any immunoglobulin (Ig), such as IgG-1, IgG-2, IgG-3 or IgG-4 hypotype, IgA (comprising IgA-1 and IgA-2), IgE, IgD or IgM.

" DR6 receptor antibody ", " DR6 antibody " or " anti-DR6 antibody " use with broad sense, refer to the antibody in conjunction with at least a form DR6 acceptor (preferred people DR6 acceptor, DR6 sequence shown in Figure 1A) or its ectodomain sequence.Optional is that DR6 antibody merges with heterologous sequence or molecule or is connected.Preferably, heterologous sequence allows or helps antibody to form more high-grade or oligomeric complex.Term " anti-DR6 antibody " and grammer equivalent thereof are specifically contained the DR6 monoclonal antibody described in the part of embodiment hereinafter.Optional is, DR6 antibodies DR6 acceptor but do not combine with any other tnf family cytokines acceptor (for example DR4, DR5, TNFR1, TNFR2, Fas) or cross reaction takes place.Optional is, according to the measurement of BIAcore binding assay, DR6 antibody of the present invention about 0.067 μ M in the concentration range of about 0.033 μ M in conjunction with the DR6 acceptor.

Term " anti-APP antibody ", " APP antibody " and grammer equivalent use with broad sense, refer to the antibody in conjunction with at least a form APP (preferred people APP is such as the concrete described APP polypeptide isoform of this paper).Preferably, APP antibody is the DR6 antagonistic antibodies.For example, the disclosed in this article method that is used for generating and/or identifying the DR6 antagonist, one or more isoforms that can use APP and/or its part come SCREENED COMPOUND library (for example recombinant antibodies storehouse) as the original immune animal of immunity (for example mouse, as the part of the process that generates monoclonal antibody) and/or as probe.Useful in embodiments of the invention typical APP polypeptide comprises following non-limitative example.Can select these illustration forms, be used for each embodiment of the present invention.In some embodiment of the present invention, the APP polypeptide comprises total length APP isoform, all APP as shown in Figure 1 ₆₉₅And/or APP ₇₅₁And/or APP ₇₇₀Isoform.In other embodiments of the present invention, the APP polypeptide comprises the translation post-treatment form of APP, for example experienced the APP polypeptide (for example solubility N-terminal fragment, such as sAPP α or sAPP β) of Secretases (such as alpha-secretase enzyme, beta-secretase or gamma-secretase) cutting.In related embodiment of the present invention, can select the APP polypeptide, comprising one or more ad hoc structures territory, (referring to for example Quast etc., FASEB is J.2003 such as the terminal extracellular domain of N-; 17 (12): 1739-41), the heparin binding domains is (referring to for example Rossjohn etc., Nat Struct Biol.1999 Apr; 6 (4): 327-31), copper II type (referring to for example Hesse etc., FEBSLetters 349 (1): 109-116 (1994)) or Kunitz proteinase inhibitor structural domain (referring to for example Ponte etc., Nature; 331 (6156): 525-7 (1988)).In some embodiment of the present invention, the APP polypeptide comprises observes the sequence that comprises the epi-position that is subjected to DR6 antagonist disclosed herein (such as antibody or DR6 immunoadhesin) identification, for example APP ₆₉₅Amino acid 22-81, a kind of sequence of monoclonal antibody 22C11 institute bonded epi-position that comprises is (referring to for example Hilbich etc., Journal of BiologicalChemistry, 268 (35): 26571-26577 (1993)).In certain embodiments of the invention, the APP polypeptide does not comprise one or more ad hoc structure territory or sequences, for example do not comprise some N-end or C-end amino acid APP polypeptide (for example among the embodiment 12 disclosed people recombinate N-APP polypeptide), do not comprise the APP polypeptide (APP for example of Kunitz proteinase inhibitor structural domain ₆₉₅) or the APP polypeptide (for example sAPP β, a kind of A β 40 and/or A β 42 polypeptide of sequence of not comprising) that do not comprise Alzheimer's beta amyloid albumen (A β) sequence (referring to for example Bond etc., J.Struct Biol.2003 Feb; 141 (2): 156-70).In other embodiments of the present invention, employed APP polypeptide comprises one or more structural domains or sequence but does not comprise other structural domain or sequence in embodiment of the present invention, for example comprises the terminal extracellular domain of N-(or at least its observe be subjected to DR6 antagonist (such as monoclonal antibody 22C11) bonded part) but does not comprise the structural domain of one or more Secretases cleavage site C ends or the APP polypeptide (for example sAPP α or sAPP β) of sequence (such as a beta amyloid albumen (A β) sequence).Optional is, anti-APP antibody can suppress the combination of APP polypeptide to DR6, and can be bonded to the APP polypeptide in the concentration of 10 μ g/ml to 50 μ g/ml, as described in this article and/or as quantitative binding assay based on cell in measured.

Term " antibody " uses with broad sense in this article, the multi-specificity antibody (for example bi-specific antibody) that clearly covers complete monoclonal antibody, polyclonal antibody, formed by at least two kinds of complete antibodies, and antibody fragment are as long as they show desired biological activity.

" antibody fragment " comprises the part of complete antibody, preferably comprises the antigen binding domain or the variable region of antibody.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂With the Fv fragment; Double antibody; Linear antibody; The single-chain antibody molecule; Reach the multi-specificity antibody that forms by antibody fragment.

" natural antibody " refers to common about 150, the 000 daltonian different tetramer glycoprotein that are made of two identical light (L) chains and two identical weights (H) chain.Every light chain is connected with heavy chain by a covalent disulfide bonds, and the number of disulfide linkage changes between the heavy chain of different immunoglobulin (Ig) isotypes.Every heavy chain and light chain also have the intrachain disulfide bond of rule at interval.Every heavy chain at one end has a variable domain (V _H), then be a plurality of constant domains.Every light chain at one end has a variable domain (V _L), and the other end is a constant domain.The constant domain of light chain is arranged in first constant domain of heavy chain, and the variable domain of light chain is arranged in the variable domain of heavy chain.Think that specified amino acid residues forms the interface between light chain and heavy chain variable domain.

Term " variable " refer in the variable domain some part at the antibody sequence differences extensively and be used for combination and the specific truth of every kind of specific antibodies to its specific antigen.Yet variability is not the whole variable domain that is uniformly distributed in antibody.It concentrates on three sections that are called hypervariable region or complementary determining region in light chain and the heavy chain variable domain.In the variable domain more the part of high conservative be called framework region (FR).Each self-contained four FR of the variable domain of natural heavy chain and light chain, they take the beta-pleated sheet conformation mostly, connect and form in some situation three hypervariable regions connections of a beta-pleated sheet structure part by the formation ring-type.Hypervariable region in every chain is by very approaching the keeping together of FR, and facilitate the formation of the antigen binding site of antibody (referring to Kabat etc. with the hypervariable region of another chain, Sequences of Proteins ofImmunological Interest, the 5th edition, Public Health Service, National Institutes ofHealth, Bethesda, MD. (1991)).Constant domain is not participated in antibody directly and is combined with antigenic, but shows multiple effector functions, such as the participation of antibody in the cytotoxicity (ADCC) of antibody dependent cellular mediation.

Produce two identical Fabs with papain digestion antibody, be called " Fab " fragment, have an antigen binding site separately, and remaining " Fc " fragment, its title has reflected that it is easy to the crystalline ability.Pepsin produces a F (ab ') ₂Fragment, it has two antigen binding sites and still can crosslinked antigen.

" Fv " is the minimum antibody fragment that comprises complete antigen recognition and antigen binding site.This zone is made up of the dimer in a heavy chain variable domain of tight, non-covalent bonded and a light chain variable territory.Just in this structure, three hypervariable regions of each variable domain interact and at V _H-V _LDefine an antigen binding site on the dimer surface.Antibody is given together with antigen-binding specificity in six hypervariable regions.Yet,, be that avidity is lower than complete binding site even single variable domain (or only comprise specific three CDR of antigen half Fv) also has the ability of identification and conjugated antigen.

The Fab fragment also comprises the constant domain of light chain and first constant domain (CH1) of heavy chain.The segmental difference of Fab ' fragment and Fab is that the C-terminal of heavy chain CH1 structural domain has increased the minority residue, comprises the one or more halfcystines from antibody hinge region.Fab '-SH is the appellation of constant domain cysteine residues wherein being carried the Fab ' of at least one free sulphur alcohol radical herein.F (ab ') ₂Antibody fragment is as there being the paired Fab ' fragment of hinge cysteine to generate in pairs between Fab ' fragment at first.Also know other chemical coupling form of antibody fragment.

According to the aminoacid sequence of its constant domain, can be included into a kind of in two kinds of distinct types from " light chain " of the antibody (immunoglobulin (Ig)) of any invertebrate species, be called card handkerchief (κ) and lambda (λ).

According to the aminoacid sequence of its heavy chain constant domain, antibody can be included into different classes.Complete antibody has five big class: IgA, IgD, IgE, IgG and IgM, and wherein some can be further divided into subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Heavy chain constant domain that will be corresponding with inhomogeneous antibody is called α, δ, ε, γ and μ respectively.The subunit structure of inhomogeneous immunoglobulin (Ig) and three-dimensional structure are well-known.

" strand Fv " or " scFv " antibody fragment comprise the V of antibody _HAnd V _LStructural domain, wherein these structural domains are present on the polypeptide chain.Preferably, the Fv polypeptide is at V _HWith V _LFurther comprise peptide linker between the structural domain, it makes scFv can form the desired structure of conjugated antigen.About the summary of scFv referring to Pl ü ckthun, in " The Pharmacology of Monoclonal Antibodies ", the 113rd volume, Rosenburg and Moore compile, Springer-Verlag, New York, 269-315 page or leaf, 1994.

Term " double antibody " refers to have the small-sized antibody fragment of two antigen binding sites, and this fragment is at same polypeptide chain (V _H-V _L) in comprise continuous heavy chain variable domain (V _H) and light chain variable territory (V _L).Can not match between two structural domains on same the chain by using too short joint to make, force the complementary structure territory pairing of these structural domains and another chain, thereby produce two antigen binding sites.Double antibody is more complete is recorded in for example EP 404,097; WO 93/11161; Hollinger etc., Proc.Natl.Acad.Sci.USA 90:6444-6448 (1993).

Term " monoclonal antibody " refers to that when being used for this paper each antibody that promptly constitutes colony is identical from a group antibody that obtains of the antibody of homogeneity basically, except may be with indivisible possible natural the existence the mutant form that exists.Monoclonal antibody is a high degree of specificity, at single antigenic site.In addition, with routine (polyclone) the antibody preparations difference that typically comprises at the different antibodies of different determinants (epi-position), every kind of monoclonal antibody is at the single determinant on the antigen.Beyond their specificity, the advantage of monoclonal antibody is that they are by hybridoma culture synthetic, are not subjected to the pollution of other immunoglobulin (Ig).Modifier " mono-clonal " indication antibody should not be construed as requirement and generates antibody by any ad hoc approach from the feature that the antibody population of homogeneity basically obtains.For example, remain to be prepared by the hybridoma method of putting down in writing by (1975) Nature 256:495 such as Kohler first according to the monoclonal antibody that the present invention uses, perhaps can prepare (referring to for example U.S. Patent No. 4,816,567) by recombinant DNA method." monoclonal antibody " also can use the technology of putting down in writing among (1991) J.Mol.Biol.222:581-597 such as (1991) Nature352:624-628 such as Clackson and Marks for example to separate from phage antibody library.

Monoclonal antibody clearly comprises " chimeric " antibody (immunoglobulin (Ig)) in this article, wherein the part of heavy chain and/or light chain with derived from specific species or belong to the identical or homology of corresponding sequence in the antibody of specific antibodies classification or subclass, and the remainder of chain with derived from another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody classification or subclass, and the fragment of this antibody-like, as long as they show desired biological activity (U.S. Patent No. 4,816,567; Morrison etc., Proc.Natl.Acad.Sci.USA 81:6851-6855 (1984)).Interested herein chimeric antibody comprises and comprising derived from non-human primate (Old World monkey class (Old World Monkey) for example, such as baboon, rhesus monkey or macaque) variable domain antigen binding sequence and " primatesization " antibody (U.S. Patent No. 5 of human constant region sequence, 693,780).

" humanization " form of inhuman (for example mouse) antibody refers to comprise chimeric antibody derived from the sequence of non-human immunoglobulin with bottom line.Largely, humanized antibody refers to the immunoglobulin (Ig) that the hypervariable region residue in the human normal immunoglobulin (receptor antibody) is replaced with the hypervariable region residue of inhuman species (donor antibody) such as mouse, rat, rabbit or non-human primate with expectation specificity, avidity and ability.In some situation, framework region (FR) residue of human normal immunoglobulin is replaced with corresponding inhuman residue.In addition, humanized antibody can be included in the receptor antibody or the residue that does not find in donor antibody.Carrying out these modifications is in order further to improve the performance of antibody.Generally speaking, humanized antibody will comprise at least one, common two whole basically following variable domains, wherein all or basically all hypermutation rings corresponding to the hypermutation ring of non-human immunoglobulin, and all or basically all FR are FR of human normal immunoglobulin sequence.Optional partial immunity immunoglobulin constant district (Fc), the normally constant region of human normal immunoglobulin at least of also will comprising of humanized antibody.More details are referring to Jones etc., Nature 321:522-525 (1986); Riechmann etc., Nature 332:323-329 (1988); And Presta, Curr.Op.Struct.Biol.2:593-596 (1992).

Term " hypervariable region " refers to that when being used for this paper antibody is responsible for antigen bonded amino-acid residue.The hypervariable region comprises from the amino-acid residue of " complementary determining region " or " CDR " (residue 31-35 (H1), 50-65 (H2) and the 95-102 (H3) in the residue 24-34 (L1) in the light chain variable territory, 50-56 (L2) and 89-97 (L3) and the heavy chain variable domain for example; Kabat etc., Sequences of Proteins of Immunological Interest, the 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, (1991)) and/or those residues from " hypermutation ring " (residue 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the residue 26-32 (L1) in the light chain variable territory, 50-52 (L2) and 91-96 (L3) and the heavy chain variable domain for example; Chothia and Lesk, J.Mol.Biol.196:901-917 (1987))." framework region " or " FR " residue refers to those residues except that the hypervariable region residue of definition herein in the variable domain.

The antigenic antibody of " combination " purpose refers to can be with enough avidity and/or avidity in conjunction with this antigen, makes this antibody can be used as the antibody that therapeutical agent or diagnostic reagent are used for this antigenic cell of targeted expression.

For the present invention, " immunotherapy " refers to the method with Antybody therapy Mammals (preferred human patients), wherein antibody can be not link coupled or " exposing " antibody, perhaps antibody can coupling or fusion heterologous molecule or reagent are arranged, such as one or more cytotoxic agents, produce thus " immune conjugate ".

" isolating " antibody refers to identify and from a kind of composition of its natural surroundings separately and/or the antibody that reclaims.The contaminative composition of its natural surroundings refers to disturb the diagnosis of this antibody or the material of therepic use, can comprise the solute of enzyme, hormone and other protein properties or nonprotein character.In preferred embodiments, with antibody purification to (1) mensuration according to the Lowry method, antibody weight surpasses 95%, most preferably weight surpasses 99%, (2) be enough to by using the rotary-cup type sequenator to obtain the N-end of at least 15 residues or the degree of internal amino acid sequence, or (3) reach homogeneity according to the SDS-PAGE under reductibility or the irreducibility condition and use Coomassie blue or preferred silver dyeing.Since at least a composition of antibody natural surroundings can not exist, isolated antibody comprises the original position antibody in the reconstitution cell so.Yet isolated antibody will prepare by at least one purification step usually.

Term " tape label " refers to comprise the chimeric molecule that antibody or polypeptide and itself and " label polypeptide " merge when being used for this paper.The label polypeptide have enough residues can prepare so that epi-position to be provided at its antibody or the ability (for example have leucine zipper motif peptide was taken place) of some other function such as oligomerizations is provided, but enough shortly make it not disturb the activity of described antibody or polypeptide.The label polypeptide is preferably still fairly individual, make the label specific antibody basically not with other epi-position generation cross reaction.Suitable label polypeptide has at least 6 amino-acid residues usually, and about usually 8 are arrived (preferred about 10 are arrived between about 20 residues) between about 50 amino-acid residues.

Term " Fc acceptor " or " FcR " are used to describe the acceptor in energy binding antibody Fc district.Preferred FcR is native sequences people FcR.In addition, preferred FcR can comprise the acceptor of Fc γ RI, Fc γ RII and Fc γ RIII subclass in conjunction with the FcR (γ acceptor) of IgG antibody, comprises the allelic variant and the alternative splicing form of these acceptors.Fc γ RII acceptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" inhibition acceptor "), and they have similar aminoacid sequence, and difference mainly is its cytoplasmic structure territory.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) of immunity receptor based on tyrosine in its cytoplasmic structure territory.Suppress acceptor Fc γ RIIB in its cytoplasmic structure territory, comprise immunity receptor based on the inhibition motif (ITIM) of tyrosine (referring to

Annu.Rev.Immunol.15:203-234 (1997)).The summary of FcR is referring to Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991); Capel etc., Immunomethods 4:25-34 (1994); And de Haas etc., J.Lab.Clin.Med.126:330-341 (1995).Other FcR contained in this article in term " FcR ", comprises what will identify those futures.This term also comprises newborn infant's acceptor, FcRn, and it is responsible for parent IgG is transferred to fetus (Guyer etc., J.Immunol.117:587 (1976) and Kim etc., J.Immuno1.24:249 (1994)).FcR comprises polymorphism in this article, causes being arranged in the gene such as coding Fc γ RIIIa being subjected to physical efficiency in conjunction with zone the 158th amino acids of IgG1 or for phenylalanine (F) or be the genetic dimorphism of Xie Ansuan (V).The Xie Ansuan Fc γ RIIIa (Fc γ IIIa-158V) that isozygotys has the ADCC that higher avidity and mediation to the human IgG1 raise external demonstrating with respect to isozygoty phenylalanine Fc γ RIIIa (Fc γ RIIIa-158F) or heterozygosis (Fc γ IIIa-158F/V) acceptor.

Term " polyol " is made a general reference the polyhydroxy-alcohol compound when being used for this paper.Polyvalent alcohol can be for example any water-soluble poly (alkylene oxide) polymkeric substance, and can have linear chain or branched chain.Preferred polyhydric alcohols comprises the polyvalent alcohol that those replace such as the alkyl with 1 to 4 carbon with chemical group in one or more hydroxy position.Be typically, polyvalent alcohol is poly-(aklylene glycol), preferably gathers (ethylene glycol) (PEG).Yet, those skilled in the art recognize that other polyvalent alcohol can utilize the coupling technology of describing about PEG to be adopted such as poly-(propylene glycol) and polyethylene-polypropylene glycol multipolymer herein.Polyvalent alcohol comprises those types well-known in the art and those types that can openly obtain, such as obtaining from commercial source, such as

Company.

Term " coupling " (conjugate) defines use according to it in this article the most widely, and finger closes or connects together.If molecule works when being bonded together or operates, then they are " couplings ".

Statement " significant quantity " refers to effectively prevention of medicament (for example DR6 antagonist etc.), alleviate or treatment the amount of the illness of discussing or illness.DR6 antagonist of the present invention can be useful slowing down or stopping in the progress of sex change neuroscience illness or strengthening the reparation of injured neurons cell or tissue and helping to recover in the correct neural function.

Term " processing ", " treatment " and " therapy " refer to therapeutic treatment, preventative processing and precaution processing when being used for this paper.Treat continuously or use finger based at least once a day, during do not interrupt the processing of a day or many days.Intermittent therapy or use or the treatment of intermittent mode or use the also discontinuous in essence but round-robin of finger and handle.

When being used for this paper, term " illness " refers generally to the illness that DR6 antagonist for treating described herein is benefited from any meeting.This comprises chronic and acute disease, and those make Mammals tend to the pathological condition of the illness of discussing.

" neuronal cell or tissue " refers generally to Dopamine HCL (DA) neurone, striatum DA neurone, cortical neuron, brain stem neurone, spinal interneuron and motor neuron, hippocampal neuron (including but not limited to the CA1 cone neurone of hippocampus) and the preceding brain neuron of motor neuron, relay cell (including but not limited to commissural neuron), Sensory neurone (including but not limited to dorsal root ganglion neurons), black substance.Term neuronal cell or be organized in refers to the neuronal cell be made up of cell paste, aixs cylinder and dendron and the aixs cylinder or the dendron that refer to form this type of neuronal cell part herein.

" neuroscience illness " is used in reference in this article and comprises neurodegenerative illness, the neuronal cell that is feature with the dysfunction of maincenter or peripheral nervous system or with the necrosis and/or the apoptosis of neuronal cell or tissue or tissue injury's damage, and deprives the relevant neuronal cell or the illness of tissue injury with nutritional factor.The example of neurodegenerative disease comprises familial and sporadic amyotrophic lateral sclerosis (being respectively FALS and ALS), familial and sporadic Parkinson's disease, Huntington disease (Huntington chorea), familial and sporadic Alzheimer's, Duchenne-Arandisease (SMA), optic neuropathy (opticalneuropathies) is (such as glaucoma or involve retinal degeneration, diabetic neuropathy, or the association disease of macular degeneration (associated disease)), because the hearing loss that inner ear sensory cell or neuronal degeneration cause, epilepsy, bell's palsy (Bell ' s palsy), the frontotemporal dementia with parkinson's syndrome (parkinsonism) (FTDP-17) chain with karyomit(e) 17, multiple sclerosis, the atrophy of diffusivity pallium, Lewy corpusculum dementia, Pick's disease (Pick disease), trinucleotide repeats sick (trinucleotiderepeat disease), Protein virus illness (prion disorder), with summer moral two Cotards (Shy-Dragersyndrome).Neuronal cell or tissue injury can be derived from the multiple different reasons of the survival that jeopardizes neuronal cell or tissue or correct function, include but not limited to: for example be derived from the acute and non-acute injury of the ischemic situation of restriction (temporarily or for good and all) blood flow, just as in globality and focal cerebral ischemia (apoplexy); Otch or cut wound to for example cerebral tissue or spinal cord; Infringement in the neuronal tissue or patch (placques); Depriving of the growth of pair cell and the needed nutritional factor of surviving; Be exposed to neurotoxin, such as chemotherapeutics; And other accidental morbid state (incidental to other disease states) is such as chronic metabolic disease such as diabetes or renal insufficiency.

" experimenter " or " patient " refers to any single experimenter of needs treatment, comprises the people.Also be intended to participate in the clinical study test and do not demonstrate any experimenter of the clinical sign of any disease, the experimenter who participates in epidemiological study or experimenter in contrast as having of comprising of experimenter.

Term " Mammals " aim when being used for this paper is gone into mammiferous any Mammals, comprises people, ox, horse, dog and cat.In a preferred embodiment of the invention, Mammals refers to the people.

II. exemplary methods of the present invention and material

Previous research checked the necrocytosis phenomenon in the nervous system development process (Hamburger etc., J. Neurosci., 1:60-71 (1981); Oppenheim, Ann.Rev.Neurosci., 14:453-501 (1991); O ' Leary etc., J.Neurosci., 6:3692-3705 (1986); Henderson etc., Nature, 363:266-270 (1993); Yuen etc., Brain Dev., 18:362-368 (1996)).Think the death of the neuronal cell (Price etc. that in the formation of multiple neuroscience illness (such as familial and sporadic amyotrophic lateral sclerosis (being respectively FALS and ALS), familial and sporadic Parkinson's disease, Huntington disease, familial and sporadic Alzheimer's and Duchenne-Arandisease (SMA)) and/or progress, play a role Science, 282:1079-1083 (1998)).

The applicant finds that unexpectedly DR6 (member of TNFR family) expresses embryo and adult's central nervous system (relay cell that comprises pallium, hippocampus, motor neuron and spinal cord) camber.Described in hereinafter embodiment, the applicant has carried out the effect that the kinds of experiments assay method checks DR6 may bring into play as the instrumentality of neuronal cell survival or death.The survival of commissural neuron relies on the nutritional support from one of target thing (base plate of spinal cord) in the middle of it.In external explant culture, the applicant finds to disturb the axonal degeneration that suppresses DR6 expression blocking-up commissural neuron by RNA.Also in dorsal part spinal cord survival assay method, tested anti-DR6 monoclonal antibody, determined that suppressing the conduction of DR6 receptor signal by DR6 specific antibody 3F4.4.8,4B6.9.7 and 1E5.5.7 stops the neuronic axonal degeneration of external explant culture mediocommissure.Reported in the literature DR6 through the activation of JNK signal (Pan etc., See above1998; Zhao etc., See above2001).Thereby, in order to investigate the effect of DR6-JNK signal conduction in axonal degeneration, carried out dorsal part spinal cord survival assay method, wherein with the JNK signal transduction path in the inhibitor peptides L-JNK-I blocking-up commissural neuron.This JNK signal conduction suppresses partly to block the axonal degeneration in the dorsal part spinal cord survival assay method.So, think that DR6 sends the axonal degeneration process signal through the JNK approach to small part.In order to understand the effect of DR6 in the adjusting of neuronal cell death in the growth course better, in full embryo culture system, conduct with anti-DR6 antibody blocking DR6 signal.Surprising is, some DR6 specific antibody is to the developmental character necrocytosis protection spinal neuron at natural generation of being suppressed in this system of DR6 signal conduction.Therefore, DR6 antagonist (such as the DR6 antagonistic antibodies) can be used for reducing the neuronal cell death that takes place in neuroscience illness (such as the neurodegenerative illness, for example ALS, SMA, Alzheimer's and Parkinson's disease, FTDP-17, Huntington disease) and the apoplexy.In order to check DR6 whether to bring into play the function of short apoptosis acceptor in vivo veritably, the applicant has analyzed the phenotype that the DR6 that is in etap E15.5 knocks out embryo.Consistent with DR6 as the effect of the down regulator of neuronal cell survival, in DR6 disappearance spinal cord and dorsal root ganglion, detect and compare with DR6 heterozygosis littermate contrast that neuronal cell is dead to reduce about 40% to 50%.

The applicant finds unexpectedly that also amyloid precursor protein (APP) is the related part of DR6 acceptor, and the APP performance triggers the function of axonal degeneration through the DR6 acceptor.Although previous hypothesis amyloid precursor protein is brought into play effect (Selkoe, the J.Biol.Chem.271:18295 (1996) that some are not understood fully in Alzheimer's; Scheuner; Deng, Nature Med.2:864 (1996); Goate, etc., Nature 349:704 (1991)).

Think that the DR6 antagonist can be to be particularly useful in the various neuroscience illnesss of treatment.The present invention thus the DR6 antagonist composition is provided and be used for suppressing Mammals, blocking-up and in and the active method of DR6, comprise the DR6 antagonist of using significant quantity.Preferably, the DR6 antagonism dosage that is adopted can be the amount of effectively blocking axonal degeneration and neuronal cell death.This can according to for example hereinafter with embodiment described in method realize.

Adoptable DR6 antagonist includes but not limited to covalent modification form, DR6 and/or APP variant, its fusion rotein, and DR6 and/or the APP antibody of DR6 and/or APP immunoadhesin, the fusion rotein that comprises DR6 and/or APP, DR6 and/or APP in the described method.The multiple technologies that can be used for generating antagonist have been described herein.For example, method and the technology that is used to prepare DR6 and APP polypeptide described.The further modification of DR6 and APP polypeptide has also been described, and at the antibody of DR6 and APP.

Method disclosed herein has many embodiments.The invention provides and suppress the method for DR6, be included under the condition that DR6 is suppressed the combination of APP DR6 polypeptide and/or APP polypeptide are exposed to one or more DR6 antagonists in conjunction with APP.Related embodiment of the present invention provides and has suppressed DR6 polypeptide that comprises SEQ ID NO:1 amino acid/11-655 and the bonded method that comprises the APP polypeptide (for example sAPP β) of SEQ ID NO:6 amino acid 66-81, this method comprises DR6 polypeptide and APP polypeptide and combines the isolating antagonist combination of DR6 or APP, and wherein said isolating antagonist is selected from antibody in conjunction with APP, in conjunction with the antibody of DR6 with comprise at least a in the solubility DR6 polypeptide of SEQ ID NO:1 amino acid/11-354; And described isolating antagonist suppresses DR6 according to it and APP bonded ability is selected; Make DR6 be suppressed to the combination of APP.

Optional is, in these class methods, one or more DR6 antagonists are selected from antibody in conjunction with DR6 (for example competitive inhibition is by the bonded of the 3F4.4.8, the 4B6.9.7 that generate with the hybridoma cell line of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively or the 1E5.5.7 monoclonal antibody antibody in conjunction with DR6), comprise the solubility DR6 polypeptide (for example DR6 immunoadhesin) of SEQ ID NO:1 amino acid/11-354 or in conjunction with the antibody (for example monoclonal antibody 22C11) of APP.In certain embodiments of the invention, the DR6 antagonist be connected to one or more antibody that are selected from down the nonprotein character polymkeric substance of group in conjunction with DR6, in conjunction with antibody or the solubility DR6 polypeptide of APP: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

In the optional embodiment of these methods, the DR6 polypeptide is to express on the cell surface of one or more mammalian cells (for example commissural neuron cell, Sensory neurone cell or motor neuron cell), and the combination of described one or more DR6 antagonists suppresses DR6 activation or signal conduction.In this type of embodiment of the present invention, described method is in external enforcement, in order to suppressing the apoptosis in one or more mammalian cells of expressing DR6, thereby strengthens growth and/or the regeneration and/or the survival of neuronal cell in the tissue culture.For example, this type of DR6 antagonist is useful as the external additive of tissue culture medium (TCM) (for example those are designed for propagation of neural unit cell culture).Particularly, as known in the art, the breeding of some neuronal cell culture may be a problem owing to the tendency of this type of cell experience apoptosis.For example, some neurone culture death when lacking exogenous factor (such as nerve growth factor).The disclosure that is provided has shown that the DR6 antagonist can be used for this type of neuronal cell culture to strengthen cell growth and/or regeneration and/or survival herein, for example with this type of culture in the similar mode of use of nerve growth factor.

In other embodiments of the present invention, can in the Mammals that suffers from neuroscience illness or illness, implement to suppress the method for DR6 in the body in conjunction with APP.Optional is that neuroscience illness or illness are amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease or Alzheimer's.Perhaps, neuroscience illness or illness comprise be derived from apoplexy, to the wound of brain or myeloid tissue or the neuronal cell or the tissue injury of the infringement in the neuronal tissue.

Other embodiment of the present invention provides treatment to suffer from the mammiferous method of neuroscience illness or illness, comprises one or more DR6 antagonists to described administration significant quantity.Be typically, in these class methods, one or more DR6 antagonists are selected from antibody in conjunction with DR6, comprise the solubility DR6 polypeptide of SEQ IDNO:1 amino acid/11-354 and in conjunction with the antibody of APP.In optional embodiment of the present invention, neuroscience illness or illness are amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease or Alzheimer's.Perhaps, neuroscience illness or illness comprise be derived from apoplexy, to the wound of brain or myeloid tissue or the neuronal cell or the tissue injury of the infringement in the neuronal tissue.In various embodiments of the present invention, to one or more other therapeutical agents of described administration.In some exemplary embodiment of the present invention, described one or more other therapeutical agents are selected from NGF, inhibitors of apoptosis, EGFR inhibitor, beta-secretase inhibitor, inhibitors of gamma-secretase, anticholinesterase, anti-amyloid beta antibodies and nmda receptor antagonist.Optional is that described one or more DR6 antagonists and/or other therapeutical agent are to be applied to mammiferous through injection, infusion or perfusion.

Other embodiment of the present invention provides the method for inhibition DR6 in conjunction with the molecules of interest of APP of identifying, this method comprises: combination DR6 and APP in the situation that has or do not exist molecules of interest, detect in having the situation of described molecules of interest then to the inhibition of DR6 in conjunction with APP.Related embodiment of the present invention provides determines whether certain composition is regulated and control to comprise the DR6 polypeptide of SEQ ID NO:1 amino acid/11-655 (with optional SEQ ID NO:1 amino acid/11-354) and comprised the APP polypeptide (APP for example of SEQ ID NO:6 amino acid 66-81 ₆₉₅, sAPP α or sAPP β) between the bonded method, this method comprises composition and DR6 and APP combination, DR6 in the time of will having said composition then and the combination between the APP when not having said composition DR6 and APP between combine and compare, determine thus whether said composition regulates and control the combination between DR6 and the APP.Optional is, measuring by surperficial plasmon (SPR) technology (for example can from Biacore Life Sciences acquisition) that resonates in conjunction with difference in these class methods.Embodiment of the present invention further comprise the molecules of interest of identifying according to these methods.

Other embodiment of the present invention comprise diagnosis suffer from the neuroscience illness or to the patient's of neuroscience illness susceptible method, comprise from described patient and obtain sample and described sample test is had the existence of the DR6 polypeptide variants of the peptide sequence different with the DR6 peptide sequence of SEQ IDNO:1.Optional is that this method further comprises identifies whether described polypeptide variants has and the avidity to APP polypeptide different to the observed avidity of DR6 peptide sequence of SEQ ID NO:1.Related embodiment of the present invention comprises the method for determining whether to exist in the Mammals DR6 polypeptide variants that comprises SEQ ID NO:1 amino acid/11-655, this method comprises the sequence of DR6 polypeptide expressed in the Mammals and SEQ ID NO:1 is compared determine whether there is the DR6 polypeptide variants in this Mammals thus.Some embodiment of these methods can comprise further step, promptly identify observe the polypeptide variants that in Mammals, exists whether be APP in conjunction with variant, wherein APP in conjunction with variant be characterized by had to amyloid precursor protein (APP) polypeptide that comprises SEQ ID NO:6 amino acid 66-81 (APP for example ₆₉₅, sAPP α or sAPP β) binding affinity be different from the DR6 polypeptide that comprises SEQ ID NO:1 binding affinity to the APP polypeptide that comprises SEQ IDNO:6 amino acid 66-81.Optional is, the difference of binding affinity is by resonate (SPR) technology (for example can from the Biacore LifeSciences acquisition) measurement of surperficial plasmon in these class methods.Some embodiment of these methods can comprise the step of the individual patients of selecting to have in amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease or Alzheimer's observed symptom or illness.

Outside total length native sequences DR6 described herein and APP polypeptide, can prepare DR6 and/or APP polypeptide variants.DR6 and/or APP variant can change be introduced coding DNA and/or prepare synthesizing of polypeptide by expectation by the Nucleotide that will suit.Those skilled in the art can understand, and amino acid changes the translation post-treatment that may change DR6 and/or APP polypeptide, such as the number that changes glycosylation site or position or change film anchor feature.

Variation in DR6 described herein and/or the APP polypeptide can for example use any technology and the guilding principle about conservative and non-conservative sudden change to produce, and for example U.S. Patent No. 5,364, is proposed in 934.Variation can be the substituting of codon, the deletion of one or more coded polypeptides or insert, and it causes comparing with the native sequences polypeptide variation in the aminoacid sequence.Randomly, variation is by realizing with at least one amino acid of any other amino acid replacement in one or more structural domains of DR6 and/or APP polypeptide.Determine which amino-acid residue can be inserted into, substitute or deletion and can influence the active guidance of expectation sharply can be by the relatively sequence and the sequence of homologous known protein molecule of DR6 polypeptide, and make the number of aminoacid sequence variation of the region generating of high homology minimize and find.Amino acid replacement can be the amino acid whose result of amino acid replacement who has analog structure and/or chemical property with another, and such as substituting leucine with Serine, promptly conserved amino acid substitutes.Randomly, inserting or delete can be in about 1 to 5 amino acid whose scope.Can followingly determine the variation of allowing, promptly in sequence, systematically produce amino acid whose insertion, deletion or substitute, and to the variant test DR6 and/or the APP antagonistic activity of gained.

DR6 and/or APP polypeptide fragment are provided herein.This type of fragment can perhaps can lack inner residue, for example when comparing with the total length native protein in N end or the brachymemma of C end.The expectation biologic activity that some fragment lacks for the DR6 polypeptide is not vital amino-acid residue.

DR6 and/or APP polypeptide can prepare by in many routine techniquess any.Can chemosynthesis the peptide fragment of expectation.Alternative methods involves by enzymatic digestion generation polypeptide fragment, for example passes through with known enzyme-treated protein in the site scinderin matter that limits by particular amino acid residue, or by realizing with suitable restriction enzyme dna digestion and separation expectation fragment.Another kind of suitable technique involves separates the also dna fragmentation of amplification coding expectation polypeptide fragment, and it is realized by polymerase chain reaction (PCR).Adopt the oligonucleotide of the expectation end that limits dna fragmentation on 5 ' and the 3 ' primer in PCR.

In specific embodiment, shown interested conservative substituting in the following table under the preferred alternate title.Cause biologic activity to change if this type of substitutes, can be called the more substantial variations of " illustration substitutes " so in the importing table, or it is further described to see below the amino acid classification, and the screening product.

Table

Original residue	Illustration substitutes	Preferred substituting
			??Ala(A)	?val；leu；ile	??val
??Arg(R)	?lys；gln；asn	??lys
			??Asn(N)	?gln；his；asp；lys；arg	??gln
??Asp(D)	?glu；asn	??glu
			??Cys(C)	?ser；ala	??ser
??Gln(Q)	?asn；glu	??asn
			??Glu(E)	?asp；gln	??asp
??Gly(G)	?ala	??ala

??His(H)	?asn；gln；lys；arg	??arg
			??Ile(I)	Leu; Val; Met; Ala; Phe; Nor-leucine	??leu
??Leu(L)	Nor-leucine; Ile; Val; Met; Ala; Phe	??ile
			??Lys(K)	?arg；gln；asn	??arg
??Met(M)	?leu；phe；ile	??leu
			??Phe(F)	?leu；val；ile；ala；tyr	??tyr
??Pro(P)	?ala	??ala
			??Ser(S)	?thr；cys	??cys
??Thr(T)	?ser	??ser
			??Trp(W)	?tyr；phe	??tyr
??Tyr(Y)	?trp；phe；thr；ser	??phe
			??Val(V)	Ile; Leu; Met; Phe; Ala; Nor-leucine	??leu

The substance of the function of DR6 and/or APP polypeptide or immunology identity is modified and can be realized by selecting substitute significantly the difference on effect of keeping following aspect: (a) structure of polypeptide main chain in the replacement area, for example as (folding) lamella or helical conformation, (b) electric charge or the hydrophobicity of target site punishment, or (c) volume of side chain.According to common side chain characteristic, the natural residue that exists can followingly divide into groups:

(1) hydrophobic: nor-leucine, met, ala, val, leu, ile;

(2) neutral, hydrophilic: cys, ser, thr;

(3) tart: asp, glu;

(4) alkalescence: asn, gln, his, lys, arg;

(5) influence the residue of chain orientation: gly, pro; And

(6) aromatic: trp, tyr, phe.

Non-conservative substitute need be replaced another classification with the member of one of these classifications.This type of alternative residue can also be introduced in the conservative alternate site, or more preferably, be introduced in residue (nonconservative) site.

Variation can use method as known in the art to produce, and scans, reaches PCR mutagenesis such as oligonucleotide mediated (fixed point) mutagenesis, L-Ala.Can implement site-directed mutagenesis [Carter etc., Nucl.Acids Res., 13:4331 (1986) to the DNA that is cloned; Zoller etc., Nucl.Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells etc., Gene, 34:315 (1985)], restricted selection mutagenesis [Wells etc., Philos.Trans.R.Soc.London SerA, 317:415 (1986)] or other known technology to generate DR6 polypeptide variants DNA.

Can also adopt scanning amino acid analysis with along the one or more amino acid of successive Sequence Identification.Less relatively, neutral amino acids is one of preferred scanning amino acid.This amino acid comprises L-Ala, glycine, Serine, reaches halfcystine.L-Ala is preferred scanning amino acid in this group normally, exceeds the outer side chain of β-carbon because it has been eliminated, and unlikely changes the main chain conformation [Cunningham and Wells, Science, 244:1081-1085 (1989)] of variant.Common also preferred L-Ala is because it is modal amino acid.Further, it is frequently found [Creighton, The Proteins, (W.H.Freeman﹠amp in sheltering position and exposure position; Co., N.Y.); Chothia, J.Mol.Biol., 150:1 (1976)].If L-Ala substitutes the variant that can not produce q.s, then can use isostere (isoteric) amino acid.

Any cysteine residues that does not relate to the correct conformation of keeping DR6 and/or APP polypeptide generally also can substitute improving the oxidative stability of molecule with Serine, and stops crosslinked unusually.On the contrary, the halfcystine key can be added into DR6 and/or APP polypeptide to improve its stability.

Each embodiment of invention disclosed herein is applicable to APP polypeptide extremely widely.For example, in certain embodiments of the invention, APP is total length shown in Figure 1B-1D 695,750 or 770APP isoform (isoform).In other embodiments of the present invention, APP comprises the N end parts (for example sAPP α or sAPP β) that has extracellular domain and translate the APP of post-treatment incident generation certainly.Optional is, for example, APP can comprise 695,750 or the soluble form of one of 770APP isoform, and it is derived from the cutting action of Secretases, the soluble form of neurone APP695 for example, and it is derived from the cutting action of beta-secretase.In a concrete exemplary embodiment, APP comprises APP ₆₉₅Amino acid 20-591 (referring to for example Jin etc., J.Neurosci., 14 (9): 5461-5470 (1994)).In another embodiment of the invention, APP comprises that having the monoclonal antibody of being subjected to 22C11 (for example can derive from Chemicon International Inc., Temecula, CA, U.S.A.) polypeptide of Shi Bie epi-position.Optional is that APP comprises APP ₆₉₅Residue 66-81, promptly comprise the zone (referring to for example Hilbrich, J.B.C. volume 268, phase 35:26571-26577 (1993)) of 22C11 epi-position.

Below describe the carrier conversion or the cells transfected that relate generally to by cultivating and generate DR6 and/or APP polypeptide through comprising DR6 peptide coding nucleic acid.Certainly, can adopt alternative approach (it is being known in the art) to prepare DR6 and/or APP polypeptide.For example, suitable aminoacid sequence or its part can generate by the direct peptide that uses solid phase technique is synthetic [referring to for example Stewart etc., Solid-PhasePeptide Synthesis, W.H.Freeman Co., San Francisco, CA (1969); Merrifield, J.Am.Chem.Soc., 85:2149-2154 (1963)].External protein synthesis can use manual technique or implement by automatization.Can realize that automatization is synthetic, (Foster City CA) uses the specification sheets of manufacturers to realize for example to use Applied Biosystems peptide synthesizer.The various piece of DR6 and/or APP polypeptide can use chemistry or enzymatic means to come chemosynthesis dividually and combination to generate the DR6 and/or the APP polypeptide of expectation.

Be applicable to the DR6 of DR6 and/or APP variant, modified forms and/or APP, and the production of DR6 and/or APP antibody like described method and the technology type.

The separation of the DNA of encoding D R6 and/or APP polypeptide

Can obtain the DNA of encoding D R6 and/or APP polypeptide from the cDNA library, but this cDNA library is to prepare from thinking to have DR6 and/or APP polypeptide mRNA and express with detection level its tissue.Thereby people DR6 and/or APP polypeptid DNA can be easily available from the cDNA libraries for preparing from people's tissue.The gene of encoding D R6 and/or APP polypeptide can also obtain available from genomic library or by known synthetic rules (for example automatic nucleic acid is synthetic).

Can use designed probe (such as oligonucleotide) screening library with the evaluation gene of interest or by its encoded protein matter at least about 20-80 base.Can use standard schedule (such as being recorded in Sambrook etc. with selected probe screening cDNA or genomic library, among the Molecular Cloning:ALaboratory Manual (New York:Cold Spring Harbor Laboratory Press, 1989)) carry out.The alternative approach of separating the gene of encoding D R6 polypeptide is to use PCR method, and [Sambrook etc. see above; Dieffenbach etc., PCR Primer:A Laboratory Manual (Cold SpringHarbor Laboratory Press, 1995)].

The technology in screening cDNA library is being known in the art.The oligonucleotide sequence that is elected to be probe should have enough length, and is enough clear and definite so that false positive is reduced to minimum.Oligonucleotide is mark preferably, make its can with just be detected after DNA in the screened library hybridization.The method of mark is being known in the art, and comprise use radioactively labelled substance (as ³²The ATP of p-mark), biotinylation or enzyme labelling.Hybridization conditions (comprise medium severity and height severity) provides in seeing above at Sambrook etc.

The sequence of identifying in this type of library screening method can compare with storage in public database (such as GenBank) or other the privately owned sequence library and obtainable other known array and compare.In the regulation zone of molecule or the sequence identity of whole full length sequence (or at amino acid levels or at nucleotide level) can use method known in the art and as herein described to measure.

The nucleic acid that can following acquisition has protein coding sequence, promptly use the disclosed first reasoning aminoacid sequence of this paper, and when needed as Sambrook etc., see above described such cDNA that conventional primer extension rules screen selection or genomic library of using detecting precursor, and handle and also not have reverse transcription to become the mRNA intermediate of cDNA.

The selection of host cell and conversion

Host cell is used for expression or cloning vector transfection or the conversion that DR6 and/or APP polypeptide generate with described herein, and in conventional nutritional medium, cultivates for evoked promoter, the gene of selecting transformant or amplification coding expectation sequence and suitably improvement.The technician need not undo experimentation just can select culture condition, such as substratum, temperature, pH etc.Generally speaking, be used to make the maximized principle of cell cultures biological productivity, scheme, and practice technology can be referring to Mammalian Cell Biotechnology:aPractical Approach, M.Butler compiles (IRL Press, 1991) and Sambrook etc., see above.

The method that eukaryotic cell transfection and prokaryotic cell prokaryocyte transform is known for those of ordinary skill, for example, and CaCl ₂, CaPO ₄, liposome-mediated and electroporation.According to the host cell that uses, use for the suitable standard technique of this type of cell and implement to transform.(as be recorded in Sambrook etc., see above) or electroporation generally are used for prokaryotic organism to adopt the calcium of calcium chloride to handle.Agrobacterium tumefaciens (Agrobacterium tumefaciens) infect and to be used for the certain plants transformation, as Shaw etc., and Gene, the WO 89/05859 of 23:315 (1983) and announcement on June 29th, 1989 is put down in writing.For the mammalian cell that does not have this type of cell walls, can adopt Graham and van der Eb, Virology, the calcium phosphate precipitation method of 52:456-457 (1978).The general aspect of mammalian cell host system transfection has been recorded in U.S. Patent No. 4,399,216.Typically, according to Van Solingen etc., J.Bact., 130:946 (1977) and Hsiao etc., Proc.Natl.Acad.Sci. (USA), the method for 76:3829 (1979) is implemented the conversion in yeast.Yet, can also use other method that is used for the DNA transfered cell, merge or polycation, for example Polybrene, polyornithine such as the bacterium protoplastis that carries out by nuclear microinjection, electroporation, with intact cell.For the various technology of transformed mammalian cell, referring to Keown etc., Methods in Enzymology, 185:527-537 (1990) and Mansour etc., Nature, 336:348-352 (1988).

The host cell that is suitable for cloning or express the DNA in this paper carrier comprises prokaryotic cell prokaryocyte, yeast cell or higher eucaryotic cells.Suitable prokaryotic organism include but not limited to eubacterium, and such as gram-negative or gram-positive organism, enterobacteriaceae for example is such as intestinal bacteria.Multiple coli strain is that the public is obtainable, such as e. coli k12 strain MM294 (ATCC 31,446); Intestinal bacteria X1776 (ATCC 31,537); Coli strain W3110 (ATCC 27,325) and K5772 (ATCC 53,635).Other suitable prokaryotic host cell comprises enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia) (for example intestinal bacteria or bacillus coli (E.coli)), enterobacter (Enterobacter), erwinia (Erwinia), Klebsiella (Klebsiella), proteus (Proteus), salmonella (Salmonella) (for example Salmonella typhimurium (Salmonellatyphimurium)), serratia (Serratia) (for example serratia marcescens (Serratiamarcescans)), and Shigella (Shigella), and bacillus (Bacilli) (such as subtilis (B.subtilis) and Bacillus licheniformis (B.licheniformis) (for example Bacillus licheniformis 41P disclosed in the disclosed DD 266,710 on April 12nd, 1989)), Rhodopseudomonas (Pseudomonas) (such as Pseudomonas aeruginosa (P.aeruginosa)), and streptomyces (Streptomyces).These examples are exemplary and nonrestrictive.Bacterial strain W3110 is a kind of special preferred host or parent host, because it is the host strain commonly used for recombinant DNA product fermentation usefulness.Preferably, the minimum proteolytic ferment of secretory host cell.For example, can modify bacterial strain W3110 to produce genetic mutation in the gene of coding host endogenous protein, this type of host's example comprises intestinal bacteria W3110 bacterial strain 1A2 (it has complete genotype tonA); Intestinal bacteria W3110 bacterial strain 9E4 (it has complete genotype tonAptr3); (it has complete genotype tonA ptr3phoA E15 (argF-lac) 169 degP ompT kan to intestinal bacteria W3110 bacterial strain 27C7 (ATCC 55,244) ^r); (it has complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan to intestinal bacteria W3110 bacterial strain 37D6 ^r); Intestinal bacteria W3110 bacterial strain 40B4 (it is the bacterial strain 37D6 that contains non-kalamycin resistance degP deletion sudden change); And the coli strain of the disclosed mutant periplasm protein enzyme of the U.S. Patent No. 4,946,783 with August 7 nineteen ninety bulletin.Perhaps, body outer clone method (for example PCR or other nucleic acid polymerase reaction) is suitable.

Outside prokaryotic organism, eukaryotic microorganisms (such as filamentous fungus or yeast) is clone or the expressive host that is suitable for DR6 peptide coding carrier.Yeast saccharomyces cerevisiae or Saccharomyces cerevisiae (Saccharomycescerevisiae) are the eucaryon host microorganisms of using always such as low.Other comprise grain wine fragmentation sugar yeast (Schizosaccharomyces pombe) (Beach and Nurse, Nature, 290:140[1981]; The EP 139,383 that on May 2nd, 1985 announced); Crewe Vickers yeast belong (Kluyveromyces) host (U.S. Patent No. 4,943,529; Fleer etc., Bio/Technology, 9:968-975 (1991)), such as for example newborn Crewe Vickers yeast (K.lactis) (MW98-8C, CBS683, CBS4574; Louvencourt etc., J.Bacteriol., 154 (2): 737-742[1983]), (ATCC 12 for crisp wall Crewe Vickers yeast (K.fragilis), 424), (ATCC 16 for Bulgarian Crewe Vickers yeast (K.bulgaricus), 045), (ATCC 24 for Brunswick Man Crewe Vickers yeast (K.wickeramii), 178), K.waltii (ATCC 56,500), fruit bat Crewe Vickers yeast (K.drosophilarum) (ATCC 36,906); Van den Berg etc., Bio/Technology, 8:135 (1990)), heat-resisting Crewe Vickers yeast (K.thermotolerans), and Marx's Crewe Vickers yeast (K.marxianus); Inferior sieve yeast belong (yarrowia) (EP 402,226); (EP 183,070 for Pichia pastoris (Pichiapastoris); Sreekrishna etc., J.Basic Microbiol., 28:265-278[1988]); Mycocandida (Candida); Rui Shi wood mould (Trichoderma reesia) (EP 244,234); Coarse arteries and veins spore mould (Neurospora crassa) (Case etc., Proc.Natl.Acad.Sci.USA, 76:5259-5263[1979]); Permitted Wang Shi yeast belong (Schwanniomyces), permitted Wang Shi yeast (Schwanniomyces occidentalis) (EP 394,538 that announce October 31 nineteen ninety) such as the west; And filamentous fungus, such as for example arteries and veins spore mould (Neurospora), Penicillium (Penicillium), the curved mould genus of neck (Tolypocladium) (WO 91/00357 that on January 10th, 1991 announced), and Aspergillus (Aspergillus) host, such as Aspergillus nidulans (A.nidulans) (Balance etc., Biochem.Biophys.Res.Commun., 112:284-289[1983]; Tilburn etc., Gene, 26:205-221[1983]; Yelton etc., Proc.Natl.Acad.Sci.USA, 81:1470-1474[1984]) and aspergillus niger (A.niger) (Kelly and Hynes, EMBO J., 4:475-479[1985]).Methylotrophic yeast is suitable in this article, and includes but not limited to be selected from down yeast dependent of dead military hero, that can grow on methyl alcohol: Hansenula anomala belongs to (Hansenula), mycocandida, Ke Lekeshi yeast belong (Kloeckera), pichia genus (Pichia), saccharomyces (Saccharomyces), torulopsis (Torulopsis), reaches Rhodotorula (Rhodotorula).The tabulation of the particular types that this class yeast is exemplary can be referring to C.Anthony, TheBiochemistry of Methylotrophs, 269 (1982).

Be suitable for expressing the host cell of glycosylation DR6 and/or APP polypeptide derived from multicellular organisms.The example of invertebral zooblast comprises insect cell (such as fruit bat S2 and noctuid (Spodoptera) Sf9), and vegetable cell, such as cotton, corn, potato, soybean, morning glory, tomato, and the cell culture of tobacco.A lot of baculovirus strains and variant and corresponding have been identified from the permission insect host cell of organizing the host down, such as fall army worm (Spodoptera frugiperda) (caterpillar), Aedes aegypti (Aedes aegypti) (mosquito), Aedes albopictus (Aedes albopictus) (mosquito), drosophila melanogaster (Drosophila melanogaster) (fruit bat), and silkworm (Bombyx mori).The multiple virus strain that is used for transfection is the public's obtainable (for example the L-1 variant of autographa california (Autographa californica) NPV and Bm-5 strain of silkworm NPV), and this viroid can be used as according to virus herein of the present invention, especially for transfection fall army worm cell.

Yet, the interest maximum be vertebrate cells, and make the vertebrate cells propagation in the cultivation (tissue culture) become conventional rules.The example of useful mammalian host cell line is the monkey kidney CV1 system (COS-7, ATCC CRL 1651) that transforms through SV40; Human embryo kidney (HEK) system (293 cells or for growth in suspension culture 293 cells of subclone, Graham etc., J.Gen Virol.36:59 (1977)); Hamster nephrocyte childhood (BHK, ATCC CCL 10); Chinese hamster ovary cell/-DHFR (CHO, Urlaub etc., Proc.Natl.Acad.Sci.USA 77:4216 (1980)); Mouse Sai Tuoli (sertoli) cell (TM4, Mather, Biol.Reprod.23:243-251 (1980)); Monkey-kidney cells (CV1ATCC CCL 70); African green monkey kidney cell (VERO-76, ATCC CRL-1587); Human cervical carcinoma cell (HELA, ATCC CCL 2); Madin-Darby canine kidney(cell line) (MDCK, ATCC CCL 34); Ox mouse (buffalo rat) liver cell (BRL 3A, ATCC CRL 1442); Human pneumonocyte (W138, ATCC CCL 75); Human liver cell (Hep G2, HB 8065); MMT (MMT 060562, ATCC CCL51); TRI cell (Mather etc., Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC 5 cells; The FS4 cell; And people's hepatoma (hepatoma) is (Hep G2).

Host cell is generated the expression or the cloning vector conversion of usefulness with mentioned above for DR6 and/or APP polypeptide, and in conventional nutritional medium, cultivate for evoked promoter, the gene of selecting transformant or amplification coding expectation sequence and suitably improvement.

The selection of replicable vector and use

The nucleic acid (for example cDNA or genomic dna) of encoding D R6 and/or APP polypeptide can be inserted in replicable vector clone's (amplification of DNA) usefulness or for expression usefulness.Variety carrier is that the public is obtainable.Carrier can be for example plasmid, clay, virion or phage form.Suitable nucleotide sequence can insert in the carrier by multiple rules.Generally speaking, use technology known in the art that DNA is inserted in the suitable restriction endonuclease site.Support element generally comprise but be not limited to following one or more: signal sequence, replication orgin, one or more marker gene, enhancer element, promotor, and transcription termination sequence.The structure that contains the suitable carrier of one or more these members adopts for standard interconnection technique known to the skilled.

DR6 and/or the APP generation of not only can directly recombinating, but also can be used as the fusion polypeptide that contains heterologous polypeptide (it can be at the signal sequence of the N of maturation protein or polypeptide end or have other polypeptide of specific cleavage site) and be re-combined into.Generally speaking, signal sequence can be the member of carrier, and perhaps it can be to insert a DR6 in the carrier and/or the part of APP peptide coding DNA.Signal sequence can be a prokaryotic signal sequence, and it is selected from for example alkaline phosphatase, penicillinase, lpp or heat-staple enterotoxin 1 I leader sequence.For yeast secretary, signal sequence can be that for example yeast invertase leader sequence, α-factor leader sequence (comprise saccharomyces and Crewe Vickers yeast belong α-factor leader sequence, the latter is recorded in U.S. Patent No. 5,010,182) or the signal put down in writing of acid phosphatase leader sequence, white candiyeast (C.albicans) glucoamylase leading (EP 362,179 that announce April 4 nineteen ninety) or the WO 90/13646 that announces November 15 nineteen ninety.In mammalian cell expression, can use the mammalian signal sequence to instruct protein secreting, such as signal sequence from the secreted polypeptide of identical or relevant species, and the viral secretory leader sequence.

Expression vector and cloning vector all contain the nucleotide sequence that makes that carrier can duplicate in one or more selected host cells.This type of sequence that is used for various bacteria, yeast and virus is known.Replication orgin from plasmid pBR322 is applicable to most of gram negative bacteriums, 2 μ plasmid starting points are applicable to yeast, and various viral starting point (SV40, polyomavirus, adenovirus, VSV or BPV) is useful for the cloning vector in the mammalian cell.

Expression and cloning vector typically can comprise selects gene (being also referred to as selection marker).The typical genes encoding following proteins of selecting, its (a) gives the resistance to microbiotic or other toxin (for example penbritin, Xin Meisu, methotrexate or tsiklomitsin), (b) extra-nutrition defective type defective, or (c) provide the crucial nutrition that can not obtain from complex medium, the gene of the genus bacillus D-alanine racemase of for example encoding.

The example that is applicable to the selection marker of mammalian cell is the selection marker that those feasible cells of having the ability picked-up DR6 and/or APP peptide coding nucleic acid are identified, such as DHFR or thymidine kinase.When adopting wild-type DHFR, appropriate host cell is the Chinese hamster ovary celI system of the active defective of DHFR, and its preparation and propagation is as Urlaub etc., Proc.Natl.Acad.Sci.USA, and 77:4216 (1980) is described.The selection gene that is fit to use in the yeast is trp1 gene [Stinchcomb etc., Nature, the 282:39 (1979) that exists among the yeast plasmid YRp7; Kingsman etc., Gene, 7:141 (1979); Tschemper etc., Gene, 10:157 (1980)].The trp1 gene provides the selection marker for the yeast mutation type bacterial strain that lacks energy for growth in tryptophane (for example ATCC No.44076 or PEP4-1[Jones, Genetics, 85:12 (1977)]) usefulness.

Expression and cloning vector comprise the promotor that can be operatively connected with DR6 and/or APP peptide coding nucleotide sequence usually and synthesize to instruct mRNA.The promotor that multiple potential host cell is discerned is known.Be suitable for comprising β-Nei Xiananmei and lactose promoter systems [Chang etc., Nature, 275:615 (1978) with the promotor that prokaryotic hosts uses; Goeddel etc., Nature, 281:544 (1979)], alkaline phosphatase, tryptophane (trp) promoter systems [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP36,776], and hybrid promoter, such as tac promotor [deBoer etc., Proc.Natl.Acad.Sci.USA, 80:21-25 (1983)].The promotor of using in the bacterial system also can comprise Shine-Dalgamo (S.D.) sequence that the DNA with encoding D R6 and/or APP polypeptide can be operatively connected.

The example that is suitable for the initiating sequence that uses with yeast host comprises the kinase whose promotor [Hitzeman etc. of 3-phoshoglyceric acid, J.Biol.Chem., 255:2073 (1980)] or other glycolytic ferment (such as Hydratase, phosphoenolpyruvate, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvic carboxylase, phosphofructokinase, G-6-P isomerase, 3-phoshoglyceric acid mutase, pyruvate kinase, triose-phosphate isomerase, glucose phosphate isomerase, and glucokinase) promotor [Hess etc., J.Adv.Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)].

Other Yeast promoter (it is to have additional advantage promptly to transcribe the inducible promoter that is subjected to growth conditions control) is the promoter region of histone matter down: alcoholdehydrogenase 2, different cell pigment (isocytochrome) C, acid phosphatase, the degrading enzyme relevant with nitrogen metabolism, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase, and the enzyme of responsible maltose and galactose utilization.Carrier that is suitable for using in yeast expression and promotor have further description in EP 73,657.

In mammalian host cell, transcribe and be subjected to following promotor control from the DR6 of carrier and/or APP polypeptide, for example viral certainly (such as polyomavirus, fowlpox virus (the UK2 that on July 5th, 1989 announced, 211,504), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcomata virus, cytomegalovirus, retrovirus, hepatitis B virus and simian virus 40 (SV40)) genome, allos mammalian promoter (for example actin promoter or immunoglobulin promoter), reach the promotor that obtains from the heat-shocked promotor, if the compatible words of this type of promotor and host cell systems.

The DNA that higher eucaryote is transcribed encoding D R6 and/or APP polypeptide can be increased by enhancer sequence is inserted in the carrier.Enhanser is that promotor is worked and increases the cis-acting DNA element that it is transcribed, and is generally about 10-300bp.At present known many enhancer sequence from mammalian genes (globin, elastoser, white protein, alpha-fetoprotein, and Regular Insulin).Typically, yet, enhanser can be used from eukaryotic cell virus.The late period that example comprises replication orgin SV40 enhanser (bp 100-270) on the side, the sub-enhanser of cytomegalovirus early promoter, replication orgin the polyomavirus enhanser of late period on the side, and adenovirus enhanser.Enhanser can be gone in the carrier in 5 ' or 3 ' position montage of DR6 and/or APP polypeptid coding sequence, but is preferably placed at the site of promotor 5 '.

The expression vector that uses in the eukaryotic host cell (yeast, fungi, insect, plant, animal, people or from the karyocyte of other multicellular organisms) also can comprise Transcription Termination and make mRNA stablize necessary sequence.This type of sequence usually can be available from the 5 ' non-translational region of Eukaryotic or viral DNA or cDNA, 3 ' non-translational region occasionally.These zones comprise the Nucleotide section of transcribing as the polyadenylation fragment in the untranslated part of the mRNA of encoding D R6 polypeptide.

Be suitable for adapting other method, carrier and the host cell that in the recombinant vertebrate cell culture, synthesize DR6 and/or APP polypeptide in the back and be recorded in Gething etc., Nature, 293:620-625 (1981); Mantei etc., Nature, 281:40-46 (1979); EP 117,060; And EP 117,058.

Cultivate host cell

The host cell that is used for generating DR6 of the present invention and/or APP polypeptide can be cultivated at multiple substratum.Commercial substratum (such as HamShi F10 (Sigma), minimum essential medium (MEM) (Sigma), RPMI-1640 (Sigma), and DulbeccoShi improvement EagleShi substratum ((DMEM) Sigma)) be suitable for cultivating host cell.In addition, Ham etc., Meth.Enz.58:44 (1979); Barnes etc., Anal.Biochem.102:255 (1980), U.S. Patent No. 4,767,704; 4,657,866; 4,927,762; 4,560,655; Or 5,122,469; WO 90/03430; WO 87/00195; Or United States Patent (USP) Re.30, any substratum described in 985 can be as the substratum of host cell.Any of these substratum when needed hormone supplemented and/or other somatomedin (such as Regular Insulin, transferrin or Urogastron), salt (such as sodium-chlor, calcium, magnesium, and phosphoric acid salt), buffer reagent (such as HEPES), Nucleotide (such as adenosine and thymidine), microbiotic be (such as GENTAMYCIN ^TMMedicine), trace elements (being defined as the mineral compound that usually exists), and glucose or be equal to the energy with the final concentration in the micro-molar range.Can also comprise that those skilled in the art are any other essential fill-ins of known proper concn.Culture condition (such as temperature, pH etc.) is exactly that those before used with the host cell of selecting to be used to express, and is conspicuous for those of ordinary skill.

Detect gene amplification/expression

Based on the sequence that this paper provided, gene amplification and/or expression can directly be measured in sample, the probe that for example uses suitable mark is by conventional Southern trace, Northern trace (in order to transcribing of quantification of mrna) [Thomas, Proc.Natl.Acad.Sci.USA, 77:52015205 (1980)], Dot blot (DNA analysis) or in situ hybridization realizes.Perhaps, can adopt the antibody that can discern specific duplex, described duplex comprises DNA duplex, RNA duplex, reaches DNA-RNA heterozygosis duplex or DNA-protein duplex.Then can traget antibody, and can implement assay method, wherein duplex is bonded to the surface, make form duplex from the teeth outwards after, can detect the existence of duplex institute bonded antibody.

Perhaps, genetic expression can be measured the expression with the direct quantitative gene product by immunological method (such as the immunohistochemical staining of cell or tissue section and the assay method of cell culture or body fluid).For immunohistochemical staining and/or the useful antibody of humoral determination method can be monoclonal or polyclonal, and can prepare in any Mammals.Easily, antibody can be at native sequences DR6 polypeptide or at the synthetic peptide that the DR6 sequence is provided based on this paper or at preparing with the exogenous array of DR6DNA fusion and coding specific antibodies epi-position.

The purifying of DR6 polypeptide

Can reclaim various forms of DR6 and/or APP polypeptide from substratum or from the host cell lysate.If membrane-bound, then can use suitable detergent solution (for example Triton-X 100) or by enzymatic cutting and it is discharged from described film.The cell that is adopted in the DR6 polypeptide expression can destroy by multiple physics or chemical means (such as the molten born of the same parents' agent of freeze thaw circulation, supersound process, Mechanical Crushing or cell).

May wish purifying DR6 and/or APP polypeptide from recombinant cell protein or polypeptide.Following rules are illustrations of suitable purifying rules: by at the ion-exchange column fractionation; Ethanol sedimentation; Reversed-phase HPLC; On the tripoli or the chromatography on the Zeo-karb (such as DEAE); Chromatofocusing; SDS-PAGE; Ammonium sulfate precipitation; Use for example gel-filtration of Sephadex G-75; Albumin A Sepharose post is to remove pollutent, such as IgG; And metal chelating column adds the DR6 and/or the APP polypeptide of epi-position label form with combination.Can adopt the several different methods of protein purification, and these class methods are well known in the art, and are recorded in for example Deutscher, Methods in Enzymology, 182 (1990); Scopes, ProteinPurification:Principles and Practice, Springer-Verlag, New York (1982).Selected purification step can depend on the character of for example employed generation method and the specific DR6 polypeptide that is generated.

Can adopt the DR6 of soluble form and/or APP as the DR6 antagonist in the method for the present invention.The DR6 of this type of soluble form and/or APP can comprise modification, (such as by merging with immunoglobulin (Ig), epi-position label or leucine zipper) as mentioned below.Further imagined the immunoadhesin molecule has been used for herein method.DR6 and/or APP immunoadhesin can comprise various forms of DR6 and/or APP, such as full-length polypeptide and DR6 and/or APP or its fragment solvable, the ectodomain form.In specific embodiment, described molecule can comprise the fusions of DR6 polypeptide and immunoglobulin (Ig) or its specific region.For the immunoadhesin of bivalent form, this type of fusions can be the fusions with IgG molecule Fc district.(membrane spaning domain deletion or deactivation) polypeptide that the Ig fusions preferably comprises with soluble form replaces substituting of at least one variable region of Ig intramolecularly.In an especially preferred embodiment, the immunoglobulin (Ig) fusions comprises hinge, CH2 and the CH3 of IgG1 molecule, perhaps hinge, CH1, CH2 and CH3 district.Generation about the immunoglobulin (Ig) fusions also can be referring to United States Patent (USP) the 5th, 428, and No. 130, June 27 nineteen ninety-five bulletin and Chamow etc., TIBTECH, 14:52-60 (1996).

A kind of optional immunoadhesin design is that the binding domains of adhesin (for example DR6 and/or APP extracellular domain) and the Fc district of heavy chain immunoglobulin are made up.Usually, in preparation during immunoadhesin of the present invention, the nucleic acid of coding adhesin binding domains is merged C-end side at the nucleic acid of coding constant region for immunoglobulin sequence N-end, yet the terminal fusions of N-also is possible.

Usually, in this type of fusions, coded chimeric polyeptides will remain with immunoglobulin heavy chain constant region hinge, the CH of functionally active at least ₂And CH ₃Structural domain.Can also merge constant region fc partial C-end, perhaps be close to heavy chain CH ₁Or the N-end in the corresponding district of light chain carries out.The accurate site of merging is not vital; Concrete site is well-known, and can select with the biologic activity of optimizing immunoadhesin, secretion or in conjunction with feature.

In a preferred embodiment, the adhesin sequence is merged at immunoglobulin G ₁(IgG ₁) the N-end in Fc district.Whole CH and adhesin sequence might be merged.Yet, more preferably, in fusion, use and in hinge area, be close to the sequence that begins in the upstream, similar site that chemically defines the papoid cleavage site of IgG Fc (being residue 216, is 114 with first residue of CH) or other immunoglobulin (Ig).In an especially preferred embodiment, with (a) hinge area, the CH of adhesin aminoacid sequence and IgG heavy chain ₂And CH ₃, perhaps (b) CH ₁, hinge, CH ₂And CH ₃Structural domain merges.

For the dual specific immunoadhesin, immunoadhesin is assembled into polymer, particularly the heterodimer or the different tetramer.Usually, these immunoglobulin (Ig)s that assemble will have known modular construction.Four basic chain structure unit are exactly the form that IgG, IgD and IgE exist.Four chain units are more repeating in the high-molecular weight immunoglobulin (Ig); IgM exists as the pentamer of four elementary cells that keep together by disulfide linkage usually.IgA sphaeroprotein and IgG sphaeroprotein once in a while also can be present in the serum with polymeric form.In polymeric situation, four unit can be identical separately or different.

Schematically enumerated the various exemplary immunoadhesin that assembles in this paper scope below:

(a)AC _L-AC _L；

(b) AC _H-(AC _H, AC _L-AC _H, AC _L-V _HC _H, or V _LC _L-AC _H);

(c) AC _L-AC _H-(AC _L-AC _H, AC _L-V _HC _H, V _LC _L-AC _H, or V _LC _L-V _HC _H)

(d) AC _L-V _HC _H-(AC _H, AC _L-V _HC _H, or V _LC _L-AC _H);

(e) V _LC _L-AC _H-(AC _L-V _HC _H, or V _LC _L-AC _H); And

(f)(A-Y)n-(V _LC _L-V _HC _H) ₂，

Wherein each A represents identical or different adhesin aminoacid sequence;

V _LIt is the immunoglobulin light chain variable territory;

V _HIt is the immunoglobulin heavy chain variable territory;

C _LIt is the immunoglobulin light chain constant territory;

C _HIt is the heavy chain immunoglobulin constant domain;

N is the integer greater than 1;

Y represents the residue of covalent crosslinking agent.

For the purpose of brief, said structure has only shown key feature; They do not indicate connection (J) or other structural domain of immunoglobulin (Ig), do not show disulfide linkage yet.Yet if need this type of structural domain in conjunction with activity, they should be present in their occupied common positions in immunoglobulin molecules when making up.

Perhaps, the adhesin sequence can be inserted between heavy chain immunoglobulin and the sequence of light chain, thereby be obtained comprising the immunoglobulin (Ig) of chimeric heavy chain.In this embodiment, with 3 ' end of the heavy chain immunoglobulin of adhesin sequence fusion in each arm of immunoglobulin (Ig), or at hinge and CH ₂Between the structural domain, or at CH ₂With CH ₃Between the structural domain.Similarly construction has been reported in Hoogenboom etc., Mol.Immunol., 28:1027-1037 (1991).

Although do not need to exist light chain immunoglobulin in the immunoadhesin of the present invention, however light chain immunoglobulin can exist with the covalently bound form of adhesin-heavy chain immunoglobulin fusion polypeptide, or directly to exist with the form of adhesin fusion.In the previous case, the DNA coexpression of the DNA of the light chain immunoglobulin of will encoding usually and coding adhesin-heavy chain immunoglobulin fusion rotein.After secretion, the heterozygosis heavy chain comprises heavy chain immunoglobulin-light chain right immunoglobulin (Ig) spline structure that two disulfide linkage link to each other with covalent attachment to provide with light chain.The method that is suitable for preparing this class formation for example is disclosed in United States Patent (USP) the 4th, 816, No. 567, is announced on March 28th, 1989.Most convenient be that cDNA sequence by the adhesin part of will encoding and immunoglobulin (Ig) cDNA sequence merge with the form that meets reading frame and make up immunoadhesin.Yet, also can use fusion with the genome immunoglobulin fragment (referring to for example Aruffo etc., Cell, 61:1303-1313 (1990); With Stamenkovic etc., Cell, 66:1133-1144 (1991)).The fusion that the back is one type requires to exist the Ig regulating and controlling sequence for expression.The cDNA of coding IgG CH can be according to reported sequence is self-derived from the separation of the cDNA library of spleen or peripheral blood lymphocyte, by hybridization or by polymerase chain reaction (PCR) technology." adhesin " of coding immunoadhesin and the cDNA series connection of immunoglobulin part are inserted in the plasmid vector that efficiently expresses in the selected host cell.

In another embodiment, the DR6 antagonist can pass through with United States Patent(USP) Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179, listed mode is one of receptor polypeptides and multiple nonprotein character polymkeric substance in 337, for example polyoxyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene, and perhaps other similar molecule such as polyglutamic acid esters/salt (polyglutamate) connects and covalent modification.This type of PEGization form can use technology known in the art to prepare.

The present invention has also imagined these molecules of leucine zipper form." leucine zipper " is the term that is rich in the leucine sequence that is used in reference to enhancing in this area, promotes or drives its fusion partner (for example leucine zipper merge with it or connect sequence or molecule) dimerization or trimerizing.Multiple leucine zipper polypeptide has been put down in writing in this area.Referring to for example Landschulz etc., Science, 240:1759 (1988); United States Patent (USP) 5,716,805; WO 94/10308; Hoppe etc., FEBS Letters, 344:1991 (1994); Maniatis etc., Nature, 341:24 (1989).Those skilled in the art will understand, and the leucine zipper sequence can merge in 5 of DR6 molecule ' or 3 ' end.

DR6 of the present invention and/or APP polypeptide can also be modified as follows, promptly pass through polypeptide and another kind of heterologous polypeptide or aminoacid sequence fusion formation chimeric molecule.Preferably, the effect of described heterologous polypeptide or aminoacid sequence is to make the chimeric molecule oligomerization.In one embodiment, this type of chimeric molecule comprises the fusion of DR6 and/or APP polypeptide and label polypeptide, and described label polypeptide provides anti-tag antibody alternative bonded epi-position.Usually the epi-position label is placed the amino or the C-terminal of polypeptide.The situation that exists of the polypeptide of this type of epi-position mark pattern can use the antibody at the label polypeptide to detect.Also have, the providing of epi-position label makes polypeptide be easy to use anti-tag antibody or carries out purifying in conjunction with the affine mechanism of other type of epi-position label by affinity purification.Multiple label polypeptide and corresponding antibody thereof are well-known in the art.Example comprises polyhistidyl (poly-his) or polyhistidyl-glycine (poly-his-gly) label; Influenza HA label polypeptide and antibody 12CA5 thereof (Field etc., Mol.Cell.Biol., 8:2159-2165 (1988)); C-myc label and antibody 8F9,3C7,6E10, G4, B7 and 9E10 (Evan etc., Molecular and Cellular Biology, 5:3610-3616 (1985)); And herpes simplex virus glycoprotein D (gD) label and antibody (Paborsky etc., Protein Engineering, 3 (6): 547-553 (1990)).Other label polypeptide comprises Flag peptide (Hopp etc., BioTechnology, 6:1204-1210 (1988)); KT3 epitope peptide (Martin etc., Science, 255:192-194 (1992)); Alpha-tubulin epitope peptide (Skinner etc., J.Biol.Chem., 266:15163-15166 (1991)); And T7 gene 10 protein peptide tags (Lutz-Freyermuth etc., Proc.Natl.Acad.Sci.USA, 87:6393-6397 (1990)).

Anti-DR6 and anti-APP antibody

Other embodiment of the present invention provides DR6 and/or APP antibody.That exemplary antibody comprises is polyclonal, monoclonal, humanized, dual specific with allos link coupled antibody.These anti-DR6 and/or APP antibody is the DR6 antagonistic antibodies preferably.

Polyclonal antibody

Antibody of the present invention can comprise polyclonal antibody.The method that is used to prepare polyclonal antibody is well known by persons skilled in the art.Polyclonal antibody can generate in Mammals, for example by agent of one or many injecting immune and adjuvant as required.Usually, immunizing agent and/or adjuvant will be expelled in the Mammals by subcutaneous or peritoneal injection repeatedly.Immunizing agent can comprise DR6 and/or APP polypeptide (for example DR6 and/or APP ECD) or its fusion rotein.With immunizing agent and known immunogenic protein coupling to be arranged in the immune Mammals may be useful.The example of this type of immunogenic protein includes but not limited to keyhole

Hemocyanin, serum albumin, bovine thyroglobulin and Trypsin inhibitor SBTI.The example of adoptable adjuvant comprises Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose two diphtherinic acid esters (dicorynomycolate)).Immunization protocol can need not too much by those skilled in the art, and experiment makes a choice.Can take a blood sample to Mammals then, and to determination of serum DR6 and/or APP antibody titers.As required, can carry out booster immunization, raise or reach stable high density (plateau) up to titre to Mammals.

Monoclonal antibody

Perhaps, antibody of the present invention can be monoclonal antibody.Monoclonal antibody can be used the preparation of hybridoma method, and such as Kohler and Milstein, Nature 256:495 (1975) is described.In the hybridoma method, mouse, hamster or other suitable host animal generate the lymphocyte that maybe can generate the antibody of specificity binding immunoassay agent with the immunizing agent immunity to cause usually.Perhaps, can be at external immune lymphocyte.

Immunizing agent will generally include DR6 and/or APP polypeptide (for example DR6 and/or APP ECD) or its fusion rotein, such as DR6ECD-IgG and/or APP sAPP-IgG fusion rotein.

Usually, if wish then to use peripheral blood lymphocyte (" PBL "), perhaps, then use splenocyte or lymph-node cell for the cell in non-human mammal source if wish for the cell of people's origin.Then, use suitable fusogen, such as polyoxyethylene glycol, lymphocyte and immortalized cell line are merged to form hybridoma (Goding, Monoclonal Antibodies:Principles and Practice, AcademicPress (1986), pp.59-103).The myeloma cell of the mammalian cell that immortalized cell line normally transforms, particularly rodents, ox and people's origin.Usually, adopt rat or mouse myeloma cell line.Hybridoma can be cultivated in suitable medium, and described substratum preferably contains immortalized cells growth that inhibition do not merge or one or more materials of survival.For example, if parental cell lacks hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the substratum that is used for hybridoma so will contain xanthoglobulin, aminopterin-induced syndrome and thymidine (" HAT substratum ") usually, and these materials stop the growth of HGPRT deficient cells.

Preferred immortalized cell line is that those efficiently merge, support selected antibody-producting cell high level ground expressing antibodies and to the clone such as substratum sensitivities such as HAT substratum stably.Preferred immortalized cell line is a mouse source myelomatosis system, can be from for example Sol gram cell distribution center (the SalkInstitute Cell Distribution Center of institute, San Diege, California, USA) and American type culture collection (American Type Culture Collection, Manassas, Virginia USA) obtains.An example of this type of rat bone marrow tumour cell system is P3X63Ag8U.1 (ATCC CRL 1580).Be used to generate the human myeloma and also existing (Kozbor, the J.Immunol.133:3001 (1984) of describing of mouse-people's allos myeloma cell line of human monoclonal antibodies; Brodeur etc., Monoclonal Antibody ProductionTechniques and Applications, Marcel Dekker, Inc., New York (1987) pp.51-63).

The substratum that can cultivate therein hybridoma is measured the existence at the monoclonal antibody of DR6 and/or APP then.Preferably, by immunoprecipitation or by external binding assay,, measure the binding specificity of the monoclonal antibody that generates by hybridoma such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).This type of technology and assay method are known in the art.The binding affinity of monoclonal antibody can be by Munson and Pollard for example, and the Scatchard of Anal.Biochem.107:220 (1980) analyzes or analyzes by BiaCore and measures.

Behind the hybridoma that evaluation obtains expecting, this clone can carry out subclone by the limiting dilution flow process, and cultivates (Goding sees above) by standard method.The substratum that is suitable for this purpose comprises the EagleShi substratum or the RPMI-1640 substratum of for example DulbeccoShi improvement.Perhaps, hybridoma can carry out culturing in vivo as ascites in Mammals.

Can pass through routine immunization sphaeroprotein purifying flow process,, subclone excretory monoclonal antibody and substratum or ascites be separated or purifying such as for example albumin A-Sepharose, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibody also can generate by recombinant DNA technology, such as United States Patent (USP) 4,816, described in 567.The DNA of coding monoclonal antibody is easy to use old process to separate and order-checking (for example use can specificity in conjunction with the oligonucleotide probe of the gene of coding monoclonal antibody heavy chain and light chain).Hybridoma is the preferred source of this type of DNA.In case separate, DNA can be placed expression vector, then with this expression vector transfection to not producing in addition in the proteinic host cell of immunoglobulin (Ig), such as Bacillus coli cells, ape and monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell, in recombinant host cell, to obtain the synthetic of monoclonal antibody.All right modifying DNA, for example by substituting, promptly the choose encoding sequence of heavy chain and constant region of light chain replaces homology mouse source sequence (Morrison etc., Proc.Nat.Acad.Sci.81:6851 (1984)), perhaps engage the whole or part encoding sequence of immunoglobulin coding sequence and NIg polypeptide by covalency." chimeric " or " heterozygosis " antibody that can prepare binding specificity herein in this mode with anti-DR6 monoclonal antibody.

Usually substitute the constant domain of antibody of the present invention with this type of NIg polypeptide, perhaps the variable domain of an antigen binding site that substitutes antibody of the present invention with their to be producing chimeric bivalent antibody, and it comprises DR6 is had a specific antigen binding site and synantigen is not had specific another antigen binding site.

Chimeric or hybrid antibody also can prepare at the currently known methods of external use synthetic protein chemistry, comprises that those relate to the method for linking agent.For example, can use disulfide exchange reaction or make up immunotoxin by forming thioether bond.The example that is suitable for the reagent of this purpose comprises imino-mercaptan ester/salt (iminothiolate) and 4-sulfydryl butyryl imidic acid methyl esters (methyl-4-mercaptobutyrimidate).

Also can generate strand Fv fragment, such as Iliades etc., FEBS Letters is described in the 409:437-441 (1997).This type of single-chain fragment uses the coupling of various terminal to be recorded in Kortt etc., ProteinEngineering, 10:423-433 (1997).The multiple technologies that are used for recombinant production and operation antibody are known in this area.Hereinafter more detailed description the il-lustrative example of normally used these type of technology of those of skill in the art.

Humanized antibody

Usually, one or more amino-acid residues have been introduced in the humanized antibody from inhuman source.These inhuman amino-acid residues are often referred to as " input " residue, and they take from " input " variable region usually.Humanization can be followed Winter and colleague's thereof method basically and carry out (Jones etc., Nature 321:522-525 (1986); Riechmann etc., Nature 332:323-327 (1988); Verhoeyen etc., Science 239:1534-1536 (1988)), promptly use the corresponding sequence of rodents CDR or CDR sequence replacing people antibody.

Therefore, this type of " humanization " antibody is chimeric antibody, wherein is less than whole people variable region basically and uses the corresponding sequence from inhuman species to substitute.In practice, humanized antibody some of them CDR residue and some possible FR residues residue alternate people antibody normally from similar site in the rodents antibody.

Importantly, antibody keeps behind humanization antigenic high-affinity and other favourable biological characteristics.In order to realize this goal, according to a kind of preferable methods, the method for analyzing parental array and each ways makes conceptual researches humanization product by the three-dimensional model that uses parent and humanization sequence prepares humanized antibody.Three-dimensional immunoglobulin (Ig) model is normally obtainable, and is familiar with by those skilled in the art.Can obtain the computer program of the possible three-dimensional conformation structure of diagram and the selected candidate's immunoglobulin sequences of demonstration.Check that these display images are allowed and analyze residue may act in candidate's immunoglobulin sequences functionating, promptly analyzing influence candidate immunoglobulin (Ig) is in conjunction with the residue of its antigenic ability.Like this, can from total and list entries, select the FR residue and make up, thereby obtain expectation antibody feature, improve such as avidity to target antigen.Generally speaking, the CDR residue directly and relating to of essence the antigen bonded is influenced.

People's antibody

Human monoclonal antibodies can generate by hybridoma method.Be used to generate the human myeloma of human monoclonal antibodies and mouse-people's allos myeloma cell line is existing describes, Kozbor for example, J.Immunol.133:3001 (1984); Brodeur etc., Monoclonal Antibody Production Techniques andApplications, pp.51-63, Marcel Dekker, Inc., New York (1987).

Might be created on the transgenic animal (for example mouse) that can when immunity, generate the complete complete or collected works of people's antibody under the situation that lacks endogenous immunoglobulin (Ig) generation now.For example, put down in writing heavy chain of antibody joining region (J in the chimeric and germ line mutation mouse _H) deletion of isozygotying of the gene inhibition fully that causes endogenous antibody to generate.Shifting a whole set of ethnic group in this type of germ line mutation mouse is that immunoglobulin gene will cause generating people's antibody when antigen is attacked.Referring to for example Jakobovits etc., Proc.Natl.Acad.Sci.USA 90:2551-255 (1993); Jakobovits etc., Nature 362:255-258 (1993).

People such as Mendez (Nature Genetics 15:146-156 (1997)) have further improved technology, generated the transgenic mice strain that is called " Xenomouse II " (heterograft mouse), it is at the antibody that is subjected to generating when antigen is attacked the complete people of high-affinity.This is as mentioned above by realizing in the mouse that people's heavy chain of millions of bases and light chain gene seat kind system are incorporated into endogenous JH section have deletion.Xenomouse II carries 1, and people's heavy chain gene seat of 020kb comprises about 66 kinds of V _HGene, complete D _HAnd J _HDistinguish, reach three kinds of different constant regions (μ, δ and χ), also carry 800kb people's kappa gene seat, comprise 32 kinds of V kappa genes, J κ section and C kappa gene.The antibody that generates in these mouse in all respects in human body, see extremely similar, comprise gene rearrangement, assembling and complete or collected works.Because endogenous J _HThe deletion of section has prevented the gene rearrangement of musculus cdna seat, so people's antibody is preferentially expressed than endogenous antibody.

Perhaps, display technique of bacteriophage (McCafferty etc., Nature 348:552-553 (1990)) is used in external from generating people's antibody and antibody fragment from the immune globulin variable region of epidemic disease donor (V) gene complete or collected works rather.According to this technology, antibody V domain gene is cloned in the main or less important coat protein gene of filobactivirus such as M13 or fd in the mode that meets reading frame, and on the phage particle surface, is shown as the functional antibodies fragment.Because the filobactivirus particle comprises the single stranded DNA copy of phage genome, be the selection that carries out on the basis also cause the encoding selection of gene of the antibody of showing those characteristics with the functional performance of antibody.So, some characteristics of phage simulation B cell.Phage display can carry out in a variety of forms; Relevant summary is referring to for example Johnson, Kevin S.and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993).Several sources of V constant gene segment C can be used for phage display.Clackson etc., Nature 352:624-628 (1991) is from obtaining a large amount of different anti-azolactone antibody derived from separating the combinatorial library at random through the small-sized V gene of immune mouse spleen.Can follow Marks etc. in essence, J.Mol.Biol.222:581-597 (1991) or Griffith etc., EMBO is (1993) described technology J.12:725-734, is made up V gene complete or collected works and is separated at the antibody of synantigen (comprising autoantigen) not in a large number by not immune people's donor.In the natural immunity was replied, antibody gene was with height ratio accumulation sudden change (somatic hypermutation).Some variation that imports will be given more high-affinity, and the B cell of displaying high-affinity surface immumoglobulin preferentially duplicates in antigen attack process subsequently and breaks up.This natural process can be called the technology of " chain reorganization " by employing and simulate (Marks etc., Bio/Technol.10:779-783 (1992)).In this method, the avidity of " initially " the people's antibody that obtains by phage display can be replaced heavy chain and light chain V district gene improves in succession by the naturally occurring variant of V domain gene (complete or collected works) that obtains with immune donor never.This technology is generated antibody and the antibody fragment of avidity in the nM scope.Waterhouse etc., Nucl.Acids Res.21:2265-2266 (1993) have put down in writing the strategy that is used to make up very large phage complete or collected works (being also referred to as " female storehouses in all libraries " (the mother-of-alllibraries)).Gene reorganization also can be used for from the rodents antibody people's antibody of deriving, and wherein people's antibody has avidity and the specificity similar to initial rodents antibody.According to this method, it is also referred to as " the epi-position marking " (epitope imprinting), and the heavy chain of the rodents antibody that obtains by display technique of bacteriophage or light chain V domain gene personnel selection V domain gene complete or collected works replace, and produce rodents-people's block polymer.The selection that antigen is carried out causes the separation of people variable region that can the restore functionality antigen binding site, i.e. epi-position decision (marking, imprint) selection of mating partner.When repeating this process, obtain people's antibody (, being disclosed on April 1st, 1993) referring to PCT patent application WO 93/06213 with replacement residue rodents V structural domain.Pass through CDR to transplant the humanization of the rodents antibody that carries out different with traditional, this technology provides complete people's antibody, and they do not contain the framework or the CDR residue of rodents origin.

As what hereinafter go through, antibody of the present invention can be chosen the multivalence form that comprises antibody monomer, antibody dimer and antibody wantonly.Those skilled in the art can and use herein DR6 and/or APP antibody to make up this type of dimer or multivalence form by technology known in the art.The method that is used to prepare univalent antibody also is well-known in the art.For example, a kind of method relates to the recombinant expressed of light chain immunoglobulin and modified heavy chain.The heavy chain arbitrfary point brachymemma in the Fc district usually is crosslinked to prevent heavy chain.Perhaps, relevant cysteine residues substitutes with another kind of amino-acid residue or deletes crosslinked to prevent.

Bi-specific antibody

Bi-specific antibody refer to at least two kinds not synantigen have the mono-clonal of binding specificity, preferred people or humanized antibody.In this case, a kind of binding specificity is to the DR6 acceptor, and another kind is to any other antigen, preferably to another kind of acceptor or receptor subunit.The method that is used to generate bi-specific antibody is known in the art.Traditionally, the reorganization of bi-specific antibody generates the coexpression based on two pairs of heavy chain immunoglobulin-light chains, and wherein two kinds of heavy chains have different specificity (Millstein and Cuello, Nature305:537-539 (1983)).Because the random assignment of heavy chain immunoglobulin and light chain, these hybridomas (four source hybridomas (quadroma)) generate the potential mixture of ten kinds of different antibodies molecules, wherein have only a kind of correct dual specific structure that has.Usually quite trouble and product productive rate are low for the purifying of the correct molecule that is undertaken by the affinity chromatography step.On May 13rd, 1993 disclosed WO 93/08829 and Traunecker etc., EMBO 10:3655-3659 has disclosed similar flow process in (1991).

According to a kind of different and preferred method, the antibody variable domains and the immunoglobulin (Ig) constant domain sequence that will have expectation binding specificity (antibody-antigen binding site) merge.Merging preferred the use comprises to small part hinge, CH ₂And CH ₃The heavy chain immunoglobulin constant domain in district.Preferably at least a fusions, exist and comprise the first CH (CH of light chain in conjunction with necessary site ₁).The heavy chain immunoglobulin fusions of will encoding reaches, and if desired, the DNA of light chain immunoglobulin inserts expression vector separately, and cotransfection is in the appropriate host organism.The embodiment of optimum yield is provided when the three peptide species chain ratios that are used for making up do not wait, and this provides great handiness for adjusting the segmental mutual ratio of three peptide species.Yet, express when causing high yield with same ratio or when this ratio does not have special meaning at least two peptide species chains, the encoding sequence of two kinds or all three peptide species chains might be inserted same expression vector.In a preferred embodiment of this method, bi-specific antibody is made of (second binding specificity is provided) heterozygosis heavy chain immunoglobulin that has first binding specificity on the arm and the heterozygosis heavy chain immunoglobulin-light chain on another arm.Because the light chain immunoglobulin only existence in half bispecific molecule provides isolating convenient approach, therefore find this unsymmetrical structure be convenient to will expectation the dual specific mixture make up with undesired immunoglobulin chain and separate.This method is disclosed in disclosed PCR application on March 3rd, 1994 WO 94/04690.

About the further details that generate bi-specific antibody referring to for example Suresh etc., Methods inEnzymology 121:210 (1986).

Allos coupling antibody

Allos coupling antibody also within the scope of the invention.Allos coupling antibody is made of two kinds of covalently bound antibody.This antibody-like suggestion for example is used for the undesired cell of immune system cell target (United States Patent (USP) 4,676,980) and is used for the treatment of HIV infecting (PCT application WO 91/00360 and WO 92/200373; EP 03089).Allos coupling antibody can use any cross-linking method easily to generate.Suitable crosslinking agent is well-known in the art, is disclosed in United States Patent (USP) 4,676,980 together with many crosslinking technologicals.

Antibody fragment

In certain embodiments, anti-DR6 and/or APP antibody (comprise mouse, the people and humanized antibody, and antibody variants) be antibody fragment.The multiple technologies that are used to generate antibody fragment have been developed.Traditionally, derive these fragments (referring to for example Morimoto etc., J.Biochem.Biophys.Methods 24:107-117 (1992) by the proteolytic digestion complete antibody; Brennan etc., Science 229:81 (1985)).Yet, can directly generate these fragments now by recombinant host cell.For example, can be directly reclaim Fab '-SH fragment and chemical coupling to form F (ab ') from intestinal bacteria ₂Fragment (Carter etc., Bio/Technology 10:163-167 (1992)).In another embodiment, F (ab ') ₂Fragment is to use leucine zipper GCN4 to form, to promote F (ab ') ₂The assembling of molecule.According to another kind of method, can directly separate Fv, Fab or F (ab ') from the recombinant host cell culture ₂Fragment.The multiple technologies that are used to generate antibody fragment will be conspicuous to skilled practitioner.For example, can use papoid to digest.The example of papain digestion is recorded in disclosed WO 94/29348 and United States Patent (USP) 4,342,566 on December 22nd, 94.Produce two identical Fabs with papain digestion antibody, be called the Fab fragment, have an antigen binding site separately, and a remaining Fc fragment.Pepsin produces a F (ab ') ₂Fragment, it has two antigen binding sites and still can crosslinked antigen.

The Fab fragment that produces in the antibody digestion also comprises the constant domain of light chain and the first constant domain (CH of heavy chain ₁).The segmental difference of Fab ' fragment and Fab is heavy chain CH ₁The C-terminal of structural domain has increased the minority residue, comprises the one or more halfcystines from antibody hinge region.Fab '-SH is the appellation of constant domain cysteine residues wherein being carried the Fab ' of free sulphur alcohol radical herein.F (ab ') ₂Antibody fragment is to generate as the paired Fab ' fragment that hinge cysteine is arranged between Fab ' fragment at first.Also know other chemical coupling of antibody fragment.

The glycosylation variant of antibody

Glycosylation (Jefferis and Lund, Chem.Immunol.65:111-128 (1997) take place in the conservative position of antibody in its constant region; Wright and Morrison, TibTECH 15:26-32 (1997)).The oligosaccharides side chain of immunoglobulin (Ig) influences proteinic function (Boyd etc., Mol.Immunol.32:1311-1318 (1996); Wittwe and Howard, Biochem.29:4175-4180 (1990)), and the intramolecular interaction between the glycoprotein each several part, it can influence the conformation of glycoprotein and the three-dimensional surface that is presented (Hefferis and Lund, supra; Wyss and Wagner, Current Opin.Biotech.7:409-416 (1996)).Oligosaccharides also can be used to make given glycoprotein target some molecule based on the specific identification structure.For example, existing report, in no galactosylation IgG, the oligosaccharides module is from CH ₂Between the space " stretch out ", terminal N-acetyl-glucosamine residue is able in conjunction with mannose-binding protein (Malhotra etc., Nature Med.1:237-243 (1995)).The oligosaccharides of removing on the CAMPATH-1H (the antigenic recombinant humanized mouse of a kind of identification human lymphocyte CDw52 mono-clonal IgG1 antibody) that is generated in Chinese hamster ovary (CHO) cell with glycopeptidase causes the cytolysis (CMCL) of complement-mediated to reduce (Boyd etc. fully, Mol.Immunol.32:1311-1318 (1996)), eliminate the forfeiture that sialic acid residues does not cause DMCL with the neuraminic acid enzyme selectivity.Also have report, the glycosylation of antibody influences the cytotoxicity of antibody dependent cellular (ADCC).Particularly, report, wherein β (1,4)-expression of N-acetyl-glucosamine transferase I II (GnTIII) (glycosyltransferases that branch GlcNAc such as catalysis forms) is subjected to the Chinese hamster ovary celI of tsiklomitsin regulation and control to have the ADCC activity (Umana etc., Mature Biotech.17:176-180 (1999)) of improvement.

The glycosylation variant of antibody refers to the variant that the glycosylation pattern of antibody wherein changes.Change and to mean one or more carbohydrate modules of finding in the deletion antibody, in antibody, add one or more carbohydrate modules, change the composition of glycosylation (glycosylation pattern), glycosylated degree, etc.The glycosylation variant can be by for example eliminating in the nucleotide sequence of encoding antibody, changing and/or add one or more glycosylation sites and prepare.

That the glycosylation of antibody is typical or N-connects or the O-connection.N-connects and refers to that the carbohydrate module is attached to the side chain of asparagine residue.Tripeptide sequence l-asparagine-X-Serine and l-asparagine-X-Threonine (wherein X is any amino acid except that proline(Pro)) is the recognition sequence that carbohydrate module enzymatic is attached to the l-asparagine side chain.So, these two kinds of arbitrary existence of tripeptide sequence have produced the potential glycosylation site in the polypeptide.The glycosylation that O-connects refers to one of carbohydrate N-acetylgalactosamine, semi-lactosi or wood sugar are attached to hydroxy-amino-acid, and modal is Serine or Threonine, but also can use 5-oxyproline or 5-hydroxylysine.

Adding glycosylation site in antibody can make it comprise one or more above-mentioned tripeptide sequences and finish (being used for the glycosylation site that N-connects) easily by changing aminoacid sequence.Described change also can be by adding in the sequence of initial antibodies or substituting one or more Serines or threonine residues is carried out (being used for the glycosylation site that O-connects).

Can also under the prerequisite that does not change basic nucleotide sequence, change the glycosylation (comprising the glycosylation pattern) of antibody.Glycosylation depends on the host cell that is used for expressing antibodies to a great extent.Since be used to express as the recombinant glycoprotein of potential therapeutical agent for example the cell type of antibody seldom be n cell, therefore can expect that the glycosylation pattern of antibody will have significant variation (referring to for example Hse etc., J.Biol.Chem.272:9062-9070 (1997)).Outside the selection of host cell, the glycosylated factor of influence comprises growth pattern, culture medium prescription, culture density, oxygen oxygen supply (oxygenation), pH, purification scheme, like that in the reorganization generative process of antibody.Proposed several different methods and be used for changing the glycosylation pattern of realizing the specific host organism, comprised importing or cross to express relating to some enzyme (United States Patent (USP) 5,047,335 that oligosaccharides generates; 5,510,261 and 5.278,299).The glycosylation of glycosylation or some type can be removed by enzymatic from glycoprotein, for example uses endoglycosidase H (Endo H).In addition, can carry out genetic engineering reorganization, for example make its defective aspect the polysaccharide of some type of processing recombinant host cell.These and similar techniques are well-known in the art.

The glycosylation structure of antibody can be easy to by the routine techniques of carbohydrate analysis analyze, comprise lectin chromatography, NMR, mass spectrum, HPLC, GPC, monose composition analysis, sequential enzymatic digestion and HPAEC-PAD, it utilizes high pH anion-exchange chromatography to separate oligosaccharides according to electric charge.The method that discharges oligosaccharides for analysis purposes also knows, include but not limited to enzymically treat (usually using peptide-N-Glycosylase F/ inscribe-beta-galactosidase enzymes to carry out), use the elimination of strong alkali environment mainly to be the O-syndeton to discharge, and the chemical process of using anhydrous hydrazine with discharge N-be connected with O-oligosaccharides the two.

Exemplary antibody

Described in hereinafter embodiment, many anti-DR6 monoclonal antibodies have been identified.In optional embodiments, DR6 antibody of the present invention will have and clear and definite disclosed any anti-DR6 and/or the identical biological property of APP antibody herein.

Term " biological property " is used in reference to the external and/or activity in vivo or the characteristic of monoclonal antibody, such as specificity in conjunction with DR6 or blocking-up, inhibition or in and DR6 activatory ability.The characteristic of DR6 and/or APP antibody and activity further describe among the embodiment hereinafter.

Optional is, monoclonal antibody of the present invention will have with embodiment hereinafter in the identical biological property of any antibody of concrete sign, and/or identical epi-position with these antibodies.This can measure by carrying out multiple assay method, described in this paper and embodiment.For example, whether have and DR6 that clearly mentions herein and/or the identical specificity of APP antibody, can in the competitive binding assay method, compare its activity in order to measure certain monoclonal antibody.In addition, specific anti-DR6 and/or APP antibody institute bonded epi-position can by DR6 and/or APP and crystallography research of mixture be discussed between antibody measure.

As described herein, DR6 and/or APP antibody will preferably have the DR6 antagonistic activity of expectation.This type of DR6 antibody can include but not limited to chimeric, humanized, the people and antibody affinity maturation.As mentioned above, DR6 and/or APP antibody can use multiple technologies to make up or reorganize to realize these expectation activity or characteristics.

Other embodiment of the present invention comprises such anti-DR6 acceptor and/or APP ligand antibody disclosed herein, and it links to each other with the polymkeric substance of one or more nonprotein character that are selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene (polyoxyalkylene).Optional is that anti-DR6 acceptor disclosed herein and/or APP ligand antibody are glycosylated or not glycosylated.

Antibody of the present invention comprises " crosslinked " DR6 and/or APP antibody.Term " crosslinked " refers to that when being used for this paper at least two IgG molecules combine to form (or single) molecule.It is crosslinked that DR6 and/or APP antibody can use multiple link molecule, and preferably, DR6 and/or APP antibody are to use anti-IgG molecule, complement, chemically modified or molecular engineering and crosslinked.In case those skilled in the art will be appreciated that the antibodies cell surface membrane, complement antagonist molecule has higher relatively avidity.Therefore, think that complement can be used as corsslinking molecular and is used to connect the two or more anti-DR6 antibody of cell surface membrane institute bonded.

The present invention also provides and has encoded the isolating nucleic acid of DR6 described herein and/or APP antibody, the carrier that comprises described nucleic acid and host cell and be used to generate the recombinant technology of described antibody.

For the generation antibody of recombinating, separate its nucleic acid of coding, and insert replicable vector, be used for further clone (DNA cloning) or expression.Can use old process easily to separate the DNA and the order-checking (for example use can with the gene specific bonded oligonucleotide probe of encoding antibody) of encoding antibody.Can obtain many carriers.Support element generally include but be not limited to following one or more: signal sequence, replication orgin, one or more marker gene, enhancer element, promotor and transcription termination sequence.

Method herein comprises and is used to generate chimeric and the anti-DR6 of reorganization and/or the method for APP antibody, comprises the steps: to provide the carrier of the dna sequence dna that comprises the anti-DR6 of coding and/or APP light chain of antibody or heavy chain (perhaps light chain and heavy chain the two); With described carrier transfection or transformed host cell; And under the condition that is enough to generate anti-DR6 of reorganization and/or APP antibody product, cultivate described host cell.

The preparaton of DR6 antagonist

When the typical preparaton for preparing herein, notice the recommendation quality of the composition that adopts or the end-use that " grade " will depend on preparaton.For therepic use, various compositions are preferably allowed (such as " GRAS ") as the grade of medicinal product additive.

In certain embodiments, the composition that comprises DR6 antagonist and one or more vehicle is provided, described vehicle provides enough ionic strengths to improve the solvability and/or the stability of DR6 antagonist, and wherein the pH of said composition is that 6 (or about 6) are to 9 (or about 9).The DR6 antagonist can prepare to realize desirable protein matter purity by any appropriate method, for example according to method above.In certain embodiments, the DR6 antagonist is recombinant expressed or prepare by chemosynthesis in host cell.The concentration of DR6 antagonist in preparaton can change according to the planned use of for example preparaton.Those skilled in the art need not the expectation concentration that too much experiment just can determine the DR6 antagonist.

Provide in the preparaton enough ionic strengths to improve the DR6 antagonist solvability and/or one or more vehicle of stability optional be the acid of polyion (polyionic) organic or inorganic, aspartic acid (salt)/(ester), sodium sulfate, sodium succinate, sodium-acetate, sodium-chlor, Captisol ^TM, Tris, arginic acid salt or other amino acid, sugar and polyvalent alcohol such as trehalose and sucrose.Preferably, providing one or more vehicle of enough ionic strengths in the preparaton is salt.Adoptable salt includes but not limited to sodium salt and arginic acid salt.The type of the salt that adopts and the concentration of salt preferably make preparaton have relative higher ionic strength, make that the DR6 antagonist in the preparaton is stable.Optional is that the concentration that salt exists in preparaton is that about 20mM is to about 0.5M.

The pH of composition is preferably 6 (or about 6) to 9 (or about 9), and more preferably about 6.5 to about 8.5, even more preferably about 7 to about 7.5.Of this embodiment preferred aspect, it is about 6 to about 8 with the pH that keeps composition at least that composition also will comprise buffer reagent.The example of adoptable buffer reagent includes but not limited to Tris, HEPES and Histidine.When adopting Tris, choose wantonly pH is transferred to about 7 to 8.5.When adopting Hepes or Histidine, choose wantonly pH is transferred to about 6.5 to 7.Optional is that the working concentration of buffer reagent in preparaton is that about 5mM is to about 50mM.

Particularly, may wish in composition, to comprise one or more tensio-active agents for liquid adjustments (or freeze-dried formulation of rebuilding).This type of tensio-active agent can comprise for example nonionogenic tenside, as TWEEN ^TMOr PLURONICS ^TM(for example polysorbate or poloxamer).Preferably, tensio-active agent comprises polysorbate20 (" TWEEN 20 ").The working concentration of tensio-active agent is optional for about 0.005% to about 0.2%.

Preparaton of the present invention can further comprise various other vehicle or composition outside DR6 antagonist and those compositions mentioned above.Optional is, can comprise pharmaceutics or parenteral acceptable carrier for the preparaton of parenteral administration, promptly the dosage that is adopted and concentration to the recipient nontoxic and with the carrier of other component compatibility of preparaton.Optional is, carrier is the parenteral carrier, such as with the isoosmotic solution of recipient's blood.The example of examples of such carriers media comprises water, salt solution or buffered soln, such as phosphate buffered saline (PBS) (PBS), woods Ge Shi (Ringer) solution and dextrose solution.Various optional pharmaceutics acceptable carrier, vehicle or stablizers further are recorded in Remington ' s PharmaceuticalSciences, 16th edition, Osol, A.ed. (1980).

Preparaton herein also can contain one or more sanitass.Example comprises octadecyl dimethyl benzyl ammonium chloride, chlorination hexane diamine, benzalkonium chloride (mixture of kelene benzyl dimethyl ammonium, wherein alkyl is a long-chain compound) and benzethonium chloride.The sanitas of other type comprises aromatic alcohol, alkyl paraben such as methyl p-hydroxybenzoate or propyl ester and meta-cresol.Antioxidant comprises xitix and methionine(Met); Sanitas is (such as octadecyl dimethyl benzyl ammonium chloride; Chlorination hexane diamine; Benzalkonium chloride, benzethonium chloride; Butanols; Alkyl paraben is such as methyl p-hydroxybenzoate or propyl ester; Pyrocatechol; Resorcinol; Hexalin; The 3-amylalcohol; And meta-cresol); Lower molecular weight (being less than about 10 residues) polypeptide; Protein is such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer is such as polyvinylpyrrolidone; Amino acid is such as glycine, glutamine, l-asparagine, Histidine, arginine or Methionin; Monose, disaccharides and other carbohydrate comprise glucose, seminose or dextrin; Carbohydrate is such as sucrose, N.F,USP MANNITOL, trehalose or sorbyl alcohol; Or polyoxyethylene glycol (PEG).

Composition of the present invention can comprise liquid adjustments (liquor or liquid suspension) and freeze-dried formulation, and the suspension preparation agent.

If liquid, the final preferred refrigerated storage of preparaton is in≤20 ℃.Perhaps, but this preparaton of freeze-drying and provide as the pulvis of rebuilding with water for injection, and this pulvis is optional can be stored in 2-30 ℃.

The preparaton that being used for the treatment of property is used must be aseptic.Can realize sterilization easily by aseptic filter membrane (for example 0.2 micron filter membrane) filtration.Usually therapeutic composition is placed container, for example have the intravenous solution bag or the medicine bottle of the stopper that available hypodermic needle pierces through with sterilization access port.

As the aqueous solution or the freeze-dried formulation that is used to rebuild, usually composition is stored in single unit or the multi-dose container, for example Mi Feng ampoule or medicine bottle.This container can be the obtainable any container in this area, and fills with ordinary method.Optional is, this preparaton injection pen device (or cartridge case of the pen device of packing into) of can packing into, and such as the obtainable device in those this areas (referring to for example United States Patent (USP) 5,370,629), their are fit to the therapeutic of preparaton and deliver.For example, can use water for injection to rebuild freeze dried DR6 antagonist preparaton and prepare injection liquid.

Use the therapy of DR6 antagonist

DR6 antagonist of the present invention has multiple effectiveness.The DR6 antagonist is useful in the diagnosis of neuroscience illness and treatment.The diagnosis of various pathology illness described herein can be undertaken by skilled practitioner in the Mammals.Diagnostic techniques is that this area can obtain, and it allows the diagnosis or the detection of various neuroscience illnesss in the Mammals for example.

Neuroscience illness by the present invention's treatment comprises familial and sporadic amyotrophic lateral sclerosis (being respectively FALS and ALS), familial and sporadic Parkinson's disease, Huntington disease, familial and sporadic Alzheimer's and Duchenne-Arandisease (SMA) (Price etc. See above).Many in these diseases is the typical case with the middle adulthood outbreak, and causes the quick sex change (degeneration) of the specific neurone subgroup of neural system, finally causes premature dead.

Amyotrophic lateral sclerosis (ALS) is the most normal progressive exercise neuronal disease of diagnosing out.This disease with the sex change of motor neuron in cortex, brain stem and the spinal cord be feature (Siddique etc., J.Neural Transm. Suppl., 49:219-233 (1997); Siddique etc., Neurology, 47:(4 Suppl 2): S27-34; Discussion S34-5 (1996); Rosen etc., Nature, 362:59-62 (1993); Gurney etc., Science, 264:1772-17751994)).

Parkinson's disease (Parkinsonism) is common neurodegenerative illness, and it comes across life mid-term usually to late period.Familial and sporadic case have generation, although the familial case only accounts for the 1-2% of viewed case.The patient usually has reactive neurogliosis (reactive gliosis) and the Lewy corpusculum (Lewy bodies) in neurocyte loss and brain stem locus coeruleus and the black substance.As a class, the nigrostriatum dopaminergic neuron seemingly the most affected (Uhl etc., Neurology, 35:1215-1218 (1985); Levine etc., Trends Neurosci., 27:691-697 (2004); Fleming etc., NeuroRx, 2:495-503 (2005)).

Near-end Duchenne-Arandisease (SMA) is common human autosomal recessive neurodegenerative disease, typically with dynamoneure loss and four limbs and muscle of trunk atrophy be feature (Monani etc., Hum.Mol.Genet., 9:2451-2457 (2000); Monani etc., J.Cell Biol., 160:41-52 (2003)).It takes place with the frequency of 1 example in 10,000 individualities, and is the most common genetic cause of infant death.According to the age of outbreak and the seriousness of disease phenotype, near-end SMA is divided into I type (severe), II type (moderate) and III type (slightly) SMA.Three kinds of forms of all of this disease all be because the sudden change of the telomere of motor neuron gene (SMN1) survival or forfeiture (Monani etc., See above, 2000; Monani etc. see above, and 2003)).

In many neurodegenerative diseases, report the neuronal cell loss, comprised Alzheimer's, Parkinson's disease, amyotrophic lateral sclerosis (ALS) and Duchenne-Arandisease (SMA).

Optional is, the diagnosis of Alzheimer's can be based on the 4th edition " diagnostic and statistical manual of mental disorder " (Diagnostic and Statistical Manual of Mental disorders among the patient, DSM-IV-TR) standard is (referring to the 4th edition revised text of diagnostic and statistical manual (American Psychiatric Association.Diagnostic and statisticalmanual of mental disorders of for example APA's mental disorder, 4th Edition-text revised) .Washington, DC:2000).In brief, the DSM-IV-TR standard comprises: (A) form multiple cognitive defect, its show as the memory impaired and with the next item down or multinomial the two: (1) aphasia; (2) appraxia; (3) agnosia; Or (4) functionating disorder; (B) this cognitive defect presents from previous function and descends and cause the remarkable infringement of society or professional function; (C) course of disease is to show effect gradually and to continue to drop to feature; (D) this cognitive defect is not the illness that causes memory and cognitive gradual defective owing to other central nervous system, systematic or material inductive; (E) this disorder can not better be explained by other psychiatric disorders.The alternative criteria that can make the Alzheimer's diagnosis comprises that those are based on national neurological and communication disorder and apoplexy association-Alzheimer's and (the National Institute of Neurological and CommunicativeDisorders and Stroke-Alzheimer ' s Disease and Related Disorder Association of associated conditions association, NINDS-ADRDA) working group (referring to for example McKhann etc., Neurology 1984 about the standard of Alzheimer's; 34:939-944).In brief, NINCDS-ADRDA comprises with atypical outbreak, performance (presentation) or progress about the standard of possible Alzheimer's and does not have the chronic brain syndrome of the known cause of disease, wherein thinks any and can cause that the disease (co-morbid disease) of dull-witted common morbidity is not a reason.NINCDS-ADRDA comprises by dementia clinical and that neuropsychological test is determined about the standard of possible Alzheimer's, and involves the gradual defective in (a) two or more understanding fields (comprising memory); (b) 40 and 90 years old between the outbreak; (c) do not exist and to cause that chronic brain syndrome comprises the systematic of delirium or other encephalopathic.NINCDS-ADRDA comprises about the standard of the Alzheimer's made a definite diagnosis and reaching about the standard of possible Alzheimer's and the histopathology evidence that has Alzheimer's through postmortem or biopsy.

Revised NINDS-ADRDA Case definition is at Dubois etc., The LancetNeurology, and Volume 6, and Issue 8, and August 2007, propose among the Pages 734-746.As what hereinafter summarize, in order to reach this standard about possible Alzheimer's, diseased individuals must satisfy at least one or the multinomial supportive biomarker standard that writes down among standard A (core clinical criteria) and B, C, D or the E.In this linguistic context, standard A is to exist early stage and significant episodic memory defective is a feature, and this defective comprises following feature: memory function gradually and progressive changed in (1) patient or insider had reported and surpassed 6 months; (2) objective evidence of significantly impaired episodic memory in the test: this is generally by significantly not improving after the efficient coding of formerly having controlled information in prompting or understanding test neutralization or not recovering normal and recall defective and constituted; When (3) AD shows effect or along with the AD development, the episodic memory defective can or be united (associated) with other cognitive variation isolated (isolated).Standard B is a feature there to be temporal atrophy placed in the middle, for example shows as: MRI and use the qualitative grading (qualitative ratings) (colony of the well-characterized age norm of reference) of vision scoring (visualscoring) and the quantitative volumetric determination (quantitative volumetry) (colony of the well-characterized age norm of reference) of area-of-interest has proved the VOLUME LOSS of hippocampus, entorhinal cortex, amygdala.Standard C is feature, for example amyloid-beta with unusual celiolymph biomarker _1-42Low, the total τ concentration of concentration raises or phosphoric acid-τ concentration raises or three's combination.Standard C is feature with the special style of the functional neuroimaging that uses PET, and for example glucose metabolism reduces in the bilateral temporoparietal region.Standard C sports feature with proof AD autosomal dominant among the close relative.If there is and the following, think that then AD makes a definite diagnosis: (1) according to the requirement of NIA-Reagan about the standard of AD paragnosis, two kinds of evidences of the clinical and histopathology of disease (biopsy of brain or postmortem); Standard must exist (referring to for example Neurobiol Aging 1997; 18:S1-S2); (2) clinical and hereditary two kinds of evidences (sudden change on the karyomit(e) 1,14 or 21) of AD; Standard must exist.

In the method for the invention, the DR6 antagonist is applied to Mammals preferably at carrier in the preferred pharmaceutical acceptable carrier.Suitable carriers and preparaton thereof are recorded in " Remington ' s PharmaceuticalSciences ", and the 16th edition, 1980, Mack Publishing Co., people such as Osol compile.Be typically, in preparaton, use the pharmaceutically acceptable salt of sufficient quantity to make preparaton etc. ooze.The example of carrier comprises salt solution, woods Ge Shi (Ringer) solution and dextrose solution.The pH of solution preferably about 5 is to about 8, and more preferably from about 7 to about 7.5.Other carrier comprises extended release preparation, and such as the solid hydrophobic polymkeric substance semipermeability matrix that contains antibody, this matrix is the form of standardized product, for example film, liposome or microcapsule.It will be apparent for a person skilled in the art that some carrier may be preferred according to the concentration of for example using the path and the DR6 antagonist of using.

The DR6 antagonist can be by injection (for example in intravenously, intraperitoneal, subcutaneous, intramuscular, the portal vein (intraportal)), by oral administration to Mammals, perhaps by guaranteeing to be delivered to other method of blood flow such as perfusion with effective form.The DR6 antagonist also can pass through isolated organ perfusion (isolated perfusion) technology (such as separating tissues perfusion), or by in the sheath, intraocular, or lumbar puncture uses, with performance topical therapeutic effect.The DR6 antagonist that is difficult for passing hemato encephalic barrier can directly give, and for example in the brain, or enters spinal cord space (spinal cord space) or alternate manner, and it can pass barrier with their transportations.Using the effective dose and the timetable of DR6 antagonist can determine by rule of thumb, makes this type of decision in the art technology scope.It will be understood by those skilled in the art that the DR6 antagonist dosage that must use will depend on the Mammals that for example will accept antagonist, use the particular type of path, used antagonist and be applied to mammiferous other medicines.Select the guidance of optimal dose for example to see document about the Antybody therapy purposes, for example " Handbook of Monoclonal Antibodies ", people such as Ferrone compile, NogesPublications, Park Ridge, NJ, 1985, the 22 chapters, the 303-357 page or leaf; Smith etc., Antibodiesin Human Diagnosis and Therapy, Haber etc., eds., Raven Press, New York (1977) pp.365-389.The typical per daily dose that DR6 antibody uses separately can every day about 1 μ g/kg body weight to the scope of 100mg/kg body weight nearly or more, this depends on above-mentioned factor.

The DR6 antagonist also can be co-administered in Mammals with one or more other therapeutical agents.The example of this type of other therapeutical agent comprises EGF-R ELISA (EGFR) inhibitor, for example in conjunction with or otherwise with EGFR direct interaction and prevention or reduce the compound of its signaling activity, such as Tarceva, antibody, as C225 (be also referred to as Cetuximab (cetuximab) and

(ImCloneSystems Inc.)), complete people's ABX-EGF (panitumumab, Abgenix Inc.), and be called E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and US 6, the complete people's of record antibody in 235,883; MDX-447 (Medarex Inc), and EGFR micromolecular inhibitor are such as US5616582, US5457105, US5475001, US5654307, US5679683, US6084095, US6265410, US6455534, US6521620, US6596726, US6713484, US5770599, US6140332, US5866572, US6399602, US6344459, US6602863, US6391874, WO9814451, WO9850038, WO9909016, WO9924037, US6344455, US5760041, US6002008, the compound of putting down in writing among the US5747498; Concrete small molecules EGFR inhibitor comprises OSI-774 (CP-358774, erlotinib, OSI Pharmaceuticals); PD 183805 (CI 1033,2-acrylamide (propenamide), N-[4-[(3-chloro-4-fluorophenyl) amino]-7-[3-(4-morpholinyl) propoxy-]-the 6-quinazolyl]-, dihydrochloride, Pfizer Inc.); Iressa (ZD1839, Gefitinib (gefitinib), 4-(3 '-chloro-4 '-fluoroanilino)-7-methoxyl group-6-(3-morpholino propoxy-) quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-aminomethyl phenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-Mi Dingbing [5,4-d] pyrimidine-2,8-diamines, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl) amino]-1H-pyrrolo-[2,3-d] pyrimidine-6-yl]-phenol); (R)-and 6-(4-hydroxy phenyl)-4-[(1-phenylethyl) amino]-7H-pyrrolo-[2,3-d] pyrimidine); CL-387785 (N-[4-[(3-bromophenyl) amino]-the 6-quinazolyl]-2-butyne acid amides (butynamide)); And EKB-569 (N-[4-[(3-chloro-4-fluorophenyl) amino]-3-cyano group-7-oxyethyl group-6-quinolyl]-4-(dimethylamino)-2-butylene acid amides (butenamide)).Adoptable other therapeutical agent comprises inhibitors of apoptosis, inhibitors of apoptosis in the born of the same parents particularly, Caspase inhibitor for example is such as Caspase-3, Caspase-6 or Caspase-8 inhibitor, Bid inhibitor, Bax inhibitor or its arbitrary combination.The example of suitable inhibitor generally has the Caspase inhibitor, two inhibitor peptides, the carbamate inhibitor, the aspartic acid acetal that replaces, heterocycle two carbonyl acid amides (heterocyclyldicarbamides), quinoline-(dipeptides, tripeptides, tetrapeptide) derivative, the 2-amino-benzene acid amides Caspase inhibitor of replacement, the a-hydroxy acid Caspase inhibitor that replaces, the restraining effect of nitrosylation; CASP-1; CASP-3: protein inhibitor, antisense molecule, nicotinoyl (nicotinyl)-aspartoyl-ketone, y-ketone acid dipeptidase derivant, CASP-8: antisense molecule, interaction protein CASP-9, CASP2: antisense molecule; CASP-6: antisense molecule; CASP-7: antisense molecule; With the CASP-12 inhibitor.Other example has the plastosome inhibitor, such as the Bcl-2 regulatory factor; Bcl-2 mutant peptide, Bad, BH3 interaction domain death agonist, Bax arrestin and BLK gene and gene product derived from Bad.Adjusting control agent has CASP9/Apaf-1 bonded antagonist, the antisense adjusting control agent of Apaf-1 expression, the peptide that is used to suppress apoptosis, the anti-apoptosis composition, MEKK1 and the fragment thereof that comprise hsv R1 subunit, the adjusting control agent of survivin (Survivin), the adjusting control agent and the HIAP2 of inhibitors of apoptosis in the suitable born of the same parents of other of apoptosis.Other example of this type of medicament comprises and suppresses the plastosome cytochrome c release and block Minocycline HCl (Minocycline) that Caspase-3mRNA raises (NeuroapoptosisLaboratory), Pifithrin alpha (UIC) as the p53 inhibitor, CEP-1346 (Cephalon Inc.) as the JNK approach restrainer, the TCH346 (Novartis) that suppresses short apoptosis GAPDH signal conduction, IDN6556 (Idun Pharmaceuticals) as general Caspase inhibitor, AZQs (AstraZeneca) as Caspase-3 inhibitor, HMR-3480 (Aventis Pharma) as Caspase-1/-4 inhibitor, Activase/TPA (Genentech) with dissolving clot (thrombolysis medicine).

The suitable medicament of other that can use except the DR6 antagonist comprises that anticholinesterase is (such as E2020 (Donepezil), lycoremine (Galantamine), profit is cut down this bright (Rivastigmine), tacrine (Tacrine)), nmda receptor antagonist (such as memantine (Memantine)), A beta peptide aggregation inhibitor, antioxidant, the gamma-secretase adjusting control agent, NGF stand-in or ngf gene therapy, the PPAR gamma agonist, HMG-CoA reductase inhibitor (statins (statins)), ampakine (ampakines), calcium channel blocker, the GABA receptor antagonist, the glycogen synthase kinase enzyme inhibitors, the intravenously immunoglobulin (Ig), Muscarmic receptor agonist (muscarinic receptor agonists), nAChR adjusting control agent (nicotinic receptor modulators), initiatively or passive A β immunization, phosphodiesterase inhibitor, serotonin receptor antagonist and anti-amyloid beta antibodies are (referring to for example WO 2007/062852; WO2007/064972; WO 2003/040183; WO 1999/06066; WO 2006/081171; WO1993/21526; EP 0276723B1; WO 2005/028511; WO 2005/082939).

The DR6 antagonist can or be used simultaneously with one or more other therapeutical agent orders.The amount of DR6 antagonist and therapeutical agent depends on for example type, the pathology illness of being treated and the timetable of using and the path of used medicine, but is usually less than the amount when using separately separately.

Behind administration DR6 antagonist, can monitor mammiferous physiology situation in the well-known multiple mode of skilled practitioner.

The result of treatment of DR6 antagonist of the present invention can be checked by animal model in the external test method and in the use body.The effect that multiple well-known assay method and animal model can be used for testing candidate therapeutic agent.Essence makes their can forecast response in human patients especially in the body of this class model.The animal model of various neurodegenerative illness and relevant technologies embodiment 14 discussion hereinafter that are used for checking the pathologic process relevant (for example when existing and not having the DR6 antagonist) with these neurodegeneration models.

The animal model of various neuroscience illnesss comprises (genetically modified) animal nonrecombinant and reorganization.Non-recombinant animal model comprises for example rodents (for example mouse) model.This class model can generate by using standard technique (for example subcutaneous injection, tail vein injection, spleen are implanted, intraperitoneal is implanted and the scrotum is implanted down) that cell is imported in the homogenic mouse.The body inner model comprises the body inner model of apoplexy/cerebral ischemic model, neurodegenerative disease, such as parkinsonian mouse model; The mouse model of Alzheimer's; The mouse model of amyotrophic lateral sclerosis ALS; The mouse model of Duchenne-Arandisease SMA; Mouse/the rat model of focal and globality cerebral ischemia, for example common carotid artery occlusion model or middle cerebral artery occlusion model; Or in stripped (ex vivo) full embryo culture thing.Can implement various assay methods to measure form in the known external or body, all as described below or as known in the art with document in put down in writing (referring to for example McGowan etc., TRENDS in Genetics, 22:281-289 (2006); Fleming etc., NeuroRx, 2:495-503 (2005); Wong etc., Nature Neuroscience, 5:633-639 (2002)).Various these type of animal models also can obtain from the commercial distribution merchant, such as Jackson Laboratory (referring to Http:// jaxmice.jax.org).

Many animal models known in the art can be used for checking DR6 antagonist disclosed herein to the activity of neuroscience illness (such as AD) (referring to for example Rakover etc., Neurodegener Dis.2007; 4 (5): 392-402; Mouri etc., FASEB is Jul J.2007; 21 (9): 2135-48; Minkeviciene etc., JPharmacol Exp Ther.2004 Nov; 311 (2): 677-82; And Yuede etc., Behav Pharmacol.2007 Sep; 18 (5-6): 347-63)).For example, DR6 antagonist disclosed herein to the effect of mouse cognitive function can use Target Recognition test (object recognition tests) check (referring to for example Ennaceur etc., Behav.Brain Res.1988; 31:47-59).DR6 antagonist disclosed herein can be checked in mouse the activity of for example encephalitis disease, by for example be designed for the ELISA scheme of measuring marker of inflammation (such as IL-1 β and TNF-α) and anti-inflammatory cytokines IL-10 level in the mice plasma fraction and tissue chemical analysis (referring to for example Rakover etc., Neurodegener Dis.2007; 4 (5): 392-402).

DR6 antagonist disclosed herein can for example be used for check (referring to for example Leber P:Guidelines for theClinical Evaluation of Antidementia Drugs by cognitive outcome measurement (cognitive outcome measure) together with making of the overall evaluation (global assessment) to the effect of neuroscience illness among the mankind (such as Alzheimer's (AD)), 1st draft, Rockville, MD, US Foodand Drug Administration, 1990).Can for example use single group or many groups standard to check to the effect of neuroscience illness (such as AD).For example, Europe medicine assessing mechanism (European MedicineEvaluation Agency, EMEA) introduced with function and whole the two activity in cognitive and stable aspect be scheduled to improvement degree corresponding response person's (responders) definition (referring to for example European MedicineEvaluation Agency (EMEA): Note for Guidelines on Medicinal Products in theTreatment of Alzheimer ' s Disease.London, EMEA, 1997).This area know many can be individually or be used in combination the test specific, that set up of assess patient to the medicament response (referring to for example Van Dyke etc., AM J Geriatr.Psychiatry 14:5 (2006).For example can use serious sick damage group (Severe Impairment Battery, SIB) assess response to medicament, this be a kind of be used for measuring the cognitive test that changes of the patient who suffers from more serious AD (referring to for example Schmitt etc., Alzheimer DisAssoc Disord 1997; 11 (suppl 2): 51-56).Also can use 19 Alzheimer's joint study-activities of daily living inventories (19-item Alzheimer ' s Disease CooperativeStudy-Activities of Daily Living inventory, ADCSADL19) measure response to medicament, this is 19 inventories measuring the independence level of carrying out activities of daily living, for dull-witted design in late period and through checking (referring to for example Galasko etc., J Int Neuropsychol Soc 2005; 11:446-453).Also can use variation impression based on clinicist's interview add nursing staff's input (Clinician ' sInterview-Based Impression of Change Plus Caregiver Input, CIBIC-Plus) measure response to medicament, this be based on the two the seven o'clock sharp body of structurizing interview of patient and nursing staff change grading (referring to for example Schneider etc., Alzheimer Dis Assoc Disord 1997; 11 (suppl2): 22-32).Also can use neuropsychiatry inventory (Neuropsychiatric Inventory, NPI) measure response to medicament, it assesses the frequency of 12 behavior symptoms and seriousness based on nursing staff's interview, and (referring to for example Cummings etc., Neurology 1994; 44:2308-2314).

Multiple anticholinesterase (E2020 (Donepezil), lycoremine (Galantamine), profit are cut down this bright (Rivastigmine) and tacrine (Tacrine)) and memantine (Memantine) (a kind of known N-methyl-D-aspartate salt/ester (NMDA) receptor antagonist) have received that administration approval is used for the treatment of Alzheimer's (referring to for example Roberson etc., Science 314:781-784 (2006)).At anticholinesterase in the slight clinical trial to the moderate seriousness AD patient, the treatment common definition of replying involve Alzheimer's assessment scale-cognitive sublist in six months (Alzheimer ' s Disease Assessment Scale-Cognitive Subscale, ADAS-cog) 4 improve on (referring to for example Winblad etc., Int J Geriatr Psychiatry 2001; 16:653-666; Cummings J., Am J Geriatr Psychiatry 2003; 11:131-145; And Lanctot etc., CMAJ2003; 169:557-564).Also these results were compared with respectively lysis being fallen back in 6 months or 1 year (referring to for example Doraiswamy etc., Alzheimer Dis Assoc Disord (2001) 15:174-183).In the clinical trial of memantine, will treat the respondent be predefined for aspect whole capability show not have decline and all show aspect function or the cognitive arbitrary ability patient that do not have to fail (referring to for example Reisberg etc., N.Engl.J.Med.2003; 348:1333-1341).Memantine another test in the patient who takes consistent dose anticholinesterase E2020 is characterized by memantine the benefit that shows surpassing placebo aspect the outcome measurement, comprise with respect to serious sick damage group (SevereImpairment Battery, SIB) and the improvement 19 AD Cognitive Study-activities of daily living inventory (modified 19-item AD Cooperative Study-Activities of Daily Living Inventory, ADCS-ADL19), variation impression based on clinicist's interview adds nursing staff's suggestion (Clinician ' sInterview-Based Impression of Change Plus Caregiver Input, CIBIC-Plus), neuropsychiatry inventory (Neuropsychiatric Inventory, NPI), with presbyatrics patient behavior grading scale (Behavioral Rating Scale for Geriatric Patients, (referring to for example Tariot etc., JAMA 2004 in the variation of baseline BGP Care DependencySubscale); 291:317-324).By between multiple SIB, ADCS-ADL19, CIBIC-Plus and NPI outcome measurement, producing the improvement of symptom and stablizing the two, memantine further has been characterized by effectively (referring to for example van Dyck etc., AM J Geriatr.Psychiatry 14:5 (2006)).

The diagnostic use of DR6 antagonist

Familial Alzheimer's (FAD) or autosomal dominant early onset Alzheimer's (ADEOAD) refer to (be defined as before 65 years old in early days at life usually, usually between between 20-65 year) the unusual Alzheimer's of invasion and attack, it can be with the heredity of autosomal dominant mode.The evidence that provides sudden change in these genes to be responsible for most of ADEOAD to observe cases about the research of amyloid precursor protein (APP), senilism albumen 1 (PSEN1) and senilism albumen 2 (PSEN2) gene (referring to for example Raux etc., Journal of Medical Genetics 2005; 42:793-795).Yet many observation cases of this type of symptom still do not obtain explaining.The polypeptide of the data prompting death receptor 6 that is presented herein and/or some cases that the polynucleotide variant may be responsible for FAD and/or other neuroscience illness.Embodiment of the present invention comprise the method that whether has the polypeptide variants of death receptor 6 (DR6) polypeptide that comprises SEQ ID NO:1 in the individuality of measuring, this method comprises the sequence and the SEQ IDNO:1 of the DR6 polypeptide that exists in the comparison individuality, to determine whether to exist in this individuality the polypeptide variants of DR6.What choose wantonly in these class methods is that this patient suffers from or suspects and suffer from FAD and/or other neuroscience illness.

In this linguistic context, can analyze DR6 polypeptide in patient's sample and/or polynucleotide (for example in order to identify the natural variant that exists of DR6) by multiple means well-known in the art, include but not limited to clinical sample and clone immunohistochemical analysis, in situ hybridization, RT-PCR analysiss, Western engram analysis, reach the tissue array analysis.The typical scenario that is used to assess the sequence of DR6 gene (for example DR65 ' and 3 ' regulates sequence, intron, exon or the like) and DR6 gene product (for example DR6 mRNA, DR6 polypeptide or the like) for example is found in volumes such as Ausubel, 2007, Current Protocols In MolecularBiology, unit 2 (Northern trace), unit 4 (Southern trace), unit 15 (immunoblotting) and unit 18 (pcr analysis).

In an exemplary embodiment of this alanysis, suffer from the patient of neuroscience illness and obtain neuronal cell from suffering from neuroscience illness or suspection, thereby can by analyze such as the rules of immunoassay, Northern trace assay method or polynucleotide sequence analysis wherein the DR6 polypeptide of expressing and/or mRNA sequence (referring to for example Lane etc., Laryngoscope.2002; 112 (7 Pt 1): 1183-9; And Silani etc., Amyotroph Lateral Scler Other Motor Neuron Disord.200; 2 Suppl 1:S69-76).In certain embodiments of the invention, can analyze the DR6 polypeptide (referring to for example Pettermann etc., J Neurosci. (10): 3624-3632 (1988)) that obtains from patient's neuronal cell (it can be chosen wantonly in vitro culture and go down to posterity) by immunoassay such as Western engram analysis.Perhaps, can analyze the part or the whole coding region of DR6 gene by the reversed transcriptive enzyme chain reaction (RT-PCR) of the mRNA that for example extracts from patient's neuronal cell.In other embodiments of the present invention, cell beyond neuronal cell obtains the DR6 genome sequence, for example inoblast or peripheral blood leucocyte, analyze then with determine these genome sequences whether encoding D R6 polypeptide variants and/or comprise the polynucleotide variant (comprise 5 ' and 3 ' adjusting sequence variants, for example influence DR6 expression level in cell) of DR6.In certain embodiments of the invention, this alanysis can imitate the analysis of amyloid precursor protein (APP), senilism albumen 1 (PSEN1) and senilism albumen 2 (PSEN2) gene (referring to for example Nagasaka etc., Proc Natl Acad SciUSA.2005; 102 (41): 14854-9; And Finckh etc., Neurogenetics.2005; 6 (2): 85-9).

Be used to identify the screening method of DR6 antagonist

Embodiment of the present invention comprise the method for inhibition DR6 in conjunction with the molecules of interest of APP of identifying, this method is included in combination DR6 and APP in the situation that has or do not exist molecules of interest, detects the inhibition of DR6 in conjunction with APP in having the situation of described molecules of interest then.Particularly, use the disclosure provided herein, can for example identify and DR6 and/or APP interaction and inhibition DR6 and APP between interacting proteins, small molecules and other molecule.In an exemplary embodiment of this method, DR6 can be immobilized on the matrix.Can in the situation that has and do not exist molecules of interest, observe the ability of free APP (for example with the APP that can detect the mark mark, the described mark that detects is such as colour developing mark, fluorescence labels, radioactively labelled substance, magnetic label or enzymatic reaction product etc.) then in conjunction with immobilization DR6.Can use APP that the bonded reduction (for example observed via the variation of level that can detect mark and/or position) of DR6 is identified that molecule is to suppress the ability of APP in conjunction with DR6 then.In alternative embodiment of the present invention, APP can be immobilized on the matrix, in the situation that has and do not exist molecules of interest, to detect the ability of APP in conjunction with free DR6 (for example with the DR6 that can detect the mark mark).Optional is that in this type of embodiment, molecules of interest can be an antibody.

The disclosure that is provided allows that being used to characterize the multiple scheme of bonded between the polypeptide (such as DR6 and APP) in this area identifies the molecule that suppresses binding interactions between DR6 and the APP herein.This type of embodiment of the present invention comprises and those adopts the ELISA assay method (for example competitiveness or sandwich style ELISA assay method are as U.S. Patent No. 6,855,508; 6,113,897; With 7,241, disclosed in 803), radioimmunoassay (volume such as Ausubel for example, Current Protocols In MolecularBiology, disclosed in 2007 unit 10.24), Western trace assay method is (for example as Pettermann etc., J Neurosci. (10): 3624-3632 (1988) and hereinafter disclosed in the embodiment 10), immunohistology assay method (for example disclosed in hereinafter embodiment 10), IAsys analysis and the analysis of CM-5 (BIAcore) sensor chip are (referring to for example U.S. Patent No. 6,720,156 and 7,101,851).In certain embodiments of the invention, identify that suppressing DR6 uses protein microarray to the method for APP bonded molecules of interest.Protein microarray is used in usually and limits position immobilized from the teeth outwards proteins of interest matter molecule (for example DR6 and/or APP), and be used to identify that small molecules is conjugated protein (referring to for example Wilson etc., Curr.Opinion in Chemical Biology 2001,6,81-85; And Zhu, H., etc., Science 2001,293,1201-2105).

Test kit and goods

In other embodiments of the present invention, provide goods and the test kit that comprises for the useful material of treatment neuroscience illness.Described goods comprise the container that has label.Suitable containers comprises for example bottle, phial and test tube.Described container can be made with multiple material, such as glass or plastics, and preferably through the sterilization.Described container is equipped with the promoting agent of effective treatment neuroscience illness (comprising Alzheimer's).Promoting agent in the composition is the DR6 antagonist, and preferably includes anti-DR6 monoclonal antibody or anti-APP monoclonal antibody.Label on the container indicates said composition and is used for the treatment of the neuroscience illness, but also can indicate in the body or the guidance of external purposes, such as described above those.The optional package insert that further comprises of goods or test kit, it refers to be usually included in treatment with the explanation in the product commercial package, other therapeutic product that it comprises indication, usage, dosage that relevant this type of treatment uses with product, uses, avoids, will unite with the packaging product and/or warning message etc.

Test kit of the present invention comprises the container mentioned above and second container, and buffer reagent wherein is housed.It can further comprise from other material of commercial and user's position needs, comprise other damping fluid, thinner, filter, pin and syringe.

Embodiment

Further described with illustration all respects of the present invention by embodiment hereinafter, none is intended to these embodiment limit the scope of the invention.

Embodiment 1: the DR6 in embryo and the adult central nervous system expresses

Carried out the RNA original position screening of TNF receptor superfamily expression pattern in the mouse embryonic tissue.In particular, use mRNA Locator test kit (Ambion, catalog number 1803) to abide by the scheme implementation hybridization in situ experiment of manufacturers.Use following DR6 cDNA primary sequence to generate the riboprobe that is used for these experiments:

GAGCAGAAACGGCTCCTTTATTACCAAAGAAAAGAAGGACACAGTGTTGCGGCAGGTCCGCCTGGACCCCTGTGACTTGCAGCCCATCTTTGATGACATGCTGCATATCCTGAACCCCGAGGAGCTGCGGGTGATTGAAGAGATTCCCCAGGCTGAGGACAAACTGGACCGCCTCTTCGAGATCATTGGGGTCAAGAGCCAAGAAGCCAGCCAGACCCTCTTGGACTCTGTGTACAGTCATCTTCCTGACCTATTGTAGAACACAGGGGCACTGCATTCTGGGAATCAACCTACTGGCGG.(SEQ?ID?NO：3)

Use Maxiscript test kit (Ambion, catalog number 1308) to carry out the external synthetic of riboprobe according to the scheme of manufacturers.

As shown in Fig. 2 A, in the developmental spinal cord and dorsal root ganglion cell that are in E10 to the E12 stage (stage of known generation neuronal cell death), find that DR6 is almost expressed specially by the neurone that has broken up, but not the my late grandfather in the propagation.

Described in Fig. 2 B, DR6 albumen is expressed on the two in neuronic cell main body and aixs cylinder.

In Fig. 2 B, two top width of cloth figure have shown the neurone from normal mouse, manifest DR6 (left side) or reference protein (right side).Two following width of cloth figure have correspondingly shown the neurone from the DR6 knock-out mice, manifest DR6 (left side) or reference protein (right side).

The material and the method that are used for generating the shown data of this figure are as follows.In order to manifest, generate DR6 specificity mouse monoclonal antibody (embodiment 3 sees below) as immunogen at the Genentech end user DR6 that recombinates as the DR6 protein expression on the sensation neurite as shown among Fig. 2 B for example.By immunofluorescence these antibody are further screened them and be identified in the total length mouse of expressing on the cell surface and the ability of people DR6.A kind of being called " RA.3 ", (be also referred to as " 3F4.8.8 " monoclonal antibody, and have among embodiment 3 and the embodiment 7 hereinafter and further describe) this antibody-like and people and mouse DR6 polypeptide the two play cross reaction, and be used to manifest as the DR6 on the aixs cylinder as shown among Fig. 2 B and express.Use standard scheme known in the art implement the immunofluorescence dyeing rules (Nikolaev etc., 2003, Cell, 112 (1), 29-40).For the DR6 that manifests on the aixs cylinder expresses, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

As shown in Fig. 2 C, DR6 mRNA is by the neuron expression in the differentiation.In Fig. 2 C, from left to right, three photos have shown the neuronic brain scans from the normal mouse that is in etap E10.5, E11.5 and E12.5 respectively.

The material and the method that are used for generating the shown data of this figure are as follows.For the DR6 mRNA that manifests in the developmental mice embryonic expresses, to organizing transverse section to implement the original position mRNA hybridization (ISH) of using the specific radiolabeled rna probe of DR6 3 ' UTR to carry out 20 microns of the chest axle horizontal collection of E10.5-E12.5 mice embryonic.Use mRNA Locator in situ hybridization test kit according to the such ISH of enforcement experiment of being summarized in the scheme of manufacturers such as the mRNA Locator instruction manual (Ambion Inc., catalog number 1803).In external translation reaction, use the MAXIscript test kit to generate the radiolabeled mRNA probe corresponding with the antisense sequences of mouse DR6 3 ' UTR according to the instruction manual (Ambion Inc., catalog number 1308-1326) of manufacturers.Kodak radioautograph emulsion (Kodak) is put on the slide glass that has the embryonic tissue transverse section manifest DR6 mRNA expression data.In dark-field, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

The primary sequence of DR6cDNA of riboprobes that is used for generating these experiments is as follows:

GAGCAGAAACGGCTCCTTTATTACCAAAGAAAAGAAGGACACAGTGTTGCGGCAGGTCCGCCTGGACCCCTGTGACTTGCAGCCCATCTTTGATGACATGCTGCATATCCTGAACCCCGAGGAGCTGCGGGTGATTGAAGAGATTCCCCAGGCTGAGGACAAACTGGACCGCCTCTTCGAGATCATTGGGGTCAAGAGCCAAGAAGCCAGCCAGACCCTCTTGGACTCTGTGTACAGTCATCTTCCTGACCTATTGTAGAACACAGGGGCACTGCATTCTGGGAATCAACCTACTGGCGG(SEQ?ID?NO：3)

Further analysis use Allen mind map volume ( Http:// www.brainatlas.org/aba/Allen mind map volume is the obtainable science resource of the public, it provides about 20,000 kinds of expression of gene maps in the mouse brain) disclosed DR6 high expression level in the pallium of brain of growing up.DR6mRNA is for example expressing in cortical neuron, hippocampus CA1-CA4 cone neurone and the dentate gyrus.Express in the neuronal cell main body of DR6 albumen in grow up cortex and hippocampus.

This expression pattern provides evidence to prove, its about developmental outside, DR6 also brings into play function in the progress of the neurodegenerative disease relevant with the neuronal cell loss.

Embodiment 2:RNA disturbs the inhibition that DR6 is expressed to stop the neuronic axonal degeneration of explant culture mediocommissure

Commissural neuron is one group of spinal interneuron in the long projection that produces in the dorsal part spinal cord between the etap E9.5 to E11.5.Think that it is the nutritional support of spinal cord base plate that the survival of commissural neuron depends on one of middle target thing from them.This dependency occurred in during the time period of a lasting a couple of days, and their aixs cylinder is extended along base plate at that time, and they form other nutritional requirement afterwards.Neurone has been to by the way providing the neuronic mechanism of quick elimination mistake projection derived from their dependency of nutritional support of middle aixs cylinder target thing, this have help prevent in neural growth course, form unusual neuronal circuit (Wang etc., Nature, 401:765-769 (1999)).

In order to check DR6 in the axonal degeneration of commissural neuron and the functional effect in the apoptosis, carried out based on the dorsal part spinal cord of RNAi survival assay method (Kennedy etc., Cell, 78:425-435 (1994); Wang etc., See above, 1999) and (see figure 3).E13 rat or E11.5 mice embryonic are placed L15 substratum (Gibco), and siRNA (IDT) is injected in the neurocele with green fluorescent protein (" GFP ") coding plasmid.By electroporation siRNA and plasmid are delivered to the dorsal part progenitor cell then.Separate and cut dorsal part spinal cord explant, be embedded in the 3D-collagen gel matrix, and lead albumen (netrin)-1 (R﹠amp containing reorganization; D Pharmaceuticals) and in the Opti-MEM/F12 substratum (Invitrogen) of 5% horse serum (Sigma) in 37 ℃ at 5%CO ₂Cultivate in the environment.In 16 hours, as chemoattractant being led replying of albumen-1, commissural neuron grows from explant, enter in the collagen gel matrix (Kennedy etc., See above, 1994).By using the observation of inverted microscope, by GFP fluorescent appear Colaesce aixs cylinder.

As shown in Fig. 4 A, in lacking situation about supporting, cultivate 48 hours derived from the nutritional factor of base plate after, commissural neuron experience apoptosis and their axonal degeneration (also can be referring to Wang etc., see above 1999).Timing under the DR6 in the commissural neuron expresses by DR6 specific siRNA molecule, such axonal degeneration is by significantly blocking-up (seeing Fig. 4, following little figure).In the control experiment of using non-target siRNA molecule to carry out, do not observe this inhibition to axonal degeneration.These Notes of Key Datas DR6 is the needed important short apoptosis acceptor of the axonal degeneration of commissural neuron when the nutritional support that the middle target thing from commissural neuron is the spinal cord base plate is broken off.

As shown in Fig. 4 B, RNAi resistance DR6 cDNA has saved the sex change phenotype of being blocked by DR6siRNA.

In Fig. 4 B, from left to right, four top photos have shown the neurone when having and the following: (1) contrast RNAi; (2) be exposed to the wild-type DR6 of DR6siRNA#3; (3) be exposed to the mispairing DR6 of DR6siRNA#2; (4) be exposed to the mispairing DR6 of DR6siRNA#3.The following little figure of two width of cloth has shown the radioautogram of and the following: (1) wild-type DR6mRNA when having contrast siRNA, siRNA#2 and siRNA#3; (2) the mispairing DR6mRNA when having contrast siRNA, siRNA#2 and siRNA#3.

The material and the method that are used for generating the shown data of this figure are as follows.In order to investigate the DR6 acceptor at the axonal degeneration of commissural neuron and the physiological role in the apoptosis, according to scheme implementation known in the art dorsal part spinal cord survival assay method (Kennedy etc., Cell, 78:425-435 (1994); Wang etc., Nature, 401:765-769 (1999)) and (data presentation is in Fig. 4 B).The E13 rat embryo is placed L15 substratum (Gibco), and following siRNA construct (Fig. 4 B) is injected in their neurocele:

Contrast DR6siRNA#3 (IDT) and DR6cDNA wild-type or mispairing and the GFP coding plasmid of DR6siRNA#2 non-target or target or target.DR6cDNA and GFPcDNA subclone are gone into can be in the pCAGGS carrier framework that BCCM/LMBP buys.By electroporation siRNA and plasmid are delivered to the dorsal part progenitor cell then.Separate then and cut dorsal part spinal cord explant, be embedded in the 3D collagen gel matrix, and lead in the Opti-MEM/F12 substratum (Invitrogen) of albumen-1 and 5% horse serum (Sigma) in 37 ℃ at 5%CO containing reorganization ₂Cultivate in the environment.In 16 hours, as chemoattractant being led replying of albumen-1, commissural neuron grows from explant, enter in the collagen gel matrix (Kennedy etc., Cell, 78:425-435 (1994)).By using the observation of inverted microscope, by GFP fluorescent appear Colaesce aixs cylinder.After in lacking situation about supporting, cultivating 48 hours derived from the nutritional factor of base plate, commissural neuron experience apoptosis and their axonal degeneration (Wang etc., Nature, 401:765-769 (1999)) and (Fig. 4 B).Yet the axonal degeneration program can be blocked (Fig. 4 B) by the importing of target DR6 specific siRNA #3.DR specific siRNA #3 specific, go up target (on-target) effect and in saving experiment, obtained further confirmation, wherein siRNA#3 resistance mispairing DR6 cDNA construct is expressed with crossing of DR6 siRNA#3 and has been recovered axonal degeneration phenotype (Fig. 4 B).The experimental evidence that is presented has confirmed that the DR6 function of receptors is is the axonal degeneration of spinal cord base plate commissural neuron when regaining and dead needed at the middle target thing from them.

Employed DR6siRNAs#2 and #3 (sense strand) and as follows in the assay method mentioned above with DR6siRNA#3 sequence complementary DR6cDNA mispairing fragments sequence:

Rat DR6siRNA#25 '-AAU CUG UUG AGU UCA UGC CUU-3 ' (SEQ IDNO:11)

Rat DR6siRNA#35 '-CAA UAG GUC AGG AAG AUG GCU-3 ' (SEQ IDNO:12)

With DR6siRNA#3 sequence complementary rat DR6cDNA mispairing fragment: 5 '-GGACTCTGTGTACAGTCACCTCCCAGATCTGTTATAG-3 ' (SEQ IDNO:13)

Embodiment 3: anti-DR6 antibody stops the neuronic axonal degeneration of explant culture mediocommissure to the inhibition of DR6 receptor signal conduction

Use anti-DR6 antibody to carry out dorsal part spinal cord survival assay method (as mentioned described in the embodiment 2).Adopt microscopic examination (using the green fluorescence passage of GFP) to manifest the Colaesce aixs cylinder.According to scheme implementation dorsal part spinal cord known in the art survival assay method (Kennedy etc., Cell, 78:425-435 (1994); Wang etc., Nature, 401:765-769 (1999)), have the modification of being summarized among the embodiment 2 above.GFP expression plasmid construct (GFP cDNA subclone is gone into can be from the pCAGGS carrier framework that BCCM/LMBP buys) is injected in the neurocele of E13 rat embryo.By electroporation the GFP expression plasmid is delivered to the dorsal part progenitor cell then.To resist DR6 blocking antibody or contrast normal mouse IgG to be added into the Colaesce explant with 40ug/ml in 24 hours behind the bed board.As hereinafter summarizing, took the photo of Colaesce explant behind the bed board in 48 hours.In order to manifest the Colaesce aixs cylinder of expressing GFP, take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss ImagingSolutions in (in the green fluorescence passage at GFP) use on the Axiovert 200Zeiss inverted microscope.

Be used for the following generation of anti-DR6 antibody of this experiment.

Using to merge has the people DR6 ectodomain sequence (hDR6-ECD-Fc) of Fc to generate anti-DR6 mouse monoclonal antibody as immunogen.The immunogenic sequence of employed hDR6-ECD-Fc is as follows:

MGTSPSSSTALASCSRIARRATATMIAGSLLLLGFLSTTTAQPEQKASNLIGTYRHVDRATGQVLTCDKCPAGTYVSEHCTNTSLRVCSSCPVGTFTRHENGIEKCHDCSQPCPWPMIEKLPCAALTDRECTCPPGMFQSNATCAPHTVCPVGWGVRKKGTETEDVRCKQCARGTFSDVPSSVMKCKAYTDCLSQNLVVIKPGTKETDNVCGTLPSFSSSTSPSPGTAIFPRPEHMETHEVPSSTYVPKGMNSTESNSSASVRPKVLSSIQEGTVPDNTSSARGKEDVNKTLPNLQVVNHQQGPHHRHILKLLPSMEATGGEKSSTPIKGPKRGHPRQNLHKHFDINEHLPWMIPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ?ID?NO：4)

Use previously described scheme generate fusion polypeptide (Ashkenazi etc., Curr Opin Immunol., 9 (2): 195-200 (1997); Haak-Frendscho etc., J Immunol., 152 (3): 1347-53 (1994)).

By injection 100ul hDR6-ECD-Fc immunogen (1mg/ animal) in about 8 time-of-week sections give 9 age in week the Balb/c mouse immune.Then with all lymphoglandula (11x10 through immune mouse ⁶Cell/ml is 5ml) with PU.1 myeloma cell's (from rat bone marrow tumour cell of ATCC) (5x10 ⁶Cell/ml 5ml) merges.With cell with 2x10 ⁶Cell/ml is dispensed in 4 blocks of plates.

Use and catch ELISA the specificity combination of hybridoma screening to hDR6-ECD-Fc polypeptide mentioned above.Plate is spent the night in 4 ℃ of bags with the anti-human IgG Fc of 50ul 2ug/ml goat specificity (Cappel catalog number 55071).Plate is added Brij with PBS clean 3 times, and plate was sealed 1 hour in room temperature with 200ul 2%BSA.Then plate being added Brij with PBS cleans 3 times.Subsequently, with plate with the 0.4ug/ml immunoadhesin in 100ul/ hole incubation 1 hour on shaking table.Then plate being added Brij with PBS cleans 3 times.Be added into the hole with 100ul one is anti-, and on shaking table incubation 1 hour.Once more plate being added Brij with PBS cleans 3 times.100ul1: 1000 sheep anti mouse IgG HRP (not intersecting Cappel catalog number 55569) antibody 1 hour with the people.Plate is added Brij with PBS to be cleaned 3 times.Add 50ul substrate (TMB Microwell peroxidase KPL#50-76-05) and with plate incubation 5 minutes.With 50ul/ hole stop buffer (KPL#50-85-05) termination reaction.Read the absorbancy at 450nm place.Employed mensuration damping fluid contains PBS, 5%BSA and 0.05%Tween 20.

Be cloned in by limiting dilution (the SCDME substratum that contains 10%HCF, 10%FCS) then and catch test in the ELISA assay method hDR6-ECD-Fc polypeptide is combined into the male hybridoma.After 10 days, take out plate and measure the hole that a colony is arranged by seizure ELISA mentioned above.Then multiple selected monoclonal antibody is tested isotype, and be shown as the IgG1 isotype.

In dorsal part spinal cord survival assay method, discriminating is tested the ability of their blocking-up axonal degenerations then for four kinds of anti-DR6 monoclonal antibodies of " 3B11.7.7 ", " 3F4.4.8 ", " 4B6.9.7 " and " 1E5.5.7 ".

Surprising is, some (3F4.4.8,4B6.9.7 and the 1E5.5.7) in these anti-DR6 monoclonal antibodies can partly suppress the axonal degeneration (see figure 5) by the deprivation induced commissural neuron of 48 hours nutrition in cultivating.Think that this antibody-like may promote neuronal survival, for example DR6 part of inferring by blocking-up and the interaction between the DR6 acceptor or by suppressing the conduction of ligand dependent DR6 signal.3B11.7.7DR6 antibody has stimulatory effect slightly in inducing axonal degeneration.

The specific peptide inhibitor of embodiment 4:Jun N-terminal kinases (JNKI) is to the inhibition of DR6 receptor signal conduction

The DR6 acceptor it is reported and signal by the activation of JNK, and observe the JNK activity DR6 lack impaired in the no mouse model (Pan etc., FEBS Lett., 431:351-356 (1998); Zhao etc., Journalof Experimental Medicine, volume 194,1441-1441,2001)).In order to check the effect of DR6-JNK signal conduction in axonal degeneration, implemented dorsal part spinal cord survival assay method (as mentioned described in the embodiment 2), just by using inhibitor peptides L-JNK-I ((the L)-HIV-TAT48-57-PP-JBD20 of 1 μ M concentration; Calbiochem) the JNK signal transduction path in the blocking-up commissural neuron.DMSO in contrast (SIGMA) and normal mouse IgG have been tested.

As shown in Figure 6, this being suppressed in the dorsal part spinal cord survival assay method of JNK signal conduction partly blocked axonal degeneration.These Notes of Key Datas DR6 sends the signal of axonal degeneration process at least in part by the JNK approach.

Embodiment 5: anti-DR6 antibody is to the neuronal cell death in the inhibition prevention mice embryonic spinal cord of DR6 receptor signal conduction

Carried out assay method, wherein in full embryo culture system, conducted with anti-DR6 monoclonal antibody blocking-up DR6 signal.Hereinafter described this system allow the full embryo of mouse in phial vitro culture 2 days, grow stage E 9.5 certainly to E11.5.Separate from the uterus and to cut the E9.5 embryo, yolk sac is attached to the embryo, and is incubated at 37 ℃ in second day in 95% oxygen in the neutralization of 65% oxygen atmosphere in first day in 100% rat blood serum (Harlan).In assay method, add anti-DR6 monoclonal antibody (above described in the embodiment) with the 10ug/ml final concentration, and use normal mouse IgG antibody in contrast with 10ug/ml concentration.

By use identification through the antibody of the Caspase-3 of cutting (at antibody, available from R﹠amp through the Caspase-3 of cutting; D Systems) immunofluorescence dyeing that carries out detects and microscopy observing apoptosis cell.The results are shown in Fig. 7.Surprising is that anti-DR6 monoclonal antibody 3F4.4.8,4B6.9.7 and 1E5.5.7 protect spinal neuron to the inhibition of DR6 at the developmental character necrocytosis of natural generation in this system.

Embodiment 6:DR6 lacks the neuronal cell death that reduces in the no mouse

Analyzed the phenotype that the DR6 that is in etap E15.5 knocks out embryo (Zhao etc., Journal ofExperimental Medicine, Vol.194,1441-1441,2001).Caspase 3 through cutting is marks of apoptotic cell, and in order to check the degree of neuronal cell death in the embryonic spinal cord, use immunostaining (at antibody, available from R﹠amp through the mouse Caspase-3 of cutting through the Caspase 3 of cutting; D Systems).Also checked DR6 heterozygosis littermate in contrast.With paraformaldehyde (PFA) fixed embryonic tissue section in confining liquid (2% heat-inactivated lowlenthal serum (Sigma)/PBS (Gibco)/0.1% Triton (Sigma)) sealing 1 hour and with one anti-(dilution in 1: 500 at antibody, available from R﹠amp through the mouse Caspase-3 that cuts; D Systems) in confining liquid, is incubated overnight together in 4 ℃.To cut into slices and last 1 hour with confining liquid in room temperature and clean 3 times, and with two anti-(the anti-rabbit Alexa 488 of goat of dilution in 1: 500, Molecular Probes Invitrogen) one arised from the room temperature incubation 1 hour.To cut into slices then and manifest in room temperature cleaning 1 hour and by the immunofluorescence in the green channel with confining liquid.

To the number of Caspase 3 positive nucleuss of each each spinal cord slice of embryo quantitatively (seeing Fig. 8 and 9A).In DR6 lacks no mouse spinal cord and dorsal root ganglion (DRG), detect and compare the dead reduction of neuronal cell about 40-50% (Fig. 8 and 9A) with DR6 heterozygosis littermate contrast.Thereby, think that DR6 signal conduction in vivo promotes the neuronal cell death in the developmental neural system.

As shown in Fig. 9 B, DR6 is that the kinematic axis mutagenicity is needed, is confirmed as the scarce no mouse of DR6.In the situation that has and do not exist brain derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3) (BDNF and NT-3 derive from Chemicon), analyze and knock out embryo (Zhao etc. from the DR6 that is in etap E13.5, Journal of Experimental Medicine, volume 194,1441-1441,2001) and the ventral cord explant (motor neuron) of fetal tissues.

In Fig. 9 B, upper left little figure has shown the ventral cord explant from normal mouse in the situation that has BDNF and NT-3, and the little figure in lower-left has shown knock out the ventral cord explant of (KO) mouse from DR6 in the situation that has BDNF and NT-3.Similarly, upper right little figure has shown the ventral cord explant from normal mouse in the situation that does not have these somatomedins, and the little figure in bottom right has shown knock out the ventral cord explant of (KO) mouse from DR6 in the situation that does not have BDNF and NT-3.

The material and the method that are used for generating the shown data of this Fig. 9 B are as follows.As Henderson etc., Nature, described in the 363:266-270 (1993) like that but have a few modifications to implement motor neuron ventral cord survival assay method.The scissors that use is handled through alcohol are separated and are cut DR6 heterozygosis or the scarce no mouse E13.5 embryo of DR6 and place warm L15 substratum (Gibco).Use same scissors and tweezers, open embryo's abdomen district, exteriorize, cut away rib, and separate and cut whole spinal cord, the meninges tissue around removing with tweezers then.Remove top board, obtained to open book type spinal cord prepared product (open book prep of spinalcord).Separate comprise MMC and LMC motor column ventral side of spinal cord half and cut away remaining base plate tissue carefully.Be used in the yellow rifle head (tip) that the bag quilt is crossed among the L15 ventral cord is transferred to the new shallow bid that L15+5%FBS (Sigma) serum is housed, be used to use the tungsten syringe needle further to be cut into explant.

Give 8 hole slide glass (Becton through PDL/ ln bag quilt, Dickinson and Company) filling 500ul every hole Neurobasal substratum (Invitrogen) adds reorganization BDNF and the NT-3 (Chemicon) that is 50ng/ml, adds B-27 fill-in X50 (Invitrogen); Add penicillin-Streptomycin sulphate-glutamine X100 (catalog number 10378-016; Gibco) add glucose X100.The ventral cord explant that cuts is placed each hole (each hole 2-3 explant) and places 48 hours with growth at 37 ℃ of incubators.After 2 days, following enforcement nutritional factor is deprived: take old substratum away, and the hole is leniently cleaned 2 times with Neurobasal substratum (not containing nutritional factor).

Add pre-warm Neurobasal substratum/B-27 (Invitrogen) add (preparation as indicated above does not contain nutritional factor) the anti-BDNF of 20ug/ml and anti-NT3 blocking antibody (Genentech, Inc.).The slide glass that will have explant then in 37 ℃ incubation 24-48 hour again.

After 2 days, will be among the 4%PFA of explant in PBS fixing, with the Net gel (Nikolaev etc., 2003, Cell, 112 (1), 29-40) 0.2%Triton in is in 0 ℃ of saturatingization 10 minutes, and with Net gel cleaning 2 times.In order to seal nonspecific binding site, slide glass is incubated overnight in 4 ℃ in containing the PBS of 1%BSA.In order to manifest the motion aixs cylinder in the sex change, implement next day with the (dilution in 1: 500 of anti-p75NTR specific antibody, Chemicon) immunostaining that carries out (one of dilution in 1: 500 resists in 4 ℃ and spends the night in 1%BSA/PBS, and two of dilution in 1: 500 resists in room temperature 1 hour).Take off the hole, and use Fluoromount-G that cover glass is contained on the slide glass.In order to manifest the motion aixs cylinder of expressing p75NTR, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

Shown in disclosed data among Fig. 9 C, in the DR6 knock-out mice, obtain postponing by the sex change of damage inductive.

In Fig. 9 C, from left to right, the top little figure of four width of cloth has shown the neurone from normal mouse respectively: in the situation that has nerve growth factor (NGF); And damaged the back 4,8 or 16 hours.In Fig. 9 C, from left to right, the following little figure of 4 width of cloth has shown the neurone from the DR6KO mouse respectively: in the situation that has external source nerve growth factor (NGF); And damaged the back 4,8 or 16 hours.

The external sensation neurite infringement assay method of following enforcement as shown in Fig. 9 C.Separate and cut DR6 heterozygosis or the scarce no mouse E12.5 embryo of DR6 and place warm L15 substratum (Gibco).Use same scissors and tweezers, open embryo's abdomen district, exteriorize, cut away rib, and separate with tweezers and to cut the dorsal root ganglion (DRG) that is attached to spinal cord.Be used in the yellow rifle head that the bag quilt is crossed among the L15 then DRG is transferred to the new shallow bid that L15+5%FBS (Sigma) serum is housed, be used to use the tungsten syringe needle further to be cut into the 1/4DRG explant.

Give the 8 hole slide glass (Becton that wrap quilt through the PDL/ ln in advance, Dickinson andCompany) filling 500ul every hole Neurobasal substratum (Invitrogen) adds 50ng/ml NGF (RocheMolecular Biochemicals), adds B-27 fill-in X50 (Invitrogen); Add penicillin-Streptomycin sulphate-glutamine X100; Add glucose X100.The DRG explant that cuts is placed each hole (each hole 2-3 DRG explant) and places 48 hours with growth at 37 ℃ of incubators.After 2 days, following enforcement aixs cylinder infringement assay method: by with micro-knife (Fine Science Tools) directly over the DRG explant with under do two parallel cuts to sensation neurite and bring out damage.The aixs cylinder of not cutting on the DRG explant left side and the right is served as endogenous harmless contrast.The slide glass that will have a DRG explant of cutting is fixing among 0,4,8,16 and 24 hour 4%PFA in PBS after damage, with Net gel (Nikolaev etc., 2003, Cell, 112 (1), 29-40) 0.2%Triton in is in 0 ℃ of saturatingization 10 minutes, and cleans 2 times with the Net gel.In order to seal nonspecific binding site, will be incubated overnight in 4 ℃ among the 1%BSA of slide glass in PBS.In order to manifest the sensation neurite in the sex change, implement next day with the (dilution in 1: 500 of neurone III class 'beta '-tubulin (TUJ1) specific antibody, Covance) immunostaining (one of dilution in 1: 500 resists in 4 ℃ and spends the night in 1%BSA/PBS, and two of dilution in 1: 500 resists in room temperature 1 hour).Take off the hole, and use Fluoromount-G to come to slide glass mounting cover slide.In order to manifest sensation neurite, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions with immunofluorescence label.

Embodiment 7: anti-DR6 antibody antagonist suppresses neuronal degeneration

As shown in Figure 10 A, anti-DR6 antibody suppresses neuronic sex change (in the axonal degeneration assay method) of having been deprived nutritional factor of all kinds.

In Figure 10 A, from left to right, two photos have shown the data from commissural neuron about two groups of the front.In four photos of this front, two top photos have shown the commissural neuron in the situation that has contrast IgG and RA.5 DR6 antibody respectively, and two following photos have shown the commissural neuron in the situation that has RA.1 DR6 antibody and RA.3 DR6 antibody respectively.Among Figure 10 A two groups of intermediary up and down two photos shown data from Sensory neurone.In these intermediary four photos, above two photos shown Sensory neurone in the situation that has and do not exist NGF respectively, and two following photos shown do not have NGF but have RA.1 DR6 antibody respectively and the situation of RA.3 DR6 antibody in Sensory neurone.Two photos have shown the data from motor neuron about two groups of Figure 10 A right side.On the right in this four photos, above two photos shown motor neuron in the situation that has and do not exist somatomedin respectively, and two following photos shown do not have somatomedin but have RA.1 DR6 antibody respectively and the situation of RA.3 DR6 antibody in motor neuron.

The material and the method that are used for generating the shown data of this figure are as follows.Mouse monoclonal RA.1-RA.5 DR6 antibody as mentioned described in the embodiment 3 by generating with DR6 extracellular domain immune mouse.The DR6 antibody that is called " RA.1 " and " RA.3 " antibody in this embodiment and accompanying drawing is respectively " 1E5.5.7 " and " 3F4.4.8 " antibody (for example using alternative name to censure simply) described in the embodiment 3.Similarly, " RA.5 " antibody of addressing in this embodiment and accompanying drawing is " 3B11.7.7 " antibody (for example having used alternative name to censure) described in the embodiment 3.

Among embodiment 2 as indicated above and the embodiment 6 like that but have following modifications ground to implement sensation, motion and the cultivation of Colaesce explant.For Colaesce explant survival assay method, behind bed board, with the 20ug/ml final concentration DR6 antibody RA.1 or RA.3 or contrast IgG were added into Colaesce explant culture (Figure 10 A) in 24 hours.For sensation explant culture, 48 hours enforcement NGF deprive assay method behind bed board.Behind bed board, will not contain NGF with the 20ug/ml final concentration in 48 hours but contain the NGF blocking antibody (Genentech, Inc.) and shown in the fresh Neurobasal substratum of DR6 antibody (RA.1 or RA.3) or contrast IgG be added into sensation explant culture (Figure 10 A).For motion explant culture, 48 hours enforcement nutritional factor are deprived assay method behind bed board.Behind bed board, will not contain NT3/BDNF with the 20ug/ml final concentration in 48 hours but contain the BDNF blocking antibody and the NT3 blocking antibody (monoclonal antibody of the function of performance blocking-up nutritional factor, Genentech, Inc.) and shown in DR6 antibody (RA.1 or RA.3) or the contrast IgG fresh Neurobasal substratum be added into the sensation explant culture (Figure 10 A).In order to manifest, on Axioplan-2 imaging Zeiss microscope, to use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions by the sensation and the motion aixs cylinder of immunofluorescence dyeing with anti-TUJ1 (Covance) and anti-p75NTR (Chemicon/Millipore) antibody labeling.In order to manifest the Colaesce aixs cylinder of expressing GFP, (in the green fluorescence passage at GFP) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of CarlZeiss Imaging So1utions on Axiovert 200 Zeiss inverted microscopes.

As shown in Figure 10 B, anti-DR6 antibody suppresses neuronic sex change (in the assay method of cell main body in the painted apoptosis of TUNEL) of having been deprived nutritional factor of all kinds.In Figure 10 B, begin from the left side, two width of cloth have shown the data from commissural neuron about in the of two groups.In four photos of this front, two top photos have shown the commissural neuron in the situation that has contrast IgG and RA.5 DR6 antibody respectively, and two following photos have shown the commissural neuron in the situation that has RA.1 DR6 antibody and RA.3DR6 antibody respectively.In the middle of among Figure 10 B two groups up and down two photos shown data from Sensory neurone.In these intermediary four photos, above two photos shown Sensory neurone in the situation that has and do not exist NGF respectively, and two following photos shown do not have NGF but have RA.1 DR6 antibody respectively and the situation of RA.3 DR6 antibody in Sensory neurone.Two photos have shown the data from motor neuron about two groups of Figure 10 B right side.In four photos on these the right, above two photos shown motor neuron in the situation that has and do not exist somatomedin respectively, and two following photos shown do not have somatomedin but have RA.1 DR6 antibody respectively and the situation of RA.3 DR6 antibody in motor neuron.

Disclosure prompting part among Figure 10 may play a significant role in the function of DR6 in axonal degeneration.

The material and the method that are used for generating the shown data of this figure are as follows.As mentioned above, mouse monoclonal RA.1-RA.5 DR6 antibody as mentioned described in the embodiment 3 by generating with DR6 extracellular domain immune mouse.Among embodiment 2 as indicated above and the embodiment 6 like that but have the modification ground of following general introduction to implement sensation, motion and Colaesce explant to cultivate.For Colaesce explant survival assay method, as mentioned described in the embodiment 3, behind bed board, respectively DR6 antibody RA.1 and RA.3, antibody RA.5 (perhaps were called " 1B11.7.7 " with the 20ug/ml final concentration in 24 hours, Genentech, Inc.) or contrast IgG (Genentech, Inc.) be added into Colaesce explant culture (Figure 10 B, the left side).

For sensation explant culture, 48 hours enforcement NGF deprive assay method behind bed board.Behind bed board, will not contain NGF with the 20ug/ml final concentration in 48 hours but contain NGF blocking antibody (Genentech, Inc.) and DR6 antibody RA.1 or RA.3 or contrast IgG (Genentech, Inc.) fresh Neurobasal substratum is added into sensation explant culture (Figure 10 B, centre).For motion explant culture, 48 hours enforcement nutritional factor are deprived assay method behind bed board.Behind bed board, will not contain NT3/BDNF with the 20ug/ml final concentration in 48 hours but contain the BDNF blocking antibody and the NT3 blocking antibody (monoclonal antibody of the function of performance blocking-up nutritional factor, Genentech, Inc.) and RA.1 or RA.3 or contrast IgG (Genentech, Inc.) fresh Neurobasal substratum is added into sensation explant culture (Figure 10 B, the right).

Explant is fixing and for to process at unicellular level detection apoptosis in 4%PFA/PBS, (catalog number 11684795910 is Roche) according to instruction manual (Roche) the marker DNA splitting of chain (TUNNEL technology) of manufacturers based on using original position necrocytosis detection kit in described detection.By the apoptosis (Figure 10 B) in the cell main body of fluorescent microscopy analysis Colaesce sensation and motion explant culture.In order to manifest fluorescently-labeled TUNNEL positive apoptotic cells main body, (in the red fluorescence passage) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axioplan-2 imaging Zeiss microscope.

Embodiment 8:DR6 immunoadhesin antagonist suppresses neuronal degeneration

As shown in Figure 11 A, hDR6-ECD-Fc postpones the Colaesce axonal degeneration.Employed hDR6-ECD-Fc immune adherence fibroin in this assay method has above been described among the embodiment 3.

In Figure 11 A, from left to right, first photos provides the contrast of the Colaesce axonal degeneration when showing 48 hours.Colaesce axonal degeneration when second photos has shown in the situation that has 30ug/ml hDR6-ECD-Fc 48 hours.Colaesce axonal degeneration when the 3rd photos has shown in the situation that has 10ug/ml hDR6-ECD-Fc 48 hours.

The material and the method that are used for generating the shown data of this figure are as follows.Prepare Colaesce explant culture as mentioned described in the embodiment 2-6 and implement the survival assay method.Employed hDR6-ECD-Fc immunoadhesin in this assay method has above been described among the embodiment 3.In order to manifest the Colaesce aixs cylinder of GFP mark, (in the green fluorescence passage at GFP) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of CarlZeiss Imaging Solutions on Axiovert 200 Zeiss inverted microscopes.

As shown in Figure 11 B, the sensation neurite sex change that the hDR6-ECD-Fc delay is broken off bringing out by nerve growth factor (NGF).In Figure 11 B, from left to right, above three photos shown the Sensory neurone of in the situation that has contrast Fc, having been deprived NGF in the time of 0,6 and 24 hour respectively, and three following photos have shown the Sensory neurone of having been deprived NGF in the situation that has the DR6-Fc construct in the time of 0,6 and 24 hour respectively.

The disclosure that is provided among Figure 11 provides further prompting, and promptly part may play a significant role in the function of DR6 in axonal degeneration.

The material and the method that are used for generating the shown data of this figure are as follows.In order to check whether part is that the function of DR6 in the sensation neurite sex change is needed, the compartment culture assays of following enforcement sensation neurite growth and sex change.Use Campenot neurocyte chamber system to separate neurone in different compartments's (different fluid environments) and give prominence to (aixs cylinder) and cell main body, this and neuronal cell main body are in a neural position and to throw their aixs cylinder similar to the far-end target thing of another location.The initial record of this assay method such as Campenot (Campenot etc. .J Neurosci.11 (4): 1126-39 (1991)) like that but the enforcement of following modification is arranged.In brief, with 35mm tissue culture dish with PDL/ ln bag by and with rake (Tyler Research) scraping producing track, as for example Campenot etc., See aboveFig. 1 and Fig. 4 shown in.

A substratum (the Neurobasal substratum that contains B27 fill-in, 25ng/ml NGF and 4g/L methylcellulose gum) is placed through on the bottom of swiping.Be placed in Teflon separation scraper (Tyler Research) on the silicone grease and a small amount of silicone grease be positioned over the oral area of central groove.Will being suspended in the substratum of methylcellulose gum thickening and being loaded in the disposable sterilized syringe of being furnished with No. 22 syringe needles derived from E12.5 mouse DRG through dissociated Sensory neurone.Under dissecting microscope, this cell suspending liquid is injected in the central groove of each compartment dish.Allow the neurone sedimentation spend the night.Give the periphery (cell main body compartment) of dish and the substratum that the filling of inner aixs cylinder compartment contains methylcellulose gum.External, in 3-5 days, aixs cylinder begins to enter the compartment on the left side and the right, as for example Campenot etc., See aboveFig. 1 and Fig. 4 shown in.

In order to trigger local axonal degeneration, with containing NGF blocking antibody (anti-NGF, Genentech, Inc., the substratum that contains NGF of the alternative aixs cylinder compartment of Neurobasal substratum 20ug/ml).NGF deprived back 0 hour, 6 hours or 24-48 hour, Sensory neurone is fixed 30 minutes in room temperature in 4%PFA also process to manifest the aixs cylinder (Figure 11 B) (embodiment 7 is described as mentioned) in the sex change by fluorescent microscopy for the immunofluorescence dyeing that carries out with aixs cylinder mark TUJ-1 (Covance, dilution in 1: 500).For the sensation neurite of immunofluorescence label in the aixs cylinder compartment that manifests the Campenot chamber, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

In order to check whether part is that the function of DR6 in the axonal degeneration program of being broken off triggering by NGF is needed, in the aixs cylinder compartment of Campenot chamber, comprise 30ug/ml hDR6-ECD-Fc immune adherence fibroin (above described in the embodiment 3) or 30ug/ml contrast Fc (Genentech, Inc.) and anti-NGF handle.NGF deprived the back 0-24 hour, aixs cylinder in the Campenot chamber fixed with 4%PFA/PBS and by using TUJ-1 (1: 500, Covance)/coupling have fluorophor Alexa 488 two anti-(MolecularProbes, BD) immunofluorescence dyeing that carries out manifests (Figure 11 B).

NGF deprives the surprising pattern that has triggered axonal degeneration, as shown in Figure 11 B.Importantly, the proteic interpolation of hDR6-ECD-Fc immunoadhesin postpones the generation (Figure 11 B, following little figure) of axonal degeneration in this system.Thereby these Notes of Key Data soluble ligands may be that the function of DR6 acceptor in the local axonal degeneration that brings out by the elimination somatomedin is needed.

Embodiment 9:NGF deprives back DR6 ligand-binding site point and comes off from aixs cylinder

As shown in Figure 12 A and 12B, use DR6-AP construct manifests the DR6 binding site on the sensation neurite.

In Figure 12 A, from left to right, above two photos shown with low and high-amplification-factor respectively and use the DR6-AP construct being apparent in the sensation neurite video picture of DR6 binding site on these aixs cylinders in the time of 48 hours that is in etap E12.5 in the situation that has NGF that and two following photos have shown the video picture of the sensation neurite that uses AP contrast construct respectively with low and high-amplification-factor.

As shown in Figure 12B, DR6 ligand-binding site point is deprived the back from feeling that aixs cylinder loses at NGF.

In Figure 12 B, from left to right, two top photos have shown the video picture of DR6 binding site on the sensation neurite, wherein first photos has shown the Sensory neurone in the situation that has NGF and BAX inhibitor, and second photos has shown that the Bax in having the situation of NGF lacks the Sensory neurone of not having.Below two photos shown respectively and do not have NGF but exist Sensory neurone and the Bax in the situation that does not have NGF in the situation of BAX inhibitor to lack the Sensory neurone of not having.In the situation that has and do not exist neurotrophin, in the motion aixs cylinder, observe the result who is equal to.

The material and the method that are used for generating the shown data of Figure 12 A and 12B are as follows.Use pRK5-AP cloning vector (referring to for example Yan etc., Nature Immunology 1,37-41 (2000)), generate DR6-AP construct (DR6-AP) by mouse DR6 extracellular domain being merged to people embryo alkaline phosphatase.PRK5 parent cloning vector can be from Becton, Dickinson and Company, and Pharmingen division obtains.The mouse DR6 extracellular domain sequence that is used to generate the DR6-AP fusion rotein is as follows:

MGTRASSITALASCSRTAGQVGATMVAGSLLLLGFLSTITAQPEQKTLSLPGTYRHVDRTTGQVLTCDKCPAGTYVSEHCTNMSLRVCSSCPAGTFTRHENGIERCHDCSQPCPWPMIERLPCAALTDRECICPPGMYQSNGTCAPHTVCPVGWGVRKKGTENEDVRCKQCARGTFSDVPSSVMKCKAHTDCLGQNLEVVKPGTKETDNVCGMRLFFSSTNPPSSGTVTFSHPEHMESHDVPSSTYEPQGMNSTDSNSTASVRTKVPSGIEEGTVPDNTSSTSGKEGTNRTLPNPPQVTHQQAPHHRHILKLLPSSMEATGEKSSTAIKAPKRGHPRQNAHKHFDINEH(SEQ?ID?NO：14)

Bax lacks no mouse system (Bax-R1) (Deckwerth etc., Neuron, volume 17,401-411,1996) before on the books, and derives from Jackson Laboratories.Use the BAX inhibiting peptide to come the necrocytosis (Bax-V5, Tocris Inc) of block nerves unit with 10uM.

In order to generate mouse DR6 extracellular domain-AP fusion rotein (DR bv6-AP), the COS-1 cell that uses FuGene transfection reagent (Roche) to cultivate in DMEM/10%FBS (Gibco) substratum with the transfection of 15ug DR6-AP amalgamation and expression construct according to the scheme of manufacturers.After the transfection 12 hours, change the COS-1 cell culture medium into OPTI-MEM (Invitrogen).After the transfection 48 hours, collect and filter and contain the proteic COS-1 cell of DR6-AP conditioning substratum.The proteic amount of DR6-AP in the following quantitative culture medium:

With 100ul 2XAP damping fluid (by with 100mg p-nitrophenyl phosphate (Sigma) and 15ul1M MgCl ₂Being added into 15ml 2M diethanolamine pH 9.8 prepares) mix through rotaring redyeing COS cell conditioning substratum or from the collating condition substratum of untransfected COS-1 cell with isopyknic.Color development to reacting in 12-15 minute, O.D. (0.1-1) in linearity range at this moment.Come the conditioned reaction volume by adding 800ul distilled water then, and measure the O.D. at 405nm absorbancy wavelength place.Concentration in nM is calculated (being used for 100ul): C (nM)=O.D.X 100X (60/ developing time)/30 according to following formula.

For original position DR6-AP sensation neurite binding assay, cultivate sensation explant wild-type or the scarce nothing of Bax among the Neurobasal substratum/B27 (Invitrogen) that in containing the 7-8 of embodiment as mentioned of 50ng/ml NGF (Roche), is summarized.Behind the bed board 2 days, DRG explant or do not handle or deprive NGF described in the embodiment 7-8 as mentioned.Add the Bax inhibiting peptide, and shown in Figure 12 B (10uM, Bax-V5, Tocris).NGF deprived back 12 hours, and (HBSS, Gibco catalog number 14175-095 contains 0.2%BSA, 0.1%NaN3,5mM CaCl with binding buffer liquid with the DRG explant ₂, 1mM MgCl ₂, 20mMHEPES pH=7.0) cleans 2 times.Implement the AP binding assay then, 1: 1 mixture that promptly prepares DR6-AP conditioning substratum and binding buffer liquid (or contrast AP conditioning substratum and binding buffer liquid), with its directly be applied to 8 holes cultivate in the slide glasss (Becton, Dickinson and Company) the DRG explant and in room temperature incubation 90 minutes.

Behind the incubation, by washing the DRG explant off unconjugated DR6-AP albumen for 5 times with the rinsing of binding buffer liquid.Then the DRG explant is fixed 12 minutes with 3.7% formaldehyde that dilutes among the PBS in room temperature.By the DRG explant is removed remaining formaldehyde 3 times with HBS damping fluid (20mM HEPES pH=7.0,150mM NaCl) rinsing.By in the HBS damping fluid, blocking endogenous AP activity in 30 minutes in 65 ℃ of hot deactivations.Then with the DRG explant at AP reaction buffer (100mM TRIS pH=9.5,100mM NaCl, 50mM MgCl ₂) in rinsing 3 times.Then by in the AP reaction buffer that contains 1/50 (by volume) NBT/BCIP stoste (Roche, catalog number 1681451), the color dyes spot on the DRG explant being manifested the DR6-AP fusion rotein (Figure 12 A and 12B) that is bonded to sensation neurite in color development at room temperature after yesterday.In parallel control experiment, the conditioning substratum of the COS cell of the AP transfection of hanging oneself in the future is used for AP aixs cylinder binding assay (Figure 12 A, following little figure).

As shown in Figure 12B, the DR6-AP binding site is deprived the back from feeling that the aixs cylinder surface loses at NGF, and prompting DR6 part is released into after nutrition is deprived in the aixs cylinder conditioning substratum.

As shown in Figure 12 C, beta-secretase (BACE) inhibitor that studies show that is in the scarce numbness aixs cylinder of BAX of etap E12.5 can be blocked NGF and break off back DR6-AP binding site from feeling that aixs cylinder disappears.In Figure 12 C, from left to right, three top photos have shown these neurones in the situation that has DMSO contrast, OM99-2 (BACE-I inhibitor) and TAP1 (alpha-secretase enzyme-I inhibitor) respectively.Following photo has shown these neurones in having the situation of NGF.

The mouse DR6 extracellular domain-AP fusion rotein that is used to generate these data has above been described.Bax lacks no mouse system (Bax-R1) (Deckwerth etc., Neuron, volume 17,401-411,1996) before on the books, and derives from Jackson Laboratories.Cultivate and DR6-AP aixs cylinder binding assay about Figure 12 A and the described such DRG explant of implementing of 12B as mentioned.In this assay method with the 1um final concentration use the BACE inhibitor (InSolution OM99-2, Calbiochem/Merck).In this assay method with the 10uM final concentration use alpha-secretase enzyme inhibitors TAPI (TAPI-1, Calbiochem).In order to manifest the positive sensation neurite of DR6-AP (dyeing), on Axioplan-2 imaging Zeiss microscope, use and take bright visual field photo from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions by the AP colorimetry staining reaction of being summarized among the embodiment 9 above.

Embodiment 10: amyloid precursor protein (APP) is the related part of DR6

As shown in Figure 13, find that N-APP is a DR6 extracellular domain associated ligands.

In Figure 13 A, from left to right, preceding two width of cloth traces provide the data from the research of using the DR6-AP construct to carry out, to detect the protein that (and existing in the situation of Bax inhibitor) felt certainly and motor neuron obtains in the situation that has and do not exist somatomedin.In these traces, in the sensation of having deprived somatomedin (and existing in the situation of Bax inhibitor) and motor neuron, all observe APP polypeptide (being included in the strong band at about 35kDA place).The central trace of Figure 13 A has shown with the anti-N-APP antibody probe of the polypeptide that obtains from the Sensory neurone of having deprived somatomedin correspondingly observes APP polypeptide (being included in the strong band at about 35kDA place).The anti-N-APP antibody of polyclone that dilution in 1: 100 is used for the Western Blot experiment derives from Thermo Scientific (catalog number RB-9023-P1).Use Bax inhibiting peptide P5 (inhibitor peptides is synthesized in the Bax plastosome transposition of cell permeability for Tocris Biosciences, catalog number 1786) with 10uM.

The data that presented in the trace on Figure 13 A the right have confirmed that further APP is the observations of DR6 extracellular domain associated ligands.Under the condition that NGF deprives, collect sensation neurite conditioning substratum from Campenot chamber aixs cylinder compartment, and use general pull-down scheme (general pull-down protocol) (Nikolaev etc. for example, 2004, BBRC, 323,1216-1222) feel aixs cylinder conditioning substratum purifying DR6-ECD extracellular domain correlation factor certainly.The NiNTA pearl (Sigma) that coupling is had a DR6-ECD-His extracellular domain (construct hereinafter described) is with 50ml sensation neurite conditioning substratum incubation under the following conditions: 150mM NaCl, 0.2%NP-40 (Calbiochem), 1X PBS damping fluid spends the night in 4 ℃.The NiNTA pearl (Sigma) that then coupling is had a DR6-ECD-His extracellular domain is with 10 times of excessive binding buffer liquid (150mM NaCl, 0.2%NP-40 (Calbiochem), in 1X PBS damping fluid) clean 5 times, and, then it separately and with anti-N-APP antibody is detected through gel electrophoresis with the conjugated protein mixture of 1X SDS sample loading damping fluid (Invitrogen) wash-out DR6-ECD.Come the data of the drop-down experiment of DR6-ECD since then correspondingly to identify APP polypeptide (being included in the strong band at 35kDA place).

Scheme (Pettmann etc., 1988, J.Neurosci., 8 (10): 3624-3632) aixs cylinder conditioning substratum is implemented DR6-AP trace assay method according to previous record.The anti-N-APP antibody of polyclone that is used for the Western Blot experiment derives from Thermo Scientific (catalog number RB-9023-P1).Employed mouse DR6 extracellular domain-AP fusion rotein is as mentioned described in the embodiment 9.Express mouse reorganization DR6-ECD-His, subsequently from Chinese hamster ovary celI culture purifying.The aminoacid sequence of mouse DR6-ECD-His is as follows:

MGTRASSITALASCSRTAGQVGATMVAGSLLLLGFLSTITAQPEQKTLSLPGTYRHVDRTTGQVLTCDKCPAGTYVSEHCTNMSLRVCS?SCPAGTFTRHENGIERCHDCSQPCPWPMIERLPCAALTDRECICPPGMYQSNGTCAPHTVCPVGWGVRKKGTENEDVRCKQCARGTFSDVPSSVMKCKAHTDCLGQNLEVVKPGTKETDNVCGMRLFFSSTNPPSSGTVTFSHPEHMESHDVPSSTYEPQGMNSTDSNSTASVRTKVPSGIEEGTVPDNTSSTSGKEGTNRTLPNPPQVTHQQAPHHRHILKLLPSSMEATGEKSSTAIKAPKRGHPRQNAHKHFDINEHHHHHH(SEQ?ID?NO：15)

Figure 13 B has shown another video picture of the DR6 part in the aixs cylinder conditioning substratum by the DR6 trace.These trace data have been identified many APP polypeptide, are included in the terminal APP of N-and the C99-APP and the C83/C89 APP polypeptide at 35kDa place.Scheme (Pettmann etc., 1988, J.Neurosci., 8 (10): 3624-3632) aixs cylinder conditioning substratum is implemented DR6-AP trace assay method according to previous record.Generate mouse DR6 extracellular domain-AP fusion rotein as mentioned described in the embodiment 8.Express mouse DR6-ECD-His, subsequently from Chinese hamster ovary celI culture purifying.The aminoacid sequence of DR6-ECD-His sees below.The anti-N-APP antibody of polyclone that is used for the Western Blot experiment derives from Thermo Scientific (catalog number RB-9023-P1).In order to manifest APP C-terminal fragment (CTF) C99-APP and the C83/C89-APP of film mooring, (mono-clonal 4G8 1: 500, Covance) implements the Western engram analysis to the molten born of the same parents' thing of aixs cylinder to use the 4G8 antibody of discerning the epi-position in the A β middle body.

The photo that Figure 14 A provides has shown after NGF deprives coming off of APP extracellular domain has been taken place soon.In Figure 14 A, in the situation that has the Bax inhibitor that adds in order to block axonal degeneration, will be in somatomedin and eliminate the N-APP polyclonal antibody dyeing of the neurone of back different time.From left to right, these photos have shown 0 hour and NGF elimination (and adding anti-ngf antibodies) back axonal degeneration in the time of 3,6,12 and 24 hours.

Be used for manifesting the anti-N-APP antibody of polyclone that surfaces A PP expresses and deriving from Thermo Scientific (catalog number RB-9023-P1) in the experiment that comes off of APP aixs cylinder.Such described in the embodiment 6 and 7 implements to feel that explant cultivates as mentioned.As mentioned described in the embodiment 7 like that but have following modifications ground enforcement NGF to deprive assay method.Fixed time after NGF deprives is fixed DRG explant culture after (0 hour, 3 hours, 6 hours, 12 hours and 24 hours) at interval in 4%PFA/PBS.Express in order to manifest surfaces A PP, as described in embodiment 6 and 7 but do not have Triton to change processing ground thoroughly to process the DRG explant for immunofluorescence dyeing, wherein use anti-N-APP one mentioned above to resist.

For the surfaces A PP that manifests on the sensation neurite expresses (with anti-N-APP antibody mediated immunity fluorescent mark, Thermo Scientific (catalog number RB-9023-P1)), (in the red fluorescence passage) use is taken pictures from Axio Vision40Release 4.5.0.0 SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axioplan-2 imaging Zeiss microscope.

The photo that Figure 14 B provides has shown that the DR6 extracellular domain is in conjunction with the expressed APP of culturing cell.In Figure 14 B, from left to right, two top photos have shown uses DR6-APP (having the DR6 extracellular domain) contrast Cos cell of detecting and the cell of expressing APP respectively.Two following photos have shown p75NTR acceptor and the DR6 expression of receptor cell of detecting with DR6-AP.DR6 extracellular domain debond p75NTR or DR6 expression of receptor cell.

The material and the method that are used for generating the shown data of this figure are as follows.For test APP whether directly with the interaction of DR6 extracellular domain, implemented AP binding assay (Figure 14 B) based on cell.In order to generate DR6 extracellular domain-AP fusion rotein (DR6-AP), the COS-1 cell that uses FuGene transfection reagent (Roche) to cultivate in DMEM/10%FBS (Gibco) substratum with the transfection of 15ug DR6-AP amalgamation and expression construct according to the scheme of manufacturers.After the transfection 12 hours, change the COS-1 cell culture medium into OPTI-MEM (Invitrogen).After the transfection 48 hours, collect and filter and contain the proteic COS-1 cell of DR6-AP conditioning substratum.

According to the proteic amount of DR6-AP in the following rules quantitative culture medium.With 100ul 2XAP damping fluid (by with 100mg p-nitrophenyl phosphate (Sigma) and 15ul 1M MgCl ₂Being added into 15ml 2M diethanolamine pH 9.8 prepares) mix through rotaring redyeing COS cell conditioning substratum or from the collating condition substratum of untransfected COS-1 cell with isopyknic.Color development to reacting in 12-15 minute, O.D. (0.1-1) in linearity range at this moment.Come the conditioned reaction volume by adding 800ul distilled water then, and measure the O.D. at 405nm absorbancy wavelength place.Concentration in nM is calculated (being used for 100ul): C (nM)=O.D.X 100X (60/ developing time)/30 according to following formula.

For APP AP binding assay, the COS-1 cell that uses FuGene transfection reagent (Roche) in DMEM/10%FBS (Gibco) substratum, to cultivate in 6 hole culture plates with the transfection of 2ug APP expression vector according to the every hole of the scheme of manufacturers.After the transfection 2 days, (HBSS, Gibco catalog number 14175-095 contained 0.2%BSA, 0.1%NaN with binding buffer liquid with cell ₃, 5mM CaCl ₂, 1mM MgCl ₂, 20mM HEPES pH=7.0) cleans twice.Implement the AP binding assay then, promptly prepare 1: 1 mixture of DR6-AP conditioning substratum and binding buffer liquid, it directly was applied to the COS-1 cell of expressing APP and in room temperature incubation 90 minutes.Behind the incubation, by washing the COS-1 cell off unconjugated DR6-AP albumen for 5 times with the rinsing of binding buffer liquid.Then cell is used in 3.7% formaldehyde that dilutes among the PBS and fixes 12 minutes in room temperature.By cell is removed remaining formaldehyde 3 times with HBS damping fluid (20mM HEPES pH=7.0,150mM NaCl) rinsing.By in the HBS damping fluid, blocking endogenous AP activity in 30 minutes in 65 ℃ of hot deactivations.Then with the COS-1 cell at AP reaction buffer (100mM TRISpH=9.5,100mM NaCl, 50mM MgCl ₂) in rinsing 3 times.Then by in the AP binding buffer liquid that contains 1/50 (by volume) NBT/BCIP stoste (Roche, catalog number 1681451), the color reaction on the COS-1 cell being manifested the combination (Figure 14 B) of DR6-AP fusion rotein to striding film APP in color development at room temperature after yesterday.In parallel control experiment, will be used for the AP binding assay from the conditioning substratum of untransfected COS cell.Do not show specificity to the DR6-AP fusion rotein in conjunction with (Figure 14 B) at stride film p75NTR and the DR6 acceptor of expressing in the COS-1 cell under the identical experiment condition, the interaction between indication DR6 extracellular domain and the APP is specific.

The photo that Figure 14 C provides has shown that DR6 is the principal recipient of the N-APP on the sensation neurite, and the APP binding site is significantly subdued in the neuronal cell of the scarce no mouse of DR6.In Figure 14 C, from left to right, top three photos shown respectively with AP contrast, N-APP-AP and Sema3A-AP detect from DR6+/-(het) neurone that obtains of mouse.Below three photos correspondingly shown respectively with AP contrast, N-APP-AP and Sema3A-AP detect from DR6-/-(KO) neurone that obtains of mouse.

The material and the method that are used for generating the shown data of Figure 14 C are as follows.Generate mouse DR6 extracellular domain-AP fusion rotein as mentioned described in the embodiment 9.As previous the record (Feiner etc., 1997, Neuron, volume 19 539-545) generates mouse Sema3A extracellular domain-AP (Sema3A-AP) fusion rotein.DR6 lacks no mouse system (DR6.KO) (Zhao etc., Journal of ExperimentalMedicine, volume 194,1441-1441,2001) before on the books.Embodiment 9 cultivates and DR6-AP aixs cylinder binding assay about Figure 12 A and the described enforcement of 12B DRG explant as mentioned.

The photo that Figure 14 D provides has shown that antagonism DR6 antibody destroys the interaction between DR6 extracellular domain and the neurone APP.In these researchs, N-APP is added into the neuronal cell of expressing DR6, manifest with anti-N-APP antibody then.From left to right, preceding four photos have shown that in the situation that has and the following respectively N-APP is in conjunction with the ability of the lip-deep DR6 of neurone: contrast IgG; The anti-DR6 antibody of RA.4; The anti-DR6 antibody of RA.3; With the anti-DR6 antibody of RA.1.Rightmost photo has shown the dyeing of DR6 on the cell that uses contrast IgG.

The material and the method that are used for generating the shown data of this figure are as follows.As previous the record (Okada etc., Nature, 2006, volume 444,369-373) but the following part binding assay based on cell that the shown data of Figure 14 D are implemented to be used for obtaining in ground of revising is arranged.In order to generate the terminal growth factor-like structural domain APP-His fusion rotein (N-APP-His) of N-, the COS-1 cell that uses FuGene transfection reagent (Roche) to cultivate in DMEM/10%FBS (Gibco) substratum with the transfection of 15ug N-APP-His amalgamation and expression construct according to the scheme of manufacturers.After the transfection 12 hours, change the COS-1 cell culture medium into OPTI-MEM (Invitrogen).After the transfection 48 hours, collect and filter and contain the proteic COS-1 cell of N-APP-His conditioning substratum.By the Western engram analysis with anti-N-APP TPPA N-APP-His concentration mentioned above.

The aminoacid sequence of employed people N-APP-His is as follows in this binding assay:

MLPGLALLLLAAWTARALEVPTDGNAGLLAEPQIAMFCGRLNMHMNVQNGKWDSDPSGTKTCIDTKEGILQYCQEVYPELQITNVVEANQPVTIQNWCKRGRKQCKTHPHFVIPYRCLVGEFVSDALLVPDKCKFLHQERMDVCETHLHWHTVAKETCSEKSTNLHDYGMLLPCGIDKFRGVEFVCCPLAEESDNVDSADAEEDHHHHHH(SEQ?ID?NO：10)

Implement the N-APP-His binding assay then, promptly prepare 1: 1 mixture of N-APP-His conditioning substratum and binding buffer liquid, it directly was applied to the COS-1 cell of expressing the DR6 acceptor and in room temperature incubation 90 minutes.As illustrated, individually add DR6 monoclonal antibody RA.1, RA.3 or RA.4 (above embodiment 3 and 7 is described) with N-APP-His conditioning substratum and binding buffer liquid with 20ug/ml.In control experiment, add normal mouse IgG (Genentech Inc) with 20ug/ml with N-APP-His conditioning substratum and binding buffer liquid.

By immunofluorescence dyeing with anti-N-APP antibody (Thermo Scientific catalog number RB-9023-P1) according to as the described known arrangement (Okada etc. of the scheme of embodiment 6 and 7, Nature, 2006, volume 444 369-373) manifests the combination of N-APP to the cell of expression DR6 acceptor.(fluorescently-labeled in order to manifest the N-APP albumen that is bonded to DR6 acceptor on the cell surface with anti-N-APP antibody mediated immunity, Thermo Scientific (catalog number RB-9023-P1)), (in the red fluorescence passage) use is taken pictures from Axio Vision40Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axioplan-2 imaging Zeiss microscope.

Embodiment 11: amyloid precursor protein (APP) activation DR6 induces axonal degeneration

The photo that Figure 15 A provides has shown at the polyclonal antibody of the terminal APP of N-blocks axonal degeneration in Colaesce aixs cylinder assay method.From left to right, the photo among Figure 15 A has shown the Colaesce axonal degeneration in the situation that has and the following respectively: contrast IgG; The anti-NAPP antibody of 30ug/ml; With the anti-NAPP antibody of 1.1ug/ml.

The material and the method that are used for generating the shown data of Figure 15 A are as follows.As described in the data that generated among the scheme of embodiment 2 and Fig. 4 B with shown in the anti-N-APP antibody of polyclone (ThermoScientific catalog number RB-9023-P1, through a large amount of dialysis) or contrast IgG (tame rabbit igg, the R﹠amp of quantity; D Systems) implements Colaesce explant survival assay method.In order to manifest the Colaesce aixs cylinder of GFP mark, (in the green fluorescence passage at GFP) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of CarlZeiss Imaging Solutions on Axiovert 200 Zeiss inverted microscopes.

The photo that Figure 15 B provides has shown that the terminal APP antibody of N-suppresses to eliminate the sensation neurite sex change of bringing out by NGF.From left to right, three photos above Figure 15 B have shown the sensation neurite in the situation that has NGF and and the following respectively: control antibodies; Anti-APP monoclonal antibody 22C11; With anti-APP polyclonal antibody.Three following photos have correspondingly shown the sensation neurite in the situation that does not have NGF (and anti-ngf antibodies) and and the following respectively: control antibodies; Anti-APP monoclonal antibody 22C11; With anti-APP polyclonal antibody.

The material and the method that are used for generating the shown data of this Figure 15 B are as follows.In the Campenot chamber, implement NGF as mentioned described in the embodiment 8 and deprive assay method.Employed antibody at the terminal APP of N-is the anti-N-APP antibody of polyclone (Thermo Scientific catalog number RB-9023-P1 in this assay method, through what dialyse in a large number) or 22C11 monoclonal antibody (22C11, Chemicon is through what dialyse in a large number).Add normal IgG (tame rabbit igg, R﹠amp; D Systems) experiment in contrast.Usefulness TUJ1 antibody described in embodiment 1,7 and 8 (1: 500, the Covance) immunofluorescence label of enforcement sensation neurite.For the sensation neurite of the immunofluorescence label in the aixs cylinder compartment that manifests the Campenot chamber, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

The photo that Figure 15 C provides has shown that interpolation N-APP can save the axonal degeneration of blocking by the active inhibition to beta-secretase (BACE).From left to right, three photos above Figure 15 C have shown observed neurone in the situation that has and the following respectively (cultivating) and axonal degeneration: DMSO contrast, BACE inhibitor and N-APP (and BACE-I) in the situation that does not have NGF.Three photos below Figure 15 C have shown and have correspondingly shown the neurone in having the situation of and the following respectively (exist and cultivate in the situation of NGF): DMSO contrast, BACE inhibitor and N-APP (and BACE-I).

The material and the method that are used for generating the shown data of this Figure 15 C are as follows.In the Campenot chamber, implement NGF as mentioned described in the embodiment 8 and deprive assay method.Employed people recombinates N-APP amino acid/11 9-306 available from Novus (Novus Biologicals, catalog number H00000351-P01) in this assay method.When NGF deprived, (1uM final concentration, InSolutionOM99-2 Calbiochem/Merck) added N-APP with 3ug/ml together with the BACE inhibitor.In this assay method with the 1uM final concentration use the BACE inhibitor (InSolution OM99-2, Calbiochem/Merck).Usefulness TUJ1 antibody described in embodiment 1,7 and 8 (1: 500, the Covance) immunofluorescence label of enforcement sensation neurite.In order to manifest the sensation neurite of the immunofluorescence label in the aixs cylinder compartment of Campenot chamber, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

The photo that Figure 15 D provides has shown the necrocytosis sensitization that RNAi makes the neuronal cell growth in the situation that has the BACE inhibitor bring out N-APP to the elimination of APP.In Figure 15 D, from left to right, three top photos have shown the neurone of cultivating in the situation that has contrast RNAi.Photo above these has shown contrast and the neurone cultivated with 3ug/ml N-APP or 0.1ug/ml N-APP respectively.Three following photos have shown the neurone of cultivating in the situation that has APP RNAi.Photo below these has shown contrast and the neurone of cultivating with 3ug/ml N-APP or 0.1ug/ml N-APP.

The material and the method that are used for generating the shown data of this Figure 15 D are as follows.Implement the APP RNAi in the Colaesce explant culture as described in example 2 above.Employed people recombinates N-APP amino acid/11 9-306 available from Novus (Novus Biologicals, catalog number H00000351-P01) in this assay method.In this assay method, use the rat specificity APPON-TARGETplus siRNA set of pre-design to express (APPON-TARGETplus siRNA set with the APP in the downward modulation E13 rat Colaesce explant according to the scheme of manufacturers, GeneID:54226, catalog number 088191, Dharmacon Inc.).In order to manifest the GFP mark and the Colaesce aixs cylinder RFP mark (described in embodiment 2 and 7), (in the green fluorescence passage at GFP) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axiovert 200 Zeiss inverted microscopes.

Embodiment 12:DR6 is that the axonal degeneration that brings out of APP is needed but be not that the sex change that triggers of A β is needed

Described in Figure 16 A, DR6 activation is that the axonal degeneration that brings out of N-APP is needed.

In Figure 16 A, from left to right, three top photos shown from DR6+/-(het) neurone that obtains of mouse.The photo of first width of cloth has shown the contrast neurone that is not exposed to A β or N-APP, and second photos has shown the neurone that is exposed to A β, and the 3rd photos has shown the neurone that is exposed to N-APP.Below three photos shown from DR6-/-(KO) neurone that obtains of mouse.From left to right, the first following photos has shown the contrast neurone that is not exposed to A β or N-APP, and second photos has shown the neurone that is exposed to A β, and the 3rd photos has shown the neurone that is exposed to N-APP.

The material and the method that are used for generating the shown data of this Figure 16 A are as follows.Implement cultivation of Colaesce explant and survival assay method as described in example 2 above.DR6 lacks no mouse system (DR6.KO) (Zhao etc., Journal of Experimental Medicine, volume 194,1441-1441,2001) before on the books.Employed people recombinates N-APP amino acid/11 9-306 available from Novus (Novus Biologicals, catalog number H00000351-P01) in this assay method.In this assay method employed recombinant human beta amyloid Argine Monohydrochloride 1-42 available from Chemicon (hyperpure people A β 1-42, catalog number AG912, Chemicon).With 3ug/ml N-APP was added into the Colaesce explant with the BACE inhibitor in 24 hours behind the bed board.With 3uM recombinant human beta amyloid Argine Monohydrochloride 1-42 was added into the Colaesce explant with the BACE inhibitor in 24 hours behind the bed board.In this assay method with the 1uM final concentration use the BACE inhibitor (InSolutionOM99-2, Calbiochem/Merck).With the Colaesce explant with the N-APP of specified amount or A β incubation 24 hours again.48 hours collection data behind Colaesce explant bed board.In order to manifest the Colaesce aixs cylinder, (in the bright field) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl ZeissImaging Solutions on Axiovert 200 Zeiss inverted microscopes.

Shown in Figure 16 B, antagonism DR6 antibody fails to block the axonal degeneration that is triggered by A β.In Figure 16 B, from left to right, three top photos shown contrast neurone, the neurone in having the situation of BACE-I and have BACE-I and the situation of A β in neurone.In Figure 16 B, two following photos have shown the neurone in the situation that has BACE-I, A β and anti-DR6 antibody RA.1, are the neurone in the situation that has BACE-I, A β and anti-DR6 antibody RA.3 then.

The material and the method that are used for generating the shown data of this Figure 16 B are as follows.Implement cultivation of Colaesce explant and survival assay method as described in example 2 above.In this assay method employed recombinant human beta amyloid Argine Monohydrochloride 1-42 available from Chemicon (hyperpure people A β 1-42, catalog number AG912, Chemicon).In this assay method with the 1uM final concentration use the BACE inhibitor (InSolution OM99-2, Calbiochem/Merck).Behind bed board, with 3uM recombinant human beta amyloid Argine Monohydrochloride 1-42 was added into the Colaesce explant with BACE inhibitor and specified anti-DR6 monoclonal antibody (40ug/ml) in 24 hours.In this assay method with the 1uM final concentration use the BACE inhibitor (InSolution OM99-2, Calbiochem/Merck).With the Colaesce explant with the A β of specified amount incubation 24 hours again.48 hours collection data behind Colaesce explant bed board.

Mouse monoclonal RA.1-RA.5DR6 antibody as mentioned described in the embodiment 3 by generating with DR6 extracellular domain immune mouse.As mentioned above, the DR6 antibody that is called RA.1 and RA.3 antibody in this article is respectively " 1E5.5.7 " and " 3F4.4.8 ", i.e. DR6 antibody described in the embodiment 3.In order to manifest the Colaesce aixs cylinder of GFP mark, (in the greenfluorescence channel for GFP) uses and takes pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axiovert 200 Zeiss inverted microscopes.

Embodiment 13: DR6 signal conduction in the born of the same parents

Caspase is that important factor in the apoptosis approach is (referring to for example Grutter etc., Curr Opin Struct Biol.10 (6): 649-55 (2000); Kuida etc., Nature 384 (6607): 368-72 (1996); And Finn etc., J Neurosci.20 (4): 1333-41 (2000)), and the conduction of the intracellular signal in some Caspase and the neurodegenerative disease (comprising Huntington disease and AD) relevant (referring to for example Wellington etc., J Neurosci.22 (18): 7862-72 (2002); Graham etc., Cell125 (6): 1179-91 (2006); Guo etc., Am J Pathol. (2): 523-31 (2004); And Horowitz etc., J Neurosci.24 (36): 7895-902 (2004)).

Figure 17 A has shown to cultivate and was exposed to the photo that various different culture condition reach 24 hours Sensory neurone in 5 days then.As shown in Figure 17 A, by to JNK and upstream Caspase-8 but the inhibition of non-downstream Caspase-3 has postponed axonal degeneration.

In Figure 17 A, two photos on the left side have shown the Sensory neurone that is exposed to NGF and anti-ngf antibodies respectively with the order that successively decreases.In Figure 17 A, four photos on the right have shown with the order that successively decreases and have been exposed to anti-ngf antibodies and jnk inhibitor respectively; Anti-ngf antibodies and Caspase-8 inhibitor; Anti-ngf antibodies and BAX inhibitor; And the Sensory neurone of anti-ngf antibodies and Caspase-3 inhibitor.

The material and the method that are used for generating the shown data of this Figure 17 A are as follows.In the Campenot chamber, implement NGF as mentioned described in the embodiment 8 and deprive assay method.In this assay method, use small molecules jnk inhibitor SP 600125, (catalog number 1496, Tocris Bioscience) with the 1uM final concentration.In this assay method with 10uM use Caspase-3 inhibitor Z-DEVD-FMK (Z-DEVD-FMK, catalog number 264155, Calbiochem).In this assay method, use Caspase-8 inhibitor Z-IETD-FMK (Z-IETD-FMK, catalog number FMK007, R﹠amp with 10uM; D Systems).Use the BAX inhibiting peptide to come the necrocytosis (Bax-V5, Tocris Inc) of block nerves unit with 10uM.Bax lacks no mouse system (Bax-R1) (Deckwerth etc., Neuron, volume 17,401-411,1996) before on the books, and derives from Jackson Lab.Usefulness TUJ1 antibody described in embodiment 1,7 and 8 (1: 500, the Covance) immunofluorescence label of enforcement sensation neurite.For the sensation neurite of the immunofluorescence label in the aixs cylinder compartment that manifests the Campenot chamber, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl ZeissImaging Solutions.

Figure 17 B provides the photo from the motor neuron of E12.5 motor neuron explant culture, shown that Caspase-3 brings into play function in the cell main body, and Caspase-6 is brought into play function in aixs cylinder.

In Figure 17 B, from left to right, four photos have shown the neurone of cultivating with and the following respectively: (1) somatomedin; (2) there is not somatomedin and do not have Caspase inhibitor (contrast); (3) do not have somatomedin but have Caspase-3 inhibitor; (4) do not have somatomedin but have Caspase-6 inhibitor.

The material and the method that are used for generating the shown data of this Figure 17 B are as follows.In this assay method with 10uM use Caspase-3 inhibitor Z-DEVD-FMK (Z-DEVD-FMK, catalog number 264155, Calbiochem).In this assay method, use Caspase-6 inhibitor Z-VEID-FMK (Z-VEID-FMK, catalog number 550379, Becton, Dickinson andCompany PHARMINGEN Division) with 10uM.Implement motor neuron ventral cord survival assay method as mentioned described in the embodiment 6.Usefulness TUJ1 antibody described in embodiment 1,7 and 8 (1: 500, the Covance) immunofluorescence label of enforcement motion aixs cylinder.In order to manifest the motion aixs cylinder of immunofluorescence label, on Axioplan-2 imaging Zeiss microscope, use and take pictures from Axio Vision40 Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions.

Figure 17 C provides to cultivate and had been exposed to the photo that various different culture condition reach 24 hours Sensory neurone in 5 days then.Though the data presentation among Figure 17 C Caspase-3 be not that axonal degeneration is needed as if, BAX is.

In Figure 17 C, from left to right, four top photos have shown with NGF respectively to be cultivated, be then in having the situation of anti-ngf antibodies (being that NGF deprives) cultivate 16,24 and 48 hours BAX+ /+neurone.Below four photos correspondingly shown with NGF respectively and cultivated, be then with anti-ngf antibodies cultivate 16,24 and 48 hours BAX-/-neurone.

The material and the method that are used for generating the shown data of this Figure 17 C are as follows.In the Campenot chamber, implement NGF as mentioned described in the embodiment 8 and deprive assay method.Bax lacks no mouse system (Bax-R1) (Deckwerth etc., Neuron, volume 17,401-411,1996) before on the books, and derives from Jackson Lab.NGF deprive in the assay method use in the aixs cylinder compartment in the Campenot chamber NGF antibody (the anti-NGF#911 of mono-clonal function interdiction, Genentech, 20ug/ml).Usefulness TUJ1 antibody described in embodiment 1,7 and 8 (1: 500, the Covance) immunofluorescence label of enforcement sensation neurite.For the sensation neurite of the immunofluorescence label in the aixs cylinder compartment that manifests the Campenot chamber, (in the green fluorescence passage) uses and takes pictures from Axio Vision40Release 4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axioplan-2 imaging Zeiss microscope.

Figure 17 D provides the photo of the culture of the E13 rat explant commissural neuron of cultivating 24 hours under different culture condition.Data presentation among Figure 17 D Caspase-3 in the cell main body, bring into play function, and Caspase-6 is brought into play function in aixs cylinder.

In Figure 17 D, from left to right, three top photos have shown that the GFP that the contrast neurone is compared with the neurone of cultivating with Caspase-3 or Caspase-6 inhibitor respectively analyzes.Three following photos have shown that correspondingly the TUNEL (necrocytosis) that the contrast neurone is compared with the neurone of cultivating with Caspase-3 or Caspase-6 inhibitor respectively analyzes.

The material and the method that are used for generating the shown data of this Figure 17 D are as follows.Implement cultivation of Colaesce explant and survival assay method as described in example 2 above.In Colaesce explant culture, manifest apoptosis in the heteromeral cells main body by TUNNEL assay method described in the embodiment 7 as mentioned.The Colaesce explant is fixed and is the detection processing of the apoptosis (apoptosis) of unicellular level in 4%PFA/PBS, this detects, and (catalog number 11 684 795910 is Roche) according to the DNA splitting of chain mark (TUNNEL technology) of the instruction manual (Roche) of manufacturers based on using original position necrocytosis detection kit.By the apoptosis (Figure 17 D) in the cell main body of fluorescence microscopy analysis Colaesce sensation and motion explant culture.In this assay method with 10uM use Caspase-3 inhibitor Z-DEVD-FMK (Z-DEVD-FMK, catalog number 264155, Calbiochem).In this assay method, use Caspase-6 inhibitor Z-VEID-FMK (Z-VEID-FMK, catalog number 550379, Becton, Dickinson and Company, PHARMINGEN Division) with 10uM.In order to manifest the Colaesce aixs cylinder of GFP mark, (in the green fluorescence passage at GFP) uses and takes pictures from Axio Vision40 Release4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axiovert 200 Zeiss inverted microscopes.In order to manifest fluorescently-labeled TUNNEL positive apoptotic cells main body, (in the red fluorescence passage of using for TUNNEL) uses and takes pictures from Axio Vision40 Release4.5.0.0SP1 (03/2006) computer software of Carl Zeiss Imaging Solutions on Axioplan-2 imaging Zeiss microscope.

Embodiment 14: the DR6 antagonistic activity in the animal model

Those of skill in the art can adopt the relevant animal model of multiple and different neurodegenerative diseases to check DR6 antagonist effect in vivo.For example, the APP/RK transgenic mice is expressed mutant amyloid precursor protein polypeptide and is represented serious neurodegeneration and apoptosis.Therefore, the APP/RK transgenic mice provides the model of Alzheimer's, it can be used for checking the DR6 antagonist, and (referring to for example Moechars etc., Neuroscience 91 (3): 819-830 (1999)) to the effect of the relevant pathologic process of observed syndrome therewith in this animal model.Multiple other transgenic mouse system (such as APP23 and JNPL3 transgenic lines) expresses mutant alzheimer's related polypeptide and further represents the neuronal cell loss.APP23 and JNPL3 transgenic mice so provide the alternative model of Alzheimer's, wherein can use DR6 antagonist (referring to for example McGowan etc., TRENDS in Genetics rolled up for 22 phases 5 (2006)).

G93A SOD1 transgenic mice expressing human superoxide-dismutase mutant polypeptides and the Caspase-3 that represents elevated levels are expressed and the motor neuron apoptosis.G93A SOD1 transgenic mice provides the model of amyotrophic lateral sclerosis, and its effect that can be used for checking the DR6 antagonist is (referring to for example Tokuda etc., Brain Res.1148:234-242 (2007); And Wang etc., Eur.J.Neurosci.26 (3): 633-641 (2007)).The R6/2 transgenic mice is expressed the exon-1 of huntington under the control of its natural promoter and the terminal many L-glutamic acid of N-of extension repeat and represent gradual neuropathology to change, it reproduces Huntington disease among mankind (referring to for example Mangarini etc., Cell, 87,493-506 (1996); Chen etc., Nat.Med.6,797-801 (2000)).The R6/2 transgenic mice provides the model of Huntington disease, its can be used for checking the DR6 antagonist to the effect of the relevant pathologic process of observed syndrome therewith in this animal model (referring to for example Wang etc., European Journal of Neuroscience, 26:633-641 (2007)).The PK-KO transgenic mice is not expressed the protein of Park-2 gene, represent with Parkinson's disease similarly unusual, and have recently from the neurone of wild-type mice the apoptosis neurone of susceptible (referring to for example Casarejos etc., J Neurochem.97 (4): 934-46 (2006)) more.The PK-KO transgenic mice provides parkinsonian model, and it can be used for characterizing the effect of DR6 antagonist to the relevant pathologic process of observed syndrome therewith in this animal model.In addition, many transgenic mices system (such as Smn-/-239 pure trinucleotide CAG multiple transgenic mices under the SMN2 mouse, carrier AP promotor and people SMN is arranged ^CDual the knocking out of natural mouse Smn gene transgenic of at least one copy performance function in the mouse background) all or not express the protein of survival motor neuron gene or expressing it changes pattern, therefore and represent with the Duchenne-Arandisease disease similarly unusual (referring to for example Hsiu etc., Nature Genetics 24,66-70 (2000); Ferri etc., Neuroreport 15 (2): 275-280 (2004); Ferri etc., Curr Biol.2003 Apr 15; 13 (8): 669-73; And Rossol etc., Journal of Cell Biology, volume 163, phase 4,801-812 (2003)).Therefore this type of transgenic mouse system provides the model of Duchenne-Arandisease, and it can be used for characterizing the effect of DR6 antagonist to the relevant pathologic process of observed syndrome therewith in this animal model.

The animal model of neurological disease or illness (comprise mentioned above those) can be used for checking the effect of DR6 antagonist disclosed herein, for example one or more antibody (for example 3F4.4.8,4B6.9.7 or 1E5.5.7 monoclonal antibody) and/or one or more soluble form DR6 (for example comprising SEQ ID NO:1 amino acid/11-354) and/or one or more antibody (for example 22C11 monoclonal antibody) and combination with one another in conjunction with APP in conjunction with APP in conjunction with DR6 and/or with these medicaments of other therapeutical agent combination known in the art.

In the exemplary scheme of the experiment test of disclosed in this article one or more DR6 antagonists, can be assigned to one of a plurality of tests and/or control group from the age of animal model and the animal of gender matched (for example 6 month female APP/RK transgenic mices) with many.Can use selected DR6 antagonist (peritoneal injection DR6 antagonist antibodies for example, per injection 20mg/kg body weight whenever biweekly, continue 6 months) to first test group of these animals according to specific application program then.The condition of other test group can secundum legem practice and changing, for example use various dose DR6 antagonist (for example 1,5,10,15mg/kg body weight), use different progress charts DR6 antagonist (for example weekly, as to continue 12 months), use different DR6 antagonist (for example DR6 immunoadhesin), make the with medicament combination DR6 antagonist of anticholinesterase combination (for example with), use different administration path (for example intravenously is used) etc.But one or more groups animal served as control is for example accepted sterile phosphate buffered saline according to the application identical with the test group of accepting the DR6 antagonist.

Some time period after accepting the DR6 antagonist can compare test group and the coupling control group of these animals then, for example checks and/or characterize DR6 antagonist effect in vivo.For example, can comprise from the sample of the neuronal cell of the particular organization of the test group of these animals and control group or organ (for example brain) with the situation of the neuronal cell in these groups relatively (referring to for example Petrik etc., NeuromolecularMed.9 (3): 216-29 (2007)) by assessing such as the technology of mr microscopy and/or immunohistochemical analysis.Perhaps, can attenuate the phenomenon of (thinning) (referring to for example Tsai etc. with proof such as the spinous process track that changes, dendritic spine loss or dendron by assess the sample that obtains from these groups such as the technology of multi-photon microscopy, Nat.Neurosci.7,1181-1183 (2004); And Spires etc., J.Neurosci.25,7278-7287 (2005)).Perhaps, can carry out the ELISA scheme with the blood that obtains from these groups or other tissue sample, it is designed for the level of the mark of measuring inflammation and/or apoptosis, such as IL-1 β, TNF-α, IL-10, p53 albumen, interferon-or NF-κ B (referring to for example Rakover etc., Neurodegener.Dis.4 (5): 392-402 (2007); And Mogi etc., NeurosciLett.414 (1): 94-7 (2007)).Perhaps, can in performance testing example known in the art, compare from test group and coupling animals of control group, for example Morris water maze or Target Recognition test (referring to for example Hsiao etc., Science 274,99-102 (1996); Janus etc., Nature 408,979-982 (2000); Morgan etc., Nature 408,982-985 (2000); And Ennaceur etc., Behav.Brain Res.1988; 31:47-59).Comparative result between test group and coupling control animals allows that those skilled in the art check the DR6 antagonist effect in animal model in vivo.

Among the embodiment 1-13 included data and therewith the relevant sign of data proved that the DR6 antagonist can for example suppress the apoptosis of neuronal cell in vivo.Particularly, above embodiment 1-13 has for example instructed: (1) DR6 is apoptosis-induced in extremely multiple neuronal cell; (2) APP is in conjunction with DR6 and triggers the related part of DR6 of DR6 mediated Apoptosis; (3) therefore the DR6 antagonist at vitro inhibition DR6/APP binding interactions suppresses the DR6 mediated Apoptosis in vivo.In view of applicant's discovery and disclosure, those skilled in the art understand rational expectation DR6 antagonist and suppress the DR6 mediated Apoptosis in vivo.For this reason, those of skill in the art understand rational expectation animal model (all as indicated above those) and correlation technique is used for checking in the biologic activity of the observed various pathologic processes of these animal models with checking DR6 antagonist, and are as described herein.

Embodiment 15: the RA.1 in the Duchenne-Arandisease animal model (" 1E5.5.7 "), RA.2, RA.3 (" 3F4.4.8 ") and RA.4 antibody treatment

Duchenne-Arandisease (SMA) is degeneration (recessive) motor neurone disease of the motor neuron in the invasion and attack ventricornu, and thinks that it is derived from SMN (survival motor neuron) proteic minimizing (reduction).The animal model of SMA is transgenic mice system with family name FVB.Cg-Tg (SMN2*delta7) 4299Ahmb Tg (SMN2) 89Ahmb SmnltmlMsd/J (JAX5025) (referring to for example Le etc., Human Molecular Genetics14 (6): 845-857 (2005)).This triple mutant mouse comprises two transgenosis allelotrope and a target is decided mutant.Tg (SMN2*delta7) 4299Ahmb allelotrope is made up of the SMA cDNA that lacks exon 7, and Tg (SMN2) 89Ahmb allelotrope is by complete people SMN2 genomic constitution.In the explanation, this strain is also referred to as delta (Δ, Delta) 7SMAKO model hereinafter.

For the fixed Smn allelotrope of target be isozygoty and represent symptom and the europathology similar for the mouse that two kinds of transgenosis allelotrope are purifying to the patient who suffers from Duchenne-Arandisease (SMA).At birth, the triple mutant body is littler significantly than normal littermate.By the 5th day, the weak sign of infantile myasthenia obviously and in a week subsequently, become more and more significant, promptly mouse represents unusual gait, hind leg trembles and be tending towards falling down.The survival average is about 13 days.Triple mutant body mouse further represents and impaired the surface is righted (surface righting), negative geotropism (negative geotaxis) and steep cliff turn to replying of (cliffaversion), but does not have impaired to replying of haptic stimulus.Self-motion is active and grip is also significantly impaired (referring to for example Butchbach etc., Neurobiol Dis.27 (2): 207-19 (2007)) in these mouse.Below conceptual design be used to check some antibody (such as the DR6 antagonistic antibodies) and dosage effect to survival, body weight and the musculartone of Δ 7SMA model mice (KO).

As mentioned above, this research in employed mouse can be Δ-7SMA (JAX 5025) KO model (smn-/-; SMN2+ /+; D7+ /+).Can wherein for example take out the male and female of equal number at birth with 10 animals of the selected at random one-tenth of nest (or some other numbers).Follow this scheme, can the first time during dosed administration (P3) with 8 mouse of the selected one-tenth of nest.Can from research, reject any nest that is less than 6 young babies that has.At birth (P0) give from Monday and the mouse of the nest of being born between Wednesday cut tail.Can implement gene type by several different methods known in the art, for example use can by commercial sources from the biopsy that molecular diagnostics company such as Transnetyx Inc obtains transgenosis, knock out and knock in the automatization gene type service screening of sudden change.Usually can obtain this genotype data in birth in back 48 hours.

Can in exemplary experiment, use the mouse that for example on Monday is born to Wednesday.Can begin dosed administration at P3 to mouse IP.Typical number in this research can be: (1) for example average 10 KO (5 male and 5 female) contrast and accept media such as aseptic PBS; (2) for example average 10 KO (5 male and 5 female) accept the corresponding antibodies of first dosage, and it comprises 20mg/kg; (3) for example average 10 KO (5 male and 5 female) accept the corresponding DR6 antibody of second dosage, and it comprises 5mg/kg.Every kind of animal can be accepted the IP agent of corresponding RA.1, RA.2, RA.3 and RA.4 antibody, twice weekly." RA.1 antibody " is corresponding to " 1E5.5.7 ", and " RA.3 antibody " is corresponding to " 3F4.4.8 "." RA.2 antibody " corresponding to " 4B6.9.7 ", and " RA.4 antibody " corresponding to " 2C7.3.7 " (Genentech, Inc., a kind of in conjunction with DR6 but the antibody of non-block function)." RA.5 antibody " corresponding to " 3B11.7.7 " (Genentech, Inc., a kind of in conjunction with DR6 but can strengthen or stimulate the active antibody of DR6).

RA.1, RA.2, RA.3 and RA.4 antibody can be stored in 4C.Where necessary, can be before dosed administration that these antibody are warm to room temperature.Can use typical media, such as PBS.Though the RA.1 among this embodiment, RA.2, RA.3 and RA.4 monoclonal antibody are to use people DR6 peptide sequence to generate as immunogen, but all these antibody all react with people and rat and mouse DR6, and are shown as the scheme of axonal degeneration described in embodiment 7 and apoptosis assay method.

In an exemplary embodiment, the DR6 antagonist of being assessed can be antagonistic antibodies: RA.1, RA.2, RA.3 and RA.4; The number of the treatment group of every kind of antibody can be 2 (every group of 10 animals); Using the path can be IP; And dosage range can be 5 and 20mg/kg.Optional is that group can be as follows: (1) RA.1:5mg/kg IP; (2) RA.1:20mg/kg IP; (3) RA.2:5mg/kg IP; (4) RA.2:20mg/kg IP; (5) RA.3:5mg/kg IP; (6) RA.3:20mg/kg IP; (7) RA.4:5mg/kg IP; (8) RA.4:20mg/kg IP; (9) media (PBS) IP.In this scheme, can weigh to mouse once a day.Going out DAB (PND) 10,12 and 14, weighing for every young baby in the nest.At PND 6,8,10,12,14 and 16, can implement musculartone assessment (referring to the exemplary phenotype somatotype scheme that is for example hereinafter provided) to every animal in the research.

Going out the birthday (P0), can use nontoxic ink to tatoo, be applied to subcutaneous time, and collecting scissors tail sample is being used for gene type (result can obtain usually) in 48 hours to the young baby.Testing that day (P3), can will have the same time of every day neonatal female beast to take the laboratory to and without disturbance staying at least 10 minutes before the test beginning.Can at first in the geotaxis test, in the pipe test, (manage test 2 these successive tests are arranged) the test young baby then.The young baby can be placed that all young babies until nest have accepted test on the heating cushion, all young babies can be returned their female beastly (after operation, the young baby can be mixed with their cage straw mattress) then to reduce to the repulsion of mother beast minimum.Can check that survival and body weight are until wean every day from birth.Usually assess the influence of medicine in the long-term MTD research process of formerly implementing to newborn infant's axis body temperature.Body temperature: can gather a reading of axis body temperature at the age of regulation.

Can check difference to test group and mice in control group by the verification scheme that comprises geotaxis.The geotaxis test animal is given own directed ability when quilt cover down is placed on the sloping platform.This thermometrically motor coordination and vestibular system.

Can use Kaplan-Meier to analyze to implement the survival assessment, wherein with Mantel-Cox as check (post-hoc test) afterwards.

In order to analyze the data that replicate measurement in time obtains, can adopt mixed effects model (MixedEffects Models) (be also referred to as and mix the ANOVA model).This method is based on likelihood estimation (likelihoodestimation) but not moment estimation (moment estimation) (in typical replicate measurement ANOVA analyzes), but it owing to mouse in time death and be easy to lose numerical value more.(NC) the PROC MIXED program in is come all models of match for SAS Institute, Cary can to use SAS 9.1.3..Processing is a most important factor in the model.It is also conceivable that sex and fate, and they and the interaction handled.

The research terminal point can be dead.

Can further assess animal by all methods of (for example histologic analysis) as described in example 14 above.In addition, can assess serum/blood to measure RA.1, RA.2, RA.3 and RA.4 serum-concentration.

The material preservation

Following material be preserved in American type culture collection (American Type CultureCollection, 10801 University Blvd., Manassas, VA 20110-2209, USA) (ATCC):

Materials A TCC preserving number preservation day

3F4.4.8 PTA-8095 on December 21st, 2006

4B6.9.7 PTA-8094 on December 21st, 2006

1E5.5.7 PTA-8096 on December 21st, 2006

This preservation is to be used for the microbial preservation budapest treaty (Budapest Treaty) of patented procedure and the regulation of (budapest treaty) detailed rules for the implementation is carried out according to international recognition.This has guaranteed to preserve from the preservation survival culture 30 years of preservation.The preservation thing can obtain by ATCC according to the clause of budapest treaty, and the agreement between obedience Genentech company and the ATCC, it guaranteed after the relevant United States Patent (USP) mandate or at any U.S. or foreign patent application after the public is open, with among both prior to the person be as the criterion, the public can be permanent and the offspring of unrestricted acquisition preservation culture, and guaranteed the individual that ratifies by United States Patent and Trademark Office head according to 35USC § 122 and according to its management article (comprise 37CFR § 1.14, will mention 886OG 638 especially) offspring with qualified acquisition preservation culture.

The application's transferee agrees, if death when the culture of preserved material is cultivated under conditions suitable, lose or destroyed, then he will be in the back that has notice rapidly with another part material replacing of same culture.The availability of institute's preserved material also is not interpreted as and implements permission of the present invention to violating any government organs according to its patent law institute granted entitlements.

Think that aforementioned written description is enough to make those skilled in the art can implement the present invention.The present invention is not limited to the scope of embodiment that this paper presents.In fact, according to top description, at this paper shown and describe outside, multiple modification of the present invention is conspicuous for those skilled in the art, and within the scope of the appended claims.

Sequence table

＜110〉Genentech Inc (GENENTECH, INC.)

＜120〉suppress DR6 antibody and the purposes in treatment neuroscience illness thereof of DR6 in conjunction with APP

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Ile?Ala?Arg?Arg?Ala?Thr?Ala?Thr?Met?Ile?Ala?Gly?Ser?Leu?Leu?Leu

20??????????????????25??????????????????30

Leu?Gly?Phe?Leu?Ser?Thr?Thr?Thr?Ala?Gln?Pro?Glu?Gln?Lys?Ala?Ser

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Asn?Leu?Ile?Gly?Thr?Tyr?Arg?His?Val?Asp?Arg?Ala?Thr?Gly?Gln?Val

50??????????????????55??????????????????60

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

65??????????????????70??????????????????75??????????????????80

Thr?Asn?Thr?Ser?Leu?Arg?Val?Cys?Ser?Ser?Cys?Pro?Val?Gly?Thr?Phe

85??????????????????90??????????????????95

Thr?Arg?His?Glu?Asn?Gly?Ile?Glu?Lys?Cys?His?Asp?Cys?Ser?Gln?Pro

100?????????????????105?????????????????110

Cys?Pro?Trp?Pro?Met?Ile?Glu?Lys?Leu?Pro?Cys?Ala?Ala?Leu?Thr?Asp

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Arg?Glu?Cys?Thr?Cys?Pro?Pro?Gly?Met?Phe?Gln?Ser?Asn?Ala?Thr?Cys

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Ala?Pro?His?Thr?Val?Cys?Pro?Val?Gly?Trp?Gly?Val?Arg?Lys?Lys?Gly

145?????????????????150?????????????????155?????????????????160

Thr?Glu?Thr?Glu?Asp?Val?Arg?Cys?Lys?Gln?Cys?Ala?Arg?Gly?Thr?Phe

165?????????????????170?????????????????175

Ser?Asp?Val?Pro?Ser?Ser?Val?Met?Lys?Cys?Lys?Ala?Tyr?Thr?Asp?Cys

180?????????????????185?????????????????190

Leu?Ser?Gln?Asn?Leu?Val?Val?Ile?Lys?Pro?Gly?Thr?Lys?Glu?Thr?Asp

195?????????????????200?????????????????205

Asn?Val?Cys?Gly?Thr?Leu?Pro?Ser?Phe?Ser?Ser?Ser?Thr?Ser?Pro?Ser

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Pro?Gly?Thr?Ala?Ile?Phe?Pro?Arg?Pro?Glu?His?Met?Glu?Thr?His?Glu

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Val?Pro?Ser?Ser?Thr?Tyr?Val?Pro?Lys?Gly?Met?Asn?Ser?Thr?Glu?Ser

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Asn?Ser?Ser?Ala?Ser?Val?Arg?Pro?Lys?Val?Leu?Ser?Ser?Ile?Gln?Glu

260?????????????????265?????????????????270

Gly?Thr?Val?Pro?Asp?Asn?Thr?Ser?Ser?Ala?Arg?Gly?Lys?Glu?Asp?Val

275?????????????????280?????????????????285

Asn?Lys?Thr?Leu?Pro?Asn?Leu?Gln?Val?Val?Asn?His?Gln?Gln?Gly?Pro

290?????????????????295?????????????????300

His?His?Arg?His?Ile?Leu?Lys?Leu?Leu?Pro?Ser?Met?Glu?Ala?Thr?Gly

305?????????????????310?????????????????315?????????????????320

Gly?Glu?Lys?Ser?Ser?Thr?Pro?Ile?Lys?Gly?Pro?Lys?Arg?Gly?His?Pro

325?????????????????330?????????????????335

Arg?Gln?Asn?Leu?His?Lys?His?Phe?Asp?Ile?Asn?Glu?His?Leu?Pro?Trp

340?????????????????345?????????????????350

Met?Ile?Val?Leu?Phe?Leu?Leu?Leu?Val?Leu?Val?Val?Ile?Val?Val?Cys

355?????????????????360?????????????????365

Ser?Ile?Arg?Lys?Ser?Ser?Arg?Thr?Leu?Lys?Lys?Gly?Pro?Arg?Gln?Asp

370?????????????????375?????????????????380

Pro?Ser?Ala?Ile?Val?Glu?Lys?Ala?Gly?Leu?Lys?Lys?Ser?Met?Thr?Pro

385?????????????????390?????????????????395?????????????????400

Thr?Gln?Asn?Arg?Glu?Lys?Trp?Ile?Tyr?Tyr?Cys?Asn?Gly?His?Gly?Ile

405?????????????????410?????????????????415

Asp?Ile?Leu?Lys?Leu?Val?Ala?Ala?Gln?Val?Gly?Ser?Gln?Trp?Lys?Asp

420?????????????????425?????????????????430

Ile?Tyr?Gln?Phe?Leu?Cys?Asn?Ala?Ser?Glu?Arg?Glu?Val?Ala?Ala?Phe

435?????????????????440?????????????????445

Ser?Asn?Gly?Tyr?Thr?Ala?Asp?His?Glu?Arg?Ala?Tyr?Ala?Ala?Leu?Gln

450?????????????????455?????????????????460

His?Trp?Thr?Ile?Arg?Gly?Pro?Glu?Ala?Ser?Leu?Ala?Gln?Leu?Ile?Ser

465?????????????????470?????????????????475?????????????????480

Ala?Leu?Arg?Gln?His?Arg?Arg?Asn?Asp?Val?Val?Glu?Lys?Ile?Arg?Gly

485?????????????????490?????????????????495

Leu?Met?Glu?Asp?Thr?Thr?Gln?Leu?Glu?Thr?Asp?Lys?Leu?Ala?Leu?Pro

500?????????????????505?????????????????510

Met?Ser?Pro?Ser?Pro?Leu?Ser?Pro?Ser?Pro?Ile?Pro?Ser?Pro?Asn?Ala

515?????????????????520?????????????????525

Lys?Leu?Glu?Asn?Ser?Ala?Leu?Leu?Thr?Val?Glu?Pro?Ser?Pro?Gln?Asp

530?????????????????535?????????????????540

Lys?Asn?Lys?Gly?Phe?Phe?Val?Asp?Glu?Ser?Glu?Pro?Leu?Leu?Arg?Cys

545?????????????????550?????????????????555?????????????????560

Asp?Ser?Thr?Ser?Ser?Gly?Ser?Ser?Ala?Leu?Ser?Arg?Asn?Gly?Ser?Phe

565?????????????????570?????????????????575

Ile?Thr?Lys?Glu?Lys?Lys?Asp?Thr?Val?Leu?Arg?Gln?Val?Arg?Leu?Asp

580?????????????????585?????????????????590

Pro?Cys?Asp?Leu?Gln?Pro?Ile?Phe?Asp?Asp?Met?Leu?His?Phe?Leu?Asn

595?????????????????600?????????????????605

Pro?Glu?Glu?Leu?Arg?Val?Ile?Glu?Glu?Ile?Pro?Gln?Ala?Glu?Asp?Lys

610?????????????????615?????????????????620

Leu?Asp?Arg?Leu?Phe?Glu?Ile?Ile?Gly?Val?Lys?Ser?Gln?Glu?Ala?Ser

625?????????????????630?????????????????635?????????????????640

Gln?Thr?Leu?Leu?Asp?Ser?Val?Tyr?Ser?His?Leu?Pro?Asp?Leu?Leu

645?????????????????650?????????????????655

<210>2

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＜213〉people's " death receptor 6 "

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ctc?ccc?gcc?tgg?gcg?gcc?gcg?ccg?ctg?ggc?agg?tgc?tga?gcg?ccc?cta????258

gag?cct?ccc?ttg?ccg?cct?ccc?tcc?tct?gcc?cgg?ccg?cag?cag?tgc?aca????306

tgg?ggt?gtt?gga?ggt?aga?tgg?gct?ccc?ggc?ccg?gga?ggc?ggc?ggt?gga????354

tgc?ggc?gct?ggg?cag?aag?cag?ccg?ccg?att?cca?gct?gcc?ccg?cgc?gcc????402

ccg?ggc?gcc?cct?gcg?agt?ccc?cgg?ttc?agc?cat?ggg?gac?ctc?tcc?gag????450

cag?cag?cac?cgc?cct?cgc?ctc?ctg?cag?ccg?cat?cgc?ccg?ccg?agc?cac????498

agc?cac?gat?gat?cgc?ggg?ctc?cct?tct?cct?gct?tgg?att?cct?tag?cac????546

cac?cac?agc?tca?gcc?aga?aca?gaa?ggc?ctc?gaa?tct?cat?tgg?cac?ata????594

ccg?cca?tgt?tga?ccg?tgc?cac?cgg?cca?ggt?gct?aac?ctg?tga?caa?gtg????642

tcc?agc?agg?aac?cta?tgt?ctc?tga?gca?ttg?tac?caa?cac?aag?cct?gcg????690

cgt?ctg?cag?cag?ttg?ccc?tgt?ggg?gac?ctt?tac?cag?gca?tga?gaa?tgg????738

cat?aga?gaa?atg?cca?tga?ctg?tag?tca?gcc?atg?ccc?atg?gcc?aat?gat????786

tga?gaa?att?acc?ttg?tgc?tgc?ctt?gac?tga?ccg?aga?atg?cac?ttg?ccc????834

acc?tgg?cat?gtt?cca?gtc?taa?cgc?tac?ctg?tgc?ccc?cca?tac?ggt?gtg????882

tcc?tgt?ggg?ttg?ggg?tgt?gcg?gaa?gaa?agg?gac?aga?gac?tga?gga?tgt????930

gcg?gtg?taa?gca?gtg?tgc?tcg?ggg?tac?ctt?ctc?aga?tgt?gcc?ttc?tag????978

tgt?gat?gaa?atg?caa?agc?ata?cac?aga?ctg?tct?gag?tca?gaa?cct?ggt????1026

ggt?gat?caa?gcc?ggg?gac?caa?gga?gac?aga?caa?cgt?ctg?tgg?cac?act????1074

ccc?gtc?ctt?ctc?cag?ctc?cac?ctc?acc?ttc?ccc?tgg?cac?agc?cat?ctt????1122

tcc?acg?ccc?tga?gca?cat?gga?aac?cca?tga?agt?ccc?ttc?ctc?cac?tta????1170

tgt?tcc?caa?agg?cat?gaa?ctc?aac?aga?atc?caa?ctc?ttc?tgc?ctc?tgt????1218

tag?acc?aaa?ggt?act?gag?tag?cat?cca?gga?agg?gac?agt?ccc?tga?caa????1266

cac?aag?ctc?agc?aag?ggg?gaa?gga?aga?cgt?gaa?caa?gac?cct?ccc?aaa????1314

cct?tca?ggt?agt?caa?cca?cca?gca?agg?ccc?cca?cca?cag?aca?cat?cct????1362

gaa?gct?gct?gcc?gtc?cat?gga?ggc?cac?tgg?ggg?cga?gaa?gtc?cag?cac????1410

gcc?cat?caa?ggg?ccc?caa?gag?ggg?aca?tcc?tag?aca?gaa?cct?aca?caa????1458

gca?ttt?tga?cat?caa?tga?gca?ttt?gcc?ctg?gat?gat?tgt?gct?ttt?cct????1506

gct?gct?ggt?gct?tgt?ggt?gat?tgt?ggt?gtg?cag?tat?ccg?gaa?aag?ctc????1554

gag?gac?tct?gaa?aaa?ggg?gcc?ccg?gca?gga?tcc?cag?tgc?cat?tgt?gga????1602

aaa?ggc?agg?gct?gaa?gaa?atc?cat?gac?tcc?aac?cca?gaa?ccg?gga?gaa????1650

atg?gat?cta?cta?ctg?caa?tgg?cca?tgg?tat?cga?tat?cct?gaa?gct?tgt????1698

agc?agc?cca?agt?ggg?aag?cca?gtg?gaa?aga?tat?cta?tca?gtt?tct?ttg????1746

caa?tgc?cag?tga?gag?gga?ggt?tgc?tgc?ttt?ctc?caa?tgg?gta?cac?agc????1794

cga?cca?cga?gcg?ggc?cta?cgc?agc?tct?gca?gca?ctg?gac?cat?ccg?ggg????1842

ccc?cga?ggc?cag?cct?cgc?cca?gct?aat?tag?cgc?cct?gcg?cca?gca?ccg????1890

gag?aaa?cga?tgt?tgt?gga?gaa?gat?tcg?tgg?gct?gat?gga?aga?cac?cac????1938

cca?gct?gga?aac?tga?caa?act?agc?tct?ccc?gat?gag?ccc?cag?ccc?gct????1986

tag?ccc?gag?ccc?cat?ccc?cag?ccc?caa?cgc?gaa?act?tga?gaa?ttc?cgc????2034

tct?cct?gac?ggt?gga?gcc?ttc?ccc?aca?gga?caa?gaa?caa?ggg?ctt?ctt????2082

cgt?gga?tga?gtc?gga?gcc?cct?tct?ccg?ctg?tga?ctc?tac?atc?cag?cgg????2130

ctc?ctc?cgc?gct?gag?cag?gaa?cgg?ttc?ctt?tat?tac?caa?aga?aaa?gaa????2178

gga?cac?agt?gtt?gcg?gca?ggt?acg?cct?gga?ccc?ctg?tga?ctt?gca?gcc????2226

tat?ctt?tga?tga?cat?gct?cca?ctt?tct?aaa?tcc?tga?gga?gct?gcg?ggt????2274

gat?tga?aga?gat?tcc?cca?ggc?tga?gga?caa?act?aga?ccg?gct?att?cga????2322

aat?tat?tgg?agt?caa?gag?cca?gga?agc?cag?cca?gac?cct?cct?gga?ctc????2370

tgt?tta?tag?cca?tct?tcc?tga?cct?gct?gta?gaa?cat?agg?gat?act?gca????2418

ttc?tgg?aaa?tta?ctc?aat?tta?gtg?gca?ggg?tgg?ttt?ttt?aat?ttt?ctt????2466

ctg?ttt?ctg?att?ttt?gtt?gtt?tgg?ggt?gtg?tgt?gtg?tgt?ttg?tgt?gtg????2514

tgt?gtg?tgt?gtg?tgt?gtg?tgt?gtg?tgt?tta?aca?gag?aat?atg?gcc?agt????2562

gct?tga?gtt?ctt?tct?cct?tct?ctc?tct?ctc?ttt?ttt?ttt?taa?ata?act????2610

ctt?ctg?gga?agt?tgg?ttt?ata?agc?ctt?tgc?cag?gtg?taa?ctg?ttg?tga????2658

aat?acc?cac?cac?taa?agt?ttt?tta?agt?tcc?ata?ttt?tct?cca?ttt?tgc????2706

ctt?ctt?atg?tat?ttt?caa?gat?tat?tct?gtg?cac?ttt?aaa?ttt?act?taa????2754

ctt?acc?ata?aat?gca?gtg?tga?ctt?ttc?cca?cac?act?gga?ttg?tga?ggc????2802

tct?taa?ctt?ctt?aaa?agt?ata?atg?gca?tct?tgt?gaa?tcc?tat?aag?cag????2850

tct?tta?tgt?ctc?tta?aca?ttc?aca?cct?act?ttt?taa?aaa?caa?ata?tta????2898

tta?cta?ttt?tta?tta?ttg?ttt?gtc?ctt?tat?aaa?ttt?tct?taa?aga?tta????2946

aga?aaa?ttt?aag?acc?cca?ttg?agt?tac?tgt?aat?gca?att?caa?ctt?tga????2994

gtt?atc?ttt?taa?ata?tgt?ctt?gta?tag?ttc?ata?ttc?atg?gct?gaa?act????3042

tga?cca?cac?tat?tgc?tga?ttg?tat?ggt?ttt?cac?ctg?gac?acc?gtg?tag????3090

aat?gct?tga?tta?ctt?gta?ctc?ttc?tta?tgc?taa?tat?gct?ctg?ggc?tgg????3138

aga?aat?gaa?atc?ctc?aag?cca?tca?gga?ttt?gct?att?taa?gtg?gct?tga????3186

caa?ctg?ggc?cac?caa?aga?act?tga?act?tca?cct?ttt?agg?att?tga?gct????3234

gtt?ctg?gaa?cac?att?gct?gca?ctt?tgg?aaa?gtc?aaa?atc?aag?tgc?cag????3282

tgg?cgc?cct?ttc?cat?aga?gaa?ttt?gcc?cag?ctt?tgc?ttt?aaa?aga?tgt????3330

ctt?gtt?ttt?tat?ata?cac?ata?atc?aat?agg?tcc?aat?ctg?ctc?tca?agg????3378

cct?tgg?tcc?tgg?tgg?gat?tcc?ttc?acc?aat?tac?ttt?aat?taa?aaa?tgg????3426

ctg?caa?ctg?taa?gaa?ccc?ttg?tct?gat?ata?ttt?gca?act?atg?ctc?cca????3474

ttt?aca?aat?gta?cct?tct?aat?gct?cag?ttg?cca?ggt?tcc?aat?gca?aag????3522

gtg?gcg?tgg?act?ccc?ttt?gtg?tgg?gtg?ggg?ttt?gtg?ggt?agt?ggt?gaa????3570

gga?ccg?ata?tca?gaa?aaa?tgc?ctt?caa?gtg?tac?taa?ttt?att?aat?aaa????3618

cat?tag?gtg?ttt?gtt?aaa?aaa?aaa?aaa?aaa?aaa?aaa?aaa?aaa?aa?????????3662

<210>3

<211>300

<212>DNA

＜213〉house mouse (Mus Musculis)

<400>3

gagcagaaac?ggctccttta?ttaccaaaga?aaagaaggac?acagtgttgc?ggcaggtccg????60

cctggacccc?tgtgacttgc?agcccatctt?tgatgacatg?ctgcatatcc?tgaaccccga????120

ggagctgcgg?gtgattgaag?agattcccca?ggctgaggac?aaactggacc?gcctcttcga????180

gatcattggg?gtcaagagcc?aagaagccag?ccagaccctc?ttggactctg?tgtacagtca????240

tcttcctgac?ctattgtaga?acacaggggc?actgcattct?gggaatcaac?ctactggcgg????300

<210>4

<211>582

<212>PRT

＜213〉artificial sequence

<220>

＜223〉mosaic

<400>4

Met?Gly?Thr?Ser?Pro?Ser?Ser?Ser?Thr?Ala?Leu?Ala?Ser?Cys?Ser?Arg

1???????????????5??????????????????10??????????????????15

Ile?Ala?Arg?Arg?Ala?Thr?Ala?Thr?Met?Ile?Ala?Gly?Ser?Leu?Leu?Leu

20??????????????????25??????????????????30

Leu?Gly?Phe?Leu?Ser?Thr?Thr?Thr?Ala?Gln?Pro?Glu?Gln?Lys?Ala?Ser

35??????????????????40??????????????????45

Asn?Leu?Ile?Gly?Thr?Tyr?Arg?His?Val?Asp?Arg?Ala?Thr?Gly?Gln?Val

50??????????????????55??????????????????60

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

65??????????????????70??????????????????75??????????????????80

Thr?Asn?Thr?Ser?Leu?Arg?Val?Cys?Ser?Ser?Cys?Pro?Val?Gly?Thr?Phe

85??????????????????90??????????????????95

Thr?Arg?His?Glu?Asn?Gly?Ile?Glu?Lys?Cys?His?Asp?Cys?Ser?Gln?Pro

100?????????????????105?????????????????110

Cys?Pro?Trp?Pro?Met?Ile?Glu?Lys?Leu?Pro?Cys?Ala?Ala?Leu?Thr?Asp

115?????????????????120?????????????????125

Arg?Glu?Cys?Thr?Cys?Pro?Pro?Gly?Met?Phe?Gln?Ser?Asn?Ala?Thr?Cys

130?????????????????135?????????????????140

Ala?Pro?His?Thr?Val?Cys?Pro?Val?Gly?Trp?Gly?Val?Arg?Lys?Lys?Gly

145?????????????????150?????????????????155?????????????????160

Thr?Glu?Thr?Glu?Asp?Val?Arg?Cys?Lys?Gln?Cys?Ala?Arg?Gly?Thr?Phe

165?????????????????170?????????????????175

Ser?Asp?Val?Pro?Ser?Ser?Val?Met?Lys?Cys?Lys?Ala?Tyr?Thr?Asp?Cys

180?????????????????185?????????????????190

Leu?Ser?Gln?Asn?Leu?Val?Val?Ile?Lys?Pro?Gly?Thr?Lys?Glu?Thr?Asp

195?????????????????200?????????????????205

Asn?Val?Cys?Gly?Thr?Leu?Pro?Ser?Phe?Ser?Ser?Ser?Thr?Ser?Pro?Ser

210?????????????????215?????????????????220

Pro?Gly?Thr?Ala?Ile?Phe?Pro?Arg?Pro?Glu?His?Met?Glu?Thr?His?Glu

225?????????????????230?????????????????235?????????????????240

Val?Pro?Ser?Ser?Thr?Tyr?Val?Pro?Lys?Gly?Met?Asn?Ser?Thr?Glu?Ser

245?????????????????250?????????????????255

Asn?Ser?Ser?Ala?Ser?Val?Arg?Pro?Lys?Val?Leu?Ser?Ser?Ile?Gln?Glu

260?????????????????265?????????????????270

Gly?Thr?Val?Pro?Asp?Asn?Thr?Ser?Ser?Ala?Arg?Gly?Lys?Glu?Asp?Val

275?????????????????280?????????????????285

Asn?Lys?Thr?Leu?Pro?Asn?Leu?Gln?Val?Val?Asn?His?Gln?Gln?Gly?Pro

290?????????????????295?????????????????300

His?His?Arg?His?Ile?Leu?Lys?Leu?Leu?Pro?Ser?Met?Glu?Ala?Thr?Gly

305?????????????????310?????????????????315?????????????????320

Gly?Glu?Lys?Ser?Ser?Thr?Pro?Ile?Lys?Gly?Pro?Lys?Arg?Gly?His?Pro

325?????????????????330?????????????????335

Arg?Gln?Asn?Leu?His?Lys?His?Phe?Asp?Ile?Asn?Glu?His?Leu?Pro?Trp

340?????????????????345?????????????????350

Met?Ile?Pro?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu

355?????????????????360?????????????????365

Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp

370?????????????????375?????????????????380

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

385?????????????????390?????????????????395?????????????????400

Val?Ser?His?Glu?Asp?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly

405?????????????????410?????????????????415

Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn

420?????????????????425?????????????????430

Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp

435?????????????????440?????????????????445

Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro

450?????????????????455?????????????????460

Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu

465?????????????????470?????????????????475?????????????????480

Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Glu?Glu?Met?Thr?Lys?Asn

485?????????????????490?????????????????495

Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile

500?????????????????505?????????????????510

Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr

515?????????????????520?????????????????525

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

530?????????????????535?????????????????540

Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys

545?????????????????550?????????????????555?????????????????560

Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu

565?????????????????570?????????????????575

Ser?Leu?Ser?Pro?Gly?Lys

580

<210>5

<211>2088

<212>DNA

＜213〉amyloid precursor protein 695cDNA

<400>5

atgctgcccg?gtttggcact?gctcctgctg?gccgcctgga?cggctcgggc?gctggaggta????60

cccactgatg?gtaatgctgg?cctgctggct?gaaccccaga?ttgccatgtt?ctgtggcaga????120

ctgaacatgc?acatgaatgt?ccagaatggg?aagtgggatt?cagatccatc?agggaccaaa????180

acctgcattg?ataccaagga?aggcatcctg?cagtattgcc?aagaagtcta?ccctgaactg????240

cagatcacca?atgtggtaga?agccaaccaa?ccagtgacca?tccagaactg?gtgcaagcgg????300

ggccgcaagc?agtgcaagac?ccatccccac?tttgtgattc?cctaccgctg?cttagttggt????360

gagtttgtaa?gtgatgccct?tctcgttcct?gacaagtgca?aattcttaca?ccaggagagg????420

atggatgttt?gcgaaactca?tcttcactgg?cacaccgtcg?ccaaagagac?atgcagtgag????480

aagagtacca?acttgcatga?ctacggcatg?ttgctgccct?gcggaattga?caagttccga????540

ggggtagagt?ttgtgtgttg?cccactggct?gaagaaagtg?acaatgtgga?ttctgctgat????600

gcggaggagg?atgactcgga?tgtctggtgg?ggcggagcag?acacagacta?tgcagatggg????660

agtgaagaca?aagtagtaga?agtagcagag?gaggaagaag?tggctgaggt?ggaagaagaa????720

gaagccgatg?atgacgagga?cgatgaggat?ggtgatgagg?tagaggaaga?ggctgaggaa????780

ccctacgaag?aagccacaga?gagaaccacc?agcattgcca?ccaccaccac?caccaccaca????840

gagtctgtgg?aagaggtggt?tcgagttcct?acaacagcag?ccagtacccc?tgatgccgtt????900

gacaagtatc?tcgagacacc?tggggatgag?aatgaacatg?cccatttcca?gaaagccaaa????960

gagaggcttg?aggccaagca?ccgagagaga?atgtcccagg?tcatgagaga?atgggaagag????1020

gcagaacgtc?aagcaaagaa?cttgcctaaa?gctgataaga?aggcagttat?ccagcatttc????1080

caggagaaag?tggaatcttt?ggaacaggaa?gcagccaacg?agagacagca?gctggtggag????1140

acacacatgg?ccagagtgga?agccatgctc?aatgaccgcc?gccgcctggc?cctggagaac????1200

tacatcaccg?ctctgcaggc?tgttcctcct?cggcctcgtc?acgtgttcaa?tatgctaaag????1260

aagtatgtcc?gcgcagaaca?gaaggacaga?cagcacaccc?taaagcattt?cgagcatgtg????1320

cgcatggtgg?atcccaagaa?agccgctcag?atccggtccc?aggttatgac?acacctccgt????1380

gtgatttatg?agcgcatgaa?tcagtctctc?tccctgctct?acaacgtgcc?tgcagtggcc????1440

gaggagattc?aggatgaagt?tgatgagctg?cttcagaaag?agcaaaacta?ttcagatgac????1500

gtcttggcca?acatgattag?tgaaccaagg?atcagttacg?gaaacgatgc?tctcatgcca????1560

tctttgaccg?aaacgaaaac?caccgtggag?ctccttcccg?tgaatggaga?gttcagcctg????1620

gacgatctcc?agccgtggca?ttcttttggg?gctgactctg?tgccagccaa?cacagaaaac????1680

gaagttgagc?ctgttgatgc?ccgccctgct?gccgaccgag?gactgaccac?tcgaccaggt????1740

tctgggttga?caaatatcaa?gacggaggag?atctctgaag?tgaagatgga?tgcagaattc????1800

cgacatgact?caggatatga?agttcatcat?caaaaattgg?tgttctttgc?agaagatgtg????1860

ggttcaaaca?aaggtgcaat?cattggactc?atggtgggcg?gtgttgtcat?agcgacagtg????1920

atcgtcatca?ccttggtgat?gctgaagaag?aaacagtaca?catccattca?tcatggtgtg????1980

gtggaggttg?acgccgctgt?caccccagag?gagcgccacc?tgtccaagat?gcagcagaac????2040

ggctacgaaa?atccaaccta?caagttcttt?gagcagatgc?agaactag?????????????????2088

<210>6

<211>695

<212>PRT

＜213〉people's amyloid precursor protein APP695

<400>6

Met?Leu?Pro?Gly?Leu?Ala?Leu?Leu?Leu?Leu?Ala?Ala?Trp?Thr?Ala?Arg

1???????????????5??????????????????10??????????????????15

Ala?Leu?Glu?Val?Pro?Thr?Asp?Gly?Asn?Ala?Gly?Leu?Leu?Ala?Glu?Pro

20??????????????????25??????????????????30

Gln?Ile?Ala?Met?Phe?Cys?Gly?Arg?Leu?Asn?Met?His?Met?Asn?Val?Gln

35??????????????????40??????????????????45

Asn?Gly?Lys?Trp?Asp?Ser?Asp?Pro?Ser?Gly?Thr?Lys?Thr?Cys?Ile?Asp

50??????????????????55??????????????????60

Thr?Lys?Glu?Gly?Ile?Leu?Gln?Tyr?Cys?Gln?Glu?Val?Tyr?Pro?Glu?Leu

65??????????????????70??????????????????75??????????????????80

Gln?Ile?Thr?Asn?Val?Val?Glu?Ala?Asn?Gln?Pro?Val?Thr?Ile?Gln?Asn

85??????????????????90??????????????????95

Trp?Cys?Lys?Arg?Gly?Arg?Lys?Gln?Cys?Lys?Thr?His?Pro?His?Phe?Val

100?????????????????105?????????????????110

Ile?Pro?Tyr?Arg?Cys?Leu?Val?Gly?Glu?Phe?Val?Ser?Asp?Ala?Leu?Leu

115?????????????????120?????????????????125

Val?Pro?Asp?Lys?Cys?Lys?Phe?Leu?His?Gln?Glu?Arg?Met?Asp?Val?Cys

130?????????????????135?????????????????140

Glu?Thr?His?Leu?His?Trp?His?Thr?Val?Ala?Lys?Glu?Thr?Cys?Ser?Glu

145?????????????????150?????????????????155?????????????????160

Lys?Ser?Thr?Asn?Leu?His?Asp?Tyr?Gly?Met?Leu?Leu?Pro?Cys?Gly?Ile

165?????????????????170?????????????????175

Asp?Lys?Phe?Arg?Gly?Val?Glu?Phe?Val?Cys?Cys?Pro?Leu?Ala?Glu?Glu

180?????????????????185?????????????????190

Ser?Asp?Asn?Val?Asp?Ser?Ala?Asp?Ala?Glu?Glu?Asp?Asp?Ser?Asp?Val

195?????????????????200?????????????????205

Trp?Trp?Gly?Gly?Ala?Asp?Thr?Asp?Tyr?Ala?Asp?Gly?Ser?Glu?Asp?Lys

210?????????????????215?????????????????220

Val?Val?Glu?Val?Ala?Glu?Glu?Glu?Glu?Val?Ala?Glu?Val?Glu?Glu?Glu

225?????????????????230?????????????????235?????????????????240

Glu?Ala?Asp?Asp?Asp?Glu?Asp?Asp?Glu?Asp?Gly?Asp?Glu?Val?Glu?Glu

245?????????????????250?????????????????255

Glu?Ala?Glu?Glu?Pro?Tyr?Glu?Glu?Ala?Thr?Glu?Arg?Thr?Thr?Ser?Ile

260?????????????????265?????????????????270

Ala?Thr?Thr?Thr?Thr?Thr?Thr?Thr?Glu?Ser?Val?Glu?Glu?Val?Val?Arg

275?????????????????280?????????????????285

Val?Pro?Thr?Thr?Ala?Ala?Ser?Thr?Pro?Asp?Ala?Val?Asp?Lys?Tyr?Leu

290?????????????????295?????????????????300

Glu?Thr?Pro?Gly?Asp?Glu?Asn?Glu?His?Ala?His?Phe?Gln?Lys?Ala?Lys

305?????????????????310?????????????????315?????????????????320

Glu?Arg?Leu?Glu?Ala?Lys?His?Arg?Glu?Arg?Met?Ser?Gln?Val?Met?Arg

325?????????????????330?????????????????335

Glu?Trp?Glu?Glu?Ala?Glu?Arg?Gln?Ala?Lys?Asn?Leu?Pro?Lys?Ala?Asp

340?????????????????345?????????????????350

Lys?Lys?Ala?Val?Ile?Gln?His?Phe?Gln?Glu?Lys?Val?Glu?Ser?Leu?Glu

355?????????????????360?????????????????365

Gln?Glu?Ala?Ala?Asn?Glu?Arg?Gln?Gln?Leu?Val?Glu?Thr?His?Met?Ala

370?????????????????375?????????????????380

Arg?Val?Glu?Ala?Met?Leu?Asn?Asp?Arg?Arg?Arg?Leu?Ala?Leu?Glu?Asn

385?????????????????390?????????????????395?????????????????400

Tyr?Ile?Thr?Ala?Leu?Gln?Ala?Val?Pro?Pro?Arg?Pro?Arg?His?Val?Phe

405?????????????????410?????????????????415

Asn?Met?Leu?Lys?Lys?Tyr?Val?Arg?Ala?Glu?Gln?Lys?Asp?Arg?Gln?His

420?????????????????425?????????????????430

Thr?Leu?Lys?His?Phe?Glu?His?Val?Arg?Met?Val?Asp?Pro?Lys?Lys?Ala

435?????????????????440?????????????????445

Ala?Gln?Ile?Arg?Ser?Gln?Val?Met?Thr?His?Leu?Arg?Val?Ile?Tyr?Glu

450?????????????????455?????????????????460

Arg?Met?Asn?Gln?Ser?Leu?Ser?Leu?Leu?Tyr?Asn?Val?Pro?Ala?Val?Ala

465?????????????????470?????????????????475?????????????????480

Glu?Glu?Ile?Gln?Asp?Glu?Val?Asp?Glu?Leu?Leu?Gln?Lys?Glu?Gln?Asn

485?????????????????490?????????????????495

Tyr?Ser?Asp?Asp?Val?Leu?Ala?Asn?Met?Ile?Ser?Glu?Pro?Arg?Ile?Ser

500?????????????????505?????????????????510

Tyr?Gly?Asn?Asp?Ala?Leu?Met?Pro?Ser?Leu?Thr?Glu?Thr?Lys?Thr?Thr

515?????????????????520?????????????????525

Val?Glu?Leu?Leu?Pro?Val?Asn?Gly?Glu?Phe?Ser?Leu?Asp?Asp?Leu?Gln

530?????????????????535?????????????????540

Pro?Trp?His?Ser?Phe?Gly?Ala?Asp?Ser?Val?Pro?Ala?Asn?Thr?Glu?Asn

545?????????????????550?????????????????555?????????????????560

Glu?Val?Glu?Pro?Val?Asp?Ala?Arg?Pro?Ala?Ala?Asp?Arg?Gly?Leu?Thr

565?????????????????570?????????????????575

Thr?Arg?Pro?Gly?Ser?Gly?Leu?Thr?Asn?Ile?Lys?Thr?Glu?Glu?Ile?Ser

580?????????????????585?????????????????590

Glu?Val?Lys?Met?Asp?Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val

595?????????????????600?????????????????605

His?His?Gln?Lys?Leu?Val?Phe?Phe?Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys

610?????????????????615?????????????????620

Gly?Ala?Ile?Ile?Gly?Leu?Met?Val?Gly?Gly?Val?Val?Ile?Ala?Thr?Val

625?????????????????630?????????????????635?????????????????640

Ile?Val?Ile?Thr?Leu?Val?Met?Leu?Lys?Lys?Lys?Gln?Tyr?Thr?Ser?Ile

645?????????????????650?????????????????655

His?His?Gly?Val?Val?Glu?Val?Asp?Ala?Ala?Val?Thr?Pro?Glu?Glu?Arg

660?????????????????665?????????????????670

His?Leu?Ser?Lys?Met?Gln?Gln?Asn?Gly?Tyr?Glu?Asn?Pro?Thr?Tyr?Lys

675?????????????????680?????????????????685

Phe?Phe?Glu?Gln?Met?Gln?Asn

690?????????????????695

<210>7

<211>751

<212>PRT

＜213〉people's amyloid precursor protein (APP751)

<400>7

Met?Leu?Pro?Gly?Leu?Ala?Leu?Leu?Leu?Leu?Ala?Ala?Trp?Thr?Ala?Arg

1???????????????5??????????????????10??????????????????15

Ala?Leu?Glu?Val?Pro?Thr?Asp?Gly?Asn?Ala?Gly?Leu?Leu?Ala?Glu?Pro

20??????????????????25??????????????????30

Gln?Ile?Ala?Met?Phe?Cys?Gly?Arg?Leu?Asn?Met?His?Met?Asn?Val?Gln

35??????????????????40??????????????????45

Asn?Gly?Lys?Trp?Asp?Ser?Asp?Pro?Ser?Gly?Thr?Lys?Thr?Cys?Ile?Asp

50??????????????????55??????????????????60

Thr?Lys?Glu?Gly?Ile?Leu?Gln?Tyr?Cys?Gln?Glu?Val?Tyr?Pro?Glu?Leu

65??????????????????70??????????????????75??????????????????80

Gln?Ile?Thr?Asn?Val?Val?Glu?Ala?Asn?Gln?Pro?Val?Thr?Ile?Gln?Asn

85??????????????????90??????????????????95

Trp?Cys?Lys?Arg?Gly?Arg?Lys?Gln?Cys?Lys?Thr?His?Pro?His?Phe?Val

100?????????????????105?????????????????110

Ile?Pro?Tyr?Arg?Cys?Leu?Val?Gly?Glu?Phe?Val?Ser?Asp?Ala?Leu?Leu

115?????????????????120?????????????????125

Val?Pro?Asp?Lys?Cys?Lys?Phe?Leu?His?Gln?Glu?Arg?Met?Asp?Val?Cys

130?????????????????135?????????????????140

Glu?Thr?His?Leu?His?Trp?His?Thr?Val?Ala?Lys?Glu?Thr?Cys?Ser?Glu

145?????????????????150?????????????????155?????????????????160

Lys?Ser?Thr?Asn?Leu?His?Asp?Tyr?Gly?Met?Leu?Leu?Pro?Cys?Gly?Ile

165?????????????????170?????????????????175

Asp?Lys?Phe?Arg?Gly?Val?Glu?Phe?Val?Cys?Cys?Pro?Leu?Ala?Glu?Glu

180?????????????????185?????????????????190

Ser?Asp?Asn?Val?Asp?Ser?Ala?Asp?Ala?Glu?Glu?Asp?Asp?Ser?Asp?Val

195?????????????????200?????????????????205

Trp?Trp?Gly?Gly?Ala?Asp?Thr?Asp?Tyr?Ala?Asp?Gly?Ser?Glu?Asp?Lys

210?????????????????215?????????????????220

Val?Val?Glu?Val?Ala?Glu?Glu?Glu?Glu?Val?Ala?Glu?Val?Glu?Glu?Glu

225?????????????????230?????????????????235?????????????????240

Glu?Ala?Asp?Asp?Asp?Glu?Asp?Asp?Glu?Asp?Gly?Asp?Glu?Val?Glu?Glu

245?????????????????250?????????????????255

Glu?Ala?Glu?Glu?Pro?Tyr?Glu?Glu?Ala?Thr?Glu?Arg?Thr?Thr?Ser?Ile

260?????????????????265?????????????????270

Ala?Thr?Thr?Thr?Thr?Thr?Thr?Thr?Glu?Ser?Val?Glu?Glu?Val?Val?Arg

275?????????????????280?????????????????285

Glu?Val?Cys?Ser?Glu?Gln?Ala?Glu?Thr?Gly?Pro?Cys?Arg?Ala?Met?Ile

290?????????????????295?????????????????300

Ser?Arg?Trp?Tyr?Phe?Asp?Val?Thr?Glu?Gly?Lys?Cys?Ala?Pro?Phe?Phe

305?????????????????310?????????????????315?????????????????320

Tyr?Gly?Gly?Cys?Gly?Gly?Asn?Arg?Asn?Asn?Phe?Asp?Thr?Glu?Glu?Tyr

325?????????????????330?????????????????335

Cys?Met?Ala?Val?Cys?Gly?Ser?Ala?Ile?Pro?Thr?Thr?Ala?Ala?Ser?Thr

340?????????????????345?????????????????350

Pro?Asp?Ala?Val?Asp?Lys?Tyr?Leu?Glu?Thr?Pro?Gly?Asp?Glu?Asn?Glu

355?????????????????360?????????????????365

His?Ala?His?Phe?Gln?Lys?Ala?Lys?Glu?Arg?Leu?Glu?Ala?Lys?His?Arg

370?????????????????375?????????????????380

Glu?Arg?Met?Ser?Gln?Val?Met?Arg?Glu?Trp?Glu?Glu?Ala?Glu?Arg?Gln

385?????????????????390?????????????????395?????????????????400

Ala?Lys?Asn?Leu?Pro?Lys?Ala?Asp?Lys?Lys?Ala?Val?Ile?Gln?His?Phe

405?????????????????410?????????????????415

Gln?Glu?Lys?Val?Glu?Ser?Leu?Glu?Gln?Glu?Ala?Ala?Asn?Glu?Arg?Gln

420?????????????????425?????????????????430

Gln?Leu?Val?Glu?Thr?His?Met?Ala?Arg?Val?Glu?Ala?Met?Leu?Asn?Asp

435?????????????????440?????????????????445

Arg?Arg?Arg?Leu?Ala?Leu?Glu?Asn?Tyr?Ile?Thr?Ala?Leu?Gln?Ala?Val

450?????????????????455?????????????????460

Pro?Pro?Arg?Pro?Arg?His?Val?Phe?Asn?Met?Leu?Lys?Lys?Tyr?Val?Arg

465?????????????????470?????????????????475?????????????????480

Ala?Glu?Gln?Lys?Asp?Arg?Gln?His?Thr?Leu?Lys?His?Phe?Glu?His?Val

485?????????????????490?????????????????495

Arg?Met?Val?Asp?Pro?Lys?Lys?Ala?Ala?Gln?Ile?Arg?Ser?Gln?Val?Met

500?????????????????505?????????????????510

Thr?His?Leu?Arg?Val?Ile?Tyr?Glu?Arg?Met?Asn?Gln?Ser?Leu?Ser?Leu

515?????????????????520?????????????????525

Leu?Tyr?Asn?Val?Pro?Ala?Val?Ala?Glu?Glu?Ile?Gln?Asp?Glu?Val?Asp

530?????????????????535?????????????????540

Glu?Leu?Leu?Gln?Lys?Glu?Gln?Asn?Tyr?Ser?Asp?Asp?Val?Leu?Ala?Asn

545?????????????????550?????????????????555?????????????????560

Met?Ile?Ser?Glu?Pro?Arg?Ile?Ser?Tyr?Gly?Asn?Asp?Ala?Leu?Met?Pro

565?????????????????570?????????????????575

Ser?Leu?Thr?Glu?Thr?Lys?Thr?Thr?Val?Glu?Leu?Leu?Pro?Val?Asn?Gly

580?????????????????585?????????????????590

Glu?Phe?Ser?Leu?Asp?Asp?Leu?Gln?Pro?Trp?His?Ser?Phe?Gly?Ala?Asp

595?????????????????600?????????????????605

Ser?Val?Pro?Ala?Asn?Thr?Glu?Asn?Glu?Val?Glu?Pro?Val?Asp?Ala?Arg

610?????????????????615?????????????????620

Pro?Ala?Ala?Asp?Arg?Gly?Leu?Thr?Thr?Arg?Pro?Gly?Ser?Gly?Leu?Thr

625?????????????????630?????????????????635?????????????????640

Asn?Ile?Lys?Thr?Glu?Glu?Ile?Ser?Glu?Val?Lys?Met?Asp?Ala?Glu?Phe

645?????????????????650?????????????????655

Arg?His?Asp?Ser?Gly?Tyr?Glu?Val?His?His?Gln?Lys?Leu?Val?Phe?Phe

660?????????????????665?????????????????670

Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys?Gly?Ala?Ile?Ile?Gly?Leu?Met?Val

675?????????????????680?????????????????685

Gly?Gly?Val?Val?Ile?Ala?Thr?Val?Ile?Val?Ile?Thr?Leu?Val?Met?Leu

690?????????????????695?????????????????700

Lys?Lys?Lys?Gln?Tyr?Thr?Ser?Ile?His?His?Gly?Val?Val?Glu?Val?Asp

705?????????????????710?????????????????715?????????????????720

Ala?Ala?Val?Thr?Pro?Glu?Glu?Arg?His?Leu?Ser?Lys?Met?Gln?Gln?Asn

725?????????????????730?????????????????735

Gly?Tyr?Glu?Asn?Pro?Thr?Tyr?Lys?Phe?Phe?Glu?Gln?Met?Gln?Asn

740?????????????????745?????????????????750

<210>8

<211>2310

<212>DNA

＜213〉people's amyloid precursor protein (APP770)

<400>8

atgctgcccg?gtttggcact?gctcctgctg?gccgcctgga?cggctcgggc?gctggaggta????60

cccactgatg?gtaatgctgg?cctgctggct?gaaccccaga?ttgccatgtt?ctgtggcaga????120

ctgaacatgc?acatgaatgt?ccagaatggg?aagtgggatt?cagatccatc?agggaccaaa????180

acctgcattg?ataccaagga?aggcatcctg?cagtattgcc?aagaagtcta?ccctgaactg????240

cagatcacca?atgtggtaga?agccaaccaa?ccagtgacca?tccagaactg?gtgcaagcgg????300

ggccgcaagc?agtgcaagac?ccatccccac?tttgtgattc?cctaccgctg?cttagttggt????360

gagtttgtaa?gtgatgccct?tctcgttcct?gacaagtgca?aattcttaca?ccaggagagg????420

atggatgttt?gcgaaactca?tcttcactgg?cacaccgtcg?ccaaagagac?atgcagtgag????480

aagagtacca?acttgcatga?ctacggcatg?ttgctgccct?gcggaattga?caagttccga????540

ggggtagagt?ttgtgtgttg?cccactggct?gaagaaagtg?acaatgtgga?ttctgctgat????600

gcggaggagg?atgactcgga?tgtctggtgg?ggcggagcag?acacagacta?tgcagatggg????660

agtgaagaca?aagtagtaga?agtagcagag?gaggaagaag?tggctgaggt?ggaagaagaa????720

gaagccgatg?atgacgagga?cgatgaggat?ggtgatgagg?tagaggaaga?ggctgaggaa????780

ccctacgaag?aagccacaga?gagaaccacc?agcattgcca?ccaccaccac?caccaccaca????840

gagtctgtgg?aagaggtggt?tcgagaggtg?tgctctgaac?aagccgagac?ggggccgtgc????900

cgagcaatga?tctcccgctg?gtactttgat?gtgactgaag?ggaagtgtgc?cccattcttt????960

tacggcggat?gtggcggcaa?ccggaacaac?tttgacacag?aagagtactg?catggccgtg????1020

tgtggcagcg?ccatgtccca?aagtttactc?aagactaccc?aggaacctct?tgcccgagat????1080

cctgttaaac?ttcctacaac?agcagccagt?acccctgatg?ccgttgacaa?gtatctcgag????1140

acacctgggg?atgagaatga?acatgcccat?ttccagaaag?ccaaagagag?gcttgaggcc????1200

aagcaccgag?agagaatgtc?ccaggtcatg?agagaatggg?aagaggcaga?acgtcaagca????1260

aagaacttgc?ctaaagctga?taagaaggca?gttatccagc?atttccagga?gaaagtggaa????1320

tctttggaac?aggaagcagc?caacgagaga?cagcagctgg?tggagacaca?catggccaga????1380

gtggaagcca?tgctcaatga?ccgccgccgc?ctggccctgg?agaactacat?caccgctctg????1440

caggctgttc?ctcctcggcc?tcgtcacgtg?ttcaatatgc?taaagaagta?tgtccgcgca????1500

gaacagaagg?acagacagca?caccctaaag?catttcgagc?atgtgcgcat?ggtggatccc????1560

aagaaagccg?ctcagatccg?gtcccaggtt?atgacacacc?tccgtgtgat?ttatgagcgc????1620

atgaatcagt?ctctctccct?gctctacaac?gtgcctgcag?tggccgagga?gattcaggat????1680

gaagttgatg?agctgcttca?gaaagagcaa?aactattcag?atgacgtctt?ggccaacatg????1740

attagtgaac?caaggatcag?ttacggaaac?gatgctctca?tgccatcttt?gaccgaaacg????1800

aaaaccaccg?tggagctcct?tcccgtgaat?ggagagttca?gcctggacga?tctccagccg????1860

tggcattctt?ttggggctga?ctctgtgcca?gccaacacag?aaaacgaagt?tgagcctgtt????1920

gatgcccgcc?ctgctgccga?ccgaggactg?accactcgac?caggttctgg?gttgacaaat????1980

atcaagacgg?aggagatctc?tgaagtgaag?atggatgcag?aattccgaca?tgactcagga????2040

tatgaagttc?atcatcaaaa?attggtgttc?tttgcagaag?atgtgggttc?aaacaaaggt????2100

gcaatcattg?gactcatggt?gggcggtgtt?gtcatagcga?cagtgatcgt?catcaccttg????2160

gtgatgctga?agaagaaaca?gtacacatcc?attcatcatg?gtgtggtgga?ggttgacgcc????2220

gctgtcaccc?cagaggagcg?ccacctgtcc?aagatgcagc?agaacggcta?cgaaaatcca????2280

acctacaagt?tctttgagca?gatgcagaac?????????????????????????????????????2310

<210>9

<211>770

<212>PRT

＜213〉people's amyloid precursor protein (APP770)

<400>9

Met?Leu?Pro?Gly?Leu?Ala?Leu?Leu?Leu?Leu?Ala?Ala?Trp?Thr?Ala?Arg

1???????????????5??????????????????10??????????????????15

Ala?Leu?Glu?Val?Pro?Thr?Asp?Gly?Asn?Ala?Gly?Leu?Leu?Ala?Glu?Pro

20??????????????????25??????????????????30

Gln?Ile?Ala?Met?Phe?Cys?Gly?Arg?Leu?Asn?Met?His?Met?Asn?Val?Gln

35??????????????????40??????????????????45

Asn?Gly?Lys?Trp?Asp?Ser?Asp?Pro?Ser?Gly?Thr?Lys?Thr?Cys?Ile?Asp

50??????????????????55??????????????????60

Thr?Lys?Glu?Gly?Ile?Leu?Gln?Tyr?Cys?Gln?Glu?Val?Tyr?Pro?Glu?Leu

65??????????????????70??????????????????75??????????????????80

Gln?Ile?Thr?Asn?Val?Val?Glu?Ala?Asn?Gln?Pro?Val?Thr?Ile?Gln?Asn

85??????????????????90??????????????????95

Trp?Cys?Lys?Arg?Gly?Arg?Lys?Gln?Cys?Lys?Thr?His?Pro?His?Phe?Val

100?????????????????105?????????????????110

Ile?Pro?Tyr?Arg?Cys?Leu?Val?Gly?Glu?Phe?Val?Ser?Asp?Ala?Leu?Leu

115?????????????????120?????????????????125

Val?Pro?Asp?Lys?Cys?Lys?Phe?Leu?His?Gln?Glu?Arg?Met?Asp?Val?Cys

130?????????????????135?????????????????140

Glu?Thr?His?Leu?His?Trp?His?Thr?Val?Ala?Lys?Glu?Thr?Cys?Ser?Glu

145?????????????????150?????????????????155?????????????????160

Lys?Ser?Thr?Asn?Leu?His?Asp?Tyr?Gly?Met?Leu?Leu?Pro?Cys?Gly?Ile

165?????????????????170?????????????????175

Asp?Lys?Phe?Arg?Gly?Val?Glu?Phe?Val?Cys?Cys?Pro?Leu?Ala?Glu?Glu

180?????????????????185?????????????????190

Ser?Asp?Asn?Val?Asp?Ser?Ala?Asp?Ala?Glu?Glu?Asp?Asp?Ser?Asp?Val

195?????????????????200?????????????????205

Trp?Trp?Gly?Gly?Ala?Asp?Thr?Asp?Tyr?Ala?Asp?Gly?Ser?Glu?Asp?Lys

210?????????????????215?????????????????220

Val?Val?Glu?Val?Ala?Glu?Glu?Glu?Glu?Val?Ala?Glu?Val?Glu?Glu?Glu

225?????????????????230?????????????????235?????????????????240

Glu?Ala?Asp?Asp?Asp?Glu?Asp?Asp?Glu?Asp?Gly?Asp?Glu?Val?Glu?Glu

245?????????????????250?????????????????255

Glu?Ala?Glu?Glu?Pro?Tyr?Glu?Glu?Ala?Thr?Glu?Arg?Thr?Thr?Ser?Ile

260?????????????????265?????????????????270

Ala?Thr?Thr?Thr?Thr?Thr?Thr?Thr?Glu?Ser?Val?Glu?Glu?Val?Val?Arg

275?????????????????280?????????????????285

Glu?Val?Cys?Ser?Glu?Gln?Ala?Glu?Thr?Gly?Pro?Cys?Arg?Ala?Met?Ile

290?????????????????295?????????????????300

Ser?Arg?Trp?Tyr?Phe?Asp?Val?Thr?Glu?Gly?Lys?Cys?Ala?Pro?Phe?Phe

305?????????????????310?????????????????315?????????????????320

Tyr?Gly?Gly?Cys?Gly?Gly?Asn?Arg?Asn?Asn?Phe?Asp?Thr?Glu?Glu?Tyr

325?????????????????330?????????????????335

Cys?Met?Ala?Val?Cys?Gly?Ser?Ala?Met?Ser?Gln?Ser?Leu?Leu?Lys?Thr

340?????????????????345?????????????????350

Thr?Gln?Glu?Pro?Leu?Ala?Arg?Asp?Pro?Val?Lys?Leu?Pro?Thr?Thr?Ala

355?????????????????360?????????????????365

Ala?Ser?Thr?Pro?Asp?Ala?Val?Asp?Lys?Tyr?Leu?Glu?Thr?Pro?Gly?Asp

370?????????????????375?????????????????380

Glu?Asn?Glu?His?Ala?His?Phe?Gln?Lys?Ala?Lys?Glu?Arg?Leu?Glu?Ala

385?????????????????390?????????????????395?????????????????400

Lys?His?Arg?Glu?Arg?Met?Ser?Gln?Val?Met?Arg?Glu?Trp?Glu?Glu?Ala

405?????????????????410?????????????????415

Glu?Arg?Gln?Ala?Lys?Asn?Leu?Pro?Lys?Ala?Asp?Lys?Lys?Ala?Val?Ile

420?????????????????425?????????????????430

Gln?His?Phe?Gln?Glu?Lys?Val?Glu?Ser?Leu?Glu?Gln?Glu?Ala?Ala?Asn

435?????????????????440?????????????????445

Glu?Arg?Gln?Gln?Leu?Val?Glu?Thr?His?Met?Ala?Arg?Val?Glu?Ala?Met

450?????????????????455?????????????????460

Leu?Asn?Asp?Arg?Arg?Arg?Leu?Ala?Leu?Glu?Asn?Tyr?Ile?Thr?Ala?Leu

465?????????????????470?????????????????475?????????????????480

Gln?Ala?Val?Pro?Pro?Arg?Pro?Arg?His?Val?Phe?Asn?Met?Leu?Lys?Lys

485?????????????????490?????????????????495

Tyr?Val?Arg?Ala?Glu?Gln?Lys?Asp?Arg?Gln?His?Thr?Leu?Lys?His?Phe

500?????????????????505?????????????????510

Glu?His?Val?Arg?Met?Val?Asp?Pro?Lys?Lys?Ala?Ala?Gln?Ile?Arg?Ser

515?????????????????520?????????????????525

Gln?Val?Met?Thr?His?Leu?Arg?Val?Ile?Tyr?Glu?Arg?Met?Asn?Gln?Ser

530?????????????????535?????????????????540

Leu?Ser?Leu?Leu?Tyr?Asn?Val?Pro?Ala?Val?Ala?Glu?Glu?Ile?Gln?Asp

545?????????????????550?????????????????555?????????????????560

Glu?Val?Asp?Glu?Leu?Leu?Gln?Lys?Glu?Gln?Asn?Tyr?Ser?Asp?Asp?Val

565?????????????????570?????????????????575

Leu?Ala?Asn?Met?Ile?Ser?Glu?Pro?Arg?Ile?Ser?Tyr?Gly?Asn?Asp?Ala

580?????????????????585?????????????????590

Leu?Met?Pro?Ser?Leu?Thr?Glu?Thr?Lys?Thr?Thr?Val?Glu?Leu?Leu?Pro

595?????????????????600?????????????????605

Val?Asn?Gly?Glu?Phe?Ser?Leu?Asp?Asp?Leu?Gln?Pro?Trp?His?Ser?Phe

610?????????????????615?????????????????620

Gly?Ala?Asp?Ser?Val?Pro?Ala?Asn?Thr?Glu?Asn?Glu?Val?Glu?Pro?Val

625?????????????????630?????????????????635?????????????????640

Asp?Ala?Arg?Pro?Ala?Ala?Asp?Arg?Gly?Leu?Thr?Thr?Arg?Pro?Gly?Ser

645?????????????????650?????????????????655

Gly?Leu?Thr?Asn?Ile?Lys?Thr?Glu?Glu?Ile?Ser?Glu?Val?Lys?Met?Asp

660?????????????????665?????????????????670

Ala?Glu?Phe?Arg?His?Asp?Ser?Gly?Tyr?Glu?Val?His?His?Gln?Lys?Leu

675?????????????????680?????????????????685

Val?Phe?Phe?Ala?Glu?Asp?Val?Gly?Ser?Asn?Lys?Gly?Ala?Ile?Ile?Gly

690?????????????????695?????????????????700

Leu?Met?Val?Gly?Gly?Val?Val?Ile?Ala?Thr?Val?Ile?Val?Ile?Thr?Leu

705?????????????????710?????????????????715?????????????????720

Val?Met?Leu?Lys?Lys?Lys?Gln?Tyr?Thr?Ser?Ile?His?His?Gly?Val?Val

725?????????????????730?????????????????735

Glu?Val?Asp?Ala?Ala?Val?Thr?Pro?Glu?Glu?Arg?His?Leu?Ser?Lys?Met

740?????????????????745?????????????????750

Gln?Gln?Asn?Gly?Tyr?Glu?Asn?Pro?Thr?Tyr?Lys?Phe?Phe?Glu?Gln?Met

755?????????????????760?????????????????765

Gln?Asn

770

<210>10

<211>210

<212>PRT

＜213〉artificial sequence

<220>

＜223〉people

<400>10

Met?Leu?Pro?Gly?Leu?Ala?Leu?Leu?Leu?Leu?Ala?Ala?Trp?Thr?Ala?Arg

1???????????????5??????????????????10??????????????????15

Ala?Leu?Glu?Val?Pro?Thr?Asp?Gly?Asn?Ala?Gly?Leu?Leu?Ala?Glu?Pro

20??????????????????25??????????????????30

Gln?Ile?Ala?Met?Phe?Cys?Gly?Arg?Leu?Asn?Met?His?Met?Asn?Val?Gln

35??????????????????40??????????????????45

Asn?Gly?Lys?Trp?Asp?Ser?Asp?Pro?Ser?Gly?Thr?Lys?Thr?Cys?Ile?Asp

50??????????????????55??????????????????60

Thr?Lys?Glu?Gly?Ile?Leu?Gln?Tyr?Cys?Gln?Glu?Val?Tyr?Pro?Glu?Leu

65??????????????????70??????????????????75??????????????????80

Gln?Ile?Thr?Asn?Val?Val?Glu?Ala?Asn?Gln?Pro?Val?Thr?Ile?Gln?Asn

85??????????????????90??????????????????95

Trp?Cys?Lys?Arg?Gly?Arg?Lys?Gln?Cys?Lys?Thr?His?Pro?His?Phe?Val

100?????????????????105?????????????????110

Ile?Pro?Tyr?Arg?Cys?Leu?Val?Gly?Glu?Phe?Val?Ser?Asp?Ala?Leu?Leu

115?????????????????120?????????????????125

Val?Pro?Asp?Lys?Cys?Lys?Phe?Leu?His?Gln?Glu?Arg?Met?Asp?Val?Cys

130?????????????????135?????????????????140

Glu?Thr?His?Leu?His?Trp?His?Thr?Val?Ala?Lys?Glu?Thr?Cys?Ser?Glu

145?????????????????150?????????????????155?????????????????160

Lys?Ser?Thr?Asn?Leu?His?Asp?Tyr?Gly?Met?Leu?Leu?Pro?Cys?Gly?Ile

165?????????????????170?????????????????175

Asp?Lys?Phe?Arg?Gly?Val?Glu?Phe?Val?Cys?Cys?Pro?Leu?Ala?Glu?Glu

180?????????????????185?????????????????190

Ser?Asp?Asn?Val?Asp?Ser?Ala?Asp?Ala?Glu?Glu?Asp?His?His?His?His

195?????????????????200?????????????????205

His?His

210

<210>11

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<223>SiRNA

<400>11

aaucuguuga?guucaugccu?u?????????????????????21

<210>12

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Rattus norvegicus (Rattus norvegicus)

<400>12

caauagguca?ggaagauggc?u?????????????????????21

<210>13

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉Rattus norvegicus

<400>13

ggactctgtg?tacagtcacc?tcccagatct?gttatag????37

<210>14

<211>349

<212>PRT

＜213〉house mouse

<400>14

Met?Gly?Thr?Arg?Ala?Ser?Ser?Ile?Thr?Ala?Leu?Ala?Ser?Cys?Ser?Arg

1???????????????5??????????????????10??????????????????15

Thr?Ala?Gly?Gln?Val?Gly?Ala?Thr?Met?Val?Ala?Gly?Ser?Leu?Leu?Leu

20??????????????????25??????????????????30

Leu?Gly?Phe?Leu?Ser?Thr?Ile?Thr?Ala?Gln?Pro?Glu?Gln?Lys?Thr?Leu

35??????????????????40??????????????????45

Ser?Leu?Pro?Gly?Thr?Tyr?Arg?His?Val?Asp?Arg?Thr?Thr?Gly?Gln?Val

50??????????????????55??????????????????60

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

65??????????????????70??????????????????75??????????????????80

Thr?Asn?Met?Ser?Leu?Arg?Val?Cys?Ser?Ser?Cys?Pro?Ala?Gly?Thr?Phe

85??????????????????90??????????????????95

Thr?Arg?His?Glu?Asn?Gly?Ile?Glu?Arg?Cys?His?Asp?Cys?Ser?Gln?Pro

100?????????????????105?????????????????110

Cys?Pro?Trp?Pro?Met?Ile?Glu?Arg?Leu?Pro?Cys?Ala?Ala?Leu?Thr?Asp

115?????????????????120?????????????????125

Arg?Glu?Cys?Ile?Cys?Pro?Pro?Gly?Met?Tyr?Gln?Ser?Asn?Gly?Thr?Cys

130?????????????????135?????????????????140

Ala?Pro?His?Thr?Val?Cys?Pro?Val?Gly?Trp?Gly?Val?Arg?Lys?Lys?Gly

145?????????????????150?????????????????155?????????????????160

Thr?Glu?Asn?Glu?Asp?Val?Arg?Cys?Lys?Gln?Cys?Ala?Arg?Gly?Thr?Phe

165?????????????????170?????????????????175

Ser?Asp?Val?Pro?Ser?Ser?Val?Met?Lys?Cys?Lys?Ala?His?Thr?Asp?Cys

180?????????????????185?????????????????190

Leu?Gly?Gln?Asn?Leu?Glu?Val?Val?Lys?Pro?Gly?Thr?Lys?Glu?Thr?Asp

195?????????????????200?????????????????205

Asn?Val?Cys?Gly?Met?Arg?Leu?Phe?Phe?Ser?Ser?Thr?Asn?Pro?Pro?Ser

210?????????????????215?????????????????220

Ser?Gly?Thr?Val?Thr?Phe?Ser?His?Pro?Glu?His?Met?Glu?Ser?His?Asp

225?????????????????230?????????????????235?????????????????240

Val?Pro?Ser?Ser?Thr?Tyr?Glu?Pro?Gln?Gly?Met?Asn?Ser?Thr?Asp?Ser

245?????????????????250?????????????????255

Asn?Ser?Thr?Ala?Ser?Val?Arg?Thr?Lys?Val?Pro?Ser?Gly?Ile?Glu?Glu

260?????????????????265?????????????????270

Gly?Thr?Val?Pro?Asp?Asn?Thr?Ser?Ser?Thr?Ser?Gly?Lys?Glu?Gly?Thr

275?????????????????280?????????????????285

Asn?Arg?Thr?Leu?Pro?Asn?Pro?Pro?Gln?Val?Thr?His?Gln?Gln?Ala?Pro

290?????????????????295?????????????????300

His?His?Arg?His?Ile?Leu?Lys?Leu?Leu?Pro?Ser?Ser?Met?Glu?Ala?Thr

305?????????????????310?????????????????315?????????????????320

Gly?Glu?Lys?Ser?Ser?Thr?Ala?Ile?Lys?Ala?Pro?Lys?Arg?Gly?His?Pro

325?????????????????330?????????????????335

Arg?Gln?Asn?Ala?His?Lys?His?Phe?Asp?Ile?Asn?Glu?His

340?????????????????345

<210>15

<211>354

<212>PRT

＜213〉artificial sequence

<220>

＜223〉house mouse

<400>15

Met?Gly?Thr?Arg?Ala?Ser?Ser?Ile?Thr?Ala?Leu?Ala?Ser?Cys?Ser?Arg

1???????????????5??????????????????10??????????????????15

Thr?Ala?Gly?Gln?Val?Gly?Ala?Thr?Met?Val?Ala?Gly?Ser?Leu?Leu?Leu

20??????????????????25??????????????????30

Leu?Gly?Phe?Leu?Ser?Thr?Ile?Thr?Ala?Gln?Pro?Glu?Gln?Lys?Thr?Leu

35??????????????????40??????????????????45

Ser?Leu?Pro?Gly?Thr?Tyr?Arg?His?Val?Asp?Arg?Thr?Thr?Gly?Gln?Val

50??????????????????55??????????????????60

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

65??????????????????70??????????????????75??????????????????80

Thr?Asn?Met?Ser?Leu?Arg?Val?Cys?Ser?Ser?Cys?Pro?Ala?Gly?Thr?Phe

85??????????????????90??????????????????95

Thr?Arg?His?Glu?Asn?Gly?Ile?Glu?Arg?Cys?His?Asp?Cys?Ser?Gln?Pro

100?????????????????105?????????????????110

Cys?Pro?Trp?Pro?Met?Ile?Glu?Arg?Leu?Pro?Cys?Ala?Ala?Leu?Thr?Asp

115?????????????????120?????????????????125

Arg?Glu?Cys?Ile?Cys?Pro?Pro?Gly?Met?Tyr?Gln?Ser?Asn?Gly?Thr?Cys

130?????????????????135?????????????????140

Ala?Pro?His?Thr?Val?Cys?Pro?Val?Gly?Trp?Gly?Val?Arg?Lys?Lys?Gly

145?????????????????150?????????????????155?????????????????160

Thr?Glu?Asn?Glu?Asp?Val?Arg?Cys?Lys?Gln?Cys?Ala?Arg?Gly?Thr?Phe

165?????????????????170?????????????????175

Ser?Asp?Val?Pro?Ser?Ser?Val?Met?Lys?Cys?Lys?Ala?His?Thr?Asp?Cys

180?????????????????185?????????????????190

Leu?Gly?Gln?Asn?Leu?Glu?Val?Val?Lys?Pro?Gly?Thr?Lys?Glu?Thr?Asp

195?????????????????200?????????????????205

Asn?Val?Cys?Gly?Met?Arg?Leu?Phe?Phe?Ser?Ser?Thr?Asn?Pro?Pro?Ser

210?????????????????215?????????????????220

Ser?Gly?Thr?Val?Thr?Phe?Ser?His?Pro?Glu?His?Met?Glu?Ser?His?Asp

225?????????????????230?????????????????235?????????????????240

Val?Pro?Ser?Ser?Thr?Tyr?Glu?Pro?Gln?Gly?Met?Asn?Ser?Thr?Asp?Ser

245?????????????????250?????????????????255

Asn?Ser?Thr?Ala?Ser?Val?Arg?Thr?Lys?Val?Pro?Ser?Gly?Ile?Glu?Glu

260?????????????????265?????????????????270

Gly?Thr?Val?Pro?Asp?Asn?Thr?Ser?Ser?Thr?Ser?Gly?Lys?Glu?Gly?Thr

275?????????????????280?????????????????285

Asn?Arg?Thr?Leu?Pro?Asn?Pro?Pro?Gln?Val?Thr?His?Gln?Gln?Ala?Pro

290?????????????????295?????????????????300

His?His?Arg?His?Ile?Leu?Lys?Leu?Leu?Pro?Ser?Ser?Met?Glu?Ala?Thr

305?????????????????310?????????????????315?????????????????320

Gly?Glu?Lys?Ser?Ser?Thr?Ala?Ile?Lys?Ala?Pro?Lys?Arg?Gly?His?Pro

325?????????????????330?????????????????335

Arg?Gln?Asn?Ala?His?Lys?His?Phe?Asp?Ile?Asn?Glu?His?His?His?His

340?????????????????345?????????????????350

His?His

Claims (65)

1. method that suppresses death receptor 6 (DR6) in conjunction with amyloid precursor protein (APP) is included under the condition that DR6 is suppressed the combination of APP DR6 polypeptide and/or APP polypeptide is exposed to one or more DR6 antagonists.

2. the process of claim 1 wherein that described one or more DR6 antagonists are selected from antibody in conjunction with DR6, comprise the solubility DR6 polypeptide of SEQ ID NO:1 amino acid/11-354 and in conjunction with the antibody of APP.

3. the method for claim 2, wherein said solubility DR6 polypeptide comprises the DR6 immunoadhesin.

4. the method for claim 3, wherein said solubility DR6 polypeptide comprises the DR6 ectodomain sequence that merges to immunoglobulin fc region.

5. the method for claim 2, wherein said antibodies in conjunction with DR6 comprises the DR6 polypeptide of Fig. 1 (SEQ ID NO:1) amino acid/11-349 or 42-349.

6. the method for claim 2, wherein said antibody in conjunction with DR6 is chimeric antibody, humanized antibody or people's antibody.

7. the method for claim 2, wherein said antibody competition inhibition in conjunction with DR6 is by the 3F4.4.8, the 4B6.9.7 that generate with the hybridoma cell line of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively or the combination of 1E5.5.7 monoclonal antibody.

8. the method for claim 2, wherein said antibody or solubility DR6 polypeptide in conjunction with DR6 is to be connected to one or more nonprotein character polymkeric substance that are selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

9. the process of claim 1 wherein that described antibody in conjunction with APP is monoclonal antibody.

10. the method for claim 9, wherein said monoclonal antibody in conjunction with APP is chimeric antibody, humanized antibody or people's antibody.

11. the method for claim 9 is wherein said in conjunction with monoclonal antibody competitive inhibition 3F4.4.8, the 4B6.9.7 of APP or the combination of 1E5.5.7 antibody.

12. the method for claim 9, wherein said antibody in conjunction with APP are to be connected to one or more polymkeric substance that are selected from down the nonprotein character of organizing: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

13. the process of claim 1 wherein that described DR6 polypeptide is conducting in conjunction with suppressing DR6 activation or signal of one or more DR6 antagonists that express and described on the cell surface of one or more mammalian cells.

14. the method for claim 13, wherein this method is in external enforcement, in order to suppress the apoptosis in one or more mammalian cells of expressing DR6.

15. the method for claim 13, wherein this method is implemented in vivo, in order to suppress the apoptosis in one or more mammalian cells of expressing DR6.

16. it is commissural neuron cell, Sensory neurone cell or motor neuron cell that the method for claim 13, wherein said one or more are expressed at least a in the mammalian cell of DR6 polypeptide on cell surface.

17. the method for claim 13, wherein this method is to implement in the Mammals with neuroscience illness or illness in vivo.

18. the method for claim 17, wherein said neuroscience illness or illness are amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease or Alzheimer's.

19. the method for claim 17, wherein said neuroscience illness or illness comprise neuronal cell or tissue injury, and it is derived from apoplexy, to the wound of brain or myeloid tissue or the infringement in the neuronal tissue.

20. the process of claim 1 wherein the combination of at least a inhibition DR6 in described one or more DR6 antagonists to the APP polypeptide that comprises SEQ ID NO:6 amino acid 66-81.

21. the process of claim 1 wherein the combination of at least a inhibition APP in described one or more DR6 antagonists to the DR6 polypeptide that comprises SEQ ID NO:1 amino acid/11-655.

22. a treatment suffers from the mammiferous method of neuroscience illness or illness, comprises one or more DR6 antagonists to described administration significant quantity.

23. the method for claim 22, wherein said one or more DR6 antagonists be selected from antibody in conjunction with DR6, comprise the solubility DR6 polypeptide of SEQ ID NO:1 amino acid/11-354 and in conjunction with the antibody of APP.

24. the method for claim 23, wherein said solubility DR6 polypeptide comprises the DR6 immunoadhesin.

25. the method for claim 23, wherein said solubility DR6 polypeptide comprise the DR6 ectodomain sequence that merges to immunoglobulin fc region.

26. the method for claim 23, wherein said antibodies in conjunction with DR6 comprise the DR6 polypeptide of Fig. 1 (SEQ IDNO:1) amino acid/11-349 or 42-349.

27. the method for claim 23, wherein said antibody in conjunction with DR6 are chimeric antibody, humanized antibody or people's antibody.

28. the method for claim 23, wherein said antibody competition inhibition in conjunction with DR6 is by the 3F4.4.8, the 4B6.9.7 that generate with the hybridoma cell line of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively or the combination of 1E5.5.7 monoclonal antibody.

29. the method for claim 23, wherein said antibody or solubility DR6 polypeptide in conjunction with DR6 is to be connected to one or more nonprotein character polymkeric substance that are selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

30. the method for claim 22, wherein said antibody in conjunction with APP is monoclonal antibody.

31. the method for claim 30, wherein said monoclonal antibody in conjunction with APP are chimeric antibody, humanized antibody or people's antibody.

32. the method for claim 30, the combination of wherein said monoclonal antibody competitive inhibition monoclonal antibody 22C11 in conjunction with APP.

33. the method for claim 30, wherein said monoclonal antibody in conjunction with APP are to be connected to one or more polymkeric substance that are selected from down the nonprotein character of organizing: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

34. the method for claim 22, at least a inhibition DR6 is to the combination of the APP polypeptide that comprises SEQ ID NO:6 amino acid 66-81 in wherein said one or more DR6 antagonists.

35. the method for claim 22, at least a inhibition APP is to the combination of the DR6 polypeptide that comprises SEQ ID NO:1 amino acid/11-655 in wherein said one or more DR6 antagonists.

36. the method for claim 22, wherein said neuroscience illness or illness are amyotrophic lateral sclerosis, Parkinson's disease, Huntington disease or Alzheimer's.

37. the method for claim 22, wherein said neuroscience illness or illness comprise neuronal cell or tissue injury, and it is derived from apoplexy, to the wound of brain or myeloid tissue or the infringement in the neuronal tissue.

38. the method for claim 22 is wherein to one or more other therapeutical agents of described administration.

39. the method for claim 22, wherein said one or more DR6 antagonists through injection, infusion or perfusion to described administration.

40. the method for claim 38, wherein said one or more other therapeutical agents are selected from NGF, inhibitors of apoptosis, EGFR inhibitor, beta-secretase inhibitor, inhibitors of gamma-secretase, anticholinesterase, anti-amyloid beta antibodies and nmda receptor antagonist.

41. identify for one kind and suppress the method for DR6 to the bonded molecules of interest of APP, this method comprises:

Combination DR6 and APP in the situation that has or do not exist molecules of interest; And

Be determined in the situation that has described molecules of interest the inhibition of DR6 in conjunction with APP.

42. the method for claim 41, wherein said molecules of interest be in conjunction with APP antibody, in conjunction with the antibody of DR6 or comprise the solubility DR6 polypeptide of SEQ ID NO:1 amino acid/11-354.

43. the method for claim 41 is wherein for for DR6 the detection of the bonded inhibition of APP being implemented in the cell-less measurement method in having the situation of molecules of interest.

44. the method for claim 41 further comprises:

Use is expressed the mammalian cell of DR6 and is implemented described method on cell surface; And detection is to the inhibition of DR6 activation or signal conduction.

45. a composition, it comprises the molecules of interest of identifying according to the method for claim 40.

46. the composition of claim 45 and carrier.

47. the composition of claim 46, wherein said carrier is a pharmaceutical acceptable carrier.

48. isolating DR6 antagonist, comprise that (a) is in conjunction with the monoclonal antibody of the DR6 polypeptide that comprises SEQ ID NO:1 or (b) solubility DR6 polypeptide or (c) in conjunction with comprising the monoclonal antibody of the APP of SEQ ID NO:6, wherein said DR6 antagonist suppresses the combination of APP to DR6.

49. the isolating DR6 antagonist of claim 48, wherein said solubility DR6 polypeptide comprises the DR6 immunoadhesin.

50. the isolating DR6 antagonist of claim 49, described solubility DR6 polypeptide comprise the DR6 ectodomain sequence that merges to immunoglobulin fc region.

51. the isolating DR6 antagonist of claim 48, wherein said antibodies in conjunction with DR6 comprise the DR6 polypeptide of Fig. 1 (SEQ ID NO:1) amino acid/11-349 or 42-349.

52. the isolating DR6 antagonist of claim 48, wherein said antibody in conjunction with DR6 are chimeric antibody, humanized antibody or people's antibody.

53. the isolating DR6 antagonist of claim 48, wherein said antibody competition inhibition in conjunction with DR6 is by the 3F4.4.8, the 4B6.9.7 that generate with the hybridoma cell line of ATCC numbering PTA-8095, PTA-8094 or PTA-8096 preservation respectively or the combination of 1E5.5.7 monoclonal antibody.

54. the isolating DR6 antagonist of claim 48, wherein said antibody or solubility DR6 polypeptide in conjunction with DR6 is to be connected to one or more nonprotein character polymkeric substance that are selected from down group: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

55. the isolating DR6 antagonist of claim 48, wherein said DR6 antagonist suppress the combination of DR6 to the APP polypeptide that comprises SEQ ID NO:6 amino acid 66-81.

56. the isolating DR6 antagonist of claim 48, wherein said antagonist is in conjunction with suppressing the bonded epi-position of DR6 to APP by steric hindrance.

57. the isolating DR6 antagonist of claim 48, wherein said monoclonal antibody in conjunction with APP are chimeric antibody, humanized antibody or people's antibody.

58. the isolating DR6 antagonist of claim 48, the combination of wherein said antibody competition inhibition 22C11 monoclonal antibody in conjunction with APP.

59. the isolating DR6 antagonist of claim 48, wherein said antibody in conjunction with APP are to be connected to one or more polymkeric substance that are selected from down the nonprotein character of organizing: polyoxyethylene glycol, polypropylene glycol and polyoxyalkylene.

60. the isolating DR6 antagonist of claim 48, wherein said antagonist suppress the combination of DR6 to the APP polypeptide that comprises SEQID NO:6 amino acid 66-81.

61. a pharmaceutical composition, it comprises DR6 antagonist and the pharmaceutical acceptable carrier of claim 47-60.

62. a diagnosis suffer from the neuroscience illness or to the patient's of neuroscience illness susceptible method, comprise from described patient and obtain sample and described sample test is had the existence of the DR6 polypeptide variants of the peptide sequence different with the DR6 peptide sequence of SEQ ID NO:1.

63. the method for claim 62 further comprises and identifies whether described polypeptide variants has and the avidity to APP polypeptide different to the observed avidity of DR6 peptide sequence of SEQ IDNO:1.

64. goods comprise:

(a) comprise the composition of DR6 antagonist of the claim 47-60 of significant quantity;

(b) container of described composition is housed; With

(c) affix to the label of described container or be included in package insert in the described container, it provides the explanation about the purposes of described DR6 antagonist in neuroscience illness or treatment of conditions.

65. a test kit comprises:

Label on first container, the described container and be contained in composition in the described container, wherein said composition comprises the promoting agent of the apoptosis in the mammalian nervous unit cell of at least a type of effective inhibition, described label on the described container or be included in package insert in the described container and indicate described composition to can be used for suppressing apoptosis in the mammalian nervous unit cell of at least a type, and the described promoting agent in the described composition comprises at least a DR6 antagonist of claim 47-60;

Pharmacy is housed accepts second container of buffer reagent; With

About using described DR6 antagonist to suppress the explanation of the apoptosis in the mammalian nervous unit cell of at least a type.

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